A Genome-Wide Screen Identifies a Critical Role For Mitochondrial NDP Kinases in Inflammasome Activation

Introduction:

While there has been a remarkable progress in understanding the mechanisms of cytosolic LPS sensing by caspase11, the cellular processes regulating non-canonical inflammasome activation are less clearly understood.

Methods:

In an effort to address this, we have conducted a genome-scale siRNA screen for the non-canonical inflammasome response to cytosolic LPS. We have used a screening-optimized HTRF assay for secreted IL-1α in RAW264.7 mouse macrophage cells. Through a combination of LDH and TNFα measurements we have filtered and prioritized hits in a secondary screen. We used siRNA knockdown in primary BMDM and CRISPR/Cas9 knock out of hit genes both in macrophage cell lines and mice to further validate and study novel mechanisms of inflammasome activation.

Results:

Among the top screen hits we identified numerous expected genes, including Myd88, Irak4, Irak2, Casp4, Gsdmd and Il1α, and also discovered numerous novel regulators. Significant mitochondrial-associated gene enrichment supported an important role for the mitochondria and cellular metabolism in inflammasome activation. Specifically, we investigated the role of NDP kinase in inflammasome activation and find NDPK^-/-macrophages have a strongly diminished IL-1α response to cytosolic-LPS, show defective ROS- and cardiolipin-dependent mitochondrial licensing, and have markedly dysregulated inflammasome priming and triggering. Metabolic analysis suggests NDPK is critical to priming-induced glycolytic commitment, however we observe normal NF-kB and MAPK activation in primed NDPK^-/-, suggesting the mitochondrial and metabolic contribution to inflammasome priming occurs independently of these signaling responses. We also find that NDPK deficient mice show substantial resistance to LPS-induced endotoxic shock.

Conclusions:

We found an unexpected role for the NDPK family of mitochondrial dinucleotide kinases in the effective priming of the inflammasome response by TLR ligands, thus establishing a link between the cellular metabolic state of the macrophage and the LPS-driven inflammatory response. Our data delineate the mitochondrial and metabolic processes critical to inflammasome activation and suggest that a critical mitochondrial fitness signal is required to induce inflammasome activation.

This work was supported by the Intramural Research Program of NIAID, NIH.