RESUMO
Intron-containing RNA expressed from the HIV-1 provirus activates type 1 interferon in primary human blood cells, including CD4+ T cells, macrophages, and dendritic cells. To identify the innate immune receptor required for detection of intron-containing RNA expressed from the HIV-1 provirus, a loss-of-function screen was performed with short hairpin RNA-expressing lentivectors targeting twenty-one candidate genes in human monocyte-derived dendritic cells. Among the candidate genes tested, only knockdown of XPO1 (CRM1), IFIH1 (MDA5), or MAVS prevented activation of the interferon-stimulated gene ISG15. The importance of IFIH1 protein was demonstrated by rescue of the knockdown with nontargetable IFIH1 coding sequence. Inhibition of HIV-1-induced ISG15 by the IFIH1-specific Nipah virus V protein, and by IFIH1-transdominant 2-CARD domain-deletion or phosphomimetic point mutations, indicates that IFIH1 (MDA5) filament formation, dephosphorylation, and association with MAVS are all required for innate immune activation in response to HIV-1 transduction. Since both IFIH1 (MDA5) and DDX58 (RIG-I) signal via MAVS, the specificity of HIV-1 RNA detection by IFIH1 was demonstrated by the fact that DDX58 knockdown had no effect on activation. RNA-Seq showed that IFIH1 knockdown in dendritic cells globally disrupted the induction of IFN-stimulated genes by HIV-1. Finally, specific enrichment of unspliced HIV-1 RNA by IFIH1 (MDA5), over two orders of magnitude, was revealed by formaldehyde cross-linking immunoprecipitation (f-CLIP). These results demonstrate that IFIH1 is the innate immune receptor for intron-containing RNA from the HIV-1 provirus and that IFIH1 potentially contributes to chronic inflammation in people living with HIV-1, even in the presence of effective antiretroviral therapy.
Assuntos
Células Dendríticas , HIV-1 , Imunidade Inata , Helicase IFIH1 Induzida por Interferon , Íntrons , Provírus , RNA Viral , Humanos , HIV-1/genética , HIV-1/imunologia , Helicase IFIH1 Induzida por Interferon/genética , Helicase IFIH1 Induzida por Interferon/metabolismo , Provírus/genética , Células Dendríticas/imunologia , Células Dendríticas/virologia , Células Dendríticas/metabolismo , Íntrons/genética , RNA Viral/genética , RNA Viral/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , Infecções por HIV/genética , Carioferinas/genética , Carioferinas/metabolismoRESUMO
Lyssavirus is a kind of neurotropic pathogen that needs to evade peripheral host immunity to enter the central nervous system to accomplish infection. NLRP3 inflammasome activation is essential for the host to defend against pathogen invasion. This study demonstrates that the matrix protein (M) of lyssavirus can inhibit both the priming step and the activation step of NLRP3 inflammasome activation. Specifically, M of lyssavirus can compete with NEK7 for binding to NLRP3, which restricts downstream apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization. The serine amino acid at the 158th site of M among lyssavirus is critical for restricting ASC oligomerization. Moreover, recombinant lab-attenuated lyssavirus rabies (rabies lyssavirus [RABV]) with G158S mutation at M decreases interleukin-1ß (IL-1ß) production in bone-marrow-derived dendritic cells (BMDCs) to facilitate lyssavirus invasion into the brain thereby elevating pathogenicity in mice. Taken together, this study reveals a common mechanism by which lyssavirus inhibits NLRP3 inflammasome activation to evade host defenses.
Assuntos
Células Dendríticas , Inflamassomos , Interleucina-1beta , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteínas da Matriz Viral , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Inflamassomos/metabolismo , Inflamassomos/imunologia , Camundongos , Proteínas da Matriz Viral/metabolismo , Humanos , Interleucina-1beta/metabolismo , Células Dendríticas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/virologia , Lyssavirus/metabolismo , Lyssavirus/imunologia , Quinases Relacionadas a NIMA/metabolismo , Ligação Proteica , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Células HEK293RESUMO
CD150, also termed signaling lymphocyte activation molecule family member 1, is a cell surface receptor expressed on T cells, B cells, dendritic cells (DCs) and some tumors. Stimulation of CD150 on immune cells induces cell proliferation and cytokine production. However, the function of CD150 in EpsteinBarr virus (EBV)infected B cells is still not fully understood. In the present study, CD150 expression on B cells increased rapidly following EBV infection, and various CD150 antibodies, measles viral proteins and recombinant CD150 proteins induced the secretion of multiple cytokines in both CD150+ EBVtransformed B cells and EBV+ lymphoma cells. Notably, the IL1α protein level showed the greatest increase among all cytokines measured. The culture supernatant containing these cytokines induced the rapid differentiation of monocytes to DCs after only 2 days in vitro, which was faster than the established DC maturation time. Furthermore, knockdown of CD150 expression led to a reduction in the secretion of multiple cytokines, and monocyte differentiation was partially inhibited by antiIL1α and antigranulocytemacrophage colonystimulating factor neutralizing antibodies. Collectively, the results of the present study suggest that CD150 activation triggers cytokine production in EBVtransformed B cells, and that measles virus coinfection might affect immune responses through the production of various cytokines in EBV+ lymphoma cells.
