Molecular Immune Mechanism Of CD300e And Its Ligand Sphingomyelin In Linked To Psoriasis

Objective:Based on the previous transcriptome sequencing data of differential expression genes profiles of Psoriasis Vulgaris(PsV)in advanced and stable stages before and after treatment,the expression of CD300e gene with the most significant difference in expression profile was verified in PsV skin lesions and peripheral blood.To analyze the role of CD300e and its ligand SM in the pathogenesis and its molecular immunological mechanism of psoriasis.Methods:(1)Collect PsV skin lesions in the progressive and stable stages,obtain differentially expressed genes and immune pathways through transcriptome sequencing.(2)Collect PsV skin lesions and blood samples,verify the mRNA expression levels of CD300e,IL-17A,IL-23A,IL-12A,and TNF-α using quantitative real-time PCR(qRT PCR);evaluate CD300e protein expression using Western blotting(WB)and flow cytometry(FCM);determine the localization of CD300e in skin lesions and its associated immune cells using fluorescent immunohistochemistry,skin lesion single-cell isolation combined with FCM.(3)Collect PsV and healthy serum,and analyze SM serum levels using liquid chromatograph mass spectrometry(LC/MS).(4)Administer U-937,peripheral blood CD14+cells,and total dendritic cells(DCs)with SM,sphingomyelin synthase inhibitor Malabaricone C,SM combined with Malabaricone C,and CD300e-siRNA interference group combined with SM stimulation,and detect changes in mRNA expression levels of CD300e,IL-23A,etc.using qRT PCR,and IL-23A and TNF-α secretion in culture supernatant using ELISA 24 hours later.(5)Under SM intervention,antigen presenting cells(APCs)(CD14+cells,total DCs)and sorted CD3+T cells co-culture for 3 days to evaluate Th cell differentiation.(6)Apply imiquimod cream continuously to the back for 6 days to establish a mouse psoriasis-like dermatitis model,and identify it through mouse PASI score and histological examination.(7)Construct cd300e-KO mouse psoriasis-like dermatitis,and detect the mRNA and protein expression of CD300e and IL-23A in skin lesions using qRT PCR and WB.Results:(1)Compared with stable stage,the expression of CD300e gene was upregulated in the progressive stage,and signal pathway analysis revealed that the upregulated genes were significantly enriched in pathways related to acquired immune response,intracellular lipid metabolism,and immune cell differentiation.(2)qRT PCR and WB results showed that levels of CD300e,IL-17A,IL-23A,and TNF-α were significantly higher in both progressive PsV lesion and blood than in uninvolved skin and normal individuals.FCM analysis showed that CD300e+cells were expressed in CD11c+ and CD14+ cells in Peripheral blood mononuclear cells(PBMCs),as well as in CD1c+ DCs(cDC2)and monocytes in the dermal papilla layer of PsV lesions.(3)LC/MS results showed that SM levels in the serum were higher in the progressive stage of PsV than in the stable stage.(4)CD300e and IL-23A were highly expressed in APCs stimulated by SM,and downregulation of inflammatory molecules was observed after CD300e silencing or inhibition of SM synthesis.(5)SM did not affect the differentiation of Th cell subsets in the co-culture system of APCs and T cells.(6)Mouse psoriasiform dermatitis showed infiltrative erythema and scaling,with pathological analysis revealing excessive epidermal keratinization,incomplete keratinization,thickened spinous layer,and inflammatory cell infiltration.(7)cd300e knockout improved inflammation and decreased inflammatory cell infiltration in mice.Conclusions:CD300e and its ligand SM are involved in the development of PsV through IL-23A axis.CD300e and its ligand SM are expected to be a breakthrough point in molecular immunology and a new therapeutic target of PsV.