Src Reduces Neutrophil Extracellular Traps Generation and Resolves Acute Organ Damage

Abstract

Neutrophil extracellular traps (NETs) are key factors mediating acute inflammatory injury. However, the underlying mechanisms and potential therapeutic targets remain unclear. Previous results suggest Src may be involved in regulating the NETs formation. Here, Src is found activated in the NETs model in vitro, in the murine- and human-derived neutrophils (acute pancreatitis and sepsis). Moreover, p-Src expression correlates with the clinical prognosis of acute pancreatitis and sepsis patients. Meanwhile, the inhibition of Src activity (gene silencing or inhibitors) inhibits NETs formation in vitro. Mechanistically, Src directly activates RAF1 by regulating phosphorylation at the Ser 621 site and mediates the RAF/MEK/ERK pathway, thereby affecting the intracellular ROS production. Alternatively, Src activates the RAF/MEK/ERK pathway by mediating PKC phosphorylation. In vivo, neutrophil Src - specific defect significantly reduces acute inflammatory response, organ damage, and the NETs formation in damaged tissue. Eventually, Src inhibitors are used and validated their pharmacological effects. These results identify Src as a key mediator in intracellular ROS production, NETs formation, and acute organ injury. Hence, Src inhibition may represent a promising therapeutic strategy for treating acute organ injury.

Keywords: NETs; ROS; Src; acute pancreatitis; sepsis.

Figure 1

Src kinase is required for the formation of NETs. A) Network analysis at 30‐ and 60‐min for PMA‐mediated NETs formation, predicting the number of transcriptional genes phosphorylated by different kinase cascades activating transcription factors. B) Representative flow cytometry plots and bar graphs depicting the expression levels of p‐Src in neutrophils. Data is presented as mean ± SD (N = 4). C) p‐Src protein abundance in neutrophils determined via western blotting (N = 3). Relative protein expression of p‐Src (N = 3); Src is used as a control for protein loading. D) Representative immunofluorescence image of p‐Src and MPO at 400 × (Scale Bar = 50 µm) and 1000 × (Scale Bar = 10 µm) magnification. Representative SEM of neutrophils between the control and PMA groups. White arrows: neutrophils, yellow arrows: NETs (N = 4). Densitometric analysis of p‐Src and MPO fluorescence (N = 4). E) Bone marrow neutrophils isolated from S100a8^cre Src^fl/fl and Src^fl/fl mice were cultured and stimulated with PMA (100 nm) for 4 h before flow cytometry analysis. Representative flow cytometry gating of bone marrow neutrophils (CD45.2⁺ CD11b⁺ Ly6G⁺). Representative flow cytometry plots and bar graphs depicting the proportion of neutrophils and the expression level of MPO. Data is presented as mean ± SD (N = 4). F) Representative immunofluorescence image of Cith3 and MPO at 400 × (Scale Bar = 50 µm) and 1000 × (Scale Bar = 10 µm) magnification. Representative SEM of neutrophils. White arrows: neutrophils, yellow arrows: NETs (n = 6). G) Densitometric analysis of Cith3 and MPO fluorescence (N = 6). H) Supernatant levels of IL6, TNF‐α, and MCP‐1 detected using ELISA (N = 6). I) Bone marrow neutrophils isolated from C57BL/6J mice cultured and stimulated with PMA (100 nm), and incubated with different doses of tirbanibulin (10, 50, and 100 nm) before flow cytometry analysis. Representative flow cytometry gating of bone marrow neutrophils (CD45.2⁺ CD11b⁺ Ly6G⁺). Representative flow cytometry plots and bar graphs depicting the proportion of neutrophils and the expression level of MPO. Data is presented as mean ± SD (N = 4). J) Representative immunofluorescence image of Cith3 and MPO at 400 × (Scale Bar = 50 µm) and 1000 × (Scale Bar = 10 µm) magnification. Representative SEM of neutrophils. White arrows: neutrophils, yellow arrows: NETs (N = 6). K) Densitometric analysis of Cith3 and MPO fluorescence (N = 6). L) Supernatant levels of IL6, TNF‐α, and MCP‐1 detected using ELISA (N = 6). Statistical significance was denoted as: ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001.

