Respiratory virus-induced EGFR activation suppresses IRF1-dependent interferon λ and antiviral defense in airway epithelium

Abstract

Viruses suppress host responses to increase infection, and understanding these mechanisms has provided insights into cellular signaling and led to novel therapies. Many viruses (e.g., Influenza virus, Rhinovirus [RV], Cytomegalovirus, Epstein-Barr virus, and Hepatitis C virus) activate epithelial epidermal growth factor receptor (EGFR), a tyrosine kinase receptor, but the role of EGFR in viral pathogenesis is not clear. Interferon (IFN) signaling is a critical innate antiviral host response and recent experiments have implicated IFN-λ, a type III IFN, as the most significant IFN for mucosal antiviral immune responses. Despite the importance of IFN-λ in epithelial antiviral responses, the role and mechanisms of epithelial IFN-λ signaling have not been fully elucidated. We report that respiratory virus-induced EGFR activation suppresses endogenous airway epithelial antiviral signaling. We found that Influenza virus- and RV-induced EGFR activation suppressed IFN regulatory factor (IRF) 1-induced IFN-λ production and increased viral infection. In addition, inhibition of EGFR during viral infection augmented IRF1 and IFN-λ, which resulted in decreased viral titers in vitro and in vivo. These findings describe a novel mechanism that viruses use to suppress endogenous antiviral defenses, and provide potential targets for future therapies.

Figure 1.

Role of EGFR in respiratory viral infection. (A) EGFR-p was measured by ELISA at 10 min in BEAS-2b cell culture lysates. Cells were treated with serum-free medium alone (control), with the selective EGFR tyrosine kinase inhibitor 10 µM Gefitinib, ROS scavenger (nPG, 100 µM), MP inhibitor (TAPI, 10 µM), with IAV and RV1b alone, or with the addition of nPG and TAPI (n = 3–5 independent experiments, mean ± SEM; *, P < 0.05 and **, P < 0.005 vs. control; #, P < 0.01 and ##, P < 0.005 vs. each virus alone). (B) BEAS-2b cells were treated with IAV, RV1b, and RV16 alone, with 10 µM Gefitinib alone, or transfected with control (C) or EGFR siRNA (E). After 24 h, cell culture homogenates were collected and virus was quantified by plaque assay (n = 3–4 independent experiments, mean ± SEM; *, P < 0.05 and **, P < 0.01 vs. virus plus Gefitinib or EGFR siRNA). BEAS-2b cells were transfected with EGFR siRNA and EGFR protein was assessed by Western blot (representative of three independent experiments). Molecular masses are provided in kilodaltons. (C) NHBE cells were treated with IAV and RV16 alone, or with 10 µM Gefitinib for 24 h and viral titers in cell culture homogenates were quantified by plaque assay (n = 4 independent experiments, mean ± SEM; **, P < 0.005 and ***, P < 0.0001 vs. virus alone). (D) C57BL/6 mice were infected (intranasal) with IAV (10^4.5 TCID50%) alone, or with 50 mg/kg Gefitinib and viral titers were quantified by plaque assay at 48 h. In a prophylaxis model (top), Gefitinib was given 16 h before viral infection and then continued daily, and in a therapeutic model (bottom), Gefitinib was given 1 h after viral infection and then continued daily (n = 7–9 mice/group repeated twice, mean ± SEM; **, P < 0.01 and ***, P < 0.001 vs. virus alone).

Figure 2.

Effect of EGFR inhibition on viral internalization and IFN-λ production. (A) NHBE cells were treated with IAV alone, or with 10 µM Gefitinib for 2 h and analyzed by flow cytometry. Cells infected with IAV alone (solid line), or IAV plus Gefitinib (dashed line), were stained with anti-HA Ab or second step Ab alone (solid histogram). Data shown are representative of four independent experiments. Similar findings were obtained with anti-NP and anti-M1 Abs (not depicted). (B) BEAS-2b cells were treated with serum-free medium (first column), DMSO (vehicle; second column), 10 µM Gefitinib (third column), IAV alone (fourth column), or IAV + Gefitinib (fifth column) for 30 min and cell culture homogenates were analyzed by Western blotting for IAV M1 protein (molecular masses are provided in kilodaltons). Data shown are representative of three independent experiments. (C) BEAS-2b cells were treated with IAV and RV alone, or with 10 µM Gefitinib. After 2 h, cell culture homogenates were collected and virus was quantified by plaque assay (n = 4 independent experiments, mean ± SEM). (D) NHBE cell cells were treated with serum-free medium alone (empty bars), 10 µM AG 1478, IAV and RV16 alone, or with AG 1478, and secreted IFN-λ was measured by ELISA at 24 h (n = 3 independent experiments, mean ± SEM; **, P < 0.005 vs. control; #, P < 0.05 vs. each virus alone). (E) C57BL/6 mice were treated with vehicle (DMSO), 50 mg/kg Gefitinib, or infected (intranasal) with IAV (10^4.5 TCID50%), or IAV plus Gefitinib. After 24 h, BAL was collected and IFN-λ was measured by ELISA in BAL (n = 6 mice/group representative of three independent experiments, mean ± SEM; *, P < 0.01 vs. vehicle alone; #, P < 0.05 vs. virus alone).

Figure 3.

