The Role And Mechanism Of Progranulin Regulating Regulatory T Cells To Affect Macrophage Polarization In Acute Lung Injury

Objective:To investigate the role of progranulin?PGRN?in the differentiation of regulatory T cells?Tregs?in an experimental acute lung injury?ALI?mouse model,and its effect on the polarization of macrophages and the development of lung injury,and its underlying mechanism.Method:1.Fifteen C57BL/6 male wild type?WT?mice were randomly divided into three groups,normal control group?WT group?,lipopolysaccharide intervene group?WT+LPS group?and LPS intervene with PGRN treatment group?WT+LPS+PGRN group?,n=5/group.Ten PGRN-deficient(PGRN^-/-)male mice with a C57/BL6 background were randomly divided into two groups,lipopolysaccharide intervene group(PGRN^-/-+LPS group)and LPS intervene with PGRN treatment group(PGRN^-/-+LPS+PGRN group),n=5/group.The LPS-induced ALI mouse model was built via intratracheal instillation.Thirty minutes after LPS instillation,the WT+LPS+PGRN group and PGRN^-/-+LPS+PGRN group was intratracheal instillation with PGRN.The mice were observed at 24 hours after LPS challenge.The blood,lung tissue,and spleen tissue were obtained.Mice lung tissues hematoxylin and eosin?H&E?staining,immunohistochemistry?IHC?of myeloperoxidase?MPO?,interleukin?IL?-10and terminal-deoxynucleoitidyl transferase mediated nick end labeling?TUNEL?and immunofluorescence?IF?of macrophage phenotypes were performed and the morphological changes were measured under microscope to evaluated the degree of injury,neutrophil aggregation,inflammation and apoptosis.The plasma expression of proinflammatory mediators IL-1?,IL-6,tumor necrosis factor?TNF?-?,IL-17A,chemokine?C-X-C motif?ligand 1 protein?CXCL1?and anti-inflammatory cytokine IL-10 were determined by enzyme linked immunosorbent assay?ELISA?.The proportion of Treg?CD4⁺CD25⁺Foxp3⁺?in peripheral blood mononuclear cells?PBMCs?and splenic mononuclear cells?MNCs?was tested by flow cytometry.2.RAW 264.7 cells from the same batch were divided into 5 groups,normal control group?Control group?,LPS stimulate group?LPS group?,LPS stimulate with IL-10 treatment group?LPS+IL-10 group?,LPS stimulate with PGRN treatment group?LPS+PGRN group?and LPS stimulate with IL-10 and PGRN co-treatment group?LPS+IL-10+PGRN group?,n=5/group.After incubation at 37°C for 24 hours,the phenotype?M1:CD86⁺,M2:CD206⁺?of macrophages was detected by flow cytometry.Result:1.Pathological changes in mouse lung tissueThe lung tissues stained with H&E were observed under optical microscope.In WT+LPS group,WT+LPS+PGRN group,PGRN^-/-+LPS group and PGRN^-/-+LPS+PGRN group,the lung tissues showed obvious edema,extensive alveolar wall thickening,alveolar collapse,alveolar hemorrhage,and a large number of inflammatory cell infiltration with statistical differences between the groups?F=151.00,P<0.0001?.But the above-mentioned lung pathological changes of WT+LPS+PGRN group and PGRN^-/-+LPS+PGRN group were significantly lighter,compared with their corresponding LPS intervene group(P_WT<0.0001,P_PGRN-/-<0.0001).No obvious lung injury and inflammation were observed in the WT group.2.The expression of cytokines in mouse plasmaThere were significant differences in the expression of mice plasma pro-inflammatory factors IL-1?,TNF-?,CXCL1,IL-17A,IL-6 and anti-inflammatory cytokine IL-10 in WT group,WT+LPS group,WT+LPS+PGRN group,PGRN^-/-+LPS group and PGRN^-/-+LPS+PGRN group(F_IL-1?=9.481,P_IL-1?=0.0006;F_TNF-?=33,08,P_TNF-?<0.0001;F_CXCL1=75.37,P_CXCL1<0.0001;F_IL-17A=42.66,P_IL-17A<0.0001;F_IL-6=25.89,P_IL-6<0.0001;F_IL-10=11.46,P_IL-10=0.0002).After PGRN treatment,the expression levels of IL-1?,TNF-?,CXCL1,IL-17A and IL-6 in WT+LPS+PGRN group and PGRN^-/-+LPS+PGRN group were significantly lower than those in WT+LPS group and PGRN^-/-+LPS group,respectively(WT mice:P_IL-1?<0.001,P_TNF-?<0.0001,P_CXCL1<0.0001,P_IL-17A<0.0001,P_IL-6<0.0001;PGRN^-/-mice:P_IL-1?<0.0001,P_TNF-?