IRF3-deficient Mice Research Articles

IRF3 Promotes Asthma Pathogenesis by Regulating Type 2 Innate Lymphoid Cells

ABSTRACT Background Allergic asthma is characterized by airway hyperresponsiveness triggered by inhaled allergens. Type 2 innate lymphoid cells (ILC2s) have been demonstrated to play a crucial role in promoting airway inflammation through the secretion of type 2 effector cytokines. However, the mechanisms underlying the functions of lung ILC2s remain unclear. Methods In this study, we investigated the expression of IRF3 in ILC2s in both human patients and mouse models of asthma. We utilized IRF3-deficient mice to assess the impact of IRF3 deficiency on ILC2 function in a model of IL33-induced asthma. Additionally, we explored the mechanisms underlying IRF3-mediated regulation of ILC2s, focusing on the involvement of the transcription factor Gata3. Results Our findings revealed elevated expression of IRF3 in ILC2s of patients and mice with asthma, suggesting a potential role for IRF3 in the pathogenesis of allergic asthma. Furthermore, we demonstrated that IRF3 deficiency impairedthe expansion and function of ILC2s in IL33-induced asthma, highlighting the importance of IRF3 in regulating ILC2-mediated responses. Importantly, we showed that the regulation of ILC2s by IRF3 was independent of Th2 cells and mediated by the transcription factor Gata3. Conclusion This study identifies IRF3 as a novel regulator of lung ILC2s and suggests its potential as a promising immunotherapeutic target for allergic asthma. These findings shed light on the intricate mechanisms underlying asthma pathogenesis and provide insights into potential strategies for the development of targeted therapies for this prevalent airway disease.

Identification and Characterization of Small-Molecule IRF3-Dependent Immune Activators for Pharmaceutical Development.

We sought to develop a small-molecule activator of interferon regulatory factor 3 (IRF3), an essential innate immune transcription factor, which could potentially be used therapeutically in multiple disease settings. Using a high-throughput screen, we identified small-molecule entities that activate a type I interferon response, with minimal off-target NFκB activation. We identified 399 compounds at a hit rate of 0.24% from singlicate primary screening. Secondary screening included the primary hits and additional compounds with similar chemical structures obtained from other library sources and resulted in 142 candidate compounds. The hit compounds were sorted and ranked to identify compound groups with activity in both human and mouse backgrounds to facilitate animal model engagement for translational development. Chemical modifications within two groups of small molecules produced leads with improved activity over original hits. Furthermore, these leads demonstrated activity in ex vivo cytokine release assays from human blood- and mouse bone marrow-derived macrophages. Dependence on IRF3 was demonstrated using bone marrow-derived macrophages from IRF3-deficient mice, which were not responsive to the molecules. To identify the upstream pathway leading to IRF3 activation, we used a library of CRISPR knockout cell lines to test the key innate immune adaptor and receptor molecules. These studies indicated a surprising toll-interleukin-1 receptor-domain-containing-adapter-inducing interferon-β-dependent but TLR3/4-independent mechanism of IRF3 activation.

Heparin fragments induce cervical inflammation by recruiting immune cells through Toll-like receptor 4 in nonpregnant mice.

