Hepatocellular carcinoma (HCC) is the fifth most common cancer in men and the second leading cause of cancer deaths globally. The high prevalence of HCC is due in part to the high prevalence of chronic HBV infection and the high mortality rate is due to the lack of biomarkers for early detection and limited treatment options for late stage HCC. The observed individual variance in development of HCC is attributable to differences in HBV genotype and mutations, host predisposing germline genetic variations, the acquisition of tumor-specific somatic mutations, as well as environmental factors. HBV genotype C and mutations in the preS, basic core promoter (BCP) or HBx regions are associated with an increased risk of HCC. Genome-wide association studies have identified common polymorphisms in KIF1B, HLA-DQ, STAT4, and GRIK1 with altered risk of HBV-related HCC. HBV integration into growth control genes (such as TERT), pro-oncogenic genes, or tumor suppressor genes and the oncogenic activity of truncated HBx promote hepatocarcinogenesis. Somatic mutations in the TERT promoter and classic cancer signaling pathways, including Wnt (CTNNB1), cell cycle regulation (TP53), and epigenetic modification (ARID2 and MLL4) are frequently detected in hepatic tumor tissues. The identification of HBV and host variation associated with tumor initiation and progression has clinical utility for improving early diagnosis and prognosis; whereas the identification of somatic mutations driving tumorigenesis hold promise to inform precision treatment for HCC patients.

We previously reported that IL-12, but not IFN-alphaA/D, induces T helper type (Th) 1 development and STAT4 phosphorylation in murine CD4+ T cells. However, a recent study reported that IFN-alphaA/D and recombinant murine IFN-alphaA can induce STAT4 phosphorylation, although more weakly than IL-12, largely in CD8+ rather than CD4+ T cells. That report did not examine Th1 development or directly demonstrate induction of IFN-gamma by IFN-alpha. To address these differences, we compared IFN-alphaA/D, murine IFN-alphaA, and IL-12 for STAT4 phosphorylation, formation of active nuclear DNA binding complexes, induction of Th1 development, and production of IFN-gamma in murine CD4+ T cells. IFN-alphaA induced detectable STAT4 phosphorylation, although at significantly lower levels than induced by IL-12. Furthermore, in contrast to IL-12, IFN-alphaA failed to induce Th1 development or the formation of DNA binding complexes or to synergize with IL-18 for induction of IFN-gamma production. STAT1-deficient CD4+ T cells showed increased IFN-alphaA-induced STAT4 phosphorylation but still exhibited significantly lower amounts of cytokine-induced IFN-gamma compared to IL-12. In summary, these results suggest that in contrast to IL-12, IFN-alphaA does not play a functionally significant role in meditating the STAT4-dependent induction of Th1 development or IFN-gamma production in CD4+ T cells.

OBJECTIVE

Although hepatitis delta is considered an immune-mediated disease, adaptive immune responses to hepatitis delta virus (HDV) are hardly detectable. Thus, the role of other immune responses, including those mediated by natural killer (NK) cells, must be considered in HDV pathogenesis and in treatments with immune-stimulating agents such as interferon (IFN)α. However, the phenotype and function of NK cells in chronic HDV infection, or in HDV-infected individuals undergoing IFNα treatment, have not been extensively studied.

METHODS

We performed an extensive analysis of NK cells in chronically HDV-infected patients before and during treatment with IFNα, and compared the results with those for patients with HBV mono-infection as well as healthy controls.

RESULTS

In untreated HDV-infected patients, a higher than normal frequency of NK cells was observed in peripheral blood with unaltered phenotypic NK cell differentiation status. In contrast, long-term IFNα treatment of HDV-infected patients caused a significant change in NK cell differentiation status, with selective loss of terminally differentiated NK cells and, in parallel, a relative enrichment in immature NK cell subsets. Treatment was associated with marked functional impairment of the NK cells, which was independent of the changes in NK cell differentiation status. Furthermore, treatment polarised NK cell IFN signalling from STAT4 towards STAT1 dependency. Strikingly, a high frequency of CD56(dim) NK cells at baseline was positively associated with IFNα treatment outcome in the patients.

CONCLUSIONS

We describe in detail how HDV infection, and IFNα treatment of this infection, affects the NK cell compartment and what consequences this has for the functional capacity of NK cells.

