RESUMO
Immune cells need to sustain a state of constant alertness over a lifetime. Yet, little is known about the regulatory processes that control the fluent and fragile balance that is called homeostasis. Here we demonstrate that JAK-STAT signaling, beyond its role in immune responses, is a major regulator of immune cell homeostasis. We investigated JAK-STAT-mediated transcription and chromatin accessibility across 12 mouse models, including knockouts of all STAT transcription factors and of the TYK2 kinase. Baseline JAK-STAT signaling was detected in CD8+ T cells and macrophages of unperturbed mice-but abrogated in the knockouts and in unstimulated immune cells deprived of their normal tissue context. We observed diverse gene-regulatory programs, including effects of STAT2 and IRF9 that were independent of STAT1. In summary, our large-scale dataset and integrative analysis of JAK-STAT mutant and wild-type mice uncovered a crucial role of JAK-STAT signaling in unstimulated immune cells, where it contributes to a poised epigenetic and transcriptional state and helps prepare these cells for rapid response to immune stimuli.
Assuntos
Homeostase , Janus Quinases , Macrófagos , Camundongos Knockout , Fatores de Transcrição STAT , Transdução de Sinais , Animais , Camundongos , Macrófagos/imunologia , Macrófagos/metabolismo , Janus Quinases/metabolismo , Fatores de Transcrição STAT/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT1/genética , Camundongos Endogâmicos C57BL , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/metabolismo , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/genética , TYK2 Quinase/metabolismo , TYK2 Quinase/genética , Regulação da Expressão GênicaRESUMO
Analysis of genetically defined immunodeficient patients allows study of the effect of the absence of specific proteins on human immune function in real-world conditions. Here we have addressed the importance of type I interferon signalling for human NK cell development by studying the phenotype and function of circulating NK cells isolated from patients suffering primary immunodeficiency disease due to mutation of either the human interferon regulatory factor 9 (IRF9) or the signal transducer and activator of transcription 2 (STAT2) genes. IRF9, together with phosphorylated STAT1 and STAT2, form a heterotrimer called interferon stimulated gene factor 3 (ISGF3) which promotes the expression of hundreds of IFN-stimulated genes that mediate antiviral function triggered by exposure to type I interferons. IRF9- and STAT2-deficient patients are unable to respond efficiently to stimulation by type I interferons and so our experiments provide insights into the importance of type I interferon signalling and the consequences of its impairment on human NK cell biology. Surprisingly, the NK cells of these patients display essentially normal phenotype and function.
Assuntos
Interferon Tipo I , Fator Gênico 3 Estimulado por Interferon, Subunidade gama , Células Matadoras Naturais , Fator de Transcrição STAT2 , Transdução de Sinais , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Fator de Transcrição STAT2/metabolismo , Fator de Transcrição STAT2/genética , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/metabolismo , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/genética , Interferon Tipo I/metabolismo , Mutação , Diferenciação Celular , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT1/genética , Células CultivadasRESUMO
OBJECTIVES: Autoreactive B cells and interferon (IFN) signature are hallmarks of primary sjögren's syndrome (pSS), but how IFN signaling pathways influence autoantibody production and clinical manifestations remain unclear. More detailed studies hold promise for improved diagnostic methodologies and personalized treatment. METHODS: We analyzed peripheral blood T and B cell subsets from 34 pSS patients and 38 healthy donors (HDs) at baseline and upon stimulation regarding their expression levels of type I and II IFN signaling molecules (STAT1/2, IRF1, IRF9). Additionally, we investigated how the levels of these molecules correlated with serological and clinical characteristics and performed ROC analysis. RESULTS: Patients showed elevated IFN pathway molecules, including STAT1, STAT2 and IRF9 among most T and B cell subsets. We found a reduced ratio of phosphorylated STAT1 and STAT2 in patients in comparison to HDs, although B cells from patients were highly responsive by increased phosphorylation upon IFN stimulation. Correlation matrices showed further interrelations between STAT1, IRF1 and IRF9 in pSS. Levels of STAT1 and IRF9 in T and B cells correlated with the IFN type I marker Siglec-1 (CD169) on monocytes. High levels of STAT1 and IRF9 within pSS B cells were significantly associated with hypergammaglobulinemia as well as anti-SSA/anti-SSB autoantibodies. Elevated STAT1 levels were found in patients with extraglandular disease and could serve as a biomarker for this subgroup (p < 0.01). Notably, IRF9 levels in T and B cells correlated with EULAR Sjögren's syndrome disease activity index (ESSDAI). CONCLUSION: Here, we provide evidence that in active pSS patients, enhanced IFN signaling incl. unphosphorylated STAT1 and STAT2 with IRFs entertain chronic T and B cell activation. Furthermore, increased STAT1 levels candidate as biomarker of extraglandular disease, while IRF9 levels can serve as biomarker for disease activity.
