Intermediate M1/M2 Polarized Macrophages Highly Express HB-EGF Promoting Lung Tissue Repair In Lung Injury

Background: Acute respiratory distress syndrome(ARDS)is a serious respiratory disorder with significant mortality in patients.Alveolar epithelial cells(AECs)injury is the main pathological basis of ARDS.Therefore,AECs proliferation is a prerequisite for the recovery of lung function in ARDS.The lung injury and repair in ARDS are closely related to alveolar macrophage(AM).It is known that AMs are activated to M1 phenotype at the acute phase of ARDS,resulting in exacerbation of lung injury.While at the resolving phase of ARDS,AMs are polarized to M2 phenotype contributing to repair lung tissue.In the development of lung injury,AMs can undergo a M1-to-M2 transition in which they exhibit a M1/M2 intermediate polarization(M1-M2)phenotype.However,the special feature and function of M1-M2 AMs are unknown.Our preliminary study showd that M1-M2 AMs expressed a high level of heparin-binding EGF-like growth factor(HB-EGF),a kind of epidermal growth factor.HB-EGF can promote tissue repair and stimulate alveolar epithelial cell type Ⅱ(AEC 2)proliferation.Therefore,we establish the hypothesis that M1-M2 AMs can promote AEC 2 proliferation and lung tissue repair through highly expressing HB-EGF in lung injury development.Objective: To investigate whether M1-M2 AMs secrete a high level of HB-EGF in ARDS development and the role and mechanism of HB-EGF in promoting lung injury repair.Methods:There are two parts of the experiments.Experiments in vivo were conducted on male C57BL/6 mice of 6 to 8 weeks old,weighing 20 to 25g.Acute lung injury was induced by intratracheal instillation of lipopolysaccharide(3 mg/kg)in male C57BL/6 mice.To investigate HB-EGF expression in this model,mice were sacrificed by abdominal aorta depletion under anesthesia at 0,1,2,3,until 7 days after LPS challenge,and taken bronchoalveolar lavage fluid(BALF),lung tissue from mice and AMs.ELISA techniques were used to detect the expression of HB-EGF in alveolar lavage fluid and in lung tissue in the process of lung injury repair.HB-EGF expression in macrophages in lung of lipopolysaccharide-induced ALI mice was determined by using immunofluorescence technique and further detected in M1,M2 and M1-M2phenotypes of both peritoneal macrophages and AMs in detection of HB-EGF and M1,M2 marker expression changes.The mice were randomly divided into the PBS group and HB-EGF group(n=5 per group).They were treated intratracheally with lipopolysaccharide(3 mg/kg).12 hours later,the PBS group was intraperitoneally injected with phosphate-buffered saline(PBS,the same volume as HB-EGF),and mice in the HB-EGF group were injected intraperitoneally with HB-EGF(5μg per mouse).Then each group was repeatedly given the same dose of the same reagent via the same route every 24 hours until the day before the mice were sacrificed.3,5,and 7 days after ALI,collect BALF and lung tissue.Lung injury scores,level of protein,Ig M,TNF-αand IL-6 in BALF of lipopolysaccharide-induced ALI mice were compared with those in mice challenged with recombinant exogenous HB-EGF and PBS.Similarly,the above experiment was repeated after administration of the EGFR signaling pathway inhibitor AG1478.Experiments in vitro were conducted on the murine alveolar macrophage(AMs)and peritoneal macrophages(PMs).AMs and PMs were incubated for 4 h or 24h with lipopolysaccharide(1μg/ml)and interferon-γ(20 ng/ml)were designated as M1,incubated for 4h or 24h with interleukin-4(20 ng/ml)were designated as M2 and treated with lipopolysaccharide(1μg/ml),interferon-γ(20 ng/ml)and interleukin-4(20ng/ml)for 4h were designated as M1-M2.AMs treated with PBS were designated as M0.RT-PCR technology was used to detect in M1,M2 and M1-M2 phenotypes of both peritoneal macrophages and AMs in detection of HB-EGF and M1,M2 marker expression changes.ELISA techniques were used to detect HB-EGF expression in AMs and PMs.Results:1.HB-EGF was highly expressed in alveolar macrophages of ALI mice,and the expression of HB-EGF on the seventh day after injury was significantly different from that on the first day(P<0.001).The content of HB-EGF in lung tissue homogenate expressed the same trend.2.After AM depleted,the expression of HB-EGF in ALI lung tissue was significantly reduced.3.HB-EGF is mainly expressed by alveolar macrophages in lipopolysaccharide-induced acute lung injury(ALI)in mice after 7 days.4.In cultured AMs,HB-EGF was mainly generated by M1-M2 AMs.5.Compared with LPS+PBS group,level of protein was significantly reduced by recombinant HB-EGF,after 3 days(P<0.001);after 5 days(P<0.001);after 7 days(P<0.001).Other indicators show the same trend,to promoted alveolar structural reconstruction and AEC 2 proliferation.6.After HB-EGF treatment,compared with the LPS+PBS group,the SPC proliferation signal increased significantly.7.At 7 days after ALI,the EGFR signaling pathway was significantly activated in the lung tissue.AG1478 can significantly inhibit the effect of HB-EGF in promoting lung injury repair.Conclusion: HB-EGF was highly expressed by M1-M2 alveolar macrophages in lipopolysaccharide-induced ALI development.HB-EGF mediate AEC 2 proliferation and alveolar structure reconstruction by activating the epidermal growth factor pathway of ACE 2 and promotes lung injury repair.