Card9 Broadly Regulates Host Immunity against Experimental Pulmonary Cryptococcus neoformans 52D Infection

Abstract

The ubiquitous soil-associated fungus Cryptococcus neoformans causes pneumonia that may progress to fatal meningitis. Recognition of fungal cell walls by C-type lectin receptors (CLRs) has been shown to trigger the host immune response. Caspase recruitment domain-containing protein 9 (Card9) is an intracellular adaptor that is downstream of several CLRs. Experimental studies have implicated Card9 in host resistance against C. neoformans; however, the mechanisms that are associated with susceptibility to progressive infection are not well defined. To further characterize the role of Card9 in cryptococcal infection, Card9^em1Sq mutant mice that lack exon 2 of the Card9 gene on the Balb/c genetic background were created using CRISPR-Cas9 genome editing technology and intratracheally infected with C. neoformans 52D. Card9^em1Sq mice had significantly higher lung and brain fungal burdens and shorter survival after C. neoformans 52D infection. Susceptibility of Card9^em1Sq mice was associated with lower pulmonary cytokine and chemokine production, as well as reduced numbers of CD4⁺ lymphocytes, neutrophils, monocytes, and dendritic cells in the lungs. Histological analysis and intracellular cytokine staining of CD4⁺ T cells demonstrated a Th2 pattern of immunity in Card9^em1Sq mice. These findings demonstrate that Card9 broadly regulates the host inflammatory and immune response to experimental pulmonary infection with a moderately virulent strain of C. neoformans.

Keywords: Cryptococcus neoformans; caspase recruitment domain-containing protein 9; innate and adaptive immunity; meningoencephalitis; respiratory infection.

Figure 1

Card9 signaling is required for survival and control of fungal burden after infection with Cryptococcus neoformans 52D. Wild-type (WT) and Card9^em1Sq mice were infected intratracheally with 10⁴ CFU of C. neoformans strain 52D. (A) Mice were observed for up to 36 days for survival analysis (n = 16 mice/strain, using a log-rank test). (B,C) Fungal burden in the lung and brain at serial time intervals was determined by plating tissue homogenates on Sabouraud dextrose agar. CFU data are shown as mean ± SEM and combine at least two independent experiments (n = 9–16 mice /strain/time point). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001.

Figure 2

Histological analysis of C. neoformans pulmonary infection. Wild-type (WT) and Card9^em1Sq mice were infected intratracheally with 10⁴ CFU of C. neoformans 52D. Lungs were harvested at day 21 post-infection; perfused with phosphate-buffered saline; embedded in paraffin; and stained with (A,B) hematoxylin–eosin (H&E), (C,D) periodic acid–Schiff (PAS) or (E,F) mucicarmine. Rectangular boxes correspond to magnified regions (Balb/c; (A,C,E) and Card9^em1Sq; (B,D,F)). Arrows identify findings that are detailed in the text of the manuscript.

Figure 3

Pulmonary inflammatory mediator expression after C. neoformans infection. Wild-type (WT) and Card9^em1Sq whole lung proteins were collected at 14 days post infection with 10⁴ CFU of C. neoformans strain 52D. Milliplex and ELISA (IFN-γ) were performed to determine the level of (A) pro-inflammatory immune mediators, (B) chemokines, (C) Type 3 and (D) Type 2 cytokines. Mediators with levels below detectable levels are not shown. Data is shown as mean ± SEM. * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001.

Figure 4

Pulmonary T-lymphocyte recruitment following C. neoformans infection. Lungs of Card9^em1Sq mice display fewer CD4⁺ and CD8⁺ T lymphocytes during the adaptive phase of immunity after Cryptococcus neoformans 52D infection. Total number of (A) CD3⁺CD4⁺ T lymphocytes and (B) CD3⁺CD8⁺ T lymphocytes in the lungs at 0, 14, 21, and 28 days post-infection. (C) Representative flow plots and (D) percentages of IFN-γ⁺CD4⁺, IL-17A⁺CD4⁺ and IL-13⁺CD4⁺ T cells. (E) percentages of IFN-γ⁺CD8⁺, IL-17A⁺CD8⁺ and IL-13⁺CD8⁺ T cells. Data are shown as mean ± SEM (n = 5–7 mice/strain/time point). * p ≤ 0.05 and ** p ≤ 0.01.

Figure 5

Pulmonary myeloid cell recruitment following C. neoformans infection. Lungs of Card9^em1Sq mice have decreased monocytes, dendritic cells, and neutrophil recruitment and increased alveolar macrophages and eosinophil recruitment to the lungs after Cryptococcus neoformans 52D infection. (A) Absolute numbers of total CD45⁺ cells in the lungs at 0,14, 21 and 28 days post-infection. Total number of (B) alveolar macrophages (CD45⁺CD11c⁺F4/80⁺SiglecF⁺), (C) monocytes (CD45⁺SiglecF⁻CD11b⁺Ly-6G⁻Ly-6C^hi), (D) macrophages (CD45⁺SiglecF⁻CD11b⁺Ly-6G⁻Ly-6C^lo-intCD11c⁻ F4/80⁺) (E) dendritic cells (CD45⁺SiglecF⁻CD11b⁺Ly-6G⁻Ly-6C^lo-intCD11c⁺) (F) neutrophils (CD45⁺SiglecF⁻CD11b⁺Ly-6G⁺Ly-6C^hi) (G) NK cells (CD45⁺SiglecF⁻CD11b⁻NK1.1⁺), and (H) eosinophils (CD45⁺CD11c⁻F4/80^int-hi) in the lungs at 14, 21 and 28 days post-infection. Data are shown as mean ± SEM (n = 5–7 mice/strain/time point). * p ≤ 0.05 and ** p ≤ 0.01.