STAT4 activation by leukemia inhibitory factor confers a therapeutic effect on intestinal inflammation.

T helper 17 (Th17)-cell differentiation triggered by interleukin-6 (IL-6) via STAT3 activation promotes inflammation in inflammatory bowel disease (IBD) patients. However, leukemia inhibitory factor (LIF), an IL-6 family cytokine, restricts inflammation by blocking Th17-cell differentiation via an unknown mechanism. Here, we report that microbiota dysregulation promotes LIF secretion by intestinal epithelial cells (IECs) in a mouse colitis model. LIF greatly activates STAT4 phosphorylation on multiple SPXX elements within the C-terminal transcription regulation domain. STAT4 and STAT3 act reciprocally on both canonical cis-inducible elements (SIEs) and noncanonical "AGG" elements at different loci. In lamina propria lymphocytes (LPLs), STAT4 activation by LIF blocks STAT3-dependent Il17a/Il17f promoter activation, whereas in IECs, LIF bypasses the extraordinarily low level of STAT4 to induce YAP gene expression via STAT3 activation. In addition, we found that the administration of LIF is sufficient to restore microbiome homeostasis. Thus, LIF effectively inhibits Th17 accumulation and promotes repair of damaged intestinal epithelium in inflamed colon, serves as a potential therapy for IBD.

Lung airway-surveilling CXCR3(hi) memory CD8(+) T cells are critical for protection against influenza A virus.

Inducing memory CD8(+) T cells specific for conserved antigens from influenza A virus (IAV) is a potential strategy for broadly protective vaccines. Here we show that memory CD8(+) T cells in the airways played an important role in early control of IAV. Expression of the chemokine receptor CXCR3 was critical for memory CD8(+) T cells to populate the airways during the steady state and vaccination approaches were designed to favor the establishment of memory CD8(+) T cells in the airways. Specifically, we found that interleukin-12 (IL-12) signaling shortly after immunization limited CXCR3 expression on memory CD8(+) T cells. Neutralization of IL-12 or adjuvants that did not induce high amounts of IL-12 enhanced CXCR3 expression, sustained airway localization of memory CD8(+) T cells, and resulted in superior protection against IAV.

Trichinella spiralis cystatin, TsCstN, modulates STAT4/IL-12 to specifically suppress IFN-γ production.

We have previously identified a cystatin, TsCstN, derived from the L1 stage of Trichinella spiralis and have shown that this protein is internalised in macrophages. Here we sought to address if this macrophage-TsCstN interaction could alter downstream T-cell priming. Using LPS-primed macrophages to stimulate T-cells in a co-culture system with or without TsCstN we assessed the resultant T-cell outcomes. IFN-γ, both protein and mRNA, but not IL-17A was negatively regulated by inclusion of TsCstN during macrophage priming. We identified a cell-cell contact independent change in the levels of IL-12 that led to altered phosphorylated STAT4 levels and translocation. TsCstN also negatively regulated the autonomous response in the myotubule cell line, C2C12. This work identifies a potential pathyway for L1 larvae to evade protective Th1 based immune responses and establish muscle-stage T. spiralis infection.

STAT4 Directs a Protective Innate Lymphoid Cell Response to Gastrointestinal Infection.

Innate lymphoid cells (ILCs) are strategically positioned at mucosal barrier surfaces where they respond quickly to infection or injury. Therefore, we hypothesized that ILCs are key contributors to the early immune response in the intestine against Listeria monocytogenes Using a modified strain of L. monocytogenes that mimics human gastrointestinal listeriosis in mice, we find ILCs to be essential for control of early replication of L. monocytogenes in the intestine as well as for restricted dissemination of bacteria to peripheral tissues. Specifically, group 1 ILCs (ILC1s) and group 3 ILCs (ILC3s) respond to infection with proliferation and IFN-γ and IL-22 production. Mechanistically, we show that the transcription factor STAT4 is required for the proliferative and IFN-γ effector response by ILC1s and ILC3s, and loss of STAT4 signaling in the innate immune compartment results in an inability to control bacterial growth and dissemination. Interestingly, STAT4 acts acutely as a transcription factor to promote IFN-γ production. Together, these data illustrate a critical role for ILCs in the early responses to gastrointestinal infection with L. monocytogenes and identify STAT4 as a central modulator of ILC-mediated protection.

