Fuzhen Li

Identification of a susceptibility locus in STAT4 for Behçet's disease in Han Chinese in a genome-wide association study

OBJECTIVE To identify susceptibility loci for Behçets disease (BD) and elucidate their functional role. METHODS A genome-wide association study (GWAS) and functional studies were conducted. A total of 149 patients and 951 controls were enrolled in the initial GWAS, and 554 patients and 1,159 controls were enrolled in the replication study. Real-time polymerase chain reaction, luciferase reporter assay, and enzyme-linked immunosorbent assay were performed. RESULTS Our GWAS and replication studies identified a susceptibility locus around STAT4 (single-nucleotide polymorphisms [SNPs] rs7574070, rs7572482, and rs897200; P = 3.36 × 10(-7) to 6.20 × 10(-9) ). Increased expression of STAT4 was observed in individuals carrying the rs897200 risk genotype AA. Consistent with the idea that STAT4 regulates the production of interleukin-17 (IL-17) and interferon-γ, IL17 messenger RNA and protein levels were increased in individuals carrying the rs897200 risk genotype AA. Interestingly, the risk allele A of rs897200 creates a putative transcription factor binding site. To test whether it directly affects STAT4 transcription, an in vitro luciferase reporter gene assay was performed. Higher transcription activity was observed in individuals carrying the risk allele A, suggesting that rs897200 is likely to directly affect STAT4 expression. Additionally, 2 SNPs, rs7574070 and rs7572482, which are tightly linked with rs897200, were cis-expression quantitative trait loci (eQTL) SNPs, suggesting that SNP rs897200 is an eQTL SNP. Most importantly, the clinical disease severity score was higher in individuals with the rs897200 risk genotype AA. CONCLUSION These findings strongly suggest that STAT4 is a novel locus underlying BD. We propose a model in which up-regulation of STAT4 expression and subsequent STAT4-driven production of inflammatory cytokines, such as IL-17, constitute a potential pathway leading to BD.

The Effects of Th17 Cytokines on the Inflammatory Mediator Production and Barrier Function of ARPE-19 Cells

Th17 cells have emerged as a key factor in the pathogenesis of uveitis as well as other autoimmune disorders. They secrete a number of cytokines including IL-17A, IL-17F and IL-22 and until now the effects of these cytokines on resident cells of the eye were not yet clear. The purpose of this study was to investigate the effects of Interleukin (IL)-17A, IL-17F and IL-22 on the production of inflammatory mediators and barrier function of retinal pigment epithelium cells. We showed that ARPE-19 cells, a spontaneously arisen cell line of retinal pigment epithelium (RPE), constitutively expressed IL-17RC and IL-22R, but not IL-17RA. IL-17A significantly enhanced the production of CXCL8, CCL2, CCL20 and IL-6 by these cells. IL-17F had a similar effect on the production of CXCL8, CCL2 and IL-6 by ARPE-19 cells, but did not influence the expression of CCL20. Both IL-17A and IL-17F significantly decreased the transepithelial electrical resistance (TER) of the ARPE-19 monolayer and increased the diffusion rate of fluorescein isothiocyanate (FITC)-dextran. They also disrupted the distribution of the junction proteins zonula occludens (ZO)-1 and occludin at the interface of adjacent cells. IL-22 did not have a detectable effect on the production of the tested inflammatory mediators by ARPE-19 cells, TER of the ARPE-19 monolayer, the diffusion rate of FITC-dextran or the distribution of ZO-1 and occludin. This study demonstrates that IL-17A and IL-17F, but not IL-22, significantly promoted ARPE-19 cells to secrete inflammatory mediators and compromised the ARPE-19 monolayer barrier function in association with a disrupted distribution of ZO-1 and occludin. These results suggest that both IL-17A and IL-17F may play a role in posterior segment inflammation of the eye.

STAT4 polymorphism in a Chinese Han population with Vogt-Koyanagi-Harada syndrome and Behçet's disease.