Assuntos
Biomarcadores , Fator Gênico 3 Estimulado por Interferon, Subunidade gama , Fator de Transcrição STAT1 , Síndrome de Sjogren , Humanos , Síndrome de Sjogren/imunologia , Síndrome de Sjogren/diagnóstico , Síndrome de Sjogren/metabolismo , Fator de Transcrição STAT1/metabolismo , Feminino , Fosforilação , Pessoa de Meia-Idade , Masculino , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/metabolismo , Idoso , Adulto , Linfócitos B/imunologia , Linfócitos B/metabolismo , Autoanticorpos/imunologia , Autoanticorpos/sangue , Transdução de Sinais , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
COVID-19 is characterized by a wide range of clinical manifestations, where aging, underlying diseases, and genetic background are related to worse outcomes. In the present study, the differential expression of seven genes related to immunity, IRF9, CCL5, IFI6, TGFB1, IL1B, OAS1, and TFRC, was analyzed in individuals with COVID-19 diagnoses of different disease severities. Two-step RT-qPCR was performed to determine the relative gene expression in whole-blood samples from 160 individuals. The expression of OAS1 (p < 0.05) and IFI6 (p < 0.05) was higher in moderate hospitalized cases than in severe ones. Increased gene expression of OAS1 (OR = 0.64, CI = 0.52-0.79; p = 0.001), IRF9 (OR = 0.581, CI = 0.43-0.79; p = 0.001), and IFI6 (OR = 0.544, CI = 0.39-0.69; p < 0.001) was associated with a lower risk of requiring IMV. Moreover, TGFB1 (OR = 0.646, CI = 0.50-0.83; p = 0.001), CCL5 (OR = 0.57, CI = 0.39-0.83; p = 0.003), IRF9 (OR = 0.80, CI = 0.653-0.979; p = 0.03), and IFI6 (OR = 0.827, CI = 0.69-0.991; p = 0.039) expression was associated with patient survival. In conclusion, the relevance of OAS1, IRF9, and IFI6 in controlling the viral infection was confirmed.
Assuntos
2',5'-Oligoadenilato Sintetase , COVID-19 , Fator Gênico 3 Estimulado por Interferon, Subunidade gama , SARS-CoV-2 , Humanos , 2',5'-Oligoadenilato Sintetase/genética , COVID-19/genética , COVID-19/imunologia , COVID-19/virologia , Masculino , Feminino , Pessoa de Meia-Idade , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/genética , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/metabolismo , Proteínas Nucleares/genética , Adulto , Idoso , Proteínas MitocondriaisRESUMO
IRF9 is a crucial component in the JAK-STAT pathway. IRF9 interacts with STAT1 and STAT2 to form IFN-I-stimulated gene factor 3 (ISGF3) in response to type I IFN stimulation, which promotes ISG transcription. However, the mechanism by which IFN signaling regulates Malabar grouper (Epinephelus malabaricus) IRF9 is still elusive. Here, we explored the nd tissue-specific mRNA distribution of the MgIRF9 gene, as well as its antiviral function in E. malabaricus. MgIRF9 encodes a protein of 438 amino acids with an open reading frame of 1317 base pairs. MgIRF9 mRNA was detected in all tissues of a healthy M. grouper, with the highest concentrations in the muscle, gills, and brain. It was significantly up-regulated by nervous necrosis virus infection and poly (I:C) stimulation. The gel mobility shift test demonstrated a high-affinity association between MgIRF9 and the promoter of zfIFN in vitro. In GK cells, grouper recombinant IFN-treated samples showed a significant response in ISGs and exhibited antiviral function. Subsequently, overexpression of MgIRF9 resulted in a considerable increase in IFN and ISGs mRNA expression (ADAR1, ADAR1-Like, and ADAR2). Co-immunoprecipitation studies demonstrated that MgIRF9 and STAT2 can interact in vivo. According to the findings, M. grouper IRF9 may play a role in how IFN signaling induces ISG gene expression in grouper species.