Fibroblast signal transducer and activator of transcription 4 drives cigarette smoke-induced airway fibrosis.

Cigarette smoke-induced emphysema and small airway remodeling are the anatomic bases of chronic obstructive pulmonary disease (COPD), but the pathogenesis of these changes is unclear, and current treatments for COPD are minimally effective. To evaluate the role of signal transducer and activator of transcription (STAT)-4 in cigarette smoke-induced small airway remodeling, we used C57BL/6J (wild type [WT]) and STAT4-/- mice exposed to air or cigarette smoke for 6 months and isolated airway and parenchymal fibroblasts. We also compared the results with those obtained with human fibroblasts. We found that STAT4-/- mice were protected against smoke-induced small airway remodeling but not emphysema. STAT4 is abundantly expressed in airway compared with parenchymal-derived fibroblasts isolated from normal human and murine lung. WT airway fibroblasts proliferate faster than STAT4-/- airway fibroblasts, whereas there is no difference between strains for parenchymal fibroblasts. IL-12 is up-regulated in the lung after smoke exposure, and IL-12 receptor B2 is expressed on airway and parenchymal fibroblasts in mouse and human lung. Treatment with IL-12 causes phosphorylation of STAT4 in WT airway fibroblasts. Exposure of WT airway, but not parenchymal, fibroblasts to IL-12 causes increased expression of collagen 1α1 and transforming growth factor ß1, factors involved in small airway remodeling, whereas STAT4-/- fibroblasts are unresponsive to IL-12. These results indicate that IL-12 can drive small airway remodeling via STAT4 signaling and suggest that treatment with clinically available anti-IL-12p40 drugs might provide a new approach to preventing small airway remodeling in cigarette smokers.

Twist1 regulates Ifng expression in Th1 cells by interfering with Runx3 function.

A transcription factor network that includes STAT4, T-bet, and Runx3 promotes the differentiation of Th1 cells and inflammatory immune responses. How additional transcription factors regulate the function of Th1 cells has not been defined. In this study we show that the negative regulatory factor Twist1 decreases expression of T-bet, Runx3, and IL-12Rß2 as it inhibits IFN-γ production. Ectopic expression of Runx3, but not T-bet or IL-12Rß2, compensates for the effects of Twist1 on IFN-γ production, and Twist1 regulation of Ifng depends on complex formation with Runx3. Twist1 decreases Runx3 and T-bet binding at the Ifng locus, and it decreases chromatin looping within the Ifng locus. These data define an IL-12/STAT4-induced negative regulatory loop that impacts multiple components of the Th1 transcriptional network and provide further insight into regulation of Th1 differentiation.

N-ethyl-N-nitrosourea-induced mutation in ubiquitin-specific peptidase 18 causes hyperactivation of IFN-αß signaling and suppresses STAT4-induced IFN-γ production, resulting in increased susceptibility to Salmonella typhimurium.

To deepen our knowledge of the natural host response to pathogens, our team undertook an in vivo screen of mutagenized 129S1 mice with Salmonella Typhimurium. One mutation affecting Salmonella susceptibility was mapped to a region of 1.3 Mb on chromosome 6 that contains 15 protein-coding genes. A missense mutation was identified in the Usp18 (ubiquitin-specific peptidase 18) gene. This mutation results in an increased inflammatory response (IL-6, type 1 IFN) to Salmonella and LPS challenge while paradoxically reducing IFN-gamma production during bacterial infection. Increased STAT1 phosphorylation correlated with impaired STAT4 phosphorylation, resulting in overwhelming IL-6 secretion but reduced IFN-gamma production during infection. The reduced IFN-gamma levels, along with the increased inflammation, rationalize the S. Typhimurium susceptibility in terms of increased bacterial load in target organs and cytokine-induced septic shock and death.

