Immune Recognition of Citrullinated Proteoglycan Aggrecan Epitopes in Mice with Proteoglycan-Induced Arthritis and in Patients with Rheumatoid Arthritis

<div><p>Background</p><p>Rheumatoid arthritis (RA) is an autoimmune inflammatory disease affecting the joints. Anti-citrullinated protein antibodies (ACPA) are frequently found in RA. Previous studies identified a citrullinated epitope in cartilage proteoglycan (PG) aggrecan that elicited pro-inflammatory cytokine production by RA T cells. We recently reported the presence of ACPA-reactive (citrullinated) PG in RA cartilage. Herein, we sought to identify additional citrullinated epitopes in human PG that are recognized by T cells or antibodies from RA patients.</p><p>Methods</p><p>We used mice with PG-induced arthritis (PGIA) as a screening tool to select citrulline (Cit)-containing PG peptides that were more immunogenic than the arginine (R)-containing counterparts. The selected peptide pairs were tested for induction of pro-inflammatory T-cell cytokine production in RA and healthy control peripheral blood mononuclear cell (PBMC) cultures using ELISA and flow cytometry. Anti-Cit and anti-R peptide antibodies were detected by ELISA.</p><p>Results</p><p>Splenocytes from mice with PGIA exhibited greater T-cell cytokine secretion in response to the Cit than the R version of PG peptide 49 (P49) and anti-P49 antibodies were found in PGIA serum. PBMC from ACPA+ and ACPA- RA patients, but not from healthy controls, responded to Cit49 with robust cytokine production. High levels of anti-Cit49 antibodies were found in the plasma of a subset of ACPA+ RA patients. Another PG peptide (Cit13) similar to the previously described T-cell epitope induced greater cytokine responses than R13 by control (but not RA) PBMC, however, anti-Cit13 antibodies were rarely detected in human plasma.</p><p>Conclusions</p><p>We identified a novel citrullinated PG epitope (Cit49) that is highly immunogenic in mice with PGIA and in RA patients. We also describe T-cell and antibody reactivity with Cit49 in ACPA+ RA. As citrullinated PG might be present in RA articular cartilage, Cit PG epitope-induced T-cell activation or antibody deposition may occur in the joints of RA patients.</p></div

Antibodies reacting with P13 and P49 peptides in plasma samples from RA patients and HC subjects.

<p>IgG antibodies reacting with Cit or R versions of P13 and P49 in the human plasma samples were detected by ELISA as described in the Methods. Cit:R ratios of (A) anti-P13 and (B) anti-P49 antibodies in plasma samples from RA and HC subjects. ΔOD 450 nm values representing plasma levels of (C) anti-Cit13 and (D) anti-Cit49 antibodies. Data shown are the mean±SEM. Sample numbers for both peptide pairs (RA all n = 46; RA ACPA+ n = 34; RA ACPA- n = 12; HC n = 9). Cit:R ratio of 1.0 is indicated by a dotted line. Statistical analysis was performed using Wilcoxon signed rank test (*p<0.05: Cit:R ratio vs 1.0) and Kruskal-Wallis test followed by Dunn’s multiple comparison test (#p<0.05: ACPA+ group vs ACPA- group).</p

Anti-PG peptide antibodies in serum samples from mice with PGIA or GIA, or from naïve mice.

<p>IgG antibodies reacting with Cit or R versions of 6 PG peptides were measured by ELISA using biotinylated Cit-R peptide pairs. Delta optical density at 450 nm (ΔOD 450 nm) was determined as described in the Methods. (A) Serum antibodies against the Cit versions of six PG peptides in mice with PGIA (red bars) or GIA (blue bars) or naïve mice (gray bars). Data are shown as the mean ΔOD 450 nm±SEM (PGIA n = 10 mice/peptide; GIA n = 3–5 mice/peptide; Naïve n = 3 mice/peptide). Inter-group comparisons were made using Kruskal-Wallis test followed by Dunn’s multiple comparison test (^#p<0.05: PGIA vs naïve mice). (B) Cit:R ratios of peptide-specific serum antibodies from mice with PGIA or GIA and from naïve mice. Data are expressed as Cit:R ratios of ΔOD 450 nm (mean±SEM) of serum antibodies specific for the peptide pairs in the groups of mice described above. Inter-group comparisons were made as described for graph A above (#p<0.05: GIA vs naïve or PGIA vs GIA mice). A Cit:R ratio of 1.0 is indicated by a dotted line. None of the Cit:R ratios were significantly different from 1.0 as determined by Wilcoxon signed rank test.</p

Proliferation of spleen cells from mice with PG-induced arthritis (PGIA) in response to stimulation with peptide pools and individual peptides.

<p>Spleen cells from mice with PGIA were cultured with (A) Cit and R pairs of PG or OVA peptide pools or (B) Cit and R pairs of selected individual peptides (P) in triplicate wells. The Cit:R ratio of SI was calculated as described in the Methods. Data are expressed as the mean of Cit:R ratio of SI±SEM (n = 12 mice). Equal response to the Cit and R version of a peptide pool or peptide (theoretical Cit:R ratio of 1.0, at which no preference for the Cit or R version is assumed) is indicated with a dotted line. Statistical analysis was performed using Wilcoxon signed rank test (*p<0.05: Cit:R ratio vs 1.0) and Kruskal-Wallis test followed by Dunn’s multiple comparison test (#p<0.05: Cit:R ratio of PG13 vs Cit:R ratio of any other peptide pool).</p

Cit:R ratios of cytokine concentrations in culture supernatants of peptide-stimulated spleen cells from mice with PGIA.

<p>Concentrations of cytokines IL-17, IFNγ, IL-6, and IL-10 in 4-day culture supernatants were measured by ELISA. Data are shown as the mean of Cit:R ratios±SEM for the concentrations of (A) IL-17, (B) IFNγ, (C) IL-6, and (D) IL-10. Cit:R ratio of 1.0 is indicated with a dotted line. Wilcoxon signed rank test was used to identify Cit:R ratios significantly higher than 1.0 (preference for a Cit pool or peptide) (*p<0.05; n = 10 mice). Cit:R ratio of the P13 pair-induced vs Cit:R ratio of any other peptide pair-induced production of cytokines in the same cultures was compared using Kruskal-Wallis test followed by Dunn’s multiple comparison test (#p<0.05).</p