Cell Fate Determination in the Innate Immune System

Abstract

Phagocytes of the innate immune system adopt cell fates that serve distinct purposes during homeostasis or inflammation. The innate immune system performs threat assessments by integrating pattern recognition receptor (PRR) sensation of microbial or host-derived ligands termed pathogen associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), respectively. Beyond these defined ligand interactions, PRRs also monitor key cellular processes for patterns of dysfunction. For example, the inflammasomes are a set of supramolecular organizing centers (SMOCs) poised to assemble in response to select cytosolic perturbations. Inflammasomes mediate the opposing inflammatory cell fates of hyperactivation and pyroptosis that differ in the binary outcome of cellular viability but share secretion of the interleukin-1 (IL-1) family of cytokines. One favored model of IL-1 secretion invokes passive secretion upon pyroptotic lysis. As hyperactivation shares similar genetic and biochemical signaling components with pyroptosis, we sought to understand how a viable cell could secrete IL-1 through interrogation of the recently discovered executioner of pyroptosis, known as gasdermin D (GSDMD). Our studies revealed GSDMD was necessary for IL-1β secretion from living macrophages exposed to inflammasome activators including bacteria, microbial ligands, and host-derived oxidized lipids. Cell- and liposome-based assays demonstrated that GSDMD pores were required for IL-1β transport across an intact lipid bilayer. These findings identify a non-pyroptotic function for GSDMD pores as conduits for the secretion of cytosolic cytokines under conditions of cell hyperactivation. The abundance and plasma membrane residence of GSDMD pores may explain why some cells survive (hyperactivation) or lyse (pyroptosis) downstream of inflammasome activation. Numerous proteins promote GSDMD cleavage, but none are known to regulate pore formation after GSDMD cleavage. Using a forward genetic screen, we identified the Ragulator-Rag complex as necessary for GSDMD pore formation and pyroptosis in macrophages. Mechanistic analysis revealed that Ragulator-Rag is not required for GSDMD cleavage upon inflammasome activation, but rather promotes GSDMD oligomerization in the plasma membrane. Defects in GSDMD oligomerization and pore formation can be rescued by mitochondrial poisons that stimulate reactive oxygen species (ROS) production. These findings reveal an unexpected link between key regulators of immunity (inflammasome-GSDMD) and metabolism (Ragulator-Rag).