Proteomic analysis of necroptotic extracellular vesicles.

Necroptosis is a regulated and inflammatory form of cell death. We, and others, have previously reported that necroptotic cells release extracellular vesicles (EVs). We have found that necroptotic EVs are loaded with proteins, including the phosphorylated form of the key necroptosis-executing factor, mixed lineage kinase domain-like kinase (MLKL). However, neither the exact protein composition, nor the impact, of necroptotic EVs have been delineated. To characterize their content, EVs from necroptotic and untreated U937 cells were isolated and analyzed by mass spectrometry-based proteomics. A total of 3337 proteins were identified, sharing a high degree of similarity with exosome proteome databases, and clearly distinguishing necroptotic and control EVs. A total of 352 proteins were significantly upregulated in the necroptotic EVs. Among these were MLKL and caspase-8, as validated by immunoblot. Components of the ESCRTIII machinery and inflammatory signaling were also upregulated in the necroptotic EVs, as well as currently unreported components of vesicle formation and transport, and necroptotic signaling pathways. Moreover, we found that necroptotic EVs can be phagocytosed by macrophages to modulate cytokine and chemokine secretion. Finally, we uncovered that necroptotic EVs contain tumor neoantigens, and are enriched with components of antigen processing and presentation. In summary, our study reveals a new layer of regulation during the early stage of necroptosis, mediated by the secretion of specific EVs that influences the microenvironment and may instigate innate and adaptive immune responses. This study sheds light on new potential players in necroptotic signaling and its related EVs, and uncovers the functional tasks accomplished by the cargo of these necroptotic EVs.

Malaria parasites both repress host CXCL10 and use it as a cue for growth acceleration.

Pathogens are thought to use host molecular cues to control when to initiate life-cycle transitions, but these signals are mostly unknown, particularly for the parasitic disease malaria caused by Plasmodium falciparum. The chemokine CXCL10 is present at high levels in fatal cases of cerebral malaria patients, but is reduced in patients who survive and do not have complications. Here we show a Pf 'decision-sensing-system' controlled by CXCL10 concentration. High CXCL10 expression prompts P. falciparum to initiate a survival strategy via growth acceleration. Remarkably, P. falciparum inhibits CXCL10 synthesis in monocytes by disrupting the association of host ribosomes with CXCL10 transcripts. The underlying inhibition cascade involves RNA cargo delivery into monocytes that triggers RIG-I, which leads to HUR1 binding to an AU-rich domain of the CXCL10 3'UTR. These data indicate that when the parasite can no longer keep CXCL10 at low levels, it can exploit the chemokine as a cue to shift tactics and escape.

Cancer-Associated Fibroblasts Promote Aggressive Gastric Cancer Phenotypes via Heat Shock Factor 1-Mediated Secretion of Extracellular Vesicles.

Gastric cancer is the third most lethal cancer worldwide, and evaluation of the genomic status of gastric cancer cells has not translated into effective prognostic or therapeutic strategies. We therefore hypothesize that outcomes may depend on the tumor microenvironment (TME), in particular, cancer-associated fibroblasts (CAF). However, very little is known about the role of CAFs in gastric cancer. To address this, we mapped the transcriptional landscape of human gastric cancer stroma by microdissection and RNA sequencing of CAFs from patients with gastric cancer. A stromal gene signature was associated with poor disease outcome, and the transcription factor heat shock factor 1 (HSF1) regulated the signature. HSF1 upregulated inhibin subunit beta A and thrombospondin 2, which were secreted in CAF-derived extracellular vesicles to the TME to promote cancer. Together, our work provides the first transcriptional map of human gastric cancer stroma and highlights HSF1 and its transcriptional targets as potential diagnostic and therapeutic targets in the genomically stable tumor microenvironment. SIGNIFICANCE: This study shows how HSF1 regulates a stromal transcriptional program associated with aggressive gastric cancer and identifies multiple proteins within this program as candidates for therapeutic intervention. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/7/1639/F1.large.jpg.

20S proteasomes secreted by the malaria parasite promote its growth.

Mature red blood cells (RBCs) lack internal organelles and canonical defense mechanisms, making them both a fascinating host cell, in general, and an intriguing choice for the deadly malaria parasite Plasmodium falciparum (Pf), in particular. Pf, while growing inside its natural host, the human RBC, secretes multipurpose extracellular vesicles (EVs), yet their influence on this essential host cell remains unknown. Here we demonstrate that Pf parasites, cultured in fresh human donor blood, secrete within such EVs assembled and functional 20S proteasome complexes (EV-20S). The EV-20S proteasomes modulate the mechanical properties of naïve human RBCs by remodeling their cytoskeletal network. Furthermore, we identify four degradation targets of the secreted 20S proteasome, the phosphorylated cytoskeletal proteins ß-adducin, ankyrin-1, dematin and Epb4.1. Overall, our findings reveal a previously unknown 20S proteasome secretion mechanism employed by the human malaria parasite, which primes RBCs for parasite invasion by altering membrane stiffness, to facilitate malaria parasite growth.

