miRNAs play a central role in the complex signaling network of cancer cells with the tumor microenvironment. Little is known on the origin of circulating miRNAs and their relationship with the tumor microenvironment in lung cancer. Here, we focused on the cellular source and relative contribution of different cell types to circulating miRNAs composing our risk classifier of lung cancer using in vitro/in vivo models and clinical samples. A cell-type specific expression pattern and topography of several miRNAs such as mir-145 in fibroblasts, mir-126 in endothelial cells, mir-133a in skeletal muscle cells was observed in normal and lung cancer tissues. Granulocytes and platelets are the major contributors of miRNAs release in blood. miRNAs modulation observed in plasma of lung cancer subjects was consistent with de-regulation of the same miRNAs observed during immunosuppressive conversion of immune cells. In particular, activated neutrophils showed a miRNA profile mirroring that observed in plasma of lung cancer subjects. Interestingly mir-320a secreted by neutrophils of high-risk heavy-smokers promoted an M2-like protumorigenic phenotype through downregulation of STAT4 when shuttled into macrophages. These findings suggest a multifactorial and non-epithelial cell-autonomous origin of circulating miRNAs associated with risk of lung cancer and that circulating miRNAs may act in paracrine signaling with causative role in lung carcinogenesis and immunosuppression. This article is protected by copyright. All rights reserved.

OBJECTIVE

Recent studies demonstrated an association of STAT4 polymorphisms with autoimmune diseases including systemic lupus erythematosus and rheumatoid arthritis, indicating multiple autoimmune diseases share common susceptibility genes. We therefore investigated the influence of STAT4 polymorphisms on the susceptibility and phenotype of type-1 autoimmune hepatitis in a Japanese National Hospital Organization (NHO) AIH multicenter cohort study.

RESULTS

Genomic DNA from 460 individuals of Japanese origin including 230 patients with type-1 autoimmune hepatitis and 230 healthy controls was analyzed for two single nucleotide polymorphisms in the STAT4 gene (rs7574865, rs7582694). The STAT4 rs7574865T allele conferred risk for type-1 autoimmune hepatitis (OR = 1.61, 95% CI = 1.23-2.11; P = 0.001), and patients without accompanying autoimmune diseases exhibited an association with the rs7574865T allele (OR = 1.50, 95%CI = 1.13-1.99; P = 0.005). Detailed genotype-phenotype analysis of type-1 autoimmune hepatitis patients with (n = 44) or without liver cirrhosis (n = 186) demonstrated that rs7574865 was not associated with the development of liver cirrhosis and phenotype (biochemical data and the presence of auto-antibodies).

CONCLUSIONS

This is the first study to show a positive association between a STAT4 polymorphism and type-1 autoimmune hepatitis, suggesting that autoimmune hepatitis shares a gene commonly associated with risk for other autoimmune diseases.

The cytokine gamma interferon (IFN-γ), with antimicrobial and immunoregulatory functions, can be produced by T cells following stimulation through their T cell receptors (TCRs) for antigen. The innate cytokines type 1 IFNs and interleukin-12 (IL-12) can also stimulate IFN-γ production by natural killer (NK) but not naive T cells. High basal expression of signal transducer and activator of transcription 4 (STAT4), used by type 1 IFN and IL-12 to induce IFN-γ as well as CD25, contributes to the NK cell responses. During acute viral infections, antigen-specific CD8 T cells are stimulated to express elevated STAT4 and respond to the innate factors with IFN-γ production. Little is known about the requirements for cytokine compared to TCR stimulation. Primary infections of mice with lymphocytic choriomeningitis virus (LCMV) demonstrated that although the elicited antigen-specific CD8 T cells acquired STAT4-dependent innate cytokine responsiveness for IFN-γ and CD25 induction ex vivo, TCR stimulation induced these through STAT4-independent pathways. During secondary infections, LCMV-immune CD8 T cells had STAT4-dependent IFN-γ expression at times of innate cytokine induction but subsequently expanded through STAT4-independent pathways. At times of innate cytokine responses during infection with the antigen-distinct murine cytomegalovirus virus (MCMV), NK and LCMV-immune CD8 T cells both had activation of pSTAT4 and IFN-γ. The T cell IFN-γ response was STAT4 and IL-12 dependent, but antigen-dependent expansion was absent. By dissecting requirements for STAT4 and antigen, this work provides novel insights into the endogenous regulation of cytokine and proliferative responses and demonstrates conditioning of innate immunity by experience. Importance: Understanding the regulation and function of adaptive immunity is key to the development of new and improved vaccines. Its CD8 T cells are activated through antigen-specific receptors to contribute to long-lasting immunity after natural infections or purposeful immunization. The antigen-receptor pathway of stimulation can lead to production of gamma interferon (IFN-γ), a cytokine having both direct antimicrobial and immunoregulatory functions. Natural killer cells can also produce IFN-γ in response to the innate cytokines type 1 IFNs and/or interleukin-12. This work demonstrates that CD8 T cells acquire parallel responsiveness to innate cytokine signaling for IFN-γ expression during their selection and development and maintain this capability to participate in innate immune responses as long-lived memory cells. Thus, CD8 T cells are conditioned to play a role in innate immunity, and their presence under immune conditions has the potential to regulate resistance to either secondary challenges or primary infections with unrelated agents.

