Upregulation Of Leptin-mediated PI3K/AKT Expression Promotes Inflammation Of Type Ⅱ Innate Lymphocytes In Allergic Rhinitis In Children

PART 1:Expression of leptin and its relationship with the proportion and function of type Ⅱ innate lymphoid cells in allergic rhinitis in children Objective:To investigate the expression of leptin and its relationship with the proportion and function of type Ⅱ innate lymphoid cells(ILC2)in allergic rhinitis(AR)in children.Methods:A total of 26 children with AR and 20 normal children were enrolled in this study and classified into AR and control groups,respectively.Peripheral blood samples were collected and serum leptin concentration was detected by enzyme-linked immunosorbent assay(ELISA).The proportions of ILC2,IL-4+ILC2,IL-5+ILC2 and IL-13+ILC2 in peripheral blood mononuclear cells(PBMCs)of AR and control group were detected by flow cytometry,and the correlation between serum leptin level and the proportion of ILC2 in PBMC was analyzed.Results:Serum leptin level in AR group was significantly higher than that in the control group(10.2±3.4ng/m L vs 3.3±0.8ng/mL),(P﹤0.05).The proportions of ILC2,IL-4+ILC2,IL-5+ILC2 and IL-13+ILC2 in PBMCs of AR group were significantly higher than those of control group(ILC2:0.097±0.068 vs 0.025±0.016,P<0.05;IL-4+ILC2:0.074±0.046 vs 0.027±0.015,P<0.05;IL-5+ILC:20.081±0.053 vs 0.017±0.006,P<0.05;IL-13+ILC2:0.078±0.056 vs 0.016±0.006,P<0.05).Correlation analysis showed that the up-regulated serum leptin level in AR was positively correlated with the proportion of ILC2 in PBMCs(r=0.59,P=0.001).Conclusion:This study showed that the expression of leptin in serum of AR patients was up-regulated and correlated with the proportion of ILC2 and the functional phenotype,suggesting that leptin may play an important role in the regulation of ILC2 cells in the pathogenesis of AR.Part Ⅱ: regulation and related mechanism of leptin on ILC2Objective: To investigate the regulation of leptin on ILC2 and its related signaling pathway in the pathogenesis of AR.Methods: ILC2 s were isolated from PBMCs of normal adult and cultured.Subsequently,ILC2 s were stimulated respectively with PBS,Cytokine combination(contains IL-33(10 ng/ml),thymic stromal lymphocytes hormone(TSLP,10ng/ml)、 IL-25(10ng/ml)and IL-2(50ng/ml)),leptin,leptin+anti-leptin,leptin+LY294002(PI3K specific inhibitor),leptin +AG490(JAK inhibitor),and leptin +SB203580(MAPK inhibitor).The proliferations of ILC2 in both group were detected by the tritiated thymidine incorporation method(3H-Tdr).The protein concentration of ILC2-related cytokines(IL-4,IL-5,IL-13)in the supernatant of both group was detected by ELISA,The difference of the gene expression of GATA3 and ROR in each group were detected by RT-PCR,and the p AKT pathway was detected by Western Bolt.AR mouse models were established and sensitized by PBS,OVA,leptin,leptin +anti-leptin and leptin + LY294002.The frequency of nose scratching and sneezing was recorded,the OVA-specific Ig E level in AR mice in each group was detected by ELISA,and the proportion of ILC2 in peripheral blood was detected by flow cytometry.Results: The results showed that the proliferation capacity of ILC2 enhanced after the stimulation of leptin(P < 0.05),and the process was only inhibited by LY294002.what’s more,after leptin stimulation,the protein concentrations of IL-4,IL-5 and IL-13 and the expressions of GATA3 and ROR in ILC2 culture solution of peripheral blood were significantly increased(P < 0.05),which was only inhibited by LY294002.The results of Western Bolt showed that p AKT expression in ILC2 medium increased significantly after leptin stimulation(P < 0.05).In the AR mouse model,the frequency of scratching nose,sneezing times and OVA-specific Ig E levels in AR group mice sensitized by leptin were significantly higher than those in the control group(P < 0.05),and this phenomenon was inhibited by LY294002.In addition,the proportion of ILC2 in peripheral blood of AR group mice stimulated by leptin was significantly higher than that of PBS group(P<0.05),and anti-leptin and LY294002 can inhibited this phenomenon.Conclusion: The leptin in the pathogenesis of AR can activate the expression of GATA3 and RORα genes and promote ILC2 proliferation and ILC2 associated inflammatory response,and this process is achieved through PI3 K / AKT pathway.