Exosomes are extracellular vesicles (EVs) secreted upon fusion of endosomal multivesicular bodies (MVBs) with the plasma membrane. The mechanisms involved in their biogenesis have not yet been fully ...identified although they could be used to modulate exosome formation and therefore are a promising tool in understanding exosome functions. We have performed an RNA interference screen targeting 23 components of the endosomal sorting complex required for transport (ESCRT) machinery and associated proteins in MHC class II (MHC II)-expressing HeLa-CIITA cells. Silencing of HRS, STAM1 or TSG101 reduced the secretion of EV-associated CD63 and MHC II but each gene altered differently the size and/or protein composition of secreted EVs, as quantified by immuno-electron microscopy. By contrast, depletion of VPS4B augmented this secretion while not altering the features of EVs. For several other ESCRT subunits, it was not possible to draw any conclusions about their involvement in exosome biogenesis from the screen. Interestingly, silencing of ALIX increased MHC II exosomal secretion, as a result of an overall increase in intracellular MHC II protein and mRNA levels. In human dendritic cells (DCs), ALIX depletion also increased MHC II in the cells, but not in the released CD63-positive EVs. Such differences could be attributed to a greater heterogeneity in size, and higher MHC II and lower CD63 levels in vesicles recovered from DCs as compared with HeLa-CIITA. The results reveal a role for selected ESCRT components and accessory proteins in exosome secretion and composition by HeLa-CIITA. They also highlight biogenetic differences in vesicles secreted by a tumour cell line and primary DCs.
Dendritic cells (DC) are professional antigen-presenting cells that orchestrate immune responses. The human DC population comprises two main functionally specialized lineages, whose origins and ...differentiation pathways remain incompletely defined. Here, we combine two high-dimensional technologies-single-cell messenger RNA sequencing (scmRNAseq) and cytometry by time-of-flight (CyTOF)-to identify human blood CD123
CD33
CD45RA
DC precursors (pre-DC). Pre-DC share surface markers with plasmacytoid DC (pDC) but have distinct functional properties that were previously attributed to pDC. Tracing the differentiation of DC from the bone marrow to the peripheral blood revealed that the pre-DC compartment contains distinct lineage-committed subpopulations, including one early uncommitted CD123
pre-DC subset and two CD45RA
CD123
lineage-committed subsets exhibiting functional differences. The discovery of multiple committed pre-DC populations opens promising new avenues for the therapeutic exploitation of DC subset-specific targeting.
Cells of the myeloid lineage, particularly macrophages, serve as primary hosts for HIV
, along with CD4 T lymphocytes. Macrophages are present in virtually every tissue of the organism, including ...locations with negligible T cell colonization, such as the brain, where HIV-mediated inflammation may lead to pathological sequelae. Moreover, infected macrophages are present in multiple other tissues. Recent evidence obtained in humanized mice and macaque models highlighted the capacity of macrophages to sustain HIV replication
in the absence of T cells. Combined with the known resistance of the macrophage to the cytopathic effects of HIV infection, such data bring a renewed interest in this cell type both as a vehicle for viral spread as well as a viral reservoir. While our understanding of key processes of HIV infection of macrophages is far from complete, recent years have nevertheless brought important insight into the uniqueness of the macrophage infection. Productive infection of macrophages by HIV can occur by different routes including from phagocytosis of infected T cells. In macrophages, HIV assembles and buds into a peculiar plasma membrane-connected compartment that preexists to the infection. While the function of such compartment remains elusive, it supposedly allows for the persistence of infectious viral particles over extended periods of time and may play a role on viral transmission. As cells of the innate immune system, macrophages have the capacity to detect and respond to viral components. Recent data suggest that such sensing may occur at multiple steps of the viral cycle and impact subsequent viral spread. We aim to provide an overview of the HIV-macrophage interaction along the multiple stages of the viral life cycle, extending when pertinent such observations to additional myeloid cell types such as dendritic cells or blood monocytes.
