Association of Long Non-coding RNA C1RL-AS1 and PTPN6 Gene Polymorphisms with Ocular Behcet's Disease in Han Chinese.

PURPOSE: To explore the association of the polymorphisms in PTPN6 and LncRNA C1RL-AS1 genes with ocular BD in Han Chinese patients. METHODS: Correlation study was performed using the iPLEX system on a cohort of ocular BD patients andcontrols. The genotyping of 7 SNPs for LncRNA C1RL-AS1 and PTPN6 genes in ocular BD patients was performed using the iPLEX Gold genotype. RESULTS: The frequencies of rs4013722 AG genotype/A allele in LncRNA C1RL-AS1 were significantly decreased in BD patients, and the frequency of GG genotype was significantly increased in BD patients. The rs4013722 was associated with ocular BD in male patients, but not in female patients. The AG and GG genotype of rs4013722 were associated with skin lesions in male patients. The gene polymorphisms of PTPN6 were not associated with BD patients. CONCLUSIONS: The LncRNA C1RL-AS1/rs4013722 polymorphism conferred susceptibility to ocular BD in Han Chinese patients, which was influenced by sex.Abbreviations: LncRNA: Long Non-coding RNA; BD: Behcet's disease; SNP: single nucleotide polymorphism; PBMCs: peripheral blood mononuclear cells; PTPs: Protein tyrosine phosphatases; PTPN6: protein tyrosine phosphatase non-receptor 6; GWAS: genome-wide association study; HWE: Hardy-Weinberg equilibrium; LD: linkage disequilibrium; OR: odds ratio; CI: confidence interval; eQTL: expression quantitative trait loci; IBD: inflammatory bowel disease; RA: rheumatoid arthritis; Padj: Bonferroni corrected P value; NS: non-significant.

A de novo missense mutation in MPP2 confers an increased risk of Vogt-Koyanagi-Harada disease as shown by trio-based whole-exome sequencing.

Vogt-Koyanagi-Harada (VKH) disease is a leading cause of blindness in young and middle-aged people. However, the etiology of VKH disease remains unclear. Here, we performed the first trio-based whole-exome sequencing study, which enrolled 25 VKH patients and 50 controls, followed by a study of 2081 VKH patients from a Han Chinese population to uncover detrimental mutations. A total of 15 de novo mutations in VKH patients were identified, with one of the most important being the membrane palmitoylated protein 2 (MPP2) p.K315N (MPP2-N315) mutation. The MPP2-N315 mutation was highly deleterious according to bioinformatic predictions. Additionally, this mutation appears rare, being absent from the 1000 Genome Project and Genome Aggregation Database, and it is highly conserved in 10 species, including humans and mice. Subsequent studies showed that pathological phenotypes and retinal vascular leakage were aggravated in MPP2-N315 mutation knock-in or MPP2-N315 adeno-associated virus-treated mice with experimental autoimmune uveitis (EAU). In vitro, we used clustered regularly interspaced short palindromic repeats (CRISPR‒Cas9) gene editing technology to delete intrinsic MPP2 before overexpressing wild-type MPP2 or MPP2-N315. Levels of cytokines, such as IL-1ß, IL-17E, and vascular endothelial growth factor A, were increased, and barrier function was destroyed in the MPP2-N315 mutant ARPE19 cells. Mechanistically, the MPP2-N315 mutation had a stronger ability to directly bind to ANXA2 than MPP2-K315, as shown by LC‒MS/MS and Co-IP, and resulted in activation of the ERK3/IL-17E pathway. Overall, our results demonstrated that the MPP2-K315N mutation may increase susceptibility to VKH disease.

Genetic association of PRKCD and CARD9 polymorphisms with Vogt-Koyanagi-Harada disease in the Chinese Han population.

