Publications

The elucidation of viral-receptor interactions and an understanding of virus-spreading mechanisms are of great importance, particularly in the era of a pandemic. Indeed, advances in computational chemistry, synthetic biology, and protein engineering have allowed precise prediction and characterization of such interactions. Nevertheless, the hazards of the infectiousness of viruses, their rapid mutagenesis, and the need to study viral-receptor interactions in a complex in vivo setup call for further developments. Here, we show the development of biocompatible genetically engineered extracellular vesicles (EVs) that display the receptor binding domain (RBD) of SARS-CoV-2 on their surface as coronavirus mimetics (EVsRBD). Loading EVsRBD with iron oxide nanoparticles makes them MRI-visible and, thus, allows mapping of the binding of RBD to ACE2 receptors noninvasively in live subjects. Moreover, we show that EVsRBD can be modified to display mutants of the RBD of SARS-CoV-2, allowing rapid screening of currently raised or predicted variants of the virus. The proposed platform thus shows relevance and cruciality in the examination of quickly evolving pathogenic viruses in an adjustable, fast, and safe manner. Relying on MRI for visualization, the presented approach could be considered in the future to map ligand-receptor binding events in deep tissues, which are not accessible to luminescence-based imaging.

Malaria is the most serious mosquito-borne parasitic disease, caused mainly by the intracellular parasite Plasmodium falciparum. The parasite invades human red blood cells and releases extracellular vesicles (EVs) to alter its host responses. It becomes clear that EVs are generally composed of sub-populations. Seeking to identify EV subpopulations, we subject malaria-derived EVs to size-separation analysis, using asymmetric flow field-flow fractionation. Multi-technique analysis reveals surprising characteristics: we identify two distinct EV subpopulations differing in size and protein content. Small EVs are enriched in complement-system proteins and large EVs in proteasome subunits. We then measure the membrane fusion abilities of each subpopulation with three types of host cellular membranes: plasma, late and early endosome. Remarkably, small EVs fuse to early endosome liposomes at significantly greater levels than large EVs. Atomic force microscope imaging combined with machine-learning methods further emphasizes the difference in biophysical properties between the two subpopulations. These results shed light on the sophisticated mechanism by which malaria parasites utilize EV subpopulations as a communication tool to target different cellular destinations or host systems.

Glycoconjugates on extracellular vesicles (EVs) play a vital role in internalization and mediate interaction as well as regulation of the host immune system by viruses, bacteria, and parasites. During their intraerythrocytic life‐cycle stages, malaria parasites, Plasmodium falciparum (Pf) mediate the secretion of EVs by infected red blood cells (RBCs) that carry a diverse range of parasitic and host‐derived molecules. These molecules facilitate parasite‐parasite and parasite‐host interactions to ensure parasite survival.
To date, the number of identified Pf genes associated with glycan synthesis and the repertoire of expressed glycoconjugates is relatively low. Moreover, the role of Pf glycans in pathogenesis is mostly unclear and poorly understood. As a result, the expression of glycoconjugates on Pf‐derived EVs or their involvement in the parasite life‐cycle has yet to be reported.
Herein, we show that EVs secreted by Pf‐infected RBCs carry significantly higher sialylated complex N‐glycans than EVs derived from healthy RBCs. Furthermore, we reveal that EV uptake by host monocytes depends on N‐glycoproteins and demonstrate that terminal sialic acid on the N‐glycans is essential for uptake by human monocytes. Our results provide the first evidence that Pf exploits host sialylated N‐glycans to mediate EV uptake by the human immune system cells.

The chemokine CXCL10 is associated with pathogenesis of cerebral malaria in Plasmodium falciparum infection. Here the authors show that P. falciparum produces extracellular vesicles laden with RNAs that are taken up by monocytes resulting in a RIG-I and HUR-1 mediated mechanism of inhibition of CXCL10 protein translation.

