Background/Purpose: The gut is a key mucosal tissue that can impact the immune system and contribute to systemic inflammation in the setting of increased intestinal permeability. The levels of serum zonulin, a surrogate marker of intestinal permeability, have been reported to be increased in patients with rheumatoid arthritis (RA) and pre-onset RA. In a model mice of RA increased zonulin was associated with immune cell migration from the gut to the synovium. We postulated that gut permeability may be a driver of pathogenic B cells involved in the pathogenesis of human RA.

Methods: Zonulin levels were measured by ELISA in sera from 17 healthy donors (HD) and 21 patients with RA. Paired PBMCs were analyzed by flow cytometry. Naïve B cells (IgD+ and CD27-) were classified into resting (rNAV, CD11c- and CD21+) and activated (acNAV, CD11c+ and CD21-). Among IgD- antigen-experienced B cells, autoimmunity/age-associated B cells (ABC) were defined as CD27-, CD11c+ and CD21-. Antibody-secreting cells (ASC) were defined as CD27++ and CD38++. Chemokine receptor expression was examined as a read-out of cell migration potential, CCR9 for gut-homing and CXCR3 for migration to inflammatory tissues. We also looked at the expression of those chemokine receptors in single cell analysis data from the Accelerating Medicines Partnership Program: Rheumatoid Arthritis and Systemic Lupus Erythematosus (AMP RA/SLE) Network.

Results: Our RA cohort was characterized by long-standing seropositive disease (70% duration > 5 years; 82 % positive for anti-CCP antibodies). RA had significantly higher levels of serum zonulin than HD, and this was inversely correlated with CCR9+ and CCR9+ IgA+ ASC. ABC and acNAV were significantly more abundant in RA than HD (ABC, 0.9% vs 0.35%, p < 0.05; acNAV, 0.78% vs 0.56%, p < 0.05), but their frequencies were not correlated with zonulin.
CXCR3 and CCR9 were exclusively expressed on IgD- subsets (CXCR3% on IgD- 15% vs rNAV 0.78%, CCR9% 7.0% vs 0.69%). Notably, CXCR3 and CCR9 expression was mutually exclusive, and their frequencies were inversely correlated in RA (p = 0.066, Spearman’s correlation test). Compared to CCR9+ B cells, both T-bet+ and IgG+ B cells were significantly enriched in CXCR3+ B cells, while IgA+ B cells were more enriched in CCR9+ B cells. Despite the low frequencies (0.73%), CCR9+ CXCR3+ B cells primarily comprise IgA+ CD11c+ T-bet+ B cells. When synovial B cells from AMP RA/SLE Network were analyzed, CCR9 expression was notably absent in most of the B cell subsets, but CXCR3 expression was enriched in ABCs.  0.05

Conclusion: Our study implicates increased gut permeability and ABC expansion in long-standing RA. Dichotomous phenotypes of CXCR3+ and CCR9+ in antigen-experienced B cells may reflect different developmental pathways and migration propensity. Given there is little evidence on the role of IgA+ B cells in RA, the ongoing studies are underway to assess the functional difference between IgG+ and IgA+ B cells in the synovium. We will also examine clonal relationships between B cell populations in the blood and synovium to capture the dynamics of their migration.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/dichotomous-expression-of-cxcr3-and-ccr9-and-relationship-to-intestinal-permeability-in-b-cells-in-rheumatoid-arthritis/