Epidermal growth factor receptor signaling governs the host inflammatory response to invasive aspergillosis

Abstract

The epidermal growth factor receptor (EGFR) has been identified as an epithelial cell receptor for Mucorales fungi and Candida albicans. Blocking EGFR with small molecule inhibitors reduces disease severity in mouse models of mucormycosis and oropharyngeal candidiasis. In contrast, cases of invasive aspergillosis have been reported in cancer patients who were treated with EGFR inhibitors, suggesting that EGFR signaling may play a protective role in the host defense against this infection. Here, we analyzed transcriptomic data from the lungs of mice with invasive aspergillosis and found evidence that Aspergillus fumigatus infection activates multiple genes that are predicted to function in the EGFR signaling pathway. We also found that A. fumigatus infection activates EGFR in both a human small-airway epithelial (HSAE) cell line and in the lungs of immunosuppressed mice. EGFR signaling in HSAE cells is required for maximal endocytosis of A. fumigatus and for fungal-induced proinflammatory cytokine and chemokine production. In a corticosteroid immunosuppressed mouse model of invasive pulmonary aspergillosis, inhibition of EGFR with gefitinib decreased whole-lung cytokine and chemokine levels and reduced accumulation of phagocytes in the lung, leading to a decrease in fungal killing, an increase in pulmonary fungal burden, and accelerated mortality. Thus, EGFR signaling is required for pulmonary epithelial cells to orchestrate the host innate immune defense against invasive aspergillosis in immunosuppressed hosts.IMPORTANCEWhen A. fumigatus infects the lungs, it invades epithelial cells that line the airways. During this process, the fungus interacts with epithelial cell receptors. This interaction stimulates epithelial cells to endocytose the fungus. It also induces these cells to secrete proinflammatory cytokines and chemokines that recruit phagocytes to the site of infection where they can kill the fungus. Here, we show that in small-airway epithelial cells, the EGFR acts as a sensor for A. fumigatus that triggers the production of chemokines in response to fungal infection. In corticosteroid-immunosuppressed mice, blocking EGFR with the kinase inhibitor gefitinib reduces chemokine production in the lungs. This leads to decreased accumulation of neutrophils and dendritic cells in the lungs, reduced A. fumigatus killing, and increased mortality. These results provide a potential explanation as to why some cancer patients who are treated with EGFR inhibitors develop invasive aspergillosis.

Keywords: Aspergillus fumigatus; alveolar epithelial cell; chemokine; cytokine; endocytosis; epidermal growth factor receptor; small-airway epithelial cell.

Fig 1

A. fumigatus infection of immunosuppressed mice activates the EGFR in the lungs. (A) Heatmap showing the results of upstream regulator analysis of the transcriptome of the lungs of mice that had been immunosuppressed with cortisone acetate and infected with A. fumigatus for 2, 4, and 6 days. Control mice were immunosuppressed but not infected. (B) Heatmap showing the effects of A. fumigatus infection on the mRNA levels of 25 genes in the EGFR pathway (C) A. fumigatus infection of immunosuppressed mice stimulates EGFR autophosphorylation, which is reduced in mice treated with the EGFR inhibitor, gefitinib. Confocal microscopic images of thin sections of the lungs of immunosuppressed mice 12 h after intratracheal inoculation with A. fumigatus. The sections were stained for A. fumigatus (green), pulmonary epithelial cells (CD326, blue), and pEGFR (red). Scale bar: 50 µm. dpi, days post-infection; EGFR, epidermal growth factor receptor; pEGFR, phosphorylated epidermal growth factor receptor.

