Juvenile idiopathic arthritis (JIA) represents the most frequent

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1 Immunology and Microbiology Elevated S100A8/A9 and S100A12 Serum Levels Reflect Intraocular Inflammation in Juvenile Idiopathic Arthritis- Associated Uveitis: Results From a Pilot Study Karoline Walscheid, 1,2 Arnd Heiligenhaus, 1,3 Dirk Holzinger, 2 Johannes Roth, 4 Carsten Heinz, 1,3 Christoph Tappeiner, 1,5 Maren Kasper, 1 and Dirk Foell 2 1 Department of Ophthalmology and Ophtha Lab, St. Franziskus-Hospital Muenster, Muenster, Germany 2 Department of Pediatric Rheumatology and Immunology, University Hospital Muenster, Muenster, Germany 3 Department of Ophthalmology, University of Duisburg-Essen, Essen, Germany 4 Institute of Immunology, University of Muenster, Muenster, Germany 5 Department of Ophthalmology, Inselspital, University of Bern, Bern, Switzerland Correspondence: Karoline Walscheid, Department of Ophthalmology at St. Franziskus-Hospital, Hohenzollernring 74, Muenster, Germany; karoline.walscheid@uveitis-zentrum. de. Submitted: April 10, 2015 Accepted: November 1, 2015 Citation: Walscheid K, Heiligenhaus A, Holzinger D, et al. Elevated S100A8/A9 and S100A12 serum levels reflect intraocular inflammation in juvenile idiopathic arthritis-associated uveitis: results from a pilot study. Invest Ophthalmol Vis Sci. 2015;56: DOI: /iovs PURPOSE. Juvenile idiopathic arthritis-associated uveitis (JIAU) is the most common uveitis entity in childhood. As S100A8/A9 and S100A12 proteins are valuable biomarkers in childhood arthritis, we investigated the occurrence of these proteins in childhood uveitis. METHODS. Serum samples from patients with JIAU (n ¼ 79) or idiopathic anterior uveitis (IAU, n ¼ 24), as well as from nonuveitic controls (n ¼ 24), were collected. Furthermore, aqueous humor samples (JIAU n ¼ 17, nonuveitic controls n ¼ 16, IAU n ¼ 12) were obtained. Samples were analyzed for S100A8/A9 and S100A12 protein levels by ELISA. Intergroup comparisons were performed, involving patient data, clinical data, and S100 levels. RESULTS. S100A8/A9 and S100A12 serum levels were elevated in IAU and JIAU patients as compared to nonuveitic controls (all P < 0.05). S100 serum levels in JIAU patients were higher in active arthritis (not significant; P ¼ for S100A8/A9 and P ¼ for S100A12) and active uveitis (P ¼ for S100A8/A9 and P ¼ for S100A12) than in controls. No significant differences in S100 levels were found in a subgroup analysis for sex, antinuclear antibody (ANA) status, disease duration, or presence of uveitis complications. In JIAU patients, S100 serum levels correlated with age and age at onset of uveitis. A longitudinal analysis in JIAU patients showed a correlation of serum S100A8/A9 and S100A12 levels with uveitis activity (both P ¼ 0.03). S100A8/A9 levels in aqueous humor of patients with JIAU (P ¼ 0.001) and IAU (P ¼ ) were increased as compared to nonuveitic controls. CONCLUSIONS. Increased S100A8/A9 and S100A12 levels are found in the serum and aqueous humor of patients with autoimmune uveitis. Serum levels reflect activity of joint and eye disease. Keywords: juvenile idiopathic arthritis, pathogenesis, monocytes, myeloid-related proteins, S100 proteins, uveitis Juvenile idiopathic arthritis (JIA) represents the most frequent rheumatic disease in childhood. 1 The disease is commonly associated with vision-threatening anterior uveitis. 2 Risk factors for uveitis occurrence include early onset of oligoarthritis and the presence of serum antinuclear antibodies (ANA). 3,4 Juvenile idiopathic arthritis-associated uveitis (JIAU) typically takes a chronic course, with complications developing frequently and limiting visual prognosis. 5 Since the disease is usually asymptomatic, 6 clinically monitoring the course of uveitis according to inflammatory activity and tissue damage constitutes an important challenge. 