The Effect And Mechanism Of IL-17 On The Differentiation And Proliferation Of MDSC In BRONJ

?Research background?Bisphosphonate-related osteonecrosis of the jaw(BRONJ)is one of the serious complications of using bisphosphonate drugs to treat patients with cancer and osteoporosis.More and more studies have shown that the occurrence of BRONJ is closely related to the suppression of immune function in inflammatory infections.As an important immune suppressor cell,the role of myeloid-derived suppressor cells(MDSC)in BRONJ development has not been systematically studied.?Research objects?This study will explore the potential role of Interleukin 17(IL-17)on the differentiation and proliferation of myeloid-derived suppressor cells(MDSC)in the pathogenesis of BRONJ,suggesting that bisphosphonates affected the immune function through IL-17,thereby promoting the occurrence of BRONJ,which has further revealed the pathogenesis of BRONJ.?Materials and Methods?The expression of IL-17 and MDSC markers at the local mucosal site of 15 normal people,20 periodontitis patients and 20 BRONJ patients was detected by immunohistochemistry,and the correlation between IL-17 and CD33 was explored.The C57BL/6 mice were intraperitoneally injected with zoledronic acid(a typical bisphosphonate)with the first molar of right upper maxillary removed to establish the BRONJ model,which was judged to be successful by observing the gingival healing of the trauma area,Micro-CT measurement of the alveolar bone,histological detection of the bone destruction and necrosis.Immunohistochemical assay was used to detect the expression of IL-17 and MDSC markers in the alveolar bone trauma area of tooth extraction + saline mice,tooth extraction + zoledronic acid unaffected mice,and tooth extraction + zoledronic acid onset mice.Enzyme linked immunosorbent assay(ELISA)was used to detect the expression of IL-17 in the peripheral blood of the above three groups.Flow cytometry was used to detect the proportion changes of MDSC(CD11b+Gr1+),M-MDSC(CD11b+Ly6Chi Ly6G-)and PMN-MDSC(CD11b+Ly6Clo Ly6G+)in peripheral blood and bone marrow to evaluate the correlation between the IL-17 levels and MDSC ratio.Finally,MDSC was induced by bone marrow cells in vitro,and stimulated with different concentrations of IL-17 for 2 days.The effect of IL-17 on MDSC and its subgroup ratio was detected by flow cytometry in vitro.Changes in STAT3 and NF-?B signaling pathways associated with MDSC proliferation were measured by western blot(WB)to verify the effect of IL-17 on the level of phosphorylation activation in these two pathways.?Results:?1.In the soft tissue around the tooth extraction wound of BRONJ patients,IL-17 levels were significantly increased and MDSC(CD33+ cells)was decreased.The high expression of IL-17 was related to the reduction of MDSC.2.By intraperitoneal injection of zoledronic acid 125 ?g/kg twice a week,and removing the first molar of right upper maxillary in the second week after injection,a stable BRONJ model could be established.The incidence is about 50%.It was characterized by poor healing of tooth extraction,rupture of surrounding gums,obvious swelling and hyperplasia,and exposure of dead bones.Micro-CT showed that the alveolar fossa was empty,and the structure of trabecula was lost.Scattered high density images can be seen in the tooth extraction fossa.Microscopically,there was a large range of inflammatory infiltration,with dense neutrophils,lymphocytes and plasma cells,obvious destruction and absorption of trabecular and the formation of dead bone.3.The level of IL-17 in the soft tissue around the non-healing tooth extraction wound was increased,while the number of MDSC(Ly6G/Gr1+ cells)decreased.The high expression of IL-17 was related to the decrease in the number of MDSC,which was consistent with clinical patients.In the BRONJ onset group,the proportion of MDSC in both bone marrow and peripheral blood decreased,while the M-MDSC subgroup with stronger immunosuppressive function decreased dramatically and the PMN-MDSC subgroup with weaker immunosuppressive function did not change significantly.The expression of IL-17 in peripheral blood was increased.The above results further indicate that the increased production of IL-17 in BRONJ mice is closely related to the decrease in the proportion of MDSC,and IL-17 mainly inhibits the proliferation of M-MDSC subpopulations.4.Vitro studies further showed that IL-17 at 30 ng/m L inhibited the expansion of MDSC,and low concentrations of IL-17 had no effect on the proliferation of MDSC.It mainly inhibited the proliferation of M-MDSC,which is consistent with the results in vivo experiments.In addition,western blot showed that 30ng/m L IL-17 inhibited the phosphorylation level of STAT3 and NF-?B signaling pathway in MDSC.?Conclusion?These findings establish a link between IL-17-mediated MDSC cell proliferation disorder and BRONJ-like lesions.No matter in clinical patients,mouse models or vitro experiments,the increase IL-17 inhibited the proliferation of MDSC,especially M-MDSC which has strong immunosuppressive function.Inhibition of phosphorylation levels of STAT3 and NF-?B signaling pathways may play an important role in this process.