Assuntos
Linfócitos B , Diferenciação Celular , Citocinas , Herpesvirus Humano 4 , Monócitos , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Humanos , Herpesvirus Humano 4/imunologia , Linfócitos B/metabolismo , Linfócitos B/imunologia , Linfócitos B/virologia , Citocinas/metabolismo , Monócitos/metabolismo , Monócitos/imunologia , Monócitos/virologia , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismo , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/virologia , Infecções por Vírus Epstein-Barr/metabolismo , Células Dendríticas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/virologia , Ativação Linfocitária/imunologiaRESUMO
During the birch pollen season an enhanced incidence of virus infections is noticed, raising the question whether pollen can affect anti-viral responses independent of allergic reactions. We previously showed that birch pollen-treatment of monocyte-derived dendritic cells (moDC) enhances human cytomegalovirus (HCMV) infection. Here we addressed how in moDC the relatively weak pollen response can affect the comparably strong response to HCMV. To this end, moDC were stimulated with aqueous birch pollen extract (APE), HCMV, and APE with HCMV, and transcriptomic signatures were determined after 6 and 24 h of incubation. Infection was monitored upon exposure of moDC to GFP expressing HCMV by flow cytometric analysis of GFP expressing cells. Principle component analysis of RNA sequencing data revealed close clustering of mock and APE treated moDC, whereas HCMV as well as APE with HCMV treated moDC clustered separately after 6 and 24 h of incubation, respectively. Communally induced genes were detected in APE, HCMV and APE with HCMV treated moDC. In APE with HCMV treated moDC, the comparably weak APE induced signatures were maintained after HCMV exposure. In particular, NF-κB/RELA and PI3K/AKT/MAPK signaling were altered upon APE with HCMV exposure. Earlier, we discovered that NF-κB inhibition alleviated APE induced enhancement of HCMV infection. Here we additionally found that impairment of PI3K signaling reduced HCMV infection in HCMV and APE with HCMV treated moDC. APE treated moDC that were exposed to HCMV show a unique host gene signature, which to a large extent is regulated by NF-κB activation and PI3K/AKT/MAPK signaling.
Assuntos
Betula , Citomegalovirus , Células Dendríticas , Pólen , Células Dendríticas/virologia , Células Dendríticas/metabolismo , Células Dendríticas/imunologia , Pólen/genética , Pólen/imunologia , Citomegalovirus/genética , Citomegalovirus/fisiologia , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Infecções por Citomegalovirus/virologia , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/imunologia , Transcriptoma , Transdução de Sinais , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição RelA/metabolismo , Fator de Transcrição RelA/genética , Células CultivadasRESUMO
Dengue virus (DENV), transmitted by infected mosquitoes, is a major public health concern, with approximately half the world's population at risk for infection. Recent decades have increasing incidence of dengue-associated disease alongside growing frequency of outbreaks. Although promising progress has been made in anti-DENV immunizations, post-infection treatment remains limited to non-specific supportive treatments. Development of antiviral therapeutics is thus required to limit DENV dissemination in humans and to help control the severity of outbreaks. Dendritic cells (DCs) are amongst the first cells to encounter DENV upon injection into the human skin mucosa, and thereafter promote systemic viral dissemination to additional human target cells. Autophagy is a vesicle trafficking pathway involving the formation of cytosolic autophagosomes, and recent reports have highlighted the extensive manipulation of autophagy by flaviviruses, including DENV, for viral replication. However, the temporal profiling and function of autophagy activity in DENV infection and transmission by human primary DCs remains poorly understood. Herein, we demonstrate that mechanisms of autophagosome formation and extracellular vesicle (EV) release have a pro-viral role in DC-mediated DENV transmission. We show that DENV exploits early-stage canonical autophagy to establish infection in primary human DCs. DENV replication enhanced autophagosome formation in primary human DCs, and intrinsically-heightened autophagosome biogenesis correlated with relatively higher rates of DC susceptibility to DENV. Furthermore, our data suggest that viral replication intermediates co-localize with autophagosomes, while productive DENV infection introduces a block at the late degradative stages of autophagy in infected DCs but not in uninfected bystander cells. Notably, we identify for the first time that approximately one-fourth of DC-derived CD9/CD81/CD63+ EVs co-express canonical autophagy marker LC3, and demonstrate that DC-derived EV populations are an alternative, cell-free mechanism by which DCs promote DENV transmission to additional target sites. Taken together, our study highlights intersections between autophagy and secretory pathways during viral infection, and puts forward autophagosome accumulation and viral RNA-laden EVs as host determinants of DC-mediated DENV infection in humans. Host-directed therapeutics targeting autophagy and exocytosis pathways thus have potential to enhance DC-driven resistance to DENV acquisition and thereby limit viral dissemination by initial human target cells following mosquito-to-human transmission of DENV.
