Inflammation and Infection in Atherosclerosis

描述(申请人提供)：冠状动脉疾病(CAD)是世界范围内发病率和死亡率的主要原因。由于包括高胆固醇血症、吸烟和高血压在内的传统危险因素不能完全解释冠心病的发病率，最近的兴趣集中在慢性感染性疾病，如牙周感染，作为冠心病的潜在贡献者。流行病学研究表明，患有牙周感染的人患冠心病的可能性要高出30%到100%。在过去的资金周期中，我们产生了大量与感染感知回路的作用以及随后的炎症细胞因子在牙龈卟啉单胞菌(P.G)相关动脉粥样硬化小鼠模型中的表达相关的数据。针对这一相互竞争的应用，我们评估了P.G侵袭在动脉粥样硬化形成中的作用。我们发现，当侵袭缺陷的P.G.DPG3菌株取代了野生型P.G，或者当包括侵袭干扰抗生素甲硝唑处理时，随后的动脉粥样硬化减少了约50%，支持存在侵袭介导的胞浆过程，这对P.G驱动的动脉粥样硬化具有重要作用。我们还开始研究感染和动脉粥样硬化之间的联系机制。核苷酸结合寡聚结构域样受体(NOD1和NOD2)代表一种免疫监视系统，它检测细胞内微生物分子的存在。最近研究表明，肽聚糖(PGN；一种TLR2配体)不依赖于其胞壁二肽(MDP)组分(NOD2配体)被细胞表面TLR2识别，也可通过涉及MyD88的独特的TLR信号通路激活NF-：B。这引入了TLR2和NOD2信号联系在一起的可能性，NOD2的一个功能是调节TLR2。然而，NOD2在TLR介导的细胞因子反应中的作用仍然存在争议。我们的初步数据显示，在P.G刺激的巨噬细胞中引入NOD2 siRNA可以增强促炎反应。然而，TLR2介导的细胞因子反应中NOD2的激活依赖于MDP的剂量：低配体刺激似乎是协同的，而高配体水平似乎是抑制的。这种调节导致了核因子：B的调节以及IL-1和肿瘤坏死因子的转录调节。我们的假设是，NOD2的激活负向调节TLR2的反应，进而减轻动脉粥样硬化；缺乏这种调节会导致免疫反应增强和动脉粥样硬化加重。为了验证我们的假设，我们在目标1中建议使用功能损失研究来确定NOD2在P.g诱导的动脉粥样硬化中的作用；在目标2中确定NOD2调节先天炎症反应和侵袭介导的胞浆过程的分子机制；在目标3中利用功能研究的增益来确定给予胞壁二肽(MDP)或NOD2阻滞剂是否保护小鼠免受P.g诱导的动脉粥样硬化的影响。这些实验将清楚地确定NOD2在P.G动脉粥样硬化形成中的作用，并为旨在减少或预防动脉粥样硬化形成的药物干预铺平道路。 公共卫生相关性：冠状动脉疾病(CAD)是全球人类发病率和死亡率的主要原因。拟议的实验结果将确定免疫感应在感染相关动脉粥样硬化形成中的作用，并为旨在减少或预防动脉粥样硬化形成的药物干预铺平道路。

DESCRIPTION (provided by applicant): Coronary artery disease (CAD) is a major cause of morbidity and mortality worldwide. Recent interest has focused on chronic infectious diseases such as periodontal infection as potential contributors to CAD since traditional risk factors, including hypercholesterolemia, smoking, and hypertension fail to fully explain the incidence of CAD. Epidemiological studies indicate that individuals with periodontal infection are 30 to 100% more likely to have CAD. During the past funding cycle, we generated substantial data related to the role of infection-sensing circuits and ensuing inflammatory cytokine expression in a mouse model of Porphyromonas gingivalis (P.g)-associated atherogenesis. Pertinent to this competing application, we evaluated the role of P.g invasion in atherogenesis. We found that when invasion-deficient P.g. strain DPG3 was substituted for wild-type P.g, or when treatment with the invasion interfering antibiotic metronidazole was included, subsequent atherogenesis was reduced by about 50%, supporting the existence of an invasion-mediated cytosolic process that contributes significantly to P.g-driven atherogenesis. We have also begun to characterize the mechanisms linking infection and atherogenesis. Nucleotide binding oligomerization domain- like receptors (NOD1 and NOD2) represent an immune surveillance system that detects the presence of microbial molecules inside the cell. Recently peptidoglycan (PGN; a TLR2 ligand) was shown to be recognized independently of its muramyl dipeptide (MDP) components (NOD2 ligands) by cell-surface TLR2, and also to activate NF-:B through a distinct TLR signaling pathway involving MyD88. This introduced the possibility that TLR2 and NOD2 signaling are linked, and that one function of NOD2 is the regulation of TLR2. However, the role of NOD2 in TLR-mediated cytokine responses remains controversial. Our preliminary data show that introducing NOD2 siRNA into P.g-stimulated macrophages heightens the pro-inflammatory response. However, NOD2 activation of TLR2-mediated cytokine response was found dependent on MDP dose: low ligand stimulation appears to be synergistic while high ligand levels appear inhibitory. This regulation leads to NF:B modulation and IL-1 and TNF transcriptional regulation. Our hypothesis is that activation of NOD2 negatively regulates TLR2 responses, which in turn reduce artherosclerosis; the absence of such regulation leads to heightened immune responses and aggravated atherosclerosis. To test our hypothesis we are proposing in Aim 1 to use loss of function studies to determine the role of NOD2 in P.g- driven atherosclerosis; in Aim 2 to determine the molecular mechanisms involved in NOD2 regulation of the innate inflammatory response and invasion-mediated cytosolic process; and in Aim 3 to determine whether administration of muramyl dipeptide (MDP) or NOD2 blockers protect mice from P. g induced atherosclerosis using gain of function studies. These experiments will clearly define the role of NOD2 in P.g atherogenesis and pave the way for pharmacological interventions aimed at reducing or preventing atherogenesis. PUBLIC HEALTH RELEVANCE: Coronary artery disease (CAD) is a major cause of morbidity and mortality in humans worldwide. The results of the proposed experiments will define the role of immune sensing in infection-associated atherogenesis and pave the way for pharmacological interventions aimed reducing or preventing atherogenesis.