The NLRP3 inflammasome is a protein complex consisting of the sensor protein NLRP3, the adaptor protein ASC and the enzyme caspase-1 which matures pro-inflammatory cytokines IL-1β and IL-18. Inflammasome components are upregulated via NF-κB activation, particularly by lipopolysaccharide (LPS), and oligomerised into a ‘speck’ when exposed to inflammasome activators from the host, environment or a pathogen such as nigericin. The NLRP3 inflammasome is linked to various inflammatory and infectious diseases, but the expression of inflammasome components and the molecular aspects of ‘speck’ formation remain unclear.
Previous studies have relied on ASC overexpression systems to study the NLRP3 inflammasome. We recently generated the world’s first fluorescently-tagged inflammasome reporter mice expressed under endogenous promoters. The NLRP3-CHCI reporter (mCitrine-NLRP3-mCherry) allows active and inactive NLRP3 to be distinguished by measuring the fluorescence resonance energy transfer (FRET) between mCitrine and mCherry. In contrast, the ASC-mCherry reporter enables detection of ASC specks. Initial experiments confirmed that bone marrow-derived macrophages (BMDMs) from both reporter mice displayed comparable IL-1β maturation to multiple inflammasome activators as WT BMDMs. NLRP3-CHCI mice also exhibited airway inflammation analogous to WT mice when intranasally exposed to nigericin. Collectively, NLRP3 inflammasome activation is not impeded in our reporter mice.
We then examined BMDMs from reporter mice to elucidate the molecular details of the NLRP3 inflammasome. Using bio-imaging and flow cytometry, we observed NLRP3 expression in resting NLRP3-CHCI BMDMs which was significantly upregulated during LPS treatment. Interestingly, we observed punctate ASC-mCherry and NLRP3-CHCI ‘specks’ following exposure to LPS and various inflammasome activators, indicating that NLRP3 inflammasome activation only requires a small amount of NLRP3 and ASC. Furthermore, we observed NLRP3 activation via FRET analysis following LPS and nigericin treatment.
Collectively, these results highlight the usage of ASC-mCherry and NLRP3-CHCI mice to study inflammasome expression, activation and therapeutic targeting in vitro and in vivo.