Temporal induction pattern of STAT4 target genes defines potential for Th1 lineage-specific programming.

STAT4 is a critical component in the development of inflammatory adaptive immune responses. It has been extensively characterized as a lineage-determining factor in Th1 development. However, the genetic program activated by STAT4 that results in an inflammatory cell type is not well defined. In this report, we use DNA isolated from STAT4-chromatin immunoprecipitation to perform chromatin immunoprecipitation-on-chip analysis of over 28,000 mouse gene promoters to identify STAT4 targets. We demonstrate that STAT4 binds multiple gene-sets that program distinct components of the Th1 lineage. Although many STAT4 target genes display STAT4-dependent IL-12-inducible expression, other genes displayed IL-12-induced histone modifications but lack induction, possibly due to high relative basal expression. In the subset of genes that STAT4 programs for expression in Th1 cells, IL-12-induced mRNA levels remain increased for a longer time than mRNA from genes that are not programmed. This suggests that STAT4 binding to target genes, while critical, is not the only determinant for STAT4-dependent gene programming during Th1 differentiation.

Cutting edge: autoimmune disease risk variant of STAT4 confers increased sensitivity to IFN-alpha in lupus patients in vivo.

Increased IFN-alpha signaling is a primary pathogenic factor in systemic lupus erythematosus (SLE). STAT4 is a transcription factor that is activated by IFN-alpha signaling, and genetic variation of STAT4 has been associated with risk of SLE and rheumatoid arthritis. We measured serum IFN-alpha activity and simultaneous IFN-alpha-induced gene expression in PBMC in a large SLE cohort. The risk variant of STAT4 (T allele; rs7574865) was simultaneously associated with both lower serum IFN-alpha activity and greater IFN-alpha-induced gene expression in PBMC in SLE patients in vivo. Regression analyses confirmed that the risk allele of STAT4 was associated with increased sensitivity to IFN-alpha signaling. The IFN regulatory factor 5 SLE risk genotype was associated with higher serum IFN-alpha activity; however, STAT4 showed dominant influence on the sensitivity of PBMC to serum IFN-alpha. These data provide biologic relevance for the risk variant of STAT4 in the IFN-alpha pathway in vivo.

Notch regulates IL-10 production by T helper 1 cells.

T helper 1 (Th1) cells mediate powerful cellular immune responses. However, if unbalanced, Th1 immunity eventually may cause pathology. Recently, it has been shown that IL-10, an antiinflammatory cytokine strongly antagonizing Th1-mediated effects, can be produced by Th1 cells and is indeed essential for self-regulation of Th1 immunity. Here, we show that Notch induces IL-10 production in newly developing and already established Th1 cells via a signal transducer and activator of transcription 4 (STAT4)-dependent process. Notch signaling in the presence of the cytokines IL-12 or IL-27 induces Th1 cells to produce large amounts of IL-10 without diminishing IFN-gamma production. Notch-modified Th1 cells completely lose their inflammatory capacity and instead are able to actively suppress a Th1 cell-induced delayed-type hypersensitivity (DTH) reaction in an IL-10-dependent fashion. IL-10 production can be elicited by active forms of all four mammalian Notch receptors but was found to be specific for the Delta-like family of Notch ligands. Dendritic cells (DC) selectively acquire Delta-like 4 expression upon stimulation with various Toll-like receptor (TLR) ligands and concomitantly induce IL-10 production by Th1 cells in vitro and in vivo. This effect can be selectively reversed by pharmacological inhibitors of Notch signaling (gamma-secretase inhibitor). Our data suggest that Notch regulates IL-10 production in Th1 cells by a STAT4-dependent process that converts proinflammatory Th1 cells into T cells with regulatory activity. This pathway may provide unique opportunities for therapeutic intervention in Th1-driven immune diseases and for Th1-associated vaccination strategies.