Recently an outbreak that emerged in Wuhan, China in December 2019, spread to the whole world in a short time and killed >1,410,000 people. It was determined that a new type of beta coronavirus called severe acute respiratory disease coronavirus type 2 (SARS-CoV-2) was causative agent of this outbreak and the disease caused by the virus was named as coronavirus disease 19 (COVID19). Despite the information obtained from the viral genome structure, many aspects of the virus-host interactions during infection is still unknown. In this study we aimed to identify SARS-CoV-2 encoded microRNAs and their cellular targets. We applied a computational method to predict miRNAs encoded by SARS-CoV-2 along with their putative targets in humans. Targets of predicted miRNAs were clustered into groups based on their biological processes, molecular function, and cellular compartments using GO and PANTHER. By using KEGG pathway enrichment analysis top pathways were identified. Finally, we have constructed an integrative pathway network analysis with target genes. We identified 40 SARS-CoV-2 miRNAs and their regulated targets. Our analysis showed that targeted genes including NFKB1, NFKBIE, JAK1-2, STAT3-4, STAT5B, STAT6, SOCS1-6, IL2, IL8, IL10, IL17, TGFBR1-2, SMAD2-4, HDAC1-6 and JARID1A-C, JARID2 play important roles in NFKB, JAK/STAT and TGFB signaling pathways as well as cells' epigenetic regulation pathways. Our results may help to understand virus-host interaction and the role of viral miRNAs during SARS-CoV-2 infection. As there is no current drug and effective treatment available for COVID19, it may also help to develop new treatment strategies.

Keywords: ACE-2, angiotensin-converting enzyme 2; AKT1, AKT serine/threonine kinase 1; BCL2, BCL2 apoptosis regulator; CDK1, cyclin dependent kinase 1; CDKL2, cyclin dependent kinase like 2; COVID19, new type corona virus disease; CTNNB1, catenin beta 1; CXCL1, C-X-C motif chemokine ligand 1; CXCL10, C-X-C motif chemokine ligand 10; CXCL11, C-X-C motif chemokine ligand 11; CXCL16, C-X-C motif chemokine ligand 16; CXCL9, C-X-C motif chemokine ligand 9; E2F1, E2F transcription factor 1; EIF4A1, eukaryotic translation initiation factor 4A1; GRB2, growth factor receptor bound protein 2; HDAC1, histone deacetylase 1; HDAC2, histone deacetylase 2; HDAC3, histone deacetylase 3; HIF1A, hypoxia inducible factor 1 subunit alpha; ICTV, International Committee on Taxonomy of Viruses; IFNGR2, interferon gamma receptor 2; IKBKE, inhibitor of nuclear factor kappa B kinase subunit epsilon; IL10, interleukin 10; IL13, interleukin 13; IL15, interleukin 15; IL16, interleukin 16; IL17A, interleukin 17 A; IL2, interleukin 2; IL21, interleukin 21; IL22, interleukin 22; IL24, interleukin 24; IL25, interleukin 25; IL33, interleukin 33; IL5, interleukin 5; IL7, interleukin 7; IL8, interleukin 8; JAK/STAT; JAK1, Janus kinase 1; JAK2, Janus kinase 2; JARID1A, lysine demethylase 5A; JARID1B, lysine demethylase 5B; JARID1C, lysine demethylase 5C; JARID2, Jumonji and AT-rich interaction domain containing 2; KEGG, Kyoto Encyclopedia of Genes and Genomes; MAPK1, mitogen-activated protein kinase 1; MAPK3, mitogen-activated protein kinase 3; MAPK4, mitogen-activated protein kinase 4; MAPK6, mitogen-activated protein kinase 6; MAPK7, mitogen-activated protein kinase 7; NFKB; NFKB1, nuclear factor kappa B subunit 1; NFKBIE, NFKB inhibitor epsilon; NOS3, nitric oxide synthase 3; PANTHER, protein analysis through evolutionary relationships; PIK3CA, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha; PTEN, phosphatase and tensin homolog; RB1, RB transcriptional corepressor 1; RHOA, ras homolog family member A; SARS-CoV-2; SARS-CoV-2, severe acute respiratory disease coronavirus type 2; SMAD2, SMAD family member 2; SMAD3, SMAD family member 3; SMAD4, SMAD family member 4; SOCS1, suppressor of cytokine signaling 1; SOCS3, suppressor of cytokine signaling 3; SOCS4, suppressor of cytokine signaling 4; SOCS5, suppressor of cytokine signaling 5; SOCS6, suppressor of cytokine signaling 6; SOS1, SOS Ras/Rac guanine nucleotide exchange factor 1; SP1, Sp1 transcription factor; STAT3, signal transducer and activator of transcription 3; STAT4, signal transducer and activator of transcription 4; STAT5B, signal transducer and activator of transcription 5B; STAT6, signal transducer and activator of transcription 6; SUMO1, small ubiquitin like modifier 1; SUMO2, small ubiquitin like modifier 2; TBP, TATA-box binding protein; TGFB; TGFBR1, transforming growth factor beta receptor 1; TGFBR2, transforming growth factor beta receptor 2; TMPRSS11A, transmembrane serine protease 11A; TMPRSS4, transmembrane serine protease 4; TNFRSF21, TNF receptor superfamily member 21; WHO, World Health Organization; miRNA.

