Progranulin Suppressed Autoimmune Uveitis and Autoimmune Neuroinflammation by Inhibiting Th1/Th17 Cells and Promoting Treg Cells and M2 Macrophages.

BACKGROUND AND OBJECTIVES: Progranulin (PGRN) is an important immune regulatory molecule in several immune-mediated diseases. The objective of this study is to investigate the role of PGRN in uveitis and its counterpart, experimental autoimmune uveitis (EAU), and experimental autoimmune encephalomyelitis (EAE). METHODS: Serum PGRN levels in patients with Behcet disease (BD) or Vogt-Koyanagi-Harada (VKH) disease and normal controls were measured by ELISA. EAE and EAU were induced in B10RIII, wild-type, and PGRN-/- mice to evaluate the effect of PGRN on the development of these 2 immune-mediated disease models. The local and systemic immunologic alterations were detected by ELISA, flow cytometry, and real-time PCR. RNA sequencing was performed to identify the hub genes and key signaling pathway. RESULTS: A significantly decreased PGRN expression was observed in patients with active BD and active VKH. Recombinant PGRN significantly reduced EAU severity in association with a decreased frequency of Th17 and Th1 cells. PGRN-/- mice developed an exacerbated EAU and EAE in association with strikingly increased frequency of Th1 and Th17 cells and reduced frequency of regulatory T (Treg) cells. In vitro studies revealed that rPGRN could inhibit IRBP161-180-specific Th1 and Th17 cell response and promote Treg cell expansion. It promoted non-antigen-specific Treg cell polarization from naive CD4+ T cells in association with increased STAT5 phosphorylation. Using RAN sequencing, we identified 5 shared hub genes including Tnf, Il6, Il1b, Cxcl2, and Ccl2 and the most significantly enriched MAPK and tumor necrosis factor signaling pathway in PGRN-/- EAU mice. The aggravated EAE activity in PGRN-/- mice was associated with a skew from M2 to M1 macrophages. DISCUSSION: Our results collectively reveal an important protective role of PGRN in EAU and EAE. These studies suggest that PGRN could serve as an immunoregulatory target in the study of prevention and treatment for the Th1/Th17-mediated diseases.

Elevated Serum Immunoglobulin E Levels are Associated with the Severity of Newly Diagnosed, Acute Vogt-Koyanagi-Harada Disease.

OBJECTIVE: To investigate the correlation between serum immunoglobulin E (IgE) levels and severity of Vogt-Koyanagi-Harada (VKH) disease. METHODS: The medical records of patients with VKH disease between 2015 and 2020 were reviewed. Serum immunoglobulins (IgA, IgE, IgG, and IgM), tumor necrosis factor α (TNFα) and C-reactive protein (CRP) were measured. Patients were divided into IgE-positive (IgE ≥ 100 IU/mL) and IgE-negative (IgE < 100 IU/mL) groups. The best-corrected visual acuity (BCVA) and macular morphologic characteristics including foveal thickness (FT), serous retinal detachment (SRD), sensory retinal thickness (SRT), central foveal thickness (CFT), cube volume (V), and cube average thickness (AT) were determined in patients in both groups. RESULTS: Of 128 patients included in the study, 35 (27.34%) patients were IgE-positive, BCVA (logMAR) was worse in the IgE-positive group. The mean CRP (P= .012) and TNFα (P≤ 0.001) levels were greater in the IgE-positive group than in the IgE-negative group. Regarding macular morphologic characteristics, FT (P= .010), SDR (P= .004), CFT (P= .008), V (P= .013), and AT (P= .006) were significantly greater in the IgE-positive group than in the IgE-negative group. CONCLUSIONS: Elevated serum IgE levels were associated with more severe macular changes in patients with VKH disease. These findings suggest that IgE may be involved in the progression of VKH disease.

Increased serum level of interleukin-33 in Vogt-Koyanagi-Harada correlates with disease activity.

