Degradation of EGFR on lung epithelial cells by neutrophil elastase contributes to the aggravation of pneumococcal pneumonia

Pneumococcus is the main cause of bacterial pneumonia. Pneumococcal infection has been shown to cause elastase, an intracellular host defense factor, to leak from neutrophils. However, when neutrophil elastase (NE) leaks extracellularly, it can degrade host cell surface proteins such as epidermal growth factor receptor (EGFR) and potentially disrupt the alveolar epithelial barrier. In this study, we hypothesized that NE degrades the extracellular domain (ECD) of EGFR in alveolar epithelial cells and inhibits alveolar epithelial repair. Using SDS-PAGE, we showed that NE degraded the recombinant EGFR ECD and its ligand epidermal growth factor, and that the degradation of these proteins was counteracted by NE inhibitors. Furthermore, we confirmed the degradation by NE of EGFR expressed in alveolar epithelial cells in vitro. We showed that intracellular uptake of epidermal growth factor and EGFR signaling was downregulated in alveolar epithelial cells exposed to NE and found that cell proliferation was inhibited in these cells These negative effects of NE on cell proliferation were abolished by NE inhibitors. Finally, we confirmed the degradation of EGFR by NE in vivo. Fragments of EGFR ECD were detected in bronchoalveolar lavage fluid from pneumococcal pneumonia mice, and the percentage of cells positive for a cell proliferation marker Ki67 in lung tissue was reduced. In contrast, administration of an NE inhibitor decreased EGFR fragments in bronchoalveolar lavage fluid and increased the percentage of Ki67-positive cells. These findings suggest that degradation of EGFR by NE could inhibit the repair of alveolar epithelium and cause severe pneumonia. Pneumococcus is the main cause of bacterial pneumonia. Pneumococcal infection has been shown to cause elastase, an intracellular host defense factor, to leak from neutrophils. However, when neutrophil elastase (NE) leaks extracellularly, it can degrade host cell surface proteins such as epidermal growth factor receptor (EGFR) and potentially disrupt the alveolar epithelial barrier. In this study, we hypothesized that NE degrades the extracellular domain (ECD) of EGFR in alveolar epithelial cells and inhibits alveolar epithelial repair. Using SDS-PAGE, we showed that NE degraded the recombinant EGFR ECD and its ligand epidermal growth factor, and that the degradation of these proteins was counteracted by NE inhibitors. Furthermore, we confirmed the degradation by NE of EGFR expressed in alveolar epithelial cells in vitro. We showed that intracellular uptake of epidermal growth factor and EGFR signaling was downregulated in alveolar epithelial cells exposed to NE and found that cell proliferation was inhibited in these cells These negative effects of NE on cell proliferation were abolished by NE inhibitors. Finally, we confirmed the degradation of EGFR by NE in vivo. Fragments of EGFR ECD were detected in bronchoalveolar lavage fluid from pneumococcal pneumonia mice, and the percentage of cells positive for a cell proliferation marker Ki67 in lung tissue was reduced. In contrast, administration of an NE inhibitor decreased EGFR fragments in bronchoalveolar lavage fluid and increased the percentage of Ki67-positive cells. These findings suggest that degradation of EGFR by NE could inhibit the repair of alveolar epithelium and cause severe pneumonia. Community-acquired pneumonia (CAP) has high morbidity and mortality rates worldwide (1File T.M. Community-acquired pneumonia.Lancet. 2003; 362: 1991-2001Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar), and severe CAP can cause sepsis and subsequent acute respiratory distress syndrome (2Cilloniz C. Ferrer M. Liapikou A. Garcia-Vidal C. Gabarrus A. Ceccato A. et al.Acute respiratory distress syndrome in mechanically ventilated patients with community-acquired pneumonia.Eur. Respir. J. 2018; 511702215Crossref Scopus (41) Google Scholar). Streptococcus pneumoniae is the primary causative microorganism of CAP (3File Jr., T.M. Streptococcus pneumoniae and community-acquired pneumonia: a cause for concern.Am. J. Med. 2004; 117: 39S-50SAbstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar). The main treatment for CAP is antimicrobial therapy; however, it