IFN-λ is required for EGFR inhibitor-induced suppression of viral infection. (A) BEAS-2b cells were treated with IAV and RV1b alone, with 10 µM Gefitinib, and Gefitinib plus two neutralizing Abs to IFN-λ receptor (columns 3 and 4), two neutralizing Abs to IFN-λ (columns 6 and 7), and isotype-matched Abs (columns 5 and 8) for 24 h and viral titers of cell culture homogenates were assessed by plaque assay (n = 3 independent experiments, mean ± SEM; *, P < 0.05 vs. virus alone; #, P < 0.05 and ##, P < 0.01 vs. virus plus Gefitinib). (B) BEAS-2b cells were treated with serum-free medium alone (empty columns), or dsRNA (gray columns), and IFN-λ mRNA was analyzed by quantitative RT-PCR (n = 3 independent experiments, mean ± SEM; **, P < 0.005 vs. serum-free medium). (C) BEAS-2b cells were treated with serum-free medium alone (empty columns), or dsRNA (gray columns), and secreted IFN-λ was measured by ELISA at 24 h (n = 3 independent experiments; *, P < 0.01 and **, P < 0.005 vs. serum-free medium). (D) BEAS-2b cells were treated with serum-free medium alone (empty columns), or dsRNA (gray columns), and IRF1 mRNA was analyzed by quantitative RT-PCR (n = 3 independent experiments; *, P < 0.05 vs. serum-free medium).

Figure 4.

IRF1-dependent IFN-λ is required for EGFR inhibitor-induced suppression of viral infection. (A) BEAS-2b cells were treated with serum-free medium alone, or transfected with IRF1 or control (C) siRNA for 24 h and treated with serum-free medium alone (empty column), or IAV (striped column), RV1b (black column), and RV16. 24 h after viral infection secreted IFN-λ was measured by ELISA (n = 6 independent experiments, mean ± SEM; *, P < 0.05 vs. serum-free medium and C siRNA; #, P < 0.05 vs. C siRNA plus virus). BEAS-2b cells were transfected with IRF1 siRNA and IRF1 protein (molecular masses are provided in kilodaltons) was assessed by Western blotting (representative of three independent experiments). (B) BEAS-2b cells were treated with serum-free medium alone, or transfected with IRF1 or control (C) siRNA for 24 h and treated with IAV, or IAV plus 10 µM Gefitinib (Gef). After 24 h, cell culture homogenates were collected and viral titer was quantitated by plaque assay (n = 4 independent experiments, mean ± SEM; **, P < 0.005 vs. C and IRF1 siRNA plus Gef). (C) C57BL/6 mice were treated with vehicle (DMSO), 50 mg/kg Gefitinib, infected (intranasal) with IAV (10^4.5 TCID50%), or IAV plus Gefitinib. After 48 h, lungs were collected and IRF1 was measured by Western blotting (representative of five independent experiments; molecular masses are provided in kilodaltons). Densitometry (bottom) was calculated from Western blots (n = 5 independent experiments, mean ± SEM; **, P < 0.01 and ***, P < 0.001 vs. vehicle; #, P < 0.01 vs. IAV alone). (D) BEAS-2b cells were treated with serum-free medium alone, or transfected with IRF3 or control (C) siRNA for 24 h and treated with serum-free medium alone, or 25 µg/ml dsRNA. At 24 h, secreted IFN-λ was measured by ELISA (n = 3–4 independent experiments, mean ± SEM; #, P < 0.05 vs. serum-free medium and C siRNA; **, P < 0.005 vs. C siRNA plus dsRNA). BEAS-2b cells were transfected with IRF3 siRNA and IRF3 protein was assessed by Western blotting (representative of three independent experiments). Molecular masses are provided in kilodaltons.

Figure 5.

EGFR activation suppresses IRF1 and IFN-λ. (A) BEAS-2b cells were transfected with IRF1 luciferase reporter (left), and after 24 h treated with serum-free medium alone (empty columns), 10 ng/ml EGF, IAV (striped columns), and IAV plus EGF for 3 h before luciferase activity was measured (n = 3 independent experiments in duplicate; *, P < 0.01 vs. serum-free medium; #, P < 0.01 vs. IAV alone). IRF1 protein was measured (right) in BEAS-2b cells by Western blot 3 h after treatment with serum-free medium, 10 ng/ml EGF, IAV, and IAV plus EGF (data shown are representative of three independent experiments). Molecular masses are provided in kilodaltons. (B) BEAS-2b cells were treated with serum-free medium alone (empty columns), 10 ng/ml EGF, IAV (striped columns) and RV16 (black columns), or virus plus 10 ng/ml EGF, and secreted IFN-λ was measured by ELISA at 24 h (n = 6 independent experiments, mean ± SEM; ***, P < 0.0005 vs. control; ###, P < 0.0005 vs. each virus alone). (C) BEAS-2b cells were infected with IAV (striped columns) and RV16 (black columns) alone, or with the addition of 10 µM Gefitinib, 10 ng/ml EGF, or both Gefitinib and EGF for 24 h and viral titers in cell culture homogenates were assessed by plaque assay (n = 5 independent experiments, mean ± SEM; **, P < 0.01; ***, P < 0.001; ****, P < 0.0005 vs. virus alone).