目的:探究口腔黏膜树突状细胞(dendritic cells,DCs)肝激酶B1(liver kinase B1,LKB1)表达对于应用黏膜疫苗佐剂反应的影响.方法:使用CD11ccre-GFP;LKB1fl/fl条件性敲除(conditional knockout,cKO)小鼠,同窝LKB1n/fl小鼠作为野生型(wild type,WT)对照组.首先选6～8周龄cKO与WT小鼠各12只,流式细胞术分选小鼠口腔黏膜DCs,进行转录组高通量测序;其次使用cKO与WT小鼠各16只,建立小鼠黏膜免疫模型,在第0、2和4周用卵清蛋白(ovalbumin,OVA)模式抗原颊黏膜下注射免疫(cKO-OVA,WT-OVA),鼠伤寒沙门氏菌来源的 FimH(FimH-S.typhimurium,FimH-ST)佐剂联合 OVA 模式抗原颊黏膜下注射免疫(cKO-OVA+FimH-ST,WT-OVA+FimH-ST).在接种后第3周收集小鼠血清和唾液,通过免疫酶联吸附试验(enzyme-linked immu-nosorbent assay,ELISA)法检测血清中OVA特异性IgG1和IgG2b、唾液中OVA特异性IgA抗体水平;在接种后第6周,使用流式细胞术检测小鼠颊黏膜和包括下颌下、颈部淋巴结的口腔引流淋巴结(draining lymph node,dLN)中各免疫细胞数目和比例.结果:cKO组口腔黏膜DCs的上调基因在基因本体论(gene ontology,GO)数据库富集到先天免疫反应、B细胞介导的免疫反应;基因集富集分析(gene set enrichment analysis,GSEA)中富集到Toll样受体信号通路和mTOR信号通路.WT-OVA+FimH-ST组相对WT-OVA组血清OVA特异性IgG1和IgG2b、唾液中OVA特异性IgA抗体水平升高;黏膜局部免疫B细胞与浆细胞数目与比例升高.cKO-OVA组血清IgG1和IgG2b、唾液中IgA抗体水平较WT-OVA组升高;dLN中滤泡辅助T细胞(follicular helper T,Tfh)及B细胞比例升高(P＜0.05).但cKO-OVA+FimH-ST组相对cKO-OVA组抗体分泌水平无差异,仅表现为dLN中Tfh和B细胞增多.结论:LKB1通过DCs调控先天免疫反应和B细胞功能;FimH-ST经小鼠口腔黏膜下注射具有佐剂效应;DCs激活口腔黏膜免疫反应依赖LKB1,DCs上LKB1缺陷导致FimH-ST佐剂效应丧失.
Influence of Dendritic Cell LKB1 Signaling on Mucosal Adjuvant Effect of FimH
Objective:To investigate the impact of liver kinase B1(LKB1)expression in dendritic cells(DCs)in o-ral mucosa on the response to mucosal vaccine adjuvants.Methods:CD11ccreGFP;LKB1fl/fl mice(conditional knock-out,cKO)were used and littermate LKB1fl/fl mice were set as wild-type(WT).Firstly,6-8-week-old cKO and WT mice(n=12 each)were selected,and flow cytometry was used to isolate DCs from mouse oral mucosa for tran-scriptome high-throughput sequencing.Secondly,using cKO and WT mice(n=16 each),a mouse mucosal im-mune model was established.Immunizations were performed with ovalbumin(OVA)via submucosal injection in the cheek after 0,2,and 4 weeks,with or without FimH protein from S.typhimurium(FimH-ST).Serum and saliva were collected at week 3 post-immunization to detect OVA-specific IgG1 and IgG2b in serum and OVA-specific IgA in saliva by enzyme-linked immunosorbent assay(ELISA).At week 6 post-immunization,flow cytometry was performed to detect the number and proportion of immune cells in mouse cheek mucosa and draining lymph nodes(dLN),inclu-ding submandibular,cervical,and neck lymph nodes.Results:The up-regulated genes of oral mucosal dendritic cells in cKO mice were enriched in innate immune responses and B cell mediated immunity in the Gene Ontology(GO)database.The Toll-like receptor signaling pathway and mTOR signaling pathway were enriched in Gene Set Enrichment Analysis(GSEA).Compared to the WT-OVA group,the WT-OVA+FimH-ST group showed in-creased levels of serum OVA-specific IgG1 and IgG2b and saliva OVA-specific IgA antibodies;and the numbers and proportions of mucosal local immune B cells and plasma cells were increased.The cKO-OVA group showed higher levels of serum IgG1 and IgG2b and saliva IgA antibodies compared to the WT-OVA group,along with increased proportions of follicular helper T cells(Tfh)and B cells in dLN(P＜0.05).However,there was no difference in antibody secretion levels between the cKO-OVA+FimH-ST group and the cKO-OVA group,only showing an in-crease in Tfh and B cells in dLN.Conclusion:LKB1 regulates innate immune responses and B cell function through DCs;FimH-ST exhibits adjuvant effects when injected submucosally in the mouse oral mucosa;the activation of o-ral mucosal immune responses by DCs depends on LKB1,and defective LKB1 on DCs leads to the loss of the adju-vant effect of FimH-ST.
万方数据
维普