Inflammation is a hallmark in the human cervix remodelling. A possible candidate inducing the inflammatory driven ripening of the cervix is the matrix component heparan sulphate, which has been shown to be elevated in late pregnancy in the cervix and uterus. Heparin and a glycol-split low molecular weight heparin (gsHep) with low anticoagulant potency has been shown to enhance myometrial contraction and interleukin (IL)-8 production by cervical fibroblasts. The aim of this study was to investigate the mechanism by which heparin promotes cervical inflammation. Wild-type, Toll-like receptor 4 (TLR4), Myeloid differentiation primary response gene 88 (MyD88) and Interferon regulatory factor 3 (IRF3)-deficient mice were treated by deposition of gsHep into the vaginas of nonpregnant mice. To identify which cells that responded to the heparin fragments, a rhodamine fluorescent construct of gsHep was used, which initially did bind to the epithelial cells and were at later time points located in the sub-mucosa. The heparin fragments induced a strong local inflammatory response in wild-type mice shown by a rapid infiltration of neutrophils and to a lesser extent macrophages into the epithelium and the underlying extracellular matrix of the cervix. Further, a marked migration into the cervical and vaginal lumen was seen by both neutrophils and macrophages. The induced mucosal inflammation was strongly reduced in TLR4- and IRF3-deficient mice. In conclusion, our findings suggest that a TLR4/IRF3-mediated innate immune response in the cervical mucosa is induced by gsHep. This low anticoagulant heparin version, a novel TLR4 agonist, could contribute to human cervical ripening during the initiation of labour.

IRF3 prevents colorectal tumorigenesis via inhibiting the nuclear translocation of β-catenin

Occurrence of Colorectal cancer (CRC) is relevant with gut microbiota. However, role of IRF3, a key signaling mediator in innate immune sensing, has been barely investigated in CRC. Here, we unexpectedly found that the IRF3 deficient mice are hyper-susceptible to the development of intestinal tumor in AOM/DSS and Apcmin/+ models. Genetic ablation of IRF3 profoundly promotes the proliferation of intestinal epithelial cells via aberrantly activating Wnt signaling. Mechanically, IRF3 in resting state robustly associates with the active β-catenin in the cytoplasm, thus preventing its nuclear translocation and cell proliferation, which can be relieved upon microbe-induced activation of IRF3. In accordance, the survival of CRC is clinically correlated with the expression level of IRF3. Therefore, our study identifies IRF3 as a negative regulator of the Wnt/β-catenin pathway and a potential prognosis marker for Wnt-related tumorigenesis, and describes an intriguing link between gut microbiota and CRC via the IRF3-β-catenin axis.

From the Editor’s desk…: May 2019

From the Editor’s desk…: May 2019

Interferon-Independent Protection by Interferon Regulatory Factor 3

Abstract BACKGROUND Independent type I/III IFN regulation, interferon-regulatory factor 3 (IRF3) can mediate cell death and suppress proliferation during stress. Novel ‘suicide necrosis’ centers around IRF3 and is one protective mechanism against intracellular pathogens. Uterine epithelial cells infected with obligate intracellular Chlamydia trachomatis are typically shed 3–5 days post-infection (dpi) by unknown mechanism(s) and exhibit new growth. We have previously shown IRF3-deficient mice exhibit decreased epithelial shedding and 10-fold greater bacterial burdens during early infection. We hypothesize that IRF3-mediated cell death restricts chlamydial infection in the genital mucosa, providing a potentially novel mechanism to reduce pathogen load. METHODS Progesterone-treated C57BL/6J and IRF3 KO mice were infected vaginally with 3e5 IFUs of C. muridarum strain “Nigg” and histologically assessed 3 and 4 dpi for chlamydia inclusions, cell death and proliferation using proliferation marker PCNA. Cell death was assessed in vivo with PI delivery by NSET device 10 minutes before sacrifice 4 dpi. RESULTS/CONCL 3 dpi, uterine epithelia in WT and IRF3 KO mice remain intact with no epithelial shedding. WT show a single layer of infected epithelial cells, while IRF3 KO have several layers of infected cells, suggesting the next layer(s) of epithelial cells are generated before the first is shed. 4 dpi we see dramatic increases in epithelial shedding in WT mice when stained in vivo with PI. In contrast, IRF3 KO epithelia show relatively few PI-stained cells. We conclude that IRF3 is playing a significant and protective role during C. muridarum infection by facilitating cell death and limiting cellular proliferation of epithelial cells.

