Background/Purpose: Axial spondyloarthritis (axSpA) is a type of inflammatory arthritis that primarily affects the entheses of the spine and/or sacroiliac joints. The cause of axSpA is unknown but pathogenetic CD4+ T cells that produce the proinflammatory cytokine IL-17 (Th17s) drive disease. Genome-wide association studies identified single nucleotide polymorphism rs4077515 (CARD9 S12N) as a risk variant for axSpA. CARD9 is an adaptor protein essential in protection against systemic candidiasis, but its role in axSpA pathogenesis is unknown. We use autoimmune-predisposed SKG mice, who develop Th17-mediated axSpA disease following a single exposure to fungal ß-glucan, to study axSpA pathogenesis. We previously demonstrated that ß-glucan-activated neutrophils signal through CARD9 to induce Th17 expansion and the onset of arthritis in SKG mice. Here we investigated a role for the CARD9 S12N mutation in controlling CARD9-NFkB signaling within neutrophils and neutrophil-mediated Th17 responses.

Methods: Neutrophils and CD4+ T cells were isolated from naïve CARD9 S12N or wildtype (WT) C57BL/6 mice. Neutrophils were stimulated with ß-glucan and lysed for RNA or cocultured with TCR-activated splenic CD4+ T cells. After 24 hours, coculture supernatants were collected and IL-17A was quantified by ELISA. Il12b, Tnfa, and Card9 transcripts were quantified by RT-qPCR. N=1-4 mice/experiment/genotype. Statistical significance was calculated by multiple unpaired parametric student T tests.

Results: Our previous studies showed that loss-of-function in CARD9 (CARD9^-/-) protects against neutrophil-driven arthritogenic Th17 responses in SKG mice. To determine how the CARD9 S12N mutation affects neutrophil responses, WT and CARD9 S12N neutrophils were activated for 1 hour with ß-glucan and CARD9 signaling was assessed. CARD9 S12N neutrophils had increased expression of Card9 mRNA and downstream NFkB-mediated transcripts (Il12b and Tnfa) compared to WT neutrophils. These data indicate that CARD9 S12N results in a gain-of-function (GOF) in canonical CARD9 signaling in neutrophils. To determine the impact of the proposed CARD9 S12N GOF mutation on neutrophil-mediated Th17 expansion, we cocultured ß-glucan-activated WT or CARD9 S12N neutrophils with CD4+ T cells for 24 hours and measured IL-17A production. We found that T cells cocultured with CARD9 S12N neutrophils produced double the amount of IL-17A compared to T cells cocultured with WT neutrophils, indicating that the CARD9 S12N mutation enhances Th17 expansion.

Conclusion: Our study suggests the axSpA-associated CARD9 S12N mutation increases CARD9 signaling downstream of ß-glucan and enhances neutrophil-mediated Th17 expansion. Understanding how neutrophil-intrinsic CARD9 controls axSpA pathogenesis will elucidate novel neutrophil and T cell targets for future therapies.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-axial-spondyloarthritis-risk-variant-card9-s12n-enhances-neutrophil-mediated-th-responses/