Role of dendritic cells and alveolar macrophages in regulating early host defense against pulmonary infection with Cryptococcus neoformans

Abstract

Successful pulmonary clearance of the encapsulated yeast Cryptococcus neoformans requires a T1 adaptive immune response. This response takes up to 3 weeks to fully develop. The role of the initial, innate immune response against the organism is uncertain. In this study, an established model of diphtheria toxin-mediated depletion of resident pulmonary dendritic cells (DC) and alveolar macrophages (AM) was used to assess the contribution of these cells to the initial host response against cryptococcal infection. The results demonstrate that depletion of DC and AM one day prior to infection results in rapid clinical deterioration and death of mice within 6 days postinfection; this effect was not observed in infected groups of control mice not depleted of DC and AM. Depletion did not alter the microbial burden or total leukocyte recruitment in the lung. Mortality (in mice depleted of DC and AM) was associated with increased neutrophil and B-cell accumulation accompanied by histopathologic evidence of suppurative neutrophilic bronchopneumonia, cyst formation, and alveolar damage. Collectively, these data define an important role for DC and AM in regulating the initial innate immune response following pulmonary infection with C. neoformans. These findings provide important insight into the cellular mechanisms which coordinate early host defense against an invasive fungal pathogen in the lung.

FIG. 1.

Lung CD11c-expressing cells are comprised of DC and AM at baseline and in response to pulmonary infection with C. neoformans. WT (C57BL/6) mice were either uninfected (day 0) or infected (i.t.) with 10⁴ C. neoformans organisms. Single-cell suspensions were generated following enzymatic digestion of lungs harvested at days 0, 4, and 7 postinfection and assessed by flow cytometric analysis. (A) Representative scatter plots of lung leukocytes obtained at day 0 (uninfected; top panels) or day 7 (postinfection; bottom panels). A gating strategy (see Materials and Methods) was used to identify large cells expressing CD11c⁺ (left scatter plots; gate R1) as either AM (right scatter plots; AM; CD11c⁺/CD11b⁻) or DC (right scatter plots; DC; CD11c⁺/CD11b⁺). (B) Kinetics of the accumulation of CD11c⁺ cells, including AM and DC subsets, in the first week following infection with C. neoformans. Absolute numbers of cells were obtained by multiplying the frequency of a cell population by the total number of lung leukocytes recovered. (n = 3 to 6 mice from three separate experiments; error bars, SEM; *, P < 0.05 versus day 0 CD11c⁺ cells; **, P < 0.01 versus day 0 DC [using analysis of variance with Dunnet's post hoc analysis]).

FIG. 2.

Depletion of resident DC and AM markedly impairs survival following pulmonary infection with C. neoformans. WT or Tg(CD11c-DTR) mice were injected (i.p.) with DT (5 ng/g BW). (A) Representative histograms displaying the CD11c⁺ population present in the lungs at 24 h following DT administration to WT (left panel) or Tg(CD11c-DTR) (right panel) mice. (B) In separate experiments, WT or Tg(CD11c-DTR) mice were infected (i.t.) with 10⁴ C. neoformans (Cneo) organisms at 24 h following saline (sham) or DT (i.p.) administration. Data represent survival at the indicated days postinfection for three treatment groups (n = 6 to 9 mice per group from three separate experiments; P < 0.05 versus WT + sham + Cneo [*] and WT + DT + Cneo [**] by log rank analysis).

FIG. 3.

Depletion of DC and AM does not alter pulmonary microbial load or serum cryptococcal antigen levels following pulmonary infection with C. neoformans. WT or Tg(CD11c-DTR) mice were injected (i.p.) with either saline (sham) or DT (5 ng/g BW) and infected (i.t.) with 10⁴ C. neoformans organisms 24 h later. At 4 days postinfection, lungs and serum were harvested and assessed for pulmonary microbial load (by CFU assay; n = 6 to 9 mice per group from three separate experiments) (A) or serum antigen levels (by antigen agglutination assay; n = 3 mice per group) (B). No significant differences were observed between treatment groups (by unpaired Student t test).

FIG. 4.

