Sex-specific circulating unconventional neutrophils determine immunological outcome of auto-inflammatory Behçet's uveitis.

Neutrophils are the most abundant immune cells that first respond to insults in circulation. Although associative evidence suggests that differences in neutrophils may be linked to the sex-specific vulnerability of inflammatory diseases, mechanistic links remain elusive. Here, we identified extensive sex-specific heterogeneity in neutrophil composition under normal and auto-inflammatory conditions at single-cell resolution. Using a combination of single-cell RNA sequencing analysis, neutrophil-specific genetic knockouts and transfer experiments, we discovered dysregulation of two unconventional (interferon-α responsive and T cell regulatory) neutrophil subsets leading to male-biased incidence, severity and poor prognosis of auto-inflammatory Behçet's uveitis. Genome-wide association study (GWAS) and exosome study revealed that male-specific negative effects of both genetic factors and circulating exosomes on unconventional neutrophil subsets contributed to male-specific vulnerability to disease. Collectively, our findings identify sex-specifically distinct neutrophil subsets and highlight unconventional neutrophil subsets as sex-specific therapeutic targets to limit inflammatory diseases.

Clinical characteristics of viral-associated Fuchs uveitis syndrome and Posner-Schlossman syndrome in a Chinese population.

PURPOSE: To identify the types of viral infection in aqueous humor (AqH) among patients diagnosed as Fuchs uveitis syndrome (FUS) or Posner-Schlossman syndrome (PSS) and investigate their relevance to clinical manifestations and visual outcome. METHODS: A total of 375 patients and 171 patients were diagnosed as FUS or PSS in our department. AqH and serum samples from 68 FUS patients and 16 PSS patients were obtained during eye surgery. The viral etiologies, clinical features, auxiliary tests and visual prognosis of patients with FUS or PSS who underwent AqH analysis were analysed and compared. RESULTS: Among 68 FUS patients, rubella virus (RV), cytomegalovirus (CMV), herpes simplex virus (HSV) and varicella-zoster virus were identified in 17, 11, 1 and 1 patients, respectively. Seven patients with CMV and 1 with HSV were identified in 16 PSS patients. In both FUS and PSS groups, virus-associated eyes had higher proportion of secondary glaucoma and worse visual prognosis as compared with non-virus-associated eyes (all P < 0.05). In FUS group, specifically, CMV infection manifested as more obvious anterior segment inflammation and lower corneal endothelial cell density (CECD). RV infection showed a higher percentage of vitritis. In PSS group, CMV-associated PSS had a lower retinal nerve fiber layer thickness and CECD, worse visual prognosis as compared with non-virus-associated PSS (all P < 0.05). CONCLUSION: Our study identified 4 types of viral infection in FUS and 2 types of viral infection in PSS. Virus-associated patients are usually associated with more obvious clinical signs and poor visual prognosis.

OR11H1 Missense Variant Confers the Susceptibility to Vogt-Koyanagi-Harada Disease by Mediating Gadd45g Expression.

Vogt-Koyanagi-Harada (VKH) disease is a severe autoimmune disease. Herein, whole-exome sequencing (WES) study are performed on 2,573 controls and 229 VKH patients with follow-up next-generation sequencing (NGS) in a collection of 2,380 controls and 2,278 VKH patients. A rare c.188T>C (p Val63Ala) variant in the olfactory receptor 11H1 (OR11H1) gene is found to be significantly associated with VKH disease (rs71235604, Pcombined = 7.83 × 10-30 , odds ratio = 3.12). Functional study showes that OR11H1-A63 significantly increased inflammatory factors production and exacerbated barrier function damage. Further studies using RNA-sequencing find that OR11H1-A63 markedly increased growth arrest and DNA-damage-inducible gamma (GADD45G) expression. Moreover, OR11H1-A63 activates the MAPK and NF-κB pathways, and accelerates inflammatory cascades. In addition, inhibiting GADD45G alleviates inflammatory factor secretion, likely due to the regulatory effect of GADD45G on the MAPK and NF-κB pathways. Collectively, this study suggests that the OR11H1-A63 missense mutation may increase susceptibility to VKH disease in a GADD45G-dependent manner.

Proteomic profiling of aqueous humor-derived exosomes in Vogt-Koyanagi-Harada disease and Behcet's uveitis.

