Gangxiang Yuan

Genetic polymorphisms of cell adhesion molecules in Behcet's disease in a Chinese Han population.

Cell adhesion molecules (CAMs) are involved in various immune-mediated diseases. This study was conducted to investigate the association of single nucleotide polymorphisms (SNPs) of CAMs with Behçet’s disease (BD) in a Chinese Han population. A two-stage association study was carried out in 1149 BD patients and 2107 normal controls. Genotyping of 43 SNPs was performed using MassARRAY System (Sequenom), polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and TaqMan SNP assays. The expression of CD6 and CD11c was examined by real-time PCR and cytokine production was measured by ELISA. A significantly higher frequency of the CT genotype, and a lower frequency of the CC genotype and C allele of CD6 rs11230563 were observed in BD as compared with controls. Analysis of CD11c rs2929 showed that patients with BD had a significantly higher frequency of the GG genotype and G allele, and a lower frequency of the AG genotype as compared with controls. Functional experiments showed an increased CD11c expression and increased production of TNF-α and IL-1beta by LPS stimulated PBMCs in GG carriers of CD11c rs2929 compared to AA/AG carriers. Our study provides evidence that CD6 and CD11c are involved in the susceptibility to BD in a Chinese Han population.

Association of Genetic Variations in TNFSF15 With Acute Anterior Uveitis in Chinese Han

PURPOSE T cells play an important role in the pathogenesis of uveitis. Recent studies have indicated that the TNFSF15 gene that encodes the TL1A protein can regulate the differentiation and activation of T cells. TNFSF15 gene polymorphisms have been found to be associated with several autoimmune disorders. A possible association of TNFSF15 with acute anterior uveitis (AAU) has not yet been reported and was therefore the purpose of our study. METHODS Eight single nucleotide polymorphisms (SNPs) were examined using TaqMan SNP Genotyping Assay or PCR-restriction fragment length polymorphism in 983 AAU patients and 1128 healthy controls. Genotype distributions and allele frequencies were compared using χ2 analysis between AAU patients and healthy controls. Stratified analysis was also performed according to ankylosing spondylitis (AS) status. The TNFSF15 mRNA expression was quantified by real-time PCR. RESULTS A significantly decreased frequency of the TT genotype in TNFSF15-rs3810936 was found in AAU patients (P = 6.36 × 10(-6), corrected P[Pc] = 1.52 × 10(-4), OR = 0.6, 95% CI = 0.5-0.8). Stratification according to AS status did not reveal a difference concerning the association with TNFSF15-rs3810936. None of the other TNFSF15 SNPs tested were associated with AAU. CONCLUSIONS This study shows an association between TNFSF15-rs3810936 and AAU and suggests that the TL1A/DR3 pathway may be implicated in the pathogenesis of this disease.

Hypermethylation of Interferon Regulatory Factor 8 (IRF8) Confers Risk to Vogt-Koyanagi-Harada Disease

Aberrant methylation change of IRF8 confers risk to various tumors, and abnormal expression of IRF8 is involved in many autoimmune diseases, including ocular Behcet’s disease. However, whether the methylation change of IRF8 is associated with Vogt-Koyanagi-Harada (VKH) disease remains unknown. In the present study, we found a decreased IRF8 mRNA expression in association with a higher methylation level in monocyte-derived dendritic cells (DCs) from active VKH patients compared with the normal and inactive subjects. DCs incubated with cyclosporin a (CsA) or dexamethasone (DEX) showed a lower methylation and higher mRNA expression of IRF8 in active VKH patients. A demethylation reagent, 5-Aza-2′-deoxycytidine (DAC) showed a notable demethylation effect as evidenced by increasing the mRNA expression and reducing the methylation level of IRF8. It also suppressed the Th1 and Th17 responses through down-regulating the expression of co-stimulatory molecules (CD86, CD80, CD40), and reducing the production of pro-inflammatory cytokines (IL-6, IL-1β, IL-23, IL-12) produced by DCs. These findings shows that hypermethylation of IRF8 in DCs confers risk to VKH disease. Demethylation of IRF8 may offer a novel therapeutic strategy protect against VKH disease.

Promoter Hypermethylation of GATA3, IL-4, and TGF-β Confers Susceptibility to Vogt-Koyanagi-Harada Disease in Han Chinese

Purpose We investigated the role of promoter methylation of transcriptional and inflammatory factors, including TBX21, GATA3, RORγt, FOXP3, IFN-γ, IL-4, IL-17A, and TGF-β in the development of Vogt-Koyanagi-Harada (VKH) disease. Methods The promoter methylation levels were detected by the Sequenom MassARRAY system in CD4+ T cells that were separated from 20 healthy individuals and 32 VKH patients (20 in the active stage without medication, 12 in inactive stage with medication). The mRNA expression level of GATA3, IL-4, and TGF-β in CD4+ T cells was analyzed by real-time RT-PCR. Results The promoter methylation levels of GATA3, IL-4, and TGF-β were significantly higher in active VKH patients than in healthy individuals (P < 0.05). A decreased mRNA expression of GATA3 and TGF-β was found in active VKH patients, which was correlated negatively with the DNA methylation of these factors. Treatment with systemic corticosteroid and cyclosporin A (CsA) decreased the methylation level of GATA3 and TGF-β in association with an increased mRNA expression of molecules and reduced disease activity. Conclusions Our findings suggest that promoter hypermethylation of GATA3 and TGF-β in CD4+ T cells confers risk to VKH disease in Han Chinese.