This study investigated the association of rs7574865 polymorphism in STAT4 with Vogt-Koyanagi-Harada (VKH) syndrome and Behçets disease (BD) in a Chinese Han population. Genotyping of rs7574865 polymorphism in the STAT4 gene was performed using polymerase chain reaction restriction fragment length polymorphisms in 379 VKH patients, 366 BD patients, and 414 controls. Of the samples, 20% were sequenced to validate polymerase chain reaction restriction fragment length polymorphism results. A binary logistic regression analysis was used to assess the influence of the gender on the association of STAT4 polymorphism with BD. A significantly increased frequency of TT genotype of the STAT4 rs7574865 was observed in VKH patients (p = 0.013). GT genotypic frequency was significantly lower in BD patients than in controls (p = 0.003) However the significance of rs7574865 was lost in all tested BD patients when adjusted for gender (p = 0.775). A significantly lower frequency of GT genotype and a significantly higher frequency of GG genotype was found in male BD patients compared with male controls (p = 0.000458 and p = 0.009, respectively). Stratification analysis according to tinnitus, alopecia, poliosis, headache, and vitiligo for VKH syndrome and oral ulceration, genital ulceration, skin lesions and arthritis for BD failed to find any association between the tested single nucleotide polymorphism and any of the extraocular findings. Our results suggest that TT genotype of rs7574865 may be a susceptible factor for VKH syndrome in a Chinese Han population, and that GG genotype of this SNP may confer susceptibility in male BD patients.

Decreased microRNA-155 Expression in Ocular Behcet's Disease but Not in Vogt Koyanagi Harada Syndrome

PURPOSE MicroRNAs (miRNAs) have emerged as a class of gene expression regulators involved in immune regulation. In the present study, we investigated the role of miRNA in two uveitis entities: Behcets disease (BD) and Vogt Koyanagi Harada syndrome (VKH). METHODS The expression of five miRNAs was studied in PBMCs, DCs, and CD4(+) T cells from BD patients with active and inactive uveitis, VKH patients with active uveitis, and healthy controls using real-time PCR. MiR-155 mimics and inhibitor were transfected to DCs to evaluate the effect on DC maturation and cytokine production by these cells and CD4(+) T cells. Luciferase reporter assays and Western blotting were performed to identify the target gene of miR-155. RESULTS Only miR-155 expression was significantly decreased in PBMCs and DCs from BD patients with active uveitis and no differences were observed in the miRNA expression in cells from patients with VKH as compared with controls. Overexpression of miR-155 in DCs was shown to inhibit the production of IL-6 and IL-1β, and to promote the expression of IL-10 by these cells. MiR-155 transfected DCs significantly inhibited intracellular IL-17 expression in allogeneic CD4(+) T cells; however, it did not influence the expression of cell surface markers CD80, CD40, CD83, CD86, and HLA-DR. Luciferase reporter assays revealed that TAB2 was a target gene of miR-155, which was confirmed by Western blotting. CONCLUSIONS The present results suggest that miR-155 expression is decreased in active BD but not in VKH patients. Downregulated miR-155 may be involved in BD pathogenesis by targeting TAB2.

Decreased IL-27 expression in association with an increased Th17 response in Vogt-Koyanagi-Harada disease.

PURPOSE IL-27 has emerged as an important regulator of proinflammatory T-cell responses in animal models. We investigated the pathophysiological role of IL-27 in Vogt-Koyanagi-Harada (VKH) disease. METHODS IL-27P28 and EBI3 mRNA expression in peripheral blood mononuclear cells (PBMCs) were assayed by RT-PCR. Cytokines in the serum and supernatants of PBMCs, naïve CD4(+) T cells and DC-T cocultures were assayed by ELISA. Flow cytometry was used to evaluate the frequencies of IL-17-producing CD4(+) T cells. RESULTS The active VKH patients showed a decreased IL-27P28 mRNA expression in PBMCs and lower IL-27 expression in the serum and supernatants of PBMCs, but higher Th17 cells in PBMCs. EBI3 mRNA expression was not different among the groups tested. Stimulation of naïve CD4(+) T cells under Th17 polarizing conditions showed a higher Th17 cell differentiation in active VKH patients. IL-27 significantly inhibited Th17 cell differentiation. IL-27-treated DCs showed a significant inhibition on Th17 differentiation. There was a significant defect in the Tr1 cell induction as measured by IL-10 in active VKH patients. Treatment with corticosteroids and cyclosporine A (CsA) resolved the intraocular inflammation in association with an upregulation of IL-27 and a downregulation of IL-17. In vitro experiments showed that corticosteroids, but not CsA, significantly upregulated the expression of IL-27. CONCLUSIONS The present study suggests that decreased IL-27 expression may result in a higher Th17 in active VKH patients, which may promote the autoimmune response observed in these patients. Manipulation of IL-27 may offer a novel target for treatment of this disease.

