You will be presented with the abstract, introduction, and discussion section from a biomedical research article. Your task is to create a summary that a layperson can understand, capturing the essence of the research article. Keep essential scientific terms, but ensure that the language remains clear and the concepts are explained in an uncomplicated manner.

Title: Disruption of glycolytic flux is a signal for inflammasome signaling and pyroptotic cell death}
Article:
Abstract:
When innate immune cells such as macrophages are challenged with environmental stresses or infection by pathogens, they trigger the rapid assembly of multi-protein complexes called inflammasomes that are responsible for initiating pro-inflammatory responses and a form of cell death termed pyroptosis. We describe here the identification of an intracellular trigger of NLRP3-mediated inflammatory signaling, IL-1β production and pyroptosis in primed murine bone marrow-derived macrophages that is mediated by the disruption of glycolytic flux. This signal results from a drop of NADH levels and induction of mitochondrial ROS production and can be rescued by addition of products that restore NADH production. This signal is also important for host-cell response to the intracellular pathogen Salmonella typhimurium, which can disrupt metabolism by uptake of host-cell glucose. These results reveal an important inflammatory signaling network used by immune cells to sense metabolic dysfunction or infection by intracellular pathogens.
Introduction:
Inflammation is an immunological process required for an organized response to infection, injury, and stress. Because excessive inflammation can be damaging, its initiation is highly regulated. Innate immune cells such as macrophages have evolved sensors of pathogens and homeostatic perturbations which, when activated, induce an immune response( Medzhitov, 2008). Amongst these sensors are Nod-like receptors( NLRs), which are activated in response to a diverse set of pathogen-associated molecular patterns( PAMPs) and danger-associated molecular patterns( DAMPs). Activated NLR proteins recruit and facilitate activation of the protease caspase-1 either directly, through caspase activation and recruitment domain( CARD) interactions, or indirectly, through the adaptor apoptosis-associated speck-like protein containing a CARD( ASC; also known as Pycard). The resulting macromolecular complex is referred to as the inflammasome( Lamkanfi and Dixit, 2014). The inactive precursor of the cytokine interleukin-1β( pro-IL-1β) is also recruited to the inflammasome complex, where proteolysis by caspase-1 induces activation and secretion of the bioactive cytokine, further promoting inflammation. In addition to cytokine maturation, inflammasome formation and caspase activation are associated with a pro-inflammatory form of cell death termed pyroptosis( Fink and Cookson, 2006). This form of cell death results in lytic release of cytosolic contents and other pro-inflammatory factors such as interleukin-1α and high-mobility group protein B1( HMGB1), which are potent inducers of inflammation( Medzhitov, 2008; Croker et al., 2014). Diverse activation signals have been reported as triggers of NLR signaling. For example, the NLR AIM2 is activated by cytosolic double-stranded DNA( Lamkanfi and Dixit, 2014; Fernandes-Alnemri et al., 2009; Hornung et al., 2009; Bürckstümmer et al., 2009), a structural feature associated with infections with pathogens and not found in healthy host cells( Fink and Cookson, 2006; Hornung et al., 2009; Jones et al., 2010). The NLR NLRP3 is a sensor of a wide variety of PAMPs and DAMPs but the unifying mechanism of its disparate activators is not understood( Sutterwala et al., 2014). Furthermore, while the NLRP3 signaling pathway can be activated by a variety of both gram-positive and gram-negative bacteria, the mechanism by which these pathogens induce inflammasome signaling through this receptor is often unclear( Storek and Monack, 2015). Specifically, effective defense against Salmonella typhimurium( S. typhimurium) requires NLRP3( Broz et al., 2010), yet the mechanism by which the pathogen activates this pathway remains unknown. Here, we report a small molecule, GB111-NH2, that induces NLRP3 inflammasome formation, caspase-1 activation, IL-1β secretion, and pyroptotic cell death in bone marrow-derived macrophages( BMDM). Using chemical proteomics, we identify the glycolytic enzymes GAPDH and α-enolase as the phenotypically relevant targets of this molecule. Facilitating TCA metabolism downstream of glycolysis by addition of pyruvate or succinate blocked the effects of the compound. We find that S. typhimurium infection, like direct chemical inhibition of the glycolytic enzymes, reduced glycolytic flux and that restoring metabolism downstream of glycolysis also prevented S. typhimurium-induced inflammasome formation, IL-1β secretion, and pyroptosis. We find that glycolytic disruption induced by either the small molecules or S. typhimurium infection impaired NADH production, resulting in the formation of mitochondrial ROS that were essential for NLRP3 inflammasome activation. Therefore, disruption of glycolytic flux is a biologically relevant trigger of NLRP3 inflammasome activation that is mediated by mitochondrial redox changes, revealing a mechanistic link between cellular metabolism and initiation of inflammation.
