The Effect And Mechanism Of FGFR3 Regulated Macrophage On Arthritis

PurposeOsteoarthritis?OA?are whole joint degenerative diseases that cause pain and disability to the patients.And there are no perfect ways to prevent and cue the disease due to the complexity of the disease until now.Previous studies reported that OA is characterized with chronic,low-degree synovial inflammatory response which plays a very important role in the development of OA.Furthermore,aberrant accumulation and activation of macrophage in synovium plays a key role in the synovial inflammatory response and cartilage destruction.We recently reported that FGFR3 signaling plays a protective role in articular cartilage of osteoarthritis.However,its role on macrophage and synovitis regulation were not reported until now.This study aims to investigate the role and mechanisms of monocytes/macrophage-FGFR3 in arthritis of mice.Methods1.Generation of mice with conditional deletion of FGFR3 gene in myeloid cellsHeterozygote FGFR3-flox(FGFR3^flox/+)mice were crossed with Lysozyme-Cre(LysM^cre)mice expression Cre enzyme specifically in the myeloid lineage to obtain the FGFR3^flox/+;LysM^cre dihybrid mice.Then the dihybrid mice were crossed with FGFR3-flox(FGFR3^flox/+)mice again to obtain homozygote FGFR3 conditional knockout(FGFR3^flox/flox;LysM^cre,R3cKO)mice and control(FGFR3^flox/flox,Control)mice.2.Gross observation of mice3,6 and 13-month-old Control and R3cKO mice were grossly observed and subjected to body weight,body length measurement and photography.The joint range of motion as well as gait behavior were evaluated with video capture.3.Imageological examination of joints in miceFaxitron MX-20?USA?X-ray scanner was used to evaluate the joint destruction in mice.Micro-computed tomography??CT?was performed on knee joint of mice using Scanco viva 40 CT instrument?Switzerland?.All scans were performed under identical conditions,with a 70-kV scanning voltage,113 mA current and 15?m scan thickness.The region of interest?ROI?was selected from periarticular osteophytes including calcified meniscus and synovium in knee joints.3D construction was performed on build-in software with the parameter 0.8/1/220.4.Histological analysis of joint sections in miceAfter fixation,the knee joints of mice were decalcified in 15%EDTA for about 1 week at 4?,subsequently,PBS rinsing overnight,dehydration using alcohol gradient,vitrification by dimethylbenzene,paraffin embedded and serially sectioned at 5?m thickness on paraffin slicing machine?Leica?.Joint sections were selected at approximately 80?m intervals,dewaxed,rehydrated,and stained with Safranin-O/Fast-Green and H&E.After mounting with neutral resins,sections were observed under microscope.The joint scoring was performed according to the recommendation of OARSI system.A maximal score was read out from each of the sections,and the sum of four highest scores were calculated for sum score,statistics were performed on maximal scores and sum scores.Synovitis scores were performed according to previously reported methods,the enlargement of the synovial lining cell layer,density of the resident cells and inflammatory infiltrate were analyzed.Histochemistry was performed to evaluated the accumulation of macrophages in synovium.5.Circulating monocyte traffickingIntravascular administration of fluorescent beads can be used to track circulating monocytes.Briefly,10-month-old Control and R3cKO mice received an injection of Fluoresbrite Polychromic Red 1.0-micron beads.Mice were sampled 3 days after the injection and knee joints were frozen sectioned and DAPI stained.Confocal microscope was used to observe the recruitment of beads labeled monocytes to the inflamed joint synovium of mice.6.RNA-seq analysis of differentially expressed genes?DEGs?in FGFR3-deficiency macrophagesRNA was extracted from primary macrophage of mice,the DEGs were compared between the Control and R3cKO mice,and GO enrichment and KEGG pathway analysis were performed,the differentially expressed CXCR7 was identified along with the role of CXCR7 in macrophage.7.The effect of FGFR3 deficiency on chemotaxis of macrophageTranswell assay was performed to investigate the effect of FGFR3 deficiency on chemotaxis of macrophage under the stimulation of CXCL12/CXCL12 with or without the treatment of CXCR7 antibody,NF-?B inhibitor and HIF-1?inhibitor.8.The therapeutic effect of neutralizing antibody on arthritis of mice9-month-old R3cKO and Control mice were intra-articularly treated with CXCR7antibody for 8 weeks,the R3cKO mice with DMM surgery were intra-articularly treated with CXCR7 antibody for 4 weeks,the alteration of arthritic phenotypes in mice were evaluated after the treatment.9.The molecular mechanisms of FGFR3 regulated chemotaxis and gene expression in macrophagesCellular