Lithocholic acid inhibits dendritic cell activation by reducing intracellular glutathione via TGR5 signaling.

Dendritic cells (DCs) are the major antigen-presenting cells and play an important role in autoimmune uveitis. Emerging evidence suggests that bile acids (BAs) regulate DCs maturation. However, the underlying mechanisms by which BAs regulate the function of DCs still need to be clarified. Here, we demonstrate that lithocholic acid (LCA) inhibits the production of pro-inflammatory cytokines and the expression of surface molecules in bone marrow-derived dendritic cells (BMDCs). LCA attenuates the severity of EAU by modulating the maturation of splenic CD11C+MHCIIhigh DCs. Notably, Takeda G-protein coupled receptor 5 (TGR5) deficiency partially reverses the inhibitory effect of LCA on DCs in vitro and in vivo. TGR5 activation also downregulates the NF-κB and MAPK pathways by inhibiting glutathione production and inducing oxidative stress in DCs, which leads to apoptosis and autophagy in DCs. In addition, LCA or INT-777 treatment increases the TGR5 expression in monocyte-derived dendritic cells (MD-DCs) of patients with active BD, whereas both LCA and TGR5 agonists inhibit the activation of MD-DCs. These results suggest that LCA and TGR5 agonists might be potential therapeutic drugs for the treatment of autoimmune uveitis.

Identification of Novel Risk Loci for Behçet's Disease-Related Uveitis in a Chinese Population in a Genome-Wide Association Study.

OBJECTIVE: To explore susceptibility loci associated with uveitis in Behçet's disease (BD). METHODS: We conducted a 2-stage study, consisting of a genome-wide association study (GWAS) stage and a replication stage, in a Chinese population. The GWAS stage included 978 cases with BD-related uveitis and 4,388 controls, and the replication stage included 953 cases with BD-related uveitis and 2,129 controls. Luciferase reporter analysis and chromatin immunoprecipitation assay were performed to explore the functional role of susceptibility genetic variants near ZMIZ1. RESULTS: Three independent HLA alleles (HLA-B51 [3.75 × 10-190 ], HLA-A26 [1.50 × 10-18 ], and HLA-C0704 [3.44 × 10-16 ]) were identified as having a genome-wide association with BD-related uveitis. In the non-HLA region, in addition to confirming 7 previously reported loci, we identified 22 novel susceptibility variants located in 16 loci. Meta-analysis of the Chinese cohort consisting of 1,931 cases and 6,517 controls and a published Japanese cohort of 611 cases and 737 controls showed genome-wide significant associations with ZMIZ1, RPS6KA4, IL10RA, SIPA1-FIBP-FOSL1, and VAMP1. Functional experiments demonstrated that genetic variants of ZMIZ1 were associated with enhanced transcription activity and increased expression of ZMIZ1. CONCLUSION: This GWAS study identified a novel set of genetic variants that are associated with susceptibility to uveitis in BD. These findings enrich our understanding of the contribution of genetic factors to the disease.

SNP-mediated binding of TBX1 to the enhancer element of IL-10 reduces the risk of Behçet's disease.

Aims: The genetic association between Behçet's disease susceptibility and IL-10 has been confirmed in multiple cohorts, but the underlying mechanism of this association remains unclear. Materials & methods: We combined public resources and laboratory experiments (electrophoretic mobility shift assays, chromatin immunoprecipitation, luciferase reporter gene and CRISPR/Cas9 genome editing) to analyze transcription factor binding and enhancer activity controlling IL-10 expression. Results & conclusion: The T allele of noncoding rs3024490 within super-enhancer elements is able to specifically bind TBX1 and, in turn, promotes the enhancer activity and increased expression of IL-10. However, a relative deficiency in TBX1 in Behçet's disease patients leads to the low expression of IL-10 and increased risk of developing Behçet's disease.

