RESUMO
Multiphoton intravital microscopy (MP-IVM) is an imaging technique used for the observation of living organisms at a microscopic resolution. The tissue of interest is exposed through a window allowing imaging of cells in real time. Using MP-IVM, the temporospatial kinetics of leukocyte transendothelial migration can be visualized and quantitated using reporter mice and cell-specific fluorophore-conjugated monoclonal antibodies to track the leukocytes within and outside of vascular beds. Here we describe a method used to study neutrophil transendothelial migration and blood-brain barrier permeability in a mouse model of herpes simplex virus I (HSV) encephalitis.
Assuntos
Barreira Hematoencefálica , Modelos Animais de Doenças , Encefalite por Herpes Simples , Microscopia Intravital , Microscopia de Fluorescência por Excitação Multifotônica , Neutrófilos , Migração Transendotelial e Transepitelial , Animais , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/virologia , Barreira Hematoencefálica/patologia , Camundongos , Microscopia Intravital/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Neutrófilos/metabolismo , Encefalite por Herpes Simples/patologia , Encefalite por Herpes Simples/virologia , Encefalite por Herpes Simples/metabolismo , Herpesvirus Humano 1/fisiologia , PermeabilidadeRESUMO
Viral myocarditis, an inflammatory disease of the heart, causes significant morbidity and mortality. Type I interferon (IFN)-mediated antiviral responses protect against myocarditis, but the mechanisms are poorly understood. We previously identified A Disintegrin And Metalloproteinase domain 9 (ADAM9) as an important factor in viral pathogenesis. ADAM9 is implicated in a range of human diseases, including inflammatory diseases; however, its role in viral infection is unknown. Here, we demonstrate that mice lacking ADAM9 are more susceptible to encephalomyocarditis virus (EMCV)-induced death and fail to mount a characteristic type I IFN response. This defect in type I IFN induction is specific to positive-sense, single-stranded RNA (+ ssRNA) viruses and involves melanoma differentiation-associated protein 5 (MDA5)-a key receptor for +ssRNA viruses. Mechanistically, ADAM9 binds to MDA5 and promotes its oligomerization and thereby downstream mitochondrial antiviral-signaling protein (MAVS) activation in response to EMCV RNA stimulation. Our findings identify a role for ADAM9 in the innate antiviral response, specifically MDA5-mediated IFN production, which protects against virus-induced cardiac damage, and provide a potential therapeutic target for treatment of viral myocarditis.
Assuntos
Proteínas ADAM , Infecções por Cardiovirus , Vírus da Encefalomiocardite , Imunidade Inata , Interferon Tipo I , Helicase IFIH1 Induzida por Interferon , Proteínas de Membrana , Miocardite , Animais , Camundongos , Proteínas ADAM/metabolismo , Proteínas ADAM/genética , Proteínas ADAM/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Infecções por Cardiovirus/imunologia , Infecções por Cardiovirus/virologia , Vírus da Encefalomiocardite/imunologia , Células HEK293 , Interferon Tipo I/metabolismo , Interferon Tipo I/imunologia , Helicase IFIH1 Induzida por Interferon/metabolismo , Helicase IFIH1 Induzida por Interferon/genética , Helicase IFIH1 Induzida por Interferon/imunologia , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocardite/imunologia , Miocardite/virologia , Transdução de Sinais/imunologiaRESUMO
Interleukin-1 receptor-associated kinases (IRAKs) -4, -2, and -1 are involved in transducing signals from Toll-like receptors (TLRs) via the adaptor myeloid differentiation primary-response protein 88 (MYD88). How MYD88/IRAK4/2/1 complexes are formed, their redundancies, and potential non-enzymatic roles are subjects of debate. Here, we examine the hierarchical requirements for IRAK proteins in the context of TLR4 activation and confirmed that the kinase activity of IRAK4 is essential for MYD88 signaling. Surprisingly, the IRAK4 scaffold is required for activation of the E3 ubiquitin ligase TNF receptor-associated factor 6 (TRAF6) by both MYD88 and TIR domain-containing adaptor protein inducing IFN-ß (TRIF), a unique adaptation in the TLR4 response. IRAK4 scaffold is, therefore, essential in integrating MYD88 and TRIF in TLR4 signaling.
