BACKGROUND

Cytokine-activated transcription factors from the STAT (Signal Transducers and Activators of Transcription) family control common and context-specific genetic programs. It is not clear to what extent cell-specific features determine the binding capacity of seven STAT members and to what degree they share genetic targets. Molecular insight into the biology of STATs was gained from a meta-analysis of 29 available ChIP-seq data sets covering genome-wide occupancy of STATs 1, 3, 4, 5A, 5B and 6 in several cell types.

RESULTS

We determined that the genomic binding capacity of STATs is primarily defined by the cell type and to a lesser extent by individual family members. For example, the overlap of shared binding sites between STATs 3 and 5 in T cells is greater than that between STAT5 in T cells and non-T cells. Even for the top 1,000 highly enriched STAT binding sites, ~15% of STAT5 binding sites in mouse female liver are shared by other STATs in different cell types while in T cells ~90% of STAT5 binding sites are co-occupied by STAT3, STAT4 and STAT6. In addition, we identified 116 cis-regulatory modules (CRM), which are recognized by all STAT members across cell types defining a common JAK-STAT signature. Lastly, in liver STAT5 binding significantly coincides with binding of the cell-specific transcription factors HNF4A, FOXA1 and FOXA2 and is associated with cell-type specific gene transcription.

CONCLUSIONS

Our results suggest that genomic binding of STATs is primarily determined by the cell type and further specificity is achieved in part by juxtaposed binding of cell-specific transcription factors.

OBJECTIVE

Subtypes of juvenile idiopathic arthritis (JIA) share phenotypic features with other autoimmune disorders. We investigated several genetic variants associated with rheumatoid arthritis (RA) and other autoimmune disorders for association with JIA to test the hypothesis that clinically distinct phenotypes share common genetic susceptibility factors.

METHODS

Cases were 445 children with JIA, and controls were 643 healthy adults. Using the TaqMan assay, subjects were genotyped for 8 single-nucleotide polymorphisms in 7 loci including rs10499194 and rs6920220 in the TNFAIP3 locus, rs6679677 in the RSBN1 locus, rs17696736 in the C12orf30 locus, rs3761847 in the TRAF1/C5 locus, rs2104286 in the IL2RA locus, rs7574865 in the STAT4 locus, and rs2542151 in the PTPN2 locus. Alleles and genotypes were analyzed for association with JIA and JIA subtypes. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated.

RESULTS

The strongest associations with JIA risk or protection were observed for TNFAIP3 variants rs10499194 (OR 0.74 [95% CI 0.61-0.91], P < 0.004) and rs6920220 (OR 1.30 [95% CI 1.05-1.61], P = 0.015). We also observed associations between JIA and both STAT4 (OR 1.24 [95% CI 1.02-1.51], P = 0.029) and C12orf30 (OR 1.20 [95% CI 1.01-1.43], P = 0.041) variants. The PTPN2 variant rs2542151 deviated from Hardy-Weinberg equilibrium and was excluded from analyses. Variants in IL2RA, TRAF1/C5, and RSBN1 were not associated with JIA. After stratification by JIA subtype, the TNFAIP3 and C12orf30 variants were associated with oligoarticular JIA, while the STAT4 variant was associated primarily with polyarticular JIA.

CONCLUSIONS

We have demonstrated associations between JIA and variants in the TNFAIP3, STAT4, and C12orf30 regions that have previously shown associations with other autoimmune diseases, including RA and systemic lupus erythematosus. Our results suggest that clinically distinct autoimmune phenotypes share common genetic susceptibility factors.

It is now well appreciated that the cytokines interleukin (IL)-12 and IL-4 are important for the generation of Th1 and Th2 cells, respectively. Only recently, however, have the molecular mechanisms by which these cytokines affect Th cell differentiation begun to be defined. Previous work from our laboratory has demonstrated that members of the signal transducer and activator of transcription (STAT) gene family are critical for the differentiation of Th cell subsets. In particular, Stat4-deficient mice show an impairment in the generation of Th1 cells, whereas Stat6-deficient animals do not generate Th2 cells. We have now generated Stat4-Stat6 double-deficient mice to determine whether STAT-independent pathways exist for the development of Th cell subsets. It is surprising that Th1 but not Th2 cells can be generated from double-deficient mice in vitro and these animals are able to mount an in vivo Th1 cell-mediated immune response. These results suggest a model of Th cell differentiation in which Stat4 and Stat6 have different roles in the development of Th cell subsets.

