Protective immunity against pulmonary cryptococcosis is associated with STAT1-mediated classical macrophage activation

Abstract

Experimental pulmonary Cryptococcus neoformans infection in BALB/c mice is associated with polarized Th2-type cytokine production, alternative macrophage activation, and severe bronchopneumonia. In contrast, pulmonary infection with a C. neoformans strain that secretes IFN-γ, H99γ, elicits Th1-type cytokine production and classical macrophage activation. Additionally, mice infected with H99γ resolve the acute infection and are subsequently protected against challenge with wild-type C. neoformans. The present study characterizes macrophage activation during the protective response to wild-type C. neoformans in mice previously immunized with H99γ. We observed increased pulmonary Th1-type cytokine production in lung homogenates and classical macrophage activation as evidenced by enhanced expression of inducible NO synthase in the lungs of H99γ-immunized mice compared with mice given a nonprotective immunization with heat-killed C. neoformans (HKCn). Furthermore, macrophages isolated from H99γ-immunized mice on day 7 postchallenge and cultured in vitro were fungistatic against C. neoformans, whereas cryptococcal growth was uncontrolled within macrophages from HKCn-immunized mice. Th2-type cytokine production and induction of alternatively activated macrophages were also observed in lungs of HKCn-immunized mice during rechallenge. Gene expression arrays showed that classical macrophage activation during challenge infection in H99γ-immunized mice was associated with induction of the transcription factor STAT1 and its downstream targets IFN regulatory factor-1, suppressor of cytokine signaling-1, CXCL9, and CXCL10. These studies demonstrate that protective responses to C. neoformans challenge in immunized mice include classical macrophage activation and enhanced macrophage fungistasis of C. neoformans yeasts. Finally, the classical activation phenotype of protective anticryptococcal macrophages is likely mediated via STAT1 signal transduction pathways.

FIGURE 1.

Th1-type cytokine production is elicited in the lungs of C. neoformans strain H99γ-immunized mice upon rechallenge with wild-type C. neoformans. BALB/c mice received an intranasal inoculum of 1 × 10⁴ CFU C. neoformans strain H99γ or HKCn in 50 μl sterile PBS. Thirty-five days later, mice received an intranasal inoculation with 1 × 10⁴ CFU C. neoformans strain H99. The lungs of each group of mice were excised at days 3, 7, and 14 after secondary inoculation and fungal burden was quantified (A) or cytokine production measured (B–D) in pulmonary homogenates. Fungal burden data are representative of three separate studies with five mice per group. Cytokine data are cumulative of three experiments using five mice per group. Fungal burden results are expressed as means ± SEM log₁₀ CFU per milliliter of lung homogenate. *p < 0.05, mice immunized with C. neoformans strain H99γ compared with HKCn-immunized mice (data are reported as means ± SEM).

FIGURE 2.

Macrophages within the lungs of H99γ-immunized mice responding to a secondary cryptococcal challenge display a classical activation phenotype. BALB/c mice received an intranasal inoculum of 1 × 10⁴ CFU C. neoformans strain H99γ or HKCn in 50 μl sterile PBS. Thirty-five days later, mice were intranasally infected with 1 × 10⁴ CFU C. neoformans strain H99. Lungs were excised on days 3, 7, and 14 postinoculation and immediately frozen in OCT medium. Lungs were subsequently cryosectioned and F4/80 (A), iNOS (B), Arg1 (C), CD206 (D), and Ym1 (E) expressions were evaluated using immunofluorescence staining. 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. Original magnification ×20.

FIGURE 3.

Real-time PCR analysis of macrophage-enriched populations show enhanced expression of transcripts indicative of classical macrophage activation during infection in H99γ-immunized mice. BALB/c mice received an intranasal inoculum of 1 × 10⁴ CFU C. neoformans strain H99γ or HKCn in 50 μl sterile PBS. Mice were subsequently infected with 1 × 10⁴ CFU C. neoformans strain H99 by intranasal inhalation or not challenged (UC). Pulmonary leukocytes were isolated from lung tissues by enzymatic digestion on days 1, 3, 7, and 14 postinoculation and macrophages were enriched for by positive selection of CD11b⁺ cells. Real-time PCR analyses of total mRNA from macrophage-enriched populations were conducted for iNOS, IL-17, and IFN-γ (A) and for Arg1, CD206, FIZZ1, Ym1, IL-4, and IL-13 (B). Bars represent the fold change in gene expression in macrophages from H99γ-immunized mice compared with nonprotected mice. Data shown are cumulative of three independent experiments utilizing pooled leukocytes of five mice per group per experiment and are reported as means ± SEM. *p < 0.05.

FIGURE 4.

Macrophages derived from H99γ-immunized during secondary challenge mice are fungistatic and produce more NO. BALB/c mice received an intranasal inoculum of 1 × 10⁴ CFU C. neoformans strain H99γ or HKCn in 50 μl sterile PBS. Mice were subsequently infected with 1 × 10⁴ CFU C. neoformans strain H99. Pulmonary leukocytes were isolated from lung tissues on days 3 and 7 postinoculation and macrophages enriched by positive selection of CD11b⁺ cells. Macrophages were lysed and plated on YPD agar for CFU determination (0 h). Additionally, macrophages were cultured in complete medium for 24 h, lysed, and subsequently plated on YPD agar for CFU determination (A). Culture supernatants were also used to determine NO production using the Griess reagent (B). Data are cumulative of three experiments utilizing three mice per group (*p < 0.05, **p < 0.01, ***p < 0.001) and are reported as means ± SEM.

FIGURE 5.

STAT1 signaling cascade is upregulated in macrophages from infected H99γ-immunized mice. BALB/c mice were intranasally immunized with 1 × 10⁴ CFU C. neoformans strain H99γ or HKCn in 50 μl sterile PBS. Thirty-five days later, mice were subsequently infected with1 × 10⁴ CFU C. neoformans strain H99. Pulmonary leukocytes were isolated from lung tissues by enzymatic digestion on days 1, 3, and 7 postinoculation and macrophages were enriched for by positive selection using CD11b⁺ magnetic beads. Real-time PCR arrays of total mRNA from macrophage-enriched populations were conducted. STAT1, CXCL9, CXCL10, IRF-1, SOCS-1, and SOCS-3 (A) are downstream of the IFN-γR, Jak1, and Jak2 (B). Bars represent the fold change in gene expression in macrophages from H99γ-immunized mice compared with nonprotected mice. Data shown are cumulative of three independent experiments utilizing pooled leukocytes of five mice per group per experiment and are reported as means ± SEM. *p < 0.05. HK, Heat-killed.

FIGURE 6.

STAT1 phosphorylation is increased in macrophages from H99γ-immunized mice during cryptococcal rechallenge. BALB/c mice were intranasally immunized with 1 × 10⁴ CFU C. neoformans strain H99γ or HKCn in 50 μl sterile PBS. Thirty-five days later, mice were intranasally infected with 1 × 10⁴ CFU C. neoformans strain H99. The lungs from each group of mice were excised on days 1, 3, and 7 postinoculation, and pulmonary leukocytes were isolated from lung tissues by enzymatic digestion and macrophages were enriched for by positive selection using CD11b⁺ magnetic beads. Total protein was extracted from cell lysates, resolved by SDS-PAGE, and blotted onto a polyvinylidene difluoride membrane. Blots were probed with an mAb against phosphorylated STAT1, then sequentially stripped and reprobed for total STAT1 and β-actin proteins. Data shown are representative of three independent experiments utilizing pooled leukocytes of five mice per group per experiment. HK, Heat-killed.