Assuntos
Quinases Associadas a Receptores de Interleucina-1 , Fator 88 de Diferenciação Mieloide , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Humanos , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Transdução de Sinais/fisiologia , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismoRESUMO
New variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to emerge, causing surges, breakthrough infections, and devastating losses-underscoring the importance of identifying SARS-CoV-2 antivirals. A simple, accessible human cell culture model permissive to SARS-CoV-2 variants is critical for identifying and assessing antivirals in a high-throughput manner. Although human alveolar A549 cells are a valuable model for studying respiratory virus infections, they lack two essential host factors for SARS-CoV-2 infection: angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). SARS-CoV-2 uses the ACE2 receptor for viral entry and TMPRSS2 to prime the SARS-CoV-2 spike protein, both of which are negligibly expressed in A549 cells. Here, we report the generation of a suitable human cell line for SARS-CoV-2 studies by transducing human ACE2 and TMPRSS2 into A549 cells. We show that subclones highly expressing ACE2 and TMPRSS2 ("ACE2plus" and the subclone "ACE2plusC3") are susceptible to infection with SARS-CoV-2, including the delta and omicron variants. These subclones express more ACE2 and TMPRSS2 transcripts than existing commercial A549 cells engineered to express ACE2 and TMPRSS2. Additionally, the antiviral drugs EIDD-1931, remdesivir, nirmatrelvir, and nelfinavir strongly inhibit SARS-CoV-2 variants in our infection model. Our data show that ACE2plusC3 cells are highly permissive to SARS-CoV-2 infection and can be used to identify anti-SARS-CoV-2 drugs.
Assuntos
Enzima de Conversão de Angiotensina 2 , COVID-19 , Células A549 , Enzima de Conversão de Angiotensina 2/genética , Antivirais/farmacologia , Humanos , Peptidil Dipeptidase A/metabolismo , SARS-CoV-2/genética , Serina Endopeptidases/genética , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
Plasmodium falciparum (P. falciparum) induces trained innate immune responses in vitro, where initial stimulation of adherent PBMCs with P. falciparum-infected RBCs (iRBCs) results in hyperresponsiveness to subsequent ligation of TLR2. This response correlates with the presence of T and B lymphocytes in adherent PBMCs, suggesting that innate immune training is partially due to adaptive immunity. We found that T cell-depleted PBMCs and purified monocytes alone did not elicit hyperproduction of IL-6 and TNF-α under training conditions. Analysis of P. falciparum-trained PBMCs showed that DCs did not develop under control conditions, and IL-6 and TNF-α were primarily produced by monocytes and DCs. Transwell experiments isolating purified monocytes from either PBMCs or purified CD4+ T cells, but allowing diffusion of secreted proteins, enabled monocytes trained with iRBCs to hyperproduce IL-6 and TNF-α after TLR restimulation. Purified monocytes stimulated with IFN-γ hyperproduced IL-6 and TNF-α, whereas blockade of IFN-γ in P. falciparum-trained PBMCs inhibited trained responses. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) on monocytes from patients with malaria showed persistently open chromatin at genes that appeared to be trained in vitro. Together, these findings indicate that the trained immune response of monocytes to P. falciparum is not completely cell intrinsic but depends on soluble signals from lymphocytes.