The H3K4me3 demethylase Kdm5a regulates gene transcription and is implicated in carcinogenesis. However, the role of Kdm5a in innate immune response remains poorly understood. Here, we demonstrate that Kdm5a deficiency impairs activation of natural killer (NK) cells, with decreased IFN-γ production. Accordingly, Kdm5a(-/-) mice are highly susceptible to Listeria monocytogenes (Lm) infection. During NK cell activation, loss of Kdm5a profoundly impairs phosphorylation and nuclear localization of STAT4, along with increased expression of suppressor of cytokine signaling 1 (SOCS1). Mechanistic studies reveal that Kdm5a associates with p50 and binds to the Socs1 promoter region in resting NK cells, leading to a substantial decrease in H3K4me3 modification and repressive chromatin configuration at the Socs1 promoter. Thus, Kdm5a is required for priming activation of NK cells by suppressing the suppressor, SOCS1. Our study provides insights into the epigenetic regulation of innate immune response of NK cells.

Saikosaponin A (SSa) is isolated from the dried root of Radix Bupleuri, an herb widely used in traditional Chinese medicine, exerting antitumor activities. The T helper cell type 1(Th1)/Th2 balance is associated with antitumor immunity in breast cancer. The present study aimed to investigate the effects of SSa on Th1/Th2 balance in breast cancer and to explore the underlying mechanisms. Breast cancer in rats was induced by intragastrical administration of 7,12-dimethyl-benz[a] anthracene once (100 mg/kg). At d₉₁, the rats suffering from tumors were randomly divided into three groups and treated with vehicle solution (control group), tamoxifen (TAM group), and SSa (SSa group) daily for 56 days, respectively. The tumor volume reduction ratio and tumor cell proliferation were detected to assess the antitumor effect of SSa. The positive staining numbers of CD8+ and CD4+ T cells infiltrated in breast tumors were measured by immunohistochemistry to evaluate the antitumor immunity of SSa. Cytokine levels in serum secreted by Th1 cells [interferon gamma (IFN-γ), interleukin (IL)-12] and Th2 cells (IL-4, IL-10) were detected to evaluate Th1/Th2 balance. The related molecules of IL-12/signal transducers and activators of transcription 4 (STAT4) pathway were detected by immunohistochemistry staining, RT-PCR, and Western blot to explore the mechanisms of SSa. The results showed that, compared with the control group, SSa significantly inhibited tumor growth and tumor cell proliferation. SSa enhanced antitumor immunity, which was demonstrated as increased CD8+ T cells and CD4+ T cells infiltrated in tumors. SSa shifted Th1/Th2 balance toward Th1, which was confirmed as increased serum IFN-γ and IL-12 levels, while decreased serum IL-4 and IL-10 levels. SSa increased IL-12, IL-12 receptor, and phosphorylated STAT4 expressions to promote Th1 differentiation. In conclusion, the present work suggested that SSa could inhibit breast cancer growth by shifting Th1/Th2 balance toward Th1. The underlying mechanism may involve activation of the IL-12/STAT4 pathway that induced Th1 differentiation.

The past 10 years have seen enormous advances in our understanding of how cytokine signals are mediated intracellularly. Of particular significance was the discovery of a family of seven Signal Transducer and Activators of Transcription (STAT) proteins. Each of these has now been studied in detail, and appropriate gene-disrupted mouse models are available for all except STAT2 (Leonard and O'Shea 1998). Fetal lethality is observed in Stat3-deficient mice, and various immunodeficiencies characterize mice with disrupted Stat1, Stat4, and Stat6 genes, which is consistent with impaired signaling from the specific cytokines that activate each of these proteins. The recent characterization of Stat5-deficient mice has led to several unanticipated findings that point to diverse biological functions for the two STAT5 forms, STAT5a and STAT5b. These include roles for one or both STAT5 forms in the immune system, hematopoiesis, sexually dimorphic growth, mammary development, hair growth, deposition of adipose tissue, and pregnancy. Here we review the hormone- and cytokine-activated signaling pathways in which STAT5 participates and the extensive evidence, from laboratory animals, that these factors are required for sex-specific aspects of development, including control of body size. Finally, we consider human growth disorders that may involve defects in STAT5-dependent signal transduction.

OBJECTIVE

To investigate the role of signal transduction and activation of transcription 4 (STAT4) in the development and progression of human hepatocellular carcinoma (HCC).

METHODS

Recent genetic investigations have identified that a genetic variant of STAT4 is associated with hepatitis B virus (HBV)-related HCC. The level of STAT4 in 90 HCC patients was examined via Western blot and immunohistochemical analyses. The correlation between STAT4 expression and the clinicopathological characteristics of the patients was analyzed. The level of STAT4 expression in the HCC liver tissues was significantly lower than that in the non-HCC liver tissues and correlated with tumor size, histological grade of HCC and serum hepatitis B surface antigen level in HCC patients. The data were statistically analyzed using SPSS. Furthermore, siRNA oligos targeting STAT4 were employed to investigate the influence of STAT4 RNA interference on HCC cell physiology. Based on Cell Counting Kit-8 and flow cytometric assays, we found that depletion of STAT4 expression significantly enhanced the proliferation of L02 cells.

RESULTS

STAT4 protein expression was significantly lower in HCC tissues than in normal liver tissues. Immunohistochemistry followed by statistical analysis revealed that the expression of STAT4 negatively correlated with Ki67 expression (r = 0.851; P < 0.05) and positively correlated with maximal tumor size (P < 0.05), HBV (P = 0.012) and histological grade (P < 0.05). Kaplan-Meier analysis revealed significant differences in the survival curves between HCC patients expressing low and high levels of STAT4 and Ki67 (P < 0.05). Based on a multivariate Cox proportional hazard model, STAT4 expression was an independent prognostic indicator for HCC patients who underwent curative resection. In vitro, following the release of L02 cell lines from serum starvation, the expression of STAT4 was downregulated, and transfection of L02 cells with siRNA targeting STAT4 inhibited cell proliferation.

CONCLUSIONS

Our data indicate that STAT4 may inhibit HCC development by modulating HCC cell proliferation.

Understanding the molecular regulation of hematopoietic stem and progenitor cell (HSPC) engraftment is paramount to improving transplant outcomes. To discover novel regulators of HSPC repopulation, we transplanted >1,300 mice with shRNA-transduced HSPCs within 24 h of isolation and transduction to focus on detecting genes regulating repopulation. We identified 17 regulators of HSPC repopulation: Arhgef5, Armcx1, Cadps2, Crispld1, Emcn, Foxa3, Fstl1, Glis2, Gprasp2, Gpr56, Myct1, Nbea, P2ry14, Smarca2, Sox4, Stat4, and Zfp251. Knockdown of each of these genes yielded a loss of function, except in the cases of Armcx1 and Gprasp2, whose loss enhanced hematopoietic stem cell (HSC) repopulation. The discovery of multiple genes regulating vesicular trafficking, cell surface receptor turnover, and secretion of extracellular matrix components suggests active cross talk between HSCs and the niche and that HSCs may actively condition the niche to promote engraftment. We validated that Foxa3 is required for HSC repopulating activity, as Foxa3(-/-) HSC fails to repopulate ablated hosts efficiently, implicating for the first time Foxa genes as regulators of HSPCs. We further show that Foxa3 likely regulates the HSC response to hematologic stress. Each gene discovered here offers a window into the novel processes that regulate stable HSPC engraftment into an ablated host.

Genes and mechanisms involved in autoimmune diseases, affecting approximately 5% of human population, remain still obscure but there is accumulating evidence that common genetic factors might predispose to multiple autoimmune disorders. STAT4, a transcription factor transmitting signals induced by several key cytokines, has recently been identified as a genetic risk factor for rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and Sjögren's disease (SD), thus indicating that multiple autoimmune diseases may share common biochemical pathways that lead to immune deregulation. Here we demonstrate for the first time, in a genetically homogeneous population, the association of the STAT4 rs7574865 G/T polymorphism, which has been shown to be associated with these autoimmune diseases, with susceptibility to type 1 diabetes (T1D). The susceptibility is associated with a significant increase of the frequency of the T allele (p = 0.0012, two-tailed chi(2), OR = 1.94, 95% CI = 1.29-2.91) in this single-nucleotide polymorphism (SNP). We also present an indication for association with Wegener's granulomatosis. These findings suggest that this variant form of STAT4 may have a putative key role in the development of a variety of autoimmune diseases, probably because of signaling defects that it causes in the IL-12 pathway.

