Characterization of extracellular inflammasome specks and their vascular effects

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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.