Uveitis is one of the devastating ocular diseases causing

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1 Genetics Complement C5 Gene Confers Risk for Acute Anterior Uveitis Dengfeng Xu, 1 Shengping Hou, 1 Yanni Jiang, 1 Jun Zhang, 1 Shuang Cao, 1 Dike Zhang, 1 Le Luo, 1 Aize Kijlstra, 2 and Peizeng Yang 1 1 The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Ophthalmology and Chongqing Eye Institute, Chongqing, People s Republic of China 2 University Eye Clinic Maastricht, Maastricht, The Netherlands Correspondence: Peizeng Yang, The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Ophthalmology and Chongqing Eye Institute, Chongqing, PR China, ; peizengycmu@126.com. Submitted: February 9, 2015 Accepted: May 20, 2015 Citation: Xu D, Hou S, Jiang Y, et al. Complement C5 gene confers risk for acute anterior uveitis. Invest Ophthalmol Vis Sci. 2015;56: DOI: /iovs PURPOSE. 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. METHODS. 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. RESULTS. The frequency of the GG genotype of rs in C5 was increased in AAU patients with or without AS compared to controls (Pc ¼ , odds ratio [OR] ¼ 1.94 and Pc ¼ ,OR¼ 1.89, respectively). The mrna and serum concentrations of C5 were significantly increased in rs GG cases as compared to that in CG or CC cases (P ¼ 0.012, P ¼ 0.002; P ¼ 0.021, P ¼ 0.006, respectively). An increased production of interleukin-17 was observed in rs GG cases compared to CG or CC cases (P ¼ , P ¼ , respectively). CONCLUSIONS. The C5 rs 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. Keywords: acute anterior uveitis, ankylosing spondylitis, C3, C5 Uveitis is one of the devastating ocular diseases causing blindness. 1 Acute anterior uveitis (AAU), including iritis or iridocyclitis, shows an acute inflammation onset occurring in the anterior segment of the eye which can lead to visual impairment. 2 Ankylosing spondylitis (AS) is a chronic inflammatory disease that involves the spine and other extra-articular organs, such as the eye, skin, and cardiovascular system. 3 Acute anterior uveitis is thought to be the most frequent extra-articular manifestation of AS. 4 Previous studies showed that nearly half of patients with HLA-B27 positive AS will experience a bout of AAU during their disease course. 5 Recently, many studies have shown that, besides HLA-B27, many other genetic factors are involved in the onset of AAU and AS, such as CTLA-4, PTPN22, and TNF in AAU, 6,7 and ERAP1 and IL-23R in AS. 8 The complement system has an important role in the innate immune system. 9 Previous studies revealed that certain genetic variants of C3 and C5 are a risk factor for several autoimmune disorders, such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) An association between C3 and C5 polymorphisms in ocular disease has been investigated in agerelated macular degeneration Furthermore, several studies have demonstrated that the complement system has a critical role in the pathogenesis of experimental autoimmune anterior uveitis (EAAU) or endotoxin-induced uveitis (EIU). 10,19 21 Although the exact pathogenesis of AS remains unknown, previous studies have shown that complement components are involved in the pathogenesis of AS However, to our knowledge the association between C3 and C5 genetic variants has not yet been addressed in other ocular diseases, such as uveitis and, therefore, was the subject of the study described here. MATERIALS AND METHODS Study Design A total of 395 AAU patients with AS, 397 AAU patients without AS, and 597 normal controls were recruited. All the participants were Chinese Han. Blood samples were taken from patients and controls visiting the First Affiliated Hospital of Chongqing Medical University (Chongqing, China) or the Zhongshan Ophthalmic Center of Sun Yat-sen University (Guangzhou, China). The diagnosis of AS was based on the modified New York criteria. 