〔Abstract〕 Abstract Objective To investigate the role of caspase recruitment domain-containing protein 9 (Card9) in As- pergillus fumigatus (A. fumigatus) keratitis and the effect of its deficiency on macrophage resistance to fungal in- fection . Methods ① C57BL/7 mice aged 6 - 8 weeks were selected and the mice pretreated Card9 siRNA and Blank siRNA , respectively , and the expression of Card9 in each group was detected by Western blot and RT-PCR. The corneal epithelium of the mice was scraped away 72 hours later , and A. fumigatus spore suspension was injec- ted into the corneal stroma. The corneal scores were recorded at 1d , 3d , 5d and 7d after infection . The expression of Card9 , nuclear factor κB (NF-κB) , interleukin 1β(IL-1β) , interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) in each group was detected by RT-PCR and immunohistochemical (IHC) . ② Human corneal epithelial cells (HCECs) and human monocytic-leukemia cells (THP-1) in vitro , RT-PCR was used to examine the expres- sion of Card9 gene in the two cells , and a stable cell line of THP-1 cells was constructed using shRNA vectors . The expression of Card9 in the cell line was detected by Western Blot and RT-PCR. The cells were induced into macro- phages and stimulation by A. fumigatus , and the expression of Card9 , NF-κB , IL-1β, IL-6 and TNF-αwas detec- ted by RT-PCR. Results Card9 expression increased in A. fumigatus keratitis , mainly distributed in cytoplasm of immune cells . The expression of Card9 in the cornea of mice treated with Card9 siRNA was significantly reduced . After inhibiting the expression of Card9 gene , the expressions of Card9 , NF-κB , IL-1β, IL-6 and TNF-αsignifi- cantly decreased and the changes of IL-1βwere most significant. In in vitro studies , Card9 exhibited negligible ex- pression in human corneal epithelial cells , contrasting with its pronounced expression in THP-1 cells . After the in- duction of macrophages , Card9 , NF-κB , IL-1β, IL-6 , and TNF-αwere significantly upregulated under the stimu- lation of A. fumigatus . After inhibiting the expression of Card9 , the stimulated expression of these factors was sig- nificantly reduced , with the most notable change observed in IL-1β. Conclusion Card9 is involved in the inflam- matory development and healing process of A. fumigatus keratitis . Card9 deficiency can cause functional impair- ment of macrophages and inhibit the expression of inflammatory factors to a certain extent , in which IL-1βhas the greatest effect.