The cytosolic DNA- and RNA-sensing pathways play important and non-redundant roles in host defense against vaccinia infection

Abstract Vaccinia virus is a prototypic poxvirus, and during the life cycle of the virus, viral DNA and RNA can be detected by the nucleic acid-sensing pathways to induce the expression of type I IFN and IFN-stimulating genes, which leads to the establishment of antiviral state. Poxviruses have evolved multiple strategies to evade host immunity. Vaccinia E3, which consists of two distinct Z-DNA-binding and dsRNA-binding domains, is a critical virulence factor. Virus lacks E3L gene (ΔE3L) is non-pathogenic in wild-type mice in an intranasal infection model. We have previously reported that ΔE3L infection of murine primary keratinocytes (KCs) induces Ifnb, Il6, Ccl5 and Ccl4 gene expression and protein secretion via a MAVS/IRF3-dependent mechanism. Here we show that MDA5, a cytosolic dsRNA sensor, is essential for the induction of innate immunity by ΔE3L in KCs, whereas PKR, another cytosolic dsRNA sensor, is not. ΔE3L gains virulence in MDA5, MAVS, or IRF3-deficient mice, but not in STING-or MyD88-deficient mice, in an intranasal infection model. The MDA5-, MAVS-, or IRF3-deficient mice lost 20% of weight but all recovered in response to ΔE3L infection. By contrast, both the MDA5 and STING-mediated sensing pathways contribute to host defense against WT vaccinia virus infection. Lastly, whereas ΔE3L virus fails to replicate in wild-type murine primary fibroblasts, it gains replication capacity in cGAS or STING-deficient murine primary fibroblasts. Taken together, these results indicate that vaccinia viral nucleic acids are detected via the cytosolic DNA- and/or RNA-sensing pathways in a cell-type dependent manner, and both pathways play important and non-redundant roles in host defense against vaccinia infection.

Cross-Regulation of Two Type I Interferon Signaling Pathways in Plasmacytoid Dendritic Cells Controls Anti-malaria Immunity and Host Mortality

SummaryType I interferon (IFN) is critical for controlling pathogen infection; however, its regulatory mechanisms in plasmacytoid cells (pDCs) still remain unclear. Here, we have shown that nucleic acid sensors cGAS-, STING-, MDA5-, MAVS-, or transcription factor IRF3-deficient mice produced high amounts of type I IFN-α and IFN-β (IFN-α/β) in the serum and were resistant to lethal plasmodium yoelii YM infection. Robust IFN-α/β production was abolished when gene encoding nucleic acid sensor TLR7, signaling adaptor MyD88, or transcription factor IRF7 was ablated or pDCs were depleted. Further, we identified SOCS1 as a key negative regulator to inhibit MyD88-dependent type I IFN signaling in pDCs. Finally, we have demonstrated that pDCs, cDCs, and macrophages were required for generating IFN-α/β-induced subsequent protective immunity. Thus, our findings have identified a critical regulatory mechanism of type I IFN signaling in pDCs and stage-specific function of immune cells in generating potent immunity against lethal YM infection.

IRF3 promotes adipose inflammation and insulin resistance and represses browning.

The chronic inflammatory state that accompanies obesity is a major contributor to insulin resistance and other dysfunctional adaptations in adipose tissue. Cellular and secreted factors promote the inflammatory milieu of obesity, but the transcriptional pathways that drive these processes are not well described. Although the canonical inflammatory transcription factor NF-κB is considered to be the major driver of adipocyte inflammation, members of the interferon regulatory factor (IRF) family may also play a role in this process. Here, we determined that IRF3 expression is upregulated in the adipocytes of obese mice and humans. Signaling through TLR3 and TLR4, which lie upstream of IRF3, induced insulin resistance in murine adipocytes, while IRF3 knockdown prevented insulin resistance. Furthermore, improved insulin sensitivity in IRF3-deficient mice was associated with reductions in intra-adipose and systemic inflammation in the high fat-fed state, enhanced browning of subcutaneous fat, and increased adipose expression of GLUT4. Taken together, the data indicate that IRF3 is a major transcriptional regulator of adipose inflammation and is involved in maintaining systemic glucose and energy homeostasis.