25 Inclusion of AAU patients was based on an anterior uveitis, characterized by sudden onset and with limited duration of less than 3 months. Diagnosis was based on clinical features, including ciliary injection, dust-like keratic precipitates, a large number of cells in the anterior chamber, and obvious aqueous flare. Typically, these patients showed recurrent episodes after first disease onset. There was no visible retinal or choroid lesion except a reactive and transient papilledema or macular edema. 26,27 Patients were excluded from this study if they suffered from other uveitis entities or if the diagnosis was not definitely made. The study was authorized by the First Affiliated Hospital of Chongqing Medical Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc. iovs.arvojournals.org j ISSN:

2 C5 Polymorphisms Are Associated With AAU IOVS j July 2015 j Vol. 56 j No. 8 j 4955 University Ethics Research Committee (Permit Number, ). All procedures in this study were based on the principles of the Declaration of Helsinki. Before accepting the samples of peripheral venous blood (EDTA-anticoagulated whole blood or serum), all the participants needed to provide written informed consent. Serum was separated at 4000g after clot retraction and then stored at 808C. Single Nucleotide Polymorphism (SNP) Selection The selection of C3 and C5 SNPs was based on previous studies. 14,15,17,18,28 34 Nine SNPs of C3, including rs7951, rs344555, rs , rs , rs171094, rs , rs , rs , and rs , were analyzed. Among them, rs , rs , and rs were monozygous in the Han Chinese population (1000 genome database) and, therefore, were not included in our study. Two pairs of SNPs (rs and rs408290, rs and rs ) are in linkage disequilibrium (D ¼ 0.91, r 2 ¼ 0.45 and D ¼ 1, r 2 ¼ 0.13, respectively). Two SNPs, including rs and rs432001, were selected as TagSNPs. Finally, six C3 SNPs, including rs408290, rs7951, rs344555, rs , rs432001, and rs , were selected for this study. Ten SNPs of C5 (rs , rs17611, rs , rs , rs , rs , rs , rs , rs25681, rs ) were analyzed as candidates for our study and, among these, rs , rs , rs , rs , and rs are in linkage disequilibrium (r ). Also, rs , rs17611, rs25681, and rs are in strong linkage disequilibrium (r 2 ¼ 1). Single nucleotide polymorphism rs was selected as a TagSNP. Finally, four C5 SNPs, including rs , rs , rs , and rs , were selected for this study. Taken together, 10 SNPs were analyzed for this study (six C3 SNPs and four C5 SNPs). DNA Extraction and SNP Genotyping Genomic DNA was extracted from peripheral blood with the Qiagen (Venlo, The Netherlands) QIAamp DNA Mini blood kit. Two SNPs, including rs and rs , were genotyped by PCR assay (TaqMan assay ID, C_ _1, C_ _10, respectively) on real-time PCR (Applied Biosystems, Carlsbad, CA, USA). The other eight SNPs were genotyped by PCR-restriction fragment length polymorphism (PCR-RFLP) method with proper primers and restriction enzymes (the primer sequences and restriction enzymes are shown in Supplementary Table S1). Four percent agarose gels were used to separate the digestion products and colored with GoldView (SBS Genetech, Beijing, China). Data were analyzed to ensure that our study population was in Hardy-Weinberg equilibrium. Ten percent of study samples were used randomly for direct sequencing to assure the validity of the SNP genotyping. Cell Isolation and Culture Peripheral blood mononuclear cells (PBMCs) from healthy genotyped individuals were isolated using Ficoll-Hypaque density-gradient centrifugation. The isolated PBMCs were seeded in 24-well plates ( cells per well) and cultured for 1 or 3 days in 100 lg/ml streptomycin, RPMI medium 1640 supplemented with 10% fetal calf serum (FCS, Belgium) and 100 