Ocular manifestations of syphilitic uveitis in Chinese patients.

Purpose: To present the manifestations of syphilitic uveitis in Chinese patients. Methods: This is a retrospective case series of 35 eyes of 19 patients with syphilitic uveitis. The data of these patients including complaints, ocular and systemic manifestations, human immunodeficiency virus status, results of auxiliary examinations, treatment, and follow-up were reviewed. Results: Nineteen consecutive Chinese patients were diagnosed with syphilitic uveitis by serologic tests. Four patients had circulating human immunodeficiency virus antibodies. Ocular involvement was found in 35 eyes. Posterior segment involvement was found in 30 eyes of 17 patients (85.7%), whereas anterior segment involvement was found in 14 eyes of 8 patients (40.0%). Thirty eyes of 17 patients (85.7%) presented with vitreous opacities and 28 eyes of 16 patients (80.0%) with retinitis. Papillitis and retinal vasculitis were found in 10 eyes of 6 patients (28.6%) and 7 eyes of 4 patients (20.0%), respectively. Multiple precipitates on the retina and posterior vitreous membrane were observed in six eyes of three patients. A large iris granuloma was observed in one eye. Conclusion: Posterior uveitis was the most common ocular finding in these investigated Chinese patients with syphilis. Coinfection of syphilis and human immunodeficiency virus was less common in these patients. Syphilis should be considered in the differential diagnosis of patients presenting with large iris granulomas.

IFN-α blocks IL-17 production by peripheral blood mononuclear cells in Behçet's disease

OBJECTIVES IFN-α has been used to treat patients with Behçets disease (BD). Recent studies have implicated the IL-23/Th-17 pathway in the pathogenesis of BD. In this study, we investigated whether IFN-α could affect this pathway. METHODS Peripheral blood mononuclear cells (PBMCs) obtained from patients with active BD and controls were cultured alone or with IFN-α and the levels of IL-17 and IL-10 in the supernatants were measured by ELISA. Similar experiments were performed with isolated CD4(+) T cells from controls. The levels of phosphorylated STAT1 (p-STAT1), p-STAT2, p-STAT3 and p-STAT5 in CD4(+) T cells from controls cultured with or without IFN-α were also evaluated by ELISA. Furthermore, an experiment using anti-IL-10 was performed to examine underlying mechanisms of action of IFN-α. RESULTS Significantly higher levels of IL-17 and IL-10 were observed in the supernatants of PBMCs from BD patients as compared with controls. IFN-α significantly decreased IL-17 production by PBMCs from both patients and controls. On the other hand, IFN-α increased IL-10 production by PBMCs from patients and controls. Similar findings were obtained when using CD4(+) T cells from controls, IFN-α significantly increased p-STAT2 expression in control CD4(+) T cells. Anti-IL-10 antibody was able to neutralize the inhibitory effect of IFN-α on IL-17 by 35% as compared with controls. CONCLUSIONS In vitro experiments showed that IFN-α could inhibit IL-17 expression and increased IL-10 production by PBMCs and CD4(+) T cells. The inhibitory role of IFN-α on IL-17 was partly mediated by IL-10. IFN-α activity was mediated via STAT2 phosphorylation.

Upregulation of interleukin 21 and promotion of interleukin 17 production in chronic or recurrent Vogt-Koyanagi-Harada disease.

