The Regulatory Effect Of Leptin On CD8~+ T Cells And Its Role In The Pathogenesis Of Vitiligo

Background:Vitiligo is a common autoimmune skin disease mainly mediated by CD8+T cells,which affects about 0.1%-2%of the world’s population.The melanocytes in the patients’ skin would be gradually destroyed,resulting in disfiguring depigmented plaques.At present,the pathogenesis of vitiligo is still not completely elucidated.In our previous literature analysis,we found that the serum leptin levels in patients with various autoimmune-related skin diseases were significantly higher than those in healthy controls,but no study has reported the expression of leptin in vitiligo patients.Leptin is an adipokine with immunoregulatory function.Many studies have shown that it can affect the disease process of autoimmune skin diseases such as systemic lupus erythematosus and psoriasis.It also has a critical role in regulating the proliferation and differentiation of T cells,including activation of CD8+T cells.CD8+T cells play an important role in the pathogenesis of vitiligo,but no studies have explored the regulatory effect and mechanism of leptin on CD8+T cells and the occurrence and development of vitiligo in patients with vitiligo.Objective:To study the expression of leptin-related genes in patients with vitiligo,and to further explore the effect of leptin on CD8+T cells and its influence and molecular mechanism on the occurrence and development of vitiligo.Methods:(1)Detection and verification of differential genes in vitiligo skin lesions:The skin lesions of 3 cases of vitiligo patients and 3 cases of healthy controls were collected,whole-exome transcriptome sequencing was performed,and functional enrichment analysis of differentially expressed genes was performed to explore whether there is a significant difference in the expression of lipid metabolism pathways between vitiligo patients and normal people;after further expanding the sample size,the real-time quantitative nucleic acid amplification detection system was used to verify the expression levels of differentially expressed genes in lipid metabolism pathways including leptin-related differentially expressed genes,leptin genes(LEP)and leptin receptor gene(LEPR)between vitiligo patients and healthy controls;(2)Detection of the expression of leptin and CD8+T cell-related cytokines in the skin tissue and peripheral blood of vitiligo:The skin lesions and peripheral blood of vitiligo patients and healthy controls were collected,and the skin lesions of vitiligo patients and normal healthy controls were detected by immunofluorescence staining.The expression and distribution of LEPR in CD8+T cells was further detected by enzyme-linked immunosorbent assay(ELISA);the peripheral blood of patients with vitiligo and healthy controls were collected and detected the serum leptin levels.The peripheral blood mononuclear cells(PBMCs)of patients and healthy controls were further detected by flow cytometry for the expression of LEPR,γ-interferon(IFN-γ),Granzyme B(Granzyme B),and Perforin(Perforin);(3)In vitro study of the effect of leptin on the function of CD8+T cells:PBMCs of 5 healthy controls were collected,and normal human PBMCs were stimulated with leptin in vitro to observe the changes of CD8+T cells after receiving leptin stimulation for 72 hours.The mRNA and protein expression levels of functional cytokines such as perforin,granzyme and IFN-y in CD8+T cells were detected;(4)Use leptin knockout C57 BL/6 mice(Lep KO mice)to establish an in vivo vitiligo model to study the effect of leptin on the occurrence and development of vitiligo:a monobenzone-induced vitiligo mouse model was established on Lep KO mice,and the results were obtained by flow cytometry.CD8+T cell whole exome sequencing,Quantitative Realtime PCR(RT-qPCR)and other methods were used to explore the effect of leptin deficiency on the function of CD8+T cells and the construction of the vitiligo model.Results:(1)Compared with the normal control group,there were a total of 557 genes with significant differences in gene expression in the vitiligo group,including 154 up-regulated genes and 403 down-regulated genes.Further GO analysis of the transcriptome sequencing data showed that the lipid metabolism pathway is likely to be closely related to the pathogenesis and disease progression of vitiligo,especially the PPAR signaling pathway in lipid metabolism;RT-qPCR further verified that LEPR was highly expressed in vitiligo patients.(2)CD8/LEPR immunofluorescence double staining results of skin tissue showed that the expression of LEPR in CD8+T cells in skin lesions of vitiligo patients was significantly higher than that in CD8+T cells in normal skin tissues of healthy controls(Vitiligo vs HC:1.400 ± 0.2449 vs 0.2000±0.2000,P<0.01);the serum leptin level of vitiligo patients was significantly lower than that of healthy controls(Vitiligo vs HC:8.453 ±1.022 vs 12.47 ± 1.511,P<0.05);The LEPR of CD8+T cells,as well as interferon gamma(IFN-y)and perforin(Perforin/Perforin)all showed an increasing trend(Vitiligo vs HC:LEPR 27.19 ± 13.43 vs 12.13 ± 3.046,P>0.05;LEPR+IFN-γ+9.196 ± 1.372 vs 4.102 ± 1.066,P<0.05;LEPR+Perforin+:14.12 ± 5.494 vs 7.528 ± 1.912,P>0.05);ELISA assay(PHA+Leptin vs PHA:Perforin 0.80 ± 0.23 vs 0.26 ± 0.08,P>0.05;Granzyme B 5.65 ± 1.89 vs 1.72 ± 0.58,P>0.05;IFN-y 0.18 ± 0.07 vs 0.03 ± 0.02,P<0.05)and flow cytometry(PHA+Leptin vs PHA:CD8+LEPR+perforin+28.62 ± 1.562 vs 20.88 ± 2.435,P<0.01;CD8+LEPR+granzyme B+42.92 ± 2.158 vs 44.30 ± 2.029,P>0.05;CD8+LEPR+IFN-γ+ 3.154 ± 0.6288 vs 1.2256 ± 0.712 P>0.05)results indicated that leptin could increase the levels of IFN-γ,Perforin and Granzyme B expressed by CD8+T cells in peripheral blood of vitiligo patients.(3)We ad ministrated monobenzone to C57 BL/6 Lep KO(N=5)mice and C57 BL/6 WT(N=5)mice for establishing vitiligo mouse model,and found that the depigmentation of hair in Lep KO mice were significantly less severe than the WT mice;The flow cytometry,CD8+T cell sequencing and RT-qPCR further proved that the Lep KO C57 BL/6 mice had vitiligo disease-related genes Cxcr3,Cxcl9,Cxcl10,Cxcll2 after modeling.The expression of Ifng,Gzmb,Prf1,Jak2,Mif,Mx1,Icam5,Cdhl,Hsp70,Il15 was lower than that in C57 BL/6 WT mice to varying degrees.Conclusion:The expression level of Leptin may promote the development of vitiligo by regulating the cytotoxic effects of CD8+T cells.It provides an experimental basis for the pathogenesis of vitiligo and the study of potential therapeutic targets.