Erosive rheumatoid arthritis is associated with antibodies that activate PAD4 by increasing calcium sensitivity

Abstract

Peptidylarginine deiminases (PADs) play a critical role in generating autoantigens in rheumatoid arthritis (RA), but the mechanisms underlying their dysregulation in this disease remain unknown. Although PADs require supraphysiologic concentrations of calcium for activity in vitro, the enzymes are active in vivo (for example, in RA synovial fluid) where calcium concentrations are much lower. We have discovered a subset of anti-PAD4 autoantibodies (identified by their cross-reactivity with PAD3) that markedly increase the catalytic efficiency of PAD4 by decreasing the enzyme's requirement for calcium into the physiologic range. Patients with these PAD3/PAD4 cross-reactive autoantibodies had higher baseline radiographic damage scores and a higher likelihood of radiographic progression compared to individuals negative for these antibodies. The ability of autoantibodies to activate an enzyme that itself generates citrullinated autoantigens identifies an important feed-forward loop, which may drive the erosive outcome observed in RA patients with these autoantibodies. PAD3 autoantibodies may therefore identify RA patients who would benefit from early aggressive treatment or addition of PAD inhibitor therapy.

Figure. 1. PAD3/PAD4 cross-reactive autoantibodies are present in a subset of RA patients

(A) Sera from the ESCAPE RA cohort and convenience samples of RA, psoriatic arthritis, and healthy controls were used to immunoprecipitate IVTT PAD3. “Normalized anti-PAD3 units” were calculated by densitometry and samples with a value ≥0.01 were considered positive. (B) Representative radiographs from 18 anti-PAD4 positive (upper panel) and 18 anti-PAD4 negative (lower panel) ESCAPE RA patient sera are shown. (C) Anti-PAD3/anti-PAD4 positive patient sera were blocked with rPAD3 or rPAD4 prior to performing IVTT PAD3 or IVTT PAD4 immunoprecipitations. (D) The “% IVTT PAD4 IP blocked by recombinant protein” was calculated by densitometry for 10 PAD3/PAD4 positive and 8 PAD4-only patients and the two groups were compared using a two-tailed t-test.

Figure. 2. Patients with cross-reactive antibodies have the most severe and progressive disease

(A) 194 patients in the ESCAPE RA cohort were divided into three groups according to their autoantibody status (Anti-PAD negative, Anti-PAD4 only, or Anti-PAD3/PAD4). The mean total baseline SvdH score for each group was determined and revealed a significant association of PAD3/PAD4 cross-reactive antibodies with Sharp score (Crude). (B) Sharp scores were available on 150 patients at follow-up, with progression depicted as the proportion of patients with any increase in SvdH score from baseline (Crude). These associations were maintained after adjusting for age, gender, RA duration, shared epitope alleles, anti-CCP status, and log CRP levels (Adjusted).

Figure. 3. PAD3/PAD4 cross-reactive antibodies increase the sensitivity of PAD4 to Ca²⁺

PAD4 was pre-incubated with no antibody, anti-PAD4 only (RA34-P4), or PAD3/PAD4 cross-reactive (RA45-XR) IgG prior to use in citrullination assays. (A) Calcium titrations were performed using a sub-saturating concentration of histone H3 (700 nM) and H3 citrullination was determined by immunoblotting. Representative data from 5 independent experiments is shown. (B) The “Fold change in H3 citrullination over no IgG control” was determined by densitometry. (C) H3 was citrullinated at 0.2mM calcium in the presence of 6 cross-reactive IgGs (XR IgG) or 6 PAD4-only IgGs. Representative data from 4 experiments is shown. (D) Citrullination was quantified by densitometry and the mean citrullination of the two groups was compared using a two-tailed t-test.(E) Anti-PAD3 antibodies were depleted prior to use in H3 citrullination assays. H3 citrullination and equal PAD4 and IgG loading were determined by immunoblotting. Representative data from 2 experiments is shown. (F) Specificity of the anti-PAD3 depletion was confirmed by performing IVTT PAD4 or IVTT PAD3 immunoprecipitations.

Figure. 4. Cross-reactive antibodies decrease the calcium-binding cooperativity of PAD4

PAD4 was pre-incubated with no IgG, control IgG, anti-PAD4 only (P4) IgG, or PAD3/PAD4 cross-reactive (XR) IgG prior to use in citrullination assays. (A) Histone H3 titrations were performed at 5 mM calcium and citrullinated H3 was detected by immunoblotting. (B) BAEE titrations were performed at 2mM calcium and citrullination was calculated using an L-citrulline standard curve. The data were fit to the Michaelis-Menten equation. (C) Calcium titrations were performed and data were fit to the Hill equation. Representative data from two experiments P4-IgG (RA27 and RA34), XR-IgG (RA45 and RA16), and no IgG is compiled and shown.

Figure. 5. Cross-reactive antibodies modify accessibility of PAD4 to protease cleavage at D³⁸⁸

(A) IVTT wild type or D³⁸⁸→A mutated PAD4 (filled arrows) was incubated with 0, 5, or 50 nM GrB for 1 hour at 37°C and dominant fragments were detected (open arrows). (B) IVTT PAD4 or the negative control IVTT B23 was pre-incubated with no IgG, PAD4-only IgG (P4), or cross-reactive IgG (XR) prior to incubating with 50 nM GrB. (C) Proteins were visualized by radiography and “% Protection from GrB cleavage” was determined by densitometry. Representative data from 3 independent experiments performed with either 5 nM or 50 nM protease is compiled and shown. A two-tailed t-test was performed and a p-value<0.05 was considered significant.