Aspergillus fumigatus is responsible for inflammation in a murine model of chronic obstructive pulmonary disease exacerbation

Abstract

Background: In patients with chronic obstructive pulmonary disease (COPD), a sensitization to A. fumigatus has been related to a decline in lung function, but the role of fungal agents in the disease pathogenesis remains unclear. The main purpose of the present study was to investigate whether cell inflammation could worsen after exposure to A. fumigatus spores in vitro and then, in mice, following chronic exposure to cigarette smoke mimicking COPD.

Methods: The inflammatory response to cigarette smoke alone or with A. fumigatus was investigated in cell culture models of murine macrophages and alveolar epithelial cells. In an animal model, mice were exposed daily to two cigarettes smoke over 14 weeks, and two intranasal instillations of 10⁵ spores at weeks 7 and 14. Then, their lungs were recovered to perform inflammatory and histopathological analyses.

Results: In co-cultures of macrophages and epithelial cells treated with both cigarette smoke extracts (CSE) and A. fumigatus compared to CSE alone there were significant inductions in TNF-α (6.2-fold) and CXCL-2 (21.5-fold) gene expression, confirmed by significant increases in the corresponding protein secretion. In the murine model, histological analyses of the lung after chronic smoke exposure showed an increase in airspace enlargement. Moreover, a Bio-Plex approach on bronchoalveolar lavage of cigarette smoke and A. fumigatus-treated mice showed significant increases in multiple inflammatory proteins secreted in the lung.

Conclusions: There was a stronger inflammatory response after cigarette smoke exposure with A. fumigatus compared to cigarette smoke alone. These findings were correlated with histopathological changes in the mouse lung in vivo.

Keywords: Aspergillus fumigatus; Chronic obstructive pulmonary disease; Cigarette smoke; Epithelial cells; Exacerbation; Inflammation; Macrophages; Murine model.

Fig. 1

Gene expression of multiple mediators by RAW 264.7 and MLE-15 cells. Cells were exposed during 1 h to cigarette smoke extract 5% (CSE) and during 4 h to A. fumigatus spores at MOI3 (CSE + AFsp). (a) Fold induction of the gene expression of inflammatory mediators in RAW 264.7 cells. (b) Fold induction of the gene expression of inflammatory mediators in MLE-15 cells (c) Fold induction of the gene expression of inflammatory mediators in co-cultured cells. The mean ± SD is presented, n = 3 in monocultures and n = 4 in co-cultures, *: p < 0.05, **: p < 0.01, ***: p < 0.001

Fig. 2

Quantification of mediators in the supernatants of co-cultured MLE-15/RAW264.7 cells exposed to CSE and CSE + AFsp: (a) TNF-α, (b) CXCL-2, (c) CXCL-1 and (d) IL-1α. The mean ± SD is presented, n = 4, *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001

Fig. 3

Tissue degradation and inflammation observed in formalin-fixed paraffin-embedded lung tissues. Representative images 10X (scale bar 300 μm) stained with hematoxylin, eosin, saffron (HES). a) Control mice exposed to filtered air, b) Cigarette-smoke (CS)-exposed mice, c) Cigarette-smoke and Aspergillus fumigatus spores (CS + AFsp)-exposed mice, d) inflammation in CS + AFsp mice – inflammatory cells are indicated by blue arrows. No fibrosis nor fungal elements were detected on the HES (or Grocott-stained slides). CS induced a notable airspace enlargement in both CS and CS + AFsp groups

Fig. 4

Marked airspace enlargement in both CS-exposed mice. Significant increases of the mean linear intercept (Lm) were observed in CS and CS + AFsp-exposed mice. The mean ± SD is presented, n = 5–7 mice per group, *: p < 0.05

Fig. 5

Assessment of cell count in BAL and fungal load in lung tissue. a) Total BAL cell count, b) cellularity in BAL and c) fungal load in lung tissue. The total cell count was performed with an automatic cell counter with trypan blue solution. Cell types were determined with cytospin slides. Data are represented by mean ± SD for a) and b). Box and whiskers indicate the median, IQR, as well as minimal and maximal values for c), n = 5–7 mice per group, *: p < 0.05, ***: p < 0.001, ****: p < 0.0001

Fig. 6

Gene expression and secretion of mediators in specimens from exposed mice. a) gene expression of inflammatory mediators in pulmonary tissue, b) concentrations (pg/mL) of some secreted cytokines in bronchoalveolar lavage (BAL) fluids, (c) CXCL-2 concentrations in BAL and (d) eotaxin concentrations in sera of mice exposed to cigarette smoke (CS) and CS + AFsp. Data are represented by mean ± SD, n = 5–7 mice per group, *: p < 0.05, **: p < 0.01, ***: p < 0.001