RESUMO
Age-related microangiopathy, also known as small vessel disease (SVD), causes damage to the brain, retina, liver, and kidney. Based on the DNA damage theory of aging, we reasoned that genomic instability may underlie an SVD caused by dominant C-terminal variants in TREX1, the most abundant 3'-5' DNA exonuclease in mammals. C-terminal TREX1 variants cause an adult-onset SVD known as retinal vasculopathy with cerebral leukoencephalopathy (RVCL or RVCL-S). In RVCL, an aberrant, C-terminally truncated TREX1 mislocalizes to the nucleus due to deletion of its ER-anchoring domain. Since RVCL pathology mimics that of radiation injury, we reasoned that nuclear TREX1 would cause DNA damage. Here, we show that RVCL-associated TREX1 variants trigger DNA damage in humans, mice, and Drosophila, and that cells expressing RVCL mutant TREX1 are more vulnerable to DNA damage induced by chemotherapy and cytokines that up-regulate TREX1, leading to depletion of TREX1-high cells in RVCL mice. RVCL-associated TREX1 mutants inhibit homology-directed repair (HDR), causing DNA deletions and vulnerablility to PARP inhibitors. In women with RVCL, we observe early-onset breast cancer, similar to patients with BRCA1/2 variants. Our results provide a mechanistic basis linking aberrant TREX1 activity to the DNA damage theory of aging, premature senescence, and microvascular disease.
Assuntos
Dano ao DNA , Exodesoxirribonucleases , Fosfoproteínas , Animais , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Humanos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Camundongos , Reparo de DNA por Recombinação , Fenótipo , Mutação , Drosophila/genética , Envelhecimento/genética , Envelhecimento/metabolismo , Feminino , Drosophila melanogaster/genética , Masculino , Doenças Retinianas , Doenças Vasculares , Doenças Desmielinizantes Hereditárias do Sistema Nervoso CentralRESUMO
Stimulator of interferon genes (STING) is a sensor of cyclic dinucleotides including cyclic GMP-AMP, which is produced by cyclic GMP-AMP synthase (cGAS) in response to cytosolic DNA. The cGAS-STING signaling pathway regulates both innate and adaptive immune responses, as well as fundamental cellular functions such as autophagy, senescence, and apoptosis. Mutations leading to constitutive activation of STING cause devastating human diseases. Thus, the cGAS-STING pathway is of great interest because of its role in diverse cellular processes and because of the potential therapeutic implications of targeting cGAS and STING. Here, we review molecular and cellular mechanisms of STING signaling, and we propose a framework for understanding the immunological and other cellular functions of STING in the context of disease.
Assuntos
Nucleotidiltransferases , Transdução de Sinais , Humanos , Transdução de Sinais/fisiologia , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Inflamação/metabolismo , DNA/metabolismo , Citosol/metabolismo , Imunidade InataRESUMO
STING gain-of-function mutations cause STING-associated vasculopathy with onset in infancy (SAVI) in humans, a disease characterized by spontaneous lung inflammation and fibrosis. Mice with STING gain-of-function mutations (SAVI mice) develop αß T cell-dependent lung disease and also lack lymph nodes. Although SAVI has been regarded as a type I interferonopathy, the relative contributions of the three interferon receptors are incompletely understood. Here, we show that STING gain of function led to upregulation of IFN-γ-induced chemokines in the lungs of SAVI mice and that deletion of the type II IFN receptor (IFNGR1), but not the type I IFN receptor (IFNAR1) or type III IFN receptor (IFNλR1), ameliorated lung disease and restored lymph node development in SAVI mice. Furthermore, deletion of IFNGR1, but not IFNAR1 or IFNλR1, corrected the ratio of effector to Tregs in SAVI mice and in mixed bone marrow chimeric mice. Finally, cultured SAVI mouse macrophages were hyperresponsive to IFN-γ, but not IFN-ß, in terms of Cxcl9 upregulation and cell activation. These results demonstrate that IFNGR1 plays a major role in autoinflammation and immune dysregulation mediated by STING gain of function.