OBJECTIVES: To measure the serum level of IL-33 in patients with Vogt-Koyanagi-Harada disease (VKH) and Behçet's uveitis (BU) in the Chinese Han population and investigate its associations with disease activity and clinical parameters. METHODS: Serum was collected from 41 VKH patients (16 active and 25 inactive patients), 60 BU patients (24 active and 36 inactive patients), and 36 healthy controls. The serum level of IL-33 was measured using the enzyme-linked immunosorbent assay (ELISA) method. Demographic features, clinical manifestations, and intraocular inflammation activity scores (anterior chamber cells score, anterior chamber flare score, and vitreal haze score) were recorded. RESULTS: The serum level of IL-33 significantly increased in all VKH patients, active VKH patients, and inactive VKH patients, as compared to healthy controls (p < 0.001, p < 0.001, and p = 0.002, respectively), and was higher in the active VKH than in the inactive VKH patients (p = 0.049). The serum level of IL-33 positively correlated with the anterior chamber cells score, vitreal haze score, and the annualized number of relapses in VKH patients (Rho = 0.359, p = 0.021; Rho = 0.344, p = 0.028; Rho = 0.537, p < 0.001, respectively). Serum IL-33 level was significantly associated with the annualized number of relapses in patients with BU (Rho = 0.361, p = 0.005). CONCLUSION: Serum IL-33 level is significantly increased in VKH patients in the Chinese Han population. IL-33 level is in positive correlation with the activity and relapses of VKH. Increased IL-33 might contribute to the pathogenesis of VKH and serve as a potential biomarker for VKH disease.

Analyses of circRNA and mRNA Profiles in Vogt-Koyanagi-Harada Disease.

Recent studies revealed that circular RNAs (circRNAs) are important in numerous biological process and involved in autoimmune diseases. However, their role in Vogt-Koyanagi-Harada (VKH) disease, a classical autoimmune disease, is not yet known. This research aimed to study the expression profile of mRNAs, microRNAs (miRNAs) and circRNAs and investigate the influence of circRNAs on the pathogenesis of VKH disease. We identified circRNAs, miRNAs, and mRNAs expression profiles in CD4+ T cells between 4 VKH patients and 3 healthy controls using the whole-transcriptome sequencing (RNA-seq) technique. We discovered that a total of 5088 mRNAs, 451 circRNAs and 433 miRNAs were differently expressed. The GO and KEGG pathway enrichment analyses were performed for significantly differentially expressed circRNAs and mRNAs. GSEA was conducted for all mRNAs. The functional enrichment suggested that the inflammatory response, the adaptive immune response, NF-kappa B signaling pathway, Th17 cell differentiation, Th1 and Th2 cell differentiation and T cell receptor signaling pathway were associated with VKH disease. In addition, based on the immune-related genes we screened, the circRNA-miRNA-mRNA ceRNA network was analyzed and constructed. Ten differently expressed mRNAs (LAT, ZAP70, ITK, ICOS, RASGRP1, PAG1, PLCG1, PRKCQ, LCK, CARD11) and 5 differently expressed circRNAs (hsa_circ_0033144, hsa_circ_0000233, hsa_circ_0000396, hsa_circ_0001924, hsa_circ_0001320) were selected to be validated by Real-time qPCR (RT-qPCR). The results of RT-qPCR turned out to be consistent with RNA-seq data. Further analysis showed that hsa_circ_0001320 and hsa_circ_0001924 may serve as crucial candidate marker genes of VKH disease. These results reveal that circRNAs may have a crucial immunomodulatory function in the pathophysiological process of VKH disease.

Proteomics Profiling of Plasma Exosomes in VKH Patients.

