EGFR activation suppresses respiratory virus-induced IRF1-dependent CXCL10 production

Abstract

Airway epithelial cells are the primary cell type involved in respiratory viral infection. Upon infection, airway epithelium plays a critical role in host defense against viral infection by contributing to innate and adaptive immune responses. Influenza A virus, rhinovirus, and respiratory syncytial virus (RSV) represent a broad range of human viral pathogens that cause viral pneumonia and induce exacerbations of asthma and chronic obstructive pulmonary disease. These respiratory viruses induce airway epithelial production of IL-8, which involves epidermal growth factor receptor (EGFR) activation. EGFR activation involves an integrated signaling pathway that includes NADPH oxidase activation of metalloproteinase, and EGFR proligand release that activates EGFR. Because respiratory viruses have been shown to activate EGFR via this signaling pathway in airway epithelium, we investigated the effect of virus-induced EGFR activation on airway epithelial antiviral responses. CXCL10, a chemokine produced by airway epithelial cells in response to respiratory viral infection, contributes to the recruitment of lymphocytes to target and kill virus-infected cells. While respiratory viruses activate EGFR, the interaction between CXCL10 and EGFR signaling pathways is unclear, and the potential for EGFR signaling to suppress CXCL10 has not been explored. Here, we report that respiratory virus-induced EGFR activation suppresses CXCL10 production. We found that influenza virus-, rhinovirus-, and RSV-induced EGFR activation suppressed IFN regulatory factor (IRF) 1-dependent CXCL10 production. In addition, inhibition of EGFR during viral infection augmented IRF1 and CXCL10. These findings describe a novel mechanism that viruses use to suppress endogenous antiviral defenses, and provide potential targets for future therapies.

Keywords: CXCL10; epidermal growth factor receptor; innate immunity; interferon regulatory factor 1.

Fig. 1.

Role of epidermal growth factor receptor (EGFR) in respiratory virus-induced inflammation. A: normal human bronchial epithelial (NHBE) cells were treated with serum-free medium alone (white bars), AG-1478 (10 μM), influenza A virus (H1N1, hatched bars), rhinovirus (RV) 1b and RV16 (black bars), and respiratory syncytial virus (RSV, gray bars) alone, or with AG-1478, and secreted interleukin (IL)-8 was measured by ELISA at 24 h (n = 6 independent experiments, means ± SE; *P < 0.05 and **P < 0.01 vs. control; #P < 0.05 vs. each virus alone). B: BEAS-2b cells were treated with serum-free medium alone or transfected with EGFR or control (C) siRNA for 24 h and treated with serum-free medium alone (white bars), or H1N1 (hatched bars), RV1b (black bars), RV16, and RSV (gray bars). After viral infection (24 h), secreted IL-8 was measured by ELISA (n = 8 independent experiments, means ± SE; **P < 0.001 and ***P < 0.0005 vs. serum-free medium and C siRNA; ##P < 0.001 and ###P < 0.0005 vs. C siRNA + virus). BEAS-2b cells were transfected with EGFR siRNA, and EGFR protein was assessed by Western blot (representative of 3 independent experiments). C: BEAS-2b cells were treated with serum-free medium alone (white bars), N-propyl galatte (nPG, 100 μM), diphenyleneiodonium chloride (DPI, 3 μM), tumor necrosis factor-α proteinase inhibitor-1 (TAPI, 10 μM), H1N1 (hatched bars), RV1b and RV16 (black bars), and RSV (gray bars) alone, or with nPG, DPI, and TAPI, and secreted IL-8 was measured by ELISA at 24 h (n = 3–5 independent experiments, means ± SE; **P < 0.005 vs. control; ##P < 0.005 and ###P < 0.0001 vs. each virus alone). D: BEAS-2b cells were treated with serum-free medium alone (white bars), H1N1 (hatched bars), RV1b and RV16 (black bars), and RSV (gray bars) alone, or with an EGFR Ab or isotype-matched control Ab, and secreted IL-8 was measured by ELISA at 24 h (n = 3–5 independent experiments, means ± SE; **P < 0.005, ####P < 0.0001 vs. control; #P < 0.05, ##P < 0.005, and ####P < 0.0001 vs. each virus alone). E: C57BL/6 mice were treated with vehicle (DMSO) or gefitinib (50 mg/kg) or infected (intranasally) with H1N1 (10^4.5 TCID50%) or H1N1 + gefitinib. After 24 h, bronchoalveolar lavage (BAL) was collected, and MIP2 was measured by ELISA in BAL (n = 5 mice/group repeated two times, means ± SE; *P < 0.01 vs. vehicle alone; ##P < 0.005 vs. virus alone).

