口腔医学研究

口腔医学研究 ›› 2024, Vol. 40 ›› Issue (5): 393-400.DOI: 10.13701/j.cnki.kqyxyj.2024.05.004

• 口腔微生物学研究 • 上一篇    下一篇

树突状细胞肝激酶B1信号对FimH黏膜佐剂效应的影响

刘馨远1, 林玉红2, 陈君兰1, 黄冰冰1*, 李宇红1*   

  1. 1.口颌系统重建与再生全国重点实验室,口腔生物医学教育部重点实验室,口腔医学湖北省重点实验室,武汉大学口腔医(学)院 湖北 武汉 430079;
    2.烟台市口腔医院牙体牙髓科 山东 烟台 264000
  • 收稿日期:2024-02-29 出版日期:2024-05-28 发布日期:2024-05-22
  • 通讯作者: * 黄冰冰,E-mail:hbbing@whu.edu.cn；李宇红,E-mail:1004809372@whu.edu.cn
  • 作者简介:刘馨远(1999~ ),女,山东枣庄人,硕士在读,主要从事口腔免疫学相关研究。
  • 基金资助:
    国家自然科学基金(编号:81670981)

Influence of Dendritic Cell LKB1 Signaling on Mucosal Adjuvant Effect of FimH

LIU Xinyuan1, LIN Yuhong2, CHEN Junlan1, HUANG Bingbing1*, LI Yuhong1*   

  1. 1. State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Key Laboratory of Oral Biomedicine Ministry of Education, Hubei Key Laboratory of Stomatology, School & Hospital of Stomatology, Wuhan University, Wuhan 430079, China;
    2. Department of Cariology and Endodontology, Yantai Stomatological Hospital, Yantai 264000, China
  • Received:2024-02-29 Online:2024-05-28 Published:2024-05-22

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摘要： 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LKB1fl/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 immunosorbent 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。

关键词: FimH, 口腔黏膜免疫, 树突状细胞, 肝激酶B1, 口腔黏膜疫苗

Abstract: Objective: To investigate the impact of liver kinase B1 (LKB1) expression in dendritic cells (DCs) in oral mucosa on the response to mucosal vaccine adjuvants. Methods: CD11ccre-GFP;LKB1fl/fl mice (conditional knockout, 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 transcriptome high-throughput sequencing. Secondly, using cKO and WT mice (n=16 each), a mouse mucosal immune 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), including 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 increased 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 increase 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 oral mucosal immune responses by DCs depends on LKB1, and defective LKB1 on DCs leads to the loss of the adjuvant effect of FimH-ST.

Key words: FimH, oral mucosal immune, dendritic cells, liver kimase B1, oral mucosal vaccine