Neither classical nor alternative macrophage activation is required for Pneumocystis clearance during immune reconstitution inflammatory syndrome

Abstract

Pneumocystis is a respiratory fungal pathogen that causes pneumonia (Pneumocystis pneumonia [PcP]) in immunocompromised patients. Alveolar macrophages are critical effectors for CD4(+) T cell-dependent clearance of Pneumocystis, and previous studies found that alternative macrophage activation accelerates fungal clearance during PcP-related immune reconstitution inflammatory syndrome (IRIS). However, the requirement for either classically or alternatively activated macrophages for Pneumocystis clearance has not been determined. Therefore, RAG2(-/-) mice lacking either the interferon gamma (IFN-γ) receptor (IFN-γR) or interleukin 4 receptor alpha (IL-4Rα) were infected with Pneumocystis. These mice were then immune reconstituted with wild-type lymphocytes to preserve the normal T helper response while preventing downstream effects of Th1 or Th2 effector cytokines on macrophage polarization. As expected, RAG2(-/-) mice developed severe disease but effectively cleared Pneumocystis and resolved IRIS. Neither RAG/IFN-γR(-/-) nor RAG/IL-4Rα(-/-) mice displayed impaired Pneumocystis clearance. However, RAG/IFN-γR(-/-) mice developed a dysregulated immune response, with exacerbated IRIS and greater pulmonary function deficits than those in RAG2 and RAG/IL-4Rα(-/-) mice. RAG/IFN-γR(-/-) mice had elevated numbers of lung CD4(+) T cells, neutrophils, eosinophils, and NK cells but severely depressed numbers of lung CD8(+) T suppressor cells. Impaired lung CD8(+) T cell responses in RAG/IFN-γR(-/-) mice were associated with elevated lung IFN-γ levels, and neutralization of IFN-γ restored the CD8 response. These data demonstrate that restricting the ability of macrophages to polarize in response to Th1 or Th2 cytokines does not impair Pneumocystis clearance. However, a cell type-specific IFN-γ/IFN-γR-dependent mechanism regulates CD8(+) T suppressor cell recruitment, limits immunopathogenesis, preserves lung function, and enhances the resolution of PcP-related IRIS.

FIG 1

Blockade of classical or alternative macrophage polarization does not affect Pneumocystis clearance or development of anti-Pneumocystis antibodies. (A) Pneumocystis burden in the lungs of RAG2^−/−, RAG2/IL-4Rα^−/−, and RAG2/IFN-γR^−/− mice was measured by real-time PCR quantitation of Pneumocystis kexin gene copy numbers in lung homogenates at days 14, 18, 21, 26, and 34 post-immune reconstitution. Values are means ± 1 standard error (n = 6 to 10 per group at each time point). Data represent results from at least two independent experiments. *, P < 0.05 compared to RAG2^−/− mice; #, P < 0.05 compared to RAG2/IL-4Rα^−/− mice. ND indicates that no signal was detected in any mice at that time. Pneumocystis-specific IgG and IgM (B), IgG1 (C), and IgG2a (D) antibodies in Pneumocystis-infected RAG2^−/−, RAG2/IL-4Rα^−/−, and RAG2/IFN-γR^−/− mouse sera were measured by an ELISA using soluble Pneumocystis antigen from infected mouse lung homogenates as the target. OD, optical density. *, P < 0.05 compared to the indicated group.

FIG 2

Impaired alveolar macrophage polarization in RAG2/IL-4Rα^−/− and RAG2/IFN-γR^−/− mice. (A and B) BAL fluid cells were collected from immune-reconstituted, Pneumocystis-infected RAG2^−/−, RAG2/IL-4Rα^−/−, and RAG2/IFN-γR^−/− mice and stimulated with PMA and ionomycin. Total numbers of CD4⁺ T cells producing IFN-γ and IL-4 were determined by intracellular cytokine staining. Values are means ± 1 standard error (n = 3 to 5 per group at each time point). (C and D) BAL fluid cells were collected from Pneumocystis-infected RAG2^−/−, RAG2/IL-4Rα^−/−, and RAG2/IFN-γR^−/− mice at day 18 post-immune reconstitution. (C) Cells were stained with antibodies specific for the alveolar macrophage marker CD11c (green) and arginase (red). (D) Cells were also stained with antibodies specific for CD11c (red) and iNOS (green). Nuclei were stained with DAPI (blue). At least 200 CD11c⁺ cells were examined for each strain. Magnification, ×400.

FIG 3

Loss of IFN-γR signaling is associated with greater pulmonary function deficits and increased lung injury. Physiological measurements of lung function in Pneumocystis-infected immune-reconstituted RAG2^−/−, RAG2/IL-4Rα^−/−, and RAG2/IFN-γR^−/− mice were performed. Body weights (A) and respiratory rates (B) were monitored noninvasively over a 27-day period postreconstitution. Specific dynamic lung compliance (C) and lung resistance (D) measurements were performed post-immune reconstitution on anesthetized mice placed in a whole-body plethysmography chamber. Values are means ± 1 standard error (n = 6 to 10 per group at each time point). Data represent results from at least two independent experiments. *, P < 0.05 compared to RAG2^−/− mice; #, P < 0.05 compared to RAG2/IL-4Rα^−/− mice.

FIG 4

Loss of IFN-γR signaling is associated with increased lung inflammation. Lung sections were collected from Pneumocystis-infected RAG2^−/−, RAG2/IL-4Rα^−/−, and RAG2/IFN-γR^−/− mice at day 18 post-immune reconstitution and stained with hematoxylin and eosin. Representative pictures were taken at a ×100 magnification. Arrows denote peribronchial regions, and stars denote alveolar regions.

FIG 5

Loss of IFN-γR signaling is associated with increased CD4⁺ T cell-mediated inflammation but decreased lung CD8⁺ T cell responses. BAL fluid cells were collected from immune-reconstituted Pneumocystis-infected RAG2^−/−, RAG2/IL-4Rα^−/−, and RAG2/IFN-γR^−/− mice. Total cells (A), CD4⁺ T cells (B), CD8⁺ T cells (C), and CD8⁺ Foxp3⁺ T cells (D) were enumerated by flow cytometry. Values are means ± 1 standard error (n = 6 to 10 per group at each time point). Data represent results from at least two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 6

Excessive IFN-γ production impairs the CD8⁺ T cell response during PcP-related IRIS. (A and B) IL-4 (A) and IFN-γ (B) levels in the lung homogenates from immune-reconstituted Pneumocystis-infected RAG2^−/−, RAG2/IL-4Rα^−/−, and RAG2/IFN-γR^−/− mice were measured (n = 6 to 10 per group at each time point). (C) In a separate experiment, Pneumocystis-infected RAG2/IFN-γR^−/− mice were immune reconstituted and treated with either anti-IFN-γ or control rat IgG beginning at day 3 postreconstitution. At day 18 postreconstitution, the mice were sacrificed, and lung CD8⁺ T cell numbers were determined by flow cytometry. Values are means ± 1 standard error (n = 4 to 5 per group at each time point). *, P < 0.05; **, P < 0.01; ***, P < 0.001.