Fig. 2.

EGFR activation suppresses respiratory virus-induced CXCL10 production. A: BEAS-2b cells were treated with serum-free medium alone or the addition of polyinosine-polycytidylic acid (poly I:C, 25 μg/ml; dsRNA), and secreted CXCL10 protein was measured by ELISA at the indicated time points (n = 3–6 independent experiments, means ± SE; ****P < 0.0001 vs. control). B: BEAS-2b (left) and NHBE (right) cells were treated with serum-free medium alone (white bars), transforming growth factor (TGF)-α (10 ng/ml), H1N1 (hatched bars), RV (black bars), and RSV (gray bars), or virus + TGF-α, and secreted CXCL10 protein was measured by ELISA at 24 h (n = 3–4 independent experiments, means ± SE; *P < 0.05, **P < 0.01, and ***P < 0.005 vs. control; #P < 0.05, ##P < 0.01, and ###P < 0.005 vs. each virus alone). C: BEAS-2b cells were treated with serum-free medium alone, epidermal growth factor (EGF, 10 ng/ml), TGF-α (10 ng/ml), or poly I:C (100 μg/ml; dsRNA) alone, or poly I:C + EGF and TGF-α. CXCL10 mRNA was analyzed by quantitative RT-PCR (n = 6 independent experiments; *P < 0.05 vs. serum-free medium; #P < 0.05 vs. dsRNA alone). D: NHBE cells were treated with serum-free medium alone (white bars), H1N1 (hatched bars), RV (black bars), RSV (gray bars), and virus + EGF (10 ng/ml) or TGF-α, and CXCL10 mRNA was analyzed at 8 h by quantitative RT-PCR (n = 4 independent experiments; *P < 0.05 vs. control; #P < 0.05 vs. each virus alone).

Fig. 3.

EGFR activation suppresses interferon regulatory factor (IRF) 1-induced CXCL10 production. 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 (white bars), or H1N1 (hatched bars), RV (black bars), and RSV (gray bars). After viral infection (24 h), secreted CXCL10 was measured by ELISA (n = 5 independent experiments, means ± SE; *P < 0.05 vs. serum-free medium; #P < 0.05 vs. serum-free medium and C siRNA; +P < 0.05 vs. C siRNA + virus). BEAS-2b cells were transfected with IRF1 siRNA, and IRF1 protein was assessed by Western blot (representative of 3 independent experiments). B: left, BEAS-2b cells were treated with serum-free medium alone, dsRNA, dsRNA + EGF (10 ng/ml) and TGF-α (10 ng/ml), and IRF1 mRNA was analyzed at 2 h by quantitative RT-PCR (n = 5 independent experiments; *P < 0.05 vs. serum-free medium; ##P < 0.005 vs. dsRNA alone). NHBE cells were treated with serum-free medium alone (white bars), H1N1 (hatched bars), RV (black bars), RSV (gray bars), and virus + EGF (10 ng/ml), and IRF1 mRNA was analyzed at 2 h by quantitative RT-PCR (n = 3 independent experiments in duplicate; *P < 0.05 vs. control; #P < 0.05 vs. each virus alone). C: BEAS-2b cells were transfected with IRF1 luciferase reporter and after 24 h treated with serum-free medium alone, EGF (10 ng/ml), dsRNA (100 μg/ml), and dsRNA + EGF for 3 h before luciferase activity was measured (n = 4 independent experiments in duplicate; ***P < 0.0005 vs. serum-free medium; ##P < 0.01 vs. dsRNA alone). D: IRF1 protein was measured in primary mouse tracheal epithelial cells (pMTECs) by Western blot 2 h after treatment with serum-free medium, dsRNA (100 μg/ml), dsRNA + EGF (10 ng/ml), and EGF alone (data shown are representative of 3 independent experiments).

Fig. 4.