Assuntos
Autofagossomos , Autofagia , Células Dendríticas , Vírus da Dengue , Dengue , Via Secretória , Replicação Viral , Humanos , Vírus da Dengue/fisiologia , Células Dendríticas/imunologia , Células Dendríticas/virologia , Células Dendríticas/metabolismo , Dengue/transmissão , Dengue/virologia , Dengue/imunologia , Autofagossomos/metabolismo , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/virologia , Células CultivadasRESUMO
Respiratory viral infections represent one of the major causes of death worldwide. The recent coronavirus disease 2019 pandemic alone claimed the lives of over 6 million people around the globe. It is therefore crucial to understand how the immune system responds to these threats and how respiratory infection can be controlled and constrained. Dendritic cells (DCs) are one of the key players in antiviral immunity because of their ability to detect pathogens. They can orchestrate an immune response that will, in most cases, lead to viral clearance. Different subsets of DCs are present in the lung and each subset can contribute to antiviral responses through various mechanisms. In this review, we discuss the role of the different lung DC subsets in response to common respiratory viruses, with a focus on respiratory syncytial virus, influenza A virus and severe acute respiratory syndrome coronavirus 2. We also review how lung DC-mediated responses to respiratory viruses can lead to the worsening of an existing chronic pulmonary disease such as asthma. Throughout the review, we discuss results obtained from animal studies as well as results generated from infected patients.
Assuntos
Células Dendríticas , Infecções Respiratórias , Células Dendríticas/imunologia , Células Dendríticas/virologia , Humanos , Animais , Infecções Respiratórias/imunologia , Infecções Respiratórias/virologia , Pulmão/imunologia , Pulmão/virologia , Interações Hospedeiro-Patógeno , COVID-19/imunologia , Viroses/imunologia , Viroses/virologia , SARS-CoV-2/imunologia , SARS-CoV-2/patogenicidadeRESUMO
The transmission electron microscopy revealed a dendritic cell in the medulla of the chicken bursal follicle. This dendritic cell has a classical secretory machinery; therefore, it has been named a bursal secretory dendritic cell (BSDC). The corticomedullary epithelial arch (CMEA) encloses lymphoid-like cells, which can proliferate and after entering the medulla, begin to differentiate to immature, then mature BSDC, which discharges glycoprotein (gp). With the exhaustion of gp production, the BSDC rapidly transforms into a macrophage-like cell (Mal), which is an activated endocytic cell of innate immunity. The Mal drifts through the follicle-associated epithelium (FAE)-supporting cells into the FAE, and via FAE, the Mal is eliminated in the bursal lumen. The infectious bursal disease virus (IBDV) infection accelerates the maturation process of BSDC precursors, which results in acute emptying of CMEA and subsequently, numerous immature BSDC(s) emerge. The IBDV infection stops the gp discharge, and the gp appears in the virus-containing Mal. The Movat pentachrome staining recognizes the gp in the extracellular spaces of the medulla and after infection in the Mal. The BSDC is the primary target of the IBDV. During IBDV infection, a large number of suddenly formed Mal actively migrate into the cortex, initiating cytokine storm and recruiting heterophil granulocytes. During embryogenesis, the vimentin-positive, possibly embryonic dendritic cells provide a microenvironment for carbohydrate switch. Around hatching, these embryonic, temporary dendritic cells get the Fc receptor, which bind maternal IgY. The posthatched forms of BSDC(s) gradually replace the embryonic ones and bind their own IgY.
Assuntos
Bolsa de Fabricius , Galinhas , Células Dendríticas , Vírus da Doença Infecciosa da Bursa , Animais , Bolsa de Fabricius/virologia , Células Dendríticas/fisiologia , Células Dendríticas/virologia , Vírus da Doença Infecciosa da Bursa/fisiologia , Infecções por Birnaviridae/veterinária , Infecções por Birnaviridae/virologia , Doenças das Aves Domésticas/virologia , Doenças das Aves Domésticas/imunologiaRESUMO
Enterovirus 71 (EV71) is a significant causative agent of hand, foot, and mouth disease, with potential serious neurologic complications or fatal outcomes. The lack of effective treatments for EV71 infection is attributed to its elusive pathogenicity. Our study reveals that human plasmacytoid dendritic cells (pDCs), the main type I IFN-producing cells, selectively express scavenger receptor class B, member 2 (SCARB2) and P-selectin glycoprotein ligand 1 (PSGL-1), crucial cellular receptors for EV71. Some strains of EV71 can replicate within pDCs and stimulate IFN-α production. The activation of pDCs by EV71 is hindered by Abs to PSGL-1 and soluble PSGL-1, whereas Abs to SCARB2 and soluble SCARB2 have a less pronounced effect. Our data suggest that only strains binding to PSGL-1, more commonly found in severe cases, can replicate in pDCs and induce IFN-α secretion, highlighting the importance of PSGL-1 in these processes. Furthermore, IFN-α secretion by pDCs can be triggered by EV71 or UV-inactivated EV71 virions, indicating that productive infection is not necessary for pDC activation. These findings provide new insights into the interaction between EV71 and pDCs, suggesting that pDC activation could potentially mitigate the severity of EV71-related diseases.
