IL-4促进大肠杆菌麦芽糖结合蛋白诱导巨噬细胞向M1型极化作用及其机制研究

We have firstly found that MBP can directly activate macrophages and polarize them into M1-type, moreover,the polarization is much more significant when MBP combined with IL-4 (a M2-type inducer). However, the mechanism is still unclear. Based on previous researches, in this study, we will investigate the effects of MBP, IL-4, and MBP combined with IL-4 on macrophage polarization both in vitro and in vivo, and analyze TLR2/4 signal transduction pathway using TLR2/4 antibodies and inhibitors of downstream molecules. Furthermore, to clarify MBP targeting molecule, TLR2/4 gene silencing in RAW264.7 cells and MBP-pull down will be performed. The studies described above will demonstrate that MBP induces macrophages toward to M1 type polarization through the activation of TLR2/4 signaling pathway, and reveal that IL-4 regulates MBP-induced M1 type polarization via promoting TLR2/4 signal activation. Eventually, we will explore PI3K-Akt signal pathway by using inhibitor or agonist of PI3K and Akt gene silencing or over-expression, therefore, illustrate that IL-4 promotes TLR2/4 signal transduction through reducing PI3K-Akt signal. This study will lay the theoretical foundation for the treatment and prevention on inflammation and cancer, and provide a novel idea for the development of new vaccine adjuvants and immunostimulants.

我们首次发现MBP能诱导巨噬细胞活化并向M1型极化，当与M2型诱导剂-IL-4联用时，显著促进巨噬细胞向M1型极化，但其机制尚不清楚。本研究拟在前期研究基础上，通过体内、体外实验探讨MBP与IL-4联合对巨噬细胞极化的影响，采用TLR2/4抗体及下游信号分子的抑制剂分析TLR2/4介导的信号传导路的变化，通过RNAi技术沉默TLR2/4基因以及MBP-pull down进一步明确MBP作用的靶分子，从分子水平阐明MBP通过活化TLR2/4信号通路诱导M1型极化的作用，揭示IL-4通过促进TLR2/4信号活化而调控M1型极化的机制。采用PI3K抑制剂及激动剂、Akt基因沉默或过表达技术探讨PI3K-Akt信号的变化，阐明IL-4通过降低PI3K-Akt信号促进TLR2/4信号传导的调控机制，为炎症及肿瘤等疾病的防治奠定理论基础，为免疫增强剂或联合佐剂的开发提供新的思路

一、MBP通过激活TLR2/4-MyD88-p38/NF-κB通路诱导小鼠腹腔巨噬细胞及RAW264.7细胞活化并向M1型极化.通过体内与体外实验，从分子水平研究MBP诱导巨噬细胞向M1型极化的信号传导机制。共聚焦显微镜分析显示MBP能够直接结合于巨噬细胞；同时，MBP能够上调巨噬细胞活化标志及M1型标志，下调M2标志。提示MBP能够直接诱导小鼠腹腔巨噬细胞及RAW264.7细胞向M1型分化。进一步，通过TLR2/4抗体预封闭等方式分析MBP结合巨噬细胞的受体，结果显示：MBP经由TLR2/4-MyD88、激活p38 MAPK、NF-κB信号传导通路从而发挥诱导巨噬细胞的活化与M1型极化的作用。以上结果阐明了MBP可作为TLR2/4激动剂的分子机制，并为免疫增强剂开发提供依据。..二、MBP体外翻转小鼠腹腔巨噬细胞及RAW264.7细胞由M2型向M1型极化，体内规划小鼠肿瘤相关巨噬细胞TAM-M2表达.研究通过体内、体外两种方式揭示MBP能够翻转M2型巨噬细胞向M1型极化。体外以小鼠乳腺癌细胞系4T1肿瘤细胞培养上清模拟肿瘤微环境与RAW264.7细胞混合培养构建体外TAM模型。研究显示MBP能够翻转TCS-induced M2型RAW向M1型极化作用。体内研究通过构建B16小鼠黑色素瘤模型进行，结果显示MBP可能通过规划肿瘤相关巨噬细胞进而对于抑制荷瘤小鼠的肿瘤生长。以上结果揭示MBP能够翻转TAM-M2巨噬细胞向TAM-M1型极化，为其免疫增强剂的开发提供依据。..三、IL-4+MBP通过TLR2/4介导的MyD88依赖途径及My88非依赖（TRIF-TRAF3/6途径）调节巨噬细胞极化.研究以TLR2/4为切入点，以MyD88依赖与TRIF依赖途径为主线，通过MBP和IL-4联合刺激小鼠腹腔巨噬细胞，探讨二者联用对TLRs通路相关信号分子的影响；并进一步利用TLR2、TLR4抗体分析TLR及其介导的信号传导分子在M1极化中的作用。 结果显示MBP联合IL-4后，通过显著上调TLR2/4介导的MyD88/TRAF6表达，下调 TLR4介导的TRIF/TRAF3表达引起高水平的M1活化。揭示在TLR信号通路中TRAF6上调可促巨巨噬细胞M1型极化，TRAF3上调则引起相反的作用。为炎症及肿瘤等疾病的防治奠定理论基础，为免疫增强剂或联合佐剂的开发提供新思路。