Disruption of glycolytic flux is a signal for inflammasome signaling and pyroptotic cell death
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 .
While screening peptide-based compounds for their effects on inflammasome signaling , we identified one compound , GB111-NH2 ( Blum et al . , 2005; Verdoes et al . , 2012 ) ( Figure 1A ) , that was sufficient to induce caspase-1 activation in LPS-primed bone marrow-derived macrophages . We measured caspase-1 activation by monitoring conversion of procaspase-1 to the mature p10 form by Western blot and , in parallel , by labeling BMDM with the caspase-1-selective activity-based probe ( ABP ) , AWP28 ( Puri et al . , 2012 ) ( Figure 1B ) . In addition to producing active caspase-1 , we found that GB111-NH2-treated BMDMs secreted the cytokine IL-1β in a dose-dependent manner ( Figure 1C ) . Western blot analysis confirmed that secreted IL-1β was primarily the bioactive p17 form ( Figure 1—figure supplement 1 ) that is generated by active caspase-1 . 10 . 7554/eLife . 13663 . 003Figure 1 . Identification of the NLRP3 inflammasome activator GB111-NH2 . ( A ) Structure of GB111-NH2 . ( B ) Western blot and activity-based probe analysis of caspase-1 activation . BMDM primed with 100 ng/mL LPS for 3 hr were then treated with GB111-NH2 . Intact cells were labeled with the caspase-1 probe AWP28 ( 1 μM ) for the last hour before lysate harvest . Whole cell lysates were separated by SDS-PAGE . AWP28 labeling was analyzed by fluorescence scan and caspase-1 processing analyzed by western blot . Gray arrowheads indicate active forms of caspase-1 labeled by AWP28 . HSP90 serves as loading control . ( C ) LPS-primed BMDM were treated with the indicated concentrations of GB111-NH2 for 2 hr . Supernatants were analyzed by ELISA . ( D ) LPS-primed BMDM were treated with 10 μM GB111-NH2 for 2 hr , labeled with AWP28 , fixed , stained for ASC and DAPI , and visualized by confocal microscopy . Scale bar 10 μm . ( E-F ) BMDM of the indicated genetic backgrounds were treated with GB111-NH2 as in ( D ) and inflammasome foci/nuclei quantified . At least 4 fields of view ( 20x objective , 0 . 5x zoom ) were taken per condition per experiment , ~2000 cells/condition . ( G ) LPS-primed BMDM were treated with the indicated compounds ( ATP: 5 mM; nigericin: 10 μM; GB111-NH2: 10 μM ) and supernatant analyzed by ELISA . ( H ) BMDM of the indicated genetic backgrounds were treated as in ( D ) and supernatant analyzed by ELISA . ( I ) BMDM were primed with LPS or vehicle , then treated with GB111-NH2 for 2 hr . Whole cell lysates were separated by SDS-PAGE , blotted for pro-IL-1β , stripped , and reblotted for HSP90 . ( J ) Cell death in LPS-primed , GB111-NH2-treated BMDM was analyzed by LDH release . ( K ) LPS-primed BMDM were treated with 10 μM GB111-NH2 for 2 hr , labeled with AWP28 , stained for Annexin V ( AnnV ) and propidium iodide ( PI ) , and visualized by microscopy . White arrowhead indicates AWP28 focus . Scale bar 10 μm . In all cases , data are representative of at least n=3 experiments and error bars indicate mean +/- sd of technical triplicate . Statistical significance was analyzed using an unpaired , two-tailed t test . DOI: http://dx . doi . org/10 . 7554/eLife . 13663 . 00310 . 7554/eLife . 13663 . 004Figure 1—figure supplement 1 . Secreted IL-1β is the bioactive , p17 form . Supernatant from BMDM treated with the indicated concentrations of GB111-NH2 were analyzed by western blot for IL-1β . Different processing variants are indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 13663 . 00410 . 7554/eLife . 13663 . 005Figure 1—figure supplement 2 . GB111-NH2 does not impair secretion of TNF-α and dose-dependently reduces IL-6 secretion . LPS-primed BMDM were treated with the indicated concentrations of GB111-NH2 for 2 hr and then analyzed for IL-6 ( left ) and TNF-α ( right ) production by ELISA . DOI: http://dx . doi . org/10 . 7554/eLife . 13663 . 005 By fluorescence microscopy , we observed formation of foci containing the inflammasome adaptor ASC and active caspase-1 in GB111-NH2-treated BMDMs ( Figure 1D ) . Formation of these foci was dependent on NLRP3 and ASC but not caspase-1 , caspase-11 , NLRC4 , or AIM2 ( Figure 1E–F ) . We observed that GB111-NH2 induced a similar level of IL-1β secretion as the NLRP3 stimuli ATP and nigericin ( Figure 1G ) and that the absence of NLRP3 completely abrogated IL-1β secretion induced by GB111-NH2 treatment . The absence of other NLRs , specifically NLRC4 and AIM2 , had no effect on IL-1β secretion ( Figure 1H ) . Taken together , these data indicate that GB111-NH2 induces caspase-1 activation and IL-1β secretion solely through the NLRP3 inflammasome , acting as an activating ‘Signal II’ for the canonical NLRP3 pathway ( Lamkanfi and Dixit , 2014 ) . In order for ‘Signal II’ to activate the NLRP3 inflammasome , BMDM must first be primed by a ‘Signal I’ such as LPS . LPS priming induces NF-κB-dependent transcription of pro-inflammatory genes such as IL-1β and inflammasome-independent secretion of pro-inflammatory cytokines such as IL-6 and TNF-α ( Lamkanfi and Dixit , 2014 ) . We measured lysate protein levels by Western blotting and supernatant cytokine levels by ELISA in BMDM treated as in previously described experiments; first primed for 3 hr with LPS and then treated for 2 hr with GB111-NH2 . We observed the appearance of pro-IL-1β upon LPS priming ( Figure 1I ) but there was no effect of GB111-NH2 on either IL-1β protein levels in BMDM that had received LPS priming ( Figure 1C , Figure 1G ) . In addition , IL-6 secretion decreased with increasing dose of GB111-NH2 and TNF-α secretion was unaffected by GB111-NH2 ( Figure 1—figure supplement 2 ) . Therefore , GB111-NH2 does not have a direct effect on Signal I , but functions predominantly as a Signal II for the NLRP3 inflammasome . Macrophages containing active inflammasome complexes often rapidly die by a pro-inflammatory process called pyroptosis ( Fink and Cookson , 2006 ) . We observed features of this form of cell death in GB111-NH2-treated BMDM , including release of the intracellular enzyme lactate dehydrogenase ( LDH ) ( Figure 1J ) , and foci of caspase-1 activity in propidium iodide ( PI ) and Annexin V ( AnnV ) positive cells ( Figure 1K ) . These data confirm that GB111-NH2 is a small molecule activator of the NLRP3 inflammasome that also triggers pyroptotic cell death . Given that GB111-NH2 is chemically distinct from other known activators of NLRP3 and easily modifiable , we wanted to use it as a tool to identify protein targets that are involved in triggering this pro-inflammatory response . To accomplish this , we first conducted a small structure-activity relationship ( SAR ) study in which we synthesized a series of analogs of GB111-NH2 to identify compounds that could be used for affinity isolation of labeled targets . We identified a number of modifications to the primary compound scaffold that resulted in loss of activity ( Figure 2A ) , suggesting that the effects of the parent compound are likely dictated by affinity to specific protein targets . Importantly , our SAR efforts identified both an inactive analog ( GB-IA ) as well as an azide-containing analog ( az-GB ) that retained the inflammasome-activating ability of GB111-NH2 ( Figure 2A , Figure 2—figure supplement 1 ) . We used this azide analog as a probe to identify potential protein targets using Click chemistry to attach a fluorescent tag ( Figure 2—figure supplement 1 ) or affinity tag ( biotin ) to labeled target proteins . The choice of this probe does limit identification to covalent binding partners . However , because removal of the acyloxymethylketone ( AOMK ) electrophile from GB111-NH2 ( compound 2 ) resulted in loss of the ability of the compound to induce IL-1β secretion , we reasoned that the compound likely acted through covalent modification of its relevant targets . 10 . 7554/eLife . 13663 . 006Figure 2 . Structure-activity relationship study and identification of GB111-NH2 targets . ( A ) Structures of GB111-NH2 analogs with structural changes highlighted in gray . LPS-primed BMDM were treated with analogs and supernatant IL-1β measured by ELISA . A dose response is shown above each analog . ( B ) Set-up of MudPIT target identification experiment . In all cases , data are representative of n=3 experiments . Error bars indicate mean +/- sd of technical triplicate . DOI: http://dx . doi . org/10 . 7554/eLife . 13663 . 00610 . 7554/eLife . 13663 . 007Figure 2—figure supplement 1 . Az-GB is a functional probe version of GB111-NH2 . ( A ) BMDM were treated with GB111-NH2 or az-GB and labeled with AWP28 . Whole cell lysates were separated by SDS-PAGE and analyzed by fluorescence scan and western blotting . Gray arrowheads indicate active caspase-1 species . HSP90 serves as loading control . ( B ) SV40-immortalized macrophages were treated with az-GB . Lysates were reacted with TAMRA-alkyne under Click reaction conditions , separated by SDS-PAGE , and analyzed by fluorescence gel scan . DOI: http://dx . doi . org/10 . 7554/eLife . 13663 . 007 We conducted a proteomic study in which we pre-treated BMDMs with either active or inactive analogs of GB111-NH2 , labeled with the az-GB probe , reacted the resulting lysates with alkyne-biotin and identified affinity isolated targets using multidimensional protein identification technology ( MudPIT ) ( Weerapana et al . , 2007 ) ( Figure 2B ) . By using active and inactive compounds in our pretreatment ( GB111-NH2 and GB-IA , respectively ) , we could identify labeled proteins that were lost by pretreatment with the active compound but not the inactive control . Employing this strategy , we obtained a short list of potentially relevant binding partners ( Supplementary files 1–3 ) . Interestingly , this list included proteins critical to cellular metabolism and homeostatic maintenance . To determine which of the potential protein targets of GB111-NH2 were responsible for its’ phenotypic effects , we first tested whether reported selective inhibitors of several of the identified targets mimicked the effects of GB111-NH2 . Based on our target list ( Supplementary file 3 ) , we selected the compounds Atpenin A5 ( Miyadera et al . , 2003 ) ( AA5; inhibitor of succinate dehydrogenase in the TCA cycle ) , 6-aminonicotinamide ( Street et al . , 1997 ) ( 6AN; inhibitor of 6-phosphogluconate dehydrogenase in the pentose phosphate pathway ) , koningic acid ( Endo et al . , 1985 ) ( KA; inhibitor of GAPDH in glycolysis ) and ENOblock ( Jung et al . , 2013 ) ( EB; inhibitor of α-enolase in glycolysis ) . We tested these compounds for their effects on LPS-primed BMDM and found that only inhibitors of the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) and α-enolase induced IL-1β secretion , inflammasome formation , and caspase-1 processing ( Figure 3A–C ) . We chose concentrations of each inhibitor based on literature descriptions of concentrations at which targets should be completely inhibited ( Miyadera et al . , 2003; Street et al . , 1997; Endo et al . , 1985; Jung et al . , 2013 ) . Mass spectrometry results demonstrated that GB111-NH2 also bound to lysosomal cysteine cathepsins ( Supplementary file 2 ) , though not in the expected pattern . To rule out a mode of action based on cathepsin inhibition , we tested the established cathepsin inhibitors leupeptin , E-64d , and Ca074Me and found that they did not induce caspase-1 activation in BMDM ( Figure 3—figure supplement 1 ) . These data indicate that the phenotypically relevant targets of GB111-NH2 are the glycolytic enzymes GAPDH and α-enolase . 10 . 7554/eLife . 13663 . 008Figure 3 . The phenotypically relevant targets of GB111-NH2 are glycolytic enzymes . ( A ) LPS-primed BMDM were treated with the indicated compounds ( GAPDH inhibitor koningic acid = KA; 10 μM , α-enolase inhibitor ENOblock = EB; 20 μM , succinate dehydrogenase inhibitor Atpenin A5 = AA5; 10 μM , 6-phosphogluconate dehydrogenase inhibitor 6-aminonicotinamide = 6-AN; 500 μM ) and IL-1β secretion was analyzed by ELISA . Whole cell lysates were separated by SDS-PAGE and blotted for pro-IL-1β . ( B ) BMDMs were treated with KA and EB and cell lysates were analyzed for caspase-1 processing by western blot . HSP90 serves as loading control . ( C ) BMDM were treated as in ( B ) , labeled with AWP28 , fixed , stained for ASC and DAPI , and analyzed by fluorescence microscopy . Scale bar 10 μm . ( D ) LPS-primed BMDM from the indicated genetic backgrounds were treated with KA or EB for 3 hr , fixed , stained for ASC and DAPI , and analyzed by confocal microscopy . At least 4 fields of view were captured per condition , ~2000 cells/condition/experiment . ( E ) LPS-primed BMDM of the indicated genetic backgrounds were treated with the indicated compounds ( GB111-NH2 – 10 μM for 2 hr , KA – 5 μM for 3 hr , EB – 20 μM for 3 hr , nigericin – 12 . 5 μM for 1 hr ) and supernatant analyzed for IL-1β production by ELISA . ( F ) BMDM were treated as in ( B ) . Whole cell lysates were separated by SDS-PAGE and blotted for pro-IL-1β , NLRP3 , and α-tubulin . DOI: http://dx . doi . org/10 . 7554/eLife . 13663 . 00810 . 7554/eLife . 13663 . 009Figure 3—figure supplement 1 . Cathepsin inhibition does not induce caspase-1 activation . BMDM were treated with GB111-NH2 or structurally dissimilar cathepsin inhibitors CA074Me , E64d , and leupeptin . Caspase-1 activation was assessed by AWP28 labeling and cathepsin inhibition by BMV109 labeling . HSP90 serves as loading control . DOI: http://dx . doi . org/10 . 7554/eLife . 13663 . 00910 . 7554/eLife . 13663 . 010Figure 3—figure supplement 2 . Koningic acid and ENOblock induce dose-dependent IL-1β secretion . LPS-primed BMDM were treated with the indicated concentrations of koningic acid or ENOblock for 3 hr and supernatants analyzed by ELISA . DOI: http://dx . doi . org/10 . 7554/eLife . 13663 . 010 The GAPDH and α-enolase inhibitors KA and EB failed to induce inflammasome formation in cells that lack Pycard or Nlrp3 ( Figure 3D ) , induced IL-1β secretion in a dose-dependent manner that was also NLRP3-dependent ( Figure 3E , Figure 3—figure supplement 2 ) , and had no effect on pro-IL-1β or NLRP3 levels in LPS-primed BMDM ( Figure 3A , Figure 3F ) . These data demonstrate that structurally dissimilar inhibitors of either GAPDH or α-enolase activate the canonical NLRP3 inflammasome pathway similarly to GB111-NH2 . To further confirm the targets of GB111-NH2 , we measured the ability of the parent compound and its analogs to covalently bind and inhibit the activity of the identified glycolytic enzyme targets . To test if our compounds covalently bound to GAPDH and α-enolase , we incubated recombinant GAPDH and α-enolase with our az-GB probe , used Click chemistry to attach TAMRA-alkyne to the az-GB probe , and analyzed reaction mixtures by fluorescent scanning of SDS-PAGE gels . We observed probe labeling of both GAPDH and α-enolase , indicating that the az-GB probe covalently binds to both enzymes ( Figure 4A–B , Figure 4—figure supplement 1 ) . Pretreatment with GB111-NH2 blocked az-GB binding to both GAPDH and α-enolase in a dose-dependent manner , indicating that both compounds bind to similar sites on their enzyme targets . The GB111-NH2 analog containing a more reactive phenoxymethylketone electrophile ( compound 6 ) , which also induced IL-1β secretion more potently in cells than GB111-NH2 , also blocked az-GB binding to both GAPDH and α-enolase at lower concentrations compared to GB111-NH2 . Importantly , the analogs that did not induce IL-1β secretion in BMDM , compound 2 ( which lacks the AOMK electrophile ) and GB-IA ( which lacks the carboxybenzyl cap of GB111-NH2 ) , did not compete for az-GB binding to GAPDH and α-enolase . We did observe some labeling of GAPDH and α-enolase by TAMRA-alkyne independent of the az-GB probe , which is potentially due to the ability of alkynes to function as cysteine electrophiles ( Ekkebus et al . , 2013 ) . 