OBJECTIVES: Vogt-Koyanagi-Harada syndrome is common autoimmune uveitis that can cause blindness. Recent studies have shown that plasma exosomes carry disease-related proteins that may serve as biomarkers. Here, we aimed to find candidate biomarkers of Vogt-Koyanagi-Harada disease using proteomic analysis of plasma exosomes. METHODS: Exosomes were isolated from the plasma of normal controls and Vogt- Koyanagi-Harada patients in the following groups: a) initial inflammatory attack (active stage), b) remission after one month of treatment (unstable stage), and c) stationary phase after three months of treatment (stable stage). Groups were analyzed by mass spectrometry using isobaric tags for relative and absolute quantitation. After functional analysis, proteins of interest were verified by ELISA. RESULTS: 463 proteins were identified in the exosomes. Forty-three were upregulated at the active inflammation stage, including inflammation-associated proteins. Thirty-one were downregulated. Gene ontology and pathway analyses revealed differential proteins related to cell adhesion, cell phagocytosis, cytoskeleton movement, leukocyte migration across endothelial cells, and platelet activation. By ELISA, Carbonic anhydrase 2 and Ras-related protein Rap-1b were verified as more plentiful at the active stage compared to the normal control and stationary phase in exosomes, but not, however, in microvesicles or plasma. CONCLUSION: Plasma exosomes of Vogt-Koyanagi-Harada patients contain many proteins related to the degree of inflammation. The levels of Carbonic anhydrase 2 and Ras-related protein Rap-1b in exosomes can be used as biomarkers for active inflammation in Vogt-Koyanagi-Harada disease. Further investigation could help study the pathogenesis of Vogt-Koyanagi-Harada disease and identify therapeutic targets.

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.

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

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

Glucocorticoid Receptor-α and MKP-1 as Candidate Biomarkers for Treatment Response and Disease Activity in Vogt-Koyanagi-Harada Disease.

PURPOSE: To investigate the potential of utilizing the expression of genes for glucocorticoid receptor (GR) and mitogen-activated protein kinase phosphatase-1 (MKP-1) as biomarkers of corticosteroid (CS) refractoriness and disease activity in patients with Vogt-Koyanagi-Harada (VKH) disease. DESIGN: Prospective cohort study. METHODS: Twenty VKH patients receiving their first cycle of CS treatment in the absence of additional systemic immunosuppressive therapy and a control group of fifteen healthy volunteers were recruited from the University of Chile (Santiago, Chile) and US National Institutes of Health (Bethesda, United States). Intraocular inflammation was clinically quantified at enrolment and all follow-up visits. CS refractoriness was defined as an ocular reactivation of VKH upon CS withdrawal at a daily oral prednisone dose of 10 mg or more. Quantitative Reverse transcription polymerase chain reaction (qRT-PCR) was performed to measure the mRNA levels of the alpha (α) and beta (ß) isoforms of GR and MKP-1 in peripheral blood mononuclear cells (PBMC) after in vitro stimulation with either anti-CD3/anti-CD28 antibodies, lipopolysaccharide (LPS), or phytohemagglutinin (PHA), in the presence or absence of dexamethasone (Dex). RESULTS: After 6 hours of stimulation in the presence of Dex, PBMC from CS-refractory VKH patients had an impaired elevation in GRα expression (P = .03). Furthermore, inactive patients showed a significant Dex-induced upregulation of MKP-1 (P = .005). CONCLUSIONS: In this pilot study, the expression of GR isoforms and MKP-1 corresponded with patients' clinical response to systemic CS treatment and disease activity, respectively. Hence, these candidate biomarkers have potential clinical utility in the early identification of CS refractoriness and subclinical inflammation in patients with VKH disease.

HLA-DRB1*04:05 in two cases of Vogt-Koyanagi-Harada disease-like uveitis developing from an advanced melanoma patient treated by sequential administration of nivolumab and dabrafenib/trametinib therapy.