In this report, we examined the molecular basis underlying the genetic difference between BALB/c and B10.D2 T cells for T helper phenotype development in vitro. We found a strain-dependent difference in early maintenance of IL-12 responsiveness by T cells developing in vitro in unmanipulated (neutral) conditions. Thus, when activated without addition of exogenous cytokines or neutralization of endogenous cytokines, B10.D2, but not BALB/c, T cells remain responsive to IL-12 when activated for 7 days. The pattern of IL-12 responsiveness correlated with expression of the IL-12R signaling subunit, IL-12R beta2, and with IL-12-induced STAT4 phosphorylation. When activated under neutral conditions, BALB/c T cells rapidly lose IL-12R beta2 expression, STAT4 phosphorylation, and functional IL-12 responsiveness. More efficient maintenance of IL-12R beta2 expression by B10.D2 T cells activated under neutral conditions may explain the previously observed increase in IFN-gamma production relative to that of BALB/c. This difference could potentially provide greater protection from certain pathogens that do not immediately elicit strong Th1-inducing conditions via activation of the innate immune system.

The crystal structures of the N-terminal domain (N-domain) and the core region of the STAT family of transcription factors have been determined previously. STATs can form cooperative higher order structures (tetramers or higher oligomers) while bound to DNA. The crystal packing in the STAT4 N-domain crystal structure, determined at 1.5 A resolution, suggests two alternate organizations of the N-domain dimer. We now present the results of site directed mutagenesis of residues predicted to be involved at each dimer interface. Our results indicate that the dimer interface suggested earlier as being physiologically relevant is, in fact, unlikely to be so. Given the alternative model for the N-domain dimer, the ability of the N-domain to mediate interactions of two STAT dimers on DNA remains unchanged.

Stat4 is required for Th1 development, although how a transiently activated factor generates heritable patterns of gene expression is still unclear. We examined the regulation of IL-18Ralpha expression to define a mechanism for Stat4-dependent genetic programming of a Th1-associated gene. Although Stat4 binds the Il18r1 promoter following IL-12 stimulation and transiently increases acetylated histones H3 and H4, patterns of histone acetylation alone in Th1 cells may not be sufficient to explain cell-type-specific patterns of gene expression. The level of DNA methylation and recruitment of Dnmt3a to Il18r1 inversely correlate with IL-18Ralpha expression, and blocking DNA methylation increases IL-18Ralpha expression. Moreover, there was decreased Il18r1-Dnmt3a association and DNA methylation following transient trichostatin A-induced histone hyperacetylation in Stat4-/-Th1 cultures. Increased association of Dnmt3a and the Dnmt3a cofactor Dnmt3L with the promoters of several Stat4-dependent genes was found in Stat4-/- Th1 cultures, providing a general mechanism for Stat4-dependent gene programming. These data support a mechanism wherein the transient hyperacetylation induced by Stat4 prevents the recruitment of DNA methyltransferases and the subsequent repression of the Il18r1 locus.

Interleukin-12 (IL-12) is a key cytokine for the development of T helper type 1 (Th1) responses; however, naïve CD4(+) T cells do not express IL-12Rbeta2, and are therefore unresponsive to IL-12. We have examined the mechanisms that control Th1-specific expression of the human IL-12Rbeta2 gene at early time points after T-cell stimulation. We have identified a Th1-specific enhancer element that binds signal transducer and activator of transcription 4 (STAT4) in vivo in developing Th1 but not Th2 cells. T-cell receptor (TCR) signaling induced histone hyperacetylation and recruitment of BRG1, the ATPase subunit of the SWI/SNF-like BAF chromatin remodeling complex, to the IL-12Rbeta2 regulatory regions and was associated with low-level gene transcription at the IL-12Rbeta2 locus. However, high-level IL-12Rbeta2 expression required TCR triggering in the presence of IL-12. Our results indicate a synergistic role of TCR-induced chromatin remodeling and cytokine-induced STAT4 activation to direct IL-12Rbeta2 expression during Th1 cell development.