STAT4 isoforms differentially regulate Th1 cytokine production and the severity of inflammatory bowel disease.

STAT4, a critical regulator of inflammation in vivo, can be expressed as two alternative splice forms, a full-length STAT4alpha, and a STAT4beta isoform lacking a C-terminal transactivation domain. Each isoform is sufficient to program Th1 development through both common and distinct subsets of target genes. However, the ability of these isoforms to mediate inflammation in vivo has not been examined. Using a model of colitis that develops following transfer of CD4(+) CD45RB(high) T cells expressing either the STAT4alpha or STAT4beta isoform into SCID mice, we determined that although both isoforms mediate inflammation and weight loss, STAT4beta promotes greater colonic inflammation and tissue destruction. This correlates with STAT4 isoform-dependent expression of TNF-alpha and GM-CSF in vitro and in vivo, but not Th1 expression of IFN-gamma or Th17 expression of IL-17, which were similar in STAT4alpha- and STAT4beta-expressing T cells. Thus, higher expression of a subset of inflammatory cytokines from STAT4beta-expressing T cells correlates with the ability of STAT4beta-expressing T cells to mediate more severe inflammatory disease.

STAT4: an immunoregulator contributing to diverse human diseases.

Signal transducer and activator of transcription 4 (STAT4) is a member of the STAT family and localizes to the cytoplasm. STAT4 is phosphorylated after a variety of cytokines bind to the membrane, and then dimerized STAT4 translocates to the nucleus to regulate gene expression. We reviewed the essential role played by STAT4 in a wide variety of cells and the pathogenesis of diverse human diseases, especially many kinds of autoimmune and inflammatory diseases, via activation by different cytokines through the Janus kinase (JAK)-STAT signaling pathway.

STAT6 inhibits T-bet-independent Th1 cell differentiation.

STAT6 plays critical roles in Th2 cell differentiation, whereas STAT4 and T-bet are important for Th1 cell differentiation. However, it is still largely unknown about the cross talk of these transcription factors during Th1/Th2 cell differentiation. To further address the regulatory mechanisms underlying Th1/Th2 cell differentiation, we generated the mice lacking both STAT6 and T-bet (STAT6(-/-)T-bet(-/-) mice). Importantly, although Th2 cell differentiation was severely and similarly decreased in STAT6(-/-)T-bet(-/-) mice and STAT6(-/-) mice, Th1 cell differentiation was rescued in part in STAT6(-/-)T-bet(-/-) mice as compared with that in T-bet(-/-) mice. While no significant difference was observed in the expression of IL-12Rbeta2 and STAT4 between STAT6(-/-)T-bet(-/-) CD4(+) T cells and T-bet(-/-) CD4(+) T cells, IL-12-induced STAT4 phosphorylation was increased in STAT6(-/-)T-bet(-/-) CD4(+) T cells as compared with that in T-bet(-/-) CD4(+) T cells. These results indicate that STAT6 inhibits T-bet-independent Th1 cell differentiation by suppressing IL-12-STAT4 signaling.

Constitutive Activation of Natural Killer Cells in Primary Biliary Cholangitis.