BACKGROUND

Mycosis fungoides (MF) is a rare, but potentially devastating malignancy. It classically presents with cutaneous patches and plaques and can progress to tumours on the skin with lymph node, blood and visceral involvement. While most patients with MF have a relatively benign disease course, a subset of patients will develop progressive disease that is often fatal.

OBJECTIVE

The aim of this study was to identify genetic markers in early MF limited to the skin (stages IA-IIA) that distinguish those patients who will have progressive disease from those who will not, so that early appropriate treatment may be instituted.

METHODS

The study includes 18 patients who were diagnosed with early stage MF at the time of biopsy and had follow-up to determine which patients developed progressive disease. RNA was extracted from skin biopsy specimens and analysed for expression of CD3, FOXP3, IFNγ, Interleukin (IL)-4, IL-13, KIR3DL2, MICB, PLS3 and STAT4 by quantitative real-time polymerase chain reaction.

CONCLUSIONS

Reduced expression of FOXP3 and STAT4 and increased expression of IL-4 relative to CD3 expression levels were significantly associated with MF progression. Further studies will be needed to fully assess the usefulness of these genetic markers to predict disease progression and guide treatment options in patients diagnosed with early MF.

Interferon (IFN)-β is a front-line therapy for the treatment of the relapsing-remitting form of multiple sclerosis. However, its immunosuppressive mechanism of function remains incompletely understood. While it has been proposed that IFN-β suppresses the function of inflammatory myelin antigen-reactive T cells by promoting the release of immunomodulatory cytokines such as IL-27 from antigen-presenting cells (APCs), its direct effects on inflammatory CD4+ Th1 cells are less clear. Here, we establish that IFN-β inhibits mouse IFN-γ+ Th1 cell function in the absence of APCs. CD4+ T cells express the type I interferon receptor, and IFN-β can suppress Th1 cell proliferation under APC-free stimulation conditions. IFN-β-treated myelin antigen-specific Th1 cells are impaired in their ability to induce severe experimental autoimmune encephalomyelitis (EAE) upon transfer to lymphocyte-deficient Rag1-/- mice. Polarized Th1 cells downregulate IFN-γ and IL-2, and upregulate the negative regulatory receptor Tim-3, when treated with IFN-β in the absence of APCs. Further, IFN-β treatment of Th1 cells upregulates phosphorylation of Stat1, and downregulates phosphorylation of Stat4. Our data indicate that IFN-γ-producing Th1 cells are directly responsive to IFN-β and point to a novel mechanism of IFN-β-mediated T cell suppression that is independent of APC-derived signals.

