Pulmonary infection with an interferon-gamma-producing Cryptococcus neoformans strain results in classical macrophage activation and protection

Abstract

Alternative macrophage activation is associated with exacerbated disease in murine models of pulmonary cryptococcosis. The present study evaluated the efficacy of interferon-gamma transgene expression by Cryptococcus neoformans strain H99gamma in abrogating alternative macrophage activation in infected mice. Macrophage recruitment into the lungs of mice after infection with C. neoformans strain H99gamma was comparable with that observed in mice challenged with wild-type C. neoformans. However, pulmonary infection in mice with C. neoformans strain H99gamma was associated with reduced pulmonary fungal burden, increased pulmonary Th1-type and interleukin-17 cytokine production, and classical macrophage activation as evidenced by increased inducible nitric oxide synthase expression, histological evidence of enhanced macrophage fungicidal activity, and resolution of inflammation. In contrast, progressive pulmonary infection, enhanced Th2-type cytokine production, and the induction of alternatively activated macrophages expressing arginase-1, found in inflammatory zone 1, Ym1, and macrophage mannose receptor were observed in the lungs of mice infected with wild-type C. neoformans. These alternatively activated macrophages were also shown to harbor highly encapsulated, replicating cryptococci. Our results demonstrate that pulmonary infection with C. neoformans strain H99gamma results in the induction of classically activated macrophages and promotes fungal clearance. These studies indicate that phenotype, as opposed to quantity, of infiltrating macrophages correlates with protection against pulmonary C. neoformans infection.

Figure 1

Resolution of pulmonary infection with C. neoformans strain H99γ is not associated with increased macrophage recruitment. BALB/c mice received an intranasal inoculum of 1 × 10⁴ CFU of C. neoformans strains H99 or H99γ in 50 μl of sterile PBS. The lungs from each group of mice were excised at days 3, 7, and 14 after secondary inoculation, and the fungal burden was quantified (A). Alternatively, pulmonary leukocytes were enzymatically dispersed and the absolute number of F4/80⁺/CD45⁺ macrophages was quantified by flow cytometry (B). Pulmonary fungal burden data are cumulative of three experiments using five mice per time point. Results are expressed as mean log₁₀ CFU per milliliter of lung homogenate ± SEM. Flow cytometry data are cumulative results of five independent experiments using pooled leukocytes from five mice per experiment. Results are expressed as the absolute number of F4/80⁺/CD45⁺ dual positive cells. Significant decreases were observed in C. neoformans strain H99γ-infected compared with wild-type C. neoformans H99-infected mice. (**P < 0.01).

Figure 2

Mice given an experimental pulmonary infection with C. neoformans strain H99γ or wild-type cryptococci have comparable macrophage recruitment into the lungs during infection. BALB/c mice were given an intranasal inoculum of 1 × 10⁴ CFU of C. neoformans strains H99 or H99γ in 50 μl of sterile PBS. Lungs were excised on days 3, 7, and 14 postinoculation and immediately frozen in OCT medium. Lungs were cryosectioned, and macrophage infiltration was evaluated using immunofluorescence staining with anti-F4/80 antibodies. Nuclei were counterstained with DAPI. Data shown are representative lung sections from three independent experiments (three mice per group and per experiment). Digital photographs show representative areas of lungs: ×20 objective.

Figure 3

Pulmonary infection with C. neoformans strain H99γ, but not wild-type C. neoformans, results in increased iNOS expression by pulmonary macrophages. BALB/c mice were given an intranasal inoculum of 1 × 10⁴ CFU of C. neoformans strains H99 or H99γ in 50 μl of sterile PBS. Lungs were excised on days 3, 7, and 14 postinoculation and immediately frozen in OCT medium. Lungs were cryosectioned, and iNOS was evaluated using immunofluorescence staining with anti-iNOS antibodies. Nuclei were counterstained with DAPI. Data shown are representative lung sections from three independent experiments (three mice per group and per experiment). Digital photographs show representative areas of lungs: ×20 objective.

Figure 4

Mice given an experimental pulmonary infection with C. neoformans show increased expression of Arg1 on lung macrophages that is not observed in C. neoformans strain H99γ-infected mice. BALB/c mice were given an intranasal inoculum of 1 × 10⁴ CFU of C. neoformans strains H99 or H99γ in 50 μl of sterile PBS. Lungs were excised on days 3, 7, and 14 postinoculation and immediately frozen in OCT medium. Lungs were subsequently cryosectioned, and Arg1 expression was evaluated using immunofluorescence staining with anti-Arg1 antibodies. Nuclei were counterstained with DAPI. Data shown are representative lung sections from three independent experiments (three mice per group and per experiment). Digital photographs show representative areas of lungs: ×20 objective.

