The Research Of SEL1L Deletion On Th1/Th17-mediated EAE Disease Progression In Mice

Objective:Multiple sclerosis（MS）and its animal model experimental autoimmune encephalomyelitis（EAE）are autoimmune demyelinating diseases which mainly mediated by activated Th1/Th17 cell subsets,but their pathogenesis remains unclear.Endoplasmic reticulum adaptor protein Sel1L is involved in the recognition,transport and degradation of unfolded or misfolded proteins in ER,and is closely related to Unfolded protein response（UPR）and autophagy.Our previous study found that the deletion of Sel1L could promoted the differentiation of Th1/Th17 cells in vitro.Therefore,this study explored the regulatory effect of Sel1L on the differentiation of Th1/Th17 subsets and the role of Sel1L molecule in EAE process by constructing T cell-specific Sel1L gene knockout mice and combining with EAE model,in order to provide a new theory for the differentiation mechanism of CD4⁺T cell subsets and find a new target for the treatment of MS-related immune diseases.Methods:1.Establishment of the mouse model of specific Sel1L knockout in T cell.The Lck-Cre^+/-mice were crossed with Se1l L^f/fmice,and the cages were bred to the third generation to obtain wild-type and knock-out mice.Polymerase chain reaction（PCR）and agarose gel electrophoresis techniques were used to identify WT and KO mice.After magnetic beads sorting CD4⁺T cells in mouse thymocytes,Western Blot technology was used to verify the expression of Se1l L protein in WT and KO mice.2.The effect on behavior and histopathology of EAE miceMOG_35-55peptide antigen,complete Freund’s adjuvant and pertussis toxin were used to induce EAE model.After immunization,the neurological score of the mice were recorded every other day from the 0th day.During the peak period of EAE,the spinal cord tissues of WT and KO mice were taken to prepare paraffin sections.HE staining to observe the number and distribution of infiltrating inflammatory cells in the spinal cord;LFB staining to observe the demyelination;immunohistochemical staining to observe the infiltrating CD4⁺T cells in the spinal cord.3.The effect of Sel1L deletion on Th1 and Th17 cell subsets in EAE mice.（1）At the peak of EAE,the brain and spinal cord tissues of WT and KO mice were taken to prepare single-cell suspensions.After mononuclear cells were separated by Pecoll,intracellular staining was used to detect the proportion of Th1 and Th17 cell subsets in the central nervous system.（2）During the peak period of EAE,spleen and draining lymph node cells of WT and KO mice were taken to prepare single cell suspension,and the ratio of Th1 and Th17cells was detected by intracellular staining.（3）In the progressive stage of EAE,spleen and draining lymph node cells of WT and KO mice were taken,cultured in vitro with MOG_35-55antigen peptide for 72h-96h,and then intracellular staining was performed to detect Th1 and Th17 subgroups.（4）In the progressive stage of EAE,the spleen and draining lymph node cells of WT and KO mice were collected,cultured in vitro with MOG_35-55for 72h-96h,and the cell supernatant was collected,and then the secretion of IFN-γand IL-17A were detected with an ELISA kit.Results:1.Successfully constructed a T cell-specific knockout Sel1L mouse model.PCR and agarose gel electrophoresis methods accurately screened wild type(WT:Lck-Cre^-Se1l L^f/f)and knockout type(KO:Lck-Cre⁺Se1l L^f/f)mice.Western Blot technology further verified that the Se1l L gene was not expressed in CD4⁺T cells sorted from thymocytes of KO mice.2.The results of the EAE process showed that,compared with WT mice,During the onset of EAE,KO mice developed neurological deficit symptoms earlier and had higher clinical scores.In addition,the results of HE staining showed that the number of inflammatory cells infiltrated in the spinal cord of KO mice was more numerous and distributed more widely than that of WT mice;LFB results showed that the loss of Sel1L aggravated myelin damage;immunohistochemistry results showed that the proportion and number of CD4⁺T cells infiltrated of KO mice are greater than that of WT mice.3.（1）At the peak of the onset of EAE mice,the absence of Sel1L can increase the proportion of Th1 and Th17 cells in the central nervous system,and the proportion of Th17 cells is higher.（2）At the peak of EAE mice,the lack of Sel1L significantly increase the proportion of Th1 and Th17 cells in the spleen and draining lymph nodes.（3）In the progressive stage of EAE mice,the loss of Sel1L significantly increase the proportion of Th1 and Th17 cells in the reactivated spleen and draining lymph nodes in vitro.（4）In the progressive stage of EAE mice,the absence of Sel1L increase the secretion of IFN-γand IL-17A in the supernatant of spleen and draining lymph nodes cultivated in an antigen-specific context.Conclusions:The specific deletion of the Endoplasmic reticulum adaptor protein Sel1L in T lymphocytes increases the ratio of Th1 and Th17 cell subsets and the secretion of related inflammatory cytokines,which further aggravates inflammatory cell infiltration and myelin damage in the spinal cord and accelerates the progress of EAE.