BACKGROUND: Protein kinase C delta (PRKCD) and caspase recruitment domain family member 9 (CARD9) are genes involved in B and T cell activation, and cytokine production, which are vital mechanisms underlying autoimmune disease development. This study aimed to explore the association of the PRKCD and CARD9 genes with Vogt-Koyanagi-Harada disease (VKH) disease. The case-control study was performed to in 912 patients with VKH and 878 normal controls. MassARRAY system, SHEsis online platform, real-time PCR, and enzyme-linked immunosorbent assay were used to detect genotyping, haplotyping, mRNA expression, and cytokine levels, respectively. RESULTS: We found that rs74437127 C allele of PRKCD, rs3812555 CC genotype, and C allele of CARD9 were associated with increased susceptibility of VKH (Pc = 0.020, OR = 1.624; Pc = 2.04 × 10-5, OR = 1.810; Pc = 2.76 × 10-5, OR = 1.698, respectively). However, the rs74437127 T allele, and rs3812555 TC genotype and T allele were linked with decreased susceptibility to VKH (Pc = 0.020, OR = 0.616; Pc = 7.85 × 10-5, OR = 0.559; Pc = 2.76 × 10-5, OR = 0.589, respectively). PRKCD ATG and CARD9 GCTTA haplotypes decreased susceptibility to VKH (Pc = 3.11 × 10-3, OR = 0.594; Pc = 5.00 × 10-3, OR = 0.639, respectively). Functional studies on rs3812555 genotyped individuals revealed that CC carriers had significantly higher CARD9 mRNA expression and tumour necrosis factor-α production than TC/TT carriers (P = 1.00 × 10-4; P = 2.00 × 10-3, respectively). CONCLUSIONS: We found an association between PRKCD rs74437127 and CARD9 rs3812555 polymorphisms and VKH susceptibility and revealed that the increased susceptibility of rs3812555 for VKH may be mediated by regulating CARD9 gene expression and the production of pro-inflammatory cytokines, such as TNF-α.

Gut microbiota-mediated secondary bile acids regulate dendritic cells to attenuate autoimmune uveitis through TGR5 signaling.

Gut microbiota-mediated secondary bile acids (BAs) play an important role in energy balance and host metabolism via G protein-coupled receptors and/or nuclear receptors. Emerging evidence suggests that BAs are important for maintaining innate immune responses via these receptors. However, the effect of BAs on autoimmune uveitis is still unknown. Here, we demonstrate decreased microbiota-related secondary BA concentration in feces and serum of animals with experimental autoimmune uveitis (EAU). Restoration of the gut BAs pool attenuates severity of EAU in association with inhibition of nuclear factor κB (NF-κB)-related pro-inflammatory cytokines in dendritic cells (DCs). TGR5 deficiency partially reverses the inhibitory effect of deoxycholic acid (DCA) on DCs. TGR5 signaling also inhibits NF-κB activation via the cyclic AMP (cAMP)-protein kinase A (PKA) pathway in DCs. Additionally, both DCA and TGR5 agonists inhibit human monocyte-derived DC activation. Taken together, our results suggest that BA metabolism plays an important role in adaptive immune responses and might be a therapeutic target in autoimmune uveitis.

The Rs12569232 SNP Association with Vogt-Koyanagi-Harada Disease and Behcet's Disease is Probably Mediated by Regulation of Linc00467 Expression.

Purpose: To investigate whether the rs12569232 SNP association with Vogt-Koyanagi-Harada disease and Behcet's disease is mediated by regulation of Linc00467 expression.Methods: The expression of linc00467 was detected by real-time PCR. Adenovirus carrying the linc00467 was transduced into CD4+T cells and the effect on cell viability was measured by the CCK-8 test. Human proteome microarray and starBase 2.0 were used to identify the binding proteins of linc00467 and RNA Immunoprecipitation (RIP) was used to confirm the identity of bound proteins.Results: The rs12569232 was associated with the expression of linc00467. The expression of linc00467 was up-regulated in PBMCs and CD4+T cells from VKH disease and BD patients. Over-expression of linc00467 increased cell viability of CD4+T cells. HUR was the common binding protein identified by the two methods and confirmed by RIP.Conclusions: The rs12569232 association with VKH disease and BD may be mediated via regulating the expression of linc00467.