10 . 7554/eLife . 13663 . 011Figure 4 . Compounds covalently bind to reactive cysteines on GAPDH and α-enolase and inhibit enzyme activity in vitro . ( A ) Recombinant human GAPDH was pretreated with GB111-NH2 and its analogs at the indicated concentrations for 1 hr in 0 . 1 M Tris-HCl pH 8 . 0 , then labeled with az-GB ( 50 μM ) for 1 hr . Mixtures were reacted with TAMRA-alkyne , separated by SDS-PAGE , and analyzed by fluorescence scan . Gels were silver stained to assess loading . % of competition was calculated as 100- ( fluor . intensitycmpd+az-GB/fluor . intensityaz-GB-only ) . ( B ) Recombinant human α-enolase was labeled as described for GAPDH in ( A ) . ( C ) GAPDH was incubated with NEM ( 5 μM ) , GB111-NH2 ( 10 μM ) or vehicle for 30 min , then labeled with iodoacetamide fluorescein ( IAF; 10 μM ) for 30 min . Reaction mixtures were separated by SDS-PAGE . Gels were analyzed by fluorescent scan and blotted for GAPDH to assess loading . ( D ) α-enolase was treated as described for GAPDH in ( C ) and blotted for α-enolase to assess loading . ( E ) Recombinant GAPDH and α-enolase were pretreated with inhibitors for 30 min and then enzyme activity assessed using substrate turnover assays . ( F ) GAPDH and α-enolase were incubated with GB111-NH2 for the indicated amounts of time and then enzyme activity assessed . Data are representative of at least n=3 experiments and error bars indicate mean +/- sd of technical triplicate . DOI: http://dx . doi . org/10 . 7554/eLife . 13663 . 01110 . 7554/eLife . 13663 . 012Figure 4—figure supplement 1 . az-GB binds to reactive cysteines on recombinant GAPDH and α-enolase in a manner that is dependent on enzyme activity . Recombinant human GAPDH or α-enolase ( rhGAPDH , rhEno1 ) were pretreated with vehicle ( veh ) , N-ethylmaleimide ( NEM , 5 μM ) , active ( GB111-NH2 , 10 μM ) or inactive ( GB-IA , 50 μM ) analogs , KA ( 5 μM ) or EB ( 20 μM ) for 30 min in PBS with 1% NP-40 and 0 . 1% SDS and then labeled with az-GB ( 50 μM ) . Reaction mixtures were reacted with TAMRA-alkyne , separated by SDS-PAGE , and analyzed by fluorescence scan and Coomassie Blue to assess loading . Black arrowhead indicates faster migrating active GAPDH species . DOI: http://dx . doi . org/10 . 7554/eLife . 13663 . 012 az-GB probe binding to both enzymes was blocked by the cysteine-alkylating compound N-ethylmaleimide ( NEM ) , and by KA and EB ( for GAPDH and α-enolase , respectively ) , suggesting that binding was dependent on enzyme activity and was mediated by reaction with key reactive cysteines ( Figure 4—figure supplement 1 ) . To further investigate the proposed covalent interaction of GB111-NH2 with reactive cysteine residues in GAPDH and α-enolase , we performed competition studies with the general cysteine reative probe iodoacetamide fluorescein ( IAF ) . IAF labeled both GAPDH and α-enolase , consistent with previous reactive cysteine profiling data demonstrating that the catalytic Cys 152 of GAPDH is highly reactive and Cys 388 , an active-site proximal cysteine of α-enolase , is also reactive ( Weerapana et al . , 2010 ) . NEM potently blocked IAF labeling , confirming that IAF was reacting with cysteine residues in GAPDH and α-enolase . Importantly , GB111-NH2 also competed for IAF labeling , indicating that it covalently binds these same reactive cysteines ( Figure 4C–D ) . Modification of the active-site cysteine of GAPDH and the active site-proximal Cys 388 of α-enolase have both been shown to potently impair enzyme activity ( Kato et al . , 1992; Ishii and Uchida , 2004 ) . To confirm that binding of our compounds to these enzymes also inhibits enzyme activity , we performed substrate assays with recombinant GAPDH and α-enolase and found that GB111-NH2 dose-dependently inhibited turnover of the respective substrates , glyceraldehyde-3-phosphate and 2-phosphoglycerate ( Figure 4E ) . GB-IA did not significantly inhibit GAPDH but did exhibit modest inhibitory activity towards α-enolase . GB111-NH2 also showed time-dependent inhibition of GAPDH and α-enolase activity ( Figure 4F ) , suggesting that it is acting as an irreversible inhibitor ( Singh et al . , 2011 ) . Taken together , these data indicate that GB111-NH2 binds covalently to reactive cysteine residues in both GAPDH and α-enolase and that binding to these cysteine residues inhibits enzyme activity . Because GAPDH activity was recently shown to determine flux through aerobic glycolysis ( Pietzke et al . , 2014 ) , we hypothesized that our compounds were activating NLRP3 by disrupting glycolytic flux . Productive glycolysis results in conversion of NAD+ to NADH , secretion of lactate , and ATP production ( Figure 5A ) . To first test the hypothesis that GB111-NH2 , KA , and EB block glycolytic flux we measured the ratio of NAD+/NADH , lactate production , and intracellular ATP concentration in inhibitor-treated BMDM . LPS stimulation , which up-regulates glycolysis in macrophages ( Krawczyk et al . , 2010 ) , resulted in an increase in NADH levels ( demonstrated by a decrease in NAD+/NADH ratio ) and an increase in lactate secretion ( Figure 5B–D ) . GB111-NH2 treatment completely blocked the lactate and NADH production induced by LPS stimulation , indicating that it directly impaired LPS-induced glycolytic flux ( Figure 5B–D ) . ATP production was also significantly impaired in LPS-primed BMDM upon GB111-NH2 treatment ( Figure 5E ) . Notably , treatment with the NLRP3 activator nigericin did not reduce NADH levels ( Figure 5B , Figure 5—figure supplement 1 ) , indicating that the metabolic disruption that we observed with GB111-NH2 is not a general feature of inflammasome activation and cell death . The GAPDH and α-enolase inhibitors KA and EB also affected metrics of glycolytic flux ( Figure 5B–D ) . Finally , GB111-NH2 suppressed the increase in extracellular acidification rate ( ECAR ) induced by glucose stimulation ( Figure 5F ) . 2DG , a glycolytic inhibitor that targets hexokinase , did not dramatically impair glycolytic flux ( as measured by NADH production and lactate secretion ) and did not induce inflammasome formation ( Figure 5—figure supplement 2 ) . This indicates that severe limitation of glycolytic flux is required to activate the NLRP3 inflammasome . Furthermore , these results are in accord with recent studies showing that inhibiting GAPDH , and not enzymes in upper glycolysis , is flux-limiting in highly glycolytic cells ( Shestov et al . , 2014 ) . Inhibitors of the TCA cycle and pentose-phosphate pathways ( AA5 and 6-AN , respectively ) , carbohydrate metabolism pathways that are closely tied to glycolysis , also had no effect on lactate secretion or NADH production in LPS-primed BMDM and did not induce IL-1β secretion ( Figure 3A , Figure 5C ) . 10 . 7554/eLife . 13663 . 013Figure 5 . Inhibition of glycolytic flux creates a unique metabolic defect that activates the NLRP3 inflammasome . ( A ) Map of relevant metabolic pathways . ( B ) BMDM were stimulated with LPS or vehicle for 3 hr and then the indicated compounds for 2 hr , after which cytosolic NAD+/NADH ratio was measured . ( C ) BMDM were treated as in ( B ) and supernatants were analyzed for lactate production . Inhibitor concentrations are those from Figure 3A . ( D ) BMDM were stimulated with LPS or vehicle for 3 hr and then with the indicated compounds for 2 hr , after which cytosolic NAD+/NADH was measured . ( E ) BMDM were treated as in ( B ) and cytosolic ATP concentration analyzed by ATP-coupled luminescence assay . ( F ) ECAR was measured in BMDM upon addition of fresh glucose-containing medium . Fresh medium contained vehicle ( DMSO; gray circles ) or 10 μM GB111-NH2 -/+ 1 mM pyruvate ( black/gray triangles ) . Error bars represent mean +/- sd of 6 technical replicates per condition . DOI: http://dx . doi . org/10 . 