Exosomes are secreted membrane vesicles that share structural and biochemical characteristics with intraluminal vesicles of multivesicular endosomes (MVEs). Exosomes could be involved in ...intercellular communication and in the pathogenesis of infectious and degenerative diseases. The molecular mechanisms of exosome biogenesis and secretion are, however, poorly understood. Using an RNA interference (RNAi) screen, we identified five Rab GTPases that promote exosome secretion in HeLa cells. Among these, Rab27a and Rab27b were found to function in MVE docking at the plasma membrane. The size of MVEs was strongly increased by Rab27a silencing, whereas MVEs were redistributed towards the perinuclear region upon Rab27b silencing. Thus, the two Rab27 isoforms have different roles in the exosomal pathway. In addition, silencing two known Rab27 effectors, Slp4 (also known as SYTL4, synaptotagmin-like 4) and Slac2b (also known as EXPH5, exophilin 5), inhibited exosome secretion and phenocopied silencing of Rab27a and Rab27b, respectively. Our results therefore strengthen the link between MVEs and exosomes, and introduce ways of manipulating exosome secretion in vivo.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Here we describe a new mouse model that exploits the pattern of expression of the high-affinity IgG receptor (CD64) and allows diphtheria toxin (DT)-mediated ablation of tissue-resident macrophages ...and monocyte-derived cells. We found that the myeloid cells of the ear skin dermis are dominated by DT-sensitive, melanin-laden cells that have been missed in previous studies and correspond to macrophages that have ingested melanosomes from neighboring melanocytes. Those cells have been referred to as melanophages in humans. We also identified melanophages in melanocytic melanoma. Benefiting of our knowledge on melanophage dynamics, we determined the identity, origin, and dynamics of the skin myeloid cells that capture and retain tattoo pigment particles. We showed that they are exclusively made of dermal macrophages. Using the possibility to delete them, we further demonstrated that tattoo pigment particles can undergo successive cycles of capture-release-recapture without any tattoo vanishing. Therefore, congruent with dermal macrophage dynamics, long-term tattoo persistence likely relies on macrophage renewal rather than on macrophage longevity.
A subset of TLRs is specialised in the detection of incoming pathogens by sampling endosomes for nucleic acid contents. Among them, TLR3 senses the abnormal presence of double-stranded RNA in the ...endosomes and initiates a potent innate immune response via activation of NF-κB and IRF3. Nevertheless, mechanisms governing TLR3 regulation remain poorly defined. To identify new molecular players involved in the TLR3 pathway, we performed a genome-wide screen using CRISPR/Cas9 technology. We generated TLR3+ reporter cells carrying a NF-κB-responsive promoter that controls GFP expression. Cells were next transduced with a single-guide RNA (sgRNA) library, subjected to sequential rounds of stimulation with poly(I:C) and sorting of the GFP-negative cells. Enrichments in sgRNA estimated by deep sequencing identified genes required for TLR3-induced activation of NF-κB. Among the hits, five genes known to be critically involved in the TLR3 pathway, including TLR3 itself and the chaperone UNC93B1, were identified by the screen, thus validating our strategy. We further studied the top 40 hits and focused on the transcription factor aryl hydrocarbon receptor (AhR). Depletion of AhR had a dual effect on the TLR3 response, abrogating IL-8 production and enhancing IP-10 release. Moreover, in primary human macrophages exposed to poly(I:C), AhR activation enhanced IL-8 and diminished IP-10 release. Overall, these results reveal AhR plays a role in the TLR3 cellular innate immune response.
Significance A major obstacle to the eradication of HIV-1 by combination antiretroviral therapy (cART) is the formation of cellular reservoirs in CD4 ⺠T lymphocytes (carrying latently integrated ...provirus) and tissue macrophages. Infected macrophages assemble new virions in subcellular vacuoles known as virus-containing compartments (VCC), hiding them from the immune system and, in part, from antiretroviral agents. Here we report that extracellular ATP is capable of inducing the rapid release of virions accumulated in VCC via interaction with the P2X7 receptor and without inducing cell death, whereas the antidepressant agent Imipramine blocks the release. Thus, our study identifies two âdruggableâ targets affecting the release of stored virions from infected human macrophages that could bear relevance for purging HIV-1 reservoirs in individuals receiving cART.