Signal transducers and activators of transcription (STAT) proteins are activated in response to many cytokines, growth factors and hormones. STAT4 mediates IL-12 signaling and regulates T helper 1 (Th1) cell differentiation. Both IL-12 and Th1 cell activation participate in the development of autoimmune diabetes. In this study, we investigated the role of STAT4 in autoimmune diabetes. We crossbred Stat4 deficient (Stat4-/-) mice with nonobese diabetic (NOD) mice to generate the Stat4-/- NOD model. In Stat4-/- NOD mice, serum levels of both IFN-gamma and IL-2 were significantly reduced as compared to the controls. Insulin secretion in pancreatic islets was preserved in Stat4-/- NOD mice. Significantly, disruption of Stat4 activation completely prevented the development of spontaneous diabetes in NOD mice. This study reveals the important role of STAT4 in autoimmune diabetes pathogenesis.

The aim of this study was to determine the influence of STAT4 (rs7574865) and TRAF1/C5 (rs10818488 and rs2900180) gene polymorphisms on the risk of developing rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) in a Colombian population. This was a case-control study in which 839 individuals with RA (N=274) and SLE (N=144) and matched healthy controls (N=421) were included. Genotyping was performed by using a polymerase chain reaction system with pre-developed TaqMan allelic discrimination assay. STAT4 rs7574865T allele disclosed a significant influence on the risk of developing SLE (P=0.0005; OR 1.62, 95% CI 1.22-2.16) and RA (P=0.008; OR 1.36; 95% CI 1.08-1.71), whereas no effect on these autoimmune diseases was observed for the TRAF1/C5 polymorphisms examined. Our data strengthen STAT4 rs7574865 polymorphism as a susceptibility factor for RA and SLE and provide further evidence for a common origin of autoimmune diseases.

The aim of this study was to explore candidate single nucleotide polymorphisms (SNPs) and candidate mechanisms of systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Two SLE genome-wide association studies (GWASs) datasets were included in this study. Meta-analysis was conducted using 737,984 SNPs in 1,527 SLE cases and 3,421 controls of European ancestry, and 4,429 SNPs that met a threshold of p < 0.01 in a Korean RA GWAS dataset was used. ICSNPathway (identify candidate causal SNPs and pathways) analysis was applied to the meta-analysis results of the SLE GWAS datasets, and a RA GWAS dataset. The most significant result of SLE GWAS meta-analysis concerned rs2051549 in the human leukocyte antigen (HLA) region (p = 3.36E-22). In the non-HLA region, meta-analysis identified 6 SNPs associated with SLE with genome-wide significance (STAT4, TNPO3, BLK, FAM167A, and IRF5). ICSNPathway identified five candidate causal SNPs and 13 candidate causal pathways. This pathway-based analysis provides three hypotheses of the biological mechanism involved. First, rs8084 and rs7192 → HLA-DRA → bystander B cell activation. Second, rs1800629 → TNF → cytokine network. Third, rs1150752 and rs185819 → TNXB → collagen metabolic process. ICSNPathway analysis identified three candidate causal non-HLA SNPs and four candidate causal pathways involving the PADI4, MTR, PADI2, and TPH2 genes of RA. We identified five candidate SNPs and thirteen pathways, involving bystander B cell activation, cytokine network, and collagen metabolic processing, which may contribute to SLE susceptibility, and we revealed candidate causal non-HLA SNPs, genes, and pathways of RA.

IL-12 exerts several regulatory effects on natural killer (NK) cells by activating IL-12 signaling. IL-12 signaling is tightly auto-regulated to control its onset and termination, with prolonged IL-12 treatment resulting in IL-12 hyporesponsiveness. However, the mechanisms underlying IL-12 auto-regulation are still unclear. In this study we report that prolonged IL-12 treatment significantly up-regulates microRNAs (miRNAs), including miR-132, -212, and -200a in primary human NK cells. This up-regulation correlates temporally with gradually decreasing STAT4 levels and decreasing IFN-γ expression, after an initial increase within the first 16 hours of IL-12 treatment. The IL-12 hyporesponsiveness is dependent on IL-12 concentration, and associated up-regulation of miR-132, -212, and -200a. Furthermore, IL-12-hyporesponsive cells regain responsiveness of IFN-γ production 24 hours after IL-12 removal, which correlates with decreases in miR-132, -212, and -200a levels. Overexpression of miR-132, -212, and -200a by transfection into NK cells mimics IL-12 priming, inducing IL-12 hyporesponsiveness, whereas transfection of miR-132, -212, and -200a inhibitors largely abolishes IL-12 induction of IL-12 hyporesponsiveness. These data suggest that miR-132, -212, and -200a up-regulation during prolonged IL-12 treatment, negatively regulates the IL-12 signaling pathway by reducing STAT4 expression in primary human NK cells.

