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

Journal name:
Nature Medicine
Volume:
21,
Pages:
263–269
Year published:
DOI:
doi:10.1038/nm.3804
Received
Accepted
Published online

The ketone bodies β-hydroxybutyrate (BHB) and acetoacetate (AcAc) support mammalian survival during states of energy deficit by serving as alternative sources of ATP1. BHB levels are elevated by starvation, caloric restriction, high-intensity exercise, or the low-carbohydrate ketogenic diet2. 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 unknown2, 3, 4, 5, 6. 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.

At a glance

Figures

  1. BHB specifically inhibits the NLRP3 inflammasome.
    Figure 1: BHB specifically inhibits the NLRP3 inflammasome.

    (a) Representative western blot analysis of caspase-1 (casp1; active subunit p20) and IL-1β (active p17) in the supernatant of BMDMs primed with LPS for 4 h and stimulated with ATP for 1 h in the presence of various concentrations of D-BHB. Procasp1, procaspase-1, biologically inactive; Pro-IL-1β, non-secreted biologically inactive form of IL-1β. (b) Western blot analysis of caspase-1 activation in BMDMs stimulated with LPS and ATP and treated with BHB (10 mM), butyrate (10 mM), AcAc (10 mM) or acetate (10 mM). Western blot analysis of caspase-1 activation in LPS-primed BMDMs stimulated with (c) MSU and treated with butyrate or D-BHB, or (d) nigericin (10 μM) for 1 h, palmitate (200 μM) for 24 h, C6 ceramide for 6 h (80 μg/ml), and sphingosine (50 μM) for 1 h and treated with BHB. (e) Western blot analysis of IL-1β activation in BMDMs primed with the TLR ligands lipid A, Pam3-CSK (Pam3) or LTA for 4 h and stimulated with ATP and increasing doses of D-BHB for 1 h. Active IL-1β (p17) was analyzed in supernatants by western blotting. IL-1β (f) or caspase-1 activation (g) in BMDMs infected with (f) F. tularensis or (g) S. typhimurium and treated with different doses of BHB. Data are expressed as mean ± sem (*P < 0.05) from cells derived from n = 12 (ad); n = 6 (e); or n = 3 (f,g) mice with each independent experiment each carried out in triplicate (ad,e) or in duplicate (f,g). All bar graphs in ae 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 analysis of variance (ANOVA) using Tukey's test.

  2. BHB inhibits the NLRP3 inflammasome independently of Gpr109a and starvation-regulated mechanisms.
    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 combinations of rotenone (10 μM), ATP (5 μM), and BHB (10 mM). (b) Western blot analysis of caspase-1 activation in BMDMs derived from control Atg5fl/fl or LysM-Cre Atg5fl/fl mice primed with LPS and stimulated with combinations of ATP and BHB (10 mM). (c) Immunoblot analysis of caspase-1 activation in LPS-primed BMDMs stimulated in the presence of ATP and different concentrations of BHB and pretreated with either 3-MA or epoxomicin for 30 min. (d) Western blot analysis of caspase-1 and IL-1β activation in LPS-primed BMDMs stimulated with ATP and BHB (10 mM) in the presence of an AMPK activator (AICAR, 2 mM) or AMPK antagonist Compound C (Comp. C; 25 μM). Capase-1 activation was analyzed in both cell supernatants and BMDM cell lysates. (e) Proliferation of BMDMs in response to increasing concentrations of BHB. (f,g) Western blot analysis of (f) IL-1β and (g) caspase-1 activation in BMDMs from control and Gpr109a-deficient mice activated with LPS and ATP and co-incubated with BHB (10 or 20 mM) and (f) TSA (50 nM) and niacin (1 mM), or (g) butyrate (10 mM) and AcAc (5 or 10 mM). (h) Western blot analysis of caspase-1 activation in BMDMs of WT and Gpr109a−/− mice treated with LPS for 4 h and stimulated with ATP in the presence of (S)-BHB for 1 h. Data are expressed as mean ± sem (*P < 0.05) from cells derived from n = 6 (a); n = 4 (b,fh); or n = 10 (ce) mice with each independent experiment carried out in triplicate. Because of space limitations, the quantitation of p20 caspase-1 and p17 IL-1β band intensity from each experiment is presented in Supplementary Figure 2a. The differences between means and the effects of treatments were determined by one-way ANOVA using Tukey's test.

