Rapid host defense against Aspergillus fumigatus involves alveolar macrophages with a predominance of alternatively activated phenotype

Abstract

The ubiquitous fungus Aspergillus fumigatus is associated with chronic diseases such as invasive pulmonary aspergillosis in immunosuppressed patients and allergic bronchopulmonary aspergillosis (ABPA) in patients with cystic fibrosis or severe asthma. Because of constant exposure to this fungus, it is critical for the host to exercise an immediate and decisive immune response to clear fungal spores to ward off disease. In this study, we observed that rapidly after infection by A. fumigatus, alveolar macrophages predominantly express Arginase 1 (Arg1), a key marker of alternatively activated macrophages (AAMs). The macrophages were also found to express Ym1 and CD206 that are also expressed by AAMs but not NOS2, which is expressed by classically activated macrophages. The expression of Arg1 was reduced in the absence of the known signaling axis, IL-4Rα/STAT6, for AAM development. While both Dectin-1 and TLR expressed on the cell surface have been shown to sense A. fumigatus, fungus-induced Arg1 expression in CD11c(+) alveolar macrophages was not dependent on either Dectin-1 or the adaptor MyD88 that mediates intracellular signaling by most TLRs. Alveolar macrophages from WT mice efficiently phagocytosed fungal conidia, but those from mice deficient in Dectin-1 showed impaired fungal uptake. Depletion of macrophages with clodronate-filled liposomes increased fungal burden in infected mice. Collectively, our studies suggest that alveolar macrophages, which predominantly acquire an AAM phenotype following A. fumigatus infection, have a protective role in defense against this fungus.

Figure 1. Infection by Aspergillus fumigatus induces markers of Alternatively Activated Macrophages in the lung.

(A) Mice were infected with 100 cfu of K. pneumoniae or 50×10⁶ resting conidia (RC) of A. fumigatus given intratracheally and lungs were harvested after 4 days (Klebsiella) or 48 hours (Aspergillus) of infection for total RNA extraction. RT-PCR was performed to measure mRNA expression of Arg1, Fizz1 and NOS2. The results shown were generated using RNA from 1 mouse (n = 4) with the PCR products in the different lanes generated with increasing dilutions of cDNA. β-actin expression was used as an internal control. mRNA expression corresponding to the various genes in infected lungs was compared with that expressed in the lungs of uninfected mice. Mice were infected with 50×10⁶ RC or given PBS intratracheally (uninfected group). The expression of Arg1, NOS2 and Fizz1 was analyzed by quantitative RT-PCR (B) at 6, 12 and 24 hours (C) at 48 and 96 hours p. i. The fold increase in expression for each gene is expressed relative to that in uninfected mice using Gus-β expression for normalization. Values shown are mean ± SEM. (D) Arginase activity expressed as U/mg protein was measured in protein extracts made from lungs 48 and 96 hours p. i. (E) Immunoblotting of YM1 was performed using protein extracts made from lungs using anti-YM1 antibody. β-actin expression was examined as loading control and the intensity of YM1 band was quantified relative to that of β-actin. Data shown are representatives of two independent experiments (n = 4–6 mice in each group).

Figure 2. Characterization of BAL cells from after A. fumigatus infection.

(A) Mice were infected with 50×10⁶ RC and BAL cells were collected at different time points after infection. Total and differential cell counts of BAL cells recovered from uninfected and infected mice were determined from stained cytospin slides. Macs: macrophages; PMNs: neutrophils; Eos: eosinophils and Lymphs: lymphocytes. Values shown are mean±SEM. (B) Representative flow cytometry plots showing different cell types in the BAL fluid by staining for various cell surface markers. Cells are gated on live CD45⁺ cells (leukocytes) and analyzed for CD3, CD19, Ly6G or CD11c expression. Numbers represent the percentages of specific cell types in the CD45⁺ population. Characterization of CD11c⁺ cells in the BAL fluid from uninfected or infected mice at 48 h p.i. based on autofluorescence and MHC II expression. Flow cytometry plots show that CD11c⁺ cells can be divided into highly autofluorescent macrophages (gate 1) and low autofluorescent DCs (gate 2). MHC II expression in each population was examined. Numbers represent the percentages of cells in gated populations (n = 4 mice per group).

Figure 3. Identification of CD11c⁺Arg1-expressing alveolar macrophages after A. fumigatus infection.

