Inactivation of dystrophin gene is the primary cause of Duchenne muscular dystrophy (DMD) in humans and mdx mice. However, the underpinning mechanisms, which govern the pathogenesis of dystrophin-deficient skeletal muscle, remain poorly understood. We have previously reported activation of mitogen-activated protein kinases (MAPK), nuclear factor-kappa B (NF-kappaB), and phosphatidyl-inositol 3-kinase/Akt (PI3K/Akt) signaling pathways in diaphragm muscle of mdx mice. In this study, using a protein-DNA array-based approach, we have investigated the activation of 345 transcription factors in diaphragm muscle of 6-week old normal and dystrophin-deficient mdx mice. Our data demonstrate increased activation of a number nuclear transcription factors including AP1, HFH-3, PPARalpha, c.myb BP, ETF, Fra-1/JUN, kBF-A, N-rasBP, lactoferrin BP, Myb(2), EBP40_45, EKLF(1), p53(2), TFEB, Myc-Max; c-Rel; E2, ISRE; NF-kB; Stat1 p84/p91, Antioxidant RE, EVI-1, Stat3, AP3, p53, Stat4, AP4, HFH-1, FAST-1, Pax-5, and Beta-RE in the diaphragm muscle of mdx mice compared to corresponding normal mice. The level of activation for p53 was highest among all the transcription factors studied. Furthermore, higher activation of p53 in diaphragm muscle of mdx mice was associated with its increased phosphorylation and nuclear translocation. Collectively, our data suggest that the primary deficiency of dystrophin leads to the aberrant activation of nuclear transcription factors which might further contribute to muscle pathogenesis in mdx mice.

Curcumin is a polyphenol derived from the dietary spice turmeric. It possesses diverse anti-inflammatory and anti-cancer properties. Curcumin has been shown to exhibit an inhibitory effect on the production of inflammatory cytokines by human monocytes and has inhibited the animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE) in association with a decrease in interleukin 12 (IL-12) production and signal transducer and activator of transcription 4 (STAT4) activation. The type I interferon (IFN) IFN-has the ability to suppress IL-12. Both IL-12 and IFN-alpha/beta signal through the activation by phosphorylation of STAT4. Our aim was to investigate the effects of curcumin on the ability of T cells to respond to IL-12 or IFN-alpha/beta. We report that curcumin decreases IL-12-induced STAT4 phosphorylation, IFN-gamma production, and IL-12 Rbeta1 and beta2 expression. IFN-beta-induced STAT4 phosphorylation, IL-10 production and IFN receptor (IFNAR) subunits 1 and 2 expression were enhanced by curcumin. Curcumin increased IFN-alpha-induced IL-10 and IFNAR1 expression. Prior exposure to curcumin decreased IFN-alpha-induced IFNAR2 expression and did not modify the level of IFN-alpha-induced pSTAT4 generation. Thus, the effect of curcumin on STAT4 activation in T cells is dependent upon the stimulus to which the T cells have been exposed.

Deregulation of STAT signaling has been implicated in the pathogenesis for a variety of cancers, including CTCL. Recent reports indicate that loss of STAT4 expression is an important prognostic marker for CTCL progression and is associated with the acquisition of T helper 2 cell phenotype by malignant cells. However, little is known about the molecular mechanism behind the downregulation of STAT4 in this cancer. In the current work we test the expression of STAT4 and STAT6 via RT-PCR and/or Western Blot in CTCL lesional skin samples and in immortalized patient-derived cell lines. In these malignant cell lines we correlate the expression of STAT4 and STAT6 with the T helper (Th) phenotype markers and test the effect of Histone Deacetylase (HDAC) inhibitors and siRNA-mediated knock down of miR-155 on STAT4 expression. Our findings demonstrate that STAT4 expression correlates with Th1 phenotype, while STAT6 is associated with the Th2 phenotype. Our results further document that STAT4 and STAT6 genes are inversely regulated in CTCL. Treatment with HDAC inhibitors upregulates STAT4 expression, while at the same time decreases STAT6 expression in MyLa cells. Also, siRNA-mediated knock down of miR-155 leads to upregulation in STAT4 expression in MyLa cells. In summary, our results suggest that loss of STAT4 expression and associated switch to Th2 phenotype during Mycosis Fungoides progression may be driven via aberrant histone acetylation and/or upregulation of oncogenic miR-155 microRNA.