Lay abstract Behçet's disease is a prominant cause of blindness. Previous reports show that genetic factors are linked with this disease, although the exact genetic mechanism is unclear. Many of these genetic factors are involved in the control of the immune response, including a large family of proteins known as cytokines. Some of the cytokines are proinflammatory while others can dampen the inflammatory response. An example of the latter is the IL-10 cytokine. We found that individuals carrying a specific site in the noncoding region of the IL-10 gene had a higher risk of Behçet's disease than other noncarriers. This study was designed to further investigate the biological mechanisms explaining the role of the specific site in the development of Behçet's disease. The results show that this specific site affects the binding of an important transcription factor, TBX1, which reduces IL-10 production. The dysregulated control of IL-10 explains why individuals with this genetic trait are more susceptible to developing Behçet's disease.

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.

Changes in the Gut Microbiome Contribute to the Development of Behcet's Disease via Adjuvant Effects.

Behcet's disease (BD) is associated with considerable gut microbiome changes. However, it still remains unknown how the composition of the gut microbiome exactly affects the development of this disease. In this study, transplantation of stool samples from patients with active ocular BD to mice via oral gavage was performed. This resulted in decreases of three short chain fatty acids (SCFAs) including butyric acid, propionic acid and valeric acid in the feces of the BD-recipient group. Intestinal barrier integrity of mice receiving BD feces was damaged as shown by a decreased expression of tight junction proteins and was associated with the release of Lipopolysaccharides (LPS) in the circulation. The mice also showed a higher frequency of splenic neutrophils as well as an enrichment of genes associated with innate immune responses in the neutrophils and CD4 + T cells as identified by single cell RNA sequencing. Analysis of neutrophils and T cells functions in these mice showed an enhanced mesenteric lymph node and splenic Th1 and Th17 cell differentiation in association with activation of neutrophils. Transplantation of BD feces to mice and subsequent induction of experimental uveitis (EAU) or encephalomyelitis (EAE) led to an exacerbation of disease in both models, suggesting a microbial adjuvant effect. These findings suggest that the gut microbiome may regulate an autoimmune response via adjuvant effects including increased gut permeability and enhancement of innate immunity.

Functional Genetic Polymorphisms in the IL1RL1-IL18R1 Region Confer Risk for Ocular Behçet's Disease in a Chinese Han Population.

Single nucleotide polymorphisms (SNPs) in the IL1RL1-IL18R1 region are associated with various immune-mediated diseases. This study was carried out to investigate the causal variant for ocular Behçet's disease (BD) and elucidate its target genes in the IL1RL1-IL18R1 region. Nine candidate functional SNPs were prioritized with bioinformatics analysis, followed by a two-stage association study in 694 ocular BD patients and 1,458 unaffected controls. Functional studies were performed in the peripheral blood mononuclear cells (PBMCs) of 45 healthy men and 16 active male BD patients. Genotyping was performed using the MassARRAY System. The mRNA expressions of IL1RL1, IL18R1, IL18RAP, and SLC9A4 were assayed by real-time PCR and secretion of cytokines was examined by ELISA. Significantly lower frequencies of the rs12987977 GG genotype/G allele (P c = 8.93 × 10-7, OR = 0.39; P c = 2.60 × 10-3, OR = 0.77, respectively), rs12999364 TT genotype/T allele (P c = 3.15 × 10-4, OR = 0.51; P c = 1.13 × 10-2, OR = 0.80, respectively), and rs4851569 AA genotype/A allele (P c = 3.29 × 10-4, OR = 0.52; P c = 9.72 × 10-3, OR = 0.80, respectively) were observed in BD patients compared with the controls. Functional experiments revealed a downregulation of IL1RL1, IL18R1, and SLC9A4 and a decreased secretion of IFN-γ in the anti-CD3/CD28 antibody-treated PBMCs as well as a decreased production of TNF-α in the lipopolysaccharide (LPS)-stimulated PBMCs in carriers of the protective homozygous rs12987977/GG genotype compared with the TT genotype. Our findings show that functional SNPs-rs12987977, rs12999364, and rs4851569-in the IL1RL1-IL18R1 region confer susceptibility to ocular BD in a Chinese Han population. And IL1RL1, IL18R1, and SLC9A4 may be the target genes of rs12987977.

Analysis of the role of palmitoleic acid in acute anterior uveitis.