Discussion:
The inflammasome is a multiprotein complex that forms in response to various pathogen- and danger-associated signals. Formation of the inflammasome leads to processing and secretion of pro-inflammatory cytokines to activate the immune system( Lamkanfi and Dixit, 2014; Biswas and Mantovani, 2012). While inflammasome formation and pyroptotic cell death are critical for fighting infection and also contribute to inflammation in diseases including type II diabetes, obesity, and atherosclerosis( Kuemmerle-Deschner et al., 2011; Wen et al., 2011; 2012), the signals that trigger caspase-1 activation remain poorly understood. In this study, we used a small molecule, GB111-NH2, to identify two glycolytic enzymes that regulate inflammasome formation. When functionally blocked, innate immune cells sense metabolic perturbation as a danger signal, resulting in inflammasome formation, caspase-1 activation, and cytokine secretion. Our results using this molecule and other established inhibitors of these enzymes suggest that disrupting glycolytic flux serves as a trigger for inflammation and cell death in macrophages. Disturbance of glycolytic flux by the intracellular pathogen S. typhimurium similarly results in inflammasome formation and pyroptotic cell death in an effort to clear the pathogen. Restoration of metabolism downstream of glycolytic disruption by GB111-NH2 or S. typhimurium infection was sufficient to abrogate the inflammasome response by restoring NADH production and preventing mitochondrial ROS production. Though the enzymes and metabolites involved in glycolysis are well established, the specific mechanisms that limit glycolytic flux are not well understood. The irreversible reactions within glycolysis, catalyzed by hexokinase, phosphofructokinase, and pyruvate kinase, were historically thought to be rate limiting. However, recent metabolite flux analyses have also suggested that flux through GAPDH, the enzyme separating upper and lower glycolysis, is rate-limiting under nutrient-rich conditions( Shestov et al., 2014). Under similar conditions, we also observed that targeting GAPDH or enzymes in lower glycolysis is sufficient to disrupt glycolytic flux and activate the NLRP3 inflammasome. Conversely, targeting entry into glycolysis using 2DG was not sufficient to disrupt glycolysis in highly glycolytic cells, as has previously been reported( Pietzke et al., 2014), or to activate NLRP3. This reaffirms previous observations that the pre-existing metabolic state of the cell and the point of intervention are equally important for effectively limiting glycolytic flux( Sengupta et al., 2013). Disruption of glycolytic flux led to an NAD+/NADH imbalance and induced mitochondrial ROS accumulation, which has been shown here and elsewhere( Tschopp and Schroder, 2010; Zhou et al., 2010) to activate the NLRP3 inflammasome( Figure 10). 10. 7554/eLife. 13663. 024Figure 10. Model of NLRP3 inflammasome activation induced by disruption of glycolytic flux. S. typhimurium and chemical inhibitors disrupt glycolytic flux in LPS-primed BMDM, resulting in an increase in NAD+/NADH, a decrease in ATP production, and a decrease in lactate secretion. TCA cycle metabolism is also impaired, potentiating the elevated NAD+/NADH ratio into the mitochondria. Mitochondrial ROS are produced by glycolytic disruption and trigger NLRP3 inflammasome formation, IL-1β production, and pyroptosis. DOI: http://dx. doi. org/10. 7554/eLife. 13663. 024 During infection with S. typhimurium, inflammasome activation is an especially important mechanism of host response because, though it kills the host cell, it initiates inflammatory signals that activate the immune system and combat infection( Storek and Monack, 2015). Two inflammasome complexes, NLRP3 and NLRC4, are required to fully combat infection( Broz et al., 2010). The NLRC4 inflammasome responds to a clear pathogen-associated molecular pattern presented by S. typhimurium—cytosolic flagellin and type 3 secretion system components( Zhao et al., 2011). Here, we provide evidence that NLRP3 activation results in response to another effect of S. typhimurium colonization of the host cell, namely disruption of host cell glycolytic metabolism. This could explain a recent study showing that mutants of S. typhimurium defective for the TCA cycle enzyme aconitase induce a more rapid NLRP3-dependent immune response in vivo( Wynosky-Dolfi et al., 2014). We reason that aconitase deficiency would force S. typhimurium