Chagas' disease is caused by the protozoan parasite Trypanosoma cruzi. The immune system plays an important role in the reduction of parasite load, but may also contribute to the development of lesions observed during the chronic phase of the disease. We analyzed cytokines produced by inflammatory heart cells in 21 autopsy samples obtained from patients with Chagas' disease divided according to the presence or absence of heart failure (HF). Left ventricular sections were analyzed by immunohistochemistry using antibodies against human IL-4, IFN-γ, TGF-β, TNF-α, and NOS2. In situ mRNA expression was quantified by a Low Density Array. The number of IFN-γ-positive cells was significantly higher than IL-4 positive cells. TNF-α, TGF-β and NOS2 were detected in 65%, 62% and 94% of samples respectively. There was an association between TNF-α-producing cells and the presence of HF. Subjects with HF presented higher levels of STAT4 mRNA, whereas FoxP3 and STAT6 levels were similar in the two groups. A Th1 cytokine pattern predominated in the cardiac inflammatory cell infiltrate of Chagas' disease patients associated with HF. High degree of fibrosis was associated with low NOS2 expression. These results support the idea that Th1 immune responses are involved in heart lesions of Chagas' disease patients.

Regulation of hematopoietic progenitor cell homeostasis is crucial for maintenance of innate immunity and the ability of the body to respond to injury and infection. In this report, we demonstrate that progenitor cell numbers and cycling status in vivo are dramatically increased in mice deficient in Stat6 and decreased in mice deficient in Stat4, targeted mutations which also alter T helper cell polarization. Experiments using mice that have T cell restricted transgenic expression of Stat4 or Stat6 or have been in vivo depleted of T cell subsets demonstrate that CD4(+) T cells regulate progenitor cell activity. Injection of the Th1 cytokine Oncostatin M but not other cytokines into Stat4-deficient mice recovers progenitor cell activity to wild-type levels. Thus, T helper cells actively regulate hematopoietic progenitor cell homeostasis.

Steatohepatitis enhances the severity of liver injury caused by acute inflammation. The purpose of this study was to test the hypothesis that fatty liver due to chronic choline-deficient diet exacerbates concanavalin A (ConA)-induced liver hepatitis, which is predominantly facilitated by T cells. Male C57BL/6 mice were fed either control choline-sufficient diet (CSD) or choline-deficient diet (CDD) for 6 weeks before ConA administration. Mice were sacrificed 3, 9, and 24 hours after ConA injection. Liver injury measured by aspartate aminotransferase (AST), alanine aminotransferase (ALT), pathology, and terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) staining was minimal in mice fed either diet before ConA exposure. However, ConA-induced liver injury was significantly greater in CDD-fed mice compared with control-fed mice. Liver cytokines were assessed by quantitative real-time polymerase chain reaction (PCR). The expression of T helper (Th) 1 cytokines tumor necrosis factor alpha (TNF-alpha), interleukin 12 (IL-12), and interferon gamma (IFN-gamma) were dramatically elevated after ConA in CDD-fed mice compared with control-fed mice. CDD also enhanced ConA-induced STAT4 activation, but not STAT6. Notably, regulators of T-cell differentiation were strongly shifted toward a predominant Th1 profile. T-bet, regulator of the Th1 response, was up-regulated in CDD-fed mice, whereas Th2 regulator GATA-3 was significantly suppressed in CDD-fed mice after ConA. Moreover, the expression of suppressor of cytokine signaling (SOCS)-1, SOCS-3, and repressor of GATA-3 (ROG) favored a predominant Th1 cytokine response in CDD-fed mice. In conclusion, these data support the hypothesis that hepatosteatosis caused by CDD is associated with more severe ConA-induced hepatitis due to a predominant shift toward Th1 response.

OBJECTIVE

Cytokines, hormones and growth factors use signal transducers and activators of transcription (STAT) signaling pathways to control various biological responses, including development, differentiation, cell proliferation and survival. Constitutive activation of STATs has been found in a wide variety of human tumors. In this study we examined the activity of STATs in primary human prostate tissues.

METHODS

STAT activity was determined in 104 human primary prostate tissues, including 42 tumors, 42 matched normal prostates adjacent to tumors and 20 normal prostates from donors without cancer by electromobility shift assay.