4,7 The mainstay of treatment is topical corticosteroids, while nonbiological and biological disease-modifying antirheumatic drugs (DMARDs) are used to treat a severe course of the disease. Therapeutic decisions related to duration and discontinuation of immunosuppressive treatment are generally uncertain with respect to the risk of disease relapse. While some statistical data from JIAU cohorts are available to assist with these decisions, individualized factors for predicting the risk of relapse after withdrawal of immunosuppression are required. 8 Reliable biomarkers, therefore, represent highly desirable tools for disease monitoring. S100 protein subtypes have proven to be valuable biomarkers in different subtypes of JIA and for other (auto)inflammatory or autoimmune diseases In humans, the S100 protein family consists of 21 subtypes that share structural similarities and are expressed cell specifically in different cell types, exerting very diverse functions, as reviewed elsewhere As some S100 protein subtypes can be released or even actively secreted from various cells, expression can be demonstrated not only in tissues, but also in body fluids as serum, saliva, or synovial fluid as well, which makes specific subtypes attractive candidates for minimally invasive disease monitoring. 14,17 19 Due to cell specificity, different S100 protein subtypes are clinically established in monitoring a spectrum of very diverse diseases, varying from S100B in melanoma to S100A12 in inflammatory bowel disease. 13,20 Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc. iovs.arvojournals.org j ISSN:

2 S100 Levels in Anterior Uveitis IOVS j December 2015 j Vol. 56 j No. 13 j 7654 The S100 subtypes S100A8 (myeloid-related protein [MRP] 8, calgranulin A), S100A9 (MRP 14, calgranulin B), and S100A12 (calgranulin C) are phagocyte specific and therefore are of interest in phagocyte-mediated inflammatory diseases, as they can be actively secreted upon cell activation. 12 S100A8/ A9 form heterocomplexes (frequently referred to as calprotectin in the literature) and are mainly expressed by monocytes and granulocytes, while S100A12 is an activation marker specific for neutrophils. 10,11 Once secreted from activated phagocytes, S100A8/A9 and S100A12 act as proinflammatory ligands of toll-like receptor (TLR)-4 or RAGE (receptor for advanced glycation end products), triggering inflammatory reactions among other functions. 12 Serum levels of these proteins reflect systemic disease activity status in patients with oligo-, polyarticular, or systemic JIA and facilitate prediction of risk for arthritis relapse after withdrawal of immunosuppressive medication. 9,21,22 One previous study reported elevated levels of serum calprotectin in adult patients with active endogenous posterior uveitis. 23 The current study investigates the value of S100A8/A9 and S100A12 serum and aqueous humor levels as biomarkers in children with noninfectious anterior uveitis. METHODS Patients All patients involved in this study were recruited in the Department of Ophthalmology at St. Franziskus-Hospital Muenster, Germany, during 2012 and The following groups were included: (1) patients with JIAU (n ¼ 79) who all fulfilled the International League of Associations for Rheumatology criteria of oligoarticular JIA 24 ; (2) pediatric patients with idiopathic, noninfectious anterior uveitis that developed in the absence of any inflammatory systemic or ocular disease (n ¼ 24); and (3) samples from nonuveitic controls obtained either from healthy volunteers (n ¼ 8; only serum) or from patients without any inflammatory/ immune-mediated ocular or systemic disease in whom cataract surgery had been performed (serum and/or aqueous humor; n ¼ 16; total number of controls: 24). Age-related macular degeneration was present in one of these patients and nonproliferative diabetic retinopathy in another one. All uveitis patients were 16 years of age at time of disease onset. Written informed consent of patients and/or parents was obtained. The study was performed according to the tenets of the Declaration of Helsinki and was approved by the local ethics committee. A standardized ophthalmic database was used for recording epidemiological patient characteristics and collecting clinical data. Uveitis was defined and anatomically classified according to the Standardization of Uveitis Nomenclature (SUN) Working Group recommendations. 