分泌型与膜结合型TGF-β调控Treg细胞/NK细胞系统在细粒棘球蚴早期免疫逃逸中的作用

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. In the body, TGF-β is a immune-inhibitory molecular that plays an important role in the process of immune escape. TGF-β exists in two forms of secreted one and membrane-bound one with the different physiological function. Recent studies unveiled that tumor growth promotes the expansion of CD4+CD25+ regulatory T (Treg) cells. An inverse correlation between natural killer (NK) cell activation and Treg cell expansion in tumor-bearing patients was confirmed. It has been shown that human Treg cells expressed membrane-bound transforming growth factor (TGF)-beta, which directly inhibited NK cell effector functions and down-regulated NKG2D receptors on the NK cell surface, which controlling innate antitumor immunity. The phenomena of upregulated levels of the secreted TGF-β and Treg cells were observed in CE patients. We therefore, hypothesize that similar to tumorgenesis, echinococcus granulosus may also achieve the early immune escape through inhibiting the NK cell activation by upregulating the Treg cells. Thus, in this study we plan to elucidate the mechanism of the Treg cell expansion inhibiting the NK cell activation, to analyse the feasibility of pharmacological modulating TGF-βactivity to inhibit the immune escape in vitro and in vivo, and finally, to explore a new therapeutic target for the disease.

细粒棘球蚴病是一种危害严重的人畜共患寄生虫病，早期免疫逃逸是其赖以寄生于宿主的前提条件，也是防治策略的核心靶点，然而其机制尚不清楚。TGF-β在体内以分泌型与膜结合型两种形式存在，是机体重要的免疫抑制分子，在肿瘤、感染等疾病的免疫逃逸过程中起着重要的作用。研究表明：肿瘤促进Treg细胞的活化，并通过表达膜结合型TGF-β直接抑制NK细胞的功能，继而实现早期免疫逃逸。我们及其他研究者观察到细粒棘球蚴感染病人存在着分泌型TGF-β上调和Treg细胞增多现象。据此假设：与肿瘤相似，细粒棘球蚴通过上调分泌型TGF-β，促进Treg细胞的活化并上调膜结合型TGF-β的表达，继而抑制NK细胞的活性，实现早期免疫逃逸。本研究拟通过一系列体内体外实验，揭示分泌型与膜结合型TGF-β调控Treg细胞/NK细胞系统在细粒棘球蚴早期免疫逃逸中的作用机制。探讨药物调控体内TGF-β信号活性抑制其免疫逃逸的可行性。

建立细粒棘球蚴感染小鼠动物模型。在感染早期，小鼠CD4+/CD8+ T细胞比值逐渐下降；随着感染时间的延长，CD4+CD25+ T细胞数量呈逐渐增高；Foxp3在基因水平上的表达呈增高的趋势。在感染早期，外周血细胞因子TGF-β1的含量均显著高于对照组，并随着感染时间的延长而增高；TGF-β1在mRNA水平的表达量均高于对照组，随着感染时间的延长，呈增高的趋势。在感染早期，小鼠NK细胞对Yac-1细胞的裂解率（NK细胞杀伤活性）与对照组比较差异均有统计学意义；随着时间的延长NK细胞的活性受体NKG2D的表达量呈下降的趋势，与对照组比较，感染后1、3、9、12天NKG2D的表达量都有统计学意义。随着时间的延长NK细胞数量呈下降的趋势，与感染前比较，感染后第1、3、9、12天NK数量差异都有统计学意义。细粒棘球蚴早期，感染小鼠NK细胞的杀伤活性与其活性受体NKG2D的表达呈正相关。早期感染小鼠，抑制剂SB-525334阻断TGF-β1受体，其下游分子Smad2/3磷酸化蛋白表达下降，NK细胞活性受体NKG2D表达增加， NK细胞对靶细胞的裂解率恢复，小鼠NK细胞的活性与其活性受体NKG2D的相关性分析的表达呈正相关，CD4+/CD8+ T细胞比值升高，CD4+CD25+ T细胞数量减少。感染早期，使CD4+/CD8+ T细胞的比值倒置，Treg细胞数量增加有利于细粒棘球蚴的免疫逃逸。TGF-β1在小鼠细粒棘球蚴感染早期表达增加，可能有利于细粒棘球蚴的免疫逃逸。小鼠感染细粒棘球蚴后，可降低NK细胞数量、NK细胞活性受体NKG2D的表达和NK细胞的杀伤活性；包虫免疫逃逸可能是通过降低NK活性受体，来降低NK细胞的杀伤活性来实现。SB-525334可使棘球蚴早期感染小鼠TGF-β1的下游分子Smad2/3磷酸化蛋白表达下降，NK细胞活性受体NKG2D表达增加， NK细胞对靶细胞的裂解率恢复，CD4+/CD8+ T细胞比值升高，CD4+CD25+ T细胞数量减少。说明Treg细胞数量增并大量分泌TGF-β1抑制NK细胞对棘球蚴的杀伤功能，从而有利于棘球蚴的免疫逃逸。