The NLRP3 inflammasome is a multi-protein complex that initiates innate immunity responses when exposed to a wide range of stimuli, including pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs). Inflammasome activation leads to the release of the pro-inflammatory cytokines interleukin (IL)-1β and IL-18 and to pyroptotic cell death. Over-activation of NLRP3 inflammasome has been associated with several chronic inflammatory diseases. A deep knowledge of NLRP3 inflammasome biology is required to better exploit its potential as therapeutic target and for the development of new selective drugs. To this purpose, in the past few years, several tools have been developed for the biological characterization of the multimeric inflammasome complex, the identification of the upstream signaling cascade leading to inflammasome activation, and the downstream effects triggered by NLRP3 activation. In this review, we will report cellular models and cellular, biochemical, and biophysical assays that are currently available for studying inflammasome biology. A special focus will be on those models/assays that have been used to identify NLRP3 inhibitors and their mechanism of action.

The NLRP3 inflammasome is formed by the sensor NLRP3, the adaptor ASC, and pro‐caspase‐1. Assembly and activation of the inflammasome trigger caspase‐1‐dependent cleavage of pro‐IL‐1β and pro‐IL‐18 into their secreted forms. Cigarette smoke is a risk factor for chronic inflammatory diseases and is associated with macrophage dysfunction. The impact of cigarette smoke on NLRP3‐dependent responses in macrophages is largely unknown. Herein, we investigated the effects of cigarette smoke extract (CSE) on the NLRP3 inflammasome in human monocyte‐derived macrophages (MDMs) and THP‐1 cells stimulated with lipopolysaccharide (LPS) and LPS plus the NLRP3 inflammasome activator ATP. We found that CSE inhibited the release of IL‐1β and IL‐18 as well as the expression of NLRP3 acting mainly at the transcriptional level. Interestingly, we found that CSE increased the caspase‐1 activity via an NLRP3‐independent and TLR4‐TRIF‐caspase‐8‐dependent pathway. Activation of caspase‐1 by CSE led to a reduction of the basal glycolytic flux and impaired glycolytic burst in response to LPS. Overall, our findings unveil novel pathways leading to immune‐metabolic alterations in human macrophages exposed to cigarette smoke. These mechanisms may contribute to macrophage dysfunction and increased risk of infection in smokers.