RESUMO
Tissue immunity and responses to injury depend on the coordinated action and communication among physiological systems. Here, we show that, upon injury, adaptive responses to the microbiota directly promote sensory neuron regeneration. At homeostasis, tissue-resident commensal-specific T cells colocalize with sensory nerve fibers within the dermis, express a transcriptional program associated with neuronal interaction and repair, and promote axon growth and local nerve regeneration following injury. Mechanistically, our data reveal that the cytokine interleukin-17A (IL-17A) released by commensal-specific Th17 cells upon injury directly signals to sensory neurons via IL-17 receptor A, the transcription of which is specifically upregulated in injured neurons. Collectively, our work reveals that in the context of tissue damage, preemptive immunity to the microbiota can rapidly bridge biological systems by directly promoting neuronal repair, while also identifying IL-17A as a major determinant of this fundamental process.
Assuntos
Interleucina-17 , Microbiota , Regeneração Nervosa , Células Th17 , Axônios , Regeneração Nervosa/fisiologia , Células Receptoras Sensoriais , Animais , Camundongos , Células Th17/citologiaRESUMO
Germinal centers (GCs) that form in mucosal sites are exposed to gut-derived factors that have the potential to influence homeostasis independent of antigen receptor-driven selective processes. The G-protein Gα13 confines B cells to the GC and limits the development of GC-derived lymphoma. We discovered that Gα13-deficiency fuels the GC reaction via increased mTORC1 signaling and Myc protein expression specifically in the mesenteric lymph node (mLN). The competitive advantage of Gα13-deficient GC B cells (GCBs) in mLN was not dependent on T cell help or gut microbiota. Instead, Gα13-deficient GCBs were selectively dependent on dietary nutrients likely due to greater access to gut lymphatics. Specifically, we found that diet-derived glutamine supported proliferation and Myc expression in Gα13-deficient GCBs in the mLN. Thus, GC confinement limits the effects of dietary glutamine on GC dynamics in mucosal tissues. Gα13 pathway mutations coopt these processes to promote the gut tropism of aggressive lymphoma.
RESUMO
Most studies of adaptive immunity to SARS-CoV-2 infection focus on peripheral blood, which may not fully reflect immune responses at the site of infection. Using samples from 110 children undergoing tonsillectomy and adenoidectomy during the COVID-19 pandemic, we identified 24 samples with evidence of previous SARS-CoV-2 infection, including neutralizing antibodies in serum and SARS-CoV-2-specific germinal center and memory B cells in the tonsils and adenoids. Single-cell B cell receptor (BCR) sequencing indicated virus-specific BCRs were class-switched and somatically hypermutated, with overlapping clones in the two tissues. Expanded T cell clonotypes were found in tonsils, adenoids and blood post-COVID-19, some with CDR3 sequences identical to previously reported SARS-CoV-2-reactive T cell receptors (TCRs). Pharyngeal tissues from COVID-19-convalescent children showed persistent expansion of germinal center and antiviral lymphocyte populations associated with interferon (IFN)-γ-type responses, particularly in the adenoids, and viral RNA in both tissues. Our results provide evidence for persistent tissue-specific immunity to SARS-CoV-2 in the upper respiratory tract of children after infection.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Criança , Pandemias , Imunidade Adaptativa , Tonsila Palatina , Anticorpos AntiviraisRESUMO
Regulatory T cells (Treg cells) can activate multiple suppressive mechanisms in vitro after activation via the T cell antigen receptor, resulting in antigen-independent suppression. However, it remains unclear whether similar pathways operate in vivo. Here we found that antigen-specific Treg cells activated by dendritic cells (DCs) pulsed with two antigens suppressed conventional naive T cells (Tnaive cells) specific for both cognate antigens and non-cognate antigens in vitro but suppressed only Tnaive cells specific for cognate antigen in vivo. Antigen-specific Treg cells formed strong interactions with DCs, resulting in selective inhibition of the binding of Tnaive cells to cognate antigen yet allowing bystander Tnaive cell access. Strong binding resulted in the removal of the complex of cognate peptide and major histocompatibility complex class II (pMHCII) from the DC surface, reducing the capacity of DCs to present antigen. The enhanced binding of Treg cells to DCs, coupled with their capacity to deplete pMHCII, represents a novel pathway for Treg cell-mediated suppression and may be a mechanism by which Treg cells maintain immune homeostasis.