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease with complex genetic inheritance. Genome-wide association studies have identified SLE susceptibility variations at the IRF7/KIAA1542 locus and with STAT4 gene in European populations. We decided to investigate the association of single-nucleotide polymorphisms (SNPs) in the IRF7/KIAA1542 region (rs4963128, rs2246614, and rs702966) and in STAT4 (rs7574865 and rs7582694) with SLE disease in a Northern Han Chinese population of 748 patients and 750 healthy controls. Our study indicated a strong association between rs7574865 (odds ratio = 0.68; 95% confidence interval 0.59-0.79; p = 1.57 × 10(-6)) and SLE and between rs7574865 and the production of anti-Sm antibodies. Additionally, rs4963128 and rs2246614 were correlated with a variety of clinical subphenotypes, such as lupus nephritis, arthritis, and the production of anti-SSA/B autoantibodies, despite a lack of significant association between these two SNPs and SLE disease susceptibility in general.

Recent genome-wide association studies (GWAS) of many complex diseases have successfully identified novel susceptibility loci, with many of them being associated with more than one condition. Taking into consideration that different autoimmune diseases may share some common pathogenetic pathways, we hypothesized that STAT4, a susceptibility gene found to be associated with increased risk for systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes, Sjögren's syndrome, Wegener's granulomatosis, Crohn's disease, and ulcerative colitis may also have a role in psoriasis. Psoriasis is an autoimmune, chronic inflammatory skin disease. Here we performed a case-control study in the population of island of Crete and demonstrated for the first time the association of a STAT4 single nucleotide polymorphism (SNP) with susceptibility to psoriasis, thus suggesting a putative key role of STAT4 in multiple autoimmune diseases. We found that mutated allele T of the STAT4 rs7574865 SNP, which previously was implicated in the predisposition to many autoimmune diseases, were more common in individuals with psoriasis than in controls (p = 0.045, odds ratio = 1.42, 95% confidence interval 1.01-2.00), thus concluding that the polymorphism examined is associated with the development of psoriasis in our population.

BACKGROUND

In experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, mice genetically deficient in the transcription factor signal transducer and activator of transcription 4 (STAT4) are resistant to disease. In contrast, deletion or inhibition of the Th1-associated cytokines IL-12 or IFNγ which act upstream and downstream of STAT4, respectively, does not ameliorate disease. These discordant findings imply that STAT4 may act in a non-canonical role during EAE. Recently, STAT4 has been shown to regulate GM-CSF production by CD4 T cells and this cytokine is necessary for the induction of EAE. However, it is not known if STAT4 controls GM-CSF production by both Th1 and Th17 effector CD4 T cells.

METHODS

This study utilized the MOG(35-55) peptide immunization model of EAE. Intracellular cytokine staining and novel mixed bone marrow chimeric mice were used to study the CD4 T cell-intrinsic role of STAT4 during disease. STAT4 chromatin-immunoprecipitation (ChIP-PCR) experiments were performed to show STAT4 directly interacts with the Csf2 gene loci.

RESULTS

Herein, we demonstrate that STAT4 controls CD4 T cell-intrinsic GM-CSF production by both Th1 and Th17 CD4 T cells during EAE as well as in vitro. Importantly, we show that STAT4 interacts with the Csf2 locus in MOG(35-55)-activated effector CD4 T cells demonstrating direct modulation of GM-CSF.

CONCLUSIONS

Overall, these studies illustrate a previously unrecognized role of STAT4 to regulate GM-CSF production by not only Th1 cells, but also Th17 effector CD4 T cell subsets during EAE pathogenesis. Critically, these data highlight for the first time that STAT4 is able to modulate the effector profile of Th17 CD4 T cell subsets, which redefines our current understanding of STAT4 as a Th1-centric factor.

C-C chemokines play an important role in recruitment of T lymphocytes to inflammatory sites. T lymphocytes secrete chemokines, but the activation requirements for chemokine production by T cells are uncertain. We studied the regulation of C-C chemokine production by CD28 costimulatory signals by murine T lymphocytes. Splenocytes from BALB/c mice cultured with anti-CD3 mAb expressed macrophage-inflammatory protein (MIP)-1alpha mRNA and secreted MIP-1alpha, which was inhibited by anti-B7-1 plus anti-B7-2 mAbs. MIP-1alpha production by Ag-stimulated T cells from DO.11.10 TCR transgenic mice was augmented by anti-CD28 mAb and increased compared with DO.11.10/CD28(-/-) cells. When T cell costimulation was provided by IL-2, MIP-1alpha was not enhanced. Studies with IL-2, IL-4, STAT4, and STAT6 knock-out mice suggested that chemokine production is controlled by pathways different from those regulating T cell differentiation. Thus, CD28 costimulation may amplify an immune response by stimulating T cell survival, proliferation, and production of chemokines that recruit T cells to inflammatory sites.