HIV type 1 (HIV-1) infects CD4 ⺠T lymphocytes and tissue macrophages. Infected macrophages differ from T cells in terms of decreased to absent cytopathicity and for active accumulation of new progeny HIV-1 virions in virus-containing compartments (VCC). For these reasons, infected macrophages are believed to act as âTrojan horsesâ carrying infectious particles to be released on cell necrosis or functional stimulation. Here we explored the hypothesis that extracellular ATP (eATP) could represent a microenvironmental signal potentially affecting virion release from VCC of infected macrophages. Indeed, eATP triggered the rapid release of infectious HIV-1 from primary human monocyte-derived macrophages (MDM) acutely infected with the CCR5-dependent HIV-1 strain. A similar phenomenon was observed in chronically infected promonocytic U1 cells differentiated to macrophage-like cells (D-U1) by costimulation with phorbol esters and urokinase-type plasminogen activator. Worthy of note, eATP did not cause necrotic, apoptotic, or pyroptotic cell death, and its effect on HIV-1 release was suppressed by Imipramine (an antidepressant agent known to inhibit microvesicle formation by interfering with membrane-associated acid sphingomyelinase). Virion release was not triggered by oxidized ATP, whereas the effect of eATP was inhibited by a specific inhibitor of the P2X7 receptor (P2X7R). Thus, eATP triggered the discharge of virions actively accumulating in VCC of infected macrophages via interaction with the P2X7R in the absence of significant cytopathicity. These findings suggest that the microvesicle pathway and P2X7R could represent exploitable targets for interfering with the VCC-associated reservoir of infectious HIV-1 virions in tissue macrophages.
Toll-like receptor (TLR) 3 is an endosomal TLR that mediates immune responses against viral infections upon activation by its ligand double-stranded RNA, a replication intermediate of most viruses. ...TLR3 is expressed widely in the body and activates both the innate and adaptive immune systems. However, little is known about how TLR3 intracellular trafficking and maturation are regulated. Here we show that newly synthesized endogenous TLR3 is transported through the ER and Golgi apparatus to endosomes, where it is rapidly cleaved. TLR3 protein expression is up-regulated by its own ligand, leading to the accumulation of its cleaved form. In agreement with its proposed role as a transporter, UNC93B1 expression is required for TLR3 cleavage and signaling. Furthermore, TLR3 signaling and cleavage are sensitive to cathepsin inhibition. Cleavage occurs between aa 252 and 346, and results in a functional receptor that signals upon activation. A truncated form of TLR3 lacking the N-terminal 345 aa also signals from acidic compartments in response to ligand activation. Screening of the human cathepsin family by RNA interference identified cathepsins B and H as key mediators of TLR3 processing. Taken together, our data indicate that TLR3 proteolytic processing is essential for its function, and suggest a mechanism of tight control of TLR3 signaling and thus immunity.
During the late stages of the HIV-1 replication cycle, the viral polyprotein Pr55(Gag) is recruited to the plasma membrane (PM), where it binds phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and ...directs HIV-1 assembly. We show that Rab27a controls the trafficking of late endosomes carrying phosphatidylinositol 4-kinase type 2 α (PI4KIIα) toward the PM of CD4(+) T cells. Hence, Rab27a promotes high levels of PM phosphatidylinositol 4-phosphate and the localized production of PI(4,5)P2, therefore controlling Pr55(Gag) membrane association. Rab27a also controls PI(4,5)P2 levels at the virus-containing compartments of macrophages. By screening Rab27a effectors, we identified that Slp2a, Slp3, and Slac2b are required for the association of Pr55(Gag) with the PM and that Slp2a cooperates with Rab27a in the recruitment of PI4KIIα to the PM. We conclude that by directing the trafficking of PI4KIIα-positive endosomes toward the PM, Rab27a controls PI(4,5)P2 production and, consequently, HIV-1 replication.
Targeting TLR3 through formulations of polyI:C is widely studied as an adjuvant in cancer immunotherapy. The efficacy of such targeting has been shown to increase in combination with anti-PD-L1 ...treatment. Nevertheless, the mechanistic details of the effect of polyI:C on DC maturation and the impact on T-DC interactions upon PD-L1 blockade is largely unknown. Here we found that although DC treatment with polyI:C induced a potent inflammatory response including the production of type I interferon, polyI:C treatment of DCs impaired activation of peptide specific CD8+ T cells mainly due to PD-L1. Interestingly, we found that PD-L1 trafficking to the cell surface is regulated in two waves in polyI:C-treated DCs. One induced upon overnight treatment and a second more rapid one, specific to polyI:C treatment, was induced upon CD40 signaling leading to a further increase in surface PD-L1 in DCs. The polyI:C-induced cell surface PD-L1 reduced the times of contact between DCs and T cells, potentially accounting for limited T cell activation. Our results reveal a novel CD40-dependent regulation of PD-L1 trafficking induced upon TLR3 signaling that dictates its inhibitory activity. These results provide a mechanistic framework to understand the efficacy of anti-PD-L1 cancer immunotherapy combined with TLR agonists.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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