<0.0001,P_CXCL1<0.0001,P_IL-17A<0.0001,P_IL-6<0.0001).In addition,the expression of IL-10 of WT+LPS+PGRN group and PGRN^-/-+LPS+PGRN group was significantly higher than that of WT+LPS group and PGRN^-/-+LPS group,accordingly(P_WT<0.0001,P_PGRN-/-<0.05).3.The expression of IL-10 in mouse lung tissueThe expressions of IL-10 in lung tissue of WT group,WT+LPS group,WT+LPS+PGRN group,PGRN^-/-+LPS group and PGRN^-/-+LPS+PGRN group was significantly different?F=108.2,P<0.0001?.Compared with WT group,the expression of IL-10 in the lung tissue of WT+LPS group and PGRN^-/-+LPS group significantly increased,severally(P_WT<0.0001,P_PGRN-/-<0.0001).Compared with the WT+LPS group and PGRN^-/-+LPS group apart,the expression of IL-1 in the lung tissue of mice significantly reduced after PGRN treatment(P_WT<0.01,P_PGRN-/-<0.0001).4.Proportion of apoptotic cells in mouse lung tissueThere were significant differences in the percentages of apoptotic cells in the lung tissue of the WT group,WT+LPS group,WT+LPS+PGRN group,PGRN^-/-+LPS group and PGRN^-/-+LPS+PGRN group?F=151.30,P<0.0001?.Compared with WT group,apoptotic cells in the lung tissue of WT+LPS group and PGRN^-/-+LPS group were significantly increased(P_WT<0.0001,P_PGRN-/-<0.0001).Apoptotic cells in the lung tissue of the WT+LPS+PGRN group and PGRN^-/-+LPS+PGRN group were significantly reduced after PGRN treatment,compared with their LPS challenge group(P_WT<0.0001,P_PGRN-/-<0.0001).5.MPO expression in mouse lung tissueThe expressions of MPO in lung tissue of WT group,WT+LPS group,WT+LPS+PGRN group,PGRN^-/-+LPS group and PGRN^-/-+LPS+PGRN group was significantly different?F=390.4,P<0.0001?.Compared with the WT group,the expression of MPO in the lung tissue of WT+LPS group and PGRN^-/-+LPS group significantly increased,separately(P_WT<0.01,P_PGRN-/-<0.0001).After PGRN treatment,the expression of MPO in lung tissue of WT+LPS+PGRN group and PGRN^-/-+LPS+PGRN group significantly reduced compared with the WT+LPS group and PGRN^-/-+LPS group,respectively(P_WT<0.05,P_PGRN-/-<0.0001).6.The proportion of Treg in mouse PBMCs and splenic MNCsThere were significant differences in the proportions of Treg in PBMCs and splenic MNCs between the WT group,WT+LPS group,WT+LPS+PGRN group,PGRN^-/-+LPS group and PGRN^-/-+LPS+PGRN group(F_PBMCs=25.52,P_PBMCs<0.0001;F_MNCs=44.90,P_MNCs<0.0001).There was no statistical difference in proportions of Treg between WT group and WT+LPS or PGRN^-/-+LPS group.And in WT+LPS group and PGRN^-/-+LPS group,splenic MNCs significantly reduced compared with the WT group(P_WT<0.01,P_PGRN-/-<0.01).After PGRN treatment,the proportion of Treg significantly increased in PBMCs and splenic MNCs both in WT mice and PGRN^-/-mice(PBMCs:P_WT<0.0001,P_PGRN-/-<0.0001;MNCs:P_WT<0.001,P_PGRN-/-<0.0001).7.The polarization of lung macrophagesThere were significant differences in the proportions of M1macrophage and M2 macrophage between the WT group,WT+LPS group,WT+LPS+PGRN group,PGRN^-/-+LPS group and PGRN^-/-+LPS+PGRN group(F_M1=39.09,P_M1<0.0001;F_M2=109.20,P_M2<0.0001).In WT+LPS group and PGRN^-/-+LPS group,the proportions of M₁ macrophage significantly increased compared with the WT group,separately(P_WT<0.0001,P_PGRN-/-<0.0001).After PGRN treatment,the proportion of M₁ macrophage significantly reduced in WT mice and PGRN^-/-mice(P_WT<0.01,P_PGRN-/-<0.0001).While in WT+LPS group and PGRN^-/-+LPS group,the proportions of M₂ macrophage increased compared with the WT group(P_WT<0.05),but there was no statistical difference between PGRN^-/-+LPS group and WT group.After PGRN treatment,the proportion of M₂ macrophage significantly increased in WT mice and PGRN^-/-mice(P_WT<0.0001,P_PGRN-/-<0.0001).8.The polarization of RAW 264.7 cells in vitroThere were significant differences in the proportions of M1macrophage and M2 macrophage between the Control group,LPS group,LPS+IL-10 group,LPS+PGRN group,LPS+IL-10+PGRN group(F_M1=33.98,P_M1<0.0001;F_M2=66.03,P_M2<0.0001).Compared with the Control group,the ratios of M1 macrophage and M2 macrophage in LPS group significantly increased(P_M1<0.0001,P_M2<0.001).After IL-10treatment or PGRN treatment or co-treatment,the M1 macrophage ratio significantly reduced compared with the LPS group,respectively(P_LPS+IL-10<0.0001,P_LPS+PGRN<0.001,P_{LPS+IL-10+PGRN}<0.001),while the M2macrophage ratio significantly increased(P_LPS+IL-10<0.0001,P_LPS+PGRN<0.001,P_{LPS+IL-10+PGRN}<0.001).Specifically,the effect of IL-10treatment was best,followed by IL-10+PGRN co-treatment,and finally PGRN treatment.Conclusion:PGRN promotes Treg differentiation,improves uncontrolled inflammation and alleviates lung injury in LPS-induced ALI mice.The effect of PGRN is related to the expression of IL-10 affected by Tregs,which promote the M2 macrophage polarization.In addition,the effect of PGRN and IL-10 on promoting the M2 macrophage polarization from RAW 264.7 cell injury model are not synergistic.