RESULTS

Significant levels of activated Stat4 and Stat6 were detected in primary prostate tissues. However, little or no expression of active Stat1, Stat2 or Stat5 was detected in primary prostate tissues. Significantly higher levels of constitutive Stat6 activity were found in prostate carcinomas compared with levels in normal tissue adjacent to tumors and normal prostates from donors without prostate cancer. There was no significant difference in Stat6 activity in normal prostate tissues adjacent to tumors and normal prostates from donors without prostate cancer. The levels of Stat4 activity varied but failed to yield statistically significant differences among tumors, matched normal prostates adjacent to tumors and normal prostates from donors without cancer.

CONCLUSIONS

We have previously shown that Stat3 is activated in prostate cancer. The results of the current study demonstrate that in addition to Stat3, Stat6 is selectively activated in prostate cancer.

Mycosis fungoides (MF) is the most frequent type of cutaneous T-cell lymphoma, whose diagnosis and study is hampered by its morphologic similarity to inflammatory dermatoses (ID) and the low proportion of tumoral cells, which often account for only 5% to 10% of the total tissue cells. cDNA microarray studies using the CNIO OncoChip of 29 MF and 11 ID cases revealed a signature of 27 genes implicated in the tumorigenesis of MF, including tumor necrosis factor receptor (TNFR)-dependent apoptosis regulators, STAT4, CD40L, and other oncogenes and apoptosis inhibitors. Subsequently a 6-gene prediction model was constructed that is capable of distinguishing MF and ID cases with unprecedented accuracy. This model correctly predicted the class of 97% of cases in a blind test validation using 24 MF patients with low clinical stages. Unsupervised hierarchic clustering has revealed 2 major subclasses of MF, one of which tends to include more aggressive-type MF cases including tumoral MF forms. Furthermore, signatures associated with abnormal immunophenotype (11 genes) and tumor stage disease (5 genes) were identified.

A role for Stat4 in IL-12-induced up-regulation of selectin ligands on Th1 cells was explored. Th1 cells generated from Stat4(-/-) mice exhibited no IL-12-inducible P-selectin ligands, no up-regulation of core 2 beta1,6-glucosaminyltransferase I (C2GlcNAcT-I), and low levels of the Th1 transcription factor T-bet. In contrast, Stat4(-/-) Th1 cells exhibited only a partial defect in expression of IL-12-inducible E-selectin ligands and expressed equivalently high levels of alpha1,3-fucosyltransferase VII (FucT-VII) as wild-type Th1 cells. FucT-VII expression was induced by T cell activation, and was enhanced by IL-12 independently of Stat4, whereas C2GlcNAcT-I up-regulation was mediated exclusively by IL-12, acting through Stat4. These data show that FucT-VII and C2GlcNAcT-I are controlled through distinct pathways and imply the existence of at least one other IL-12-inducible glycosyltransferase required for E-selectin and possibly P-selectin ligand formation in Th1 cells.

OBJECTIVE

Natural killer (NK) cells play an important role in the immune response against virus infection. Interferon (IFN)-alpha, an essential component in therapy against hepatitis C virus (HCV) infection, regulates NK cell function. However, it remains obscure how chronic HCV infection (CHC) modifies intracellular IFN-alpha signaling in NK cells. We investigated IFN-alpha signaling in NK cells in patients with CHC.

METHODS

Peripheral blood mononuclear cells were obtained from patients with CHC and healthy subjects (HS) as controls.

RESULTS

The expression level of signal transducer and activator of transcription (STAT) 1, a key molecule of IFN-alpha signaling, was clearly higher in NK cells from the CHC patients than in those from HS. The phosphorylation level of STAT1 with IFN-alpha stimulation was significantly greater in NK cells from the CHC patients than in those from the HS, while that of STAT4 was significantly less. These phosphorylation levels of STAT1 and STAT4 positively and negatively correlated with the STAT1 level in NK cells, respectively. The IFN-alpha induced messenger RNA level of the suppressor of cytokine signaling 1, which is a downstream gene of phosphorylated-STAT1, was clearly greater in NK cells from the CHC patients than in those from the HS, while that of IFN-gamma, which is a downstream gene of phosphorylated-STAT4, was clearly lower.

CONCLUSIONS

These results indicate altered IFN-alpha signaling in NK cells in CHC patients, suggesting that this alteration is associated with the persistence of HCV infection and resistance to IFN-alpha therapy.