Background: The role of epicardial fat (eFat)-derived extracellular vesicles (EVs) in the pathogenesis of atrial fibrillation (AF) has never been studied. We tested the hypothesis that eFat-EVs transmit proinflammatory, profibrotic, and proarrhythmic molecules that induce atrial myopathy and fibrillation. Methods: We collected eFat specimens from patients with (n=32) and without AF (n=30) during elective heart surgery. eFat samples were grown as organ cultures, and the culture medium was collected every 2 days. We then isolated and purified eFat-EVs from the culture medium, and analyzed the EV number, size, morphology, specific markers, encapsulated cytokines, proteome, and microRNAs. Next, we evaluated the biological effects of unpurified and purified EVs on atrial mesenchymal stromal cells and endothelial cells in vitro. To establish a causal association between eFat-EVs and vulnerability to AF, we modeled AF in vitro using induced pluripotent stem cell–derived cardiomyocytes. Results: Microscopic examination revealed excessive inflammation, fibrosis, and apoptosis in fresh and cultured eFat tissues. Cultured explants from patients with AF secreted more EVs and harbored greater amounts of proinflammatory and profibrotic cytokines, and profibrotic microRNA, as well, than those without AF. The proteomic analysis confirmed the distinctive profile of purified eFat-EVs from patients with AF. In vitro, purified and unpurified eFat-EVs from patients with AF had a greater effect on proliferation and migration of human mesenchymal stromal cells and endothelial cells, compared with eFat-EVs from patients without AF. Last, whereas eFat-EVs from patients with and without AF shortened the action potential duration of induced pluripotent stem cell–derived cardiomyocytes, only eFat-EVs from patients with AF induced sustained reentry (rotor) in induced pluripotent stem cell–derived cardiomyocytes. Conclusions: We show, for the first time, a distinctive proinflammatory, profibrotic, and proarrhythmic signature of eFat-EVs from patients with AF. Our findings uncover another pathway by which eFat promotes the development of atrial myopathy and fibrillation.

Extracellular vesicles (EVs) are cell-derived membrane-bound structures that are believed to play a major role in intercellular communication by allowing cells to exchange proteins and genetic cargo between them. In particular, pathogens, such as the malaria parasite Plasmodium (P.) falciparum, utilize EVs to promote their growth and to alter their host's response. Thus, better characterization of these secreted organelles will enhance our understanding of the cellular processes that govern EVs' biology and pathological functions. Here we present a method that utilizes a high-end flow cytometer system to characterize small EVs, i.e., with a diameter less than 200 nm. Using this method, we could evaluate different parasite-derived EV populations according to their distinct cargo by using antibody-free labeling. It further allows to closely monitor a sub-population of vesicles carrying parasitic DNA cargo. This ability paves the way to conducting a more 'educated' analysis of the various EV cargo components.

During the chronic stage of Schistosoma infection, the female lays fertile eggs, triggering a strong anti-parasitic type 2 helper T-cell (Th2) immune response. It is unclear how this Th2 response gradually declines even though the worms live for years and continue to produce eggs. Here, we show that Schistosoma mansoni downregulates Th2 differentiation in an antigen-presenting cell-independent manner, by modulating the Th2-specific transcriptional program. Adult schistosomes secrete miRNA-harboring extracellular vesicles that are internalized by Th cells in vitro. Schistosomal miRNAs are found also in T helper cells isolated from Peyer's patches and mesenteric lymph nodes of infected mice. In T helper cells, the schistosomal miR-10 targets MAP3K7 and consequently downmodulates NF-kappa B activity, a critical transcription factor for Th2 differentiation and function. Our results explain, at least partially, how schistosomes tune down the Th2 response, and provide further insight into the reciprocal geographic distribution between high prevalence of parasitic infections and immune disorders such as allergy. Furthermore, this worm-host crosstalk mechanism can be harnessed to develop diagnostic and therapeutic approaches for human schistosomiasis and Th2-associated diseases.

All living organisms secrete molecules for intercellular communication. Recent research has revealed that extracellular vesicles (EVs) play an important role in inter-organismal cell-to-cell communication by transporting diverse messenger molecules, including RNA, DNA, lipids and proteins. These discoveries have raised fundamental questions regarding EV biology. How are EVs biosynthesized and loaded with messenger/cargo molecules? How are EVs secreted into the extracellular matrix? What are the EV uptake mechanisms of recipient cells? As EVs are produced by all kind of organisms, from unicellular bacteria and protists, filamentous fungi and oomycetes, to complex multicellular life forms such as plants and animals, basic research in diverse model systems is urgently needed to shed light on the multifaceted biology of EVs and their role in inter-organismal communications. To help catalyse progress in this emerging field, a mini-symposium was held in Munich, Germany in August 2018. This report highlights recent progress and major questions being pursued across a very diverse group of model systems, all united by the question of how EVs contribute to inter-organismal communication.