The haplotypes of various TNF related genes associated with scleritis in Chinese Han.

BACKGROUND: Several studies have stated that TNF-α participates in the pathogenesis of scleritis, but also in several systemic autoimmune diseases and vasculitis, of which some are associated with scleritis. Earlier GWAS and SNP studies have confirmed that multiple SNPs of TNF related genes are associated with many immune-mediated disorders. The purpose of this study was to examine the association of TNF related gene polymorphisms with scleritis in Chinese Han. A case-control study was carried out in 556 non-infectious scleritis cases and 742 normal controls. A total of 28 single-nucleotide polymorphisms (SNPs) were genotyped by the iPLEXGold genotyping assay. RESULTS: No significant correlations were seen between the individual SNPs in the TNF related genes and scleritis. Haplotype analysis showed a significantly decreased frequency of a TNFAIP3 TGT haplotype (order of SNPs: rs9494885, rs3799491, rs2230926) (Pc = 0.021, OR = 0.717, 95% CI = 0.563-0.913) and a significantly increased frequency of a TNFSF4 GT haplotype (order of SNPs: rs3850641, rs704840) (Pc = 0.004, OR = 1.691, 95% CI = 1.205-2.372) and TNFSF15 CCC haplotype (order of SNPs: rs6478106, rs3810936, rs7865494) (Pc = 0.012, OR = 1.662, 95% CI = 1.168-2.363) in patients with scleritis as compared with healthy volunteers. CONCLUSIONS: This study reveals that a TGT haplotype in TNFAIP3 may be a protective factor for the development of scleritis and that a GT haplotype in TNFSF4 and a CCC haplotype in TNFSF15 may be risk factors for scleritis in Chinese Han.

Weak association of a TNFRSF1A polymorphism with Behcet's disease in Chinese Han.

The purpose of this study was to investigate whether single nucleotide polymorphisms (SNPs) of the tumor necrosis factor receptor superfamily (TNFRSF) and their ligand (TNFSF) gene are associated with susceptibility to Behcet's Disease (BD) in Chinese Han. A two-phase case-control study was performed in 1055 BD patients and 1829 healthy controls. A total of 27 SNPs was tested using MassARRAY iPLEX® technology. Data were analyzed using a Chi-square (χ2) test and Fisher's exact calibration test. The Bonferroni correction was applied for multiple testing. A statistically significant higher frequency of the A allele and a lower frequency of the G allele of rs1800692 was found in BD (Pc = 0.013, OR = 1.233, 95% CI = 1.103-1.379: Pc = 0.013, OR = 0.811, 95% CI = 0.725-0.907, respectively). Our findings indicate that TNFRSF1A might confer genetic susceptibility to BD in a Chinese Han population.

Altered gut microbiome composition in patients with Vogt-Koyanagi-Harada disease.

BACKGROUND: Vogt-Koyanagi-Harada (VKH) disease is a multisystemic autoimmune disorder characterized by granulomatous panuveitis. Gut microbiome has been considered to play a role in the pathogenesis of this disease but whether the alternation of gut microbiome was involved is unclear. This study was set up to identify abnormalities of gut microbiome composition in VKH disease. RESULTS: Depleted butyrate-producing bacteria, lactate-producing bacteria and methanogens as well as enriched Gram-negative bacteria were identified in the active VKH patients, as well as in VKH patients of Mix enterotype and Bacteroides enterotype. Changes of gut microbiome in the VKH patients were partially restored after an immunosuppressive treatment. The disease susceptibility genotype HLA-DRA was associated with Bacteroides sp.2.1.33B, Paraprevotella clara, Alistipes finegoldii and Eubacterium eligens. A microbial marker profile including 40 disease-associated species was established to differentiate patients from controls. Another microbial marker profile including 37 species was found to be associated with the response to treatment. An animal experiment showed that transfer of gut microbiome from VKH patients could significantly exacerbate disease activity clinically and pathologically in the recipient mice. CONCLUSION: Our results revealed a distinct gut microbiome signature in VKH patients and showed an exacerbating effect of this gut microbiome on experimental autoimmune uveitis (EAU). We also developed two microbial marker profiles in differentiating VKH patients from healthy controls as well as predicting the effectiveness of treatment.

