RESUMEN
HIV integrates into the host genome to create a persistent viral reservoir. Stimulation of CD4+ memory T lymphocytes with common γc-chain cytokines renders these cells more susceptible to HIV infection, making them a key component of the reservoir itself. IL-15 is up-regulated during primary HIV infection, a time when the HIV reservoir established. Therefore, we investigated the molecular and cellular impact of IL-15 on CD4+ T-cell infection. We found that IL-15 stimulation induces SAM domain and HD domain-containing protein 1 (SAMHD1) phosphorylation due to cell cycle entry, relieving an early block to infection. Perturbation of the pathways downstream of IL-15 receptor (IL-15R) indicated that SAMHD1 phosphorylation after IL-15 stimulation is JAK dependent. Treating CD4+ T cells with Ruxolitinib, an inhibitor of JAK1 and JAK2, effectively blocked IL-15-induced SAMHD1 phosphorylation and protected CD4+ T cells from HIV infection. Using high-resolution single-cell immune profiling using mass cytometry by TOF (CyTOF), we found that IL-15 stimulation altered the composition of CD4+ T-cell memory populations by increasing proliferation of memory CD4+ T cells, including CD4+ T memory stem cells (TSCM). IL-15-stimulated CD4+ TSCM, harboring phosphorylated SAMHD1, were preferentially infected. We propose that IL-15 plays a pivotal role in creating a self-renewing, persistent HIV reservoir by facilitating infection of CD4+ T cells with stem cell-like properties. Time-limited interventions with JAK1 inhibitors, such as Ruxolitinib, should prevent the inactivation of the endogenous restriction factor SAMHD1 and protect this long-lived CD4+ T-memory cell population from HIV infection.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Interleucina-15/inmunología , Linfocitos T CD4-Positivos/patología , Linfocitos T CD4-Positivos/virología , Susceptibilidad a Enfermedades , Femenino , Células HEK293 , Infecciones por VIH/patología , Humanos , Janus Quinasa 1/inmunología , Janus Quinasa 2/inmunología , Masculino , Memoria a Corto Plazo , Nitrilos , Pirazoles/farmacología , Pirimidinas , Proteína 1 que Contiene Dominios SAM y HD/inmunologíaRESUMEN
The influenza A virus (IAV) nonstructural protein 1 (NS1) contributes to disease pathogenesis through the inhibition of host innate immune responses. Dendritic cells (DCs) release interferons (IFNs) and proinflammatory cytokines and promote adaptive immunity upon viral infection. In order to characterize the strain-specific effects of IAV NS1 on human DC activation, we infected human DCs with a panel of recombinant viruses with the same backbone (A/Puerto Rico/08/1934) expressing different NS1 proteins from human and avian origin. We found that these viruses induced a clearly distinct phenotype in DCs. Specifically, viruses expressing NS1 from human IAV (either H1N1 or H3N2) induced higher levels of expression of type I (IFN-α and IFN-ß) and type III (IFN-λ1 to IFNλ3) IFNs than viruses expressing avian IAV NS1 proteins (H5N1, H7N9, and H7N2), but the differences observed in the expression levels of proinflammatory cytokines like tumor necrosis factor alpha (TNF-α) or interleukin-6 (IL-6) were not significant. In addition, using imaging flow cytometry, we found that human and avian NS1 proteins segregate based on their subcellular trafficking dynamics, which might be associated with the different innate immune profile induced in DCs by viruses expressing those NS1 proteins. Innate immune responses induced by our panel of IAV recombinant viruses were also characterized in normal human bronchial epithelial cells, and the results were consistent with those in DCs. Altogether, our results reveal an increased ability of NS1 from avian viruses to antagonize innate immune responses in human primary cells compared to the ability of NS1 from human viruses, which could contribute to the severe disease induced by avian IAV in humans.IMPORTANCE Influenza A viruses (IAVs) cause seasonal epidemics which result in an important health and economic burden. Wild aquatic birds are the natural host of IAV. However, IAV can infect diverse hosts, including humans, domestic poultry, pigs, and others. IAVs circulating in animals occasionally cross the species barrier, infecting humans, which results in mild to very severe disease. In some cases, these viruses can acquire the ability to be transmitted among humans and initiate a pandemic. The nonstructural 1 (NS1) protein of IAV is an important antagonist of the innate immune response. In this study, using recombinant viruses and primary human cells, we show that NS1 proteins from human and avian hosts show intrinsic differences in the modulation of the innate immunity in human dendritic cells and epithelial cells, as well as different cellular localization dynamics in infected cells.