Assuntos
Células Dendríticas , Enterovirus Humano A , Interferon-alfa , Proteínas de Membrana Lisossomal , Glicoproteínas de Membrana , Células Dendríticas/imunologia , Células Dendríticas/virologia , Humanos , Enterovirus Humano A/imunologia , Enterovirus Humano A/fisiologia , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana Lisossomal/metabolismo , Proteínas de Membrana Lisossomal/imunologia , Interferon-alfa/metabolismo , Interferon-alfa/imunologia , Receptores Depuradores/metabolismo , Infecções por Enterovirus/imunologia , Infecções por Enterovirus/virologia , Replicação ViralRESUMO
Zika virus (ZIKV) is a mosquito-borne flavivirus that caused an epidemic in the Americas in 2016 and is linked to severe neonatal birth defects, including microcephaly and spontaneous abortion. To better understand the host response to ZIKV infection, we adapted the 10× Genomics Chromium single-cell RNA sequencing (scRNA-seq) assay to simultaneously capture viral RNA and host mRNA. Using this assay, we profiled the antiviral landscape in a population of human monocyte-derived dendritic cells infected with ZIKV at the single-cell level. The bystander cells, which lacked detectable viral RNA, expressed an antiviral state that was enriched for genes coinciding predominantly with a type I interferon (IFN) response. Within the infected cells, viral RNA negatively correlated with type I IFN-dependent and -independent genes (the antiviral module). We modeled the ZIKV-specific antiviral state at the protein level, leveraging experimentally derived protein interaction data. We identified a highly interconnected network between the antiviral module and other host proteins. In this work, we propose a new paradigm for evaluating the antiviral response to a specific virus, combining an unbiased list of genes that highly correlate with viral RNA on a per-cell basis with experimental protein interaction data. IMPORTANCE: Zika virus (ZIKV) remains a public health threat given its potential for re-emergence and the detrimental fetal outcomes associated with infection during pregnancy. Understanding the dynamics between ZIKV and its host is critical to understanding ZIKV pathogenesis. Through ZIKV-inclusive single-cell RNA sequencing (scRNA-seq), we demonstrate on the single-cell level the dynamic interplay between ZIKV and the host: the transcriptional program that restricts viral infection and ZIKV-mediated inhibition of that response. Our ZIKV-inclusive scRNA-seq assay will serve as a useful tool for gaining greater insight into the host response to ZIKV and can be applied more broadly to the flavivirus field.
Assuntos
Células Dendríticas , Análise de Célula Única , Infecção por Zika virus , Zika virus , Humanos , Zika virus/fisiologia , Infecção por Zika virus/virologia , Infecção por Zika virus/imunologia , Células Dendríticas/virologia , Células Dendríticas/imunologia , RNA Viral/metabolismo , RNA Viral/genética , Interferon Tipo I/metabolismo , Interações Hospedeiro-Patógeno , Análise de Sequência de RNARESUMO
Human immunodeficiency virus type 1 (HIV-1) disease manifestations differ between cisgender women and men, including better control of viral replication during primary infection and less frequent residual HIV-1 replication on antiretroviral therapy (ART) in cisgender women with HIV-1 (WWH). Investigating plasmacytoid dendritic cell (pDC) functions and HIV-1 reservoir sizes in 20 WWH on stable ART, we observed inverse correlations between interferon-α and tumor necrosis factor responses of pDCs to Toll-like receptor 7/8 stimulation and intact/total proviral HIV-1 DNA levels. Additionally, ISG15 mRNA levels in peripheral blood mononuclear cells correlated with cytokine responses of pDCs. These findings demonstrate an association between higher type I interferon responses and lower HIV-1 reservoir sizes in WWH on ART, warranting studies to identify the underlying mechanisms.
Assuntos
Células Dendríticas , Infecções por HIV , HIV-1 , Interferon Tipo I , Receptor 7 Toll-Like , Humanos , Células Dendríticas/imunologia , Células Dendríticas/virologia , Feminino , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Infecções por HIV/virologia , Adulto , Pessoa de Meia-Idade , Replicação Viral/efeitos dos fármacos , Carga Viral , Antirretrovirais/uso terapêutico , Leucócitos Mononucleares/virologia , Leucócitos Mononucleares/imunologiaRESUMO
Respiratory syncytial virus (RSV) primarily infects the respiratory epithelium, but growing evidence suggests that it may also be responsible for neurologic sequelae. In 3-dimensional microphysiologic peripheral nerve cultures, RSV infected neurons, macrophages, and dendritic cells along 2 distinct trajectories depending on the initial viral load. Low-level infection was transient, primarily involved macrophages, and induced moderate chemokine release with transient neural hypersensitivity. Infection with higher viral loads was persistent, infected neuronal cells in addition to monocytes, and induced robust chemokine release followed by progressive neurotoxicity. In spinal cord cultures, RSV infected microglia and dendritic cells but not neurons, producing a moderate chemokine expression pattern. The persistence of infection was variable but could be identified in dendritic cells as long as 30 days postinoculation. This study suggests that RSV can disrupt neuronal function directly through infection of peripheral neurons and indirectly through infection of resident monocytes and that inflammatory chemokines likely mediate both mechanisms.
