The Circadian Clock Protein BMAL1 Acts as a Metabolic Sensor In Macrophages to Control the Production of Pro IL-1β

Abstract

The transcription factor BMAL1 is a clock protein that generates daily or circadian rhythms in physiological functions including the inflammatory response of macrophages. Intracellular metabolic pathways direct the macrophage inflammatory response, however whether the clock is impacting intracellular metabolism to direct this response is unclear. Specific metabolic reprogramming of macrophages controls the production of the potent pro-inflammatory cytokine IL-1β. We now describe that the macrophage molecular clock, through Bmal1, regulates the uptake of glucose, its flux through glycolysis and the Krebs cycle, including the production of the metabolite succinate to drive Il-1β production. We further demonstrate that BMAL1 modulates the level and localisation of the glycolytic enzyme PKM2, which in turn activates STAT3 to further drive Il-1β mRNA expression. Overall, this work demonstrates that BMAL1 is a key metabolic sensor in macrophages, and its deficiency leads to a metabolic shift of enhanced glycolysis and mitochondrial respiration, leading to a heightened pro-inflammatory state. These data provide insight into the control of macrophage driven inflammation by the molecular clock, and the potential for time-based therapeutics against a range of chronic inflammatory diseases.

Keywords: IL-1β; macrophage inflammation; metabolism; molecular clock; pSTAT3.

Figure 1

Mitochondrial respiration and Krebs cycle glucose flux is altered in Bmal1^-/- macrophages. Bmal1^+/+ and Bmal1^-/- BMDMs were stimulated with LPS (100 ng/ml), subjected to a Seahorse XF mitochondrial stress test. (A) OCR of BMDMs was measured, and the metabolic trace illustrates changes in OCR following injection of the mitochondrial stress test compounds oligomycin, FCCP, and rotenone/antimycin a. (B) Measures of basal respiration, ATP production, maximal respiration, and proton leak were derived from (A). Assay results are presented +/- SEM and are representative of n=3 independent experiments. BMDMs were isolated, seeded in 10 mM U-13C6 glucose, and stimulated with LPS for 8 hours. BMDMs were lysed and metabolites were quenched and measured via GC-MS to trace Krebs cycle flux of labelled glucose. (C) A schematic of U-¹³C₆ glucose-derived carbon Krebs cycle flux and incorporation into metabolic intermediates. Relative abundance of U-¹³C₆-labelled (D) pyruvate, (F) citrate, (J) itaconate, (K) α-Ketoglutarate, and (M) succinate, and mass isotopologue distribution (MID) of (E) pyruvate, (G) citrate, and (L) α-Ketoglutarate, and (N) succinate were measured. MID values of low abundance isotopologues are excluded for clarity. (I) Ratio of m+2 citrate/m+3 pyruvate representative of U-¹³C₆ glucose-derived carbon flux through pyruvate dehydrogenase. Data presented is n=4 +/- SEM. (H) BMDMs were stimulated with LPS and protein expression of PDH was analysed by Western blot using β-Actin as a loading control. Densitometry is relative to the Bmal1^+/+ control band. This band is indicated by a * symbol. Data presented is representative of n=3 independent experiments. Statistical analysis was performed for Seahorse XF data and U-¹³C₆ relative abundance values by one-way ANOVA with Sidak’s multiple comparisons test and for MID values by multiple student’s t-tests with Holm-Sidak correction for multiple comparisons (*p < 0.05, **p < 0.01 ***p < 0.001, ****p < 0.0001).

