The Discovery Of A New Ligand For CD6 And Investigation Of The Role And Mechanism Of CDCP1 In Kawasaki Disease Mouse Model Induced By CAWS

Objective:CD6 is a glycoprotein mainly expressed on the surface of T cells and some B cells,which is involved in the regulation of lymphocyte development,selection,activation and differentiation.Two known mammalian CD6 ligands,CD166 and CDCP1,are widely expressed in immune cells and many other cell types,including epithelial cells,mesenchymal cells and tumor cells.More and more evidence shows that CD6 and its ligands play a role in the pathophysiological process of immune related diseases such as multiple sclerosis and psoriasis,and can also be used as potential therapeutic targets.Many studies have suggested that there are unknown receptors of CD6 except for CD166and CDCP1.After CDCP1 is confirmed to be a CD6 ligand,it suggests that it may have immunomodulatory function,but there are few reports in related fields.The aim of this study was to explore the new ligands of CD6 except CD166 and CDCP1,and the role and mechanism of CDCP1 in Kawasaki disease(KW)arteritis model induced by Candida albicans water soluble fraction(CAWS).Methods:The adjacent protein of CD6 on h TERT RPE cell surface was labeled by SPPLAT method.After cell lysis,the biotin labeled proteins were isolated and purified by streptavidin magnetic beads.The labeled proteins were identified by mass spectrometry to screen potential CD6 ligands.The potential ligands were identified by ELISA,immunoprecipitation,competitive inhibition method and surface plasmon resonance(SPR).Finally,the specific si RNA of the new ligand was used to knock down the ligand expression,which confirmed the interaction between CD6 and the new ligand.In the second part of the study,we used C57BL/6J wild-type mice and CDCP1^-/-mice to construct Kawasaki disease arteritis model by CAWS,and evaluated the effects of knockout CDCP1 on the formation of aortic arteritis,expression of inflammatory factors,splenomegaly,and composition of spleen and peripheral blood cells.Immunofluorescence and Western blot were used to detect the expression of CDCP1 in different tissues and organs of mice.Cardiac fibroblasts and bone marrow-derived dendritic cells(BMDCs)of mice were isolated and cultured to study the effects of CAWS stimulation on the expression of CDCP1 and the secretion of inflammatory factors.Finally,Western blot was used to detect the effect of CDCP1 on dectin-2 signaling pathway in mouse BMDCs stimulated by CAWS.Results:h TERT RPE cells expressed two known ligands of CD6,CD166 and CDCP1,and there was no endogenous CD6 expression.Recombinant human CD6 molecule could specifically bind to h TERT RPE cells,and CD44 was the adjacent protein of recombinant human CD6 binding to the surface of h TERT RPE cells;ELISA and SPR experiments confirmed that recombinant human CD44 could directly bind to recombinant human CD6;Recombinant human CD44 could precipitate CD6 in cell protein lysate,On the contrary,hyaluronic acid,the ligand of CD44,can competitively inhibit CD6 in the protein lysate of recombinant human CD44 precipitated cells;The binding of recombinant human CD6to the surface of h TERT RPE cells can be competitively inhibited by recombinant human CD44;The specific binding of recombinant human CD6 to h TERT RPE cells causes CD166,CDCP1 and CD44 molecules on the surface of RPE cells clustered together and caused the cytoskeleton remodeling at the same time;After the expression of CD44 was knocked down by si RNA,the cytoskeleton remodeling was inhibited.The arteritis model of Kawasaki disease was established by intraperitoneal injection of CAWS into wild-type and CDCP1 gene knockout mice.It was found that CDCP1^-/-mice showed significantly mild cardiac inflammatory reaction and serum antibody levels;the serum IL-6 level of CDCP1^-/-mice was significantly lower than that of wild-type mice 28 days after injection of CAWS;The mechanism study found that BMDCs expressed and could induce the synthesis of CDCP1 under the stimulation of CAWS;The secretion of IL-6 by CAWS was significantly decreased when CDCP1 was absent,which was related to Syk MAPK signaling pathway.Conclusion:CD44 is the third ligand of CD6 besides CD166 and CDCP1.The interaction between CD6 and CD44 can cause cytoskeleton remodeling.CDCP1 can affect the secretion of IL-6 through Syk-MAPK signaling pathway on DCs,and then affect the development of arteritis in KW mice model induced by CAWS,suggesting that CDCP1and IL-6 are potential targets for KW treatment.