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: 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.

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.

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.

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.

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.

Type 1 Interferons Potentiate Human CD8+ T-Cell Cytotoxicity Through a STAT4- and Granzyme B-Dependent Pathway.

Events defining the progression to human type 1 diabetes (T1D) have remained elusive owing to the complex interaction between genetics, the immune system, and the environment. Type 1 interferons (T1-IFN) are known to be a constituent of the autoinflammatory milieu within the pancreas of patients with T1D. However, the capacity of IFNα/ß to modulate human activated autoreactive CD8+ T-cell (cytotoxic T lymphocyte) responses within the islets of patients with T1D has not been investigated. Here, we engineer human ß-cell-specific cytotoxic T lymphocytes and demonstrate that T1-IFN augments cytotoxicity by inducing rapid phosphorylation of STAT4, resulting in direct binding at the granzyme B promoter within 2 h of exposure. The current findings provide novel insights concerning the regulation of effector function by T1-IFN in human antigen-experienced CD8+ T cells and provide a mechanism by which the presence of T1-IFN potentiates diabetogenicity within the autoimmune islet.

STAT4 Polymorphisms are Associated with Neuromyelitis Optica Spectrum Disorders.

STAT4 plays a crucial role in the functioning of the innate and adaptive immune cells and has been identified as a susceptibility gene in numerous autoimmune disorders. However, its association with neuromyelitis optica spectrum disorders (NMOSD) remains uncertain. Here, we performed a case-control study to determine whether STAT4 contributed to the risk of NMOSD. We tested five STAT4 SNPs in 233 patients with established NMOSD and 492 healthy controls. Chi-square tests and logistic regression analyses were performed with four genetic models, including allelic, additive, dominant, and recessive models, to identify associations with NMOSD. The results of multiple test comparisons were corrected using the Benjamini and Hochberg false discovery rate (FDR-BH). After correcting for multiple test comparisons, the minor alleles of four STAT4 SNPs exhibited significant association with increased risk of NMOSD (rs7574865 T, odds ratio [OR] = 1.66, 95% confidence interval [CI] 1.32-2.08, P corr = 0.000; rs10181656 G, OR = 1.62, 95% CI 1.29-2.03, P corr = 0.000; rs10168266 T, OR = 1.59, 95% CI 1.27-2.00, P corr = 0.001; and rs13426947 A, OR = 1.51, 95% CI 1.21-1.90, P corr = 0.004). Identical results were observed in the dominant, recessive, and additive models. In contrast, the G allele of rs7601754 displayed a protective effect against NMOSD (OR = 0.53, 95% CI 0.36-0.76, P corr = 0.006). Our study indicates that STAT4 polymorphisms are associated with the risk of NMOSD, which provides novel insights into the underlying mechanisms of this disease.

The pro-inflammatory signalling regulator Stat4 promotes vasculogenesis of great vessels derived from endothelial precursors.

Vasculogenic defects of great vessels (GVs) are a major cause of congenital cardiovascular diseases. However, genetic regulators of endothelial precursors in GV vasculogenesis remain largely unknown. Here we show that Stat4, a transcription factor known for its regulatory role of pro-inflammatory signalling, promotes GV vasculogenesis in zebrafish. We find stat4 transcripts highly enriched in nkx2.5+ endothelial precursors in the pharynx and demonstrate that genetic ablation of stat4 causes stenosis of pharyngeal arch arteries (PAAs) by suppressing PAAs 3-6 angioblast development. We further show that stat4 is a downstream target of nkx2.5 and that it autonomously promotes proliferation of endothelial precursors of the mesoderm. Mechanistically, stat4 regulates the emerging PAA angioblasts by inhibiting the expression of hdac3 and counteracting the effect of stat1a. Altogether, our study establishes a role for Stat4 in zebrafish great vessel development, and suggests that Stat4 may serve as a therapeutic target for GV defects.

Complement Activation and STAT4 Expression Are Associated with Early Inflammation in Diabetic Wounds.

