A Study On The Mechanisms Of M2 Macrophages Differentiating Into Osteoclasts

RA patients often suffered from various degrees of bone damage,such as joint stiffness and deformation,and osteoclasts plays an important role in this process.Monocytes,macrophages,immature DCs and MDSCs,as progenitor cells,could differentiate into osteoclasts through RANK-RANKL signaling pathway.Macrophages,as one of the most important immune cells in the body,could polarize into different phenotypes under different microenvironments,including the classical activated macrophages(M1)and the alternatively activated macrophage(M2).It is reported that there are high proportion of CD163+CD206+M2 macrophages in the synovial inflammatory cells of RA patients,but the effects of M2 macrophages in RA are not clear and whether M2 macrophages could differentiate into osteoclasts has been unknown.Our study aims to explore the bone destruction effects and its molecular mechanism of M2 macrophages derived-osteoclasts in RA.Firstly,we differentiated human and mous M2 macrophages with M-CSF and IL-4 in vitro and induced M2 differentiating in the presence of M-CSF and RANKL for 6-9 days,then we used osteoclasts detection methods,such as TRAP staining,F actin-ring staining,Q-PCR and bone destruction test,to verify that M-CSF and RANKL could induce M2 macrophages differentiating into functional osteoclasts.Secondly,we compared human and mouse M0,M1,M2 macrophages differentiating into osteoclasts by M-CSF and RANKL,which results showed that M2 macrophage are stronger for differentiating in osteoclast than M0,M1.In addition,we sorted M1 and M2 macrophages from normal and CIA mice and cultured with M-CSF and RANKL,which results showed that the ability of M2 differentiating to osteoclasts are stronger than M1 macrophages.And the expression of CD206 and RANK on M2 were higher than M0 and M1 by flow cytometry,which may be related to M2 differentiating into osteoclasts.Thirdly,CD206 ligand,mannan,can significantly block the differentiation of M2 macrophages to osteoclasts.Previous results in our laboratory showed that the level of the lactoferrin-containing immunocomplex(LTF-IC)increased obviously in the serum of rheumatoid arthritis(RA)patients,and LTF-IC can induce monocytes differentiating into functional osteoclasts through CD32 a expressed on the surface of the cell.It's reported that CD32 a is also highly expressed on the surface of M2 macrophages,therefore,we supposed that LTF-IC may activate M2 macrophages through the above pathway.The results indicated that LTF-IC could induce M2 macrophages to differentiate into functional osteoclasts and anti-CD32 a antibodies and the inhibitor of its downstream molecular Syk could block LTF-IC induced osteoclasts differentiation.In conclusion,we have found that M-CSF+RANKL and LTF-IC can induce CD206 high expressed M2 macrophages to differentiate into osteoclasts.Our studies provide new signal pathway for the formation of osteoclasts and provides new directions and targets for the diagnosis and treatment of rheumatoid arthritis.