Sel1L Regulates The Phenotype,Function And Autophagy In Bone Marrow Derived Dendritic Cells

Objective:Dendritic cell is an important antigen-presenting cell that initiate T cell responses by processing and presenting antigens,playing a key role in innate and adaptive immunity.In the process of dendritic cell functioning,a large amount of new proteins are synthesized in the endoplasmic reticulum（ER）.The rapid clearance of intracellular non-normally folded proteins by ERAD,which is composed of Sel1L molecule,is an important mechanism for ensuring the normal folding of these newly synthesized proteins.It may be involved in the regulation of dendritic cell phenotype and function,as well as affecting autophagy in dendritic cell.Therefore,to elucidate the regulation of Sel1L molecule on dendritic cell phenotype,function and autophagy,which important for the correct understanding of dendritic cell and immune response.Methods:1.Establishment of DCs directed knockout Sel1L mouse model using Cre-loxP recombination system.CD11c-Cre^-/-Sel1L^f/f and CD11c-Cre^+/-Sel1L^f/f mice were screened by PCR technology in the F3 generation,which were wild type（WT）mice and knockout type（KO）mice.2.Bone marrow stem cells were stimulated by GM-CSF and IL-4 to induce the culture of BMDCs,and the cells and supernatant were collected after LPS stimulation.Extract RNA from BMDCs for sequencing analysis.Fluorescent antibody staining of MHC-I,MHC-II,CD80 and CD86 for BMDCs and phenotypic analysis by flow cytometry.Detection of IL-6 and IL-12 expression levels in supernatant by ELISA.Flow cytometry was used to detect the ability of BMDCs to induce proliferation of antigen-specific CD4⁺T cells.3.Bone marrow stem cells were stimulated by GM-CSF and IL-4 to induce the culture of BMDCs,and the cells were collected after LPS stimulation.Confirmation of autophagosomes in BMDCs by transmission electron microscopy.Analysis of LC3expression levels in BMDCs by immunofluorescence.Analysis of the expression levels of BiP,LC3 and p62 in DMDCs by Western Blot.4.The mTOR signaling pathway-related proteins of DMDCs were analyzed by Western Blot.The mTOR signaling pathway-related proteins of DMDCs were analyzed by Western Blot.Results:1.Compared with WT LPS,the RNA-Seq results of KO LPS showed an increase in MHC class I complexes,an increase in phagosomes,and enhanced antigen processing and presentation.Flow cytometry showed that the expression of MHC class I molecules was increased in Sel1L-deficient BMDCs,the expression of MHC class II molecules was decreased,and the expressions of CD80 and CD86 were not significantly different.The results of ELISA showed that in immature DMDCs,IL-6expression increased and IL-12 did not change significantly compared with WT.In mature BMDCs,there was no significant change in IL-6 expression and increased IL-12 expression in KO compared with WT.Flow cytometry showed that KO LPS decreased the proliferation of antigen-specific CD4⁺T cells compared with WT LPS.2.KO compared with WT,and KO LPS compared with WT LPS,the expression of BiP increased,the number of autophagosomes increased,the expression of LC3increased,and the expression of p62 decreased.3.The expression of Akt and mTOR decreased and the expression of p70S6K increased in BMDCs with Sel1L gene deletion.Conclusions:Sel1L selectively regulates DC phenotype and function.After specific deletion in DC,Sel1L enhances the autophagy activity of DC by regulating the Akt/mTOR signaling pathway.