7 Patients were tested for bestcorrected visual acuity (BCVA) and received a slit-lamp examination, Goldmann applanation tonometry, funduscopy, and additional examinations such as fluorescein angiography or optical coherence tomography (OCT), when appropriate. An anterior chamber cell grade 1þ was defined as active uveitis. Arthritis activity was evaluated by a pediatric rheumatologist and patient s evaluation. A specific clinical scoring system for arthritis activity was not applied. Any treatments (topical and systemic anti-inflammatory medication), the presence of uveitis-related complications (e.g., cataract, ocular hypertension, glaucoma, synechiae formation, central band keratopathy, vitreous opacities, macular edema, or macular atrophy), and any prior ocular surgery were documented in accordance with the previously proposed outcome measures. 25 In addition to consultation with a pediatric rheumatologist, laboratory tests (e.g., ANA, rheumatoid factor [RF], and human leukocyte antigen [HLA]-B27) were performed as well as additional chest x-ray, if necessary. Common infectious diseases were ruled out (serological tests for EBV [Epstein-Barr virus], CMV [cytomegalovirus], HSV [herpes simplex virus], VZV [varicella zoster virus], treponema pallidum, and tuberculosis [IFNc release assay]). Patients were classified as having idiopathic uveitis only if all tests were without any findings indicating associated systemic disease. Sample Collection and Laboratory Testing Venous blood samples were obtained and centrifuged at 1500g for 10 minutes within 2 hours after acquisition; serum aliquots were stored at 808C until examination. Aqueous humor samples were obtained during cataract surgery, immediately aliquoted, and stored in sterile tubes at 808C until analysis. Preoperatively, topical corticosteroid treatment was intensified in uveitis patients in order to minimize the risk of inflammatory complications. Surgery was performed only if uveitis was clinically inactive. Concentrations of S100A8/A9 and S100A12 were determined by specific double-sandwich enzyme-linked immunosorbent assay (ELISA) systems as previously described. 21 The readers of the laboratory assay were blinded to the study groups. Serum samples with particularly high S100A8/A9 and S100A12 levels were analyzed for serum potassium (K þ ) and lactate dehydrogenase (LDH) levels. Patients with elevated LDH and/or K þ levels probably related to hemolysis were excluded from the study (n ¼ 4), as hemolysis can nonspecifically elevate the S100 serum concentration. Those patients were therefore not included in our study cohort (serum sample n ¼ 129). All remaining serum samples were tested for C-reactive protein (CRP) to check for systemic infections/inflammation potentially accounting for elevated S100A8/A9 and S100A12 serum levels. In four serum samples from patients with clinically active arthritis, CRP levels were elevated (values between 2.9 and 9.4 mg/dl; normal range, <0.5 mg/dl). These four samples were included in the statistical analysis. Otherwise, CRP serum levels were in the normal range. Statistics Differences in S100A8/A9 and S100A12 levels and clinical characteristics between different patient groups were analyzed by the v 2 test, Fisher s exact test, Wilcoxon matched-pairs test, Mann-Whitney U test, and one-way ANOVA, as appropriate. Correlation of clinical characteristics and S100 levels was analyzed by Pearson s r. 26 P values resulting from multiple intergroup comparisons by ANOVA were corrected for multiple investigations by post hoc Tukey s hones significant difference (HSD). P values reported are uncorrected unless stated otherwise. For statistical analysis, we used SPSS PASWStatistics version 20.0 (SPSS, Inc., Chicago, IL, USA). Graphs were prepared using GraphPad Prism Version 5.0a (GraphPad Software, Inc., San Diego, CA, USA). P < 0.05 was considered statistically significant. Data are expressed as median and mean values 6 standard deviation (SD). RESULTS Study Patients All patient data are displayed in Table 1. Children with JIAU and idiopathic anterior uveitis (IAU) were of similar age. The

