RESUMO
Efferocytic clearance of apoptotic cells in general, and T cells in particular, is required for tissue and immune homeostasis. Transmembrane mucins are extended glycoproteins highly expressed in the cell glycocalyx that function as a barrier to phagocytosis. Whether and how mucins may be regulated during cell death to facilitate efferocytic corpse clearance is not well understood. Here we show that normal and transformed human T cells express a subset of mucins which are rapidly and selectively removed from the cell surface during apoptosis. This process is mediated by the ADAM10 sheddase, the activity of which is associated with XKR8-catalyzed flipping of phosphatidylserine to the outer leaflet of the plasma membrane. Mucin clearance enhances uptake of apoptotic T cells by macrophages, confirming mucins as an enzymatically-modulatable barrier to efferocytosis. Together these findings demonstrate a glycocalyx regulatory pathway with implications for therapeutic intervention in the clearance of normal and transformed apoptotic T cells.
Assuntos
Eferocitose , Mucinas , Humanos , Linfócitos T/metabolismo , Apoptose , Fagocitose , Proteína ADAM10/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Secretases da Proteína Precursora do AmiloideRESUMO
While tuberculosis (TB) is a risk factor in HIV-1-infected individuals, the mechanisms by which Mycobacterium tuberculosis (Mtb), the agent of TB in humans, worsens HIV-1 pathogenesis still need to be fully elucidated. Recently, we showed that HIV-1 infection and spread are exacerbated in macrophages exposed to TB-associated microenvironments. Transcriptomic analysis of macrophages conditioned with medium of Mtb-infected human macrophages (cmMTB) revealed an up-regulation of the typeI interferon (IFN-I) pathway, characterized by the overexpression of IFN-inducible genes. Historically, IFN-I are well known for their antiviral functions, but our previous work showed that this is not the case in the context of coinfection with HIV-1. Here, we show that the IFN-I response signature in cmMTB-treated macrophages matches the one observed in the blood of active TB patients, and depends on the timing of incubation with cmMTB. This suggests that the timing of macrophage's exposure to IFN-I can impact their capacity to control HIV-1 infection. Strikingly, we found that cmMTB-treated macrophages are hyporesponsive to extrastimulation with exogenous IFN-I, used to mimic HIV-1 infection. Yet, depleting STAT1 by gene silencing to block the IFN-I signaling pathway reduced TB-induced exacerbation of HIV-1 infection. Altogether, by aiming to understand why TB-derived IFN-I preexposure of macrophages did not induce antiviral immunity against HIV-1, we demonstrated that these cells are hyporesponsive to exogenous IFN-I, a phenomenon that prevents macrophage activation against HIV-1.
Mycobacterium tuberculosis induces hyporesponsiveness of the IFN-I signaling pathway in macrophages, leading to the exacerbation of HIV-1 replication.
Assuntos
Coinfecção , Infecções por HIV , Interferon Tipo I , Macrófagos , Mycobacterium tuberculosis , Tuberculose , Humanos , HIV-1 , Macrófagos/metabolismo , Macrófagos/virologia , Transdução de Sinais , Tuberculose/metabolismo , Interferon Tipo I/metabolismoRESUMO
Macrophage distribution density is tightly regulated within the body, yet the importance of macrophage crowding during in vitro culture is largely unstudied. Using a human induced pluripotent stem cell (iPSC)-derived macrophage model of tissue resident macrophages, we characterize how increasing macrophage culture density changes their morphology and phenotype before and after inflammatory stimulation. In particular, density drives changes in macrophage inflammatory cytokine and chemokine secretion in both resting and activated states. This density regulated inflammatory state is also evident in blood monocyte derived-macrophages, the human monocytic THP-1 immortalized cell line, and iPSC-derived microglia. Density-dependent changes appear to be driven by a transferable soluble factor, yet the precise mechanism remains unknown. Our findings highlight cell plating density as an important but frequently overlooked consideration of in vitro macrophage research relevant to a variety of fields ranging from basic macrophage cell biology to disease studies.
