PDX regulates inflammatory cell infiltration via resident macrophage in LPS-induced lung injury

Abstract

Inflammatory cell infiltration contributes to the pathogenesis of acute respiratory distress syndrome (ARDS). Protectin DX (PDX), an endogenous lipid mediator, shows anti-inflammatory and proresolution bioactions. In vivo, the mice were intraperitoneally injected with PDX (0.1 µg/mouse) after intratracheal (1 mg/kg) or intraperitoneal (10 mg/kg) LPS administration. Flow cytometry was used to measure inflammatory cell numbers. Clodronate liposomes were used to deplete resident macrophages. RT-PCR, and ELISA was used to measure MIP-2, MCP-1, TNF-α and MMP9 levels. In vitro, sorted neutrophils, resident and recruited macrophages (1 × 10⁶ ) were cultured with 1 μg/mL LPS and/or 100 nmol/L PDX to assess the chemokine receptor expression. PDX attenuated LPS-induced lung injury via inhibiting recruited macrophage and neutrophil recruitment through repressing resident macrophage MCP-1, MIP-2 expression and release, respectively. Finally, PDX inhibition of neutrophil infiltration and transmembrane was associated with TNF-α/MIP-2/MMP9 signalling pathway. These data suggest that PDX attenuates LPS-stimulated lung injury via reduction of the inflammatory cell recruitment mediated via resident macrophages.

Keywords: acute respiratory distress syndrome; neutrophil; protectin DX; recruited macrophage; resident macrophage.

FIGURE 1

PDX attenuated LPS‐induced lung tissue damage. Mice received LPS by intratracheal atomization (ih) (1 mg/kg) or intraperitoneal injection (ip) (10 mg/kg) and then received PDX (0.1 µg/mouse) by intraperitoneal injection. Lung histological changes were assessed 24 h later by haematoxylin and eosin staining (A) and acute lung injury scoring (B). Aerosol inhalation and intraperitoneal injection of LPS both significantly increased the TNF‐α concentration in lung tissue homogenates, and this effect was markedly attenuated by PDX treatment. Data are presented as the mean ± SEM. n = 6‐8. *P < .05, **P < .01, ***P < .001

FIGURE 2

PDX reduced recruited macrophage and neutrophil infiltration in LPS‐induced lung injury in vivo. F4/80^‐Ly6c⁺Ly6g⁺ neutrophils, F4/80⁺Ly6C^‐CD11c^hiCD11b^int resident macrophages and F4/80⁺Ly6c⁺CD11c^loCD11b^hi recruited macrophages in the BALF were separated by flow cytometry (A). The numbers of neutrophils, resident macrophages and recruited macrophages in the BALF were determined by flow cytometry after LPS inhalation (1 mg/kg) (B) or intraperitoneal injection (10 mg/kg) (C). Next, PDX (0.1 µg/mouse) was administered to mice 10 min after LPS (1 mg/kg) inhalation; 24 h later, the three cell types were counted by flow cytometry (D). Data are presented as the mean ± SEM. n = 6‐8. *P < .05, **P < .01, ***P < .001

FIGURE 3

The inhibition of LPS‐induced inflammatory cell infiltration by PDX was dependent on resident macrophages. Resident macrophages were depleted in the lungs 72 h after intratracheal administration of 50 µl clodronate liposomes, and the administration of PDX (0.1 µg/mouse) occurred 10 min after LPS (1 mg/kg) stimulation (A). The numbers of resident macrophages (B, C), recruited macrophages (B, D) and neutrophils (B, E) in the BALF were measured by flow cytometry. CL = clodronate liposome, PB = PBS liposome. The data are presented as the mean ± SEM. n = 6‐8. *P < .05, **P < .01, ***P < .001