Vogt-Koyanagi-Harada (VKH) disease and Behcet's uveitis (BU) are the two major vision-threatening uveitis entities. This study performed the first label-free quantitative proteomics on aqueous humor-derived exosomes from 84 patients with VKH or BU to determine their potential roles. Sixty-five differentially expressed proteins (DEPs) and 40 DEPs were detected in the VKH and BU groups, respectively. GO and KEGG analysis showed that DEPs were mainly enriched in the complement-related pathways. The complement C1q subcomponent subunit B (C1QB) was identified as a key exosomal protein, and its expression was significantly increased by western blotting in both diseases. Additionally, the integrated analysis based on the published scRNA-seq data showed that C1QB-containing exosomes were mainly produced by mononuclear macrophages in the anterior segment tissue. Overall, our proteomic profiling highlights that complement-related pathways may be actively involved in the pathogenesis of these two diseases. These pathways may also serve as treatment targets for both diseases.

4-Octyl Itaconate Inhibits Proinflammatory Cytokine Production in Behcet's Uveitis and Experimental Autoimmune Uveitis.

4-octyl itaconate (4-OI) is an anti-inflammatory metabolite that activates the nuclear-factor-E2-related factor 2 (NRF2) signaling. In the current work, we investigated whether 4-OI could affect the production of proinflammatory cytokines in Behcet's uveitis (BU) and experimental autoimmune uveitis (EAU). Peripheral blood mononuclear cells (PBMCs) of active BU patients and healthy individuals with in vitro 4-OI treatment were performed to assess the influence of 4-OI on the proinflammatory cytokine production. EAU was induced and used for investigating the influence of 4-OI on the proinflammatory cytokine production in vivo. The flow cytometry, qPCR, and ELISA were performed to detect proinflammatory cytokine expression. NRF2 signaling activation was evaluated by qPCR and western blotting (WB). Splenic lymphocyte transcriptome was performed by RNA sequencing. The NRF2 expression by BU patients-derived PBMCs was lower than that by healthy individuals. After treatment with 4-OI, the proportion of Th17 cells, along with the expression of proinflammatory cytokines (IL-17, TNF-α, MCP-1, and IL-6) by PBMCs, were downregulated, and anti-inflammatory cytokine (IL-10) expression was upregulated, although IFN-γ expression was unaffected. The EAU severity was ameliorated by 4-OI in association with a lower splenic Th1/Th17 cell proportion and increased nuclear NRF2 expression. Additionally, 4-OI downregulated a set of 248 genes, which were enriched in pathways of positive regulation of immune responses. The present study shows an inhibitory effect of 4-OI on the proinflammatory cytokine production in active BU patients and EAU mice, possibly mediated through activating NRF2 signaling. These findings suggest that 4-OI could act as a potential therapeutic drug for the treatment and prevention of BU in the future study.

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.

Global Research Status Regarding Uveitis in the Last Decade.

PURPOSE: To provide an overview on global uveitis research in the last decade in terms of countries/regions, organizations, scholars, journals, trending topics, and fundings. METHODS: This cross-sectional bibliometric analysis yielded 10656 uveitis publications in English for subsequent bibliometric analysis. RESULTS: In terms of the number of publications, the leading country/region was the USA (3007). The most productive organization was the University College London (420). The most productive research team was Professor Yang's group (146). A higher h-index was noted in University College London (48). Professor Rosenbaum was the first h-index holder (32). Keywords of interest included topics such as biologics, COVID and OCT. Publications by Ocular Immunology and Inflammation (968) ranked the first position. CONCLUSIONS: The USA is the leading force in uveitis study. Asian countries/regions, such as China (mainland) and India, are exerting a substantial role worldwide. Trendy topics cover COVID-19, OCTA.

Molecular diagnostic yield for Blau syndrome in previously diagnosed juvenile idiopathic arthritis with uveitis or cutaneous lesions.