Assuntos
Quimiocinas , Infecções por Vírus Respiratório Sincicial , Medula Espinal , Quimiocinas/metabolismo , Medula Espinal/virologia , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/virologia , Animais , Neurônios/virologia , Neurônios/metabolismo , Humanos , Nervos Periféricos/virologia , Macrófagos/virologia , Macrófagos/imunologia , Macrófagos/metabolismo , Carga Viral , Células Dendríticas/virologia , Células Dendríticas/imunologia , Monócitos/virologia , Monócitos/imunologia , Monócitos/metabolismo , Células Cultivadas , Vírus Sinciciais Respiratórios/imunologia , Microglia/virologia , Microglia/metabolismoRESUMO
The DNA sensor cyclic GMP-AMP synthase (cGAS) is critical in host antiviral immunity. Vaccinia virus (VACV) is a large cytoplasmic DNA virus that belongs to the poxvirus family. How vaccinia virus antagonizes the cGAS-mediated cytosolic DNA-sensing pathway is not well understood. In this study, we screened 80 vaccinia genes to identify potential viral inhibitors of the cGAS/Stimulator of interferon gene (STING) pathway. We discovered that vaccinia E5 is a virulence factor and a major inhibitor of cGAS. E5 is responsible for abolishing cGAMP production during vaccinia virus (Western Reserve strain) infection of dendritic cells. E5 localizes to the cytoplasm and nucleus of infected cells. Cytosolic E5 triggers ubiquitination of cGAS and proteasome-dependent degradation via interacting with cGAS. Deleting the E5R gene from the Modified vaccinia virus Ankara (MVA) genome strongly induces type I IFN production by dendritic cells (DCs) and promotes DC maturation, and thereby improves antigen-specific T cell responses.
Assuntos
Células Dendríticas , Nucleotidiltransferases , Vaccinia virus , Proteínas Virais , Camundongos Endogâmicos C57BL , Animais , Camundongos , Camundongos Knockout , Feminino , Nucleotidiltransferases/imunologia , Células Dendríticas/imunologia , Células Dendríticas/virologia , Vaccinia virus/patogenicidade , Fatores de Virulência/imunologia , Ubiquitinação , Proteínas Virais/genética , Proteínas Virais/imunologia , Complexo de Endopeptidases do Proteassoma , Interferon Tipo I/imunologia , Células HEK293 , Humanos , Proteínas de Membrana/imunologia , Linfócitos T/imunologiaRESUMO
Viruses have evolved numerous strategies to impair immunity so that they can replicate more efficiently. Among those, the immunosuppressive effects of morbillivirus infection can be particularly problematic, as they allow secondary infections to take hold in the host, worsening disease prognosis. In the present work, we hypothesized that the highly contagious morbillivirus peste des petits ruminants virus (PPRV) could target monocytes and dendritic cells (DC) to contribute to the immunosuppressive effects produced by the infection. Monocytes isolated from healthy sheep, a natural host of the disease, were able be infected by PPRV and this impaired the differentiation and phagocytic ability of immature monocyte-derived DC (MoDC). We also assessed PPRV capacity to infect differentiated MoDC. Ovine MoDC could be productively infected by PPRV, and this drastically reduced MoDC capacity to activate allogeneic T cell responses. Transcriptomic analysis of infected MoDC indicated that several tolerogenic DC signature genes were upregulated upon PPRV infection. Furthermore, PPRV-infected MoDC could impair the proliferative response of autologous CD4+ and CD8+ T cell to the mitogen concanavalin A (ConA), which indicated that DC targeting by the virus could promote immunosuppression. These results shed new light on the mechanisms employed by morbillivirus to suppress the host immune responses. IMPORTANCE Morbilliviruses pose a threat to global health given their high infectivity. The morbillivirus peste des petits ruminants virus (PPRV) severely affects small-ruminant-productivity and leads to important economic losses in communities that rely on these animals for subsistence. PPRV produces in the infected host a period of severe immunosuppression that opportunistic pathogens exploit, which worsens the course of the infection. The mechanisms of PPRV immunosuppression are not fully understood. In the present work, we demonstrate that PPRV can infect professional antigen-presenting cells called dendritic cells (DC) and disrupt their capacity to elicit an immune response. PPRV infection promoted a DC activation profile that favored the induction of tolerance instead of the activation of an antiviral immune response. These results shed new light on the mechanisms employed by morbilliviruses to suppress the immune responses.
