Background/Purpose: Caspase recruitment domain-containing protein 9 (Card9) is a C-type lectin receptor known for its function in protection against fungal infection and association with human diseases including ankylosing spondylitis and inflammatory bowel disease. Recently, polymorphisms in Card9 were linked to the pathogenesis of a prevalent type of autoimmune arthritis, Rheumatoid arthritis (RA). Here we sought to investigate the role of Card9 in the pathogenesis of arthritis using the genetically susceptible strain of mice (SKG) that develop a T cell-mediated arthritis in response to microbial triggers. Understanding how Card9 affects the pathogenesis of T cell-mediated arthritis will contribute to our understanding of the etiology of RA and development of future therapeutics.

Methods: SKG and Card9^-/-SKG mice housed under specific pathogen-free conditions were intraperitoneally injected with zymosan (1.5 mg) at 6-8 wk age, clinically monitored for arthritis, and analyzed histologically for joint pathology. CD4⁺ T cells from ankle-draining (popliteal) lymph nodes were stimulated in vitro with PMA/ionomycin and production of pro-inflammatory cytokines was quantified by intracellular cytokine staining and flow cytometry. For adoptive T cell transfers, splenic CD4⁺ T cells (4×10⁶) purified from na•ve mice by magnetic positive selection were intravenously injected into lymphopenic nude (nu/nu) recipients who were injected 24h later with zymosan. Three independent experiments were performed (n=5-6 mice/genotype), and data analyzed using non-parametric statistics.

Results: To investigate the role of Card9 in arthritis we created Card9-deficient SKG (Card9^-/- SKG) mice. In stark contrast to SKG mice that develop chronic arthritis following zymosan injection, Card9^-/-SKG mice developed no detectable arthritis. Further examination of the popliteal lymph nodes showed reduced numbers of CD4⁺, CD8⁺, and CD4^–CD8^– T cells and decreased total numbers of pro-inflammatory CD4⁺ T effector cells including; Th17, Th1, and Th22. Although Card9^-/-SKG had reduced T cell responses and no arthritis there was still a substantial induction in Th17, Th22, and Th1 responses relative to na•ve SKG and Card9^-/-SKG mice. Furthermore, Card9-deficiency did not affect the arthritogenic capacity of SKG T cells as adoptive transfer of CD4⁺ T cells from na•ve Card9^-/-SKG or na•ve SKG mice into T cell-deplete nude recipients resulted in equal levels of arthritis. These data indicate that Card9 expression is required for the development of arthritis in SKG mice but not for the induction of a pro-inflammatory T effector response.

Conclusion: These findings identify Card9 as an essential mediator of arthritis induced by environmental microbial β-glucans (zymosan) in SKG mice and imply that Card9 positively regulates the magnitude of pathogenic T cell responses and ankle/joint inflammation in SKG mice. Understanding the immunological mechanism by which Card9 controls arthritis will contribute to our understanding of how the environment influences development of autoimmunity in genetically predisposed individuals.