The ketone metabolite β-hydroxybutyrate blocks NLRP3 inflammasome-mediated inflammatory disease

Abstract

The ketone bodies β-hydroxybutyrate (BHB) and acetoacetate (AcAc) support mammalian survival during states of energy deficit by serving as alternative sources of ATP. BHB levels are elevated by starvation, caloric restriction, high-intensity exercise, or the low-carbohydrate ketogenic diet. Prolonged fasting reduces inflammation; however, the impact that ketones and other alternative metabolic fuels produced during energy deficits have on the innate immune response is unknown. We report that BHB, but neither AcAc nor the structurally related short-chain fatty acids butyrate and acetate, suppresses activation of the NLRP3 inflammasome in response to urate crystals, ATP and lipotoxic fatty acids. BHB did not inhibit caspase-1 activation in response to pathogens that activate the NLR family, CARD domain containing 4 (NLRC4) or absent in melanoma 2 (AIM2) inflammasome and did not affect non-canonical caspase-11, inflammasome activation. Mechanistically, BHB inhibits the NLRP3 inflammasome by preventing K(+) efflux and reducing ASC oligomerization and speck formation. The inhibitory effects of BHB on NLRP3 are not dependent on chirality or starvation-regulated mechanisms like AMP-activated protein kinase (AMPK), reactive oxygen species (ROS), autophagy or glycolytic inhibition. BHB blocks the NLRP3 inflammasome without undergoing oxidation in the TCA cycle, and independently of uncoupling protein-2 (UCP2), sirtuin-2 (SIRT2), the G protein-coupled receptor GPR109A or hydrocaboxylic acid receptor 2 (HCAR2). BHB reduces NLRP3 inflammasome-mediated interleukin (IL)-1β and IL-18 production in human monocytes. In vivo, BHB or a ketogenic diet attenuates caspase-1 activation and IL-1β secretion in mouse models of NLRP3-mediated diseases such as Muckle-Wells syndrome, familial cold autoinflammatory syndrome and urate crystal-induced peritonitis. Our findings suggest that the anti-inflammatory effects of caloric restriction or ketogenic diets may be linked to BHB-mediated inhibition of the NLRP3 inflammasome.

Figures

Figure 1. BHB specifically inhibits the NLRP3 inflammasome

(a) Representative Western blot analysis of caspase-1 (active subunit p20) and IL-1β (active p17) in the supernatant of BMDMs primed with LPS for 4 hours and stimulated with ATP for 1 hour in the presence of various concentrations of D-BHB. (b) Western blot analysis of caspase-1 activation in BMDMs stimulated with LPS and ATP and treated with BHB (10mM), butyrate (10mM), acetoacetate (10mM) and acetate (10mM). (c) Western blot analysis of caspase-1 activation in LPS- primed BMDMs stimulated with MSU and treated with butyrate and D-BHB (d) nigericin (10 μM) for 1h, palmitate (200μM) for 24h, C6 ceramide for 6h (80μg/ml), and sphingosine (50 μM) for 1hour. (e) Western blot analysis of IL-1β activation (active subunit p17) in BMDMs primed with TLR ligands lipid A, Pam3-CSK and LTA for 4h and stimulated with ATP and increasing doses of D-BHB for 1h. Active IL1β (p17) was analysed in supernatants by western blot. BMDMs were infected with (f)F. tularensis and (g) S. typhimurium and treated with different doses of BHB and IL1β activation (p17 active form) was analyzed. Data are expressed as mean ± S.E.M (*P < 0.05) from cells derived from twelve (a-d) or six (e), three (f, g), mice with each independent experiment each carried out in triplicate (a-d, e) and duplicate (f g). All bar graphs in (a-e) represent quantitation of p20 caspase-1 band intensity as fold-change by normalizing to inactive p48 procaspase-1, or p17 IL-1β band intensity as fold change by normalizing to inactive p37 pro-IL-1β. The differences between means and the effects of treatments were determined by one-way ANOVA using Tukey's test.

Figure 2. BHB inhibits the NLRP3 inflammasome independently of Gpr109a and starvation-regulated mechanisms