MicroRNA-20a-5p suppresses IL-17 production by targeting OSM and CCL1 in patients with Vogt-Koyanagi-Harada disease

Aim To elucidate the role of microRNA-20a-5p (miR-20a-5p) in the pathogenesis of Vogt-Koyanagi-Harada (VKH) disease. Methods Quantitative real-time PCR was used to quantify miR-20a-5p expression in CD4+ T cells from patients with active VKH and normal controls. The promoter methylation status of miR-20a-5p was detected by bisulfite sequencing PCR. Targets were evaluated by a luciferase reporter assay. The functional effects of miR-20a-5p on CD4+ T cells from patients with active VKH were assessed by upregulation or downregulation of its expression using liposomes. Results The miR-20a-5p level was significantly decreased in CD4+ T cells from patients with active VKH as compared with normal controls. The two genes, oncostatin M (OSM) and C-C motif chemokine ligand 1 (CCL1), were identified as targets of miR-20a-5p. The upregulation of miR-20a-5p significantly suppressed interleukin 17 (IL-17) production in CD4+ T cells from patients with active VKH, whereas downregulation of miR-20a-5p exhibited an inverse effect. In addition, overexpression of OSM and CCL1 could rescue the effect of the upregulation of miR-20a-5p. Moreover, the level of miR-20a-5p was reduced in response to hypermethylation of the promoter. Further study showed that miR-20a-5p suppressed the activity of the phosphoinositide 3-kinase-AKT pathway. Conclusions Our findings indicate that downregulation of miR-20a-5p is caused by promoter hypermethylation. MiR-20a-5p could also suppress the production of IL-17 by targeting OSM and CCL1 production in CD4+ T cells in patients with active VKH.

ERAP1/ERAP2 and RUNX3 polymorphisms are not associated with ankylosing spondylitis susceptibility in Chinese Han: ERAP1/ERAP2 and RUNX3 polymorphisms and AS susceptibility

Previous studies show that endoplasmic reticulum‐associated aminopeptidase (ERAP1/ERAP2) and runt‐related transcription factor 3 (RUNX3) gene polymorphisms are associated with AS (ankylosing spondylitis) in European Caucasians. However, contradictory results were reported in different Asian populations. The purpose of this study was to determine whether eleven candidate single nucleotide polymorphisms (SNPs) in ERAP1/ERAP2 and six in RUNX3 genes confer susceptibility to AS with or without acute anterior uveitis (AAU) [AS+AAU+ or AS+AAU–] in Chinese Han. Therefore, a case–control association study was performed in 882 AS+AAU–, 884 AS+AAU+ and 1727 healthy controls. Genotyping was performed using the iPLEXGold genotyping assay. A meta‐analysis was performed to assess the association of polymorphisms of ERAP1 with AS susceptibility in Asian populations. No association was found between SNPs of ERAP1/ERAP2/RUNX3 and susceptibility of AS with or without AAU. A case–control study between patients with human leucocyte antigen HLA‐B27‐positive and healthy controls also failed to demonstrate an association of the tested SNP with AS with or without AAU. Moreover, a meta‐analysis showed that there was no association of rs30187, rs27037, rs27980, rs27434 and rs27582 in ERAP1 with AS in Chinese Han. Taken together, 17 SNPs in ERAP1/ERAP2 and RUNX3 genes did not confer disease susceptibility to AS in Chinese Han.

Genetic polymorphisms of C-type lectin receptors in Behcet's disease in a Chinese Han population

C-type lectin receptors (CLRs) have been demonstrated to be involved in several autoimmune diseases. The role of CLRs in Behcet’s disease (BD) is unknown and thus was the purpose of this study. A two-stage association study was carried out and a total of 766 BD patients and 1674 healthy controls were recruited. Genotyping of 14 SNPs of 13 genes in CLRs was carried out by iPLEX Gold genotyping or polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. The expression of mannose binding lectin 2 (MBL2) and killer cell lectin like receptor C4 (KLRC4) was measured by Real-time PCR. Significantly increased frequencies of the A allele as well as AA genotype of rs1800450 in MBL2 (Pc = 2.50 × 10−6, OR = 1.494; Pc = 2.24 × 10−6,OR = 2.899; respectively) and TT genotype of rs2617170 in KLRC4 (Pc = 2.53 × 10−6, OR = 1.695) and decreased frequencies of GG genotype of rs1800450 (Pc = 1.56 × 10−3, OR = 0.689) and C allele as well as CC genotype of rs2617170 (Pc = 2.05 × 10−9,OR = 0.664; Pc = 1.20 × 10−5, OR = 0.585; respectively) were observed in BD. Two variants, p.Gly54Asp (rs1800450) and p.Asn104Ser (rs2617170) affect MBL2 and KLRC4 protein stability and expression. Our study demonstrates that the MBL2/rs1800450 and KLRC4/rs2617170 are susceptibility factors for BD in a Chinese Han population.

