Autoantibody epitope spreading in the pre-clinical phase predicts progression to rheumatoid arthritis

Abstract

Rheumatoid arthritis (RA) is a prototypical autoimmune arthritis affecting nearly 1% of the world population and is a significant cause of worldwide disability. Though prior studies have demonstrated the appearance of RA-related autoantibodies years before the onset of clinical RA, the pattern of immunologic events preceding the development of RA remains unclear. To characterize the evolution of the autoantibody response in the preclinical phase of RA, we used a novel multiplex autoantigen array to evaluate development of the anti-citrullinated protein antibodies (ACPA) and to determine if epitope spread correlates with rise in serum cytokines and imminent onset of clinical RA. To do so, we utilized a cohort of 81 patients with clinical RA for whom stored serum was available from 1-12 years prior to disease onset. We evaluated the accumulation of ACPA subtypes over time and correlated this accumulation with elevations in serum cytokines. We then used logistic regression to identify a profile of biomarkers which predicts the imminent onset of clinical RA (defined as within 2 years of testing). We observed a time-dependent expansion of ACPA specificity with the number of ACPA subtypes. At the earliest timepoints, we found autoantibodies targeting several innate immune ligands including citrullinated histones, fibrinogen, and biglycan, thus providing insights into the earliest autoantigen targets and potential mechanisms underlying the onset and development of autoimmunity in RA. Additionally, expansion of the ACPA response strongly predicted elevations in many inflammatory cytokines including TNF-α, IL-6, IL-12p70, and IFN-γ. Thus, we observe that the preclinical phase of RA is characterized by an accumulation of multiple autoantibody specificities reflecting the process of epitope spread. Epitope expansion is closely correlated with the appearance of preclinical inflammation, and we identify a biomarker profile including autoantibodies and cytokines which predicts the imminent onset of clinical arthritis.

Figure 1. The number of elevated autoantibodies and cytokines increase as individuals in the preclinical period approach the clinical diagnosis of RA.

A–C, Mean titers of CCP2 (A), mean total number of ACPAs (B), and mean total number of cytokines (C) were evaluated at each time preclinical timepoint demonstrating a parallel rise in number of ACPA epitopes with rise in anti-CCP2 titer. D, The percent of subjects with elevated levels of each cytokine was evaluated in relation to number of ACPA subtypes present (representative examples of 48 measured cytokines) E, The proportion of subjects positive for each ACPA subtype was evaluated over the preclinical period. The X axis represents days relative to the diagnosis of RA. The Y axis represents the proportion of pre-clinical RA patients with positive value for each marker relative to total number of specimens available for analysis at that timepoint. A–E, Anti-CCP2 antibody titers were measured by CCP2 ELISA (A), ACPA subtypes were measured using a custom multiplex autoantigen bead array, serum cytokine concentrations were measured using commercial bead based multiplex cytokine kits.

Figure 2. Accumulation of ACPA fine specificity before and concurrent with anti-CCP2 antibody seroconversion.

A, Paired SAM analysis was performed on serum specimens from pre-clinical RA patients from whom at least 2 specimens were available prior to anti-CCP2 antibody seroconversion. B, Paired SAM analysis was performed on serum specimens from pre-clinical RA patients for whom a serum specimen was available both prior to and after anti-CCP2 antibody seroconversion. The heatmap represents absolute change in Z-score* from the first to the second timepoint. *Z-score represents the number of standard deviations above or the below the mean level observed in control subjects for each cytokine or autoantibody thus increase in Z-score represents increased change from normal population.

Figure 3. Prediction of imminent RA using multiplex biomarkers.

Multiple logistic regression was performed to identify markers from the reduced set of 21 antibodies and 38 cytokines which could classify pre-clinical RA subjects as being within 2 years of the onset of clinical RA. 5-fold cross validation was performed and common markers selected for final validation. A, Demonstrated is a receiver operating characteristic (ROC) curve using the panel of markers listed in table 3. B–C, Mean and standard deviation of values for each individual autoantibody (B) or cytokine (C) contributing to prediction of imminent onset RA as measured among controls, those RA patients at a timepoint greater than 2 years prior to RA onset, or those within 2 years of clinical RA onset.