Publications

Macrophages in the meninges contribute to immune defense of the central nervous system (CNS), yet their site-specific origin and function remain poorly understood. Using an intravenous model of streptococcal meningoencephalitis in mice, we found bacteria predominantly in the leptomeninges and dura. Nevertheless, monocyte infiltration into the leptomeninges and parenchyma strongly correlated with disease severity. In the dura, infection triggered activation and loss of resident macrophages, followed by rapid engraftment of inflammatory monocytes that transiently replenished the macrophage niche. Under homeostasis, dural monocytes were supplied CCR2 independently from adjacent skull bone marrow. During infection, this local source was insufficient, necessitating recruitment from peripheral bone marrow. Infection further reshaped monocyte ontogeny, increasing monocyte?dendritic cell progenitor?derived monocytes, which expressed higher major histocompatibility complex class II levels and persisted in the brain alongside CD4+ T cells during resolution. Together, these findings reveal dynamic, compartment-specific remodeling of monocyte recruitment and differentiation across CNS borders during bacterial meningoencephalitis. Streptococcal meningoencephalitis disrupts homeostatic, skull bone marrow?derived monocyte and macrophage trajectories in the dura. Meningeal macrophages contribute to central nervous system (CNS) homeostasis and immune responses, but how infection shapes their development and function remains unclear. Using a mouse model of streptococcal meningoencephalitis, Gres et al. found that bacterial infection induced activation and loss of resident dural macrophages. During infection, macrophage replenishment shifted toward monocytes derived from monocyte?dendritic cell progenitors (MDPs), which had greater capacity to activate T cells. Bacterial infection also depleted myeloid progenitors from local reservoirs in the skull bone marrow, necessitating monocyte recruitment from peripheral sources. These findings demonstrate that bacterial meningoencephalitis reshapes monocyte recruitment and macrophage ontogeny in the CNS borders. ?Claire Olingy

Brain metastases (BrM) are a leading cause of morbidity and mortality, arising in multiple brain compartments. BrM colonization and progression are shaped by interactions with distinct tumor-associated macrophage (TAM) subsets, including microglia (MG), monocyte-derived macrophages (MDM), and border-associated macrophages (BAM). While transcriptomes of TAM have been characterized in detail, their spatial distribution, abundance, and compartment-specific composition -particularly in relation to BrM size and the cancer originremain poorly defined. Here, we performed a comprehensive spatial analysis of TAM subtypes across brain regions using experimental BrM models of lung and breast cancer, as well as melanoma. We distinguished TAM subsets by both origin and location, employing genetically traceable mouse models. We observed expansion of MG and BAM, as well as MDM infiltration associated with BrM, albeit with distinct compositions. Parenchymal BrM contained both MG and MDM, whereas ventricular and leptomeningeal BrM contained BAM and MDM but lacked MG. TAM abundance varied with BrM size, compartment, and cancer type: MG predominated in early parenchymal lesions, with MDM becoming dominant as tumors grew. Notably, melanoma BrM exhibited markedly reduced MDM infiltration compared with lung and breast cancer BrM. These findings highlight the need to tailor TAM-targeted therapies not only to the primary tumor type but also to the brain compartment affected. A deeper understanding of TAM dynamics across compartments may improve the precision and efficacy of BrM treatments. (Figure presented.)

The molecular details of macrophage-fibroblast crosstalk during the onset and resolution of inflammatory disease remain incompletely understood. Here, we apply a bioinformatic modeling approach based on single-cell RNA sequencing and single-cell assay for transposase-accessible chromatin using sequencing to map heterocellular signaling circuits of synovial macrophage and synovial fibroblast (SF) subsets during various stages of inflammatory arthritis. While SFs function as key pacemakers of synovial inflammation, individual subsets of synovial macrophages support both the perpetuation and the resolution of arthritis. Pro-inflammatory Il1b+ macrophages dominate the early stages of inflammation and retain a substantial intrinsic plasticity that is characterized by chromatin remodeling and an eventual differentiation into Spp1+ macrophages. These cells display a terminally differentiated phenotype, suppress the activation of pro-inflammatory SFs, and initiate the resolution of arthritis by secretion of regulatory mediators, including osteopontin. Our data highlight the dichotomous character of macrophage-fibroblast crosstalk and define the cellular and molecular checkpoints that control the onset and resolution of immune-mediated inflammatory diseases.

The array of tissue macrophages characterized is ever-expanding, underscoring the importance of these cells in physiology and pathology. A population of specialized gland-associated macrophages termed adenophages has now been discovered that support efficient saliva secretion along with possible other immunological functions.

Macrophages are critical players in physiology and pathophysiology, and an in-depth understanding of their diverse origins and specific functions holds promise for therapeutic intervention. Mammalian macrophages can arise from consecutive waves of hematopoietic development. Two of these pathways that commence in extra-embryonic tissue remain confined to the fetus and cease before birth, although some of their macrophage products persist throughout life. The third wave of macrophages derives from hematopoietic stem cells (HSC) that arise in the embryo proper and sustain lifelong myeloid hematopoiesis. These latter cells rely on blood monocytes for dissemination and progressively replace existing macrophage compartments to a varying extent. In this chapter, we discuss the current understanding of macrophage ontogeny and its physiological impact and promise for therapeutic strategies.

The timing of endotoxin administration in mice matters and is associated with diurnal variation in survival; however, underlying mechanisms remain poorly understood. Here, we report that afternoon LPS challenges in mice induce a robust inflammatory response involving increased neutrophil activation and release of cytotoxic mediators, causing higher mortality compared with challenges at midnight. Mechanistically, the cyclic patterns of corticosterone and melatonin hormones differentially modulate neutrophil responses. The afternoon corticosterone peak was associated with heightened incidence and severity of LPS-induced hyperinflammation. Conversely, higher melatonin levels at midnight conferred protection to challenged mice by restraining the magnitude of inflammation. High cortisol and low melatonin profiles detected in septic patients mirror those observed in mice and suggest a novel prognostic marker for sepsis. Our study unveils a regulatory network that links light/dark signals and circadian-regulated hormones to the intensity of the host's inflammatory response to infection.

Summary Microglia are parenchymal brain macrophages that are established during embryogenesis and form a self-containing cellular compartment that resists seeding with cells derived from adult definitive hematopoiesis. We report that monocyte-derived macrophages (MoMΦs) accumulate in the brain of aging mice with distinct topologies, including the nigrostriatum and medulla but not the frontal cortex. Parenchymal MoMΦs adopt bona fide microglia morphology and expression profiles. Due to their hematopoietic stem cell (HSC) derivation, monocyte-derived microglia (MoMg) are unlike yolk-sac-derived cells, targets of clonal hematopoiesis (CH). Indeed, using a chimeric transfer model, we show that the hematopoietic expression of DNMT3AR882H, a prominent human CH variant, renders MoMg pathogenic and promotes motor deficits resembling atypical Parkinsonian disorders. Collectively, we establish that MoMg progressively seed the brain of healthy aging mice, accumulate in selected areas, and, when carrying a somatic mutation associated with CH, can cause brain pathology.

Concussions can cause debilitating symptoms despite no evidence of structural changes on diagnostic imaging. The cellular events occurring in the brain parenchyma following concussion, especially repetitive concussion, are not well elucidated. We developed a concussion model to induce a confined area of injury without causing frank hemorrhage. Using intravital microscopy, we observe activation of the vasculature that supported neutrophil rolling and platelet adhesion but no overt cellular recruitment from blood into brain parenchyma. Activated resident, not monocyte-derived, macrophages relocated to the injury site via Cx3cr1 and phagocytosed dysfunctional/ detached astrocytes via scavenger receptors and TLR4, particularly after repetitive concussion. Additionally, microglia sealed areas of bloodbrain barrier (BBB) disruption via purinergic pathways. Using a splitCre approach to dissect microglia and perivascular macrophages, we show that microglial invasion into the injury site is key to reducing BBB disruption. Our data suggest that microglia repair the BBB following concussion, but in doing so significantly alter the cellular ultrastructure of the brain milieu.

Langerhans cells (LCs) are distinct among phagocytes, functioning both as embryo-derived, tissue-resident macrophages in skin innervation and repair and as migrating professional antigen-presenting cells, a function classically assigned to dendritic cells (DCs). Here, we demonstrate that both intrinsic and extrinsic factors imprint this dual identity. Using ablation of embryo-derived LCs in the murine adult skin and tracking differentiation of incoming monocyte-derived replacements, we found intrinsic intraepidermal heterogeneity. We observed that ontogenically distinct monocytes give rise to LCs. Within the epidermis, Jagged-dependent activation of Notch signaling, likely within the hair follicle niche, provided an initial site of LC commitment before metabolic adaptation and survival of monocyte-derived LCs. In the human skin, embryo-derived LCs in newborns retained transcriptional evidence of their macrophage origin, but this was superseded by DC-like immune modules after postnatal expansion. Thus, adaptation to adult skin niches replicates conditioning of LC at birth, permitting repair of the embryo-derived LC network.

Since the classical studies of Pío del Río-Hortega, microglia research has come a long way. In particular, recent advances in bulk and single-cell (sc) transcriptomics have yielded many fascinating new insights into these intriguing immune cells at the interface with the central nervous system (CNS), both in small animal models and human samples. In parallel, tools developed by advanced mouse genetics have revealed the unique ontogeny of microglia and their striking dynamic interactions with other cells in the brain parenchyma. In this chapter, we will discuss various applications of the Cre/loxP-based approach that have enabled the study of microglia in their physiological context of the mouse brain. We will highlight selected key findings that have shaped our current understanding of these cells and discuss the technical intricacies of the Cre/loxP approach and some remaining challenges.

Summary Classical monocytes (CMs) are ephemeral myeloid immune cells that circulate in the blood. Emerging evidence suggests that CMs can have distinct ontogeny and originate from either granulocyte-monocyte- or monocyte-dendritic-cell progenitors (GMPs or MDPs). Here, we report surface markers that allowed segregation of murine GMP- and MDP-derived CMs, i.e., GMP-Mo and MDP-Mo, as well as their functional characterization, including fate definition following adoptive cell transfer. GMP-Mo and MDP-Mo yielded an equal increase in homeostatic CM progeny, such as blood-resident non-classical monocytes and gut macrophages; however, these cells differentially seeded various other selected tissues, including the dura mater and lung. Specifically, GMP-Mo and MDP-Mo differentiated into distinct interstitial lung macrophages, linking CM dichotomy to previously reported pulmonary macrophage heterogeneity. Collectively, we provide evidence for the existence of two functionally distinct CM subsets in the mouse that differentially contribute to peripheral tissue macrophage populations in homeostasis and following challenge.

The mycobiota are a critical part of the gut microbiome, but hostfungal interactions and specific functional contributions of commensal fungi to host fitness remain incompletely understood. Here, we report the identification of a new fungal commensal, Kazachstania heterogenica var. weizmannii, isolated from murine intestines. K. weizmannii exposure prevented Candida albicans colonization and significantly reduced the commensal C. albicans burden in colonized animals. Following immunosuppression of C. albicans colonized mice, competitive fungal commensalism thereby mitigated fatal candidiasis. Metagenome analysis revealed K. heterogenica or K. weizmannii presence among human commensals. Our results reveal competitive fungal commensalism within the intestinal microbiota, independent of bacteria and immune responses, that could bear potential therapeutic value for the management of C. albicansmediated diseases.

Kazachstania heterogenica is a member of the K. telluris complex, where all members to date are reported to be pathogenic fungi. We have isolated a strain, K. heterogenica var. weizmannii, from the gut of mice that seems to be a commensal strain and sequenced its genome.

Macrophages have emerged as critical cellular components of the central nervous system (CNS), promoting development, maintenance, and immune defense of the CNS. Here we will review recent advances in our understanding of brain macrophage heterogeneity, including microglia and border-associated macrophages, focusing on the mouse. Emphasis will be given to the discussion of strengths and limitations of the experimental approaches that have led to the recent insights and hold promise to further deepen our mechanistic understanding of brain macrophages that might eventually allow to harness their activities for the management of CNS pathologies.

Monocytes are short-lived myeloid immune cells that arise from adult hematopoiesis and circulate for a short time in the blood. They comprise two main subsets, in mice defined as classical Ly6Chigh and non-classical Ly6Clow monocytes (CM, NCM). Recent fate mapping and transcriptomic analyses revealed that CM themselves are heterogeneous. Here, we report surface markers that allow segregation of murine GMP- and MDP-derived CM in the BM and blood. Functional characterization, including fate definition following adoptive cell transfer, established that GMP-Mo and MDP-Mo could equal rise to homeostatic CM progeny, such as NCM in blood and gut macrophages, but differentially seeded selected other tissues. Specifically, GMP-Mo and MDP-Mo gave rise to distinct interstitial lung macrophages, thus linking CM dichotomy to previously reported pulmonary macrophage heterogeneity. Collectively, we provide comprehensive evidence for the existence of two functionally distinct CM subsets in the mouse, which differentially contribute to peripheral tissue macrophage populations in homeostasis and following challenge. Our findings are indicative of impact of monocyte ontogeny on in situ differentiation.Competing Interest StatementThe authors have declared no competing interest.

Microglia are established in embryogenesis forming a self-containing cellular compartment resisting seeding with cells derived from adult definitive hematopoiesis. We report that monocyte-derived macrophages (MoMΦ) accumulate in the brain of aging mice with distinct topology, including the nigrostriatum and medulla, but not the frontal cortex. Parenchymal MoMΦ adopt bona fide microglia expression profiles. Unlike microglia, these monocyte-derived microglia (MoMg) are due to their hematopoietic origin targets of clonal hematopoiesis (CH). Using a chimeric transfer model, we show that hematopoietic expression of DNMT3AR822H, a prominent mutation in human CH, renders MoMg pathogenic promoting motor deficits resembling atypical Parkinsonian disorders. Collectively, these data establish in a mouse model that MoMg progressively seed the brains of aging healthy mice, accumulate in selected areas, and, when carrying a somatic mutation associated with CH, can contribute to brain pathology.

Macrophages represent a broad spectrum of distinct, but closely related tissue-resident immune cells. This presents a major challenge for the study of functional aspects of these cells using classical Cre recombinase-mediated conditional mutagenesis in mice, since single promoter-driven Cre transgenic models often display limited specificity toward their intended target. The advent of CRISPR/Cas9 technology has now provided a time- and cost-effective method to explore the full potential of binary transgenic, intersectional genetics. Specifically, the use of two promoters driving inactive Cre fragments that, when co-expressed, dimerize and only then gain recombinase activity allows the characterization and manipulation of genetically defined tissue macrophage subpopulations. Here, we will elaborate on the use of this protocol to capitalize on these recent technological advances in mouse genetics and discuss their strengths and pitfalls to improve the study of tissue macrophage subpopulations in physiology and pathophysiology.

Global gene expression profiling has provided valuable insights into the specific contributions of different cell types to various physiological processes. Notably though, both bulk and single-cell transcriptomics require the prior retrieval of the cells from their tissue context to be analyzed. Isolation protocols for tissue macrophages are, however, notoriously inefficient and, moreover, prone to introduce considerable bias and artifacts. Here, we will discuss a valuable alternative, originally introduced by Amieux and colleagues. This so-called RiboTag approach allows, in combination with respective macrophage-specific Cre transgenic lines, to retrieve macrophage translatomes from crude tissue extracts. We will review our experience with this ingenious method, focusing on the study of brain macrophages, including microglia and border-associated cells. We will elaborate on the advantages of the RiboTag approach that render it a valuable complement to standard cell sorting-based profiling strategies, especially for the investigation of tissue macrophages.

Gliomas are the most frequent primary tumors of the brain. Glioma progression is regulated by the tumor microenvironment, which is mainly composed of tumor-associated microglia (TA-MG) and monocyte-derived macrophages (MDM). Recent studies have highlighted the distinct properties of these cells in glioma progression. However, their spatiotemporal alteration during tumor progression has not been fully explored. Using a genetic lineage tracing approach, we show that TA-MG and MDMs differ in their spatiotemporal distribution and interaction with other components of the glioma microenvironment. MDM were present only inside the tumor, whereas TA-MG accumulated both outside and inside the tumor. However, TA-MG was eliminated from the tumor mass as the tumor progressed. Depletion of MDM led to enhanced occupancy of TA-MG in the tumor core, indicating that TA-MG elimination was regulated by MDM. TA-MG and MDM are heterogeneous cell populations whose compositions and properties can change during tumor progression. Finally, MG, TA-MG and MDM were enriched in the perivascular area (PVA) compared to more distal blood vessel-associated areas. However, inside the tumor, the MDM enrichment in PVA was higher than that in TA-MG. Collectively, we established that TA-MG and MDM exhibit different spatiotemporal features in glioma, suggesting distinctive roles during tumor progression.

Alzheimers disease (AD) is characterized by synaptic loss, which can result from dysfunctional microglial phagocytosis and complement activation. However, what signals drive aberrant microglia-mediated engulfment of synapses in AD is unclear. Here we report that secreted phosphoprotein 1 (SPP1/osteopontin) is upregulated predominantly by perivascular macrophages and, to a lesser extent, by perivascular fibroblasts. Perivascular SPP1 is required for microglia to engulf synapses and upregulate phagocytic markers including C1qa, Grn and Ctsb in presence of amyloid-β oligomers. Absence of Spp1 expression in AD mouse models results in prevention of synaptic loss. Furthermore, single-cell RNA sequencing and putative cellcell interaction analyses reveal that perivascular SPP1 induces microglial phagocytic states in the hippocampus of a mouse model of AD. Altogether, we suggest a functional role for SPP1 in perivascular cells-to-microglia crosstalk, whereby SPP1 modulates microglia-mediated synaptic engulfment in mouse models of AD.

Microglia, the parenchymal brain macrophages of the central nerve system (CNS), have emerged as critical players in brain development and homeostasis. However, immune functions of these cells remain less well defined. We investigated contributions of microglia in a relapsing remitting (RR) multiple sclerosis paradigm, experimental autoimmune encephalitis (RR-EAE) in C57BL/6 / SJL F1 mice. Fate mapping-assisted translatome profiling during the RR disease course revealed the potential of microglia to interact with T cells through antigen presentation, co-stimulation and co-inhibition. Abundant microglia / T cell aggregates, as observed by histology and flow cytometry, supported the notion of functional interactions during remission, with a bias towards T regulatory cells. Finally, microglia ablation and microglia-restricted Ifnγ receptor mutagenesis, while compatible with EAE onset, significantly affected the T cell compartment of the animals and remission. Collectively, our data establish critical interactions of microglia with T cells during autoimmune neuro-degeneration.

Microglia, the parenchymal brain macrophages of the central nervous system, have emerged as critical players in brain development and homeostasis. The immune functions of these cells, however, remain less well defined. We investigated contributions of microglia in a relapsingremitting multiple sclerosis paradigm, experimental autoimmune encephalitis in C57BL/6 x SJL F₁ mice. Fate mapping-assisted translatome profiling during the relapsingremitting disease course revealed the potential of microglia to interact with T cells through antigen presentation, costimulation and coinhibition. Abundant microgliaT cell aggregates, as observed by histology and flow cytometry, supported the idea of functional interactions of microglia and T cells during remission, with a bias towards regulatory T cells. Finally, microglia-restricted interferon-γ receptor and major histocompatibility complex mutagenesis significantly affected the functionality of the regulatory T cell compartment in the diseased central nervous system and remission. Collectively, our data establish critical non-redundant cognate and cytokine-mediated interactions of microglia with CD4⁺ T cells during autoimmune neuroinflammation.

Microglial research has advanced considerably in recent decades yet has been constrained by a rolling series of dichotomies such as \u201cresting versus activated\u201d and \u201cM1 versus M2.\u201d This dualistic classification of good or bad microglia is inconsistent with the wide repertoire of microglial states and functions in development, plasticity, aging, and diseases that were elucidated in recent years. New designations continuously arising in an attempt to describe the different microglial states, notably defined using transcriptomics and proteomics, may easily lead to a misleading, although unintentional, coupling of categories and functions. To address these issues, we assembled a group of multidisciplinary experts to discuss our current understanding of microglial states as a dynamic concept and the importance of addressing microglial function. Here, we provide a conceptual framework and recommendations on the use of microglial nomenclature for researchers, reviewers, and editors, which will serve as the foundations for a future white paper.

The tongue is a unique muscular organ situated in the oral cavity where it is involved in taste sensation, mastication, and articulation. As a barrier organ, which is constantly exposed to environmental pathogens, the tongue is expected to host an immune cell network ensuring local immune defence. However, the composition and the transcriptional landscape of the tongue immune system are currently not completely defined. Here, we characterised the tissue-resident immune compartment of the murine tongue during development, health and disease, combining single-cell RNA-sequencing with in situ immunophenotyping. We identified distinct local immune cell populations and described two specific subsets of tongue-resident macrophages occupying discrete anatomical niches. Cx3cr1⁺ macrophages were located specifically in the highly innervated lamina propria beneath the tongue epidermis and at times in close proximity to fungiform papillae. Folr2⁺ macrophages were detected in deeper muscular tissue. In silico analysis indicated that the two macrophage subsets originate from a common proliferative precursor during early postnatal development and responded differently to systemic LPS in vivo. Our description of the under-investigated tongue immune system sets a starting point to facilitate research on tongue immune-physiology and pathology including cancer and taste disorders.

Monocyte-derived macrophages (Mφs) are crucial regulators during muscularis inflammation. However, it is unclear which micro-environmental factors are responsible for monocyte recruitment and anti-inflammatory Mφ differentiation in this paradigm. Here, we investigate Mφ heterogeneity at different stages of muscularis inflammation and determine how environmental cues can attract and activate tissue-protective Mφs. Results showed that muscularis inflammation induced marked alterations in mononuclear phagocyte populations associated with a rapid infiltration of Ly6c⁺ monocytes that locally acquired unique transcriptional states. Trajectory inference analysis revealed two main pro-resolving Mφ subpopulations during the resolution of muscularis inflammation, i.e. Cd206⁺ MhcII^hi and Timp2⁺ MhcII^lo Mφs. Interestingly, we found that damage to the micro-environment upon muscularis inflammation resulted in EGC activation, which in turn stimulated monocyte infiltration and the consequent differentiation in anti-inflammatory CD206⁺ Mφs via CCL2 and CSF1, respectively. In addition, CSF1-CSF1R signaling was shown to be essential for the differentiation of monocytes into CD206⁺ Mφs and EGC proliferation during muscularis inflammation. Our study provides a comprehensive insight into pro-resolving Mφ differentiation and their regulators during muscularis inflammation. We deepened our understanding in the interaction between EGCs and Mφs, thereby highlighting pro-resolving Mφ differentiation as a potential novel therapeutic strategy for the treatment of intestinal inflammation.

Consecutive exposures to different pathogens are highly prevalent and often alter the host immune response. However, it remains unknown how a secondary bacterial infection affects an ongoing adaptive immune response elicited against primary invading pathogens. We demonstrated that recruitment of Sca-1+ monocytes into lymphoid organs during Salmonella Typhimurium (STm) infection disrupted pre-existing germinal center (GC) reactions. GC responses induced by influenza, plasmodium, or commensals deteriorated following STm infection. GC disruption was independent of the direct bacterial interactions with B cells and instead was induced through recruitment of CCR2-dependent Sca-1+ monocytes into the lymphoid organs. GC collapse was associated with impaired cellular respiration and was dependent on TNFα and IFNγ, the latter of which was essential for Sca-1+ monocyte differentiation. Monocyte recruitment and GC disruption also occurred during LPS-supplemented vaccination and Listeria monocytogenes infection. Thus, systemic activation of the innate immune response upon severe bacterial infection is induced at the expense of antibody-mediated immunity.

Macrophage infiltration in mammary tumors is associated with enhanced tumor progression, metastasis, and poor clinical outcome, and considered as target for therapeutic intervention. By using different genetic mouse models, the authors show that ablation of the tyrosine kinase PYK2, either in breast cancer cells, only in the tumor microenvironment, or in both, markedly reduces the number of infiltrating tumor macrophages and concomitantly inhibits tumor angiogenesis and tumor growth. Strikingly, PYK2 ablation only in macrophages is sufficient to induce similar effects. These phenotypic changes are associated with reduced monocyte recruitment and a substantial decrease in tumor-associated macrophages (TAMs). Mechanistically, the authors show that PYK2 mediates mutual communication between breast cancer cells and macrophages through critical effects on key receptor signaling. Specifically, PYK2 ablation inhibits Notch1 signaling and consequently reduces CCL2 secretion by breast cancer cells, and concurrently reduces the levels of CCR2, CXCR4, IL-4Rα, and Stat6 activation in macrophages. These bidirectional effects modulate monocyte recruitment, macrophage polarization, and tumor angiogenesis. The expression of PYK2 is correlated with infiltrated macrophages in breast cancer patients, and its effects on macrophage infiltration and pro-tumorigenic phenotype suggest that PYK2 targeting can be utilized as an effective strategy to modulate TAMs and possibly sensitize breast cancer to immunotherapy.

T cells undergo rigorous selection in the thymus to ensure self-tolerance and prevent autoimmunity, with this process requiring innocuous self-antigens (Ags) to be presented to thymocytes. Self-Ags are either expressed by thymic stroma cells or transported to the thymus from the periphery by migratory dendritic cells (DCs); meanwhile, small blood-borne peptides can access the thymic parenchyma by diffusing across the vascular lining. Here we describe an additional pathway of thymic Ag acquisition that enables circulating antigenic macromolecules to access both murine and human thymi. This pathway depends on a subset of thymus-resident DCs, distinct from both parenchymal and circulating migratory DCs, that are positioned in immediate proximity to thymic microvessels where they extend cellular processes across the endothelial barrier into the blood stream. Transendothelial positioning of DCs depends on DC-expressed CX3CR1 and its endothelial ligand, CX3CL1, and disrupting this chemokine pathway prevents thymic acquisition of circulating proteins and compromises negative selection of Ag-reactive thymocytes. Thus, transendothelial DCs represent a mechanism by which the thymus can actively acquire blood-borne Ags to induce and maintain central tolerance.

The etiology of inflammatory bowel disorders (IBDs) involves an interplay of genetic and environmental factors. While human genomic analyses and preclinical animal models have revealed critical IBD risk genes and circuits, environmental effects remain poorly defined. In this issue, He et al. (2021) begin to fill this gap by reporting a striking potential of widely used food colorants to cause IBD-like colitis in mice.

The developmental and molecular heterogeneity of tissue macrophages is unravelling, as are their diverse contributions to physiology and pathophysiology. Moreover, also given tissues harbor macrophages in discrete anatomic locations. Functional contributions of specific cell populations can in mice be dissected using Cre recombinase-mediated mutagenesis. However, single promoter-based Cre models show limited specificity for cell types. Focusing on macrophages in the brain, we establish here a binary transgenic system involving complementation-competent NCre and CCre fragments whose expression is driven by distinct promoters: Sall1ncre: Cx3cr1ccre mice specifically target parenchymal microglia and compound transgenic Lyve1ncre: Cx3cr1ccre animals target vasculature-associated macrophages, in the brain, as well as other tissues. We imaged the respective cell populations and retrieved their specific translatomes using the RiboTag in order to define them and analyze their differential responses to a challenge. Collectively, we establish the value of binary transgenesis to dissect tissue macrophage compartments and their functions.

Systemic inflammation is associated with alterations in complex brain functions such as learning and memory. However, diagnostic approaches to functionally assess and quantify inflammation-associated alterations in synaptic plasticity are not well-established. In previous work, we demonstrated that bacterial lipopolysaccharide (LPS)-induced systemic inflammation alters the ability of hippocampal neurons to express synaptic plasticity, i.e., the long-term potentiation (LTP) of excitatory neurotransmission. Here, we tested whether synaptic plasticity induced by repetitive magnetic stimulation (rMS), a non-invasive brain stimulation technique used in clinical practice, is affected by LPS-induced inflammation. Specifically, we explored brain tissue cultures to learn more about the direct effects of LPS on neural tissue, and we tested for the plasticity-restoring effects of the anti-inflammatory cytokine interleukin 10 (IL10). As shown previously, 10 Hz repetitive magnetic stimulation (rMS) of organotypic entorhino-hippocampal tissue cultures induced a robust increase in excitatory neurotransmission onto CA1 pyramidal neurons. Furthermore, LPS-treated tissue cultures did not express rMS-induced synaptic plasticity. Live-cell microscopy in tissue cultures prepared from a novel transgenic reporter mouse line [C57BL/6-Tg(TNFa-eGFP)] confirms that ex vivo LPS administration triggers microglial tumor necrosis factor alpha (TNFα) expression, which is ameliorated in the presence of IL10. Consistent with this observation, IL10 hampers the LPS-induced increase in TNFα, IL6, IL1β, and IFNγ and restores the ability of neurons to express rMS-induced synaptic plasticity in the presence of LPS. These findings establish organotypic tissue cultures as a suitable model for studying inflammation-induced alterations in synaptic plasticity, thus providing a biological basis for the diagnostic use of transcranial magnetic stimulation in the context of brain inflammation.