7554/eLife . 13663 . 01310 . 7554/eLife . 13663 . 014Figure 5—figure supplement 1 . A dose of nigericin that induces cell death with similar kinetics to GB111-NH2 does not effect NADH production . LPS-primed BMDM were treated with nigericin ( 1 μM ) or GB111-NH2 ( 10 μM ) for the indicated amounts of time ( A ) % LDH release was measured to assess the extent of cell death . ( B ) NADH production was measured in cell lysates . Error bars are mean +/- sd of technical triplicate . Statistical difference between conditions was assessed using an unpaired , two–tailed t test . DOI: http://dx . doi . org/10 . 7554/eLife . 13663 . 01410 . 7554/eLife . 13663 . 015Figure 5—figure supplement 2 . 2DG does not dramatically impair glycolytic flux or induce inflammasome formation . ( A ) Lactate production from 2-deoxyglucose ( 2DG at 10 mM ) –treated LPS-primed BMDM was measured . ( B ) BMDM were treated as in ( A ) and analyzed for cytosolic NAD+/NADH . ( C ) LPS-primed BMDM were treated with GB111-NH2 ( 10 μM ) or 2DG ( 10 mM ) for 2 hr and labeled with AWP28 . BMDM were then fixed , stained for ASC and with DAPI , and inflammasome foci were quantified . DOI: http://dx . doi . org/10 . 7554/eLife . 13663 . 015 We hypothesized that , due to the dependence of macrophages on glycolytic metabolism ( Tavakoli et al . , 2013 ) , disruption of this pathway would create a metabolic signal that is responsible for activating NLRP3 . We hypothesized that supplementation of downstream metabolites of glycolysis would restore partial metabolic function and block the NLRP3-activating signal . When we cultured GB111-NH2-treated BMDMs with cell-permeable versions of the terminal metabolite of glycolysis , pyruvate , or the TCA cycle metabolite succinate , we observed a dramatic reduction in the number of inflammasome foci that formed ( Figure 6A ) . A structurally related metabolite , lactate , which does not fuel the TCA cycle , did not reduce GB111-NH2-induced NLRP3 inflammasome formation ( Figure 6A ) . Doubling the media concentration of L-glutamine , a metabolite that can be converted into succinate via anaplerosis ( Tannahill et al . , 2013 ) , significantly reduced the number of inflammasomes that formed . Complete removal of L-glutamine from media sensitized BMDM to GB111-NH2-induced inflammasome formation ( Figure 6B ) . Taken together , this indicates that levels of glycolytic products that fuel downstream metabolism mediate inflammasome induction in response to glycolytic disruption . 10 . 7554/eLife . 13663 . 016Figure 6 . Addition of metabolites downstream of glycolysis prevents NLRP3 inflammasome activation induced by glycolytic disruption . ( A ) LPS-primed BMDM were treated with GB111-NH2 for 2 hr in the presence of pyruvate ( pyr; 1 mM ) or cell-permeable esters of lactate ( lac; 1 mM ) and succinate ( succ; 10 mM ) . Cells were fixed , stained for ASC and DAPI , and inflammasome foci/nuclei quantified . At least four fields of view were quantified per condition per experiment , ~2000 cells/condition . Error bars represent mean +/- sd of fields of view analyzed . ( B ) BMDM were primed with LPS and then treated with 10 μM GB111-NH2 for 2 hr in the presence of the indicated concentrations of L-glutamine or succinate . Cells were fixed , stained for ASC and DAPI , and quantified by microscopy . Four fields of view ( ~2000 cells ) were analyzed per condition . Error bars represent mean +/- sd of separate fields of view . ( C ) LPS-primed BMDM were treated with the indicated compounds in the presence or absence of pyruvate and analyzed as in ( A ) . ( D ) BMDM were treated as in ( C ) and supernatants were analyzed for IL-1β by ELISA . ( E ) BMDM were treated as in ( C ) and cell death was measured by LDH release . ( F ) BMDM were treated with the indicated inhibitors , stained for ASC and DAPI , and quantified by microscopy as in ( B ) . ( G ) BMDM were treated with GB111-NH2 for 2 hr in the presence or absence of pyruvate ( 1 mM ) , after which cytosolic NAD+/NADH was measured . ( H ) BMDM were treated as in ( G ) and cytosolic ATP measured by ATP-coupled luminescence assay . For ELISA and LDH release data , error bars represent mean +/- sd of technical triplicate . Data were analyzed for statistical significance using an unpaired , two-tailed t test . DOI: http://dx . doi . org/10 . 7554/eLife . 13663 . 016 In addition to preventing inflammasome formation , supplementation of the glycolytic product pyruvate resulted in significant reductions in caspase-1 activation , IL-1β secretion , and cell death induced by GB111-NH2 . Pyruvate supplementation had no effect on inflammasome signaling induced by the NLRP3 activators ATP and nigericin ( Figure 6C–E ) , indicating that pyruvate does not impair NLRP3 inflammasome signaling by a nonspecific mechanism . Pyruvate treatment also blocked inflammasome formation induced by KA and EB ( Figure 6F ) and restored NADH and ATP production in the treated cells ( Figure 6G–H ) . We hypothesized that changes in the NAD+/NADH ratio or a drop in ATP concentration could serve as a secondary signal that connects glycolytic disruption to NLRP3 inflammasome formation . To test whether either of these signals is important , we manipulated the NAD+/NADH and ATP levels downstream of glycolysis by chemically blocking specific components of the TCA cycle and oxidative phosphorylation . We first treated LPS-primed BMDM with GB111-NH2 and pyruvate to block glycolysis and stimulate downstream metabolism . We then added the succinate dehydrogenase ( TCA cycle enzyme ) inhibitor AA5 ( Miyadera et al . , 2003 ) to reduce NADH levels , the Complex I inhibitor rotenone to increase NADH levels and reduce ATP production , or the ATP synthase inhibitor Oligomycin A to only inhibit ATP synthesis by oxidative phosphorylation ( Figure 7A–B ) . We found that AA5 addition partially reversed the protection conferred by pyruvate , as demonstrated by an increase in the number of inflammasome complexes . Rotenone treatment suppressed inflammasome formation more than pyruvate alone . Oligomycin A induced a small but statistically insignificant increase in the number of inflammasomes that formed ( Figure 7C ) . The number of inflammasomes positively correlated with a drop in NADH production ( an increase in NAD+/NADH ratio ) ( Figure 7D ) , while ATP concentration exhibited no correlation with the numbers of inflammasomes ( Figure 7—figure supplement 1 ) . Interestingly , rotenone treatment was sufficient to completely abrogate inflammasome formation induced by GB111-NH2 ( Figure 7E ) , conditions under which we also observed a significant decrease in the NAD+/NADH ratio ( Figure 7F ) . These data suggest that the inability to produce NADH , and not ATP , is predictive of NLRP3 inflammasome formation upon glycolytic disruption by GB111-NH2 . It should be noted , however , that the α-enolase inhibitor EB did not induce a significant NAD+/NADH ratio defect ( Figure 5 ) , suggesting that either EB induces inflammasome activation through a distinct mechanism from GB111-NH2 and KA , or that there are additional universal signals responsible for inflammasome activation downstream of glycolytic disruption . 10 . 7554/eLife . 13663 . 