Assuntos
Células-Tronco Pluripotentes Induzidas , Humanos , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , Citocinas/metabolismoRESUMO
Human cells respond to infection by SARS-CoV-2, the virus that causes COVID-19, by producing cytokines including type I and III interferons (IFNs) and proinflammatory factors such as IL6 and TNF. IFNs can limit SARS-CoV-2 replication but cytokine imbalance contributes to severe COVID-19. We studied how cells detect SARS-CoV-2 infection. We report that the cytosolic RNA sensor MDA5 was required for type I and III IFN induction in the lung cancer cell line Calu-3 upon SARS-CoV-2 infection. Type I and III IFN induction further required MAVS and IRF3. In contrast, induction of IL6 and TNF was independent of the MDA5-MAVS-IRF3 axis in this setting. We further found that SARS-CoV-2 infection inhibited the ability of cells to respond to IFNs. In sum, we identified MDA5 as a cellular sensor for SARS-CoV-2 infection that induced type I and III IFNs.
Assuntos
COVID-19/imunologia , Interferon Tipo I/imunologia , Helicase IFIH1 Induzida por Interferon/imunologia , Interferons/imunologia , SARS-CoV-2/imunologia , Linhagem Celular , Humanos , Imunidade Inata , RNA/imunologia , Interferon lambdaRESUMO
The identification of individuals with null alleles enables studying how the loss of gene function affects infection. We previously described a non-functional variant in SIGLEC1, which encodes the myeloid-cell receptor Siglec-1/CD169 implicated in HIV-1 cell-to-cell transmission. Here we report a significant association between the SIGLEC1 null variant and extrapulmonary dissemination of Mycobacterium tuberculosis (Mtb) in two clinical cohorts comprising 6,256 individuals. Local spread of bacteria within the lung is apparent in Mtb-infected Siglec-1 knockout mice which, despite having similar bacterial load, developed more extensive lesions compared to wild type mice. We find that Siglec-1 is necessary to induce antigen presentation through extracellular vesicle uptake. We postulate that lack of Siglec-1 delays the onset of protective immunity against Mtb by limiting antigen exchange via extracellular vesicles, allowing for an early local spread of mycobacteria that increases the risk for extrapulmonary dissemination.
Assuntos
Vesículas Extracelulares/imunologia , Mycobacterium tuberculosis/imunologia , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Animais , Apresentação de Antígeno/imunologia , Humanos , Imunidade/genética , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Mycobacterium tuberculosis/patogenicidade , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Tuberculose dos Linfonodos/microbiologia , Tuberculose dos Linfonodos/patologia , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/patologiaAssuntos
Coinfecção , HIV-1/patogenicidade , Mycobacterium tuberculosis/patogenicidade , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/fisiologia , Coinfecção/microbiologia , Coinfecção/virologia , Infecções por HIV/complicações , Infecções por HIV/genética , Infecções por HIV/imunologia , HIV-1/imunologia , Humanos , Macrófagos/fisiologia , Mycobacterium tuberculosis/imunologia , Receptores Virais/fisiologia , Tuberculose/complicações , Tuberculose/genética , Tuberculose/imunologiaRESUMO
Many pathogens infect macrophages as part of their intracellular life cycle. This is particularly true for viruses, of which HIV-1 is one of the best studied. HIV-1 infection of macrophages has important consequences for viral persistence and pathogenesis, but the mechanisms of macrophage infection remain to be fully elucidated. Despite expressing viral entry receptors, macrophages are inefficiently infected by cell-free HIV-1 virions, whereas direct cell-cell spread is more efficient. Different modes of cell-cell spread have been described, including the uptake by macrophages of infected T cells and the fusion of infected T cells with macrophages, both leading to macrophage infection. Cell-cell spread can also transmit HIV-1 between macrophages and from macrophages to T cells. Here, we describe the current state of the field concerning the cell-cell spread of HIV-1 to and from macrophages, discuss mechanisms, and highlight potential in vivo relevance.