Diabetic non-healing wounds are a major clinical problem. The mechanisms leading to poor wound healing in diabetes are multifactorial but unresolved inflammation may be a major contributing factor. The complement system (CS) is the most potent inflammatory cascade in humans and contributes to poor wound healing in animal models. Signal transducer and activator of transcription 4 (STAT4) is a transcription factor expressed in immune and adipose cells and contributes to upregulation of some inflammatory chemokines and cytokines. Persistent CS and STAT4 expression in diabetic wounds may thus contribute to chronic inflammation and delayed healing. The purpose of this study was to characterize CS and STAT4 in early diabetic wounds using db/db mice as a diabetic skin wound model. The CS was found to be activated early in the diabetic wounds as demonstrated by increased anaphylatoxin C5a in wound fluid and C3-fragment deposition by immunostaining. These changes were associated with a 76% increase in nucleated cells in the wounds of db/db mice vs. CONTROLS: The novel classical CS inhibitor, Peptide Inhibitor of Complement C1 (PIC1) reduced inflammation when added directly or saturated in an acellular skin scaffold, as reflected by reduced CS components and leukocyte infiltration. A significant increase in expression of STAT4 and the downstream macrophage chemokine CCL2 and its receptor CCR2 were also found in the early wounds of db/db mice compared to non-diabetic controls. These studies provide evidence for two new promising targets to reduce unresolved inflammation and to improve healing of diabetic skin wounds.

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.

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.

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.

HemoHIM ameliorates the persistent down-regulation of Th1-like immune responses in fractionated γ-irradiated mice by modulating the IL-12p70-STAT4 signaling pathway.

Whole body irradiated mice appear to experience a down-regulation of the helper T (Th)1-like immune response, and maintain a persistent immunological imbalance. In the current study, we evaluated the effect of HemoHIM (an herbal product made from Angelica Radix, Cnidium officinale , and Paeonia japonica cultivated in Korea) to ameliorate the immunological imbalance induce in fractionated γ-irradiated mice. The mice were exposed to γ rays twice a week (0.5 Gy fractions) for a total dose of 5 Gy, and HemoHIM was administrated orally from 1 week before the first irradiation to 1 week before the final analysis. All experiments were performed 4 and 6 months after their first exposure. HemoHIM ameliorated the Th1- and Th2-related immune responses normally occur in irradiated mice with or without dinitrophenylated keyhole limpet hemocyanin immunization. HemoHIM also restored the natural killer cell activities without changing the percentage of natural killer cells in irradiated mice. Furthermore, the administration of HemoHIM prevented the reduction in levels of interleukin-12p70 in irradiated mice. Finally, we found that HemoHIM enhanced the phosphorylation of signal transducer and activator of transcription (STAT) 4 that was reduced in irradiated mice. Our findings suggest that HemoHIM ameliorates the persistent down-regulation of Th1-like immune responses by modulating the IL-12p70/pSTAT4 signaling pathway.

Activation mechanisms of natural killer cells during influenza virus infection.

During early viral infection, activation of natural killer (NK) cells elicits the effector functions of target cell lysis and cytokine production. However, the cellular and molecular mechanisms leading to NK cell activation during viral infections are incompletely understood. In this study, using a model of acute viral infection, we investigated the mechanisms controlling cytotoxic activity and cytokine production in response to influenza (flu) virus. Analysis of cytokine receptor deficient mice demonstrated that type I interferons (IFNs), but not IL-12 or IL-18, were critical for the NK cell expression of both IFN-γ and granzyme B in response to flu infection. Further, adoptive transfer experiments revealed that NK cell activation was mediated by type I IFNs acting directly on NK cells. Analysis of signal transduction molecules showed that during flu infection, STAT1 activation in NK cells was completely dependent on direct type I IFN signaling, whereas STAT4 activation was only partially dependent. In addition, granzyme B induction in NK cells was mediated by signaling primarily through STAT1, but not STAT4, while IFN-γ production was mediated by signaling through STAT4, but not STAT1. Therefore, our findings demonstrate the importance of direct action of type I IFNs on NK cells to mount effective NK cell responses in the context of flu infection and delineate NK cell signaling pathways responsible for controlling cytotoxic activity and cytokine production.

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.

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.

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.

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.