| Background Bone cancer pain is present in many patients with primary bone tumors and bone metastasis,which significantly compromises quality of life.Drug therapies for bone cancer pain are still unsatisfactory currently,as the adverse effect of central nervous system and drug tolerance,thus there are still great challenges for cancer pain treatment.Recent studies have highlighted that spinal cord infiltrated T lymphocytes play an important role in the evolution of neuropathic pain.T helper type 17(Th17)cells,a subpopulation of T cells,which can produce IL-17/IL-17A specifically,have been more recognized recent years.Th17 cells have been shown to manifest a higher migratory capability to transmigrate across the blood-brain barrier/blood-spinal cord barrier compared with other T cell subpopulations,and they possess the ability to promote inflammation in the central nervous system.In contrast,transcription factor forkhead box P3(Foxp3)-expressing regulatory T(Treg)cells represent a unique subpopulation of T cells that has been shown to be effective in suppressing neuropathic pain recently.In addition,induced Treg cells and Th17 cells are regulated by the level of transforming growth factor-?(TGF-?),IL-6 and IL-23 to develop from antigen-naive T cells.However,there is no report about whether T cells infiltrate into the spinal cord during bone cancer pain development currently,and it is still unclear whether Th17/Treg cells participate in bone cancer pain progress.As the primary immune cells in the central nervous system,microglia have been proved to play a critical role in bone cancer pain development.The alteration of microglial M1/M2 polarization in the spinal cord has also been observed in the model of neuropathic pain by numerous studies.Moreover,studies have indicated that the immune responses of T cells may induce the microglia activation and M1/M2 polarization.However,more data about the interactions between T cells and microglial M/M2 polarization in the spinal cord during the development of bone cancer pain need to be provided.Based on the previous evidence,the purposes of the present study were:(1)To establish the mouse model of bone cancer pain,and determine whether T cells infiltrate into the spinal cord during the development of bone cancer pain;(2)To investigate the expression of Th17/Treg cells in the spinal cord during the development of bone cancer pain;(3)To explore the alteration of microglial M1/M2 polarization during the development of bone cancer pain;(4)To clarify the role of Th17/Treg regulated microglial polarization in the spinal cord in the pathogenesis of bone cancer pain.Methods(1)Male C3H/HeN mice and sarcoma NCTC 2472 cells were used to establish the bone cancer pain model in this study.Paw withdrawal mechanical threshold(PWMT)and the number of spontaneous flinches(NSF)were used to assess pain behaviors,and HE staining of the ipsilateral femur was used to observe the tumor growth.Expression of T cells in the spinal cord was detected by using immunofluorescence.(2)Immunofluorescence,western blotting,and/or ELISA were used to detect the alteration of Th17 and Treg cells in the spinal cord,the levels of IL-17?IL-17A?IL-17A receptor(IL-17AR),and the concentrations of TGF-?,IL-6,and IL-23.(3)Immunofluorescence,western blotting,and/or ELISA were used to characterize the microglial M1/M2 polarization and to detect the expression of iNOS and Arg-1.The levels of IL-1? and IL-10 in the spinal cord and blood were also detected.(4)Tumor group mice were intrathecally injected with a monoclonal mouse IL-17/IL-17A antibody and a monoclonal mouse IL-17RA/IL-17R antibody separately on day 14 post-operation.We determined the effects of IL-17/IL-17A antibody and IL-17RA/IL-17R antibody on PWMT and NSF.Then we observed the alteration of T cell infiltration,Th17/Treg expression,and microglial M1/M2 polarization in the spinal cord.The inflammatory mediators in the spinal cord and blood were also examined.Results(1)Compared with naive and sham group mice,the tumor group mice presented significantly decreased PWMT and increased NSF since day 14 after sarcoma cells impalantation,and HE staining showed abundant tumor cells infiltration and invasion into the medullary cavity,accompanied by large numbers of T cells relocating to the spinal cord.(2)The imbalance of Th17/Treg shifted to Th17 after a transient increased Treg cells in the spinal cord,with significantly increased expression of IL-17 and IL-17A and elevated level of TGF-?,IL-6 and IL-23 during bone cancer pain development.(3)IL-17AR expressed on Microglia and the M1/M2 polarization shifted to M1 during bone cancer pain development,accompanied by the increased inflammatory response both in the spinal cord and blood.(4)Intrathecal injection of IL-17/IL-17A antibody alleviated bone cancer pain,blocked T cell infiltration,inhibited microglial polarization,and decreased the inflammatory response in the spinal cord and blood.(5)Intrathecal injection of IL-17RA/IL-17R antibody alleviated bone cancer pain.T cell infiltration was not significantlly blocked,while Treg cells increased and Th17 cells decreased after IL-17RA/IL-17R antibody administration.Microglial polarization was also inhibited,accompanied by decreased inflammatory response in the spinal cord and blood.Conclusion This study provides the first report of T cell infiltration in the spinal cord of bone cancer pain mice,and shows that the Th17/Treg imbalance in the spinal cord shifted towards Th17 after a transient increased Treg expression during the development of bone cancer pain.This promoted more production of IL-17 and IL-17A and further polarized microglia via the IL-17AR pathway.The microglial polarization showed increased Ml and decreased M2 in the spinal cord,promoted more production of pro-inflammatory factors,and accelerated the development of bone cancer pain.Intrathecally injection of IL-17/IL-17A and IL-17RA/IL-17R antibody could ameliorate bone cancer pain,companied by inhibited T cell infiltration,altered Th17/Treg expression and suppressed microglia polarization. |
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