Although uveitis is reported as a rare adverse event (AE) associated with dabrafenib/trametinib therapy or nivolumab, the occurrence of severe uveitis is extremely rare. We describe two cases of Vogt-Koyanagi-Harada (VKH)-like uveitis developing after the sequential administration of nivolumab and dabrafenib/trametinib therapy. Interestingly, both cases had HLA-DRB1*04:05, which is strongly associated with VKH disease, and achieved biologically complete remission after the treatment for uveitis. Our cases suggest a possible correlation between VKH-like uveitis as an AE and the clinical outcomes of sequential administration of nivolumab and dabrafenib/trametinib therapy for the treatment of advanced melanoma.

Increased Complement 3a Receptor is Associated with Behcet's disease and Vogt-Koyanagi-Harada disease.

Behcet's disease (BD) and Vogt-Koyanagi-Harada disease (VKH) are systemic and recurrent autoimmune diseases associated with abnormal T cell immune response. Complement 3a receptor (C3aR) and complement 5a receptor (C5aR) have been reported to be involved in T cell mediated autoimmune disease. This study aimed to investigate the role of C3aR and C5aR in these two diseases. The C3aR expression in PBMCs was increased in patients with active BD (aBD) and active VKH (aVKH). No statistical difference was found concerning the expression of C5aR in PBMCs between patients with aBD or aVKH and normal controls. After the intraocular inflammation in BD and VKH patients was controlled, the C3aR expression returned back to normal levels. The serum from patients with aBD and aVKH significantly induced C3aR expression by PBMCs. C3a induced IL-6, IL-1ß and TNF-α secretion, while inhibited the production of IL-10 by monocytes. Activation of C3aR in CD4+T cells could upregulate IL-17 production and inhibit IL-10 production, but had no detectable influence on IFN-γ production. Our data indicates that increased C3aR expression may lead to over activation of the Th17 cell response and may therefore contribute to the pathogenesis of BD and VKH disease.

Sequence analysis of four vitamin D family genes (VDR, CYP24A1, CYP27B1 and CYP2R1) in Vogt-Koyanagi-Harada (VKH) patients: identification of a potentially pathogenic variant in CYP2R1.

BACKGROUND: VKH is a rare autoimmune disease. Decreased level of vitamin D has recently been found to be involved in the pathogenesis of Vogt-Koyanagi-Harada (VKH) disease. This study was designed to screen the vitamin D pathway genes for pathogenic mutations, if any, in VKH patients. METHODS: Genomic DNA was extracted from blood samples collected from patients with VKH disease and healthy controls. Entire coding region, exon-intron junctions of four genes were sequenced in DNA from 39 Saudi VKH patients and 50 ethnically matched healthy individuals. All patients and controls were unrelated. RESULTS: Vitamin D levels in VKH patients were found either insufficient (21-29 ng/mL) or deficient (<20 ng/mL). Sequencing analysis of the VDR, CYP24A1, CYP27B1 and CYP2R1 detected twelve nucleotide changes in these genes in our cohort of 39 patients; 4 of which were non-coding, 6 were synonymous coding and 2 were non-synonymous coding sequence changes. All synonymous coding variants were benign polymorphisms with no apparent clinical significance. A non-synonymous coding sequence variant (c.2 T > C; p.1Met?) found in VDR is an initiation coding change and was detected in control individuals as well, while another variant (c.852G > A; p.284 M > I) found in CYP2R1 is predicted to be disease causing by mutationtaster software. This potentially pathogenic variant was found in 17 out of 39 VKH patients. CONCLUSIONS: Screening of four Vitamin D pathway genes in 39 VKH patients shows that a potentially pathogenic sequence variant in CYP2R1 may cause VKH in a subset of patients. These findings support the previous observation that low vitamin D levels might play a role in VKH pathogenesis and mutations in genes involved in vitamin D anabolism and catabolism might be of importance in VKH pathobiology.

High C4 gene copy numbers protects against Vogt-Koyanagi-Harada syndrome in Chinese Han.

