CD147 Facilitates The Pathogenesis Of Psoriasis By Regulating Metabolic Reprogramming In Keratinocytes

Objective:Psoriasis is a chronic inflammatory disease characterized by rapid proliferation of keratinocytes（KCs）,dermal inflammatory cell infiltration and vascular proliferation.The mechanism of the role of these metabolic alterations in the pathogenesis of psoriasis remains unclear.CD147（also known as Basigin or Bsg）,a transmembrane glycoprotein belonging to the immunoglobulin superfamily,is widely expressed in epithelial cells,endothelial cells and immune cells.It is involved in key aspects of inflammation and immune disease pathogenesis.Our team has previously demonstrated that CD147 is a susceptibility gene for psoriasis.CD147 is highly expressed in human psoriasis skin lesions and imiquimod（IMQ）-induced psoriasis-like skin lesions in mice,and plays a pro-inflammatory role in IL-22-mediated signaling pathways.However,the metabolic and immunological mechanisms of epidermal CD147 in the pathogenesis of psoriasis are not clear.Therefore,this study will elucidate the metabolic mechanisms by which epidermal CD147 promotes psoriasis progression in the context of clinical and in vivo and in vitro experiments,and provide new metabolic targets and therapeutic ideas for psoriasis treatment.Methods:1.CD147 expression and inflammatory cell infiltration in the skin samples of patients with psoriasis vulgaris and healthy controls were detected by multicolor flow cytometry and correlation analysis was performed.2.Epidermis-specific knockout CD147 gene mice(K14.Bsg^fl/flmice)were constructed,and the ears were molded with IMQ to observe the effect of epidermis-specific knockout CD147 on IMQ-induced psoriasis-like dermatitis phenotype,and the infiltration of inflammatory cells in skin lesions and spleen was detected by flow cytometry,and the expression of psoriasis-related inflammatory mediators in skin lesions was detected by RT-PCR.3.The extracellular acidification rate（ECAR）of full-skin cells from K14.Bsg^fl/flmice and control mice induced by IMQ was detected using Seahorse energy metabolism meter to detect the difference in their glycolytic levels;RT-PCR was performed to detect the altered transcript expression of key metabolic enzymes of glycolysis in skin lesions.4.Glucose uptake in IMQ-induced K14.Bsg^fl/flmice and control mice was examined by[¹⁸F]-fluorodeoxyglucose positron emission tomography-x-ray computed tomography(¹⁸F-FDG PET-CT);using the fluorescentglucoseanalogue2-（N-7-nitro-2,1,3-benzoxadiazol-4-amino）-2-deoxy-D-glucose（2-NBDG）uptake to determine the effect of specific knockdown of CD147on glucose uptake in primary KCs.5.Glycolytic stress assay,lactate assay and mitochondrial stress assay on KCs from K14.Bsg^fl/fltransgenic mice treated with IMQ or IL-17A were performed.6.Co-transfection of plasmid CD147-Myc and SLC2A-Flag into293T cells for exogenous immunoprecipitation（Co-IP）;endogenous immunoprecipitation（IP）in Ha Ca T cells.7.RT-PCR to detect altered inflammatory factor gene expression in primary KCs of K14.Bsg^fl/flmice and control mice under IL-17A induction and after inhibition of glycolysis of primary KCs with2-deoxy-D-glucose（2-DG）.8.To study the effect of epidermal-specific knockdown of CD147 on the epidermal metabolic profile of IMQ-induced psoriasis-like lesions in mice using non-targeted metabolomics and targeted metabolomics analysis.9.To identify the differential plasma metabolites in patients with psoriasis using non-targeted metabolomics and targeted metabolomics approaches,and to study the correlation between the levels of differential metabolites and the severity of psoriasis in humans.10.Validation of metabolomic results and study of the effect of in vivo supplementation with L-carnitine LC（C0）on IMQ-induced psoriasis-like dermatitis in mice.11.Analysis of the epidermal gene expression profile of IMQ-induced psoriasis-like lesions in mice by RNA-seq with epidermal-specific knockdown of CD147,and to further compare the screened differential genes with those in the public database as well as correlation analysis with CD147 expression.12.Validation of differential gene Bbox1 expression in IL-17A-induced primary KCs of K14.Bsg^fl/flmice,and detection of changes in Bbox1 expression after inhibition of glycolysis with 2-DG.13.Level ofα-KG,a key metabolite of the TCA cycle,andγ-butyrobetaine dioxygenase（γ-BBD）activity were measured in primary KCs of K14.Bsg^fl/flmice treated with IMQ or IL-17A,respectively.14.Histone methylation expression of H3K9me3,H3K4me3,H2K27me3 and H3K36me3 in KCs from