has been severely hampered by the increase in antimicrobial-resistant strains of S. pneumoniae (4Mandell L.A. Bartlett J.G. Dowell S.F. File Jr., T.M. Musher D.M. Whitney C. Update of practice guidelines for the management of community-acquired pneumonia in immunocompetent adults.Clin. Infect. Dis. 2003; 37: 1405-1433Crossref PubMed Scopus (924) Google Scholar). Current pneumococcal vaccines have limited efficacies against specific serotypes. Owing to the diversity of pneumococcal serotypes and serotype replacement, CAP cannot be completely prevented (5Masomian M. Ahmad Z. Gew L.T. Poh C.L. Development of next generation Streptococcus pneumoniae vaccines conferring broad protection.Vaccines (Basel). 2020; 8: 132Crossref PubMed Scopus (85) Google Scholar). In addition, it is not highly effective in protecting against pneumococcal infection, since the antigens are not proteins but polysaccharides (6Musher D.M. Anderson R. Feldman C. The remarkable history of pneumococcal vaccination: an ongoing challenge.Pneumonia (Nathan). 2022; 14: 5Crossref PubMed Google Scholar). Therefore, elucidating the mechanisms of pathogenesis and severity of pneumococcal disease and developing alternative treatment and prevention methods for antimicrobial agents and current vaccines should be emphasized. Pneumococcal pneumonia is characterized by an excessive infiltration of neutrophils into the lung tissue (7Palmer C.S. Kimmey J.M. Neutrophil recruitment in pneumococcal pneumonia.Front. Cell. Infect. Microbiol. 2022; 12894644Crossref Scopus (10) Google Scholar). Neutrophils eliminate pathogens; however, S. pneumoniae is not killed by neutrophils. S. pneumoniae lyses neutrophils with its toxin pneumolysin and leaks elastase from neutrophils (8Domon H. Oda M. Maekawa T. Nagai K. Takeda W. Terao Y. Streptococcus pneumoniae disrupts pulmonary immune defence via elastase release following pneumolysin-dependent neutrophil lysis.Sci. Rep. 2016; 638013Crossref PubMed Scopus (45) Google Scholar). Neutrophil elastase (NE) degrades constituent proteins of lung tissue, such as elastin (9Mižíková I. Ruiz-Camp J. Steenbock H. Madurga A. Vadász I. Herold S. et al.Collagen and elastin cross-linking is altered during aberrant late lung development associated with hyperoxia.Am. J. Physiol. Lung Cell. Mol. Physiol. 2015; 308: L1145-L1158Crossref PubMed Scopus (54) Google Scholar), collagen (9Mižíková I. Ruiz-Camp J. Steenbock H. Madurga A. Vadász I. Herold S. et al.Collagen and elastin cross-linking is altered during aberrant late lung development associated with hyperoxia.Am. J. Physiol. Lung Cell. Mol. Physiol. 2015; 308: L1145-L1158Crossref PubMed Scopus (54) Google Scholar), and E-cadherin (10Boxio R. Wartelle J. Nawrocki-Raby B. Lagrange B. Malleret L. Hirche T. et al.Neutrophil elastase cleaves epithelial cadherin in acutely injured lung epithelium.Respir. Res. 2016; 17: 129Crossref PubMed Scopus (47) Google Scholar), and acts as an exacerbating factor in acute and chronic pulmonary diseases, including acute respiratory distress syndrome (11Wang Z. Chen F. Zhai R. Zhang L. Su L. Lin X. et al.Plasma neutrophil elastase and elafin imbalance is associated with acute respiratory distress syndrome (ARDS) development.PLoS One. 2009; 4e4380Google Scholar), cystic fibrosis (12Kelly E. Greene C.M. McElvaney N.G. Targeting neutrophil elastase in cystic fibrosis.Expert Opin. Ther. Targets. 2008; 12: 145-157Crossref PubMed Scopus (73) Google Scholar), and chronic obstructive pulmonary disease (13Demkow U. van Overveld F.J. Role of elastases in the pathogenesis of chronic obstructive pulmonary disease: implications for treatment.Eur. J. Med. Res. 2010; 15: 27-35Crossref PubMed Scopus (40) Google Scholar). Loss of epithelial cell integrity and shedding are common in these diseases, including pneumococcal pneumonia; however, the mechanisms of epithelial destruction by NE are not completely understood. Since NE degrades receptor proteins, such as the C5a receptor (14van den Berg C.W. Tambourgi D.V. Clark H.W. Hoong S.J. Spiller O.B. McGreal E.P. Mechanism of neutrophil dysfunction: neutrophil serine proteases cleave and inactivate the C5a receptor.J. Immunol. 2014; 192: 1787-1795Crossref PubMed Scopus (57) Google Scholar) and Toll-like receptor (15Domon H. Nagai K. Maekawa T. Oda M. Yonezawa D. Takeda W. et al.Neutrophil elastase subverts the immune response by cleaving toll-like receptors and cytokines in pneumococcal pneumonia.Front. Immunol. 