U/mL penicillin. Peripheral blood mononuclear cells were stimulated with a mixture of anti-cd3 (5 lg/ml; ebioscience, San Diego, CA, USA) and anti-cd28 antibodies (1 lg/ml; ebioscience) for 72 hours, then the production of IL-17, IFN-c, and IL-10 was measured in the culture supernatants. To measure the level of IL-1b, IL-8, TNF-a, IL-6, and MCP-1 following an inflammatory stimulus, we cultured PBMCs with 100 ng/ml lipopolysaccharide (100 ng/ml; Sigma-Aldrich Corp., St. Louis, MO, USA) for 24 hours and then collected supernatants for cytokine measurements. Real-Time PCR TRIzol (Invitrogen, Carlsbad, CA, USA) and transcriptase kit (Applied Biosystems) were used for total RNA extraction and cdna reverse transcription, respectively. The primer sequences for C3 and C5 were as follows: forward, 5 0 -GGAGCAGTC AAGGTCTAC GC-3 0 ; reverse, 5 0 -CACAGTTCATCACGGCAG AG- 3 0 ; and forward, 5 0 -GGCACAAA GTCCTCCAAATG-3 0 ; reverse, 5 0 -CCAAACCA AGTCTCCAGTGA-3 0, respectively. b-actin was selected as the internal reference gene and the 2 DDCt method was used to calculate the relative expression for each gene. ELISA Assay The human Duoset ELISA development kit (R&D Systems, Minneapolis, MN, USA) was used to measure the C5 concentration in serum and the concentration of IL-17, IFN-c, IL-10, IL-8, TNF-a, IL-6, IL-1b, and MCP-1 in PBMC culture supernatants according to the manufacturer s protocols. Statistical Analysis To analyze the frequencies of genotypes and alleles, the v 2 test (SPSS version 17.0; SPSS, Inc., Chicago, IL, USA) was applied to compare the differences between patients and controls. The mrna expression, C5 serum concentrations and various cytokines were analyzed by 1-way ANOVA or a nonparametric test for independent samples among three genotype cases. Bonferroni correction was used for multiple comparisons. A total of 10 SNPs, including 20 alleles and 27 genotypes, were analyzed in this study. As the two alleles of one SNP showed the same result, the P values of allele comparisons were corrected by a factor 10. Similarly, there were 27 different genotypes of all the studied SNPs, so the P values for the frequency of genotypes was corrected by a factor 27. Significance was tested by 1-tailed P values < 0.05 (SPSS version 17.0). RESULTS Clinical Characteristics of AAU Patients With or Without AS The details of clinical features, age, and sex distribution in the AAU patient group and controls are shown in Supplementary Table S2. Increased Frequency of C5 GG Genotypes in AAU Patients The 10 SNPs were genotyped in 395 AAU patients with AS, 397 AAU patients without AS, and 597 normal controls. The genotype frequencies of the 10 SNPs did not deviate from HWE in normal controls (P > 0.05). The results showed that the frequencies of the C5 rs GG genotype and G allele in AAU patients with AS (AAU þ AS þ ) was significantly increased compared to controls (Pc ¼ , odds ratio [OR] ¼ 1.94 and Pc ¼ ,OR¼ 1.66, respectively). Similar to AAU þ AS þ, the frequencies of GG genotypes and G alleles of rs in AAU þ AS patients were also higher than that in controls (Table 1). The genotype and allele frequencies of the other three SNPs of C5 and the six SNPs of C3 were not

3 C5 Polymorphisms Are Associated With AAU IOVS j July 2015 j Vol. 56 j No. 8 j 4956 TABLE 1. Frequencies of Genotypes and Alleles of C5 Polymorphisms in AAU Patients With AS, AAU Patients Without AS, and Normal Control AAU þ AS vs. Control AAU þ AS þ vs. Control P Value Pc OR (95% CI) P Value Pc OR (95% CI) Genes SNPs Allele Control AAU þ AS AAU þ AS þ C5 rs G 898 (0.753) 644 (0.832) 647 (0.836) ( ) ( ) CC 29 (0.049) 15 (0.039) 14 (0.036) NS 0.78 ( ) NS 0.74 ( ) CG 236 (0.396) 100 (0.258) 99 (0.256) ( ) ( ) GG 331 (0.555) 272 (0.703) 274 (0.708) ( ) ( ) rs A 692 (0.582) 460 (0.591) 461 (0.584) NS 1.03 ( ) NS 1.00 ( ) AA 