Microglia, the resident macrophages of the brain parenchyma, are key players in central nervous system (CNS) development, homeostasis, and disorders. Distinct brain pathologies seem associated with discrete microglia activation modules. How microglia regain quiescence following challenges remains less understood. Here, we explored the role of the interleukin-10 (IL-10) axis in restoring murine microglia homeostasis following a peripheral endotoxin challenge. Specifically, we show that lipopolysaccharide (LPS)-challenged mice harboring IL-10 receptor-deficient microglia displayed neuronal impairment and succumbed to fatal sickness. Addition of a microglial tumor necrosis factor (TNF) deficiency rescued these animals, suggesting a microglia-based circuit driving pathology. Single cell transcriptome analysis revealed various IL-10 producing immune cells in the CNS, including most prominently Ly49D+ NK cells and neutrophils, but not microglia. Collectively, we define kinetics of the microglia response to peripheral endotoxin challenge, including their activation and robust silencing, and highlight the critical role of non-microglial IL-10 in preventing deleterious microglia hyperactivation.

Protective MHC class I-dependent immune responses require an overlap between repertoires of proteins directly presented on target cells and cross-presented by professional APC, specifically dendritic cells. How stable proteins that rely on defective ribosomal proteins for direct presentation are captured for cell-to-cell transfer remains enigmatic. In this study, we address this issue using a combination of in vitro (C57BL/6-derived mouse cell lines) and in vivo (C57BL/6 mouse strains) approaches involving stable and unstable versions of OVA model Ags displaying defective ribosomal protein-dependent and -independent Ag presentation, respectively. Apoptosis, but not necrosis, of donor cells was found associated with robust global protein aggregate formation and captured stable proteins permissive for cross-presentation. Potency of aggregates to serve as Ag source was directly demonstrated using polyglutamine-equipped model substrates. Collectively, our data implicate global protein aggregation in apoptotic cells as a mechanism that ensures the overlap between MHC class I epitopes presented directly or cross-presented by APC and demonstrate the unusual ability of dendritic cells to process stable protein aggregates.

Monocytes are circulating myeloid immune precursor cells that are generated in the bone marrow. Mature monocytes are released into the circulation and, in case of need, recruited to peripheral sites of inflammation to differentiate into monocyte-derived effector cells. In absence of overt inflammation, monocytes also extravasate into selected tissues, where they complement tissue-resident macrophage compartments. Adjustment of these homeostatic monocyte infiltrates to local environment is critical to maintain health, as best established for the intestine. Defined gene expression changes that differ between gut segments presumably help strike the fine balance between the crucial function of these monocyte-derived macrophages as tissue rheostats and their detrimental hyperactivation. Environmental factors that dictate local monocyte differentiation remain incompletely understood. Definition of the latter could aid our general understanding of in vivo monocyte functions and their relation to inflammatory disorders. In this review, we summarize recent advances in our understanding of monocyte subsets, their differentiation into tissue macrophages, and selected contributions of monocyte-derived cells to steady-state physiology. Moreover, we will discuss emerging evidence for an intriguing bifurcation of monocyte development in the bone marrow and potential functional implications. Emphasis will be given to points of controversies, but we will largely focus on the healthy organism. For a discussion of monocyte and macrophage contributions to inflammatory conditions, we refer the reader to other dedicated reviews.

Microglia are the principal phagocytes that clear cell debris in the central nervous system (CNS). This raises the question, which cells remove cell debris when microglial phagocytic activity is impaired. We addressed this question using Siglechdtr mice, which enable highly specific ablation of microglia. Non-microglial mononuclear phagocytes, such as CNS-associated macrophages and circulating inflammatory monocytes, did not clear microglial debris. Instead, astrocytes were activated, exhibited a pro-inflammatory gene expression profile, and extended their processes to engulf microglial debris. This astrocytic phagocytosis was also observed in Irf8-deficient mice, in which microglia were present but dysfunctional. RNA-seq demonstrated that even in a healthy CNS, astrocytes express TAM phagocytic receptors, which were the main astrocytic phagocytic receptors for cell debris in the above experiments, indicating that astrocytes stand by in case of microglial impairment. This compensatory mechanism may be important for the maintenance or prolongation of a healthy CNS.

Microglia and central nervous system (CNS)-associated macrophages (CAMs), such as perivascular and meningeal macrophages, are implicated in virtually all diseases of the CNS. However, little is known about their cell-type-specific roles in the absence of suitable tools that would allow for functional discrimination between the ontogenetically closely related microglia and CAMs. To develop a new microglia gene targeting model, we first applied massively parallel single-cell analyses to compare microglia and CAM signatures during homeostasis and disease and identified hexosaminidase subunit beta (Hexb) as a stably expressed microglia core gene, whereas other microglia core genes were substantially downregulated during pathologies. Next, we generated HexbtdTomato mice to stably monitor microglia behavior in vivo. Finally, the Hexb locus was employed for tamoxifen-inducible Cre-mediated gene manipulation in microglia and for fate mapping of microglia but not CAMs. In sum, we provide valuable new genetic tools to specifically study microglia functions in the CNS.

Mildner and colleagues characterize two subsets (Cxcl10(+) and Saa3(+)) of monocytes with pathogenic potential in the central nervous system of mice with experimentally induced autoimmune encephalomyelitis and show these pathogenic cells are not derived from Ly6C(+) monocytes, but from early myeloid cell progenitors.Multiple sclerosis (MS) is characterized by pathological inflammation that results from the recruitment of lymphoid and myeloid immune cells from the blood into the brain. Due to subset heterogeneity, defining the functional roles of the various cell subsets in acute and chronic stages of MS has been challenging. Here, we used index and transcriptional single-cell sorting to characterize the mononuclear phagocytes that infiltrate the central nervous system from the periphery in mice with experimentally induced autoimmune encephalomyelitis, a model of MS. We identified eight monocyte and three dendritic cell subsets at acute and chronic disease stages in which the defined transcriptional programs pointed toward distinct functions. Monocyte-specific cell ablation identified Cxcl10(+) and Saa3(+) monocytic subsets with a pathogenic potential. Transfer experiments with different monocyte and precursor subsets indicated that these Cxcl10(+) and Saa3(+) pathogenic cells were not derived from Ly6C(+) monocytes but from early myeloid cell progenitors. These results suggest that blocking specific pathogenic monocytic subsets, including Cxcl10(+) and Saa3(+) monocytes, could be used for targeted therapeutic interventions.

Acute graft-versus-host disease (GVHD) can affect the central nervous system (CNS). The role of microglia in CNS-GVHD remains undefined. In agreement with microglia activation, we found that profound morphological changes and MHC-II and CD80 upregulation occurred upon GVHD induction. RNA sequencing-based analysis of purified microglia obtained from mice with CNS-GVHD revealed TNF upregulation. Selective TNF gene deletion in microglia of Cx3cr1creER Tnffl/- mice reduced MHC-II expression and decreased CNS T cell infiltrates and VCAM-1+ endothelial cells. GVHD increased microglia TGF-β-activated kinase-1 (TAK1) activation and NF-κB/p38 MAPK signaling. Selective Tak1 deletion in microglia using Cx3cr1creER Tak1fl/fl mice resulted in reduced TNF production and microglial MHC-II and improved neurocognitive activity. Pharmacological TAK1 inhibition reduced TNF production and MHC-II expression by microglia, Th1 and Th17 T cell infiltrates, and VCAM-1+ endothelial cells and improved neurocognitive activity, without blocking graft-versus-leukemia effects. Consistent with these findings in mice, we observed increased activation and TNF production of microglia in the CNS of GVHD patients. In summary, we prove a role for microglia in CNS-GVHD, identify the TAK1/TNF/MHC-II axis as a mediator of CNS-GVHD, and provide a TAK1 inhibitor-based approach against GVHD-induced neurotoxicity.

Chronic lymphocytic leukemia (CLL) is a malignancy of mature B lymphocytes. The microenvironment of the CLL cells is a vital element in the regulation of the survival of these malignant cells. CLL cell longevity is dependent on external signals, originating from cells in their microenvironment including secreted and surface-bound factors. Dendritic cells (DCs) play an important part in tumor microenvironment, but their role in the CLL bone marrow (BM) niche has not been studied. We show here that CLL cells induce accumulation of bone marrow dendritic cells (BMDCs). Depletion of this population attenuates disease expansion. Our results show that the support of the microenvironment is partly dependent on CD84, a cell surface molecule belonging to the Signaling Lymphocyte Activating Molecule (SLAM) family of immunoreceptors. Our results suggest a novel therapeutic strategy whereby eliminating BMDCs or blocking the CD84 expressed on these cells may reduce the tumor load.

Recruited blood monocytes contribute to the establishment, perpetuation, and resolution of tissue inflammation. Specifically, in the inflamed intestine, monocyte ablation was shown to ameliorate colitis scores in preclinical animal models. However, the majority of intestinal macrophages that seed the healthy gut are also monocyte derived. Monocyte ablation aimed to curb inflammation would therefore likely interfere with intestinal homeostasis. In this study, we used a TLR2 trans-membrane peptide that blocks TLR2 dimerization that is critical for TLR2/1 and TLR2/6 heterodimer signaling to blunt inflammation in a murine colitis model. We show that although the TLR2 peptide treatment ameliorated colitis, it allowed recruited monocytes to give rise to macrophages that lack the detrimental proinflammatory gene signature and reduced potentially damaging neutrophil infiltrates. Finally, we demonstrate TLR blocking activity of the peptide on in vitro cultured human monocyte-derived macrophages. Collectively, we provide a significantly improved anti-inflammatory TLR2 peptide and critical insights in its mechanism of action toward future potential use in the clinic.

Monocytes are circulating short-lived macrophage precursors that are recruited on demand from the blood to sites of inflammation and challenge. In steady state, classical monocytes give rise to vasculature-resident cells that patrol the luminal side of the endothelium. In addition, classical monocytes feed macrophage compartments of selected organs, including barrier tissues, such as the skin and intestine, as well as the heart. Monocyte differentiation under conditions of inflammation has been studied in considerable detail. In contrast, monocyte differentiation under non-inflammatory conditions remains less well understood. Here we took advantage of a combination of cell ablation and precursor engraftment to investigate the generation of gut macrophages from monocytes. Collectively, we identify factors associated with the gradual adaptation of monocytes to tissue residency. Moreover, comparison of monocyte differentiation into the colon and ileum-resident macrophages revealed the graduated acquisition of gut segment-specific gene expression signatures.

The small intestine hosts specialized lymphoid structures, the Peyer's patches, that face the gut lumen and are overlaid with unique epithelial cells, called microfold (M) cells. M cells are considered to constitute an important route for antigen uptake in the mucosal immune system. Here, we used intravital microscopy to define immune cell populations, which are in close contact with M cells and potentially sample antigen. We present live evidence that DCs enter M cell pockets and highlight the abundance of mononuclear phagocytes in these structures. Taking advantage of the respective reporter animals, we focused on classical DCs that express Zbtb46 and analyzed how these cells interact with M cells in steady state and sample antigen for T cell activation in the Peyer's patches following challenge.

Conditional mutagenesis and fate mapping have contributed considerably to our understanding of physiology and pathology. Specifically, Cre recombinase-based approaches allow the definition of cell type-specific contributions to disease development and of inter-cellular communication circuits in respective animal models. Here we compared Cx(3)cr1(CreER) and Sall1(CreER) transgenic mice and their use to decipher the brain macrophage compartment as a showcase to discuss recent technological advances. Specifically, we highlight the need to define the accuracy of Cre recombinase expression, as well as strengths and pitfalls of these particular systems that should be taken into consideration when applying these models.

Microglia were first recognized as a distinct cell population in the CNS one century ago. For a long time, they were primarily considered to be phagocytes responsible for removing debris during CNS development and disease. More recently, advances in imaging and genetics and the advent of single-cell technology provided new insights into the much more complex and fascinatir; biology of microglia. The ontogeny of microglia was identified, and their functions in health and disease were better defined. Although many qL lions about microglia and their roles in human diseases remain unanswered, the prospect of targeting microglia for the treatment of neurological and psychiatric disorders is tantalizing.

Hematopoiesis is well known to be affected by environmental factors, adjusting the balance of lymphoid and myeloid output according to peripheral needs.(1,2) Specifically, the bone marrow (BM), as a site of adult blood cell generation, has been shown to sense the gut microbiome composition and respond to dysbiosis associated with antibiotics treatment and numerous gastrointestinal disorders. In this issue of Blood, Lee et al(3) describe how this remote sensing is achieved and how the microbiota educate the immune system while maintaining critical steady-state myelopoiesis.(4)

A low response rate, acquired resistance and severe side effects have limited the clinical outcomes of immune checkpoint therapy. Here, we show that combining cancer nanovaccines with an anti-PD-1 antibody (alpha PD-1) for immunosuppression blockade and an anti-OX40 antibody (alpha OX40) for effector T-cell stimulation, expansion and survival can potentiate the efficacy of melanoma therapy. Prophylactic and therapeutic combination regimens of dendritic cell-targeted mannosylated nanovaccines with alpha PD-1/alpha OX40 demonstrate a synergism that stimulates T-cell infiltration into tumours at early treatment stages. However, this treatment at the therapeutic regimen does not result in an enhanced inhibition of tumour growth compared to alpha PD-1/alpha OX40 alone and is accompanied by an increased infiltration of myeloid-derived suppressor cells in tumours. Combining the double therapy with ibrutinib, a myeloid-derived suppressor cell inhibitor, leads to a remarkable tumour remission and prolonged survival in melanoma-bearing mice. The synergy between the mannosylated nanovaccines, ibrutinib and alpha PD-1/alpha OX40 provides essential insights to devise alternative regimens to improve the efficacy of immune checkpoint modulators in solid tumours by regulating the endogenous immune response.

Cytokines maintain intestinal health, but precise intercellular communication networks remain poorly understood. Macrophages are immune sentinels of the intestinal tissue and are critical for gut homeostasis. Here, we show that in a murine inflammatory bowel disease (IBD) model based on macrophage-restricted interleukin-10 (IL-10) receptor deficiency (Cx3cr1^Cre:Il10ra^fl/fl mice), proinflammatory mutant gut macrophages cause severe spontaneous colitis resembling the condition observed in children carrying IL-10R mutations. We establish macrophage-derived IL-23 as the driving factor of this pathology. Specifically, we report that Cx3cr1^Cre:Il10ra^fl/fl:Il23a^fl/fl mice harboring macrophages deficient for both IL-10R and IL-23 are protected from colitis. By analyzing the epithelial response to proinflammatory macrophages, we provide evidence that T cells of colitic animals produce IL-22, which induces epithelial chemokine expression and detrimental neutrophil recruitment. Collectively, we define macrophage-specific contributions to the induction and pathogenesis of colitis, as manifested in mice harboring IL-10R deficiencies and human IBDs.

Dendritic cells (DC) are unrivaled in their potential to prime naive T cells by presenting antigen and providing costimulation. DC are furthermore believed to decode antigen context by virtue of pattern recognition receptors and to polarize T cells through cytokine secretion toward distinct effector functions. Diverse polarized T helper (T-H) cells have been explored in great detail. In contrast, studies of instructing DC have to date largely been restricted to in vitro settings or adoptively transferred DC. Here we report efforts to unravel the DC response to cognate T cell encounter in antigen-challenged lymph nodes (LN). Mice engrafted with antigen-specific T cells were immunized with nanoparticles (NP) entrapping adjuvants and absorbed with antigen to study the immediate DC response to T cell encounter using bulk and single cell RNA-seq profiling. NP induced robust antigen-specific T(H)1 cell responses with minimal bystander activation. Fluorescent-labeled NP allowed identification of antigen-carrying DC and focus on transcriptional changes in DC that encounter T cells. Our results support the existence of a bi-directional crosstalk between DC and T cells that promotes T(H)1 responses, including involvement of the ubiquitin-like molecule Isg15 that merits further study.

A characteristic subset of microglia expressing CD11c appears in response to brain damage. However, the functional role of CD11c(+) microglia, as well as the mechanism of its induction, are poorly understood. Here we report that the genetic ablation of signal regulatory protein alpha (SIRP alpha), a membrane protein, induced the emergence of CD11c(+) microglia in the brain white matter. Mice lacking CD47, a physiological ligand of SIRP alpha, and microglia-specific SIRP alpha-knockout mice exhibited the same phenotype, suggesting that an interaction between microglial SIRP alpha and CD47 on neighbouring cells suppressed the emergence of CD11c(+) microglia. A lack of SIRP alpha did not cause detectable damage to the white matter, but resulted in the increased expression of genes whose expression is characteristic of the repair phase after demyelination. In addition, cuprizone-induced demyelination was alleviated by the microglia-specific ablation of SIRP alpha. Thus, microglial SIRP alpha suppresses the induction of CD11c(+) microglia that have the potential to accelerate the repair of damaged white matter.

The involvement of macrophages in the pathogenesis of obesity has been recognized since 2003. Early studies mostly focused on the role of macrophages in adipose tissue (AT) and in obesity-associated chronic low-grade inflammation. Lately, AT macrophages were shown to undergo intrinsic metabolic changes that affect their immune function (i.e., immunometabolism), corresponding to their unique properties along the range of pro- versus anti-inflammatory activity. In parallel, recent studies in mice revealed critical neuronal-macrophage interactions, both in the CNS and in peripheral tissues, including in white and brown AT. These intercellular activities impinge on energy and metabolic homeostasis, partially by also engaging adipocytes in a neuronal-macrophage-adipocyte menage a trois. Finally, neuropeptides (NP), such as NPY and appetite-reducing NPFF, may prove as mediators in such intercellular network. In this concise review, we highlight some of these recent insights on adipose macrophage immunometabolism, as well as central and peripheral neuronal-macrophage interactions with emphasis on their impact on adipocyte biology and whole-body metabolism. We also discuss the expanding view on the role of the NP, NPY and NPFF, in obesity.

Microglia are yolk sac-derived macrophages residing in the parenchyma of brain and spinal cord, where they interact with neurons and other glial. After different conditioning paradigms and bone marrow (BM) or hematopoietic stem cell (HSC) transplantation, graft-derived cells seed the brain and persistently contribute to the parenchymal brain macrophage compartment. Here we establish that graft-derived macrophages acquire, over time, microglia characteristics, including ramified morphology, longevity, radio-resistance and clonal expansion. However, even after prolonged CNS residence, transcriptomes and chromatin accessibility landscapes of engrafted, BM-derived macrophages remain distinct from yolk sac-derived host microglia. Furthermore, engrafted BM-derived cells display discrete responses to peripheral endotoxin challenge, as compared to host microglia. In human HSC transplant recipients, engrafted cells also remain distinct from host microglia, extending our finding to clinical settings. Collectively, our data emphasize the molecular and functional heterogeneity of parenchymal brain macrophages and highlight potential clinical implications for HSC gene therapies aimed to ameliorate lysosomal storage disorders, microgliopathies or general monogenic immuno-deficiencies.

Background: Fractalkine (CX(3)CL1) and its receptor (CX(3)CR1) play an important role in regulating microglial function. We have previously shown that Cx(3)cr1 deficiency exacerbated tau pathology and led to cognitive impairment. However, it is still unclear if the chemokine domain of the ligand CX(3)CL1 is essential in regulating neuronal tau pathology.Methods: We used transgenic mice lacking endogenous Cx(3)cl1 (Cx(3)cl1(-/-)) and expressing only obligatory soluble form (with only chemokine domain) and lacking the mucin stalk of CX(3)CL1 (referred to as Cx(3)cl1(105 Delta) mice) to assess tau pathology and behavioral function in both lipopolysaccharide (LPS) and genetic (hTau) mouse models of tauopathy.Results: First, increased basal tau levels accompanied microglial activation in Cx(3)cl1(105 Delta) mice compared to control groups. Second, increased CD45(+) and F4/80(+) neuroinflammation and tau phosphorylation were observed in LPS, hTau/Cx(3)cl1(-/-), and hTau/Cx(3)cl1(105 Delta) mouse models of tau pathology, which correlated with impaired spatial learning. Finally, microglial cell surface expression of CX(3)CR1 was reduced in Cx(3)cl1(105 Delta) mice, suggesting enhanced fractalkine receptor internalization (mimicking Cx(3)cr1 deletion), which likely contributes to the elevated tau pathology.Conclusions: Collectively, our data suggest that overexpression of only chemokine domain of CX(3)CL1 does not protect against tau pathology.

Microglia are resident immune cells in the CNS, strategically positioned to clear dead cells and debris, and orchestrate CNS inflammation and immune defense. In steady state, these macrophages lack MHC class II (MHCII) expression, but microglia activation can be associated with MHCII induction. Whether microglial MHCII serves antigen presentation for critical local T-cell restimulation in CNS auto-immune disorders or modulates microglial signaling output remains under debate. To probe for such scenarios, we generated mice harboring an MHCII deficiency in microglia, but not peripheral myeloid cells. Using the CX(3)CR1(CreER)-based approach we report that microglial antigen presentation is obsolete for the establishment of EAE, with disease onset, progression, and severity unaltered in mutant mice. Antigen presentation-independent roles of microglial MHCII were explored using a demyelination model induced by the copper chelator cuprizone. Absence of microglial I-A(b) did not affect the extent of these chemically induced white matter alterations, nor did it affect microglial proliferation or gene expression associated with locally restricted de- and remyelination.

Cell ablation is a valuable complement to mutagenesis for experimentally defining specific cell functions in physiology and pathophysiology in small animal models. One of the most popular ablation strategies involves transgenic expression of a primate diphtheria toxin receptor (DTR) on murine cells that are otherwise resistant to the bacterial exotoxin. The efforts of many laboratories using the DTR approach over the years have yielded numerous valuable insights into specific cell functions. Here, we will discuss the technical aspects of the DTR approach, including the strengths, pitfalls, and future strategies to overcome the shortcomings, highlighting a recent paper published in the European Journal of Immunology [El Hachem etal. Eur. J. Immunol. 2018 ]. A particular focus will be given to the application of DTR approach to decipher in vivo functions of the murine myeloid cell compartment.

Transcriptome profiling is widely used to infer functional states of specific cell types, as well as their responses to stimuli, to define contributions to physiology and pathophysiology. Focusing on microglia, the brain's macrophages, we report here a side-by-side comparison of classical cell-sorting-based transcriptome sequencing and the 'RiboTag' method, which avoids cell retrieval from tissue context and yields translatome sequencing information. Conventional whole-cell microglial transcriptomes were found to be significantly tainted by artifacts introduced by tissue dissociation, cargo contamination and transcripts sequestered from ribosomes. Conversely, our data highlight the added value of RiboTag profiling for assessing the lineage accuracy of Cre recombinase expression in transgenic mice. Collectively, this study indicates method-based biases, reveals observer effects and establishes RiboTag-based translatome profiling as a valuable complement to standard sorting-based profiling strategies.

Microglia, the mononuclear phagocytes of the central nervous system (CNS), are important for the maintenance of CNS homeostasis, but also critically contribute to CNS pathology. Here we demonstrate that the nuclear factor kappa B (NF-kappa B) regulatory protein A20 is crucial in regulating microglia activation during CNS homeostasis and pathology. In mice, deletion of A20 in microglia increases microglial cell number and affects microglial regulation of neuronal synaptic function. Administration of a sublethal dose of lipopolysaccharide induces massive microglia activation, neuroinflammation, and lethality in mice with microgliaconfined A20 deficiency. Microglia A20 deficiency also exacerbates multiple sclerosis (MS) like disease, due to hyperactivation of the NIrp3 inflammasome leading to enhanced interleukin-113 secretion and CNS inflammation. Finally, we confirm a NIrp3 inflammasome signature and IL-1 beta expression in brain and cerebrospinal fluid from MS patients. Collectively, these data reveal a critical role for A20 in the control of microglia activation and neuroinflammation.

Nitric oxide (NO) plays an established role in numerous physiological and pathological processes, but the specific cellular sources of NO in disease pathogenesis remain unclear, preventing the implementation of NO-related therapy. Argininosuccinate lyase (ASL) is the only enzyme able to produce arginine, the substrate for NO generation by nitric oxide synthase (NOS) isoforms. Here, we generated cell-specific conditional ASL knockout mice in combination with genetic and chemical colitis models. We demonstrate that NO derived from enterocytes alleviates colitis by decreasing macrophage infiltration and tissue damage, whereas immune cell-derived NO is associated with macrophage activation, resulting in increased severity of inflammation. We find that induction of endogenous NO production by enterocytes with supplements that upregulate ASL expression and complement its substrates results in improved epithelial integrity and alleviation of colitis and of inflammation-associated colon cancer.

Nanoparticulate vaccines are promising tools to overcome cancer immune evasion. However, a deeper understanding on nanoparticle-immune cell interactions and treatments regime is required for optimal efficacy. We provide a comprehensive study of treatment schedules and mode of antigen-association to nanovaccines on the modulation of T cell immunity in vivo, under steady-state and tumor-bearing mice. The coordinated delivery of antigen and two adjuvants (Monophosphoryl lipid A, oligodeoxynucleotide cytosine-phosphate-guanine motifs (CpG)) by nanoparticles was crucial for dendritic cell activation. A single vaccination dictated a 3-fold increase on cytotoxic memory-T cells and raised antigen-specific immune responses against B16.M05 melanoma. It generated at least a 5-fold increase on IFN-gamma cytokine production, and presented over 50% higher lymphocyte count in the tumor microenvironment, compared to the control. The number of lymphocytes at the tumor site doubled with triple immunization. This lymphocyte infiltration pattern was confirmed in mammary huHER2 carcinoma, with significant tumor reduction. (c) 2018 Elsevier Inc. All rights reserved.

Protective immune responses depend on the formation of immune synapses between T cells and antigen-presenting cells (APCs). The two main LFA-1 ligands, ICAM-1 and ICAM-2, are co-expressed on many cell types, including APCs and blood vessels. Although these molecules were suggested to be key players in immune synapses studied in vitro, their contribution to helper T cell priming in vivo is unclear. Here, we used transgenic mice and intravital imaging to examine the role of dendritic cell (DC) ICAM-1 and ICAM-2 in naive CD4 T cell priming and differentiation in skin-draining lymph nodes. Surprisingly, ICAM deficiency on endogenous CD40-stimulated lymph node DCs did not impair their ability to arrest and prime CD4 lymphocyte activation and differentiation into Th1 and Tfh effectors. Thus, functional T cell receptor (TCR)-specific helper T cell synapses with antigen-presenting DCs and subsequent proliferation and early differentiation into T effectors do not require LFA-1-mediated T cell adhesiveness to DC ICAMs.

Objective Postoperative ileus (POI), the most frequent complication after intestinal surgery, depends on dendritic cells (DCs) and macrophages. Here, we have investigated the mechanism that activates these cells and the contribution of the intestinal microbiota for POI induction.Design POI was induced by manipulating the intestine of mice, which selectively lack DCs, monocytes or macrophages. The disease severity in the small and large intestine was analysed by determining the distribution of orally applied fluorescein isothiocyanate-dextran and by measuring the excretion time of a retrogradely inserted glass ball. The impact of the microbiota on intestinal peristalsis was evaluated after oral antibiotic treatment.Results We found that Cd11c-Cre(+) Irf4(flox/flox) mice lack CD103(+) CD11b(+) DCs, a DC subset unique to the intestine whose function is poorly understood. Their absence in the intestinal muscularis reduced pathogenic inducible nitric oxide synthase (iNOS) production by monocytes and macrophages and ameliorated POI. Pathogenic iNOS was produced in the jejunum by resident Ly6C(-) macrophages and infiltrating chemokine receptor 2-dependent Ly6C(+) monocytes, but in the colon only by the latter demonstrating differential tolerance mechanisms along the intestinal tract. Consistently, depletion of both cell subsets reduced small intestinal POI, whereas the depletion of Ly6C(+) monocytes alone was sufficient to prevent large intestinal POI. The differential role of monocytes and macrophages in small and large intestinal POI suggested a potential role of the intestinal microbiota. Indeed, antibiotic treatment reduced iNOS levels and ameliorated POI.Conclusions Our findings reveal that CD103(+) CD11b(+) DCs and the intestinal microbiome are a prerequisite for the activation of intestinal monocytes and macrophages and for dysregulating intestinal motility in POI.