Assuntos
Linfócitos , Malária Falciparum , Monócitos , Cromatina , Humanos , Interleucina-6/genética , Linfócitos/imunologia , Malária Falciparum/imunologia , Monócitos/imunologia , Plasmodium falciparum , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Recurrent respiratory syncytial virus (RSV) infection requiring hospitalization is rare and the underlying mechanism is unknown. We aimed to determine the role of CD14-mediated immunity in the pathogenesis of recurrent RSV infection. METHODS: We performed genotyping and longitudinal immunophenotyping of the first patient with a genetic CD14 deficiency who developed recurrent RSV infection. We analyzed gene expression profiles and interleukin (IL)-6 production by patient peripheral blood mononuclear cells in response to RSV pre- and post-fusion (F) protein. We generated CD14-deficient human nasal epithelial cells cultured at air-liquid interface (HNEC-ALI) of patient-derived cells and after CRISPR-based gene editing of control cells. We analyzed viral replication upon RSV infection. RESULTS: Sanger sequencing revealed a homozygous single-nucleotide deletion in CD14, resulting in absence of the CD14 protein in the index patient. In vitro, viral replication was similar in wild-type and CD14-/- HNEC-ALI. Loss of immune cell CD14 led to impaired cytokine and chemokine responses to RSV pre- and post-F protein, characterized by absence of IL-6 production. CONCLUSIONS: We report an association of recurrent RSV bronchiolitis with a loss of CD14 function in immune cells. Lack of CD14 function led to defective immune responses to RSV pre- and post-F protein without a change in viral replication.
Assuntos
Infecções por Vírus Respiratório Sincicial , Citocinas , Humanos , Leucócitos Mononucleares/metabolismo , Receptores de Lipopolissacarídeos/deficiência , Vírus Sincicial Respiratório HumanoRESUMO
SARS coronavirus 1 (SARS-CoV-1) causes a respiratory infection that can lead to acute respiratory distress characterized by inflammation and high levels of cytokines in the lung tissue. In this study we constructed a herpes simplex virus 1 replication-defective mutant vector expressing SARS-CoV-1 spike protein as a potential vaccine vector and to probe the effects of spike protein on host cells. The spike protein expressed from this vector is functional in that it localizes to the surface of infected cells and induces fusion of ACE2-expressing cells. In immunized mice, the recombinant vector induced antibodies that bind to spike protein in an ELISA assay and that show neutralizing activity. The spike protein expressed from this vector can induce the expression of cytokines in an ACE2-independent, MyD88-dependent process. These results argue that the SARS-CoV-1 spike protein intrinsically activates signaling pathways that induce cytokines and contribute directly to the inflammatory process of SARS.
Assuntos
Anticorpos Neutralizantes/imunologia , Herpesvirus Humano 1/genética , Imunidade Inata , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Fusão Celular , Linhagem Celular , Citocinas/imunologia , Vetores Genéticos , Humanos , Camundongos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Transdução de Sinais , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas Virais/imunologiaRESUMO
Herpes simplex virus (HSV)-1 encephalitis has significant morbidity partly because of an over-exuberant immune response characterized by leukocyte infiltration into the brain and increased blood-brain barrier (BBB) permeability. Determining the role of specific leukocyte subsets and the factors that mediate their recruitment into the brain is critical to developing targeted immune therapies. In a murine model, we find that the chemokines CXCL1 and CCL2 are induced in the brain following HSV-1 infection. Ccr2 (CCL2 receptor)-deficient mice have reduced monocyte recruitment, uncontrolled viral replication, and increased morbidity. Contrastingly, Cxcr2 (CXCL1 receptor)-deficient mice exhibit markedly reduced neutrophil recruitment, BBB permeability, and morbidity, without influencing viral load. CXCL1 is produced by astrocytes in response to HSV-1 and by astrocytes and neurons in response to IL-1α, and it is the critical ligand required for neutrophil transendothelial migration, which correlates with BBB breakdown. Thus, the CXCL1-CXCR2 axis represents an attractive therapeutic target to limit neutrophil-mediated morbidity in HSV-1 encephalitis.