PURPOSE: To study the role of palmitoleic acid (PA) in the pathogenesis of acute anterior uveitis (AAU). METHODS: PA levels in feces from AAU patients were measured by gas chromatography coupled with a mass spectrometer (GC-MS) and compared with samples obtained from healthy individuals. Enzyme linked immunosorbent assay (ELISA) and flow cytometry (FCM) were used to assess the effect of PA on dendritic cells (DCs) and CD4+T cells obtained from mice, AAU patients and healthy individuals. C57BL/6 mice were fed with PA or vehicle and experimental autoimmune uveitis (EAU) was induced with a human retinal IRBP651-670 peptide. Disease severity of EAU was evaluated by clinical manifestation and histology. Differentiation of splenic Type 1 helper T cells (Th1) and Th17 cells was evaluated by FCM. Tandem mass tag (TMT)-based proteomics analysis was used to identify differentially expressed proteins following incubation of DCs with PA. RESULTS: The fecal concentration of PA was increased in AAU patients as compared with healthy individuals. In vitro, PA promoted apoptosis of DCs and inhibited the secretion of TNF-α from mouse bone-marrow-derived dendritic cells (BMDCs) as well as in DCs from AAU patients and healthy individuals. It only decreased DCs surface marker expression and IL-12p70 secretion in BMDCs and healthy individuals DCs but not in AAU patient DCs. PA-treated BMDCs inhibited Th cell differentiation from mouse naïve CD4+T cells and IL-17 and IFN-γ secretion in co-culture supernatants. PA also inhibited the differentiation of Th cells and secretion of IFN-γ and IL-17 in CD4+T cells from mice, AAU patients and healthy individuals. In vivo, PA-treated EAU mice showed milder clinical and histopathological intraocular manifestations as compared with the control group. PA feeding inhibited differentiation of splenic Th17 cells, whereas Th1 cells were not affected. Up to 30 upregulated and 77 downregulated proteins were identified when comparing PA-treated DCs with controls. CONCLUSION: An increased expression of fecal PA was observed in AAU patients. PA was shown to have immunoregulatory effects on DCs and CD4+T cells and attenuated disease severity in EAU mice.

Increased Expression of Indoleamine 2,3-Dioxygenase (IDO) in Vogt-Koyanagi-Harada (VKH) Disease May Lead to a Shift of T Cell Responses Toward a Treg Population.

Previous studies have pointed out that indoleamine 2,3-dioxygenase (IDO), the rate-limiting enzyme initiating tryptophan metabolism, plays a role in the regulation of the immune system. This project was designed to investigate the potential role of IDO in monocyte-derived dendritic cells (moDCs) obtained from active Vogt-Koyanagi-Harada (VKH) disease patients. In this study, we found that the IDO mRNA expression and enzyme activity were increased in active VKH patients as compared with healthy controls and patients in remission. To investigate the role of IDO in immune regulation, an effective inhibitor 1-methyl-L-tryptophan (1-MT) was used to suppress its activity in DCs. The results showed that inhibition of IDO with 1-MT significantly decreased the expression of DC marker CD86. IDO inhibition did not affect the cytokine production of IL-6, IL-1ß, TNF-α, IL-10, and TGF-ß in DCs. Downregulation of IDO in DCs also led to the reduction of regulatory T (Treg) cells and an increased CD4+ T cell proliferation. Treatment with 1-MT did not affect the phosphorylation of the MAPK pathway in DCs. In general, our study suggests that IDO may play an important role in the pathogenesis of VKH disease by regulating DC and CD4+ T cell function. Tryptophan deficiency and kynurenine accumulation may account for the complicated effects of IDO. Further research is needed to study the precise tryptophan metabolites that may limit immune responses in VKH disease.

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.

Identification of Ribosomal Protein S4, Y-Linked 1 as a cyclosporin A plus corticosteroid resistance gene.