Hematopoietic-specific miR-142 is a critical regulator of various blood cell lineages including CD4+ dendritic cells1 and platelet biogenesis in megakaryocytes.2 Furthermore, we recently reported that miR-142 is required in order to maintain the biconcave shape of erythrocytes, their structural resilience and lifespan, through a mechanism that involves actin filament homeostasis.3 Here, we used a mouse loss of function allele to characterize a new axis, where miR-142 functions upstream of Rac1 in regulating erythropoiesis.

Vaccination is a promising strategy to trigger and boost immune responses against cancer or infectious disease. We have designed, synthesized and characterized aliphatic-polyester (poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NP) to investigate how the nature of protein association (adsorbed versus entrapped) and polymer/surfactant concentrations impact on the generation and modulation of antigen-specific immune responses. The ability of the NP formulations to target dendritic cells (DC), be internalized and activate the T cells was characterized and optimized in vitro and in vivo using markers of DC activation and co-stimulatory molecules. Ovalbumin (OVA) was used as a model antigen in combination with the engraftment of CD4⁺ and CD8⁺ T cells, carrying a transgenic OVA-responding T cell receptor (TCR), to trace and characterize the activation of antigen-specific CD4⁺ and CD8⁺ lymph node T cells upon NP vaccination. Accordingly, the phenotype and frequency of immune cell stimulation induced by the NP loaded with OVA, isolated or in combination with synthetic unmethylated cytosine-phosphate-guanine (CpG) oligodeoxynucleotide (ODN) motifs, were characterized. DC-NP interactions increased with incubation time, presenting internalization values between 50 and 60% and 3040%, in vitro and in vivo, respectively. Interestingly, animal immunization with antigen-adsorbed NP up-regulated major histocompatibility complex (MHC) class II (MHCII), while NP entrapping the antigen up-regulated MHCI, suggesting a more efficient cross-presentation. On the other hand, rather surprisingly, the surfactant used in the NP formulation had a major impact on the activation of antigen presenting cells (APC). In fact, DC collected from lymph nodes of animals immunized with NP prepared using poly(vinil alcohol) (PVA), as a surfactant, expressed significantly higher levels of CD86, MHCI and MHCII. In addition, those NP prepared with PVA and co-entrapping OVA and the toll-like receptor (TLR) ligand CpG, induced the most profound antigen-specific T cell response, by both CD4⁺ and CD8⁺ T cells, in vivo. Overall, our data reveal the impact of NP composition and surface properties on the type and extension of induced immune responses. Deeper understanding on the NP-immune cell crosstalk can guide the rational development of nano-immunotherapeutic systems with improved and specific therapeutic efficacy and avoiding off-target effects.

Tissue macrophages arise during embryogenesis from yolk-sac (YS) progenitors that give rise to primitive YS macrophages. Until recently, it has been impossible to isolate or derive sufficient numbers of YS-derived macrophages for further study, but data now suggest that induced pluripotent stem cells (iPSCs) can be driven to undergo a process reminiscent of YS-hematopoiesis in vitro. We asked whether iPSC-derived primitive macrophages (iMacs) can terminally differentiate into specialized macrophages with the help of growth factors and organ-specific cues. Co-culturing human or murine iMacs with iPSC-derived neurons promoted differentiation into microglia-like cells in vitro. Furthermore, murine iMacs differentiated in vivo into microglia after injection into the brain and into functional alveolar macrophages after engraftment in the lung. Finally, iPSCs from a patient with familial Mediterranean fever differentiated into iMacs with pro-inflammatory characteristics, mimicking the disease phenotype. Altogether, iMacs constitute a source of tissue-resident macrophage precursors that can be used for biological, pathophysiological, and therapeutic studies. Yolk-sac (YS) embryonic macrophages contribute to tissue-resident macrophages but remain difficult to study because of their stage-dependent limited availability. Takata et al. demonstrate that iPSCs can generate YS macrophage-like cells (iMacs) that differentiate into functional tissue-resident macrophage-like cells upon receiving organ-specific cues, thus providing a platform for modeling tissue-resident macrophages.

T-cell development is a spatially and temporally regulated process, orchestrated by well-defined contributions of transcription factors and cytokines. Here, we identify the noncoding RNA miR-142 as an additional regulatory layer within murine thymocyte development and proliferation. MiR-142 deficiency impairs the expression of cell cycle-promoting genes in mature mouse thymocytes and early progenitors, accompanied with increased levels of cyclin-dependent kinase inhibitor 1B (Cdkn1b, also known as p27^Kip1). By using CRISPR/Cas9 technology to delete the miR-142-3p recognition element in the 3UTR of cdkn1b, we confirm that this gene is a novel target of miR-142-3p in vivo. Increased Cdkn1b protein expression alone however was insufficient to cause proliferation defects in thymocytes, indicating the existence of additional critical miR-142 targets. Collectively, we establish a key role for miR-142 in the control of early and mature thymocyte proliferation, demonstrating the multifaceted role of a single miRNA on several target genes.

Microglia seed the embryonic neuro-epithelium, expand and actively sculpt neuronal circuits in the developing central nervous system, but eventually adopt relative quiescence and ramified morphology in the adult. Here, we probed the impact of post-transcriptional control by microRNAs (miRNAs) on microglial performance during development and adulthood by generating mice lacking microglial Dicer expression at these distinct stages. Conditional Dicer ablation in adult microglia revealed that miRNAs were required to limit microglial responses to challenge. After peripheral endotoxin exposure, Dicer-deficient microglia expressed more pro-inflammatory cytokines than wild-type microglia and thereby compromised hippocampal neuronal functions. In contrast, prenatal Dicer ablation resulted in spontaneous microglia activation and revealed a role for Dicer in DNA repair and preservation of genome integrity. Accordingly, Dicer deficiency rendered otherwise radio-resistant microglia sensitive to gamma irradiation. Collectively, the differential impact of the Dicer ablation on microglia of the developing and adult brain highlights the changes these cells undergo with time. Microglia of developing and adult brain differ in activation state and function. Here, Varol and colleagues ablated microglial Dicer expression at distinct times. Adult microglia tolerated the perturbation but became hyper-responsive to challenge compromising hippocampus functions. Dicer and microRNA absence during development caused spontaneous microglia activation and impaired genome integrity.

Microglia constitute a highly specialized network of tissue-resident immune cells that is important for the control of tissue homeostasis and the resolution of diseases of the CNS. Little is known about how their spatial distribution is established and maintained in vivo. Here we establish a new multicolor fluorescence fate mapping system to monitor microglial dynamics during steady state and disease. Our findings suggest that microglia establish a dense network with regional differences, and the high regional turnover rates found challenge the universal concept of microglial longevity. Microglial self-renewal under steady state conditions constitutes a stochastic process. During pathology this randomness shifts to selected clonal microglial expansion. In the resolution phase, excess disease-associated microglia are removed by a dual mechanism of cell egress and apoptosis to re-establish the stable microglial network. This study unravels the dynamic yet discrete self-organization of mature microglia in the healthy and diseased CNS.

Monocyte-derived macrophages (MoMF) play a pivotal role in the resolution of acetaminophen-induced liver injury (AILI). Timely termination of neutrophil activity and their clearance are essential for liver regeneration following injury. Here, we show that infiltrating Ly6C^hi monocytes, their macrophage descendants, and neutrophils spatially and temporally overlap in the centrilobular necrotic areas during the necroinflammatory and resolution phases of AILI. At the necroinflammatory phase, inducible ablation of circulating Ly6C^hi monocytes resulted in reduced numbers and fractions of reactive oxygen species (ROS)-producing neutrophils. In alignment with this, neutrophils sorted from monocyte-deficient livers exhibited reduced expression of NADPH oxidase 2. Moreover, human CD14⁺ monocytes stimulated with lipopolysaccharide or hepatocyte apoptotic bodies directly induced ROS production by cocultured neutrophils. RNA-seq-based transcriptome profiling of neutrophils from Ly6C^hi monocyte-deficient versus normal livers revealed 449 genes that were differentially expressed with at least twofold change (p ≤ 0.05). In the absence of Ly6C^hi monocytes, neutrophils displayed gene expression alterations associated with decreased innate immune activity and increased cell survival. At the early resolution phase, Ly6C^hi monocytes differentiated into ephemeral Ly6C^lo MoMF and their absence resulted in significant accumulation of late apoptotic neutrophils. Further gene expression analysis revealed the induced expression of a specific repertoire of bridging molecules and receptors involved with apoptotic cell clearance during the transition from Ly6C^hi monocytes to MoMF. Collectively, our findings establish a phase-dependent task division between liver-infiltrating Ly6C^hi monocytes and their MoMF descendants with the former regulating innate immune functions and cell survival of neutrophils and the later neutrophil clearance.

Tissue macrophages provide immunological defense and contribute to the establishment and maintenance of tissue homeostasis. Here we used constitutive and inducible mutagenesis to delete the nuclear transcription regulator Mecp2 in macrophages. Mice that lacked the gene encoding Mecp2, which is associated with Rett syndrome, in macrophages did not show signs of neurodevelopmental disorder but displayed spontaneous obesity, which was linked to impaired function of brown adipose tissue (BAT). Specifically, mutagenesis of a BAT-resident Cx 3 Cr1 + macrophage subpopulation compromised homeostatic thermogenesis but not acute, cold-induced thermogenesis. Mechanistically, malfunction of BAT in pre-obese mice with mutant macrophages was associated with diminished sympathetic innervation and local titers of norepinephrine, which resulted in lower expression of thermogenic factors by adipocytes. Mutant macrophages overexpressed the signaling receptor and ligand PlexinA4, which might contribute to the phenotype by repulsion of sympathetic axons expressing the transmembrane semaphorin Sema6A. Collectively, we report a previously unappreciated homeostatic role for macrophages in the control of tissue innervation. Disruption of this circuit in BAT resulted in metabolic imbalance.

Monocytes are circulating, short-lived mononuclear phagocytes, which in mice and man comprise two main subpopulations. Murine Ly6C+ monocytes display developmental plasticity and are recruited to complement tissue-resident macrophages and dendritic cells on demand. Murine vascular Ly6C monocytes patrol the endothelium, act as scavengers, and support vessel wall repair. Here we characterized population and single cell transcriptomes, as well as enhancer and promoter landscapes of the murine monocyte compartment. Single cell RNA-seq and transplantation experiments confirmed homeostatic default differentiation of Ly6C+ into Ly6C monocytes. The main two subsets were homogeneous, but linked by a more heterogeneous differentiation intermediate. We show that monocyte differentiation occurred through de novo enhancer establishment and activation of pre-established (poised) enhancers. Generation of Ly6C monocytes involved induction of the transcription factor C/EBPb and C/EBPb-deficient mice lacked Ly6C monocytes. Mechanistically, C/EBPb bound the Nr4a1 promoter and controlled expression of this established monocyte survival factor.

Adaptive thermogenesis is the process of heat generation in response to cold stimulation. It is under the control of the sympathetic nervous system, whose chief effector is the catecholamine norepinephrine (NE). NE enhances thermogenesis through beta 3-adrenergic receptors to activate brown adipose tissue and by 'browning' white adipose tissue. Recent studies have reported that alternative activation of macrophages in response to interleukin (IL)-4 stimulation induces the expression of tyrosine hydroxylase (TH), a key enzyme in the catecholamine synthesis pathway, and that this activation provides an alternative source of locally produced catecholamines during the thermogenic process. Here we report that the deletion of Th in hematopoietic cells of adult mice neither alters energy expenditure upon cold exposure nor reduces browning in inguinal adipose tissue. Bone marrow-derived macrophages did not release NE in response to stimulation with IL-4, and conditioned media from IL-4-stimulated macrophages failed to induce expression of thermogenic genes, such as uncoupling protein 1 (Ucp1), in adipocytes cultured with the conditioned media. Furthermore, chronic treatment with IL-4 failed to increase energy expenditure in wild-type, Ucp1(-/-) and interleukin-4 receptor-alpha double-negative (Il4ra(-/-)) mice. In agreement with these findings, adipose-tissue-resident macrophages did not express TH. Thus, we conclude that alternatively activated macrophages do not synthesize relevant amounts of catecholamines, and hence, are not likely to have a direct role in adipocyte metabolism or adaptive thermogenesis.

Monocytes are circulating mononuclear phagocytes, poised to extravasate to sites of inflammation and differentiate into monocyte-derived macrophages and dendritic cells. Tumor necrosis factor (TNF) and its receptors are up-regulated during monopoiesis and expressed by circulating monocytes, as well as effector monocytes infiltrating certain sites of inflammation, such as the spinal cord, during experimental autoimmune encephalomyelitis (EAE). In this study, using competitive in vitro and in vivo assays, we show that monocytes deficient for TNF or TNF receptors are outcompeted by their wild-type counterpart. Moreover, monocyte-autonomous TNF is critical for the function of these cells, as TNF ablation in monocytes/macrophages, but not in microglia, delayed the onset of EAE in challenged animals and was associated with reduced acute spinal cord infiltration of Ly6C^hi effector monocytes. Collectively, our data reveal a previously unappreciated critical cell-autonomous role of TNF on monocytes for their survival, maintenance, and function.

Hematopoietic-specific microRNA-142 is a critical regulator of various blood cell lineages, but its role in erythrocytes is unexplored. Herein, we characterize the impact of microRNA-142 on erythrocyte physiology and molecular cell biology, using a mouse loss-of-function allele. We report that microRNA-142 is required for maintaining the typical erythrocyte biconcave shape and structural resilience, for the normal metabolism of reactive oxygen species, and for overall lifespan. microRNA-142 further controls ACTIN filament homeostasis and membrane skeleton organization. The analyses presented reveal previously unappreciated functions of microRNA-142 and contribute to an emerging view of small RNAs as key players in erythropoiesis. Finally, the work herein demonstrates how a housekeeping network of cytoskeletal regulators can be reshaped by a single micro-RNA denominator in a cell type specific manner.

Monocytes are a conserved population of leukocytes that are present in all vertebrates, with some evidence of a parallel cell population in fly hemolymph (1). Monocytes are defined by their location in the bloodstream, their phenotype and nuclear morphology, as well as by their characteristic gene and microRNA expression signatures (2-5). In mice, monocytes represent 4% of the nucleated cells in the blood, with considerable marginal pools in the spleen and lungs that can be mobilized on demand (6,7). Within the blood, monocytes, and in particular the classical Ly6C+mouse subset, exhibit a characteristically short half-life of 20 h (8), akin to that of similar ephemer neutrophils (9).

Myelin is synthesized as a multilamellar membrane, but the mechanisms of membrane turnover are unknown. We found that myelin pieces were gradually released from aging myelin sheaths and were subsequently cleared by microglia. Myelin fragmentation increased with age and led to the formation of insoluble, lipofuscin-like lysosomal inclusions in microglia. Thus, age-related myelin fragmentation is substantial, leading to lysosomal storage and contributing to microglial senescence and immune dysfunction in aging.

To assess the role of alveolar macrophages (AMs) during a pulmonary Aspergillus fumigatus infection AMs were depleted by intratracheal application of diphtheria toxin (DTX) to transgenic CD11c.DTR mice prior to fungal infection. Unexpectedly, all CD11c.DTR mice treated with DTX died within 45 days, whether being infected with A. fumigatus or not. Despite measurable impact of DTX on lung functional parameters, these constrictions could not explain the high mortality rate. Instead, DTX-treated CD11c.DTR animals developed fulminant myocarditis (FM) characterized by massive leukocyte infiltration and myocardial cell destruction, including central parts of the heart's stimulus transmission system. In fact, standard limb lead ECG recordings of diseased but not healthy mice showed a \u201cBrugada\u201d-like pattern with an abnormally high ST segment pointing to enhanced susceptibility for potential lethal arrhythmias. While CD11c.DTR mice are extensively used for the characterization of CD11c⁺ cells, including dendritic cells, several studies have already mentioned adverse side effects following DTX treatment. Our results demonstrate that this limitation is based on severe myocarditis but not on the expected lung constrictions, and has to be taken into consideration if this animal model is used. Based on these properties, however, the CD11c.DTR mouse might serve as useful animal model for FM.

Microglia, the resident CNS macrophages, have been implicated in the pathogenesis of Rett Syndrome (RTT), an X-linked neurodevelopmental disorder. However, the mechanism by which microglia contribute to the disorder is unclear and recent data suggest that microglia do not play a causative role. Here, we use the retinogeniculate system to determine if and how microglia contribute to pathogenesis in a RTT mouse model, the Mecp2 null mouse (Mecp2^tm1.1Bird/y). We demonstrate that microglia contribute to pathogenesis by excessively engulfing, thereby eliminating, presynaptic inputs at end stages of disease (≥P56 Mecp2 null mice) concomitant with synapse loss. Furthermore, loss or gain of Mecp2 expression specifically in microglia (Cx3cr1^CreER; Mecp2^fl/yor Cx3cr1^CreER; Mecp2^LSL/y) had little effect on excessive engulfment, synapse loss, or phenotypic abnormalities. Taken together, our data suggest that microglia contribute to end stages of disease by dismantling neural circuits rendered vulnerable by loss of Mecp2 in other CNS cell types.

Perivascular, subdural meningeal and choroid plexus macrophages are non-parenchymal macrophages that mediate immune responses at brain boundaries. Although the origin of parenchymal microglia has recently been elucidated, much less is known about the precursors, the underlying transcriptional program and the dynamics of the other macrophages in the central nervous system (CNS). It was assumed that they have a high turnover from blood-borne monocytes. However, using parabiosis and fate-mapping approaches in mice, we found that CNS macrophages arose from hematopoietic precursors during embryonic development and established stable populations, with the notable exception of choroid plexus macrophages, which had dual origins and a shorter life span. The generation of CNS macrophages relied on the transcription factor PU.1, whereas the MYB, BATF3 and NR4A1 transcription factors were not required.

Astrocytes have important roles in the central nervous system (CNS) during health and disease. Through genome-wide analyses we detected a transcriptional response to type I interferons (IFN-Is) in astrocytes during experimental CNS autoimmunity and also in CNS lesions from patients with multiple sclerosis (MS). IFN-I signaling in astrocytes reduces inflammation and experimental autoimmune encephalomyelitis (EAE) disease scores via the ligand-Activated transcription factor aryl hydrocarbon receptor (AHR) and the suppressor of cytokine signaling 2 (SOCS2). The anti-inflammatory effects of nasally administered interferon (IFN)-β are partly mediated by AHR. Dietary tryptophan is metabolized by the gut microbiota into AHR agonists that have an effect on astrocytes to limit CNS inflammation. EAE scores were increased following ampicillin treatment during the recovery phase, and CNS inflammation was reduced in antibiotic-Treated mice by supplementation with the tryptophan metabolites indole, indoxyl-3-sulfate, indole-3-propionic acid and indole-3-Aldehyde, or the bacterial enzyme tryptophanase. In individuals with MS, the circulating levels of AHR agonists were decreased. These findings suggest that IFN-Is produced in the CNS function in combination with metabolites derived from dietary tryptophan by the gut flora to activate AHR signaling in astrocytes and suppress CNS inflammation.

The study of the intestinal dendritic cell (DC) compartment, its homeostasis, regulation, and response to challenges calls for the investigation within the physiological tissue context comprising the unique anatomic constellation of the epithelial single cell layer and the luminal microbiota, as well as neighboring immune and nonimmune cells. Here we provide protocols we developed that use a combination of conditional cell ablation, conditional compartment mutagenesis, and adoptive precursor transfers to study DC and other intestinal mononuclear phagocytes in in vivo context. We will highlight pitfalls and strengths of these approaches.

Background The developmental origin of the c-kit expressing progenitor cell pool in the adult heart has remained elusive. Recently, it has been discovered that the injured heart is enriched with c-kit⁺ cells, which also express the hematopoietic marker CD45. Methods and results In this study, we characterize the phenotype and transcriptome of the c-kit +/CD45 +/CD11b +/Flk-1 +/Sca-1 ± (B-type) cell population, originating from the left atrial appendage. These cells are defined as cardiac macrophage progenitors. We also demonstrate that the CD45 + progenitor cell population activates heart development, neural crest and pluripotency-associated pathways in vitro, in conjunction with CD45 down-regulation, and acquire a c-kit +/CD45 -/CD11b -/Flk-1 -/Sca-1 + (A-type) phenotype through cell fusion and asymmetric division. This putative spontaneous reprogramming evolves into a highly proliferative, partially myogenic phenotype (C-type). Conclusions Our data suggests that A-type cells and cardiac macrophage precursor cells (B-type) have a common lineage origin, possibly resolving some current conundrums in the field of cardiac regeneration.

Monocytes have emerged as critical driving force of acute inflammation. Here, we show that inhibition of Toll-like receptor 2(TLR2) dimerization by a TLR2 transmembrane peptide (TLR2-p) ameliorated DSS-induced colitis by interfering specifically with the activation of Ly6C⁺ monocytes without affecting their recruitment to the colon. We report that TLR2-p directly interacts with TLR2 within the membrane, leading to inhibition of TLR2-TLR6/1 assembly induced by natural ligands. This was associated with decreased levels of extracellular signal-regulated kinases (ERK) signaling and reduced secretion of pro-inflammatory cytokines, such as interleukin (IL)-6, IL-23, IL-12, and IL-1β. Altogether, our study provides insights into the essential role of TLR2 dimerization in the activation of pathogenic pro-inflammatory Ly6C^hi monocytes and suggests that inhibition of this aggregation by TLR2-p might have therapeutic potential in the treatment of acute gut inflammation. Synopsis Here, we utilize a novel strategy to neutralized TLR2 activation by inhibiting its dimerization by TLR2 transmembrane-derived peptide (TLR2-p). We show that TLR-2 peptide ameliorated DSS-induced colitis by interfering specifically with the activation of Ly6C⁺ monocytes without affecting their recruitment to the colon. The TLR2 transmembrane-derived peptide (TLR2-p) inhibits TLR2 signaling by interacting with its reciprocal receptors within the membrane. TLR2-p inhibits the dimerization of TLR2-TLR6/1 induced by natural ligands, resulting in attenuation of pro-inflammatory downstream signaling. Inhibition of TLR2 dimerization ameliorates acute colitis. TLR2-p inhibits TLR2 signaling in pathogenic pro-inflammatory Ly6C^hi monocytes without affecting their recruitment to the inflamed gut. A novel approach for blocking TLR2 signaling modulates immune cell activation and reduces disease severity during acute gut inflammation.

Macrophages provide a critical systemic network cells of the innate immune system. Emerging data suggest that in addition, they have important tissue-specific functions that range from clearance of surfactant from the lungs to neuronal pruning and establishment of gut homeostasis. The differentiation and tissue-specific activation of macrophages require precise regulation of gene expression, a process governed by epigenetic mechanisms such as DNA methylation, histone modification and chromatin structure. We argue that epigenetic regulation of macrophages is determined by lineage- and tissue-specific transcription factors controlled by the built-in programming of myeloid development in combination with signaling from the tissue environment. Perturbation of epigenetic mechanisms of tissue macrophage identity can affect normal macrophage tissue function and contribute to pathologies ranging from obesity and autoimmunity to neurodegenerative diseases.

Multiple sclerosis is the most frequent chronic inflammatory disease of the CNS. The entry and survival of pathogenic T cells in the CNS are crucial for the initiation and persistence of autoimmune neuroinflammation. In this respect, contradictory evidence exists on the role of the most potent type of antigen-presenting cells, dendritic cells. Applying intravital two-photon microscopy, we demonstrate the gatekeeper function of CNS professional antigen-presenting CD11c⁺ cells, which preferentially interact with Th17 cells. IL-17 expression correlates with expression of GM-CSF by T cells and with accumulation of CNS CD11c⁺ cells. These CD11c⁺ cells are organized in perivascular clusters, targeted by T cells, and strongly express the inflammatory chemokines Ccl5, Cxcl9, and Cxcl10. Our findings demonstrate a fundamental role of CNS CD11c⁺ cells in the attraction of pathogenic T cells into and their survival within the CNS.

Homo and heterozygote cx₃cr1 mutant mice, which harbor a green fluorescent protein (EGFP) in their cx₃cr1 loci, represent a widely used animal model to study microglia and peripheral myeloid cells. Here we report that microglia in the dentate gyrus (DG) of cx₃cr1^-/- mice displayed elevated microglial sirtuin 1 (SIRT1) expression levels and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) p65 activation, despite unaltered morphology when compared to cx₃cr1+/- or cx₃cr1+/+ controls. This phenotype was restricted to the DG and accompanied by reduced adult neurogenesis in cx₃cr1^-/- mice. Remarkably, adult neurogenesis was not affected by the lack of the CX3CR1-ligand, fractalkine (CX₃CL1). Mechanistically, pharmacological activation of SIRT1 improved adult neurogenesis in the DG together with an enhanced performance of cx₃cr1^-/- mice in a hippocampusdependent learning and memory task. The reverse condition was induced when SIRT1 was inhibited in cx₃cr1^-/- mice, causing reduced adult neurogenesis and lowered hippocampal cognitive abilities. In conclusion, our data indicate that deletion of CX3CR1 from microglia under resting conditions modifies brain areas with elevated cellular turnover independent of CX₃CL1.

Monocytes are short-lived mononuclear phagocytes that circulate in the bloodstream and comprise two main subpopulations that in the mouse are best defined by the Ly6C marker. Intravascular functions of "classical" Ly6C⁺ monocytes and their interactions with other lymphoid and myeloid leukocytes in the circulation remain poorly understood. Rather, these cells are known to efficiently extravasate into tissues. Indeed, Ly6C⁺ monocytes and their descendants have emerged as a third, highly plastic and dynamic cellular system that complements the two classical, tissue-resident mononuclear phagocyte compartments, i.e., macrophages and dendritic cells, on demand. Following recruitment to injured tissue, Ly6C⁺ monocytes respond to local cues and can critically contribute to the initiation and resolution of inflammatory reactions. The second main murine monocyte subset, Ly6C^- cells, derive in steady state from Ly6C⁺ monocytes and remain in the vasculature, where the cells act as scavengers. Moreover, a major fraction of Ly6C^- monocytes adheres to the capillary endothelium and patrols the vessel wall for surveillance. Given the central role of monocytes in homeostasis and pathology, in-depth study of this cellular compartment can be highly informative on the health state of the organism and provides an attractive target for therapeutic intervention.

Within the bone marrow, stem cells differentiate and give rise to diverse blood cell types and functions. Currently, hematopoietic progenitors are defined using surface markers combined with functional assays that are not directly linked with in vivo differentiation potential or gene regulatory mechanisms. Here, we comprehensively map myeloid progenitor sub-populations by transcriptional sorting of single cells from the bone marrow. We describe multiple progenitor subgroups, showing unexpected transcriptional priming toward seven differentiation fates but no progenitors with a mixed state. Transcriptional differentiation is correlated with combinations of known and previously undefined transcription factors, suggesting that the process is tightly regulated. Histone maps and knockout assays are consistent with early transcriptional priming, while traditional transplantation experiments suggest that in vivo priming may still allow for plasticity given strong perturbations. These data establish a reference model and general framework for studying hematopoiesis at single-cell resolution.