Recent advances in endocrine therapy have improved the prospects for estrogen receptor-positive breast cancer. Tamoxifen is an effective drug for patients with estrogen receptor-positive breast cancer, but the development of resistance is common. Therefore, discovering ways to enhance the sensitivity of cancer cells to tamoxifen may help improve breast cancer treatment. We studied the biological role of sirtuin 4 (SIRT4) in tamoxifen-treated MCF7 and T47D cells. The levels of the MYC proto-oncogene (MYC) and cyclin D1 (CCND1) were detected by western blotting and quantitative reverse transcription-polymerase chain reaction in breast cancer cells with SIRT4 overexpression or depletion. Immunofluorescence and western blotting were used to assess the phosphorylation status of signal transducer and activator of transcription 3 (STAT3). SIRT4 overexpression decreased the half maximal inhibitory concentration of tamoxifen in MCF7 and T47D cells, while its depletion increased it. Thus, SIRT4 enhances the sensitivity of breast cancer cells to tamoxifen. Moreover, western blotting revealed decreased STAT3 phosphorylation after SIRT4 transfection. The transcription and translation of MYC and CCND1, target genes of the STAT3 pathway, were also blocked. Immunofluorescence revealed that pathway activation and nuclear STAT4 translocation were suppressed when SIRT4 was overexpressed. Furthermore, the effects of SIRT4 overexpression or depletion on proliferation could be offset by STAT3 activation or inhibition. Taken together, these results demonstrate that SIRT4 enhances the tamoxifen sensitivity of breast cancer cells by inhibiting the STAT3 signaling pathway. With this knowledge, therapeutic strategies with reduced drug resistance risk may be developed.

Diabetic nephropathy is one of the most significant microvascular complications associated with diabetes. Until now, there is no effective treatment and the gene mechanism of diabetic nephropathy is still unclear. Tangshen formula is a traditional Chinese medicine, and has been shown to have good clinical efficacy in diabetic nephropathy treatment. The objective of this study was to investigate the changes of gene expression profiling and explore the molecular mechanism using a db/db mice model treated by Tangshen formula. After administration for 12 weeks, a microarray was applied to detect the gene expression of db/db mice kidney tissues. Quantitative real-time PCR was used to confirm the differential gene expression and carry out a JAK/STAT/SOCS signaling pathway study. Treatment with Tangshen formula reduced the levels of serum glucose and urinary albumin in db/db mice, and the effects of Tangshen formula on db/db mice were significantly different from the positive control (Losartan potassium tablets) on microarray data. It also showed that the JAK/STAT/SOCS signaling pathway played an important role in the treatment process. The expressions of JAK1, JAK2, and STAT3 were upregulated, and STAT4 was downregulated in Tangshen formula-treated db/db mice. SOCS1, 3, and 7 were all activated, while negative feedback regulated other related genes in the JAK/STAT/SOCS pathway. Our study suggested that Tangshen formula has beneficial effects on diabetic nephropathy treatment via regulating the JAK/STAT/SOCS signaling pathway. This study will help to provide evidence-based recommendations for Tangshen formula clinical treatment.

Many genes implicated in Th1 and Th2 differentiation have been identified in both human and mouse. However, the functional roles and hierarchy of these factors in the signaling pathways leading to either Th1 or Th2 responses are less clear. To explore at which stage of polarization the differences between Th1 and Th2 cells occur, we have studied the expression of 23 key genes implicated in the process during the first week of polarization from human precursor T helper cells using quantitative real-time reverse transcription-PCR. According to our results, 14 of the genes were clearly regulated differentially in Th1 and Th2 conditions in distinct time-dependent patterns, either during the first 2 days or after 1 week of polarization. Furthermore, 6 of these genes were identified to be targets of STAT4/6 regulation. Thus, for the first time we demonstrate expression kinetics of a number of key genes involved in Th1 and Th2 differentiation during the first week of polarization in both human and mouse. In addition, our study shows for the first time that the genes BCL-6 and TRADD are differentially regulated during the polarization of human Th1 and Th2 cells.

Hematopoietic progenitor cell (HPC) homeostasis is critical in maintaining innate immunity and healing processes. Recently, we demonstrated that Th1 cells regulate HPC homeostasis, partly based on altered homeostasis in Stat4- and Stat6-deficient mice. To explore changes in HPC responsiveness in altered T helper cell environments, we directly examined growth factor-stimulated colony formation and chemokine-induced myelosuppression of HPC in Stat4- and Stat6-deficient bone marrow cells. Stat6-deficient cells have increased responses to the synergy between granulocyte-macrophage colony-stimulating factor (GM-CSF) and steel factor (SLF), compared to wild-type and Stat4-deficient cells. Increased responses are eliminated by in vivo depletion of CD4 cells. Whereas Stat6-deficient bone marrow cells respond to chemokine-mediated myelosuppression, Stat4-deficient bone marrow cells are refractory to the suppressive effects of chemokines. Thus, T helper cell development affects HPC homeostasis through several mechanisms, including the sensitivity to growth factor stimulation and chemokine suppression of HPC colony formation. Since Stat4 and Stat6 regulate opposing programs of T helper differentiation, there are distinct requirements for Stat4 and Stat6 in regulation of growth factor and chemokine responses of HPC.

