Origin Of Synovial Macrophages And The Phenotypic Study Of Synovial Macrophages With Different Origins In Rheumatoid Arthritis

Rheumatoid arthritis(RA)is a joint synovial malignant lesions as the main pathological features of systemic autoimmune diseases(AID),its main performance for small joint swelling,joint synovial inflammation caused by invasive symmetry,which in turn leads to the destruction of the cartilage and bone,lead to joint rigidity deformity and dysfunction,its specific pathogenesis has not been elucidated.However,many studies have shown that macrophages are one of the key immune cells that cause RA and play an important role in the occurrence and development of RA.Macrophages are the body's first line of defense against exogenous damage.The conventional view is that macrophages are derived from monocytes.However,recent studies have found that macrophages in different tissues or organs do not come entirely from circulating monocytes in which yolk sac and fetal liver are the two main sources of macrophages in the embryonic stage.Macrophages in articular synovial membrane(SM)although have been found,however,the ontogenesis of SM is still not get system research.Importantly,different sources of SM including embryonic source synovial macrophages(ESM)and the source of the bone marrow synovial macrophages(BMSM)in patients with RA and the collagen induced arthritis(CIA)the role of animal models have not been systematic research,therefore,this study mainly discusses the SM ontogenesis and the role of different sources of SM in arthritisObjective:To investigate the different origins and ontogeny of SM,including the dynamic changes in embryonic perinatal neonatal and adult mice,and to compare the differences in cell function and phenotype of SM of different origins,as well as the phenotype in synovium of RA patients.Methods:1.Immunohistochemical method was used to determine the specific time of CX3CR1+/GFP mouse embryo SM implantation at the joint.2.The ontogenetic,phagocytosis and in situ proliferative capacity of ESM and BMSM was detected by flow cytometry.3.Expression of IL-4(Interleukin-4),IL-10(Interleukin-10),TNF-?(Tumor necrosis factor-?),IL-1?(Interleukin-4)in ESM and BMSM was detected by qRT-PCR.4.Bone marrow chimera model was established to detect the effect of bone marrow cells on ESM and BMSM ratio.5.The origin of BMSM was detected by CCR2 gene tool mice.6.Flow cytometry and immunofluorescence were used to observe the colonization of ESM and BMSM in mice at different ontogenetic stages.7.The CIA model was prEPared with chicken collagen and complete freuiden adjuvant,and the proportion of ESM BMSM and polarization phenotype in the disease course of CIA model were detected by flow cytometry.8.The change of ESM,BMSM proportion and polarization phenotype were verified by synovium of RA patients.Results:1.I dentification of SM in joints.Fluorescence results of identification of SM in CX3CR1+/GFP gene tool mice showed that F4/80'SM(ESM)appeared in E12.5.In addition,this result was also confirmed by immunohistochemistry and flow cytometry.With the development of embryo,ESM gradually increased,and F4/80-CD11b+SM(BMSM)was detected in E20.5.2.The ontogeny of ESM and BMSM.To explore different sources of SM ontogenesis,we tested in different periods of the development of mice after including the embryonic period perinatal newborn period and the adult in four stages the ESM and BMSM results show that the proportion of cells with the embryonic development of F4/80+cells in E12.5 appear,in perinatal(E20.5)CD11b+cells appear,in the synovial membrane of newborn mice and adult mice,we found F4/80+CD11b-(ESM)gradually increased with the F4/80-CD11b+(BMSM)gradually reduce.At the same time,the mixed cell population of F4/80+CD11b+was also found,and these cells gradually increased as the ontogeny reached adulthood.3.Study on the origin of F4/80+CD11b+mixed cell population.In order to explore the source of this mixed cell population,we used Ly6C and MHC to identify three subsets of F4/80+CD11b+SM cells in adult wild-type mice,Ly6C+,Ly6C+MHCII-,Ly6C+MHC+ and used CCR2-/-tool mice to evaluate the contribution of classical circulating monocytes to Ly6C+SM.The results showed that the proportion of Ly6C+SM in CCR2-/-mice decreased significantly compared with adult wild-type mice,while the proportion from Ly6C-SM was similar to that from wild-type mice,suggesting that CCR2 deficiency did not affect ESM4.Comparison of cell characteristics and functions of ESM and BMSM.In order to further reveal the difference cell characteristics and functions of ESM,BMSM,we tested the two respectively of the cell to gobble up ability proliferation ability and the use of RT-qPCR technology test anti-inflammatory and pro-inflammatory factor expression.Using the FITC labeled glucan respectively to detect the phagocytosis of ESM and BMSM,results show that the ESM has increasing consuming activity.Further detected using Ki67 in prenatal postpartum proliferation ability according to the results of in situ in E16.5 ESM has high proliferative capacity,along with the embryonic development by give birth to postpartum its proliferation ability gradually decline RT-qPCR results showed that IL-4,IL-10 in ESM had a high expression level,while IL-1?,TNF-? had a high expression level in BMSM.In addition,bone marrow chimera model was used to further reveal the contribution of bone marrow cells to ESM BMSM.The results showed that most of the cells after BMSM transplantation were rEPlaced,and ESM had a certain resistance to radiation.5.The role of ESM and BMSM in CIA mouse inflammatory model and synovial rEPlacement model in RA patients.In order to further study the role of ESM and BMSM in arthritis,we establish the CIA inflammation model in mice and patients with RA synovial alternative model and extract the SM respectively in the CIA mice at different stages of the test the dynamic expression patterns.The results showed that the proportion of ESM inflammation in the initial stage to the gradual decline in the peak stage,gradually rise in the disease recovery phase,however,BMSM rendering instead of expression patterns in addition to the differences in detection of polarization mode,the results show that the ESM tended to the polarization of M2 subtype anti-inflammatory and BMSM proinflammatory M1 subtype.This suggests that ESM plays an anti-inflammatory role in arthritis while BMSM plays a pro-inflammatory role and the results are rEProduced using the synovial membrane rEPlacement model in RA patients.Conclusion:1.SM was found that have two distinct origins:embryonic and bone marrow2.With the ontogeny of mice,the proportion of ESM decreased from embryo stage to perinatal stage.The expression pattern of BMSM is reversed by the rising trend from infancy to adulthood.3.ESM has a high ability of proliferation before birth,but it decreases with ontogeny.4.ESM exhibits a phenotype that inhibits inflammation,while BMSM exhibits a phenotype that promotes inflammation.5.CIA mice and RA patients showed a tendency toward the M2 subtype of ESM polarization in the synovial membrane,while BMSM showed a tendency toward the M1 subtype and the ESM ratio decreased to the peak stage in the initial stage of the disease course,and increased in the remission stage,while BMSM showed an opposite expression pattern.6.Compared with the synovium of OA patients,different SM subtypes similar to ESM and BMSM also exist in the synovium of RA patients,where ESM tends to be an anti-inflammatory phenotype,while BMSM tends to be a pro-inflammatory phenotype.