Towards an in vitro model of NLRP3 inflammasome activation via refining measures of IL-1β secretion and inflammasome assembly

Abstract

The formation of the (NOD)-like receptor protein 3 (NLRP3) inflammasome, an intracellular multiprotein oligomer, drives the secretion of pro-inflammatory cytokines such as IL-1β and IL-18 and inflammatory modes of cell death in response to microbial infection and cellular damage. As aberrant inflammasome function is implicated in a wide range of diseases, the proper characterization of its underlying mechanisms and regulation is key to understanding its potential as a therapeutic target. Here, we present an updated in vitro cell culture model of the NLRP3 inflammasome using lipopolysaccharide (LPS)-primed, nigericin-activated J774A.1 macrophages. Phase-contrast microscopy, Western blotting and ELISA were used to evaluate changes in cell morphology and mature IL-1β expression, respectively. Immunocytochemistry was used to detect apoptosis-associated speck-like protein containing a CARD (ASC) speck formation in nigericin-treated macrophages. While experimental approaches to mature IL-1β measurement require further development, we observed distinctive, irregular nigericin-induced morphological changes and the formation of ASC specks as likely indicators of NLRP3 inflammasome activation, making our cell culture model suitable for future research.