Assuntos
Linfócitos T CD4-Positivos/virologia , Infecções por HIV/virologia , HIV-1/fisiologia , Macrófagos/virologia , Animais , Linfócitos T CD4-Positivos/citologia , Comunicação Celular , Infecções por HIV/fisiopatologia , HIV-1/genética , Humanos , Macrófagos/citologiaRESUMO
While tuberculosis (TB) is a risk factor in HIV-1-infected individuals, the mechanisms by which Mycobacterium tuberculosis (Mtb) worsens HIV-1 pathogenesis remain scarce. We showed that HIV-1 infection is exacerbated in macrophages exposed to TB-associated microenvironments due to tunneling nanotube (TNT) formation. To identify molecular factors associated with TNT function, we performed a transcriptomic analysis in these macrophages, and revealed the up-regulation of Siglec-1 receptor. Siglec-1 expression depends on Mtb-induced production of type I interferon (IFN-I). In co-infected non-human primates, Siglec-1 is highly expressed by alveolar macrophages, whose abundance correlates with pathology and activation of IFN-I/STAT1 pathway. Siglec-1 localizes mainly on microtubule-containing TNT that are long and carry HIV-1 cargo. Siglec-1 depletion decreases TNT length, diminishes HIV-1 capture and cell-to-cell transfer, and abrogates the exacerbation of HIV-1 infection induced by Mtb. Altogether, we uncover a deleterious role for Siglec-1 in TB-HIV-1 co-infection and open new avenues to understand TNT biology.
Assuntos
HIV-1/patogenicidade , Interferon Tipo I/imunologia , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/virologia , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Tuberculose Pulmonar/imunologia , Animais , Células Cultivadas , Coinfecção/imunologia , Feminino , Perfilação da Expressão Gênica , Infecções por HIV , Humanos , Macaca mulatta , Masculino , Nanotubos , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/imunologiaAssuntos
HIV-1/fisiologia , Macrófagos Alveolares/virologia , Mycobacterium tuberculosis/fisiologia , Nanotubos , Animais , Diferenciação Celular , Microambiente Celular , Coinfecção/microbiologia , Coinfecção/virologia , Progressão da Doença , Infecções por HIV/virologia , Humanos , Interleucina-10/fisiologia , Macaca , Ativação de Macrófagos/fisiologia , Macrófagos Alveolares/microbiologia , Macrófagos Alveolares/ultraestrutura , Monócitos/fisiologia , Receptores de Superfície Celular/metabolismo , Tuberculose/microbiologia , Replicação ViralRESUMO
Dendritic cells (DCs) and macrophages as well as osteoclasts (OCs) are emerging as target cells of HIV-1 involved in virus transmission, dissemination, and establishment of persistent tissue virus reservoirs. While these myeloid cells are poorly infected by cell-free viruses because of the high expression levels of cellular restriction factors such as SAMHD1, we show here that HIV-1 uses a specific and common cell-to-cell fusion mechanism for virus transfer and dissemination from infected T lymphocytes to the target cells of the myeloid lineage, including immature DCs (iDCs), OCs, and macrophages, but not monocytes and mature DCs. The establishment of contacts with infected T cells leads to heterotypic cell fusion for the fast and massive transfer of viral material into OC and iDC targets, which subsequently triggers homotypic fusion with noninfected neighboring OCs and iDCs for virus dissemination. These two cell-to-cell fusion processes are not restricted by SAMHD1 and allow very efficient spreading of virus in myeloid cells, resulting in the formation of highly virus-productive multinucleated giant cells. These results reveal the cellular mechanism for SAMHD1-independent cell-to-cell spreading of HIV-1 in myeloid cell targets through the formation of the infected multinucleated giant cells observed in vivo in lymphoid and nonlymphoid tissues of HIV-1-infected patients.IMPORTANCE We demonstrate that HIV-1 uses a common two-step cell-to-cell fusion mechanism for massive virus transfer from infected T lymphocytes and dissemination to myeloid target cells, including dendritic cells and macrophages as well as osteoclasts. This cell-to-cell infection process bypasses the restriction imposed by the SAMHD1 host cell restriction factor for HIV-1 replication, leading to the formation of highly virus-productive multinucleated giant cells as observed in vivo in lymphoid and nonlymphoid tissues of HIV-1-infected patients. Since myeloid cells are emerging as important target cells of HIV-1, these results contribute to a better understanding of the role of these myeloid cells in pathogenesis, including cell-associated virus sexual transmission, cell-to-cell virus spreading, and establishment of long-lived viral tissue reservoirs.
