Effect Of Progranulin On P.g-LPS Induced Macrophage M1 Polarization And Underlying Mechanism

Background and PurposePeriodontitis is an inflammatory and infectious disease.Bacterial plaque initiates periodontal inflammation and host immune response plays a significant role in breakdown and resorption of tissues.Macrophages differentiate to pro-inflammatory M1 phenotype and anti-inflammatory M2 phenotype.When macrophages are recruited to diseased tissues,they are primed into different phenotypes depending on their exposure to different stimuli.When stimulated by lipopolysaccharide(LPS)or/and interferon gamma(IFN-γ),macrophages differentiate into M1 phenotype,involved in pro-inflammatory activity and in host defense against bacteria and viruses,secreting pro-inflammatory factors,resulting in breakdown of tissues.On the contrary,IL-4 or/and IL-13 induces macrophage to differentiate into M2 phenotype,behaving anti-inflammatory and pro-healing functions,secreting anti-inflammatory factors,resulting in regeneration of tissues.Progranulin(PGRN)is identified to be competitive antagonist binding to tumor necrosis-factor receptors(TNFRs)and plays significant role in anti-inflammation and immunoregulation.Our previous studies verified that concentrations of PGRN in gingival crevicular fluid of periodontitis patients was higher than that in normal gingival crevicular fluid.It was also revealed that PGRN enhanced osteogenesis differentiation of human periodontal ligament fibroblasts in vitro.Additionally,rPGRN inhibited inflammation and bone breakdown of periodontitis in experimental rats,promoting regeneration of bone defect.However,the role and mechanism of PGRN in modulating macrophage differentiation are not very clear.The current study is to investigate the effect of PGRN on macrophage M1 polarization and its underlying mechanism.Materials and Methods1.The effect of P.g-LPS on macrophages Ml polarizationRAW264.7 cells were treated in medium with or without 100 ng/ml P.g-LPS for 24 h.Surface marker of M1(CD86)was detected by flow cytometry.Expressions of TNF-αand iNOS were determined by qRT-PCR and Western Blot.Secretion of TNF-α was evaluated by ELISA.2.The effect of PGRN on phenotype and function of LPS-induced M1 macrophagesFirstly,RAW264.7 cells were divided into 1)Blank control group;2)P.g-LPS(100 ng/ml)group;3)100 ng/ml P.g-LPS plus various dosage(5,10,20,40 and 80 ng/ml)rPGRN groups.RAW264.7 cells were treated in each group medium respectively for 24 h.Surface markers of M1/M2(CD86/CD206)was detected by flow cytometry.Expressions of TNF-α and iNOS were determined by qRT-PCR and Western Blot.Secretion of TNF-α was evaluated by ELISA.Then,THP-1 and bone marrow derived macrophages(BMDMs)were derived into 1)Blank control group;2)P.g-LPS(100 ng/ml)group;3)100 ng/ml P.g-LPS+10 ng/ml rPGRN group.THP-1 and BMDMs cells were treated in each group medium respectively for 24 h.Expressions of TNF-α and iNOS were determined by qRT-PCR and Western Blot.Secretion of TNF-α was evaluated by ELISA.3.The mechanisms by which PGRN exerts effect on LPS-induced macrophage Ml polarizationIt is well known that TNF-α is one of the main effector molecules of macrophage M1 polarization and anti-inflammatory effects of PGRN are related to tumor necrosis factor receptor(TNFRs).Therefore,the role of TNF-α in LPS-induced macrophages M1 polarization was first evaluated,providing a reference for speculating whether the mechanism of PGRN inhibiting macrophages M1 polarization is related to TNFRs.Experiment Grouping:1)Blank control group;2)P.g-LPS(100 ng/ml)group;3)100 ng/ml P.g-LPS+anti-TNF-α(1:200)group;4)20 ng/ml rTNF-α group;5)30 ng/ml rTNF-α group;6)40 ng/ml rTNF-α group.RAW264.7 cells were treated in each group medium respectively for 24 h.Surface marker of M1(CD86)was detected by flow cytometry.Expressions of TNF-α and iNOS were determined by q-PCR and Western Blot.To further determine mechanism of effects of PGRN on LPS induced macrophages M1 polarization,RAW264.7 cells were divided into 1)Blank control group;2)10 ng/ml rPGRN group;3)P.g-LPS(100 ng/ml)group;3)100 ng/ml P.g-LPS+10 ng/ml rPGRN group.RAW264.7 cells were treated in each group medium respectively for 1 h.Phosphorylations of NF-κB and MAPK signal associated protein(iκκα/β,iκBα,p65,JNK,p38 and ERK)were determined by Western Blot.Nuclear translocation of NF-κB p65 was detected by immunofluorescence.Results1.P.g-LPS induced macrophage M1 polarizationCompared with blank control group,P.g-LPS stimulated significantly higher expression of CD86,TNF-α and iNOS in RAW264.7 cells.2.PGRN inhibited phenotype and function changes of M1 macrophage in RAW264.7Compared with blank control group,P.g-LPS stimulated significantly higher ratio of CD86/CD206,expression of TNF-α and iNOS,but lower when stimulated by P.g-LPS plus 5,10,20 ng/ml rPGRN in RAW264.7 cells.3.PGRN inhibited phenotype and function changes of M1 macrophage in THP-1 and BMDMsCompared with blank control group,P.g-LPS stimulated significantly higher expression of CD86,TNF-α and iNOS,but lower when stimulated by P.g-LPS plus 10 ng/ml rPGRN in THP-1 and BMDMs.4.TNF-α promoted pro-inflammatory cytokines and anti-TNF-α inhibited LPS-induced pro-inflammatory cytokines in RAW264.7In RAW264.7 cells,compared with blank control group,40 ng/ml rTNF-α stimulated significantly higher expression of TNF-α and iNOS,but no difference of M1 surface marker CD86.Similarly,anti-TNF-α inhibited P.g-LPS-stimulated expressions of TNF-α and iNOS,but no difference of CD86.5.PGRN inhibited LPS-induced activation of NF-κB and MAPK signal pathwayCompared with blank control group,P.g-LPS enhanced phosphorylation of iκκα/β,iKBa,p65,JNK and p38,but no difference of ERK.However,10 ng/ml rPGRN inhibited P.g-LPS-stimulated phosphorylation of iκκα/β,iκBα,p65,JNK and p38.ConclusionPGRN inhibits LPS-induced phenotype and function changes of M1 macrophage and its mechanism is involved in suppression of NF-κB and MAPK signal pathway and may also be related to the blocking of autocrine TNF-α signaling pathways.