  3. BHB inhibits ASC oligomerization and speck formation without undergoing mitochondrial oxidation.
    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 from Oxctfl/fl and LysM-Cre Oxctfl/fl mice treated with LPS for 4 h and stimulated with ATP in the presence of BHB and AcAc. p48 indicates the molecular weight of inactive procaspase-1. (b) Western blot analysis of caspase-1 activation in LPS-primed BMDMs treated with ATP alone and in the presence of BHB (10 mM), Sirt2 antagonist AGK2 (10 μM) or NAD+ (10 μM). (c,d) Western blot analysis of caspase-1 activation or IL-1β in BMDMs of WT (c,d), Sirt2−/− (c), or Ucp2−/− (d) mice treated with LPS for 4 h and stimulated with ATP alone or in the presence of BHB (10 mM). UnRx, untreated. (e) Intracellular potassium levels in BMDMs unstimulated (unstim) or stimulated with LPS and ATP in the presence or absence of BHB (10 mM), as measured by inductively coupled mass spectrometry (ICP-MS). (f,g) Intracellular potassium levels in LPS-primed BMDMs treated with BHB and (f) ATP or (g) MSU for 1 h as assessed using an APG-1 dye that selectively binds potassium and has an excitation emission spectrum of 488–540 nm. (h) Representative immunoblot analysis of disuccinimidyl suberate (DSS)–cross-linked ASC in the Nonidet P-40–insoluble pellet of BMDMs that were primed with LPS (4 h) and stimulated with ATP and BHB for 1 h. The bar graph represents the quantification of band intensity of the ASC dimer compared to LPS + ATP stimulation. (i) Representative immunofluorescence images of ASC speck formation in LPS-primed BMDMs stimulated with ATP in the presence or absence of BHB (10 mM). Scale bars, 200 μm in panel and 20 μm in inset. Data are expressed as mean ± sem (*P < 0.05) from cells derived from n = 5 (a); n = 6 (b,d); n = 8 (eg); or n = 4 (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.

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

    (a) Analysis of IL-1β and IL-18 secretion in culture supernatants of human monocytes stimulated with vehicle (not shown) or LPS (1 μg/mL) for 4 h in the presence of increasing concentrations of BHB. n = 6 per treatment; symbols (circles, triangles, etc.) are data points from individual subjects. (b) BHB-complexed nanolipogels (nLGs) block 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 of body weight), as assessed by FACS (n = 6 per group). (d,e) IL-1β secretion from peritoneal cells cultured overnight (d) and serum IL-1β levels in mice challenged with MSU and treated with BHB–nLGs (n = 6 per group) (e). (f,g) Western blot analysis of caspase-1 and IL-1β activation in BMDM cells stimulated with LPS in the presence of BHB–nLGs from mice harboring the human (f) MWS NLRP3 (A350V) or (g) FCAS NLRP3 (L351P) 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 mouse models (n = 6) that were primed with LPS (4 h) and treated with increasing concentrations of BHB–nLGs. (i) Neutrophil numbers in the peritoneum of FCAS mouse models fed a chow or ketone diester (KD; 1,3-butanediol) diet for 1 week. (n = 6 per group). Data are expressed as mean ± sem (*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).

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Author information

  1. These authors contributed equally to this work.

    • Yun-Hee Youm &
    • Kim Y Nguyen

Affiliations

  1. Section of Comparative Medicine and Program on Integrative Cell Signaling and Neurobiology of Metabolism, Yale School of Medicine, New Haven, Connecticut, USA.

    • Yun-Hee Youm,
    • Kim Y Nguyen,
    • Emily L Goldberg,
    • Tamas L Horvath &
    • Vishwa Deep Dixit
  2. Department of Nutrition Sciences, Purdue University, West Lafayette, Indiana.

    • Ryan W Grant
  3. Laboratory of Molecular Biology and Immunology, National Institute on Aging, National Institutes of Health (NIH), Baltimore, Maryland, USA.

    • Monica Bodogai &
    • Arya Biragyn
  4. Department of Biomedical Engineering, Yale University, New Haven, Connecticut, USA.

    • Dongin Kim &
    • Tarek M Fahmy
  5. Department of Molecular Pharmacology and Physiology, University of South Florida, Tampa, Florida, USA.

    • Dominic D'Agostino
  6. Department of Geology and Geophysics, Yale University, New Haven, Connecticut, USA.

    • Noah Planavsky
  7. Department of Immunology, St. Jude Children's Hospital, Memphis, Tennessee, USA.

    • Christopher Lupfer &
    • Thirumala D Kanneganti
  8. Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, USA.

    • Seokwon Kang &
    • Emad Alnemri
  9. Diabetes and Obesity Research Center, Sanford-Burnham Medical Research Institute, Orlando, Florida, USA.

    • Peter A Crawford
  10. Department of Immunobiology, Yale School of Medicine, New Haven, Connecticut, USA.

    • Vishwa Deep Dixit

Contributions

Y. H.Y. and K.Y.N. designed and conducted the majority of in vitro and all in vivo experiments, analyzed and interpreted the data, and participated in writing the manuscript. R.W.G. participated in design and conduct of inflammasome activation experiments. E.L.G. performed ASC speck and neutrophil assays. M.B. and A.B. performed the human monocytes experiments. D.K. and T.M.F. synthesized the BHB–nanolipogels and conducted control experiments to determine the dose response. D.D'A. formulated the ketone diester diet. N.P. conducted the ICP-MS experiments to determine K+ efflux. C.L. and T.D.K. conducted the F. tularensis and S. typhimurium infection experiments. T.L.H. designed the experiments and provided essential reagents for experiments involving mitochondrial ROS and UCP2. P.A.C. generated the macrophage-specific, Scot-deficient mice and contributed to experiment design. S.K. and E.A. designed and conducted the ASC oligomerization experiments. V.D.D. conceived and supervised the project, interpreted the data, and wrote the manuscript.

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The authors declare no competing financial interests.

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