Background/Purpose: Anti-citrullinated protein antibodies (ACPA) and inflammation characterized by neutrophil extracellular trap (NET)osis have been detected at mucosal sites such as the lung and periodontium in individuals at-risk for RA. We postulated that the intestine is an additional site for inflammation and ACPA generation in subjects at-risk for future RA.  

Methods: Stool was collected from 16 healthy controls, 13 subjects with early RA (eRA) (<1 year), and 34 subjects at-risk for the future development of RA. At-risk subjects included individuals without inflammatory arthritis at or prior to the study visit and were first-degree relatives (FDRs) of RA probands (10 serum CCP+ and 9 CCP-) or non-FDRs who were CCP+ (N=15). Stool was tested by ELISA for CCP3 IgA, total IgA, myeloperoxidase (MPO)-DNA NET remnants, and calprotectin. Isotype-specific antibody coating of stool bacteria was determined by flow cytometry. The murine collagen-induced arthritis (CIA) model was treated with broad-spectrum antibiotics after induction of disease (day 21) to interrogate the microbiome’s role in stimulating intestinal inflammation and autoantibodies. Antibodies to collagen (CII) were measured by ELISA, and tissue neutrophils were assessed by flow cytometry.

Results:  Stool IgA CCP3 was detected in 30% of at-risk subjects and 38% of eRA, and was significantly increased in serum CCP3 IgG+ FDRs, while those in the eRA group demonstrated significantly increased IgA-coating of bacteria in the stool samples compared to the other groups (Table 1). Neutrophil-driven mucosal inflammation was present in at-risk subjects, reflected by high calprotectin and NET-remnant levels. MPO-containing NET remnants, which are reported to be induced by bacteria, significantly positively correlated with stool IgA CCP3 (r=0.3, P=0.004). In mice with CIA, antibiotic treatment significantly reduced disease severity >95%, neutrophil recruitment to the intestine, and mucosal anti-CII IgA. Mucosal anti-CII IgA significantly correlated with serum anti-CII IgG (r=0.33, P=0.03).

Conclusion: These data implicate a role of intestinal microbiota and inflammation in the induction of mucosal ACPA in at-risk subjects and anti-CII antibodies in CIA. Local neutrophil-associated inflammation strongly correlates with the generation of ACPA IgA in the intestine of at-risk subjects. In eRA, IgA coating of bacteria is increased, suggesting that the earlier generation of intestinal ACPA may lead to increased targeting of specific bacteria. The CIA model confirms the necessity of bacteria in driving the mucosal autoantibody response as mice depleted of bacteria using antibiotics resulted in reduced neutrophils and autoantibodies. While more specific mechanisms need to be defined, we propose that intestinal mucosal responses in at-risk individuals are significantly involved in the progression towards RA in a subset of individuals.