Combination of corticosteroids (CS) with cyclosporin A (CsA) is widely used in the treatment of autoimmune diseases, autoinflammatory diseases and transplantation rejection. However, some patients fail to respond or develop resistance to the combination regimen. In Vogt-Koyanagi-Harada (VKH) disease model, we performed RNA sequencing (RNA-seq) based transcriptomics, isobaric tags for relative and absolute quantification (iTRAQ) based proteomics and assays in vitro to screen and validate potential resistant molecules. We found that a total of 1697 differentially expressed genes (DEGs) and 21 differentially expressed proteins (DEPs) in CD4+ T cells between CsA & CS-resistant and -sensitive VKH patients. Ribosomal Protein S4, Y-Linked 1 (RPS4Y1) was verified to regulate the resistance of CD4+ T cells from male VKH patients to CsA & CS. Importantly, we showed that chlorambucil (CLB) could reverse the resistance by RPS4Y1 suppression. Taken together, we identify RPS4Y1 as an important CsA & CS resistance gene in VKH disease. Researchers should consider validating the resistant effect of RPS4Y1 in other autoimmune diseases or organ transplantation.

Effect of berberine on spleen transcriptome and gut microbiota composition in experimental autoimmune uveitis.

BACKGROUND: Berberine (BBR) was reported to have immunoregulatory and anti-inflammatory properties. In this study, we investigated whether BBR could exert its effects on the development of experimental autoimmune uveitis (EAU), and if so, what was the underlying mechanism? METHODS: EAU was induced in B10R.III mice by immunization with IRBP 161-180, followed by 100 mg/kg/d BBR intragastric administration. Disease severity was assessed by evaluation of clinical and histopathological scores. Blood-retinal barrier (BRB) breakdown was tested by Evans blue. Effector and regulatory T (Treg) cell balance was evaluated by quantitative real-time PCR and flow cytometry. Spleen transcriptome was characterized by RNA sequencing (RNA-seq). Gut microbiota composition was investigated by 16S rRNA analysis. RESULTS: BBR treatment significantly blocked EAU as shown by the decrease of the clinical and histological scores, as well as the inhibition of BRB breakdown. The frequency of splenic Th1 and Th17 cells was decreased, whereas Treg cells were increased in the BBR-treated group. RNA-seq of the spleen revealed 476 differentially expressed genes (DEGs) between the EAU and EAU-BBR group. GO functional classification, as well as KEGG analysis demonstrated that BBR treatment markedly influences genes belonging to chromatin remodeling and immune-related pathways. Intervention with BBR modified the gut microbiome in EAU mice, increasing the number of bacteria with immunomodulatory capacity. Depletion of gut microbiota affected the efficacy of BBR on EAU. Moreover, the altered bacterial strains showed a significant correlation with the expression of histones. CONCLUSIONS: BBR inhibited IRBP induced EAU, which was associated with a significant change in the spleen transcriptome and intestinal microbial composition.

Different Methylation of CpG-SNPs in Behcet's Disease.

PURPOSE: We recently performed an Epigenome-Wide Association Studies (EWAS) study in Behcet's disease (BD) and identified various cytosine-phosphate-guanine (CpG) loci that were aberrantly methylated. In the current study, we wanted to investigate whether these sites contained genetic polymorphisms and whether the frequency of these polymorphisms was altered in BD. METHODS: A two-stage study was performed. The first stage involved 358 BD patients and 704 healthy controls to investigate genetic variants of 10 CpG-SNPs (rs10454134, rs176249, rs3808620, rs10176517, rs11247118, rs78016579, rs9461624, rs10492166, rs34929465, and rs6507921) using an iPLEX Gold genotyping assay and a Sequenom MassARRAY. In the second stage, an additional 172 independent BD patients and 330 healthy individuals are to confirm trends found in the first stage. RESULTS: A higher frequency of both the rs10454134 AG genotypes (p = 0.008, OR = 1.413, 95% CI = 1.094-1.826) and a lower GG genotype frequency (p = 0.003, OR = 0.630, 95% CI = 0.465-0.854) were found in BD patients compared to the controls in the first stage. However, after correcting for multiple comparisons, all associations identified in the first stage lost statistical significance. The frequencies of the other CpG-SNPs investigated were not different between BD patients and controls. The second stage was designed using an additional cohort to confirm the association with CpG-SNP, rs10454134. The data failed to confirm the association between this CpG-SNP and BD. CONCLUSIONS: This study did not show an association between BD and CpG-SNPs in gene sites that were earlier shown to be aberrantly methylated.

Epigenome-wide association study identifies Behçet's disease-associated methylation loci in Han Chinese.

