A genome-wide screen uncovers multiple roles for mitochondrial nucleoside diphosphate kinase D in inflammasome activation

Abstract

Noncanonical inflammasome activation by cytosolic lipopolysaccharide (LPS) is a critical component of the host response to Gram-negative bacteria. Cytosolic LPS recognition in macrophages is preceded by a Toll-like receptor (TLR) priming signal required to induce transcription of inflammasome components and facilitate the metabolic reprograming that fuels the inflammatory response. Using a genome-scale arrayed siRNA screen to find inflammasome regulators in mouse macrophages, we identified the mitochondrial enzyme nucleoside diphosphate kinase D (NDPK-D) as a regulator of both noncanonical and canonical inflammasomes. NDPK-D was required for both mitochondrial DNA synthesis and cardiolipin exposure on the mitochondrial surface in response to inflammasome priming signals mediated by TLRs, and macrophages deficient in NDPK-D had multiple defects in LPS-induced inflammasome activation. In addition, NDPK-D was required for the recruitment of TNF receptor–associated factor 6 (TRAF6) to mitochondria, which was critical for reactive oxygen species (ROS) production and the metabolic reprogramming that supported the TLR-induced gene program. NDPK-D knockout mice were protected from LPS-induced shock, consistent with decreased ROS production and attenuated glycolytic commitment during priming. Our findings suggest that, in response to microbial challenge, NDPK-D–dependent TRAF6 mitochondrial recruitment triggers an energetic fitness checkpoint required to engage and maintain the transcriptional program necessary for inflammasome activation.

Empagliflozin attenuates cardiac microvascular ischemia/reperfusion through activating the AMPKα1/ULK1/FUNDC1/mitophagy pathway

“…The transfected cells were incubated at 37 °C for 48 h, followed by extraction of cellular DNA, RNA and proteins. A non-targeting negative stealth siRNA (scrambled, sc-37007, Santa Cruz) was used as a negative control [ 36 ].…”

Dual specificity phosphatase 1 attenuates inflammation-induced cardiomyopathy by improving mitophagy and mitochondrial metabolism

“…Depending on the total RNA yield, 50–500 ng of total RNA was reverse-transcribed using Superscript II Reverse Transcriptase (Invitrogen) [ 46 ]. The reaction mixture for each well was prepared using KAPA SYBR FAST (Kapa Biosystems), forward and reverse primers, nuclease-free water and cDNA, per the manufacturer's recommendations [ 47 ]. Each reaction was performed in technical duplicate.…”

Reactive Oxygen Species: Not Omnipresent but Important in Many Locations

“…Typically used inhibitors are rotenone (Stowe and Camara, 2009;Heinz et al, 2017;Scialo et al, 2017), which inhibits complex I and increases ROS production inside the mitochondrial matrix (St-Pierre et al, 2002;Lambert and Brand, 2004;Panov et al, 2005;Stowe and Camara, 2009;Sena et al, 2013) and antimycin A (Murphy, 2009;Bleier and Drose, 2013), which inhibits complex III and increases ROS production into the intermembrane space (IMS) (Chen et al, 2003;Han et al, 2003;Al-Mehdi et al, 2012;Quinlan et al, 2012;Herb et al, 2019a). The most commonly used ROS probe for detection of mitochondrial ROS is MitoSOX, which measures O 2 − exclusively inside the mitochondrial matrix (Robinson et al, 2006;Mukhopadhyay et al, 2007;Ernst et al, 2021). However, since the ETC complexes show compartment-specific differences concerning ROS production (Fridovich, 1997;Murphy, 2009;Brand, 2010;West et al, 2011b;Herb and Schramm, 2021), this probe can only be used to measure ROS production inside mitochondria and, therefore, other cellular compartments should always be analyzed in addition.…”