Assuntos
Pneumopatias , Doenças Vasculares , Animais , Mutação com Ganho de Função , Humanos , Pulmão , Proteínas de Membrana/genética , Camundongos , Linfócitos T , Doenças Vasculares/genéticaRESUMO
T follicular helper cells (TFH) participate in germinal center (GC) development and are necessary for B cell production of high-affinity, isotype-switched antibodies. In a forward genetic screen, we identified a missense mutation in Prkd2, encoding the serine/threonine kinase protein kinase D2, which caused elevated titers of immunoglobulin E (IgE) in the serum. Subsequent analysis of serum antibodies in mice with a targeted null mutation of Prkd2 demonstrated polyclonal hypergammaglobulinemia of IgE, IgG1, and IgA isotypes, which was exacerbated by the T cell-dependent humoral response to immunization. GC formation and GC B cells were increased in Prkd2-/- spleens. These effects were the result of excessive cell-autonomous TFH development caused by unrestricted Bcl6 nuclear translocation in Prkd2-/- CD4+ T cells. Prkd2 directly binds to Bcl6, and Prkd2-dependent phosphorylation of Bcl6 is necessary to constrain Bcl6 to the cytoplasm, thereby limiting TFH development. In response to immunization, Bcl6 repressed Prkd2 expression in CD4+ T cells, thereby committing them to TFH development. Thus, Prkd2 and Bcl6 form a mutually inhibitory positive feedback loop that controls the stable transition from naïve CD4+ T cells to TFH during the adaptive immune response.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Proteínas Quinases/imunologia , Proteínas Proto-Oncogênicas c-bcl-6/imunologia , Animais , Linfócitos B/imunologia , Transplante de Medula Óssea , Diferenciação Celular , Feminino , Centro Germinativo/imunologia , Células HEK293 , Humanos , Imunoglobulinas/sangue , Imunoterapia Adotiva , Masculino , Camundongos Transgênicos , Mutação , Proteína Quinase D2 , Proteínas Quinases/genéticaRESUMO
BACKGROUND: Zika virus (ZIKV) has become a global concern because infection of pregnant mothers was linked to congenital birth defects. Zika virus is unique from other flaviviruses, because it is transmitted vertically and sexually in addition to by mosquito vectors. Prior studies in mice, nonhuman primates, and humans have shown that ZIKV targets the testis in males, resulting in persistent infection and oligospermia. However, its effects on the corresponding female gonads have not been evaluated. METHODS: In this study, we assessed the effects of ZIKV on the ovary in nonpregnant mice. RESULTS: During the acute phase, ZIKV productively infected the ovary causing accumulation of CD4+ and virus-specific CD8+ T cells. T cells protected against ZIKV infection in the ovary, as higher viral burden was measured in CD8-/- and TCRßδ-/- mice. Increased cell death and tissue inflammation in the ovary was observed during the acute phase of infection, but this normalized over time. CONCLUSIONS: In contrast to that observed with males, minimal persistence and no long-term consequences of ZIKV infection on ovarian follicular reserve or fertility were demonstrated in this model. Thus, although ZIKV replicates in cells of the ovary and causes acute oophoritis, there is rapid resolution and no long-term effects on fertility, at least in mice.
Assuntos
Fertilidade , Ooforite/fisiopatologia , Ooforite/virologia , Infecção por Zika virus/fisiopatologia , Infecção por Zika virus/virologia , Zika virus/fisiologia , Animais , Biomarcadores , Modelos Animais de Doenças , Feminino , Infertilidade Feminina/etiologia , Camundongos , Camundongos Knockout , Ooforite/complicações , Ooforite/patologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Carga Viral , Tropismo Viral , Infecção por Zika virus/complicações , Infecção por Zika virus/patologiaRESUMO
Arthritogenic alphaviruses comprise a group of enveloped RNA viruses that are transmitted to humans by mosquitoes and cause debilitating acute and chronic musculoskeletal disease 1 . The host factors required for alphavirus entry remain poorly characterized 2 . Here we use a genome-wide CRISPR-Cas9-based screen to identify the cell adhesion molecule Mxra8 as an entry mediator for multiple emerging arthritogenic alphaviruses, including chikungunya, Ross River, Mayaro and O'nyong nyong viruses. Gene editing of mouse Mxra8 or human MXRA8 resulted in reduced levels of viral infection of cells and, reciprocally, ectopic expression of these genes resulted in increased infection. Mxra8 bound directly to chikungunya virus particles and enhanced virus attachment and internalization into cells. Consistent with these findings, Mxra8-Fc fusion protein or anti-Mxra8 monoclonal antibodies blocked chikungunya virus infection in multiple cell types, including primary human synovial fibroblasts, osteoblasts, chondrocytes and skeletal muscle cells. Mutagenesis experiments suggest that Mxra8 binds to a surface-exposed region across the A and B domains of chikungunya virus E2 protein, which are a speculated site of attachment. Finally, administration of the Mxra8-Fc protein or anti-Mxra8 blocking antibodies to mice reduced chikungunya and O'nyong nyong virus infection as well as associated foot swelling. Pharmacological targeting of Mxra8 could form a strategy for mitigating infection and disease by multiple arthritogenic alphaviruses.
Assuntos
Vírus Chikungunya/metabolismo , Imunoglobulinas/metabolismo , Proteínas de Membrana/metabolismo , Vírus O'nyong-nyong/metabolismo , Receptores Virais/metabolismo , Células 3T3 , Animais , Anticorpos Bloqueadores/imunologia , Sistemas CRISPR-Cas/genética , Vírus Chikungunya/patogenicidade , Condrócitos/metabolismo , Fibroblastos/metabolismo , Humanos , Imunoglobulinas/imunologia , Masculino , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/imunologia , Camundongos , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Vírus O'nyong-nyong/patogenicidade , Osteoblastos/metabolismo , Receptores Fc/metabolismo , Receptores Virais/deficiência , Receptores Virais/genéticaRESUMO
Although the outcome of flavivirus infection can vary from asymptomatic to lethal, environmental factors modulating disease severity are poorly defined. Here, we observed increased susceptibility of mice to severe West Nile (WNV), Dengue, and Zika virus infections after treatment with oral antibiotics (Abx) that depleted the gut microbiota. Abx treatment impaired the development of optimal T cell responses, with decreased levels of WNV-specific CD8+ T cells associated with increased infection and immunopathology. Abx treatments that resulted in enhanced WNV susceptibility generated changes in the overall structure of the gut bacterial community and in the abundance of specific bacterial taxa. As little as 3 days of treatment with ampicillin was sufficient to alter host immunity and WNV outcome. Our results identify oral Abx therapy as a potential environmental determinant of systemic viral disease, and they raise the possibility that perturbation of the gut microbiota may have deleterious consequences for subsequent flavivirus infections.
