Background/Purpose: Caspase recruitment domain-containing protein 9 (CARD9) is an adaptor protein downstream of C-type lectin receptor signaling within myeloid cells. CARD9 is critical for anti-fungal immunity; yet also linked to inflammatory diseases including ankolysing spondylitis and Crohn’s Disease. Here, we investigated the role of Card9 in autoimmunity as modeled in SKG mice, which develop T cell-mediated arthritis in response to microbial triggers.

Methods: SKG and Card9^-/-SKG mice,were injected with zymosan (1.5 mg), clinically monitored for arthritis, and histologically analyzed for joint pathology. At 8 wk post-zymosan, CD4⁺ T cells from draining lymph nodes were stimulated in vitro with PMA/ionomycin and Th effector subsets were quantified by intracellular cytokine staining and flow cytometry. For adoptive T cell transfers, splenic CD4⁺ T cells purified from naïve mice by immunomagnetic selection were intravenously injected (4×10⁶) into nude (nu/nu) recipients who were injected 24h later with zymosan. For lavage studies, peritoneal fluid collected 4h post-zymosan was analyzed for cellular (ie. neutrophils) and non-cellular (ie. chemokine/cytokine) content by flow cytometry and ELISA. Neutrophils were depleted in SKG mice with anti-Ly6G (1A8) or isotype control (2A3) beginning 24h prior to zymosan and every 2d for 11d. For all studies, three independent experiments were performed (n=5-6 mice/genotype), and data analyzed using non-parametric statistics.

Results: After zymosan injection, SKG mice developed chronic arthritis while Card9^-/-SKG mice developed no detectable arthritis. Card9^-/-SKG mice had reduced numbers of T cells in pLN including reduced CD4⁺ T effector cells (Th17, Th1, Th22), and decreased IL-17A in synovial fluid. The mechanism by which Card9 suppresses arthritis is T cell-extrinsic since Card9-deficient and –sufficient T cells had similar abilities to induce arthritis. Given the known anti-fungal role for Card9 within myeloid cells we examined acute myeloid cell responses to zymosan. Within 4h of zymosan injection Card9^-/-SKG mice had dramatically reduced proportions of peritoneal neutrophils, whereas the dendritic cells and macrophage responses were comparable in Card9^-/-SKG vs. SKG mice.   Neutrophil chemoattractants (CXLC1, CXCL2) or inflammatory cytokines (IL-1b, IL-6, IL-23) levels in the peritoneum were not altered by Card9-deficiency. However, Card9-deficient neutrophils had significant reduction in activation status (CD62L) and in their ability to undergo degranulation as determined by mobilization of secondary (CD66b) and primary granules (CD63). Neutrophil depletion during the priming stage delayed arthritis onset.

Conclusion: These data reveal a previously unconsidered role for Card9-signaling within neutrophils in response to environmental triggers in conferring susceptibility to arthritis in SKG mice.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-card9-neutrophil-signaling-axis-is-critical-to-induction-of-th17-mediated-arthritis-in-skg-mice/