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Interferon regulatory factors (IRFs) play key roles during viral and bacterial infections. However, their regulation by inflammatory cytokines, including interleukin (IL)-1β and tumor necrosis factor (TNF) α, remains underexplored. As airway epithelial cells (AECs) modulate lung inflammation, IRF expression was characterized in pulmonary A549 and bronchial BEAS-2B epithelial cells along with primary AECs grown in submersion, or air-liquid interface, culture. While, IRF6 mRNA was only highly expressed in primary cells, IRF4 and IRF8 mRNAs were consistently low across the models. All the other IRF mRNAs were expressed in each model. IRF3 and IRF9 mRNAs were highly expressed, but their proteins remained primarily cytoplasmic post-IL-1β treatment in A549 cells. IRF2 showed moderate/high mRNA expression and was constitutively nuclear. However, RNA silencing did not support roles for IRF2 or IRF3, with only a modest role for IRF9, in the IL-1β-induced activation of an IRF reporter. IRF1 mRNA was highly induced by IL-1β in A549 and primary cells. Similarly, IRF1 protein was increased by IL-1β and TNFα in A549 cells, and by TNFα in BEAS-2B cells. In A549 cells, IL-1β-induced IRF1 protein localized to the nucleus and since IRF1 silencing prevented IRF reporter activity, a major transcriptional role was indicated. Mechanistically, the inflammatory transcription factor, nuclear factor (NF)-κB, was necessary for IL-1β- and TNFα-induced IRF1 expression. Further, four novel enhancer regions 5' to IRF1 bound the NF-κB subunit, p65, and their IL-1β/TNFα-induced reporter activity required consensus NF-κB motifs. Three such regions recruited RNA polymerase-2 and were flanked by the active chromatin mark, histone 3 lysine 27 acetylation, supporting enhancer involvement in IRF1 transcription. Finally, IRF1 expression, transcription rate, and enhancer activity induced by IL-1β, or TNFα, were relatively unaffected by glucocorticoid. IRF1-dependent gene expression may therefore show insensitivity to glucocorticoid and could contribute to glucocorticoid-resistance in diseases that include severe asthma.

The transcription factors nuclear factor of activated T cells (NFAT) and activator protein 1 (AP-1; Fos–Jun) cooperate to promote the effector functions of T cells, but NFAT in the absence of AP-1 imposes a negative feedback program of T cell hyporesponsiveness (exhaustion). Here, we show that basic leucine zipper ATF-like transcription factor (BATF) and interferon regulatory factor 4 (IRF4) cooperate to counter T cell exhaustion in mouse tumor models. Overexpression of BATF in CD8 + T cells expressing a chimeric antigen receptor (CAR) promoted the survival and expansion of tumor-infiltrating CAR T cells, increased the production of effector cytokines, decreased the expression of inhibitory receptors and the exhaustion-associated transcription factor TOX and supported the generation of long-lived memory T cells that controlled tumor recurrence. These responses were dependent on BATF–IRF interaction, since cells expressing a BATF variant unable to interact with IRF4 did not survive in tumors and did not effectively delay tumor growth. BATF may improve the antitumor responses of CAR T cells by skewing their phenotypes and transcriptional profiles away from exhaustion and towards increased effector function. Chronic antigen stimulation leads to CD8 + T cell exhaustion, which is mediated by persistent activation of the transcription factor NFAT in the absence of AP-1. Seo, González-Avalos and colleagues show that overexpressed BATF cooperates with IRF4 to counteract NFAT-induced exhaustion and promote better tumor control by CAR T cells in mouse models.

The Interferon regulatory factors (IRFs) are a family of transcription factors that play pivotal roles in many aspects of the immune response, including immune cell development and differentiation and regulating responses to pathogens. Three family members, IRF3, IRF5, and IRF7, are critical to production of type I interferons downstream of pathogen recognition receptors that detect viral RNA and DNA. A fourth family member, IRF9, regulates interferon-driven gene expression. In addition, IRF4, IRF8, and IRF5 regulate myeloid cell development and phenotype, thus playing important roles in regulating inflammatory responses. Thus, understanding how their levels and activity is regulated is of critical importance given that perturbations in either can result in dysregulated immune responses and potential autoimmune disease. This review will focus the role of IRF family members in regulating type I IFN production and responses and myeloid cell development or differentiation, with particular emphasis on how regulation of their levels and activity by ubiquitination and microRNAs may impact autoimmune disease.