Assuntos
Antibacterianos/efeitos adversos , Flavivirus/isolamento & purificação , Infecção por Zika virus/tratamento farmacológico , Administração Oral , Aedes , Ampicilina/efeitos adversos , Ampicilina/farmacologia , Animais , Antibacterianos/farmacologia , Ceco/efeitos dos fármacos , Ceco/microbiologia , Chlorocebus aethiops , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microbiota/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Células Vero , Infecção por Zika virus/imunologia , Infecção por Zika virus/microbiologia , Infecção por Zika virus/patologiaRESUMO
The innate immune system protects cells against viral pathogens in part through the autocrine and paracrine actions of alpha/beta interferon (IFN-α/ß) (type I), IFN-γ (type II), and IFN-λ (type III). The transcription factor interferon regulatory factor 1 (IRF-1) has a demonstrated role in shaping innate and adaptive antiviral immunity by inducing the expression of IFN-stimulated genes (ISGs) and mediating signals downstream of IFN-γ. Although ectopic expression experiments have suggested an inhibitory function of IRF-1 against infection of alphaviruses in cell culture, its role in vivo remains unknown. Here, we infected Irf1 -/- mice with two distantly related arthritogenic alphaviruses, chikungunya virus (CHIKV) and Ross River virus (RRV), and assessed the early antiviral functions of IRF-1 prior to induction of adaptive B and T cell responses. IRF-1 expression limited CHIKV-induced foot swelling in joint-associated tissues and prevented dissemination of CHIKV and RRV at early time points. Virological and histological analyses revealed greater infection of muscle tissues in Irf1 -/- mice than in wild-type mice. The antiviral actions of IRF-1 appeared to be independent of the induction of type I IFN or the effects of type II and III IFNs but were associated with altered local proinflammatory cytokine and chemokine responses and differential infiltration of myeloid cell subsets. Collectively, our in vivo experiments suggest that IRF-1 restricts CHIKV and RRV infection in stromal cells, especially muscle cells, and that this controls local inflammation and joint-associated swelling.IMPORTANCE Interferon regulatory factor 1 (IRF-1) is a transcription factor that regulates the expression of a broad range of antiviral host defense genes. In this study, using Irf1 -/- mice, we investigated the role of IRF-1 in modulating pathogenesis of two related arthritogenic alphaviruses, chikungunya virus and Ross River virus. Our studies show that IRF-1 controlled alphavirus replication and swelling in joint-associated tissues within days of infection. Detailed histopathological and virological analyses revealed that IRF-1 preferentially restricted CHIKV infection in cells of nonhematopoietic lineage, including muscle cells. The antiviral actions of IRF-1 resulted in decreased local inflammatory responses in joint-associated tissues, which prevented immunopathology.
RESUMO
In 2013, chikungunya virus (CHIKV) transmission was documented in the Western Hemisphere, and the virus has since spread throughout the Americas with more than 1.8 million people infected in more than 40 countries. CHIKV targets the joints, resulting in symmetric polyarthritis that clinically mimics rheumatoid arthritis and can endure for months to years. At present, no approved treatment is effective in preventing or controlling CHIKV infection or disease. We treated mice with eight different disease-modifying antirheumatic drugs and identified CLTA4-Ig (abatacept) and tofacitinib as candidate therapies based on their ability to decrease acute joint swelling. CTLA4-Ig reduced T cell accumulation in the joints of infected animals without affecting viral infection. Whereas monotherapy with CTLA4-Ig or a neutralizing anti-CHIKV human monoclonal antibody provided partial clinical improvement, therapy with both abolished swelling and markedly reduced levels of chemokines, proinflammatory cytokines, and infiltrating leukocytes. Thus, combination CTLA4-Ig and antiviral antibody therapy controls acute CHIKV infection and arthritis and may be a candidate for testing in humans.