IRF3 deficiency contributes to impaired memory T cell function in response to influenza infection (IRM14P.453)

Abstract The role of IRF3 in initiating the type I interferon response in innate immune cells has been well studied in the context of viral infection. However, it remains unclear what impact IRF3 has in shaping and maintaining adaptive immune responses. To address this, IRF3 deficient (IRF3 KO) mice were infected with influenza A virus (IAV) and T cell responses measured. At day 9 post infection, the frequency of IFN-γ and Granzyme B (GrB) positive T cells were similar between IRF3 KO and wildtype mice. Despite similar primary responses, IRF3 KO mice exhibited significant deficits in recall memory T cell responses to IAV with reduced expression of IFN-γ and GrB. Since IRF3 has been implicated in the expression of cytokines that influence effector and memory T cells responses, cytokine supplementation studies were performed. Addition of IL-12 was the only cytokine tested that was able to rescue the IFN-γ deficiencies in IRF3 KO T cells. Interestingly, peptide specific GrB re-expression could not be restored by any cytokines tested. These results suggest that IRF3 plays a role in the maintenance of antigen specific memory T cell responses and that IRF3 deficiency has a substantial impact on the cytotoxic potential of memory T cells and recall responses. Together, this work provides further insight into the molecular regulation of adaptive immune responses to viral infections and may serve to inform future vaccine strategies that require the development of memory T cells.

Interferon regulatory factor 3 deficiency leads to interleukin-17-mediated liver ischemia-reperfusion injury

Interferon regulatory factor 3 (IRF3) is an important transcription factor in Toll-like receptor 4 (TLR4) signaling, a pathway that is known to play a critical role in liver ischemia-reperfusion injury. In order to decipher the involvement of IRF3 in this setting, we first compared the intensity of hepatic lesions in IRF3-deficient versus wildtype mice. We found increased levels of blood transaminases, enhanced liver necrosis, and more pronounced neutrophil infiltrates in IRF3-deficient mice. Neutrophil depletion by administration of anti-Ly6G monoclonal antibody indicated that neutrophils play a dominant role in the development of severe liver necrosis in IRF3-deficient mice. Quantification of cytokine genes expression revealed increased liver expression of interleukin (IL)-12/IL-23p40, IL-23p19 messenger RNA (mRNA), and IL-17A mRNA in IRF3-deficient versus wildtype (WT) mice, whereas IL-27p28 mRNA expression was diminished in the absence of IRF3. The increased IL-17 production in IRF3-deficient mice was functionally relevant, as IL-17 neutralization prevented the enhanced hepatocellular damages and liver inflammation in these animals. Evidence for enhanced production of IL-23 and decreased accumulation of IL-27 cytokine in M1 type macrophage from IRF3-deficient mice was also observed after treatment with lipopolysaccharide, a setting in which liver gamma-delta T cells and invariant natural killer T cells were found to be involved in IL-17A hyperproduction. IRF3-dependent events downstream of TLR4 control the IL-23/IL-17 axis in the liver and this regulatory role of IRF3 is relevant to liver ischemia-reperfusion injury.

Essential contribution of IRF3 to intestinal homeostasis and microbiota-mediated Tslp gene induction

The large intestinal epithelial cells and immune cells are exposed to a variety of molecules derived from commensal microbiota that can activate innate receptors, such as Toll-like receptors (TLRs) and retinoic acid-inducible gene-I-like receptors (RLRs). Although the activation of these receptors is known to be critical for homeostasis of the large intestine, the underlying gene regulatory mechanisms are not well understood. Here, we show that IFN regulatory factor (IRF)3 is critical for the suppression of dextran sulfate sodium-induced colitis. IRF3-deficient mice exhibited lethal defects in the inflammatory and recovery phases of the colitis, accompanied by marked defects in the gene induction for thymic stromal lymphopoietin (TSLP), a cytokine known to be essential for protection of the large intestine. We further provide evidence that DNA and RNA of the large intestinal contents are critical for Tslp gene induction via IRF3 activation by cytosolic nucleic acid receptors. We also demonstrate that IRF3 indeed activates the gene promoter of Tslp via IRF-binding sequences. This newly identified intestinal gene regulatory mechanism, wherein IRF3 activated by microbiota-derived nucleic acids plays a critical role in intestinal homeostasis, may have clinical implication in colonic inflammatory disorders.