FIGURE 4

PDX down‐regulated LPS‐stimulated resident macrophage MIP‐2 and MCP‐1 expression and release to inhibit inflammatory cell infiltration. Mice received 1 mg/kg LPS by intratracheal atomization and then received PDX (0.1 µg/mouse) by intraperitoneal injection. MIP‐2 and MCP‐1 mRNA expression in lung tissue homogenates (A) and protein levels in the BALF (B) were measured 24 h later. Next, the sorted neutrophils (1 × 10⁶) and recruited macrophages (1 × 10⁶) were incubated with LPS (1 μg/mL) in the presence or absence of PDX (100 nmol/mL) for 24 h, respectively. CXCR2 mRNA expression on neutrophils and CCR2 mRNA level on recruited macrophages were measured by real‐time PCR (C,F). In addition, mice received a CXCR2 inhibitor (2 mg/kg) or CCR2 inhibitor (30 mg/kg) in the presence or absence of PDX via intraperitoneal injection 10 min after LPS administration. The number of neutrophils (G) and recruited macrophages (H) in the BALF was evaluated by flow cytometry. In addition, the mean fluorescence intensity (MFI) of MIP‐2 (I) and MCP‐1 (J) was assessed by flow cytometry. MIP‐2 and MCP‐1 were mainly expressed on resident macrophages, but this expression was strongly down‐regulated by PDX (I, J). CXCR2i = CXCR2 inhibitor, CCR2i = CCR2 inhibitor. Data are presented as the mean ± SEM. n = 6‐8. *P < .05, **P < .01

FIGURE 5

PDX inhibited neutrophil infiltration via recruited macrophage TNF‐α/MIP‐2 signalling pathway. Mice received 1 mg/kg LPS by intratracheal atomization and then received PDX (0.1 µg/mouse) by intraperitoneal injection. Lung homogenates were collected and processed into single‐cell suspensions 24 h later. The mean fluorescence intensity (MFI) of TNF‐α was assessed by flow cytometry (A). TNF‐α was mainly expressed on resident macrophages, but this expression was strongly down‐regulated by PDX (B). TNF‐α mRNA expression in lung tissue homogenates (B) and TNF‐α protein levels in the BALF (C) were measured 24 h later. Then, TNF‐α expression in different kinds of cells was assessed by flow cytometry (D). Next, the sorted resident macrophages (1 × 10⁶) were incubated with LPS (1 μg/mL) in the presence or absence of PDX (100 nmol/mL) for 24 h. TNF‐α mRNA expression on resident macrophages was measured by real‐time PCR (E). In addition, mice received a TNFR inhibitor (15 mg/kg) in the presence or absence of PDX via intraperitoneal injection 10 min after LPS administration. The number of neutrophils (F) in the BALF was evaluated by flow cytometry, and the MIP‐2, MCP‐1 level was measured by ELISA (G). TNFRi = TNFR inhibitor. Data are presented as the mean ± SEM. n = 6‐8. *P < .05, **P < .01

FIGURE 6

PDX regulated neutrophil transmigration into the alveolar space in connection with TNF‐α/MIP‐2/MMP9 signalling pathway. A CXCR2 inhibitor (2 mg/kg), TNFR inhibitor (15 mg/kg) or MMP9 inhibitor (15 mg/kg) in the presence or absence of PDX was administered via intraperitoneal injection after LPS administration. MMP9 expression in the BALF was measured by ELISA (A). MMP9 expression on cells was assessed by flow cytometry (B). Neutrophil numbers in the BALF and lung tissues (C) were measured by flow cytometry. Data are presented as the mean ± SEM. n = 6‐8. *P < .05, **P < .01, ***P < .001

FIGURE 7

Graphical summary of the sequence of events. Our findings document the following sequence of events (Figure 7): 1) resident macrophages sensed LPS stimulation and produced the chemokine MIP‐2 and MCP‐1, which recruited neutrophils and recruited macrophages; 2) recruited macrophages produced TNF‐α; 3) TNF‐α and MIP‐2 caused MMP9 expression in neutrophils, which allowed these cells to transmigrate into the alveolar space; and 4) these processes were regulated by PDX