DNA methylation, a major epigenetic mechanism, may regulate coordinated expression of multiple genes at specific time points during alveolar septation in lung development. The objective of this study was to identify genes regulated by methylation during normal septation in mice and during disordered septation in bronchopulmonary dysplasia. In mice, newborn lungs (preseptation) and adult lungs (postseptation) were evaluated by microarray analysis of gene expression and immunoprecipitation of methylated DNA followed by sequencing (MeDIP-Seq). In humans, microarray gene expression data were integrated with genome-wide DNA methylation data from bronchopulmonary dysplasia versus preterm and term lung. Genes with reciprocal changes in expression and methylation, suggesting regulation by DNA methylation, were identified. In mice, 95 genes with inverse correlation between expression and methylation during normal septation were identified. In addition to genes known to be important in lung development (Wnt signaling, Angpt2, Sox9, etc.) and its extracellular matrix (Tnc, Eln, etc.), genes involved with immune and antioxidant defense (Stat4, Sod3, Prdx6, etc.) were also observed. In humans, 23 genes were differentially methylated with reciprocal changes in expression in bronchopulmonary dysplasia compared with preterm or term lung. Genes of interest included those involved with detoxifying enzymes (Gstm3) and transforming growth factor-β signaling (bone morphogenetic protein 7 [Bmp7]). In terms of overlap, 20 genes and three pathways methylated during mouse lung development also demonstrated changes in methylation between preterm and term human lung. Changes in methylation correspond to altered expression of a number of genes associated with lung development, suggesting that DNA methylation of these genes may regulate normal and abnormal alveolar septation.

A significant source of variability in the literature on systemic lupus erythematosus (SLE) susceptibility genes has been the inability to replicate genetic findings across different racial or ethnic groups. We investigated whether a single nucleotide polymorphism (SNP) of the STAT4 (rs7574865), PTPN22 (rs2476601), TRAF1/C5 (rs10818488), and C1q (rs292001) genes as well as the 27-bp VNTR polymorphism on intron 4 of eNOS, previously associated with SLE in other populations, are also associated with SLE risk in Turkey. A group of 158 SLE patients and 155 healthy controls were included in this study. A genetic association of the TRAF1/C5, C1q, and eNOS gene polymorphism, but not of STAT4 and PTPN22, was found to confer a degree of risk for SLE. These data highlight the importance of comparative studies in different populations to confirm the previously detected genetic associations.

In this study we examined mechanisms that regulate T-helper lymphocyte 1 (Th1) commitment in Helicobacter pylori-infected human gastric mucosa. The levels of gamma interferon (IFN-gamma), interleukin-4 (IL-4), and IL-12 in total extracts of gastric biopsies taken from H. pylori-infected and uninfected patients were determined by an enzyme-linked immunosorbent assay. The levels of signal transducer and activator of transcription 4 (STAT4), STAT6, and T-box expressed in T cells (T-bet) in total proteins extracted from gastric biopsies were determined by Western blotting. Finally, the effect of a neutralizing IL-12 antibody on expression of Th1 transcription factors and the levels of IFN-gamma in organ cultures of H. pylori-infected biopsies was examined. Increased levels of IFN-gamma and IL-12 were found in gastric biopsy samples of H. pylori-infected patients compared to the levels in uninfected patients. In addition, H. pylori-infected biopsies exhibited high levels of expression of phosphorylated STAT4 and T-bet. Higher levels of IFN-gamma and expression of Th1 transcription factors were associated with greater infiltration of mononuclear cells in the gastric mucosa. By contrast, production of IL-4 and expression of phosphorylated STAT6 were not associated with the intensity of mononuclear cell infiltration. In ex vivo organ cultures of H. pylori-infected biopsies, neutralization of endogenous IL-12 down-regulated the expression of phosphorylated STAT4 and T-bet and reduced IFN-gamma production. Our data indicated that IL-12 contributes to the Th1 cell commitment in H. pylori-infected human gastric mucosa.