to rely even more heavily on glycolysis to survive within the host. These S. typhimurium mutants would likely disrupt cellular glycolysis more quickly and thus activate NLRP3 more rapidly. It is also interesting that, in long-term models of S. typhimurium infection, the bacteria preferentially resides within alternatively activated or ‘M2’ macrophages, which primarily utilize oxidative metabolism rather than glycolysis( Eisele et al., 2013). Thus, the macrophages in which Salmonella survives the longest are those in which host metabolic pathways are minimally perturbed, enabling prolonged infection without invoking an inflammasome response. These findings additionally shed light on recent work highlighting the connection between metabolic changes and immune system activation( Blatnik et al., 2008; Chawla et al., 2011; Young et al., 1984). For example, metabolic sensing by immune cells has been recently shown to drive NLRP3-dependent IL-1β release and inflammation in diseases ranging from type II diabetes and obesity to Muckle-Wells syndrome( Kuemmerle-Deschner et al., 2011; Strowig et al., 2012), though the specific mechanisms driving macrophage and NLRP3 activation in these diseases have remained unclear. We speculate that, since glucose metabolism is often impaired in these diseases, glycolytic impairment may be the mechanism driving NLRP3-dependent inflammation. Restoring glycolysis or downstream TCA cycle metabolism through supplementation with specific metabolites or activation of glycolytic enzymes could be therapeutically useful for dampening inflammation and associated immunopathology. In summary, our results suggest that inhibition of glycolysis creates a unique metabolic state that activates the NLRP3 inflammasome. They suggest that innate immune cells sense perturbed metabolite production and flux through the glycolytic pathway, in turn activating NLRP3 to initiate inflammatory responses. Inhibitors of flux-limiting enzymes and S. typhimurium effectively limited glycolysis through distinct mechanisms, each resulting in NLRP3-mediated inflammasome formation and pyroptosis. Glycolytic disruption may be a broadly relevant mechanism of NLRP3 activation triggered in response to metabolic parasitism by microbes. Moreover, this pathway may also provide novel avenues for treating diseases in which NLRP3-driven inflammation results in pathology.
Cells of the innate immune system , such as macrophages , are the body’s first line of defense against infection . Macrophages can sense a wide variety of danger signals associated with the presence of infectious microbes , and some of these signals cause macrophages to form protein complexes called inflammasomes inside the cell . Inflammasomes produce molecules that stimulate inflammation and trigger the death of the macrophage . This attracts other immune cells to the infection site to help combat the source of danger . Inflammasome complexes form around an activated receptor molecule called NLRP3 . NLRP3 is activated by a range of danger signals , including those produced by Salmonella bacteria . However , the sequence of events that leads to NLRP3 activation is still not well understood . Sanman et al . have now identified a small molecule that unexpectedly causes the formation of inflammasomes via NLRP3 and so triggers the death of macrophages . Further investigation revealed that this molecule disrupts glycolysis , a process macrophages use to produce energy . The energy imbalance caused by disrupting glycolysis triggers a stress response in macrophages , which ultimately activates the NLRP3 receptor and hence the inflammasome . Sanman et al . then found that Salmonella bacteria also activate the inflammasome by disrupting glycolysis when they invade macrophages . This occurs because the bacteria use up the macrophage’s supply of glycolysis precursor molecules . Replenishing the macrophage with products of glycolysis restored partial energy production and prevented the inflammasome from being activated . Overall , Sanman et al . have identified a previously unknown trigger of inflammation and cell death in macrophages whereby cells can respond to infectious bacteria by sensing a change in energy levels . A next step will be to define the signaling molecules that activate NLRP3 to trigger the construction of the inflammasome . Sanman et al . also hope to uncover other infections and diseases where changes in energy balance might trigger inflammation and cell death .