Characterization of a distinct host response profile to Pneumocystis murina asci during clearance of pneumocystis pneumonia

Abstract

Pneumocystis spp. are yeast-like fungi that cause pneumocystis pneumonia (PcP) in immunocompromised individuals and exacerbate chronic lung diseases in immunocompetent individuals. The Pneumocystis life cycle includes trophic forms and asci (cyst forms). The cell walls of Pneumocystis asci contain β-1,3-D-glucan, and treatment of PcP with β-1,3-D-glucan synthase inhibitors, such as anidulafungin, results in depletion of asci, but not trophic forms. The pulmonary host response during immune reconstitution (IR)-mediated clearance of PcP in anidulafungin-treated and untreated mice was characterized to identify ascus-specific responses. During IR, similar numbers of trophic forms were present in the anidulafungin-treated and untreated mice; however, asci were only present in the untreated mice. IR resulted in a significant reduction of trophic forms from the lungs in both groups and asci in the untreated group. The presence of asci in untreated mice correlated with increased β-glucan content in the lungs. The untreated mice mounted immune responses associated with a deleterious host inflammatory response, including increased CD8(+) T cell influx and expression of macrophage inflammatory response markers. A more robust cellular response was also observed in the untreated mice, with increased numbers of macrophages and neutrophils that were associated with greater lung damage. Markers of a Th17 response were also elevated in the untreated mice. These results suggest that the host mounts unique responses to asci and trophic forms. That these 2 life cycle stages provoked distinct host response profiles has significant implications for clearance and interpretation of the host immune responses to PcP.

Fig 1

Ascus and trophic burdens in untreated (Un Tx) and anidulafungin-treated (Tx) mice during immune reconstitution. At 1, 7, and 14 days postcessation of dexamethasone treatment, average ascus and trophic form burdens were determined by microscopic enumeration of histologically stained mouse lung homogenates using cresyl echt violet (A) or a rapid Wright-Giemsa stain (B). Gray bars indicate the organism burden in untreated (UnTx) mice that did not receive anidulafungin (ascus replete). Black bars indicate the organism burden in mice treated (Tx) with anidulafungin throughout the experiment (to suppress ascus formation). Black brackets (*) indicate statistically significant differences between the 2 untreated and treated groups at a given time point (1, 7, or 14 days of immune reconstitution [IR]), as determined by the unpaired two-tailed t test (P < 0.05). Gray dashed brackets (#) indicate significant differences among the 3 time points within the untreated (Un Tx) or treated (Tx) groups, as determined by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test (P < 0.05). Error bars represent the standard errors of the mean within each group. Data are expressed as the log₁₀ asci or trophic forms per lung. There were 8 mice in the untreated groups at days 1 and 7 of IR, 7 mice at day 14, and 5, 5, and 4 mice in the anidulafungin-treated groups at the same time points.

Fig 2

β-1,3-d-Glucan levels in the lungs of untreated (Un Tx) and anidulafungin-treated (Tx) mice during immune reconstitution. Glucatell assay kits (Associates of Cape Cod, East Falmouth, MA) were used to measure the content of β-1,3-d-glucan in the lung tissue 1, 7, and 14 days postcessation of dexamethasone treatment. Gray bars indicate β-1,3-glucan levels in untreated (UnTx) mice that did not receive anidulafungin (ascus replete). Black bars indicate β-1,3-glucan levels in mice treated (Tx) with anidulafungin throughout the experiment (to suppress ascus formation. Black lines (*) indicate statistically significant differences between the 2 untreated and treated groups at a given time point (1, 7, or 14 days of immune reconstitution [IR]), as determined by the unpaired two-tailed t test (P < 0.05). Gray dashed brackets (#) indicate significant differences among the 3 time points within the untreated (Un Tx) or treated (Tx) groups, as determined by one-way analysis of variance (ANOVA) followed by the Tukey's multiple comparison test (P < 0.05). Error bars represent the standard errors of the mean within each group. Data are expressed as pg/ml of β-1,3-d-glucan per lung. There were 8 mice in the untreated groups at days 1 and 7 of IR, 7 at day 14, and 4, 5, and 3 mice in the anidulafungin-treated groups at the same time points.

Fig 3

Pulmonary CD4⁺ and CD8⁺ T cell responses in untreated (Un Tx) and anidulafungin-treated (Tx) mice during immune reconstitution. Pulmonary T cell populations were characterized by flow cytometry at 7 and 14 days postcessation of dexamethasone treatment. Gray bars indicate organism burden in untreated (Un Tx) mice that did not receive anidulafungin (ascus replete). Black bars indicate organism burden in mice treated (Tx) with anidulafungin throughout the experiment (to suppress ascus formation). (A) Total number of CD4⁺ T cells; (B) total number of CD8⁺ T cells; (C) percentage of CD4⁺ T cells; (D) percentage of CD8⁺ T cells. Black brackets (*) indicate statistically significant differences between the 2 untreated and treated groups at a given time point (7 or 14 days of immune reconstitution [IR]), as determined by the unpaired two-tailed t test (P < 0.05). Gray dashed brackets (#) indicate significant differences within either the untreated or treated groups at either 7 or 14 days IR, as determined by the unpaired two-tailed t test (P < 0.05). Error bars represent the standard errors of the mean within each group. Data are expressed as the total number of CD4⁺ or CD8⁺ T cells and as the percentage of CD3⁺ T cells per lung. There were 8 and 7 mice in the untreated groups at days 7 and 14, respectively, and 5 and 4 mice in the treated groups at the same time points.

