To the Editor:

Chronic obstructive pulmonary disease (COPD) is associated with a characteristic underlying inflammation that comprises macrophages, neutrophils, and T-lymphocytes. Despite the presence of increased numbers of pulmonary innate immune cells, patients with COPD are prone to infective exacerbations. These are predominantly attributed to viral and/or bacterial infection; however, fungal pathogens are increasingly being recognized as contributors to COPD exacerbations, with Aspergillus fumigatus being the most common species detected in the sputum of patients with COPD both at steady state and during exacerbation (1). In a previous study, filamentous A. fumigatus was cultured in sputum from approximately half of the subjects with COPD and was associated with the use of higher doses of inhaled corticosteroid (2). In addition, sensitization to A. fumigatus was shown to correlate with poorer lung function and prognosis, although the exact nature of these relationships requires further study (2, 3). Why these fungal spores persist in the airways of patients with COPD is not known, but it suggests a failure of clearance by alveolar macrophages. In COPD, alveolar macrophages exhibit reduced phagocytosis of bacteria and apoptotic cells (4, 5), and it is postulated that this contributes to the pathogenesis of disease. Furthermore, this defect also occurs in macrophages that have been differentiated from circulating monocytes (monocyte-derived macrophages [MDMs]) (5), suggesting that these cells can be used to model this aspect of macrophage function in COPD. Whether this defect extends to fungal spores such as A. fumigatus is unknown. Therefore, we used the MDM model to differentiate monocytes into proinflammatory and resolving macrophages from nonsmokers, smokers, and patients with COPD (Table E1 in the data supplement), using granulocyte–macrophage colony–stimulating factor (GM-CSF; G-Mφ) and M-CSF (M-Mφ), respectively (6). We then measured the phagocytic potential and cytokine output of these cells in response to A. fumigatus (for details, see this article’s Methods section in the data supplement). These models are considered to represent polarized macrophages; therefore, by using both macrophage phenotypes, one can investigate whether any changes in the innate response to A. fumigatus are due to differences in the macrophage phenotype, and whether the disease state is contributing to these differences (6, 7).

Using GFP-expressing A. fumigatus, we confirmed that uptake of conidia by both G-Mφ (Figure 1A) and M-Mφ (Figure 1B) was time dependent, but that cells derived from smokers and patients with COPD showed a significant reduction in the percentage of cells that had phagocytosed these spores compared with cells derived from healthy controls. There was no statistical difference in the responses of cells from smokers compared with patients with COPD in either macrophage phenotype. We also assessed the capacity of each cell to phagocytose by measuring the number of conidia taken up per cell (measured by median fluorescence intensity; Figures E1A and E1B), and observed similar suppressed responses by cells from smokers and patients with COPD. Internalization of conidia was confirmed by confocal microscopy (Figures 1C and 1D). No correlations were observed between age, smoking history, or any lung-function parameters and the uptake of conidia by either G-Mφ or M-Mφ. However, when comparing cells from the same patient, there was a weak positive correlation between phagocytosis of G-Mφ and that of M-Mφ (Figure E1E). These data suggest that the clearance of fungal spores is diminished in COPD macrophages, similar to what is observed for bacteria and apoptotic cells. To determine whether these defective immune responses could be attributed to reduced expression of Aspergillus-sensing receptors, we examined the expression of dectin-1, a C-type lectin receptor that recognizes β-glucan on A. fumigatus cell walls (8). However, there were no differences in dectin-1 expression on the surface of G-Mφ or M-Mφ from patients with COPD compared with cells from other control groups at baseline or after phagocytosis (Figures E1C and E1D).

Figure 1. Decreased phagocytosis of Aspergillus fumigatus by chronic obstructive pulmonary disease (COPD) monocyte-derived macrophages (MDMs). MDMs were differentiated in granulocyte–macrophage colony–stimulating factor (G-Mφ) or macrophage colony–stimulating factor (M-Mφ) from nonsmokers (solid circle; n = 11), smokers (open circle; n = 7), or patients with COPD (solid square; n = 9), and phagocytosis of GFP-labeled conidia of A. fumigatus was measured by flow cytometry (A and B, respectively) and confirmed by confocal microscopy (C and D, respectively). Red, cytoplasm; blue, nuclei; green, conidia. Release of IL-10 (E and F), TNF-α (G and H), and CXCL8 (I and J) was measured by ELISA. Data are means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001 for differences between nonsmokers and smokers; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 for differences between nonsmokers and patients with COPD. US = unstimulated cells.

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Having found that MDMs from patients with COPD and smokers showed defective uptake of A. fumigatus spores, we aimed to determine whether the inflammatory response to this pathogen was also suppressed by measuring cytokine release. A time-dependent increase was observed in the expression of IL-10, TNF-α, and CXCL8 measured in both G-Mφ (Figures 1E, 1G, and 1I) and M-Mφ (Figures 1F, 1H, and 1J), with a greater cytokine output from M-Mφ. Between subject groups, no differences were observed in the release of IL-10 in response to A. fumigatus in M-Mφ, but G-Mφ from smokers released significantly more IL-10 at 24 hours than cells from controls or patients with COPD. However, both G-Mφ and M-Mφ from smokers and patients with COPD produced significantly less TNF-α in response to A. fumigatus compared with cells from nonsmokers at 24 hours. Similarly, G-Mφ from smokers and patients with COPD produced significantly less CXCL8 when exposed to A. fumigatus for 24 hours compared with cells from nonsmokers, although this was not observed for M-Mφ. The mechanism underlying this response remains unclear, but decreased detection of fungal spores by intracellular receptors in COPD macrophages may contribute to this reduction in cytokine release.

This study shows that MDMs from smokers and patients with COPD display defective phagocytic and proinflammatory cytokine responses to A. fumigatus. This reduced immune defense does not appear to be due to altered expression of dectin-1, a key receptor in conidia recognition. This failure to recognize and clear A. fumigatus may lead to fungal germination and dissemination, infection, and lung damage, contributing to the increased incidence of Aspergillus-related disease in patients with COPD.

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