Abstract 118: Assembly of the Nlrp3 Inflammasome Regulates NET Formation and is Promoted by the Vimentin Intermediate Filament Cytoskeletal System

Neutrophils and neutrophil extracellular traps (NETs) play a pivotal role in innate immunity and inflammation, thus significantly contributing to the development and progression of autoimmune diseases and thrombo-occlusive disorders. One of the major signaling eventsduring the innate immune response and inflammation is the secretion of the pro-inflammatory cytokines IL-1ß and IL-18 after inflammasome activation. The most prominent inflammasome in the innate immune system is composed of the NLR Family Pyrin Domain-Containing 3 (NLRP3) protein. Although the NLRP3 inflammasome is highly expressed in neutrophils, nothing is known about its possible role in NET formation (NETosis). Neutrophil stimulation with nigericin, a K + ionophore and a well-known activator of the NLRP3 inflammasome, results in assembly of the NLRP3 inflammasome, observed as ASC/speck formation, and subsequent NETosis. To investigate the contribution of the NLRP3 inflammasome in NETosis, MCC950 was used to inhibit NLRP3 signaling. This resulted insignificantly decreased NET formation in both murine and human neutrophils after activation with nigericin and monosodium urate crystals (MSU) or ionomycin, a common NETosis stimulant. Since NLRP3 activation in macrophages is significantly promoted by the vimentin intermediate filament network, we investigated NET formation in vimentin null mice. Neutrophils from vimentin null mice show a significant decrease in NETosis after stimulation with nigericin, MSU, or ionomycin, as well as a reduction in the assembly of ASC/speck compared to neutrophils from wild-type mice. Taken together, our results demonstrate that the NLRP3 inflammasome activation plays an important role in the process of NETosis and may, in part, explain why many diseases aggravated by NETs are also accompanied by increased activation of NLRP3 inflammasome.