Figure 2

Src‐specific deletion inhibits NETs formation and protects against acute organ damage. A) The AP model is established by intraperitoneal injection of caerulein to S100a8^cre Src^fl/fl and Src^fl/fl mice. B) Protein levels of p‐Src in peripheral blood neutrophils of AP mice were determined using Western blotting (N = 3). C) Relative protein expression of p‐Src; Src is used as a control for protein loading (N = 3). Representative flow cytometry plots and bar graphs depicting the expression levels of p‐Src. Data is presented as mean ± SD (N = 6). D) Representative H&E staining of pancreatic tissues at 100 × (Scale Bar = 100 µm) and 400 × (Scale Bar = 50 µm) magnification. Representative immunofluorescence image of Cith3 and MPO at 1000 × magnification (Scale Bar = 10 µm) (N = 6 group⁻¹). E) Pathological scores of pancreatic tissues (N = 6 group⁻¹). F) Serum levels of amylase and lipase (N = 6 group⁻¹). G) Serum levels of IL6, TNF‐α, and MCP‐1 detected by ELISA (N = 6 group⁻¹). H) Densitometric analysis of Cith3 and MPO fluorescence (N = 6 group⁻¹). I) Sepsis model is established by CLP to S100a8^cre Src^fl/fl and Src^fl/fl mice. J) Protein levels of p‐Src in peripheral blood neutrophils of sepsis mice were determined using Western blotting (N = 3). Relative protein expression of p‐Src; Src is used as a control for protein loading (N = 3). K) Representative flow cytometry plots and bar graphs depicting the expression levels of p‐Src. Data is presented as mean ± SD (N = 6). L) Representative H&E staining of lung tissues at 100 × (Scale Bar = 100 µm) and 400 × (Scale Bar = 50 µm) magnification. Representative immunofluorescence image of Cith3 and MPO at 1000 × magnification (Scale Bar = 10 µm; N = 6 group⁻¹). M) Pathological scores of lung tissues (N = 6 group⁻¹). N) Serum levels of IL6, TNF‐α, and MCP‐1 detected by ELISA (N = 6 group⁻¹). O) Densitometric analysis of Cith3 and MPO fluorescence (N = 6 group⁻¹). P) Survivorship curve of mice in sepsis model induced by CLP (N = 10 group⁻¹). Statistical significance was denoted as: ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001.

Figure 3

Src regulates intracellular ROS production through the RAF/MEK/ERK signaling pathway. A) Reactome enrichment analysis of the differential genes of S100a8^cre Src^fl/fl + PMA and Src^fl/fl + PMA mice (N = 3). B) The protein levels of p‐RAF1, p‐MEK, and p‐ERK in neutrophils of Src^fl/fl, S100a8^cre Src^fl/fl, PMA+Src^fl/fl, and PMA+S100a8^cre Src^fl/fl groups determined by western blotting (N = 3). The protein levels of p‐RAF1, p‐MEK, and p‐ERK in neutrophils of four groups (Control, Control + Tirbanibulin, PMA, PMA + Tirbanibulin) assessed using western blotting (N = 3). C) Bone marrow neutrophils isolated from S100a8^cre Src^fl/fl and Src^fl/fl mice cultured and stimulated with PMA (100 nm) for 4 h before flow cytometry analysis. Representative bar graphs depicting the proportion of neutrophils and the expression level of ROS. Data is presented as mean ± SD (N = 4). D) Bone marrow neutrophils isolated from C57BL/6J mice cultured and stimulated with PMA (100 nm), and incubated with different doses of tirbanibulin (10, 50, and 100 nm) before flow cytometry analysis. Representative bar graphs depicting the proportion of neutrophils and the expression level of ROS. Data is presented as mean ± SD (N = 4). E) Representative bar charts of ROS expression level in five groups: Control, PMA, PMA+Tirbanibulin, PMA+GW5074, and PMA+Tirbanibulin+GW5074. Data is presented as mean ± SD (N = 4). F) Representative bar charts of ROS expression level in five groups: Control, PMA, PMA+Tirbanibulin, PMA+NAC, and PMA+Tirbanibulin+NAC. Data is presented as mean ± SD (N = 4). G) Supernatant levels of IL‐6, TNF‐α, and MCP‐1 detected by ELISA (N = 6). Statistical significance was denoted as: ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001.