Assuntos
Bass , Fator Gênico 3 Estimulado por Interferon, Subunidade gama , Animais , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/metabolismo , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/genética , Bass/genética , Bass/imunologia , Bass/metabolismo , Nodaviridae , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Doenças dos Peixes/virologia , Doenças dos Peixes/imunologia , Sequência de Aminoácidos , Poli I-C/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Antivirais/farmacologia , Regiões Promotoras Genéticas , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
IRF9 can play an antibacterial role by regulating the type I interferon (IFN) pathway. Streptococcus iniae can cause many deaths of yellowfin seabream, Acanthopagrus latus in pond farming. Nevertheless, the regulatory mechanism of type I IFN signalling by A. latus IRF9 (AlIRF9) against S. iniae remains elucidated. In our study, AlIRF9 has a total cDNA length of 3200 bp and contains a 1311 bp ORF encoding a presumed 436 amino acids (aa). The genomic DNA sequence of AlIRF9 has nine exons and eight introns, and AlIRF9 was expressed in various tissues, containing the stomach, spleen, brain, skin, and liver, among which the highest expression was in the spleen. Moreover, AlIRF9 transcriptions in the spleen, liver, kidney, and brain were increased by S. iniae infection. By overexpression of AlIRF9, AlIRF9 is shown as a whole-cell distribution, mainly concentrated in the nucleus. Moreover, the promoter fragments of -415 to +192 bp and -311 to +196 bp were regarded as core sequences from two AlIFNa3s. The point mutation analyses verified that AlIFNa3 and AlIFNa3-like transcriptions are dependent on both M3 sites with AlIRF9. In addition, AlIRF9 could greatly reduce two AlIFNa3s and interferon signalling factors expressions. These results showed that in A. latus, both AlIFNa3 and AlIFNa3-like can mediate the regulation of AlIRF9 in the process of infection with S. iniae.
Assuntos
Doenças dos Peixes , Proteínas de Peixes , Fator Gênico 3 Estimulado por Interferon, Subunidade gama , Dourada , Infecções Estreptocócicas , Streptococcus iniae , Animais , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Infecções Estreptocócicas/imunologia , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/genética , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/metabolismo , Dourada/genética , Dourada/imunologia , Dourada/microbiologia , Streptococcus iniae/fisiologia , Regiões Promotoras Genéticas/genética , Transdução de Sinais , Regulação da Expressão Gênica , Imunidade Inata/genéticaRESUMO
Deciphering the intricate dynamic events governing type I interferon (IFN) signaling is critical to unravel key regulatory mechanisms in host antiviral defense. Here, we leverage TurboID-based proximity labeling coupled with affinity purification-mass spectrometry to comprehensively map the proximal human proteomes of all seven canonical type I IFN signaling cascade members under basal and IFN-stimulated conditions. This uncovers a network of 103 high-confidence proteins in close proximity to the core members IFNAR1, IFNAR2, JAK1, TYK2, STAT1, STAT2, and IRF9, and validates several known constitutive protein assemblies, while also revealing novel stimulus-dependent and -independent associations between key signaling molecules. Functional screening further identifies PJA2 as a negative regulator of IFN signaling via its E3 ubiquitin ligase activity. Mechanistically, PJA2 interacts with TYK2 and JAK1, promotes their non-degradative ubiquitination, and limits the activating phosphorylation of TYK2 thereby restraining downstream STAT signaling. Our high-resolution proximal protein landscapes provide global insights into the type I IFN signaling network, and serve as a valuable resource for future exploration of its functional complexities.