3 S100 Levels in Anterior Uveitis IOVS j December 2015 j Vol. 56 j No. 13 j 7655 TABLE 1. General Characteristics of the Study Population JIA-Associated Uveitis Idiopathic Anterior Uveitis Nonuveitic Controls P Value Patients, n Serum samples, n Anterior chamber samples, n Patient age, mean 6 SD * <0.001 Male sex, n (%) 17 (21.5) 5 (20.8) 6 (25) Age at uveitis diagnosis <0.001 Systemic immunosuppression, n (%) 69 (87.3) 14 (58.3) Topical corticosteroids, n (%) 57 (72.2) 18 (75) 1.0 Any ocular complications, n (%) 69 (87.3) 23 (95.8) Any ocular surgery, n (%) 48 (60.8) 11 (45.8) Active uveitis, n (%) 18 (22.8) 6 (25) Active arthritis, n (%) 21 (26.9) Mann-Whitney U test, v 2 test, Fisher s exact test. * Comparison of uveitis groups. Comparison of uveitis groups with nonuveitic controls. Cell grade þ1; according to Jabs et al. 7 (2005) and Heiligenhaus et al. 25 (2012). Active arthritis according to rheumatologist s and patient s assessment. nonuveitic controls were older on average because samples from cataract surgery were included. Most children with JIAU suffered from persistent oligoarthritis (73.4%, and 26.6% from extended oligoarthritis). Antinuclear antibody positivity was observed frequently in the JIAU subgroup (91.1%). In four patients with clinically active arthritis, CRP serum levels were elevated (values between 2.9 and 9.4 mg/dl; normal range, <0.5 mg/dl); the remaining patients had normal CRP values. S100 Serum Levels in the Study Groups In comparison to healthy controls, the S100A8/A9 and S100A12 serum levels were significantly elevated in both uveitis groups (P < 0.05 each; Fig. 1). The S100A8/9 and S100A12 levels correlated well in all study groups (P < 0.001; correlation coefficient 0.929). No significant differences in S100A8/A9 or S100A12 levels were found between JIAU and IAU (Fig. 1). S100 Protein Levels in Different JIAU Subgroups Serum levels of S100A8/A9 were elevated in patients with either clinically active arthritis (not significant, P ¼ 0.289) or active uveitis (P ¼ 0.010), and with both active arthritis and uveitis (P ¼ 0.139; Fig. 2) in comparison to nonuveitic controls. Serum levels were slightly higher even when articular and ocular manifestations were both clinically inactive. S100A8/A9 and S100A12 levels from patients with both clinically active arthritis and uveitis were slightly higher than in patients with completely inactive disease. However, the differences did not reach statistical significance (S100A8/A9 P ¼ 0.469, S100A12 P ¼ 0.978). Serum S100A12 values were elevated in patients with clinically active arthritis or active uveitis and in patients with both active arthritis and uveitis as compared to nonuveitic controls (Fig. 2). S100A12 serum levels were significantly increased when only ocular inflammation was present (P ¼ 0.026), and the levels were higher than in patients with active arthritis. S100 Protein Levels and Uveitis Activity We then determined the mean S100 serum levels in the subgroups of patients with active and inactive uveitis. While significantly higher S100A8/A9 levels were detected in JIAU patients (patients with inactive arthritis only) if uveitis was active, the levels did not differ between IAU patients with active and inactive eye disease. S100A12 did not differ significantly between patients with active or inactive uveitis (Fig. 3). Mean serum S100A8/A9 and S100A12 levels did not vary depending on anterior chamber cell grade (1þ to 3þ). When combining all IAU and JIAU patients without arthritis activity in one uveitis group, we found that mean serum FIGURE 1. S100 proteins in juvenile idiopathic arthritis-associated uveitis (JIAU) and idiopathic anterior uveitis (IAU) as compared to nonuveitic controls. Serum S100 protein levels. Mann-Whitney U test.