Fig 2

EGFR interacts with A. fumigatus and regulates fungal endocytosis by HSAE cells. (A) Confocal micrographs showing EGFR accumulation around A. fumigatus in A549 and HSAE cells after 2.5 h of infection. Hollow arrows indicate the organisms in the magnified images in the lower right of each panel. Scale bar, 20 µm. (B) A. fumigatus binds to EGFR in membrane protein extracts of both A549 and HSAE cells. Representative immunoblots (right). Densitometric analysis of three immunoblots (left). Results are mean ± SD. (C) Western blots showing that A. fumigatus infection for 2.5 h inhibits EGFR phosphorylation in A549 cells but stimulates EGFR phosphorylation in HSAE cells. (D) Densitometric analysis of four phospho-EGFR Western blots such as the ones in panel C. (E and F) Effects of the EGFR inhibitor, gefitinib, on the endocytosis of A. fumigatus conidia (E) and germlings (F) by A549 and HSAE cells. (G and H) Effects of gefitinib on the cell association (a measure of adherence) of A. fumigatus conidia (G) and germlings (H) with A549 and HSAE cells. (I) Endocytosis and cell association of A. fumigatus by wild-type NIH/3T3 cells or NIH/3T3 cells that expressed human EGFR. Results in panels E–I are mean ± SD of three independent experiments, each performed in triplicate. *P < 0.05, ***P < 0.001, ****P < 0.0001 by unpaired Student’s t-test (D) or one-way analysis of variance with Dunnett’s test for multiple comparisons (E–I). A. fum, A. fumigatus; cell-assoc, cell-associated; HSAE, human small-airway epithelial; ns, not significant; orgs/HPF, organisms per high-powered field.

Fig 3

EGFR is required for maximal cytokine and chemokine release in HSAE cells infected with A. fumigatus. (A–F) HSAE cells were treated with gefitinib or dimethyl sulfoxide (DMSO) control and infected with A. fumigatus for 16 h, and then levels of the indicated cytokines were measured. Results are mean ± SD of three independent experiments, each performed in duplicate. *P < 0.05, ***P < 0.001, ****P < 0.0001 by one-way analysis of variance with Dunnett’s test for multiple comparisons. A. fum, A. fumigatus; ns, not significant; Uninfect, uninfected.

Fig 4

Gefitinib worsens outcome in immunosuppressed mice infected with A. fumigatus. (A) Effects of gefitinib on the survival of mice infected with A. fumigatus by aerosol inhalation. Results are the combined data from two experiments (n = 24 mice per group). (B) Effects of gefitinib on pulmonary fungal burden after 4 days of infection, measured by the relative fungal DNA content in the infected mouse lungs. (C–H) Effects of gefitinib on the levels of the indicated inflammatory mediators in homogenates of the lungs of mice after 4 days of infection. Results in panels B–H are median ± interquartile range of 12 mice per group in a single experiment. **P < 0.01, ***P < 0.001, ****P < 0.0001 by the log-rank test (A) or the Mann-Whitney test (B–H). ns, not significant.

Fig 5

Gefitinib inhibits A. fumigatus killing by immune cells during invasive aspergillosis. (A–C) Corticosteroid-immunosuppressed mice were infected intratracheally with A. fumigatus conidia expressing dsRed and labeled with AlexaFluor 633. After 12 h, the tissue macrophages (A), neutrophils (B), and dendritic cells (C) were analyzed by flow cytometry to determine the number of cells in the samples, the percentage of cells with phagocytosed conidia, and the percentage of phagocytosed conidia that had been killed. Results are the median ± interquartile range of six mice per group in a single experiment. **P < 0.01 by the Mann-Whitney test. ns, not significant.

Fig 6

Gefitinib treatment of HSAE cells infected with A. fumigatus inhibits human neutrophil chemotaxis. Effects of gefitinib on neutrophil migration across transwell inserts induced by medium alone, and medium conditioned by uninfected HSAE cells and HSAE cells infected with live Af; HSAE cells incubated with paraformaldehyde killed A. fumigatus; live A. fumigatus or paraformaldehyde killed A. fumigatus. Results are the mean ± SD of four experiments, each performed in duplicate. *P < 0.05, **P < 0.01 , ****P < 0.0001 by one-way analysis of variance with Sidak’s multiple comparisons test. Af, A. fumigatus; ns, not significant.