Assuntos
Apresentação de Antígeno/imunologia , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Tolerância Imunológica/imunologia , Linfócitos T Reguladores/imunologia , Animais , Efeito Espectador/imunologia , Células Cultivadas , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Peptídeos/imunologia , Cultura Primária de Células , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T Reguladores/metabolismoRESUMO
B cells are activated by two temporally distinct signals, the first provided by the binding of antigen to the B cell antigen receptor (BCR), and the second provided by helper T cells. Here we found that B cells responded to antigen by rapidly increasing their metabolic activity, including both oxidative phosphorylation and glycolysis. In the absence of a second signal, B cells progressively lost mitochondrial function and glycolytic capacity, which led to apoptosis. Mitochondrial dysfunction was a result of the gradual accumulation of intracellular calcium through calcium response-activated calcium channels that, for approximately 9 h after the binding of B cell antigens, was preventable by either helper T cells or signaling via the receptor TLR9. Thus, BCR signaling seems to activate a metabolic program that imposes a limited time frame during which B cells either receive a second signal and survive or are eliminated.
Assuntos
Linfócitos B/fisiologia , Mitocôndrias/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Receptor Toll-Like 9/metabolismo , Animais , Apoptose , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Citocinas/metabolismo , Glicólise , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células NIH 3T3 , Fosforilação Oxidativa , Receptores de Antígenos de Linfócitos B/genética , Transdução de Sinais , Receptor Toll-Like 9/genéticaRESUMO
Key events in T cell-dependent antibody responses, including affinity maturation, are dependent on the B cell's presentation of antigen to helper T cells at critical checkpoints in germinal-center formation in secondary lymphoid organs. Here we found that signaling via Toll-like receptor 9 (TLR9) blocked the ability of antigen-specific B cells to capture, process and present antigen and to activate antigen-specific helper T cells in vitro. In a mouse model in vivo and in a human clinical trial, the TLR9 agonist CpG enhanced the magnitude of the antibody response to a protein vaccine but failed to promote affinity maturation. Thus, TLR9 signaling might enhance antibody titers at the expense of the ability of B cells to engage in germinal-center events that are highly dependent on B cells' capture and presentation of antigen.
Assuntos
Formação de Anticorpos/imunologia , Apresentação de Antígeno/genética , Ativação Linfocitária/imunologia , Receptor Toll-Like 9/imunologia , Animais , Afinidade de Anticorpos , Centro Germinativo/imunologia , Humanos , Vacinas Antimaláricas , Camundongos , Receptor Toll-Like 9/agonistasRESUMO
The somatosensory nervous system surveils external stimuli at barrier tissues, regulating innate immune cells under infection and inflammation. The roles of sensory neurons in controlling the adaptive immune system, and more specifically immunity to the microbiota, however, remain elusive. Here, we identified a mechanism for direct neuroimmune communication between commensal-specific T lymphocytes and somatosensory neurons mediated by the neuropeptide calcitonin gene-related peptide (CGRP) in the skin. Intravital imaging revealed that commensal-specific T cells are in close proximity to cutaneous nerve fibers in vivo. Correspondingly, we observed upregulation of the receptor for the neuropeptide CGRP, RAMP1, in CD8+ T lymphocytes induced by skin commensal colonization. The neuroimmune CGRP-RAMP1 signaling axis functions in commensal-specific T cells to constrain Type 17 responses and moderate the activation status of microbiota-reactive lymphocytes at homeostasis. As such, modulation of neuroimmune CGRP-RAMP1 signaling in commensal-specific T cells shapes the overall activation status of the skin epithelium, thereby impacting the outcome of responses to insults such as wounding. The ability of somatosensory neurons to control adaptive immunity to the microbiota via the CGRP-RAMP1 axis underscores the various layers of regulation and multisystem coordination required for optimal microbiota-reactive T cell functions under steady state and pathology.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina , Neuroimunomodulação , Peptídeo Relacionado com Gene de Calcitonina/genética , Proteína 1 Modificadora da Atividade de Receptores/genética , Receptores de Peptídeo Relacionado com o Gene de Calcitonina , Imunidade AdaptativaRESUMO