Macrophages can be of dual origin. Most tissue-resident macrophage compartments are generated before birth and subsequently maintain themselves independently from each other locally in healthy tissue. Under inflammatory conditions, these cells can however be complemented by macrophages derived from acute monocyte infiltrates. Due to the lack of suitable experimental systems, differential functional contributions of central nervous system (CNS)-resident microglia and monocyte-derived macrophages (MoMF) to CNS inflammation, such as experimental autoimmune encephalomyelitis (EAE), the mouse model of multiple sclerosis (MS), remain poorly understood. Here, we will review recent progress in this field that suggest distinct roles of microglia and MoMF in disease induction and progression, capitalizing on novel transgenic mouse models. The latter finding could have major implications for the rationale development of therapeutic approaches to the management of brain inflammation and MS therapy.

Emerging evidence suggests that immunological mechanisms underlie metabolic control of adipose tissue. Here, we have shown the regulatory impact of a rare subpopulation of dendritic cells, rich in perforin-containing granules (perf-DCs). Using bone marrow transplantation to generate animals selectively lacking perf-DCs, we found that these chimeras progressively gained weight and exhibited features of metabolic syndrome. This phenotype was associated with an altered repertoire of T cells residing in adipose tissue and could be completely prevented by T cell depletion in vivo. A similar impact of perf-DCs on inflammatory T cells was also found in a well-defined model of multiple sclerosis, experimental autoimmune encephlalomyelitis (EAE). Thus, perf-DCs probably represent a regulatory cell subpopulation critical for protection from metabolic syndrome and autoimmunity.

Microglia are macrophages of the central nervous system (CNS) that continuously scrutinize their environment for damage. They colonize the cephalic mesenchyme during embryogenesis and actively shape the developing neuronal network by immune-mediated mechanisms. Upon CNS maturation, microglia drastically change phenotype and function. During health, adult microglia contribute to homeostasis, but also the establishment and resolution of inflammatory conditions. Fulfillment of these distinct tasks requires these long-lived cells to accurately adjust to their changing environment. Deciphering microglia responsiveness to divergent stimuli is central to understanding this cell type and for eventual microglia manipulation to potentially reduce disease burden. Here we discuss new aspects of myeloid cell biology in general with special emphasis on the shifting role of microglia during establishment and protection of CNS integrity.

Recent technological advances have enabled researchers to accurately and efficiently assay the chromatin dynamics of scarce cell populations. In this Opinion article, we advocate the application of these technologies to central questions in immunology. Unlike changes to other molecular structures in the cell, chromatin features can reveal the past (developmental history), present (current activity) and future (potential response to challenges) of a given immune cell type; chromatin profiling is therefore an important new tool for studying the immune-regulatory networks of health and disease.

Localization of memory CD8⁺ T cells to lymphoid or peripheral tissues is believed to correlate with proliferative capacity or effector function. Here we demonstrate that the fractalkine-receptor/CX 3 CR1 distinguishes memory CD8⁺ T cells with cytotoxic effector function from those with proliferative capacity, independent of tissue-homing properties. CX₃ CR1-based transcriptome and proteome-profiling defines a core signature of memory CD8⁺ T cells with effector function. We find CD62L hi CX³ CR1⁺ memory T cells that reside within lymph nodes. This population shows distinct migration patterns and positioning in proximity to pathogen entry sites. Virus-specific CX₃CR1⁺ memory CD8⁺ T cells are scarce during chronic infection in humans and mice but increase when infection is controlled spontaneously or by therapeutic intervention. This CX₃ CR1-based functional classification will help to resolve the principles of protective CD8⁺ T-cell memory.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has emerged as a crucial cytokine produced by auto-reactive T helper (Th) cells that initiate tissue inflammation. Multiple cell types can sense GM-CSF, but the identity of the pathogenic GM-CSF-responsive cells is unclear. By using conditional gene targeting, we systematically deleted the GM-CSF receptor (Csf2rb) in specific subpopulations throughout the myeloid lineages. Experimental autoimmune encephalomyelitis (EAE) progressed normally when either classical dendritic cells (cDCs) or neutrophils lacked GM-CSF responsiveness. The development of tissue-invading monocyte-derived dendritic cells (moDCs) was also unperturbed upon Csf2rb deletion. Instead, deletion of Csf2rb in CCR2⁺Ly6C^hi monocytes phenocopied the EAE resistance seen in complete Csf2rb-deficient mice. High-dimensional analysis of tissue-infiltrating moDCs revealed that GM-CSF initiates a combination of inflammatory mechanisms. These results indicate that GM-CSF signaling controls a pathogenic expression signature in CCR2⁺Ly6C^hi monocytes and their progeny, which was essential for tissue damage.

During early embryogenesis, microglia arise from yolk sac progenitors that populate the developing central nervous system (CNS), but how the tissue-resident macrophages are maintained throughout the organism's lifespan still remains unclear. Here, we describe a system that allows specific, conditional ablation of microglia in adult mice. We found that the microglial compartment was reconstituted within 1week of depletion. Microglia repopulation relied on CNS-resident cells, independent from bone-marrow-derived precursors. During repopulation, microglia formed clusters of highly proliferative cells that migrated apart once steady state was achieved. Proliferating microglia expressed high amounts of the interleukin-1 receptor (IL-1R), and treatment with an IL-1R antagonist during the repopulation phase impaired microglia proliferation. Hence, microglia have the potential for efficient self-renewal without the contribution of peripheral myeloid cells, and IL-1R signaling participates in this restorative proliferation process.

Mutations in MECP2, encoding the epigenetic regulator methyl-CpG-binding protein 2, are the predominant cause of Rett syndrome, a disease characterized by both neurological symptoms and systemic abnormalities. Microglial dysfunction is thought to contribute to disease pathogenesis, and here we found microglia become activated and subsequently lost with disease progression in Mecp2-null mice. Mecp2 was found to be expressed in peripheral macrophage and monocyte populations, several of which also became depleted in Mecp2-null mice. RNA-seq revealed increased expression of glucocorticoid- and hypoxia-induced transcripts in Mecp2-deficient microglia and peritoneal macrophages. Furthermore, Mecp2 was found to regulate inflammatory gene transcription in response to TNF stimulation. Postnatal re-expression of Mecp2 using Cx3cr1(creER) increased the lifespan of otherwise Mecp2-null mice. These data suggest that Mecp2 regulates microglia and macrophage responsiveness to environmental stimuli to promote homeostasis. Dysfunction of tissue-resident macrophages might contribute to the systemic pathologies observed in Rett syndrome.

Macrophages are myeloid immune cells that are strategically positioned throughout the body tissues, where they ingest and degrade dead cells, debris, and foreign material and orchestrate inflammatory processes. Here we review two major recent paradigm shifts in our understanding of tissue macrophage biology. The first is the realization that most tissue-resident macrophages are established prenatally and maintained through adulthood by longevity and self-renewal. Their generation and maintenance are thus independent from ongoing hematopoiesis, although the cells can be complemented by adult monocyte-derived macrophages. Second, aside from being immune sentinels, tissue macrophages form integral components of their host tissue. This entails their specialization in response to local environmental cues to contribute to the development and specific function of their tissue of residence. Factors that govern tissue macrophage specialization are emerging. Moreover, tissue specialization is reflected in discrete gene expression profiles of macrophages, as well as epigenetic signatures reporting actual and potential enhancer usage.

Gut homeostasis and mucosal immune defense rely on the differential contributions of dendritic cells (DC) and macrophages. Here we show that colonic CX 3 CR1 + mononuclear phagocytes are critical inducers of the innate response to Citrobacter rodentium infection. Specifically, the absence of IL-23 expression in macrophages or CD11b + DC results in the impairment of IL-22 production and in acute lethality. Highlighting immunopathology as a death cause, infected animals are rescued by the neutralization of IL-12 or IFNγ. Moreover, mice are also protected when the CD103 + CD11b - DC compartment is rendered deficient for IL-12 production. We show that IL-12 production by colonic CD103 + CD11b - DC is repressed by IL-23. Collectively, in addition to its role in inducing IL-22 production, macrophage-derived or CD103 - CD11b + DC-derived IL-23 is required to negatively control the otherwise deleterious production of IL-12 by CD103 + CD11b - DC. Impairment of this critical mononuclear phagocyte crosstalk results in the generation of IFNγ-producing former TH17 cells and fatal immunopathology.

Intestinal mononuclear phagocytes find themselves in a unique environment, most prominently characterized by its constant exposure to commensal microbiota and food antigens. This anatomic setting has resulted in a number of specializations of the intestinal mononuclear phagocyte compartment that collectively contribute the unique steady state immune landscape of the healthy gut, including homeostatic innate lymphoid cells, B, and T cell compartments. As in other organs, macrophages and dendritic cells (DCs) orchestrate in addition the immune defense against pathogens, both in lymph nodes and mucosa-associated lymphoid tissue. Here, we will discuss origins and functions of intestinal DCs and macrophages and their respective subsets, focusing largely on the mouse and cells residing in the lamina propria.

Macrophages are critical for innate immune defense and also control organ homeostasis in a tissue-specific manner. They provide a fitting model to study the impact of ontogeny and microenvironment on chromatin state and whether chromatin modifications contribute to macrophage identity. Here, we profile the dynamics of four histone modifications across seven tissue-resident macrophage populations. We identify 12,743 macrophage-specific enhancers and establish that tissue-resident macrophages have distinct enhancer landscapes beyond what can be explained by developmental origin. Combining our enhancer catalog with gene expression profiles and open chromatin regions, we show that a combination of tissue- and lineage-specific transcription factors form the regulatory networks controlling chromatin specification in tissue-resident macrophages. The environment is capable of shaping the chromatin landscape of transplanted bone marrow precursors, and even differentiated macrophages can be reprogramed when transferred into a new microenvironment. These results provide a comprehensive view of macrophage regulatory landscape and highlight the importance of the microenvironment, along with pioneer factors in orchestrating identity and plasticity.

Several Alzheimer's disease (AD) risk genes are specifically expressed by microglia within the CNS. However, the mechanisms by which microglia regulate the pathological hallmarks of ADextracellular deposition of β-amyloid (Aβ) and intraneuronal hyperphosphorylation of microtubule-associated protein tau (MAPT)remain to be established. Notably, deficiency for the microglial CX3CR1 receptor has opposing effects on Aβ and MAPT pathologies. CX3CL1, the neuronally derived cognate ligand for CX3CR1, signals both in membrane-anchored and soluble forms. In this study, we sought to determine the relative contribution on membrane-anchored versus soluble CX3CL1 in regulating the microglia-mediated amelioration of Aβ pathology, as well as provide insight into the potential downstream microglial-based mechanisms. As expected, CX3CL1 deficiency reduced Aβ deposition in APPPS1 animals in a similar manner to CX3CR1 deficiency. Surprisingly, however, CX3CL1-deficient APPPS1 animals exhibited enhanced neuronal MAPT phosphorylation despite reduced amyloid burden. Importantly, neither of these phenotypes was altered by transgenic expression of the soluble CX3CL1 isoform, suggesting that it is the membrane-anchored version of CX3CL1 that regulates microglial phagocytosis of Aβ and neuronal MAPT phosphorylation. Analysis of transcript levels in purified microglia isolated from APPPS1 mice with the various CX3CL1/CX3CR1 genotypes revealed increased expression of inflammatory cytokines and phagocytic markers, which was associated with activation of p38 mitogen-activated protein kinase and Aβ internalization within microglia. Together, these studies challenge the \u201cfrustrated phagocytosis\u201d concept and suggest that neuronalmicroglial communication link the two central AD pathologies.

Microglia are highly specialized tissue macrophages of the brain with dedicated functions in neuronal development, homeostasis and recovery from pathology Despite their unique localization in the central nervous system (CNS), microglia are ontogenetically and functionally related to their peripheral counterparts of the mononuclear phagocytic system in the body, namely tissue macrophages and circulating myeloid cells. Recent developments provided new insights into the myeloid system in the body with microglia emerging as intriguing unique archetypes. Similar to other tissue macrophages, microglia develop early during embryogenesis from immature yolk sac progenitors. But in contrast to most of their tissue relatives microglia persist throughout the entire life of the organism without any significant input from circulating blood cells due to their longevity and their capacity of self-renewal. Notably, microglia share some features with short-lived blood monocytes to limit CNS tissue damage in pathologies, but only bone marrow-derived cells display the ability to become permanently integrated in the parenchyma. This emphasizes the therapeutic potential of bone marrow-derived microglia-like cells. Further understanding of both fate and function of microglia during CNS pathologies and considering their uniqueness among other tissue macrophages will be pivotal for potential manipulation of immune cell function in the CNS, thereby reducing disease burden. Here, we discuss new aspects of myeloid cell biology in general with special emphasis on the brain-resident macrophages and microglia.

Chromatin modifications are crucial for development, yet little is known about their dynamics during differentiation. Hematopoiesis provides a well-defined model to study chromatin state dynamics; however, technical limitations impede profiling of homogeneous differentiation intermediates. We developed a high-sensitivity indexing-first chromatin immunoprecipitation approach to profile the dynamics of four chromatin modifications across 16 stages of hematopoietic differentiation. We identify 48,415 enhancer regions and characterize their dynamics.We find that lineage commitment involves de novo establishment of 17,035 lineage-specific enhancers. These enhancer repertoire expansions foreshadow transcriptional programs in differentiated cells. Combining our enhancer catalog with gene expression profiles, we elucidate the transcription factor network controlling chromatin dynamics and lineage specification in hematopoiesis. Together, our results provide a comprehensive model of chromatin dynamics during development.

Monocytes and macrophages have crucial and distinct roles in tissue homeostasis and immunity, but they also contribute to a broad spectrum of pathologies and are thus attractive therapeutic targets. Potential intervention strategies that aim to manipulate these cells will require an in-depth understanding of their origins and the mechanisms that ensure their homeostasis. Recent evidence shows that monocytes do not substantially contribute to most tissue macrophage populations in the steady state or during certain types of inflammation. Rather, most tissue macrophage populations in mice are derived from embryonic precursors, are seeded before birth and can maintain themselves in adults by self-renewal. In this Review, we discuss the evidence that has dramatically changed our understanding of monocyte and macrophage development, and the maintenance of these cells in the steady state.

Genome-encoded microRNAs (miRNAs) provide a posttranscriptional regulatory layer that controls the differentiation and function of various cellular systems, including hematopoietic cells. miR-142 is one of the most prevalently expressed miRNAs within the hematopoietic lineage. To address the in vivo functions of miR-142 we utilized a novel reporter and loss-of-function mouse allele that we have recently generated. Here, we show that miR-142 is broadly expressed in the adult hematopoietic system. Our data further reveal that miR-142 is critical for megakaryopoiesis. Thus, genetic miR-142 ablation caused impaired megakaryocyte maturation, inhibition of polyploidization, abnormal proplatelet formation, and thrombocytopenia. Finally, we characterize a network of miR-142-3p targets which collectively controls actin filament homeostasis, thereby ensuring proper execution of actin-dependent proplatelet formation. Our study reveals a pivotal role for miR-142 activity in megakaryocyte maturation and function, and demonstrates a critical contribution of a single miRNA in orchestrating cytoskeletal dynamics and normal haemostasis.

Classical dendritic cells (cDCs) form a critical interface between innate and adaptive immunity. As myeloid immune cell sentinels, cDCs are specialized in the sensing of pathogen challenges and cancer. They translate the latter for Tcells into peptide form. Moreover, cDCs provide additional critical information on the original antigen context to trigger a diverse spectrum of appropriate protective responses. Here we review recent progress in our understanding of cDC subsets in mice. We will discuss cDC subset ontogeny and transcription factor dependencies, as well as emerging functional specializations within the cDC compartment in lymphoid and nonlymphoid tissues.

Interleukin-10 (IL-10) is a pleiotropic anti-inflammatory cytokine produced and sensed by most hematopoietic cells. Genome-wide association studies and experimental animal models point at a central role of the IL-10 axis in inflammatory bowel diseases. Here we investigated the importance of intestinal macrophage production of IL-10 and their IL-10 exposure, as well as the existence of an IL-10-based autocrine regulatory loop in the gut. Specifically, we generated mice harboring IL-10 or IL-10 receptor (IL-10Rα) mutations in intestinal lamina propria-resident chemokine receptor CX₃CR1-expressing macrophages. We found macrophage-derived IL-10 dispensable for gut homeostasis and maintenance of colonic T regulatory cells. In contrast, loss of IL-10 receptor expression impaired the critical conditioning of these monocyte-derived macrophages and resulted in spontaneous development of severe colitis. Collectively, our results highlight IL-10 as a critical homeostatic macrophage-conditioning agent in the colon and define intestinal CX₃CR1^hi macrophages as a decisive factor that determines gut health or inflammation.

Eosinophilia is a hallmark characteristic of T helper type 2 (T _H2) cell-associated diseases and is critically regulated by the central eosinophil growth factor interleukin 5 (IL-5). Here we demonstrate that IL-5 activity in eosinophils was regulated by paired immunoglobulin-like receptors PIR-A and PIR-B. Upon self-recognition of β₂- microglobulin (β₂M) molecules, PIR-B served as a permissive checkpoint for IL-5-induced development of eosinophils by suppressing the proapoptotic activities of PIR-A, which were mediated by the Grb2-Erk-Bim pathway. PIR-B-deficient bone marrow eosinophils underwent compartmentalized apoptosis, resulting in decreased blood eosinophilia in naive mice and in mice challenged with IL-5. Subsequently, Pirb^-/- mice displayed impaired aeroallergen-induced lung eosinophilia and induction of lung TH₂ cell responses. Collectively, these data uncover an intrinsic, self-limiting pathway regulating IL-5-induced expansion of eosinophils, which has broad implications for eosinophil-associated diseases.

In multicellular organisms, biological function emerges when heterogeneous cell types form complex organs. Nevertheless, dissection of tissues into mixtures of cellular subpopulations is currently challenging. We introduce an automated massively parallel single-cell RNA sequencing (RNA-seq) approach for analyzing in vivo transcriptional states in thousands of single cells. Combined with unsupervised classification algorithms, this facilitates ab initio cell-type characterization of splenic tissues. Modeling single-cell transcriptional states in dendritic cells and additional hematopoietic cell types uncovers rich cell-type heterogeneity and gene-modules activity in steady state and after pathogen activation. Cellular diversity is thereby approached through inference of variable and dynamic pathway activity rather than a fixed preprogrammed cell-type hierarchy. These data demonstrate single-cell RNA-seq as an effective tool for comprehensive cellular decomposition of complex tissues.

Communication between different immune cells of the intestinal mucosa acts as a rheostat that maintains tolerance to gut microbes.

Cardiac macrophages (cMΦ) are critical for early postnatal heart regeneration and fibrotic repair in the adult heart, but their origins and cellular dynamics during postnatal development have not been well characterized. Tissue macrophages can be derived from embryonic progenitors or from monocytes during inflammation. We report that within the first weeks after birth, the embryo-derived population of resident CX3CR1+ cMΦ diversifies into MHCII+ and MHCII- cells. Genetic fate mapping demonstrated that cMΦ derived from CX3CR1⁺ embryonic progenitors persisted into adulthood but the initially high contribution to resident cMΦ declined after birth. Consistent with this, the early significant proliferation rate of resident cMΦ decreased with age upon diversification into subpopulations. Bone marrow (BM) reconstitution experiments showed monocyte-dependent quantitative replacement of all cMΦ populations. Furthermore, parabiotic mice and BM chimeras of nonirradiated recipient mice revealed a slow but significant donor contribution to cMΦ. Together, our observations indicate that in the heart, embryo-derived cMΦ show declining self-renewal with age and are progressively substituted by monocyte-derived macrophages, even in the absence of inflammation.

Adult neovascularization relies on the recruitment of monocytes to the target organ or tumor and functioning therein as a paracrine accessory. The exact origins of the recruited monocytes and the mechanisms underlying their plasticity remain unclear. Using a VEGFbased transgenic system in which genetically tagged monocytes are conditionally summoned to the liver as part of a VEGF-initiated angiogenic program, we show that these recruited cells are derived from the abundant pool of circulating Ly6Chi monocytes. Remarkably, however, upon arrival at the VEGF-induced organ, but not the naive organ, monocytes undergo multiple phenotypic and functional changes, endowing them with enhanced proangiogenic capabilities and, importantly, with a markedly increased capacity to remodel existing small vessels into larger conduits. Notably, monocytes do not differentiate into long-lived macrophages, but rather appear as transient accessory cells. Results from transfers of presorted subpopulations and a novel tandem transfer strategy ruled out selective recruitment of a dedicated preexisting subpopulation or onsite selection, thereby reinforcing active reprogramming as the underlying mechanism for improved performance. Collectively, this study uncovered a novel function of VEGF, namely, on-site education of recruited "standard" monocytes to become angiogenic and arteriogenic professional cells, a finding that may also lend itself for a better design of angiogenic therapies.

Microglia are brain macrophages and, as such, key immune-competent cells that can respond to environmental changes. Understanding the mechanisms of microglia-specific responses during pathologies is hence vital for reducing disease burden. The definition of microglial functions has so far been hampered by the lack of genetic in vivo approaches that allow discrimination of microglia from closely related peripheral macrophage populations in the body. Here we introduce a mouse experimental system that specifically targets microglia to examine the role of a mitogen-associated protein kinase kinase kinase (MAP3K), transforming growth factor (TGF)-β-activated kinase 1 (TAK1), during autoimmune inflammation. Conditional depletion of TAK1 in microglia only, not in neuroectodermal cells, suppressed disease, significantly reduced CNS inflammation and diminished axonal and myelin damage by cell-autonomous inhibition of the NF-κB, JNK and ERK1/2 pathways. Thus, we found TAK1 to be pivotal in CNS autoimmunity, and we present a tool for future investigations of microglial function in the CNS.

Classical dendritic cells (cDC) are specialized antigen-presenting cells mediating immunity and tolerance. cDC cell-lineage decisions are largely controlled by transcriptional factor regulatory cascades. Using an in vivo cell-specific targeting of Runx3 at various stages of DC lineage development we show that Runx3 is required for cell-identity, homeostasis and function of splenic Esam^hi DC. Ablation of Runx3 in DC progenitors led to a substantial decrease in splenic CD4⁺/CD11b⁺ DC. Combined chromatin immunoprecipitation sequencing and gene expression analysis of purified DC-subsets revealed that Runx3 is a key gene expression regulator that facilitates specification and homeostasis of CD11b⁺Esam^hi DC. Mechanistically, loss of Runx3 alters Esam^hi DC gene expression to a signature characteristic of WT Esam^low DC. This transcriptional reprogramming caused a cellular change that diminished phagocytosis and hampered Runx3^-/- Esam^hi DC capacity to prime CD4⁺ T cells, attesting to the significant role of Runx3 in specifying Esam^hi DC identity and function.

Over the past 25 years, genetically engineered mouse models have become an integral and invaluable research tool to develop our understanding of mammalian physiology and pathology. This unit describes methods for generating transgenic mice, focusing on reporter animals relevant to chemokine receptor and ligand expression. Specifically, we describe the use of bacterial artificial chromosome (BAC) engineering and embryonic stem cell manipulation to generate "knock in" and transgenic mice.

Macrophages are the most abundant mononuclear phagocytes in the healthy intestinal lamina propria and have emerged as crucial sentinels for the maintenance of tissue homeostasis. Matching the dynamic mucosal landscape, CX₃C chemokine receptor (CX₃CR)1-expressing macrophages are relatively short lived, and as opposed to most other tissue macrophages, are continuously replaced from blood monocytes that acquire in the healthy tissue context a robust noninflammatory gene expression signature. By contrast, during gut inflammation, monocytes differentiate in the gut into proinflammatory effector cells, as well as migratory antigen-presenting cells. Manipulation of monocyte fates in the intestine might hold promise for the disease management of inflammatory bowel disorders.

Monocyte-derived macrophages are essential for recovery after spinal cord injury, but their homing mechanism is poorly understood. Here, we show that although of common origin, the homing of proinflammatory (M1) and the " alternatively activated" anti-inflammatory (M2) macrophages to traumatized spinal cord (SC) was distinctly regulated, neither being through breached blood-brain barrier. The M1 macrophages (Ly6c^hiCX3CR1^lo) derived from monocytes homed in a CCL2 chemokine-dependent manner through the adjacent SC leptomeninges. The resolving M2 macrophages (Ly6c^loCX3CR1^hi) derived from monocytes trafficked through a remote blood-cerebrospinal-fluid (CSF) barrier, the brain-ventricular choroid plexus (CP), via VCAM-1-VLA-4 adhesion molecules and epithelial CD73 enzyme for extravasation and epithelial transmigration. Blockage of these determinants, or mechanical CSF flow obstruction, inhibited M2 macrophage recruitment and impaired motor-function recovery. The CP, along with the CSF and the central canal, provided an anti-inflammatory supporting milieu, potentially priming the trafficking monocytes. Overall, our finding demonstrates that the route of monocyte entry to central nervous system provides an instructional environment to shape their function.

Microglial cells in brain and spinal cord are characterized by high expression of the chemokine receptor CX₃CR1. Expression of the sole CX₃CR1 ligand, the membrane-tethered and sheddable chemokine CX₃CL1/fractalkine, is restricted in the brain parenchyma to selected neurons. Here we summarize our current understanding of the physiological role of CX₃CR1 for microglia function and the CX₃C axis in microglial/neuronal crosstalk in homeostasis and under challenge. Moreover, we will discuss the efforts of our laboratory and others to exploit CX₃CR1 promoter activity for the visualization and genetic manipulation of microglia to probe their functional contributions in the central nerve system (CNS) context.

Intestinal mononuclear phagocytes have collectively emerged as key players in the maintenance of gut homeostasis, the development of gut inflammation and its resolution. Moreover, recent intense research efforts of many laboratories have revealed evidence for critical labor division between lamina propria-resident CD103 + dendritic cells and CX3CR1 + macrophages. In depth understanding of the respective activities of these cells in the mucosal landscape might pave the way for novel treatments of inflammatory bowel disorders (IBD).

The mononuclear phagocyte system comprises cells as diverse as monocytes, macrophages, and dendritic cells (DCs), which collectively play key roles in innate immune responses and the triggering of adaptive immunity. Recent studies have highlighted the role of growth and transcription factors in defining developmental pathways and lineage relations within this cellular compartment. However, contributions of miRNAs to the development of mononuclear phagocytes remain largely unknown. In the present study, we report a comprehensive map of miRNA expression profiles for distinct myeloid populations, including BM-resident progenitors, monocytes, and mature splenic DCs. Each of the analyzed cell populations displayed a distinctive miRNA profile, suggesting a role for miRNAs in defining myeloid cell identities. Focusing on DC development, we found miR-142 to be highly expressed in classic FLT3-L-dependent CD4⁺ DCs, whereas reduced expression was observed in closely related CD8α ⁺ or CD4^-CD8α^- DCs. Moreover, mice deficient for miR-142 displayed an impairment of CD4⁺ DC homeostasis both in vitro and in vivo. Furthermore, loss of miR-142-dependent CD4 ⁺ DCs was accompanied by a severe and specific defect in the priming of CD4⁺ T cells. The results of our study establish a novel role for miRNAs in myeloid cell specification and define miR-142 as a pivotal genetic component in the maintenance of CD4⁺ DCs.

Mononuclear phagocytes, including monocytes, macrophages, and dendritic cells, contribute to tissue integrity as well as to innate and adaptive immune defense. Emerging evidence for labor division indicates that manipulation of these cells could bear therapeutic potential. However, specific ontogenies of individual populations and the overall functional organization of this cellular network are not well defined. Here we report a fate-mapping study of the murine monocyte and macrophage compartment taking advantage of constitutive and conditional CX₃CR1 promoter-driven Cre recombinase expression. We have demonstrated that major tissue-resident macrophage populations, including liver Kupffer cells and lung alveolar, splenic, and peritoneal macrophages, are established prior to birth and maintain themselves subsequently during adulthood independent of replenishment by blood monocytes. Furthermore, we have established that short-lived Ly6C⁺ monocytes constitute obligatory steady-state precursors of blood-resident Ly6C^- cells and that the abundance of Ly6C⁺ blood monocytes dynamically controls the circulation lifespan of their progeny.

