Qingyun Zhou

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.

MicroRNA-146a and Ets-1 gene polymorphisms in ocular Behçet's disease and Vogt–Koyanagi–Harada syndrome

Aim MicroRNA-146a (miR-146a) is involved in certain immune-mediated diseases. Transcription factor Ets-1 strongly affects miR-146a promoter activity and directly regulates miR-146a expression. This study was performed to investigate the association of miR-146a and Ets-1 gene polymorphisms with Behçets disease (BD) and Vogt–Koyanagi–Harada (VKH) disease in a Chinese Han population. Methods A total of 809 patients with BD, 613 patients with VKH and 1132 normal controls were genotyped for miR-146a/rs2910164, rs57095329 and rs6864584, Ets-1/rs1128334 and rs10893872 using a PCR restriction fragment length polymorphism assay. miR-146a expression was examined in peripheral blood mononuclear cells (PBMCs) by real-time PCR. Cytokine production by PBMCs was measured by ELISA. Results A significantly decreased frequency of the homozygous rs2910164 CC genotype and C allele was observed in patients with BD compared with controls (pca=1.24×10−5, OR 0.61; pca=1.33×10−4, OR 0.75, respectively). MiR-146a expression in GG cases was 2.45-fold and 1.99-fold higher, respectively, than that in CC cases and GC cases. There was no association of the other four single nucleotide polymorphisms (SNPs) with BD. There was also no association of these five SNPs with its main clinical features. No associations were found with the five SNPs tested or with its clinical manifestations in VKH disease. Interleukin (IL)-17, tumour necrosis factor (TNF)α and IL-1β production from rs2910164 CC cases was markedly lower than that in GG cases. No effect of genotype was observed on IL-6 and monocyte chemoattractant protein (MCP)-1 production and IL-8 expression was slightly higher in CC cases. Conclusions Our study identified a strong association of rs2910164 of miR-146a with BD in a Chinese population and decreased expression of miR-146a and certain proinflammatory cytokines in individuals carrying the CC genotype.

IL-1β triggered by peptidoglycan and lipopolysaccharide through TLR2/4 and ROS-NLRP3 inflammasome-dependent pathways is involved in ocular Behçet's disease.

PURPOSE Behçets disease (BD) is a chronic systemic inflammatory disorder of unknown etiology. Toll-like receptors (TLRs) are critical in the innate immune response to microbial invaders. In this study we investigated the role of TLRs in the pathogenesis of BD. METHODS TLR2/4 expression and IL-1β and reactive oxygen species (ROS) production were studied in monocyte-derived macrophages (MDMs) obtained from BD patients, acute anterior uveitis (AAU) patients, and healthy controls using real-time PCR, flow cytometry, and ELISA. The NLRP3 inflammasome of MDMs was downregulated by RNA interference. The levels of phosphorylated P38, Erk1/2, and JNK MAPK were evaluated using flow cytometry. RESULTS TLR2/4 expression was significantly increased in MDMs from active BD patients. IL-1β and ROS production of peptidoglycan (PGN)/lipopolysaccharide (LPS)-induced MDMs from active BD patients was significantly increased compared with inactive BD patients, AAU patients, and healthy controls. ROS activator and inhibitor significantly increased and decreased the production of IL-1β, respectively. The production of IL-1β was significantly decreased after the NLRP3 inflammasome was downregulated. The phosphorylation levels of p38 and ERK1/2 in MDMs from BD patients and controls were increased following stimulation with either PGN or LPS. Both SB203580 (p38 inhibitor) and PD98059 (ERK1/2 inhibitor) significantly decreased the production of IL-1β. CONCLUSIONS The results suggest that TLR2/4 expression in MDMs from active BD patients is significantly increased. Interaction of TLR2/4 with their ligands PGN/LPS is involved in BD pathogenesis, possibly by the induction of IL-1β through a ROS-NLRP3-dependent pathway.

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.

Increased Expression of IL-22 Is Associated with Disease Activity in Behcet’s Disease

Objective Interleukin (IL)-22 has been reported to be involved in the development of autoimmune diseases. This study aimed to analyze the expression and potential role of IL-22 in the pathogenesis of Behcet’s disease (BD). Methods The levels of IL-22 in patient sera or supernatants of cultured peripheral blood mononuclear cells (PBMCs) and CD4+T cells were detected by enzyme-linked immunosorbent assay (ELISA). Flow cytometry was used to evaluate the frequency of IL-22–producing CD4+ T cells. IL-22 mRNA from erythema nodosum skin lesions was examined using real time quantitative RT-PCR. Results BD patients with active uveitis showed a significantly higher expression of IL-22 in the supernatants of stimulated PBMCs and CD4+T cells compared with BD patients without active uveitis and normal controls. An increased frequency of IL-22-producing CD4+T cells was also found in BD patients with active uveitis. IL-22 mRNA expression was elevated in erythema nodosum skin lesions. In BD patients, a high IL-22 level in the supernatant of stimulated PBMCs correlated with the presence of retinal vasculitis and erythema nodosum. Conclusions IL-22 was associated with disease activity in BD and correlated with the presence of small vessel inflammation, suggesting that it may be involved in its pathogenesis.

