Hongsong Yu

Genome-wide association analysis of Vogt-Koyanagi-Harada syndrome identifies two new susceptibility loci at 1p31.2 and 10q21.3

To identify new genetic risk factors for Vogt-Koyanagi-Harada (VKH) syndrome, we conducted a genome-wide association study of 2,208,258 SNPs in 774 cases and 2,009 controls with follow-up in a collection of 415 cases and 2,006 controls and a further collection of 349 cases and 1,588 controls from a Han Chinese population. We identified three loci associated with VKH syndrome susceptibility (IL23R-C1orf141, rs117633859, Pcombined = 3.42 × 10−21, odds ratio (OR) = 1.82; ADO-ZNF365-EGR2, rs442309, Pcombined = 2.97 × 10−11, OR = 1.37; and HLA-DRB1/DQA1, rs3021304, Pcombined = 1.26 × 10−118, OR = 2.97). The five non-HLA genes were all expressed in human iris tissue. IL23R was also expressed in the ciliary body, and EGR2 was expressed in the ciliary body and choroid. The risk G allele of rs117633859 in the promoter region of IL23R exhibited low transcriptional activation in a cell-based reporter assay and was associated with diminished IL23R mRNA expression in human peripheral blood mononuclear cells.

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.

Predisposition to Behçet’s disease and VKH syndrome by genetic variants of miR-182

Previous studies have identified miR-182, miR-27a, FoxO1, and IL2RA as regulatory factors for Treg cell development and function. In order to investigate the association of miR-182, miR-27a, FoxO1, and IL2RA gene polymorphisms with Behçet’s disease (BD) and Vogt–Koyanagi–Harada (VKH) syndrome in a Chinese Han population, a two-stage association study was performed in 820 BD, 900 VKH patients, and 1,800 controls using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assay. In the first stage study, association analysis of 10 single nucleotide polymorphisms (SNPs) was performed in 400 BD, 400 VKH patients, and 600 controls. The results showed significantly decreased frequencies of the miR-182/rs76481776 CC genotype and C allele in BD (P = 3.36 × 10−4, OR = 0.55; P = 4.74 × 10−4, OR = 0.59) and VKH patients (P = 1.11 × 10−4, OR = 0.53; P = 1.26 × 10−4, OR = 0.56). No significant association of the other nine SNPs with BD or VKH was observed. In the second stage study, association analysis of miR-182/rs76481776 was performed in 420 BD, 500 VKH patients, and 1,200 controls. The second stage and combined studies confirmed the association of miR-182/rs76481776 with BD (CC genotype: P = 3.25 × 10−7, OR = 0.58; C allele: P = 1.81 × 10−7, OR = 0.60) and VKH (CC genotype: P = 7.89 × 10−8, OR = 0.57; C allele: P = 2.52 × 10−8, OR = 0.59). Real-time PCR analysis showed a significantly increased expression of miR-182 in TT/CT cases compared to CC cases in anti-CD3/CD28 antibodies-stimulated CD4+ T cells (P = 2.1 × 10−2). In conclusion, this study suggests that miR-182, but not miR-27a, FoxO1, and IL2RA, contributes to the genetic susceptibility of BD and VKH.Key MessageMiR-182 contributes to genetic susceptibility of BD and VKH.No significant association of miR-27a, FoxO1, and IL2RA with BD or VKH was observed.Significantly increased expression of miR-182 in TT/CT cases compared to CC cases was observed.

Berberine Suppresses Th17 and Dendritic Cell Responses

PURPOSE Berberine (BBR) has been shown to exert immunosuppressive and anti-inflammatory effects in several autoimmune diseases. This study was designed to investigate the possible effect of BBR on Th17 and dendritic cell (DC) responses. METHODS Twelve patients with active VKH disease and 20 healthy individuals were enrolled in this study. CD4(+) T cells and CD14(+) monocytes were isolated from peripheral blood mononuclear cells using magnetic-activated cell sorting. Monocyte-derived DCs were generated by culturing monocytes with GM-CSF and IL-4. Proinflammatory cytokines secreted by CD4(+) T cells and LPS induced DCs when exposed to BBR or only vehicle were detected by ELISA. The frequency of IL-17-producing CD4(+) T cells and the surface markers of BBR-treated DCs were measured by flow cytometry. RESULTS Activation of CD4(+) T cells using anti-CD3 and anti-CD28 showed a higher Th17 response in active VKH patients. BBR showed a direct suppression of the Th17 response both in active VKH patients and healthy donors. It also suppressed the Th17 response indirectly by influencing DC maturation. On the one hand, BBR downregulated the expression of costimulatory molecules (CD40, CD80, and CD86) and, on the other hand, it inhibited IL-6, IL-1β, and IL-23 secretion by DCs. CONCLUSIONS These findings suggest that the inhibitory effect of BBR on the Th17 response was mediated by a direct action on T cells as well as an indirect effect via DCs. This study provides new evidence that the natural compound BBR is of great value in the search for novel therapeutic agents in the treatment of T-cell-mediated autoimmune diseases.