017Figure 7 . NAD+/NADH ratio elevation and mitochondrial ROS accumulation are signals for NLRP3 inflammasome formation downstream of glycolytic disruption . ( A ) LPS-primed BMDM were treated with the indicated compounds ( GB111-NH2 - 10 μM , sodium pyruvate - 1 mM , AA5 - 10 μM , oligomycin A - 1 μM , rotenone - 5 μM ) for 2 hr , after which cells were fixed , stained for ASC and DAPI , and visualized by microscopy . ( B ) Cells were treated as in ( A ) and cytosolic NAD+/NADH measured . ( C ) Cells were treated as in ( A ) and cytosolic ATP measured by ATP-coupled luminescence assay . ( D ) % Cells with ASC foci values from ( A ) are plotted against NAD+/NADH values from ( B ) . Error bars are representative of mean +/- sd of technical triplicate from ( A ) and ( B ) . ( E ) LPS-primed BMDM were treated with vehicle or 10 μM GB111-NH2 in the presence or absence of 5 μM rotenone for 2 hr . Cells were fixed , stained for ASC and DAPI , and analyzed by microscopy . Four fields of view were collected per condition ( ~2000 cells ) . ( F ) Cells were treated as in ( E ) and cytosolic NAD+/NADH analyzed . Error bars represent mean +/- sd of technical triplicate . ( G ) BMDM were treated with 10 μM GB111-NH2 or vehicle in the presence or absence of 1 mM pyruvate ( pyr ) and stained with MitoSOX ( 2 . 5 μM ) . Cells were analyzed for MitoSOX uptake by flow cytometry . ( H ) LPS-primed BMDMs were treated with GB111-NH2 in the presence or absence of 4-hydroxyTEMPO ( 4-HT ) . Whole cell lysates and cell supernatants ( sup ) were separated by SDS-PAGE and analyzed by western blot to detect the active p10 form of caspase-1 . HSP90 serves as loading control . ( I ) BMDM were treated with nigericin ( 12 . 5 μM ) or GB111-NH2 ( 10 μM ) in Ringer’s buffer with increasing concentrations of K+ . Cells were fixed , stained for ASC and DAPI , and inflammasome foci/nuclei quantified . DOI: http://dx . doi . org/10 . 7554/eLife . 13663 . 01710 . 7554/eLife . 13663 . 018Figure 7—figure supplement 1 . ATP concentration does not correlate with inflammasome numbers . ATP concentration values from Figure 7A were plotted against % Cells with ASC foci from Figure 7B . DOI: http://dx . doi . org/10 . 7554/eLife . 13663 . 01810 . 7554/eLife . 13663 . 019Figure 7—figure supplement 2 . K+ efflux is not required for GB111-NH2-induced pyroptotic cell death . BMDM were treated with nigericin ( 12 . 5 μM ) or GB111-NH2 ( 10 μM ) in Ringer’s buffer with increasing concentrations of K+ . After 3 hr , cell death was assessed by measuring % LDH release . Error bars are mean +/- sd of technical triplicate . DOI: http://dx . doi . org/10 . 7554/eLife . 13663 . 019 Mitochondrial ROS and K+ efflux are proposed to be unifying signals preceding NLRP3 inflammasome formation ( Tschopp and Schroder , 2010; Muñoz-Planillo et al . , 2013 ) . Therefore , we wanted to determine whether either of these signals is relevant to NLRP3 inflammasome activation induced by disruption of glycolysis . We stained BMDM with MitoSOX , a dye that reports accumulation of mitochondrial ROS , and observed that GB111-NH2 induced an increase in cellular MitoSOX fluorescence that was abrogated by addition of pyruvate ( Figure 7G ) . We also found that the ROS scavenger 4-hydroxyTEMPO ( 4-HT ) prevented GB111-NH2-induced caspase-1 cleavage and activation ( Figure 7H ) . Addition of extracellular K+ , in contrast , did not reduce the number of inflammasome foci in GB111-NH2-treated BMDMs ( Figure 7I ) or impair GB111-NH2-induced cell death ( Figure 7—figure supplement 2 ) , indicating that mitochondrial ROS , but not K+ efflux , is required for GB111-NH2-induced NLRP3 activation and pyroptosis . We and others have shown that the intracellular pathogen Salmonella typhimurium ( S . typhimurium ) requires glucose and its own glycolytic enzymes for intracellular replication ( Bowden et al . , 2009; 2014 ) . In addition , host defense against S . typhimurium requires the NLRC4 and NLRP3 inflammasomes ( Broz et al . , 2010 ) . While it is clear that S . typhimurium flagellin and type 3 secretion system proteins activate NLRC4 via NAIP proteins ( Zhao et al . , 2011 ) , the mechanism by which NLRP3 is activated is not well understood . We hypothesized that S . typhimurium infection may stimulate NLRP3 through disruption of host cell metabolic pathways by co-opting cellular resources during intracellular replication . To test this notion , we infected naïve BMDM with S . typhimurium grown to stationary phase ( conditions that lead to NLRP3-dependent inflammasome activation [Broz et al . , 2010] ) . In this infection model , inflammasome complexes begin forming at ~11 hr post-infection and progressively accumulate . We confirmed that infection with either wildtype S . typhimurium or S . typhimurium lacking the SPI-1 secretion system ( which genetically limits S . typhimurium to activate NLRP3 ) induced inflammasome formation ( Figure 8A–B ) . In addition , the percentage of cells with ASC foci was similar in magnitude to GB111-NH2 and alum treatment but lower than nigericin , ATP , or log phase S . typhimurium stimulation ( Figure 8C ) . Using this infection model , we assessed the extent to which intracellular S . typhimurium utilize host-cell glucose by culturing infected BMDM with the fluorescent glucose analog 2- ( N- ( 7-nitrobenz-2-oxa-1 , 3-diazol-4-yl ) amino ) -2-deoxyglucose ( 2-NBDG ) . We lysed infected BMDM at 5 hr post-infection ( while bacteria are still intracellular ) , harvested the bacterial fraction of BMDM lysates , and measured 2-NBDG fluorescence . We observed fluorescent signal that was dependent on infection in 2-NBDG-treated macrophages , indicating that the bacterial fraction took up the fluorescent glucose analog from the host cell ( Figure 8D ) . We also fixed uninfected and infected 2-NBDG-treated BMDM and analyzed the pattern of 2-NBDG fluorescence by confocal microscopy . We observed the presence of strongly 2-NBDG fluorescent punctae that resemble S . typhimurium and colocalize with Hoechst stain ( which stains both host cell and bacterial DNA ) in infected BMDM . Furthermore , we quantified the portions of cytosol that were not Hoechst-positive and observed a significant decrease in 2-NDBG fluorescence in the cytosol of infected BMDM compared to uninfected BMDM ( Figure 8E ) . These data indicate that intracellular S . typhimurium derive glucose from host macrophages and reduce host glucose availability . 10 . 7554/eLife . 13663 . 020Figure 8 . Salmonella typhimurium disrupts host cell glycolysis . ( A ) BMDMs were infected with S . typhimurium strain SL1344 grown to stationary phase and infected at the indicated multiplicity of infection ( MOI; 25:1 and 100:1 ) . At the indicated timepoints , cells were fixed and stained for ASC and DAPI . Inflammasome foci/nuclei were quantified . ( B ) BMDM were infected with △orgA ( SPI-1 deficient ) S . typhimurium grown to stationary phase . Cells were fixed at the indicated timepoints , stained for ASC and DAPI , and foci/nuclei quantified . ( C ) Cells were treated with the indicated compounds or infected with S . typhimurium grown to stationary phase ( 100:1 MOI ) or log phase ( 10:1 MOI ) . Cells were fixed , stained for ASC , and ASC foci/nuclei were quantified . ( D ) BMDM were infected with 100:1 MOI stationary phase S . typhimurium for 5 hr . 