Research On The Role And Mechanism Of PD-1/PD-L1 Pathway In Mouse Osteoarthritis

BackgroundOsteoarhritis(OA)is a chronic,degenerative joint disease of articular cartilage degeneration and pain caused by local joint damage,inflammation,chronic strain,and activity disorder.The main pathological change is synovial inflammation with varying degrees of cartilage degeneration.Cartilage degeneration is the result of a combination of many factors,such as the apoptosis of chondrocytes,inflammatory environment and expression of metalloproteinase.Among them,inflammatory environment is closely related to cartilage degeneration.Studies have shown that various immune cells,pro-inflammatory cytokines,complement and other parts of the immune system are involved in the inflammatory process of OA.Inflammatory environment destroys chondrocytes and extracellular matrix and promotes cartilage degeneration,which can further stimulate inflammatory response.Therefore,osteoarthritis has been identified as an immunopathological disease.The development of osteoarthritis is an inflammatory activation process of synovial cells,chondrocytes and other cells.Studies have shown that during the development and progression of osteoarthritis,the innate immune system is first activated,which leads to the activation of the adaptive immune system and further promotes the development of inflammation and osteoarthritis.Activation of innate and adaptive immune systems also leads to the release of VEGF and cyclooxygenase-2,and the increased infiltration of CD4+T cells,macrophages and other immune cells.However,there is no definite conclusion on how the inflammatory process is generated,what the relationships inimmune cells are and what the molecular mechanisms are.Programmed cell death receptor 1(PD-1)is a protein expressed on the surface of cells such as sputum lymphocytes,B cells,macrophages,etc.It belongs to the CD28 superfamily.It is involved in the control of the mouse immune system and induces programmed cell death through stimulation.Programmed death ligand-1(PD-L1)is a member of the B7 family and is expressed in various cells such as macrophages and tumor cells,which is a newly discovered negative co-stimulatory molecule.The combination of Pd-1 and pd-llinhibits the activation of immune cells by blocking phosphorylation signals pathwayand plays an important role in a variety of autoimmune diseases.When the pd-1/pd-11 immune checkpoint is blocked,immune cells can be activated and proliferated in order to promote the occurrence of inflammation in vivo.However,it is still unknown whether the immune checkpoint is involved in the regulation of inflammatory process in osteoarthritis There are few studies on the role and mechanism of pd-1/pd-11 in osteoarthritisThe Purpose of ResearchThe purpose of this study is to investigate the changes in the model of osteoarthritis in mice by blocking the pd-1/pd-11 immune checkpoint,explore the role and mechanism of this pathway in osteoarthritis,and find a new research direction for the pathogenesis of osteoarthritis,which can set up a theoretical foundation for finding new targets for clinical treatment of osteoarthritis.In order to study the role and mechanism of pd-1/pd-11 pathway in osteoarthritis in mice,it is mainly studied from the following aspects:1.The role of immune checkpoint pd-1/pd-11 in osteoarthritis:.A mouse model of osteoarthritis is established and pd-11 antibody is injected into the articular cavity to block the pd-1/pd-11 pathway.And then through studying the changes in the severity of osteoarthritis,it indicates whether the immune checkpoint was involved in the occurrence of osteoarthritis.Then,through measuring the levels of inflammatory cytokines IL-6 and TNF-ain synovial fluid and synovial tissue,detected by ELISA and real-time PCR,it indicates that the pd-1/pd-11 pathway is involved in the development of osteoarthritis by inhibiting the expression of IL-6 and TNF-?.2.The role of immune response mediated by blocking pd-1/pd-11 pathway in vitro in osteoarthritis:.Immune cells from articular cavity synovial fluid are conducted and treated with pd-11 antibody in vitro,in order to observe whether the changes in IL-6 and TNF-? levels in culture supernatane areconsistent with the results in vivo,thus verify the effect of this pathway on osteoarthritis.Then,flow cytometry is used to analyze the types of immune cells in the joint,aiming to study how the immune response mediated by the pd-1/pd-11 