OBJECTIVE: Diagnostic pitfalls often arise in the community because of potentially misleading similarities between juvenile idiopathic arthritis (JIA) and Blau syndrome, an immune-related disorder caused by NOD2 gene mutations. It remains unclear in which population and to which extent next-generation sequencing techniques can aid in diagnosis. METHODS: We evaluated clinical usefulness of targeted next-generation sequencing in previously diagnosed JIA. Participants were required to have symptoms and signs suspected of Blau syndrome, including at least uveitis or cutaneous lesions in addition to arthritis. Targeted sequencing was conducted on NOD2 gene to detect diagnostic variants classified as pathogenic or likely pathogenic for Blau syndrome. We assessed the molecular diagnostic yield and clinical implications on patient care. RESULTS: Between May 1, 2008, and June 1, 2021, sequencing data were accrued from 123 previously diagnosed JIA (median age: 5 years; female: 62.6%). Targeted NOD2 sequencing yielded a positive molecular diagnosis of Blau syndrome in 21.1% (95% CI, 14.9%-29.2%), encompassing six heterozygous missense mutations classified as pathogenic variants. Among those receiving a molecular diagnosis, changes in clinical management and treatment were considered as having occurred in 38.5%. Nine predictors were identified to be associated with a higher diagnostic yield, providing clinical clues to suspect the possibility of Blau syndrome. CONCLUSION: Among some patients with pediatric-onset arthritis complicated with uveitis or cutaneous lesions, reassessing their diagnosis of JIA may be warranted. Targeted NOD2 sequencing established the molecular diagnosis of Blau syndrome in nearly one fifth of these cases and provided clinically relevant information for patient-care decisions.

IL-8 Triggers Neutrophil Extracellular Trap Formation Through an Nicotinamide Adenine Dinucleotide Phosphate Oxidase- and Mitogen-Activated Protein Kinase Pathway-Dependent Mechanism in Uveitis.

Purpose: To explore the mechanism underlying IL-8-induced neutrophil extracellular trap (NET) formation in patients with ocular-active Behçet's disease (BD) and the effect of inhibiting NET formation on the severity of inflammation in experimental autoimmune uveitis (EAU) mice. Methods: The serum extracellular DNA and neutrophil elastase (NE) and IL-8 levels in patients with ocular-active BD, the expression of myeloperoxidase, NE, and histone H3Cit in IL-8-induced neutrophils isolated from healthy controls, and the effects of NETs on HMC3 cells were detected. Female C57BL/6J mice were immunized with IRBP651-670 to induce EAU and EAU mice received intravitreal injection of the CXCR2 (IL-8 receptor) antagonist SB225002 or PBS. The serum levels of extracellular DNA, NE, and keratinocyte-derived chemokine (the mouse ortholog of human IL-8) and expression of myeloperoxidase, NE, and histone H3Cit in mouse retinas were detected. Disease severity was evaluated by clinical and histopathological scores. Results: Serum keratinocyte-derived chemokine expression levels in EAU mice and IL-8 expression levels in patients with ocular-active BD increased. IL-8 notably increased NET formation in a dose-dependent manner through an nicotinamide adenine dinucleotide phosphate oxidase and mitogen-activated protein kinase pathway dependent mechanism. IL-8-induced NET formation damaged HMC3 cells in vitro. Pretreatment with SB225002 notably ameliorated the production of NETs in EAU mice. Conclusions: Our data confirm that NET formation is induced by IL-8. IL-8-induced NET formation was found to be related to mitogen-activated protein kinase and nicotinamide adenine dinucleotide phosphate pathways. Pretreatment with the CXCR2 antagonist SB225002 alleviated neutrophil infiltration and suppressed NET formation in EAU mice.

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.

Identification of FCER1G as a cyclosporin A plus corticosteroid sensitization gene in female patients with Vogt-Koyanagi-Harada disease.

The resistance development of the combination regimen of corticosteroids (CS) with cyclosporin A (CsA) leads to therapeutic failure of some patients with autoimmune diseases. In the male patients with Vogt-Koyanagi-Harada (VKH) disease, we have identified RPS4Y1 as an important resistance gene of the regimen and a functional mediator of chlorambucil (CLB). However, it remains unclear what is responsible for the resistance in female patients. In the present study, we performed RNA sequencing, tandem mass tag (TMT) proteomics, gain- and loss-of-function assays and rescue assays to screen and validate potential resistant mediators. The results showed that only Fc epsilon receptor Ig (FCER1G) exhibited significantly differential expression in CD4+ T cells among female CsA & CS resistant, sensitive and CLB & CsA & CS treated patients at transcription and protein levels. Inhibition of FCER1G was demonstrated to modulate CD4+ T cell resistance to CsA & CS in female patients. Importantly, the inhibition was mediated by elevated DNA methylation in the promoter region of the FCER1G gene. Moreover, we found that the salvage effect of CLB on CsA & CS resistance was mediated by an increased FCER1G expression via DNA demethylation in female patients. Taken together, the downregulation of FCER1G due to DNA hypermethylation is responsible for the resistance to CsA & CS and CLB reverses this resistance by inducing FCER1G expression via DNA demethylation in female patients. Modulation of FCER1G would be a promising sensitization strategy in female patients with resistance to CsA & CS.