OBJECTIVES To analyze the expression and potential role of interleukin (IL) 21 in the pathogenesis of Vogt-Koyanagi-Harada (VKH) disease. METHODS Blood samples were obtained from patients with VKH disease and from healthy control subjects. Serum IL-21 level and IL-21 messenger RNA (mRNA) expression by peripheral blood mononuclear cells (PBMCs) were determined by enzyme-linked immunosorbent assay and by reverse transcriptase-polymerase chain reaction, respectively. Interleukin 17 and interferon γ levels in the supernatants of PBMCs and CD4(+) T cells cultured with anti-CD3 and anti-CD28 antibodies in the presence or absence of recombinant IL-21 were detected by enzyme-linked immunosorbent assay. RESULTS The results showed a significantly increased serum IL-21 level, as well as higher IL-21 mRNA expression by PBMCs, in patients having chronic or recurrent active VKH disease compared with patients having inactive VKH disease and with controls. In vitro experiments showed that recombinant IL-21 significantly increased IL-17 production by PBMCs and by CD4(+) T cells from patients and from controls. However, recombinant IL-21 did not affect interferon γ expression by PBMCs or by CD4(+) T cells. CONCLUSION Interleukin 21 may be involved in the pathogenesis of chronic or recurrent VKH disease, possibly by promoting IL-17 secretion. CLINICAL RELEVANCE Findings from the present study suggest that IL-21 may be a potential target in the development of therapy for VKH disease.

Polymorphisms of IL23R and Vogt-Koyanagi-Harada syndrome in a Chinese Han population.

Polymorphisms of interleukin-23 receptor (IL23R) gene have recently been reported to be associated with the susceptibility to several immune-related diseases. The aim of this study was to determine the association of IL23R polymorphisms with Vogt-Koyanagi-Harada (VKH) syndrome, a disease presumably mediated by autoimmune response. A total of 382 Chinese Han patients with VKH syndrome and 407 healthy controls were genotyped by the polymerase chain reaction-restriction fragment length polymorphism method. Data were analyzed by chi(2) analysis. All genotype distributions in healthy controls were in Hardy-Weinberg equilibrium. There was no difference among the investigated four single nucleotide polymorphisms concerning the linkage disequilibrium between the tested samples and those available in the international HapMap. The genotype and allele frequencies of rs17375018, rs7517847, rs11209032, and rs1343151 were not different between patients with VKH syndrome and healthy controls. Analysis according to gender and clinical findings did not show any association of the four polymorphisms with these parameters. In conclusion, the tested IL23R gene polymorphisms are not associated with the susceptibility to VKH syndrome in the Chinese Han population.

Elevated Serum Osteopontin Levels and Genetic Polymorphisms of Osteopontin Are Associated with Vogt-Koyanagi-Harada Disease

PURPOSE Osteopontin (OPN) is a proinflammatory cytokine involved in chronic inflammatory diseases. This study aimed to analyze the role of OPN in the pathogenesis of Vogt-Koyanagi-Harada (VKH) disease. METHODS Serum levels of OPN in VKH patients and healthy controls were assayed by enzyme-linked immunosorbent assay (ELISA). Peripheral blood mononuclear cells (PBMCs) or CD4+ T cells were cultured with anti-CD3 and anti-CD28 antibodies in the absence or presence of recombinant OPN for the determination of cell proliferation and cytokines. Cell proliferation was detected using a cell counting kit. Levels of interferon (IFN)-γ and interleukin (IL)-17 were detected by ELISA. Four single nucleotide polymorphisms (SNPs) of OPN and four SNPs of OPN receptors were genotyped in 601 VKH patients and 605 healthy controls using a polymerase chain reaction-restriction fragment length polymorphism assay. RESULTS OPN serum levels were significantly higher in patients with active VKH than in patients with inactive VKH and in healthy controls. PBMCs or CD4+ T cells cultured with recombinant OPN induced a marked cell proliferation and profound secretion of IFN-γ and IL-17 from patients with active VKH. A significantly increased frequency of the OPN rs4754 TT genotype (P = 0.004, pc = 0.048) was observed in VKH patients compared with healthy controls. No association could be detected among the four selected SNPs of OPN receptors and VKH. CONCLUSIONS OPN may be relevant to the pathogenesis of VKH disease. The TT genotype of rs4754 may be a susceptible factor for VKH disease in a Chinese Han population.