NAIP/NLRC4 inflammasome dynamics in murine enteroids are tuned by NAIP ligand concentration and epithelial cell differentiation

Abstract

Inflammasomes are central components of mammalian innate immunity activated upon the detection of danger signals such as pathogen ligands. This activation generally leads to pyroptosis and interleukin release, but specific responses vary between cell types. Here, we study nucleotide-binding, leucine-rich repeat (NLR) family apoptosis inhibitory protein (NAIP)/NLR CARD-containing protein 4 (NLRC4) inflammasome dynamics of intestinal epithelial cells of murine enteroids at the single-cell level. Using an apoptosis-associated speck-like protein containing a CARD (ASC)::GFP reporter and a cell-membrane-permeable NAIP/NLRC4 agonist, we observe pyroptosis with or without ASC polymerization. We use fluidic force microscopy to inject NAIP ligands into cells, revealing a dose-dependent response. High doses promote ASC polymerization, while lower doses lead to survival or NLRC4-dependent cell death in the absence of ASC polymerization. Using a fluorescence resonance energy transfer (FRET) reporter as a proxy for interleukin maturation, we reveal that single epithelial cell caspase-1 dynamics are affected by the differentiation state of cells and NAIP ligand dosing. Our results indicate an inflammasome response that is tuned at multiple levels in intestinal epithelial cells.