Cytokines are central regulatory elements in peripheral lymphocyte differentiation, but their role in T cell ontogeny is poorly defined. In the present study, we evaluated the role of IL-12 in thymocyte selection more directly by determining its role in two models of in vivo negative selection. In initial studies we demonstrated that abundant intrathymic IL-12 synthesis occurs during OVA peptide-induced negative selection of thymocytes in neonatal OVA-TCR transgenic mice, and such synthesis is associated with increased IL-12R beta2-chain expression as well as STAT4 intracellular signaling. In further studies, we showed that this form of negative selection was occurring at the alphabetaTCRlowCD4lowCD8low stage and was prevented by the coadministration of anti-IL-12. In addition, the IL-12-dependent thymocyte depletion was occurring through an intrathymic apoptosis mechanism, also prevented by administration of anti-IL-12. Finally, we showed that IL-12 p40-/- mice displayed aberrant negative selection of double positive CD4+CD8+ thymocytes when injected with anti-CD3 mAb. These studies suggest that intact intrathymic IL-12 production is necessary for the negative selection of thymocytes occurring in relation to a high "self" Ag load, possible through its ability to induce the thymocyte maturation and cytokine production necessary for such selection.

T cells in the Peyer's patches (PP) of the human ileum are exposed to a myriad of dietary and bacterial Ags from the gut lumen. Recall proliferative responses to common dietary Ags are readily demonstrable by PP T cells from healthy individuals, and the cytokine response is dominated by IFN-gamma. Consistent with Th1 skewing, PP cells spontaneously secrete IL-12p70, and IL-12p40 protein can be visualized underneath the PP dome epithelium. In this study, we have analyzed IL-12 signaling in PP and investigated whether IL-12 plays a functional role. CD3+ T lymphocytes isolated from PP and adjacent ileal mucosa spontaneously secrete IFN-gamma with negligible IL-4 or IL-5. RNA transcripts for IL-12Rbeta2, the signaling component of the IL-12R, are present in purified CD4+ and CD8+ T PP lymphocytes. Active STAT4, a transcription factor essential for IL-12-mediated Th1 differentiation, is readily detectable in biopsies from PP and ileal mucosa and STAT4-DNA binding activity is demonstrable by EMSA. Nuclear proteins from CD3+ T PP lymphocytes contain STAT4 and T-bet, a transcription factor selectively expressed in Th1 cells. Stimulation of freshly isolated PP cells with staphylococcal enterotoxin B dramatically enhanced the production of IFN-gamma, an effect which was largely inhibited by neutralizing anti-IL-12 Ab. These data show that IL-12 in human PP is likely to be responsible for the Th1-dominated cytokine response of the human mucosal immune system.

In this report, the role of STAT4 and local production of interleukin (IL)-12 in the central nervous system (CNS) were examined during experimental vesicular stomatitis virus (VSV) encephalitis. We have previously shown that IL-12 treatment is beneficial both in vitro and in vivo during experimental VSV infection. This inhibition of VSV replication was dependent on the production of nitric oxide (NO) by the neuronal isoform of nitric oxide synthase (NOS-1). In vitro, IL-12 induces the phosphorylation and nuclear localization of STAT4 in neuroblastoma cell lines. STAT4 expression was not required for host survival or clearance of virus during experimental VSV encephalitis. Taken together, these data suggest that while neurons can respond directly to IL-12 in vitro by signaling through STAT4, STAT4 is not required for survival. It is likely that redundant innate host inflammatory cytokine responses compensate for the absence of IL-12 signaling.

The aim of this study was to investigate whether the JAK-STAT pathway participates in the processes of proliferation and differentiation induced by connective tissue growth factor (CTGF) in human hypertrophic scar fibroblasts (hHSF). hHSF were grown as primary cultures, then treated with or without CTGF. Western blotting was used to detect JAK1, JAK2, JAK3, TYK2, STAT1, STAT2, STAT3, STAT4, STAT5 and STAT6 protein expression in the hHSF at various time points after stimulation with CTGF. Immunofluorescence and the electrophoretic mobility shift assay (EMSA) were used to identify the interacting signalling molecules and to investigate their activation, respectively. After the signalling molecules of interest were selected, a specific inhibitor (STAT1 ASODN) was used to block the JAK-STAT pathway. The MTT assay was used to detect the proliferation of hHSF, and differentiation was assessed by evaluating changes in α-SMA expression by RT-PCR. Based on the results of Western blotting, immunofluorescence and EMSA, proliferation and differentiation were much higher in hHSF treated with CTGF (p<0.05). After blocking the pathway with STAT1 ASODN, hHSF proliferation was markedly, though not entirely, inhibited, while α-SMA expression was not significantly altered. JAK1 and STAT1 are therefore likely to participate in the proliferation and differentiation of hHSF induced by CTGF. However, though the STAT1 signalling pathway plays a key role in the CTGF-induced proliferation of hHSF, it is not the only pathway controlling this process. JAK1 may be an upstream element of STAT1, and may also participate in the CTGF-induced proliferation of hHSF. These results elucidate the signal transduction mechanism of CTGF-induced hHSF proliferation, and may aid in the development of a novel method for the inhibition of scar fibrosis and contraction.

