RESUMO
The adaptor molecule stimulator of IFN genes (STING) is central to production of type I IFNs in response to infection with DNA viruses and to presence of host DNA in the cytosol. Excessive release of type I IFNs through STING-dependent mechanisms has emerged as a central driver of several interferonopathies, including systemic lupus erythematosus (SLE), Aicardi-Goutières syndrome (AGS), and stimulator of IFN genes-associated vasculopathy with onset in infancy (SAVI). The involvement of STING in these diseases points to an unmet need for the development of agents that inhibit STING signaling. Here, we report that endogenously formed nitro-fatty acids can covalently modify STING by nitro-alkylation. These nitro-alkylations inhibit STING palmitoylation, STING signaling, and subsequently, the release of type I IFN in both human and murine cells. Furthermore, treatment with nitro-fatty acids was sufficient to inhibit production of type I IFN in fibroblasts derived from SAVI patients with a gain-of-function mutation in STING. In conclusion, we have identified nitro-fatty acids as endogenously formed inhibitors of STING signaling and propose for these lipids to be considered in the treatment of STING-dependent inflammatory diseases.
Assuntos
Ácidos Graxos/metabolismo , Herpes Simples/metabolismo , Herpesvirus Humano 2/metabolismo , Proteínas de Membrana/metabolismo , Transdução de Sinais , Animais , Doenças Autoimunes do Sistema Nervoso/genética , Doenças Autoimunes do Sistema Nervoso/metabolismo , Doenças Autoimunes do Sistema Nervoso/patologia , Herpes Simples/genética , Herpes Simples/patologia , Humanos , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Lipoilação , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/metabolismo , Lúpus Eritematoso Sistêmico/patologia , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/metabolismo , Malformações do Sistema Nervoso/patologia , Células RAW 264.7RESUMO
Understanding the principles governing mammalian gene regulation has been hampered by the difficulty in measuring in vivo binding dynamics of large numbers of transcription factors (TF) to DNA. Here, we develop a high-throughput Chromatin ImmunoPrecipitation (HT-ChIP) method to systematically map protein-DNA interactions. HT-ChIP was applied to define the dynamics of DNA binding by 25 TFs and 4 chromatin marks at 4 time-points following pathogen stimulus of dendritic cells. Analyzing over 180,000 TF-DNA interactions we find that TFs vary substantially in their temporal binding landscapes. This data suggests a model for transcription regulation whereby TF networks are hierarchically organized into cell differentiation factors, factors that bind targets prior to stimulus to prime them for induction, and factors that regulate specific gene programs. Overlaying HT-ChIP data on gene-expression dynamics shows that many TF-DNA interactions are established prior to the stimuli, predominantly at immediate-early genes, and identified specific TF ensembles that coordinately regulate gene-induction.
Assuntos
Imunoprecipitação da Cromatina/métodos , Células Dendríticas/metabolismo , Regulação da Expressão Gênica , Ensaios de Triagem em Larga Escala , Animais , DNA/genética , DNA/metabolismo , Camundongos , Fatores de Transcrição/metabolismoRESUMO
Stimulator of Interferon Genes (STING) is essential for the inflammatory response to cytosolic DNA. Despite that aberrant activation of STING is linked to an increasing number of inflammatory diseases, the development of inhibitors has been challenging, with no compounds in the pipeline beyond the preclinical stage. We previously identified endogenous nitrated fatty acids as novel reversible STING inhibitors. With the aim of improving the specificity and efficacy of these compounds, we developed and tested a library of nitroalkene-based compounds for in vitro and in vivo STING inhibition. The structure-activity relationship study revealed a robustly improved electrophilicity and reduced degrees of freedom of nitroalkenes by conjugation with an aromatic moiety. The lead compounds CP-36 and CP-45, featuring a ß-nitrostyrene moiety, potently inhibited STING activity in vitro and relieved STING-dependent inflammation in vivo. This validates the potential for nitroalkene compounds as drug candidates for STING modulation to treat STING-driven inflammatory diseases, providing new robust leads for preclinical development.
