Background/Purpose: Caspase recruitment domain-containing protein 9 (CARD9) is an intracellular signal transduction molecule that mediates antimicrobial responses following activation of C-type lectin receptors (ie. Dectin-1) by fungal ligands including β -glucans (zymosan). CARD9-deficient patients have defects in Th17 immunity and succumb to systemic candidiasis, and single nucleotide polymorphisms in CARD9 have been associated with susceptibility to Th17-mediated autoimmune diseases including ankylosing spondylitis. However, how dysregulation of CARD9 may regulate autoimmunity remains unknown. Here we sought to investigate the role of CARD9 in arthritis and spondylitis (spinal inflammation) as modeled in SKG mice.

Methods: Arthritis was induced in SKG vs. Card9^-/- SKG mice by intraperitoneal injection of 1.5mg β-glucan (zymosan) and evaluated clinically for 8 weeks, by near-infrared imaging, and histologically. 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 lavage studies, peritoneal fluid collected 4h post-zymosan was analyzed for immune cellular composition 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 2 d for 5 d. For all studies, three independent experiments were performed (n=5-6 mice/genotype), and data analyzed using non-parametric statistics.

Results: In stark contrast to SKG mice that developed chronic arthritis and features of spondylitis by 8 weeks post zymosan, Card9^-/-SKG mice were completely protected from both diseases. Cell composition analysis of the ankle-draining lymph nodes at both 5 d and 8 wk post-zymosan revealed reduced numbers of neutrophils and effector Th17 responses in Card9^-/-SKG mice. CARD9 promoted acute (pre-arthritic) neutrophil responses in SKG mice as Card9^-/-SKG mice had reduced numbers of neutrophils, but not dendritic cells or macrophages, in lavage fluid as early as 4 h post-zymosan. Furthermore, neutrophils from Card9^-/-SKG lavage fluid had similar surface expression of Dectin-1 to neutrophils from SKG mice, yet had decreased activation status (retention of CD62L) and reduced degranulation of primary (CD63) and secondary (CD66b) granules. These data suggest that Card9 may function downstream of Dectin-1 ligation to promote activation and degranulation of neutrophils. Akin to Card9^-/-SKG mice, neutrophil-deplete SKG mice had delayed onset of arthritis and decreased Th17 responses 5 d post-zymosan, supporting a pathogenic function for early neutrophil responses in induction of arthritis. Cumulatively, these data support an integral role for Card9 in regulation of early neutrophil responses that promote Th17 cells and induction of arthritis.

Conclusion: These data show a direct link between Card9 signaling and neutrophil-intrinsic induction of autoreactive Th17 responses in arthritis. Further investigation of how Card9 functions within neutrophils to drive autoimmunity may serve to unveil novel therapeutic avenues for human Th17-mediated diseases including ankylosing spondylitis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/card9-promotes-th17-mediated-arthritis-and-spondylitis-via-control-of-acute-pathogenic-neutrophil-responses-in-skg-mice/