Active Vogt-Koyanagi-Harada Patients Research Articles

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.

Identification of Urine Metabolic Biomarkers for Vogt-Koyanagi-Harada Disease.

The diagnosis of Vogt-Koyanagi-Harada (VKH) disease is mainly based on a complex clinical manifestation while it lacks objective laboratory biomarkers. To explore the potential molecular biomarkers for diagnosis and disease activity in VKH, we performed an untargeted urine metabolomics analysis by ultra-high-performance liquid chromatography equipped with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS). Through univariate and multivariate statistical analysis, we found 9 differential metabolites when comparing VKH patients with healthy controls, and 26 differential metabolites were identified when comparing active VKH patients with inactive VKH patients. Pathway enrichment analysis showed that glycine, serine and threonine metabolism, and arginine and proline metabolism were significantly altered in VKH versus healthy controls. Lysine degradation and biotin metabolism pathways were significantly altered in active VKH versus inactive VKH. Furthermore, the receiver operating characteristic (ROC) curve analysis revealed that the combination of acetylglycine and gamma-glutamylalanine could differentiate VKH from healthy controls with an area under the curve (AUC) of 0.808. A combination of ureidopropionic acid and 5′-phosphoribosyl-5-amino-4-imidazolecarboxamide (AICAR) had an excellent AUC of 0.958 for distinguishing active VKH from inactive VKH. In summary, this study identified abnormal metabolites in urine of patients with VKH disease. Further studies are needed to confirm whether these metabolites are specific for this disease.

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-1β, TNF-α, IL-10, and TGF-β 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.

Plasma metabolomics study of Vogt-Koyanagi-Harada disease identifies potential diagnostic biomarkers

Vogt-Koyanagi-Harada (VKH) disease is a common type of uveitis in China, but the diagnosis criteria of VKH disease is controversial. The aim of this study was to investigate potential diagnostic plasma biomarkers for VKH disease. A case-control study including 55 VKH patients (28 active patients and 27 inactive VKH patients) and 30 healthy controls in a tertiary referral center was performed. The metabolic phenotype of VKH patients showed a significant difference compared to healthy controls. Fifteen differentially expressed metabolites (DEMs) were identified between active VKH patients and healthy controls and nine DEMs were found between inactive VKH patients and healthy controls after controlling variable importance in the projection (VIP) value > 1 and false discovery rate (FDR) < 0.05. D-mannose, stearic acid and L-lysine were shown to be potential diagnostic biomarkers which can discriminate active VKH patients from healthy controls with a diagnostic performance with AUC = 0.965, 0.936 and 0.910 respectively in independent diagnosis and an AUC = 0.999 when combined. Sarcosine was recognized as an independent potential biomarker which could distinguish inactive VKH patients from healthy controls. This study reveals a significant difference of plasma metabolic phenotype and identifies diagnostic biomarkers for VKH disease. Changes in the metabolic profile may provide clues towards the pathophysiology of VKH disease.

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

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

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

ABSTRACTPurpose: 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.

Th lymphocyte subsets in patients with Vogt-Koyanagi-Harada disease.

To assess helper T (Th) lymphocyte subset balance in patients with Vogt-Koyanagi-Harada (VKH) disease. Sixty-eight active VKH patients and seventy-two inactive VKH patients were included in this study. One hundred healthy individuals served as controls. Peripheral blood was obtained from VKH patients and healthy controls. Th lymphocyte subsets were analyzed by flow cytometry. Plasma concentration of interleukin (IL)-17, IL-10, transforming growth factor (TGF)-β, IL-23 and IL-6 was examined by enzyme-linked immunosorbent assay (ELISA). VKH patients with active uveitis had significantly higher percentages of both Th1 and Th17 cells and lower percentages of regulatory T (Treg) cells as compared with inactive VKH patients and healthy controls. Th1/Th2 and Th17/Treg ratios were also significantly elevated in active VKH patients. The percentages of Th1, Th17 and Treg cells and the Th1/Th2, Th17/Treg ratio did not differ between inactive VKH patients and healthy controls. There was no difference concerning the percentage of Th2 cells among all the groups. VKH patients with active uveitis showed an elevated level of peripheral Th17 related cytokines levels (TGF-β, IL-6, IL-23, and IL-17) and a decreased level of Treg related cytokines (IL-10) compared with inactive VKH patients and healthy controls. Inactive VKH patients showed no differences in peripheral Th17 related cytokines (TGF-β, IL-6, IL-23, and IL-17) and Treg related cytokines (IL-10) levels compared with healthy controls. Th1 and Th17 cells are significantly increased and Treg cells significantly decreased in active VKH compared with inactive VKH or healthy controls. Therefore, Th lymphocyte subset analysis may serve as a disease biomarker for VKH.