4 S100 Levels in Anterior Uveitis IOVS j December 2015 j Vol. 56 j No. 13 j 7656 FIGURE 2. S100 proteins in juvenile idiopathic arthritis-associated uveitis (JIAU) patients with clinically active or inactive disease. Serum S100 protein levels. One-way ANOVA with post hoc Tukey s HSD, P values corrected for multiple comparisons. S100A8/A9 levels were significantly higher in active than in clinically inactive uveitis (mean vs , P ¼ 0.038). S100A12 serum levels did not differ significantly (132.1 vs , P ¼ 0.16). Longitudinal Analysis of Serum S100 Protein Levels During the Course of Uveitis In six JIAU patients who did not present with clinical arthritis activity during the entire follow-up period, repeated serum samples were available for analyzing individual serum S100 levels during uveitis course. While uveitis was inactive at baseline, it recurred afterward. In all six patients, the mean S100A8/A9 and S100A12 serum levels increased in conjunction with uveitis recurrence from a mean of 1040 ng/ml (S100A8/ A9) and 118 ng/ml (S100A12) to 1960 ng/ml (S100A8/A9) and 294 ng/ml (S100A12), respectively (both P ¼ 0.03; Fig. 4). Comparison of Serum S100 Protein Levels With Diverse Clinical and Laboratory Parameters The S100A8/A9 and S100A12 serum levels in patients with ocular complications related to uveitis were higher than in patients without such complications (P > 0.05). S100 serum levels were not significantly different between male and female patients (see Table 2). There was no significant difference of S100A8/A9 or S100A12 values between ANA-positive and -negative uveitis patients. In patients treated with systemic immunosuppressive medication, we found higher serum levels of S100A8/A9 and S100A12 than in patients not on systemic medication, but the differences were not significant either (P ¼ 0.44 and 0.406, see Table 2). In JIAU patients, the S100A8/A9 serum levels increased with age (P ¼ 0.004, Pearson s r ¼ 0.301) or age at uveitis diagnosis (P ¼ 0.03, Pearson s r ¼ 0.232), respectively. This was also observed for S100A12 serum levels (age: P ¼ 0.005, Pearson s r ¼ 0.303; age at uveitis diagnosis: P ¼ 0.009; Pearson s r ¼ 0.283). No such correlation was found for IAU patients or healthy controls (see Table 3). S100 Aqueous Humor Levels In total, we analyzed 45 anterior chamber (AC) samples. Due to the small sample volume, we were only able to measure S100A8/A9 (Fig. 5). In 10 of the 17 JIAU patients (58.8%), in 9 of 12 IAU patients (75%), but in only 1 of 16 nonuveitic controls (6.3%) were the AC S100A8/A9 levels above the detection limit (>5 ng/ml; Fig. FIGURE 3. S100 proteins in juvenile idiopathic arthritis-associated uveitis (JIAU) and idiopathic anterior uveitis (IAU) patients with active or inactive ocular disease. All JIAU patients had clinically inactive arthritis. Serum S100 protein levels. Mann-Whitney U test.