ABSTRACT: Mutations in the small Rho-family guanosine triphosphate hydrolase RAC2, critical for actin cytoskeleton remodeling and intracellular signal transduction, are associated with neonatal severe combined immunodeficiency (SCID), infantile neutrophilic disorder resembling leukocyte adhesion deficiency (LAD), and later-onset combined immune deficiency (CID). We investigated 54 patients (23 previously reported) from 37 families yielding 15 novel RAC2 missense mutations, including one present only in homozygosity. Data were collected from referring physicians and literature reports with updated clinical information. Patients were grouped by presentation: neonatal SCID (n = 5), infantile LAD-like disease (n = 5), or CID (n = 44). Disease correlated to RAC2 activity: constitutively active RAS-like mutations caused neonatal SCID, dominant-negative mutations caused LAD-like disease, whereas dominant-activating mutations caused CID. Significant T- and B-lymphopenia with low immunoglobulins were seen in most patients; myeloid abnormalities included neutropenia, altered oxidative burst, impaired neutrophil migration, and visible neutrophil macropinosomes. Among 42 patients with CID with clinical data, upper and lower respiratory infections and viral infections were common. Twenty-three distinct RAC2 mutations, including 15 novel variants, were identified. Using heterologous expression systems, we assessed downstream effector functions including superoxide production, p21-activated kinase 1 binding, AKT activation, and protein stability. Confocal microscopy showed altered actin assembly evidenced by membrane ruffling and macropinosomes. Altered protein localization and aggregation were observed. All tested RAC2 mutant proteins exhibited aberrant function; no single assay was sufficient to determine functional consequence. Most mutants produced elevated superoxide; mutations unable to support superoxide formation were associated with bacterial infections. RAC2 mutations cause a spectrum of immune dysfunction, ranging from early onset SCID to later-onset combined immunodeficiencies depending on RAC2 activity. This trial was registered at www.clinicaltrials.gov as #NCT00001355 and #NCT00001467.
Assuntos
Síndromes de Imunodeficiência , Síndrome da Aderência Leucocítica Deficitária , Doenças da Imunodeficiência Primária , Imunodeficiência Combinada Severa , Humanos , Recém-Nascido , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/metabolismo , Neutrófilos/metabolismo , Doenças da Imunodeficiência Primária/genética , Doenças da Imunodeficiência Primária/metabolismo , Proteínas rac de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína RAC2 de Ligação ao GTP , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/metabolismo , Superóxidos/metabolismoRESUMO
Autophagosomes delivers cytoplasmic constituents to lysosomes for degradation, whereas inflammasomes are molecular platforms activated by infection or stress that regulate the activity of caspase-1 and the maturation of interleukin 1ß (IL-1ß) and IL-18. Here we show that the induction of AIM2 or NLRP3 inflammasomes in macrophages triggered activation of the G protein RalB and autophagosome formation. The induction of autophagy did not depend on the adaptor ASC or capase-1 but was dependent on the presence of the inflammasome sensor. Blocking autophagy potentiated inflammasome activity, whereas stimulating autophagy limited it. Assembled inflammasomes underwent ubiquitination and recruited the autophagic adaptor p62, which assisted their delivery to autophagosomes. Our data indicate that autophagy accompanies inflammasome activation to temper inflammation by eliminating active inflammasomes.
Assuntos
Autofagia , Inflamassomos/imunologia , Interleucina-1beta/biossíntese , Transdução de Sinais , Ubiquitinação , Animais , Proteínas de Transporte/imunologia , Linhagem Celular , Proteínas de Ligação a DNA , Humanos , Inflamassomos/metabolismo , Inflamação/imunologia , Inflamação/metabolismo , Interleucina-1beta/imunologia , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteínas Nucleares/imunologia , Proteínas ral de Ligação ao GTP/imunologiaRESUMO
Older age at the time of infection with hepatitis viruses is associated with an increased risk of liver fibrosis progression. We hypothesized that the pace of fibrosis progression may reflect changes in gene expression within the aging liver. We compared gene expression in liver specimens from 54 adult donors without evidence of fibrosis, including 36 over 40 y old and 18 between 18 and 40 y old. Chitinase 3-like 1 (CHI3L1), which encodes chitinase-like protein YKL-40/CHI3L1, was identified as the gene with the greatest age-dependent increase in expression in liver tissue. We investigated the cellular source of CHI3L1 in the liver and its function using liver tissue specimens and in vitro models. CHI3L1 expression was significantly higher in livers of patients with cirrhosis of diverse etiologies compared with controls represented by patients who underwent liver resection for hemangioma. The highest intrahepatic CHI3L1 expression was observed in cirrhosis due to hepatitis D virus, followed by hepatitis C virus, hepatitis B virus, and alcohol-induced cirrhosis. In situ hybridization of CHI3L1 messenger RNA (mRNA) identified hepatocytes as the major producers of CHI3L1 in normal liver and in cirrhotic tissue, wherein hepatocytes adjacent to fibrous septa showed higher CHI3L1 expression than did those in more distal areas. In vitro studies showed that recombinant CHI3L1 promotes proliferation and activation of primary human hepatic stellate cells (HSCs), the major drivers of liver fibrosis. These findings collectively demonstrate that CHI3L1 promotes liver fibrogenesis through a direct effect on HSCs and support a role for CHI3L1 in the increased susceptibility of aging livers to fibrosis progression.
Assuntos
Proteína 1 Semelhante à Quitinase-3/metabolismo , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Adolescente , Adulto , Envelhecimento/fisiologia , Biomarcadores/metabolismo , Proteína 1 Semelhante à Quitinase-3/fisiologia , Quitinases/metabolismo , Feminino , Expressão Gênica/genética , Hepacivirus/patogenicidade , Células Estreladas do Fígado/patologia , Hepatite C/metabolismo , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Fígado/citologia , MasculinoRESUMO
The diverse composition of mammalian tissues poses challenges for understanding the cell-cell interactions required for organ homeostasis and how spatial relationships are perturbed during disease. Existing methods such as single-cell genomics, lacking a spatial context, and traditional immunofluorescence, capturing only two to six molecular features, cannot resolve these issues. Imaging technologies have been developed to address these problems, but each possesses limitations that constrain widespread use. Here we report a method that overcomes major impediments to highly multiplex tissue imaging. "Iterative bleaching extends multiplexity" (IBEX) uses an iterative staining and chemical bleaching method to enable high-resolution imaging of >65 parameters in the same tissue section without physical degradation. IBEX can be employed with various types of conventional microscopes and permits use of both commercially available and user-generated antibodies in an "open" system to allow easy adjustment of staining panels based on ongoing marker discovery efforts. We show how IBEX can also be used with amplified staining methods for imaging strongly fixed tissues with limited epitope retention and with oligonucleotide-based staining, allowing potential cross-referencing between flow cytometry, cellular indexing of transcriptomes and epitopes by sequencing, and IBEX analysis of the same tissue. To facilitate data processing, we provide an open-source platform for automated registration of iterative images. IBEX thus represents a technology that can be rapidly integrated into most current laboratory workflows to achieve high-content imaging to reveal the complex cellular landscape of diverse organs and tissues.
Assuntos
Células/metabolismo , Imagem Óptica/métodos , Animais , Corantes Fluorescentes/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Imunização , Linfonodos/diagnóstico por imagem , Camundongos , Especificidade de Órgãos , FenótipoRESUMO
BACKGROUND & AIMS: Non-alcoholic steatohepatitis (NASH) represents the fastest growing underlying cause of hepatocellular carcinoma (HCC) and has been shown to impact immune effector cell function. The standard of care for the treatment of advanced HCC is immune checkpoint inhibitor (ICI) therapy, yet NASH may negatively affect the efficacy of ICI therapy in HCC. The immunologic mechanisms underlying the impact of NASH on ICI therapy remain unclear. METHODS: Herein, using multiple murine NASH models, we analysed the influence of NASH on the CD8+ T-cell-dependent anti-PD-1 responses against liver cancer. We characterised CD8+ T cells' transcriptomic, functional, and motility changes in mice receiving a normal diet (ND) or a NASH diet. RESULTS: NASH blunted the effect of anti-PD-1 therapy against liver cancers in multiple murine models. NASH caused a proinflammatory phenotypic change of hepatic CD8+ T cells. Transcriptomic analysis revealed changes related to NASH-dependent impairment of hepatic CD8+ T-cell metabolism. In vivo imaging analysis showed reduced motility of intratumoural CD8+ T cells. Metformin treatment rescued the efficacy of anti-PD-1 therapy against liver tumours in NASH. CONCLUSIONS: We discovered that CD8+ T-cell metabolism is critically altered in the context of NASH-related liver cancer, impacting the effectiveness of ICI therapy - a finding which has therapeutic implications in patients with NASH-related liver cancer. LAY SUMMARY: Non-alcoholic steatohepatitis represents the fastest growing cause of hepatocellular carcinoma. It is also associated with reduced efficacy of immunotherapy, which is the standard of care for advanced hepatocellular carcinoma. Herein, we show that non-alcoholic steatohepatitis is associated with impaired motility, metabolic function, and response to anti-PD-1 treatment in hepatic CD8+ T cells, which can be rescued by metformin treatment.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Metformina , Hepatopatia Gordurosa não Alcoólica , Animais , Linfócitos T CD8-Positivos/metabolismo , Carcinoma Hepatocelular/metabolismo , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Fígado/patologia , Neoplasias Hepáticas/etiologia , Metformina/farmacologia , Metformina/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/metabolismoRESUMO
Flow cytometry allows highly quantitative analysis of complex dissociated populations at the cost of neglecting their tissue localization. In contrast, conventional microscopy methods provide spatial information, but visualization and quantification of cellular subsets defined by complex phenotypic marker combinations is challenging. Here, we describe an analytical microscopy method, "histo-cytometry," for visualizing and quantifying phenotypically complex cell populations directly in tissue sections. This technology is based on multiplexed antibody staining, tiled high-resolution confocal microscopy, voxel gating, volumetric cell rendering, and quantitative analysis. We have tested this technology on various innate and adaptive immune populations in murine lymph nodes (LNs) and were able to identify complex cellular subsets and phenotypes, achieving quantitatively similar results to flow cytometry, while also gathering cellular positional information. Here, we employ histo-cytometry to describe the spatial segregation of resident and migratory dendritic cell subsets into specialized microanatomical domains, suggesting an unexpected LN demarcation into discrete functional compartments.
Assuntos
Células Dendríticas/citologia , Citometria de Fluxo/métodos , Imuno-Histoquímica/métodos , Linfonodos/citologia , Microscopia Confocal/métodos , Animais , Antígenos CD/análise , Quimera/imunologia , Células Dendríticas/imunologia , Humanos , Linfonodos/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência/métodos , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Hepatitis B virus (HBV)-associated acute liver failure (ALF) is a dramatic clinical syndrome leading to death or liver transplantation in 80% of cases. Due to the extremely rapid clinical course, the difficulties in obtaining liver specimens, and the lack of an animal model, the pathogenesis of ALF remains largely unknown. Here, we performed a comprehensive genetic and functional characterization of the virus and the host in liver tissue from HBV-associated ALF and compared the results with those of classic acute hepatitis B in chimpanzees. In contrast with acute hepatitis B, HBV strains detected in ALF livers displayed highly mutated HBV core antigen (HBcAg), associated with increased HBcAg expression ex vivo, which was independent of viral replication levels. Combined gene and miRNA expression profiling revealed a dominant B cell disease signature, with extensive intrahepatic production of IgM and IgG in germline configuration exclusively targeting HBcAg with subnanomolar affinities, and complement deposition. Thus, HBV ALF appears to be an anomalous T cell-independent, HBV core-driven B cell disease, which results from the rare and unfortunate encounter between a host with an unusual B cell response and an infecting virus with a highly mutated core antigen.
Assuntos
Anticorpos Antivirais/imunologia , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Imunidade Humoral , Falência Hepática Aguda/imunologia , Adulto , Animais , Linfócitos B/imunologia , Feminino , Hepatite B/imunologia , Hepatite B/patologia , Hepatite B/virologia , Antígenos do Núcleo do Vírus da Hepatite B/genética , Vírus da Hepatite B/genética , Humanos , Fígado/imunologia , Fígado/virologia , Falência Hepática Aguda/patologia , Falência Hepática Aguda/virologia , Masculino , Pessoa de Meia-Idade , Pan troglodytes , Linfócitos T/imunologiaRESUMO
Human respiratory syncytial virus (RSV) is a major pediatric respiratory pathogen. The attachment (G) and fusion (F) glycoproteins are major neutralization and protective antigens. RSV G is expressed as membrane-anchored (mG) and -secreted (sG) forms, both containing a central fractalkine-like CX3C motif. The CX3C motif and sG are thought to interfere with host immune responses and have been suggested to be omitted from a vaccine. We used a chimeric bovine/human parainfluenza virus type 3 (rB/HPIV3) vector to express RSV wild-type (wt) G and modified forms, including sG alone, mG alone, mutants with ablated CX3C, and G with enhanced packaging into vector virions. In hamsters, these viruses replicated to similar titers. When assayed with a complement-enhanced neutralization assay in Vero cells, sG did not reduce the serum RSV- or PIV3-neutralizing antibody (NAb) responses, whereas ablating CX3C drastically reduced the RSV NAb response. Protective efficacy against RSV challenge was not reduced by sG but was strongly dependent on the CX3C motif. In ciliated human airway epithelial (HAE) cells, NAbs induced by wt G, but not by wt F, completely blocked RSV infection in the absence of added complement. This activity was dependent on the integrity of the CX3C motif. In hamsters, the rB/HPIV3 expressing wt G conferred better protection against RSV challenge than that expressing wt F. Codon optimization of the wt G further increased its immunogenicity and protective efficacy. This study showed that ablation of the CX3C motif or sG in an RSV vaccine, as has been suggested previously, would be ill advised.IMPORTANCE Human RSV is the leading viral cause of severe pediatric respiratory illness. An RSV vaccine is not yet available. The RSV attachment protein G is an important protective and neutralization antigen. G contains a conserved fractalkine-like CX3C motif and is expressed in mG and sG forms. sG and the CX3C motif are thought to interfere with host immune responses, but this remains poorly characterized. Here, we used an attenuated chimeric bovine/human parainfluenza virus type 3 (rB/HPIV3) vector to express various modified forms of RSV G. We demonstrated that strong antibody and protective responses could be induced by G alone, and that this was highly dependent on the integrity of the CX3C motif. There was no evidence that sG or the CX3C motif impaired immune responses against RSV G or the rB/HPIV3 vector. rB/HPIV3 expressing wt RSV G provides a bivalent vaccine against RSV and HPIV3.
Assuntos
Vetores Genéticos/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Respirovirus/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Bovinos , Criança , Chlorocebus aethiops , Feminino , Humanos , Macaca mulatta , Mesocricetus , Infecções por Vírus Respiratório Sincicial/virologia , Células Vero , Proteínas Virais de Fusão/imunologia , Vírion/imunologia , Replicação Viral/imunologiaRESUMO
Entry of hepatitis C virus (HCV) into hepatocytes is a complex process that involves numerous cellular factors, including the scavenger receptor class B type 1 (SR-B1), the tetraspanin CD81, and the tight junction (TJ) proteins claudin-1 (CLDN1) and occludin (OCLN). Despite expression of all known HCV-entry factors, in vitro models based on hepatoma cell lines do not fully reproduce the in vivo susceptibility of liver cells to primary HCV isolates, implying the existence of additional host factors which are critical for HCV entry and/or replication. Likewise, HCV replication is severely impaired within hepatocellular carcinoma (HCC) tissue in vivo, but the mechanisms responsible for this restriction are presently unknown. Here, we identify tumor-associated calcium signal transducer 2 (TACSTD2), one of the most downregulated genes in primary HCC tissue, as a host factor that interacts with CLDN1 and OCLN and regulates their cellular localization. TACSTD2 gene silencing disrupts the typical linear distribution of CLDN1 and OCLN along the cellular membrane in both hepatoma cells and primary human hepatocytes, recapitulating the pattern observed in vivo in primary HCC tissue. Mechanistic studies suggest that TACSTD2 is involved in the phosphorylation of CLDN1 and OCLN, which is required for their proper cellular localization. Silencing of TACSTD2 dramatically inhibits HCV infection with a pan-genotype effect that occurs at the level of viral entry. Our study identifies TACSTD2 as a novel regulator of two major HCV-entry factors, CLDN1 and OCLN, which is strongly downregulated in malignant hepatocytes. These results provide new insights into the complex process of HCV entry into hepatocytes and may assist in the development of more efficient cellular systems for HCV propagation in vitro.
Assuntos
Antígenos de Neoplasias/metabolismo , Carcinoma Hepatocelular/virologia , Moléculas de Adesão Celular/metabolismo , Claudina-1/metabolismo , Hepacivirus/patogenicidade , Hepatite C/virologia , Neoplasias Hepáticas/virologia , Ocludina/metabolismo , Antígenos de Neoplasias/genética , Carcinoma Hepatocelular/epidemiologia , Carcinoma Hepatocelular/metabolismo , Moléculas de Adesão Celular/genética , Claudina-1/genética , Regulação para Baixo , Hepatite C/complicações , Hepatite C/metabolismo , Hepatócitos/metabolismo , Hepatócitos/patologia , Hepatócitos/virologia , Humanos , Neoplasias Hepáticas/epidemiologia , Neoplasias Hepáticas/metabolismo , Ocludina/genética , Internalização do Vírus , Replicação ViralRESUMO
Early secretion of IL-12 by mouse dendritic cells (DCs) instructs T cells to make IFN-γ. However, only activated, but not naive T cells are able to license DCs for IL-12 production. We hypothesized that it might be due to different levels of CD40L expression on the surface of these cells, as CD40 signals are required for IL-12 production. Using quantitative cell-free systems incorporating CD40L in lipid bilayers combined with total internal reflection fluorescence microscopy and flow cytometry, we show that as low as â¼200 CD40L molecules/µm2 in combination with IL-4 is sufficient to induce IL-12 production by DCs. Remarkably, CD40L alone is adequate to induce IL-23 secretion by DCs. Thus, although activated T cells have somewhat higher levels of CD40L, it is the combination of CD40L and the cytokines they secrete that licenses DCs and influences the effector class of the immune response.
Assuntos
Ligante de CD40/imunologia , Células Dendríticas/imunologia , Interleucina-12/biossíntese , Interleucina-23/biossíntese , Ativação Linfocitária/imunologia , Animais , Células Dendríticas/metabolismo , Interleucina-12/imunologia , Interleucina-23/imunologia , Camundongos , Camundongos TransgênicosRESUMO
Activation of CD4+ T cells to proliferate drives cells toward aerobic glycolysis for energy production while using mitochondria primarily for macromolecular synthesis. In addition, the mitochondria of activated T cells increase production of reactive oxygen species, providing an important second messenger for intracellular signaling pathways. To better understand the critical changes in mitochondria that accompany prolonged T cell activation, we carried out an extensive analysis of mitochondrial remodeling using a combination of conventional strategies and a novel high-resolution imaging method. We show that for 4 d following activation, mouse CD4+ T cells sustained their commitment to glycolysis facilitated by increased glucose uptake through increased expression of GLUT transporters. Despite their limited contribution to energy production, mitochondria were active and showed increased reactive oxygen species production. Moreover, prolonged activation of CD4+ T cells led to increases in mitochondrial content and volume, in the number of mitochondria per cell and in mitochondrial biogenesis. Thus, during prolonged activation, CD4+ T cells continue to obtain energy predominantly from glycolysis but also undergo extensive mitochondrial remodeling, resulting in increased mitochondrial activity.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Proteínas de Transporte de Glutamato da Membrana Plasmática/metabolismo , Mitocôndrias/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Fatores de Tempo , Animais , Células Cultivadas , Metabolismo Energético , Feminino , Glicólise , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transdução de SinaisRESUMO
B cells express the innate receptor, TLR9, which signals in response to unmethylated CpG sequences in microbial DNA. Of the two major classes of CpG-containing oligonucleotides, CpG-A appears restricted to inducing type 1 IFN in innate immune cells and CpG-B to activating B cells to proliferate and produce Abs and inflammatory cytokines. Although CpGs are candidates for adjuvants to boost innate and adaptive immunity, our understanding of the effect of CpG-A and CpG-B on B cell responses is incomplete. In this study we show that both CpG-B and CpG-A activated B cells in vitro to proliferate, secrete Abs and IL-6, and that neither CpG-B nor CpG-A alone induced type 1 IFN production. However, when incorporated into the cationic lipid, DOTAP, CpG-A, but not CpG-B, induced a type 1 IFN response in B cells in vitro and in vivo. We provide evidence that differences in the function of CpG-A and CpG-B may be related to their intracellular trafficking in B cells. These findings fill an important gap in our understanding of the B cell response to CpGs, with implications for the use of CpG-A and CpG-B as immunomodulators.
Assuntos
Linfócitos B/imunologia , Interferon Tipo I/biossíntese , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/imunologia , Animais , Formação de Anticorpos , Linfócitos B/efeitos dos fármacos , Cátions/imunologia , Citocinas/genética , Citocinas/imunologia , Imunidade Inata , Fatores Imunológicos/metabolismo , Interferon Tipo I/imunologia , Interleucina-6/biossíntese , Interleucina-6/imunologia , Lipídeos/administração & dosagem , Lipídeos/química , Lipídeos/farmacologia , Ativação Linfocitária , Camundongos , Oligodesoxirribonucleotídeos/administração & dosagem , Oligodesoxirribonucleotídeos/farmacologia , Receptor Toll-Like 9/agonistasRESUMO
The incidence of pulmonary nontuberculous mycobacteria (NTM) disease is increasing, but host responses in respiratory epithelium infected with NTM are not fully understood. In this work, we aimed to identify infection-relevant gene expression signatures of NTM infection of the respiratory epithelium. We infected air-liquid interface (ALI) primary respiratory epithelial cell cultures with Mycobacterium avium subsp. avium (MAC) or Mycobacterium abscessus subsp. abscessus (MAB). We used cells from four different donors to obtain generalizable data. Differentiated respiratory epithelial cells at the ALI were infected with MAC or MAB at a multiplicity of infection of 100:1 or 1,000:1, and RNA sequencing was performed at Days 1 and 3 after infection. In response to infection, we found down-regulation of ciliary genes but upregulation of genes associated with cytokines/chemokines, such as IL-32, and cholesterol biosynthesis. Inflammatory response genes tended to be more upregulated by MAB than by MAC infection. Primary respiratory epithelial cell infection with NTM at the ALI identified ciliary function, cholesterol biosynthesis, and cytokine/chemokine production as major host responses to infection. Some of these pathways may be amenable to therapeutic manipulation.