Signal transducer and activator of transcription 4 (STAT4) has been recently identified as a susceptibility gene for multiple autoimmune diseases. Here we performed a comprehensive analysis of the association between STAT4 and several different autoimmune disorders to identify potential common inflammatory principles behind this association. Our meta-analysis revealed that the STAT4 rs7574865 polymorphism is associated with four autoimmune diseases with systemic pathology, including systemic lupus erythematosus (OR = 1.52; 95% CI = 1.48 - 1.56, P<1.0 × 10(-16)), rheumatoid arthritis (OR = 1.27; 95% CI = 1.21 - 1.33, P < 1.00 × 10(-16)), systemic sclerosis (OR = 1.38; 95% CI = 1.27 - 1.50, P < 1.44 × 10(-14)), and primary Sjogren's syndrome (OR = 1.32; 95% CI = 1.01 - 1.73, P = 4.40 × 10(-2)), while no association was found with type I diabetes, juvenile idiopathic arthritis, ulcerative colitis and Crohn's disease. Furthermore, the stratified meta-analysis also demonstrate that the STAT4 rs7574865 polymorphism is associated with the presence of autoantibodies with systemic reactivity (anti-ds-DNA antibodies) in SLE patients (OR = 1.37; 95% CI = 1.21 - 1.56, P = 1.12 × 10(-6)). However, no such specific association was seen in RA with regard to the presence of non-systemically reacting antibodies, including rheumatoid factor and anti-cyclic citrullinated peptide antibodies. Taken together, these results suggest that STAT4 polymorphisms are associated with autoimmune diseases which are characterized by a systemic pathology and anti-dsDNA antibody.

BACKGROUND

This study investigated five confirmed rheumatoid arthritis (RA) susceptibility genes/loci (HLA-DRB1, PTPN22, STAT4, OLIG3/TNFAIP3 and TRAF1/C5) for association with susceptibility and severity in an inception cohort.

METHODS

The magnitude of association for each genotype was assessed in 1,046 RA subjects from the Yorkshire Early RA cohort and in 5,968 healthy UK controls. Additional exploratory subanalyses were undertaken in subgroups defined by autoantibody status (rheumatoid factor and anti-cyclic citrullinated peptide) or disease severity (baseline articular erosions, Health Assessment Questionnaire (HAQ) score and swollen joint count (SJC)).

RESULTS

In the total RA inception cohort, the HLA-DRB1 shared epitope (per-allele odds ratio (OR) = 2.1, trend P < 0.0001), PTPN22 (per-allele OR = 1.5, trend P < 0.0001), OLIG3/TNFAIP3 locus (per-allele OR = 1.2, trend P = 0.009) and TRAF1/C5 locus (per-allele OR = 1.1, trend P = 0.04) were associated with RA. The magnitude of association for these loci was increased in those patients who were autoantibody-positive. PTPN22 was associated with autoantibody-negative RA (per-allele OR = 1.3, trend P = 0.04). There was no evidence of association between these five genetic loci and baseline erosions or SJC in the total RA cohort, after adjustment for symptom duration. TRAF1/C5 was significantly associated with baseline HAQ, however, following adjustment for symptom duration (P trend = 0.03).

CONCLUSIONS

These findings support the mounting evidence that different genetic loci are associated with autoantibody-positive and autoantibody-negative RA, possibly suggesting that many of the genes identified to date are associated with autoantibody production. Additional studies with a specific focus on autoantibody-negative RA will be needed to identify the genes predisposing to this RA subgroup. The TRAF1/C5 locus in particular warrants further investigation in RA as a potential disease severity locus.

Polymorphisms in the transcription factor Stat4 gene have been implicated as risk factors for systemic lupus erythematosus. Although some polymorphisms have a strong association with autoantibodies and nephritis, their impact on pathophysiology is still unknown. To explore this further we used signal transducers and activators of transcription 4 (Stat4) knockout MRL/MpJ-Fas(lpr)/Fas(lpr) (MRL-Fas(lpr)) mice and found that they did not differ in survival or renal function from Stat4-intact MRL-Fas(lpr) mice. Circulating interleukin (IL)-18 levels, however, were elevated in Stat4-deficient compared to Stat4-intact mice, suggesting that this interleukin might contribute to the progression of lupus nephritis independent of Stat4. In a second approach, Stat4 antisense or missense oligonucleotides or vehicle were given to MRL-Fas(lpr) mice with advanced nephritis. Each of these treatments temporarily ameliorated disease, although IL-18 was increased in each setting. Based on these findings, studies using gene transfer to overexpress IL-18 in MRL-Fas(lpr) and IL-12p40/IL-23 knockout MRL-Fas(lpr) mice reveal a critical role for IL-18 in mediating disease. Thus, the Stat4 and IL-12 (an activator of Stat4)-independent factor, IL-18, can drive autoimmune lupus nephritis in MRL-Fas(lpr) mice. Temporarily blocking Stat4 during advanced nephritis ameliorates disease, suggesting a time-dependent compensatory proinflammatory mechanism.

