An arrayed genome-wide RNAi screen for the discovery of novel inflammasome regulators

Abstract

Abstract Bacterial lipopolysaccharide (LPS) is a prominent PAMP implicated as the primary causative agent of septic shock in a wide range of bacterial infections. LPS is known to activate two receptors: the plasma membrane receptor, TLR4, and the cytosolic receptor, Caspase 11. We have conducted a genome-wide siRNA screen to identify novel gene products involved in the non-canonical inflammasome response to cytosolic LPS. A ‘prime-trigger’ assay was established in which macrophages were primed with Pam3CSK4, triggered by transfection of the active component of LPS, Lipid A, and IL-1α secretion measured by a screening-optimized HTRF assay, and pyroptosis was evaluated using an LDH assay. The screen was designed to identify and distinguish regulators at multiple stages of the non-canonical inflammasome activation process, from TLR priming, to cytosolic LPS detection, pyroptosis and IL-1α release. Initial analysis of the primary screen data identified a high canonical pathway hit rate in both the TLR priming step and the response to cytosolic LPS, with Irak4, Irak2, Casp4, Gsdmd and Gsdmc among the strongest gene hits. Among the genes not previously linked to regulation of the non-canonical inflammasome, we observed an enrichment for mitochondrial proteins. In order to further validate this finding, we have created a CRISPR/Cas9 knock-out macrophage cell line and knock-out mouse for one of the mitochondrial screen hits, confirming the effect of this novel hit on IL-1α release in response to cytosolic LPS. Our results suggest a critical role for mitochondria in regulation of the non-canonical inflammasome response. This work was generously supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases.