Evidence of Site-Specific Mucosal Autoantibody Secretion in Rheumatoid Arthritis

Abstract

Objective: Anti-citrullinated protein antibodies (ACPA) have been detected in sputum and saliva, indicating that anti-modified protein antibodies (AMPA) can be produced at mucosal sites in patients with rheumatoid arthritis (RA). However, the body's largest mucosal compartment, the gut, has not yet been examined. We therefore investigated the presence of several AMPA (ACPA, anti-carbamylated protein antibodies [anti-CarP], and anti-acetylated protein antibodies [AAPA]) at different mucosal sites, including the intestinal tract.

Methods: Paired fecal/ileal wash, saliva, and serum samples of patients with RA and healthy volunteers were collected in two independent cohorts. Data involving feces were replicated in a third cohort. In these secretions, AMPA were analyzed using in-house enzyme-linked immunosorbent assay with unmodified peptides as control. In fecal samples, total IgA and anti-Escherichia coli IgA were measured.

Results: ACPA, anti-CarP, and AAPA IgA were measurable in saliva of seropositive patients with RA (prevalence 9%-40%). No AMPA could be detected in feces. IgA was present because total IgA and anti-E. coli IgA were detectable in feces of ACPA-positive patients with RA and healthy donors. Results were confirmed in another cohort using colonoscopically collected ileal wash samples.

Conclusion: Our study shows the presence of ACPA, anti-CarP, and AAPA IgA in saliva of ACPA-seropositive patients with RA. However, no AMPA could be detected in feces/ileal wash samples of these patients, although our assays were able to measure other antigen-specific antibodies. These data suggest that mucosal autoantibody secretion may occur in the oral mucosa of patients with RA, whereas no evidence could be found for this process in the lower gastrointestinal tract.

Figure 1

Autoantibody measurements in saliva. (A) ACPA IgA in saliva in the MUCOSA study, (B) ACPA IgA in saliva in the IntestRA study, and (C) anti‐CarP IgA, (D) AAPA IgA, and (E) RF IgA in saliva in the MUCOSA study are shown. Delta OD (difference in OD between modified peptide and unmodified peptide) for AMPAs and aU/mL (arbitrary units per milliliter) for RF are depicted. Groups on x axis are based on diagnosis and seropositivity (panel E uses RF IgM seropositivity to define groups). The number (%) of positive patients for that specific autoantibody is given. AAPA, anti–acetylated protein antibody; ACPA, anti–citrullinated protein antibody; AMPA, anti–modified protein antibody; CarP, carbamylated protein; IBD, inflammatory bowel disease; MUCOSA, MUCosal Origin of Serum Autoantibodies in rheumatoid arthritis; OD, optical density; RA, rheumatoid arthritis; RF, rheumatoid factor.

Figure 2

Paired OD values on the modified and unmodified peptide for each AMPA in saliva (A–C) and serum (D–F) of patients and healthy donors in the MUCOSA study and in saliva (G) of patients and controls in the IntestRA study. (A–C) First group of each graph shows all seropositive patients with RA who tested positive for that specific AMPA in saliva, and the second group depicts all seropositive patients with RA who tested negative for that specific AMPA in saliva. (D–F) First group shows patients who were positive for that specific AMPA IgA in serum, and the second group shows patients negative for that specific AMPA IgA in serum. AAPA, anti–acetylated protein antibody; ACPA, anti–citrullinated protein antibody; AMPA, anti–modified protein antibody; CarP, carbamylated protein; IBD, inflammatory bowel disorder; MUCOSA, MUCosal Origin of Serum Autoantibodies in rheumatoid arthritis; OD, optical density; RA, rheumatoid arthritis.

Figure 3

Autoantibody profile in serum (S) and saliva (Sa) in the MUCOSA study. Positivity for autoantibodies in serum and saliva is shown. Each row depicts a study participant. Black field indicates no saliva available due to hyposalivation. AAPA, anti–acetylated protein antibody; ACPA, anti–citrullinated protein antibody; CarP, carbamylated protein; MUCOSA, MUCosal Origin of Serum Autoantibodies in rheumatoid arthritis; RA, rheumatoid arthritis; RF, rheumatoid factor.

Figure 4

AMPA Ig in feces and ileal lavage of patients with RA. (A–C) ACPA, anti‐CarP, and AAPA Ig, respectively, in feces from patients and healthy donors in the MUCOSA study are shown. Groups on x axis are based on diagnosis and seropositivity. (D–F) AMPA Ig in feces of the Plants for Joints cohort is shown. (G–J) Total IgA levels in μg/mL (G and I) and OD (H and J) on the anti–E. coli IgA ELISA in the same feces samples. (K) ACPA IgA in ileal lavage samples from the IntestRA study is shown. (L) Total IgA levels and (M) anti–E. coli IgA OD in the same ileal lavage samples are shown. For AMPAs, the number (%) of positive patients is given. For AMPAs, the y axis depicts difference in OD (delta OD) between the modified and unmodified peptide. Red bars show the median and interquartile range. *P < 0.05. AAPA, anti–acetylated protein antibody; ACPA, anti–citrullinated protein antibody; AMPA, anti–modified protein antibody; CarP, carbamylated protein; ELISA, enzyme‐linked immunosorbent assay; IBD, inflammatory bowel disease; MUCOSA, MUCosal Origin of Serum Autoantibodies in rheumatoid arthritis; OA, osteoarthritis; OD, optical density; RA, rheumatoid arthritis. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/doi/10.1002/art.43036/abstract.

Figure 5

(A–G) Paired OD values on the modified and unmodified peptide for each AMPA Ig in feces of patients and controls in the MUCOSA study (A, C, E) and in the Plants for Joints trial (B, D, F) and ACPA IgA in ileal wash in patients and controls in the IntestRA study (G). AAPA, anti–acetylated protein antibody; ACPA, anti–citrullinated protein antibody; AMPA, anti–modified protein antibody; CarP, carbamylated protein; IBD, inflammatory bowel disease; MUCOSA, MUCosal Origin of Serum Autoantibodies in rheumatoid arthritis; OA, osteoarthritis; OD, optical density; RA, rheumatoid arthritis.