β-Hydroxybutyrate Deactivates Neutrophil NLRP3 Inflammasome to Relieve Gout Flares

Abstract

Aging and lipotoxicity are two major risk factors for gout that are linked by the activation of the NLRP3 inflammasome. Neutrophil-mediated production of interleukin-1β (IL-1β) drives gouty flares that cause joint destruction, intense pain, and fever. However, metabolites that impact neutrophil inflammasome remain unknown. Here, we identified that ketogenic diet (KD) increases β-hydroxybutyrate (BHB) and alleviates urate crystal-induced gout without impairing immune defense against bacterial infection. BHB inhibited NLRP3 inflammasome in S100A9 fibril-primed and urate crystal-activated macrophages, which serve to recruit inflammatory neutrophils in joints. Consistent with reduced gouty flares in rats fed a ketogenic diet, BHB blocked IL-1β in neutrophils in a NLRP3-dependent manner in mice and humans irrespective of age. Mechanistically, BHB inhibited the NLRP3 inflammasome in neutrophils by reducing priming and assembly steps. Collectively, our studies show that BHB, a known alternate metabolic fuel, is also an anti-inflammatory molecule that may serve as a treatment for gout.

Keywords: IL-1; NLRP3 inflammasome; aging; gout; inflammation; neutrophil; β-hydroxybutyrate.

Figure 1. Ketogenic diet protects rats from MSU-induced gouty flare

(A) AFM height image of S100A9 amyloid fibrils; scale bar=120nm. (B) Western blot of caspase-1 and IL-1β activation in BMDM. (C) Blood BHB levels after 1 week of ketogenic diet feeding, prior to injection with MSU. (D) Serum IL-1β 48hr post-MSU injection. (E) Change in knee thickness 48hr post-MSU injection. (F) Knee thickness was measured daily. (G) Representative sections of femoro-tibial joint. Local tissue reaction (black asterisks) and intrasynovial exudate (white asterisks); scale bar=500μm. (H) Representative images of synovial inflammation. (Hi, ii) Regions of macrophage infiltration (black asterisk) and neutrophil infiltration (black arrows); scale bar=20μm. (Hiii) Higher magnification of MSU-induced inflammatory response; black arrow points to infiltrating neutrophil; scale bar=10μm. (Hiv) Local tissue reaction in control PBS-injected joint; focal fibrin exudate (white asterisk) with surrounding macrophages (black arrows); macrophages within granular amorphous injected material (dashed arrows); scale bar=20μm. (I) Western blot of IL-1β secretion from stimulated BMDM. (J) Body weights and (K) bacterial load after S. aureus infection. Data are pooled from at least 3 independent experiments. Statistical differences calculated by t-test (C–E) or 2-way ANOVA (F). See also Figure S1.

Figure 2. BHB regulates neutrophil NLRP3 inflammasome activation

IL-1β secretion by stimulated (A) mouse and (B) human neutrophils. (C) Secretion of S100A8 from stimulated human neutrophils. Different colored symbols connected by dotted line represents a unique individual’s response to all treatments. Columns indicate the mean for each treatment. (D, E) Western blot analysis of IL-1β secretion of adult and old murine neutrophils. (F) Depiction of insulin-induced hypoglycemia in vivo; arrow indicates blood collection time point. (G) Neutrophil IL-1β secretion from hypoglycemic subjects. Lines connect the responses of each individual for each treatment condition. Statistical differences were calculated by paired 1-way ANOVA. See also Figure S2.

Figure 3. Elevated BHB protects against MSU-induced peritonitis in aged mice

(A) IL-1β and (B) TNFα were measured in culture supernatants after LPS+ATP stimulation of isolated neutrophils from mice of indicated ages and genotypes. Statistical differences were calculated by age-specific t-test (3 months) or 1-way ANOVA (24 months). Data are pooled from 2 independent experiments. (C) Body weights and (D) blood BHB concentrations were measured daily in old mice fed KD, prior to MSU injection. (E–G) Gene expression of Ilb, Nlrp3 and Tnfa within peritoneal cells 4hr post-MSU. Data are represented as fold change relative to sham, and are pooled from 3 independent experiments. Statistical differences were calculated by t-test between MSU-injected groups. See also Figure S3.

Figure 4. BHB inhibits inflammasome priming and assembly

(A) Western blot of culture supernatants from LPS-primed WT and Caspase 1/11^−/− neutrophils after ATP, Silica, or ceramide stimulation as indicated. (B, C) Elastase and Cathepsin G activity; n=4. (D–F) Culture supernatants were analyzed for neutrophil IL-1β secretion by Western blot after indicated treatments. (G) Total and acetylated H3 expression in cell lysates. (H) NFκB phosphorylation in neutrophil cell lysates. (I) IL-1β secretion from neutrophils derived from mouse model of FCAS bearing activating mutation of Nlrp3. For all blots, each sample is pooled from at least n=4 mice per experiment. Each blot is representative of at least two independent experiments. See also Figure S4.

Figure 5. Hypothetical model for dual role of BHB as a regulatory metabolite

(A) The metabolic role of BHB as an alternative energy source during limited energy availability, coordinated with the anti-inflammatory role of BHB upon NLRP3 inflammasome. (B) BHB inhibits both the priming signal and the secondary assembling signal in neutrophils. (C) Inhibition of IL-1β secretion by BHB does not involve inhibition of serine protease activity, autophagy, oxidation in the TCA cycle, nor the surface receptor GPR109a, although additional experiments will be needed to further clarify the mechanism.