Fig 4

Cellular pulmonary inflammatory responses in untreated (Un Tx) and anidulafungin-treated (Tx) mice during immune reconstitution. The numbers of leukocytes present in the bronchoalveolar lavage fluid (BALF) were enumerated at 7 and 14 days postcessation of dexamethasone treatment by differential counting. Gray bars indicate organism burden in untreated (Un Tx) mice that did not receive anidulafungin (ascus replete). Black bars indicate organism burden in mice treated (Tx) with anidulafungin throughout the experiment (to suppress ascus formation). (A) Percentage of neutrophils; (B) percentages of macrophages; (C) percentage of lymphocytes; (D) total number of neutrophils; (E) total number of macrophages; (F) total number of lymphocytes. Black brackets (*) indicate statistically significant differences between the 2 untreated and treated groups at a given time point (7 or 14 days of immune reconstitution [IR]), as determined by the unpaired two-tailed t test (P < 0.05). Gray dashed brackets (#) indicate significant differences within either the untreated or treated groups at either 7 or 14 days IR, as determined by the unpaired two-tailed t test (P < 0.05). Error bars represent the standard errors of the mean within each group. Data are expressed as the total number of neutrophils, macrophages, and lymphocytes or as the percentage of the total number of cells per lung. There were 8 and 7 mice in the untreated groups at days 7 and 14, respectively, and 5 and 4 mice in the treated groups at the same time points.

Fig 5

Expression of macrophage inflammatory response markers in the lungs of untreated (Un Tx) and anidulafungin-treated (Tx) mice during immune reconstitution. Multianalyte profiling of cytokine and chemokine levels in the bronchoalveolar lavage fluid (BALF) was performed with the Luminex-100 system. Gray bars indicate untreated (UnTx) mice that did not receive anidulafungin (ascus replete). Black bars indicate mice treated (Tx) with anidulafungin throughout the experiment (to suppress ascus formation). (A) G-CSF; (B) TNF-α; (C) MIP-2; (D) IL-2. Black brackets (*) indicate statistically significant differences between the 2 untreated and treated groups at a given time point (7 or 14 days of immune reconstitution [IR]), as determined by the unpaired two-tailed t test (P < 0.05). Gray dashed brackets (#) indicate significant differences within either the untreated or treated groups at either 7 or 14 days IR, as determined by the unpaired two-tailed t test (P < 0.05). Error bars represent the standard errors of the mean within each group. Data are expressed as pg/ml of BALF. There were 8 and 7 mice in the untreated groups at days 7 and 14, respectively, and 4 mice in the treated groups at the same time points.

Fig 6

Expression of IL-17 in the lungs of untreated (Un Tx) and anidulafungin-treated (Tx) mice during immune reconstitution. Multianalyte profiling of IL-17 levels in the bronchoalveolar lavage fluid (BALF) was performed with the Luminex-100 system. Gray bars indicate untreated (Un Tx) mice that did not receive anidulafungin (ascus replete). Black bars indicate mice treated (Tx) with anidulafungin throughout the experiment (to suppress ascus formation). Black brackets (*) indicate statistically significant differences between the 2 untreated and treated groups at a given time point (7 or 14 days of immune reconstitution [IR]), as determined by the unpaired two-tailed t test (P < 0.05). Gray dashed brackets (#) indicate significant differences within either the untreated or treated groups at either 7 or 14 days IR, as determined by the unpaired two-tailed t test (P < 0.05). Error bars represent the standard errors of the mean within each group. Data are expressed as pg/ml of BALF. There were 8 and 7 mice in the untreated groups at days 7 and 14, respectively, and 4 mice in the treated groups at the same time points.

Fig 7

Albumin concentrations in the bronchoalveolar lavage fluid (BALF) of untreated (Un Tx) and anidulafungin-treated (Tx) mice during immune reconstitution. Albumin concentrations were measured in BALF by ELISA. Gray bars, untreated (UnTx) mice that did not receive anidulafungin (ascus replete); black bars, mice treated (Tx) with anidulafungin throughout the experiment (to suppress ascus formation). Black brackets (*) indicate statistically significant differences between the 2 untreated and treated groups at a given time point (7 or 14 days of immune reconstitution [IR]), as determined by the unpaired two-tailed t test (P < 0.05). Gray dashed brackets (#) indicate significant differences within either the untreated or treated groups at either 7 or 14 days IR, as determined by the unpaired two-tailed t test (P < 0.05). Error bars represent the standard errors of the mean within each group. Data are expressed as pg/ml of BALF. There were 5 and 4 mice in the untreated groups at days 7 and 14, respectively, and 4 mice in the treated groups at the same time points.

Fig 8

P. murina isolated from untreated mice stimulates expression of MIP-2 by alveolar macrophages through Dectin-1. Alveolar macrophages isolated from BALF were stimulated overnight with 2 × 10⁵ trophic forms of P. murina isolated from untreated (Un Tx) or anidulalfungin-treated (Tx) mice in the absence (cont) or presence (+Ab) of 3 μg per ml of a rat anti-mouse Dectin-1 antibody. P. murina isolated from the untreated mice also contained 1.2 × 10^5. No asci were detected in the P. murina isolated from the treated mice. MIP-2 was measured in the culture supernatants by ELISA. Asterisks indicate statistically significant differences between the untreated control group, as determined by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test (P < 0.05). Error bars represent the standard errors of the mean within each group of triplicate wells. Data are expressed as pg/ml of culture supernatant. Results are representative of two separate experiments performed in triplicate.