The aim of this study was to determine whether the rs7574865 polymorphism of STAT4 (signal transducers and activators of transcription 4) confers susceptibility to rheumatoid arthritis (RA) in populations with different ethnicities. A meta-analysis was conducted on the T allele of the STAT4 rs7574865 polymorphism in 15 studies containing 16,088 RA patients and 16,509 normal control subjects. Meta-analysis revealed an association between RA and the STAT4 rs7574865 T allele in all subjects (OR = 1.271, 95% CI = 1.197-1.350, P < 0.001). Furthermore, the STAT4 rs7574865 T allele was found to be significantly associated with RA in Europeans and Asians (OR = 1.300, 95% CI = 1.195-1.414, P < 0.001; OR = 1.216, 95% CI = 1.135-1.303, P < 0.001). Stratification of RA patients according to the presence of anti-CCP antibody revealed a statistically significant association between the T allele and RA in both anti-CCP-positive and -negative RA patients versus controls. Europeans had the lowest (21.4%) and Asians had the highest (32.0%) prevalence of the T allele among the populations studied. In conclusion, this meta-analysis confirms that the STAT4 rs7574865 polymorphism is associated with RA susceptibility in different ethnic groups, and that its prevalence is ethnicity dependent.

Chronic nasal and skin colonization with superantigen (SAg)-producing Staphylococcus aureus is well documented in humans. Given that trans-mucosal and trans-cutaneous absorption of SAgs can occur, we determined whether chronic exposure to small amounts of SAg per se could activate autoreactive CD4(+) and CD8(+) T cells and precipitate any autoimmune disease without further external autoantigenic stimulation. Because HLA class II molecules present SAg more efficiently than do mouse MHC class II molecules, HLA-DQ8 transgenic mice were implanted s.c. with mini-osmotic pumps capable of continuously delivering the SAg, staphylococcal enterotoxin B (total of 10 μg/mouse), or PBS over 4 wk. Chronic exposure to staphylococcal enterotoxin B resulted in a multisystem autoimmune inflammatory disease with features similar to systemic lupus erythematosus. The disease was characterized by mononuclear cell infiltration of lungs, liver, and kidneys, accompanied by the production of anti-nuclear Abs and deposition of immune complexes in the renal glomeruli. The inflammatory infiltrates in various organs predominantly consisted of CD4(+) T cells bearing TCR Vβ8. The extent of immunopathology was markedly reduced in mice lacking CD4(+) T cells and CD28, indicating that the disease is CD4(+) T cell mediated and CD28 dependent. The absence of disease in STAT4-deficient, as well as IFN-γ-deficient, HLA-DQ8 mice suggested the pathogenic role of Th1-type cytokines, IL-12 and IFN-γ. In conclusion, our study suggests that chronic exposure to extremely small amounts of bacterial SAg could be an etiological factor for systemic lupus erythematosus.

CD4(+) T helper 1 (Th1) cells and Th2 cells are distinguished based on the pattern of cytokines they are able to produce. Selectin ligands and chemokine receptors are differentially expressed in Th1 and Th2 cells, providing a basis for tissue-specific recruitment of helper T-cell subsets. However, the modes and mechanisms regulating tissue-specific localization of Th1 and Th2 cells are still largely unknown. Here, we show the preferential expression on Th1 cells of the integrin alpha6/beta1, which is distinctly regulated by the Th1-inducing cytokines interleukin-12 (IL-12) and interferon-alfa (IFN-alpha). The pattern of integrin alpha6/beta1 regulation closely mirrors that of the chemokine receptor CCR1. Analysis of signal transducer and activator of transcription 4 (Stat4) activation by IL-12 and IFN-alpha shows distinct signaling kinetics by these cytokines, correlating with the pattern of CCR1 and integrin alpha6/beta1 expression. Unlike IFN-alpha, the ability of IL-12 to generate prolonged intracellular signals appears to be critical for inducing integrin alpha6/beta1 upregulation in Th1 cells. The expression and upregulation of CCR1 and alpha6/beta1 integrin promotes the migration of Th1 cells. These findings suggest that the exquisite regulation of integrin alpha6/beta1 and CCR1 may play an important role in tissue-specific localization of Th1 cells.