Increased Notch pathway activation in Behçet’s disease

OBJECTIVE Behçets disease (BD) is a refractory inflammatory disorder with unknown causes. Since the Notch pathway is critically involved in the immune response, the present study was undertaken to investigate the role of this pathway in BD. METHODS Hes-1, Notch 1-4, Jagged-1, DLL-1 and DLL-4 expression, frequency of IFN-γ and IL-17 expressing Th cells, Notch intracellular domain (NICD), phosphorylation of signal transducer and activator of transcription 3 (STAT3) and the production of IFN-γ and IL-17 were examined by real-time PCR, flow cytometry and ELISA. Notch blockade was performed using the γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenylglycine t-butyl ester (DAPT). Transfection with miR-23b mimics and inhibitor was used to examine the effect of miR-23b on Notch pathway activation. RESULTS Active BD patients showed an increased activation of the Notch pathway in association with a higher Th17 response. Notch blockade preferentially inhibited Th17 responses. The effect of Notch blockade on the Th17 response was associated with a lower level of STAT3 phosphorylation. miR-23b was significantly decreased in CD4(+) T cells from active BD patients. CD4(+) T cells transfected with miR-23b showed a reduced expression of NICD and a reduced frequency of IL-17- and IFN-γ-expressing T cells. CONCLUSION The present study suggests that an increased activation of the Notch pathway may contribute to the pathogenesis of BD. Decreased expression of miR-23b may be involved in activation of the Notch pathway in BD. Manipulation of the Notch pathway may offer a novel therapeutic approach for BD.

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.

FoxO1 gene confers genetic predisposition to acute anterior uveitis with ankylosing spondylitis

PURPOSE Recent studies have shown that a decrease of regulatory T (Treg) cells may contribute to the activity of acute anterior uveitis (AAU) and ankylosing spondylitis (AS). A number of immunogenetic factors including IL2RA, miR-27a, miR-182, and FoxO1 are associated with Treg cell function. In this study, we investigated the association between polymorphisms of these genes and AAU with or without AS in a Chinese Han population. METHODS Using PCR-restricted fragment length polymorphism (RFLP) assay, a two-stage association study was performed in 680 AAU patients with or without AS and 1280 controls. Gene expression was quantified by real-time PCR. RESULTS In the first stage study, an association analysis of 10 single nucleotide polymorphisms (SNPs) was performed in 230 AAU patients with AS, 240 AAU patients without AS, and 650 controls. The results showed significantly increased frequencies of the FoxO1/rs2297626 AA genotype and A allele in AAU patients with AS (AA genotype: P = 6.23 × 10(-5), odds ratio [OR] = 1.86; A allele: P = 2.17 × 10(-4), OR = 1.53). No significant association of the other 9 SNPs with AAU with or without AS was observed. In the second stage study, an association analysis of FoxO1/rs2297626 was performed in 210 AAU patients with AS and 630 controls. The second stage and combined studies confirmed the association of FoxO1/rs2297626 with AAU with AS (AA genotype: P = 3.45 × 10(-8), OR = 1.85; A allele: P = 1.55 × 10(-7), OR = 1.55). CONCLUSION This study suggests that FoxO1, but not miR-27a, miR-182, and IL2RA, contributes to the genetic susceptibility of AAU with AS, but none of the tested polymorphisms confer risk to AAU without AS.

A functional variant of PTPN22 confers risk for Vogt-Koyanagi-Harada syndrome but not for ankylosing spondylitis.

