A Preliminary Study On The Role And Mechanism Of Macrophage-associated Immune Checkpoint Molecular Imbalance In Heterotopic Ossification

?Background?Heterotopic Ossification?HO?is a true osteogenesis outside the normal skeletal system involving a variety of soft tissues including muscle,fascia and ligament.It is mainly divided into two types:hereditary HO and acquired HO.Hereditary HO,including progressive osseous heteroplasia?POH?and fibrodysplasia ossificans progressiva?FOP?is caused by genes mutations.That is to say,the former is mainly result from a mutation that inactivates GNAS gene^[1].The latter is caused by acquired mutation of ACVR1gene^[2].Acquired HO lesions are mainly caused by severe burn,war injury,spinal cord injury,craniocerebral injury and joint replacement operation^[3,4],which seriously affect the patients'joint activity and quality of life.Whether hereditary or acquired HO,its pathogenesis is still unclear,and at present,the therapeutic effects of HO?including anti-inflammatory drug therapy,radiotherapy,surgical resection and so on?are also quite limited,so it is very important to explore the pathogenesis of HO in depth.Previous results of our research group and other scholars suggest that HO formation needs to meet the following three key conditions:osteogenic stem/progenitor cells,activation of osteogenic-related signaling pathways and appropriate microenvironment?i.e.,HO formation is co-regulated by the immune system,nervous system and hematopoietic system?^[5].The focus of this study is on immune factors in the microenvironment of HO.A large number of literatures suggest that innate immunity and adaptive immunity participate in the production of HO.Among them,macrophage is very important for the occurrence and development of HO.It's reported that inflammatory monocytes infiltrate the injured muscles of SCI mice developing NHO at significantly higher levels compared to mice without SCI and macrophage-derived oncostatin M?OSM?is a key mediator of both human and mouse NHO^[6];CD14+primary monocytes from FOP subjects treated with the TLR4 activator LPS showed increased CCL5,CCR7,and CXCL10;abnormal cytokine/chemokine secretion;and prolonged activation of the NF-?B pathway^[7].In addition,in terms of adaptive immunity,the incidence and size of HO in mice with T,B cell dysfunction were significantly lower than those in normal mice^[8].What can not be ignored is that the immune response is closely regulated by the immune checkpoints?ICs?.ICs proteins are a series of molecules that produce costimulatory or inhibitory signals in the immune response,which are divided into stimulatory ICs?e.g.CD27,CD28,CD278,CD40,ICOS?and inhibitory ICs?e.g.PD1,PDL1,TIM3,CD152,CD47?.They regulate the immune response through different regulatory axes,and play an important role in the immune system.The stimulating ICs protein can promote the immune response,while the inhibitory ICs can inhibit the immune response process.At present,there are many researches on ICs protein in cancer therapy,and remarkable results have been obtained^[9-11].However,the impact of them in HO is still unclear.Therefore,our research group selected some related ICs by reading literatures,and their Immunofluorescence staining were carried out in the early,middle and late stages of the occurrence of HO,and it was co-stained with T cells,B cells and macrophages.The results showed that the expression of ICs protein was unbalanced during the development of HO,that is,the expression of stimulating ICs?CD27,CD28,CD278,CD40?in the process of HO first increased and then decreased,while the inhibitory ICs?PD1,PDL1,TIM3,CD152?gradually increased in this process,and these imbalance ICs co-located with macrophages largely.So does the ICs protein play a role in the formation of HO?We used the neutralizing antibody of CD40L/CD134L directly to interfere with the effect of stimulatory ICs protein CD40/CD134.The results showed that the neutralizing antibody of stimulatory ICs could promote the development of HO.Not surprisingly,the neutralizing antibody of inhibitory ICs could reduce the production of HO.And,the mechanism of how macrophage-related immune checkpoint proteins participate in the process of HO is further studied in this paper.In this study,based on the HO mouse model?that is,NSE-BMP4 transgenic mice,which can induce HO by high expression of BMP4,?the