Assuntos
Abatacepte/uso terapêutico , Antivirais/uso terapêutico , Artrite Infecciosa/tratamento farmacológico , Febre de Chikungunya/tratamento farmacológico , Animais , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/uso terapêutico , Artrite Infecciosa/virologia , Quimiocinas/imunologia , Citocinas/imunologia , Leucócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Carga ViralRESUMO
UNLABELLED: The interferon-induced transmembrane protein (IFITM) family of proteins inhibit infection of several different enveloped viruses in cell culture by virtue of their ability to restrict entry and fusion from late endosomes. As few studies have evaluated the importance of Ifitm3 in vivo in restricting viral pathogenesis, we investigated its significance as an antiviral gene against West Nile virus (WNV), an encephalitic flavivirus, in cells and mice. Ifitm3(-/-) mice were more vulnerable to lethal WNV infection, and this was associated with greater virus accumulation in peripheral organs and central nervous system tissues. As no difference in viral burden in the brain or spinal cord was observed after direct intracranial inoculation, Ifitm3 likely functions as an antiviral protein in nonneuronal cells. Consistent with this, Ifitm3(-/-) fibroblasts but not dendritic cells resulted in higher yields of WNV in multistep growth analyses. Moreover, transcomplementation experiments showed that Ifitm3 inhibited WNV infection independently of Ifitm1, Ifitm2, Ifitm5, and Ifitm6. Beyond a direct effect on viral infection in cells, analysis of the immune response in WNV-infected Ifitm3(-/-) mice showed decreases in the total number of B cells, CD4(+) T cells, and antigen-specific CD8(+) T cells. Finally, bone marrow chimera experiments demonstrated that Ifitm3 functioned in both radioresistant and radiosensitive cells, as higher levels of WNV were observed in the brain only when Ifitm3 was absent from both compartments. Our analyses suggest that Ifitm3 restricts WNV pathogenesis likely through multiple mechanisms, including the direct control of infection in subsets of cells. IMPORTANCE: As part of the mammalian host response to viral infections, hundreds of interferon-stimulated genes (ISGs) are induced. The inhibitory activity of individual ISGs varies depending on the specific cell type and viral pathogen. Among ISGs, the genes encoding interferon-induced transmembrane protein (IFITM) have been reported to inhibit multiple families of viruses in cell culture. However, few reports have evaluated the impact of IFITM genes on viral pathogenesis in vivo In this study, we characterized the antiviral activity of Ifitm3 against West Nile virus (WNV), an encephalitic flavivirus, using mice with a targeted gene deletion of Ifitm3 Based on extensive virological and immunological analyses, we determined that Ifitm3 protects mice from WNV-induced mortality by restricting virus accumulation in peripheral organs and, subsequently, in central nervous system tissues. Our data suggest that Ifitm3 restricts WNV pathogenesis by multiple mechanisms and functions in part by controlling infection in different cell types.
Assuntos
Imunidade Inata , Proteínas de Membrana/metabolismo , Febre do Nilo Ocidental/imunologia , Febre do Nilo Ocidental/patologia , Vírus do Nilo Ocidental/imunologia , Vírus do Nilo Ocidental/patogenicidade , Estruturas Animais/virologia , Animais , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Fibroblastos/imunologia , Fibroblastos/virologia , Teste de Complementação Genética , Predisposição Genética para Doença , Camundongos , Camundongos Knockout , Análise de Sobrevida , Carga ViralRESUMO
UNLABELLED: Host cells respond to viral infections by producing type I interferon (IFN), which induces the expression of hundreds of interferon-stimulated genes (ISGs). Although ISGs mediate a protective state against many pathogens, the antiviral functions of the majority of these genes have not been identified. IFITM3 is a small transmembrane ISG that restricts a broad range of viruses, including orthomyxoviruses, flaviviruses, filoviruses, and coronaviruses. Here, we show that alphavirus infection is increased in Ifitm3(-/-) and Ifitm locus deletion (Ifitm-del) fibroblasts and, reciprocally, reduced in fibroblasts transcomplemented with Ifitm3. Mechanistic studies showed that Ifitm3 did not affect viral binding or entry but inhibited pH-dependent fusion. In a murine model of chikungunya virus arthritis, Ifitm3(-/-) mice sustained greater joint swelling in the ipsilateral ankle at days 3 and 7 postinfection, and this correlated with higher levels of proinflammatory cytokines and viral burden. Flow cytometric analysis suggested that Ifitm3(-/-) macrophages from the spleen were infected at greater levels than observed in wild-type (WT) mice, results that were supported by experiments with Ifitm3(-/-) bone marrow-derived macrophages. Ifitm3(-/-) mice also were more susceptible than WT mice to lethal alphavirus infection with Venezuelan equine encephalitis virus, and this was associated with greater viral burden in multiple organs. Collectively, our data define an antiviral role for Ifitm3 in restricting infection of multiple alphaviruses. IMPORTANCE: The interferon-induced transmembrane protein 3 (IFITM3) inhibits infection of multiple families of viruses in cell culture. Compared to other viruses, much less is known about the antiviral effect of IFITM3 on alphaviruses. In this study, we characterized the antiviral activity of mouse Ifitm3 against arthritogenic and encephalitic alphaviruses using cells and animals with a targeted gene deletion of Ifitm3 as well as deficient cells transcomplemented with Ifitm3. Based on extensive virological analysis, we demonstrate greater levels of alphavirus infection and disease pathogenesis when Ifitm3 expression is absent. Our data establish an inhibitory role for Ifitm3 in controlling infection of alphaviruses.