2-NBDG ( 10 μM ) or vehicle was added to media 2 hr post-infection . Cells were washed , lysed , intracellular bacteria sedimented from whole cell lysate via centrifugation , resuspended , and bacterial fluorescence ( abs/em 465/540 ) analyzed by plate reader . ( E ) BMDM were treated as in ( D ) , fixed , stained for DAPI , and visualized by confocal microscopy . Left: Representative image . White arrowheads indicate cytosolic S . typhiurium positive for 2-NBDG and DAPI . Scale bar 10 μM . right: 2-NBDG signal in areas of cytosol negative for DAPI ( S . typhimurium negative ) was measured . Cytosolic regions from ~200 discrete cells from 4 fields of view were measured per condition . Error bars represent mean +/- sd of different fields of view . ( F ) BMDMs were infected with stationary phase S . typhimurium and analyzed for cytosolic NAD+/NADH . △NAD+/NADH indicates the difference between the ratio measured at 11 hr and 5 hr post-infection . ( G ) BMDMs were infected with stationary phase S . typhimurium and levels of lactate in the supernatant analyzed at the indicated timepoints . ( H ) Quantification of the difference between lactate secretion measured at 11 hr and 5 hr post-infection . DOI: http://dx . doi . org/10 . 7554/eLife . 13663 . 020 We measured glycolytic flux in infected BMDM to assess the effect of limited glucose availability on the host macrophages . Importantly , as observed for GB111-NH2 treatment , we observed reduced production of NADH and lactate ( Figure 8F–H ) that correlated with the multiplicity of infection and magnitude of inflammasome formation in host cells ( Figure 8A , Figure 8F–H ) . These metabolic defects appeared on a similar timescale as initiation of NLRP3 inflammasome formation , suggesting that infection with S . typhimurium has a direct effect on glycolytic flux in host cells . Consistent with our findings using glycolytic inhibitors , we also observed that supplementation of cells with the glycolytic end product pyruvate significantly reduced inflammasome formation , IL-1β secretion , and cell death induced by S . typhimurium infection ( Figure 9A–D ) . Pyruvate was effective at blocking inflammasome formation in BMDMs infected with both wildtype S . typhimurium and S . typhimurium defective for the SPI-1 secretion system ( Figure 9—figure supplement 1 ) . Notably , pyruvate did not completely block ASC focus formation , IL-1β secretion , and death induced by S . typhimurium infection , which could be due to compensatory activation of other inflammasomes such as the non-canonical caspase-11 inflammasome ( Broz et al . , 2012 ) , or because S . typhimurium could also partially co-opt host pyruvate . 10 . 7554/eLife . 13663 . 021Figure 9 . Disruption of glycolysis by Salmonella typhimurium activates the NLRP3 inflammasome . ( A–D ) BMDMs were infected with S . typhimurium grown to stationary phase in the presence or absence of 1 mM pyruvate and ( A ) cells were fixed and stained for ASC and DAPI . ASC foci/nuclei were quantified . At least four fields of view ( ~2000 cells ) were analyzed per condition . ( B ) IL-1β secretion was analyzed by ELISA , ( C ) Cell death was measured by LDH release . ( D ) Representative image from ( A ) . White arrowheads indicate inflammasome foci . Scale bar 30 μm . ( E ) BMDM of the indicated genetic backgrounds were infected with stationary phase S . typhimurium ( 100:1 MOI ) in the presence or absence of pyruvate . Inflammasome foci were quantified at 17 hr post-infection . ( F ) BMDMs were infected with stationary phase S . typhimurium 12 , 023 ( 25:1 ) expressing a replication plasmid . Generations were quantified at the indicated timepoints post-infection . Data are representative of n=3 experiments . ( G ) BMDMs were infected with stationary phase S . typhimurium 12 , 023 ( 100:1 MOI ) constitutively expressing EGFP . Cells were fixed at 17 hr post-infection , stained for ASC , and infection visualized by confocal microscopy . Scale bar 15 μm . ( H ) Minimal medium containing vehicle , 2 mM glucose , or 2 mM pyruvate was inoculated with wildtype S . typhimurium . Bacterial growth was measured by analyzing OD600 . ( I ) Cytosolic NAD+/NADH was analyzed at 5 and 11 hr post infection with S . typhimurium ( 100:1 MOI ) or vehicle ( uninf . ) in the presence or absence of 1 mM pyruvate ( pyr ) . NAD+ consumption rate indicates the difference in NAD+/NADH ratio between 5 and 11 hr post-infection . Data are representative of n=3 experiments . For LDH release , ELISA , and metabolic assays , error bars indicate mean +/- sd of technical triplicate . Data were analyzed for statistical significance using an unpaired , two-tailed t test . DOI: http://dx . doi . org/10 . 7554/eLife . 13663 . 02110 . 7554/eLife . 13663 . 022Figure 9—figure supplement 1 . Pyruvate prevents S . typhimurium-induced inflammasome formation in a dose-dependent manner . ( A ) BMDM were infected with stationary phase S . typhimurium in the presence of increasing concentrations of pyruvate and inflammasome foci quantified . ( B ) BMDM were infected with △orgA ( SPI-1 deficient ) S . typhimurium grown to stationary phase at 25:1 MOI in the presence or absence of 1 mM pyruvate . Inflammasome foci were quantified at 14 hr post-infection . DOI: http://dx . doi . org/10 . 7554/eLife . 13663 . 02210 . 7554/eLife . 13663 . 023Figure 9—figure supplement 2 . Inflammasome formation and cell death induced by log phase S . typhimurium infection are unaffected by pyruvate . ( A ) BMDM were infected with S . typhimurium grown to log phase ( 10:1 MOI ) in the presence or absence of pyruvate and inflammasome foci were quantified after 1 hr . ( B ) BMDMs were treated as in ( A ) and cell death was quantified by LDH release at the indicated timepoints . DOI: http://dx . doi . org/10 . 7554/eLife . 13663 . 023 We did not observe inflammasome focus formation in Nlrp3 -/- BMDMs upon infection with stationary phase S . typhimurium . In contrast , Nlrc4 -/- BMDMs had a similar number of inflammasome foci upon infection as wildtype macrophages ( Figure 9E ) . Pyruvate did not prevent inflammasome formation or cell death induced by infection with S . typhimurium in log phase growth ( Figure 9—figure supplement 2 ) , an infection model that activates the NLRC4 inflammasome . We also verified that pyruvate was not blocking inflammasome formation by inhibition of bacterial replication using both an intracellular replication reporter plasmid ( Helaine et al . , 2010 ) and by monitoring bacterial replication by microscopy ( Figure 9F–G ) . Reporter plasmid expression over the course of the intracellular replication assay indicates that intracellular S . typhimurium are viable in BMDM cultured in DMEM with or without pyruvate ( Helaine et al . , 2010 ) . In vitro replication assays demonstrated that S . typhimurium grew at a similar rate in minimal media with glucose or pyruvate as a carbon source ( Figure 9H ) , further indicating that pyruvate supplementation affects host cell recognition of intracellular bacteria rather than bacterial dynamics . Importantly , we found that the NAD+ consumption rate increased upon pyruvate treatment ( Figure 9I ) , indicating induction of productive metabolism downstream of glycolysis in infected BMDMs . Taken together , these data indicate that glycolytic perturbation is a mechanism by which innate immune cells sense altered homeostasis during S . typhimurium infection and induce a pro-inflammatory response via NLRP3 inflammasome formation and pyroptotic cell death .
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 .
