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

Youm YH, Nguyen KY, Grant RW, Goldberg EL, Bodogai M, Kim D, D'Agostino D, Planavsky N, Lupfer C, Kanneganti TD, Kang S, Horvath TL, Fahmy TM, Crawford PA, Biragyn A, Alnemri E, Dixit VD - Nat. Med. (2015)

Bottom Line: Mechanistically, BHB inhibits the NLRP3 inflammasome by preventing K(+) efflux and reducing ASC oligomerization and speck formation.BHB reduces NLRP3 inflammasome-mediated interleukin (IL)-1β and IL-18 production in human monocytes.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.

View Article: PubMed Central - PubMed

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

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.

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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).
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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).

Mentions: Next we investigated whether delivery of BHB can inhibit the NLRP3 inflammasome in human monocytes and mouse models of NLRP3-driven inflammation in vivo. BHB dose-dependently inhibited IL-1β and IL-18 (Fig. 4a) secretion in LPS-stimulated human monocytes without significantly affecting the TNFα production (Supplementary Figure 5b). In vivo administration of BHB is insufficient to achieve sustained high serum concentration due to rapid clearance1,2,30. To reduce clearance BHB was complexed with nanolipogels (nLGs) to improve its bioavailability.33 BHB nLGs inhibited NLRP3 inflammasome activation in macrophages (Fig. 4b). In mice the NLRP3 inflammasome was activated following intraperitoneal (i.p.) injection of MSU crystal, resulting in the influx of neutrophils into the peritoneum and increased secretion of IL-1β 4 h after injection34. Compared to mice given nLGs alone, BHB nLGs reduced neutrophil infiltration into the peritoneum (Fig. 4c and Supplementary Figure 5c, 5d and 5e) without directly impairing neutrophil migration (Supplementary Figure 5f) suggesting direct effects in vivo on NLRP3-driven neutrophil influx. Peritoneal cells from MSU-injected mice treated with BHB-nLGs produced less IL-1β (Fig. 4d) and the concentration of IL-1β in the serum was reduced following BHB nLG treatment (Fig. 4e).


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

Youm YH, Nguyen KY, Grant RW, Goldberg EL, Bodogai M, Kim D, D'Agostino D, Planavsky N, Lupfer C, Kanneganti TD, Kang S, Horvath TL, Fahmy TM, Crawford PA, Biragyn A, Alnemri E, Dixit VD - Nat. Med. (2015)

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).
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Related In: Results  -  Collection

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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).
Mentions: Next we investigated whether delivery of BHB can inhibit the NLRP3 inflammasome in human monocytes and mouse models of NLRP3-driven inflammation in vivo. BHB dose-dependently inhibited IL-1β and IL-18 (Fig. 4a) secretion in LPS-stimulated human monocytes without significantly affecting the TNFα production (Supplementary Figure 5b). In vivo administration of BHB is insufficient to achieve sustained high serum concentration due to rapid clearance1,2,30. To reduce clearance BHB was complexed with nanolipogels (nLGs) to improve its bioavailability.33 BHB nLGs inhibited NLRP3 inflammasome activation in macrophages (Fig. 4b). In mice the NLRP3 inflammasome was activated following intraperitoneal (i.p.) injection of MSU crystal, resulting in the influx of neutrophils into the peritoneum and increased secretion of IL-1β 4 h after injection34. Compared to mice given nLGs alone, BHB nLGs reduced neutrophil infiltration into the peritoneum (Fig. 4c and Supplementary Figure 5c, 5d and 5e) without directly impairing neutrophil migration (Supplementary Figure 5f) suggesting direct effects in vivo on NLRP3-driven neutrophil influx. Peritoneal cells from MSU-injected mice treated with BHB-nLGs produced less IL-1β (Fig. 4d) and the concentration of IL-1β in the serum was reduced following BHB nLG treatment (Fig. 4e).

Bottom Line: Mechanistically, BHB inhibits the NLRP3 inflammasome by preventing K(+) efflux and reducing ASC oligomerization and speck formation.BHB reduces NLRP3 inflammasome-mediated interleukin (IL)-1β and IL-18 production in human monocytes.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.

View Article: PubMed Central - PubMed

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

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.

Show MeSH
Related in: MedlinePlus