Association of apoptosis genes in PDCD1 but not PDCD1LG2, FAS, and FASLG with pediatric idiopathic uveitis in Han Chinese.

BACKGROUND: Previous studies have shown that aberrant T lymphocyte apoptosis is involved in the pathogenesis of uveitis. Genetic variants of apoptotic pathway-related factors (including PDCD1, PDCD1LG2, FAS, and FASLG) may affect apoptosis and in turn predict susceptibility to autoimmune disease. This has not yet been studied in pediatric idiopathic uveitis (PIU) and juvenile idiopathic arthritis (JIA)-associated uveitis and was therefore the subject of the study presented here. METHODS: Fourteen single-nucleotide polymorphisms (SNPs) of several apoptosis-related pathway genes were analyzed in 1238 PIU patients, 128 JIA-associated uveitis patients and 1114 healthy controls using the iPLEX Gold Assay and MassARRAY platform. RESULTS: A lower frequency of the PDCD1/rs6710479 CC genotype in PIU patients was found when compared to controls (Pc = 3.42 × 10-3). A higher frequency of the PDCD1/rs7421861 A allele (Pc = 4.85 × 10-3) was observed in PIU patients as compared with controls. Stratification analysis showed a positive association of band keratopathy with the PDCD1/rs7565639 CT genotype (Pc = 1.05 × 10-2) and a negative association of this parameter with the PDCD1/rs7565639 C allele (Pc = 3.76 × 10-2). CONCLUSIONS: This study revealed that rs6710479 and rs7421861 in the PDCD1 gene confer susceptibility to PIU in Han Chinese. A stratified analysis showed that PDCD1/rs7565639 is associated with band keratopathy in PIU patients.

Aqueous cytokine levels in four common uveitis entities.

To investigate aqueous cytokine profiles in acute anterior uveitis (AAU), Fuchs' syndrome, Vogt-Koyanagi-Harada (VKH) disease and Behcet's disease (BD), we assayed the concentrations of 17 cytokines by multiplex immunoassay in aqueous humor (AqH) collected during cataract surgery from 24 AAU, 29 Fuchs' syndrome, 29 VKH disease, 30 BD and 30 senile cataract control patients. Aqueous cytokine levels were compared between the five groups and analysed by logistic regression. Cytokine levels were then compared between uveitis patients who underwent cataract surgery within 3 months and those who underwent this surgery more than 3 months after complete control of intraocular inflammation. The results showed that aqueous levels of interferon (IFN)-γ, monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1ß and tumour necrosis factor (TNF)-α levels in AqH from patients with Fuchs' syndrome were significantly higher than those in the other four groups. Using multivariate analysis, MIP-1ß was found to be significantly associated with Fuchs' syndrome. There was no difference in aqueous cytokine levels between cases having cataract surgery within 3 months compared to those after 3 months of complete control of their intraocular inflammation. The current study shows that Chinese patients with Fuchs' syndrome appear to have a specific cytokine profile. MIP-1ß is an important chemokine in the intraocular environment of Fuchs' syndrome. Aqueous cytokine profiles support the performance of cataract surgery in uveitis within 3 months after intraocular inflammation control.

Macular Abnormalities in Vogt-Koyanagi-Harada Disease.