The induction and action of type I interferon (IFN) is of fundamental importance in human immune defenses toward microbial pathogens, particularly viruses. Basic discoveries within the molecular and cellular signaling pathways regulating type I IFN induction and downstream actions have shown the essential role of the IFN regulatory factor (IRF) and the signal transducer and activator of transcription (STAT) families, respectively. However, the exact biological and immunological functions of these factors have been most clearly revealed through the study of inborn errors of immunity and the resultant infectious phenotypes in humans. The spectrum of human inborn errors of immunity caused by mutations in IRFs and STATs has proven very diverse. These diseases encompass herpes simplex encephalitis (HSE) and severe influenza in IRF3- and IRF7/IRF9 deficiency, respectively. They also include Mendelian susceptibility to mycobacterial infection (MSMD) in STAT1 deficiency, through disseminated measles infection associated with STAT2 deficiency, and finally staphylococcal abscesses and chronic mucocutaneous candidiasis (CMC) classically described with Hyper-IgE syndrome (HIES) in the case of STAT3 deficiency. More recently, increasing focus has been on aspects of autoimmunity and autoinflammation playing an important part in many primary immunodeficiency diseases (PID)s, as exemplified by STAT1 gain-of-function causing CMC and autoimmune thyroiditis, as well as a recently described autoinflammatory syndrome with hypogammaglobulinemia and lymphoproliferation as a result of STAT3 gain-of-function. Here I review the infectious, inflammatory, and autoimmune disorders arising from mutations in IRF and STAT transcription factors in humans, highlightning the underlying molecular mechanisms and immunopathogenesis as well as the clinical/therapeutic perspectives of these new insights.

Microglial activation plays a central role in poststroke inflammation and causes secondary neuronal damage; however, it also contributes in debris clearance and chronic recovery. Microglial pro- and antiinflammatory responses (or so-called M1-M2 phenotypes) coexist and antagonize each other throughout the disease progress. As a result of this balance, poststroke immune responses alter stroke outcomes. Our previous study found microglial expression of interferon regulatory factor 5 (IRF5) and IRF4 was related to pro- and antiinflammatory responses, respectively. In the present study, we genetically modified the IRF5 and IRF4 signaling to explore their roles in stroke. Both in vitro and in vivo assays were utilized; IRF5 or IRF4 small interfering RNA (siRNA), lentivirus, and conditional knockout (CKO) techniques were employed to modulate IRF5 or IRF4 expression in microglia. We used a transient middle cerebral artery occlusion model to induce stroke and examined both acute and chronic stroke outcomes. Poststroke inflammation was evaluated with flow cytometry, RT-PCR, MultiPlex, and immunofluorescence staining. An oscillating pattern of the IRF5-IRF4 regulatory axis function was revealed. Down-regulation of IRF5 signaling by siRNA or CKO resulted in increased IRF4 expression, enhanced M2 activation, quenched proinflammatory responses, and improved stroke outcomes, whereas down-regulation of IRF4 led to increased IRF5 expression, enhanced M1 activation, exacerbated proinflammatory responses, and worse functional recovery. Up-regulation of IRF4 or IRF5 by lentivirus induced similar results. We conclude that the IRF5-IRF4 regulatory axis is a key determinant in microglial activation. The IRF5-IRF4 regulatory axis is a potential therapeutic target for neuroinflammation and ischemic stroke.

Background Macrophage proinflammatory activation contributes to the pathology of severe acute pancreatitis (SAP) and, simultaneously, macrophage functional changes, and increased pyroptosis/necrosis can further exacerbate the cellular immune suppression during the process of SAP, where cyclic GMP–AMP synthase (cGAS)–stimulator of interferon genes (STING) plays an important role. However, the function and mechanism of cGAS–STING in SAP-induced lung injury (LI) remains unknown. Methods Lipopolysaccharide (LPS) was combined with caerulein-induced SAP in wild type, cGAS −/− and sting −/− mice . Primary macrophages were extracted via bronchoalveolar lavage and peritoneal lavage. Ana-1 cells were pretreated with LPS and stimulated with nigericin sodium salt to induce pyroptosis in vitro. Results SAP triggered NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome activation-mediated pyroptosis of alveolar and peritoneal macrophages in mouse model. Knockout of cGAS / STING could ameliorate NLRP3 activation and macrophage pyroptosis. In addition, mitochondrial (mt)DNA released from damaged mitochondria further induced macrophage STING activation in a cGAS- and dose-dependent manner. Upregulated STING signal can promote NLRP3 inflammasome-mediated macrophage pyroptosis and increase serum interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α levels and, thus, exacerbate SAP-associated LI (SAP-ALI). Downstream molecules of STING, IRF7, and IRF3 connect the mtDNA–cGAS–STING axis and the NLRP3–pyroptosis axis. Conclusions Negative regulation of any molecule in the mtDNA–cGAS–STING–IRF7/IRF3 pathway can affect the activation of NLRP3 inflammasomes, thereby reducing macrophage pyroptosis and improving SAP-ALI in mouse model.