A large number of studies demonstrate that cell mechanics and pathology are intimately linked. In particular, deformability of red blood cells (RBCs) is key to their function and is dramatically altered in the time course of diseases such as anemia and malaria. Due to the physiological importance of cell mechanics, many methods for cell mechanical probing have been developed. While single-cell methods provide very valuable information, they are often technically challenging and lack the high data throughput needed to distinguish differences in heterogeneous populations, while fluid-flow high-throughput methods miss the accuracy to detect subtle differences. Here we present a new method for multiplexed single-cell mechanical probing using acoustic force spectroscopy (AFS). We demonstrate that mechanical differences induced by chemical treatments of known effect can be measured and quantified. Furthermore, we explore the effect of extracellular vesicles (EVs) uptake on RBC mechanics and demonstrate that EVs uptake increases RBC deformability. Our findings demonstrate the ability of AFS to manipulate cells with high stability and precision and pave the way to further new insights into cellular mechanics and mechanobiology in health and disease, as well as potential biomedical applications.

Metabolic changes within the cell and its niche affect cell fate and are involved in many diseases and disorders including cancer and viral infections. Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma (KS). KSHV latently infected cells express only a subset of viral genes, mainly located within the latency-associated region, among them 12 microRNAs. Notably, these miRNAs are responsible for inducing the Warburg effect in infected cells. Here we identify a novel mechanism enabling KSHV to manipulate the metabolic nature of the tumour microenvironment. We demonstrate that KSHV infected cells specifically transfer the virus-encoded microRNAs to surrounding cells via exosomes. This flow of genetic information results in a metabolic shift toward aerobic glycolysis in the surrounding non-infected cells. Importantly, this exosome-mediated metabolic reprogramming of neighbouring cells supports the growth of infected cells, thereby contributing to viral fitness. Finally, our data show that this miRNA transfer-based regulation of cell metabolism is a general mechanism used by other herpesviruses, such as EBV, as well as for the transfer of non-viral onco-miRs. This exosome-based crosstalk provides viruses with a mechanism for non-infectious transfer of genetic material without production of new viral particles, which might expose them to the immune system. We suggest that viruses and cancer cells use this mechanism to shape a specific metabolic niche that will contribute to their fitness.

Necroptosis is a regulated, nonapoptotic form of cell death initiated by receptor-interacting protein kinase-3 (RIPK3) and mixed lineage kinase domain-like (MLKL) proteins. It is considered to be a form of regulated necrosis, and, by lacking the "find me" and "eat me" signals that are a feature of apoptosis, necroptosis is considered to be inflammatory. One such "eat me" signal observed during apoptosis is the exposure of phosphatidylserine (PS) on the outer plasma membrane. Here, we demonstrate that necroptotic cells also expose PS after phosphorylated mixed lineage kinase-like (pMLKL) translocation to the membrane. Necroptotic cells that expose PS release extracellular vesicles containing proteins and pMLKL to their surroundings. Furthermore, inhibition of pMLKL after PS exposure can reverse the process of necroptosis and restore cell viability. Finally, externalization of PS by necroptotic cells drives recognition and phagocytosis, and this may limit the inflammatory response to this nonapoptotic form of cell death. The exposure of PS to the outer membrane and to extracellular vesicles is therefore a feature of necroptotic cell death and may serve to provide an immunologically-silent window by generating specific "find me" and "eat me" signals.

Malaria is the most devastating parasitic disease of humans, caused by the unicellular protozoa of the Plasmodium genus, such as Plasmodium falciparum (Pf) and is responsible for up to a million deaths each year. Pf life cycle is complex, with transmission of the parasite between humans via mosquitos involving a remarkable series of morphological transformations. In the bloodstream, the parasites undergo asexual multiplications inside the red blood cell (RBC), where they mature through the ring (R), trophozoite (T) and schizont (S) stages, and sexual development, resulting in gametocytes (G). All symptoms of malaria pathology are caused by the asexual blood stage parasites. Flow cytometry methods were previously used to detect malaria infected (i) RBCs, in live or fixed cells, using DNA (Hoechst) and RNA (Thiazole Orange) stains. Here, by using imaging flow cytometry, we developed improved methods of identifying and quantifying each of the four parasite blood stages (R, T, S and G). This technique allows multi-channel, high resolution imaging of individual parasites, as well as detailed morphological quantification of Pf-iRBCs cultures. Moreover, by measuring iRBC morphological properties, we can eliminate corrupted and extra cellular (dying) parasites from the analysis, providing accurate quantification and robust measurement of the parasitemia profile. This new method is a valuable tool in malaria molecular biology research and drug screen assays. (C) 2016 Elsevier Inc. All rights reserved.