pathway participates in the occurrence of osteoarthritis.3.The mechanism of pd-1/pd-11 pathway in osteoarthritis in mice.By activating normal mouse bone marrow macrophages(BMDM)and detecting the expression level of cytokines,they were injected into the OA mouse model to observe the change of cartilage damage degree of OA,in order to show whether macrophages secrete inflammatory factors such as IL-6 and TNF-? by activation,resulting in aggravation in OA.Then,macrophages in the synovial fluid of OA mice were isolated and PD-L1 levels were detected.The anti-PD-L1 treatment of macrophages and intraperitoneal injection of inflammatory factor antibody were used to observe the changes of OA cartilage damage to analyze the mechanism of action of the PD-1/PD-L1 pathway in mouse OA.Research Methods1.The role of immune checkpoint pd-1/pd-11 in osteoarthritis Mice were induced osteoarthritis by injecting collagenase or anterior cruciate ligament transection(ACLT);anti-pd-11 antibodies were injected into the joint regularly to block the signal of pd-11;the control group was injected with rat IgG.Mice in the collagenase-induced osteoarthritis group were sacrificed after 4 weeks,and mice in the ACLT-induced osteoarthritis group were sacrificed after 10 weeks.The damage degree of cartilage tissues in the knee tissue sections stained by Safranin-O was evaluated by histology and OARSI scores.Inflammatory cytokines(IL-6 and TNF-?)were detected by ELISA method,and the mRNA levels of cytokines(IL-6,TNF-?,IL-10,TGF-?)were detected by real time-PCR.2.The role of immune response mediated by blocking pd-1/pd-11 pathway in vitro in osteoarthritis:The immune cells were collected from articular synovial fluid in mouse osteoarthritis models and treated with pd-11 antibody in vitro.Then the levels of IL-6 and TNF-ain supernatant were detected by ELISA.The immune cells were sorted by flow cytometry,and the macrophages and T cells were obtained respectively.The expressions of IL-6 and TNF-ain the PD-L1 antibody treatment group and the control group were detected by Western blot method respectively.3.The mechanism of pd-1/pd-11 pathway in osteoarthritis in mice:(1)Isolate the normal mouse BMDM,add 1ng/ml Pam3CSK4 and incubate it in vitro for 6h to activate.Then detect the levels of IL-6 and TNF-ain supernatant by ELISA,and detect the mRNA expressions of IL-6 and TNF-? in cells by Real time PCR.The in vitro activated BMDM was injected into a mouse model of osteoarthritis.After 4 weeks,the cartilage tissue sections of the knee joint were stained with Safranin-O,and the damage degree of the cartilage was evaluated by OARSI score.(2)Macrophages were isolated from synovial fluid of osteoarthritis mice and their pd-11 levels were detected by flow cytometry.The macrophages were treated with pd-11 antibody in vitro and injected into the articular cavity of the osteoarthritis mouse model.Meanwhile,the mice were intraperitoneally injected with IL-6 and TNF-aantibodies.After 4 weeks,the mice were sacrificed,the cartilage tissue sections of the knee joint were stained with Safranin-O,and the damage degree of the cartilage was evaluated by histology and OARSI scores.Results1.The role of immune checkpoint pd-1/pd-11 in osteoarthritis:The results showed that after the blocking of pd-11 antibody,the tissue staining showed more serious degree of articular cartilage damage,and the higher OARSI score also indicated that the cartilage condition of the knee joint was worse after the blocking of pd-11 antibody(p<0.05).The serum and synovial fluid levels of inflammatory cytokines IL-6 and TNF-? were detected by ELISA,and the results showed that the serum and synovial fluid levels of IL-6 and TNF-awere significantly increased after the blocking of pd-11 in the collagenase-treated mice(p<0.05).The mRNA expression levels of IL-6,TNF-??IL-10 and TGF-?in synovial tissue were detected by real time-PCR,and the results showed that the mRNA expression of IL-6(p<0.01)and TNF-?