Recent insights into discrete myeloid developmental pathways have provided critical information about the organization of the murine mononuclear phagocyte compartment. Short-lived dendritic cells (DCs) have been shown to continuously arise from dedicated bone marrow-derived precursors. In contrast, it is now appreciated that most tissue macrophage populations are established before birth and subsequently maintain themselves throughout adulthood by longevity and limited self-renewal. Both of these classical tissue-resident mononuclear phagocyte compartments can be complemented on demand by monocyte infiltrates giving rise to macrophage or DC-like cells, depending on the tissue context they encounter upon extravasation. Monocytes hence have emerged as a versatile emergency squad that can be rapidly recruited to sites of injury to provide a transient supplement with proinflammatory or resolving activities for local mononuclear phagocytes.

The bone marrow (BM) hosts memory lymphocytes and supports secondary immune responses against blood-borne antigens, but it is unsettled whether primary responses occur there and which cells present the antigen. We used 2-photon microscopy in the BM of live mice to study these questions. Naïve CD8+ T cells crawled rapidly at steady state but arrested immediately upon sensing antigenic peptides. Following infusion of soluble protein, various cell types were imaged ingesting the antigen, while antigen-specific T cells decelerated, clustered, upregulated CD69, and were observed dividing in situ to yield effector cells. Unlike in the spleen, T-cell responses persisted when BM-resident dendritic cells (DCs) were ablated but failed when all phagocytic cells were depleted. Potential antigen-presenting cells included monocytes and macrophages but not B cells. Collectively, our results suggest that the BM supports crosspresentation of blood-borne antigens similar to the spleen; uniquely, alongside DCs, other myeloid cells participate in crosspresentation.

Ly6C^hi monocytes seed the healthy intestinal lamina propria to give rise to resident CX₃CR1⁺ macrophages that contribute to the maintenance of gut homeostasis. Here we report on two alternative monocyte fates in the inflamed colon. We showed that CCR2 expression is essential to the recruitment of Ly6C^hi monocytes to the inflamed gut to become the dominant mononuclear cell type in the lamina propria during settings of acute colitis. In the inflammatory microenvironment, monocytes upregulated TLR2 and NOD2, rendering them responsive to bacterial products to become proinflammatory effector cells. Ablation of Ly6C^hi monocytes ameliorated acute gut inflammation. With time, monocytes differentiated into migratory antigen-presenting cells capable of priming naive T cells, thus acquiring hallmarks reminiscent of dendritic cells. Collectively, our results highlight cellular dynamics in the inflamed colon and the plasticity of Ly6C^hi monocytes, marking them as potential targets for inflammatory bowel disease (IBD) therapy.

Hematopoietic stem and progenitor cells (HSPCs) are regulated by various bone marrow stromal cell types. Here we identified rare activated bone marrow monocytes and macrophages with high expression of α-smooth muscle actin (α-SMA) and the cyclooxygenase COX-2 that were adjacent to primitive HSPCs. These myeloid cells resisted radiation-induced cell death and further upregulated COX-2 expression under stress conditions. COX-2-derived prostaglandin E 2 (PGE 2) prevented HSPC exhaustion by limiting the production of reactive oxygen species (ROS) via inhibition of the kinase Akt and higher stromal-cell expression of the chemokine CXCL12, which is essential for stem-cell quiescence. Our study identifies a previously unknown subset of α-SMA + activated monocytes and macrophages that maintain HSPCs and protect them from exhaustion during alarm situations.

Although most genes are expressed biallelically, a number of key genomic sites-including immune and olfactory receptor regions-are controlled monoallelically in a stochastic manner, with some cells expressing the maternal allele and others the paternal allele in the target tissue. Very little is known about how this phenomenon is regulated and programmed during development. Here, using mouse immunoglobulin-κ (Igκ) as a model system, we demonstrate that although individual haematopoietic stem cells are characterized by allelic plasticity, early lymphoid lineage cells become committed to the choice of a single allele, and this decision is then stably maintained in a clonal manner that predetermines monoallelic rearrangement in B cells. This is accompanied at the molecular level by underlying allelic changes in asynchronous replication timing patterns at the κ locus. These experiments may serve to define a new concept of stem cell plasticity.

Mature dendritic cells (DCs) are established as unrivaled antigen-presenting cells (APCs) in the initiation of immune responses, whereas steady-state DCs induce peripheral T cell tolerance. Using various genetic approaches, we depleted CD11c⁺ DCs in mice and induced autoimmune CNS inflammation. Unexpectedly, mice lacking DCs developed aggravated disease compared to control mice. Furthermore, when we engineered DCs to present a CNS-associated autoantigen in an induced manner, we found robust tolerance that prevented disease, which coincided with an upregulation of the PD-1 receptor on antigen-specific T cells. Additionally, we showed that PD-1 was necessary for DC-mediated induction of regulatory T cells. Our results show that a reduction of DCs interferes with tolerance, resulting in a stronger inflammatory response, and that other APC populations could compensate for the loss of immunogenic APC function in DC-depleted mice.

Immature dendritic cells (imDCs) can have a tolerizing effect under normal conditions or after transplantation. However, because of the significant heterogeneity of this cell population, it is extremely difficult to study the mechanisms that mediate the tolerance induced or to harness the application of imDCs for clinical use. In the present study, we describe the generation of a highly defined population of imDCs from hematopoietic progenitors and the direct visualization of the fate of TCR-transgenic alloreactive CD4+ and CD8+ T cells after encountering cognate or noncognate imDCs. Whereas CD4+ T cells were deleted via an MHC-independent mechanism through the NO system, CD8+ T-cell deletion was found to occur through a unique MHC-dependent, perforin-based killing mechanism involving activation of TLR7 and signaling through Triggering Receptor-1 Expressed on Myeloid cells (TREM-1). This novel subpopulation of perforinexpressing imDCs was also detected in various lymphoid tissues in normal animals and its frequency was markedly enhanced after GM-CSF administration.

Microglia are myeloid-derived cells that colonize the central nervous system (CNS) at early stages of development and constitute up to 20% of the glial populations throughout life. While extensive progress has been recently made in identifying the cellular origin of microglia, the mechanism whereby the cells acquire the unique ramified and quiescent phenotype within the CNS milieu remains unknown. Here, we show that upon co-culturing of either CD117⁺/Lin^- hematopoietic progenitors or CD11c⁺ bone marrow derived cells with organotypic hippocampal slices or primary glia, the cells acquire a ramified morphology concomitant with reduced levels of CD86, MHCII, and CD11c and up-regulation of the microglial cell-surface proteins CX₃CR1 and Iba-1. We further demonstrate that the transforming growth factor beta (TGF-β) signaling pathway via SMAD2/3 phosphorylation is essential for both primary microglia and myeloid-derived cells in order to acquire their quiescent phenotype. Our study suggests that the abundant expression of TGF-β within the CNS during development and various inflammatory processes plays a key role in promoting the quiescent phenotype of microglia and may thus serve as a target for therapeutic strategies aimed at modulating the function of microglia in neurodegenerative diseases such as Alzheimer's and prion.

Mice are exceedingly sensitive to intra-peritoneal (IP) challenge with some virulent pneumococci (LD50 = 1 bacterium). To investigate how peripheral contact with bacterial capsular polysaccharide (PS) antigen can induce resistance, we pulsed bone marrow dendritic cells (BMDC) of C57BL/6 mice with type 4 or type 3 PS, injected the BMDC intra-foot pad (IFP) and challenged the mice IP with supra-lethal doses of pneumococci. We examined the responses of T cells and B cells in the draining popliteal lymph node and measured the effects on the bacteria in the peritoneum and blood. We now report that: 1) The PS co-localized with MHC molecules on the BMDC surface; 2) PS-specific T and B cell proliferation and IFNγ secretion was detected in the draining popliteal lymph nodes on day 4; 3) Type-specific resistance to lethal IP challenge was manifested only after day 5; 4) Type-specific IgM and IgG antibodies were detected in the sera of only some of the mice, but B cells were essential for resistance; 5) Control mice vaccinated with a single injection of soluble PS did not develop a response in the draining popliteal lymph node and were not protected; 6) Mice injected with unpulsed BMDC also did not resist challenge: In unprotected mice, pneumococci entered the blood shortly after IP inoculation and multiplied exponentially in both blood and peritoneum killing the mice within 20 hours. Mice vaccinated with PS-pulsed BMDC trapped the bacteria in the peritoneum. The trapped bacteria proliferated exponentially IP, but died suddenly at 18-20 hours. Thus, a single injection of PS antigen associated with intact BMDC is a more effective vaccine than the soluble PS alone. This model system provides a platform for studying novel aspects of PS-targeted vaccination.

BID, a BH3-only BCL2 family member, functions in apoptosis as well as the DNA-damage response. Our previous data demonstrated that BID is an ATM effector acting to induce cell-cycle arrest and inhibition of apoptosis following DNA damage. Here we show that ATM-mediated BID phosphorylation plays an unexpected role in maintaining the quiescence of haematopoietic stem cells (HSCs). Loss of BID phosphorylation leads to escape from quiescence of HSCs, resulting in exhaustion of the HSC pool and a marked reduction of HSC repopulating potential in vivo. We also demonstrate that BID phosphorylation plays a role in protecting HSCs from irradiation, and that regulating both quiescence and survival of HSCs depends on BID's ability to regulate oxidative stress. Moreover, loss of BID phosphorylation, ATM knockout or exposing mice to irradiation leads to an increase in mitochondrial BID, which correlates with an increase in mitochondrial oxidative stress. These results show that the ATM-BID pathway serves as a critical checkpoint for coupling HSC homeostasis and the DNA-damage stress response to enable long-term regenerative capacity.

The CCL2 CCR2 axis is likely to contributes to the development and progression of cancer diseases by two major mechanisms; autocrine effect of CCL2 as a survival/growth factor for CCR2+ cancer cells and, the attraction of CCR2+ CX₃CR1+tumor associated macrophages that in the absence of CCR2 hardly migrate. Thus far no in vivo system has been set up to differentiate the selective contribution of each of these features to cancer development. Here we employed a chimera animal model in which all non-malignant cells are CCR2-/-, but all cancer cells are CCR2+, combined with an adoptive transfer system of bone marrow (BM) CX₃CR1+ cells from CCR2+ mice harboring a targeted replacement of the CX₃CR1gene by an enhanced green fluorescent protein (EGFP) reporter gene (cx₃cr1^gfp), together with the CD45.1 congene. Using this system we dissected the selective contribution of CX₃CR1+CCR2+ cells, which comprise only about 7% of CD11b+ BM cells, to tumor development and angiogenesis. Showing that aside for their direct pro-angiogenic effect they are essential for the recruitment of other CD11b+ cells to the tumor site. We further show that the administration of CCR2-Ig, that selectively and specifically neutralize CCL2, to mice in which CCR2 is expressed only on tumor cells, further suppressed tumor development, implicating for the key role of this chemokine supporting tumor survival in an autocrine manner. This further emphasizes the important role of CCL2 as a target for therapy of cancer diseases.

The brain has been commonly regarded as a "tissue behind walls." Appearance of immune cells in the brain has been taken as a sign of pathology. Moreover, since infiltrating monocyte-derived macrophages and activated resident microglia were indistinguishable by conventional means, both populations were considered together as inflammatory cells that should be mitigated.Yet, because the microglia permanently reside in the brain, attributing to them negative properties evoked an ongoing debate; why cells that are supposed to be the brain guardians acquire only destructive potential? Studies over the last two decades in the immune arena in general, and in the context of central nervous system pathology in particular, have resulted in a paradigm shift toward a more balanced appreciation of the contributions of immune cells in the context of brain maintenance and repair, and toward the recognition of distinct roles of resident microglia and infiltrating monocyte-derived macrophages.

Group A Streptococcus (GAS) causes diverse infections in humans, ranging from mild to life-threatening invasive diseases, such as necrotizing fasciitis (NF), a rapidly progressing deep tissue infection. Despite prompt treatments, NF remains a significant cause of morbidity and mortality, even in previously healthy individuals. The early recruitment of leukocytes is crucial to the outcome of NF; however, although the role of polymorphonuclear neutrophils (PMNs) in host defense against NF is well established, the role of recruited macrophages remains poorly defined. Using a cutaneous murine model mimicking human NF, we found that mice deficient in TNF-α were highly susceptible to s.c. infections with GAS, and a paucity of macrophages, but not PMNs, was demonstrated. To test whether the effects of TNF-α on the outcome of infection are mediated by macrophages/monocytes, we systemically depleted C57BL/6 mice of monocytes by pharmacological and genetic approaches. Systemic monocyte depletion substantially increased bacterial dissemination from soft tissues without affecting the number of recruited PMNs or altering the bacterial loads in soft tissues. Enhanced GAS dissemination could be reverted by either i.v. injection of monocytes or s.c. administration of peritoneal macrophages. These experiments demonstrated that recruited macrophages play a key role in defense against the extracellular pathogen GAS by limiting its spread from soft tissues.

Becoming covered in platelets rescues complement-opsonized blood-borne bacteria from rapid clearance by macrophages and redirects them to dendritic cells. Although this allows priming of T cells and the generation of immune memory, bacteria can exploit this route as a beachhead and disseminate throughout host tissues.

The CX₃C chemokine family is composed of only one member, CX₃CL1, also known as fractalkine, which in mice is the sole ligand of the G protein-coupled, 7-transmembrane receptor CX₃CR1. Unlike classic small peptide chemokines, CX₃CL1 is synthesized as a membrane-anchored protein that can promote integrin-independent adhesion. Subsequent cleavage by metalloproteases, either constitutive or induced, can generate shed CX₃CL1 entities that potentially have chemoattractive activity. To study the CX₃C interface in tissues of live animals, we generated transgenic mice (CX₃CL1^cherry:CX ₃CR1^gfp), which express red and green fluorescent reporter genes under the respective control of the CX₃CL1 and CX ₃CR1 promoters. Furthermore, we performed a structure/function analysis to differentiate the in vivo functions of membrane-tethered versus shed CX₃CL1 moieties by comparing their respective ability to correct established defects in macrophage function and leukocyte survival in CX ₃CL1-deficient mice. Specifically, expression of CX ₃CL1^105Δ, an obligatory soluble CX₃CL1 isoform, reconstituted the formation of transepithelial dendrites by intestinal macrophages but did not rescue circulating Ly6C^lo CX ₃CR1^hi blood monocytes in CX₃CR1 ^gfp/gfp mice. Instead, monocyte survival required the full-length membrane-anchored CX₃CL1, suggesting differential activities of tethered and shed CX₃CL1 entities.

Dendritic cells (DCs) in tissues and lymphoid organs comprise distinct functional subsets that differentiate in situ from circulating progenitors. Tissue-specific signals that regulate DC subset differentiation are poorly understood. We report that DC-specific deletion of the Notch2 receptor caused a reduction of DC populations in the spleen. Within the splenic CD11b ⁺ DC subset, Notch signaling blockade ablated a distinct population marked by high expression of the adhesion molecule Esam. The Notch-dependent Esam ^hi DC subset required lymphotoxin beta receptor signaling, proliferated in situ, and facilitated CD4 ⁺ T cell priming. The Notch-independent Esam ^lo DCs expressed monocyte-related genes and showed superior cytokine responses. In addition, Notch2 deletion led to the loss of CD11b ⁺CD103 ⁺ DCs in the intestinal lamina propria and to a corresponding decrease of IL-17-producing CD4 ⁺ T cells in the intestine. Thus, Notch2 is a common differentiation signal for T cell-priming CD11b ⁺ DC subsets in the spleen and intestine.

The olfactory epithelium is a site of sustained adult neurogenesis where olfactory sensory neurons are continuously replaced from endogenous stem/progenitor cells. Epithelial macrophages have been implicated in the phagocytosis of degenerating cells but the molecular mechanisms allowing for their recruitment and activation while maintaining a neurogenic microenvironment are poorly understood. We have previously shown that the chemokine fractalkine (CX₃CL1) is expressed by olfactory sensory neurons and ensheathing cells in the olfactory epithelium. In turn, the fractalkine receptor, CX₃CR1, is expressed on macrophages and dendritic cells within the olfactory epithelium. We report that a selective cell death of olfactory sensory neurons in the epithelium of CX₃CR1-deficient mice via target ablation (i.e. olfactory bulbectomy) results in an exacerbated loss of olfactory sensory neurons compared to wild-type mice. In addition, reduced proliferation of intraepithelial stem/progenitor cells was observed in lesioned CX₃CR1-deficient mice, suggesting an impaired regenerative response. Importantly, a lack of CX₃CL1-signaling caused increased recruitment of macrophages into the olfactory epithelium, which in turn contained higher levels of pro-inflammatory cytokines (e.g. TNF-α and IL-6) as determined by qPCR. We also present novel data showing that, relative to wild-type, CX₃CR1-deficient macrophages have diminished phagocytic activity following stimulation with CX₃CL1. Collectively, our data indicate that signaling through the CX₃CR1 receptor modulates macrophage activity, resulting in an environment conducive to olfactory sensory neuron clearance and targeted replacement from endogenous stem/progenitor cells.

Inflammatory bowel disease is a chronic inflammatory disorder of the gastrointestinal tract associated with alterations and dysfunction of the intestinal microvasculature. The goal of this work was to develop a preclinical protocol for quantitative functional characterization of the colonic microvasculature in a murine colitis model. Experimental colitis was induced in mice by addition of dextran sodium sulfate to the drinking water. Histopathologic analysis revealed severe multifocal colitis. Dynamics of intravenously injected macromolecular dextran-FITC and biotin-BSA-GdDTPA in the colonic microvasculature were imaged using fluorescent confocal endomicroscopy and MRI (9.4 T), respectively. Both MRI and fluorescent confocal endomicroscopy revealed a substantial increase in the permeability of the colonic microvasculature associated with colitis, resulting in extravascular accumulation of the macromolecular contrast agent in the lumen of the colon. MRI data were validated by immunohistochemical staining of the contrast agent and leakage of fluorescently labeled BSA-FAM coinjected with the MRI contrast agent. Leakage of plasma proteins and deposition of a provisional matrix can support inflammation and stimulate remodeling of the colonic vasculature. Thus, the plasma protein leakage from the colonic microvasculature at the focal inflammatory patches could be quantified by MRI, providing a biomarker for assessment of disease progression. Magn Reson Med, 2011. © 2011 Wiley-Liss, Inc.

Immune mechanisms are known to control the pathogenesis of atherosclerosis. However, the exact role of DCs, which are essential for priming of immune responses, remains elusive. We have shown here that the DC-derived chemokine CCL17 is present in advanced human and mouse atherosclerosis and that CCL17 ⁺ DCs accumulate in atherosclerotic lesions. In atherosclerosis-prone mice, Ccl17 deficiency entailed a reduction of atherosclerosis, which was dependent on Tregs. Expression of CCL17 by DCs limited the expansion of Tregs by restricting their maintenance and precipitated atherosclerosis in a mechanism conferred by T cells. Conversely, a blocking antibody specific for CCL17 expanded Tregs and reduced atheroprogression. Our data identify DC-derived CCL17 as a central regulator of Treg homeostasis, implicate DCs and their effector functions in atherogenesis, and suggest that CCL17 might be a target for vascular therapy.

The mature gut renews continuously and rapidly throughout adult life, often in a damage-inflicting micro-environment. The major driving force for self-renewal of the intestinal epithelium is the Wnt-mediated signalling pathway, and Wnt signalling is frequently hyperactivated in colorectal cancer. Here we show that casein kinase Iα (CKIα), a component of the β-catenin-destruction complex, is a critical regulator of the Wnt signalling pathway. Inducing the ablation of Csnk1a1 (the gene encoding CKIα) in the gut triggers massive Wnt activation, surprisingly without causing tumorigenesis. CKIα-deficient epithelium shows many of the features of human colorectal tumours in addition to Wnt activation, in particular the induction of the DNA damage response and cellular senescence, both of which are thought to provide a barrier against malignant transformation. The epithelial DNA damage response in mice is accompanied by substantial activation of p53, suggesting that the p53 pathway may counteract the pro-tumorigenic effects of Wnt hyperactivation. Notably, the transition from benign adenomas to invasive colorectal cancer in humans is typically linked to p53 inactivation, underscoring the importance of p53 as a safeguard against malignant progression; however, the mechanism of p53-mediated tumour suppression is unknown. We show that the maintenance of intestinal homeostasis in CKIα-deficient gut requires p53-mediated growth control, because the combined ablation of Csnk1a1 and either p53 or its target gene p21 (also known as Waf1, Cip1, Sdi1 and Cdkn1a) triggered high-grade dysplasia with extensive proliferation. Unexpectedly, these ablations also induced non-proliferating cells to invade the villous lamina propria rapidly, producing invasive carcinomas throughout the small bowel. Furthermore, in p53-deficient gut, loss of heterozygosity of the gene encoding CKIα caused a highly invasive carcinoma, indicating that CKIα functions as a tumour suppressor when p53 is inactivated. We identified a set of genes (the p53-suppressed invasiveness signature, PSIS) that is activated by the loss of both p53 and CKIα and which probably accounts for the brisk induction of invasiveness. PSIS transcription and tumour invasion were suppressed by p21, independently of cell cycle control. Restraining tissue invasion through suppressing PSIS expression is thus a novel tumour-suppressor function of wild-type p53.

Classical DC (cDC) are required for efficient protective T-cell immunity. Moreover, recent data indicate that cDC also play a critical role in mediating homeostatic proliferation and maintenance of peripheral Treg. Here, we corroborate these findings by defining CD80/CD86 costimulation as an essential molecular component required for the cDC-Treg interactions. In contrast to earlier reports, the reduced Treg compartment of mice lacking cDC or selective CD80/86 expression on cDC, as such, did not render the respective animals prone to systemic lymphocyte hyperactivation or autoimmunity. Rather, we provide evidence that elevated immunoglobulin titers, as well as changes in T-cell subset prevalence and activation status are strictly associated with the nonmalignant myeloproliferative disorder triggered by the absence of cDC.

Inflammatory bowel diseases (IBDs) including Crohn's disease and ulcerative colitis represent a major challenge to clinicians and immunologists trying to understand why in certain individuals the peaceful coexistence of the commensal microflora and its host breaks down and results in chronic inflammation. Here we summarize the recent progress in our understanding of the organization of the intestinal mononuclear phagocytes with dendritic cells and macrophages of distinct phenotype, origin and function. Finally, we discuss potential strategies to translate the recent findings into the management of chronic inflammation in animal models of IBD.

The death of retinal ganglion cells (RGCs) is a hallmark of many retinal neuropathies. Neuroprotection, axonal regeneration, and cell renewal are vital for the integrity of the visual system after insult but are scarce in the adult mammalian retina. We hypothesized that monocytederived macrophages, known to promote healing in peripheral tissues, are required after an insult to the visual system, where their role has been largely overlooked. We found that after glutamate eye intoxication, monocyte-derived macrophages infiltrated the damaged retina of mice. Inhibition of this infiltration resulted in reduced survival of RGCs and diminished numbers of proliferating retinal progenitor cells (RPCs) in the ciliary body. Enhancement of the circulating monocyte pool led to increased RGC survival and RPC renewal. The infiltrating monocyte-derived macrophages skewed the milieu of the injured retina toward an antiinflammatory and neuroprotective one and down-regulated accumulation of other immune cells, thereby resolving local inflammation. The beneficial effect on RGC survival depended on expression of interleukin 10 and major histocompatibility complex class II molecules by monocyte-derived macrophages. Thus, we attribute to infiltrating monocyte-derived macrophages a novel role in neuroprotection and progenitor cell renewal in the injured retina, with far-reaching potential implications to retinal neuropathies and other neurodegenerative disorders.

Transcriptional responses to extracellular stimuli involve tuning the rates of transcript production and degradation. Here, we show that the time-dependent profiles of these rates can be inferred from simultaneous measurements of precursor mRNA (pre-mRNA) and mature mRNA profiles. Transcriptome-wide measurements demonstrate that genes with similar mRNA profiles often exhibit marked differences in the amplitude and onset of their production rate. The latter is characterized by a large dynamic range, with a group of genes exhibiting an unexpectedly strong transient production overshoot, thereby accelerating their induction and, when combined with time-dependent degradation, shaping transient responses with precise timing and amplitude.

Background: Colorectal-cancer (CRC) research has greatly benefited from the availability of small animal tumor models. Spontaneous and chemically-induced CRC models are widely used yet limited in their resemblance to human disease and are often prolonged, not accurately repetitive, and associated with inflammatory side effects. In-situ murine or human tumor implantation in the gastrointestinal tract of mice is extremely challenging, and limited by inter-animal variability and procedure-related complications and mortality. As a result, in frequent studies CRC is implanted in distal sites, most commonly the subcutaneous region, an approach that is greatly limited by the absence of normal gastrointestinal tumor milieu and has substantial effects on tumor development. Aims: In this study we aimed to develop a well-tolerated repetitive tool to study CRC in small animals by adapting the murine colonoscopy system to serve as a platform for colonic sub-mucosal orthotopic implantation of human and murine CRC tumor cells. Results: We report the establishment of a novel small-animal CRC model that is minimally invasive, rapid, well-tolerated, highly reproducible, and confers precise control of tumor number, location and growth rate. Moreover, we show that this model uniquely allows the side-by-side induction of distinct genetically manipulated tumors, enabling the mechanistic study of tumor interaction and cross-talk within the native intestinal microenvironment. Conclusions: Employment of this new approach may represent a major technical advance for the in-vivo study of CRC.

Natural killer (NK) cells serve as a crucial first line of defense against tumors, viral and bacterial infections. We studied the involvement of a principal activating natural killer cell receptor, natural cytotoxicity receptor 1 (NCR1), in the innate immune response to S. pneumoniae infection. Our results demonstrate that the presence of the NCR1 receptor is imperative for the early clearance of S. pneumoniae. We tied the ends in vivo by showing that deficiency in NCR1 resulted in reduced lung NK cell activation and lung IFNγ production at the early stages of S. pneumoniae infection. NCR1 did not mediate direct recognition of S. pneumoniae. Therefore, we studied the involvement of lung macrophages and dendritic cells (DC) as the mediators of NK-expressed NCR1 involvement in response to S. pneumoniae. In vitro, wild type BM-derived macrophages and DC expressed ligands to NCR1 and co-incubation of S. pneumoniae-infected macrophages/DC with NCR1-deficient NK cells resulted in significantly lesser IFNγ levels compared to NCR1-expressing NK cells. In vivo, ablation of lung macrophages and DC was detrimental to the early clearance of S. pneumoniae. NCR1-expressing mice had more potent alveolar macrophages as compared to NCR1-deficient mice. This result correlated with the higher fraction of NCR1-ligand ^high lung macrophages, in NCR1-expressing mice, that had better phagocytic activity compared to NCR1-ligand ^dull macrophages. Overall, our results point to the essential contribution of NK-expressed NCR1 in early response to S. pneumoniae infection and to NCR1-mediated interaction of NK and S. pneumoniae infected-macrophages and -DC.

Macrophages in the olfactory neuroepithelium are thought to play major roles in tissue homeostasis and repair. However, little information is available at present about possible heterogeneity of these monocyte-derived cells, their turnover rates, and the role of chemokine receptors in this process. To start addressing these issues, this study used Cx₃cr1^gfp mice, in which the gene sequence for eGFP was knocked into the CX₃CR1 gene locus in the mutant allele. Using neuroepithelial whole-mounts from Cx ₃cr1^gfp/+ mice, we show that eGFP⁺ cells of monocytic origin are distributed in a loose network throughout this tissue and can be subdivided further into two immunophenotypically distinct subsets based on MHC-II glycoprotein expression. BM chimeric mice were created using Cx ₃cr1^gfp/+ donors to investigate turnover of macrophages (and other monocyte-derived cells) in the olfactory neuroepithelium. Our data indicate that the monocyte-derived cell population in the olfactory neuroepithelium is actively replenished by circulating monocytes and under the experimental conditions, completely turned over within 6 months. Transplantation of Cx₃cr1^gfp/gfp (i.e., CX₃CR1-deficient) BM partially impaired the replenishment process and resulted in an overall decline of the total monocyte-derived cell number in the olfactory epithelium. Interestingly, replenishment of the CD68^lowMHC-II⁺ subset appeared minimally affected by CX₃CR1 deficiency. Taken together, the established baseline data about heterogeneity of monocyte-derived cells, their replenishment rates, and the role of CX₃CR1 provide a solid basis to further examine the importance of different monocyte subsets for neuroregeneration at this unique frontier with the external environment.

