TLR2/4在姬松茸多糖阻断肿瘤相关巨噬细胞的免疫抑制作用及机制研究

Tumor-associated macrophages has been shown to play a key role in promoting tumor progression. The exact mechanism has not been elucidated. The study found, in tumor microenvironment macrophages obtain to specific phenotype and promote tumor development, the activation of TLR induced immune tolerance. Agaricus blazei is a precious fungi,our data showed that by using Agaricus blazei Murill polysaccharides (a TLR2/4 agonist) to treat bone marrow precursors together with NTC-Ms in vivo, the up-regulation of IL-2, IL-12 and IFN-γ; induced by NF-κB and TLR2/4 and IRF-3 were largely inhibited. The treatment by Agaricus blazei Murill polysaccharides retards tumor growth and prolongs the survival of tumor-bearing mice. We have found that monocytes expressing F4/80+CD11b+ from tumor tissue, bone marrow and peripheral blood also express Gr-1 and TLR2 and TLR4. The TLRs signaling pathway activates several different signaling elements, including IRF-3 and NF-κB, which indicate that NTC-Ms (molecule from necrotic tumor cells) could regulate bone marrow development through TRAF-IRF-3-NF-κB pathway and promotes the differentiation of macrophages toward M2-like phenotype. Moreover, NTC-Ms treated TAMs can develop an inhibit phenotype, which expressed Arginase 1, Inducible nitricoxide synthase, interleukin-10, interleukin-12 and induce apoptosis of activated T cells. Supposedly " blocking TLR2/4 activation is inhibited macrophages to key factors of TAM differentiation, tumor immune therapy is a new target ". This paper shows that TLRs and TAM cell differentiation relations and Agaricus blazei Murill polysaccharide promoting tumor immunotherapy mechanism of inhibition macrophage differentiation. Accurate target and further cells, subcellular level of Agaricus blazei Murill polysaccharides inhibited macrophages to mechanism of TAM differentiation. Therefore, blockade of TLR2/4 signaling in local microenvironment should be considered in Agaricus blazei Murill polysaccharide inhibited macrophages to mechanism of TAM differentiation in organ, cell and molecular level, and provide a new way for tumor immunotherapy.

肿瘤相关巨噬细胞已被证实已被证实能调控微环境中各种免疫细胞的代谢机能，并加速肿瘤生长与转移发挥关键的作用。其确切机制尚未阐明。研究发现,巨噬细胞在肿瘤微环境作用下获得特殊的表型并促进肿瘤发展，其TLR活化诱导免疫耐受。姬松茸是一种珍贵的真菌，初步证实姬松茸多糖显著提高肿瘤免疫应答；检测微环境中巨噬细胞的TLRs受体表达情况结果显示TLR2/4表达明显下调；并抑制TLR2/4下游的IRF-3及NF-κB信号活化，它们的活化与TLR2/4信号活化呈现明显的相关性。另外本课题组分析肿瘤相关巨噬细胞和荷瘤小鼠巨噬细胞的表型，发现其均表达Gr-1+、TLR2和TLR4。据此推测“阻断TLR2/4活化是抑制巨噬细胞向TAM分化的关键因素，也是肿瘤免疫治疗一个新靶点”。本课题拟阐明TLRs与TAM细胞分化内在联系及姬松茸多糖抑制巨噬细胞分化的促免疫应答机制。并进一步细胞、亚细胞水平上探讨姬松茸多糖发挥作用的准确效应靶点。本课题首次在器官、细胞及分子水平上探讨TLRs与M2表型TAM细胞分化内在联系及姬松茸多糖阻断TLR2/4抑制巨噬细胞向TAM分化机制，为肿瘤的免疫治疗提供一种新思路。

肿瘤相关巨噬细胞（TAM）已被证实能调控微环境中各种免疫细胞的代谢机能，在加速肿瘤生长与转移过程中发挥关键的作用。TAM是由骨髓单核细胞系干细胞发育分化，以不成熟的形式从骨髓中释放入血，继而从外周血迁移至肿瘤微环境，并在那里发育成熟。作为TAM的前体细胞，骨髓来源的单核细胞(MDSCs)如何诱导分化成肿瘤相关巨噬细胞及促瘤功能的机理至今尚不清楚。.在我们的研究中，我们证实肿瘤微环境可重塑MDSCs使其具有TAM的表型及功能，诱导免疫耐受。流式细胞术分析肿瘤微环境中的MDSCs细胞表面标志，发现MDSCs和TAMs中Gr-1+细胞均表达CD11b+，其M2特性的Arg1、Nos2、IL-10表达明显上调，并抑制活化的T细胞增殖。.姬松茸是一种珍贵的真菌，我们已经分离得到纯化的姬松茸多糖，并且证实姬松茸活性多糖是TLR2的高效激动剂，通过活化MDSCs及TAM的TLR2下游的NF-κB信号，刺激MDSCs分泌IL-12、TNF-α和IL-1β，抑制肿瘤微环境的巨噬细胞向M2型的TAM转化，调节MDSCs诱导抗肿瘤Th1细胞免疫应答。体内抗肿瘤实验也证实姬松茸多糖通过活化TLR2信号明显提高抗肿瘤免疫应答，该组的IL-2、IL-12及IFN-γ表达上调及具有明显抗肿瘤效果。.我们建立基于TLR2受体抗肿瘤活性多糖体外筛选模型，阐明姬松茸多糖逆转TAM分化促免疫应答抗肿瘤的活性均一多糖的化学结构，并为寻找抗肿瘤新药提供关键靶点。.本课题已经取得如下进展①将肿瘤微环境巨噬细胞的来源、影响、TLRs在MDSCs向TAM转化途径中发挥重要作用等独特的生物学功能做了初步探讨；②初步阐明TLRs与TAM分化内在联系及姬松茸多糖抑制巨噬细胞分化的促免疫应答机制。在细胞、亚细胞水平上探讨姬松茸多糖发挥作用的准确效应靶点。③探讨TLRs与TAM分化内在联系及姬松茸多糖活化TLR2抑制巨噬细胞向TAM分化机制，为肿瘤的免疫治疗提供一种新思路。在项目执行期内，研究经费使用合理，除完成既定目标外还充实了研究内容，为所在实验室开创了新的技术平台。研究结果发表SCI收录论文1篇，尚有2篇SCI论文待发表。