OBJECTIVE: The aetiology of Behçet's disease (BD), known as a systemic vasculitis, is not completely understood. Increasing evidence suggests that aberrant DNA methylation may contribute to the pathogenesis of BD. The aim of this epigenome-wide association study was to identify BD-associated methylation loci in Han Chinese. METHODS: Genome-wide DNA methylation profiles were compared between 60 BD patients and 60 healthy controls using the Infinium Human Methylation 450 K Beadchip. BD-associated methylation loci were validated in 100 BD patients and 100 healthy controls by pyrosequencing. Gene expression and cytokine production was quantified by real-time PCR and ELISA. RESULTS: A total of 4332 differentially methylated CpG sites were associated with BD. Five differentially methylated CpG sites (cg03546163, cg25114611, cg20228731, cg23261343 and cg14290576) revealed a significant hypomethylation status across four different genes (FKBP5, FLJ43663, RUNX2 and NFIL3) and were validated by pyrosequencing. Validation results showed that the most significant locus was located in the 5'UTR of FKBP5 (cg03546163, P = 3.81E-13). Four CpG sites with an aberrant methylation status, including cg03546163, cg25114611, cg23261343 and cg14290576, may serve as a diagnostic marker for BD (area under the receiver operating curve curve = 83.95%, 95% CI 78.20, 89.70%). A significantly inverse correlation was found between the degree of methylation at cg03546163 as well as cg25114611 and FKBP5 mRNA expression. Treatment with a demethylation agent, 5-Aza-2'-deoxycytidine resulted in an increase of FKBP5 mRNA expression and a stimulated IL-1ß production. CONCLUSION: Our findings suggest that aberrant DNA methylation, independently of previously known genetic variants, plays a vital role in the pathogenesis of BD. TRIAL REGISTRATION: Chinese Clinical Trial Registry, chictr.org.cn, ChiCTR-CCC-12002184.

PRKCQ rs4750316 is associated with Vogt-Koyanagi-Harada syndrome in a Han Chinese population.

Purpose: The PRKCQ and REL genes are said to be associated with multiple autoimmune diseases. This study investigated the association between these genes and Vogt-Koyanagi-Harada (VKH) syndrome in Han Chinese. Methods: A two-stage case-control study was performed on three single nucleotide polymorphisms ([SNPs] rs4750316, rs11258747, and rs947474) of the PRKCQ gene and three SNPs (rs842647, rs702873, and rs13031237) of the REL gene using PCR-restriction fragment length polymorphism (PCR-RFLPs) in a total of 859 patients with VKH syndrome and 1,542 healthy controls. Variables such as extraocular presentations were assessed. The data were analyzed using chi-square analysis, and corrected for multiple comparisons with the Bonferroni method. Results: We found a decreased frequency of the GC genotype and the C allele of rs4750316 in patients with VKH syndrome when the GG genotype or G allele was used as a reference, respectively (GC genotype: P =2.45e-10, odds ratio [OR]=0.37, 95% confidence interval [CI]=0.28-0.51; C allele: P=8.79e-10, OR=0.41, 95% CI=0.31-0.55). The genotypic and allelic frequencies of rs11258747, rs947474, rs842647, rs702873, and rs13031237 were not statistically significantly different between patients with VKH syndrome and controls. Stratification analysis indicated that the PRKCQ rs4750316 polymorphism was associated with patients with VKH syndrome experiencing headache, alopecia, poliosis, tinnitus, and dysacusia, but no statistically significant association of the other five SNPs was found. Conclusions: The PRKCQ rs4750316 polymorphism may be a susceptibility factor for VKH syndrome pathogenesis and extraocular presentations, indicating that PRKCQ may be involved in the pathogenesis and extraocular presentations of VKH syndrome through the T-cell receptor (TCR) signaling pathway.

Case-Control Study and Meta-Analysis Show a Weak Association between ANTXR2 Polymorphisms and Ankylosing Spondylitis in Chinese Han.