Risk factors, clinical features and treatment of Behçet's disease uveitis.

Behçet's disease is a systemic vasculitis frequently associated with intraocular inflammation. Recent findings identified independent clinical clusters in Behçet's disease, each involving distinct combinations of affected organs. Ocular Behçet's disease, mainly manifested as uveitis, is characterized as an independent cluster with a low likelihood of association with other system involvements, such as intestinal, cardiovascular, or central nervous system. A prevailing theory suggests that the pathogenesis of the disease is multifactorial, where a variety of genetic and infectious agents may interact with each other to cause the disease. Among sporadic cases, the human leukocyte antigen (HLA) genes, including HLA-B51, HLA-A26, HLA-B15, and HLA-B5701, have been found to be a key component conferring genetic susceptibility. Outside the HLA region, a set of susceptibility variants are identified, closely related to interleukin (IL)-23/IL-17 pathway, tumor necrosis factor (TNF) signaling, and pattern recognition receptor systems. Microbial infections, such as Streptococcus sanguinis, Mycobacterium tuberculosis, and Herpes simplex virus (HSV), are linked to play the triggering of disease in immunogenetically predisposed individuals. Clinically, due to the notable relapsing-remitting course of ocular Behçet's disease, the prevention of recurrent attack would be the primary treatment goal. Combination of corticosteroids and immunomodulatory drugs, such as anti-TNF agents, interferon, and conventional immunosuppressants (e.g. cyclosporine, azathioprine), have been the mainstream regimen for the disease. Future research may focus on comparing the effectiveness of immunomodulatory drugs and identifying the most suitable subgroups for a specific drug on the basis of the knowledge of the molecular heterogeneity of the disease.

Comprehensive evaluation of functional vision, quality of life, and cognitive ability in pediatric uveitis.

BACKGROUND: Pediatric uveitis may cause severe impairment of vision in children and affect their quality of life as well as cognitive ability. This study aims to evaluate the functional vision, visual-related and health-related quality of life, and cognitive ability in pediatric uveitis. METHODS: Children with uveitis aged 5-16 years old completed six validated instruments to assess functional visual ability with Cardiff Visual Ability Questionnaire for Children (CVAQC), vision-related quality of life with Impact of Vision Impairment for Children (IVI-C), health-related quality of life with Pediatric Quality of Life Inventory (PedsQL), cognitive ability with Chinese Wechsler Intelligence Scale for Children (C-WISC), and depression and anxiety evaluation with Hospital Anxiety and Depression Scale (HAD). RESULTS: The CVAQC, IVI-C, and PedsQL scores of pediatric uveitis were significantly lower than that of normal levels. Full-scale intelligence quotient (IQ) and performance IQ were significantly lower in pediatric uveitis patients with impaired vision in their best eye (visual acuity < 0.3) compared to those with a vision equal to or better than 0.3. Verbal IQ was significantly lower in male pediatric uveitis patients with impaired vision compared to those with a vision equal to or better than 0.3. Additionally, parents of pediatric uveitis patients with impaired vision generally had lower educational levels than parents of those with a vision equal to or better than 0.3. CONCLUSIONS: Impaired vision caused by pediatric uveitis has a significant impact on children's functional visual ability and quality of life. The development of cognitive function in pediatric uveitis is also significantly hindered.

Epigenome-wide association study identifies Vogt-Koyanagi-Harada disease-associated DNA methylation loci in Chinese.