See below for synthesis and characterization of GB111-NH2 and analogs . NMR spectra were recorded on a Varian 400 MHz ( 400/100 ) or a Varian Inova 500 MHz ( 500/126 MHz ) equipped with a pulsed field gradient accessory . Chemical shifts ( ∂ ) are reported in parts per million ( ppm ) downfield from tetramethylsilane and are reference to the residual protium signal in the NMR solvents . Data are reported as follows: chemical shift , multiplicity ( s=singlet , d=doublet , t=triplet , m=multiplet and q=quartet ) , coupling constant ( J ) in Hertz ( Hz ) and integration . E64d ( Enzo Lifesciences , Farmingdale NY ) , leupeptin ( Sigma Aldrich , St . Louis MO ) , CA074Me ( EMD Millipore , Hayward CA ) , LPS from E . coli 0111:B4 ( Sigma Aldrich ) , 6-aminonicotinamide ( Santa Cruz Biotech , Santa Cruz CA ) , Imject Alum ( Pierce Biotechnology , Rockford IL ) , Atpenin A5 ( Santa Cruz Biotech ) , N-ethylmaleimide ( Sigma ) , rotenone ( Sigma ) , oligomycin A ( Cayman Chemical , Ann Arbor MI ) , nigericin ( Cayman Chemical ) , MitoSOX ( Life Technologies , Carlsbad CA ) , ATP ( Sigma ) , and koningic acid ( Adipogen , Switzerland ) were purchased from commercial sources , dissolved in vendor-recommended solvents , and used without further purification . ENOblock ( Jung et al . , 2013 ) was a generous gift from Dr . Darren Williams . Strains used in this study were Salmonella typhimurium NCTC 12023 and ATCC SL1344 . Bacteria were grown in LB at 37°C with aeration and supplemented with 0 . 2% arabinose if needed to induce expression of fluorescent proteins . Mice lacking Pycard , Nlrp3 , Nlrc4 , and Aim2 have been previously described ( Jones et al . , 2010; Broz et al . , 2010; Kayagaki et al . , 2011; Mariathasan et al . , 2004 ) . Mice were maintained following guidelines approved by the Stanford University School of Medicine Administrative Panel on Laboratory Animal Care . BMDM were isolated by culturing mouse bone marrow in DMEM with 2 mM L-glutamine , 10% FBS , and 10 ng/mL recombinant mouse M-CSF ( eBioscience , San Diego CA ) for 5 days in petri dishes . After 5 days , the cell monolayer was washed several times with sterile PBS to remove cell debris and then the BMDM harvested using CellStripper ( Corning CellGro , Manassas VA ) . BMDM were then plated for experiments , frozen , or cultured for up to a week . One day prior to treatment , cells were seeded in 6 well plates at a density of 1-2x106 cells/well ( or 2x105 cells/well of 24-well dish , or 3x104 cells/well of 96-well dish ) . C57BL/6 SV40-immortalized macrophages were cultured in RPMI with 10% FBS and 2 mM L-glutamine and were a generous gift from Petr Broz . In this study , biological replicates indicate replicates of the same experiment conducted upon separately seeded cultures on separate days . Technical replicates indicate separate measurements made on cells seeded on the same day and treated simultaneously . The number of biological replicates is indicated in the figure legends and was generally n=3 . For plate reader-based assays , experiments were generally conducted in technical triplicate as recommended by assay manufacturers . For microscopy experiments , at least four fields of view were generally analyzed – covering the four quadrants of the cover slip . Within each quadrant , a field was chosen at random using the DAPI channel ( to simply find a region that contained cells ) . Each field of view was counted as a technical replicate because it was a separate measurement of a singly seeded culture . When ascertaining whether differences between samples were statistically significant , an unpaired , two-tailed t test was used . This makes the assumptions that the two samples under analysis were approximately normally distributed and had equal variances . p<0 . 05 was considered significant . Because measurements were taken within linear range of the detection method ( i . e . , below saturation and above noise for absorbance-based plate reader assay , within linear range of detector for flow cytometry measurements ) , etc , technical replicates should be normally distributed around the mean . BMDM were primed with 100 ng/mL LPS-EK ( Invivogen , San Diego CA ) or LPS 0111:B4 ( Sigma ) in DMEM for 3 hr before addition of inflammasome activating agents . GB111-NH2 was added to LPS-primed BMDM at 10 μM ( unless otherwise indicated ) for inflammasome activation . The canonical NLRP3 activators ATP and nigericin were added to LPS-primed BMDM at 5 mM and 12 . 5 μM , respectively , typically for 1 hr . Alum ( Pierce ) was used at a concentration of 100 μg/mL for 5 hr . For stationary phase infections , S . typhimurium grown to stationary phase ( typically overnight culture in LB ) were centrifuged onto BMDM for 10min at 500g . After 1 hr , medium was switched to DMEM with 100 μg/mL gentamicin sulfate ( Sigma ) to kill extracellular bacteria . After 1 hr , cells were washed with plain DMEM and then incubated in DMEM with 10 μg/mL gentamicin sulfate for the remainder of the infection . For log phase infections , S . typhimurium in log phase growth ( typically a 4 hr subculture of a 1:50 dilution of an overnight culture ) were centrifuged onto BMDM for 10min at 500g in antibiotic-free DMEM . Unless otherwise stated , samples were analyzed after 1 hr of log phase infection . Probes were diluted to the desired final concentration ( 1 μM for AWP28 , 250 nM for BMV109 ) from a 1000x stock solution in DMSO directly in the media of the cell monolayer . Cells were labeled for the final hour of treatment at 37°C prior to sample preparation and analysis . For gel labeling experiments , labeled cell monolayers were washed in PBS and lysed directly with 50 μL sample buffer . For harvested supernatants , following treatment the supernatant was removed and proteins precipitated by adding 4 equivalents of cold acetone . Samples were incubated in acetone overnight at -20°C , then proteins pelleted by centrifugation for 5 min at 2000 rpm . Acetone was aspirated and protein pellets dried for 30 min at 37°C before addition of sample buffer . Samples were resolved by SDS-PAGE and visualized on a Typhoon flatbed fluorescent laser scanner ( GE Healthcare , United Kingdom ) . Following separation of samples by SDS-PAGE and transfer to 0 . 2 μM nitrocellulose resin ( BioRad , Hercules CA ) , the following antibodies were used . For cell lysates: anti-caspase-1 p10 ( 1:200 , Santa Cruz Biotechnology #514 ) , anti-HSP-90 ( 1:1000 , BD Biosciences , San Jose CA ) , anti-NLRP3 ( 1:500 , R&D Systems , Minneapolis MN ) , anti-α-tubulin ( 1:10000 , Sigma ) , anti-GAPDH ( 1:1000 , Santa Cruz Biotechnology C-9 ) , anti-α-enolase ( 1:1000 , Cell Signaling Technology , Danvers MA ) . For cell supernatant: anti-IL-1β ( 1:200 , Cell Signaling Technology ) . HRP-conjugated α-mouse and α-rabbit secondary antibodies were from GE Healthcare . BMDMs were seeded in triplicate in 96-well plates at a density of 3x104 cells/well . Following treatment , the supernatant was removed and IL-1β , IL-6 , or TNF-α release was measured using a Mouse IL-1β , IL-6 , or TNF-α READY-SET-GO ELISA kits ( eBioscience ) according to the manufacturer’s instructions . BMDMs were seeds in triplicate in 96-well plates at a density of 3x104 cells/well . Following treatment , the supernatant was removed , and the cells were lysed with 2% Triton-X-100 in D-PBS . The lysate was diluted in culture media to the original volume . LDH release was calculated as supernatant LDH activity/total LDH activity using the CytoTox 96 Assay ( Promega , Madison WI ) . BMDMs were seeded on poly-L-lysine coated glass coverslips in 24 well plates at a density of 2x105 cells/well . Following treatment and labeling with AWP28 ( 1 μM for final hour of treatment ) , the cell monolayer was rinsed 3x with warm D-PBS and then fixed with 4% paraformaldehyde in PBS for 15 min at 37°C . The cells were washed with PBS and incubated with anti-ASC ( 1:200 , Santa Cruz Biotechnology N-15 ) primary antibody in blocking buffer ( 3% BSA , 0 . 1% saponin , 0 . 