Insulin resistance (IR) is associated with an increased risk of cardiovascular diseases. The obese Zucker rat (ZR) is a model of IR that shows markedly increased insulin and triglyceride concentrations without major changes in glucose. In this study, we evaluated the response of obese and lean ZR to carotid balloon injury and determined potential mechanisms and treatments. The neointima-to-media ratio of obese ZR was greater than that of lean ZR, starting at 14 days after injury, and persisted until at least day 30. An enhanced inflammatory response to balloon injury in the obese ZR was reflected by significantly higher ED1-positive macrophage cells in the injured vessel wall compared with that in lean ZR at 3, 7, and 14 days after balloon injury. Inflammatory mediators 12-lipoxygenase (12-LO) and STAT4 were studied in neointimal lesions. Expression of 12-LO RNA was increased beginning at day 7 and showed increases of 4.3-fold on day 14 and 7-fold on day 30 in obese ZR compared with lean animals. Staining of phosphorylated STAT4 (PSTAT4), the activated form of STAT4, in lesions from obese ZR was also increased compared with that in leans. We tested the effects of a novel anti-inflammatory agent, lisofylline (LSF), in the obese ZR. LSF markedly reduced neointimal formation in the obese ZR. LSF also reduced monocyte/macrophage infiltration into the vessel wall and the activation of PSTAT4. These studies suggest both the presence of an exaggerated injury response in the insulin-resistant obese ZR model and that inflammation plays a major role in mediating neointimal growth.

Photodynamic therapy (PDT) is a local treatment of cancers. The principle of PDT is the production of reactive oxygen species, in particular singlet oxygen, by light activation of a photosensitizer introduced into the target cells. The direct photochemical and subsequent redox reactions can lead to cell death. This study sought to identify effects occurring during PDT and some of their consequences in surviving cells. Using epithelial cells in tissue culture and in tumors, several distinct PDT-mediated reactions were found, including global dephosphorylation of proteins, induced phosphorylation of a 71-kDa protein, initiation of cellular stress responses, structural modification and loss of epidermal growth factor receptor, and cross-linking of proteins. Specific covalent cross-linking of nonactivated signal transducer and activator of transcription (STAT)-3, and to a lesser extent of STAT1 and STAT4, correlated with PDT dose. Cross-linked STAT3 was primarily localized to the cytoplasm and failed to bind to DNA. The combination of STAT cross-linking and inactivation of receptor functions rendered PDT-treated cells refractory for at least 24 hours to interleukin-6 and oncostatin M, cytokines known to be elevated at site of tissue damage and inflammation. It is suggested that the loss of responsiveness to these inflammatory cytokines in the PDT-treated field assists tumor cells in evading the growth-suppressive activity of these mediators expected to be present at tissue sites after PDT.

Inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), are chronic inflammatory diseases with unsolved pathogenesis. Imbalanced Th1/Th2 may play a role in the sustained inflammation of IBD. In China, CD is rare but the incidence of UC has been rising steadily in the last two decades. We investigated the expression of IL-12 (p40) and IFN-gamma, and the activational state of Stat4 signaling in mucosal tissues at the site of disease from 30 active UC patients in comparison with 30 healthy controls. RT-PCR analyses revealed increased mRNA expression of IL-12 (p40) but not IFN-gamma in UC patients. Western blot analyses discovered, for the first time, increased levels of constitutive Stat4 in the cytoplasm and phosphorylated Stat4 in the nucleus of mucosal cells from UC patients. We conclude that a heightened, perhaps persistent, activational state of IL-12/Stat4, and/or IL-23/Stat4 signaling may be present in active Chinese UC patients, and possibly involved in chronic inflammation in UC.

