Summary
Senescent cells are present in premalignant lesions and sites of tissue damage and accumulate in tissues with age. In vivo identification, quantification and characterization of senescent ...cells are challenging tasks that limit our understanding of the role of senescent cells in diseases and aging. Here, we present a new way to precisely quantify and identify senescent cells in tissues on a single‐cell basis. The method combines a senescence‐associated beta‐galactosidase assay with staining of molecular markers for cellular senescence and of cellular identity. By utilizing technology that combines flow cytometry with high‐content image analysis, we were able to quantify senescent cells in tumors, fibrotic tissues, and tissues of aged mice. Our approach also yielded the finding that senescent cells in tissues of aged mice are larger than nonsenescent cells. Thus, this method provides a basis for quantitative assessment of senescent cells and it offers proof of principle for combination of different markers of senescence. It paves the way for screening of senescent cells for identification of new senescence biomarkers, genes that bypass senescence or senolytic compounds that eliminate senescent cells, thus enabling a deeper understanding of the senescent state in vivo.
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 (
), 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
-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.
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 β-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.
Extracellular vesicles (EVs) transfer bioactive molecules between cells in a process reminiscent of enveloped viruses. EV cargo delivery is thought to occur by protein-mediated and pH-dependent ...membrane fusion of the EV and the cellular membrane. However, there is a lack of methods to identify the fusion proteins and resolve their mechanism. We developed and benchmarked an
biophysical assay to investigate EV membrane fusion. The assay was standardized by directly comparing EV and viral fusion with liposomes. We show that EVs and retroviruses fuse with liposomes mimicking the membrane composition of the late endosome in a pH- and protein-dependent manner. Moreover, we directly visualize the stages of membrane fusion using cryo-electron tomography. We find that, unlike most retroviruses, EVs remain fusogenic after acidification and reneutralization. These results provide novel insights into the EV cargo delivery mechanism and an experimental approach to identify the EV fusion machinery.
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.
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.
Synopsis
Plasmodium falciparum invades human red blood cells and releases two extracellular vesicle subsets secreted by infected cells. These EV subpopulations harbor different protein cargo and have specific mechanical membrane properties, suggesting distinct host cell targets.
Two distinct subsets of malaria‐derived EVs with different sizes are identified using asymmetric flow field‐flow fractionation.
Small EVs are rich in complement system proteins, whereas large EVs contain 20S proteasome subunits.
Small EVs are more efficient in fusing under endosomal conditions as compared to the large subset.
The EV subpopulations possess distinct membrane mechanical properties, suggesting different lipid compositions.
Plasmodium falciparum invades human red blood cells and releases two extracellular vesicle subsets secreted by infected cells. These EV subpopulations harbor different protein cargo and have specific mechanical membrane properties, suggesting distinct host cell targets.
Tuberculosis remains one of the deadliest infectious diseases worldwide. Mycobacterium tuberculosis (M.tb) has developed various mechanisms to manipulate the human host, in particular by disrupting ...the host phagosome and the immune response. It is becoming evident that secreted extracellular vesicles (EVs) are involved in the dynamic crosstalk between M.tb and the host cells. These vesicles shuttle different cargo components, such as RNA, lipids, and proteins, between cells. In this issue of EMBO Reports, Cheng and Schorey describe a previously unknown EV‐mediated process, regulating M.tb RNA loading into EVs and their internalization by naïve macrophages. They identify the mycobacterial Sec2 secretion system as involved in RNA loading into EVs and show that secreted vesicles contain bacterial RNA that not only promotes IFN‐β production upon entry into target cells, but also leads to M.tb clearance via the activation of the host's RIG‐I/MAVS signaling pathway. Importantly, combined treatment with secreted EVs and antibiotics decreases bacterial load in a mouse model, improving lung pathology compared to treatment with antibiotics alone.
The mycobacterial Sec2 secretion system regulates the loading of bacterial RNA into macrophage‐derived extracellular vesicles. Secreted vesicles with mycobacterial RNA promote IFN‐β production upon entry into target cells, and induce bacterial clearance.
Protozoan pathogens secrete nanosized particles called extracellular vesicles (EVs) to facilitate their survival and chronic infection. Here, we show the inhibition by Plasmodium berghei NK65 blood ...stage‐derived EVs of the proliferative response of CD4+ T cells in response to antigen presentation. Importantly, these results were confirmed in vivo by the capacity of EVs to diminish the ovalbumin‐specific delayed type hypersensitivity response. We identified two proteins associated with EVs, the histamine releasing factor (HRF) and the elongation factor 1α (EF‐1α) that were found to have immunosuppressive activities. Interestingly, in contrast to WT parasites, EVs from genetically HRF‐ and EF‐1α‐deficient parasites failed to inhibit T cell responses in vitro and in vivo. At the level of T cells, we demonstrated that EVs from WT parasites dephosphorylate key molecules (PLCγ1, Akt, and ERK) of the T cell receptor signalling cascade. Remarkably, immunisation with EF‐1α alone or in combination with HRF conferred a long‐lasting antiparasite protection and immune memory. In conclusion, we identified a new mechanism by which P. berghei‐derived EVs exert their immunosuppressive functions by altering T cell responses. The identification of two highly conserved immune suppressive factors offers new conceptual strategies to overcome EV‐mediated immune suppression in malaria‐infected individuals.
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
, 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.
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.
PDF