(a) Western blot analysis of caspase-1 activation in LPS- primed BMDMs treated with rotenone (10μM), ATP (5μM) together with BHB (10mM). (b) Western blot analysis of caspase-1 activation in BMDMs derived from control Atg5fl/fl and LysM:Cre Atg5fl/fl mice primed with LPS and stimulated in presence of ATP and BHB (10mM) (c) The BMDMs were primed with LPS and pretreated with 3MA and epoxomicin for 30min and stimulated in presence of ATP and BHB. The caspase-1 activation was measured by immunoblot analysis. (d) Western blots of caspase-1 and IL-1β activation in LPS-primed BMDM stimulated with ATP and BHB (10mM) in presence of AMPK activator (AICAR, 2mM) and AMPK antagonist Compound C (25 μM). (e) Proliferation of BMDMs in response to increasing concentrations of BHB. (f ,g) Western blot analysis of caspase-1 and IL-1β activation in BMDMs from control and Gpr109a deficient mice activated with LPS and ATP and co-incubated with TSA (50nM), niacin (1mM), butyrate (10mM), acetoacetate (10mM) and BHB (10, 20mM) (h) Western blot analysis of caspase-1 activation in BMDMs of WT and Gpr109a-/- mice treated with LPS for 4h and stimulated with ATP in presence of BHB chiral enantiomer (S) BHB for 1h. Data are expressed as mean ± S.E.M (*P < 0.05) from cells derived from six (a) 4 (b) ten (c, d, e) and four (f-h) mice with each independent experiment each carried out in triplicate. Due to space limitations the quantitation of p20 caspase1 and p17 IL1β band intensity from each experiment are presented in the Supplementary Fig. 2A. The differences between means and the effects of treatments were determined by one-way ANOVA using Tukey's test.

Figure 3. BHB inhibits ASC oligomerization and speck formation without undergoing mitochondrial oxidation

(a) Western blot analysis of caspase-1 activation, SCOT and actin in BMDMs of Oxctfl/fl and LysM:Cre Oxct5fl/fl mice treated with LPS for 4h and stimulated with ATP in presence of BHB and acetoacetate. (b) Western blot analysis of caspase-1 activation in LPS-primed BMDM treated with ATP in presence of BHB (10mM), Sirt2 antagonist AGK2 (10μM) and NAD+ (10 μM).(c, d) Western blot analysis of caspase-1 activation in BMDMs of WT, Sirt2-/- and Ucp2-/- mice treated with LPS for 4h and stimulated with ATP in presence of BHB (10mM) (e) Intracellular Potassium levels in BMDMs stimulated with LPS and ATP in the presence of BHB (10 mM), as measured by Inductively Coupled Mass Spectrometry (ICP-MS). Intracellular potassium levels in LPS-primed BMDMs treated with (f) ATP, (g) MSU and BHB for 1 h. as assessed using a APG-1 dye that selectively binds potassium with an excitation emission spectra of 488-540nm. (h) Representative immunoblot analysis of disuccinimidyl suberate (DSS) cross-linked ASC in the Nonidet P-40-insoluble pellet of BMDM that were primed with LPS (4 h) and stimulated with ATP and BHB for 1 h. The bar graphs represent the quantification of band intensity of the ASC dimer compared to LPS+ATP stimulation (i) Representative immunofluroescent images of ASC speck formation in BMDMs in the presence of BHB (10mM). Data are expressed as mean ± S.E.M (*P < 0.05) from cells derived from five (a) six (b,d), eight (e-g) and four (h) mice with each independent experiment carried out in triplicate. The differences between means and the effects of treatments were determined by one-way ANOVA using Tukey's test.(i) Data are shown as mean ± SEM and are representative of two independent experiments. Statistical differences were calculated by student's t-test.

Figure 4. BHB suppresses NLRP3-mediated inflammatory disease in vivo and the inflammasome in in human monocytes

(a) Analysis of IL-1β and IL-18 secretion in culture supernatants of human monocytes stimulated with vehicle or LPS (1μg/mL) for 4h in presence of increasing concentrations of BHB (n =6/treatment). (b) BHB-complexed nanolipogels (nLGs) block the NLRP3 inflammasome activation and caspase-1 cleavage (n=3, repeated twice). (c) Frequency of CD45+ and Gr1+ immune cells in the peritoneum of mice treated with MSU (3 mg) and BHB-nLGs (125 mg/kg/bw), as assessed by FACS (N =6/group). (d) IL-1β secretion from peritoneal cells cultured overnight and (e) serum IL-1β levels from mice challenged with MSU and treated with BHB-nLGs (n =6/group) (f) Western blot analysis of caspase-1 and IL-1β activation in the BM cells stimulated in presence of LPS and BHB-nLGs from mice harbouring the MWS mutation NLRP3A350V and (g) FCAS mutation (n = 6, repeated twice) (h)Representative immunoblot analysis of disuccinimidyl suberate (DSS) cross-linked ASC in the Nonidet P-40-insoluble pellet of BMDM from FCAS mice (n = 6) that were primed with LPS (4 h) and treated with increasing concentrations of BHB-nLGs. (i) Neutrophil numbers in peritoneum of FCAS mice fed a chow or ketone diester diet (1,3-butanediol) for one week. (n =6/group).Data are expressed as mean ± S.E.M (*P < 0.05) and statistical differences between means and the effects of treatments were determined by one-way ANOVA using Tukey's test (a,d, e) and t-test (k).