Assuntos
Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Tropismo Viral , Replicação Viral , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Células Dendríticas/metabolismo , Células Dendríticas/virologia , Humanos , Macrófagos/metabolismo , Macrófagos/virologia , Células Mieloides/metabolismo , Células Mieloides/virologiaRESUMO
The tuberculosis (TB) bacillus, Mycobacterium tuberculosis (Mtb), and HIV-1 act synergistically; however, the mechanisms by which Mtb exacerbates HIV-1 pathogenesis are not well known. Using in vitro and ex vivo cell culture systems, we show that human M(IL-10) anti-inflammatory macrophages, present in TB-associated microenvironment, produce high levels of HIV-1. In vivo, M(IL-10) macrophages are expanded in lungs of co-infected non-human primates, which correlates with disease severity. Furthermore, HIV-1/Mtb co-infected patients display an accumulation of M(IL-10) macrophage markers (soluble CD163 and MerTK). These M(IL-10) macrophages form direct cell-to-cell bridges, which we identified as tunneling nanotubes (TNTs) involved in viral transfer. TNT formation requires the IL-10/STAT3 signaling pathway, and targeted inhibition of TNTs substantially reduces the enhancement of HIV-1 cell-to-cell transfer and overproduction in M(IL-10) macrophages. Our study reveals that TNTs facilitate viral transfer and amplification, thereby promoting TNT formation as a mechanism to be explored in TB/AIDS potential therapeutics.
Assuntos
Infecções por HIV/complicações , Interleucina-10/metabolismo , Macrófagos/patologia , Nanotubos , Fator de Transcrição STAT3/metabolismo , Tuberculose Pulmonar/complicações , Adulto , Idoso , Animais , Células Cultivadas , Coinfecção/patologia , Coinfecção/virologia , Feminino , Infecções por HIV/imunologia , Infecções por HIV/patologia , Infecções por HIV/virologia , Humanos , Macaca mulatta , Ativação de Macrófagos , Macrófagos/virologia , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis , Transdução de Sinais , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/patologia , Replicação Viral , Adulto JovemRESUMO
Trigonelline (TG; N-methylnicotinate) is a ubiquitous osmolyte. Although it is known that it can be degraded, the enzymes and metabolites have not been described so far. In this work, we challenged the laboratory model soil-borne, gram-negative bacterium Acinetobacter baylyi ADP1 (ADP1) for its ability to grow on TG and we identified a cluster of catabolic, transporter, and regulatory genes. We dissected the pathway to the level of enzymes and metabolites, and proceeded to in vitro reconstruction of the complete pathway by six purified proteins. The four enzymatic steps that lead from TG to methylamine and succinate are described, and the structures of previously undescribed metabolites are provided. Unlike many aromatic compounds that undergo hydroxylation prior to ring cleavage, the first step of TG catabolism proceeds through direct cleavage of the C5-C6 bound, catalyzed by a flavin-dependent, two-component oxygenase, which yields (Z)-2-((N-methylformamido)methylene)-5-hydroxy-butyrolactone (MFMB). MFMB is then oxidized into (E)-2-((N-methylformamido) methylene) succinate (MFMS), which is split up by a hydrolase into carbon dioxide, methylamine, formic acid, and succinate semialdehyde (SSA). SSA eventually fuels up the TCA by means of an SSA dehydrogenase, assisted by a Conserved Hypothetical Protein. The cluster is conserved across marine, soil, and plant-associated bacteria. This emphasizes the role of TG as a ubiquitous nutrient for which an efficient microbial catabolic toolbox is available.
Assuntos
Acinetobacter , Alcaloides/metabolismo , Genoma Bacteriano , Anotação de Sequência Molecular , Família Multigênica , Acinetobacter/enzimologia , Acinetobacter/genética , Cromatografia Líquida , Espectrometria de MassasRESUMO
The ability of Mycobacterium tuberculosis (Mtb) to persist in its human host relies on numerous immune evasion strategies, such as the deregulation of the lipid metabolism leading to the formation of foamy macrophages (FM). Yet, the specific host factors leading to the foamy phenotype of Mtb-infected macrophages remain unknown. Herein, we aimed to address whether host cytokines contribute to FM formation in the context of Mtb infection. Our approach is based on the use of an acellular fraction of tuberculous pleural effusions (TB-PE) as a physiological source of local factors released during Mtb infection. We found that TB-PE induced FM differentiation as observed by the increase in lipid bodies, intracellular cholesterol, and expression of the scavenger receptor CD36, as well as the enzyme acyl CoA:cholesterol acyl transferase (ACAT). Importantly, interleukin-10 (IL-10) depletion from TB-PE prevented the augmentation of all these parameters. Moreover, we observed a positive correlation between the levels of IL-10 and the number of lipid-laden CD14+ cells among the pleural cells in TB patients, demonstrating that FM differentiation occurs within the pleural environment. Downstream of IL-10 signaling, we noticed that the transcription factor signal transducer and activator of transcription 3 was activated by TB-PE, and its chemical inhibition prevented the accumulation of lipid bodies and ACAT expression in macrophages. In terms of the host immune response, TB-PE-treated macrophages displayed immunosuppressive properties and bore higher bacillary loads. Finally, we confirmed our results using bone marrow-derived macrophage from IL-10-/- mice demonstrating that IL-10 deficiency partially prevented foamy phenotype induction after Mtb lipids exposure. In conclusion, our results evidence a role of IL-10 in promoting the differentiation of FM in the context of Mtb infection, contributing to our understanding of how alterations of the host metabolic factors may favor pathogen persistence.