BACKGROUND

Over the last five years, there have been numerous reports of association of juvenile idiopathic arthritis with single nucleotide polymorphisms (SNPs) at various loci outside the major histocompatibility complex (MHC) region. However, the majority of these association findings have been generated using a limited number of international cohorts, and thus there is benefit in further independent replication. To address this, we examined a total of 56 SNPs in 42 non-MHC gene regions previously reported to be associated with JIA, in the ChiLdhood Arthritis Risk factor Identification sTudY (CLARITY), a new Australian collection of cases and healthy child controls.

RESULTS

Genotyping was performed on a total of 324 JIA cases (mean age 9.7 years, 67.3% female) and 568 controls (mean age 7.8 years, 40.7% female). We demonstrated clear evidence for replication of association of JIA with SNPs in or around c12orf30, c3orf1, PTPN22, STAT4, and TRAF1-C5, confirming the involvement of these loci in disease risk. Further, we generated evidence supportive of replication of association of JIA with loci containing AFF3, CD226, MBL2, PSTPIP1, and RANTES (CCL5). These results were robust to sensitivity analyses for ethnicity.

CONCLUSIONS

We have provided valuable independent data as to the underlying genetic architecture of this understudied pediatric autoimmune disease, further confirming five loci outside the MHC, and supporting a role for a further five loci in determining disease risk.

OBJECTIVE

Rheumatoid arthritis (RA) and ankylosing spondylitis (AS) are common systemic autoimmune diseases with genetic and environmental predisposing factors. Signal transducer and activator of transcription 4 (STAT4) transmits signals induced by interleukin-12, interleukin-23 and interferon-γ, which are key cytokines and play important roles in the development of autoimmune diseases. Previous studies confirmed the STAT4 rs7574865 G/T locus to be associated with RA. Thus we conducted a replication study to investigate STAT4 rs7574865 G/T polymorphism and RA/AS susceptibility in a Chinese population.

METHODS

We studied STAT4 rs7574865 G/T gene polymorphism in 520 patients with RA, 100 AS patients and 520 controls in a Chinese population. Genotyping was done using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).

RESULTS

When the STAT4 rs7574865 GG homozygote genotype was used as the reference group, the GT or GT/TT genotypes were associated with the risk for RA. After stratification analyses, a significantly increased risk for RA associated with the STAT4 rs7574865 GT genotype was evident among the rheumatoid factor (RF)-positive patients, patients with higher erythrocyte sedimentation rate (ESR) level and patients with higher RA disease activity score (DAS28) compared with the STAT4 rs7574865 GG genotype. A significantly increased risk for RA associated with the STAT4 rs7574865 TT genotype was evident among older patients and RF-negative patients compared with the STAT4 rs7574865 GG genotype. STAT4 rs7574865 G/T was not associated with susceptibility to AS.

CONCLUSIONS

This replication study confirmed that STAT4 rs7574865 G/T polymorphism was associated with the risk of RA.

CONCLUSIONS

STAT4 polymorphisms are associated with rheumatoid arthritis risk.

BACKGROUND

The genome-wide association study era has made great progress in identifying susceptibility genes and genetic loci for rheumatoid arthritis (RA) in populations of White European ancestry. However, few studies have tried to dissect disease aetiopathogenesis in other ethnic populations.

OBJECTIVE

To investigate these associations in the Han Chinese population.

METHODS

Haplotypes from the HapMap database Chinese population were used to select tag-single-nucleotide polymorphisms (SNPs) (r(2)=0.8) across 19 distinct RA genomic regions. A two phase case-control association study was performed, with 169 SNPs genotyped in phase I (n=571 cases, n=880 controls), and 64 SNPs achieving p<0.2 in the first phase being genotyped in phase II (n=464 cases, n=822 controls). Association statistics were calculated using permutation tests both unadjusted and adjusted for the number of markers studied.

RESULTS

Robust association was detected for MMEL1 and CTLA4, and modest association was identified for another six loci: PADI4, STAT4, PRDM1, CDK6, TRAF1-C5 and KIF5A-PIP4K2C. All three markers genotyped in MMEL1 demonstrated association, with peak signal for rs3890745 (p=2.6 × 10(-5) unadjusted, p=0.003 adjusted, OR=0.79). For CTLA4, significance was detected for three of five variants showing association, with peak association for marker rs12992492 (p=4.3 × 10(-5) unadjusted, p=0.0021 adjusted, OR=0.77). Lack of association of common variants in PTPN22 with RA in Han Chinese was confirmed.