Purpose: To investigate the prevalence of macular abnormalities in Chinese Vogt-Koyanagi-Harada (VKH) patients.Methods: Clinical characteristics, therapeutic effectiveness and visual outcome were reviewed and analyzed.Results: The most common macular abnormality was macular edema (ME), followed by macular choroidal neovascularization (CNV). Macular abnormalities were associated with recurrent episodes, disease course and visual acuity ≤20/50 at first visit. The prevalence of macular abnormalities in patients who were not treated according to our regular treatment regimen with corticosteroids combined with immunosuppressive agents and who were followed-up for at least one year (13.1%) was significantly higher than in patients receiving the regular treatment (5.7%). Visual improvement was found in 66.7% of eyes with macular abnormalities after regular treatment.Conclusion: Macular abnormalities were associated with recurrent uveitis, course of disease and lower visual acuity at first visit. Regular treatment could prevent the development of macular abnormalities and improved visual outcome in most patients.

Long-Term Efficacy and Safety of Interferon Alpha-2a in the Treatment of Chinese Patients with Behçet's Uveitis Not Responding to Conventional Therapy.

PURPOSE: To investigate the long-term efficacy and safety of interferon alpha-2a (IFNα-2a) in Chinese patients with Behçet's uveitis (BU) refractory to conventional therapy. METHODS: In a prospective observational cohort study, 127 patients were treated with an initial dosage of 3 million units per day in the first three months, followed by gradual tapering of the dose. RESULTS: After 3 months of treatment, IFNα-2a was shown to be effective in 115 cases (91%). At the end of the 1-year follow-up, the frequency of ocular relapses decreased to 1.59 ± 1.68 per year (ranging 0-6) (p < 0.001), as compared to 5.09 ± 2.51 per year (ranging 3-15). Moreover, the frequency of oral ulcer relapses also decreased to 2.49 ± 1.84 per year (ranging 0-6) (p < 0.001), as compared to 8.20 ± 3.72 per year (ranging 2-10). Visual improvement or stability was observed in 32 patients (59%) in these 54 patients. During a mean follow-up of 11 months (range 3-33), the mean final VA (logMAR) had progressed from 1.0 logMAR to 0.8 logMAR in all treated patients. CONCLUSIONS: Long-term low dose of IFNα-2a is useful in treating Chinese BU patients who do not respond adequately to conventional therapy.

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.

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 Behcet's 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-1ß, 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.

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-1ß, 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.

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.

FAS Gene Copy Numbers are Associated with Susceptibility to Behçet Disease and VKH Syndrome in Han Chinese.

Previous studies have identified that disturbed apoptosis was involved in the pathogenesis of Behçet disease (BD) and Vogt-Koyanagi-Harada (VKH) syndrome. This study aims to investigate whether copy number variations of apoptosis-related genes, including FAS, CASPASE8, CASPASE3, and BCL2, are associated with BD and VKH syndrome in Han Chinese. A two-stage association study was performed in 1,014 BD patients, 1,051 VKH syndrome patients, and 2,076 healthy controls. TaqMan(®) Copy Number Assays and real-time PCR were performed. The first-stage study showed that increased frequency of high FAS copy number (>2) was found in BD (P = 1.05 × 10(-3) ) and VKH syndrome (P = 2.56 × 10(-3) ). Replication and combined study confirmed the association of high copy number (>2) of FAS with BD (P = 3.35 × 10(-8) ) and VKH syndrome (P = 9.77 × 10(-8) ). A significant upregulated mRNA expression of FAS was observed in anti-CD3/CD28 antibodies-stimulated CD4(+) T cells from individuals carrying a high gene copy number (>2) as compared to normal diploid 2 copy number carriers (P = 0.004). Moreover, the mRNA expression of FAS both in active patients with BD and VKH syndrome was significantly higher than that in controls (P = 0.001 and P = 0.007, respectively). Our findings suggest that a high copy number of FAS gene confers risk for BD and VKH syndrome.