Ocular Behcet’s disease is associated with aberrant methylation of interferon regulatory factor 8 (IRF8) in monocyte-derived dendritic cells

Aberrant methylation of interferon regulatory factor 8 (IRF8) has been noted in various tumors. IRF8 has also been reported to be involved in many autoimmune diseases, including Behcets disease (BD). However, the methylation status of IRF8 in BD has not been reported. To address this issue, we investigated whether the degree of methylation of IRF8 in dendritic cells (DCs) plays a role in the development of BD. We found a lower mRNA expression and a higher methylation level of IRF8 in active ocular BD patients as compared to normal subjects and inactive patients. Treatment with a demethylation agent, 5-Aza-2’-deoxycytidine (DAC) resulted in an increase of mRNA expression and a reduction of the IRF8 methylation level. It also down-regulated the expression of the co-stimulatory molecules CD86, CD80, CD40, and reduced the production of IL-6, IL-1β, IL-23 and IL-12. An inhibition of Th1/Th17 responses was observed as evidenced by a decreased production of IFN-γ, IL-17, and a reduction of IFN-γ/IL-17- producing CD4+ T cells following treatment with DAC. This study shows that active ocular BD patients have an aberrant IRF8 methylation status. These findings suggest that epigenetic control of IRF8 expression may offer a future target in the treatment of ocular BD.

Disabled-2 (DAB2) Overexpression Inhibits Monocyte-Derived Dendritic Cells' Function in Vogt-Koyanagi-Harada Disease

Purpose Recent studies reported that the tumor suppressor disabled-2 (DAB2) is a negative regulator of immune function. In this study, we investigated the role of DAB2 in monocyte-derived dendritic cells (DCs) from Vogt-Koyanagi-Harada disease (VKH) patients. Methods The mRNA and protein levels of DAB2 were quantified by quantitative real-time PCR and Western blot. The Sequenom MassARRAY system was used to detect the promoter methylation level. An adenovirus carrying the DAB2 gene was transduced into immature DCs, isolated, and induced from active VKH patients. The surface markers of DCs, the frequency of T helper (Th) type 1 (Th1) and Th17 cells in CD4+T cells, which were cocultured with DCs, were tested by flow cytometry. ELISA was used to analyze the inflammatory cytokines produced by DC and CD4+T cell cocultures. Results The mRNA and protein expression levels of DAB2 in DCs obtained from active VKH patients were decreased, while the DAB2 promoter methylation level was marginally increased when compared with inactive VKH patients and normal controls. The expression of CD86 on DCs was significantly downregulated by DAB2 overexpression. The DC-related inflammatory factors IL-6 and TNF-α were also decreased. The frequency of Th1 and Th17 cells and their related cytokines were reduced significantly after coculture with DAB2 overexpressing DCs. DAB2 overexpression did not affect autophagy in DCs from VKH patients. Conclusions These results suggest that the decreased expression of DAB2 in DCs plays a role in the pathogenesis of VKH disease. DAB2 overexpression inhibits DC function, but this is not mediated via autophagy.

Decreased expression of A20 is associated with ocular Behcet’s disease (BD) but not with Vogt-Koyanagi-Harada (VKH) disease

Purpose A20 is a ubiquitously expressed and inducible cytosolic protein, which plays an important role in the negative regulation of inflammation and immunity. In this study, we investigated the role of A20 in Behcet’s disease (BD) and Vogt-Koyanagi-Harada (VKH) disease. Methods The levels of A20 in peripheral blood mononuclear cells (PBMCs) and dendritic cells (DCs) were detected in BD patients with active and inactive uveitis, VKH patients with active and inactive uveitis, and normal subjects, respectively, by real-time PCR. The effect of A20 silencing was performed by transduction of DCs with adenovirus containing an A20 shRNA vector. The effect of A20 silencing on the maturation of DCs was measured by flow cytometry. The effect of A20 silencing of DCs on cytokine production by DCs and CD4+ T cells was analysed by ELISA. The phosphorylation levels of JNK, p38 and ERK1/2 were detected by flow cytometry. Results The expression of A20 was markedly decreased in PBMCs and DCs obtained from BD patients with active uveitis, but not in patients with VKH disease as compared with normal controls. Silencing of A20 significantly increased the levels of interleukin (IL)-1β and IL-6 and suppressed the expression of the anti-inflammatory cytokines IL-10 and IL-27. Downregulation of A20 also led to an increase in IL-17 production by CD4+ T cells. However, downregulation of A20 in DCs did not have an effect on cell surface markers such as CD40, CD80, CD83, CD86 and HLA-DR. Silencing of A20 caused an increased expression of phospho-JNK and phospho-MAPK p38 but not phospho-ERK1/2. Conclusions This study showed that the expression of A20 was decreased in BD patients with active uveitis but not in VKH disease. Decreased expression of A20 may lead to an enhanced activation of proinflammatory Th17 cells, causing a reactivation of BD.