191 (0.322) 126 (0.324) 111 (0.281) NS 1.01 ( ) NS 0.82 ( ) AG 310 (0.522) 208 (0.535) 239 (0.605) NS 1.05 ( ) NS 1.40 ( ) GG 93 (0.157) 55 (0.141) 45 (0.114) NS 0.88 ( ) NS 0.69 ( ) rs C 83 (0.070) 67 (0.084) 49 (0.062) NS 1.21 ( ) NS 0.87 ( ) CC 6 (0.010) 4 (0.010) 4 (0.010) NS 0.99 ( ) NS 0.99 ( ) CT 71 (0.120) 59 (0.149) 41 (0.104) NS 1.27 ( ) NS 0.85 ( ) TT 513 (0.869) 334 (0.841) 349 (0.886) NS 0.79 ( ) NS 1.16 ( ) rs C 32 (0.027) 30 (0.039) 27 (0.035) NS 1.46 ( ) NS 1.30 ( ) CT 32 (0.054) 30 (0.078) 27 (0.070) NS 1.48 ( ) NS 1.31 ( ) TT 562 (0.946) 354 (0.922) 361 (0.930) NS 0.67 ( ) NS 0.76 ( ) significantly different in the AAU þ AS þ and AAU þ AS group compared to controls after Bonferroni correction (Tables 1, 2). Increased C5 mrna Expression and Serum C5 Protein Levels in Individuals With rs GG Genotype The aforementioned results revealed an obvious association of C5 rs with AAU. We next investigated mrna expression of C5 in PBMCs from healthy individuals with a known SNP genotype. The mrna expression of C5 rs in GG cases was significantly increased compared to CC and CG cases (P ¼ 0.002, P ¼ 0.012, respectively; Fig. 1). A separate set of healthy genotyped individuals was tested for their circulating C5 serum protein level. The C5 serum concentration also was elevated in GG cases compared to that in CC and CG cases (P ¼ 0.006, P ¼ 0.021, respectively; Fig. 1). The Influence of C5 rs on Cytokine Production A further experiment was performed to examine whether different genotypes of C5 affected cytokine production. The level of IL-17, IFN-c, TNF-a, IL-10, IL-1b, MCP-1, IL-6, and IL-8 was tested in the culture supernatants of stimulated PBMCs from genotyped healthy controls. The results showed that the production of IL-17 in C5 rs GG cases was higher than that in CG and CC cases (P ¼ , P ¼ , respectively; Fig. 2). The production of the other seven cytokines was not associated with C5 genotype. DISCUSSION In the present study, we investigated the association of six C3 SNPs and four C5 SNPs with AAU in a Han Chinese population. The results showed that C5 rs was a risk factor for AAU. Functional studies showed that C5 mrna and protein expression in the C5 GG genotype cases were increased compared to the other two genotype cases. Additionally, the production of IL-17 was significantly increased in the C5 rs GG genotype cases. The complement system has been considered as an important element in the innate as well as in the adaptive immune response. 9,35 An earlier study in experimental animals showed that complement was critical in the development of EAAU. 10 Others have demonstrated that the blockade of the complement alternative pathway could suppress EAAU via a modulation of the T cell response. 36 Blocking the formation of the C3 convertase, resulted in the inhibition of EAAU by reducing several cytokines, such as IFN-c, IP-10, and ICAM-1, in a Lewis rats model. 37 In the present study, we did not find evidence for an involvement of C3 polymorphisms in the risk for AAU development. This may be due to differences between the animal models of uveitis and human AAU. Recent studies showed that suppression of C5 could protect against tissue damage and reduce disease severity during the autoimmune response in mouse models of EAU. 38 Bora et al. 39,40 found that EAAU could be induced by antigenspecific CD4 þ T cells directed against antigens derived from iris and ciliary body in a Lewis rat models. Of interest is that a significantly increased expression of CD4 þ IL-17 þ Th17 cells and CD4 þ IFN-c þ Th1 cells was found in the peripheral blood of patients with HLA-B27-positive AAU. 41 These results suggested that C5 activation in combination with a CD4 þ T cell response, including the Th17 cell and Th1 cell subsets, are both critical for the development of EAAU.