Increased Expression of IL-23 Receptor (IL-23R) in Vogt–Koyanagi–Harada (VKH) Disease

ABSTRACTObjective: Vogt–Koyanagi–Harada (VKH) disease is an autoimmune disease mediated by T cells that target melanocytes. It has been shown that IL-23 receptor (IL-23R) signaling promotes the generation of pathogenic T helper 17 cells. The aim of this study was designed to detect the possible role of IL-23R in VKH disease.Methods: Subjects were divided into an active and inactive VKH patient group and a normal control group. The IL-23R level in peripheral blood mononuclear cells (PBMCs) was measured by flow cytometry and real-time polymerase chain reaction. PBMCs were stimulated with serum from patients or controls to detect the influence of serum from VKH patients on IL-23R expression.Results: The IL-23R mRNA level was markedly increased in PBMCs from the active VKH patient group as compared to normal controls. Flow cytometry analysis showed that there was also an elevated IL-23R protein level in PBMCs in active VKH patients. The IL-23R protein level was higher in PBMCs obtained from healthy controls when they were cultured with serum from active VKH patient as compared to cell cultured with serum from normal controls. After the intraocular inflammation in VKH patients was controlled, the IL-23R gene expression returned back to normal levels.Conclusion: Our study suggests that an elevated IL-23R level may participate in the development of VKH disease.

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

ABSTRACTPurpose: 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-1β 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.

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

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

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

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

TLR3 and TLR4 But not TLR2 are Involved in Vogt-Koyanagi- Harada Disease by Triggering Proinflammatory Cytokines Production Through Promoting the Production of Mitochondrial Reactive Oxygen Species.

Vogt-Koyanagi-Harada (VKH) disease is considered to be an autoimmune disease possibly triggered by an abnormal response to infection. Activation of TLRs signaling pathways by microbial products can drive inflammatory responses and adaptive immunity. In the present study, we investigated the role of TLRs in the pathogenesis of VKH disease. We showed that the expression of TLR3 and TLR4, but not TLR2, was significantly increased in monocyte-derived macrophages (MDMs) from VKH patients with active uveitis compared to controls. VKH patients with active uveitis showed an elevated level of IL-1β, IL-6, IL-8, TNF-α and reactive oxygen species (ROS) in MDMs. IL-1β, IL-6, IL-8 and TNF-α production could be significantly upregulated and downregulated by a ROS activator or inhibitor, respectively. Downregulation of the NLRP3 inflammasome significantly inhibited the production of IL-1β but not IL-6, IL-8 and TNF-α. The phosphorylation levels of p38 and ERK1/2 were significantly higher in MDMs from active VKH patients compared to controls. Inhibition of p38 or ERK1/2 significantly decreased IL-1β, IL-6, IL-8 and TNF-α expression. These results suggest that the increased expression of TLR3/4 in MDMs may be involved in the pathogenesis of VKH disease by the induction of inflammatory cytokines which is mediated by enhanced production of ROS.

Decreased interleukin-37 expression in Vogt-Koyanagi-Harada disease and upregulation following immunosuppressive treatment.

Interleukin-37 (IL-37) is emerging as an important inhibitor of immune response. This study was set up to investigate the expression of IL-37 in Vogt-Koyanagi-Harada (VKH) disease and to explore its possible regulatory role during inflammation. Twenty-four untreated active VKH patients, 10 VKH patients receiving corticosteroids and cyclosporin A (CsA), and 35 healthy controls were included in this study. IL-37 expression in lipopolysaccharides (LPS)-stimulated peripheral blood mononuclear cells (PBMCs) from these 3 groups was assayed by real-time polymerase chain reaction (RT-PCR) and flow cytometry. Cytokines in the supernatants of stimulated PBMCs and CD4(+) T cells were assayed by enzyme-linked immunosorbent assay (ELISA). Mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) activation were measured by flow cytometry. VKH patients showed a decreased IL-37 and IL-27 expression and increased IL-1β, IL-6, and tumor necrosis factor-alpha (TNF-α) levels in PBMC culture supernatants. IL-37 significantly inhibited the production of IL-1β, IL-6, and TNF-α, but induced IL-27 expression. VKH patients treated with corticosteroids combined with CsA showed a regression of the intraocular inflammation, and treatment was associated with an enhanced IL-37 production. IL-37 did not affect the production of IL-17, interferon-gamma (IFN-γ), or IL-10 from CD4(+) T cells. The present study suggests that a decreased IL-37 expression in VKH patients is associated with a reduced control of the inflammatory response. Treatment of VKH patients with corticosteroids and CsA is associated with an increased expression of IL-37, which suggests that corticosteroids and CsA may partly exert their immunosuppressive effect by upregulating IL-37 production.