IRF7-dependent IFNβ production in response to RANK facilitates medullary thymic epithelial cell development. (60.3)

Abstract The signal transducer and activator of transcription 1 (STAT1) is an essential part of signaling cascades induced by type I and II interferons. Deficiency of STAT1 results in heightened susceptibility to infections and autoimmunity in mouse and humans. While the role of IFNγ in T cell development is well known, the role of type I interferons in thymic development has largely remained unexplored. Here we show that STAT1 and IFNAR-deficient mice harbor a defect in medullary compartment characterized by a significant reduction in self-antigen presenting, AIRE expressing medullary thymic epithelial cells. Using a novel reporter mouse we demonstrate that constitutive IFNAR signaling is taking place in the thymic medulla in the absence of any infection or inflammatory signals. In addition, we found that RANK stimulation in thymic stromal cells results in the upregulation of IFNβ which in turn is inhibits RANK signaling thereby facilitating AIRE expression. Finally, we show that IRF7 is required for the induction of IFNβ by RANKL, and that IRF7-, but not IRF3-deficient mice display a defect in thymic architecture and medullary thymic epithelial cell differentiation similar to that observed in IFNAR- or STAT1-deficient animals. These results illustrate that a spatially and temporally coordinated crosstalk between the RANKL/RANK signaling pathway and the IRF7/IFNβ/IFNAR/STAT1 axis is essential for the differentiation of AIRE-expressing medullary thymic epithelial cells.

Protective Role of Interferon Regulatory Factor 3-Mediated Signaling against Prion Infection

Abnormal prion protein (PrP(Sc)) generated from the cellular isoform of PrP (PrP(C)) is assumed to be the main or sole component of the pathogen, called prion, of transmissible spongiform encephalopathies (TSE). Because PrP is a host-encoded protein, acquired immune responses are not induced in TSE. Meanwhile, activation of the innate immune system has been suggested to partially block the progression of TSE; however, the mechanism is not well understood. To further elucidate the role of the innate immune system in prion infection, we investigated the function of interferon regulatory factor 3 (IRF3), a key transcription factor of the MyD88-independent type I interferon (IFN) production pathway. We found that IRF3-deficient mice exhibited significantly earlier onset with three murine TSE strains, namely, 22L, FK-1, and murine bovine spongiform encephalopathy (mBSE), following intraperitoneal transmission, than with wild-type controls. Moreover, overexpression of IRF3 attenuated prion infection in the cell culture system, while PrP(Sc) was increased in prion-infected cells treated with small interfering RNAs (siRNAs) against IRF3, suggesting that IRF3 negatively regulates PrP(Sc) formation. Our findings provide new insight into the role of the host innate immune system in the pathogenesis of prion diseases.

IRF3 regulates cardiac fibrosis but not hypertrophy in mice during angiotensin II‐induced hypertension