2018; 9: 732Crossref PubMed Scopus (50) Google Scholar), expressed on the plasma membrane, we hypothesized that NE might also degrade epidermal growth factor receptor (EGFR) that is expressed in the alveolar epithelium and is involved in maintaining epithelial tissue. EGFR is a transmembrane protein with a molecular weight of 170 kDa and is responsible for mammalian epithelial maintenance; it consists of one each of extracellular domain (ECD), transmembrane domain, and intracellular domain (16Ferguson K.M. Structure-based view of epidermal growth factor receptor regulation.Annu. Rev. Biophys. 2008; 37: 353-373Crossref PubMed Scopus (268) Google Scholar). Binding of epidermal growth factor (EGF) to the EGFR results in receptor dimerization, autophosphorylation of two domains of cytoplasmic tyrosine kinase of the receptor, and subsequent induction of cell proliferation (17Wieduwilt M.J. Moasser M.M. The epidermal growth factor receptor family: biology driving targeted therapeutics.Cell. Mol. Life Sci. 2008; 65: 1566-1584Crossref PubMed Scopus (536) Google Scholar). Aberrations in EGFR signaling are associated with respiratory diseases, such as pulmonary fibrosis (18Ma X. Liu A. Liu W. Wang Z. Chang N. Li S. et al.Analyze and identify peiminine target EGFR improve lung function and alleviate pulmonary fibrosis to prevent exacerbation of chronic obstructive pulmonary disease by Phosphoproteomics analysis.Front. Pharmacol. 2019; 10: 737Crossref PubMed Scopus (12) Google Scholar), cancer (19Guo G. Gong K. Wohlfeld B. Hatanpaa K.J. Zhao D. Habib A.A. Ligand-independent EGFR signaling.Cancer Res. 2015; 75: 3436-3441Crossref PubMed Scopus (133) Google Scholar), and asthma (20Puddicombe S.M. Polosa R. Richter A. Krishna M.T. Howarth P.H. Holgate S.T. et al.Involvement of the epidermal growth factor receptor in epithelial repair in asthma.FASEB J. 2000; 14: 1362-1374Crossref PubMed Google Scholar). Therefore, abnormal EGFR signaling might be involved in exacerbating pneumococcal pneumonia. In this study, we examined whether NE degrades EGFR expressed in cells, using alveolar epithelial cells and a mouse model of pneumococcal pneumonia. EGFR exposes its ECD on the plasma membrane. We hypothesized that NE leaked from neutrophils targets the ECD of EGFR and degrades it. Therefore, we analyzed whether NE degraded the ECD of recombinant EGFR (rEGFR ECD). rEGFR ECD was exposed to NE (100–300 mU/ml) in the presence or the absence of NE inhibitor for 3 h and separated using SDS-PAGE to quantify the intensity of EGFR bands (Figs. 1, A and B and S1A). The intensity of EGFR bands significantly decreased in a dose-dependent manner with the addition of NE, whereas significantly higher band intensity of EGFR was detected in EGFR treated with NE inhibitor along with NE (300 mU/ml) (Fig. 1B). We also analyzed whether NE also degrades EGF, one of the ligands for EGFR. Recombinant EGF was exposed to NE (100–300 mU/ml) for 3 h in the presence or the absence of NE inhibitors and separated by SDS-PAGE to quantify the intensity of EGF bands (Figs. 1, C and D and S1B). The band intensity of EGF was significantly decreased in a dose-dependent manner with the addition of NE, whereas significantly higher band intensity of EGF was detected in EGF treated with NE inhibitor along with NE (300 mU/ml). These results suggest that NE degrades both EGFR ECD and EGF. Whether EGFR expressed in the alveolar epithelial cell line A549 was also degraded was analyzed in vitro. After NE treatment of A549 cells, total proteins were extracted, and EGFR was detected in cell extracts by Western blotting. The band intensity of EGFR significantly reduced in a dose-dependent manner by the addition of NE (Fig. 2, A and B and S2A). Significantly higher band intensity of EGFR was detected in A549 cells treated with NE inhibitor along with NE (300 mU/ml) than in cells treated with NE alone (Fig. 2, C and D and S2B). To confirm that the decrease in EGFR in NE-treated cells was not because of a decreased mRNA expression, mRNA was extracted from the cells, and real-time PCR was performed (Fig. 2E). No significant difference in EGFR mRNA levels was observed between NE-treated and untreated A549 cells. These results indicated that NE degraded EGFR expression in living cells without affecting the mRNA level of EGFR. When EGF binds to the ECD of EGFR, the EGF–EGFR complex is internalized into the cell for subsequent signaling (21Carpenter G. The EGF receptor: a nexus for trafficking and signaling.Bioessays. 