Defect of CARD9 leads to impaired accumulation of gamma interferon-producing memory phenotype T cells in lungs and increased susceptibility to pulmonary infection with Cryptococcus neoformans

Abstract

Caspase recruitment domain-containing protein 9 (CARD9) is an adaptor molecule signal that is critical for NF-κB activation and is triggered through C-type lectin receptors (CLRs), which are pattern recognition receptors that recognize carbohydrate structures. Previous studies have reported that Cryptococcus neoformans, a fungal pathogen that causes meningoencephalitis in AIDS patients, is recognized through some CLRs, such as mannose receptors or DC-SIGN. However, the role of CARD9 in the host defense against cryptococcal infection remains to be elucidated. In the present study, we analyzed the role of CARD9 in the host defense against pulmonary infection with C. neoformans. CARD9 gene-disrupted (knockout [KO]) mice were highly susceptible to this infection, as shown by the reduced fungal clearance in the infected lungs of CARD9 KO mice, compared to that in wild-type (WT) mice. Gamma interferon (IFN-γ) production was strongly reduced in CARD9 KO mice during the innate-immunity phase of infection. Reduced IFN-γ synthesis was due to impaired accumulation of NK and memory phenotype T cells, which are major sources of IFN-γ innate-immunity-phase production; a reduction in the accumulation of these cells was correlated with reduced CCL4, CCL5, CXCL9, and CXCL10 synthesis. However, differentiation of Th17 cells, but not of Th1 cells, was impaired at the adaptive-immunity phase in CARD9 KO mice compared to WT mice, although there was no significant difference in the infection susceptibility between interleukin 17A (IL-17A) KO and WT mice. These results suggest that CARD9 KO mice are susceptible to C. neoformans infection probably due to the reduced accumulation of IFN-γ-expressing NK and memory phenotype T cells at the early stage of infection.

FIG 1

C. neoformans infection in CARD9 KO mice. WT and CARD9 KO mice were infected intratracheally with C. neoformans. The lung/body weight ratio was measured and the number of live colonies in lungs was counted on day 14 after infection. Each column represents the mean and SD for eight mice. Experiments were repeated twice with similar results. *, P < 0.05.

FIG 2

Effect of CARD9 deficiency on the histological changes after infection with C. neoformans. WT and CARD9 KO mice were infected intratracheally with C. neoformans. Sections of lungs on day 14 postinfection were stained with H-E or PAS and observed under a light microscope at magnifications of ×200 (A) and ×1,000 (B). Representative pictures from three mice are shown. (C) The lung leukocytes prepared on day 14 postinfection were stained with Diff-Quick and observed under a light microscope. Representative pictures from three mice are shown. Magnification, ×200. The number of cells in each leukocyte fraction was counted. Each column represents the mean and SD for three mice. NS, not significant; *, P < 0.05.

FIG 3

Production of IFN-γ and iNOS after infection with C. neoformans. WT and CARD9 KO mice were infected intratracheally with C. neoformans. (A) IFN-γ production in lungs was measured at various time points. Data are means and SD for five or six mice. Experiments were repeated twice with similar results. *, P < 0.05. (B) Expression of iNOS mRNA in lungs was measured on days 3 and 7. Data are means and SD for five mice. Experiments were repeated twice with similar results. NS, not significant; *, P < 0.05.

FIG 4

Role of early produced IFN-γ on the host defense against cryptococcal infection. (A) WT and CARD9 KO mice were infected intratracheally with C. neoformans, and the number of live colonies in lungs was counted on day 5 after infection. Data are means and SD for five mice. Experiments were repeated twice with similar results. *, P < 0.05. (B) WT mice were infected intratracheally with C. neoformans and injected intraperitoneally with anti-IFN-γ MAb or control rat IgG at 200 μg/mouse 2 h before infection and intratracheally with the same Ab at 25 μg/mouse when they were infected. The number of live colonies in lungs was counted on day 14 after infection. Data are means and SD for five mice. Experiments were repeated twice with similar results. *, P < 0.05; NS, not significant.

FIG 5

Early producers of IFN-γ after infection with C. neoformans. WT and CARD9 KO mice were infected intratracheally with C. neoformans, and the lung leukocytes were prepared on day 3 postinfection. (A) IFN-γ expression in NK, NKT, γδ T, and T cells was analyzed using flow cytometry. Gray areas show isotype-matched IgG; solid lines correspond to anti-IFN-γ MAb. Representative histograms from five WT and four CARD9 KO mice are shown. (B) The number of each subset expressing IFN-γ was calculated. Data are means and SD for five WT and four CARD9 KO mice. Experiments were repeated twice with similar results. NS, not significant; *, P < 0.05.

FIG 6

IFN-γ production by memory phenotype T cells. WT and CARD9 KO mice were infected intratracheally with C. neoformans, and the lung leukocytes were prepared on day 3 postinfection. (A) Expression of IFN-γ in CD4⁺ or CD8⁺ naive and memory phenotype T cells, identified as CD44^dull+ and CD44^bright+ cells, was analyzed in a using flow cytometry. Representative histograms from three WT mice are shown. (B) The number of each subset expressing IFN-γ was calculated. Data are means and SD for three mice. Experiments were repeated twice with similar results. MPT cells, memory phenotype T cells. NS, not significant; *, P < 0.05.

FIG 7

Effect of CARD9 deficiency on the chemokine production. (A) WT and CARD9 KO mice were infected intratracheally with C. neoformans. Expression of CCL3, CCL4, CCL5, CXCL9, and CXCL10 in lungs was measured on day 3. Each column represents the mean ± SD of five mice. (B) BM-DCs were cultured with C. neoformans (MOI, 1) or LPS (1 μg/ml) for 24 h, and expression of CCL3, CCL4, and CCL5 was measured. Data are means and SD from triplicate cultures. Experiments were repeated twice with similar results. NS, not significant; *, P < 0.05.

FIG 8

Effect of CARD9 deficiency on the Th1/Th17-related response. WT and CARD9 KO mice were infected intratracheally with C. neoformans. (A) Expression of IL-12p35, IL-6, TGF-β, and IL-23p19 on day 3 and of T-bet, ROR-γt, IFN-γ, and IL-17A on day 7 was measured in the lung homogenates by real-time PCR. Data are means and SD for five mice. (B) LN cells obtained on day 7 postinfection were stimulated with indicated doses of C. neoformans or ConA (1 μg/ml) for 48 h, and production of IFN-γ and IL-17A was measured. Data are means and SD of triplicate cultures. Experiments were repeated twice with similar results. NS, not significant; *, P < 0.05.

FIG 9

Effect of IL-17A deficiency on the clearance of C. neoformans in lungs. WT and IL-17A KO mice were infected intratracheally with C. neoformans. The number of live colonies in lungs was counted on day 14 after infection. Data are means and SD for six WT and seven IL-17A KO mice. Experiments were repeated three times with similar results. *, P < 0.05.