[Show abstract][Hide abstract] ABSTRACT: Oxidative stress and inflammation are two interrelated biological events implicated in the pathogenesis of many diseases. Reactive oxygen species (ROS) produced under oxidative stress play a key role in pathological conditions. Inhibition of p22phox, an indispensable component of the NADPH oxidase (NOX) complex comprising the main source of ROS, plays a protective role in many ocular conditions by inhibiting the activation of NOXs and the generation of ROS. However, little is understood regarding the role of p22phox in oxidative stress-related inflammation in the eye. We used a p22phox small interfering RNA (siRNA) to transfect the retinal pigment epithelium (RPE)-derived cell line ARPE-19, and human primary RPE (hRPE) cells, then stimulated with Ang II. We observed a potent anti-inflammatory effect and studied the underlying mechanism. Downregulating p22phox resulted in decreased ROS generation, a reduction of NOXs (NOX1, 2, 4) and a decrease in inflammatory cytokine. In addition, p22phox downregulation reduced the activation of the MAPK and NF- B signaling pathways. We conclude that inhibition of p22phox has an anti-inflammatory effect in Ang II-induced oxidative stress. Suppressing the MAPK and NF- B pathways is involved in this protective effect. These results suggest that p22phox may provide a promising therapeutic target for oxidative stress-induced ocular inflammation.
[Show abstract][Hide abstract] ABSTRACT: Purpose:
We examined whether MIF could exaggerate inflammatory response in a mouse model of experimental autoimmune uveitis (EAU) and explored the underlying mechanism.
Mutant AAV8 (Y733F)-CBA-MIF or AAV8 (Y733F)-CBA-eGFP vector was delivered subretinally into B10.RIII mice respectively. Three weeks after vectors delivery, EAU was induced with a subcutaneous injection of a mixture of IRBP peptide with CFA. The levels of proinflammatory cytokines were detected by real-time PCR and ELISA. Retinal function was evaluated with electroretinography (ERG).
We found that expression of MIF and its two receptors CD74 and CD44 was increased in the EAU mouse retina. Compared to AAV8.CBA.eGFP injected and untreated EAU mice, the level of proinflammatory cytokines, the expression of Notch1, Notch4, Dll4, NICD and Hes-1 increased, but the ERG a- and b-wave amplitudes decreased in AAV8.CBA.MIF injected EAU mice. The Notch inhibitor DAPT reduced the expression of NICD, Hes-1 and proinflammatory cytokines. Further, a MIF antagonist ISO-1 attenuated intraocular inflammation, and inhibited the differentiation of Th1 and Th17 in EAU mice. We demonstrated that over-expression of MIF exaggerated ocular inflammation, which was associated with the activation of the Notch signaling.
The expression of both MIF and its receptors are elevated in EAU mice. Over-expression of MIF exaggerates ocular inflammation, and this exaggerated inflammation is associated with the activation of the Notch signaling pathway. Our data suggest that the MIF-Notch axis may play an important role in the pathogenesis of EAU. Both the MIF and Notch signaling pathways may be promising targets for developing novel therapeutic interventions for uveitis. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Purpose:
Behçet's disease (BD) is a common uveitis entity in China. The endoplasmic reticulum aminopeptidase 1 (ERAP1), has a significant influence on the stability and immunological properties of MHC-I loaded peptides. In the present study, we investigated the association of ERAP1 gene polymorphisms with BD in a Chinese Han population.
A two-stage case-control study was carried out in 930 BD patients and 1704 healthy controls. Seven single nucleotide polymorphisms (SNPs) of the ERAP1 gene were determined using a PCR restriction fragment length polymorphism (PCR-RFLP) assay and one SNP was genotyped by TaqMan SNP genotyping assay. Furthermore, ERAP1 expression in peripheral blood mononuclear cells (PBMCs) was examined in genotyped individuals by real-time PCR.
