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.

The secondary hypothesis is that PDX reduces recruited macrophage and neutrophil recruitment via repressing resident macrophage MCP-1, MIP-2 expression and release, respectively. Finally, we have been suggested that PDX inhibits neutrophil infiltration and transmembrane was associated with TNF-α/MIP-2/MMP9 signalling pathway.

| Animal preparation
C57BL/6 mice (20-25 g) were obtained from Slac Laboratory Animal (Shanghai, China). Mice were caged with free access to food and fresh water in a temperature-controlled room on a standard day-night cycle. The use of animals in the present study was ap-

| Flow cytometry
Freshly collected and isolated BALF cells were incubated with anti- Macrophage was identified by high expression of F4/80. Resident macrophage was defined by high expression of CD11b, and recruited macrophage was defined by high expression of CD11c, Ly6c.
Neutrophil was identified by high expression of Ly6c and Ly6g.

| Resident macrophages depletion
To deplete resident macrophages, clodronate liposome was given intratracheal in a volume of 50 µl (5 mg/mL) 72 hours before LPS challenge. PBS liposome was used as control. Next, mice were stimulated with LPS (1 mg/ kg) with or without PDX (0.1 µg/mouse) for 24 hours. Then BALF was harvested.

| Quantitative real-time RT-PCR
Total RNA from lung tissues was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA), according to the manufacturer's protocol.
The cDNA was synthesized using a reverse transcription Kit. Gene expression was detected using SYBR green super-mix PCR kit. Then MIP-2, MCP-1 and TNF-α mRNA level were measured. Next, Neutrophils, resident and recruited macrophages were sorted and stimulated with 1 μg/mL LPS and/or 100 nmol/L PDX for 24 hours, then CXCR2, CCR2 and TNFR expression were assayed by real-time PCR.

| Statistical analysis
Data are presented as the mean ± SEM. All data were analysed by oneway ANOVA, followed by Tukey test for post hoc comparison. Significance was considered at the P < .05. Statistical analyses were performed using Prism 6.0 software (GraphPad Software, San Diego, CA, USA).

| PDX protected lung tissue from LPS-induced lung injury
As shown in Figure 1A, the lung tissues were seriously injured in both LPS (ih) and LPS (ip) groups. Treatment with PDX alleviated LPS-induced lung injury. Acute lung injury scores were in line with pathophysiological changes ( Figure 1B). In addition, the lung tissue homogenate TNF-α level was significantly higher in both the LPS (ih) and LPS (ip) groups than in the CTR group and was lower in the PDX treatment group ( Figure 1C). But there were no significant differences between the CTR group and PDX group (P > .05).

| PDX reduced inflammatory cell infiltration in LPS-induced lung injury via resident macrophages
Clodronate liposome was used to eliminate resident macrophage ( Figure 3A). As shown in Figure 3B, clodronate liposome depleted the vast majority of resident macrophages. Compared with LPS + PBS liposome group, the recruited macrophages and neutrophils were decreased in LPS + PDX+PBS liposome group (P < .05), but not resident macrophages (P > .05) ( Figure 3C-E), suggesting that PDX reduced inflammatory cells infiltration after LPS challenge, but had no effect on resident macrophage numbers. Compared with LPS + PBS liposome group, the resident macrophages, recruited macrophages and neutrophils were decreased in LPS + clodronate liposome group (P < .05), indicating that while clodronate liposome eliminates resident macrophages, recruited macrophage and neutrophils numbers also reduced (P < .05) ( Figure 3C-E). However, PDX had no effect on inflammatory cell numbers after clodronate liposome stimulation ( Figure 3D,E), indicating that PDX reduced inflammatory cell infiltration in LPS-induced lung injury via resident macrophages.

| PDX reduced resident macrophage MIP-2 and MCP-1 production and release in LPS-induced lung injury
LPS application not only increased MIP-2 and MCP-1 mRNA expression in tissues, but also up-regulated MIP-2 and MCP-1 level in the BALF compared with CTR treatment (P < .05). Treatment with PDX observably weakened the MIP-2 and MCP-1 concentration in tissues and BALF compared with LPS group (P < .05) ( Figure 4A,B,D,E).
However, there was no difference in the CXCR2 expression on neutrophils and CCR2 expression on recruited macrophages among these groups (P > .05) ( Figure 4C,F).
Next, CXCR2i (MIP-2 receptor inhibitor) and CCR2i (MCP-1 receptor inhibitor) were administered via intraperitoneal injection. As shown in Figure 4G,H, with or without PDX, treatment with CXCR2i and CCR2i reduced the number of neutrophils and recruited macrophages, respectively, indicating that the basic function of PDX was disappeared after using CXCR2 and CCR2 inhibitors.
In addition, Figure 4I,J showed that MIP-2 and MCP-1 were mostly presented on resident macrophages, and LPS stimulation increased the MIP-2 and MCP-1 mean fluorescence intensity (MFI).
Furthermore, the up-regulation of MIP-2 and MCP-1 expression induced by LPS was reduced by PDX.