Assuntos
Células Dendríticas , Ativação Linfocitária , Peste dos Pequenos Ruminantes , Vírus da Peste dos Pequenos Ruminantes , Animais , Antivirais , Diferenciação Celular , Concanavalina A/genética , Concanavalina A/imunologia , Células Dendríticas/citologia , Células Dendríticas/virologia , Cabras , Terapia de Imunossupressão , Ativação Linfocitária/imunologia , Mitógenos/imunologia , Peste dos Pequenos Ruminantes/imunologia , Peste dos Pequenos Ruminantes/virologia , Fenótipo , Ovinos , Linfócitos T/imunologia , Linfócitos T/virologiaRESUMO
Interleukin-7 (IL-7) is a cytokine known for its importance in T cell development and survival. How IL-7 shapes CD8 T cell responses during an acute viral infection is less understood. We had previously shown that IL-7 signaling deficient mice have reduced accumulation of influenza-specific CD8 T cells following influenza infection. We sought to determine whether IL-7 affects early CD8 T cell expansion in the mediastinal lymph node and effector function in the lungs. Using IL-7Rα signaling deficient mice, we show that IL-7 is required for a normal sized mediastinal lymph node and the early clonal expansion of influenza-specific CD8 T cells therein. We show that IL-7 plays a cell-intrinsic role in the accumulation of NP366-374 and PA224-233-specific CD8 T cells in the lymph node. We also found that IL-7 shapes terminal differentiation, degranulation and cytokine production to a greater extent in PA224-233-specific than NP366-374-specific CD8 T cells. We further demonstrate that IL-7 is induced in the lung tissue by viral infection and we characterize multiple cellular sources that contribute to IL-7 production. Our findings on IL-7 and its effects on lower respiratory diseases will be important for expanding the utility of therapeutics that are currently available.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Interleucina-7/metabolismo , Pulmão/metabolismo , Linfonodos/metabolismo , Infecções por Orthomyxoviridae/metabolismo , Células A549 , Animais , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/virologia , Modelos Animais de Doenças , Feminino , Interações Hospedeiro-Patógeno , Humanos , Alphainfluenzavirus/imunologia , Alphainfluenzavirus/patogenicidade , Interleucina-7/genética , Subunidade alfa de Receptor de Interleucina-7/genética , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Pulmão/imunologia , Pulmão/virologia , Linfonodos/imunologia , Linfonodos/virologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/metabolismo , Transdução de SinaisRESUMO
Due to their ability to trigger strong immune responses, adenoviruses (HAdVs) in general and the serotype5 (HAdV-5) in particular are amongst the most popular viral vectors in research and clinical application. However, efficient transduction using HAdV-5 is predominantly achieved in coxsackie and adenovirus receptor (CAR)-positive cells. In the present study, we used the transduction enhancer LentiBOOST® comprising the polycationic Polybrene to overcome these limitations. Using LentiBOOST®/Polybrene, we yielded transduction rates higher than 50% in murine bone marrow-derived dendritic cells (BMDCs), while maintaining their cytokine expression profile and their capability to induce T-cell proliferation. In human dendritic cells (DCs), we increased the transduction rate from 22% in immature (i)DCs or 43% in mature (m)DCs to more than 80%, without inducing cytotoxicity. While expression of specific maturation markers was slightly upregulated using LentiBOOST®/Polybrene on iDCs, no effect on mDC phenotype or function was observed. Moreover, we achieved efficient HAdV5 transduction also in human monocytes and were able to subsequently differentiate them into proper iDCs and functional mDCs. In summary, we introduce LentiBOOST® comprising Polybrene as a highly potent adenoviral transduction agent for new in-vitro applications in a set of different immune cells in both mice and humans.