5 S100 Levels in Anterior Uveitis IOVS j December 2015 j Vol. 56 j No. 13 j 7657 FIGURE 4. S100 proteins in juvenile idiopathic arthritis-associated uveitis during individual course of disease. All patients had clinically inactive arthritis. Serum S100 proteins. Matched symbols represent one patient each (n ¼ 6). Wilcoxon matched-pairs test. 5, Table 4). Mean AC S100A8/A9 levels were higher in uveitis patients than in nonuveitic controls (JIAU P ¼ 0.001, IAU P ¼ ). In all JIAU and IAU patients, uveitis was clinically inactive. In JIAU patients (n ¼ 12), the AC S100A8/A9 levels did not vary with arthritis activity. In another 5 JIAU patients, arthritis activity was unknown at the time of sample collection (n ¼ 2 with AC S100A8/A9 levels above detection limit). No correlation was noted between serum and AC S100 levels or between systemic immunosuppressive therapy and AC S100A8/A9 levels. DISCUSSION S100A8/A9 and S100A12 proteins are well-established clinical biomarkers for disease activity in several JIA subtypes like systemic as well as oligo- or polyarticular JIA. 12 Serum S100A8/ A9 and S100A12 levels are helpful for predicting relapse and for making therapeutic decisions, for example, time point for withdrawing methotrexate in patients with clinically inactive JIA, with elevated serum levels reflecting subclinical inflammatory disease activity. 27,28 As some JIA subtypes, especially JIA oligoarthritis, are frequently accompanied by a chronic ocular inflammation, 2 we sought to investigate the potential use of S100 proteins as biomarkers in childhood uveitis. Our data demonstrate that S100A8/A9 and S100A12 levels are elevated in serum and aqueous humor in children with JIAU and in IAU. We observed an association between serum S100A8/A9/A12 levels and uveitis activity. Significantly increased S100A8/A9 levels were found in the aqueous humor in more than half of the JIA uveitis patients, but in only one of the noninflammatory controls. This implies that aqueous humor S100 values reflect intraocular inflammation. Increased aqueous humor values of S100A8/A9 were also found in patients with IAU, and also in patients with herpetic anterior uveitis (data not shown), which suggests that this finding is not a specific marker for JIAU but rather reflects intraocular/local neutrophil and monocyte activation in general. Intraocular expression of S100A8/A9 was probably influenced by the intensive anti-inflammatory treatment (e.g., topical steroids) that all patients received preoperatively. The presence of elevated S100A8/A9 aqueous humor levels in selected uveitis patients despite clinical inactivity suggests a remaining subclinical uveitis activity in the AC that cannot be detected by slit-lamp examination. No constant correlation was found between intraocular and systemic S100 levels. As elevation of serum S100 proteins in JIAU, of course, might be due to active joint inflammation in those children, we analyzed JIAU subgroups according to activity of articular or ocular disease. We found that S100A8/A9 and S100A12 protein levels were markedly elevated not only in patients with clinically active joint inflammation, but also in patients with clinically active uveitis and inactive joint situation as well, with TABLE 2. Clinical Characteristics and S100A8/A9 and S100A12 Serum Levels in Uveitis Patients n (%) Serum S100A8/A9 Serum S100A12 P Value Sex Male 24 (21.4) * Female 88 (78.6) Any ocular complications Yes 101 (90.2) * No 11 (9.8) ANA positivity Yes 86 (90.5) * No 9 (9.5) Immunosuppressive therapy Yes 91 (81.3) * No 21 (18.7) Patients with juvenile idiopathic arthritis-associated uveitis and idiopathic anterior uveitis (total serum sample n ¼ 112). Data are expressed as mean 6 standard deviation. Mann-Whitney U test. * P value serum S100A8/A9. P value serum S100A12.