We used signal transducer and activator of transcription 4 (STAT4) and STAT6 gene knockout (-/-) mice as recipients of fully mismatched cardiac allografts to study the role of T-cell costimulatory pathways in regulating allogeneic T-helper 1 (Th1) versus Th2 responses in vivo. STAT4(-/-) mice have impaired Th1 responses, whereas STAT6(-/-) mice do not generate normal Th2 responses. Cardiac allografts from C57BL/6 mice were transplanted into normal wild-type (WT), STAT4(-/-), and STAT6(-/-) BALB/c recipients. STAT4(-/-) and STAT6(-/-) mice rejected their grafts with the same tempo as untreated WT recipients. CD28-B7 blockade by a single injection of CTLA4Ig induced long-term engraftment and donor-specific tolerance in all three groups of recipients. CD154 blockade by a single injection of MR1 was effective in prolonging allograft survival and inducing tolerance in STAT4(-/-) mice but was only marginally effective in STAT6(-/-) recipients and WT controls. In addition, a similar protocol of MR1 was ineffective in prolonging graft survival in CD28(-/-) BALB/c recipients, suggesting that the lack of efficacy seen in WT and STAT6(-/-) mice is not due to the presence of a functional CD28-B7 pathway. Furthermore, there was a similar differential effect of CD28-B7 versus CD154-CD40 blockade in inhibiting immune responses in animals immunized with ovalbumin and complete Freund's adjuvant. These novel data indicate that Th1 and Th2 cells are differentially regulated by CD28-B7 versus CD154-CD40 costimulation pathways in vivo and may have potential implications for the development of therapeutic strategies such as T-cell costimulatory blockade in humans.

Underlying viral infections can heighten sensitivity and worsen cytokine-mediated disease following secondary inflammatory challenges. Mechanisms for this are poorly understood. The impact of the innate response to lymphocytic choriomeningitis virus (LCMV) infection on sensitivity to endotoxin (LPS) was investigated. Compared with uninfected mice, infection with LCMV for 2-days-sensitized mice to LPS by approximately 2-fold for lethality and by 2- to 6-fold for serum TNF-alpha levels. Priming for LPS-induced TNF-alpha was also seen with splenic and peritoneal leukocytes isolated from infected mice and challenged with LPS ex vivo. The effect on TNF-alpha production was present in the absence of IFN-gamma, its major producers NK and T cells, and the major pathways for its induction through IL-12 and the signal transducer and activator of transcription 4 (STAT4), and therefore was IFN-gamma independent. Early LCMV infection induces high concentrations of the type 1 IFNs, IFN-alphabeta. Administration of recombinant IFN-alpha alone heightened the TNF-alpha response to LPS. Innate IFN-alphabeta and IFN-gamma responses to LCMV exist in a delicate balance. To reduce priming for LPS-induced TNF-alpha during LCMV, deficiencies in both the IFN-alphabeta and IFN-gamma receptors or STAT1, a transcription factor downstream to both IFNs, were required. These data demonstrate that early viral infection can enhance sensitivity to bacterial products, and that this sensitization can occur in part as a result of endogenously expressed IFN-alphabeta. This work also raises issues about potential complications associated with IFN-alphabeta therapies.

Repetitive TTAGGG motifs present at high frequency in mammalian telomeres can suppress Th1-mediated immune responses. Synthetic oligonucleotides (ODN) containing TTAGGG motifs mimic this activity and have proven effective in the prevention/treatment of certain Th1-dependent autoimmune diseases. This work explores the mechanism by which suppressive ODN block the induction of Th1 immunity. Findings indicate that these ODN inhibit IFN-gamma-induced STAT1 phosphorylation and IL-12-induced STAT3 and STAT4 phosphorylation. As a result, T-bet expression is reduced as is the maturation of naive CD4+ cells into Th1 effectors. These changes indirectly support the generation of Th2-dominated immune responses. Suppressive ODN may thus represent a novel approach to influence the Th1:Th2 balance in vivo.

