利用人源化过敏小鼠研究TGF-β/Smad7调控Tregs/Thl7平衡分化及Tregs/Thl7相关因子表达的机制和作用

The discovery of Tregs and Thl7 makes up for the insufficiency of the mechanism of immune tolerance deficiency in allergy. Th17 and IL-17 play an important role in allergy, while TGF-β is a crucial factor for differentiation of Tregs/Th17 and expression of IL-17. However, the mechanism mediating this effect has not been fully elucidated. In our previous study, we developed a novel humanized murine model of peanut allergy that recapitulates the human anaphylactic response to peanuts, confirmed that IL-17 levels in humanized allergic mice displayed increased levels of IL-17 after allergen challenge compared with those in non-allergic mice. Moreover, blocking the signaling of TGF-β reversed the increased levels of IL-17 and reduced accumulation of eosinophils and neutrophils in lungs and small intestines. Therefore, we hypothesized that, in humanized allergic mice, the signaling of TGF-β and its negative feedback regulating factor Smad7 could influence Tregs/Th17 balance and cause allergic reactions by promoting Th17 differentiation and expression of IL-17. To validate the hypothesis, we will investigate the effect of TGF-β/Smad7 on allergy, Tregs/Th17 balance and expression of Tregs/Th17-related factors, and confirm the role of TGF-β/Smad7 in the regulation of Tregs/Th17 balance and Tregs/Th17-related factors. We will also explore the effect of anti-TGF-β receptor and anti-IL-17 in the humanized murine model of allergy. Our research would provide a new strategy and target for induction of peripheral immune tolerance and treatment of allergy.

Tregs和Thl7的发现弥补了过敏患者免疫耐受缺陷机制的不足。Th17及IL-17在过敏疾病中发挥重要作用，而TGF-β是调控 Tregs/Thl7分化及 IL-17 表达的重要因子，但具体机制不明。前期研究构建人源化小鼠模型成功模拟人体过敏特征，证实过敏状态下IL-17水平明显升高，且阻断TGF-β信号通路可降低IL-17表达，并减弱炎症细胞浸润。因此我们假设，TGF-β信号通路通过促进Th17分化及IL-17表达影响Tregs/Th17平衡，加重过敏反应，而Smad7可能在此发挥重要作用。为验证假说，我们在体内外探讨TGF-β/Smad7在过敏进程中的作用及其对Tregs/Th17分化和相关因子表达的影响，明确TGF-β/Smad7对 Tregs/Th17分化及相关因子的调节机制及拮抗TGF-β受体和IL-17在过敏疾病中的治疗作用，为诱导外周免疫耐受和探索新型治疗靶点提供重要依据。

Tregs和Thl7的发现弥补了过敏患者免疫耐受缺陷机制的不足。Th17及IL-17在过敏疾病中发挥重要作用，而TGF-β是调控Tregs/Thl7平衡分化及IL-17表达的重要因子，但具体机制及参与过敏进程作用不明。我们前期研究构建人源化小鼠模型成功模拟人体过敏特征，证实过敏状态下IL-17水平明显升高，且阻断TGF-β信号通路可减弱炎症细胞浸润。因此我们推测，TGF-β信号通路通过促进Th17分化及IL-17表达影响Tregs/Th17平衡，加重过敏反应，而Smad7可能在此过程中发挥重要作用。基于此，本项目构建PBMCs重组人源化过敏小鼠模型，结合过敏患者通过体内外实验检测Tregs、Th17比例，Tregs/Thl7相关细胞因子及TGF-β 1、Smad7表达，并利用TGF-β 受体抗体和IL-17抗体进行干预研究，进一步明确 TGF-β/Th17/IL-17在过敏进程中的作用。结果显示，粗花生提取物CPE可成功致敏人源化小鼠，相比于PBMCs非过敏供者对照组，PBMCs花生过敏供者组于CPE连续致敏后可于血清中检测到人过敏原特异性IgE表达。PBMCs过敏供者组肺组织TGF-β1表达增高，Smad7表达降低，脾细胞CD4+T细胞中Thl7比例升高，肺泡灌洗液BALF中IL-17表达显著升高，肺组织炎症细胞浸润及血浆组胺水平释放均明显增加；相比于非过敏对照组，PBMCs过敏供者组小鼠具有更高的系统性过敏症状评分。进一步通过拮抗TGF-β受体发现，重组过敏模型组小鼠脾细胞CD4+T细胞Thl7比例下调，且BALF中IL-17表达水平下降；联合拮抗TGF-β受体和IL-17后，重组过敏模型组小鼠肺组织炎症浸润减轻，血浆组胺水平释放下降且系统性过敏症状评分降低。本项目研究结果确证人源化过敏小鼠模型有效性，有助于明确TGF-β/Smad7调控Tregs/Th17平衡分化及相关因子表达参与过敏进程中的作用，将为过敏疾病治疗基础研究临床化提供重要依据。