IMQ or IL-17A-treated K14.Bsg^fl/flmice and control mice were detected by WB.15.The expression profile of H3K9me3 in the Bbox1 promoter region was analyzed using public databases,and CHIP was performed to detect whether CD147 regulates Bbox1 expression through H3K9me3 in KCs.Results:1.The expression of CD147 in non-immune cells of skin lesions was significantly increased in psoriasis patients compared with healthy controls,the proportion of infiltrating inflammatory cells in skin lesions was significantly increased,and the degree of inflammatory cell infiltration in skin lesions was positively correlated with CD147abundance.2.Epidermal knockdown of CD147 significantly reduced IMQ-induced psoriasis-like dermatitis and epidermal thickening,attenuated infiltration of CD45⁺CD11b⁺Gr-1⁺inflammatory cells,Th17cells and Treg cells in skin lesions and spleen,and significantly reduced inflammatory factors Cxcl1,Cxcl2,Il-1α,Il-1β,S100A8,S100A9,Il-6 and Vegf transcript expression.3.Epidermal knockdown of CD147 significantly reduced IMQ-induced glycolysis levels and transcript expression of key enzymes of glycolytic metabolism（Hk1,Hk2,Hk3 and Pkm）in murine psoriasis-like full-skin cells.4.In IMQ-induced mice,¹⁸F-FDG PET SUVmax was significantly higher than in controls,epidermal knockdown of CD147 significantly reduced IMQ-induced ¹⁸F-FDG uptake levels,and ¹⁸F-FDG PET SUVmax values were positively correlated with CD147 expression in skin lesions;in vitro experiments also demonstrated that epidermal knockdown of CD147 significantly reduced glucose uptake in primary KCs.5.Epidermal knockdown of CD147 significantly inhibited IMQ-or IL-17A-induced glycolysis levels in mouse epidermal KCs,while basal mitochondrial oxygen consumption rate（OCR）was significantly increased.6.Co-IP assay in 293T cells and IP assay in Ha Ca T cells both confirmed GLUT1 as an interacting protein of CD147 in KCs.7.Both the deletion of CD147 and the inhibition of glycolysis by2-DG in primary mice KCs significantly attenuated the expression of IL-17A-induced inflammatory factors,including Cxcl1,Cxcl2,IL-1αand IL-1β.8.Metabolomic analysis of mouse epidermis showed a significant decrease in carnitine metabolism in IMQ-induced mouse epidermal KCs and a significant increase in carnitine metabolism after epidermal knockout of CD147.9.Plasma metabolomic analysis of psoriasis patients identified 14significantly downregulated acylcarnitines;and plasma carnitine levels in psoriasis patients were negatively correlated with the severity of psoriasis.Plasma metabolomic analysis of IMQ-induced psoriatic mice identified11 significantly downregulated acylcarnitines.10.L-carnitine LC（C0）supplementation significantly attenuated IMQ-induced psoriasis-like dermatitis phenotype in mice,inhibited epidermal thickening,and suppressed Th17 and Gr-1⁺inflammatory cell infiltration in skin lesions.11.RNA-seq analysis showed that IMQ-induced expression of Bbox1,a key gene for carnitine anabolism,was significantly elevated in the epidermis of K14.Bsg^fl/flmice;public database-based analysis revealed that the expression of BBOX1 in psoriatic lesions was negatively correlated with that of CD147.12.Transcriptional expression of Bbox1 is significantly downregulated in IL-17A-stimulated primary mice KCs,whereas Bbox1expression is restored after knockdown of CD147 or inhibition of glycolysis with 2-DG.13.Theα-KG levels andγ-BBD activity were significant increased in the epidermis of K14.Bsg^fl/flmice induced by IMQ as well as in IL-17A-stimulated primary KCs of K14.Bsg^fl/flmice.14.Epidermal knockdown of CD147 inhibited IMQ-induced expression of H3K9me3 in mouse epidermis as well as in primary mice KCs.15.The expression of H3K9me3 in the Bbox1 promoter area was significantly enhanced in KCs isolated from K14.Bsg^fl/flmice.Conclusion:1.Epidermal knockout of CD147 significantly attenuated IMQ-induced psoriasis-like dermatitis.2.Epidermal deletion of CD147 abrogated glucose uptake and glycolytic capacity in IMQ-induced psoriatic dermatitis.3.CD147 played a critical role in the switch from oxidative phosphorylation to glycolysis in KCs.4.Epidermal CD147 in KCs promotes glucose uptake by interacting with GLUT1.5.Epidermal knockdown of CD147 promotes BBOX1 expression to enhance carnitine metabolism in KCs.6.Down-regulation of carnitine metabolism is one of the metabolic signatures in psoriasis.7.L-carnitine LC（C0）supplementation significantly attenuated IMQ-induced psoriasis-like dermatitis in mice.8.Epidermal CD147 regulated BBOX1 expression via H3K9me3 in KCs.Figures:24;Tables:9;References:100.