Previous studies have demonstrated associations of ANTXR2 gene polymorphisms with ankylosing spondylitis (AS). These associations differ depending on the ethnic populations and AS subgroups studied. Purposes of the current study were to evaluate the associations of 4 single nucleotide polymorphisms (SNPs) of the ANTXR2 gene with susceptibility to AS alone or AS in combination with acute anterior uveitis (AAU) in Chinese Han. Therefore, a case-control association study was performed in 880 AS+AAU-, 860 AS+AAU+, and 1700 healthy controls. Genotyping was performed using the iPLEXGold genotyping assay. Our results showed a weak association of rs6534639 AA genotype with AS+AAU+ patients (p=0.042), which was lost after correction for multiple comparisons. No other association was found between SNPs of ANTXR2 and susceptibility of AS+AAU- or AS+AAU+. A meta-analysis was performed to evaluate the associations of polymorphisms in the ANTXR2 gene with AS. Results showed a weak association of rs4389526 with AS susceptibility in all studies but failed to show an association of rs6534639 with AS in Chinese Han. Taken together, this study shows no association between ANTXR2 polymorphisms and AS susceptibility in a Chinese Han population, but meta-analysis showed that rs4389526 in the ANTXR2 gene was weakly associated with AS susceptibility in both Caucasian and Chinese Han patients.

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.

Association of LACC1, CEBPB-PTPN1, RIPK2 and ADO-EGR2 with ocular Behcet's disease in a Chinese Han population.

BACKGROUND: An Immunochip study recently identified the association of a number of new genetic loci with Behcet's disease (BD). OBJECTIVE: To confirm the association between new genetic loci reported in an Immunochip study and BD in a Han Chinese population. METHODS: A two-stage association study was carried out in 1238 patients with BD and 1458 healthy controls. Twenty-two candidate single nucleotide polymorphisms (SNPs) were selected for genotyping by iPLEXGold genotyping or TaqMan SNP assays and a meta-analysis was performed for significantly associated markers. RESULTS: The results showed that four SNPs (LACC1/rs9316059, CEBPB-PTPN1/rs913678, ADO-EGR2/rs224127 and RIPK2/rs10094579) were associated with BD in an allelic association test (rs9316059 T allele: pc=4.95×10-8, OR=0.687; rs913678 C allele: pc=3.01×10-4, OR=1.297; rs224127 A allele: pc=3.77×10-4, OR=1.274; rs10094579 A allele: pc=6.93×10-4, OR=1.302). For four SNPs tested by meta-analysis, the association with BD was strengthened and all exceeded genome-wide significance (rs9316059: p=2.96×10-16; rs913678: p=2.09×10-16; rs224127: p=5.28×10-13; rs10094579: p=9.21×10-11). CONCLUSIONS: Our findings confirmed the association of four loci (LACC1, CEBPB-PTPN1, ADO-EGR2 and RIPK2) in Chinese Han patients with BD.

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)-1ß 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.

Interleukin-17 induces angiogenesis in vitro via CXCL8 and CCL2 in retinal pigment epithelium.

Interleukin-17 (IL-17) is a major pro-inflammatory cytokine involved in choroidal endothelial cell (CEC) angiogenesis. Proteins expressed by the retinal pigment epithelium (RPE) may contribute to CEC angiogenesis. The ability of IL­17 to promote proliferation, migration and capillary­like structure formation in CECs was investigated by stimulating the RPE in vitro. CECs were cultured in a conditioned medium (CM) with IL­17 (IL­17­CM) or without IL­17 (CM) obtained from the supernatant of an ARPE­19 cell line. The pro­angiogenic role of IL­17­CM on CECs was investigated with water­soluble tetrazolium 1 analysis, wound healing and Matrigel matrix tube formation assays. The expression level of vascular endothelial growth factor was detected by enzyme­linked immunosorbent assay in RPE cells treated with or without IL­17. Ras­related C3botulinum toxin substrate 1 (Rac1) and Ras homolog gene family member A (RhoA) activities were analyzed by pull­down assays. IL­17­CM significantly enhanced tube formation and increased the migration distance in CECs in comparison with CM. This effect was diminished by neutralizing C­C motif chemokine 2 (CCL2) and C­X­C motif chemokine ligand 8 (CXCL8) expression in IL­17­CM, with a concomitant downregulation of Rac1 and RhoA activity in CECs. In conclusion, it was demonstrated that IL­17 mediated the expression of CCL2 and CXCL8 in RPE cells, resulting in increased migration and tube formation in human CECs.

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.