Plasma-derived exosomal protein SHP2 deficiency induces neutrophil hyperactivation in Behcet's uveitis.

To investigate the effect of plasma-derived exosomal proteins on neutrophil hyperactivation in Behcet's uveitis (BU), we treated neutrophils from healthy controls with plasma-derived exosomes from active BU patients, and determined the level of neutrophil activation by real-time quantitative PCR (RT-qPCR) and cytokine detection assay. The results revealed that exosomes from active BU patients could activate neutrophils as shown by increasing the expression levels of pro-inflammatory cytokines (IL-17 and IL-6), chemokines (IL-8 and MCP-1), and NETs (MPO and ELANE). Label-free quantitative proteomic analysis of plasma-derived exosomes from patients and healthy controls found a remarkably distinct protein profile and identified differentially expressed proteins (DEPs) between the two groups. The results of GO, KEGG, and GSEA enrichment analysis showed that DEPs were enriched in innate immune-mediated and neutrophil hyperactivation-related signaling pathways. The protein-protein interaction (PPI) analysis determined that SHP2 was a downregulated key hub protein in the exosomes of active BU patients. Knockdown of SHP2 in human neutrophil cell lines (NB4 cells) was shown to promote the secretion of pro-inflammatory cytokines, chemokines, and NETs. The converse effects were observed following SHP2 overexpression. In conclusion, we highlighted a pathogenic role of plasma-derived exosomal SHP2 deficiency in facilitating neutrophil activation and suggested that SHP2 might be an immunoprotective factor in BU pathologic process.

Linoleic acid inhibits in vitro function of human and murine dendritic cells, CD4+T cells and retinal pigment epithelial cells.

PURPOSE: Increased linoleic acid (LA) was observed in acute anterior uveitis (AAU) patient feces in our previous study. To investigate the immunoregulatory effect of LA, we studied the effect of LA on human and murine dendritic cells (DCs), CD4+T cells, and retinal pigment epithelial (RPE) cells in vitro. METHODS: The level of LA in feces from AAU patients and healthy individuals was measured by gas chromatography coupled with a mass spectrometer (GC-MS). The immunoregulatory effect of LA on human and murine DCs, CD4+ T cells, and RPE cells was evaluated by enzyme linked immunosorbent assay (ELISA) and flow cytometry (FCM). The effect of LA on DCs was evaluated by Tandem mass tag (TMT)-based proteomics analysis. RESULTS: Increased LA was observed in feces from AAU patients (1018.35 ± 900.01 mg/kg) as compared with healthy individuals (472.55 ± 365.49 mg/kg, p = 0.0136). LA attenuated the antigen-presenting function of human and murine DCs by decreasing the expression of CD40, the secretion of IL-6 and IL-12p70, and the ability to shift naïve T cells towards T helper type 1 (Th1) and Th17 cells. LA also inhibited the secretion of MCP-1 and IL-8 from RPE cells. Proteomics analysis showed differential expression of 28 proteins, including squalene epoxidase (SQLE), farnesyl-diphosphate farnesyltransferase 1 (FDFT1), and cytochrome P450 family 51 subfamily A member 1 (CYP51A1), in LA-treated DCs compared with controls. LA also accelerated the apoptosis of DCs from healthy individuals. CONCLUSION: LA inhibited the function of human and murine DCs, CD4+T cells, and RPE cells, regulated the expression of proteins, and promoted the apoptosis of human DCs. These results collectively suggest that LA might decrease the function of immune cells in vitro, and further studies are needed to investigate its role in the pathogenesis of AAU.

UVEOGENE: An SNP database for investigations on genetic factors associated with uveitis and their relationship with other systemic autoimmune diseases.

Uveitis is an intraocular inflammatory disease which can lead to serious visual impairment. Genetic factors have been shown to be involved in its development. However, few databases have focused on the information of associations between single nucleotide polymorphisms (SNPs) and uveitis. To discover the exact genetic background of uveitis, we developed an SNP database specific for uveitis, "UVEOGENE," which includes 370 genes and 918 SNPs covering 14 uveitis entities and 40 populations from 286 PubMed English-language papers. Stratification analyses by gender, HLA status, and different clinical features were also extracted from the publications. As a result, 371 associations were judged as "statistically significant." These associations were also shared with Global Variome shared Leiden Open Variation Database (LOVD) (https://databases.lovd.nl/shared/genes). Based on these associations, we investigated the genetic relationship among three widely studied uveitis entities including Behcet's disease (BD), Vogt-Koyanagi-Harada (VKH) disease, and acute anterior uveitis (AAU). Furthermore, "UVEOGENE" can be used as a reliable and informative resource to identify similarities as well as differences in the genetic susceptibility among uveitis and other autoimmune diseases. UVEOGENE is freely accessible at http://www.uvogene.com.

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.

Evaluation of childhood developing via optical coherence tomography-angiography in Qamdo, Tibet, China: A prospective cross-sectional, school-based study.

