AB0063 AMPA CANNOT BE DETECTED IN FAECES OF SEROPOSITIVE RA PATIENTS: NO EVIDENCE FOR LOCAL AMPA PRODUCTION IN THE LOWER GASTRO-INTESTINAL TRACT

Abstract

BackgroundRheumatoid arthritis (RA) patients harbor antibodies against several post-translational modifications (AMPA), for example anti-citrullinated protein antibodies (ACPA) and anti-acetylated protein antibodies (AAPA). The exact mechanism underlying the development of these autoantibodies is currently unclear, but the mucosal immune system has been hypothesized to play a role. ACPA IgA have been detected in sputum and saliva of seropositive RA patients (1-2), suggesting local production of autoantibodies in the lung and oral mucosa. This raises the question whether ACPA can also be produced at other mucosal sites. The intestines are an interesting candidate, as microbiome dysbiosis has been described in RA patients and intestinal content may represent a source of post-translationally modified antigens. However, it is currently unknown whether AMPA are also produced locally in the intestine.ObjectivesTo determine whether AMPA are produced locally in the intestine, we set out to detect ACPA and AAPA in faeces samples of seropositive RA patients. These findings were compared to the ACPA/AAPA levels found in paired serum and saliva samples of the same patients.MethodsPaired faeces, saliva and serum samples of 16 established seropositive RA patients and 16 healthy volunteers were collected. All patients fulfilled the ACR/EULAR 2010 criteria. Saliva was collected using the passive drooling method. Faeces was self-collected by participants at home and immediately frozen. Faeces samples were homogenized in PBS containing protease inhibitors and supernatants were used in ELISA. Saliva was diluted 1:4. Total IgA and anti-E. Coli IgA were measured using commercial and in-house ELISA, respectively. ACPA/AAPA IgA (serum, saliva) and ACPA/AAPA Ig (faeces) were detected using in-house ELISAs, coated with citrullinated and acetylated CCP2 or the unmodified arginine and lysine variants as control. Saliva samples were considered AMPA positive when the value was above the cut-off (mean + 2 times standard deviation of healthy donors) and the delta OD between the modified and unmodified peptide was larger than 0.1.Results9/16 ACPA IgG-positive RA patients were also ACPA IgA positive in serum. In the faecal supernatants total IgA was clearly detectable by ELISA: the supernatants contained on average 85 μg/ml total IgA, only slightly less compared to the mean 117 μg/ml total IgA found in the pre-diluted saliva. However, in none of the faecal supernatants ACPA Ig could be detected. Differences in optical density (OD) between the CCP2 and unmodified arginine peptide were close to 0 (Figure 1A). On the contrary, 8 RA patients were positive for ACPA IgA in saliva (Figure 1B). Similar results were found for AAPA (Figure 1C-D). To control whether the lack of an AMPA signal is explained by technical difficulties associated with measuring IgA in an antigen-specific manner in faeces, we determined anti-E. Coli IgA in the same samples. Anti-E. Coli IgA was clearly detectable in 11/32 faeces samples, indicating intact antigen-specific IgA was present in the faecal samples (Figure 1E).Figure 1.ConclusionNo ACPA or AAPA Ig antibodies could be detected in faeces of RA patients, even though these patients were positive for ACPA/AAPA IgA in both serum and saliva. The fact that it was possible to detect other antibodies in faeces, indicates that the absence of an AMPA signal is not due to inherent methodological issues. These findings indicate that the lower gastro-intestinal tract does not contain measurable levels of AMPA IgA and suggest that it is not a main site of ACPA production in RA patients.