CONCLUSIONS

This study identifies MMEL1 and CTLA4 as RA susceptibility genes, provides suggestive evidence of association for a further six loci in the Han Chinese population and confirms lack of PTPN22 association in Asian populations. It also confirms the value of multiethnic population studies to help dissect disease aetiopathogenesis.

Acute graft-versus-host disease (aGVHD) is a serious complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Our previous study found that the novel anti-inflammatory cytokine IL-35 could suppress aGVHD in patients after allo-HSCT. In this study, we used C57BL/6 (B6, H-2b) mice as donors and (B6×DBA/2) F1 (BDF1, H-2b×d) mice as recipients to create a model of aGVHD and explore the relationship between IL-35 and aGVHD. The mice receiving IL-35 survived longer than did the control mice. We observed that treatment with IL-35 and RAPA could reduce the incidence of aGVHD. Additionally, this treatment inhibited intestinal and thymic epithelial cell apoptosis and liver infiltration by the donor T-cells, thereby ameliorating the enteropathy and liver injury caused by aGVHD. We found that IL-35 and RAPA also markedly suppressed TNF-α and IL-17A expression and enhanced IFN-γ expression in the intestine and liver. We measured Tregs in spleen and found that IL-35 and RAPA treatment expanded the number of Tregs in spleen. We found that the phosphorylation of STAT1 and STAT4 were inhibited in mice with aGVHD. In contrast, STAT1 and STAT4 were phosphorylated when the mice were treated with IL-35. IL-35 may have therapeutic potential in the treatment of aGVHD after allo-HSCT.

Bile acids are established signaling molecules next to their role in the intestinal emulsification and uptake of lipids. We here aimed to identify a potential interaction between bile acids and CD4+ Th cells, which are central in adaptive immune responses. We screened distinct bile acid species for their potency to affect T cell function. Primary human and mouse CD4+ Th cells as well as Jurkat T cells were used to gain insight into the mechanism underlying these effects. We found that unconjugated lithocholic acid (LCA) impedes Th1 activation as measured by i) decreased production of the Th1 cytokines IFNγ and TNFαα, ii) decreased expression of the Th1 genes T-box protein expressed in T cells (T-bet), Stat-1 and Stat4, and iii) decreased STAT1α/β phosphorylation. Importantly, we observed that LCA impairs Th1 activation at physiological relevant concentrations. Profiling of MAPK signaling pathways in Jurkat T cells uncovered an inhibition of ERK-1/2 phosphorylation upon LCA exposure, which could provide an explanation for the impaired Th1 activation. LCA induces these effects via Vitamin D receptor (VDR) signaling since VDR RNA silencing abrogated these effects. These data reveal for the first time that LCA controls adaptive immunity via inhibition of Th1 activation. Many factors influence LCA levels, including bile acid-based drugs and gut microbiota. Our data may suggest that these factors also impact on adaptive immunity via a yet unrecognized LCA-Th cell axis.

Major surgery increases the risk for infectious complications due to the development of immunosuppression. CD56 bright NK cells play a key role in the defense against bacterial infections through the release of Interferon (IFN) γ upon stimulation with monocyte-derived Interleukin (IL) 12. We investigated whether invasive visceral surgery interferes with the IFN-γ synthesis of human NK cells in response to Staphylococcus aureus. In a prospective pilot study, peripheral blood mononuclear cells (PBMC) were isolated from 53 patients before and 1 to 7 d after elective visceral surgery. The release of IL-12 and IFN-γ from PBMC upon exposure to S. aureus in vitro was quantified. The expression of the IL-12 receptor β1 chain on the surface, the phosphorylation of signal transducer and activator of transcription (STAT) 4, and the synthesis of IFN-γ on/in individual CD56 bright NK cells were investigated using flow cytometry. The modulatory effect of IL-12 on the S. aureus-induced IFN-γ production in CD56 bright NK cells was analyzed. The IFN-γ secretion from purified CD56 bright NK cells was quantified after stimulation with IL-12 and IL-18. After surgery, CD56 bright NK cells among total PBMC were impaired in the release of IFN-γ for at least 5 d. Likewise, the IL-12-induced release of IFN-γ from purified CD56 bright NK cells was abolished. Upon stimulation with S. aureus, PBMC secreted less IL-12 but supplementation with recombinant IL-12 did not restore the capacity of CD56 bright NK cells to produce IFN-γ. CD56 bright NK cells displayed reduced levels of the IL-12Rβ1 chain whereas the phosphorylation of STAT4, the key transcription factor for the Ifng gene was not diminished. In summary, after invasive visceral surgery, CD56 bright NK cells are impaired in S. aureus-induced IFN-γ production and might contribute to the enhanced susceptibility to opportunistic infections.