Trypanosoma cruzi-Infected Human Macrophages Shed Proinflammatory Extracellular Vesicles That Enhance Host-Cell Invasion via Toll-Like Receptor 2.

Extracellular vesicles (EVs) shed by trypomastigote forms of Trypanosoma cruzi have the ability to interact with host tissues, increase invasion, and modulate the host innate response. In this study, EVs shed from T. cruzi or T.cruzi-infected macrophages were investigated as immunomodulatory agents during the initial steps of infection. Initially, by scanning electron microscopy and nanoparticle tracking analysis, we determined that T. cruzi-infected macrophages release higher numbers of EVs (50-300 nm) as compared to non-infected cells. Using Toll-like-receptor 2 (TLR2)-transfected CHO cells, we observed that pre-incubation of these host cells with parasite-derived EVs led to an increase in the percentage of infected cells. In addition, EVs from parasite or T.cruzi-infected macrophages or not were able to elicit translocation of NF-κB by interacting with TLR2, and as a consequence, to alter the EVs the gene expression of proinflammatory cytokines (TNF-α, IL-6, and IL-1ß), and STAT-1 and STAT-3 signaling pathways. By proteomic analysis, we observed highly significant changes in the protein composition between non-infected and infected host cell-derived EVs. Thus, we observed the potential of EVs derived from T. cruzi during infection to maintain the inflammatory response in the host.

Schistosomal extracellular vesicle-enclosed miRNAs modulate host T helper cell differentiation.

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-κB 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.

Nanomechanics of Extracellular Vesicles Reveals Vesiculation Pathways.

Extracellular vesicles (EVs) are emerging as important mediators of cell-cell communication as well as potential disease biomarkers and drug delivery vehicles. However, the mechanical properties of these vesicles are largely unknown, and processes leading to microvesicle-shedding from the plasma membrane are not well understood. Here an in depth atomic force microscopy force spectroscopy study of the mechanical properties of natural EVs is presented. It is found that several natural vesicles of different origin have a different composition of lipids and proteins, but similar mechanical properties. However, vesicles generated by red blood cells (RBC) at different temperatures/incubation times are different mechanically. Quantifying the lipid content of EVs reveals that their stiffness decreases with the increase in their protein/lipid ratio. Further, by maintaining RBC at "extreme" nonphysiological conditions, the cells are pushed to utilize different vesicle generation pathways. It is found that RBCs can generate protein-rich soft vesicles, possibly driven by protein aggregation, and low membrane-protein content stiff vesicles, likely driven by cytoskeleton-induced buckling. Since similar cortical cytoskeleton to that of the RBC exists on the membranes of most mammalian cells, our findings help advancing the understanding of the fundamental process of vesicle generation.

Monitoring Extracellular Vesicle Cargo Active Uptake by Imaging Flow Cytometry.

Extracellular vesicles are essential for long distance cell-cell communication. They function as carriers of different compounds, including proteins, lipids and nucleic acids. Pathogens, like malaria parasites (Plasmodium falciparum, Pf), excel in employing vesicle release to mediate cell communication in diverse processes, particularly in manipulating the host response. Establishing research tools to study the interface between pathogen-derived vesicles and their host recipient cells will greatly benefit the scientific community. Here, we present an imaging flow cytometry (IFC) method for monitoring the uptake of malaria-derived vesicles by host immune cells. By staining different cargo components, we were able to directly track the cargo's internalization over time and measure the kinetics of its delivery. Impressively, we demonstrate that this method can be used to specifically monitor the translocation of a specific protein within the cellular milieu upon internalization of parasitic cargo; namely, we were able to visually observe how uptaken parasitic Pf-DNA cargo leads to translocation of transcription factor IRF3 from the cytosol to the nucleus within the recipient immune cell. Our findings demonstrate that our method can be used to study cellular dynamics upon vesicle uptake in different host-pathogen and pathogen-pathogen systems.

Probing cellular mechanics with acoustic force spectroscopy.

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.

Malaria parasite DNA-harbouring vesicles activate cytosolic immune sensors.

STING is an innate immune cytosolic adaptor for DNA sensors that engage malaria parasite (Plasmodium falciparum) or other pathogen DNA. As P. falciparum infects red blood cells and not leukocytes, how parasite DNA reaches such host cytosolic DNA sensors in immune cells is unclear. Here we show that malaria parasites inside red blood cells can engage host cytosolic innate immune cell receptors from a distance by secreting extracellular vesicles (EV) containing parasitic small RNA and genomic DNA. Upon internalization of DNA-harboring EVs by human monocytes, P. falciparum DNA is released within the host cell cytosol, leading to STING-dependent DNA sensing. STING subsequently activates the kinase TBK1, which phosphorylates the transcription factor IRF3, causing IRF3 to translocate to the nucleus and induce STING-dependent gene expression. This DNA-sensing pathway may be an important decoy mechanism to promote P. falciparum virulence and thereby may affect future strategies to treat malaria.