2000; 22: 697-707Crossref PubMed Scopus (307) Google Scholar). The effect of EGFR degradation by NE on EGF internalization into the cell was analyzed using glutathione-S-transferase (GST)-fused EGF (GST-EGF). First, to confirm that the GST tag itself did not activate EGFR, A549 cells were stimulated with EGF, GST-EGF, or GST, and phosphorylated EGFR (pEGFR) was detected by Western blotting (Fig. S3A). The band intensity of pEGFR was similar in GST-stimulated and untreated cells but increased in cells stimulated with EGF or GST-EGF. Next, A549 cells exposed to NE in the presence or the absence of NE inhibitors were stimulated with GST-EGF. The uptake of GST-EGF by A549 cells was detected by Western blotting of A549 cell lysates using anti-EGF and anti-GST antibodies (Fig. 3A). The intensity of bands specific to anti-EGF antibody was reduced in NE-treated cells compared with that in untreated cells (lanes 3 and 5 in Figs 3, A and B and S3B). However, in cells treated with NE and NE inhibitors, bands specific to GST-EGF were detected at significantly higher intensities than those in cells treated with NE (lanes 5 and 6 in Figs 3, A and B and S3, B and C). Western blotting images of A549 cell lysate with anti-EGF antibody showed a band of approximately 37 kDa in addition to GST-EGF. EGF has been reported to have a precursor of 20 to 165 kDa in a study using mouse kidney (22Valcarce C. Björk I. Stenflo J. The epidermal growth factor precursor. A calcium-binding, beta-hydroxyasparagine containing modular protein present on the surface of platelets.Eur. J. Biochem. 1999; 260: 200-207Crossref PubMed Scopus (12) Google Scholar). Since A549 cells also express EGF (23Ikari A. Sato T. Watanabe R. Yamazaki Y. Sugatani J. Increase in claudin-2 expression by an EGFR/MEK/ERK/c-Fos pathway in lung adenocarcinoma A549 cells.Biochim. Biophys. Acta. 2012; 1823: 1110-1118Crossref PubMed Scopus (78) Google Scholar), it is possible that cell-derived precursors were detected with anti-EGF antibody. The intensity of the band of approximately 37 kDa was decreased in NE-treated cells compared with untreated A549 cells. In contrast, the intensity increased in cells treated with NE and NE inhibitors. Figure 1, B and C demonstrates that NE degrades EGF and that EGF precursors expressed in A549 cells can be degraded by NE. In addition, internalization of EGF bound to EGFR into A549 cells was visualized using pHrodo-EGF (24Suprynowicz F.A. Krawczyk E. Hebert J.D. Sudarshan S.R. Simic V. Kamonjoh C.M. et al.The human papillomavirus type 16 E5 oncoprotein inhibits epidermal growth factor trafficking independently of endosome acidification.J. Virol. 2010; 84: 10619-10629Crossref PubMed Scopus (54) Google Scholar). A549 cells treated with NE in the presence or the absence of NE inhibitor were incubated with pHrodo-EGF, and fluorescence of internalized pHrodo-EGF was microscopically observed and quantified. The fluorescence intensity of pHrodo-EGF was significantly lower in NE-treated cells than in untreated cells. However, consistent with the results in Figure 4, A and B, the fluorescence intensity was significantly higher in cells treated with NE and NE inhibitors than in cells treated with NE alone (Fig. 3, C and D). Therefore, when EGFR expressed on the surface of A549 cells is degraded by NE, the amount of EGF bound to and internalized by EGFR is reduced.Figure 4Neutrophil elastase (NE) exposure downregulates the phosphorylation of epidermal growth factor receptor (EGFR) and AKT. A549 cells were exposed to NE (300 mU/ml) in the presence or the absence of NE inhibitor (100 mg/ml) for 20 min. The culture medium was replaced with fresh Dulbecco’s modified Eagle's medium (DMEM), and cells were stimulated with epidermal growth factor (EGF) (50 ng/ml). A, expression of EGFR, phosphorylated EGFR (pEGFR), AKT, and phosphorylated AKT (pAKT) was determined by Western blotting. Representative Western blotting images are shown. B, C, quantification of band intensities of pEGFR and pAKT. Data represent mean ± SD