T helper 17 (Th17) cells are pivotal in the immune pathogenesis of EAE. Glatiramer acetate (GA) can enhance Treg FOXp3 expression. We demonstrate that GA downregulates the expression of both IL-17 and IL-6 in two different EAE models. Increased mRNA expression in CNS for ROR gamma t, IL-17, IL-12/IL-23, IL-6, TNF-alpha, STAT4 and Th1 cytokines were significantly reduced by GA with a concomitant rise in SMAD3. The increased expression of TNF-alpha, IL-6, and IL-17 in CNS of CD25+ depleted animals was suppressed by GA treatment. This study demonstrates that both Th1 polarization and Th17 expression are modulated by GA.

Murine T cells exposed to rapamycin maintain flexibility towards Th1/Tc1 differentiation, thereby indicating that rapamycin promotion of regulatory T cells (Tregs) is conditional. The degree to which rapamycin might inhibit human Th1/Tc1 differentiation has not been evaluated. In the presence of rapamycin, T cell costimulation and polarization with IL-12 or IFN-α permitted human CD4+ and CD8+ T cell differentiation towards a Th1/Tc1 phenotype; activation of STAT1 and STAT4 pathways essential for Th1/Tc1 polarity was preserved during mTOR blockade but instead abrogated by PI3 kinase inhibition. Such rapamycin-resistant human Th1/Tc1 cells: (1) were generated through autophagy (increased LC3BII expression; phenotype reversion by autophagy inhibition via 3-MA or siRNA for Beclin1); (2) expressed anti-apoptotic bcl-2 family members (reduced Bax, Bak; increased phospho-Bad); (3) maintained mitochondrial membrane potentials; and (4) displayed reduced apoptosis. In vivo, type I polarized and rapamycin-resistant human T cells caused increased xenogeneic graft-versus-host disease (x-GVHD). Murine recipients of rapamycin-resistant human Th1/Tc1 cells had: (1) persistent T cell engraftment; (2) increased T cell cytokine and cytolytic effector function; and (3) T cell infiltration of skin, gut, and liver. Rapamycin therefore does not impair human T cell capacity for type I differentiation. Rather, rapamycin yields an anti-apoptotic Th1/Tc1 effector phenotype by promoting autophagy.

Liver regeneration following partial hepatectomy is regulated by hepatotrophic factors whose precise roles are still elusive. In cell culture studies, some of them have been shown to activate members of the family of the signal transducers and activators of transcription (Stat). To test this contention in vivo, nuclear extracts were isolated from livers of partially hepatectomized and sham-operated mice killed at 30 different time points between zero and 108 h after surgery. Stat3 DNA binding is rapidly induced after surgery in both partially hepatectomized and sham-operated mice. Maximum activation of Stat3 is achieved 4-6 h after resection, and elevated Stat3 activation is detected as late as 60 h after surgery in both groups. Activated Stat5 is found sporadically in both sham-operated and resected mice but appears to be absent in the first 12 h after partial hepatectomy. Neither Stat1, Stat2, Stat4 nor Stat6 is induced during the time of observation. In contrast, AP-1 DNA binding activity is specifically induced in regenerating mouse liver.

Mice homozygous for the STAT4-null mutation were sensitized to cockroach Ag, challenged intratracheally 21 days later, and compared with STAT4-competent allergic mice. The STAT4(-/-) mice showed significant decreases in airway hyperreactivity (AHR) and peribronchial eosinophils compared with wild-type controls. In addition, pulmonary levels of chemokines were decreased in the STAT4(-/-) mice, including CC chemokine ligand (CCL)5, CCL6, CCL11, and CCL17. However, levels of Th2-type cytokines, such as IL-4 and IL-13, as well as serum IgE levels were similar in the two groups. Transfer of splenic lymphocytes from sensitized wild-type mice into sensitized STAT4(-/-) mice did not restore AHR in the mutant mice. Furthermore, chemokine production and peribronchial eosinophilia were not restored during the cellular transfer experiments. Thus, it appears that STAT4 expression contributes to a type 2 process such as allergen-induced chemokine production and AHR. In additional studies, competent allergic mice were treated with anti-IL-12 locally in the airways at the time of allergen rechallenge. These latter studies also demonstrated a decrease in AHR. Altogether, these data suggest that STAT4-mediated pathways play a role locally within the airway for the exacerbation of the allergen-induced responses.