Assuntos
Alcenos , Inflamação , Proteínas de Membrana , Nitrocompostos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Animais , Inflamação/tratamento farmacológico , Humanos , Camundongos , Alcenos/química , Alcenos/farmacologia , Nitrocompostos/química , Nitrocompostos/farmacologia , Relação Estrutura-AtividadeRESUMO
Pathogenic viruses induce metabolic changes in host cells to secure the availability of biomolecules and energy to propagate. Influenza A virus (IAV) and severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) both infect the human airway epithelium and are important human pathogens. The metabolic changes induced by these viruses in a physiologically relevant human model and how this affects innate immune responses to limit viral propagation are not well known. Using an ex vivo model of pseudostratified primary human airway epithelium, we here demonstrate that infection with both IAV and SARS-CoV-2 resulted in distinct metabolic changes including increases in lactate dehydrogenase A (LDHA) expression and LDHA-mediated lactate formation. Interestingly, LDHA regulated both basal and induced mitochondrial anti-viral signaling protein (MAVS)-dependent type I interferon (IFN) responses to promote IAV, but not SARS-CoV-2, replication. Our data demonstrate that LDHA and lactate promote IAV but not SARS-CoV-2 replication by inhibiting MAVS-dependent induction of type I IFN in primary human airway epithelium.
RESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Antiviral strategies to inhibit Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2) and the pathogenic consequences of COVID-19 are urgently required. Here, we demonstrate that the NRF2 antioxidant gene expression pathway is suppressed in biopsies obtained from COVID-19 patients. Further, we uncover that NRF2 agonists 4-octyl-itaconate (4-OI) and the clinically approved dimethyl fumarate (DMF) induce a cellular antiviral program that potently inhibits replication of SARS-CoV2 across cell lines. The inhibitory effect of 4-OI and DMF extends to the replication of several other pathogenic viruses including Herpes Simplex Virus-1 and-2, Vaccinia virus, and Zika virus through a type I interferon (IFN)-independent mechanism. In addition, 4-OI and DMF limit host inflammatory responses to SARS-CoV2 infection associated with airway COVID-19 pathology. In conclusion, NRF2 agonists 4-OI and DMF induce a distinct IFN-independent antiviral program that is broadly effective in limiting virus replication and in suppressing the pro-inflammatory responses of human pathogenic viruses, including SARS-CoV2.
Assuntos
Anti-Inflamatórios/farmacologia , Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Fumarato de Dimetilo/agonistas , Fator 2 Relacionado a NF-E2/metabolismo , Pneumonia Viral/tratamento farmacológico , Succinatos/agonistas , Adulto , Antioxidantes/farmacologia , Betacoronavirus/metabolismo , COVID-19 , Infecções por Coronavirus/virologia , Fumarato de Dimetilo/farmacologia , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Interferon Tipo I , Pulmão/patologia , Masculino , Fator 2 Relacionado a NF-E2/genética , Pandemias , Pneumonia Viral/virologia , SARS-CoV-2 , Transdução de Sinais/efeitos dos fármacos , Succinatos/farmacologia , Replicação Viral/efeitos dos fármacosRESUMO
Herpes simplex virus-2 (HSV-2) is a leading cause of sexually transmitted infections for which no effective vaccines or prophylactic treatment currently exist. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor involved in the detoxification of reactive oxygen species (ROS) and has been more recently shown to regulate inflammatory and antiviral responses. Here, we evaluated the importance of Nrf2 in the control of HSV-2 genital infection, and its role in the regulation of HSV-induced innate antiviral immunity. Comparison of antiviral gene expression profile by RNA-sequencing analysis of wild type and Nrf2-mutant (Nrf2AY/AY ) murine macrophages showed an upregulation at the basal level of the type I interferon-associated gene network. The same basal increased antiviral profile was also observed in the spleen of Nrf2-/- mice. Interestingly, the lack of Nrf2 in murine cells was sufficient to increase the responsiveness to HSV-derived dsDNA and protect cells from HSV-2 infection in vitro. Surprisingly, there was no indication of an alteration in STING expression in murine cells as previously reported in cells of human origin. Additionally, genetic activation of Nrf2 in Keap1-/- mouse embryonic fibroblasts increased HSV-2 infectivity and replication. Finally, using an in vivo vaginal herpes infection model, we showed that Nrf2 controlled early innate immune responses to HSV-2 without affecting STING expression levels. Nrf2-/- mice exhibited reduced viral replication that was associated with higher level of type I interferons in vaginal washes. Nrf2-/- mice also displayed reduced weight loss, lower disease scores, and higher survival rates than wild type animals. Collectively, these data identify Nrf2 as a negative regulator of the interferon-driven antiviral response to HSV-2 without impairing STING mRNA and protein expression levels in murine cells.