CD4(+) T cells regulate adaptive responses to pathogens by secreting unique subsets of cytokines that mediate inflammatory processes. The innate cytokines IL-12 and IFN-alpha/beta regulate type I responses and promote acute IFN-gamma secretion through the activation of the STAT4 transcription factor. Although IL-12-induced STAT4 activation is a conserved pathway across species, IFN-alpha/beta-dependent STAT4 phosphorylation does not occur as efficiently in mice as it does in human T cells. In order to understand this species-specific pathway for IFN-alpha/beta-dependent STAT4 activation, we have examined the molecular basis of STAT4 recruitment by the human IFNAR. In this report, we demonstrate that the N-domain of STAT4 interacts with the cytoplasmic domain of the human, but not the murine IFNAR2 subunit. This interaction mapped to a membrane-proximal segment of the hIFNAR2 spanning amino acids 299-333. Deletion of this region within the hIFNAR2 completely abolishes IFN-alpha/beta-dependent STAT4 tyrosine phosphorylation when expressed in human IFNAR2-deficient fibroblasts. Thus, the human IFNAR2 cytoplasmic domain serves to link STAT4 to the IFNAR as a pre-assembled complex that facilitates cytokine-driven STAT4 activation.

Rheumatoid arthritis (RA) is one of the most common autoimmune diseases. As with other complex traits, genome-wide association studies (GWASs) have tremendously enhanced our understanding of the complex etiology of RA. In this review, we describe the genetic architecture of RA as determined through GWASs and meta-analyses. In addition, we discuss the pathologic mechanism of the disease by examining the combined findings of genetic and functional studies of individual RA-associated genes, including HLA-DRB1, PADI4, PTPN22, TNFAIP3, STAT4, and CCR6. Moreover, we briefly examine the potential use of genetic data in clinical practice in RA treatment, which represents a challenge in medical genetics in the post-GWAS era.

Recently we reported the results of a genome-wide disease phenocode analysis interrogating the relationships between structural features and gene expression patterns of disease-linked SNPs, microRNAs and mRNAs of protein-coding genes in association to phenotypes of 15 common human disorders (Cell Cycle 2008;7:2570-83; 3564-76). One of the main implications of this analysis is that transcriptionally co-regulated SNP sequence-bearing RNAs are more likely to exert a cumulative effect in trans on phenotypes. In this work, we test the validity of a disease phenocode concept within a genomic context of distinct continuously spaced sets of disease-linked SNPs and mRNAs of relevant proteincoding genes. We report a sequence homology profiling of two sets of disease-linked SNPs which are located within continuous genomic regions associated with individual protein-coding genetic loci (NLRP1 and STAT4) and are likely to exhibit common profiles of transcriptional activity. Most of microRNAs (15 of 19; 79%) homologous to the NLPRP1-associated disease-linked SNPs have potential protein-coding mRNA targets among the principal components of the nuclear import pathway (NIP) and/or inflammasome pathways, including KPNA1, NLRP1 and NLRP3 genes. We demonstrate that estimates of cumulative targeting effects of microRNAs on mRNAs within distinct allelic contexts of disease-linked SNPs are in agreement with microarray analysis-defined gene expression phenotypes associated with human genotypes of Crohn's disease (CD) and rheumatoid arthritis (RA) populations. Microarray experiments and disease phenocode analysis identify ten-gene expression signatures which seem to reflect the activated status of disease-linked SNPs/microRNAs/mRNAs axis in peripheral blood mononuclear cells (PBMC) of 66% CD patients and 80% RA patients. Comparisons of ten-gene signature expression profiles and NLRP3/NLRP1 mRNA expression ratios in PBMC of individual CD and RA patients and control subjects indicate that measurements of these markers may be useful for diagnostic applications. Our findings demonstrate that NLPRP1- and STAT4-associated disease-linked SNPs have common sequence-defined features which are recapitulating the essential phenotype-affecting features of genome-wide disease-linked SNPs, suggesting that NLRP1 (NALP1) and STAT4 genetic loci may constitute "master" disease genes. We conclude that both genome-wide SNP variations and SNP polymorphisms associated with "master" disease genes may cause similar genetically-defined malfunctions of the NIP and inflammasome/innate immunity pathways which are likely to contribute to pathogenesis of multiple common human disorders.