4 C5 Polymorphisms Are Associated With AAU IOVS j July 2015 j Vol. 56 j No. 8 j 4957 TABLE 2. Frequencies of Genotypes and Alleles of C3 Polymorphisms in AAU Patients With AS, AAU Patients Without AS, and Normal Control AAU þ AS vs. Control AAU þ AS þ vs. Control Genes SNPs Allele Control AAU þ AS AAU þ AS þ P Value Pc OR (95% CI) P Value Pc OR (95% CI) C3 rs7951 C 1069 (0.906) 704 (0.912) 702 (0.900) NS 1.07 ( ) NS 0.93 ( ) CC 482 (0.817) 319 (0.826) 321 (0.823) NS 1.06 ( ) NS 1.04 ( ) CT 105 (0.178) 66 (0.171) 60 (0.154) NS 0.95 ( ) NS 0.84 ( ) TT 3 (0.005) 1 (0.003) 9 (0.023) NS 0.51 ( ) NS 4.62 ( ) rs A 950 (0.801) 653 (0.829) 635 (0.816) NS 1.17 ( ) NS 1.07 ( ) AA 373 (0.629) 265 (0.673) 257 (0.661) NS 1.15 ( ) NS 1.08 ( ) AG 204 (0.344) 123 (0.312) 121 (0.311) NS 0.91 ( ) NS 0.93 ( ) GG 16 (0.027) 6 (0.015) 11 (0.028) NS 0.46 ( ) NS 1.20 ( ) rs A 1075 (0.925) 701 (0.901) 694 (0.887) NS 0.73 ( ) NS 0.63 ( ) AA 494 (0.850) 312 (0.802) 303 (0.775) 0.05 NS 0.71 ( ) NS 0.60 ( ) AG 87 (0.150) 77 (0.198) 88 (0.225) 0.05 NS 1.40 ( ) NS 1.64 ( ) rs G 850 (0.712) 555 (0.699) 523 (0.674) NS 0.94 ( ) NS 0.83 ( ) AA 50 (0.084) 30 (0.076) 45 (0.116) NS 0.91 ( ) NS 1.43 ( ) AG 244 (0.409) 179 (0.451) 163 (0.420) NS 1.16 ( ) NS 1.04 ( ) GG 303 (0.508) 188 (0.474) 180 (0.464) NS 0.88 ( ) NS 0.84 ( ) rs C 1109 (0.932) 716 (0.935) 716 (0.927) NS 1.04 ( ) NS 0.93 ( ) CC 514 (0.864) 333 (0.869) 330 (0.855) NS 1.05 ( ) NS 0.92 ( ) GG 81 (0.136) 50 (0.131) 56 (0.145) NS 0.95 ( ) NS 1.07 ( ) rs C 826 (0.701) 547 (0.692) 529 (0.689) NS 0.95 ( ) NS 0.94 ( ) CC 283 (0.480) 201 (0.509) 191 (0.497) NS 1.12 ( ) NS 1.07 ( ) CG 260 (0.441) 145 (0.367) 147 (0.383) NS 0.73 ( ) NS 0.78 ( ) GG 46 (0.078) 49 (0.124) 46 (0.120) NS 1.67 ( ) NS 1.60 ( ) CI, confidence interval; NS, nonsignificant; Pc, P Bonferroni correction. Only few clinical studies have shown an association between complement C5 with AAU. Complement C5-derived chemotactic activity was present in inflamed aqueous humor in an endotoxin-induced rabbit model of AAU and the chemotactic activity could be inhibited by antibodies to C5. 21 This was confirmed in a clinical study whereby it was shown that the levels of C5a in inflamed aqueous humor from patients with AAU was significantly higher than that in normal controls or patients without inflamed aqueous humor. 42 Only few studies have shown an association between C5 polymorphisms with autoimmune or autoinflammatory disorders. Chang et al. 15 found that C5 rs was weakly associated with RA (OR: 1.27). To our knowledge the association between genetic variants of C5 and uveitis has not yet been reported. Genetic studies in mice have shown that C5 variants have an important role in the pathogenesis of a collagen induced arthritis model How C5 SNP rs influences the risk for AAU is not yet clear. Preliminary data showed that the C5 mrna expression was enhanced in the rs GG genotype cases. We furthermore showed that C5 concentration in serum was elevated in rs GG genotype cases. Liu et al. 46 found that C5a could promote the production of IL-17 by human T cells in AMD patients. Of interest was our finding that the production of IL-17 by stimulated PBMCs was increased in the GG genotype cases. It is possible that the C5 GG rs genotype may increase the production of IL-17 through C5 serum protein, thereby influencing the development of AAU. This is a speculative hypothesis and further studies are needed to examine the exact role of C5 genotypes and IL-17 production in the pathogenesis of AAU. Therefore, whether modulation of C5 could have therapeutic implications in our AAU patients also deserves further basic investigation. Of interest is that anti-c5 therapy has been shown to reduce the severity of disease in mouse models of EAU. 38 FIGURE 1. The mrna expression and serum concentration of C5 in different genotype cases. mrna expression (a) of C5 rs by PBMCs and C5 concentration (b) in serum from healthy controls with known C5 genotype (GG, n ¼ 15; CG, n ¼ 15; CC, n ¼ 14; GG, n ¼ 8; CG, n ¼ 8; CC, n ¼ 7, respectively). Samples for mrna expression were collected on a separate occasion as the serum samples for C5 protein levels and are from different individuals. Significance was tested by SPSS two independent samples nonparametric test.