Herpesviruses shape tumour microenvironment through exosomal transfer of viral microRNAs.

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.

Phosphatidylserine externalization, "necroptotic bodies" release, and phagocytosis during necroptosis.

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.

Pathogen-derived extracellular vesicles coordinate social behaviour and host manipulation.

Infectious diseases are the leading cause of death of children worldwide, causing a tenacious and major public-health burden. The dynamic interplay between pathogens and their host is one of the most complicated themes of the disease progression. Pathogens excel in developing different means to facilitate cell-cell communication via secreted vesicles, among others. The released vesicles are involved in the transfer of biologically active molecules that induce phenotypic changes in the recipient cells. The messages within the vesicles are delivered to coordinate diverse processes, including virulence factor expression, differentiation state and control of their population density. Importantly, production of such vesicles promotes pathogen survival, as it provides a secure means of pathogen-pathogen communication and an ability to manipulate host responses for their own benefits. This review highlights intriguing findings, which show the important role of EVs in the social activity of pathogens, within and in between their communities. We further present examples of how pathogens use EVs to alter host immune and non-immune responses. Advancing our understanding of cell-cell communication in infectious diseases will be particularly useful to decipher the complexity of the cross-talk between pathogens themselves and their hosts, leading to the development of therapeutic strategies for fighting infectious agents.

Identification and classification of the malaria parasite blood developmental stages, using imaging flow cytometry.

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 extracellular (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.

Structural switch of lysyl-tRNA synthetase between translation and transcription.

Lysyl-tRNA synthetase (LysRS), a component of the translation apparatus, is released from the cytoplasmic multi-tRNA synthetase complex (MSC) to activate the transcription factor MITF in stimulated mast cells through undefined mechanisms. Here we show that Ser207 phosphorylation provokes a new conformer of LysRS that inactivates its translational function but activates its transcriptional function. The crystal structure of an MSC subcomplex established that LysRS is held in the MSC by binding to the N terminus of the scaffold protein p38/AIMP2. Phosphorylation-created steric clashes at the LysRS domain interface disrupt its binding grooves for p38/AIMP2, releasing LysRS and provoking its nuclear translocation. This alteration also exposes the C-terminal domain of LysRS to bind to MITF and triggers LysRS-directed production of the second messenger Ap(4)A that activates MITF. Thus our results establish that a single conformational change triggered by phosphorylation leads to multiple effects driving an exclusive switch of LysRS function from translation to transcription.

Transcription factor E3, a major regulator of mast cell-mediated allergic response.

BACKGROUND: Microphthalmia transcription factor, an MiT transcription family member closely related to transcription factor E3 (TFE3), is essential for mast cell development and survival. TFE3 was previously reported to play a role in the functions of B and T cells; however, its role in mast cells has not yet been explored. OBJECTIVE: We sought to explore the role played by TFE3 in mast cell function. METHODS: Mast cell numbers were evaluated by using toluidine blue staining. FACS analysis was used to determine percentages of Kit and FcεRI double-positive cells in the peritoneum of wild-type (WT) and TFE3 knockout (TFE3(-/-)) mice. Cytokine and inflammatory mediator secretion were measured in immunologically activated cultured mast cells derived from either knockout or WT mice. In vivo plasma histamine levels were measured after immunologic triggering of these mice. RESULTS: No significant differences in mast cell numbers between WT and TFE3(-/-) mice were observed in the peritoneum, lung, and skin. However, TFE3(-/-) mice showed a marked decrease in the number of Kit(+) and FcεRI(+) peritoneal and cultured mast cells. Surface expression levels of FcεRI in TFE3(-/-) peritoneal mast cells was significantly lower than in control cells. Cultured mast cells derived from TFE3(-/-) mice showed a marked decrease in degranulation and mediator secretion. In vivo experiments showed that the level of plasma histamine in TFE3(-/-) mice after an allergic trigger was substantially less than that seen in WT mice. CONCLUSION: TFE3 is a novel regulator of mast cell functions and as such could emerge as a new target for the manipulation of allergic diseases.

Importin beta plays an essential role in the regulation of the LysRS-Ap(4)A pathway in immunologically activated mast cells.

We recently reported that diadenosine tetraphosphate hydrolase (Ap(4)A hydrolase) plays a critical role in gene expression via regulation of intracellular Ap(4)A levels. This enzyme serves as a component of our newly described lysyl tRNA synthetase (LysRS)-Ap(4)A biochemical pathway that is triggered upon immunological challenge. Here we explored the mechanism of this enzyme's translocation into the nucleus and found its immunologically dependent association with importin beta. Silencing of importin beta prevented Ap(4)A hydrolase nuclear translocation and affected the local concentration of Ap(4)A, which led to an increase in microphthalmia transcription factor (MITF) transcriptional activity. Furthermore, immunological activation of mast cells resulted in dephosphorylation of Ap(4)A hydrolase, which changed the hydrolytic activity of the enzyme.