EGFR inhibition increases IRF1-induced CXCL10 production in vitro and in vivo. A: BEAS-2b cells were treated with serum-free medium alone (white bars), gefitinib (Gef; 10 μM), AG-1295 (10 μM), H1N1 (hatched bars), RV (black bars), and RSV (gray bars), or virus + Gef and AG-1295, and secreted CXCL10 protein was measured by ELISA at 24 h (n = 3–6 independent experiments, means ± SE; *P < 0.05, **P < 0.005, and ***P < 0.0005 vs. control; ##P < 0.005 and ###P < 0.0005 vs. each virus alone). B: NHBE cells were treated with serum-free medium alone (white bars), AG-1478 (10 μM), H1N1 (hatched bars), RV (black bars), and RSV (gray bars), or virus + AG-1478, and secreted CXCL10 protein was measured by ELISA at 24 h (n = 3 independent experiments, means ± SE; *P < 0.05, vs. control; #P < 0.05 and ##P < 0.005 vs. each virus alone). C: BEAS-2b cells were transfected with IRF1 luciferase reporter and after 24 h treated with serum-free medium alone (white bars), gefitinib (Gef; 10 μM), dsRNA (100 μg/ml), and dsRNA + Gef for 3 h before luciferase activity was measured (n = 4 independent experiments in duplicate; *P < 0.05, **P < 0.005, and ***P < 0.0005 vs. control; ##P < 0.005 vs. dsRNA alone). D: C57BL/6 mice were treated with vehicle (DMSO) or gefitinib (50 mg/kg) or infected (intranasally) with influenza A virus (IAV, 10^4.5 TCID50%) or IAV + gefitinib. After 24 h, BAL was collected, and CXCL10 was measured by ELISA in BAL (n = 5 mice/group representative of 2 independent experiments, means ± SE; *P < 0.05 vs. vehicle alone; ##P < 0.005 vs. virus alone). E: C57BL/6 mice were treated with vehicle (DMSO) or gefitinib (50 mg/kg) or infected (intranasally) with IAV (10^4.5 TCID50%) or IAV + gefitinib, and viral titers were quantified by plaque assay at 48 h (n = 6 mice/group representative of 2 independent experiments, means ± SE; ***P < 0.001 vs. virus alone). F: C57BL/6 mice were treated with vehicle (DMSO) or gefitinib (50 mg/kg) or infected (intranasally) with IAV (10^4.5 TCID50%) or IAV + gefitinib, and total cell counts in BAL were measured (n = 3–4 mice/group repeated two times, means ± SE; *P < 0.05 vs. virus alone). G: C57BL/6 mice were treated with vehicle (DMSO) or gefitinib (50 mg/kg) or infected (intranasally) with IAV (10^4.5 TCID50%) or IAV + gefitinib, and percent neutrophils (N), monocyte/macrophages (M), and lymphocytes (L) was measured (n = 3–4 mice/group repeated two times, means ± SE; *P < 0.05, #P < 0.05, ##P < 0.005 vs. virus alone).

Fig. 5.

EGFR signaling affects virus-induced perforin and NK cell migration. 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 (white bars), H1N1 (hatched bars), RV (black bars), and RSV (gray bars), or each virus + gefitinib (Gef; 10 μM). After viral infection (24 h), secreted CXCL10 was measured by ELISA (n = 6 independent experiments, means ± SE; *P < 0.05 vs. control; #P < 0.05 vs. C siRNA + virus). B: C57BL/6 mice were treated with vehicle (DMSO) or gefitinib (50 mg/kg) or infected (intranasally) with IAV (10^4.5 TCID50%) or IAV + gefitinib. After 48 h, lung was collected, and perforin mRNA was measured by quantitative RT-PCR (n = 7 mice/group representative of 2 independent experiments, means ± SE; ***P < 0.001 vs. vehicle alone; ##P < 0.005 vs. virus alone). C: BEAS-2b cells were treated for 24 h with serum-free medium alone, dsRNA (25 μg/ml) alone, or with dsRNA and the addition of AG-1478 (10 μM), and TGF-α (10 ng/ml). Cell culture supernatants were collected and added to NKL cells in a standard cell migration assay for 3 h. Data are expressed as a percentage of serum-free medium (n = 5 independent experiments, means ± SE; *P < 0.05 and **P < 0.01 vs. control; #P < 0.05 vs. dsRNA alone).

Fig. 6.

Effect of EGFR signaling on IRF1-induced CXCL10 production. Top, respiratory viruses (e.g., influenza virus, RV, and RSV) stimulate airway epithelial NADPH oxidase (Nox), metalloproteinase (MP), and ligand-induced activation of EGFR, which leads to IL-8 production. EGFR activation suppresses IRF1-induced CXCL10. Bottom, in the presence of EGFR inhibition (e.g., gefitinib and AG-1478), IRF1-induced CXCL10 is increased.