The suppressive effect of rapamycin on T cells has been extensively studied, but its influence on the function of APC is less clear. The data in this study demonstrated that immunostimulatory activity of B10 (H2(b)) dendritic cells (DC) exposed to rapamycin (rapa-DC) was markedly suppressed as evidenced by the induction of low proliferative responses and specific CTL activity in allogeneic (C3H, H2(k)) T cells. Administration of rapa-DC significantly prolonged survival of B10 cardiac allografts in C3H recipients. Treatment with rapamycin did not affect DC expression of MHC class II and costimulatory molecules or IL-12 production. Rapamycin did not inhibit DC NF-kappaB pathway, however, IL-12 signaling through Janus kinase 2/Stat4 activation was markedly suppressed. Indeed, Stat4(-/-) DC similarly displayed poor allostimulatory activity. The Stat4 downstream product, IFN-gamma, was also inhibited by rapamycin, but DC dysfunction could not solely be attributed to low IFN-gamma production as DC deficient in IFN-gamma still exhibited vigorous allostimulatory activity. Rapamycin did not affect DC IL-12R expression, but markedly suppressed IL-18Ralpha and beta expression, which may in turn down-regulate DC IL-12 autocrine activation.

IFN-alpha and IL-12 are macrophage-derived cytokines that enhance innate and Th1 immune responses. However, there is little information regarding IFN-alpha and IL-12 target genes that would be involved in mediating the immunostimulatory effects of these cytokines. The interferon regulatory factor (IRF) family of transcription factors is known to be involved in controlling lymphocyte differentiation and functions. In this work we have studied the effect of IFN-alpha and IL-12 on the expression of IRF transcription factors in human NK and T cells. Both IFN-alpha and IL-12 strongly up-regulated IRF-1, IRF-4, and IRF-8 mRNA and protein expression. The binding of IRF-4 and IRF-8 to the lambdaB gene enhancer sequence was also increased following IFN-alpha- and IL-12-treatment of NK and T cells. A GAS element from the promoter region of the IRF-4 gene was identified. Following stimulation of cells with IFN-alpha or IL-12, Stat4 was found to bind to this IRF-4 GAS element, as detected by EMSA and DNA affinity binding, implying that the IRF-4 gene is directly activated by both cytokines. Our results suggest that IFN-alpha and IL-12 may enhance innate and Th1 immune responses by inducing IRF-1, IRF-4, and IRF-8 gene expression.

BACKGROUND

The transcription factors interferon regulatory factor 5 (IRF5) and signal transducer and activator of transcription 4 (STAT4) are encoded by two of the strongest susceptibility genes for systemic lupus erythematosus (SLE).

OBJECTIVE

To investigate the target genes and functional roles of IRF5 and STAT4 in human peripheral blood mononuclear cells (PBMCs).

METHODS

Chromatin immunoprecipitation-sequencing (ChIP-seq) was performed in PBMCs stimulated to activate IRF5 and STAT4. The expression of the target genes of IRF5 and STAT4 was investigated in a publicly available dataset generated from PBMCs from patients with SLE and healthy controls. The genomic regions bound by the transcription complexes mediated by IRF5 and STAT4 were examined for transcription factor binding motifs and SLE-associated sequence variants.

RESULTS

More than 7000 target genes for IRF5 and STAT4 were identified in stimulated PBMCs. These genes were enriched to functional pathways in the type I interferon system, and have key roles in the inflammatory response. The expression patterns of the target genes were characteristic for patients with SLE. The transcription factors high mobility group-I/Y, specificity protein 1, and paired box 4 may function cooperatively with IRF5 and STAT4 in transcriptional regulation. Eight of the target regions for IRF5 and STAT4 contain SLE-associated sequence variants.

CONCLUSIONS

By participating in transcription complex with other co-factors, IRF5 and STAT4 harbour the potential of regulating a large number of target genes, which may contribute to their strong association with SLE.

Rheumatoid arthritis (RA) is the most common cause of adult inflammatory arthritis. Recent genome-wide association scans have disclosed several single-nucleotide polymorphisms associated with RA susceptibility. The aim of this study was to determine whether the polymorphisms of TRAF1/C5 (tumor necrosis factor (TNF)-receptor associated factor 1)/(complement component 5) and STAT4 (signal transducers and activators of transcription 4) confer susceptibility, activity and severity to RA in Egyptian populations. One hundred and seventy-two RA patients and 160 controls were enrolled in the study. Polymorphisms of TRAF1/C5 and STAT4 genes were determined using restriction fragment length polymorphism-polymerase chain reaction. The TRAF1/C5 A and STAT4 T alleles were significantly associated with RA in Egyptian population. TRAF1/C5 A allele and STAT4 TT genotype were significantly associated with RA severity. In conclusion the mutant alleles or genotypes of both examined polymorphisms are associated with the development of RA in Egyptian population.