of four individual experiments and were evaluated using ANOVA with Tukey’s multiple comparison tests. Unless otherwise indicated, no significant differences were observed between groups. Differences in letters between bars (a, b) indicate statistically significant differences between groups (p < 0.05). (1) versus (2): p > 0.9999, (1) versus (3): p < 0.0001, (1) versus (4): p < 0.0001, (1) versus (5): p < 0.0001, (2) versus (3): p < 0.0001, (2) versus (4): p < 0.0001, (2) versus (5): p < 0.0001, (3) versus (4): p < 0.0001, (3) versus (5): p < 0.0001, (4) versus (5): p < 0.0001, (6) versus (7): p > 0.9999, (6) versus (8): p < 0.0001, (6) versus (9): p < 0.0001, (6) versus (10): p < 0.0001, (7) versus (8): p < 0.0001, (7) versus (9): p < 0.0001, (7) versus (10): p < 0.0001, (8) versus (9): p < 0.0001, (8) versus (10): p < 0.0001, and (9) versus (10): p < 0.0001.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To analyze the effect of EGFR degradation by NE on EGF–EGFR signaling, we analyzed the activation of EGFR and AKT that is involved in essential downstream pathways of EGFR signaling (25Oda K. Matsuoka Y. Funahashi A. Kitano H. A comprehensive pathway map of epidermal growth factor receptor signaling.Mol. Syst. Biol. 2005; 1: 2005Crossref PubMed Scopus (875) Google Scholar). NE-treated A549 cells were stimulated by EGF, and pEGFR and phosphorylated AKT (pAKT) levels in cell lysates were detected by Western blotting (Fig. 4A). Upon EGF stimulation, the band intensities of pEGFR and pAKT were significantly lower in NE-treated cells than in untreated cells (lanes 3 and 4 in Figs. 4, A–C and S4). However, significantly higher band intensities of pEGFR and pAKT were noticed in cells cotreated with NE and NE inhibitor than in NE-treated cells (lanes 4 and 5 in Figs. 4, A–C and S4). These results indicate that EGFR degradation by NE hampers the activation of EGFR signaling induced by EGF. Activation of EGFR by ligands, such as EGF, promotes cell proliferation and migration, thereby promoting the repair of damaged epithelium (26Crosby L.M. Waters C.M. Epithelial repair mechanisms in the lung.Am. J. Physiol. Lung Cell. Mol. Physiol. 2010; 298: L715-L731Crossref PubMed Scopus (540) Google Scholar). We performed a wound healing assay to analyze whether the suppression of signaling owing to EGFR degradation by NE inhibited wound healing in lung epithelial tissues (Fig. 5, A and B). Because fetal bovine serum (FBS) present in the growth medium contains growth factors, we first confirmed that the addition of EGF further promoted cell proliferation. The rate of wound closure after stimulation with EGF for 24 h was quantified by image analysis. EGF-stimulated A549 cells showed a significantly higher rate of wound closure than did the unstimulated cells (lanes 1, 2, and 3 in Fig. 5, A and B). NE-treated cells stimulated with EGF for 24 h showed approximately 13% lower rates of wound closure than did non–NE-treated cells (lanes 2 and 4 in Fig. 5, A and B). In addition, the rate was significantly higher in NE-treated cells by approximately 8% than in cells treated with NE and NE inhibitors (lanes 4 and 5 in Fig. 5, A and B). These findings suggested that EGFR degradation by NE inhibited EGF-mediated cell proliferation. We have shown that NE degrades EGFR on the surface of alveolar epithelial cells in vitro. Therefore, we analyzed whether degraded EGFR fragments are detected in the BALF of pneumococcus-infected mice and whether NE is involved in this process. After intratracheal administration of S. pneumoniae, NE inhibitor or vehicle control (PBS) was intraperitoneally administered thrice every 6 h immediately after infection. BALF samples were collected and subjected to Western blotting using an antibody against the full-length of EGFR (Figs. 6A and S5A). EGFR-specific bands were detected in the BALF of mice in the infected group but not in the uninfected group. The intensity of the EGFR bands in BALF was reduced in mice administered NE inhibitor after infection compared with the infected group. Clear bands were detected when the same antibody was used against rEGFR ECD and mouse lung tissue lysate used as control. Western blotting was performed on the same samples using an antibody that specifically recognizes the intracellular domain of EGFR (Figs. 6B and S6B). No bands were