AIMS: Considering the phenotypical consequences and association of C4 copy number variation (CNV) with various autoimmune diseases, we aimed to examine C4 CNVs for 1027 patients with Vogt-Koyanagi-Harada (VKH) syndrome and 2083 controls. METHODS: C4 CNVs were examined by real-time PCR for 1027 patients with VKH and 2083 controls. Peripheral blood mononuclear cells (PBMC) were prepared from venous blood by Ficoll-Hypaque density-gradient centrifugation for cell culture. Cytokine production was examined by ELISA. RESULTS: The expression of total C4 in serum was significantly decreased in patients with VKH as compared with controls (p=0.0010). A significant positive association between C4 expression with C4 CNVs was found (p=0.0023, r(2)=0.92). CNV analysis identified significantly decreased frequencies of more than two copies of C4A or more than four copies of total C4 in patients with VKH (Pc=1.42×10(-3) to 3.56×10(-4), OR=0.67 to 0.70). Linkage analysis showed the independent association of C4 with VKH syndrome from human leucocyte antigen (HLA)-DR4. No significant association was observed concerning type 1 T helper cell (Th1) cytokines and Th17 cytokine production by stimulated PBMCs and C4A copy number. CONCLUSIONS: Our findings indicate a decreased expression of serum C4 and a decreased frequency of high C4 gene copy number in patients with VKH. TRIAL REGISTRATION NUMBER: Chinese Clinical Trial Registration Number: ChiCTR-CCC-12002184.

Amelioration of experimental autoimmune uveoretinitis by inhibition of glyceraldehyde-derived advanced glycation end-product formation.

AGEs are permanently modified macromolecule derivatives that form through nonenzymatic glycation of amino groups of proteins. Glycer-AGEs are highly toxic and play an important role in the pathogenesis of chronic inflammatory diseases. However, the contribution of glycer-AGEs to the pathogenesis of uveitis is unclear. In this study, we measured serum levels of glycer-AGEs in 100 patients with endogenous uveitis (22 with HLA-B27-associated uveitis, 20 with VKH disease, 14 with Behçet's disease, and 44 with sarcoidosis) and 33 healthy volunteers. We then examined the effect of the AGE inhibitor in a mouse model of human endogenous uveitis (EAU) by continuous oral administration of pyridoxamine at 200 or 400 mg/kg/day. Regardless of the etiology, serum glycer-AGE levels were significantly higher in patients with uveitis than in healthy subjects. Treatment with 400 mg/kg pyridoxamine significantly reduced the clinical and histological severity of EAU and was accompanied by a significant decrease in serum and retinal glycer-AGE levels and suppression of translocation of NF-κB p65 into the nucleus of retinal cells. Serum glycer-AGE levels may therefore serve as a biomarker of human uveitis, as well as systemic inflammation, and may contribute to the progression of uveitis, including diabetic iritis, via the activation of NF-κB.

A functional variant of PTPN22 confers risk for Vogt-Koyanagi-Harada syndrome but not for ankylosing spondylitis.

BACKGROUND: Protein tyrosine phosphatase non-receptor 22 (PTPN22) is a key negative regulator of T lymphocytes and has emerged as an important candidate susceptibility factor for a number of immune-related diseases. This study aimed to examine the predisposition of PTPN22 SNPs to Vogt-Koyanagi-Harada (VKH) syndrome and acute anterior uveitis (AAU) associated with ankylosing spondylitis (AS). METHODS: A total of 1005 VKH syndrome, 302 AAU+AS+ patients and 2010 normal controls among the Chinese Han population were enrolled in the study. Genotyping, PTPN22 expression, cell proliferation, cytokine production and cell activation were examined by PCR-RFLP, Real-time PCR, CCK8, ELISA and Flow cytometry. RESULTS: The results showed significantly increased frequencies of the rs2488457 CC genotype and C allele but a decreased frequency of the GG genotype in VKH syndrome patients (PBonferroni correction (Pc) = 3.47×10(-7), OR = 1.54; Pc = 3.83×10(-8), OR = 1.40; Pc = 6.35×10(-4), OR = 0.62; respectively). No significant association of the tested SNPs with AAU+AS+ patients was observed. Functional studies showed a decreased PTPN22 expression, impaired cell proliferation and lower production of IL-10 in rs2488457 CC cases compared to GG cases (Pc = 0.009, Pc = 0.015 and Pc = 0.048 respectively). No significant association was observed concerning T cell activation and rs2488457 genotype. CONCLUSIONS: The study showed that a functional variant of PTPN22 confers risk for VKH syndrome but not for AAU+AS+ in a Chinese Han population, which may be due to a modulation of the PTPN22 expression, PBMC proliferation and IL-10 production.