5 C5 Polymorphisms Are Associated With AAU IOVS j July 2015 j Vol. 56 j No. 8 j 4958 FIGURE 2. The influence of C5 rs on cytokine production. Cytokine production (a h) by anti-cd3/cd28 (a, e, h) or LPS (b d, f, g) stimulated PBMCs from healthy controls with a known C5 rs genotype (GG, n ¼ 13 20; CG, n ¼ 13 21; CC, n ¼ 11 14). Significance was examined by two independent samples nonparametric test or a 1-way ANOVA test. Data are shown as mean 6 SD.

6 C5 Polymorphisms Are Associated With AAU IOVS j July 2015 j Vol. 56 j No. 8 j 4959 Some limitations in our study should be taken into account. This study was performed only in a Chinese Han population, and replication in an independent sample would strengthen our findings. Our findings also must be confirmed in other ethnic populations. All patients used in our study were recruited from an ophthalmology department, only including uveitis patients. Whether the association we found also exists between the C5 genotypes and AS patients without uveitis remains unclear and must be further studied. We studied only six C3 SNPs and four C5 SNPs, and cannot exclude a possible association with other as yet unknown SNPs. The effect of C5 genotypes on C5 and IL17 expression was performed in healthy genotyped individuals and not in patients, to exclude a confounding effect of the inflammatory response or installed therapy. Future studies could be planned to investigate how disease activity affects the C5 expression in genotyped patients. It should be noted that the C5 mrna expression in the GG genotype individuals showed a large variability in this study for which we do not yet have an explanation. Healthy individuals were nonselectively collected for C5 mrna examination over a period ranging from November 2014 to January 2015 and did not suffer from eye diseases or any immune-related disorder. A variety of cells can produce IL-17, such as Th17 cells, NK cells, gamma/delta T cells, macrophages, and neutrophils We only studied IL-17 production in a total PBMC sample and further subset analysis is necessary to investigate which cell type is responsible for the augmented IL17 response in individuals with a certain C5 genotype. In conclusion, our results revealed that the C5 rs confers a risk for AAU in a Chinese Han population. The risk genotype affects the production of C5 and of proinflammatory cytokines, such as IL-17. Acknowledgments The authors thank all the participants in this study. Supported by Natural Science Foundation Major International (Regional) Joint Research Project ( ), Key Project of Natural Science Foundation ( ), National Natural Science Foundation Project ( , ), Basic Research Program of Chongqing (cstc2013jcyjc10001), Chongqing Outstanding Youth Grant (cstc2014jcyjjq10005), Chongqing Key Laboratory of Ophthalmology (CSTC, 2008CA5003), National Key Clinical Specialties Construction Program of China, and Fund for PAR-EU Scholars Program. Disclosure: D. Xu, None; S. Hou, None; Y. Jiang, None; J. Zhang, None; S. Cao, None; D. Zhang, None; L. Luo, None; A. Kijlstra, None; P. Yang, None References 1. Suttorp-Schulten MS, Rothova A. The possible impact of uveitis in blindness: a literature survey. Br J Ophthalmol. 1996;80: Yang P, Zhang Z, Zhou H, et al. Clinical patterns and characteristics of uveitis in a tertiary center for uveitis in China. Curr Eye Res. 2005;30: Gouveia EB, Elmann D, Morales MS. Ankylosing spondylitis and uveitis: overview. Rev Bras Reumatol. 2012;52: Braun J, Sieper J. Ankylosing spondylitis. Lancet. 2007;369: Braun J, Baraliakos X, Listing J, Sieper J. Decreased incidence of anterior uveitis in patients with ankylosing spondylitis treated with the anti-tumor necrosis factor agents infliximab and etanercept. Arthritis Rheum. 