The objective of this study was to determine if genistein (GEN) modulation of the immune responses might contribute to the increased host resistances to tumors. A time-course study was performed in adult female B6C3F1 mice that had been exposed to GEN for 1-4 weeks at the dose level of 20 mg/kg by gavage. A significant increase in ex vivo cytotoxic T lymphocyte (CTL) activity was observed in the periods of 2 weeks and 4 weeks. Moreover, increased activities of CTLs were associated with a decrease in the percentage of CD4(+)CD25(+) T cells and an increase in the production of interferon-gamma and activation of STAT1 (signal transducer and activator of transcription 1) and STAT4. Additionally, exposure of mice to GEN increased the activities of in vivo CTLs. An increased activity of natural killer (NK) cells was also observed. Further study in the B16F10 tumor model suggested that GEN-mediated enhancement in host resistance to B16F10 tumor was partially related to its potentiating effect on NK cells. Finally, 7,12-dimethylbenz[a]anthracene (DMBA)-induced tumor model was employed to determine the chemopreventive effect of oral GEN treatment. Mice pretreated with GEN for 2 weeks by gavage, the time when an enhanced CTL activity had been produced, had a decreased susceptibility toward DMBA-mediated carcinogenesis, while treatment with GEN after tumor induction conferred no protection. In conclusion, pretreatment with GEN by gavage could enhance host resistances to the B16F10 tumor and DMBA-induced carcinogenesis, suggesting that GEN modulation of immune response was, at least partially, responsible for the antitumor effect of this compound.

IL-12 promotes T(h)1 development/IFN-gamma expression by activating STAT4. However, it is still unclear how STAT4 elicits IFN-gamma promoter activation. Here, we investigated the mechanism by which IL-12-activated STAT4 functions for IFN-gamma induction in TCR-triggered T cells. TCR stimulation induced high levels of IFN-gamma production depending on co-stimulation with IL-12. IL-12 stimulation greatly enhanced the promoter-binding activity of c-Jun/AP-1, a critical transcription factor for IFN-gamma gene expression in wild-type T cells, but not in STAT4-deficient (STAT4(-/-)) T cells. Comparable amounts of c-Jun were induced by TCR stimulation in both wild-type and STAT4(-/-) T cells irrespective of IL-12 co-stimulation. However, c-Jun bound to STAT4 in IL-12-co-stimulated wild-type T cells. c-Jun forming a complex with STAT4 efficiently interacted with the AP-1-related sequence of the IFN-gamma promoter. Such an enhanced c-Jun binding did not occur in STAT4(-/-) T cells. These results show that STAT4 contributes to enhancing IFN-gamma expression by up-regulating the binding of TCR signal-induced AP-1 to the relevant promoter sequence.

BACKGROUND

The aim of the study was to interrogate the genetic architecture and autoimmune pleiotropy of scleroderma susceptibility in the Australian population.

METHODS

We genotyped individuals from a well-characterized cohort of Australian scleroderma patients with the Immunochip, a custom array enriched for single nucleotide polymorphisms (SNPs) at immune loci. Controls were taken from the 1958 British Birth Cohort. After data cleaning and adjusting for population stratification the final dataset consisted of 486 cases, 4,458 controls and 146,525 SNPs. Association analyses were conducted using logistic regression in PLINK. A replication study was performed using 833 cases and 1,938 controls.

RESULTS

A total of eight loci with suggestive association (P <10-4.5) were identified, of which five showed significant association in the replication cohort (HLA-DRB1, DNASE1L3, STAT4, TNP03-IRF5 and VCAM1). The most notable findings were at the DNASE1L3 locus, previously associated with systemic lupus erythematosus, and VCAM1, a locus not previously associated with human disease. This study identified a likely functional variant influencing scleroderma susceptibility at the DNASE1L3 locus; a missense polymorphism rs35677470 in DNASE1L3, with an odds ratio of 2.35 (P = 2.3 × 10(-10)) in anti-centromere antibody (ACA) positive cases.

CONCLUSIONS

This pilot study has confirmed previously reported scleroderma associations, revealed further genetic overlap between scleroderma and systemic lupus erythematosus, and identified a putative novel scleroderma susceptibility locus.

