Fig 2: Inhibition of EGFR phosphorylation promote M2 polarization by regulating glutamine metabolism through activation of PPAR?.A–F RAW264.7 macrophages were treated with LPS (1 µg/mL) for 24 h, with or without Erlotinib (20 µM) pretreatment for 30 min. A RT-qPCR analysis of mRNA expression of M2-related genes Mcr1 (n = 3). B RT-qPCR analysis of mRNA expression of M2-related genes Ym1 (n = 3). C Representative western blot of Arg1. D Flow cytometry analysis showing the level of M2 macrophage-associated markers CD206. E Percentage of CD206-positive RAW264.7 is shown (n = 3). I Mean fluorescence intensity (MFI) is shown (n = 3). G–I Macrophages were collected from bronchoalveolar lavage fluid of C57BL/6 mice subjected to CLP and were divided into sham-operated, CLP and CLP plus Erlotinib (100 mg/kg, gavage) pretreatmend for 2 h, and alveolar macrophages were identified with CD45 + CD11b + F4/80high. G CD206 expression on the surface of alveolar macrophage was analyzed by flow cytometry. H Percentage of CD206-positive alveolar macrophage is shown (n = 9). I Mean fluorescence intensity (MFI) is shown (n = 9). J Immunoblot analysis of p-PPAR? (Ser112), t-PPAR? in RAW264.7 cells treated with LPS (1 µg/mL) for 30 min with or without indicated concentration of PD168393 (10 µM) pretreatment for 30 min. K Fluorescence images depicting PPAR? translocation (left panel, scale bar, 50 µm; right panel, scale bar, 5 µm). L–N RAW264.7 cells were treated with LPS (1 µg/mL) for 24 h with or without Erlotinib (10 µM) or Rosiglitazone (Rosi (20 µM) pretreatment. L Cell surface CD206 were analyzed by flow cytometry. M Percentage of CD206-positive RAW264.7 is shown (n = 3). N Mean fluorescence intensity (MFI) is shown (n = 3). O–Q Macrophages were collected from bronchoalveolar lavage fluid of C57BL/6 mice subjected to CLP and were divided into Sham-operated, CLP and CLP plus Erlotinib (100 mg/kg, gavage) pretreatmend for 2 h, and alveolar macrophages were identified with CD45 + CD11b + F4/80high. O CD206 expression on the surface of alveolar macrophage was analyzed by flow cytometry. P Percentage of CD206-positive alveolar macrophage is shown (n = 9). Q Mean fluorescence intensity (MFI) is shown (n = 9). R–V RAW264.7 cells were treated with LPS (1 µg/mL) for 30 min with or without PD168393 (10 µM) pretreatment for 30 min. R Flow cytometry analysis of JC-1 for the detecting the change of mitochondrial membrane potential (??m) (left panel, JC-1 aggregates; right panel, JC-1 monomers). S The ratio of JC-1 aggregates /JC-1 monomers was calculated as ??m. T Total cellular ATP level was detected (n = 3). U Immunoblot analysis of IRG1, ATP5A, SDHA, Tubulin as a loading control. V Immunoblot analysis of SDHA and IRG1 in Control or IRG1 silenced RAW264.7 cells, Tubulin as a loading control. The graphs depict mean ± SD based on three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3: LPS promotes cell surface expression of EGFR in macrophage.A–F Macrophages were treated with LPS (1 µg/mL) for 6, 12, or 24 h with or without PD168393 (PD, 10 µM)) pretreatment for 30 min. A Flow cytometry analysis of EGFR cell surface expression in BMDM. B Percentage of EGFR-positive macrophage is shown. C Mean fluorescence intensity (MFI) is shown (n = 3). D Flow cytometry analysis of EGFR cell surface expression in RAW264.7. E Percentage of EGFR-positive macrophage is shown. F Mean fluorescence intensity (MFI) is shown (n = 3). G–I Macrophages were collected from peritoneal lavage of C57BL/6 mice subjected to CLP and were divided into Sham-operated, CLP and CLP plus Erlotinib (100 mg/kg, gavage) pretreatmend for 2 h, and peritoneal macrophages were identified with CD45 + CD11b + F4/80high. G EGFR intensity on the surface of peritoneal macrophage was analyzed by flow cytometry. H Percentage of EGFR-positive peritoneal macrophage is shown (n = 9). I Mean fluorescence intensity (MFI) is shown (n = 9). J, K Blood was collected from patients with clinical sepsis or health donors, and blood mononuclear cells were identified with CD14 and CD11c. J EGFR intensity on the surface of Mononuclear cells were analyzed by flow cytometry (20 Healthy donors and 33 Septic patients). K Percentage of EGFR-positive CD14 + CD11c + mononuclear cell is shown. The graphs depict mean ± SD based on three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 4: Inhibition of EGFR phosphorylation switches M1 phenotype to M2 phenotype and alleviates acute lung injury induced by sepsis.A–E Macrophages were collected from bronchoalveolar lavage fluid of C57BL/6 mice subjected to CLP and were divided into sham-operated, CLP and CLP plus Erlotinib (100 mg/kg, gavage) pretreatmend for 2 h, and alveolar macrophages were identified with CD45 + CD11b + F4/80high. A iNOS and CD206 expression on the surface of alveolar macrophage were analyzed by flow cytometry. B Percentage of iNOS-positive alveolar macrophage is shown (n = 9). C Mean fluorescence intensity (MFI) of iNOS is shown (n = 9). D Percentage of CD206-positive alveolar macrophage is shown (n = 9). E Mean fluorescence intensity (MFI) of CD206 is shown (n = 9). F–J Mice were injected intraperitoneally with LPS (20 mg/kg) or an equal volume of PBS, after pretreatment with Erlotinib (100 mg/kg) for 2 h. F The BALF neutrophils were analyzed by flow cytometry with neutrophils markers (CD11b and Ly6G). G Percentage of CD11b + Ly6G+ positive alveolar macrophage is shown (n = 9). H Mean fluorescence intensity (MFI) of CD11b + Ly6G+ is shown (n = 9). I Immune-staining of iNOS and CD206 in Lung sections. J Lung sections were stained with hematoxylin and eosin (H&E). Scale bar, 50 µm (upper panel) and 5 µm (lower panel). The graphs depict mean ± SD based on three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.