Genetic Variations of IL-12B, IL-12Rβ1, IL-12Rβ2 in Behcet's Disease and VKH Syndrome

Purpose To investigate the associations of single nucleotide polymorphisms (SNPs) of three genes (IL-12B, IL-12Rβ1 and IL-12Rβ2) in Behcets disease (BD) and Vogt-Koyanagi-Harada (VKH) syndrome in a Chinese Han population. Methods A total of 806 BD cases, 820 VKH patients, and 1600 healthy controls were involved in this study. The first investigation included 400 BD patients, 400 VKH cases, and 600 healthy individuals. A second confirmatory study included a separate set of 406 BD patients, 420 VKH cases and another 1000 normal controls. Genotyping was carried out by PCR-restriction fragment length polymorphism assay and results were validated by using direct sequencing. The χ2 test was performed to compare the allele and genotype frequencies between cases and healthy controls. Results This study comprised two phases. In the first phase study, a significantly increased frequency of the rs3212227/IL-12B genotype CC and C allele was found in BD patients as compared to controls (Bonferroni corrected p value (pc) = 0.009, OR 1.8; pc = 0.024, OR 1.3, respectively). Moreover, the frequency of the C allele of rs3212227/IL-12B was also significantly increased in VKH patients (pc = 0.012, OR 1.3, 95% CI 1.1 to 1.6). No associations were found for the other seven tested SNPs either in BD or VKH disease. The second study as well as the combined data confirmed the significant association of rs3212227/IL-12B with BD (CC genotype: combined pc = 6.3×10−7, OR = 1.8; C allele: combined pc = 2.0×10−5, OR = 1.3, respectively) and the C allele frequency of rs3212227/IL-12B as the risk factor to VKH patients (combined pc = 2.5×10−5, OR 1.3, 95% CI 1.2 to 1.5). Conclusions Our study revealed that the IL-12B gene is involved both in the susceptibility to BD as well as VKH syndrome.

MicroRNA-146a and Ets-1 Gene Polymorphisms Are Associated with Pediatric Uveitis

Background MicroRNA-146a (miR-146a) was a key negative regulator of autoimmunity. V-Ets oncogene homolog 1 (Ets-1) was demonstrated to bind to the miR-146a promoter region and markedly affects miR-146a promoter activity. This study aimed to investigate the association of miR-146a and Ets-1 gene polymorphisms with pediatric uveitis in a Han Chinese population. Methodology/Principal Findings A total of 520 patients and 1204 healthy controls were included in the present study. Five single-nucleotide polymorphisms (SNPs), miR-146a/rs2910164, miR-146a/rs57095329, miR-146a/rs6864584, ets-1/rs1128334 and ets-1/rs10893872 were genotyped using a polymerase chain reaction-restriction fragment length polymorphism assay. The expression of Ets-1 in peripheral blood mononuclear cells from genotyped healthy controls was tested by real-time PCR. Two SNPs (rs2910164 and rs10893872) were associated with pediatric uveitis in this study. The frequencies of the rs2910164 GG genotype and G allele were significantly increased (Pc = 3.11×10−4; Pc = 2.75×10−6) while the CC genotype and C allele were significantly decreased (Pc = 0.001; Pc = 2.75×10−6) in patients compared with normal controls. The frequencies of the rs10893872 CC genotype and C allele were significantly increased (Pc = 3.89×10−4; Pc = 0.01) while the CT genotype and T allele were significantly decreased (Pc = 0.004; Pc = 0.01) in patients compared with normal controls. The SNP rs2910164 GG genotype and G/C allele were also associated with the presence of microvascular leakage as detected by fundus fluorescein angiography in pediatric uveitis (Pc = 0.01; Pc = 0.005, respectively). Ets-1 expression in rs10893872 CC carriers was significantly higher than in CT and TT individuals (Pc = 0.013). There was no association of the other three SNPs with pediatric uveitis. Conclusions This study shows that miR-146a and Ets-1 are both associated with pediatric uveitis in Han Chinese. SNP rs10893872 may affect the genetic predisposition to pediatric uveitis by modulating expression of Ets-1.

