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

Abstract

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’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 . Key words: Toll-like receptor; Neuroglial cells; IL-17 receptor; Expression