VDAC interacts with hexokinase and is implicated

VDAC interacts with hexokinase and is implicated in NLRP3 activation in response to noxious stimuli including monosodium urate, silica, and alum (Zhou et al., 2011). Hexokinase dissociation may allow VDAC to carry out this role. It will be informative to block or silence VDAC to explore its function in NLRP3 inflammasome activation in response to PGN, and if hexokinase dissociation regulates mitochondrial ROS or mtDNA release (Zhou et al., 2011). In macrophages, NLRP3 relocates to mitochondria following stimulation with alum or nigericin, possibly to be in closer proximity to activating signals from mitochondria (Zhou et al., 2011). Mitochondria may facilitate assembly of the inflammasome, and mitochondrial structure is key for its activation. Inducing mitochondrial fission might restrict NLRP3 inflammasome assembly/activation and NLRP3 has been shown to associate with the mitochondrial fusion protein mitofusin 2 (Ichinohe et al., 2013, Park et al., 2015). It would be intriguing to investigate whether such alterations in mitochondrial dynamics affect hexokinase dissociation. NAG is an atypical microbial danger signal as it is derived from PGN but is also synthesized in the glycosylation pathway as uridine diphosphate (UDP-NAG), which can then be released following degradation of glycosylated proteins. Since intrinsically produced NAG is likely to be present at low concentrations, higher concentrations are likely required for NLRP3 inflammasome activation. Wolf et al. (2016) showed that NAG in the low millimolar range inhibited purified hexokinase. However, enhanced IL-1β secretion was only observed in RO4956371 treated with much higher concentrations, likely suggesting that NAG was not readily entering the cell or that hexokinase dissociation from the mitochondria occurs at a higher concentration than hexokinase inhibition. Determining whether the concentration of PGN-derived NAG in the cytoplasm after bacterial infection is sufficient to dissociate hexokinase from VDAC could be a focus of future work. Wolf et al. (2016) found that disruption of hexokinase activity by high concentrations of 2-DG also enhanced IL-1β secretion. Although previously observed (Nomura et al., 2015), this result contrasts with studies demonstrating that blocking glycolysis with 2-DG can inhibit IL-1β activation (O’Neill and Pearce, 2016, Tannahill et al., 2013). It is conceivable that differences in stimulation conditions, 2-DG concentration, level of cell death, or the timing of hexokinase inhibition could explain these differential effects. Metabolic reprograming of macrophages and DCs occurs after PRR stimulation, and aerobic glycolysis is essential in the development of subsequent inflammatory responses (Everts et al., 2014, O’Neill and Pearce, 2016). In the current study, macrophages exposed to NAG or PGN were previously stimulated with LPS, which induces a classical inflammatory phenotype. Given the importance of aerobic glycolysis in classical macrophage activation, it would be informative to observe what effect NAG has on bioenergetics. It is possible that in a system using live Gram-positive bacteria, initial inflammatory signals would come from other bacterial components, such as muramyl dipeptide, and that NAG induced NLRP3 activation would occur at a later time point, when sufficient NAG has been digested and released into the cytosol. Comparing mitochondrial respiration and aerobic glycolysis in macrophages after exposure to PGN and NAG versus exposure to whole bacteria would help address these questions. The NLRP3 inflammasome is dysregulated in a variety of metabolic and inflammatory diseases, including obesity, type 2 diabetes, and Alzheimer’s disease. Thus, NLRP3 is an appealing therapeutic target and understanding what regulates or activates it is of vital importance. The exciting results of Wolf et al. (2016) demonstrate that the glycolytic enzyme hexokinase acts as a sensor of Gram-positive bacteria, highlighting a surprising discovery in the link between innate immune responses and metabolism.