mechanism of HO was investigated,and the ICs protein was used as the entry point and combined with macrophages for the first time.We found that in this process,ICs protein imbalance,and it is closely related to macrophages.This study on the pathogenesis of HO has a further understanding,and provides a new way of thinking for the treatment of HO.?Research methods?1.The left leg of Nse-Bmp4 mice was intramuscularly injected with CTX of100ng/100?l,and the 1 week,2 weeks and 4 weeks after the injury corresponding to the early,middle and late stages of HO,respectively.Blood was sampled from mouse eyes to detect the white blood cell count in peripheral blood of Nse-Bmp4 model mice during early,middle and late stages of HO production.Immunofluorescence was used to detect the expression of macrophages,T cells and B cells in the early,middle and late stage of HO formation.Detection of the expression of pro-inflammatory factors?IFN-?,IL-6,TNF-??and anti-inflammatory factor?IL-4,IL-10,IL-13?in injured area is by RT-qPCR.2.Immunofluorescence and Western Blot were used to detect the expression of stimulatory ICs?CD27,CD28,CD278,CD40?and inhibitory ICs?CD152,TIM3,PD1,PD-L1?in the early,middle and late stages of HO production.3.CD40L/CD134L/PD1 neutralizing antibody was injected into the tail vein of Nse-Bmp4 mice as the experimental group,and the control group was injected with PBS.After 1-2 months,the size of HO in each group was quantified by Brooke SkyScan1176 low dose CT imager.4.Magnetic activated cell sorting?MACS?was used to detect the number of macrophages?F4/80+?at 1 week/2 weeks/4 weeks after injury in each group?WT without injury,BMP4 without injury,WT with injury,and BMP4 with injury?.The expression of CD206 and F480 were detected by immunofluorescence co-staining in the early,middle and late stage of HO,and the ratio of CD206+/F480+was calculated.After 1 week/2 weeks/4 weeks of BMP4 injury,the percentage of CD206+cells in the bone marrow of BMP4 mice was detected by flow cytometry.Bone marrow cells of BMP4 mice were treated with GM-CSF,M-CSF and BMP4+GM-CSF,respectively,and then the proportion of CD206+cells was detected.5.Werstern Blot was used to detect the expression of various IC proteins?CD40,PD-1and TIM3?in bone marrow cells of BMP4 mice treated with GM-CSF,BMP4+GM-CSF.Flow cytometry was used to detect the proportion of CD206+cells in GM-CSF,GM-CSF+BMP4,GM-CSF+BMP4+Anti-PD1 and GM-CSF+BMP4+Anti-TIM3groups.The expression of pro-inflammatory factor?IFN-?,IL-6,TNF-??and anti-inflammatory factor?IL-4,L-10,L-13?in the above groups were detected by RT-qPCR.?Research results?1.During the formation of HO,the number of immune cells increased in the early phase while decreased in the later phase,and the expression of pro-inflammatory factors increased at the early stage and decreased at the later stage.2.In the process of HO,inhibitory ICs were up-regulated gradually,while stimulating ICs were up-regulated in 1-2 weeks post injury and then down-regulated afterward.3.In Nse-bmp4 mouse model,neutralizing inhibitory IC PD1 could effectively inhibit HO formation,while neutralizing CD40L or CD134L could promote HO formation.4.BMP4 protein induces polarization of M1 macrophage derived from bone marrow of Bmp4 mice to M2 type.5.In vitro assays also inidicated BMP4 can promote the expression of inhibitory ICs?P D1 and TIM3?but repressed the stimulatory IC expression?CD40?.6.Neutralization of PD1 or TIM3 can reverse the effect of BMP4 protein on macrophage transition to M2.?Research conclusions?1.Abnormal immune response?AIR?was observed during HO formation in Nse-BMP4mice.2.Immune checkpoint proteins were unbalanced in HO lesions,That is,during this process,the inhibitory ICs gradually increased,while the stimulating ICs increased first and then decreased.3.Neutralizing PD1 in Nse-BMP4 mice after injury can effectively inhibit HO formation,while neutralizing CD40L and CD134L in Nse-BMP4 mice can promote HO formation.4.BMP4 protein can induce polarization of macrophages derived from bone marrow of BMP4 mice to immunosuppressive M2 type macrophages.5.BMP4 protein can promote the expression of inhibitory ICs?PD1 and TIM3?but re press the stimulatory IC expression?CD40?in macrophages derived from bone marr ow of BMP4 mice.6.Inhibitory IC?PD1?neutralizing antibody can alleviate HO by inhibiting BMP4-mediated abnormal macrophage polarization.