To determine the temporal relationship between alcohol-induced changes in cytokines and chemokines, development of liver pathology and stimulation of hepatocyte proliferation, male Sprague-Dawley rats were intragastrically fed low carbohydrate-containing ethanol (EtOH) diets via total enteral nutrition (TEN) for up to 49 d. Induction of EtOH metabolism and appearance of steatosis preceded development of oxidative stress, inflammation, and cell death. A transitory peak of tumor necrosis factor (TNFalpha) and interferon gamma (IFN gamma) was observed at 14 d followed by reduced expression of TNFalpha, IFN gamma and another Th1 cytokine IL-12 accompanied by reduced expression of the Th1 regulators T-bet and STAT4. After 35-49 d of EtOH, at a time when hepatocyte proliferation was stimulated, IL-12 returned to control values and a second peak of TNFalpha occurred. The Th2 cytokine IL-4 remained suppressed throughout the study and was accompanied by reductions in the Th2 regulator GATA3. There was no temporal effect of EtOH on expression of IL-6 or TGFbeta. IL-5 and IL-13 mRNA were undetectable. Chemokine CXCL-2 expression increased progressively up to 35 d and preceded the appearance of inflammatory infiltrates. These data suggest that steatosis, increased ethanol metabolism, a transient induction of the innate immune response and suppression of Th2 responses were acute consequences of ethanol treatment and were followed by suppression of Th1 responses. However, the majority of necrosis, apoptosis and a late peak of TNFalpha only occurred after 6-7 weeks of ethanol, coincided with the appearance of inflammatory infiltrates and were associated with stimulation of hepatocyte proliferation.

Intestinal adhesions are bands of fibrous tissue that connect the loops of the intestine to each other, to other abdominal organs or to the abdominal wall. Fibrous tissue formation is regulated by the balance between plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (tPA), which reciprocally regulate fibrin deposition. Several components of the inflammatory system, including cytokines, chemokines, cell adhesion molecules and neuropeptide substance P, have been reported to participate in adhesion formation. We have used cecal cauterization to develop a unique experimental mouse model of intestinal adhesion. Mice developed severe intestinal adhesion after this treatment. Adhesion development depended upon the interferon-gamma (IFN-gamma) and signal transducer and activator of transcription-1 (STAT1) system. Natural killer T (NKT) cell-deficient mice developed adhesion poorly, whereas they developed severe adhesion after reconstitution with NKT cells from wild-type mice, suggesting that NKT cell IFN-gamma production is indispensable for adhesion formation. This response does not depend on STAT4, STAT6, interleukin-12 (IL-12), IL-18, tumor necrosis factor-alpha, Toll-like receptor 4 or myeloid differentiation factor-88-mediated signals. Wild-type mice increased the ratio of PAI-1 to tPA after cecal cauterization, whereas Ifng(-/-) or Stat1(-/-) mice did not, suggesting that IFN-gamma has a crucial role in the differential regulation of PAI-1 and tPA. Additionally, hepatocyte growth factor, a potent mitogenic factor for hepatocytes, strongly inhibited intestinal adhesion by diminishing IFN-gamma production, providing a potential new way to prevent postoperative adhesions.

IL-12 is a macrophage-derived cytokine that induces proliferation, cytokine production, and cytotoxic activity of T and NK cells. Signaling through its receptor, IL-12 induces these cellular responses by tyrosine phosphorylation and activation of Janus kinase-2 (Jak-2), Tyk-2, Stat3, and Stat4. We have used tyrphostin B42 (AG490), a Jak-2 inhibitor, to determine the role of Jak-2 kinase in IL-12 signaling and IL-12-induced T cell functions. Treatment of activated T cells with tyrphostin B42 inhibited the IL-12-induced tyrosine phosphorylation and activation of Jak-2 without affecting Tyk-2 kinase. In contrast, treatment with tyrphostin A1 inhibited the tyrosine phosphorylation of Tyk-2 but not that of Jak-2 kinase. Inhibition of either Jak-2 or Tyk-2 leads to a decrease in the IL-12-induced tyrosine phosphorylation of Stat3, but not of Stat4, protein. While inhibition of Jak-2 lead to programmed cell death, the inhibition of Jak-2 or Tyk-2 resulted a decrease in IFN-gamma production. We have further tested the in vivo effects of tyrphostin B42 in experimental allergic encephalomyelitis, a Th1 cell-mediated autoimmune disease. In vivo treatment with tyrphostin B42 decreased the proliferation and IFN-gamma production of neural Ag-specific T cells. Treatment of mice with tyrphostin B42 also reduced the incidence and severity of active and passive EAE. These results suggest that tyrphostin B42 prevents EAE by inhibiting IL-12 signaling and IL-12-mediated Th1 differentiation in vivo.