detected in either BALF sample. An obvious band was detected in mouse lung tissue lysate but not in rEGFR ECD. These results suggest that the EGFR ECD is degraded and released into the BALF of pneumococcus-infected mice and that NE may be involved in this process. To confirm that EGFR degradation by NE occurs in lung tissue, we analyzed the amount of EGFR in lung tissues from model mice of pneumococcal pneumonia. Mice were infected with S. pneumoniae and treated with NE inhibitor or PBS, and lung tissue was harvested. Immunofluorescent staining was performed to detect EGFR expression in lung tissue (Fig. 7, A and B). EGFR expression reduced in infected mice treated with PBS compared with that in uninfected mice. In addition, EGFR expression increased in lung tissues of infected mice treated with NE inhibitor compared with that in infected mice treated with PBS. EGFR levels in lung tissues were detected by Western blotting also (Figs. 7, C and D and S6). Consistent with the immunofluorescence staining results, band intensities of EGFR reduced in the lung tissues of infected mice treated with PBS compared with those of uninfected mice. Furthermore, the band intensity of EGFR increased in the lung tissues of infected mice treated with NE inhibitor compared with that in mice treated with PBS. These results suggest that NE leakage by pneumococcal infection is involved in degrading EGFR on the surface of lung tissue cells. Since activation of EGFR promotes cell proliferation, it is predicted that a decrease in the amount of EGFR in lungs of mice infected with pneumococcus would result in a decrease in the expression of cell proliferation markers. Therefore, we finally detected Ki67, one of the cell proliferation markers in the lung tissue (27El-Zammar O. Rosenbaum P. Katzenstein A.L. Proliferative activity in fibrosing lung diseases: a comparative study of Ki-67 immunoreactivity in diffuse alveolar damage, bronchiolitis obliterans-organizing pneumonia, and usual interstitial pneumonia.Hum. Pathol. 2009; 40: 1182-1188Crossref PubMed Scopus (28) Google Scholar), by immunofluorescence staining and calculated the percentage of Ki67-positive cells (Fig. 8, A and B). The percentage of Ki67-positive cells in the infected mice treated with PBS was reduced compared with that in uninfected mice. In addition, the percentage of Ki67-positive cells in lung tissues of infected mice treated with NE inhibitor was increased compared with that of infected mice treated with PBS. These results suggest that degradation of EGFR by pneumococcal infection inhibits cell proliferation in lung tissue. Alveolar epithelial cells contribute to host defense by forming an epithelial barrier that provides physical defense against bacterial and viral stimuli and by producing surfactant proteins that promote phagocytosis and death of microbes (28Johnston S.L. Goldblatt D.L. Evans S.E. Tuvim M.J. Dickey B.F. Airway epithelial innate immunity.Front. Physiol. 2021; 12749077Crossref Scopus (14) Google Scholar). Loss of integrity of the epithelial barrier that is composed of alveolar epithelial cells is implicated in the development of pneumonia (29Brune K. Frank J. Schwingshackl A. Finigan J. Sidhaye V.K. Pulmonary epithelial barrier function: some new players and mechanisms.Am. J. Physiol. Lung Cell. Mol. Physiol. 2015; 308: L731-L745Crossref PubMed Scopus (106) Google Scholar), and initiating tissue repair and restoring barrier function in response to epithelial damage are important. Therefore, we hypothesized that aberrations in molecules related to tissue repair contribute to the exacerbation of pneumococcal pneumonia and analyzed the interaction between EGFR and NE. We observed that NE degraded the ECD of EGFR. EGFR degradation by NE reduced the binding of EGF to EGFR and subsequently inhibited EGFR signaling and epithelial cell proliferation. These results suggest that EGFR degradation by NE leaked from neutrophils owing to pneumococcal infection can inhibit the repair of the epithelial barrier. A schematic diagram of the mechanism through which EGFR degradation by NE inhibits alveolar epithelial repair is shown in Figure 9. Previous studies have revealed the role of NE in pneumococcal pneumonia focusing on direct tissue destruction (30Domon H. Terao Y. 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