Toll样受体信号通路可促进IL-17RA在神经胶质细胞中的表达

Toll like receptors signaling pathways directly increase the expression of functional IL-17RA in neu-roglial cells

目的：研究IL-17R在神经胶质细胞中的表达是否会受到Toll样受体（TLR）信号通路的调节及可能的调节方式。方法免疫诱导B6小鼠发生实验性自身免疫性脑脊髓炎（ experimental autoimmune encephalomyelitis ， EAE），real-time PCR检测Il17ra、Il17rc在脑及脊髓组织中的表达。组织化学染色显示脊髓组织的脱髓鞘情况、IL-17 RA阳性及CD3阳性细胞的分布。在Rag1-/-小鼠中进行相同测定，以判断IL-17RA在EAE小鼠脊髓组织中的高表达是否与CD3细胞的侵润有关。以不完全弗氏佐剂（IFA）与TLR的配体相配伍替代CFA免疫B6小鼠，检测Il17ra在中枢神经系统（CNS）中的表达。自新生小鼠脑组织分离星形胶质细胞、小胶质细胞及少突胶质细胞进行体外培养，以TLR的配体进行干预，qPCR及Western blot测定其中IL-17RA的表达。进而以IL-17A及LPS协同干预体外培养的星形胶质细胞，ELISA检测培养基中CCL2、CXCL8及IP-10等趋化因子的含量。结果 EAE小鼠CNS中存在Il17 rc的高表达，脊髓组织有明显的脱髓鞘病变、大量IL-17阳性细胞分布及CD3细胞侵润。在Rag1-/-小鼠中的测定结果则提示，CNS中IL-17 RA的高表达与T淋巴细胞的侵润无关。IFA与TLRs配体相协同即可体内促进Il17rc在小鼠CNS中的表达。体外实验也证实，某些TLR配体，特别是LPS及polyI∶C，可直接上调Il17rc在星形胶质细胞、小胶质细胞及少突胶质细胞中的表达。 LPS与IL-17RA相协同可显著刺激体外培养的星形胶质细胞分泌趋化因子，其效力远高于两者的单独作用。结论某些TLR信号通路可直接刺激IL-17 RA在神经胶质细胞中的表达。所表达的IL-17R为功能性受体，可刺激胶质细胞分泌趋化因子。

Objective To investigate whether toll like receptor （ TLR） signaling pathways can in-crease the expression of IL-17 R in neuralglial cells , and if they can whether the increased IL-17 R is func-tional.Methods Experimental autoimmune encephalomyelitis （EAE） was induced in B6 mice by immuni-zation with an emulsion of myelin oligodendrocyte glycoprotein 35-55 （ MOG35-55 ） in complete Freund＆#39;s adju-vant （CFA）.The expression of Il17ra and Il17rc in the brains and spinal cords of mice with EAE were de-tected by real-time PCR.Luxol fast blue （ LFB） staining was performed to the spinal cord sections to detect tissue demyelination.Immunohistological staining against IL-17RA and CD3 were undertook to visualize IL-17RA＋and CD3 ＋ cells.Same approaches were also applied to immunized Rag1 -/- mice to figure out whether T cells infiltration is necessary for increasing IL-17RA expression in the central nervous system （ CNS） .Then B6 mice were immunized with incomplete Freund′s adjuvant （ IFA） plus different TLRs ago-nists to measure the expression of Il17ra in the brains and spinal cords by qPCR .The purified astrocytes , microglia and oligodendrocytes isolated from neonatal mice brains were cultured in vitro for two weeks , and then treated with different TLRs agonists .The expression of Il17ra at mRNA and protein levels in the cells were determined by qPCR and Western blot respectively .The astrocytes were treated with IL-17A and LPS individually or in the combination to detect the level of CCL 2, CXCL8 and IP-10 in the supernatant by ELISA.Results B6 mice with induced EAE showed significantly increased Il17ra expressions in the brain＆amp;nbsp;and spinal cord , which was also detected in immunized Rag1 -/-mice.Although no spinal cord demyeliza-tion and CD3 cells infiltration were detected in Rag1 -/-mice, significantly increased number of IL-17RA positive cells could still be visualized .In vivo TLRs agonist participated immunization and in vitro treatment of purified neuroglial cells demonstrated that TLRs agonists could directly evoke IL -17RA expression in the CNS or cultured astrocytes , microglia and oligodendrocytes with high efficiency .Both IL-17 A and LPS could stimulate astrocytes to secrete CCL2, CXCL8 and IP-10, however, a combined use of IL-17A and LPS fur-ther augmented the production of these chemokines to a large extend .Conclusion Taken together , we con-cluded that TLRs agonists could directly stimulate neuroglial cells to express IL -17RA which functionally re-spond to IL-17A by secreting chemokines .