The Role of Interleukin-1 Receptor-Associated Kinases in Vogt-Koyanagi-Harada Disease

PurposeTo explore whether IRAK1 and IRAK4 are involved in the pathogenesis of Vogt-Koyanagi-Harada (VKH) disease.MethodsThirty-nine VKH patients and thirty-two healthy controls were included in this study. The mRNA levels of IRAK1 and IRAK4 from active VKH patients, inactive VKH patients, and normal controls in peripheral blood mononuclear cells (PBMCs) were detected using real-time quantitative PCR. CD4+T cells were purified from PBMCs obtained from active VKH patients and normal controls. The effect of IRAK1/4 inhibition on CD4+T cell proliferation following stimulation with IL-18 or IL-1β was measured using a modified MTT assay. CD4+T cell expression of IFN-γ and IL-17 were detected by flow cytometry (FCM) and enzyme-linked immunosorbent assay (ELISA). The effect of IRAK1/4 inhibition on NF-κB, STAT1, and STAT3 activation was detected by FCM.ResultsThe mRNA levels of IRAK1 and IRAK4 were both significantly increased in active VKH patients compared to inactive VKH patients and healthy controls. No difference in the IRAK1 or IRAK4 mRNA level could be detected between inactive patients and healthy controls. After incubation with IRAK1/4 inhibitor, the proliferation of CD4+T cells was inhibited both in the active VKH patients and in the healthy controls. IRAK1/4 inhibition was also associated with a decreased expression of IFN-γ and IL-17. Phosphorylation of NF-κB, STAT1, and STAT3 in CD4+T from healthy controls was significantly decreased after inhibition of IRAK1/4.ConclusionsHigh mRNA levels of IRAK1 and IRAK4 correlated with VKH disease activity. IRAK1 and IRAK4 play a role in the activation and proliferation of CD4+T cells and the higher expression observed in VKH may contribute to the pathogenesis of this blinding condition.

Decreased IL-27 Expression in Association with an Increased Th17 Response in Vogt-Koyanagi-Harada Disease

IL-27 has emerged as an important regulator of proinflammatory T-cell responses in animal models. We investigated the pathophysiological role of IL-27 in Vogt-Koyanagi-Harada (VKH) disease. IL-27P28 and EBI3 mRNA expression in peripheral blood mononuclear cells (PBMCs) were assayed by RT-PCR. Cytokines in the serum and supernatants of PBMCs, naïve CD4(+) T cells and DC-T cocultures were assayed by ELISA. Flow cytometry was used to evaluate the frequencies of IL-17-producing CD4(+) T cells. The active VKH patients showed a decreased IL-27P28 mRNA expression in PBMCs and lower IL-27 expression in the serum and supernatants of PBMCs, but higher Th17 cells in PBMCs. EBI3 mRNA expression was not different among the groups tested. Stimulation of naïve CD4(+) T cells under Th17 polarizing conditions showed a higher Th17 cell differentiation in active VKH patients. IL-27 significantly inhibited Th17 cell differentiation. IL-27-treated DCs showed a significant inhibition on Th17 differentiation. There was a significant defect in the Tr1 cell induction as measured by IL-10 in active VKH patients. Treatment with corticosteroids and cyclosporine A (CsA) resolved the intraocular inflammation in association with an upregulation of IL-27 and a downregulation of IL-17. In vitro experiments showed that corticosteroids, but not CsA, significantly upregulated the expression of IL-27. The present study suggests that decreased IL-27 expression may result in a higher Th17 in active VKH patients, which may promote the autoimmune response observed in these patients. Manipulation of IL-27 may offer a novel target for treatment of this disease.

Label-Free Proteomics Reveals Decreased Expression of CD18 and AKNA in Peripheral CD4+ T Cells from Patients with Vogt-Koyanagi-Harada Syndrome

Vogt-Koyanagi-Harada (VKH) syndrome is a systemic autoimmune disease. CD4+ T cells have been shown to be involved in autoimmune diseases including VKH syndrome. To screen aberrantly expressed membrane proteins in CD4+ T cell from patients with active VKH syndrome, blood samples were taken from five patients with active VKH syndrome and five healthy individuals. A label-free quantitative proteomic strategy was used to identify the differently expressed proteins between the two groups. The results revealed that the expression of 102 peptides was significantly altered (p<0.05) between two groups and matched amino acid sequences of proteins deposited in the international protein index (ipi.HUMAN.v3.36.fasta). The identified peptides corresponded to 64 proteins, in which 30 showed more than a 1.5-fold difference between the two groups. The decreased expression of CD18 and AKNA transcription factor (AKNA), both being three-fold lower than controls in expression identified by the label-free method, was further confirmed in an additional group of five active VKH patients and six normal individuals using the Western blot technique. A significantly decreased expression of CD18 and AKNA suggests a role for both proteins in the pathogenesis of this syndrome.

Inhibitory effect of rapamycin and dexamethasone on production of IL-17 and IFN-γ in Vogt–Koyanagi–Harada patients

Aims:To evaluate the effect of rapamycin (RAPA) and dexamethasone (DEX) on the production of IL-17 and IFN-γ by peripheral blood mononuclear cells (PBMCs) from Vogt–Koyanagi–Harada (VKH) patients and healthy individuals.Methods:Blood...

Leptin increases in Vogt–Koyanagi–Harada (VKH) disease and promotes cell proliferation and inflammatory cytokine secretion

Backgroud/aims:Leptin has recently been found to play an important role in the development of autoimmune diseases. Our study is designed to investigate the expression and possible role of leptin in...