Figure 5

Pulmonary infection of mice with wild-type C. neoformans strain H99, but not C. neoformans strain H99γ, results in increased CD206 expression on lung macrophages. BALB/c mice were given an intranasal inoculum of 1 × 10⁴ CFU of C. neoformans strains H99 or H99γ in 50 μl of sterile PBS. The lungs were excised on days 3, 7, and 14 postinoculation and immediately frozen in OCT medium. Lungs were subsequently cryosectioned, and CD206 expression was evaluated using immunofluorescence staining with anti-CD206 antibodies. Nuclei were counterstained with DAPI. Data shown are representative lung sections from three independent experiments (three mice per group and experiment). Digital photographs show representative areas of lungs: ×20 objective.

Figure 6

Pulmonary Ym1 expression is increased in mice after intranasal infection with wild-type C. neoformans but is abrogated in C. neoformans strain H99γ-infected mice. BALB/c mice were given an intranasal inoculum of 1 × 10⁴ CFU of C. neoformans strains H99 or H99γ in 50 μl of sterile PBS. Lungs were excised on days 3, 7, and 14 postinoculation and immediately frozen in OCT medium. Lungs were subsequently cryosectioned, and the level of Ym1 expression was evaluated using immunofluorescence staining with anti-Ym1 antibodies. Nuclei were counterstained with DAPI. Data shown are representative lung sections from three independent experiments (three mice per group and per experiment). Digital photographs show representative areas of lungs: ×20 objective.

Figure 7

Real-time PCR analysis of macrophage-enriched populations show enhanced expression of aaMac and caMac transcripts in C. neoformans strains H99- and H99γ-infected mice, respectively. BALB/c mice were given an intranasal inoculation with 1 × 10⁴ CFU of C. neoformans strain H99 or H99γ in 50 μl of sterile PBS. Pulmonary leukocytes were isolated by enzymatic digestion on days 7 and 14 postinoculation, and macrophages were enriched for by positive selection of CD11b⁺ cells. Real-time PCR analyses of total mRNA from macrophage populations were conducted for iNOS, IFN-γ, CD206, Ym1, FIZZ1, ARG, IL-4, IL-13, IL-17, and GAPDH. Bars represent the log₁₀ fold change in gene expression during infection with C. neoformans strain H99γ compared with infection with wild-type C. neoformans strain H99. Data shown are cumulative from three independent experiments using pooled leukocytes of five mice per group per experiment. Significant differences were observed at *P < 0.05; ***P < 0.001.

Figure 8

Lung macrophages are positive for IL-4 and IL-13 protein expression during pulmonary infection with C. neoformans. BALB/c mice received an intranasal inoculum of 1 × 10⁴ CFU of C. neoformans strains H99 or H99γ in 50 μl of sterile PBS. The lungs from each group of mice were excised at days 7 and 14 after secondary inoculation, and a single cell suspension was generated using enzymatic digestion. The leukocytes were stained with anti-mouse F4/80⁺ antibodies, fixed, permeabilized, and incubated with anti-mouse antibodies specific for IL-4 and IL-13 and quantified by flow cytometry. Flow cytometry data are representative results of three independent experiments using pooled leukocytes from five mice per experiment. Results shown are the percentage of leukocytes expressing the indicated surface markers.

Figure 9

Pulmonary infection with C. neoformans H99γ results in rapid clearance of the cryptococci and rapid resolution of inflammation, whereas the lungs of mice challenged with wild-type C. neoformans strain H99 show exacerbated disease and inflammation. Lungs were collected on day 21 postinoculation from uninfected mice and mice inoculated with C. neoformans strains H99 or H99γ, frozen in OCT medium, and processed for histological analysis. Tissue sections were stained with H&E and mucicarmine and examined under a light microscope. Digital photographs show representative areas of lungs from uninfected mice (×10 objective, A), C. neoformans strain H99-infected mice (×10, ×40, and ×100 objectives, B–D), and C. neoformans H99γ-infected mice (×10 and ×40 objectives, E and F). Note widespread intra- and extracellular cryptococci (orange arrowheads, B–D), extended macrophages with proliferating cryptococci and Ym1/Ym2 crystal formation (green arrow [Ym], C), and eosinophilic infiltrate (black arrows [Eo], D) in C. neoformans strain H99-infected lungs. Note limited inflammatory response (E), few internalized yeasts (orange arrowheads, F), and small inclusion bodies (purple arrows, F) consistent with elements of destroyed fungi within the macrophages in C. neoformans strain H99γ-infected lungs.

Figure 10

Pulmonary infection with C. neoformans strain H99γ results in the generation of a predominantly Th1-type and IL-17 cytokine environment in the lungs. BALB/c mice were given an intranasal inoculation with 1 × 10⁴ CFU of C. neoformans strain H99 or H99γ in 50 μl of sterile PBS. Lung homogenates were prepared from lungs excised on days 7 and 14 postinoculation and assayed for IL-4 (A), IL-5 (B), IL-13 (C), IL-2 (D), IL-12 (E), IL-17 (F), and IFN-γ (G) cytokine production. Data are cumulative of two experiments using five mice each. Significant differences were observed at *P < 0.05; **P < 0.01; ***P < 0.001.