The result demonstrated that the frequencies of the A allele of rs1065407 and C allele of rs10050860 were significantly decreased in BD patients (Pc = 8.5 × 10-8, OR = 0.51; Pc = 1.1 × 10-5, OR = 0.54, respectively). No significant association was observed for the other six SNPs. ERAP1 expression in AA carriers of rs1065407 and CC carriers of rs10050860 was higher than that observed in AC/CC carriers (P = 0.022) or CT/TT carriers (P = 0.018) by LPS-stimulated PBMCs, respectively. In addition, the expression of ERAP1 in active BD patients not receiving immunosuppression was significantly lower than that in healthy controls (P = 3.8 × 10-4).
Our study showed that rs1065407 and rs10050860 of the ERAP1 gene may contribute to the genetic susceptibility of BD by modulating the expression of ERAP1.
[Show abstract][Hide abstract] ABSTRACT: Complement is involved in many immune-mediated diseases. However, the association of its copy number variations (CNVs) and polymorphisms with Behcet's disease (BD) and Vogt-Koyanagi-Harada syndrome (VKH) is unknown. We examined copy number and mRNA expression by real-time PCR. Cytokine production by stimulated peripheral blood mononuclear cells (PBMCs) in genotyped individuals was measured by ELISA. The frequencies of having more than two copies of C3 were significantly increased in BD and VKH, whereas CNV of C5 was only associated with BD. Increased frequencies of the GG genotype of C3 rs408290 and C5 rs2269067 were found in BD. No association was observed between C3 and C5 SNPs and VKH. mRNA expression in the high CNV group and GG cases of C3 and C5 was significantly higher compared to other genotypes. Increased interleukin-17 and IFN-γ was observed in the high CNV group and GG genotype cases of C3. Interleukin-17 but not IFN-γ was increased in the high CNV group and GG genotype cases of C5. No effect of C3 or C5 genetic variants was seen on the production of TNF-α, IL-10, IL-1β, MCP-1, IL-6 and IL-8. Our study thus provides further evidence for a role of complement in the pathogenesis of uveitis.
[Show abstract][Hide abstract] ABSTRACT: Previous studies have identified that disturbed apoptosis was involved in the pathogenesis of Behçet disease (BD) and VKH syndrome. This study aims to investigate whether copy number variations (CNVs) of apoptosis-related genes, including FAS, CASPASE8, CASPASE3 and BCL2, are associated with BD and VKH syndrome in Han Chinese. A two-stage association study was performed in 1014 BD patients, 1051 VKH syndrome patients and 2076 healthy controls. TaqMan(®) Copy Number Assays and real-time PCR were performed. The first-stage study showed that increased frequency of high FAS copy number (>2) was found in BD (P = 1.05×10(-3) ) and VKH syndrome (P = 2.56×10(-3) ). Replication and combined study confirmed the association of high copy number (>2) of FAS with BD (P = 3.35×10(-8) ) and VKH syndrome (P = 9.77×10(-8) ). A significant up-regulated mRNA expression of FAS was observed in anti-CD3/CD28 antibodies-stimulated CD4(+) T cells from individuals carrying a high gene copy number (>2) as compared to normal diploid 2 copy number carriers (P = 0.004). Moreover, the mRNA expression of FAS both in active patients with BD and VKH syndrome was significantly higher than that in controls (P = 0.001 and P = 0.007, respectively). Our findings suggest that a high copy number of FAS gene confers risk for BD and VKH syndrome. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
Human Mutation 08/2015; 36(11):1064-9. DOI:10.1002/humu.22829 · 5.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Purpose: T cells play an important role in the pathogenesis of uveitis. Recent studies have indicated that the TNFSF15 gene that encodes the TL1A protein can regulate the differentiation and activation of T cells. TNFSF15 gene polymorphisms have been found to be associated with several autoimmune disorders. A possible association of TNFSF15 with acute anterior uveitis (AAU) has not yet been reported and was therefore the purpose of our study.
Methods: Eight single nucleotide polymorphisms (SNPs) were examined using TaqMan SNP Genotyping Assay or PCR-restriction fragment length polymorphism in 983 AAU patients and 1128 healthy controls. Genotype distributions and allele frequencies were compared using χ2 analysis between AAU patients and healthy controls. Stratified analysis was also performed according to ankylosing spondylitis (AS) status. The TNFSF15 mRNA expression was quantified by real-time PCR.