Signal transducer and activator of transcription 3 (STAT3) is a latent cytoplasmic transcription factor that can be activated by cytokines and growth factors. It plays important roles in cell growth, apoptosis and cell transformation, and is constitutively active in a variety of tumor cells. In this study, we provide evidence that zipper-interacting protein kinase (ZIPK) interacts physically with STAT3. ZIPK specifically interacted with STAT3, and did not bind to STAT1, STAT4, STAT5a, STAT5b or STAT6. ZIPK phosphorylated STAT3 on serine 727 (Ser727) and enhanced STAT3 transcriptional activity. Small interfering RNA-mediated reduction of ZIPK expression decreased leukemia inhibitory factor (LIF)- and IL-6-induced STAT3-dependent transcription. Furthermore, LIF- and IL-6-mediated STAT3 activation stimulated ZIPK activity. Taken together, our data suggest that ZIPK interacts with STAT3 within the nucleus to regulate the transcriptional activity of STAT3 via phosphorylation of Ser727.

Natural killer (NK) cells are involved in innate immune responses to viral infections either via direct cytotoxicity which destroys virus-infected cells or production of immunoregulatory cytokines which modulate adaptive immunity and directly inhibit virus replication. These functions are mediated by different NK subpopulations, with cytotoxicity being generally performed by CD56(dim) NK cells, whereas CD56(bright) NK cells are mainly involved in cytokine secretion. NK functional defects are usually combined so that impaired degranulation is often associated with deficient cytokine production. Innate immunity is thought to be relevant in the control of hepatitis virus infections such as hepatitis B virus (HBV) and hepatitis C virus (HCV), and recent findings reproducibly indicate that NK cells in chronic viral hepatitis are characterized by a functional dichotomy, featuring a conserved or enhanced cytotoxicity and a reduced production of interferon (IFN)-γ and tumor necrosis factor-α. In chronic HCV infection this appears to be caused by altered IFN-α signaling resulting from increased signal transducer and activator of transcription 1 (STAT1) phosphorylation, which polarizes NK cells toward cytotoxicity, and a concomitantly reduced IFN-α induced STAT4 phosphorylation yielding reduced IFN-γ mRNA levels. These previously unappreciated findings are compatible on the one hand with the inability to clear HCV and HBV from the liver and on the other they may contribute to understand why these patients are often resistant to IFN-α-based therapies.

IL-12 is a key immunoregulatory cytokine that promotes Th1 differentiation and cell-mediated immune responses. IL-12 stimulation results in the activation of Janus kinase 2 and tyrosine kinase 2 and, subsequently, STAT4 and STAT3. In addition, mitogen-activated protein kinase kinase 6/p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt pathways have been recently demonstrated to be activated by IL-12 and play an important role in IL-12 signaling. To further elucidate the molecular mechanism underlying IL-12 signaling, we have performed a yeast two-hybrid screening and identified mouse sphingosine kinase 2 (SPHK2) as a molecule associating with the mouse IL-12Rbeta1 cytoplasmic region. Analyses of various mutants of each molecule revealed that the region including the proline-rich domain in SPHK2 is probably responsible for the binding to IL-12Rbeta1, while the regions including the carboxyl terminus and Box II in the IL-12Rbeta1 cytoplasmic region appear to be involved in the binding to SPHK2. Transient expression of wild-type SPHK2 in T cell hybridoma augmented IL-12-induced STAT4-mediated transcriptional activation. Ectopic expression of dominant-negative SPHK2 in Th1 cell clone significantly reduced IL-12-induced IFN-gamma production, while that of wild-type SPHK2 enhanced it. In contrast, the expression minimally affected IL-12-induced proliferation. A similar decrease in IL-12-induced IFN-gamma production was observed when dominant-negative SPHK2 was expressed in activated primary T cells using a retroviral expression system. These results suggest that SPHK2 associates with the IL-12Rbeta1 cytoplasmic region and probably plays a role in modulating IL-12 signaling.