The Effects Of Suppressor Enhancer Of Lin-12-like (Sel1L) On The Properties Of Bone Marrow Derived Dendritic Cells

Object:Sel1L is an adaptor protein of the E3 ubiquitin ligase HRD1,the mammalian Sel1L-Hrd1 complex regulates its function by regulating the protein synthesis of immune cells.Studies have shown that Sel1L is involved in autoimmune diseases and tumorigenesis of many cancer types,and dendritic cells play important roles in stimulating immune responses and immune tolerance.Here,we construct a Sel1L^f/fCD11c-Cre^+/-knockout mouse model to study the effect of Sel1L on the function of bone marrow-derived dendritic cells.Methods:1.The DCs-specific Sel1L-null mice model was constructed using the Cre-Loxp recombination system.The Sel1L^f/f mice were bred with CD11c-Cre^+/-mice（both C57BL/6 background）.The genotypes of the offspring mice were identified by PCR,and Sel1L^f/fCD11c-Cre^+/-mice were selected.2.Take appropriate age of wild-type（WT）mice and knockout（KO）mice to analyze body weight,spleen weight and take pictures respectively.3.The histology of spleen was examined by H&E staining.The percentage of CD3⁺T cells,CD4⁺T cells and CD8⁺T cells,CD11c⁺cells and cDC subsets in spleen of two groups was analyzed by flow cytometry.The expression of co-stimulatory molecules and antigen presenting molecules on the surface of CD11c⁺cells was detected by flow cytometry.4.BM cells were cultured with GM-CSF and IL-4 for 7 days,and stimulated with TLR agonists LPS.Western blotting was used to detect the expression of Sel1L in BMDCs.Flow cytometry was used to detect the population of CD11c⁺cells and the change of CFSE.The expression of CD80,CD86,MHC-I and MHC-II on the surface of BMDCs was analyzed by flow cytometry.ELISA and qRT-PCR were used to detect the expression of IL-6 and IL-12.The capability of BMDCs in priming OVA specific CD4⁺T cells and CD8⁺T cells proliferation was detected by flow cytometry.Results:（1）Compared with the wild-type mice,the spleen weight of the knock-out mice was significantly increased,and swelling of the spleen was significantly increased with the naked eye.The histological section showed that the white pulp region of the spleen was relatively small.（2）The loss of Sel1L significantly decreased the percentage of CD8⁺T cells in the spleen.Interestingly,we found that the population of CD11c⁺splenic cells was increased in KO mice,and the percentage of cDC1s in cDCs subsets was significantly decreased while the cDC2s ratio was increased.Meanwhile,the proliferation efficiency of BMDCs in the process of differentiation induction was inhibited.（3）Compared with WT mice,KO mice promote the secretion of inflammatory cytokines,increase the expression of MHC-I,enhance the activation of CD8⁺T cells,and significantly reduce the expression of MHC-II,inhibiting the proliferation of CD4⁺T cells.Conclusions:Specific knockout of the Sel1L in dendritic cells can inhibit the differentiation efficiency of BMDCs,promote the secretion of inflammatory factors,and regulate the antigen presentation process of DCs,thereby playing an important role in stimulating the immune response.