Inhibition of proinflammatory cytokine by IL-25 in Vogt-Koyanagi-Harada syndrome.

PURPOSE: Vogt-Koyanagi-Harada (VKH) syndrome is a multisystem disorder presumed to be mediated by an autoimmune response. Recent studies have shown that interleukin (IL) 25 was involved in the T-cell immune response. This study analyzed the expression and potential role of IL-25 in the pathogenesis of VKH syndrome. METHODS: The IL-25 serum levels were determined by enzyme-linked immunosorbent assay (ELISA). The IL-1ß, IL-6, and TNF-α level in supernatants of PBMCs cultured with LPS in the absence or presence of recombinant(r) IL-25 was detected by ELISA. RESULTS: A significantly decreased serum IL-25 level was found in VKH patients. In vitro experiments showed that rIL-25 was able to significantly inhibit the production of IL-1ß, IL-6, and TNF-α by PBMCs from active VKH patients. CONCLUSIONS: IL-25 may be involved in the development of VKH syndrome, possibly by inhibiting the expression of proinflammatory cytokines.

Behcet's disease with active uveitis: detection of serum protein biomarkers using MALDI-TOF-MS.

Behcet's Disease (BD) is a multisystem autoimmune disorder that lacks sensitive and specific diagnostic methods. The aim of this study was to identify potential biomarkers specific for BD and to establish a diagnostic model. Serum samples from patients with BD, Vogt-Koyanagi-Harada syndrome (VKH), and healthy controls (HC) were randomly divided into a training set (49 BD, 31 VKH, and 48 HC) and a testing set (13 BD, 10 VKH, and 11 HC). Proteomic mass spectra were generated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Thirty-nine differential m/z peaks associated with BD were identified, and the m/z peaks at 1,644, 1,711, 2,023, 4,347, 6,628, and 8,559 were used to construct a model for the diagnosis of BD. This diagnostic model can distinguish BD from non-BD controls with a sensitivity of 83.67% (41/49) and a specificity of 89.87% (71/79). BD was detected in our blinded testing set with good sensitivity and specificity of 84.6 and 90.48%, respectively. The results suggested that proteomic fingerprint technology combining magnetic beads with MALDI-TOF-MS has potential for the diagnosis of BD. The biomarker classification model was suitable for preliminary identification of BD and could potentially serve as a useful tool for BD diagnosis and differential diagnosis.

Increased IL-7 expression in Vogt-Koyanagi-Harada disease.

PURPOSE: IL-7/IL-7R has been found to be involved in the pathogenesis of several autoimmune diseases. This study was designed to investigate the potential role of IL-7/IL-7R in the pathogenesis of Vogt Koyanagi-Harada (VKH), an organ-specific autoimmune disease. METHODS: IL-7 was measured with an enzyme-linked immunosorbent assay (ELISA) in serum obtained from patients with active or inactive VKH and from healthy individuals. The expression of IL-7R was measured by flow cytometry (FCM). Cell proliferation was determined after exposure of peripheral blood mononuclear cells (PBMCs) and CD4(+) T cells to recombinant IL-7. The levels of IL-17 and IFN-γ levels were detected by ELISA after these cells were cocultured with recombinant IL-7. The influence of recombinant IL-7 on the expansion of Th1 and Th17 cells was evaluated by using FCM. RESULTS: IL-7 was significantly increased in the serum of patients with active VKH compared with those with inactive VKH (P < 0.001) and normal controls (P < 0.001). However, there was no difference between VKH patients and normal controls in the expression of IL-7Rα on CD4(+) T cells. Recombinant IL-7 induced significant cell proliferation and secretion of IL-17 and IFN-γ by PBMCs and CD4(+) T cells. It furthermore promoted the expansion of both Th1 and Th17 cells. CONCLUSIONS: The findings suggest that IL-7 is involved in the pathogenesis of VKH disease.

