肝脏M2型巨噬细胞分泌CCL22/CCL17募集Tregs促进泡球蚴免疫逃逸的机制研究

Alveolar echinococcosis (AE) is a serious zoonoses, which caused by Echinococcus multilocularis (E.m) infection, and invasive growth causes most patients to be difficult to cure. So far, there is no effective treatment. Tregs is closely related to the immunosuppressive function of the body. In our previous study, a large number of Tregs was found around the AE liver lesions, The expression of M2 type macrophage derived chemokine CCL22 and CCL17 was significantly increased in the liver and was positively correlated to the expression of Foxp3 and their high affinity receptor CCR4. So we speculate that CCL22/ CCL17-CCR4 chemokine axis mediated liver local recruitment of Tregs in the inflammatory microenvironment, which cause a immunosuppression status contribute to E.m escape the immune attack. However, the mechanism is unknown. In this work, we first plan to elucidate the role of the key chemokines CCL22/CCL17 secreted by M2 type macrophage in Tregs local recruitment through using the THP-1 cell line, E.m infection animal models and AE clinical specimens. Secondly, using recombinant protein (CCL22/17) induction and their neutralizing antibody blockage in vitro to identify the molecular mechanism of Tregs recruitment. Finally, we explore the feasibility and effectiveness for AE treatment by inhibit Tregs local recruitment through using the chemokines neutralizing antibody or M1 type macrophage adoptive transfer in the E.m infected mice model in vivo. These studies have important scientific significance to reveal the mechanism of growth, development and pathogenicity of E.m and to develop new drugs and new therapeutic method for AE.

泡型包虫病（AE）又称“虫癌”，侵袭性生长导致多数病人难以根治。AE病人病灶侵袭转移与免疫抑制状态密切相关，而Tregs是诱导免疫抑制形成的关键因素。我们研究发现AE肝脏局部募集大量Tregs，M2型巨噬细胞来源的趋化因子CCL22和CCL17高表达，与Tregs表面受体CCR4的表达呈正相关，推测炎症微环境中可能通过CCL22/CCL17-CCR4趋化轴介导肝脏局部Tregs募集，造成微环境的免疫抑制状态，促成E.m免疫逃逸和高侵袭性，但机制不明。本项目拟从AE临床标本、E.m动物模型和体外细胞三个层面明确该趋化轴在介导Tregs向病灶局部募集的作用；利用细胞趋化和E.m小鼠模型，通过正向诱导和反向干预手段阐明该趋化轴介导Tregs募集的分子机制；探讨靶向干预CCL22/CCL17-CCR4途径阻断Tregs募集用于AE治疗的有效性，对揭示AE致病机理和研发新的免疫治疗策略具有重要意义。

泡型包虫病（Alveolar Echinococcoisis，AE）是由多房棘球绦虫（Echinococcus multilocularis，E.m）幼虫寄生于人体所致的一种致死性寄生虫病。AE疾病恶性进展与宿主免疫状态密切相关，肝脏中募集大量Tregs是造成微环境免疫抑制状态，促进E.m侵袭性生长的关键因素，具体机制不明。本项目从AE临床标本、体外细胞和动物模型三个层面明确Tregs向病灶募集的分子机制，探讨阻断Tregs肝脏募集用于AE治疗的可行性和有效性。首先，利用AE临床标本鉴定病灶周围形成“ 炎症微环境”，免疫组化（IHC）检测发现病灶近端肝组织中Tregs（FoxP3）和巨噬细胞(Mφ：CD68、CD163和S100A9)相关指标的表达显著高于远端肝组织，且以CD163+M2型Mφ浸润占优势；通过对获得的4例AE患者组织标本的单细胞转录组数据进行分析，证实近端募集大量Tregs和M2型Mφ，且CCR4主要在CD4+Treg上高表达；IHC和qRT-PCR验证CCR4及其配体M2型Mφ分泌的趋化因子CCL17和CCL22在近端高表达，且CCL22和CCL17表达与CCR4和FoxP3表达分别呈正相关；利用E.m虫体蛋白体外刺激Mφ系发现能够诱导Mφ向M2型极化，细胞迁移实验证实虫体蛋白刺激后的巨噬细胞上清可诱导人PBMC的迁移。其次，建立肝门静脉E.m小鼠模型，流式细胞术和IHC检测发现感染早期肝脏募集大量单核细胞来源Mφ，且优先分化为M1型，在慢性感染阶段逐渐极化为M2型，CCL22和CCL17在感染小鼠肝脏中的mRNA表达水平显著高于对照组；利用氯磷酸盐脂质体清除Mφ建立E.m小鼠模型，发现Mφ在促进E.m感染早期清除和晚期慢性寄生中发挥双重作用；感染晚期肝脏中Tregs比例和绝对数均显著高于对照组，CCR4在肝脏Tregs、MDSC、Mφ和B细胞上均有表达。最后，建立野生型和CCL22、CCL17和CCR4缺陷型小鼠模型，发现CCR4缺失肝脏中MDSC和Mφ比例降低，T细胞功能恢复，病灶生长被抑制；使用CCR4拮抗剂（C021）对模型进行干预也能显著抑制病灶生长。本研究揭示了CCL22/CCL17-CCR4趋化轴介导Tregs肝脏募集进而发挥免疫抑制作用的分子机制，明确了C021对小鼠模型治疗的有效性和可行性，对寻找AE新的临床治疗药物具有重要意义。