Assuntos
Astrócitos/metabolismo , Barreira Hematoencefálica/patologia , Quimiocina CXCL1/metabolismo , Encefalite Viral/patologia , Neurônios/metabolismo , Neutrófilos/patologia , Migração Transendotelial e Transepitelial , Aciclovir/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Barreira Hematoencefálica/efeitos dos fármacos , Feminino , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 1/fisiologia , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Permeabilidade , Receptores de Interleucina-8B/deficiência , Receptores de Interleucina-8B/metabolismo , Migração Transendotelial e Transepitelial/efeitos dos fármacosRESUMO
Alcoholic liver disease results from a combination of immune and metabolic pathogenic events. In addition to liver injury, chronic alcohol consumption also causes adipose tissue inflammation. The specific immune mechanisms that drive this process are unknown. Here, we sought to determine the role of the innate immune receptor Toll-like receptor 4 (TLR4) in alcohol-induced adipose tissue inflammation. Using a model of chronic, multiple-binge alcohol exposure, we showed that alcohol-mediated accumulation of proinflammatory adipose tissue macrophages was absent in global TLR4 knockout mice. Proinflammatory macrophage accumulation did not depend on macrophage TLR4 expression; LysMCre-driven deletion of Tlr4 from myeloid cells did not affect circulating endotoxin or the accumulation of M1 macrophages in adipose tissue following alcohol exposure. Proinflammatory cytokine/chemokine production in the adipose stromal vascular fraction also occurred independently of TLR4. Finally, the levels of other adipose immune cells, such as dendritic cells, neutrophils, B cells, and T cells, were modulated by chronic, multiple-binge alcohol and the presence of TLR4. Together, these data indicate that TLR4 expression on cells, other than myeloid cells, is important for the alcohol-induced increase in proinflammatory adipose tissue macrophages.
Assuntos
Adipócitos/efeitos dos fármacos , Tecido Adiposo/efeitos dos fármacos , Etanol/farmacologia , Macrófagos/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismo , Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Animais , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Macrófagos/metabolismo , Camundongos Transgênicos , Obesidade/tratamento farmacológico , Obesidade/metabolismoRESUMO
Influenza infection increases the incidence of myocardial infarction but the reason is unknown. Platelets mediate vascular occlusion through thrombotic functions but are also recognized to have immunomodulatory activity. To determine if platelet processes are activated during influenza infection, we collected blood from 18 patients with acute influenza infection. Microscopy reveals activated platelets, many containing viral particles and extracellular-DNA associated with platelets. To understand the mechanism, we isolate human platelets and treat them with influenza A virus. Viral-engulfment leads to C3 release from platelets as a function of TLR7 and C3 leads to neutrophil-DNA release and aggregation. TLR7 specificity is confirmed in murine models lacking the receptor, and platelet depletion models support platelet-mediated C3 and neutrophil-DNA release post-influenza infection. These findings demonstrate that the initial intrinsic defense against influenza is mediated by platelet-neutrophil cross-communication that tightly regulates host immune and complement responses but can also lead to thrombotic vascular occlusion.