(p<0.05)was significantly enhanced after the blocking of pd-11 in collagenase-induced osteoarthritis,but the mRNA levels of IL-10 and TGF-? were not affected.2.The role of immune response mediated by blocking pd-1/pd-11 pathway in vitro in osteoarthritis:The immune cells in the synovial fluid of osteoarthritis mouse model were isolated and treated with pd-11 antibody in vitro.The levels of IL-6 and TNF-ain the culture supernatant were detected by ELISA.The results showed that the levels of IL-6 and TNF-ain the supernatant of the immune cells were significantly increased after pd-11 antibody treatment(p<0.05).With flow cytometry instrument to phenotypic analysis,it is found that macrophages accounted for 81.6%,T cells(9.2%).After the separation,flow cytometry instrument is used to detect phenotype,and the purity is over 90%.Then macrophages and T cells were treated with PD-L1 antibody,and the expression of protein levels of IL-6 and TNF-awere detected respectively in macrophages and T cells by Western blot method.The results show that after treating by the PD-L1 antibody,the expression of protein levels of IL-6 and TNF-alevels were only elevated in macrophages but not in T cells.3.The mechanism of pd-1/pd-11 pathway in osteoarthritis in mice:BMDM was isolated from normal mice and Pam3CSK4 was added to activate it in vitro.The expression levels of IL-6 and TNF-ain mRNA and supernatant were detected by Real time PCR and ELISA respectively.The results showed that the mRNA levels of IL-6 and TNF-awere significantly increased after BMDM activation(p<0.05,p<0.001)and supernatant(p<0.01).The activated BMDM was injected into the collagenase-induced osteoarthritis mouse model.After 4 weeks,the knee tissue sections were stained with Safranin-O in order to observe the degree of cartilage damage,which is evaluated by OARSI score.It was found that the degree of osteoarthritis treated with Pam3CSK4 activated BMDM was significantly increased(p<0.05).The macrophages in the synovial fluid of osteoarthritis mice were isolated and the level of pd-11 was detected by flow cytometry.The expression of pd-11 was significantly increased than that of the control group.The macrophages were treated by pd-11 antibodies in vitro,then were injected into the articular of osteoarthritis mice model,with injecting IL-6 and TNF-? antibody into the mice at the same time.Safranin-O staining and OARSI scores show that macrophages vaccinated PD-L1 antibody produce more severe articular cartilage injury(p<0.01),while blocking the IL-6 and TNF-? can reduce the degree of joint damage caused by PD-L1 processing macrophages(p<0.05).Conclusion1.Blocking pd-1/pd-l1 signal promotes the development of osteoarthritis in mice.2.Blocked pd-11 signal up-regulates inflammatory response,and increases il-6 and tnf-alpha levels in the body.3.The PD-1/PD-L1 pathway has a negative regulatory effect on macrophages in OA joints.Blocking PD-L1 in vitro can promote macrophage activation and produce high levels of IL-6 and TNF-?.4.The infiltrating immune cells in the synovial fluid of osteoarthritis mice were mainly macrophages,and the surface is highly expressed PD-L1.5.Activated macrophages produce high levels of IL-6 and TNF-?,which can aggravate osteoarthritis.6.The mechanism of the PD-1/PD-L1 pathway involved in the development of OA is that blocking the PD-1/PD-L1 pathway leads to activation of macrophages in the joint,thereby activating the innate immune system and promoting IL-6 and TNF-?secretion,up-regulates inflammatory response,exacerbates OA development.7.Blocking IL-6 and TNF-acan reduce joint damage aggravated by anti-pd-11 treated macrophages.Innovation and Significance of Research1.This study innovatively put forward the idea of blocking the development of osteoarthritis by pd-11,and analyzed for the first time the role of pd-1/pd-11 immune checkpoint in osteoarthritis.2.This study innovatively found that pd-11 was highly expressed by macrophages in mouse osteoarthritis through the study of molecular mechanism,which proved for the first time that the mechanism of blocking pd-11 to aggravate the development of osteoarthritis was by up-regulating the expression of inflammatory cytokines IL-6 and TNF-ain macrophages.3.This study pointed out a new research direction for exploring the pathogenesis of osteoarthritis and provided a potential diagnostic standard and therapeutic target for the clinical treatment of osteoarthritis patients.Through the detection of PD-L1 expression in macrophages of OA patients and timely reflection of the pathological changessuch as early degeneration of articular cartilage,it will play a positive role in the early diagnosis,treatment and prognosis evaluation of osteoarthritis.LimitationsThe mechanism is not detailed enough.Futher study will continue to explore the specific signaling pathway of pd-1/pd-11 pathway regulating the production of inflammatory cytokines by macrophages,so as to find the downstream target of the pd-1/pd-11 pathway.