Background Protein tyrosine phosphatase non-receptor 22 (PTPN22) is a key negative regulator of T lymphocytes and has emerged as an important candidate susceptibility factor for a number of immune-related diseases. This study aimed to examine the predisposition of PTPN22 SNPs to Vogt-Koyanagi-Harada (VKH) syndrome and acute anterior uveitis (AAU) associated with ankylosing spondylitis (AS). Methods A total of 1005 VKH syndrome, 302 AAU+AS+ patients and 2010 normal controls among the Chinese Han population were enrolled in the study. Genotyping, PTPN22 expression, cell proliferation, cytokine production and cell activation were examined by PCR-RFLP, Real-time PCR, CCK8, ELISA and Flow cytometry. Results The results showed significantly increased frequencies of the rs2488457 CC genotype and C allele but a decreased frequency of the GG genotype in VKH syndrome patients (PBonferroni correction (Pc) = 3.47×10−7, OR = 1.54; Pc = 3.83×10−8, OR = 1.40; Pc = 6.35×10−4, OR = 0.62; respectively). No significant association of the tested SNPs with AAU+AS+ patients was observed. Functional studies showed a decreased PTPN22 expression, impaired cell proliferation and lower production of IL-10 in rs2488457 CC cases compared to GG cases (Pc = 0.009, Pc = 0.015 and Pc = 0.048 respectively). No significant association was observed concerning T cell activation and rs2488457 genotype. Conclusions The study showed that a functional variant of PTPN22 confers risk for VKH syndrome but not for AAU+AS+ in a Chinese Han population, which may be due to a modulation of the PTPN22 expression, PBMC proliferation and IL-10 production.

Polymorphisms in genetics of vitamin D metabolism confer susceptibility to ocular Behçet disease in a Chinese Han population.

PURPOSE To test whether single nucleotide polymorphisms (SNPs) of the 4 vitamin D family genes (DHCR7, CYP2R1, CYP27B1, and CYP24A1) previously associated with several autoimmune diseases are associated with ocular Behçet disease, Vogt-Koyanagi-Harada (VKH) syndrome, acute anterior uveitis (AAU) with ankylosing spondylitis, or pediatric uveitis in the Chinese Han population. DESIGN Prospective case-control study. METHODS Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism, and the genotypes were verified with direct sequencing. The first-stage study comprised 400 ocular Behçet disease patients, 400 VKH syndrome patients, 218 AAU with ankylosing spondylitis patients, 400 pediatric uveitis patients, and 600 healthy subjects from Chinese Han populations. The second stage included 427 ocular Behçet disease patients and 1000 healthy Chinese Han subjects. Allele and genotype frequencies were compared between patients and controls using the χ(2) test. RESULTS In the first-stage study, only the frequencies of the rs12785878 DHCR7 genotype TT and T allele were significantly higher in ocular Behçet disease patients (P = .036 and P = .008 with Bonferroni correction, respectively) compared with controls among 6 SNPs. No associations could be detected for VKH, AAU with ankylosing spondylitis, or pediatric uveitis. A second stage and combined study confirmed the association of rs12785878 DHCR7 TT genotype and T allele with ocular Behçet disease (P = 3.28E-04 with Bonferroni correction; odds ratio, 1.506; 95% confidence interval, 1.248 to 1.818; and P = 2.82E-05 with Bonferroni correction; odds ratio, 1.339; 95% confidence interval, 1.188 to 1.508, respectively). CONCLUSIONS This study provides evidence that the DHCR7 gene is involved in the susceptibility to ocular Behçet disease.

Association of ATG5 Gene Polymorphisms With Behçet's Disease and ATG10 Gene Polymorphisms With VKH Syndrome in a Chinese Han Population.

PURPOSE This study was conducted to explore the association of autophagy-related genes (ATGs) single nucleotide polymorphisms (SNPs) with Behçets disease (BD) and Vogt-Koyanagi-Harada (VKH) syndrome in a Chinese Han population. METHODS A two-stage association study was carried out in 940 BD, 1061 VKH, and 2007 healthy controls. Genotyping for genetic variants of 10 autophagy family genes (ATG5, ATG7, ATG10, ATG16L1, IRGM, LKKR2, ATG2A, DAP, ULK1, and TSC1) was performed using PCR-restriction fragment length polymorphism (PCR-RFLP) or TaqMan SNP assays. Gene expression was quantified by real-time PCR. RESULTS In the cohort of BD patients, we observed that the TT genotype of rs573775/ATG5 decreased susceptibility to BD (Pc = 8.35 × 10-6, OR = 0.490). In the case of VKH patients, the AC genotype of rs4703863/ATG10 increased susceptibility to VKH syndrome (Pc = 9.94 × 10-5, OR = 1.444), whereas the A allele and AA genotype of rs4703863 (Pc = 7.06 × 10-5, OR = 0.745; Pc = 6.34 × 10-6, OR = 0.669, respectively) acted as protective factors for VKH. Functional experiments showed an increased ATG5 expression by LPS stimulated PBMCs in TT cases of rs573775 compared with controls. The level of ATG5 mRNA in active BD patients not receiving immunosuppression was significantly higher than that in healthy controls. CONCLUSIONS This study demonstrated an association of ATG5 rs573775 with BD and ATG10 rs4703863 with VKH syndrome in a Chinese Han population. Furthermore, a variant of the ATG5 gene was shown to be correlated with ATG5 expression.