Results: A significantly decreased frequency of the TT genotype in TNFSF15-rs3810936 was found in AAU patients (P = 6.36 × 10−6, corrected P[Pc] = 1.52 × 10−4, OR = 0.6, 95% CI = 0.5–0.8). Stratification according to AS status did not reveal a difference concerning the association with TNFSF15-rs3810936. None of the other TNFSF15 SNPs tested were associated with AAU.
Conclusions: This study shows an association between TNFSF15-rs3810936 and AAU and suggests that the TL1A/DR3 pathway may be implicated in the pathogenesis of this disease.
[Show abstract][Hide abstract] ABSTRACT: Polymorphisms in the genes encoding C3 and C5 are associated with several immune-mediated diseases. However, the association of C3 and C5 SNPs with acute anterior uveitis (AAU) has not yet been investigated and was the purpose of the study described.
Genotyping was performed for six SNPs in C3 and four SNPs in C5 in 395 AAU patients with ankylosing spondylitis (AS), 397 AAU patients without AS, and 597 healthy controls by PCR-restriction fragment length polymorphism (PCR-RFLP) or TaqMan SNP assay. The mRNA expression was detected by real-time PCR. Cytokine production and total C5 serum concentrations were measured by ELISA.
The frequency of the GG genotype of rs2269067 in C5 was increased in AAU patients with or without AS compared to controls (Pc = 4.0 × 10-5, odds ratio [OR] = 1.94 and Pc = 9.4 × 10-5, OR = 1.89, respectively). The mRNA and serum concentrations of C5 were significantly increased in rs2269067 GG cases as compared to that in CG or CC cases (P = 0.012, P = 0.002; P = 0.021, P = 0.006, respectively). An increased production of interleukin-17 was observed in rs2269067 GG cases compared to CG or CC cases (P = 5.1 × 10-4, P = 1.4 × 10-4, respectively).
The C5 rs2269067 GG genotype confers risk for AAU in a Chinese population and is associated with an elevated C5 serum concentration and an increased IL-17 production.
[Show abstract][Hide abstract] ABSTRACT: To determine the genetic lesions and to modify the clinical diagnosis for a Chinese family with significant intrafamilial phenotypic diversities and unusual presentations.
Three affected patients and the asymptomatic father were included and received comprehensive systemic examinations. Whole exome sequencing (WES) was performed for mutation detection. Structural modeling test was applied to analyze the potential structural changes caused by the missense substitution.
The proband showed a wide spectrum of systemic anomalies, including bilateral ectopia lentis, atrial septal defect, ventricular septal defect, widening of tibial metaphysis with medial bowing, and dolichostenomelia in digits, while her mother and elder brother only demonstrated similar skeletal changes. A recurrent mutation, PHEX p.R291*, was found in all patients, while a de novo mutation, FBN1 p.C792F, was only detected in the proband. The FBN1 substitution was also predicted to cause significant conformational change in fibrillin-1 protein, thus changing its physical and biological properties.
Taken together, we finalized the diagnosis for this family as X-linked hypophosphatemia (XLH), and diagnosed this girl as Marfan syndrome combined with XLH, and congenital heart disease. Our study also emphasizes the importance of WES in assisting the clinical diagnosis for complicated cases when the original diagnoses are challenged.
Journal of Translational Medicine 06/2015; 13(1). DOI:10.1186/s12967-015-0534-9 · 3.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study aimed to investigate the association of interleukin (IL)-10 gene polymorphisms with Behcet's disease (BD) and Vogt-Koyanagi-Harada (VKH) syndrome in the Chinese Han population.
A two-stage association study was performed on 718 BD patients, 300 VKH patients, and 1,753 controls. Genotyping of the IL-10 gene was performed for six single nucleotide polymorphisms (SNPs), including rs1800871, rs1800872, rs1800896, rs3021094, rs3790622, and rs1554286 using PCR-restricted fragment length polymorphism or TaqMan SNP assays. Real-time PCR was performed to test the IL-10 mRNA expression of the associated polymorphisms.