FoxP3 + regulatory T (Treg) cells restrict excessive immune responses and immunopathology as well as reactivity to self or environmental antigens and thus are crucial for peripheral immune tolerance. The transcription factor Interferon Regulatory Factor 4 (IRF4) controls differentiation and function of T cells. In Treg cells, IRF4 is required for peripheral activation and maturation to effector Treg (eTreg) cells with enhanced suppressive function. However, the mechanisms of Treg cell regulation by IRF4 are not fully understood. Here, we analyze the role of IRF4 in differentiation and maintenance of Treg cells using IRF4-deficient mice and a T cell transfer model, that allows Irf4 inactivation in peripheral T cells. We demonstrate that loss of one Irf4 allele already results in impaired eTreg cell differentiation and decreased Treg cell homeostasis, indicating that IRF4 controls peripheral Treg cell differentiation in a gene dosage dependent mode. Peripheral Irf4 inactivation was also associated with enhanced production of inflammatory but also inhibitory cytokines by Treg cells. ATAC sequencing of Treg cells after mutation of one or both Irf4 alleles revealed regions with altered accessibility in genes involved in Treg cell function. In the FoxP3 gene, Irf4 inactivation resulted in reduced ATAC signals in the promoter region and in the conserved non-coding sequence (CNS) 2, required for stability of FoxP3 expression in peripheral Treg cells in response to TCR stimulation. IRF4-deficient Treg cells also displayed a reduction in open chromatin in several Treg cell specific super enhancers, mainly located in proximity to potential IRF4 binding sites. In conclusion, our results demonstrate that IRF4 controls peripheral Treg cell differentiation and homeostasis in a gene dosage dependent manner.

The transcription factor interferon regulatory factor 4 (IRF4) belongs to the IRF family and has several important functions for the adaptive immune response. Mutations affecting IRF family members IRF1, IRF3, IRF7, IRF8, or IRF9 have been described in patients presenting with inborn errors of immunity (IEI) highlighting the importance of these factors for the cellular host defense against mycobacterial and/or viral infections. IRF4 deficiency and haploinsufficiency have been associated with IEI. More recently, two novel IRF4 disease-causing mechanisms have been described due to the characterization of IEI patients presenting with cellular immunodeficiency associated with agammaglobulinemia. Here, we review the phenotypes and physiopathological mechanisms underlying IEI of IRF family members and, in particular, IRF4.

Tumor-associated macrophages (TAMs) usually express an M2 phenotype, which enables them to perform immunosuppressive and tumor-promoting functions. Reprogramming these TAMs toward an M1 phenotype could thwart their pro-cancer activities and unleash anti-tumor immunity, but efforts to accomplish this are nonspecific and elicit systemic inflammation. Here we describe a targeted nanocarrier that can deliver in vitro-transcribed mRNA encoding M1-polarizing transcription factors to reprogram TAMs without causing systemic toxicity. We demonstrate in models of ovarian cancer, melanoma, and glioblastoma that infusions of nanoparticles formulated with mRNAs encoding interferon regulatory factor 5 in combination with its activating kinase IKKβ reverse the immunosuppressive, tumor-supporting state of TAMs and reprogram them to a phenotype that induces anti-tumor immunity and promotes tumor regression. We further establish that these nanoreagents are safe for repeated dosing. Implemented in the clinic, this immunotherapy could enable physicians to obviate suppressive tumors while avoiding systemic treatments that disrupt immune homeostasis. The previous efforts to reprogramme tumour-associated macrophages (TAMs) to M1 phenotype have caused undesired side-effects. Here, the authors report targeted nanocarriers for delivering mRNA encoding M1-polarizing transcription factors to TAMs and show their efficacy in multiple mouse tumour models.

Background Interferon regulatory factors (IRFs) are a family of transcription factors with important functions in immunity. The genomes of most vertebrates encode ten IRF genes. IRF3 and IRF9 have key roles in interferon (IFN) induction and signaling. Most of our knowledge about the IFN pathways originates from the study of the mammalian IFN system, and the description of the corresponding avian components is not as complete. Both IRF3 and IRF9 were considered missing from the chicken genome and from the genomes of other avian species. Results Here we describe multiple avian IRF3 and IRF9 genes, all with difficult GC-rich sequence context that prevented their earlier characterization. IRF3 orthologs are narrowly distributed and are present in the avian infraclass Palaeognathae. In contrast, IRF9 orthologs were found in most avian species, with the exception of the order Galliformes. In about half of the avian orders, IRF9 was located in noncanonical chromosomal positions, indicating past translocations. Phylogenetic analysis confirmed the correct orthology of all newly described IRFs. We further performed experiments using duck IRF9, confirming its role in the IFN pathway. IRF9 knockout in duck fibroblasts decreases the induction of IFN-stimulated genes (ISGs). Full induction of ISGs in duck cells requires both an intact IRF9 and a canonical IFN-stimulated response element. Lastly, intact IRF9 is needed for IFN-mediated protection of duck cells against the vesicular stomatitis virus-induced cytopathicity. Conclusions The identification of avian IRFs fills an important gap in our understanding of avian immunology and brings new questions related to the evolution of the IRF family.