Identification of novel autoantigens as potential biomarkers in juvenile idiopathic arthritis associated uveitis

Abstract

Background: Many children with juvenile idiopathic arthritis (JIA) have autoantibodies, targeting nuclear components (anti-nuclear antibodies, ANA). ANA in JIA is associated with uveitis, an eye inflammation which may cause permanent vision impairment if not detected and treated. However, ANA-testing is neither specific nor sensitive enough to be a clinically reliable predictor of uveitis risk, and the precise autoantigens targeted by ANA in JIA are largely unknown. If identified, specific autoantibodies highly associated with uveitis could be used as biomarkers to facilitate identification of JIA patients at risk.

Methods: Antibodies from six ANA-positive, oligoarticular JIA patients, with and without uveitis, were explored by two large-scale methods: (1) screening against 42,100 peptides on an autoimmunity profiling planar array, and (2) immunoprecipitations from cell lysates with antigen identification by mass spectrometry. Three hundred thirty-five peptide antigens, selected from proteins identified in the large-scale methods and the scientific literature were investigated using a bead-based array in a cohort of 56 patients with oligoarticular- or RF-negative polyarticular JIA, eight of which were having current or previous uveitis.

Results: In the planar array, reactivity was detected against 332 peptide antigens. The immunoprecipitations identified reactivity towards 131 proteins. Only two proteins were identified by both methods. In the bead-based array of selected peptide antigens, patients with uveitis had a generally higher autoreactivity, seen as higher median fluorescence intensity (MFI) across all antigens, compared to patients without uveitis. Reactivity towards 17 specific antigens was significantly higher in patients with uveitis compared to patients without uveitis. Hierarchical clustering revealed that patients with uveitis clustered together.

Conclusion: This study investigated autoantigens in JIA and uveitis, by combining two exploratory methods and confirmation in a targeted array. JIA patients with current or a history of uveitis had significantly higher reactivity towards 17 autoantigens and a generally higher autoreactivity compared to JIA patients without uveitis. Hierarchical clustering suggests that a combination of certain autoantibodies, rather than reactivity towards one specific antigen, is associated with uveitis. Our analysis of autoantibodies associated with uveitis in JIA could be a starting point for identification of prognostic biomarkers useful in JIA clinical care.

Keywords: anti nuclear antibody (ANA); autoantibodies; autoantigen; biomarker; juvenile idiopathic arthiritis; juvenile idiopathic arthiritis associated uveitis; uveitis.

Figure 1

Schematic methodology. (A) JIA plasma is analyzed for reactivity towards 42,100 peptide antigens on a glass slide in a planar array. (B) JIA plasma is incubated with whole cell protein extracts, proteins captured by JIA IgG are immunoprecipitated (IP) and identified by mass spectrometry. (C) Hits from A and B are coupled to beads and reactivity analyzed in serum from a cohort of 56 patients with oligoarticular- or RF-negative polyarticular JIA, 8 of which with current or history of uveitis, to identify uveitis-related autoantigens.

Figure 2

Peptides and proteins detected in large-scale screenings and selection for bead array. (A) Flow chart of number of PrEST antigens analyzed on planar array in each pool after quality control (QC) exclusions. (B) Venn diagram of PrEST antigen hits in the planar array in the two plasma pools. (C) Venn diagram of proteins precipitated by samples from patients in the two plasma pools. (D) Venn diagram of the 335 peptides included in bead array, based on source for selection.

Figure 3

JIA patients with uveitis have increased autoreactivity. (A) Histogram of distribution of MFI across all 335 antigens in the bead array, grouped by uveitis. (B) Reactivity towards the 17 antigens which were significantly associated with uveitis in the bead array, presented as MFI values. Each dot represents a patient.

Figure 4

JIA patients with uveitis cluster together based on their autoreactivities. Hierarchical clustering analysis of reactivity towards the 17 peptides significantly different between patients with and without uveitis. Hierarchical clustering is based on MFI distances. Bottom row indicates uveitis (0 = no, 1 = yes).