| PDX reduced neutrophil recruitment via recruited macrophage TNF-α/MIP-2 signalling pathway
As shown in Figure 5A, TNF-α was mostly presented on recruited macrophages, and treatment with PDX suppressed the TNF-α MFI compared with LPS group (P < .05). Up-regulation of lung tissue TNF-α mRNA and BALF TNF-α concentration in the LPS group could be eliminated by PDX treatment (Figure 5B,C). Figure 5D showed that TNFR was mainly presented on macrophages, but there was no significant difference among these groups (P > .05) ( Figure 5E).
In addition, TNFRi (a TNF-α receptor inhibitor) was administered via intraperitoneal injection. With or without PDX, treatment with TNFRi reduced neutrophil numbers and MIP-2 level, but not MCP-1, indicating that the basic function of PDX was disappears after using TNFR inhibitors ( Figure 5F,G).

F I G U R E 2 PDX reduced recruited macrophage and neutrophil infiltration in LPS
In addition, the neutrophil numbers in the BALF were much lower than those in the lung homogenate in the LPS + PDX+MMP9i group (P < .05) ( Figure 6C). Compared with those in the LPS + PDX group, the neutrophil numbers in the LPS + PDX + MMP9i group were decreased in the BALF and increased in the lung homogenate (P < .05) ( Figure 6C).

| D ISCUSS I ON
In the present study, two LPS-induced lung injury models were created, one established by atomization inhalation of LPS and the other established by intraperitoneal injection of LPS. We found that both approaches could significantly damage the lungs and increase lung TNF-α expression. Moreover, 2 hours after LPS stimulation, neutrophils started infiltrating into the lungs and recruited macrophages followed at 6 hours after LPS stimulation.
PDX effectively alleviated lung injury, down-regulated TNF-α concentration and inhibited inflammatory cells recruitment. However, the data were much more stable after LPS inhalation; therefore, the LPS inhalation-induced lung injury model was used in our experiments.
In the present study, we showed that recruited macrophage and neutrophil numbers decreased when the resident macrophages were depleted, indicating that resident macrophages are associated with inflammatory cell accumulation. Our work was consistent with previous report that resident macrophages recruit helper macrophages into the infected bladder. 22 PDX had no effect on inflammatory cell numbers when the resident macrophages were depleted, suggesting that PDX reducing inflammatory cell accumulation depends on resident macrophages.

F I G U R E 3 The inhibition of LPS-induced inflammatory cell infiltration by
MIP-2 and MCP-1 play crucial roles in inflammatory cell infiltration, and increased expression of MIP-2 and MCP-1 has been reported in various pulmonary diseases, including chronic obstructive pulmonary disease, 23 ARDS 24 and asthma. 25 We have reported that MIP-2 recruited neutrophils to the damaged lung. 26 A previous report also demonstrated that MCP-1 recruited inflammatory monocytes to facilitate breast tumour metastasis. 27 Here, we showed that the expression of MIP-2 and MCP-1 was TNF-a can result in severe tissue damage and underlies a number of disease states, such as rheumatoid arthritis, ARDS and malignancy. 28,29 In the present study, we found that PDX could inhibit TNF-α expression to protect lung tissues, which was consistent with a previous study showing that PDX abolished zymosan-A-induced TNF-α production. 30 We also found that TNF-α was mainly expressed on recruited macrophages and worked by binding with TNFR, which was expressed on resident macrophages. Our work was consistent with previous research, which showed that chemical or genetic depletion of macrophages suggested that early recruited macrophages expressed TNF-α 31 and indicated that recruited exudative macrophages produced TNF-α after stimulation with LPS. 32 We also found that PDX inhibiting the MIP-2 level and neutrophil numbers was associated with TNF-α.
MMP9 derived from neutrophils during the inflammatory process can change alveolar capillary permeability and mediate neutrophil transmigration into the alveolar space. 33 In the present study, we found that MMP9 was mainly expressed on Ly6g + cells, which were identified as neutrophils. PDX reduced MMP9 expression, which was consistent with a previous study showing that  expression was reduced in the corneas of HSV-1-infected mice by treatment with AT-PDX. 34  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 F I G U R E 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 transmigration into the alveolar space in connection with TNF-α/ MIP-2/MMP9 signalling pathway.
In conclusion, we have shown that PDX alleviates lung injury through inhibition of MIP-2 and MCP-1 production and release by resident macrophages in LPS-induced lung injury. PDX inhibited recruited macrophage TNF-α expression. We also found that PDX inhibited neutrophil infiltration in connection with TNF-α/MIP-2/ MMP9 signalling pathway (Figure 7). Our findings suggest that PDX may provide a new therapy for the treatment of ARDS.

ACK N OWLED G EM ENTS
This work was sponsored by grants from the National Natural

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.