细粒棘球蚴通过STAT6/KLF4/PPARγ轴诱导M2型细胞极化的免疫逃逸机制研究

Hydatid disease caused by Echinococcus granulosus, Cystic echinococcosis (CE), is an endemic problem in many countries of the world. Early immune escape is a prerequisite upon which the parasites are able to evade the host immune surveillance, and therefore, a key therapeutic target for the control strategies. However, the exact mechanism is still not fully understood. Macrophage is the first defense line in the body anti-infection, and can be classically or alternatively activated. Recent studies unveiled that nuclear transcription factor highly transcribed in macrophages mediates the alternative activation with microenvironment change, which contribute to occurrence and development of many diseases. Interestingly, the phenomenon of high levels of IL-4 and TGF-β, and upregulated levels of KLF4 and PPARγwere observed after the parasite infection by us. We therefore, hypothesize that echinococcus granulosus may achieve the early immune escape through increasing IL-4 and TGF-β expression levels, stimulating macrophages KLF4 and PPARγexpression, and subsequently inducing alternative activation of macrophages. Thus, in this study we plan to investigate the role of STAT6/KLF4/PPARγaxis in polarization and function of macrophage through a series of experiments in vitro and in vivo including establishing CE animal model and analyzing samples of hydatid disease patients, to explain significance of the axis in the establishment of early immune escape of hydatid infection. We will further analyze and evaluate the feasibility of pharmacologically blocking STAT6/KLF4/PPARγ axis or IL-4 and TGF-β activity to mediate the activation pattern of macrophages and finally, to promote immune response and pathogen clearance.

细粒棘球蚴病（包虫病）是危害严重的人畜共患病，早期免疫逃逸是其赖以生存的前提，也是防治策略的核心，而其机制尚不清楚。巨噬细胞是机体抗感染第一道防线，在不同微环境中可以极化为M1型和M2型。各种刺激改变微环境促发巨噬细胞转录水平的调控，而诱导M2型极化参与多种疾病的发生发展。我们研究发现：宿主感染包虫后可出现IL-4、TGF-β分泌增加，巨噬细胞KLF4、PPARγ转录上调。据此假设：包虫感染上调微环境IL-4、TGF-β表达，通过STAT6/KLF4/PPARγ轴诱导巨噬细胞M2型极化，继而产生免疫逃逸。我们将通过体外实验、包虫病小鼠模型及临床患者样本分析，考察STAT6/KLF4/PPARγ轴对巨噬细胞极化及功能的影响，阐述其在包虫感染早期免疫逃逸中的意义；在此基础上，通过阻断STAT6/KLF4/PPARγ轴或IL-4、TGF-β功能，改变巨噬细胞极化，从而促进免疫应答和病原体清除。

通过建立细粒棘球蚴感染小鼠模型以及体外原头蚴与巨噬细胞共培养体系，探讨KLF4、PPARs对巨噬细胞极化的影响，及在包虫病免疫逃逸过程中的作用。①原头蚴刺激RAW264.7细胞，诱导巨噬细胞KLF4高表达，巨噬细胞上调M2相关因子，下调M1相关因子。小鼠细粒棘球蚴感染早期，腹腔巨噬细胞KLF4及IκB表达上调，巨噬细胞从M1型转向M2型，对棘球蚴的杀伤力降低，有利于包虫逃避宿主免疫杀伤。干扰KLF4，M1巨噬细胞相关因子表达升高，M2相关因子表达下降，且下调IκB。推测细粒棘球蚴感染早期，由于KLF4表达上调，从而抑制了促炎因子活化，使炎症水平降低，降低对病原体的清除，从而使细粒棘球蚴逃避宿主的免疫杀伤。②RAW264.7细胞/腹腔巨噬细胞与原头蚴共培养，PPARα/γ和M2型相关因子Arg-1、TGF-β、Fizz-1的mRNA水平均逐渐升高；巨噬细胞M1型相关因子TNF-α、MCP-1、IL-1β 的mRNA先升高后降低，Arg-1和MR的蛋白水平也逐渐增高。加入PPARα/γ抑制剂后，与未阻断前相比，PPARα/γ和M2型相关因子Arg-1、TGF-β、Fizz-1的mRNA水平明显降低；M1型相关因子TNF-α、MCP-1、IL-1β的mRNA水平明显升高。蛋白水平和mRNA水平相一致。建立小鼠细粒棘球蚴感染模型，实验显示原头蚴促进巨噬细胞M2极化。体内外实验均证明原头蚴感染小鼠早期，PPARα/γ的表达逐渐升高，促进巨噬细胞由M1型向M2型极化，从而在虫体免疫逃逸中发挥一定作用。