Role of IL-37 Genetic Variants in Modulating Innate Immune Resp to Periodontal Pa

描述（由申请人提供）：牙周炎是一种对口腔共生菌群的炎症反应，是人类最普遍的感染之一。大约90%的人口表现出某种形式的早期疾病（牙龈炎），其中48%的美国成年人患有牙周炎，严重形式的疾病影响15-20%。牙周病是一种多基因疾病，与对共生生物膜的过度炎症反应有关，这种反应导致生物膜-牙龈界面的慢性生物膜失调。牙龈沟液（GCF）白细胞介素1 β (IL-1 β)水平升高已被确定为高炎症表型和介导严重炎症、骨质流失和牙周病进展的强有力的生物标志物。我们最近完成了一项4910名已知GCF-IL-1 -水平的白种人的全基因组关联研究（GWAS），以确定与高水平GCF-IL-1 -相关的位点。我们在抗炎基因IL37编码区内发现了两个新的数量性状位点，它们具有错义多态性，这两个位点都与高GCF-IL-1水平（定义为上四分位数或连续变量）具有统计学意义，p=6.8 X10-21。在30%和8.5%的人群中存在两个IL-37位点的错义多态性。这两个基因座不仅与局部高IL-1水平密切相关，而且还与更严重的牙周病有关。在正常情况下，IL-37激活Smad3并减弱TLR激动剂的先天免疫反应，包括抑制IL-1 α、IL-1 α、TNF α、IL-6、MIP-2 （CXCL2）和GM-CSF。因此，我们的中心假设是，这些IL37 SNP变异导致IL-37的功能缺陷（通过改变mRNA剪接、蛋白质合成、激活和/或生物活性），导致对口腔共生生物过度的促炎先天免疫反应，从而诱导更大的临床炎症、骨质流失和更严重的临床牙周病。由于小鼠不表达IL-37的小鼠同源物，我们在小鼠模型中提出了两个目的，以了解两种IL-37变体是否表现出IL-37功能受损，即SMAD3激活和先天免疫反应抑制。首先，我们将用人类野生型（IL37wt）或两种IL37变体（IL37v1或IL37v2）转染小鼠单核细胞RAW细胞，以评估这些突变对IL-37功能的影响，并使用一系列TLR激动剂刺激细胞。这些实验转染IL37v1或IL37v2将在人单核细胞系THP-1中得到证实。我们预计，与hIL-37wt相比，这些变体将具有高度炎症的特征，测量上述介质的水平，作为读数。我们还将创建IL37变异转基因小鼠，使用皮下腔模型和口腔骨质流失模型来测量对牙龈P攻击的反应。最后，我们将建立一个新的IL-37基因分型队列，利用分离的人PBMC和hTERT永生化细胞来研究和确认这两种IL-37变体对IL-37功能和对TLR攻击的反应的作用。预计了解这种新变异作为与严重牙周病相关的潜在高炎症特征的潜在原因的作用将最终改善诊断、预防和治疗。

DESCRIPTION (provided by applicant): Periodontitis is an inflammatory response to the commensal oral bacterial flora and represents one of the most prevalent infections in humans. Approximately 90% of the population exhibits some form of early disease (gingivitis), with 48% of the US adult population having periodontitis and the severe form of the disease affecting 15-20%. Periodontal disease is a polygenic condition that is associated with an exaggerated inflammatory response to the commensal biofilm that drives chronic biofilm dysbiosis at the biofilm-gingival interface. An elevated level of gingival crevicular fluid (GCF) interleukin 1beta (IL-1�) has previously been established as a robust biomarker for a hyper- inflammatory phenotype and for mediating severe inflammation, bone loss and periodontal disease progression. We have recently completed a genome-wide association study (GWAS) of 4910 Caucasians with known levels of GCF-IL-1� to identify loci that are associated with high levels of GCF-IL-1�. We have identified two novel quantitative trait loci with missense polymorphisms within the coding region of the anti-inflammatory gene IL37 that are both statistically significanty associated with high GCF-IL-1� levels (defined as upper quartile or as a continuous variable), p=6.8 X10-21. These missense polymorphisms at two IL-37 loci, are present in 30% and 8.5% of the population. Not only are both loci strongly associated with high local IL-1� levels, but the are also associated with more severe periodontal disease. Under normal conditions IL-37 activates Smad3 and attenuates the innate immune response to TLR agonists to include suppression of IL-1�, IL-1�, TNF�, IL-6, MIP-2 (CXCL2) and GM-CSF. Thus, our central hypothesis is that these IL37 SNP variants cause a functional defect in IL-37 (either via altered mRNA splicing, protein synthesis, activation and/or bioactivity) that results in an excessive pro-inflammatory innate immune response to the commensal organisms of the oral cavity, thereby inducing greater clinical inflammation, bone loss and more severe clinical periodontal disease. Since mice do not express a murine homologue to hIL-37 we propose two aims in murine models to understand whether the two IL37 variants demonstrate impaired IL-37 function I.e. SMAD3 activation and suppression of the innate immune response. First we will transfect murine monocytic RAW cells with the human wild-type [IL37wt] or the two IL37 variants, IL37v1 or IL37v2, to assess the effects of these mutations on IL-37 function, stimulating cells with a range of TLR agonists. These experiments transfecting with IL37v1 or IL37v2 will be confirmed in the human monocytic line THP-1. We expect that the variants will have a hyper-inflammatory trait compared to hIL-37wt, measuring the levels of the mediators listed above, as the read-out. We will also create IL37 variant transgenic mice to measure the response to challenge with P gingivalis using a subcutaneous chamber model and an oral bone loss model. Finally, we will establish a new IL-37 genotyped cohort of subjects to study and confirm the role of these two IL37 variants on IL-37 function and response to TLR challenge using isolated human PBMC and hTERT immortalized cells. It is expected that understanding the role of this novel variant as a potential cause of the underlying hyper-inflammatory trait associated with severe periodontal disease, will ultimately improve diagnosis, prevention and treatment.