Fasting and refeeding differentially regulate NLRP3 inflammasome activation in human subjects

Abstract

Background: Activation of the NLRP3 inflammasome is associated with metabolic dysfunction, and intermittent fasting has been shown to improve clinical presentation of NLRP3 inflammasome-linked diseases. As mitochondrial perturbations, which function as a damage-associated molecular pattern, exacerbate NLRP3 inflammasome activation, we investigated whether fasting blunts inflammasome activation via sirtuin-mediated augmentation of mitochondrial integrity.

Methods: We performed a clinical study of 19 healthy volunteers. Each subject underwent a 24-hour fast and then was fed a fixed-calorie meal. Blood was drawn during the fasted and fed states and analyzed for NRLP3 inflammasome activation. We enrolled an additional group of 8 healthy volunteers to assess the effects of the sirtuin activator, nicotinamide riboside, on NLRP3 inflammasome activation.

Results: In the fasting/refeeding study, individuals showed less NLRP3 inflammasome activation in the fasted state compared with that in refed conditions. In a human macrophage line, depletion of the mitochondrial-enriched sirtuin deacetylase SIRT3 increased NLRP3 inflammasome activation in association with excessive mitochondrial ROS production. Furthermore, genetic and pharmacologic SIRT3 activation blunted NLRP3 activity in parallel with enhanced mitochondrial function in cultured cells and in leukocytes extracted from healthy volunteers and from refed individuals but not in those collected during fasting.

Conclusions: Together, our data indicate that nutrient levels regulate the NLRP3 inflammasome, in part through SIRT3-mediated mitochondrial homeostatic control. Moreover, these results suggest that deacetylase-dependent inflammasome attenuation may be amenable to targeting in human disease.

Trial registration: ClinicalTrials.gov NCT02122575 and NCT00442195.

Funding: Division of Intramural Research, NHLBI of the NIH.

Figures

Figure 6. A SIRT3 agonist blunts inflammasome activation and improves mitochondrial function.

(A) Evaluation of IL-1β release in response to inflammasome activation in control and SIRT3 siRNA–treated THP-1–derived macrophages to assess the effect of NR on IL-1β secretion in the presence of SIRT3 or its depletion (n = 4). (B) Flow diagram of studies performed on blood draw from the normal volunteer blood draw protocol. (C–H) NR administration to PBMCs from healthy volunteers to evaluate the effect on (C) inflammasome induction–mediated IL-1β secretion (n = 8), (D and E) SIRT3 substrate acetylation levels (n = 6), (F) mitochondrial superoxide dismutase activity (n = 6), and (G and H) ATP-induced mitochondrial ROS levels (n = 3). (I and J) NR effect on PBMCs extracted in the refed state, showing an augmentation of maximal respiratory capacity. A representative oxygen consumption rate (OCR) tracing and a histogram are shown, including the data from all the subject cells studied (n = 5). Maximal respiration was assessed in response to dinitrophenol (DNP) and proton leak as the difference in oxygen consumption between inhibition of ATP synthase with oligomycin and the inhibition of mitochondrial respiration with antimycin A (Ant) and rotenone (Rot). Statistical analyses of experiments in this figure were performed using paired 2-tailed t tests.

Figure 4. Refeeding initiates NLRP3 inflammasome priming via NF-κB signaling.

(A) Transcript levels encoding TNFA and inflammasome components IL1B, IL18, and NLRP3. Refeeding increases the expression of all the mRNAs (n = 8). (B) Representative protein levels of signaling molecules activating the NLRP3 inflammasome. Increased phosphorylation of NF-κB and IκBα shows their activation in the fed state. The NLRP3 complex component — NLRP3 and IL-1β — levels are also increased in the fed state. The relative quantitative changes (n = 5–7) are shown in C. Note, the refeeding effects on gene transcript, phosphoprotein, and protein levels are evident at baseline and do not require exogenous inflammasome priming. The fasting to 3-hour–refed transcript and steady-state protein levels were analyzed using paired 2-tailed t tests. (D) Serum levels of soluble CD14, as a marker of endotoxin activity (n = 11). (E) Histogram showing changes in IL-1β release from THP-1–derived macrophages supplemented with fasted and refed sera in the presence and absence of the NF-κB pathway inhibitor PS-1145 (n = 11). The inhibition of NF-κB signaling abolished the excess IL-1β secretion using the refed sera, whether it was administered during the serum incubation alone and/or when maintained through the priming and activation of the inflammasome. Statistical testing for changes in IL-1β levels was performed using 2-way ANOVA.