6 S100 Levels in Anterior Uveitis IOVS j December 2015 j Vol. 56 j No. 13 j 7658 TABLE 3. Correlation of Clinical Characteristics and S100A8/A9 and S100A12 Serum Levels in Uveitis Patients and Healthy Controls P Value Serum S100A8/A9 Pearson s r P Value Serum S100A12 Pearson s r JIAU Age Age at onset of uveitis Age at onset of arthritis Disease duration, uveitis IAU Age Age at onset of uveitis Disease duration, uveitis Controls Age Patients with juvenile idiopathic arthritis-associated uveitis (JIAU, serum sample n ¼ 89), idiopathic anterior uveitis (IAU, n ¼ 23), and healthy controls (n ¼ 17). Pearson s r. mean S100A8/A9 and S100A12 values reaching those observed in active arthritis. This confirms previous observations on those markers as not disease specific, but rather as indicating an ongoing systemic immune response predominantly characterized by activated neutrophils. 9 We can only speculate about the underlying mechanisms of elevated systemic inflammation markers in (clinically) apparently isolated ocular inflammatory activity. Previously, it was shown that JIA patients with clinically inactive arthritis and normal serum CRP levels who display elevated serum S100A8/ A9 or S100A12 levels are at risk for an imminent relapse of articular disease. 21,27 Consequently, subclinical arthritis activity cannot be entirely ruled out in our patients, and we can, in a clinical setting, not definitely prove that elevation of serum S100 proteins in active uveitis is independent from joint disease. Nevertheless, we observe a rise of serum S100 proteins in patients with longstanding inactive joint disease in a situation when uveitis becomes clinically active and no other inflammation or infection is present. We believe that this observation is supporting the assumption that ocular inflammation is reflected by increase in serum S100 levels. Additionally, even children with IAU with an established diagnosis of isolated uveitis without underlying systemic disease show elevated S100A8/A9 and S100A12 serum levels, although there are no clinical signs of systemic inflammation and CRP values are normal. This raises the question about the localization of the S100A8/A9 and S100A12 producing phagocytes. Theoretically, it might be possible that systemically measurable S100 proteins are produced by phagocytes located in the ocular tissues and, due to breakdown of the blood ocular barrier, are washed out into the circulation. This would account for systemically elevated S100A8/A9/A12 levels even in the case of idiopathic uveitis without clinical or laboratory signs of systemic inflammation. Regarding the sparse histopathological data on enucleated eyes from JIAU patients, 29,30 which mainly demonstrated that lymphocytic infiltration outnumbered the granulocytes and monocytes, it seems unlikely that the latter would produce such high levels of protein. As the systemic S100A8/A9 and S100A12 levels in all uveitis subtypes hardly differed between patients with active arthritis of several joints (who would, naturally, display rather high serum levels) and patients with only active inflammation in the rather small eyeball, it seems more likely that elevated S100 serum levels in uveitis patients reflect a subclinical systemic inflammation, with proteins being produced by cells located extraocularly. Data on systemic alterations in adaptive and innate immune cell function or cell subsets in pediatric uveitis are scarce; but it has been shown, for example, that c/dt cells in the synovial fluid of JIA patients are found more frequently if uveitis is present, especially when uveitis is also active. 31 A study from Deschenes et al. 32 found elevated numbers of IL-2 receptor- FIGURE 5. S100 proteins in juvenile idiopathic arthritis-associated uveitis (JIAU, n ¼ 17), idiopathic anterior uveitis (IAU, n ¼ 12) and nonuveitic controls (n ¼ 16). S100A8/A9 levels measured in aqueous humor. Mann-Whitney U test.

7 S100 Levels in Anterior Uveitis IOVS j December 2015 j Vol. 56 j No. 13 j 7659 TABLE 4. S100A8/A9 Protein Levels in the Aqueous Humor of Uveitis Patients and Nonuveitic Controls JIA-Associated Uveitis Idiopathic Anterior Uveitis Nonuveitic Controls P Value Patients, n S100A8/A9 level, ng/ml, mean 6 SD * S100A8/A9 > detection level, n (%) 10 (58.8) 9 (75) 1 (6.3) Topical corticosteroids n.a. Active uveitis 0 0 n.a. Mann-Whitney U test, v 2 test. n.a., not applicable. * JIA-associated uveitis versus control. Idiopathic anterior uveitis versus control. Anterior chamber cell grade 1þ; according to Jabs et al. 7 (2005) and Heiligenhaus et al. 25 (2012). positive T cells in the peripheral blood of patients with autoimmune uveitis with and without underlying systemic disease. However, the patients studied were adults. Additionally, numerous studies have demonstrated systemically circulating autoantibodies