Natural killer (NK) cells are innate immune cells that interface with the adaptive immune system to generate a pro-inflammatory immune environment. Primary Biliary Cholangitis (PBC) is a hepatic autoimmune disorder with extrahepatic associations including systemic sclerosis, Sjogren's syndrome and thyroiditis. Immunogenetic studies have identified polymorphisms of the IL-12/STAT4 pathway as being associated with PBC. As this pathway is important for NK cell function we investigated NK cells in PBC. Circulating NK cells from individuals with PBC were constitutively activated, with higher levels of CD49a and the liver-homing marker, CXCR6, compared to participants with non-autoimmune chronic liver disease and healthy controls. Stimulation with minimal amounts of IL-12 (0.005 ng/ml) led to significant upregulation of CXCR6 (p < 0.005), and enhanced IFNγ production (p < 0.02) on NK cells from PBC patients compared to individuals with non-autoimmune chronic liver disease, indicating dysregulation of the IL-12/STAT4 axis. In RNAseq studies, resting NK cells from PBC patients had a constitutively activated transcriptional profile and upregulation of genes associated with IL-12/STAT4 signaling and metabolic reprogramming. Consistent with these findings, resting NK cells from PBC patients expressed higher levels of pSTAT4 compared to control groups (p < 0.001 vs. healthy controls and p < 0.05 vs. liver disease controls). In conclusion NK cells in PBC are sensitive to minute quantities of IL-12 and have a "primed" phenotype. We therefore propose that peripheral priming of NK cells to express tissue-homing markers may contribute to the pathophysiology of PBC.

Silencing of STAT4 Protects Against Autoimmune Myocarditis by Regulating Th1/Th2 Immune Response via Inactivation of the NF-κB Pathway in Rats.

Signal transducer and activator of transcription 4 (STAT4) has been implicated in the progression of myocarditis. The aim of the current study was to investigate the role by which STAT4 influences autoimmune myocarditis in an attempt to identify a theoretical therapeutic perspective for the condition. After successful establishment of an autoimmune myocarditis rat model, the expression patterns of STAT4, NF-κB pathway-related genes, Th1 inflammatory cytokines (IFN-γ and IL-2), and Th2 inflammatory cytokines (IL-6 and IL-10) were subsequently determined. The rats with autoimmune myocarditis were treated with oe-STAT4 or sh-STAT4 lentiviral vectors to evaluate the role of STAT4 in autoimmune myocarditis, or administrated with 1 mL 10 µmol/L of BAY11-7082 (the NF-κB pathway inhibitor) via tail vein to investigate the effect of the NF-κB pathway on autoimmune myocarditis. Finally, cell apoptosis was evaluated. The serum levels of IFN-γ and IL-2, extent of IκBα and P65 phosphorylation, and the expression of STAT4 were elevated, while the serum levels of IL-6 and IL-10 as well as the expression of IκBα were reduced among the rats with autoimmune myocarditis, which was accompanied by an increase in the apoptotic cells. More importantly, the silencing of STAT4 or the inhibition of the NF-κB pathway was detected to result in a decrease in the serum levels of IFN-γ and IL-2 and an elevation of the serum levels of IL-6 and IL-10, and inhibited myocardial cell apoptosis in rats with autoimmune myocarditis. Moreover, STAT4 silencing was also observed to decrease the extent of IκBα and P65 phosphorylation while acting to elevate the expression of IκBα. Taken together, silencing of STAT4 could hinder the progression of autoimmune myocarditis by balancing the expression of Th1/Th2 inflammatory cytokines via the NF-κB pathway, which may provide a novel target for experimental autoimmune myocarditis (EAM) treatment.

Interleukin (IL)-23 receptor is a major susceptibility gene for Graves' ophthalmopathy: the IL-23/T-helper 17 axis extends to thyroid autoimmunity.

