The Study Of Osteopontin Regulating Macrophage Polarization And Fusion In Vitro

Background:Macrophages belong to the mononuclear phagocytic system and play an important role in eliminating pathogens,mediating inflammatory responses and tissue repair.Macrophages show significant heterogeneity depending on the microenvironment.Under the stimulation of LPS,macrophages are polarized to M1 phenotype,which is also known as classical activated macrophage.M1 phenotype macrophages can produce inflammatory cytokines such as TNF-α,IL-1β,IL-6,IL-12 and express iNOS molecules,participating in Th1 cell immunity.Under the stimulation of IL-4,macrophages are polarized to M2 phenotype,which is called alternative activated macrophage.M2 phenotype macrophages can express mannose receptor(CD206),Arg1,IL-10 and other molecules,which are involved in tissue repair.Macrophages are highly plastic cells,and the polarized macrophages can undergo phenotype shift.In fact,macrophage phenotypes can be regulated by the local microenvironment.Osteopontin(OPN)is widely expressed in various immune cells such as T cells,macrophages and dendritic cells.As a potential immunomodulatory factor,OPN is considered as a Thl-type proinflammatory cytokine mediating cell immunity,which can regulate cell adhesion,migration,survival and participate in granulation tissue formation.In recent years,a large number of studies have found that OPN also plays an important role in the anti-inflammatory mechanism,which may have both proinflammatory and anti-inflammatory effects.Foreign body giant cells(FBGCs)are multinuclear giant cells formed by the fusion of some macrophages.It was previously believed that the long-term existence of FBGCs in the tissue around implants was the main cause of foreign body rejection.However,more and more evidences show that the exact role of FBGCs in the foreign body reaction caused by biological materials still needs to be further explored.Some studies have suggested that FBGCs are also heterogeneous and have polarized phenotypes such as M1/M2 phenotype.FBGCs may have an indispensable positive effect on the formation of bone integration between implant and bone interface.Objective:Part I:Macrophage polarization experimentMouse macrophage line RAW264.7 was selected as the cell model,exogenous OPN was added.OPN was also co-cultured with polarization inducer(LPS,IL-4).qRT-PCR,Western blot,ELISA and immunofluorescence methods were used to detect polarization markers,so as to explore the effect of OPN on macrophage polarization.Part II:Macrophage fusion experimentMouse macrophage line RAW264.7 was selected as the cell model,and exogenous OPN was added.OPN and fusion inducer IL-4 were co-cultured for 4-5 days.FBGCs formation was observed by MGG staining.Fusion gene expression was detected by qRT-PCR and Western blot,so as to explore the effect of OPN on macrophage fusion into FBGCs.Methods:Part I:To explore the effect of exogenous OPN on macrophage polarization.Mouse RAW264.7 macrophages were cultured in vitro,and then were divided into groups as follows:①OPN dose dependent experiment:set control group,different concentrations of OPN(0.1,0.5,1μg/ml)were added to the experimental group.②M1 polarization experiment:set control group,LPS(100ng/ml)group,OPN(1μg/ml)group,LPS+OPN group.③M2 polarization experiment:set control group,IL-4(20ng/ml)group,OPN(1μg/ml)group,IL-4+OPN group.The mRNA levels of M1-type genes iNOS,TNF-α,IL-1β and M2-type genes CD206,Arg-1,IL-10 were detected by qRTPCR after 24 hours.Western blot was used to analyze iNOS,Arg-1 protein expression.The concentrations of TNF-α,IL-1β,IL-10 in culture supernatant were detected by ELISA assay.iNOS and CD206 expression was also detected by immunofluorescence assay.Part II:To explore the effect of exogenous OPN on macrophage fusion into foreign body giant cells.Mouse RAW264.7 macrophages were cultured in vitro,and then were divided into groups as follows:set control group,the OPN group was treated with different concentrations of OPN(0.1,0.5,1μg/ml),the IL-4(20ng/ml)group,the IL-4+OPN group.FBGCs were observed by MGG staining after 4-5days.The mRNA levels of fusion genes DC-STAMP and ATP6V0D2 were detected by qRT-PCR after 2 days.Western blot was used to analyze ATP6V0D2 protein expression.Results:Part Ⅰ:macrophage polarization experiment results1.qRT-PCR results showed mRNA levels of M1-type genes iNOS,TNF-α,IL-1β on OPN group were up-regulated(P<0.05),compared with control group;the upregulated level was the highest on LPS+OPN group(P<0.05).mRNA levels of M2type genes CD206,Arg-1 and IL-10 were also up-regulated on OPN group(P<0.05);the up-regulated level was the highest on IL-4+OPN group(P<0.05).2.Western blot results showed iNOS,Arg-1 expression on OPN group was increased,iNOS expression on LPS+OPN group was the highest;Arg-1 expression on IL4+OPN group was the highest.3.ELISA results showed cytokines TNF-α,IL-1β and IL-10 were up-regulated on OPN group(P<0.05);concentrations of TNF-α,IL-1β were the highest on LPS+OPN group(P<0.05);IL-10 concentration on IL-4+OPN group was the highest(P<0.05).4.Immunofluorescence results showed iNOS and CD206 expression was enhanced on OPN stimulation.iNOS expression was the highest on LPS+OPN group,and CD206 expression was the highest on IL-4+OPN group.Part Ⅱ:macrophage fusion experiment resultsRAW264.7 cells were cultured with IL-4(20ng/ml)for 4-5 days in vitro,multinuclear giant cell formation could be observed by MGG staining.After stimulated by OPN,some FBGCs were formed.IL-4+OPN group induced the largest number of FBGCs.qRT-PCR showed that mRNA levels of fusion genes DC-STAMP,ATP6V0D2 were up-regulated on OPN group,and the highest up-regulated level was found on IL4+OPN group.Western blot showed that ATP6V0D2 protein expression was upregulated on OPN group,and was up-regulated highest on IL-4+OPN group.Conclusion:The experimental results showed that OPN could stimulate the expression of M1 phenotype and M2 phenotype molecules in the absence of polarization inducers(LPS,IL-4),with a dose dependent mode within a certain concentration range(0.1-1μg/ml).Further studies showed that OPN could work with LPS and IL-4 synergistically.Therefore,it was believed that OPN which could activate quiescent macrophages to secrete a series of cytokines,may stimulate macrophages to a mixed M1-M2 phenotype.OPN may have both pro-inflammatory and anti-inflammatory effects,depending on the microenvironment in which the macrophages were located.We also found that OPN could induce macrophage fusion into multinuclear FBGCs,and had a synergistic effect with IL-4.Fusion genes DC-STAMP and ATP6V0D2 expression were up-regulated.