(A) Mice were infected with 10×10⁶ RC and cells in the BAL fluid were recovered from uninfected and infected mice. Stained cytospins of CD11c⁺ and CD11c^- fractions 48 hours p.i. CD11c⁺ macrophages before and after infection exhibited different morphology when compared to those isolated from naïve controls. (B) mRNA expression of AAM markers measured by quantitative (left panel) and semi-quantitative RT-PCR (right panel) in BAL CD11c⁺ cells isolated 48 hours p.i. The fold increase shown is relative to genes expressed in CD11c⁺cells from the uninfected group after normalization to Gus-β. Values shown are mean±SEM. For semi-quantitative RT-PCR, β-actin expression was used as an internal control. Data shown were generated using RNA from 1 mouse (n = 4) with the PCR products in the different lanes generated with increasing dilutions of cDNA. The experiment was repeated three times with similar results. (C) Arg1 and (D) NOS2 expression in the BAL CD11c⁺ cells was examined at 48 hours p.i. by flow cytometry using intracellular staining techniques. Gray (filled) and black (open) histograms denote staining with isotype control and specific anti-Arg1 or anti-NOS2 antibody respectively (n = 4–6 mice in each group). The frequency of Arg1 expression in uninfected and infected cells was 2.2% and 52% respectively while that of NOS2 was 0.68% and 2.54% in the same cells. (E) Arginase activity expressed as U/mg protein was measured in protein extracts made from CD11c⁺ cells isolated by BAL from PBS-treated uninfected controls or fungus-infected mice at 48 hours p.i.

Figure 4. CD11c⁺Arg1 expressing AAMs isolated after A. fumigatus infection carry fungal load.

Mice were infected with 10×10⁶ RC and CD11c⁺ cells were isolated by BAL from PBS-treated uninfected controls or fungus-infected mice at 48 hours p.i. Fungal uptake in the BAL CD11c⁺ cells was assessed by quantitative PCR of DNA corresponding to fungal 18S rRNA and expressed as Conidia Equivalents/lung (n = 4 mice in each group). Values shown are mean±SEM.

Figure 5. IL-4Rα/STAT6 partly controls Arg1 expression in alveolar macrophages isolated from A. fumigatus-infected mice.

WT, IL-4Rα^-/- and Stat6^-/- mice were infected with 10×10⁶ RC and CD11c⁺ cells were isolated by BAL at 48 hours p.i. (A) Quantitative RT-PCR was performed to measure Arg1 mRNA expression in CD11c⁺ cells from infected WT, IL-4Rα^-/- and Stat6^-/- at 48 hours p.i. and the fold increase shown are relative to that in CD11c⁺ cells from uninfected group. Values shown are mean±SEM (B) Semi-quantitative RT-PCR analysis of expression of indicated genes CD11c⁺ cells isolated from uninfected and infected mice. The data were obtained using RNA isolated from the cells of one mouse with the bands in the 3 lanes in each group depicting PCR products obtained with increasing dilution of cDNA. Similar results were obtained in two independent experiments (n = 4–6 mice in each group).

Figure 6. Dectin-1- or MyD88-deficiency in alveolar macrophages does not affect Arginase1 expression but impairs fungal clearance.

(A) WT, Dectin-1^-/- and MyD88^-/- mice were infected with 10×10⁶ RC and CD11c⁺ cells were isolated by BAL at 48 hours p.i. Quantitative RT-PCR was performed to measure Arg1 mRNA expression in CD11c⁺ cells from infected WT, Dectin-1^-/- and MyD88^-/- and the fold increase shown are relative to that in CD11c⁺ cells from uninfected group. Values shown are mean±SEM (B) Fungal burden expressed as CFU per lung was measured in lungs harvested from WT, Dectin-1^-/- and MyD88^-/- mice at 48 hours p.i. Values shown are mean±SEM. Data shown are representative of two independent experiments (n = 6-8 mice in each group). (C) Phagocytosis of FITC-labeled A. fumigatus conidia by CD11c⁺ alveolar macrophages as examined by confocal microscopy. Images show overlay of FITC (green for conidia) and Hoechst stain (blue for nuclei) and Cell tracker (red for cell cytoplasm). The upper panel shows images captured at 60X in single optical plane and the lower panel shows 3X digital zoom images. Quantification of conidia present intracellularly was done using Metamorph and results are expressed as percentage of cells with FITC-conidia. Labeled conidia were easily identified in the macrophages isolated from both WT and MyD88-deficient mice but were rare in cells isolated from Dectin-1^-/- mice.

Figure 7. Clodronate-mediated depletion of alveolar macrophages increases A. fumigatus burden in the lung.

Mice were treated with clodronate-filled liposomes or PBS-liposomes intratracheally at 48 hours prior to A. fumigatus infection and then infected with 50×10⁶ RC. (A) Cytospins of cells present in BAL fluid recovered from PBS-liposome or clodronate-liposome treated mice at 48 and 96 hours p.i. (B) Total and differential cell counts of BAL-derived cells from PBS-liposome and clodronate-liposome treated mice at 48 and 96 hours p.i. Results shown are mean±SEM. (C and D) Fungal burden was assessed by quantitative PCR of DNA corresponding to fungal 18S rRNA and expressed as Conidia Equivalents/lung. The experiment was repeated twice with similar results (n = 4–8 mice in each group). Values shown are mean±SEM.

Figure 8. Alveolar macropjages expressing Arginase 1 dominate after A. fumigatus infection and role in fungal clearance.

Infection by A. fumigatus rapidly induces Arg1 expression in alveolar macrophages. Arg1 expression is partly dependent on the IL-4Rα/STAT6 signaling axis. Furthermore, Arg1 expression is independent of Dectin-1 and MyD88 signaling pathways. Clodronate-mediated depletion of alveolar macrophages prior to fungal infection results in increased fungal burden in the lungs.