Lymphoid organs are characterized by a complex network of phenotypically distinct dendritic cells (DC) with potentially unique roles in pathogen recognition and immunostimulation. Classical DC (cDC) include two major subsets distinguished in the mouse by the expression of CD8α. Here we describe a subset of CD8α⁺ DC in lymphoid organs of naïve mice characterized by expression of the CX3CR1 chemokine receptor. CX ₃CR1⁺ CD8α⁺ DC lack hallmarks of classical CD8α⁺ DC, including IL-12 secretion, the capacity to crosspresent antigen, and their developmental dependence on the transcriptional factor BatF3. Gene-expression profiling showed that CX₃CR1 ⁺ CD8α⁺ DC resemble CD8α^- cDC. The microarray analysis further revealed a unique plasmacytoid DC (PDC) gene signature of CX₃CR1⁺ CD8α⁺ DC. A PDC relationship of the cells is supported further by the fact that they harbor characteristic D-J Ig gene rearrangements and that development of CX ₃CR1⁺ CD8α⁺ DC requires E2-2, the critical transcriptional regulator of PDC. Thus, CX₃CR1⁺ CD8α⁺ DC represent a unique DC subset, related to but distinct from PDC. Collectively, the expression-profiling data of this study refine the resolution of previous DC definitions, sharpen the border of classical CD8α⁺ and CD8α^- DC, and should assist the identification of human counterparts of murine DC subsets.

The intestinal landscape comprises the host's own tissue and immune cells, as well as a diverse intestinal microbiota. Intricate regulatory mechanisms have evolved to maintain peaceful coexistence at this site, the breakdown of which can result in devastating inflammatory bowel diseases (IBDs). Mononuclear phagocytes promote both innate and adaptive immune responses in the gut and, as such, are essential for the maintenance of intestinal homeostasis. Here, we review the origins and functions of the mononuclear phagocytes found in the intestinal lamina propria, highlighting the problems that have arisen from their classification. Understanding these cells in their physiological context will be important for developing new therapies for IBDs.

Dendritic cells (DCs) arise from dedicated precursors and monocytes; however, the impact of these differential origins remain unclear. In this issue of Immunity, Siddiqui et al. (2010) describe a population of colitogenic monocyte-derived DCs that propagate intestinal inflammation.

Recent years have seen a major advance in our understanding of the organization of the dendritic cell (DC) compartment. Particularly rewarding in this respect have been studies investigating DC origins, based on the identification of transcription factor and growth factor requirements, as well as direct demonstrations of precursor/progeny relationships by adoptive cell transfers. However, to fully understand the organization of the DC compartment, functional definitions of DC subsets must be provided and potential task divisions revealed that distinguish DC from other immune cells, including the closely related mononuclear phagocytes, such as macrophages. In fact, functional definitions might eventually replace the current distinction between DC and macrophages, which is in part based on arbitrary historic considerations, i.e. mononuclear phagocytes identified before the advent of DC in the mid 1970s generally termed macrophages. In this article, we review recent insight in the functions of classical DC in the mouse, focusing on our own work involving conditional and constitutive cell ablation.

Monocytes and macrophages are critical effectors and regulators of inflammation and the innate immune response, the immediate arm of the immune system. Dendritic cells initiate and regulate the highly pathogen-specific adaptive immune responses and are central to the development of immunologic memory and tolerance. Recent in vivo experimental approaches in the mouse have unveiled new aspects of the developmental and lineage relationships among these cell populations. Despite this, the origin and differentiation cues for many tissue macrophages, monocytes, and dendritic cell subsets in mice, and the corresponding cell populations in humans, remain to be elucidated.

Aspergillus fumigatus is an opportunistic fungal pathogen responsible for invasive aspergillosis in immunocompromised individuals. The high morbidity and mortality rates as well as the poor efficacy of antifungal agents remain major clinical concerns. Allicin (diallyl-dithiosulfinate), which is produced by the garlic enzyme alliinase from the harmless substrate alliin, has been shown to have wide-range antifungal specificity. A monoclonal antibody (MAb) against A. fumigatus was produced and chemically ligated to the enzyme alliinase. The purified antibody-alliinase conjugate bound to conidia and hyphae of A. fumigatus at nanomolar concentrations. In the presence of alliin, the conjugate produced cytotoxic allicin molecules, which killed the fungus. In vivo testing of the therapeutical potential of the conjugate was carried out in immunosuppressed mice infected intranasally with conidia of A. fumigatus. Intratracheal (i.t.) instillation of the conjugate and alliin (four treatments) resulted in 80 to 85% animal survival (36 days), with almost complete fungal clearance. Repetitive intratracheal administration of the conjugate and alliin was also effective when treatments were initiated at a more advanced stage of infection (50 h). The fungi were killed specifically without causing damage to the lung tissue or overt discomfort to the animals. Intratracheal instillation of the conjugate without alliin or of the unconjugated monoclonal antibody significantly delayed the death of the infected mice, but only 20% of the animals survived. A limitation of this study is that the demonstration was achieved in a constrained setting. Other routes of drug delivery will be investigated for the treatment of pulmonary and extrapulmonary aspergillosis.

Purpose of review: The term mono-cyte suggests this population of cells consists of a single homogenous fraction. However, evidence from a number of laboratories indicates that monocytes are composed of several subsets, which differ in phenotype, size, nuclear morphology, granularity and gene profiles. Most importantly, recent data suggest that monocyte subsets are also functionally distinct. Here we summarize the recent advances in our understanding of monocyte subsets and their origins, fates and functions. Recent findings: The recent past has seen major progress in our understanding of myeloid differentiation. Specifically, the published literature now suggests a dichotomy that starts at the stage of a novel clonotypic bone marrow resident precursor, the macrophage dendritic cell progenitor (MDP). Insights into differential origins of macrophages and dendritic cells, linked with functional specifications, are likely to significantly change our current view of the mononuclear phagocyte system. Summary: Contemporary studies have demonstrated that two subsets of monocytes reside in the peripheral circulation. These subsets are surprisingly distinct; with regard to their functions and fates, for example, one subset might be dedicated to generate macrophages upon extravasation from the peripheral circulation, whereas, the other subset under inflammatory conditions may differentiate into inflammatory dendritic cells. The tissue response during pathogenesis seems to differentially mobilize these cells, thereby manipulating the local mononuclear phagocyte composition according to acute needs.

The study of dendritic cell involvement in complex phenomena that rely oil multi-cellular interactions, Rich as immune homeostasis, stimulation, and tolerization, called for the investigation of dendritic cell functions within physiological context. To this end we have developed a conditional cell ablation strategy that is based on dendritic cell-restricted expression of a Diphtheria Toxin receptor (DTR) using the CD11c/Itgax promoter. Here, we provide basic protocols that describe the use of this prototypic dendritic cell ablation model and highlight pitfalls and strengths of the approach.

The intestinal immune system discriminates between tolerance toward the commensal microflora and robust responses to pathogens. Maintenance of this critical balance is attributed to mucosal dendritic cells (DCs) residing in organized lymphoid tissue and dispersed in the subepithelial lamina propria. In situ parameters of lamina propria DCs (lpDCs) remain poorly understood. Here, we combined conditional cell ablation and precursor-mediated in vivo reconstitution to establish that lpDC subsets have distinct origins and functions. CD103⁺ CX₃CR1^- lpDCs arose from macrophage-DC precursors (MDPs) via DC-committed intermediates (pre-cDCs) through a Flt3L growth-factor-mediated pathway. CD11b⁺ CD14⁺ CX₃CR1⁺ lpDCs were derived from grafted Ly6C^hi but not Ly6C^lo monocytes under the control of GM-CSF. Mice reconstituted exclusively with CX₃CR1⁺ lpDCs when challenged in an innate colitis model developed severe intestinal inflammation that was driven by graft-derived TNF-α-secreting CX₃CR1⁺ lpDCs. Our results highlight the critical importance of the lpDC subset balance for robust gut homeostasis.

Background: Although macrophages (MΦ) are known as essential players in wound healing, their contribution to recovery from spinal cord injury (SCI) is a subject of debate. The difficulties in distinguishing between different MΦ subpopulations at the lesion site have further contributed to the controversy and led to the common view of MΦ as functionally homogenous. Given the massive accumulation in the injured spinal cord of activated resident microglia, which are the native immune occupants of the central nervous system (CNS), the recruitment of additional infiltrating monocytes from the peripheral blood seems puzzling. A key question that remains is whether the infiltrating monocyte-derived MΦ contribute to repair, or represent an unavoidable detrimental response. The hypothesis of the current study is that a specific population of infiltrating monocyte-derived MΦ is functionally distinct from the inflammatory resident microglia and is essential for recovery from SCI. Methods and Findings: We inflicted SCI in adult mice, and tested the effect of infiltrating monocyte-derived MΦ on the recovery process. Adoptive transfer experiments and bone marrow chimeras were used to functionally distinguish between the resident microglia and the infiltrating monocyte-derived MΦ. We followed the infiltration of the monocyte-derived MΦ to the injured site and characterized their spatial distribution and phenotype. Increasing the naïve monocyte pool by either adoptive transfer or CNS-specific vaccination resulted in a higher number of spontaneously recruited cells and improved recovery. Selective ablation of infiltrating monocyte-derived MΦ following SCI while sparing the resident microglia, using either antibody-mediated depletion or conditional ablation by diphtheria toxin, impaired recovery. Reconstitution of the peripheral blood with monocytes resistant to ablation restored the lost motor functions. Importantly, the infiltrating monocyte-derived MΦ displayed a local anti-inflammatory beneficial role, which was critically dependent upon their expression of interleukin 10. Conclusions: The results of this study attribute a novel anti-inflammatory role to a unique subset of infiltrating monocyte-derived MΦ in SCI recovery, which cannot be provided by the activated resident microglia. According to our results, limited recovery following SCI can be attributed in part to the inadequate, untimely, spontaneous recruitment of monocytes. This process is amenable to boosting either by active vaccination with a myelin-derived altered peptide ligand, which indicates involvement of adaptive immunity in monocyte recruitment, or by augmenting the naïve monocyte pool in the peripheral blood. Thus, our study sheds new light on the long-held debate regarding the contribution of MΦ to recovery from CNS injuries, and has potentially far-reaching therapeutic implications.

Rationale. Although the contribution of alveolar type II epithelial cells (AECIIs) in respiratory immunity has become increasingly appreciated, their precise function in the induction and regulation of T-cell reactivity to self-antigen remains poorly understood. Objectives: To investigate the role of AECII in the initiation of T-cell reactivity to alveolar self-antigen, and to clarify their function in the peripheral induction of Foxp3⁺ regulatory CD4⁺ T cells. Methods. To dissect the complex cellular and molecular functions of AECIIs in lung inflammation and immune regulation, we use a transgenic mouse model for CD4⁺ T-cell-mediated pulmonary inflammation. Measurements and Main Results: Here we report that AECIIs present endogenously expressed antigen on major histocompatibility complex class II molecules to CD4⁺ T cells. Epithelial antigen display was sufficient to induce primary T-cell activation and pulmonary inflammation. Upon inflammation, AECIIs induce the differentiation of Foxp3⁺ regulatory T cells by a mechanism involving antiproliferative soluble factors, including transforming growth factor (TGF)-β. Whereas, in acute inflammation, TGF-β appears to be the dominant factor to induce regulatory T cells, other AECII-derived factors can substitute for and/or synergize with TGF-β in chronic pulmonary inflammations. Conclusions. AECIIs are capable of priming naive CD4⁺ T cells, demonstrating an active participation of these cells in respiratory immunity. Moreover, AECIIs display as yet unrecognized functions in balancing inflammatory and regulatory T-cell responses in the lung by connecting innate and adaptive immune mechanisms to establish peripheral T-cell tolerance to respiratory self-antigen.

Background: Regulatory T cells (T_regs) have potential anti-inflammatory effects and are likely to be important in the pathogenesis of chronic inflammatory bowel disease (IBD). However, the induction and expansion of T_regs) at sites of mucosal inflammation are not yet fully understood and may involve antigen presentation by local dendritic cells (DCs) and/or intestinal epithelial cells (lECs). Methods: To determine the unique ways in which the gut induces or expands T_regs), a transgenic mouse model that is based on the specific expression of a model autoantigen (influenza haemagglutinin (HA)) in the intestinal epithelium (VILLIN-HA) was used. Gut-associated DCs and lECs isolated from these mice were phenotypically and functionally characterised for the potential to interact with HA-specific T _regs) in vitro and in vivo. Results: Intestinal self-antigen expression leads to peripheral expansion of antigen-specific CD4 ⁺Foxp3⁺ T_regs). Although gut-associated DCs can induce antigen-specific CD4⁺Foxp3⁺ T cell proliferation, in vivo depletion of DCs did not preclude proliferation of these cells. Interestingly, antigen presentation by primary lECs is sufficient to expand antigen-specific CD4⁺Foxp3⁺ T_regs efficiently. This is dependent on major histocompatibility complex class II, but, in contrast to DCs, is unlikely to require transforming growth factor β and retinoic acid. Conclusion: This study provides experimental evidence for a new concept in mucosal immunity: in contrast to current thinking, expansion of T _regs can be achieved independently of local DCs through antigen-specific IEC-T cell interactions.

Dendritic cells (DCs) and macrophages (M Phi s), collectively termed mononuclear phagocytes (MP), are crucial for homeostatic tissue maintenance as well as the innate and adaptive host defense. These pleiotropic functions are likely to be covered by distinct DC and M Phi subsets, defined by anatomic location and molecular make-up. However, task division within the MP system remains poorly defined. A key to Understanding of this issue, Which might have important implications for the development of future therapeutic strategies, is the elucidation of the in vivo origins of DCs and M Phi s, whose study recently gained striking momentum. Here we present methods to investigate the role of MP progenitors, such as monocytes and M Phi/DC precursors (MDPs), in the replenishment of the peripheral MP system.

CX₃CR1 is a chemokine receptor with a single ligand, the membrane-tethered che- mokine CX₃CL1 (fractalkine). All blood monocytes express CX₃CR1, but its lev- els differ between the main 2 subsets, with human CD16⁺ and murine Gr1^lowmonocytes being CX₃CR1^hi. Here, we re- port that absence of either CX₃CR1 or CX₃CL1 results in a significant reduction of Gr1^low blood monocyte levels under both steady-state and inflammatory con- ditions. Introduction of a Bc12 transgene restored the wild-type phenotype, sug- gesting that the CX₃C axis provides an essential survival signal. Supporting this notion, we show that CX₃CL1 specifically rescues cultured human monocytes from induced cell death. Human CX₃CR1 gene polymorphisms are risk factors for athero- sclerosis and mice deficient for the CX₃C receptor or ligand are relatively protected from atherosclerosis development. How- ever, the mechanistic role of CX₃CR1 in atherogenesis remains unclear. Here, we show that enforced survival of mono- cytes and plaque-resident phagocytes, including foam cells, restored atherogen- esis in CX₃CR1-deficent mice. The fact that CX₃CL1-CX₃CR1 interactions confer an essential survival signal, whose ab- sence leads to increased death of mono- cytes and/or foam cells, might provide a mechanistic explanation for the role of the CX ₃C chemokine family in atherogen- esis.

Peripheral blood monocytes play a central role in the mononuclear phagocyte system by providing a critical link between the bone marrow (BM), as major site of adult hematopoiesis, and peripheral, terminally differentiated mononuclear phagocyte populations, as represented macrophages and dendritic cells. Moreover, recent experimental evidence highlights the plasticity of these ephemeral mobile cells and their direct involvement in the establishment and resolution of inflammatory reactions. Here we summarize the recent advance in our understanding of monocyte origins, subset dynamics and monocyte fates. In particular, we will focus on emerging evidence for monocyte recirculation to the BM and discuss its potential implications in health and disease.

Vascular-targeted photodynamic therapy (VTP) takes advantage of intravascular excitation of a photosensitizer (PS) to produce cytotoxic reactive oxygen species (ROS). These ROS are potent mediators of vascular damage inducing rapid local thrombus formation, vascular occlusion, and tissue hypoxia. This light-controlled process is used for the eradication of solid tumors with Pd-bacteriochlorophyll derivatives (Bchl) as PS. Unlike classical photodynamic therapy (PDT), cancer cells are not the primary target for VTP but instead are destroyed by treatment-induced oxygen deprivation. VTP initiates acute local inflammation inside the illuminated area accompanied by massive tumor tissue death. Consequently, in the present study, we addressed the possibility of immune response induction by the treatment that may be considered as an integral part of the mechanism of VTP-mediated tumor eradication. The effect of VTP on the host immune system was investigated using WST11, which is now in phase II clinical trials for age-related macular degeneration and intended to be evaluated for cancer therapy. We found that a functional immune system is essential for successful VTP. Long-lasting systemic antitumor immunity was induced by VTP involving both cellular and humoral components. The antitumor effect was cross-protective against mismatched tumors, suggesting VTP-mediated production of overlapping tumor antigens, possibly from endothelial origin. Based on our findings we suggest that local VTP might be utilized in combination with other anticancer therapies (e.g., immunotherapy) for the enhancement of host antitumor immunity in the treatment of both local and disseminated disease.

Dendritic cells are critically involved in the promotion and regulation of T cell responses. Here, we report a mouse strain that lacks conventional CD11c^hi dendritic cells (cDCs) because of constitutive cell-type specific expression of a suicide gene. As expected, cDC-less mice failed to mount effective T cell responses resulting in impaired viral clearance. In contrast, neither thymic negative selection nor T regulatory cell generation or T cell homeostasis were markedly affected. Unexpectedly, cDC-less mice developed a progressive myeloproliferative disorder characterized by prominent extramedullary hematopoiesis and increased serum amounts of the cytokine Flt3 ligand. Our data identify a critical role of cDCs in the control of steady-state hematopoiesis, revealing a feedback loop that links peripheral cDCs to myelogenesis through soluble growth factors, such as Flt3 ligand.

The olfactory epithelium (OE) is a site of massive adult neurogenesis where olfactory sensory neurons (OSNs) are continuously turned over. Tissue macrophages have been implicated in phagocytosis of degenerating cells but the molecular mechanisms that allow for their recruitment while maintaining a neurogenic microenvironment are poorly understood. This study reports that the neuroprotective chemokine CX3CL1 is expressed by OSNs and olfactory ensheathing cells. Monocyte-derived cells in the OE depend on CX3CL1-signalling for intraepithelial migration and apical dendrite expression. These observations are first to demonstrate phenotypic differences in appearance and distribution of monocyte-derived cells in nervous tissue due to CX3CR1 deficiency.

Implantation is a key stage during pregnancy, as the fate of the embryo is often decided upon its first contact with the maternal endometrium. Around this time, DCs accumulate in the uterus; however, their role in pregnancy and, more specifically, implantation, remains unknown. We investigated the function of uterine DCs (uDCs) during implantation using a transgenic mouse model that allows conditional ablation of uDCs in a spatially and temporally regulated manner. Depletion of uDCs resulted in a severe impairment of the implantation process, leading to embryo resorption. Depletion of uDCs also caused embryo resorption in syngeneic and T cell-deficient pregnancies, which argues against a failure to establish immunological tolerance during implantation. Moreover, even in the absence of embryos, experimentally induced deciduae failed to adequately form. Implantation failure was associated with impaired decidual proliferation and differentiation. Dynamic contrast-enhanced MRI revealed perturbed angiogenesis characterized by reduced vascular expansion and attenuated maturation. We suggest therefore that uDCs directly fine-tune decidual angiogenesis by providing two critical factors, sFlt1 and TGF-β1, that promote coordinated blood vessel maturation. Collectively, uDCs appear to govern uterine receptivity, independent of their predicted role in immunological tolerance, by regulating tissue remodeling and angiogenesis. Importantly, our results may aid in understanding the limited implantation success of embryos transferred following in vitro fertilization.

Dendritic cells (DC) represent a rather heterogeneous cell population with regard to morphology, phenotype, and function and, like most cells of the immune system, are subjected to a continuous renewal process. CD103⁺ (integrin α_E) DC have been identified as a major mucosal DC subset involved in the induction of tissue-specific homing molecules on T cells, but little is known about progenitors able to replenish this DC subset. Herein we report that lineage (lin)^-CX₃CR1⁺c-kit ⁺ (GFP⁺c-kit⁺) bone marrow cells can differentiate to either CD11c⁺CD103^- or CD11c ⁺CD103⁺ DC in vitro and in vivo. Gene expression as well as functional assays reveal distinct phenotypical and functional properties of both subsets generated in vitro. CD103⁺ DC exhibit enhanced phagocytosis and respond to LPS stimulation by secreting proinflammatory cytokines, whereas CD103⁺ DC express high levels of costimulatory molecules and efficiently induce allogeneic T cell proliferation. Following adoptive transfer of GFP⁺c-kit⁺ bone marrow cells to irradiated recipients undergoing allergic lung inflammation, we identified donor-derived CD103⁺ DC in lung and the lung-draining bronchial lymph node. Collectively, these data indicate that GFP⁺c-kit⁺ cells contribute to the replenishment of CD103⁺ DC in lymphoid and nonlymphoid organs.

Aspergillus fumigatus is an opportunistic fungal pathogen responsible for invasive aspergillosis in immunocompromised individuals. The inefficiency of antifungal agents and high mortality rate resulting from invasive aspergillosis remain major clinical concerns. Recently, we reported on a new family of ultrashort cationic lipopeptides active in vitro against fungi. Mode of action studies supported a membranolytic or a detergent-like effect. Here, we screened several lipopeptides in vitro for their anti-A fumigatus activity. To investigate the therapeutic properties of the selected peptides in vivo, we challenged immunosuppressed C57BL/6 wild-type mice intranasals with DsRed-labeled A. fumigatus conidia and subsequently treated the animals locally with the lipopeptides. Confocal microscopic analysis revealed the degradation of DsRed-labeled hyphal forms and residual conidia in the lungs of the mice. The most efficient peptide was tested further using a survival assay and was found to significantly prolong the life of the treated animals, whereas no mice survived with the current standard antifungal treatment with amphotericin B. Moreover, as opposed to the drug-treated lungs, the peptide-treated lungs did not display any toxicity of the peptide. Our results highlight the potential of this family of lipopeptides for the treatment of pulmonary invasive aspergillosis.

Dendritic cells (DCs) play a central role in T-cell activation and the control of the inherent autoreactivity of the T-cell compartment. Pleiotropic DC functions are likely associated with discrete DC subsets. However, the latter remain largely defined by phenotype and unique anatomic location, rather than function. The investigation of DC involvement in complex phenomena that rely on multicellular interactions, such as immuno-stimulation and tolerization calls for an assessment of DC functions within physiological context. Given the highly dynamic DC compartment, the method of choice to study in vivo DC functions is their conditional ablation in the intact organism. Here, we summarize the recent progress in this field highlighting pitfalls and prospects of the approach.

Alum (aluminum hydroxide) is the most widely used adjuvant in human vaccines, but the mechanism of its adjuvanticity remains unknown. In vitro studies showed no stimulatory effects on dendritic cells (DCs). In the absence of adjuvant, Ag was taken up by lymph node (LN)-resident DCs that acquired soluble Ag via afferent lymphatics, whereas after injection of alum, Ag was taken up, processed, and presented by inflammatory monocytes that migrated from the peritoneum, thus becoming inflammatory DCs that induced a persistent Th2 response. The enhancing effects of alum on both cellular and humoral immunity were completely abolished when CD11c⁺ monocytes and DCs were conditionally depleted during immunization. Mechanistically, DC-driven responses were abolished in MyD88-deficient mice and after uricase treatment, implying the induction of uric acid. These findings suggest that alum adjuvant is immunogenic by exploiting "nature's adjuvant," the inflammatory DC through induction of the endogenous danger signal uric acid. JEM

Beyond its established function in hematopoiesis, the bone marrow hosts mature lymphocytes and acts as a secondary lymphoid organ in the initiation of T cell and B cell responses. Here we report the characterization of bone marrow-resident dendritic cells (bmDCs). Multiphoton imaging showed that bmDCs were organized into perivascular clusters that enveloped blood vessels and were seeded with mature B lymphocytes and T lymphocytes. Conditional ablation of bmDCs in these bone marrow immune niches led to the specific loss of mature B cells, a phenotype that could be reversed by overexpression of the antiapoptotic factor Bcl-2 in B cells. The presence of bmDCs promoted the survival of recirculating B cells in the bone marrow through the production of macrophage migration-inhibitory factor. Thus, bmDCs are critical for the maintenance of recirculating B cells in the bone marrow.

Intestinal dendritic cells (DCs) are believed to sample and present commensal bacteria to the gut-associated immune system to maintain immune homeostasis. How antigen sampling pathways handle intestinal pathogens remains elusive. We present a murine colitogenic Salmonella infection model that is highly dependent on DCs. Conditional DC depletion experiments revealed that intestinal virulence of S. Typhimurium SL1344 ΔinvG mutant lacking a functional type 3 secretion system-1 (ΔinvG) critically required DCs for invasion across the epithelium. The DC-dependency was limited to the early phase of infection when bacteria colocalized with CD11c⁺CX3CR1⁺ mucosal DCs. At later stages, the bacteria became associated with other (CD11c^-CX3CR1^-) lamina propria cells, DC depletion no longer attenuated the pathology, and a MyD88-dependent mucosal inflammation was initiated. Using bone marrow chimeric mice, we showed that the MyD88 signaling within hematopoietic cells, which are distinct from DCs, was required and sufficient for induction of the colitis. Moreover, MyD88-deficient DCs supported transepithelial uptake of the bacteria and the induction of MyD88-dependent colitis. These results establish that pathogen sampling by DCs is a discrete, and MyD88-independent, step during the initiation of a mucosal innate immune response to bacterial infection in vivo. JEM

Previously, we reported that a peptide, p458, from the sequence of the mammalian 60-kDa heat shock protein (hsp60) molecule can serve as a carrier in conjugate vaccines with capsular polysaccharide (CPS) molecules of various bacteria. These conjugate vaccines were effective injected in PBS without added adjuvants. We now report that p458 conjugated to pneumococcal CPS type 4 (PS4) manifests innate adjuvant effects: it stimulated mouse macrophages to secrete IL-12 and induced the late appearance of PS4 on the macrophage surface in a TLR4-dependent manner; PS4 alone or conjugated to other carriers did not stimulate macrophages in vitro. The injection of macrophages manifesting PS4 on the surface into mice induced long-term resistance to lethal Streptococcus pneumoniae challenge. The TLR4 ligand LPS could also induce the late appearance on the surface of unconjugated PS4 and resistance to challenge in injected mice. Resistance was not induced by macrophages containing only internalized PS4 or by pulsed macrophages that had been lysed. Glutaraldehyde-fixed macrophages pulsed with PS4 did induce resistance to lethal challenge. Moreover, bone marrow-derived dendritic cells activated by LPS and pulsed with unconjugated CPS were also effective in inducing resistance to lethal challenge. Resistance induced by the PS4-pulsed bone marrow-derived dendritic cell was specific for pneumococcal CPS serotypes (type 3 or type 4) and was associated with the induction of CPS-specific IgG and IgM Abs.

Background. Infection with Streptococcus pyogenes remains a significant health care problem. The identification of immune components required for host defenses against S. pyogenes constitutes an important area of research. Methods. Here, we have investigated the role played by dendritic cells (DCs) during infection with S. pyogenes by use of a murine infection model. Results. Our results show that S. pyogenes induced the maturation of murine DCs, which involved the upregulation of CD40, CD80, CD86, and major histocompatibility complex class II molecules and the production of interleukin (IL)-12 and tumor necrosis factor-α. After subcutaneous infection of mice, S. pyogenes disseminated systemically via the draining lymph nodes. The contribution of DCs to bacterial dissemination was negligible, because most microorganisms were found free in lymph nodes. The contribution of DCs to host defenses against S. pyogenes was investigated using CD11c-diphtheria toxin (DT) receptor (DTR) transgenic mice, in which CD11c^high cells (conventional DCs) can be transiently depleted in vivo by treatment with low doses of DT. We show that ablation of DCs led to increased bacterial dissemination into draining lymph nodes and systemic organs. Furthermore, ablation of DCs abolished IL-12 production, which is required for effective control of infection. Conclusions. These data demonstrate that DCs contribute to host defenses against S. pyogenes, likely through the production of IL-12.