DNA methylation is one of the important epigenetic mechanisms for modulating gene expression. By performing a genome-wide methylation association analysis of whole peripheral blood from 60 Vogt-Koyanagi-Harada disease (VKH) patients and 60 healthy controls, we depicted the global DNA methylation status of VKH disease. Further pyrosequencing validation in 160 patients and 159 controls identified 3 aberrant CpG sites in HLA gene regions including cg04026937 and cg18052547 (located in HLA-DRB1 region), and cg13778567 (HLA-DQA1). We also identified 9 aberrant CpG sites in non-HLA gene regions including cg13979407, cg21075643, cg24290586, cg10135747 and cg22707857 (BTNL2), cg22155039 (NOTCH4), cg02605387 (TNXB), cg06255004 (AGPAT2) and cg18855195 (RIBC2). Increased mRNA levels of BTNL2, NOTCH4 and TNXB were identified in VKH patients when compared with healthy controls, consistent with the hypomethylated CpG status in these gene regions. Moreover, seven aberrantly methylated CpG sites may serve as a diagnostic marker for VKH disease (AUC = 84.95%, 95%CI: 79.49%-90.41%).

A randomized non-inferiority trial of therapeutic strategy with immunosuppressants versus biologics for Vogt-Koyanagi-Harada disease.

Biologics are increasingly used to treat Vogt-Koyanagi-Harada disease, but head-to-head comparisons with conventional immunosuppressants are lacking. Here in this randomized trial (Chinese Clinical Trial Registry, ChiCTR2100043061), we assigned 110 patients (27 early-phase and 83 late-phase) to cyclosporine-based immunosuppressant strategy (N = 56) or adalimumab-based biologic strategy (N = 54), each combined with a modified corticosteroid regimen. The primary outcome is change from baseline in best-corrected visual acuity at week 26. The margin of non-inferiority for cyclosporine is -7 letters. The primary outcome is 11.2 letters (95% CI, 7.5 to 14.9) in the cyclosporine group and 6.3 letters (95% CI, 3.1 to 9.6) in the adalimumab group (difference, 4.9; 95% CI, 0.2 to 9.5; P < 0.001 for non-inferiority). The between-group difference is -0.8 letters (95% CI, -6.1 to 4.5) in early-phase disease and 5.7 letters (95% CI, 0.2 to 11.2) in late-phase. Serious adverse events are reported less frequently in the cyclosporine group than in the adalimumab group (0.70 vs. 1.21 events per patient-year). Here, we report that combined with a non-standard corticosteroid regimen, cyclosporine-based immunosuppressant strategy is non-inferior to adalimumab-based biologic strategy by 26 weeks for visual improvement in a cohort of patients with Vogt-Koyanagi-Harada disease, 75% of whom have a late-phase disease.

Association of ZC3HAV1 single nucleotide polymorphisms with the susceptibility of Vogt-Koyanagi-Harada Disease.

BACKGROUND: Polymorphisms of genes related to the immune response have been reported to confer susceptibility to Vogt-Koyanagi-Harada (VKH) disease. This study was carried out to determine whether zinc finger CCCH-type containing antiviral 1 (ZC3HAV1) and tripartite motif-containing protein 25 (TRIM25) genetic polymorphisms are associated with this disease. METHODS: A total of 766 VKH patients and 909 healthy individuals were enrolled in this two-stage case-control study. Thirty-one tag single nucleotide polymorphisms (SNPs) of ZC3HAV1 and TRIM25 were genotyped by MassARRAY System and iPLEX Gold Genotyping Assay. Allele and genotype frequencies were analyzed by the χ2 test or Fisher's exact test. Cochran-Mantel-Haenszel test was used to assess the pooled odds ratio (OR) in the combined study. A stratified analysis was performed in terms of the major clinical features of VKH disease. RESULTS: We found a statistically significant increased frequency of the minor A allele of ZC3HAV1 rs7779972 (P = 1.50 × 10- 4, pooled OR = 1.332, 95%CI = 1.149-1.545) in VKH disease as compared with controls by using the Cochran-Mantel-Haenszel test. The GG genotype of rs7779972 showed a protective association with VKH disease (P = 1.88 × 10- 3, OR = 0.733, 95%CI = 0.602-0.892). There was no difference regarding the frequency of the remaining SNPs between VKH cases and controls (all P > 2.08 × 10- 3). The stratified analysis showed no significant association of rs7779972 with the major clinical characteristics of VKH disease. CONCLUSION: Our study indicated that the ZC3HAV1 variant rs7779972 might confer susceptibility to VKH disease in Han Chinese.