BACKGROUND: Optical coherence tomography angiography (OCTA) is a new and reliable machine used to evaluate retinal structure and macular perfusion in children. The use of OCTA under bad condition such as high altitude, low atmospheric oxygen, and low humidity, in children is rarely. AIM: To quantify the macular micro-vasculature in healthy children of various ages using OCTA in Qamdo. METHODS: Design: Prospective cross-sectional, school-based study. Three hundred and forty-seven normal students from 9 schools in 4 different areas in Qamdo were included. OCTA was performed on a 3 mm × 3 mm area centered on the macular region and macular cube 512 × 128 showed details in macular. Early treatment of diabetic retinopathy study Vessel Flow Density (VD) of the macular central vascular plexus density (CVD), inner vascular plexus density (IVD), full vascular plexus density (FVD), and the size of the foveal avascular zone (FAZ) were measured. All these results corrected by t/s = 3.382 × 0.01306 × (axial length-1.82). The differences were compared among various ages, sexes and living environments. RESULTS: The mean FAZ area in all eyes was 0.27 mm2 ± 0.12 mm2. The mean foveal thickness (MFT) in the macular cube was 227.64 µm ± 23.51 µm. Compared with girls, boys had a lager FAZ (P = 0.0029). Among the different age groups, MFT (P < 0.001) and FVD (P < 0.0001), IVD (P < 0.0001), and CVD (P = 0.0050) increased with age. FAZ areas were not correlated with age (P = 0.8853) or others (MFT, area). CONCLUSION: OCTA can use to evaluate macular perfusion in children. Our data bridge the gap between structural OCT and perfusion density in children in high altitude. Even though these were not a longitudinal study, it may provide us with hints about retina development during puberty and clinical implications of OCTA in children.

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.

Myopia progression and associated factors of refractive status in children and adolescents in Tibet and Chongqing during the COVID-19 pandemic.

Objectives: To investigate myopia progression and associated factors of refractive status among children and adolescents in Tibet and Chongqing in China during the COVID-19 pandemic. Methods: A population-based cross-sectional study was conducted to compare rates of myopia and high myopia, axial length (AL), spherical equivalent (SE), outdoor activity time, digital device use, and frequency of visual examinations for children and adolescents affected by myopia in Chongqing and Tibet in 2021. Results: A total of 2,303 students from Chongqing and 1,687 students from Tibet were examined. The overall prevalence of myopia and high myopia in these two groups were 53.80 and 7.04% vs. 43.86 and 1.30%, respectively in each case. The Chongqing students had a longer AL than the group from Tibet (23.95 vs. 23.40 mm, respectively; p < 0.001). The mean SE of the students with myopic parents in Tibet was lower than that of the students in Chongqing with myopic parents (-2.57 ± 2.38 diopters (D) vs. -2.30 ± 2.34 D, respectively) (p < 0.001). Conversely, the mean SE of the students from urban areas in Chongqing was lower than that of the students in Tibet (-2.26 ± 2.25 D vs. -1.75 ± 1.96 D, respectively; p < 0.001). The Chongqing students exhibited lower SE (-2.44 ± 2.22 D) than their Tibetan counterparts (mean SE: -1.78 ± 1.65 D (p = 0.0001) when spending more than 2.5 h outdoors. For example, 61.35% of the students in Tibet spent more than 2.5 h outdoors daily, compared with 43.04% of the students in Chongqing. Correspondingly, the proportion of students using digital devices in Tibet (64.43%) was lower than that in Chongqing (100%). For the latter, 38.62% of the students in Chongqing spent more than 2.5 h online using digital devices compared to 10.49% of the students in Tibet. Greater monitoring of visual status was observed for the Chongqing students (mean SE: -1.90 ± 1.98 D) compared with students in Tibet (mean SE: -2.68 ± 1.85 D) (p = 0.0448), with the frequency of optimal examinations being every 6 months. Outdoor activity time was identified as a common risk factor for myopia in both of the populations examined, with odds ratios (ORs) of 1.84 (95% CI: 1.79-1.90) in Chongqing and 0.84 (95% CI: 0.73-0.96) in Tibet. Digital screen time was associated with myopia and high myopia in Chongqing, with ORs of 1.15 (95% CI: 1.08-1.22) and 1.06 (95% CI: 0.94-1.77), respectively. Digital screen time was also found to be a risk factor for high myopia in Tibet (OR: 1.21, 95% CI: 0.77-1.61). The type of digital devices used was also associated with myopia and high myopia in Tibet (OR: 1.33, 95% CI: 1.06-1.68 and OR: 1.49, 95% CI: 0.84-2.58, respectively). Finally, examination frequency was found to correlate with high myopia in the Tibet group (OR: 1.79, 95% CI: 0.66-2.71). Conclusion: Based on our data, we observed that the prevalence of refractive errors in children and adolescents was significantly lower in Tibet than in Chongqing. These results are potentially due to prolonged outdoor activity time, and the type and time of use for digital devices that characterize the group of children and adolescents from Tibet. It is recommended that parents and children in Chongqing would benefit from increased awareness regarding myopia progression and its prevention.

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.

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.

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.

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.

Survey on the Progression of Myopia in Children and Adolescents in Chongqing During COVID-19 Pandemic.