We explored the physiological role of conventional dendritic cells (cDCs) in acute colitis induced by a single cycle of dextran sodium sulfate administration. Depending on their mode of activation and independently of T cells, cDCs can enhance or attenuate the severity of dextran sodium sulfate-induced colitis. The latter beneficial effect was achieved, in part, by IFN-1 induced by Toll-like receptor 9-activated cDCs. IFN-1 inhibits colonic inflammation by regulating neutrophil and monocyte trafficking to the inflamed colon and restraining the inflammatory products of tissue macrophages. These data highlight a novel role of cDCs in the regulation of other innate immune cells and position them as major players in acute colonic inflammation.

Alveolar macrophages are a unique type of mononuclear phagocytes that populate the external surface of the lung cavity. Early studies have suggested that alveolar macrophages originate from tissue-resident, local precursors, whereas others reported their derivation from blood-borne cells. However, the role of circulating monocytes as precursors of alveolar macrophages was never directly tested. In this study, we show through the combined use of conditional cell ablation and adoptive cell transfer that alveolar macrophages originate in vivo from blood monocytes. Interestingly, this process requires an obligate intermediate stage, the differentiation of blood monocytes into parenchymal lung macrophages, which subsequently migrate into the alveolar space. We also provide direct evidence for the ability of both lung and alveolar macrophages to proliferate.

Plasmacytoid dendritic cells (PDCs) play a pivotal role as cytokine-secreting accessory cells in the antimicrobial immune defense. In contrast, the capacity of PDCs to act as antigen-presenting cells in naive T cell priming remains unclear. By studying T cell responses in mice that lack conventional DCs (cDCs), and by the use of a PDC-specific antigen-targeting strategy, we show that PDCs can initiate productive naive CD4⁺ T cell responses in lymph nodes, but not in the spleen. PDC-triggered CD4⁺ T cell responses differed from cDC-driven responses in that they were not associated with concomitant CD8⁺ T cell priming. Our results establish PDCs as a bonafide DC subset that initiates unique CD4⁺ Th cell-dominated primary immune responses. JEM

Mastitis, an inflammatory response of the mammary tissue to invading pathogenic bacteria, is a common disease in breast-feeding women and dairy animals. Escherichia coli is a leading cause of mastitis in dairy animals. During the course of the disease the host mounts a strong inflammatory response, but specific bacterial virulence factors have not yet been identified. Here we report the use of a murine mastitis model to investigate the innate inflammatory reaction of the mammary gland. We show that lipopolysaccharide (LPS) infusion induces mastitis in wild-type mice (C3H/HeN), but not in mice expressing mutated Toll-like receptor 4 (TLR4) (C3H/HeJ). The wild-type phenotype was restored by adoptive transfer of TLR4-expressing macrophages into the alveolar milk space of C3H/HeJ mice. In contrast to the LPS treatment, infection with E. coli P4 (ECP4) resulted in inflammation even in the absence of LPS/ TLR4 signalling, indicating that additional factors play a role in the pathogenesis of the intact bacteria. Furthermore, in the absence of functional TLR4 the infecting ECP4 invade the epithelial cells with high efficiency, forming intracellular microcolonies. However, adoptive transfer with TLR4-expressing macrophages drastically reduced the epithelial invasion. Taken together, these results indicate that ECP4 has an invasive potential, which is restricted by alveolar macrophages in response to the LPS/TLR4 signalling.

Microglia are resident cells in the central nervous system (CNS), of hematopoietic origin with a high plasticity. In this study, we examined whether adaptive immune system, involving in CNS maintenance and repair, can induce microglia to express markers of neural cells. We show that long exposure (above 10 days) of microglia to low doses (10 ng/ml) of the 'proinflammatory' T-cell derived cytokine, IFN-γ, induced them to express neuronal markers including γ-aminobutyric acid (GABA) and glutamic acid decarboxylase (GAD-67). In contrast, exposure of microglia to low doses (10 ng/ml) of the 'anti-inflammatory' T-cell derived cytokine, IL-4, induced the expression of oligodendrocyte markers and dendritic cell (DC) marker, CD11c. The microglial origin of the neural-like cells was confirmed using microglia from transgenic mice expressing GFP under promoter of the chemokine fractalkine receptor CX₃CR1, and diphtheria toxin receptor, under CD11c promoter. This study emphasizes that microglial plasticity includes their ability to give rise to neural-like cells and shows that cytokines produced by the adaptive immune system are involved in these processes.

Monoallelic demethylation and rearrangement control allelic exclusion of the immunoglobulin κ-chain locus (Igk locus) in B cells. Here, through the introduction of pre-rearranged Igk genes into their physiological position, the critical rearrangement step was bypassed, thereby generating mice producing B cells simultaneously expressing two different immunoglobulin-κ light chains. Such 'double-expressing' B cells still underwent monoallelic demethylation at the Igk locus, and the demethylated allele was the 'preferred' substrate for somatic hypermutation in each cell. However, methylation itself did not directly inhibit the activation-induced cytidine-deaminase reaction in vitro. Thus, it seems that the epigenetic mechanisms that initially bring about monoallelic variable-(diversity)-joining rearrangement continue to be involved in the control of antibody diversity at later stages of B cell development.

Inhalational anthrax is a life-threatening infectious disease of considerable concern, especially because anthrax is an emerging bioterrorism agent. The exact mechanisms leading to a severe clinical form through the inhalational route are still unclear, particularly how immobile spores are captured in the alveoli and transported to the lymph nodes in the early steps of infection. We investigated the roles of alveolar macrophages and lung dendritic cells (LDC) in spore migration. We demonstrate that alveolar macrophages are the first cells to phagocytose alveolar spores, and do so within 10 min. However, interstitial LDCs capture spores present in the alveoli within 30 min without crossing the epithelial barrier suggesting a specific mechanism for rapid alveolus sampling by transepithelial extension. We show that interstitial LDCs constitute the cell population that transports spores into the thoracic lymph nodes from within 30 min to 72 h after intranasal infection. Our results demonstrate that LDCs are central to spore transport immediately after infection. The rapid kinetics of pathogen transport may contribute to the clinical features of inhalational anthrax.

The uptake of immune complexes by FcRs on APCs augments humoral and cellular responses to exogenous Ag. In this study, CD11c⁺ dendritic cells are shown to be responsible in vivo for immune complex-triggered priming of T cells. We examine the conseqnence of Ab-mediated uptake of self Ag by dendritic cells in the rat insulin promoter-membrane OVA model and identify a role for the inhibitory FcγRIIB in the maintenance of peripheral CD8 T cell tolerance. Effector differentiation of diabetogenic OT-I CD8⁺ T cells is enhanced in rat insulin promoter-membrane OVA mice lacking FcγRIIB, resulting in a high incidence of diabetes. FcγRIIB-mediated inhibition of CD8 T cell priming results from suppression of both DC activation and cross-presentation through activating FcγRs. Further FcγRIIB on DCs inhibited the induction of OVA-specific Th1 effectors, limiting Th1-type differentiation and memory T cell accumulation. In these MHC II-restricted responses, the presence of FcγRIIB only modestly affected initial CD4 T cell proliferative responses, suggesting that FcγRIIB limited effector cell differentiation primarily by inhibiting DC activation. Thus, FcγRIIB can contribute to peripheral tolerance maintenance by inhibiting DC activation alone or by also limiting processing of esogenously acquired Ag.

We have developed a model of autoimmunity to investigate autoantibody-mediated cross-presentation of self antigen. RIP-mOVA mice, expressing OVA in pancreatic β cells, develop severe autoimmune diabetes when given OT-I cells (OVA-specific CD8⁺ T cells) and anti-OVA IgG but not when given T cells alone. Anti-OVA IgG is not directly injurious to the islets but rather enhances cross-presentation of apoptotic islet antigen to the OT-I cells, leading to their differentiation into potent effector cells. Antibody-driven effector T cell activation is dependent on the presence of activating Fc receptors for IgG (FcγRs) and cross-priming DCs. As a consequence, diabetes incidence and severity was reduced in mice lacking activating FcγRs. An intact complement pathway was also required for disease development, as C3 deficiency was also partially protective. C3-deficient animals exhibited augmented T cell priming overall, indicating a proinflammatory role for complement activation after the T cell priming phase. Thus, we show that autoreactive antibody can potently enhance the activation of effector T cells in response to cross-presented self antigen, thereby contributing to T cell-mediated autoimmunity.

PURPOSE. Recent investigations have revealed that populations of macrophages and dendritic cells (DCs) are present in the stroma and epithelium of the cornea, although the precise phenotype and distribution are still controversial. CX₃CR1, the sole receptor for the chemokine fractalkine, is expressed by these monocyte-derived cells. Transgenic CX ₃CR1^GFP mice, in which either one (heterozygous) or both (homozygous) copies of the CX₃CR1 gene were replaced by enhanced green fluorescent protein (eGFP), were used to characterize monocyte-derived cells in the mouse cornea and to determine whether the expression of this receptor influences the recruitment of these cells into the normal cornea. METHODS. Wholemount corneas were immunostained with anti-leukocyte antibodies to the phenotypic markers major histocompatibility complex (MHC) class II, CD169, CD68, CD11b, and CD45 and analyzed by epifluorescence and confocal microscopy. The density of intraepithelial MHC class II⁺ cells was quantified in wild-type, CX₃CR1^+/GFP heterozygous, CX ₃CR1^GFP/GFP homozygous, and CX₃CR1-knockout mice. RESULTS. There was a significant reduction in the number of MHC class H⁺ cells (putative DCs) in the corneal epithelium of CX ₃CR1-deficient mice (P < 0.009) compared with wild-type mice, and the few cells that were present did not possess classic dendriform morphology. No GFP⁺ MHC class II^- cells were noted in the epithelium. Dual immunostaining of corneas in both heterozygous and homozygous (CX ₃CR1-deficient) mice revealed GFP⁺ cells with a more pleomorphic morphology throughout the entire corneal stroma that were CD11b ⁺ CD169⁺, and had variable degrees of expression of CD68 and MHC class II. The immunophenotype and morphology of these intrastromal cells is strongly indicative of a macrophage phenotype. CONCLUSIONS. This study has identified a role for CX₃CR1 in the normal recruitment of MHC class II⁺ putative DCs into the corneal epithelium and establishes a model for investigating monocyte-derived cells and fractalkine/CX₃CR1 interactions during corneal disease.

Peripheral blood monocytes are a population of circulating mononuclear phagocytes that harbor potential to differentiate into macrophages and dendritic cells. As in humans, monocytes in the mouse comprise two phenotypically distinct subsets that are Gr1^highCX₃CR1^int and Gr1^lowCX₃CR1^high, respectively. The question remains whether these populations contribute differentially to the generation of peripheral mononuclear phagocytes. In this study, we track the fate of adoptively transferred, fractionated monocyte subsets in the lung of recipient mice. We show that under inflammatory and noninflammatory conditions, both monocyte subsets give rise to pulmonary dendritic cells. In contrast, under the conditions studied, only Gr1^lowCX₃CR1^high monocytes, but not Gr1^highCX₃CR1^int cells, had the potential to differentiate into lung macrophages. However, Gr1 ^highCX₃CR1^int monocytes could acquire this potential upon conversion into Gr1^lowCX₃CR1^high cells. Our results therefore indicate an intrinsic dichotomy in the differentiation potential of the two main blood monocyte subsets.

The mononuclear phagocyte (MP) system is a body-wide macrophage (MΦ) and dendritic cell (DC) network, which contributes to tissue homeostasis, inflammation, and immune defense. The in vivo origins of MPs remain poorly understood. Here, we use an adoptive precursor cell transfer strategy into MP-depleted mice to establish the in vivo differentiation sequence from a recently identified MΦ/DC-restricted bone marrow (BM) precursor (MDP) via BM and blood intermediates to peripheral MΦs and DCs. We show that MDPs are in vivo precursors of BM and blood monocytes. Interestingly, grafted Gr1 ^high "inflammatory" blood monocytes shuttle back to the BM in the absence of inflammation, convert into Gr1^low monocytes, and contribute further to MP generation. The grafted monocytes give rise to DCs in the intestinal lamina propria and lung, but not to conventional CD11c ^high DCs in the spleen, which develop during homeostasis from MDPs without a monocytic intermediate. JEM

Dendritic cells (DC) play an important role as antigen-presenting cells in T cell stimulation. Interestingly, a number of recent studies also imply DC as critical accessory cells in B cell activation, isotype switching and plasma blast maintenance. Here we use the conditional in vivo ablation of CD11c^high DC to investigate the role of these cells in T-independent type 2 immune responses. We show that CD11c^high DC are dispensable for the initiation and maintenance of a primary immune response against the T-independent type 2 antigen (4-hydroxy-3-nirophenyl)acetyl-Ficoll. Our results suggest that support for plasma cell formation in T cell-independent immune responses can be provided by non-DC such as stromal cells, or is independent of external signals. Interestingly, we found plasma blasts to express CD11c and to be diphtheria toxin-sensitive in CD11c-diphtheria toxin receptor-transgenic mice, providing a unique tool for future analysis of in vivo aspects of plasma cell biology.

The role of dendritic cells (DC) in urinary tract infections (UTI) is unknown. These cells contribute directly to the innate defense against various viral and bacterial infections. Here, we studied their role in UTI using an experimental model induced by transureihral instillation of the uropathogenic Escherichia coli (UPEC) strain 536 into C57BL/6 mice. While few DC were found in the uninfected bladder, many had been recruited after 24 h, mostly to the submucosa and uroepithelium. They expressed markers of activation and maturation and exhibited the CD11b⁺ F4/80⁺ CD8^- Gr-1 ^- myeloid subtype. Also, tumor necrosis factor alpha (TNF-α)- and inducible nitric oxide synthase (iNOS)-producing CD11b^INT DC (Tip-DC) were detected, which recently were proposed to be critical in the defense against bacterial infections. However, Tip-DC-deficieni CCR2 ^-/- mice did not show reduced clearance of UPEC from the infected bladder. Moreover, clearance was also unimpaired in CD11c-DTR mice depleted of all DC by injection of diphtheria toxin. This may be explained by the abundance of granulocytes and of iNOS- and TNF-α-producing non-DC that were able to replace Tip-DC functionality. These findings demonstrate that some of the abundant DC recruited in UTI contributed innate immune effector functions, which were, however, dispensable in the microenvironment of the bladder.

Background: In allergic rhinitis (AR) CD4 ⁺ T _H2 lymphocytes control inflammation by secreting T _H2 cytokines, but little is known about how these cells are activated to cause disease. Objective: We sought to study the contribution of antigen-presenting dendritic cells (DCs) in activating T _H2 cells and controlling allergic inflammation. Methods: Nasal mucosal biopsy specimens were taken from patients with house dust mite allergy and perennial AR and healthy control subjects. DC numbers were evaluated by using immunohistochemistry. The functional role of DCs was studied in a novel mouse model for AR using BALB/c mice and CD11c-diphtheria toxin (DT) receptor transgenic mice. Results: In symptomatic patients with perennial AR, the number of CD1a ⁺ and CD11c ⁺ MHCII ⁺ DCs was higher in the epithelium and lamina propria of the nasal mucosa compared with that seen in healthy control subjects. In patients with AR, DCs had a more mature (CD86 ⁺) phenotype and were found in close approximation with T lymphocytes. Similarly, in a mouse model of ovalbumin (OVA)-induced AR, CD11c ⁺ DCs accumulated in areas of nasal eosinophilic inflammation and clustered with CD4 ⁺ T lymphocytes. CD11c ⁺ DCs were conditionally depleted during allergen challenge by means of systemic administration of DT to CD11c-diphtheria toxin receptor transgenic mice to address the functional role of DCs in maintaining inflammation. In the absence of CD11c ⁺ DCs, nasal OVA challenge in OVA-sensitized mice did not induce nasal eosinophilia and did not boost OVA-specific IgE levels or T _H2 cytokine production in the cervical lymph nodes. Conversely, when OVA-pulsed DCs were administered intranasally to sensitized mice, they strongly enhanced OVA-induced nasal eosinophilia and T _H2 cytokine production. Conclusions: These data in human subjects and mice suggest an essential role for nasal DCs in activation of effector T _H2 function leading to AR. Clinical implications: Nasal DCs play an essential role in AR and therefore constitute a novel target for therapeutic intervention.

In addition to their bridging function between innate and adaptive immunity, dendritic cells (DCs) may also contribute to primary resistance against infection. Here we analyzed the role of DCs during infection with Listeria monocytogenes by performing systemic in vivo depletion of these cells. We showed that CD8α⁺ DCs were crucial for L. monocytogenes spreading and proliferation in the spleen. Efficient and rapid uptake of L. monocytogenes by CD8α⁺ DCs required the small GTPase Rac1 and is a general characteristic of this DC subpopulation in filtering particles out of the blood. Thus, CD8α⁺ DCs appear to play an important role for efficient bacterial entry into the spleen, which is of relevance for subsequent immune responses.

Microglia, the resident inflammatory cells of the CNS, are the only CNS cells that express the fractalkine receptor (CX3CR1). Using three different in vivo models, we show that CX3CR1 deficiency dysregulates microglial responses, resulting in neurotoxicity. Following peripheral lipopolysaccharide injections, Cx3cr1^-/- mice showed cell-autonomous microglial neurotoxicity. In a toxic model of Parkinson disease and a transgenic model of amyotrophic lateral sclerosis, Cx3cr1^-/- mice showed more extensive neuronal cell loss than Cx3cr1⁺ littermate controls. Augmenting CX3CR1 signaling may protect against microglial neurotoxicity, whereas CNS penetration by pharmaceutical CX3CR1 antagonists could increase neuronal vulnerability.

Leukocyte trafficking to the central nervous system (CNS), regulated in part by chemokines, determines severity of the demyelinating diseases multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). To examine chemokine receptor CX3CR1 in EAE, we studied CX3CR1GFP/GFP mice, in which CX3CR1 targeting by insertion of Green Fluorescent Protein (GFP) allowed tracking of CX3CR1+ cells in CX3CR1^+/GFP animals and cells destined to express CX3CR1 in CX3CR1^GFP/GFP knockouts. NK cells were markedly reduced in the inflamed CNS of CX3CR1-deficient mice with EAE, whereas recruitment of T cells, NKT cells and monocyte/macrophages to the CNS during EAE did not require CX3CR1. Impaired recruitment of NK cells in CX3CR1^GFP/GFP mice was associated with increased EAE-related mortality, nonremitting spastic paraplegia and hemorrhagic inflammatory lesions. The absence of CD1d did not affect the severity of EAE in CX3CR1^GFP/GFP mice, arguing against a role for NKT cells. Accumulation of NK cells in livers of wild-type (WT) and CX3CR1 ^GFP/GFP mice with cytomegalovirus hepatitis was equivalent, indicating that CX3CL1 mediated chemoattraction of NK cells was relatively specific for the CNS. These results are the first to define a chemokine that governs NK cell migration to the CNS, and the findings suggest novel therapeutic manipulation of CX3CR1+ NK cells.

The non-toxic B-subunit of Shiga toxin (STxB) interacts with the glycolipid Gb₃, which is preferentially expressed on dendritic cells (DC) and B cells. After administration of STxB chemically coupled to OVA (STxB-OVA) in mice, we showed that the immunodominant OVA_257-264 peptide restricted by k^b molecules is specifically presented by CD11c⁺CD8α ^- DC, some of them displaying a mature phenotype. Using mice carrying a transgene encoding a diphtheria toxin receptor (DTR) under the control of the murine CD11c promoter, which allows inducible ablation of DC, we showed that DC are required for efficient priming of CTL after STxB-OVA vaccination. Immunization of mice with STxB-OVA induced OVA-specific CD8⁺ T cells detected ex vivo; these cells were long lasting, since they could be detected even 91 days after the last immunization and were composed of both central and memory T cells. Vaccination of mice with STxB-OVA and STxB coupled to E7, a protein derived from HPV16, inhibited tumor growth in prophylactic and therapeutic experiments. This effect was mainly mediated by CD8⁺ T cells. STxB therefore appears to be a powerful carrier directly targeting DC in vivo, resulting in a strong and durable CTL response associated with tumor protection.

The role of activated microglia (MG) in demyelinating neurodegenerative diseases such as multiple sclerosis is controversial. Here we show that high, but not low, levels of IFN-γ (a cytokine associated with inflammatory autoimmune diseases) conferred on rodent MG a phenotype that impeded oligodendrogenesis from adult neural stem/progenitor cells. IL-4 reversed the impediment, attenuated TNF-α production, and overcame blockage of IGF-I production caused by IFN-γ. In rodents with acute or chronic EAE, injection of IL-4-activated MG into the cerebrospinal fluid resulted in increased oligodendrogenesis in the spinal cord and improved clinical symptoms. The newly formed oligodendrocytes were spatially associated with MG expressing MHC class II proteins and IGF-I. These results point to what we believe to be a novel role for MG in oligodendrogenesis from the endogenous stem cell pool.

The lamina propria that underlies and stabilizes the gut lining epithelium is densely populated with strategically located mononuclear phagocytes. Collectively, these lamina propria macrophages and dendritic cells (DC) are believed to be crucial for tissue homeostasis as well as the innate and adaptive host defense. Lamina propria DC were recently shown to gain direct access to the intestinal lumen by virtue of epithelium-penetrating dendrites. However, the role of these structures in pathogen uptake remains under debate. In this study, we report that entry of a noninvasive model pathogen (Aspergillus fumigatus conidia) into the murine small intestinal lamina propria persists in the absence of either transepithelial dendrites or lamina propria DC and macrophages. Our results suggest the existence of multiple pathogen entry pathways and point at the importance of villus M cells in the uptake of gut lumen Ags. Interestingly, transepithelial dendrites seem altogether absent from the small intestine of BALB/c mice suggesting that the function of lamina propria DC extensions resides in their potential selectivity for luminal Ags, rather than in general uptake or gut homeostasis.

Burn wound healing is a complex process consisting of an inflammatory phase, the formation of granulation tissue, and remodeling. The role of the CXCL12/CXCR4 pathway in the recovery of skin following burns is unknown. We found that CXCL12 is similarly expressed in human, swine, and rat skin by pericyte and endothelial cells, fibrous sheet, fibroblasts, and axons. Following burns, the levels of CXCL12 were markedly increased in human burn blister fluids. One day after injury, there was a gradual increase in the expression of CXCL12 in the hair follicles and in blood vessel endothelium surrounding the burn. Three to 11 days following burns, an increased number of fibroblasts expressing CXCL12 were observed in the recovering dermis of rat, swine, and human skin. In contrast to CXCL12, CXCR4 expression was detected in proliferating epithelial cells as well as in eosinophils and mononuclear cells infiltrating the skin. In vitro, CXCL12 was expressed by primary human skin fibroblasts, but not by keratinocytes, and was stimulated by wounding a confluent cell layer of these fibroblasts. Blocking the CXCR4/CXCL12 axis resulted in the significant reduction in eosinophil accumulation in the dermis and improved epithelialization. Thus, blocking CXCR4/CXCL12 interaction may significantly improve skin recovery after burns.

Macrophages and dendritic cells (DCs) are crucial for immune and inflammatory responses and belong to a network of cells that has been termed the mononuclear phagocyte system (MPS). However, the origin and lineage of these cells remain poorly understood. Here, we describe the isolation and clonal analysis of a mouse bone marrow progenitor that is specific for monocytes, several macrophage subsets, and resident spleen DCs in vivo. It was also possible to recapitulate this differentiation in vitro by using treatment with the cytokines macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor. Thus, macrophages and DCs appear to renew from a common progenitor, providing a cellular and molecular basis for the concept of the MPS.

Adult neovascularization relies on the recruitment of circulating cells, but their angiogenic roles and recruitment mechanisms are unclear. We show that the endothelial growth factor VEGF is sufficient for organ homing of circulating mononuclear myeloid cells and is required for their perivascular positioning and retention. Recruited bone marrow-derived circulating cells (RBCCs) summoned by VEGF serve a function distinct from endothelial progenitor cells. Retention of RBCCs in close proximity to angiogenic vessels is mediated by SDF1, a chemokine induced by VEGF in activated perivascular myofibroblasts. RBCCs enhance in situ proliferation of endothelial cells via secreting proangiogenic activities distinct from locally induced activities. Precluding RBCCs strongly attenuated the proangiogenic response to VEGF and addition of purified RBCCs enhanced angiogenesis in excision wounds. Together, the data suggest a model for VEGF-programmed adult neovascularization highlighting the essential paracrine role of recruited myeloid cells and a role for SDF1 in their perivascular retention.

The peripheral lymphocyte pool size is governed by homeostatic mechanisms. Thus, grafted T cells expand and replenish T cell compartments in lymphopenic hosts. Lymphopenia-driven proliferation of naive CD8⁺ T cells depends on self-peptide/MHC class I complexes and the cytokine IL-7. Lymphopenia-driven proliferation and maintenance of memory CD8⁺ T cells are MHC independent, but are believed to require IL-7 and contact with a bone marrow-derived cell that presents the cytokine IL-15 by virtue of its high affinity receptor (IL-15Rα). In this study we show that optimal spontaneous proliferation of grafted naive and memory CD8⁺ T cells in mice rendered lymphopenic through gene ablation or irradiation requires the presence of CD11c^high dendritic cells. Our results suggest a dual role of CD11c^high dendritic cells as unique APC and cytokine-presenting cells.

The existence of macrophages (MΦ) of yolk-sac (YS) origin has been reported in all vertebrate models. However, the nature of their precursors and pathways of differentiation have not been elucidated. Phenotypic and differentiation potential analyses of YS at 7.5 to 10 postcoital days (dpc), performed in CX₃CR1^GFP embryos, allowed us to discern 3 independent MΦ populations. A first transient wave consisted of mature, maternal-derived MΦ present as early as 7.5 to 8 dpc. A second wave of committed MΦ precursors arose at 8 dpc (2-4 somite stage) and was followed by a third wave of erythromyeloid precursors (4-6 somite stage). Both types of precursors displayed similar phenotypes and gave rise to CX₃CR1/green fluorescent protein (GFP)-positive MΦ, but differed by their differentiation potential, at the clonal level. The combined data of phenotypic, gene-expression, and in situ analyses allowed us to conclude that the previously named "primitive MΦ" corresponded to a mixture of the first transient wave and committed MΦ precursors. Both YS-derived precursors followed a developmental pathway common to adult MΦ and could be qualified as definitive.

Mononuclear phagocytes enter the lungs both constitutively to maintain alveolar macrophage and dendritic cell homeostasis, as well as during lung inflammation, where the role of these cells is less well defined. We used a transgenic mouse strain (CX₃CR1^+/ GFP) that harbors a GFP label in circulating monocytes to identify and sort these cells from the vascular and alveolar compartments under both constitutive and acute lung inflammatory conditions. Using nylon arrays combined with real-time RT-PCR for gene expression profiling, we found that flow-sorted, highly purified mononuclear phagocytes recruited to acutely inflamed mouse lungs showed strongly up-regulated mRNA levels of the neutrophil chemoattractants KC, MIP-2, and IP-10, which contrasted with alveolar mononuclear phagocytes that immigrated in steady state. Similar observations were made for the lysosomal cathepsins B, L, and K being strongly up-regulated in mononuclear phagocytes upon recruitment to inflamed lungs but not during constitutive alveolar immigration. Inflammatory elicited mononuclear phagocytes also demonstrated significantly increased mRNA levels of the cytokine TNF-α and the PRR-associated molecules CD14, TLR4, and syndecan-4. Together, inflammatory elicited mononuclear phagocytes exhibit strongly increased neutrophil chemoattractants, lysosomal proteases, and LPS signaling mRNA transcripts, suggesting that these cells may play a major role in acute lung inflammatory processes.

Parenchymal microglia are the principal immune cells of the brain. Time-lapse two-photon imaging of GFP-labeled microglia demonstrates that the fine termini of microglial processes are highly dynamic in the intact mouse cortex. Upon traumatic brain injury, microglial processes rapidly and autonomously converge on the site of injury without cell body movement, establishing a potential barrier between the healthy and injured tissue. This rapid chemotactic response can be mimicked by local injection of ATP and can be inhibited by the ATP-hydrolyzing enzyme apyrase or by blockers of G protein-coupled purinergic receptors and connexin channels, which are highly expressed in astrocytes. The baseline motility of microglial processes is also reduced significantly in the presence of apyrase and connexin channel inhibitors. Thus, extracellular ATP regulates microglial branch dynamics in the intact brain, and its release from the damaged tissue and surrounding astrocytes mediates a rapid microglial response towards injury.