Gut microbial signatures and their functions in Behcet's uveitis and Vogt-Koyanagi-Harada disease.

BACKGROUND: A number of public metagenomic studies reveal an association between the gut microbiome and various immune-mediated diseases including Behcet's uveitis (BU) and Vogt-Koyanagi-Harada disease (VKH). Integrated-analysis and subsequent validation of these results could be a potentially powerful way to understand the microbial signatures and their functions in these two uveitis entities. METHODS: We integrated the sequencing data of our previous metagenomic studies on two major uveitis entities, BU and VKH as well as four other publicly available immune-mediated diseases datasets, including Ankylosing Spondylitis (AS), Rheumatoid Arthritis (RA), Crohn's disease (CD) and Ulcerative Colitis (UC). Alpha-diversity and beta-diversity analysis were used to compare the gut microbiome signatures between both uveitis entities and other immune-mediated diseases and healthy controls. Amino acid homology between microbial proteins and a uveitogenic peptide of the interphotoreceptor retinoid-binding protein (IRBP)161-180 was investigated using a similarity search in the NCBI protein BLAST program (BLASTP). Enzyme-linked Immunosorbent Assay (ELISA) was performed to evaluate the cross-reactive responses of experimental autoimmune uveitis (EAU)-derived lymphocytes and BU patients-derived peripheral blood mononuclear cells (PBMCs) against homologous peptides. The area under the curve (AUC) analysis was used to test the sensitivity and specificity of gut microbial biomarkers. RESULTS: Depleted Dorea, Blautia, Coprococcus, Erysipelotrichaceae and Lachnospiraceae as well as enriched Bilophila and Stenotrophomonas were identified in BU patients. An enriched Alistipes along with a lower level of Dorea were observed in VKH patients. A peptide antigen (SteTDR) encoded by BU specifically enriched Stenotrophomonas was identified to share homology with IRBP161-180. In vitro experiments showed that lymphocytes from EAU or PBMCs from BU patients reacted to this peptide antigen as shown by the production of IFN-γ and IL-17. Addition of the SteTDR peptide to the classical IRBP immunization protocol exacerbated EAU severity. Gut microbial marker profiles consisted of 24 species and 32 species respectively differentiated BU and VKH from each other as well as from the other four immune-mediated diseases and healthy controls. Protein annotation identified 148 and 119 specific microbial proteins associated with BU and VKH, respectively. For metabolic function analysis, 108 and 178 metabolic pathways were shown to be associated with BU and VKH, respectively. CONCLUSIONS: Our study revealed specific gut microbial signatures and their potentially functional roles in BU and VKH pathogenesis that differ significantly from other immune-mediated diseases as well as healthy controls.

TET2-mediated upregulation of 5-hydroxymethylcytosine in LRRC39 promoter promotes Th1 response in association with downregulated Treg response in Vogt-Koyanagi-Harada disease.

DNA 5-Hydroxymethylcytosine (5-hmC), an oxidative reaction mediated by the ten-eleven translocation (TET) family, has been reported to play an essential role in the progression of auto-inflammatory and autoimmune diseases. By far, little is known about the effect of DNA 5-hmC and the TET family on the development of Vogt-Koyanagi-Harada (VKH) disease. In this study, we discovered that the global DNA 5-hmC level and the TET activity were elevated in association with the up-regulated expression of TET2 at both mRNA and protein levels in CD4+T cells from active VKH patients compared to healthy controls. Integrated analysis of DNA 5-hmC pattern and transcription profile of CD4+ T cells revealed that 6 candidate target genes were involved in the development of VKH disease. The promoter 5-hmC and mRNA levels of leucine rich repeat containing 39 (LRRC39) were verified to be elevated in active VKH patients. Functional experiments showed that TET2 could up-regulate LRRC39 mRNA expression by increasing the promoter 5-hmC level of LRRC39 in CD4+ T cells from active VKH patients. Up-regulated LRRC39 expression could increase the frequencies of IFN-γ+ and IL-17+ CD4+ T cells as well as the secretions of IFN-γ and IL-17 in association with the decreased frequency of CD4+CD25+FOXP3+ regulatory T (Treg) cells and the reduced production of IL-10. Additionally, restoration of LRRC39 rescued TET2-silencing-mediated reduced frequency of IFN-γ+ CD4+ T cells and increased frequency of CD4+CD25+FOXP3+ Treg cells. Collectively, our study reveals a novel axis, the TET2-5-hmC-LRRC39-Th1/Treg responses axis, in the pathogenesis of VKH and provides a potential target for further investigation into the epigenetic therapy of this disease.