Asunto(s)
Células Epiteliales/inmunología , Interacciones Huésped-Patógeno/genética , Inmunidad Innata , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H5N1 del Virus de la Influenza A/genética , Proteínas no Estructurales Virales/genética , Animales , Aves , Células Dendríticas/inmunología , Células Dendríticas/virología , Perros , Células Epiteliales/virología , Regulación de la Expresión Génica , Especificidad del Huésped , Interacciones Huésped-Patógeno/inmunología , Humanos , Subtipo H1N1 del Virus de la Influenza A/clasificación , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H3N2 del Virus de la Influenza A/clasificación , Subtipo H3N2 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/inmunología , Subtipo H5N1 del Virus de la Influenza A/clasificación , Subtipo H5N1 del Virus de la Influenza A/inmunología , Subtipo H7N2 del Virus de la Influenza A/clasificación , Subtipo H7N2 del Virus de la Influenza A/genética , Subtipo H7N2 del Virus de la Influenza A/inmunología , Subtipo H7N9 del Virus de la Influenza A/clasificación , Subtipo H7N9 del Virus de la Influenza A/genética , Subtipo H7N9 del Virus de la Influenza A/inmunología , Interferón-alfa/genética , Interferón-alfa/inmunología , Interferón beta/genética , Interferón beta/inmunología , Interferón gamma/genética , Interferón gamma/inmunología , Interleucina-6/genética , Interleucina-6/inmunología , Células de Riñón Canino Madin Darby , Filogenia , Cultivo Primario de Células , Virus Reordenados/genética , Virus Reordenados/inmunología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología , Proteínas no Estructurales Virales/clasificación , Proteínas no Estructurales Virales/inmunologíaRESUMEN
Focal segmental glomerulosclerosis (FSGS) is a leading cause of nephrotic syndrome and end-stage renal disease worldwide. Although the mechanisms underlying this important disease are poorly understood, the glomerular podocyte clearly plays a central role in disease pathogenesis. In the current work, we demonstrate that the homophilic adhesion molecule sidekick-1 (sdk-1) is up-regulated in podocytes in FSGS both in rodent models and in human kidney biopsy samples. Transgenic mice that have podocyte-specific overexpression of sdk-1 develop gradually progressive heavy proteinuria and severe FSGS. We also show that sdk-1 associates with the slit diaphragm linker protein MAGI-1, which is already known to interact with several critical podocyte proteins including synaptopodin, alpha-actinin-4, nephrin, JAM4, and beta-catenin. This interaction is mediated through a direct interaction between the carboxyl terminus of sdk-1 and specific PDZ domains of MAGI-1. In vitro expression of sdk-1 enables a dramatic recruitment of MAGI-1 to the cell membrane. Furthermore, a truncated version of sdk-1 that is unable to bind to MAGI-1 does not induce podocyte dysfunction when overexpressed. We conclude that the up-regulation of sdk-1 in podocytes is an important pathogenic factor in FSGS and that the mechanism involves disruption of the actin cytoskeleton possibly via alterations in MAGI-1 function.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Glomeruloesclerosis Focal y Segmentaria/etiología , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Inmunoglobulina G/metabolismo , Proteínas de la Membrana/metabolismo , Podocitos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Línea Celular , Membrana Celular/metabolismo , Células Cultivadas , Glomeruloesclerosis Focal y Segmentaria/genética , Guanilato-Quinasas , Humanos , Inmunoglobulina G/genética , Inmunoprecipitación , Técnicas In Vitro , Proteínas de la Membrana/genética , Ratones , Ratones Transgénicos , Reacción en Cadena de la Polimerasa , Unión ProteicaRESUMEN
Zika and dengue virus (ZIKV and DENV) are two flaviviruses responsible for important vector-borne emerging infectious diseases. While there have been multiple DENV epidemics in the last decades, there have been fewer documented epidemics caused by ZIKV until recent years. Thus, our current knowledge about the biology of ZIKV, the disease, and the immune responses in humans is limited. Here, we used mass cytometry (CyTOF) to perform a detailed characterization of the innate immune responses elicited by ZIKV and DENV in human peripheral blood mononuclear cells (PBMCs) from healthy donors infected ex vivo. We found that ZIKV and DENV exposure of human PBMCs induces