A new system for lineage ablation is based on transgenic expression of a diphtheria toxin receptor (DTR) in mouse cells and application of diphtheria toxin (DT). To streamline this approach, we generated Cre-inducible DTR transgenic mice (iDTR) in which Cre-mediated excision of a STOP cassette renders cells sensitive to DT. We tested the iDTR strain by crossing to the T cell- and B cell-specific CD4-Cre and CD19-Cre strains, respectively, and observed efficient ablation of T and B cells after exposure to DT. In MOGi-Cre/iDTR double transgenic mice expressing Cre recombinase in oligodendrocytes, we observed myelin loss after intraperitoneal DT injections. Thus, DT crosses the blood-brain barrier and promotes cell ablation in the central nervous system. Notably, we show that the developing DT-specific antibody response is weak and not neutralizing, and thus does not impede the efficacy of DT. Our results validate the use of iDTR mice as a tool for cell ablation in vivo.

Langerhans cells (LC) form a unique subset of dendritic cells (DC) in the epidermis but so far their in vivo functions in skin immunity and tolerance could not be determined, in particular in relation to dermal DC (dDC). Here, we exploit a novel diphtheria toxin (DT) receptor (DTR)/DT-based system to achieve inducible ablation of LC without affecting the skin environment. Within 24 h after intra-peritoneal injection of DT into Langerin-DTR mice LC are completely depleted from the epidermis and only begin to return 4 wk later. LC deletion occurs by apoptosis in the absence of inflammation and, in particular, the dDC compartment is not affected. In LC-depleted mice contact hypersensitivity (CHS) responses are significantly decreased, although ear swelling still occurs indicating that dDC can mediate CHS when necessary. Our results establish Langerin-DTR mice as a unique tool to study LC function in the steady state and to explore their relative importance compared with dDC in orchestrating skin immunity and tolerance.

Immunization of mice with live or heat-killed Listeria monocytogenes (HKLM) efficiently primes pathogen-specific CD8⁺ T cells. T lymphocytes primed by HKLM, however, undergo attenuated proliferation and do not fully differentiate. Thus, only infection with live bacteria induces long-term, CD8⁺ T cell-mediated protective immunity. In this study we demonstrate that live and heat-killed bacteria, while both associating with Mac-3⁺CD11b^hi cells, localize to distinct splenic areas following intravenous inoculation. While HKLM localize to the marginal zone and the splenic red pulp, live L. monocytogenes are carried to the T cell zone of splenic white pulp. Despite these differences, in vivo depletion of CD11c-expressing cells prevents priming of naive T cells by either HKLM or live L. monocytogenes. Analysis of CD11c^hi dendritic cells (DC) reveals that infection with live L. monocytogenes induces higher levels of CD40, CD80 and CD86 expression than immunization with HKLM. Our results suggest that CD8⁺ T cell priming following HKLM immunization or live infection is mediated by DC and that the disparate outcomes of priming can be attributed to suboptimal conditioning of DC in the absence of live, cytosol-invasive bacteria.

Although dendritic cells (DCs) play an important role in sensitization to inhaled allergens, their function in ongoing T helper (Th)2 cell-mediated eosinophilic airway inflammation underlying bronchial asthma is currently unknown. Here, we show in an ovalbumin (OVA)-driven murine asthma model that airway DCs acquire a mature phenotype and interact with CD4⁺ T cells within sites of peribronchial and perivascular inflammation. To study whether DCs contributed to inflammation, we depleted DCs from the airways of CD11c-diphtheria toxin (DT) receptor transgenic mice during the OVA aerosol challenge. Airway administration of DT depleted CD11c⁺ DCs and alveolar macrophages and abolished the characteristic features of asthma, including eosinophilic inflammation, goblet cell hyperplasia, and bronchial hyperreactivity. In the absence of CD11c⁺ cells, endogenous or adoptively transferred CD4⁺ Th2 cells did not produce interleukin (IL)-4, IL-5, and IL-13 in response to OVA aerosol. In CD11c-depleted mice, eosinophilic inflammation and Th2 cytokine secretion were restored by adoptive transfer of CD11c⁺ DCs, but not alveolar macrophages. These findings identify lung DCs as key proinflammatory cells that are necessary and sufficient for Th2 cell stimulation during ongoing airway inflammation.

To become accessible for rearrangement, the immunoglobulin κ locus must undergo a series of epigenetic changes. This begins in pro-B cells with the relocation of both immunoglobulin κ alleles from the periphery to the center of the nucleus. In pre-B cells, one allele became preferentially packaged into an active chromatin structure characterized by histone acetylation and methylation of histone H3 lysine 4, while the other allele was recruited to heterochromatin, where it was associated with heterochromatin protein-γ and Ikaros. These events in cis made only one allele accessible to trans-acting factors, such as RelB, which mediated DNA demethylation, to facilitate rearrangement. These results suggest that early B lymphoid epigenetic changes generate differential structures that serve as the basis for allelic exclusion.

Dendritic cells (DCs) and macrophages are critical to innate and adaptive immunity to the intestinal bacterial microbiota. Here, we identify a myeloid-derived mucosal DC in mice, which populates the entire lamina propria of the small intestine. Lamina propria DCs were found to depend on the chemokine receptor CX₃CR1 to form transepithelial dendrites, which enable the cells to directly sample luminal antigens. CX₃CR1 was also found to control the clearance of entero-invasive pathogens by DCs. Thus, CX ₃CR1-dependent processes, which control host interactions of specialized DCs with commensal and pathogenic bacteria, may regulate immunological tolerance and inflammation.

Studying the influence of chemokine receptors (CCRs) on monocyte fate may reveal information about which subpopulations of monocytes convert to dendritic cells (DCs) and the migration pathways that they use. First, we examined whether prominent CCRs on different monocyte subsets, CCR2 or CX₃CR1, mediated migration events upstream of the accumulation of monocyte-derived DCs in lymph nodes (LNs). Monocytes were labeled and traced by uptake of latex microspheres in skin. Unexpectedly, neither CCR2 nor CX₃CR1 were required. However, absence of CCR2 led to an increased labeling of the minor Gr-1^int monocyte population, and the number of latex⁺ DCs that emigrated to LNs was correspondingly increased. Characterization of Gr-1^int monocytes revealed that they selectively expressed CCR7 and CCR8 mRNA in blood. CCR7 and CCR8 pathways were used by monocyte-derived DCs during mobilization from skin to LNs. The role of CCR8 in emigration from tissues also applied to human monocyte-derived cells in a model of transendothelial trafficking. Collectively, the data suggest that Gr-1 ^int monocytes may be most disposed to become a lymphatic-migrating DCs. When these monocyte-derived DCs exit skin to emigrate to LNs, they use not only CCR7 but also CCR8, which was not previously recognized to participate in migration to LNs.

Runx3 transcription factor regulates cell lineage decisions in thymopoiesis and neurogenesis. Here we report that Runx3 knockout (KO) mice develop spontaneous eosinophilic lung inflammation associated with airway remodeling and mucus hypersecretion. Runx3 is specifically expressed in mature dendritic cells (DC) and mediates their response to TGF-β. In the absence of Runx3, DC become insensitive to TGF-β-induced maturation inhibition, and TGF-β-dependent Langerhans cell development is impaired. Maturation of Runx3 KO DC is accelerated and accompanied by increased efficacy to stimulate T cells and aberrant expression of β2-integrins. Lung alveoli of Runx3 KO mice accumulate DC characteristic of allergic airway inflammation. Taken together, abnormalities in DC function and subset distribution may constitute the primary immune system defect, which leads to the eosinophilic lung inflammation in Runx3 KO mice. These data may help elucidate the molecular mechanisms underlying the pathogenesis of allergic airway inflammation in humans.

Autoimmunity results from a deficiency in tolerance establishment, i.e. a failure of the organism to eliminate or functionally neutralize auto-reactive lymphocytes. Dendritic cells (DC) are specialized migratory antigen presenting cells (APC), found as sentinels in peripheral tissues and lymphoid organs that allow the organism to mount rapid and efficient protective immune responses against pathogenic invaders. However, the antigenic spectrum presented by mature, highly immunogenic DC is not only of foreign origin, but includes self-proteins. As powerful stimulators of naïve T cells, DC are therefore a constant challenge to the organism emphasizing the need for trans-acting mechanisms that keep auto-reactive T cells under control. Paradoxically, recent studies indicate that DC, i.e. the same cells which represent this major threat, also play a crucial role in the establishment of both central and peripheral tolerance. In the light of a number of recent excellent reviews on the topic [1,2], this article summarizes the current knowledge obtained from the murine system highlighting hallmark experiments and open questions.

Peripheral blood monocytes are a heterogeneous population of circulating leukocytes. Using a murine adoptive transfer system to probe monocyte homing and differentiation in vivo, we identified two functional subsets among murine blood monocytes: a short-lived CX₃CR1^loCCR2⁺Gr1⁺ subset that is actively recruited to inflamed tissues and a CX₃CR1^hiCCR2^-Gr1^- subset characterized by CX₃CR1-dependent recruitment to noninflamed tissues. Both subsets have the potential to differentiate into dendritic cells in vivo. The level of CX₃CR1 expression also defines the two major human monocyte subsets, the CD14⁺CD16^- and CD14^loCD16⁺ monocytes, which share phenotype and homing potential with the mouse subsets. These findings raise the potential for novel therapeutic strategies in inflammatory diseases.

We studied whether circulating activated platelets and platelet-leukocyte aggregates cause the development of atherosclerotic lesions in apolipoprotein-E-deficient (Apoe^-/-) mice. Circulating activated platelets bound to leukocytes, preferentially monocytes, to form platelet-monocyte/leukocyte aggregates. Activated platelets and platelet-leukocyte aggregates interacted with atherosclerotic lesions. The interactions of activated platelets with monocytes and atherosclerotic arteries led to delivery of the platelet-derived chemokines CCL5 (regulated on activation, normal T cell expressed and secreted, RANTES) and CXCL4 (platelet factor 4) to the monocyte surface and endothelium of atherosclerotic arteries. The presence of activated platelets promoted leukocyte binding of vascular cell adhesion molecule-1 (VCAM-1) and increased their adhesiveness to inflamed or atherosclerotic endothelium. Injection of activated wild-type, but not P-selectin-deficient, platelets increased monocyte arrest on the surface of atherosclerotic lesions and the size of atherosclerotic lesions in Apoe^-/- mice. Our results indicate that circulating activated platelets and platelet-leukocyte/monocyte aggregates promote formation of atherosclerotic lesions. This role of activated platelets in atherosclerosis is attributed to platelet P-selectin-mediated delivery of platelet-derived proinflammatory factors to monocytes/leukocytes and the vessel wall.

Cytotoxic T lymphocytes (CTL) respond to antigenic peptides presented on MHC class I molecules. On most cells, these peptides are exclusively of endogenous, cytosolic origin. Bone marrow-derived antigen-presenting cells, however, harbor a unique pathway for MHC I presentation of exogenous antigens. This mechanism permits cross-presentation of pathogen-infected cells and the priming of CTL responses against intracellular microbial infections. Here, we report a novel diphtheria toxin-based system that allows the inducible, short-term ablation of dendritic cells (DC) in vivo. We show that in vivo DC are required to cross-prime CTL precursors. Our results thus define a unique in vivo role of DC, i.e., the sensitization of the immune system for cell-associated antigens. DC-depleted mice fail to mount CTL responses to infection with the intracellular bacterium Listeria monocytogenes and the rodent malaria parasite Plasmodium yoelii.

The cytolytic activity of NK cells is tightly regulated by inhibitory receptors specific for MHC class I Ags. We have investigated the composition of signal transduction molecules in the supramolecular activation clusters in the MHC class I-regulated cytolytic and noncytolytic NK cell immune synapses. KIR2DL3-positive NK clones that are specifically inhibited in their cytotoxicity by HLA-Cw*0304 and polyclonal human NK cells were used for conjugate formation with target cells that are either protected or are susceptible to NK cell-mediated cytotoxicity. Polarization of talin, microtubule-organizing center, and lysosomes occurred only during cytolytic interactions. The NK immune synapses were analyzed by three-dimensional immunofluorescence microscopy, which showed two distinctly different synaptic organizations in NK cells during cytolytic and noncytolytic interactions. The center of a cytolytic synapse with MHC class I-deficient target is comprised of a complex of signaling molecules including Src homology (SH)2-containing protein tyrosine phosphatase-1 (SHP-1). Closely related molecules with overlapping functions, such as the Syk kinases, SYK, and ZAP-70, and adaptor molecules, SH2 domain-containing leukocyte protein of 76 kDa and B cell linker protein, are expressed in activated NK cells and are all recruited to the center of the cytolytic synapse. In contrast, the noncytolytic synapse contains SHP-1, but is lacking other components of the central supramolecular activation cluster. These findings indicate a functional role for SHP-1 in both the cytolytic and noncytolytic interactions. We also demonstrate, in three-cell conjugates, that a single NK cell forms a cytolytic synapse with a susceptible target cell in the presence of both susceptible and nonsusceptible target cells.

In a reconstituted flow chamber system, preincubation with chemokines can trigger the arrest of rolling monocytes, suggesting that this interaction could help recruit these cells to early atherosclerotic lesions. To date, however, the contribution of endothelium-derived chemokines found in these lesion to monocyte arrests has not been investigated. The endothelium of lesion-prone carotid arteries from apolipoprotein E-deficient (ApoE^-/-) mice, but not control mice, presents the chemokines KC (mouse GRO-α) and JE (mouse monocyte chemoattractant protein-1 [MCP-1]). Arrest of a monocytic cell line or mouse blood monocytes perfused through carotid arteries of ApoE^-/- mice was reduced by treating with either pertussis toxin, an antagonist of CXCR2, or an antibody to KC, but this process was insensitive to agents that blocked CCR-2 or JE. Conversely, monocyte accumulation more than doubled upon pre-perfusion of the carotid artery with KC but not with mouse MCP-1. Blockade of α₄β₁ integrin (VLA-4) or vascular cell adhesion molecule-1, but not CD18 or intercellular adhesion molecule-1, almost completely inhibited the arrest of monocytes. We conclude that when presented by early atherosclerotic lesions, KC but not murine MCP-1 triggers VLA-4-dependent monocyte recruitment.

Interstitial fluid is constantly drained into lymph nodes (LNs) via afferent lymph vessels. This conduit enables monocyte-derived macrophages and dendritic cells to access LNs from peripheral tissues. We show that during inflammation in the skin, a second recruitment pathway is evoked that recruits large numbers of blood-borne monocytes to LNs via high endothelial venules (HEVs). Inhibition of monocyte chemoattractant protein (MCP)-1 blocked this inflammation-induced monocyte homing to LNs. MCP-1 mRNA in inflamed skin was over 100-fold upregulated and paralleled MCP-1 protein levels, whereas in draining LNs MCP-1 mRNA induction was much weaker and occurred only after a pronounced rise in MCP-1 protein. Thus, MCP-1 in draining LNs was primarily derived from inflamed skin. In MCP-1^-/- mice, intracutaneously injected MCP-1 accumulated rapidly in the draining LNs where it enhanced monocyte recruitment. Intravital microscopy showed that skin-derived MCP-1 was transported via the lymph to the luminal surface of HEVs where it triggered integrin-dependent arrest of rolling monocytes. These findings demonstrate that inflamed peripheral tissues project their local chemokine profile to HEVs in draining LNs and thereby exert "remote control" over the composition of leukocyte populations that home to these organs from the blood.

The cytoplasmic protein tyrosine kinase Tec has been proposed to have important functions in hematopoiesis and lymphocyte signal transduction. Here we show that Tec-deficient mice developed normally and had no major phenotypic alterations of the immune system. To reveal potential compensatory roles of other Tec kinases such as Bruton's tyrosine kinase (Btk), Tec/Btk double-deficient mice were generated. These mice exhibited a block at the B220⁺CD43⁺ stage of B cell development and displayed a severe reduction of peripheral B cell numbers, particularly immunoglobulin (Ig)M(lo)IgD(hi) B cells. Although Tec/Btk(null) mice were able to form germinal centers, the response to T cell-dependent antigens was impaired Thus, Tec and Btk together have an important role both during B cell development and in the generation and/or function of the peripheral B cell pool. The ability of Tec to compensate for Btk may also explain phenotypic differences in X-linked immunodeficiency (xid) mice compared with human X-linked agammaglobulinemia (XLA) patients.

Proper lymph node (LN) development requires tumor necrosis factor-related activation-induced cytokine (TRANCE) expression. Here we demonstrate that the defective LN development in TRANCE(-/-) mice correlates with a significant reduction in lymphotoxin (LT)αβ⁺α₄β₇⁺CD45⁺CD4⁺CD3^- cells and their failure to form clusters in rudimentary mesenteric LNs. Transgenic TRANCE overexpression in TRANCE(-/-) mice results in selective restoration of this cell population into clusters, and results in full LN development. Transgenic TRANCE-mediated restoration of LN development requires LTαβ expression on CD45⁺CD4⁺CD3^- cells, as LNs could not be induced in LTα(-/-) mice. LTα(-/-) mice also showed defects in the fate of CD45⁺CD4⁺CD3^- cells similar to TRANCE(-/-) mice. Thus, we propose that both TRANCE and LTαβ regulate the colonization and cluster formation by CD45⁺CD4⁺CD3^- cells in developing LNs, the degree of which appears to correlate with the state of LN organogenesis.

The seven-transmembrane receptor CX₃CR1 is a specific receptor for the novel CX₃C chemokine fractalkine (FKN) (neurotactin). In vitro data suggest that membrane anchoring of FKN, and the existence of a shed, soluble FKN isoform allow for both adhesive and chemoattractive properties. Expression on activated endothelium and neurons defines FKN as a potential target for therapeutic intervention in inflammatory conditions, particularly central nervous system diseases. To investigate the physiological function of CX₃CR1-FKN interactions, we generated a mouse strain in which the CX₃CR1 gene was replaced by a green fluorescent protein (GFP) reporter gene. In addition to the creation of a mutant CX₃CR1 locus, this approach enahled us to assign murine CX₃CR1 expression to monocytes, subsets of NK and dendritic cells, and the brain microglia. Analysis of CX₃CR1-deficient mice indicates that CX₃CR1 is the only murine FKN receptor. Yet, defying anticipated FKN functions, absence of CX₃CR1 interferes neither with monocyte extravasation in a peritonitis model nor with DC migration and differentiation in response to microbial antigens or contact sensitizers. Furthermore, a prominent response of CX₃CR1-deficient microglia to peripheral nerve injury indicates unimpaired neuronal-glial cross talk in the absence of CX₃CR1.

Based on the essential involvement of NF-κB in immune and inflammatory responses and its apoptosis-rescue function in normal and malignant cells, inhibitors of this transcription factor are potential therapeutics for the treatment of a wide range of diseases, from bronchial asthma to cancer. Yet, given the essential function of NF-κB in the embryonic liver, it is important to determine its necessity in the liver beyond embryogenesis. NF- κB is normally retained in the cytoplasm by its inhibitor IκB, which is eliminated upon cell stimulation through phosphorylation-dependent ubiquitin degradation. Here, we directed a degradation-resistant IκBα transgene to mouse hepatocytes in an inducible manner and showed substantial tissue specificity using various means, including a new method for live-animal imaging. Transgene expression resulted in obstruction of NF-κB activation, yet produced no signs of liver dysfunction, even when implemented over 15 months. However, the transgene-expressing mice were very vulnerable both to a severe immune challenge and to a systemic bacterial infection. Despite having intact immunocytes and inflammatory cells, these mice were unable to clear Listeria monocytogenes from the liver and succumbed to sepsis. These findings indicate the essential function of the hepatocyte through NF-κB activation in certain systemic infections, possibly by coordinating innate immunity in the liver.

Leukocytes respond to complex patterns of chemoattractant cytokine (chemokine) gradients that guide them to their destinations in secondary lymphoid organs. This directed movement of multiple cell types requires the choreographed expression of specific G-protein-coupled chemokine receptors and both positive and negative regulation of the signal transduction pathways emanating from them.

To study the relative contributions of clonal deletion, clonal anergy, and receptor editing to tolerance induction in autoreactive B cells and their dependence on B cell receptor affinity, we have constructed 'knock in' mice in which germline encoded or somatically mutated, rearranged anti-DNA heavy (H) chains were targeted to the H chain locus of the mouse. The targeted H chains were expressed on the vast majority of bone marrow (BM) and splenic B cells and were capable of Ig class switching and the acquisition of somatic mutations. A quantitative analysis of B cell populations in the BM as well as of Jκ utilization and DNA binding of hybridoma Abs suggested that immature B cell deletion and light (L) chain editing were the major mechanisms affecting tolerance. Unexpectedly, these mechanisms were less effective in targeted mice expressing the somatically mutated, anti-DNA H chain than in mice expressing the germline-encoded H chain, possibly due to the greater abundance of high affinity, anti-DNA immature B cells in the BM. Consequently, autoreactive B cells that showed features of clonal anergy could be recovered in the periphery of these mice. Our results suggest that clonal deletion and receptor editing are interrelated mechanisms that act in concert to eliminate autoreactive B cells from the immune system. Clonal anergy may serve as a back-up mechanism for central tolerance, or it may represent an intermediate step in clonal deletion.

Previously we implicated c-Jun N-terminal kinase (JNK) as an element that is involved in signal integration during co-stimulation of T lymphocytes. This pathway has now been traced to an upper level, comprising MAPKK SEK1/MKK4/JNKK1 which, similarly to JNK, must receive input both from the TCR and CD28. A large portion of this input is probably integrated at the level of the Rho-family protein CDC42 which, here, activates SEK1 and JNK to the level reached by TCR and CD28 stimulation. We have identified another putative SEK/JNK pathway regulator, PKCθ, which in contrast to CDC42, activates SEK and JNK maximally only in conjunction with a calcium signal delivered through calcineurin. Signals originating at the TCR and CD28 may travel down the JNK pathway via PKCθ, calcineurin, CDC42, MEKK1 and SEK1.

Based primarily on the analysis of B cell lines, mature B cells are considered distinct from non-B cells and immature B cells by having constitutive nuclear NF-κB activity. By their comparison to splenic non-B cells or activated B cells we show here that primary resting B cells lack cell-autonomous NF-κB activity. This finding indicates that the role of the transcription factor in B cells is similar to that in other cells, namely a common mediator of activation and stress signals. Whereas the absence of constitutive NF-κB activity in mature B cells does not argue against a role of NF-κB in B cell development, it does not support the notion of the essential function of the factor in maintaining the unique transcriptional activities in B cells.

Mice whose IgH alleles are engineered to encode two distinct antibody heavy (H) chains generate a normal-sized B cell compartment in which most cells stably express the two heavy chains. This demonstrates that 'toxicity' of bi-allelic H chain expression and cell-autonomous mechanisms of silencing in-frame IgH gene rearrangements do not significantly contribute to allelic exclusion at the IgH locus. Notwithstanding, the stability of the various engineered IgH loci during B cell development in the bone marrow differed substantially from each other.

Activation of the transcription factor NF-κB is a paradigm for signal transduction through the ubiquitin-proteasome pathway: ubiquitin-dependent degradation of the transcriptional inhibitor IκB in response to cell stimulation. A major issue in this context is the nature of the recognition signal and the targeting enzyme involved in the proteolytic process. Here we show that following a stimulus-dependent phosphorylation, and while associated with NF-κB, IκB is targeted by a specific ubiquitin-ligase via direct recognition of the signal-dependent phosphorylation site; phosphopeptides corresponding to this site specifically inhibit ubiquitin conjugation of IκB and its subsequent degradation. The ligase recognition signal is functionally conserved between IκBα and IκBβ, and does not involve the nearby ubiquitination site. Microinjection of the inhibitory peptides into stimulated cells abolished NF-κB activation in response to TNFα and the consequent expression of E-selectin, an NF-κB-dependent cell-adhesion molecule. Inhibition of NF-κB function by specific blocking of ubiquitin ligase activity provides a novel approach for intervening in cellular processes via regulation of unique proteolytic events.

B lymphocytes can alter the class of antibody they produce by immunoglobulin class switch recombination. This recombination is targeted by distinct cytokines to particular switch regions. Prior to switch recombination, the same cytokines induce transcription through the targeted switch regions and generate IH "switch" transcripts. To show whether the two events are functionally related, we have replaced the endogenous interleukin-4 (IL-4) dependent promoter of murine I gamma 1 switch transcripts by an heterologous promoter, the human metallothionein IIA (hMT) promoter. Indeed, switch recombination can be targeted to IgG1 by the hMT promoter. In mutant mice, which cannot generate processed switch transcripts, switch recombination cannot be targeted to IgG1 by the hMT promoter. Thus, IL-4 targets switch recombination to IgG1 by induction of processed switch transcripts.

B cells can exchange gene segments for the constant region of the immunoglobulin heavy chain, altering the class and effector function of the antibodies that they produce. Class switching is directed to distinct classes by cytokines, which induce transcription of the targeted DNA sequences. These transcripts are processed, resulting in spliced "switch" transcripts. Switch recombination can be directed to immunoglobulin G1 (IgG1) by the heterologous human metallothionein II_A promoter in mutant mice. Induction of the structurally conserved, spliced switch transcripts is sufficient to target switch recombination to IgG1, whereas transcription alone is not.

NF-κB is a major inducible transcription factor in many immune and inflammatory reactions. Its activation involves the dissociation of the inhibitory subunit 1κB from cytoplasmic NF-κB/Rel complexes, following which the Rel proteins are translocated to the nucleus, where they bind to DNA and activate transcription. Phosphorylation of 1κB in cell-free experiments results in its inactivation and release from the Rel complex, but in vivo NF-κB activation is associated with IκB degradation. in vivo phosphorylation of IκBα was demonstrated in several recent studies, but its role is unknown. Our study shows that the T-cell activation results in rapid phosphorylation of IκBα and that this event is a physiological one, dependent on appropriate lymphocyte costimulation. Inducible IκBα phosphorylation was abolished by several distinct NF-κB blocking reagents, suggesting that it plays an essential role in the activation process. However, the in vivo induction of IκBα phosphorylation did not cause the inhibitory subunit to dissociate from the Rel complex. We identified several protease inhibitors which allow phosphorylation of IκBα but prevent its degradation upon cell stimulation, presumably through inhibition of the cytoplasmic proteasome. In the presence of these inhibitors, phosphorylated IκBα remained bound to the Rel complex in the cytoplasm for an extended period of time, whereas NF-κB activation was abolished. It appears that activation of NF-κB requires degradation of IκBα while it is a part of the Rel cytoplasmic complex, with inducible phosphorylation of the inhibitory subunit influencing the rate of degradation.

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Both, in humans and in mice, a major fraction of immunoglobulin E (IgE)- expressing B lymphocytes develops by sequential Ig class switching from IgM via IgG to IgE. This sequential class switch might have functional implications for the frequency and repertoire of IgE⁺ cells. Here we show that in mutant mice, in which sequential switching to IgE via IgG1 is blocked, the frequency of cells switching to IgE is not affected. Thus, sequential class switching to IgE merely reflects the simultaneous accessibility of two acceptor switch regions for switch recombination, induced by one cytokine, but with markedly distinct efficiency. Analysis of switch recombination on both IgH alleles of switched cells shows that the low frequency of switching to IgE is an inherent feature of the Sε switch region and its control elements.

Upon activation, B lymphocytes can change the class of the antibody they express by immunoglobulin class switch recombination. Cytokines can direct this recombination to distinct classes by the specific activation of repetitive recombinogenic DNA sequences, the switch regions. Recombination to a particular switch region (sγ1) was abolished in mice that were altered to lack sequences that are 5 to the sγ1 region. This result directly implicates the functional importance of 5 switch region flanking sequences in the control of class switch recombination. Mutant mice exhibit a selective agammaglobulinemia and may be useful in the assessment of the biological importance of immunoglobulin G1.