Depletion of DC and AM does not alter the absolute number of lung leukocytes at 4 days following pulmonary infection with C. neoformans. WT or Tg(CD11c-DTR) mice were injected (i.p.) with either saline (sham) or DT (5 ng/g BW) and infected (i.t.) with 10⁴ C. neoformans organisms 24 h later. At 4 days postinfection, lungs were harvested and leukocytes were enumerated by visual inspection (n = 6 to 9 mice per group from three separate experiments; error bars, SEM; P < 0.05 versus WT + sham + Cneo [*] and WT + DT + Cneo [**] by unpaired Student t test).

FIG. 5.

Depletion of DC and AM enhances the accumulation of lung neutrophils and B cells following pulmonary infection with C. neoformans. WT or Tg(CD11c-DTR) mice were injected (i.p.) with either saline (sham) or DT (5 ng/g BW) and infected (i.t.) with 10⁴ C. neoformans organisms 24 h later. At 4 days postinfection, lungs were harvested and the following lung leukocyte populations were assessed: PMNs and eosinophils (assessed by visual identification) (A) and lung lymphocytes (assessed by flow cytometric analysis as CD4⁺ T cells, CD8⁺ T cells, or B220⁺ B cells) (B). Total cell numbers were obtained by multiplying the frequency of each population by the total cell count (n = 6 to 9 mice per group from three separate experiments; error bars, SEM; P < 0.05 versus WT + sham + Cneo [*] and WT + DT + Cneo [**] by unpaired Student t test).

FIG. 6.

Depletion of DC and AM in the absence of cryptococcal infection results in minimal lung pathology. Tg(CD11c-DTR) mice were injected (i.p.) with DT (5 ng/g BW). Lungs were harvested thereafter and assessed by light microscopy. Representative photomicrographs (hematoxylin and eosin stained) of lungs removed 1 day (A, magnification of ×100; B, magnification of ×200) and 6 days (C, magnification of ×100; D, magnification of ×200) after DT administration are shown. Note the relatively preserved architecture of both airways and alveoli and the absence of inflammation.

FIG. 7.

Depletion of DC and AM results in diffuse lung inflammation following pulmonary infection with C. neoformans. WT (A, C, and E) or Tg(CD11c-DTR) (B, D, and F) mice were injected (i.p.) with DT (5 ng/g BW) and infected (i.t.) with 10⁴ C. neoformans organisms 24 h later. At 5 days postinfection, lungs were harvested and the morphological pattern of inflammation was assessed by light microscopy. Representative photomicrographs (hematoxylin and eosin stained) of lungs taken from WT (A, C, and E) and Tg(CD11c-DTR) (B, D, and F) mice are shown (A and B, magnification of ×100; C and D, magnification of ×200; E and F, magnification of ×400). Note that the magnitude of inflammation appears to be similar in both lungs (A and B). However, in WT [compared with Tg(CD11c-DTR)] mice, the airways appear to be spared (C versus D; AW), inflammation is more organized, and adjacent alveolar architecture is preserved (E versus F; arrows identify approximate boundaries between foci of inflammation and alveolar structures).

FIG. 8.

Depletion of DC and AM results in distinct patterns of lung damage following pulmonary infection with C. neoformans. Tg(CD11c-DTR) mice were injected (i.p.) with DT (5 ng/g BW) and infected (i.t.) with 10⁴ C. neoformans organisms 24 h later. At 5 days postinfection, lungs were harvested and the morphological pattern of inflammation was assessed by light microscopy. Representative high-power photomicrographs (hematoxylin and eosin stained; magnification, ×400) demonstrating three distinct patterns of lung pathology are shown. (A) Neutrophilic bronchopneumonia; note the foci of inflammation within the airway (AW) containing numerous neutrophils, individual cryptococci, and exudative debris. (B) Cyst formation; note the numerous extracellular cryptococci within the cyst (Cy) and the protein-rich (pink) exudate surrounded by neutrophils and macrophages immediately superior to the cyst. (C) Alveolar exudates containing protein and hemorrhage; note the protein-rich (pink) exudates and evidence of parenchymal hemorrhage (arrows identify collections of red blood cells).