Elevated serum osteopontin levels and genetic polymorphisms of osteopontin are associated with Vogt-Koyanagi-Harada disease.

PURPOSE: Osteopontin (OPN) is a proinflammatory cytokine involved in chronic inflammatory diseases. This study aimed to analyze the role of OPN in the pathogenesis of Vogt-Koyanagi-Harada (VKH) disease. METHODS: Serum levels of OPN in VKH patients and healthy controls were assayed by enzyme-linked immunosorbent assay (ELISA). Peripheral blood mononuclear cells (PBMCs) or CD4+ T cells were cultured with anti-CD3 and anti-CD28 antibodies in the absence or presence of recombinant OPN for the determination of cell proliferation and cytokines. Cell proliferation was detected using a cell counting kit. Levels of interferon (IFN)-γ and interleukin (IL)-17 were detected by ELISA. Four single nucleotide polymorphisms (SNPs) of OPN and four SNPs of OPN receptors were genotyped in 601 VKH patients and 605 healthy controls using a polymerase chain reaction-restriction fragment length polymorphism assay. RESULTS: OPN serum levels were significantly higher in patients with active VKH than in patients with inactive VKH and in healthy controls. PBMCs or CD4+ T cells cultured with recombinant OPN induced a marked cell proliferation and profound secretion of IFN-γ and IL-17 from patients with active VKH. A significantly increased frequency of the OPN rs4754 TT genotype (P = 0.004, pc = 0.048) was observed in VKH patients compared with healthy controls. No association could be detected among the four selected SNPs of OPN receptors and VKH. CONCLUSIONS: OPN may be relevant to the pathogenesis of VKH disease. The TT genotype of rs4754 may be a susceptible factor for VKH disease in a Chinese Han population.

Decreased 1,25-Dihydroxyvitamin D3 level is involved in the pathogenesis of Vogt-Koyanagi-Harada (VKH) disease.

PURPOSE: 1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] has recently been found to be involved in the development of autoimmune diseases. This study was to investigate the expression and potential role of 1,25(OH)(2)D(3) in the pathogenesis of Vogt-Koyanagi-Harada (VKH) disease. METHODS: Blood samples were obtained from VKH patients and healthy individuals. Serum 1,25(OH)(2)D(3) levels were measured using ELISA. Peripheral blood mononuclear cells (PBMCs) or cluster of differentiation (CD) 4(+) T cells were cultured with or without 1,25(OH)(2)D(3) in the presence of anti-CD3 and anti-CD28 for the measurement of cell proliferation and cytokines. The cell proliferation was detected using the Cell Counting Kit. The levels of interleukin (IL)-17 and interferon (IFN)-γ levels in the supernatants of PBMCs or CD4(+) T cells were detected by ELISA. RESULTS: 1,25(OH)(2)D(3) was significantly decreased in the serum of active VKH patients as compared with inactive VKH patients and controls. It significantly inhibited PBMCs proliferation and CD4(+) T cell proliferation. It was also able to significantly inhibit the production of IL-17 and IFN-γ by both PBMCs and CD4(+) T cells from VKH patients and controls. CONCLUSIONS: These findings suggest that decreased expression of 1,25(OH)(2)D(3) may be involved in the development of VKH disease. 1,25(OH)(2)D(3) may be potentially used in the treatment of this disease.