Figure 4

p‐Src phosphorylates RAF1 at the Ser 621 site. A) Co‐IP of the interaction between Src and p‐Src with RAF1 in C57BL/6J mouse bone marrow neutrophils before and after PMA stimulation. B) Co‐IP of the interaction between p‐Src and RAF1. Neutrophil lysates isolated from the bone marrow of C57BL/6J mice assessed by Co‐IP with anti‐Src Family (photo Y418) antibody or anti‐RAF1 antibody. C) Co‐IP of the interaction between p‐Src and RAF1 in HEK‐293T cells. D) SDS‐PAGE separation and silver staining of protein Co‐IP with anti‐Src Family (photo Y418) antibody. E) Mass spectrometric analysis identified phosphorylated RAF1 at S619 and S621. Secondary spectra for the phosphorylated peptides are shown. F) Immunoprecipitation analysis of the interaction between anti‐Src Family (photo Y418) antibody and RAF1 in HEK‐293T cells transfected with RAF1 (WT, Mut‐Ser 621‐Ala, Mut‐Ser 621‐Asp) plasmids. Immunoprecipitation analysis of interaction between anti‐Flag immunomagnetic beads and p‐Src in HEK‐293T cells transfected with RAF1 (WT, Mut‐Ser 621‐Ala, Mut‐Ser 621‐Asp) plasmids. G) Immunoprecipitation analysis of the interaction between anti‐Src Family (photo Y418) antibody and RAF1 in HEK‐293T cells transfected with RAF1 (WT, Mut‐Ser 619‐Ala, Mut‐Ser 619‐Asp) plasmids. Immunoprecipitation analysis of interaction between anti‐Flag immunomagnetic beads and p‐Src in HEK‐293T cells transfected with RAF1 (WT, Mut‐Ser 619‐Ala, Mut‐Ser 619‐Asp) plasmids. H) Representative immunofluorescence image of p‐Src and p‐RAF1 at 2500 × (Scale Bar = 5 µm) magnification.

Figure 5

Tirbanibulin represses the release of NETs and acute organ damage in AP and sepsis murine models. A) The AP model is established via intraperitoneal injection of caerulein to C57BL/6J mice. Three hours after the first injection, mice are gavaged with tirbanibulin (20 mg kg⁻¹). B) Representative H&E staining of pancreatic tissues at 100 × (Scale Bar = 100 µm) and 400 × (Scale Bar = 50 µm) magnification. Representative immunofluorescence image of Cith3 and MPO at 1000 × magnification (Scale Bar = 10 µm; N = 6 group⁻¹). C) Pathological scores of pancreatic tissues (N = 6 group⁻¹). D) Serum levels of amylase and lipase (N = 6 group⁻¹). E) Serum levels of IL‐6, TNF‐α, and MCP‐1 detected by ELISA (N = 6 group⁻¹). F) Densitometric analysis of Cith3 and MPO fluorescence (N = 6 group⁻¹). G) The sepsis model is established by CLP in C57BL/6J mice. Three hours after surgery, mice were given tirbanibulin (40 mg kg⁻¹) by gavage. H) Representative H&E staining of lung tissues at 100 × (Scale Bar = 100 µm) and 400 × (Scale Bar = 50 µm) magnification. Representative immunofluorescence image of Cith3 and MPO at 1000 × magnification (Scale Bar = 10 µm; N = 6 group⁻¹). I) Pathological scores of lung tissues (N = 6 group⁻¹). J) Serum levels of IL‐6, TNF‐α, and MCP‐1 detected by ELISA (N = 6 group⁻¹). K) Densitometric analysis of Cith3 and MPO fluorescence (N = 6 group⁻¹). L) Survivorship curve of mice in sepsis model induced by CLP (N = 9 group⁻¹). Statistical significance was denoted as: ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001.

Figure 6

Phosphorylation of Src is significantly increased in peripheral blood neutrophils and is associated with disease prognosis. A) Representative flow cytometry gating of human peripheral blood neutrophil cells (CD45⁺ CD11b⁺ CD16⁺). Representative flow cytometry plots and bar graphs depicted the expression levels of p‐Src in neutrophils. Data is presented as mean ± SD. Neutrophils isolated from the peripheral blood of controls (N = 20), AP patients with non‐local complications (Non‐LC AP, N = 41), AP patients with local complications (LC AP, N = 33), survivors sepsis patients (N = 23), and Non‐survivors sepsis patients (N = 17). B) Plasma NETs levels (MPO‐DNA Complexes and dsDNA) across all groups: control (N = 20), non‐LC AP patients (N = 41), LC AP patients (N = 33). The y‐axis depicts plasma MPO‐DNA complexes expressed as a percentage of controls ± SD, arbitrarily set at 100%. C) Plasma IL‐6 and TNF‐α levels across all groups: control (N = 20), non‐LC AP patients (N = 41), LC AP patients (N = 33). D) Heatmap of correlation coefficients between serum p‐Src levels and other clinical indices in AP patients. E) Plasma NETs levels (MPO‐DNA complexes and dsDNA) across all groups: control (N = 20), survivors sepsis patients (N = 23), Non‐survivors sepsis patients (N = 17). The y‐axis depicts plasma MPO‐DNA complexes expressed as a percentage of controls ± SD, arbitrarily set at 100%. F) Plasma IL‐6 and TNF‐α levels across all groups: control (N = 20), survivors sepsis patients (N = 23), Non‐survivors sepsis patients (N = 17). G) Heatmap of correlation coefficients between serum p‐Src levels and other clinical indices in sepsis patients. Statistical significance was denoted as: ^** P < 0.01, ^*** P < 0.001.