Analysis of the interplay between digestive microbiota and immune system in the development of rheumatoid arthritis – BactRiA

Background: Recent investigations have suggested a mucosal origin of rheumatoid arthritis (RA) because of its remarkable association with several mucosal inflammatory conditions. These findings are consistent with the hypothesis of a cross-reaction between auto-immune antibodies directed at both microbial and auto-antigens. However, the primary target of RA-specific auto-antibodies in vivo, in particular with respect to intestinal microbes, is as yet unknown.
As part of a SNSF-funded project, Partner 1 (P1) has established a sizable serum and stool sample collection from a cohort of individuals at risk for developing RA. Currently, ~300 stool and serum samples have been collected, with the final target set to obtain 350 to 400 samples at the end of 2021.

Hypothesis: We hypothesize that the onset of RA is preceded by dysbiosis, mucosal inflammation, and an aberrant immune response against mucosal bacteria resulting in the production of pathologic auto-antibodies. There is strong evidence to indicate that human and bacterial glycans might be good candidates as cross-reactive structures targeted by RA-specific auto-antibodies.

Objective: An integrative and comprehensive analysis of the adaptive immune responses of RA at risk individuals against commensal intestinal bacteria.

Design: A longitudinal, nested case-control, study in a population consisting of:
1) Asymptomatic individuals “control” group (n = 120), seronegative for the presence of rheumatoid factor (RF) and anti-citrullinated protein/peptide antibodies (ACPA).
2) “intermediate-risk”, asymptomatic, but ACPA or RF seropositive individuals (n = 50).
3) “high-risk”, symptomatic, untreated, individuals with inflammatory arthralgia (n = 50).

Methodology: The project is divided between 4 work-packages (WP).
WP1: Sample preparation and 16S sequencing. Standardized preparation of stool samples (n = 220). Standardized 16S sequencing of pre- and post-sorting fecal bacterial fractions targeted by (or coated with) IgA and/or IgG antibodies, retrived from workflow in WP2. 16S-related bio-informatics and identification of immunologically relevant bacteria.
WP2: Secretory & Systemic Immunomics. Using state of the art flow-cytometry to analyze fecal microbiota suspensions and isolate bacteria strongly coated with IgA. Additional assessment of serum IgG reactivity against autologous microbiota and cultured bacterial species, as well as isolation of IgG-coated bacteria. Complementary assessment of the reactivity of monoclonal antibodies (mAbs: n >100), generated from RA patients’ B cells, against whole microbiota and cultured species. Profiling of potential bacterial cross-reactivity at the clonal level after sorting and sequencing (in collaboration with WP1 &
WP3).
WP3: Glycomics. Profiling of the overall glycan-specific IgA and IgG serum repertoires using pooled serum from different risk-groups on printed glycan micro-arrays. Creation of a multiplex glycan bead array, for at least 30 immune-relevant glycans, and extended profiling of glycan-specific reactivity of all individual sera, as well as a panel of ~100 selected RA-patient derived mAbs. Comparison of glycan-reactivity profiles obtained from RA-patients’ mAbs versus those of at-risk individuals.
WP4: Integrated data analysis. Integrative data management and extended analysis of the various components of the anti-bacterial immune response against commensal intestinal bacteria and cross-reactive glycans (WP1-3), in relation to the development of autoimmunity.

Novelty: Comprehensive analysis and and multidisciplinary approach to characterize bacteria targeted by adaptive immune reactions in early RA development, permitting the identification of RA-associated biomarkers pertinent to early disease diagnosis.