TNFAIP3 Gene Polymorphisms in a Chinese Han Population with Vogt–Koyanagi–Harada Syndrome

Background This study was performed to evaluate the potential association of TNFAIP3 polymorphisms with Vogt–Koyanagi–Harada (VKH) disease in a Chinese Han population. Methodology/Principal Findings Five single-nucleotide polymorphisms (SNPs), rs10499194, rs610604, rs7753873, rs5029928 and rs9494885 of TNFAIP3 were genotyped in 834 VKH disease patients and 1415 healthy controls using a PCR-restriction fragment length polymorphism assay. An increased frequency of the C allele and CT genotype for rs9494885 were found in VKH patients in the Guangzhou and Chongqing cohorts (pc = 0.015, OR = 1.6, pc = 0.036, OR = 1.7; pc = 2.36×10−4, OR = 1.5, pc = 0.012, OR = 1.5, respectively). Meanwhile, a decreased frequency of the TT genotype for rs9494885 was observed in VKH patients in the Guangzhou and Chongqing cohorts (pc = 0.026, OR = 0.6, pc = 0.0074, OR = 0.7, respectively). The combined analysis showed that a significantly increased prevalence of the rs9494885 TC genotype and C allele were found in VKH disease patients compared with controls (pc = 2.26×10−5, OR = 1.7; pc = 1.09× 10−5, OR = 1.6, respectively). The frequency of the TT genotype of rs9494885 was markedly lower in VKH disease patients as compared with that in controls (pc = 1.12×10−5, OR = 0.6; pc = 1.09×10−5, OR = 0.6, respectively). No association was found between rs10499194, rs610604, rs7753873 and rs5029928 polymorphisms and VKH disease. To our knowledge this is the first report describing the association of a TNFAIP3 gene polymorphism with VKH disease in a Chinese Han population. Conclusions/Significance The results suggest that the rs9494885 TC genotype and C allele may be predisposing factors to VKH disease, whereas the rs9494885 TT genotype and T allele may provide protection against this disease.

miRNA Copy Number Variants Confer Susceptibility to Acute Anterior Uveitis With or Without Ankylosing Spondylitis

Purpose To investigate the association of microRNA (miRNA) copy number variants (CNVs) with acute anterior uveitis (AAU) with or without ankylosing spondylitis (AS) and to assess underlying disease mechanisms. Methods This study included 768 patients with AAU+AS+ or AAU+AS- and 660 controls from a Chinese Han population. Genotyping of CNVs was performed by TaqMan PCR. The expression of miRNAs, transfection efficiency of miR-9-3, and cytokine production were measured by real-time PCR, flow cytometry, or ELISA. Results The frequency of low copy numbers of miR-143, miR-146a, miR-9-3, and miR-205 and of high copy numbers of miR-301a and miR-23a was increased in patients with AAU+AS+ (P = 3.725 × 10-5 to 8.033 × 10-9). Additionally, the frequency of a low copy number of miR-146a and a high copy number of miR-23a and miR-205 was significantly increased in AAU+AS- patients (P = 0.002-0.001). The frequency of low copy number of miR-205 was increased in AAU+AS+ compared with AAU+AS- (P = 0.001). The mRNA expression of miR-9-3 was significantly decreased in patients with AAU+AS+ compared with controls and positively associated with its copy number. Additionally, the production of IL-1β and IL-6 was shown to be regulated by miR-9-3 in human primary retinal pigment epithelial cells. Conclusions Low gene copy numbers of miR-143, miR-146a, miR-9-3, miR-205 and high gene copy numbers of miR-301a and miR-23a were associated with susceptibility to AAU+AS+. A low copy number of miR-146a and a high copy number of miR-23a and miR-205 were associated with AAU+AS-.

Uveitis in Chinese Patients with Psoriasis

ABSTRACT Purpose: To investigate the clinical features of 51 uveitis patients with psoriasis in China. Methods: The psoriasis type, demographics, ocular findings, auxiliary examination findings, complications, and therapeutic effects were analyzed. Results: A total of 37 male and 14 female uveitis patients with psoriasis were classified into four groups: psoriasis vulgaris (29 cases); psoriatic arthritis (15 cases); psoriatic erythroderma (6 cases); and pustular psoriasis (1 case). The onset age of psoriasis was younger than for uveitis (p < 0.001). Anterior uveitis, panuveitis, and posterior uveitis was observed in 58.8%, 35.3%, and 5.9% of the patients, respectively. Hypopyon was more frequently noted in the group with psoriatic arthritis (p = 0.007). Optic disc staining was more frequently noted in the group with psoriatic erythroderma (p = 0.029). Significant visual improvement was observed in 17 patients. Conclusions: Uveitis can be associated with various types of psoriasis in China, but was most frequently observed in patients with psoriasis vulgaris and psoriatic arthritis.