Assuntos
Plaquetas/imunologia , Vírus da Influenza A/imunologia , Influenza Humana/imunologia , Ativação Plaquetária/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Complemento C3/imunologia , Complemento C3/metabolismo , Modelos Animais de Doenças , Armadilhas Extracelulares/imunologia , Armadilhas Extracelulares/metabolismo , Feminino , Humanos , Vírus da Influenza A/isolamento & purificação , Influenza Humana/sangue , Influenza Humana/virologia , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Neutrófilos/imunologia , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/imunologia , Receptor 7 Toll-Like/metabolismoRESUMO
Encephalomyocarditis virus (EMCV) is a picornavirus that produces lytic infections in murine and human cells. Employing a genome-wide CRISPR-Cas9 knockout screen to find host factors required for EMCV infection, we identified a role for ADAM9 in EMCV infection. CRISPR-mediated deletion of ADAM9 in multiple human cell lines rendered the cells highly resistant to EMCV infection and cell death. Primary fibroblasts from ADAM9 KO mice were also strongly resistant to EMCV infection and cell death. In contrast, ADAM9 KO and WT cells were equally susceptible to infection with other viruses, including the picornavirus Coxsackie virus B. ADAM9 KO cells failed to produce viral progeny when incubated with EMCV. However, bypassing EMCV entry into cells through delivery of viral RNA directly to the cytosol yielded infectious EMCV virions from ADAM9 KO cells, suggesting that ADAM9 is not required for EMCV replication post-entry. These findings establish that ADAM9 is required for the early stage of EMCV infection, likely for virus entry or viral genome delivery to the cytosol.IMPORTANCE Viral myocarditis is a leading cause of death in the United States, contributing to numerous unexplained deaths in people ≤35 years old. Enteroviruses contribute to many cases of human myocarditis. Encephalomyocarditis virus (EMCV) infection causes viral myocarditis in rodent models, but its receptor requirements have not been fully identified. CRISPR-Cas9 screens can identify host dependency factors essential for EMCV infection and enhance our understanding of key events that follow viral infection, potentially leading to new strategies for preventing viral myocarditis. Using a CRISPR-Cas9 screen, we identified adisintegrin and metalloproteinase 9 domain (ADAM9) as a major factor required for the early stages of EMCV infection in both human and murine infection.
Assuntos
Proteínas ADAM/metabolismo , Infecções por Cardiovirus/genética , Resistência à Doença , Vírus da Encefalomiocardite/crescimento & desenvolvimento , Proteínas de Membrana/metabolismo , Animais , Linhagem Celular , Técnicas de Inativação de Genes , Testes Genéticos , Humanos , Camundongos , Camundongos Knockout , Modelos BiológicosRESUMO
Viral infections associate with disease risk and select families of viruses encode miRNAs that control an efficient viral cycle. The association of viral miRNA expression with disease in a large human population has not been previously explored. We sequenced plasma RNA from 40 participants of the Framingham Heart Study (FHS, Offspring Cohort, Visit 8) and identified 3 viral miRNAs from 3 different human Herpesviridae. These miRNAs were mostly related to viral latency and have not been previously detected in human plasma. Viral miRNA expression was then screened in the plasma of 2763 participants of the remaining cohort utilizing high-throughput RT-qPCR. All 3 viral miRNAs associated with combinations of inflammatory or prothrombotic circulating biomarkers (sTNFRII, IL-6, sICAM1, OPG, P-selectin) but did not associate with hypertension, coronary heart disease or cancer. Using a large observational population, we demonstrate that the presence of select viral miRNAs in the human circulation associate with inflammatory biomarkers and possibly immune response, but fail to associate with overt disease. This study greatly extends smaller singular observations of viral miRNAs in the human circulation and suggests that select viral miRNAs, such as those for latency, may not impact disease manifestation.
Assuntos
Vírus de DNA/genética , Herpesviridae/genética , MicroRNAs/sangue , MicroRNAs/genética , RNA Viral/genética , Perfilação da Expressão Gênica , HumanosRESUMO
Innate immune receptors have a key role in the sensing of malaria and initiating immune responses. As a consequence of infection, systemic inflammation emerges and is directly related to signs and symptoms during acute disease. We have previously reported that plasmodial DNA is the primary driver of systemic inflammation in malaria, both within the phagolysosome and in the cytosol of effector cells. In this article, we demonstrate that Plasmodium falciparum genomic DNA delivered to the cytosol of human monocytes binds and activates cyclic GMP-AMP synthase (cGAS). Activated cGAS synthesizes 2'3'-cGAMP, which we subsequently can detect using liquid chromatography-tandem mass spectrometry. 2'3'-cGAMP acts as a second messenger for STING activation and triggers TBK1/IRF3 activation, resulting in type I IFN production in human cells. This induction of type I IFN was independent of IFI16. Access of DNA to the cytosolic compartment is mediated by hemozoin, because incubation of purified malaria pigment with DNase abrogated IFN-ß induction. Collectively, these observations implicate cGAS as an important cytosolic sensor of P. falciparum genomic DNA and reveal the role of the cGAS/STING pathway in the induction of type I IFN in response to malaria parasites.