2005;52: Martin TM, Bye L, Modi N, et al. Genotype analysis of polymorphisms in autoimmune susceptibility genes, CTLA-4 and PTPN22, in an acute anterior uveitis cohort. Mol Vis. 2009;15: Kuo NW, Lympany PA, Menezo V, et al. TNF-857T, a genetic risk marker for acute anterior uveitis. Invest Ophthalmol Vis Sci. 2005;46: Reveille JD. Recent studies on the genetic basis of ankylosing spondylitis. Curr Rheum Rep. 2009;11: Muller-Eberhard HJ. Molecular organization and function of the complement system. Ann Rev Biochem. 1988;57: Jha P, Sohn JH, Xu Q, et al. The complement system plays a critical role in the development of experimental autoimmune anterior uveitis. Invest Ophthalmol Vis Sci. 2006;47: Biro E, Nieuwland R, Tak PP, et al. Activated complement components and complement activator molecules on the surface of cell-derived microparticles in patients with rheumatoid arthritis and healthy individuals. Ann Rheum Dis. 2007;66: Kenyon KD, Cole C, Crawford F, et al. IgG autoantibodies against deposited C3 inhibit macrophage-mediated apoptotic cell engulfment in systemic autoimmunity. J Immunol. 2011; 187: Chen M, Daha MR, Kallenberg CG. The complement system in systemic autoimmune disease. J Autoimmun. 2010;34:J Rhodes B, Hunnangkul S, Morris DL, et al. The heritability and genetics of complement C3 expression in UK SLE families. Genes Immun. 2009;10: Chang M, Rowland CM, Garcia VE, et al. A large-scale rheumatoid arthritis genetic study identifies association at chromosome 9q33.2. PLoS Genet. 2008;4:e Hacihamdioglu DO, Demiriz M, Sobaci G, Kocaoglu M, Demirkaya E, Gok F. Cerebral vein thrombosis in a four year old with Behcet s disease. Reumatol Clin. 2014;10: Seddon JM, Yu Y, Miller EC, et al. Rare variants in CFI, C3 and C9 are associated with high risk of advanced age-related macular degeneration. Nat Genet. 2013;45: Baas DC, Ho L, Ennis S, et al. The complement component 5 gene and age-related macular degeneration. Ophthalmology. 2010;117: Bora NS, Jha P, Bora PS. The role of complement in ocular pathology. Semin Immunopath. 2008;30: Jha P, Sohn JH, Xu Q, et al. Suppression of complement regulatory proteins (CRPs) exacerbates experimental autoimmune anterior uveitis (EAAU). J Immunol. 2006;176: Rosenbaum JT, Wong K, Perez HD, Raymond W, Howes EL Jr. Characterization of endotoxin-induced C5-derived chemotactic activity in aqueous humor. Invest Ophthalmol Vis Sci. 1984;25: Wang C, Liao Q, Hu Y, Zhong D. T lymphocyte subset imbalances in patients contribute to ankylosing spondylitis. Exp Ther Med. 2015;9: Brinch L, Vinje O, Teisberg P, Mellbye OJ, Aakesson I. The invivo metabolism of C3 in ankylosing spondylitis. Ann Rheum Dis. 1982;41: Sturrock RD, Barret AJ, Versey J, Rennolds P. Proceedings: raised levels of complement inactivation products in ankylosing spondylitis. Ann Rheum Dis. 1975;34: van der Linden S, Valkenburg HA, Cats A. Evaluation of diagnostic criteria for ankylosing spondylitis. A proposal for modification of the New York criteria. Arthritis Rheum. 1984; 27: Rothova A, van Veenedaal WG, Linssen A, Glasius E, Kijlstra A, de Jong PT. Clinical features of acute anterior uveitis. Am J Ophthalmol. 1987;103:

7 C5 Polymorphisms Are Associated With AAU IOVS j July 2015 j Vol. 56 j No. 8 j Jabs DA, Nussenblatt RB, Rosenbaum JT. Standardization of uveitis nomenclature for reporting clinical data. Results of the First International Workshop. Am J Ophthalmol. 2005;140: Miyagawa H, Yamai M, Sakaguchi D, et al. Association of polymorphisms in complement component C3 gene with susceptibility to systemic lupus erythematosus. Rheumatology (Oxford). 