肿瘤细胞释放分泌型自噬小体诱导M2型巨噬细胞极化及其免疫调节作用的机制研究

Our previous studies have demonstrated the tumor-released secretory autophagosoms (TRAP) could induce B cells to differentiate into IL-10-producing regulatory B cells via the HMBG1-TLR2-MyD88-NF-κB pathway, which could potently inhibit T cell responses in IL-10-depedent manner. Our recent data showed that TRAPs could induce M2 polarization of macrophage, however, the involved mechanisms have not been studied so far. .In this study, we will investigate that: (A) the molecular mechanism of M2 polarization of macrophage induced by TRAPs, involved macrophage recognition, DAMPs on TRAP and signal transduction pathway of macrophage polarization. (B) the molecular mechanism of immunosuppressive functions TRAPs-induced M2 macrophage on antitumor immune responses or/and tumor cell growth invasion, and metastasis. (C) the correlation between functional polarization of macrophage and secretory autophagosoms released tumor cells in the tumor microenvironment..It is hoped that seeking effect of TRAPs on M2 macrophage would improve the understanding about development and differentiation of tumor-associated macrophages (TAMs) in the tumor microenvironment.

肿瘤相关巨噬细胞(TAM)是肿瘤微环境中重要的免疫抑制细胞，但其形成及功能机制尚未完全清楚。我们已结题项目证实:肿瘤细胞能释放分泌型自噬小体(TRAP)并诱导B细胞成为具有免疫抑制功能的Breg细胞。近期实验发现:TRAP也能诱导单核细胞极化成为具有M2型特性的巨噬细胞(上调CD206/IL-10/PD-L1表达)，其机制有待深入研究。. 本项目拟研究:①TRAP诱导M2型巨噬细胞极化的分子机制(通过何种配体/受体、活化途径诱导巨噬细胞极化?缺氧/缺营养条件来源TRAP是否促进极化?);②TRAP诱导M2型巨噬细胞的免疫调节作用及其机制(PD-L1/IL-10?接触/非接触?对免疫细胞/肿瘤细胞分别具有哪些调节作用?);③肿瘤微环境TRAP与M2型巨噬细胞极化的关系(动物模型和临床研究)。阐明TRAP与巨噬细胞极化的关系将为研究TAM的形成及功能机制提出新的思路。

一、阐明肿瘤细胞释放分泌性自噬小体(TRAP)是诱导肿瘤相关巨噬细胞表达PD-L1和IL-10的重要物质基础和新机制.1.肿瘤细胞释放分泌性自噬小体(TRAP)能通过TLR4-MyD88-p38-Stat3信号通路、在体内外活化巨噬细胞，进而诱导巨噬细胞成为PD-L1hiIL-10+表型的M2巨噬细胞；.2.TRAP诱导巨噬细胞主要依赖PD-L1在体外抑制T细胞增殖和在体内促进肿瘤生长；体内抑制TRAP形成可下调M2型TAM比例，增强免疫应答，减缓荷瘤小鼠肿瘤生长；.3.癌症患者胸/腹水中自噬小体与单核细胞PD-L1、胸/腹水中IL-10呈显著正相关；癌症患者外周血中含自噬小体且显著高于健康人；患者来源TRAP可诱导HLA-DRlo CD163hi PD-L1hi IL-10+的M2型单核细胞，并直接接触抑制T细胞活化和产生IFN-γ。.二、阐明肿瘤细胞释放分泌性自噬小体(TRAP)诱导抑制性CD4+T细胞和IL-10+Breg的重要物质基础和新机制.4.TRAPs表面的膜结合型Hsp90α能与CD4+T细胞TLR2结合，并通过TLR2-MyD88-NF-κB信号通路诱导CD4+T细胞分泌IL-6，另外，p38和PI3K/Akt信号通路也参与了其中的调控。TRAPs诱导CD4+T细胞自分泌或旁分泌的IL-6又通过IL-6/STAT3通路诱导CD4+T细胞分泌IL-10和IL-21。.5.TRAPs诱导的CD4+T细胞通过直接分泌IL-6和IL-10或间接诱导IL-10+Breg细胞发挥抑制T细胞免疫应答，最终促进肿瘤的生长与转移。敲低肿瘤细胞自噬相关基因Becn1或敲除小鼠体内Il6基因可以抑制肿瘤生长，也可以抑制IL-10+、IL-21+CD4+T细胞和IL-10+Breg细胞的分化。