Assuntos
Infecções por Alphavirus/imunologia , Vírus Chikungunya/imunologia , Vírus da Encefalite Equina Venezuelana/imunologia , Fatores Imunológicos/metabolismo , Proteínas de Membrana/metabolismo , Infecções por Alphavirus/patologia , Infecções por Alphavirus/virologia , Animais , Vírus Chikungunya/fisiologia , Citocinas/metabolismo , Modelos Animais de Doenças , Vírus da Encefalite Equina Venezuelana/fisiologia , Fibroblastos/imunologia , Fibroblastos/virologia , Deleção de Genes , Teste de Complementação Genética , Fatores Imunológicos/deficiência , Macrófagos/virologia , Proteínas de Membrana/deficiência , Camundongos , Camundongos Knockout , Carga Viral , Internalização do Vírus/efeitos dos fármacosRESUMO
Photosynthetic organisms synthesize carotenoids for harvesting light energy, photoprotection, and maintaining the structure and function of photosynthetic membranes. A light-sensitive, phytoene-accumulating mutant, pds1-1, was isolated in Chlamydomonas reinhardtii and found to be genetically linked to the phytoene desaturase (PDS) gene. PDS catalyzes the second step in carotenoid biosynthesis--the conversion of phytoene to ζ-carotene. Decreased accumulation of downstream colored carotenoids suggested that the pds1-1 mutant is leaky for PDS activity. A screen for enhancers of the pds1-1 mutation yielded the pds1-2 allele, which completely lacks PDS activity. A second independent null mutant (pds1-3) was identified using DNA insertional mutagenesis. Both null mutants accumulate only phytoene and no other carotenoids. All three phytoene-accumulating mutants exhibited slower growth rates and reduced plating efficiency compared to wild-type cells and white phytoene synthase mutants. Insight into amino acid residues important for PDS activity was obtained through the characterization of intragenic suppressors of pds1-2. The suppressor mutants fell into three classes: revertants of the pds1-1 point mutation, mutations that changed PDS amino acid residue Pro64 to Phe, and mutations that converted PDS residue Lys90 to Met. Characterization of pds1-2 intragenic suppressors coupled with computational structure prediction of PDS suggest that amino acids at positions 90 and 143 are in close contact in the active PDS enzyme and have important roles in its structural stability and/or activity.
Assuntos
Chlamydomonas reinhardtii/enzimologia , Chlamydomonas reinhardtii/genética , Elementos Facilitadores Genéticos/genética , Mutação , Oxirredutases/genética , Supressão Genética/genética , Alelos , Sequência de Aminoácidos , Carotenoides/metabolismo , Chlamydomonas reinhardtii/crescimento & desenvolvimento , Chlamydomonas reinhardtii/metabolismo , DNA de Plantas/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Insercional , Oxirredutases/química , Oxirredutases/metabolismo , Conformação ProteicaRESUMO
Histidine residues have been hypothesized to function as sensors of environmental pH that can trigger the activity of viral fusion proteins. We investigated a requirement for histidine residues in the envelope (E) protein of West Nile virus during pH-dependent entry into cells. Each histidine was individually replaced with a nonionizable amino acid and tested functionally. In each instance, mutants capable of orchestrating pH-dependent infection were identified. These results do not support a requirement for any single histidine as a pH-sensing "switch," and they suggest that additional features of the E protein are involved in triggering pH-dependent steps in the flavivirus life cycle.