Lysophosphatidylcholine Alleviates Acute Lung Injury by Regulating Neutrophil Motility and Neutrophil Extracellular Trap Formation

Abstract

Sepsis is predominantly initiated by bacterial infection and can cause systemic inflammation, which frequently leads to rapid death of the patient. However, this acute systemic inflammatory response requires further investigation from the perspectives of clinical judgment criteria and early treatment strategies for the relief of symptoms. Lysophosphatidylcholine (LPC) 18:0 may relieve septic symptoms, but the relevant mechanism is not clearly understood. Therefore, we aimed to assess the effectiveness of LPC as a therapeutic treatment for acute inflammation in the lung induced by lipopolysaccharide in mice. Systemic inflammation of mice was induced by lipopolysaccharide (LPS) inoculation to investigate the role of LPC in the migration and the immune response of neutrophils during acute lung injury. By employing two-photon intravital imaging of the LPS-stimulated LysM-GFP mice and other in vitro and in vivo assays, we examined whether LPC alleviates the inflammatory effect of sepsis. We also tested the effect of LPC to human neutrophils from healthy control and sepsis patients. Our data showed that LPC treatment reduced the infiltration of innate immune cells into the lung. Specifically, LPC altered neutrophil migratory patterns and enhanced phagocytic efficacy in the damaged lung. Moreover, LPC treatment reduced the release of neutrophil extracellular trap (NET), which can damage tissue in the inflamed organ and exacerbate disease. It also reduced human neutrophil migration under inflammatory environment. Our results suggest that LPC can alleviate sepsis-induced lung inflammation by regulating the function of neutrophils. These findings provide evidence for the beneficial application of LPC treatment as a potential therapeutic strategy for sepsis.

Keywords: NET; imaging; inflammation; lysophosphatidylcholine; sepsis.

FIGURE 1

LPC obstructs mouse neutrophil migration during LPS stimulation. (A,B) Migration of mouse neutrophils on a fibronectin-coated confocal dish in the presence or absence of LPC under inflammatory conditions. Given conditions were treated with LPS (0.1 μg/ml) and LPS + LPC (LPS 0.1 μg/ml co-treated with 30 µM LPC) and incubated for 1 h on serum-starved neutrophils. Length unit, μm. (C) Velocity, (D) Displacement, and (E) Meandering index. (F) Mean fluorescence intensity of CD11a and (G) CD11b adhesion molecules on neutrophils. All experiments were independently repeated at least three times. ***p < 0.001, **p < 0.01, *p < 0.05.

FIGURE 2

LPC enhances the bactericidal activity of neutrophils. (A) Images taken from 30 min to 2 h pHrodo E.coli BioParticles were removed by neutrophils over time. When the phagosome was subjected to acidic conditions through phagocytosis, pHrodo E.coli BioParticles emitted bright green fluorescence inside the neutrophils. Scale bars, 20 μm. (B,C) Representative (B) and quantitative demonstration (C) of the effect of phagocytosis of E. coli particles on neutrophils by flow cytometry. All experiments were independently repeated at least three times. *p < 0.05.

FIGURE 3

LPC reduces the formation of mouse neutrophil extracellular trap (NET). (A) Fluorescent images showing NET formation. Representative images were acquired by staining for Sytox-orange (red), CitH3 (green) and nuclei (blue). (B) Western blots of MPO and CitH3; β-actin was used as a control. (C,D) The expression of MPO and CitH3 was measured by Western blot in lung tissues of LPS group and LPS + LPC group. Results were normalized to loading control (β-actin). (E) NET localization in the lung of a mouse with LPS-induced sepsis. Immunofluorescence staining of lung sections and NET formation. MPO (green) and CitH3 (red) labeling were used to detect NET release. Nuclei were stained with DAPI (blue). White lines indicate examples of double-stained NETs. (F) Representative images of ROS release (green) in mouse neutrophils. Scale bars, 30 μm. A representative data was shown from at least three times independent experiments.

FIGURE 4

LPC inhibits NETs in human neutrophils from sepsis patients. (A,B) Representative images of NET formation. Sytox (red) and CitH3 (green) in human neutrophils from a patient with sepsis and a healthy control with and without LPC treatment under inflammation conditions. Nuclei were stained with DAPI (blue). Scale bars, 30 μm. A representative data was shown from at least three times independent experiments. (C) The total number of DAPI-stained adherent neutrophil in field with healthy control compared to sepsis patient. (D,E) Quantification of CitH3-labeled NETs in healthy control and sepsis patient.

FIGURE 5

LPC reduces lung tissue damage and decreased innate immune cell infiltration in the inflammatory lung. (A) Schematic diagram of in vivo experiment (B) Mouse lung was imaged to identify neutrophils (green) and bloodstream (red). Scale bar, 50 µm. (C) Clustered neutrophils were less abundant in LPC-treated mice. (D) Confirmation of immune cells in inflamed lungs by flow cytometry. Innate immune cells were sorted using CD11b, Ly6G, and F4/80. (E) Quantitative confirmation by flow cytometry. (F) Representative images showing H&E staining in LPS-inflamed lung tissue for 48 h with and without LPC administration. (G) Histological score of lung injury. A representative data was shown from three times independent experiments. **p < 0.01 compared with LPS-only group.

FIGURE 6

LPC restores the adhesion process inhibited by the Mac-1 blockade. (A) Neutrophil migration was confirmed in the human blood neutrophils of a healthy donor stimulated with LPS (1 μg/ml) and concurrently treated with LPC (30 µM). Adhesion of neutrophils via Mac-1 blockade was also confirmed. Scale bars, 50 μm, Length unit, μm. (B) Velocity, (C) displacement, and (D) meandering index were checked according to each LPS and LPC treatment group. **p < 0.005, *p < 0.05. A representative data was shown from three times independent experiments.

FIGURE 7

Proposed role of LPC in neutrophil immune response during bacterial infection. Infection occurs by the invasion of pathogen like bacteria. Upon bacterial infection, LPC may act as a find-me signal. The find-me signal enhances the recognition of bacteria by neutrophils, which results in fast phagocytosis by forming phagolysosome with granules such as neutrophil granule myeloperoxidase (MPO). Consequently, neutrophils become activated to highly express phagocytic receptors. In this regard, the LPC-aided activated neutrophils efficiently phagocytize bacteria and reduce NET formation to mitigate tissue damage.