Plumbagin (PL), a herbal compound derived from roots of the medicinal plant Plumbago zeylanica, has been shown to have immunosuppressive properties. Present report describes that PL is a potent novel agent in control of encephalitogenic T cell responses and amelioration of mouse experimental autoimmune encephalomyelitis (EAE), through down-regulation of JAK-STAT pathway. PL was found to selectively inhibit IFN-γ and IL-17 production by CD4(+) T cells, which was mediated through abrogated phosphorylation of JAK1 and JAK2. Consistent with IFN-γ and IL-17 reduction was suppressed STAT1/STAT4/T-bet pathway which is critical for Th1 differentiation, as well as STAT3/ROR pathway which is essential for Th17 differentiation. In addition, PL suppressed pro-inflammatory molecules such as iNOS, IFN-γ and IL-6, accompanied by inhibition of IκB degradation as well as NF-κB phosphorylation. These data give new insight into the novel immune regulatory mechanism of PL and highlight the great value of this kind of herb compounds in probing the complex cytokine signaling network and novel therapeutic targets for autoimmune diseases.

OBJECTIVE

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with a strong genetic component. We undertook the present work to perform the first genome-wide association study on individuals from the Americas who are enriched for Native American heritage.

METHODS

We analyzed 3,710 individuals from the US and 4 countries of Latin America who were diagnosed as having SLE, and healthy controls. Samples were genotyped with HumanOmni1 BeadChip. Data on out-of-study controls genotyped with HumanOmni2.5 were also included. Statistical analyses were performed using SNPtest and SNPGWA. Data were adjusted for genomic control and false discovery rate. Imputation was performed using Impute2 and, for classic HLA alleles, HiBag. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated.

RESULTS

The IRF5-TNPO3 region showed the strongest association and largest OR for SLE (rs10488631: genomic control-adjusted P [Pgcadj ] = 2.61 × 10(-29), OR 2.12 [95% CI 1.88-2.39]), followed by HLA class II on the DQA2-DQB1 loci (rs9275572: Pgcadj = 1.11 × 10(-16), OR 1.62 [95% CI 1.46-1.80] and rs9271366: Pgcadj = 6.46 × 10(-12), OR 2.06 [95% CI 1.71-2.50]). Other known SLE loci found to be associated in this population were ITGAM, STAT4, TNIP1, NCF2, and IRAK1. We identified a novel locus on 10q24.33 (rs4917385: Pgcadj = 1.39 × 10(-8)) with an expression quantitative trait locus (eQTL) effect (Peqtl = 8.0 × 10(-37) at USMG5/miR1307), and several new suggestive loci. SLE risk loci previously identified in Europeans and Asians were corroborated. Local ancestry estimation showed that the HLA allele risk contribution is of European ancestral origin. Imputation of HLA alleles suggested that autochthonous Native American haplotypes provide protection against development of SLE.

CONCLUSIONS

Our results demonstrate that studying admixed populations provides new insights in the delineation of the genetic architecture that underlies autoimmune and complex diseases.

We have previously shown that Toll-like receptor (TLR) agonists contribute to the control of viral infection by augmenting virus-specific CD8(+) T-cell responses. It is also well established that signaling by TLRs results in the production of pro-inflammatory cytokines such as interleukin 6 (IL-6). However, how these pro-inflammatory cytokines influence the virus-specific CD8(+) T-cell response during the TLR agonist stimulation remained largely unknown. Here, we investigated the role of TLR-induced IL-6 in shaping virus-specific CD8(+) T-cell responses in the Friend retrovirus (FV) mouse model. We show that the TLR agonist induced IL-6 counter-regulates effector CD8(+) T-cell responses. IL-6 potently inhibited activation and cytokine production of CD8(+) T cells in vitro. This effect was mediated by a direct stimulation of CD8(+) T cells by IL-6, which induced upregulation of STAT3 phosphorylation and SOCS3 and downregulated STAT4 phosphorylation and T-bet. Moreover, combining TLR stimulation and IL-6 blockade during an acute FV infection resulted in enhanced virus-specific CD8(+) T-cell immunity and better control of viral replication. These results have implications for our understanding of the role of TLR induced pro-inflammatory cytokines in regulating effector T cell responses and for the development of therapeutic strategies to overcome T cell dysfunction in chronic viral infections.