Extracellular inflammasomes as novel endogenous danger signals that exert pro-inflammatory signaling on vascular cells

Abstract

Abstract Background and aims As part of the innate immune response, NLRP3 inflammasomes are involved in the process of sterile inflammation, IL-1β release and are key mediators of inflammation-related vascular diseases, such as atherosclerosis. Recent data showed the existence of extracellular inflammasomes released from monocytes during pyroptotic cell death. Their biological function in the vascular system is still not known. Here, we established a method to detect extracellular inflammasomes and tested the hypothesis that extracellular NLRP3 inflammasomes can be internalized by vascular cells, such as macrophages, endothelial cells and coronary smooth muscle cells and induce pro-inflammatory signaling. Methods and result Stimulation of THP1 monocytes and of isolated primary human monocytes with Lipopolysaccharide and Nigericin activated the NLRP3 inflammasome and induced pyroptosis and the release of inflammasome complexes. Extracellular inflammasomes were isolated from cell-free supernatant and identified as inflammasome complexes (oligomers) by immunoblot. For functional characterization, isolated fluorescent-labeled NLRP3 inflammasome complexes were shown to be internalized by THP1 macrophages (19.7±9.7% pos. cells), primary endothelial cells (HUVEC: 9.0±2.3% pos. cells, coronary artery endothelial cells: 11.0% ± 2.3% pos. cells) and coronary smooth muscle cells (42.8±9.9% pos.cells) using immunofluorescence staining, Z-stacks and imaging flow cytometric analysis. Extracellular NLRP3 inflammasomes (eNLRP3) induced pro-inflammatory signaling in macrophages by increasing IL1b and Tnfa gene expression (3.0- fold) as well as IL-1β release (con: 1.9 pg/ml vs. eNLRP3: 191.0 pg/ml). In coronary smooth muscle cells, treatment with extracellular inflammasomes increased endogenous Nlrp3 and IL1b gene expression as well as upregulation of Cell adhesion molecule 1 (Cadm1). Coronary artery endothelial cells showed also increased protein level of surface adhesion marker Intercellular Adhesion Molecule 1 (ICAM1). Conclusion Upon canonical NLRP3 inflammasome activation, mononuclear cells release inflammasome complexes into the extracellular space. Macrophages, endothelial cells and smooth muscle cells are able to internalize these extracellular inflammasome complexes that exert pro-inflammatory effects. These findings support the concept that cell-free NLRP3 inflammasomes act as extracellular signal molecules triggering pro-atherogenic signaling mechanisms in vascular cells. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): Leipzig University, Medical Faculty