Assuntos
Adenovírus Humanos/genética , Células Dendríticas/virologia , Monócitos/virologia , Transdução Genética , Adenovírus Humanos/fisiologia , Animais , Diferenciação Celular , Proliferação de Células , Células Dendríticas/imunologia , Eletroporação , Vetores Genéticos , Brometo de Hexadimetrina , Especificidade de Hospedeiro , Humanos , Ativação Linfocitária , Teste de Cultura Mista de Linfócitos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Monócitos/imunologia , Fenótipo , Internalização do VírusRESUMO
Common to all cytomegalovirus (CMV) genomes analyzed to date is the presence of G protein-coupled receptors (GPCR). Animal models of CMV provide insights into their role in viral fitness. The mouse cytomegalovirus (MCMV) GPCR, M33, facilitates dendritic cell (DC)-dependent viremia, the extravasation of blood-borne infected DCs to the salivary gland, and the frequency of reactivation events from latently infected tissue explants. Constitutive G protein-coupled M33 signaling is required for these phenotypes, although the contribution of distinct biochemical pathways activated by M33 is unknown. M33 engages Gq/11 to constitutively activate phospholipase C ß (PLCß) and downstream cyclic AMP response-element binding protein (CREB) in vitro. Identification of a MCMV M33 mutant (M33ΔC38) for which CREB signaling was disabled but PLCß activation was preserved provided the opportunity to investigate their relevance in vivo. Following intranasal infection with MCMV M33ΔC38, the absence of M33 CREB Gq/11-dependent signaling correlated with reduced mobilization of lytically-infected DCs to the draining lymph node high endothelial venules (HEVs) and reduced viremia compared with wild type MCMV. In contrast, M33ΔC38-infected DCs within the vascular compartment extravasated to the salivary glands via a pertussis toxin-sensitive, Gi/o-dependent, and CREB-independent mechanism. In the context of MCMV latency, spleen explants from M33ΔC38-infected mice were markedly attenuated for reactivation. Taken together, these data demonstrate that key features of the MCMV life cycle are coordinated in diverse tissues by distinct pathways of the M33 signaling repertoire. IMPORTANCE G protein-coupled receptors (GPCRs) act as cell surface molecular "switches" that regulate the cellular response to environmental stimuli. All cytomegalovirus (CMV) genomes analyzed to date possess GPCR homologs with phylogenetic evidence for independent gene capture events, signifying important in vivo roles. The mouse CMV (MCMV) GPCR homolog, designated M33, is important for cell-associated virus spread and the establishment and/or reactivation of latent MCMV infection. The signaling repertoire of M33 is distinct from cellular GPCRs and little is known of the relevance of component signaling pathways for in vivo M33 function. In this report, we showed that temporal and tissue-specific M33 signaling was required to facilitate in vivo infection. Understanding the relevance of the viral GPCR signaling profiles for in vivo function will provide opportunities for future targeted interventions.
Assuntos
Infecções por Herpesviridae/virologia , Muromegalovirus/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Virais/metabolismo , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Células Dendríticas/virologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Infecções por Herpesviridae/metabolismo , Linfonodos/virologia , Camundongos , Camundongos Endogâmicos BALB C , Muromegalovirus/genética , Muromegalovirus/metabolismo , Mutação , Fosfolipase C beta/metabolismo , Receptores Acoplados a Proteínas G/genética , Glândulas Salivares/virologia , Transdução de Sinais , Proteínas Virais/genética , Viremia/metabolismo , Viremia/virologia , Ativação Viral/genéticaRESUMO
Dendritic cells (DCs) play a pivotal role in the functional differentiation of CD4+ T cells in response to pathogens. In CD4+ T cells, HIV-1 replicates efficiently, while HIV-2, a related virus of reduced pathogenicity, is better controlled. How the DC response to HIV-1 vs HIV-2 contributes to programming an antiviral state in CD4+ T cells is not known. Here, we identify a transcriptional signature associated with progressive resistance to HIV infection in CD4+ T cells. We developed a model of naïve CD4+ T cell priming by DCs stimulated with a panel of seven viruses or synthetic ligands for the viral nucleic acid sensors cGAS and TLRs. DCs produced a cytokine response to HIV-2 infection more similar to the response to cGAS ligands than TLR ligands. In response to these signals, naive CD4+ T cells acquired a gradual antiviral resistance to subsequent HIV infection. The antiviral state was concomitant with the induction of the TH1 cytokine IFNγ and the type I interferon-stimulated gene (ISG) MX1, while the TFH cytokine IL-21 was not increased. By performing a transcriptional network analysis in T cells, we identified five distinct gene modules with characteristic ISG, TH1, TFH, IFN-I and proliferative signatures. Finally, we leverage this module to assemble a T antiviral signature of 404 genes that correlate with the antiviral state in T cells. Altogether, the study illuminates the programming of the antiviral state in T cells. The T antiviral gene signature in human CD4+ lymphocytes constitutes a resource for genetic screens and genomics analysis.
