Poly (I:C) and hyaluronic acid directly interact with NLRP3, resulting in the assembly of NLRP3 and ASC in a cell-free system

In the NLR family, pyrin domain containing 3 (NLRP3) is an intracellular pattern recognition receptor that activates pro-caspase-1, leading to IL-1β and IL-18 processing and activation in a large complex called the NLRP3 inflammasome. Since various pathogens or endogenous metabolites have been reported to stimulate NLRP3 inflammasome, the interaction between NLRP3 and ASC induced by these stimulants may be an attractive drug target for NLRP3-related diseases, called inflammasomopathies. However, the endogenous ligand that directly interacts with NLRP3, leading to binding to ASC, remains unclear. Therefore, we developed a cell-free system consisting of NLRP3, ASC, and pro-caspase-1 or ASC and NLRP3 with an amplified luminescent proximity homogeneous assay (ALPHA). ALPHA signals of the interaction between NLRP3 and ASC were not enhanced following an incubation without any ligand, whereas strong ALPHA signals for the interaction between NLRP3 and ASC and between NLRP3 and pro-caspase-1 with the adaptor ASC were observed upon an incubation with poly (I:C) and hyaluronic acid (HA). Poly (I:C) and HA both directly interacted with NLRP3 within a specific concentration. These results suggest that NLRP3 directly interacts with intrinsic RNA and HA, which is followed by the activation of NLRP3 inflammasome, and the cell-free system consisting of NLRP3 and ASC, or NLRP3, ASC, and pro-caspase-1 may be a useful tool for elucidating the pathogenesis of inflammasomopathies and developing target therapeutics.