2008;47: Yates JR, Sepp T, Matharu BK, et al. Complement C3 variant and the risk of age-related macular degeneration. New Engl J Med. 2007;357: Kim SJ, Lee SJ, Kim NR, Chin HS. Association of polymorphisms in C2, CFB and C3 with exudative age-related macular degeneration in a Korean population. Exp Eye Res. 2012;96: Bergeron-Sawitzke J, Gold B, Olsh A, et al. Multilocus analysis of age-related macular degeneration. Eur J Hum Genet. 2009; 17: Pei XT, Li XX, Bao YZ, et al. Association of c3 gene polymorphisms with neovascular age-related macular degeneration in a chinese population. Curr Eye Res. 2009;34: Yanagisawa S, Kondo N, Miki A, et al. A common complement C3 variant is associated with protection against wet age-related macular degeneration in a Japanese population. PLoS One. 2011;6:e Fagerness JA, Maller JB, Neale BM, Reynolds RC, Daly MJ, Seddon JM. Variation near complement factor I is associated with risk of advanced AMD. Eur J Hum Genet. 2009;17: Sohn JH, Bora PS, Suk HJ, Molina H, Kaplan HJ, Bora NS. Tolerance is dependent on complement C3 fragment ic3b binding to antigen-presenting cells. Nat Med. 2003;9: Manickam B, Jha P, Matta B, Liu J, Bora PS, Bora NS. Inhibition of complement alternative pathway suppresses experimental autoimmune anterior uveitis by modulating T cell responses. J Biol Chem. 2011;286: Manickam B, Jha P, Hepburn NJ, et al. Suppression of complement activation by recombinant Crry inhibits experimental autoimmune anterior uveitis (EAAU). Mol Immunol. 2010;48: Copland DA, Hussain K, Baalasubramanian S, et al. Systemic and local anti-c5 therapy reduces the disease severity in experimental autoimmune uveoretinitis. Clin Exp Immunol. 2010;159: Bora NS, Kim MC, Kabeer NH, et al. Experimental autoimmune anterior uveitis. Induction with melanin-associated antigen from the iris and ciliary body. Invest Ophthalmol Vis Sci. 1995;36: Bora NS, Woon MD, Tandhasetti MT, Cirrito TP, Kaplan HJ. Induction of experimental autoimmune anterior uveitis by a self-antigen: melanin complex without adjuvant. Invest Ophthalmol Vis Sci. 1997;38: Zou W, Wu Z, Xiang X, Sun S, Zhang J. The expression and significance of T helper cell subsets and regulatory T cells CD(4)(þ) CD(2)(5)(þ) in peripheral blood of patients with human leukocyte antigen B27-positive acute anterior uveitis. Graefe s Arch Clin Exp Ophthalmol. 2014;252: Mondino BJ, Sumner H. Anaphylatoxin levels in human aqueous humor. Invest Ophthalmol Vis Sci. 1986;27: Linton SM, Morgan BP. Complement activation and inhibition in experimental models of arthritis. Mol Immunol. 1999;36: Wang Y, Kristan J, Hao L, Lenkoski CS, Shen Y, Matis LA. A role for complement in antibody-mediated inflammation: C5- deficient DBA/1 mice are resistant to collagen-induced arthritis. J Immunol. 2000;164: Ji H, Gauguier D, Ohmura K, et al. Genetic influences on the end-stage effector phase of arthritis. J Exp Med. 2001;194: Liu B, Wei L, Meyerle C, et al. Complement component C5a promotes expression of IL-22 and IL-17 from human T cells and its implication in age-related macular degeneration. J Trans Med. 2011;9: Sun D, Liang D, Kaplan HJ, Shao H. The role of Th17-associated cytokines in the pathogenesis of experimental autoimmune uveitis (EAU). Cytokine. 2015;74: Al Omar S, Flanagan BF, Almehmadi M, Christmas SE. The effects of IL-17 upon human natural killer cells. Cytokine. 2013;62: Ma S, Cheng Q, Cai Y, et al. IL-17A produced by gammadelta T cells promotes tumor growth in hepatocellular carcinoma. Cancer Res. 2014;74: Coffelt SB, Kersten K, Doornebal CW, et al. IL-17-producing gammadelta T cells and neutrophils conspire to promote breast cancer metastasis. Nature. 2015;522: Chan CC, Ardeljan D. Molecular pathology of macrophages and interleukin-17 in age-related macular degeneration. Adv Exp Med Biol. 2014;801: Huppler AR, Conti HR, Hernandez-Santos N, Darville T, Biswas PS, Gaffen SL. Role of neutrophils in IL-17-dependent immunity to mucosal candidiasis. J Immunol. 2014;192: