Changes Of Osteoclastic Microenvironment And Bone Marrow Edema During Collagen-induced Arthritis Model Development

ObjectiveAccording to the traditional paradigm,synovitis is the fundamental cause of rheumatoid arthritis(RA)bone erosion.However,the common anti-inflammatory factor biologics treatment are only effective in 70%of RA.It was already shown that in spite of clinical suppression of synovitis,joint destruction in RA might persist at a histological level.Indicating that synovitis is not enough to explain the pathogenesis of RA bone erosion.Magnetic resonance imaging(MRI)was widely used in RA,accumulated evidences have proved that bone marrow edema(BME)is the strongest predictor of bone erosion for RA.BME is also known as osteitis,indicating that the adipose tissue of bone marrow is replaced by a large number of inflammatory cells.At present,the mechanism of BME and how it mediate bone erosion is unclear.In our study,we utilised collagen-induced arthritis(CIA)model in order to assess the relationship between BME,bone marrow microenvironment and synovitis.From these works we try to identify the mechanisms of BME in RA bone erosion and find a new therapeutic target.Methods1.Changes of BME during the development of CIA modelDBA/1J mice were immunized with bovine type Ⅱ collagen and Freund’s adjuvant(at day 0 and 21).BME in CIA model was detected by 7.0T MRI.The relationship between joint synovitis and BME in CIA mice was observed by HE staining.2.Changes of bone marrow microenvironment during the development of CIA model2.1 Changes of osteoclasts and RANKL expression in bone marrow(1)The expression of CD11b+Gr-1low+med osteoclasts in bone marrow of CIA mice were analyzed by flow cytometry.The marker of osteoclast TRAP expression were detected in different stages of CIA mice by immunofluorescence.The changes of trabecula bone and the formation of bone channel were observed by HE and TRAP staining.Identify the distribution of osteoclasts in the bone marrow of CIA mice by HE staining.(2)The expression of RANKL on the surface of bone marrow cells in CIA mice were analyzed by flow cytometry and immunofluorescence.The source of RANKL expression in bone marrow cells were also analyzed by flow cytometry.(3)To define the changes of osteoclastogenesis after BME appearance,total bone marrow cells were cultured with different doses of RANKL(25 ng/ml,50 ng/ml,100 ng/ml)in vitro and OCs was confirmed by TRAP staining.2.2 Changes of immune cells and inflammatory factors in bone marrowTotal bone marrow cells were obstained at different stages of CIA mice.T cell,B cell,monocyte and plasma cell were detected by flow cytometry.The changes of IL-17,TNF-α,CCL3 and other related inflammatory gene expression in bone marrow cells were observed by quantitative PCR.3.A preliminary study on the related genes involved in the microenvironment of bone marrow3.1 Analysis of gene expression profile of RA bone marrowThe gene expression profile of active RA and osteoarthritis(OA)were analyzed by the common gene chip database(GEO).3.2 Expression of transcription factor SOX5 in bone marrow cells of CIA miceThe expression of SOX5 in bone marrow cells were detected by flow cytometry immunofluorescence and quantitative PCR at different stages of CIA model.Results1.Changes of BME during the development of CIA modelMRI showed BME can be detected at day 25 after the first immunization of CIA mice.At that time,there was no obvious clinical symptoms of CIA mice.The pathology of synovial was negative.Indicating that the occurrence of CIA BME is earlier than the pathological changes of synovitis.2.Changes of bone marrow microenvironment during the development of CIA model2.1 Changes of osteoclasts and RANKL expression in bone marrow(1)The results of immunofluorescence and flow cytometry showed the number of osteoclasts in the bone marrow increased at day 25.TRAP staining of the joint tissue showed the osteoclasts in the bone marrow were changed earlier than the joint tissue.Osteoclasts attached to the endometrium in the CIA mice was significantly increased after BME appearance,especially at day 28 and 35.Bone trabecular was significantly reduced in CIA mice after BME appearance.There was no obvious bone channel formation at day 25.(2)The expression of RANKL was also increased significantly during CIA development and mainly expressed by T cells and monocytes.(3)In vitro study indicated that bone marrow cells from day 28-45(with BME)could be differentiated to TRAP(+)cells at low dose RANKL.2.2 Changes of immune cells and inflammatory factors in bone marrowT cells and monocytes in bone marrow of CIA mice were significantly increased at the early stage of BME(day 25)and sustained at a high level to day 45.Plasma cells were also higher than those of healthy controls at the peak of BME.No significant difference was found in B cells.The expression of IL-17,TNF-α,CCL12 and CCL3 in bone marrow of CIA mice were elevated significantly.3.A preliminary study on the related genes involved in the microenvironment at bone marrow3.1 Changes of gene expression profile in RA bone marrowAccording to the bone marrow microarray of RA and OA patients 33 genes were up-regulated more than 1.5 times(LogFC>1.5),46 genes were down-regulated more than 1.5 times(logFC<-1.5).The expression of SOX5 gene was increased about 1.676 times(logFC=1.676),P value and adjusted P value were less than 0.05.3.2 Expression of transcription factor SOX5 in bone marrow cells of CIA miceThe expression of SOX5 in bone marrow cells of CIA mice was significantly increased.Considering that the function of SOX5 may be related to the mechanism of BME and bone erosion.We will further study the funcion of SOX5 in the mechanism of BME by SOX5 knockdown mice.Conclusion1.We found that during the development of CIA mice,BME and the the changes of bone marrow microenvironment were earlier than clinical symptoms and the histopathology of synovium.2.A special microenvironment of bone marrow was formed during the emergence of BME.With the activation of T,B immune cells,a series of genes and inflammatory factors which involved in activation(RANKL gene)and migration(CCL12,TNF-α,IL-17,IL-6)of osteoclast in bone marrow were upregulated.Besides,the differentiation of osteoclast precursor cells into the mature osteoclasts in the medullary cavity was significantly increased.3.Osteoclasts attached to the endometrium in the CIA mice was significantly increased after BME appearance and the bone trabecular was significantly reduced.In some parts osteoclasts penetrate the cortex,formed the "bone channel" between medullary cavity and the joint.4.Transcription factor SOX5 was markedly increased after BME appearance,may be involved in the development of RA bone marrow microenvironment.