CONTEXT: IL-23 and its receptor (IL-23R) guide T cells toward the T-helper 17 phenotype. IL-23R single nucleotide polymorphisms (SNPs) have been associated with several autoimmune diseases, including Crohn's disease and rheumatoid arthritis. OBJECTIVE: Our objective was to determine whether variants in the IL-23R gene are associated with Graves' disease (GD) and Graves' ophthalmopathy (GO). DESIGN AND PARTICIPANTS: A total of 216 North American Caucasian GD patients and 368 healthy controls were genotyped for four SNPs spanning the IL-23R gene. SNPs rs11209026 and rs7530511 were genotyped using the TaqMan allelic discrimination assays (Applied Biosystems, Foster City, CA), and SNPs rs2201841 and rs10889677 were genotyped using a fluorescent-based restriction fragment length polymorphism method. RESULTS: The A allele of rs2201841 was present in 78.8% of GD patients with GO and 64.7% of controls [P=1.1x10(-4); odds ratio (OR)=2.04]; the AA genotype was also significantly increased in GO patients compared with controls (62.5 and 41%, respectively; P=1.0x10(-4); OR=2.4). The C allele of rs10889677 was present in 78.6% of GO patients and 64.5% of controls (P=1.3x10(-4); OR=2.03), and the CC genotype was also significantly increased in GO patients vs. controls (62.1 and 41.0%, respectively; P=1.4x10(-4); OR=2.36). The TT genotype of rs7530511 was significantly associated with GD, but not specifically with GO; it was present in 2.5% of GD patients and 0.3% of controls (P=0.02; OR=9.4). The rs11209026 SNP, which is the most strongly associated with Crohn's disease, was not associated with GD or GO in our data set. CONCLUSIONS: Variants in the IL-23R gene are strongly associated with GO. These variants may predispose to GO by changing the expression and/or function of IL-23R, thereby promoting a proinflammatory signaling cascade.

Gene expression profiling of chicken lymphoid cells after treatment with Lactobacillus acidophilus cellular components.

Lactobacillus acidophilus has been shown to exert immunostimulating activities in a number of species, including the chicken. To examine the molecular mechanisms of this phenomenon, we investigated spatial and temporal expression of immune system genes in chicken cecal tonsil and spleen mononuclear cells in response to structural constituents of L. acidophilus. Using a low-density chicken immune system microarray, we found that cecal tonsil cells responded more rapidly than spleen cells to the bacterial stimuli, with the most potent stimulus for cecal tonsil cells being DNA and for splenocytes being the bacterial cell wall components. We also discovered that in both splenocytes and cecal tonsil cells, STAT2 and STAT4 genes were highly induced. Given the close interactions between cecal tonsil cells and commensal bacteria, we further examined the involvement of STAT2 and STAT4 signaling pathways in cellular responses to bacterial DNA. Our results revealed that the expression of STAT2, STAT4, IL-18, MyD88, IFN-alpha, and IFN-gamma genes were up-regulated in cecal tonsil cells after treatment with L. acidophilus DNA.

Role of Stat4-mediated signal transduction events in the generation of aggressor CD4+ T cells in herpetic stromal keratitis pathogenesis.

Ocular infection with herpes simplex virus (HSV) causes a vision-impairing inflammatory reaction called herpetic stromal keratitis. In murine models, herpetic stromal keratitis lesions appear to be immunopathologic, mediated by CD4(+) T cells of Th1 phenotype. To provide insight about cytokine networks and signaling events involved in the development of aggressor CD4(+) T cells, ocular HSV infection was followed in mice deficient in Stat4 (Stat4(-/-) mice), the signal transducer for the cytokine interleukin-12 (IL-12). After ocular HSV infection of Stat4(-/-) and control BALB/c mice, clinical, histologic, and immunologic analyses were carried out. Further, to evaluate the involvement of Stat4 in the development of this aggressor population, naive CD4(+) T cells from Stat4(-/-) and BALB/c mice were adoptively transferred to C.B-17 SCID mice 1 day after corneal infection. Although Stat4(-/-) mice demonstrated increased susceptibility to lethal encephalitis and facial lesions, interestingly, these mice had less severe stromal keratitis in comparison to control animals. Adoptive transfer of naive CD4(+) T cells from Stat4(-/-) mice failed to produce disease in infected SCID recipients. The data imply a significant role of Stat4-mediated signaling events in the generation of an aggressor CD4(+) T cell population in stromal keratitis pathogenesis.