presumably produced by extraocularly located plasma cells, and it is known that systemically circulating ANA are a major risk factor for developing uveitis in JIA In line with those findings, our data support the notion of uveitis being an organ-specific manifestation of a systemic immune dysregulation, which can be accompanied by (subclinical) systemic inflammation. This hypothesis was raised previously by Olson et al. 23 reporting on elevated serum calprotectin levels in six adult patients with active endogenous posterior uveitis. Nevertheless, we can only speculate about the pathogenic implications of systemically increased S100 levels in uveitis. Recent data point toward an additional mechanism for the stimulation of cells of the adaptive immune system via TLRs. 39 It has been demonstrated that potentially autoimmunogenic T cells express TLR-4, which is the most important receptor for S100A8/A9 complexes, and a receptor for S100A12 as well. This might explain the finding of elevated S100 proteins as markers of the innate immune system in an intraocular disease that is considered to be driven by cells of the adaptive immune response via antigen-specific inflammatory reactions. 40 It has also been shown that TLR-4 is expressed by several cell types in the ocular tissues, among which uvea (resident APCs, iris endothelial cells) and retina are of particular relevance in the initiation and perpetuation of inflammatory eye diseases. 41 There is some controversy about whether iris endothelial cells express TLR-4 in vivo, but it has been shown that cultured human iris endothelial cells express TLR-4 and respond to lipopolysaccharide (LPS) treatment in vitro. 41,42 It is likely that systemically circulating S100 proteins enter the eye due to breakdown of the blood ocular barrier and participate in perpetuating ocular inflammation by stimulating resident cells. Nevertheless, the role of S100 proteins in the pathogenesis of JIAU is far from clear. In addition to the established proinflammatory role of those proteins, recent data point toward an immunoregulatory function of S100A8/A9. 43 As we examined a rather heterogeneous group of patients regarding state of disease, clinical disease activity, disease duration, and immunosuppressive medication, the pathogenetic relevance of S100 levels in individuals can probably not be estimated by summarizing cohort results. Additionally, it would be desirable to have patients classified according to a standardized disease activity score (e.g., Juvenile Arthritis Disease Activity Score, JADAS) and to quantify uveitis activity as well in order to relate elevated S100 values to extent of disease activity. This was not possible within the limits of our current study because such scoring systems were not applied routinely at the time of patient recruitment, and our subgroup numbers are rather small. Additionally, one has to consider the fact that S100A8/A9 and S100A12 serum levels can vary considerably between individual patients, and even in healthy controls. It seems that there is a broad range of baseline or physiological S100A8/ A9 and S100A12 values, and several factors can lead to elevation of those levels, including (degree of) arthritis activity, number of involved joints, and uveitis activity. To properly establish S100A8/9 or S100A12 as biomarkers in pediatric uveitis, one would have to conduct a controlled study in a much larger cohort, with consideration of the JADAS score for the validation of these results in the next step. Nevertheless, the longitudinal data in our small cohort of JIAU patients with inactive arthritis, but with uveitis flares and concomitant rise of serum S100A8/A9 and S100A12 levels, suggest that a longitudinal monitoring of individual S100 serum profiles might be valuable. Corresponding to the data from Foell et al. 28 on MTX withdrawal in JIA patients, measuring S100A8/ A9 and S100A12 serum levels in clinically inactive uveitis patients might help to assess the risk for a subsequent relapse. Our data suggest a potential role for S100 proteins in autoimmune uveitis. Further investigation of serum protein kinetics during stages of ocular disease activity is required to determine whether they might be useful as biomarkers for ocular inflammation. Acknowledgments We thank Christina Elisabeth Arnold, Dirk Bauer, Martin Busch, Melanie Saers, Susanne Schleifenbaum, Susanne Wasmuth, and Lena Wildschuetz for their technical support during the study. JR has a patent, Method of diagnosis of inflammatory diseases using Calgranulin C (EP ). None of the other authors has a financial or proprietary interest in any of the materials or the method presented. Disclosure: K. Walscheid, None; A. Heiligenhaus, None; D. Holzinger, None; J. Roth, P; C. Heinz, None; C. 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