Assuntos
DNA de Protozoário/metabolismo , Interferon Tipo I/metabolismo , Nucleotidiltransferases/metabolismo , Plasmodium falciparum/genética , Adolescente , Adulto , Células Cultivadas , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Feminino , Humanos , Fator Regulador 3 de Interferon/metabolismo , Malária Falciparum/metabolismo , Malária Falciparum/parasitologia , Masculino , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Nucleotídeos Cíclicos/metabolismo , Fosforilação , Transdução de Sinais , Adulto JovemRESUMO
Innate immune responses play a major role in the control of herpes simplex virus (HSV) infections, and a multiplicity of mechanisms have emerged as a result of human evolution to sense and respond to HSV infections. HSV in turn has evolved a number of ways to evade immune detection and to blunt human innate immune responses. In this review, we summarize the major host innate immune mechanisms and the HSV evasion mechanisms that have evolved. We further discuss how disease can result if this equilibrium between virus and host response is disrupted.
Assuntos
Herpes Simples/imunologia , Herpes Simples/virologia , Herpesvirus Humano 1/fisiologia , Imunidade Inata , Animais , Interações Hospedeiro-Patógeno/imunologia , Humanos , Evasão da Resposta ImuneRESUMO
Bacterial sepsis involves a complex interaction between the host immune response and bacterial LPS. LPS binds Toll-like receptor (TLR) 4, which leads to the release of proinflammatory cytokines that are essential for a potent innate immune response against pathogens. The innate immune system is tightly regulated, as excessive inflammation can lead to organ failure and death. MicroRNAs have recently emerged as important regulators of the innate immune system. Here we determined the function of miR-718, which is conserved across mammals and overlaps with the 5' UTR of the interleukin 1 receptor-associated kinase (IRAK1) gene. As IRAK1 is a key component of innate immune signaling pathways that are downstream of most TLRs, we hypothesized that miR-718 helps regulate the innate immune response. Activation of TLR4, but not TLR3, induced the expression of miR-718 in macrophages. miR-718 expression was also induced in the spleens of mice upon LPS injection. miR-718 modulates PI3K/Akt signaling by directly down-regulating phosphatase and tensin homolog (PTEN), thereby promoting phosphorylation of Akt, which leads to a decrease in proinflammatory cytokine production. Phosphorylated Akt induces let-7e expression, which, in turn, down-regulates TLR4 and further diminishes TLR4-mediated proinflammatory signals. Decreased miR-718 expression is associated with bacterial burden during Neisseria gonorrhoeae infection and alters the infection dynamics of N. gonorrhoeae in vitro Furthermore, miR-718 regulates the induction of LPS tolerance in macrophages. We propose a role for miR-718 in controlling TLR4 signaling and inflammatory cytokine signaling through a negative feedback regulation loop involving down-regulation of TLR4, IRAK1, and NF-κB.
Assuntos
Regiões 5' não Traduzidas , Citocinas/metabolismo , Macrófagos/metabolismo , MicroRNAs/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Transdução de Sinais , Animais , Citocinas/genética , Gonorreia/genética , Gonorreia/metabolismo , Humanos , Quinases Associadas a Receptores de Interleucina-1/genética , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Macrófagos/microbiologia , Macrófagos/patologia , Camundongos , Camundongos Knockout , MicroRNAs/genética , Neisseria gonorrhoeae/metabolismo , PTEN Fosfo-Hidrolase/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismoRESUMO
Fibrosis, driven by inflammation, marks the transition from benign to progressive stages of chronic liver diseases. Although inflammation promotes fibrogenesis, it is not known whether other events, such as hepatocyte death, are required for the development of fibrosis. Interferon regulatory factor 3 (IRF3) regulates hepatocyte apoptosis and production of type I IFNs. In the liver, IRF3 is activated via Toll-like receptor 4 (TLR4) signaling or the endoplasmic reticulum (ER) adapter, stimulator of interferon genes (STING). We hypothesized that IRF3-mediated hepatocyte death is an independent determinant of chemically induced liver fibrogenesis. To test this, we performed acute or chronic CCl4 administration to WT and IRF3-, Toll/Interleukin-1R (TIR) domain-containing adapter-inducing interferon-ß (TRIF)-, TRIF-related adaptor molecule (TRAM)-, and STING-deficient mice. We report that acute CCl4 administration to WT mice resulted in early ER stress, activation of IRF3, and type I IFNs, followed by hepatocyte apoptosis and liver injury, accompanied by liver fibrosis upon repeated administration of CCl4 Deficiency of IRF3 or STING prevented hepatocyte death and fibrosis both in acute or chronic CCl4 In contrast, mice deficient in type I IFN receptors or in TLR4 signaling adaptors, TRAM or TRIF, upstream of IRF3, were not protected from hepatocyte death and/or fibrosis, suggesting that the pro-apoptotic role of IRF3 is independent of TLR signaling in fibrosis. Hepatocyte death is required for liver fibrosis with causal involvement of STING and IRF3. Thus, our results identify that IRF3, by its association with STING in the presence of ER stress, couples hepatocyte apoptosis with liver fibrosis and indicate that innate immune signaling regulates outcomes of liver fibrosis via modulation of hepatocyte death in the liver.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/etiologia , Estresse do Retículo Endoplasmático , Hepatócitos/patologia , Fator Regulador 3 de Interferon/fisiologia , Cirrose Hepática/etiologia , Proteínas de Membrana/fisiologia , Receptor de Interferon alfa e beta/fisiologia , Animais , Tetracloreto de Carbono/toxicidade , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Feminino , Hepatócitos/metabolismo , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismoRESUMO
Induction of type I interferon (IFN) in response to microbial pathogens depends on a conserved cGAS-STING signaling pathway. The presence of DNA in the cytoplasm activates cGAS, while STING is activated by cyclic dinucleotides (cdNs) produced by cGAS or from bacterial origins. Here, we show that Group B Streptococcus (GBS) induces IFN-ß production almost exclusively through cGAS-STING-dependent recognition of bacterial DNA. However, we find that GBS expresses an ectonucleotidase, CdnP, which hydrolyzes extracellular bacterial cyclic-di-AMP. Inactivation of CdnP leads to c-di-AMP accumulation outside the bacteria and increased IFN-ß production. Higher IFN-ß levels in vivo increase GBS killing by the host. The IFN-ß overproduction observed in the absence of CdnP is due to the cumulative effect of DNA sensing by cGAS and STING-dependent sensing of c-di-AMP. These findings describe the importance of a bacterial c-di-AMP ectonucleotidase and suggest a direct bacterial mechanism that dampens activation of the cGAS-STING axis.
Assuntos
Fosfatos de Dinucleosídeos/metabolismo , Evasão da Resposta Imune , Interferon Tipo I/metabolismo , Proteínas de Membrana/metabolismo , Pirofosfatases/metabolismo , Streptococcus agalactiae/imunologia , Streptococcus agalactiae/metabolismo , Biotransformação , Streptococcus agalactiae/enzimologiaRESUMO
The innate immune system is the first line of defense against Neisseria gonorrhoeae (GC). Exposure of cells to GC lipooligosaccharides induces a strong immune response, leading to type I interferon (IFN) production via TLR4/MD-2. In addition to living freely in the extracellular space, GC can invade the cytoplasm to evade detection and elimination. Double-stranded DNA introduced into the cytosol binds and activates the enzyme cyclic-GMP-AMP synthase (cGAS), which produces 2'3'-cGAMP and triggers STING/TBK-1/IRF3 activation, resulting in type I IFN expression. Here, we reveal a cytosolic response to GC DNA that also contributes to type I IFN induction. We demonstrate that complete IFN-ß induction by live GC depends on both cGAS and TLR4. Type I IFN is detrimental to the host, and dysregulation of iron homeostasis genes may explain lower bacteria survival in cGAS(-/-) and TLR4(-/-) cells. Collectively, these observations reveal cooperation between TLRs and cGAS in immunity to GC infection.