02% sodium azide in PBS ) for 30 min . The cells were washed 3x with PBS and incubated with Alexa 647 or Alexa 594-conjugated secondary antibody ( both 1:1000 , Invitrogen , Carlsbad CA ) for 30min . The cells were washed with D-PBS , mounted in Vectashield with DAPI ( Vector Labs , Burlingame CA ) , and imaged on a Zeiss LSM700 confocal microscope . Snapshots of fields were taken at random ( at least 4 fields/condition using a 10x or 20x air objective , typically ~2000 cells/condition ) . Nuclei were counted using the ITCN plug-in in ImageJ and inflammasome ( ASC and/or AWP28 positive ) foci were counted using the ‘Analyze Particles’ function in ImageJ after automated thresholding . Replicates indicate cells plated and treated on separate days . For Annexin V and propidium iodide staining , AWP28 labeled cells on coverslips were washed with Annexin V binding buffer ( 10 mM HEPES pH 7 . 4 , 150 mM NaCl , 2 . 5 mM CaCl2 ) and then incubated with 1 μg/mL propidium iodide ( ImmunoChemistry , Bloomington MN ) and 1:50 Alexa 647 conjugated Annexin V ( Invitrogen ) in Annexin V-binding buffer on ice for 30 min . Cells were washed with Annexin V-binding buffer and mounted in Vectashield ( Vector Labs ) for immediate imaging . Recombinant GAPDH ( ScienCell , Carlsbad CA ) , α-enolase ( BioVision , Milpitas CA ) were diluted into assay buffer ( 50 mM Tris-HCl pH 7 . 4 , 1 . 5 mM MgCl2 ) and incubated with inhibitor or vehicle for 30 min at 37°C . After this , az-GB ( 50 μM ) was added for 2 hr at 37°C . TAMRA-alkyne was then added under previously described Click reaction conditions ( Child et al . , 2013 ) to visualize az-GB-labeled protein . Reaction mixtures were separated by SDS-PAGE and visualized on Typhoon scanner . For ECAR measurements , BMDM were analyzed using a Seahorse XF96 Analyzer . On the day prior to the assay 8x103 BMDM were plated per well of a 96-well Seahorse Analyzer plate . The next day , cells were washed with and then immersed in 180 mL Assay Medium ( RPMI at pH 7 . 4 with 2 mM L-glutamine and without HEPES or sodium bicarbonate ) . Cells were incubated in a CO2-free incubator for 1 hr at 37°C . At initiation of assay , the plate was loaded into the Seahorse Analyzer , allowed to equilibrate , and compounds injected in Assay Medium with fresh glucose . ECAR was measured for 2 hr after compound injection . Cells were stained with Hoechst and counted after conclusion of assay . Measured ECAR values were normalized to cell number and averaged across each condition . The fluorescent glucose analog 2-NBDG ( Cayman Chemical; Abs/Em 465/540 nm ) was used to monitor glucose uptake by both infected and uninfected BMDM . BMDM were infected with SL1344 Salmonella typhimurium grown to stationary phase at an MOI of 100:1 . After 1 hr , media was changed to DMEM with high gentamicin ( 100 μg/mL ) to kill extracellular bacteria . After 1 hr , BMDM were washed with plain DMEM and then incubated in DMEM with low gentamicin and 10 μM 2-NBDG . For microscopy analysis , cells were fixed and mounted in Vectashield with DAPI after 4 hr of infection . 2-NDBG was imaged using ‘FITC’ absorption/emission settings in ZenBlack software on a Zeiss LSM700 microscope . Quantification of average cytosolic 2-NBDG fluorescence was done using ImageJ software . In the uninfected condition , cytosol was identified as 2-NBDG ( + ) areas proximal to nuclei . In the infected condition , cytosol was identified as areas proximal to nuclei that were not Salmonella typhimurium ( + ) . 4 fields per sample were quantified and the average and standard deviation of average cytosolic 2-NDBG fluorescence measurements reported . For measurement of 2-NBDG uptake into S . typhimurium , after 7 hr of infection BMDM were lysed in 0 . 1% Triton-X-100 in PBS for 10 min . Lysates were centrifuged at 5000 g/10 min/4°C . Supernatant was aspirated and the resulting bacterial pellet resuspended in PBS , transferred to an opaque 96-well plate , and measured in triplicate on a plate reader at Abs/Em 465/540 nm . Salmonella typhimurium ( strain 12023 ) expressing a replication plasmid were grown overnight in LB containing 0 . 2% arabinose . BMDM were plated in 12-well dishes at 500 k cells/well and infected with Salmonella typhimurium strain NCTC 12 , 023 at MOI 25:1 . At 12 , 16 , and 24 hr post-infection , BMDM were lysed and bacterial samples analyzed by flow cytometry . Generations of bacteria were calculated as previously described by Helaine et al . For in vitro growth curves , S . typhimurium were grown in MgM-MES minimal media supplemented with 2 mM glucose , 2 mM pyruvate , or vehicle ( ddH2O ) . OD600 was measured at various timepoints after inoculation of culture . BMDM were plate in 12-well dishes , primed for 3 hr with 100 ng/mL LPS , and then stimulated in the presence or absence of pyruvate . BMDM were labeled for the last 15min of treatment with 2 . 5 μM MitoSOX Red ( Life Technologies ) , collected , centrifuged for 5min at 2000 rpm at 4°C , then resuspending in ice cold PBS with 0 . 5% BSA and analyzed by flow cytometry ( 488 nm excitation , PE channel collection for MitoSOX Red ) . >25 , 000 cells were analyzed per condition . LPS-primed BMDM were treated with NLRP3-activating compound in Ringer’s buffer with varying concentrations of K+ . Osmolarity was kept constant by varying NaCl concentration accordingly . Rink resin ( 1g , 0 . 59 mmol ) was taken up in DMF and deprotected in 20% piperidine in DMF for 45 min at room temperature . The resin was washed with DMF . Fmoc-Lys ( Boc ) -OH ( 829 mg , 3 eq , 1 . 77 mmol ) , HOBt ( 239 mg , 3 eq , 1 . 77 mmol ) , and DIC ( 277 μL , 3 eq , 1 . 77 mmol ) were added and the reaction mixture nutated for four hours . The resin was washed with DCM and DMF and the Fmoc group removed by incubation with 20% piperidine in DMF for 45 min . The resin was washed with DMF and Z-Phe-OH ( 530 mg , 3 eq , 1 . 77 mmol ) , HOBt ( 239 mg , 3eq , 1 . 77 mmol ) , and DIC ( 277 μL , 3 eq , 1 . 77 mmol ) were added and the reaction mixture nutated overnight at room temperature . The resin was washed with DCM and DMF . The product NR-GB111 was cleaved from the Rink resin using 95% TFA , 2 . 5% triisopropylsilane , and 2 . 5% H2O for 30 min . The crude was purified by HPLC ( reverse phase C18 column , CH3CN/H2O 0 . 1% TFA , 5:95 to 80:20 over 9 column volumes ( CVs ) Pure fractions were lyophilized and 5 . 55 mg ( 0 . 013 mmol , 2 . 2% yield ) NR-GB111 ( 3 ) were afforded as a white powder . 1H NMR ( 500 MHz , CD3OD ) δ 7 . 36 – 7 . 19 ( m , 10H ) , 5 . 03 ( q , J = 12 . 6 Hz , 2H ) , 4 . 38 – 4 . 27 ( m , 2H ) , 3 . 08 ( dd , J = 13 . 7 , 6 . 5 Hz , 1H ) , 2 . 92 ( dd , J = 13 . 7 , 8 . 6 Hz , 1H ) , 2 . 86 ( t , J = 7 . 6 Hz , 2H ) , 1 . 93 – 1 . 79 ( m , 1H ) , 1 . 69 – 1 . 53 ( m , 3H ) , 1 . 47 – 1 . 32 ( m , 2H ) . HRMS ( ES+ ) : [M+H+]+ calculated for C23H30N4O4 expected mass 427 . 2345 found 427 . 2345 . LCMS ( ES+ ) : retention time 5 . 57 min . Intermediate 10 ( 25 . 6 mg , 0 . 05 mmol , 1 eq ) was converted to the AOMK following the general procedure . The crude was purified by HPLC ( reverse phase C18 column , CH3CN/H2O 0 . 1% TFA , 20:80 to 60:40 in x column volumes ) . Pure fractions were pooled and lyophilized . The lyophilized fractions were taken up in 50% TFA in DCM and stirred for 1 hr at room temperature . The reaction was concentrated with toluene in vacuo to yield 4 . 68 mg ( 9 μmol , 5 . 6% yield ) of white crystalline solid , GB-IA ( 4 ) . 1H NMR ( 400 MHz , CD3OD/CDCl3 1/1 ) δ 7 . 32 – 7 . 24 ( m , 4H ) , 7 . 23 – 7 . 15 ( m , 2H ) , 7 . 06 – 7 . 01 ( m , 2H ) , 4 . 61 – 4 . 41 ( m , 4H ) , 3 . 10 ( dd , J = 13 . 6 , 8 . 4 Hz , 1H ) , 3 . 00 ( dd , J = 13 . 6 , 7 . 4 Hz , 1H ) , 2 . 89 ( t , J = 7 . 4 Hz , 2H ) , 2 . 44 – 2 . 39 ( m , 4H ) , 2 . 35 ( s , 6H ) , 2 . 16 ( t , J = 2 . 2 Hz , 1H ) , 2 . 01 – 1 . 84 ( m , 1H ) , 1 . 72 – 1 . 53 ( m , 3H ) , 1 . 51 – 1 . 34 ( m , 2H ) . HRMS ( ES+ ) : [M+H+]+ calculated for C30H37N3O5 expected mass 520 . 2811 found 520 . 2797 . LCMS ( ES+ ) : retention time 6 . 55 min .
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 .
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 .