Assuntos
Acetil-CoA C-Acetiltransferase/imunologia , Regulação Enzimológica da Expressão Gênica/imunologia , Interleucina-10/imunologia , Mycobacterium tuberculosis/imunologia , Derrame Pleural/imunologia , Fator de Transcrição STAT3/imunologia , Esterol O-Aciltransferase , Tuberculose Pleural/imunologia , Regulação para Cima/imunologia , Acetil-CoA C-Acetiltransferase/genética , Animais , Feminino , Células Espumosas , Humanos , Interleucina-10/genética , Masculino , Camundongos , Camundongos Knockout , Mycobacterium tuberculosis/genética , Derrame Pleural/genética , Derrame Pleural/patologia , Fator de Transcrição STAT3/genética , Tuberculose Pleural/genética , Tuberculose Pleural/patologiaRESUMO
Tunneling nanotubes (TNT) are dynamic connections between cells, which represent a novel route for cell-to-cell communication. A growing body of evidence points TNT towards a role for intercellular exchanges of signals, molecules, organelles, and pathogens, involving them in a diverse array of functions. TNT form among several cell types, including neuronal cells, epithelial cells, and almost all immune cells. In myeloid cells (e.g., macrophages, dendritic cells, and osteoclasts), intercellular communication via TNT contributes to their differentiation and immune functions. Importantly, TNT enable myeloid cells to communicate with a targeted neighboring or distant cell, as well as with other cell types, therefore creating a complex variety of cellular exchanges. TNT also contribute to pathogen spread as they serve as "corridors" from a cell to another. Herein, we addressed the complexity of the definition and in vitro characterization of TNT in innate immune cells, the different processes involved in their formation, and their relevance in vivo. We also assess our current understanding of how TNT participate in immune surveillance and the spread of pathogens, with a particular interest for HIV-1. Overall, despite recent progress in this growing research field, we highlight that further investigation is needed to better unveil the role of TNT in both physiological and pathological conditions.
Assuntos
Comunicação Celular/fisiologia , Vigilância Imunológica/imunologia , Células Mieloides/imunologia , Células Mieloides/metabolismo , Nanotubos , Animais , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , Humanos , Imunidade Inata/imunologia , Camundongos , RatosRESUMO
Bone deficits are frequent in HIV-1-infected patients. We report here that osteoclasts, the cells specialized in bone resorption, are infected by HIV-1 in vivo in humanized mice and ex vivo in human joint biopsies. In vitro, infection of human osteoclasts occurs at different stages of osteoclastogenesis via cell-free viruses and, more efficiently, by transfer from infected T cells. HIV-1 infection markedly enhances adhesion and osteolytic activity of human osteoclasts by modifying the structure and function of the sealing zone, the osteoclast-specific bone degradation machinery. Indeed, the sealing zone is broader due to F-actin enrichment of its basal units (i.e., the podosomes). The viral protein Nef is involved in all HIV-1-induced effects partly through the activation of Src, a regulator of podosomes and of their assembly as a sealing zone. Supporting these results, Nef-transgenic mice exhibit an increased osteoclast density and bone defects, and osteoclasts derived from these animals display high osteolytic activity. Altogether, our study evidences osteoclasts as host cells for HIV-1 and their pathological contribution to bone disorders induced by this virus, in part via Nef.