An alteration of immune recognition and killing of misplaced endometrial cells, refluxed with menstrual debris in ectopic sites, has been claimed to be responsible for the initiation and progression of endometriosis. In particular, current evidence emphasizes the role of natural killer (NK) cells as potential effectors of peritoneal immune surveillance. Interleukin-12 (IL-12), a heterodimeric cytokine composed of p40 and p35 chains, has potent regulatory effects on NK cell growth and function. The purpose of this study was to evaluate whether this cytokine may also have a role in the specific cytolytic NK cell response toward endometrial antigens. To this aim, concentrations of IL-12 and its free p40 subunit were determined in peritoneal fluid of 33 patients with endometriosis and 40 women without laparoscopic evidence of the disease. Similar concentrations of IL-12, but significantly higher levels of free p40, were present in peritoneal fluid of patients with endometriosis compared to those in women without the disease. We also observed that the IL-12 plus free p40/IL-12 ratio increased with the severity of the disease. Moreover, we investigated whether incubation of NK cells with heterodimeric IL-12 and/or p40 has any effect on NK cell-mediated lysis of endometrial cells. NK cells pretreated with heterodimeric IL-12 exhibited an enhanced cytotoxic response toward endometrial targets. This IL-12-induced cytotoxicity could be abrogated by the p40 subunit in a specific and dose-dependent manner. The p40 inhibitory effect was mediated by down-regulation of IL-12 high affinity binding sites on NK cells, as we observed inhibition of surface IL-12 receptor beta1-chain expression, a decrease in IL-12-binding capacity, and inhibition of phosphorylation of STAT4 (signal transducer and activator of transcription) protein. These data suggest that the excess of p40 present in peritoneal fluid of patients with endometriosis may be related to the NK cell defect associated with the disease. Moreover, IL-12 could be a potential specific agent able to correct the p40-induced defect in vivo.

BACKGROUND

Deer mice (Peromyscus maniculatus) are the most common mammals in North America and are reservoirs for several zoonotic agents, including Sin Nombre virus (SNV), the principal etiologic agent of hantavirus cardiopulmonary syndrome (HCPS) in North America. Unlike human HCPS patients, SNV-infected deer mice show no overt pathological symptoms, despite the presence of virus in the lungs. A neutralizing IgG antibody response occurs, but the virus establishes a persistent infection. Limitations of detailed analysis of deer mouse immune responses to SNV are the lack of reagents and methods for evaluating such responses.

RESULTS

We developed real-time PCR-based detection assays for several immune-related transcription factor and cytokine genes from deer mice that permit the profiling of CD4+ helper T cells, including markers of Th1 cells (T-bet, STAT4, IFNgamma, TNF, LT), Th2 cells (GATA-3, STAT6, IL-4, IL-5) and regulatory T cells (Fox-p3, IL-10, TGFbeta1). These assays compare the expression of in vitro antigen-stimulated and unstimulated T cells from individual deer mice.

CONCLUSIONS

We developed molecular methods for profiling immune gene expression in deer mice, including a multiplexed real-time PCR assay for assessing expression of several cytokine and transcription factor genes. These assays should be useful for characterizing the immune responses of experimentally- and naturally-infected deer mice.

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease with complex genetic inheritance. Genetic association of signal transducer and activator of transcription 4 (STAT4) with SLE susceptibility has been convincingly established in multiple populations including Asians, whereas studies of genetic relations between STAT4 polymorphisms and subphenotypes of SLE were rarely conducted. In this study, we selected Chinese female population and investigated genetic association between a polymorphism of STAT4 gene (rs7582694) and SLE. Furthermore, genetic association tests based on different subsets classified by 11 clinical manifestations were also performed. A total of 675 SLE female patients and 678 healthy controls were enrolled into this study, and SNP genotyping was performed using Sequenom's MassArray system (Sequenom iPLEX assay). Our study showed strong evidence for genetic predisposition of rs7582694 to SLE (X ( 2 ) = 23.7, OR = 0.68, 95% CI: 0.58-0.79, P = 1.13 × 10(-6)), while no association was observed between rs7582694 and any clinical presentations. The results of our study demonstrated that STAT4 rs7582694 SNP was significantly associated with SLE, and these results were in accordance with previous studies.