Assuntos
Interferon Tipo I , Janus Quinase 1 , Receptor de Interferon alfa e beta , Fator de Transcrição STAT2 , Transdução de Sinais , TYK2 Quinase , Ubiquitinação , Humanos , Células HEK293 , Interferon Tipo I/metabolismo , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/metabolismo , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/genética , Janus Quinase 1/metabolismo , Fosforilação , Proteoma/metabolismo , Receptor de Interferon alfa e beta/metabolismo , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT2/metabolismo , TYK2 Quinase/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The essential and redundant functions of human type I and II interferons (IFNs) have been delineated over the last three decades by studies of patients with inborn errors of immunity or their autoimmune phenocopies, but much less is known about type III IFNs. Patients with cells that do not respond to type III IFNs due to inherited IL10RB deficiency display no overt viral disease, and their inflammatory disease phenotypes can be explained by defective signaling via other interleukine10RB-dependent pathways. Moreover, patients with inherited deficiencies of interferon-stimulated gene factor 3 (ISGF-3) (STAT1, STAT2, IRF9) present viral diseases also seen in patients with inherited deficiencies of the type I IFN receptor (IFNAR1/2). Finally, patients with autoantibodies neutralizing type III IFNs have no obvious predisposition to viral disease. Current findings thus suggest that type III IFNs are largely redundant in humans. The essential functions of human type III IFNs, particularly in antiviral defenses, remain to be discovered.
Assuntos
Interferon lambda , Interferons , Viroses , Humanos , Interferons/metabolismo , Interferons/imunologia , Viroses/imunologia , Animais , Transdução de Sinais/imunologia , Fator de Transcrição STAT2/metabolismo , Fator de Transcrição STAT2/genética , Fator de Transcrição STAT2/imunologia , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/genética , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/imunologia , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/metabolismo , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/imunologia , Subunidade beta de Receptor de Interleucina-10/genética , Subunidade beta de Receptor de Interleucina-10/imunologia , Subunidade beta de Receptor de Interleucina-10/metabolismoRESUMO
Poly(ADP-ribose) polymerase (PARP) inhibitors are targeted therapies that accumulate DNA damage by interfering with DNA repair mechanisms and are approved for treating several cancers with BRCA1/2 mutations. In this study, we utilized CRISPR-dCas9 interference screening to identify genes regulating sensitivity to PARP inhibitors in breast cancer cell lines. Our findings indicated that the interferon (IFN) signaling gene IRF9 was critically involved in modulating sensitivity to these inhibitors. We revealed that the loss of IRF9 leads to increased resistance to the PARP inhibitor in MDA-MB-468 cells, and a similar desensitization was observed in another breast cancer cell line, MDA-MB-231. Further analysis indicated that while the basal expression of IRF9 did not correlate with the response to the PARP inhibitor olaparib, its transcriptional induction was significantly associated with increased sensitivity to the DNA-damaging agent cisplatin in the NCI-60 cell line panel. This finding suggests a mechanistic link between IRF9 induction and cellular responses to DNA damage. Additionally, data from the METABRIC patient tissue study revealed a complex network of IFN-responsive gene expressions postchemotherapy, with seven upregulated genes, including IRF9, and three downregulated genes. These findings underscore the intricate role of IFN signaling in the cellular response to chemotherapy. Collectively, our CRISPR screening data and subsequent bioinformatic analyses suggest that IRF9 is a novel biomarker for sensitivity to DNA-damaging agents, such as olaparib and platinum-based chemotherapeutic agents. Our findings for IRF9 not only enhance our understanding of the genetic basis of drug sensitivity, but also elucidate the role of IRF9 as a critical effector within IFN signaling pathways, potentially influencing the association between the host immune system and chemotherapeutic efficacy.