Assuntos
Interferon Tipo I/metabolismo , Neisseria gonorrhoeae/fisiologia , Nucleotidiltransferases/metabolismo , Receptor 4 Toll-Like/metabolismo , Sistemas de Secreção Bacterianos , Linhagem Celular , DNA Bacteriano/metabolismo , Humanos , Ferro/metabolismo , Proteínas de Membrana/metabolismo , Viabilidade Microbiana , Nucleotídeos Cíclicos/metabolismo , TransfecçãoRESUMO
Innate sensing of nucleic acids lies at the heart of antiviral immunity. During viral infection, dying cells may also release nucleic acids into the tissue microenvironment. It is unknown what effect such host signals have on the quality or duration of the immune response to viruses. In this study, we uncovered an immune-regulatory pathway that tempers the intensity of the host response to influenza A virus (IAV) infection. We found that host-derived DNA accumulates in the lung microenvironment during IAV infection. Ablation of DNA in the lung resulted in increased mortality, increased cellular recruitment, and increased inflammation following IAV challenge. The released DNA, in turn, was sensed by the DNA receptor absent in melanoma 2. Aim2(-/-) mice showed similarly exaggerated immune responses to IAV. Taken together, our results identify a novel mechanism of cross-talk between pathogen- and damage-associated molecular pattern-sensing pathways, wherein sensing of host-derived DNA limits immune-mediated damage to infected tissues.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , DNA/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Infecções por Orthomyxoviridae/imunologia , Animais , Microambiente Celular/imunologia , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata/imunologia , Inflamação/imunologia , Pulmão/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Streptococcus pyogenes, also known as Group A Streptococcus (GAS), is an important human bacterial pathogen that can cause invasive infections. Once it colonizes its exclusively human host, GAS needs to surmount numerous innate immune defense mechanisms, including opsonization by complement and consequent phagocytosis. Several strains of GAS bind to human-specific complement inhibitors, C4b-binding protein (C4BP) and/or Factor H (FH), to curtail complement C3 (a critical opsonin) deposition. This results in diminished activation of phagocytes and clearance of GAS that may lead to the host being unable to limit the infection. Herein we describe the course of GAS infection in three human complement inhibitor transgenic (tg) mouse models that examined each inhibitor (human C4BP or FH) alone, or the two inhibitors together (C4BPxFH or 'double' tg). GAS infection with strains that bound C4BP and FH resulted in enhanced mortality in each of the three transgenic mouse models compared to infection in wild type mice. In addition, GAS manifested increased virulence in C4BPxFH mice: higher organism burdens and greater elevations of pro-inflammatory cytokines and they died earlier than single transgenic or wt controls. The effects of hu-C4BP and hu-FH were specific for GAS strains that bound these inhibitors because strains that did not bind the inhibitors showed reduced virulence in the 'double' tg mice compared to strains that did bind; mortality was also similar in wild-type and C4BPxFH mice infected by non-binding GAS. Our findings emphasize the importance of binding of complement inhibitors to GAS that results in impaired opsonization and phagocytic killing, which translates to enhanced virulence in a humanized whole animal model. This novel hu-C4BPxFH tg model may prove invaluable in studies of GAS pathogenesis and for developing vaccines and therapeutics that rely on human complement activation for efficacy.