Background: The Covid-19 pandemic restricts children and adolescents from doing normal daily activities such as playing outdoors and going to school. The incidence and prevalence of myopia have increased during the COVID-19 pandemic. The aim of this study was to investigate and evaluate the impact of the home confinement during the COVID-19 pandemic on the progression of myopia among children and adolescents in Chongqing, China. Methods: The survey was conducted by using stratified samplings. Samples were randomly selected from the 2019 National Student Physique and Health Survey database, and their visual function and refractive data were compared with those in 2020. Vision-related behavior questionnaire including digital screen exposure was applied to investigate the correlation between eye parameter and eye health-related behavior. Results: A total of 1,733 and 1,728 students were enrolled in 2020 and 2019, respectively. The percentage of myopia students was 55.02% in 2020, which was higher than that in 2019 (44.62%). The mean uncorrected visual acuity (UCVA, LogMAR, 0.35 ± 0.42) in 2020 was higher than that in 2019 (0.27 ± 0.36, P < 0.001). The mean spherical equivalent (SE) refraction (-1.94 ± 2.13 D) in 2020 was lower than that in 2019 (-1.64 ± 5.49 D, P < 0.001). For students who used digital devices for online courses, the mean SE in the television group (-1.10 ± 1.49 D) was better than that in the computer group (-2.03 ± 2.37 D, P = 0.0017) and in the cell phone group (-2.02 ± 2.09 D, P = 0.0028). The average duration of online classes (r = -0.27, P < 0.0001), the number of online classes per day (r = -0.33, P < 0.0001), as well as digital screen exposure time (r = -0.20, P < 0.0001) were negatively correlated with SE, and the average time of outdoor activity (r = 0.20, P < 0.0001) was positively correlated with SE. Conclusions: Increased digital screen exposure contributes to myopic progression in children and adolescents of Chongqing during the COVID-19 pandemic. Suitable digital devices should be provided for online classes and outdoor activity should be advocated to prevent myopic pandemic.

A20 Inhibits Intraocular Inflammation in Mice by Regulating the Function of CD4+T Cells and RPE Cells.

A20 is a negative regulator of inflammation and immunity and plays a role in several autoimmune and inflammatory diseases. Here, we demonstrate that A20 overexpression significantly ameliorates severity of EAU by inhibiting the infiltration of Th1 and Th17 cells, and by protecting integrity of the blood retinal barrier. In vitro studies showed that A20 silencing could promote CD4+T cells toward a Th1 and Th17 phenotype. A decreased expression of A20 in CD4+T cells was noticed in active BD patients but not in VKH patients. Furthermore, silencing of A20 in hRPE cells induced the production of IL-6, IL-8, and MCP-1 and downregulated ZO-1 and occludin expression which is mediated by inhibition of MAPK and NF-κB pathways. This study reveals a mechanism by which A20 prevents autoimmune uveitis.

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.

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.

Decreased Expression of TGR5 in Vogt-Koyanagi-Harada (VKH) Disease.

Purpose: To investigate the role of G-protein-coupled bile acid receptor-1, Gpbar1 (TGR5) in the pathogenesis of Vogt-Koyanagi-Harada (VKH) disease.Methods: The mRNA level of TGR5, iNOS, Arg1, CD16, and CD206 in macrophages was assayed by real-time PCR. ELISA was used to detect the production of cytokines in cell culture supernatants. The frequencies of CD4+IFN-γ+ and CD4+ IL-17+ T cells were tested by flow cytometry.Results: A decreased expression of TGR5 in M1 macrophages was observed in active VKH patients as compared with normal controls. TGR5 stimulation of M1 macrophages with INT-777 caused a shift of the inflammatory M1 toward the anti-inflammatory M2 macrophage subtype. TGR5 activation of macrophages co-cultured with CD4+ T cells inhibited Th1 and Th17 polarization, as well as the release of IFN-γ and IL-17 in the culture supernatant.Conclusion: Our results show that a decreased TGR5 expression might contribute to the pathogenesis of VKH disease.

Decreased interleukin(IL)-35 Expression is Associated with Active Intraocular Inflammation in Vogt-Koyanagi-Harada (VKH) Disease.

Purpose: Recent studies have reported that IL-35 has a protective effect in autoimmune disease. In this study, we explored the role of IL-35 in the pathogenesis of Vogt-Koyanagi-Harada (VKH) disease. Methods: The IL-35/EBI3 and IL-35/P35 mRNA level was assayed by Real-Time PCR. The level of IL-35 in serum was detected by ELISA. PBMCs and monocyte-derived DCs were cultured with or without IL-35 and the concentration of IL-17, IL-10, IFN-γ, IL-6, TNF-α, and IL-1ß in supernatants was tested by ELISA. Results: The serum level of IL-35 is reduced in active VKH patients. The mRNA expression of the two subunits IL-35/EBI3 and IL-35/P35 in PBMCs from patients with active VKH was also decreased. IL-35 significantly inhibited IFN-γ and IL-17 expression and induced IL-10 production by PBMCs and inhibited IL-6 production by monocyte-derived DCs. Conclusion: The current study suggests that a decreased IL-35 expression may be involved in the pathogenesis of VKH disease.

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.

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.

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.