Assuntos
Herpes Genital/imunologia , Interferon Tipo I/imunologia , Fator 2 Relacionado a NF-E2/imunologia , Animais , Feminino , Herpesvirus Humano 2/imunologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The transcription factor Nrf2 is a critical regulator of inflammatory responses. If and how Nrf2 also affects cytosolic nucleic acid sensing is currently unknown. Here we identify Nrf2 as an important negative regulator of STING and suggest a link between metabolic reprogramming and antiviral cytosolic DNA sensing in human cells. Here, Nrf2 activation decreases STING expression and responsiveness to STING agonists while increasing susceptibility to infection with DNA viruses. Mechanistically, Nrf2 regulates STING expression by decreasing STING mRNA stability. Repression of STING by Nrf2 occurs in metabolically reprogrammed cells following TLR4/7 engagement, and is inducible by a cell-permeable derivative of the TCA-cycle-derived metabolite itaconate (4-octyl-itaconate, 4-OI). Additionally, engagement of this pathway by 4-OI or the Nrf2 inducer sulforaphane is sufficient to repress STING expression and type I IFN production in cells from patients with STING-dependent interferonopathies. We propose Nrf2 inducers as a future treatment option in STING-dependent inflammatory diseases.
Assuntos
Proteínas de Membrana/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Vírus de DNA/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Interferon Tipo I/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Proteínas de Membrana/genética , Camundongos , Fator 2 Relacionado a NF-E2/genética , Células RAW 264.7 , RNA Mensageiro/metabolismo , Succinatos/farmacologiaRESUMO
Genome sequencing studies have shown that human malignancies often bear mutations in four or more driver genes, but it is difficult to recapitulate this degree of genetic complexity in mouse models using conventional breeding. Here we use the CRISPR-Cas9 system of genome editing to overcome this limitation. By delivering combinations of small guide RNAs (sgRNAs) and Cas9 with a lentiviral vector, we modified up to five genes in a single mouse hematopoietic stem cell (HSC), leading to clonal outgrowth and myeloid malignancy. We thereby generated models of acute myeloid leukemia (AML) with cooperating mutations in genes encoding epigenetic modifiers, transcription factors and mediators of cytokine signaling, recapitulating the combinations of mutations observed in patients. Our results suggest that lentivirus-delivered sgRNA:Cas9 genome editing should be useful to engineer a broad array of in vivo cancer models that better reflect the complexity of human disease.
Assuntos
Neoplasias da Medula Óssea/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Animais , Modelos Animais de Doenças , CamundongosRESUMO
[This corrects the article DOI: 10.7554/eLife.00603.].
Assuntos
Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Saccharomyces cerevisiae/genética , Evolução Molecular , Modelos Genéticos , Saccharomyces cerevisiae/metabolismo , Transcrição GênicaRESUMO
Divergence in gene regulation can play a major role in evolution. Here, we used a phylogenetic framework to measure mRNA profiles in 15 yeast species from the phylum Ascomycota and reconstruct the evolution of their modular regulatory programs along a time course of growth on glucose over 300 million years [corrected]. We found that modules have diverged proportionally to phylogenetic distance, with prominent changes in gene regulation accompanying changes in lifestyle and ploidy, especially in carbon metabolism. Paralogs have significantly contributed to regulatory divergence, typically within a very short window from their duplication. Paralogs from a whole genome duplication (WGD) event have a uniquely substantial contribution that extends over a longer span. Similar patterns occur when considering the evolution of the heat shock regulatory program measured in eight of the species, suggesting that these are general evolutionary principles. DOI:http://dx.doi.org/10.7554/eLife.00603.001.