Protectin D1 protects against lipopolysaccharide-induced acute lung injury through inhibition of neutrophil infiltration and the formation of neutrophil extracellular traps in lung tissue

Abstract

Protectin D1 (PD1), a DHA-derived lipid mediator, has recently been shown to possess anti-inflammatory and pro-resolving properties. To date, little is known about the effect of PD1 on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. The aim of the present study was to investigate the therapeutic effects of PD1 on LPS-induced ALI and its potential mechanisms of action. ALI was induced via an intraperitoneal injection of LPS, where PD1 (2 ng/mouse) was administered intravenously 30 min after LPS challenge. Mice were sacrificed 24 h after modeling. Lung histopathological changes were assessed using hematoxylin and eosin staining and myeloperoxidase (MPO) activity was tested using immunohistochemistry. Tumor necrosis-α and interleukin-6 levels in the bronchoalveolar lavage fluid (BALF) and serum were measured using ELISA. To detect neutrophil extracellular traps produced by infiltrated neutrophils in the lung tissue, immunofluorescence staining was performed using anti-MPO and anti-histone H3 antibodies. The results indicated that PD1 significantly attenuated histological damage and neutrophil infiltration in lung tissue, reduced the lung wet/dry weight ratio, protein concentration and proinflammatory cytokine levels in BALF and decreased proinflammatory cytokine levels in serum. Moreover, neutrophil citrullinated histone H3 (CitH3) expression was also reduced after PD1 administration. In conclusion, PD1 attenuated LPS-induced ALI in mice via inhibition of neutrophil extracellular trap formation in lung tissue. Therefore, PD1 administration may serve to be a new strategy for treating ALI.

Keywords: acute lung injury; neutrophil extracellular trap; neutrophil infiltration; protectin D1.

Figure 1

PD1 attenuates LPS-induced lung histopathological changes and pulmonary edema. (A) Representative H&E staining of lung tissues. Scale bars: 100 µm. (B) Histopathological scores of lung tissues. (C) Wet/dry weight ratios of lung tissues. n=7 in each group. PD1, protectin D1; LPS, lipopolysaccharide. ^****P<0.0001.

Figure 2

PD1 alleviates protein leakage and reduces proinflammatory cytokine levels in BALF. (A) Protein concentration in BALF. (B) Concentration of TNF-α in BALF. (C) Concentration of IL-6 in BALF. n=7 in each group. PD1, protectin D1; LPS, lipopolysaccharide; BALF, bronchoalveolar lavage fluid; TNF-α, tumor necrosis factor-α; IL-6, interleukin 6. ^***P<0.001 and ^****P<0.0001.

Figure 3

PD1 reduces serum proinflammatory cytokine levels. (A) Serum concentration of TNF-α. (B) Serum concentration of IL-6. n=7 in each group. PD1, protectin D1; LPS, lipopolysaccharide; TNF-α, tumor necrosis factor-α; IL-6, interleukin 6. ^**P<0.05 and ^****P<0.001.

Figure 4

PD1 reduces neutrophil infiltration in lung tissue. (A) Representative immunohistochemical images of MPO expression in the lung tissues. Red arrows represent infiltrating neutrophils. Scale bars: 50 µm. (B) Quantitative summary of MPO expression. n=7 each group. PD1, protectin D1; LPS, lipopolysaccharide; MPO, myeloperoxidase. ^***P<0.001 and ^****P<0.0001.

Figure 5

PD1 reduces neutrophil extracellular trap formation in mouse lungs. (A) Colocalization of CitH3 (green) with neutrophils (MPO, red) in the lung tissues. Scale bars: 100 µm. (B) Quantitative summary of neutrophil CitH3 expression. n=7 each group. PD1, protectin D1; LPS, lipopolysaccharide; CitH3, citrullinated histone 3; MPO, myeloperoxidase. ^**P<0.01 and ^****P<0.001.