Figure 2. Nutrient status modulates the NLRP3 inflammasome.

(A) Fasting/refeeding study flow diagram. (B) ELISA assay measurement of IL-1β secretion from primary PBMCs exposed to 3 mM ATP (n = 11). (C) ELISA assay measurement of IL-1β secretion from primary monocytes exposed to 3 mM ATP (n = 8). Statistical testing for changes in IL-1β levels was performed using randomized block design ANOVA. As the refeeding response was more robust at 3 hours, the data at this time point were used as the index postprandial time for all subsequent analyses.

Figure 5. SIRT3 levels modify NLRP3 activation and acetylation of SOD2.

(A) Representative immunoblot of steady-state levels of SIRT1, SIRT2, SIRT3, and SIRT5 following the siRNA knockdown of each isoform in THP-1 cells. The relative changes represent the values from 3 separate experiments. (B) IL-1β release in response to inflammasome activation in the sirtuin knockdown THP-1 cells (n = 11). (C) Inflammasome activation with wild-type and deacetylase mutant SIRT3 overexpression in THP-1 cells (n = 4). *P < 0.05. (D) Mitochondrial superoxide dismutase activity in PBMC mitochondria isolated in the fasted and fed states (n = 4). (E) Representative immunoblot analysis of relative SOD2 protein acetylation on K68 in the fed and fasted states. The relative quantitative change in SOD2 acetylation is shown in F (n = 4). (G) Histogram showing relative release of IL-1β in control and SIRT3-overexpressing cells in response to inflammasome activation in the absence of or presence of ROS inhibition with mitoTEMPO and DPI (n = 5). (H) Histogram showing the same studies as performed in F, with the exception of testing the effect of SIRT3 knockdown rather than overexpression (n = 5). Statistical analysis of changes in IL-1β secretion levels was performed using 2-way ANOVA and analysis of changes in SOD2 activity and acetylation was performed using a paired 2-tailed t test.

Figure 3. Nutrient status modulates the NLRP3 inflammasome.

(A) ELISA assay measurement of IL-1β secretion from primary monocytes exposed to 3 mM ATP (n = 8). (B) IL-18 release in the same monocyte studies, showing that both NLRP3 cytokine levels are more robustly secreted in the refed state versus fasted state. (C) TNF-α release in the same monocytes. (D) IL-1β and (E) IL-18 secretion from monocytes primed with 1 ng/ml LPS followed by 5 μM nigericin administration as a second inflammasome trigger (n = 8). The fasting to 3-hour–refed cytokine levels were analyzed using paired 2-tailed t tests. (F) Representative immunoblot analysis showing secretion of mature caspase-1 and mature IL-1β in the supernatants of monocytes treated with LPS and with and without nigericin.

Figure 1. Clinical protocol and intervention characteristics.

(A) Schematic of the protocol, showing the intervals between the fixed caloric meals and temporal drawing of research blood. (B–D) Data points and means (horizontal bars) of subjects’ sera (B) insulin, (C) glucose, and (D) growth hormone levels at the end of the 24-hour fast and 1 and 3 hours following the fixed caloric meal (n = 19). The fasting insulin level was found to be less than 50% of the insulin level 3 hours after the 500 caloric meal in this young healthy cohort, supporting the integrity of the fast. (E and F) Percentage of subjects that displayed ketones in (E) sera (n = 13) or (F) urine (n = 18). Statistical testing for changes in metabolite and hormone levels were performed using randomized block design ANOVA.