Characterization of extracellular inflammasome specks and their vascular effects

无菌炎症参与动脉粥样硬化性心血管疾病(ASCVD)的发生和发展，是治疗干预的一个有趣的新研究领域。NLRP3炎症体在ASCVD中起重要作用，其激活受外源性和内源性应激信号的调节，与ASCVD的转归有关。NLRP3炎症体最初被描述为细胞内感受性蛋白复合体，激活后导致白细胞介素1β的分泌和嗜热性细胞死亡。因此，炎性小体本身可以被释放到细胞外空间。然而，细胞外炎性小体斑点的血管效应及其在动脉粥样硬化形成过程中的作用尚未被探讨。我们的初步数据显示，在人类单核细胞中，NLRP3炎性小体激活导致依赖于NLRP3的焦磷酸细胞死亡，并将炎性小体斑点释放到细胞外空间。我们的发现表明，细胞外炎性小体能够被周围的巨噬细胞、人冠状动脉内皮细胞和平滑肌细胞内化，在那里它们发挥促炎信号。这导致了我们的假设，细胞外炎症小体作为循环的危险信号，使炎症信号从细胞到细胞持续存在，并发挥促动脉粥样硬化的功能。本项目的目的是研究体外培养的人冠状动脉内皮细胞和平滑肌细胞中细胞外炎性小体对血管的影响，并确定阻断细胞外ASC斑点的形成和成核是否对靶细胞有有益的影响(工作包(WP)1)。此外，我们将确定炎症体的翻译后修饰(PTM)是否影响组装、激活和细胞外炎症体功能(WP2)。为了研究体内动脉粥样硬化的形成和下垂是否依赖于NLRP3炎性小体的激活，我们将使用炎性小体缺陷小鼠建立PCSK9-AAV8诱导的动脉粥样硬化模型(WP3)。最后，我们将探索急性心肌梗死患者血清炎症小体水平与外周血单个核细胞(PBMC)嗜热细胞死亡的临床相关性，并将它们与健康对照组(WP4)进行比较。综上所述，这些项目结果将首次对细胞外炎症小体的血管效应、它们的调节和临床相关性以及它们如何推动体内动脉粥样硬化的进展提供新的见解。我们的发现将有助于对炎症小体作为细胞外致动脉粥样硬化因子的新理解，并将支持针对动脉粥样硬化性心血管疾病患者炎症的创新治疗策略的开发。

Sterile inflammation is involved in the development and progression of atherosclerotic cardiovascular diseases (ASCVD) and represents an interesting novel research field for therapeutic interventions. The NLRP3 inflammasome plays a crucial role in ASCVD as its activation is regulated by exogenous and endogenous stress signals and is associated with the outcome of ASCVD. The NLRP3 inflammasome was originally described as intracellular sensor protein complex that upon its activation it leads to Interleukin-1β secretion and pyroptotic cell death. Thereby, inflammasomes itself can be released into extracellular space. However, the vascular effects of extracellular inflammasome specks and their function during atherogenesis are not yet explored.Our preliminary data revealed that NLRP3 inflammasome activation in human monocytes leads to NLRP3- dependent pyroptotic cell death and release of inflammasome specks into extracellular space. Our findings revealed that extracellular inflammasomes are able to be internalized by surrounding macrophages, human coronary endothelial cells and smooth muscle cells where they exert pro- inflammatory signaling. This led us to the hypothesis that extracellular inflammasomes act as circulating danger signals that perpetuate inflammatory signaling from cell-to-cell and exert pro-atherogenic function. The aim of this project proposal is to investigate the vascular effects of extracellular inflammasomes in-vitro in human coronary endothelial cell and smooth muscle cells and determine whether a blockade of extracellular ASC speck formation and nucleation has beneficial effects on target cells (work package (WP) 1). Further, we will determine whether post-translational modifications (PTMs) of inflammasomes affect assembly, activation and extracellular inflammasome function (WP2). To study whether atherogenesis and pyroptosis in-vivo is dependent on NLRP3 inflammasome activation, we will use inflammasome deficient mice in a Pcsk9-AAV8 induced atherosclerosis model (WP3). And finally, we will explore the clinical association of serum inflammasome levels and pyroptotic cell death of peripheral blood mononuclear cells (PBMCs) in patients with acute myocardial infarct and compare them with healthy controls (WP4).Taken together, these project results will deliver for the first time new insights about the vascular effects of extracellular inflammasomes, their regulation and clinical relevance, and how they drive progression of atherogenesis in-vivo. Our findings will contribute to a novel understanding of inflammasomes as extracellular atherogenic factors and will support the development of innovative therapeutic strategies targeting inflammation in patients with atherosclerotic cardiovascular diseases.