Vogt-Koyanagi-Harada disease in pediatric, adult and elderly: clinical characteristics and visual outcomes.

PURPOSE: To depict a whole spectrum of clinical feartures and visual prognosis among pediatric, adult, and elderly Vogt-Koyanagi-Harada disease (VKH) patients. METHODS: Retrospective chart review was conducted in 2571 VKH patients diagnosed from April 2008 to January 2022. Based on age of disease onset, patients were divided into pediatric (age ≤ 16 years), adult (16 < age < 65 years), and elderly (age ≥ 65 years) VKH group. Ocular and extraocular manifestations were compared among these patients. Visual outcomes and complications were evaluated using logistic regression models and restricted cubic splines analysis. RESULTS: The median follow-up time was 48 (IQR, 12-60) months. Pediatric, adult and elderly VKH were found in 106 (4.1%), 2355 (91.6%), and 110 (4.3%) patients, respectively. All of the patients showed similar ocular manifestations in the context of disease phasing. The proportion of neurological and auditory manifestations in pediatric (42.3% and 7.5%) VKH patients was significantly lower than that in adults (66.5% and 47.9%) and elderly (68.2% and 50%) (both p < 0.0001). An increased risk of macular abnormalities was seen in adults (OR, 3.43; 95% CI, 1.62-7.29) compared with elderly VKH. An inverted-U-shaped pattern was observed between disease onset age and a poor visual outcome (visual acuity 6/18 or worse) according to OR value in VKH patients. The highest risk of BCVA ≤ 6/18 was observed in 32 years at disease onset (OR, 1.51; 95% CI, 1.18-1.94). A higher risk of visual loss was observed in adult VKH patients (OR, 9.06; 95% CI, 2.18-37.6) compared with elderly VKH patients. And stratified by macular abnormalities, the interaction test was not significant (P = 0.634). CONCLUSION: Our study identified, for the first time, a whole spectrum of clinical features of VKH based on a large cohort of Chinese patients. Adult VKH patients have an increased risk of poor visual outcomes, possibly due to increased frequency of macular abnormalities.

Effects of Plasma-Derived Exosomal miRNA-19b-3p on Treg/T Helper 17 Cell Imbalance in Behçet's Uveitis.

Purpose: To explore the potential role of plasma-derived exosomal microRNAs (miRNAs) in the development of regulatory T cell (Treg)/T helper 17 (Th17) cell imbalances in Behçet's uveitis (BU). Methods: The exosome treatment was conducted to evaluate the effects of plasma exosomes from patients with active BU and healthy controls on the Treg/Th17 cell balance. miRNA sequencing analysis of plasma exosomes was conducted to identify differentially expressed miRNAs between patients with active BU and healthy controls. miRTarBase analysis and dual-luciferase reporter assays were conducted to identify the target genes of miR-19b-3p. CD4+T cells were transfected with miR-19b-3p mimic or inhibitor to evaluate its regulation of the Treg/Th17 cell balance. The Treg/Th17 cell balance in CD4+T cells was evaluated by flow cytometry and enzyme-linked immunosorbent assay. Results: Exosomes from patients with active BU promoted Th17 cell differentiation and inhibited Treg cell differentiation. MiRNA sequencing analysis revealed 177 upregulated and 274 downregulated miRNAs in plasma exosomes of patients with active BU. Among them, miR-19b-3p was significantly elevated, and its target genes were identified as being involved in T-cell differentiation. miR-19b-3p overexpression downregulated CD46 expression and the Treg/Th17 cell ratio in CD4+T cells from healthy controls, whereas miR-19b-3p inhibition reversed these regulatory effects and restored the Treg/Th17 cell balance of CD4+T cells from patients with active BU. Conclusions: Plasma-derived exosomes from patients with active BU showed a markedly differential miRNA expression in comparison to healthy controls. Highly expressed miRNA-19b-3p could induce a Treg/Th17 cell imbalance, probably by downregulating CD46 expression.