The first-stage result showed significantly increased frequencies of the rs1800871 T allele, rs1800872 A allele, and rs1554286 T allele in BD patients compared with controls (Pcorrected (Pcorr) = 1.82×10(-5), OR = 1.837; Pcorr = 6.1×10(-5), OR = 1.780; Pcorr = 3.15×10(-5), OR = 1.794, respectively). There was no association of the tested six SNPs with VKH syndrome. A second-stage study was therefore performed in BD patients to validate the result of the first stage, showing a significantly increased frequency of the rs1800871 T allele (Second stage, Pcorr = 5.59×10(-5), OR = 1.493; Combined data, Pcorr = 3.65×10(-11), OR = 1.632). Compared to the controls, an increased frequency of the rs1800871 T allele was observed in BD patients with extraocular findings, including genital ulcers, skin lesions, and a positive pathergy test. No difference was found among the mRNA expressions of IL-10 in the peripheral blood mononuclear cells (PBMCs) of controls with different genotypes of rs1800871 after stimulation of lipopolysaccharide (LPS) or anti-CD3/CD28 antibodies.
The findings showed that IL-10 is a risk gene for BD but not for VKH syndrome.
[Show abstract][Hide abstract] ABSTRACT: This study aimed to investigate the role of genetic variants including single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) of TBX21, GATA3, Rorc and Foxp3 genes in Behcet's disease (BD) and Vogt-Koyanagi-Harada (VKH) syndrome in a Chinese Han population. Genotyping of 25 SNPs was performed by iPLEX system (Sequenom) or polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). TaqMan real time PCR was used to assess CNVs. The expression of Rorc and Foxp3 were examined by real-time PCR and cytokine production was measured by ELISA. High Rorc CNV was associated with the susceptibility to BD (P = 8.99 × 10(-8), OR = 3.0), and low Foxp3 CNV predisposed to BD in female patients (P = 1.92 × 10(-5), OR = 3.1). CNVs for the investigated genes were not altered in VKH syndrome. Further functional studies demonstrated that the relative mRNA expression levels of Rorc were increased in individuals with high Rorc copy number, but not for Foxp3. Increased production of IL-1β and IL-6 was found in individuals carrying a high CNV of Rorc. Our study showed that high CNVs of Rorc and low CNVs of Foxp3 confer risk for BD but not for VKH syndrome. The tested 25 SNPs in TBX21, GATA3, Rorc and Foxp3 did not associate with BD and VKH syndrome.
[Show abstract][Hide abstract] ABSTRACT: Bach2 was reported to play a key role in T lymphocyte development and maturation to mediate immunological homeostasis. Several autoimmune and immune-related diseases were shown to be associated with Bach2 gene polymorphisms. The current study was designed to explore the association between Bach2 gene polymorphism with Vogt-Koyanagi-Harada (VKH) syndrome and Behcet's disease (BD) in a Chinese Han population.
-427 patients with BD, 422 patients with VKH and 623 controls were recruited for the first stage from a Chinese Han population. The second stage included another set of 388 patients with BD and 460 healthy subjects. PCR fragment length polymorphism methodology was used for genotyping. Frequencies of genotypes and alleles were measured by direct counting and compared between cases and controls by χ(2) test.
No difference could be detected between patients suffering from BD or VKH with healthy controls concerning allele and genotype frequencies of rs11755527, rs3757247, rs12212193 and rs2474619. Although in the first stage the frequencies of genotype CC and AC of rs2474619 showed a weak statistical difference between BD and the control group (Pc=0.02), the difference was lost after the second stage and combined stage experiment.
The investigated Bach2 gene polymorphisms (rs11755527, rs3757247, rs12212193 and rs2474619) are not related to the susceptibility to either VKH or BD in our investigated Chinese Han population.
This project was registered in the Chinese Clinical Trial Registry (registration number: ChiCTR-CCC-12002184).