global phenotypic changes in myeloid cells, characterized mainly by upregulation of costimulatory molecules (CD86 and CD40), CD38, and the type I interferon-inducible protein CD169, a marker for phagocytic function and cross-priming potential in myeloid cells. We also found that ZIKV induces expansion of nonclassical monocytes in cell culture. The analysis of the phenotype of the three monocyte subtypes (classical, intermediate, and nonclassical) at the single-cell level identified differences in their expression of CD86, CD38, CXCL8, and CXCL10 during ZIKV and DENV infection. Overall, using CyTOF, we found that ex vivo infections of PBMCs with ZIKV and DENV reproduced many aspects of the profile found in blood from patients in previously described cohort studies, which highlights the suitability of this system for the study of the human host responses to these viruses. IMPORTANCE Zika and dengue viruses are emergent arboviruses of great public health impact. Both viruses are responsible for important diseases, yet there is currently no vaccine or specific treatment available. Immune cells play critical roles in the virus cycle as well as in the innate and adaptive immune response elicited in the host; therefore, it is critical to understand the changes induced by virus infection in peripheral blood mononuclear cells (PBMCs). In this study, we used a model of ex vivo infection of PBMCs and CyTOF technology to profile the early innate immune changes induced by Zika virus and dengue virus in blood.
Asunto(s)
Virus del Dengue/inmunología , Interferón Tipo I/inmunología , Leucocitos Mononucleares/clasificación , Leucocitos Mononucleares/virología , Fenotipo , Lectina 1 Similar a Ig de Unión al Ácido Siálico/genética , Virus Zika/inmunología , Estudios de Cohortes , Técnicas Citológicas/métodos , Humanos , Inmunidad Innata , Regulación hacia ArribaRESUMEN
Dengue virus (DENV) is the most prevalent mosquito-borne virus causing human disease. Of the 4 DENV serotypes, epidemiological data suggest that DENV-2 secondary infections are associated with more severe disease than DENV-4 infections. Mass cytometry by time-of-flight (CyTOF) was used to dissect immune changes induced by DENV-2 and DENV-4 in human DCs, the initial targets of primary infections that likely affect infection outcomes. Strikingly, DENV-4 replication peaked earlier and promoted stronger innate immune responses, with increased expression of DC activation and migration markers and increased cytokine production, compared with DENV-2. In addition, infected DCs produced higher levels of inflammatory cytokines compared with bystander DCs, which mainly produced IFN-induced cytokines. These high-dimensional analyses during DENV-2 and DENV-4 infections revealed distinct viral signatures marked by different replication strategies and antiviral innate immune induction in DCs, which may result in different viral fitness, transmission, and pathogenesis.
RESUMEN
Injury to the specialized epithelial cells of the glomerulus (podocytes) underlies the pathogenesis of all forms of proteinuric kidney disease; however, the specific genetic changes that mediate podocyte dysfunction after injury are not fully understood. Here, we performed a large-scale insertional mutagenic screen of injury-resistant podocytes isolated from mice and found that increased expression of the gene Rap1gap, encoding a RAP1 activation inhibitor, ameliorated podocyte injury resistance. Furthermore, injured podocytes in murine models of disease and kidney biopsies from glomerulosclerosis patients exhibited increased RAP1GAP, resulting in diminished glomerular RAP1 activation. In mouse models, podocyte-specific inactivation of Rap1a and Rap1b induced massive glomerulosclerosis and premature death. Podocyte-specific Rap1a and Rap1b haploinsufficiency also resulted in severe podocyte damage, including features of podocyte detachment. Over-expression of RAP1GAP in cultured podocytes induced loss of activated ß1 integrin, which was similarly observed in kidney biopsies from patients. Furthermore, preventing elevation of RAP1GAP levels in injured podocytes maintained ß1 integrin-mediated adhesion and prevented cellular detachment. Taken together, our findings suggest that increased podocyte expression of RAP1GAP contributes directly to podocyte dysfunction by a mechanism that involves loss of RAP1-mediated activation of ß1 integrin.