牙周共生菌和病原菌促进NLRP3依赖的IL-1β差异性表达的分子机制研究

It is believed that the balance of commensal microbes and periodontal tissue is essential to periodontal health. NLRP3 inflammasome regulates the activation and secretion of IL-1β, and recently its role in the periodontitis starts being taken seriously. Currently, it was found: 1) NLRP3 was differentially expressed in the gingival tissue of periodontal health and disease. Furthermore, a positive correlation was revealed between the expression of NLRP3 and IL-1β; 2) NLRP3 was differentially regulated by supragingival and subgingival microflora, and Porphyromonas gingivalis played a key regulatory role. Our preliminary study showed: periodontal pathogens could more significantly promoted IL-1β expressions than the commensals. Thus, our hypothesis is that NLRP3-driven IL-1β production is a special immune response, which discriminates between pathogenic and beneficial bacteria and regulates immune pathology of periodontitis. Based on our preliminary study, this project will testify our hypothesis through utilizations of clinical samples and biofilm-cell co-culture in vitro models, together with siRNA, Co-IP and MS, etc. techniques. Moreover, it will further identify the roles of TLRs-MyD88, TRIF-type I IFN-Caspase-11, etc. signaling pathways played in the above immune response. The expected results may provide a new explantaion for clarifying the molecular immune mechanisms and clinical immune intervention of periodontal diseases.

共生菌与牙周组织间的平衡关乎牙周健康。NLRP3炎性小体调控IL-1β活化与分泌，其在牙周病中的作用初被重视。目前仅知：NLRP3在健康人群和牙周炎患者的牙龈组织中有差异表达，且其表达与IL-1β的表达正相关；龈上和龈下菌群对NLRP3的调节有差异性，且牙龈卟啉单胞菌在此起着关键作用。我们前期发现：牙周致病菌比共生菌更能有效促进IL-1β分泌。因此假设：依赖NLRP3炎性小体产生IL-1β是一种特殊的免疫应答，能通过相应机制区别对待益生菌和病原菌，调节牙周病的免疫病理损害。在前期研究基础上，本课题将通过临床样本和菌斑生物膜-细胞共培养的体外模型联合siRNA、免疫共沉淀、蛋白质谱分析等实验手段验证上述假设，并明确TLRs-MyD88、TRIF-I型IFN-Caspase-11等信号传导通路在上述免疫应答中所扮演的角色。该研究结果将为阐明牙周病分子免疫机制和临床免疫干预提供新的理论依据。

已知NLRP3在健康人群和牙周炎患者的牙龈组织中有差异表达，并与IL-1β的表达正相关，且牙周致病菌比共生菌更能有效促进IL-1β分泌。我们假设依赖NLRP3炎性小体产生IL-1β是一种特殊的免疫应答，能通过相应机制区别对待益生菌和病原菌，调节牙周病的免疫病理损害。临床样本研究发现，相比于正常组织，IL-1β及NLRP3炎性小体在牙周炎组织中高表达。体外试验模型发现牙龈卟啉单胞菌相比轻链球菌更能有效促进IL-1β的合成与分泌，且存在显著的时间和细菌浓度依赖性。尽管两种细菌均能够在感染早期上调NLRP3的基因水平，但仅在牙龈卟啉单胞菌刺激下呈现显著的细菌浓度依赖性，且在该病原菌感染下，Caspase-1基因水平的变化趋势与IL-1β一致。而炎性小体的另一成分ASC，则在两种细菌感染下随刺激时间延长而不断下调。为探究两种细菌在NLRP3信号传导通路上的差异，以及是否与Caspase-4依赖的非经典信号通路存在联系，用siRNA分别敲除NLRP3、Caspase-1和Caspase-4三种关键蛋白。NLRP3基因敲除后宿主细胞在分别受到两种细菌感染时所分泌的成熟IL-1β相比于对照组均有所下调，进一步表明两种细菌诱导的IL-1β的分泌在一定程度上都依赖于NLRP3炎性小体。而敲除CASP1基因后，牙龈卟啉单胞菌引起的IL-1β的表达与分泌反而上调，表明在牙龈卟啉单胞菌感染下，宿主细胞中可能存在其他的信号通路调控IL-1β的表达。此外，当CASP1基因敲除后，Caspase-4的表达水平下调，尤其在牙龈卟啉单胞菌感染时更明显，表明Caspase-4与Caspase-1之间可能存在某种联系。敲除CASP4基因后，牙龈卟啉单胞菌感染下的细胞NLRP3蛋白表达水平显著上调，但轻链球菌组无明显差异。表明牙龈卟啉单胞菌感染时，Caspase-4可上调NLRP3的表达参与炎性信号通路，而不参与轻链球菌的信号传导，这为今后进一步研究宿主差异性识别牙周共生菌和病原菌的分子机制提供了重要思路。

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