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The British journal of ophthalmology 04/2015; 99(8). DOI:10.1136/bjophthalmol-2014-306163 · 2.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Conbercept (KH902), a novel recombinant, soluble vascular endothelial growth factor (VEGF) receptor-IgG fusion protein, has been developed as a new drug for ocular neovascularization and macular edema. The present study aims to clarify the changes in conbercept levels, VEGF, and intraocular pressure (IOP) after the intravitreal injection of conbercept into diabetic mouse eyes.
Five-week-old C57BL/6 mice were injected with streptozotocin to induce diabetes. Total VEGF and conbercept levels in the eyes were detected using an ELISA kit at -2 h, 1 h, 1 d, 4 d, 8 d, 16 d, 28 d, and 34 d after intravitreal injection of conbercept into diabetic and control mice. IOP was measured with a noninvasive TonoLab tonometer 7 d after intravitreal conbercept injection.
The concentration of conbercept in the treated eyes increased immediately after injection and remained at high levels for 4 d (29.77±27.19 ng/ml, 20.28±28.85 ng/ml, and 42.43±36.51 ng/ml for days 1, 2, and 4, respectively). The concentration of conbercept in the untreated fellow eyes increased from day 2 to day 4 after injection with a level of about 1% of that in the injected eyes. Conbercept concentrations in both the treated and fellow eyes decreased from day 7 after intravitreal injection. The concentration of VEGF in the treated eyes increased significantly 1 h after injection when compared with the baseline measured 2 h before injection in both the diabetic and control mice (645.91±86.47 pg/ml versus 296.10±76.11 pg/ml and 860.50±201.47 pg/ml versus 377.69±70.72 pg/ml, respectively). VEGF concentration reached its peak 24 h after injection and then decreased thereafter. At day 7 after intravitreal injection, the difference in IOP between mice that received conbercept and mice that received PBS injections was not significant (p>0.05).
Conbercept and total VEGF levels in the mouse eyes were elevated after intravitreal injection of conbercept. Increased VEGF levels likely reflect VEGF sequestered by conbercept. These data could be helpful in understanding the metabolism of anti-VEGF drugs in the eye and for determining the protocol of multiple intravitreal injections of conbercept in patients.
[Show abstract][Hide abstract] ABSTRACT: Purpose:
Recent studies have shown that a decrease of regulatory T (Treg) cells may contribute to the activity of acute anterior uveitis (AAU) and ankylosing spondylitis (AS). A number of immunogenetic factors including IL2RA, miR-27a, miR-182, and FoxO1 are associated with Treg cell function. In this study, we investigated the association between polymorphisms of these genes and AAU with or without AS in a Chinese Han population.
Using PCR-restricted fragment length polymorphism (RFLP) assay, a two-stage association study was performed in 680 AAU patients with or without AS and 1280 controls. Gene expression was quantified by real-time PCR.
In the first stage study, an association analysis of 10 single nucleotide polymorphisms (SNPs) was performed in 230 AAU patients with AS, 240 AAU patients without AS, and 650 controls. The results showed significantly increased frequencies of the FoxO1/rs2297626 AA genotype and A allele in AAU patients with AS (AA genotype: P = 6.23 × 10(-5), odds ratio [OR] = 1.86; A allele: P = 2.17 × 10(-4), OR = 1.53). No significant association of the other 9 SNPs with AAU with or without AS was observed. In the second stage study, an association analysis of FoxO1/rs2297626 was performed in 210 AAU patients with AS and 630 controls. The second stage and combined studies confirmed the association of FoxO1/rs2297626 with AAU with AS (AA genotype: P = 3.45 × 10(-8), OR = 1.85; A allele: P = 1.55 × 10(-7), OR = 1.55).
This study suggests that FoxO1, but not miR-27a, miR-182, and IL2RA, contributes to the genetic susceptibility of AAU with AS, but none of the tested polymorphisms confer risk to AAU without AS.
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Interferon & Cytokine Research 10/2014; 35(4). DOI:10.1089/jir.2014.0042 · 2.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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, r2=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.
British Journal of Ophthalmology 09/2014; 98(12). DOI:10.1136/bjophthalmol-2014-305596 · 2.98 Impact Factor