Assuntos
Linfócitos T CD4-Positivos , Células Dendríticas , Infecções por HIV , HIV-2 , Transcriptoma , Linfócitos T CD4-Positivos/imunologia , Citocinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/virologia , Infecções por HIV/genética , Infecções por HIV/imunologia , HIV-2/genética , HIV-2/fisiologia , Humanos , Ligantes , Nucleotidiltransferases/metabolismo , Replicação ViralRESUMO
BACKGROUND: Multisystem inflammatory syndrome in children (MIS-C) is an acute, febrile, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-associated syndrome, often with cardiohemodynamic dysfunction. Insight into mechanism of disease is still incomplete. OBJECTIVE: Our objective was to analyze immunologic features of MIS-C patients compared to febrile controls (FC). METHODS: MIS-C patients were defined by narrow criteria, including having evidence of cardiohemodynamic involvement and no macrophage activation syndrome. Samples were collected from 8 completely treatment-naive patients with MIS-C (SARS-CoV-2 serology positive), 3 patients with unclassified MIS-C-like disease (serology negative), 14 FC, and 5 MIS-C recovery (RCV). Three healthy controls (HCs) were used for comparisons of normal range. Using spectral flow cytometry, we assessed 36 parameters in antigen-presenting cells (APCs) and 29 in T cells. We used biaxial analysis and uniform manifold approximation and projection (UMAP). RESULTS: Significant elevations in cytokines including CXCL9, M-CSF, and IL-27 were found in MIS-C compared to FC. Classic monocytes and type 2 dendritic cells (DCs) were downregulated (decreased CD86, HLA-DR) versus HCs; however, type 1 DCs (CD11c+CD141+CLEC9A+) were highly activated in MIS-C patients versus FC, expressing higher levels of CD86, CD275, and atypical conventional DC markers such as CD64, CD115, and CX3CR1. CD169 and CD38 were upregulated in multiple monocyte subtypes. CD56dim/CD57-/KLRGhi/CD161+/CD38- natural killer (NK) cells were a unique subset in MIS-C versus FC without macrophage activation syndrome. CONCLUSION: Orchestrated by complex cytokine signaling, type 1 DC activation and NK dysregulation are key features in the pathophysiology of MIS-C. NK cell findings may suggest a relationship with macrophage activation syndrome, while type 1 DC upregulation implies a role for antigen cross-presentation.
Assuntos
COVID-19/complicações , Células Dendríticas/imunologia , Células Dendríticas/virologia , SARS-CoV-2/imunologia , Síndrome de Resposta Inflamatória Sistêmica/imunologia , Síndrome de Resposta Inflamatória Sistêmica/virologia , ADP-Ribosil Ciclase 1/sangue , Adolescente , Antígenos Virais/imunologia , COVID-19/imunologia , COVID-19/virologia , Estudos de Casos e Controles , Criança , Pré-Escolar , Apresentação Cruzada , Citocinas/sangue , Células Dendríticas/classificação , Feminino , Antígenos HLA-DR/sangue , Humanos , Imunofenotipagem , Interferon gama/sangue , Interleucinas/sangue , Células Matadoras Naturais/imunologia , Masculino , Glicoproteínas de Membrana/sangue , Modelos Imunológicos , Monócitos/imunologia , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/sangue , Linfócitos T/imunologia , Linfócitos T/virologia , Regulação para CimaRESUMO
Dendritic cells (DCs) are the most potent antigen-presenting cells, and their function is essential to configure adaptative immunity and avoid excessive inflammation. DCs are predicted to play a crucial role in the clinical evolution of the infection by the severe acute respiratory syndrome (SARS) coronavirus (CoV)-2. DCs interaction with the SARS-CoV-2 Spike protein, which mediates cell receptor binding and subsequent fusion of the viral particle with host cell, is a key step to induce effective immunity against this virus and in the S protein-based vaccination protocols. Here we evaluated human DCs in response to SARS-CoV-2 S protein, or to a fragment encompassing the receptor binding domain (RBD) challenge. Both proteins increased the expression of maturation markers, including MHC molecules and costimulatory receptors. DCs interaction with the SARS-CoV-2 S protein promotes activation of key signaling molecules involved in inflammation, including MAPK, AKT, STAT1, and NFκB, which correlates with the expression and secretion of distinctive proinflammatory cytokines. Differences in the expression of ACE2 along the differentiation of human monocytes to mature DCs and inter-donor were found. Our results show that SARS-CoV-2 S protein promotes inflammatory response and provides molecular links between individual variations and the degree of response against this virus.
Assuntos
Células Dendríticas/patologia , Células Dendríticas/virologia , Receptores Virais/metabolismo , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Moléculas de Adesão Celular/metabolismo , Diferenciação Celular , Citocinas/biossíntese , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Inflamação/patologia , Lectinas Tipo C/metabolismo , Domínios Proteicos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Superfície Celular/metabolismo , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais , Doadores de TecidosRESUMO
Plasmacytoid dendritic cells (pDC) are important innate immune cells during the onset of viral infections as they are specialized in the production of massive amounts of antiviral type I interferon (IFN). Alphaherpesviruses such as herpes simplex virus (HSV) or pseudorabies virus (PRV) are double stranded DNA viruses and potent stimulators of pDC. Detailed information on how PRV activates porcine pDC is lacking. Using PRV and porcine primary pDC, we report here that PRV virions, so-called heavy (H-)particles, trigger IFNα production by pDC, whereas light (L-) particles that lack viral DNA and capsid do not. Activation of pDC requires endosomal acidification and, importantly, depends on the PRV gD envelope glycoprotein and O-glycosylations. Intriguingly, both for PRV and HSV-1, we found that L-particles suppress H-particle-mediated activation of pDC, a process which again depends on viral gD. This is the first report describing that gD plays a critical role in alphaherpesvirus-induced pDC activation and that L-particles directly interfere with alphaherpesvirus-induced IFNα production by pDC.