Caspase-11裂解Gasdermin D促细胞焦亡对牙龈卟啉单胞菌致慢性牙周炎发生发展的分子机制研究

Porphyromonas gingivalis is a keystone periodontal pathogen, but its underlying pathogenesis is still need to be elucidated. It is known that Caspase-11 could trigger pyroptosis through cleavage of Gasdermin D, then rapidly mediated release of the pro-inflammatory cytokine IL-1β. Interesting, our preliminary study showed: Porphyromonas gingivalis could activate Caspase-11 and could trigger pyroptosis through cleavage of Gasdermin D; After inhibition of Caspase-11, the expression of active Caspase-1 and mature IL-1β were both decreased significantly. Thus, we hypothesize that Porphyromonas gingivalis activated pyroptosis, which depends on Caspase-11 to cleave Gasdermin D, plays a vital role in periodontal inflammatory effects. This project will testify our hypothesis through utilization of clinical samples, in vitro cellular and gene knockout mouse models. It will further identify how Porphyromonas gingivalis activates Caspase-11 via TLRs-TRIF-type I interferon pathway or LPS intracellular pathway. Moreover, we will focus on the mechanism of how pyroptosis, which is induced by Caspase-11-dependent Gasdermin D cleavage, affects the maturation and secretion of IL-1β, clears periodontal pathogens or initiates an uncontrollable inflammatory response. This project aims to provide a new theoretical explanation to periodontal pro-inflammatory mechanism caused by Porphyromonas gingivalis.

牙龈卟啉单胞菌是牙周关键致病菌，其致病机制复杂。已知Caspase-11裂解Gasdermin D后触发细胞焦亡，快速介导促炎因子IL-1β的释放。有趣的是，我们前期发现：牙龈卟啉单胞菌可激活Caspase-11，又引起Gasdermin D裂解触发细胞焦亡; 抑制Caspase-11后Caspase-1和成熟IL-1β分泌显著下降。因此假设：牙龈卟啉单胞菌依赖Caspase-11裂解Gasdermin D后触发的细胞焦亡在牙周致炎效应中起着重要作用。本项目拟从临床样本、细胞和基因敲除动物模型等不同层面深入研究牙龈卟啉单胞菌如何经TLRs-TRIF-I型干扰素通路或LPS胞内途径激活Caspase-11，重点关注Caspase-11裂解Gasdermin D介导的细胞焦亡如何影响IL-1β的成熟和分泌，清除病原菌或爆发不可控的炎症反应。以期为牙龈卟啉单胞菌引起的牙周致炎机制提供新理论解释。

细胞焦亡是一种通过炎性小体介导，并伴有大量炎性细胞因子释放的程序性细胞死亡方式。牙龈卟啉单胞菌作为牙周炎的关键致病菌，可以通过激活牙周组织中巨噬细胞核苷酸结合寡聚化结构域样受体蛋白3炎症小体（NLRP3），促进细胞焦亡爆发，破坏牙周菌群稳态，进而导致牙周炎症的进展。我们假设牙龈卟啉单胞菌依赖 Caspase-11 裂解 Gasdermin D 介导的细胞焦亡在牙周促炎效应中起着关键作用，且在慢性牙周炎发病机制中占有重要意义。我们在临床样本研究发现，相比于牙周健康人群的牙龈组织，慢性牙周炎患者牙龈组织中焦亡相关基因及蛋白，包括焦亡核心基因IL-1β、焦亡功能调节基因GBP2、GBP5、DDX3X、CFLAR、DHX9及焦亡炎症基因NLRP3、MEFV的mRNA显著高表达。体外模型中发现牙龈卟啉单胞菌能够促发细胞焦亡，促进促炎因子 IL-1β成熟和分泌，存在时间依赖性，且在牙龈卟啉单胞菌感染后，Caspase-11、Caspase-1、NLRP3、ASC表达水平的变化趋势与IL-1β一致。为进一步研究是否细胞焦亡关键分子Caspase-11在牙周炎发病中的作用，我们建立了Caspase-11基因敲除小鼠，发现其原代骨髓来源巨噬细胞在受到牙龈卟啉单胞菌感染后Caspase-1、NLRP3、ASC及分泌的成熟IL-1β较对照组显著下调。通过建立野生型、Caspase-11基因敲除、Caspase-1基因敲除小鼠实验性牙周炎模型，我们发现Caspase-11、Caspase-1敲除小鼠的牙周炎症显著缓解，牙槽骨吸收程度减小，并且相较于野生型小鼠，基因敲除小鼠牙周菌群多样性显著增加。进一步通过同笼饲养野生型和不同基因型小鼠，我们发现野生型牙周炎小鼠牙周炎症减轻，且牙周菌群多样性显著降低。这为菌斑与宿主免疫之间的相互关系提供新的理论基础，为临床治疗牙周炎探索新的可能干预靶点，从而维持健康的牙周生理稳态提供了重要思路。