Hypertension is a typical modern lifestyle-related disease that is closely associated with the development of cardiovascular disorders. Elevation of angiotensin II (ANG II) is one of several critical factors for hypertension and heart failure; however, the mechanisms underlying the ANG II-mediated pathogenesis are still poorly understood. Here, we show that ANG II-mediated cardiac fibrosis, but not hypertrophy, is regulated by interferon regulatory factor 3 (IRF3), which until now has been exclusively studied in the innate immune system. In a ANG II-infusion mouse model (3.0 mg/kg/d), we compared IRF3-deficient mice (Irf3(-/-)/Bcl2l12(-/-)) with matched wild-type (WT) controls. The development of cardiac fibrosis [3.95 ± 0.62% (WT) vs. 1.41 ± 0.46% (Irf3(-/-)/Bcl2l12(-/-)); P<0.01] and accompanied reduction in left ventricle end-diastolic dimension [2.89 ± 0.10 mm (WT) vs. 3.51 ± 0.15 mm (Irf3(-/-)/Bcl2l12(-/-)); P=0.012] are strongly suppressed in Irf3(-/-)/Bcl2l12(-/-) mice, whereas hypertrophy still develops. Further, we provide evidence for the activation of IRF3 by ANG II signaling in mouse cardiac fibroblasts. Unlike the activation of IRF3 by innate immune receptors, IRF3 activation by ANG II is unique in that it is activated through the canonical ERK signaling pathway. Thus, our present study reveals a hitherto unrecognized function of IRF3 in cardiac remodeling, providing new insight into the progression of hypertension-induced cardiac pathogenesis.

IFN Regulatory Factor 3 Contributes to the Host Response during Pseudomonas aeruginosa Lung Infection in Mice

Pseudomonas aeruginosa is a major opportunistic pathogen. However, host defense mechanisms involved in P. aeruginosa lung infection remain incompletely defined. The transcription factor IFN regulatory factor 3 (IRF3) is primarily associated with host defense against viral infections, and a role of IRF3 in P. aeruginosa infection has not been reported previously. In this study, we showed that IRF3 deficiency led to impaired clearance of P. aeruginosa from the lungs of infected mice. P. aeruginosa infection induced IRF3 translocation to the nucleus, activation of IFN-stimulated response elements (ISRE), and production of IFN-beta, suggesting that P. aeruginosa activates the IRF3-ISRE-IFN pathway. In vitro, macrophages from IRF3-deficient mice showed complete inhibition of CCL5 (RANTES) and CXCL10 (IP-10) production, partial inhibition of TNF, but no effect on CXCL2 (MIP-2) or CXCL1 (keratinocyte-derived chemokine) in response to P. aeruginosa stimulation. In vivo, IRF3-deficient mice showed complete inhibition of CCL5 production and partial or no effects on production of other cytokines and chemokines in the bronchoalveolar lavage fluids and lung tissues. Profiling of immune cells in the airways revealed that neutrophil and macrophage recruitment into the airspace was reduced, whereas B cell, T cell, NK cell, and NKT cell infiltration was unaffected in IRF3-deficient mice following P. aeruginosa lung infection. These data suggest that IRF3 regulates a distinct profile of cytokines and chemokines and selectively modulates neutrophil and macrophage recruitment during P. aeruginosa infection. Thus, IRF3 is an integral component in the host defense against P. aeruginosa lung infection.

Essential role of IRF-3 in lipopolysaccharide-induced interferon- β gene expression and endotoxin shock

Type I interferons (IFN-α/β) affect many aspects of immune responses. Many pathogen-associated molecules, including bacterial lipopolysaccharide (LPS) and virus-associated double-stranded RNA, induce IFN gene expression through activation of distinct Toll-like receptors (TLRs). Although much has been studied about the activation of the transcription factor IRF-3 and induction of IFN- β gene by the LPS-mediated TLR4 signaling, definitive evidence is missing about the actual role of IRF-3 in LPS responses in vitro and in vivo. Using IRF-3 deficient mice, we show here that IRF-3 is indeed essential for the LPS-mediated IFN- β gene induction. Loss of IRF-3 also affects the expression of profile of other cytokine/chemokine genes. We also provide evidence that the LPS/TLR4 signaling activates IRF-7 to induce IFN- β, if IRF-7 is induced by IFNs prior to LPS simulation. Finally, the IRF-3-deficient mice show resistance to LPS-induced endotoxin shock. These results place IRF-3 as a molecule central to LPS/TLR4 signaling.