signaling pathway were enriched from the DEGs detected by RNA-seq,and NF-?B,HIF-1?inhibition experiments were performed,western blot was used to detect the activation of NF-?B as well as the expression of CXCR7 in FGFR3-deficient macrophages.Results1.3 and 6-month-old R3cKO mice show no remarkable abnormityFGFR3 myeloid lineage specific knockout mice were obtained through crossbreeding.The 3-month-old R3cKO mice had similar size,and the body weight and body length were not significantly different between the two groups.X-ray images showed that the skeletons of 6-month-old R3cKO mice were not different from the littermate controls.2.13-month-old R3cKO mice exhibit remarkable joint malformation and gait abnormityThe 13-month-old R3cKO mice developed grossly detectable joint malformation and gait abnormity,X-ray and?CT analysis showed that the 13-month-old R3cKO mice showed osteoarthritic phenotype in multiple synovial joints,and remarkable osteophyte formation were observed in the shoulder,hip joints and ankle joints,especially the knee joints.3.R3cKO mice develop aggravated joint destructionHistological staining showed severe cartilage erosion of knee joints in 13-month-old R3cKO mice,as well as loss of cartilage matrix,subchondral bone exposure and subchondral osteosclerosis due to abnormal stress.And lot of osteophyte formation were observed beside the tibial plateau along with significant synovial swelling,a sight of classical osteoarthritic pathological changes.In the meantime,the age-matched Control mice had not remarkable changes in their knee joint.However,the 3 and 6-month-old Control and R3cKO mice showed no significant different changes in their knee joints.Moreover,the cartilage destruction was also more severe in the DMM model of R3cKO mice compared with the Control mice.4.R3cKO mice develop remarkable synovitis and macrophage accumulationHistological analysis revealed that the 13-month-old R3cKO mice showed remarkable synovitis and macrophage accumulation in the synovium.Furthermore,obvious synovitis and macrophage accumulation were observed without remarkable destruction of articular cartilage in 9-month-old R3cKO mice.Which were not observed in the 3 and 6-month-old Control and R3cKO mice.Monocyte trafficking assay showed that plenty of beads labeled cells were recruited into the inflamed synovium of R3cKO mice.In addition,Ki67 staining of proliferating cells in synovium showed that there was no significant difference in proliferating cells between Control and R3cKO mice.5.FGFR3 deficiency in macrophage results in CXCR7 upregulationThe Cytokine-cytokine receptor interaction pathway was significantly changed in the RNA-seq analysis.Furthermore,CXCR7 was identified to be upregulated in the FGFR3-deficient macrophages.In addition,the level of FGFR3 in peripheral blood-derived monocytes of mice was downregulated with advancing age,while the expression of CXCR7was upregulated,which was closely related to the development of age-related osteoarthritis.The downregulation of FGFR3 level and upregulation of CXCR7 level was also observed in the peripheral blood-derived monocytes of OA patients.6.CXCR7 upregulation promotes the chemotaxis of FGFR3-deficient macrophageThe chemotaxis of FGFR3-deficient macrophage was significantly enhanced under the stimulation of CXCL12,which could be inhibited by CXCR7 neutralizing antibodies.7.CXCR7 neutralizing antibody could attenuate arthritic phenotype of R3cKO mice.Intra-articular injection of CXCR7 antibody significantly attenuated the age-related arthritic phenotype including the cartilage destruction and synovitis in R3cKO mice.CXCR7 antibody treatment also relieved DMM-induced phenotype of synovitis and the accumulation of macrophage in R3cKO mice.8.HIF-1?and NF-?B signaling pathways activation promote CXCR7upregulation and enhance macrophage chemotaxisThe NF-?B and HIF-1?pathways were significantly enriched in the RNA-seq analysis.Activation of NF-?B could upregulate CXCR7 both in mRNA and protein level,and NF-?B and HIF-1?inhibitors could significantly inhibit the activation of NF-?B and the expression of CXCR7 as well as the enhanced chemotaxis in FGFR3-deficient macrophage.In addition,the enhanced transcription of CXCR7 in FGFR3-deficient macrophage was inhibited with the treatment of actinomycin D.ConclusionsBased on the above data,we concluded that:1.Mice with FGFR3 deficiency in the myeloid lineage develop aggravated phenotype of joint destruction.2.Mice with FGFR3 deficiency in the myeloid lineage exhibit increased macrophage in the synovium.3.FGFR3 deficiency in monocyte/macrophage positively regulates CXCR7 expression,which is associated with the activation of HIF-1?and NF-?B.4.Targeting CXCR7 attenuates FGFR3-deficiency-mediated synovial joint destruction in mice.