Enhanced innate immune responsiveness to pulmonary Cryptococcus neoformans infection is associated with resistance to progressive infection

Abstract

Genetically regulated mechanisms of host defense against Cryptococcus neoformans infection are not well understood. In this study, pulmonary infection with the moderately virulent C. neoformans strain 24067 was used to compare the host resistance phenotype of C57BL/6J with that of inbred mouse strain SJL/J. At 7 days or later after infection, C57BL/6J mice exhibited a significantly greater fungal burden in the lungs than SJL/J mice. Characterization of the pulmonary innate immune response at 3 h after cryptococcal infection revealed that resistant SJL/J mice exhibited significantly higher neutrophilia, with elevated levels of inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) and keratinocyte-derived chemokine (KC)/CXCL1 in the airways, as well as increased whole-lung mRNA expression of chemokines KC/CXCL1, MIP-1alpha/CCL3, MIP-1beta/CCL4, MIP-2/CXCL2, and MCP-1/CCL2 and cytokines interleukin 1beta (IL-1beta) and IL-1Ra. At 7 and 14 days after infection, SJL/J mice maintained significantly higher levels of TNF-alpha and KC/CXCL1 in the airways and exhibited a Th1 response characterized by elevated levels of lung gamma interferon (IFN-gamma) and IL-12/IL-23p40, while C57BL/6J mice exhibited Th2 immunity as defined by eosinophilia and IL-4 production. Alveolar and resident peritoneal macrophages from SJL/J mice also secreted significantly greater amounts of TNF-alpha and KC/CXCL1 following in vitro stimulation with C. neoformans. Intracellular signaling analysis demonstrated that TNF-alpha and KC/CXCL1 production was regulated by NF-kappaB and phosphatidylinositol 3 kinase in both strains; however, SJL/J macrophages exhibited heightened and prolonged activation in response to C. neoformans infection compared to that of C57BL/6J. Taken together, these data demonstrate that an enhanced innate immune response against pulmonary C. neoformans infection in SJL/J mice is associated with natural resistance to progressive infection.

FIG. 1.

Fungal burden following C. neoformans infection. Inbred male C57BL/6J and SJL/J mice underwent intratracheal infection with 10⁴ CFU of a serotype D C. neoformans isolate. (A) Fungal burden in the lung was determined at 0, 3, 7, 14, and 28 days after infection. Each point represents the mean ± standard error of the mean of CFU (n = 5; ***, P ≤ 0.001). (B) Fungal burden in the spleen and brain was determined 28 days after infection. Bars represent the means of CFU (n ≥ 4; ***, P ≤ 0.001).

FIG. 2.

Inflammatory response in the lungs of resistant and susceptible mice following intratracheal C. neoformans infection. Photomicroscopy of PAS-stained lung sections from individual C57BL/6J (A, C, E, and G) or SJL/J (B, D, F, H) inbred mice that were uninfected (A and B) or underwent intratracheal infection with 10⁴ CFU of a serotype D C. neoformans isolate for 7 (C and D), 14 (E and F), and 28 (G and H) days. Goblet cells and mucus stain dark pink (magnification, ×100). Each image is representative of n = 3 mice/group/time point.

FIG. 3.

Early release of airway cytokines and chemokines following C. neoformans lung infection. The secretion of TNF-α (A), KC/CXCL1 (B), and IL-6 (C) in the BALF of C57BL/6J and SJL/J mice was measured at 3 h and 24 h after intranasal administration of 10⁶ CFU of a serotype D C. neoformans isolate. Each bar represents the mean ± standard error of the mean (n ≥ 8 mice/group/time point; *, P < 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).

FIG. 4.

Analysis of airway cytokine/chemokine expression in C57BL/6J and SJL/J mice at 7 and 14 days after C. neoformans lung infection. The levels of TNF-α (A), KC/CXCL1 (B), IL-6 (C), IFN-γ (D), IL-12/IL-23p40 (E), and IL-4 (F) were measured in the BALF of C57BL6J and SJL/J mice that had been infected with a serotype D C. neoformans isolate for 7 or 14 days. Each bar represents the mean ± standard error of the mean (n ≥ 5 mice/group/time point; *, P < 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ND, not detected). (G) RT-PCR analysis of lung IFN-γ mRNA expression in C57BL/6J and SJL/J mice at 7 days after infection with a serotype D C. neoformans isolate. Amplification of mouse β-actin was performed as a control.

FIG. 5.

Macrophage inflammatory mediator release following in vitro stimulation with C. neoformans. Alveolar (A and C) and resident peritoneal macrophages (B and D) isolated from C57BL/6J and SJL/J mice were stimulated or not with a serotype D C. neoformans isolate at an MOI of 5:1. Supernatants were collected 24 h later and assayed by ELISA for TNF-α (A and B) and KC/CXCL1 (C and D) production. Each bar represents the mean ± standard error of the mean of triplicate determinations (**, P ≤ 0.01; ***, P ≤ 0.001; ND, not detected). Results shown are representative of three independent experiments.

FIG. 6.

Regulation of macrophage TNF-α production in response to stimulation with C. neoformans by PI3K and NF-κB. (A) TNF-α production by resident peritoneal macrophages from C57BL/6J and SJL/J mice stimulated with a serotype D C. neoformans isolate at an MOI of 5:1 for 6 and 24 h. (B and C) Roles of NF-κB and PI3K/Akt-dependent signaling in the activation of resident peritoneal macrophages by C. neoformans. Macrophages from C57BL/6J (B) and SJL/J (C) mice were pretreated for 45 min with inhibitors of PI3K (LY294002, 50 μM) and NF-κB (BAY11-7082, 10 μM) and then stimulated with C. neoformans at an MOI of 5:1 for 24 h. Supernatants were collected and assayed for TNF-α production by ELISA. Data are the means ± standard error of the means of triplicate determinations (*, P < 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). Results are representative of three independent experiments.

FIG. 7.

Macrophage signal transduction induced by C. neoformans infection. Activation of NF-κB and the PI3K-dependent kinase Akt. (A) Resident peritoneal macrophages from C57BL/6J and SJL/J mice were stimulated or not with a serotype D C. neoformans isolate at an MOI of 5:1 for increasing durations (0, 15, 30, 60, and 300 min). Cells were lysed, and the total and phosphorylated forms of Akt and NF-κB were determined by immunoblotting with specific antibodies. To confirm equal protein loading, the membranes were reprobed with anti-α-tubulin antibody. (B and C) Relative quantification of Akt and NF-κB phosphorylation. Bands were digitized, quantified, and expressed in arbitrary units as the ratio between the phosphorylated and total forms of each protein. (D) Alveolar macrophages from C57BL/6J and SJL/J mice were stimulated (+) or not stimulated (−) with a serotype D C. neoformans isolate at an MOI of 5:1 for 1 hour. Cells were lysed and analyzed for phospho-Akt and the phospho-p65 subunit of NF-κB. (E) Relative quantification of phospho-Akt and phospho-p65 NF-κB was performed as described above.