Figure 2

SDH-derived ROS are driving increased inflammation in macrophages with deletion of Bmal1. Bmal1^+/+ and Bmal1^-/- BMDMs were incubated in an isosmotic, ADP-supplemented mitochondrial assay solution and permeabilized with digitonin. (A) Seahorse XF analysis was used to analyse the change in OCR in response to injection of succinate (1.25 mM). (B) Response to succinate was measured in terms of %change in OCR directly following injection of succinate. Assay results are presented +/- SEM and are representative of n=3 independent experiments. BMDMs were stimulated with LPS (100 ng/ml) and (C) stained with CellROX to measure levels of reactive oxygen species by flow cytometry or (D) lysed for analysis of pro IL-1β protein expression by Western blot using β-Actin as a loading control. (E) Reactive oxygen species and (F) pro IL-1β protein expression were measured following pretreatment with DMM before stimulation with LPS. Western immunoblot data presented is representative of n=3 independent experiments. Pro IL-1β time course densitometry is relative to the Bmal1^+/+ control band. Densitometry for Bmal1^+/+ and Bmal1^-/DMM/LPS bands is relative to their LPS bands. These bands are indicated by * symbols. Flow cytometry data presented is at least n=3 independent experiments +/- SEM with each data point representative of at least 5,000 events from one sample. Statistical analysis was performed for Seahorse XF data by unpaired student’s t test and for flow cytometry data by two-way ANOVA with Tukey’s multiple comparisons test (*p < 0.05, ****p < 0.0001).

Figure 3

Bmal1^-/- macrophages display heightened glucose metabolism which is driving increased pro IL-1β expression. Bmal1^+/+ and Bmal1^-/- BMDMs were stimulated with LPS (100 ng/ml) and lysed for analysis of (A) HIF-1α and (B) GLUT1 protein expression by Western blot using β-Actin as a loading control. (G) Pro IL-1β protein expression was measured following pretreatment with 2DG (1 mM) before stimulation with LPS. Expression of (H) pro IL-1β was measured following incubation of BMDMs with different concentrations of glucose before LPS stimulation. HIF-1α time course and glucose/2DG IL-1β Western immunoblot data presented is representative of n=3 independent experiments. GLUT1 time course Western immunoblot data presented is representative of n=2 independent experiments. Time course densitometry is relative to the Bmal1^+/+ control band. Densitometry for Bmal1^+/+ and Bmal1^-/2DG/LPS bands is relative to their LPS bands. Glucose/LPS IL-1β densitometry is relative to the 0 mM glucose/LPS Bmal1^+/+ band. These bands are indicated by * symbols. (C) BMDMs were stimulated with LPS and glucose uptake was analysed via luminescent 2-DG uptake assay. Data presented is n=3 +/- SEM. Following LPS stimulation, BMDMs were subjected to a Seahorse XF glycolytic rate assay. ECAR of BMDMs was measured and converted into PER. (D) Metabolic trace illustrating changes in PER following injection of the glycolytic rate assay compounds rotenone/antimycin a and 2-DG. Measures of (E) basal glycolysis and (F) compensatory glycolysis were derived from (D). Assay results are presented +/- SEM and are representative of n=3 independent experiments. Statistical analysis was performed for glucose uptake and Seahorse XF data by one-way ANOVA with Sidak’s multiple comparisons test (*p < 0.05, ***p < 0.001, ****p < 0.0001).

Figure 4

Phosphorylation of PKM2 and STAT3 promote heightened IL-1β expression in macrophages with deletion of Bmal1. WT and Bmal1 ^-/- BMDMs were stimulated with LPS (100 ng/ml), RNA was isolated, and gene expression of (A) PKM2 and (F) IL-1β were analysed by RT-qPCR. Samples were normalized to their expression of the housekeeping gene 18S. Data presented is n=3 +/- SEM. Statistical analysis was performed by one-way ANOVA with Sidak’s multiple comparisons test (*p < 0.05, ****p < 0.0001). Protein expression of (B) PKM2 and (E) pSTAT3 was analysed by Western blot using β-Actin as a loading control. (C) PKM2 tetramers, dimers, and monomers were resolved by crosslinking samples after LPS stimulation before Western blot analysis. Pro IL-1β protein expression was measured following pretreatment with (D) DASA-58 (25 µM) or (G) STATTIC (2.5 µM) before stimulation with LPS for 8 hours. Western immunoblot data presented is representative of n=3 independent experiments. PKM2 and pSTAT3 time course densitometry is relative to the Bmal1^+/+ control band. Densitometry for Bmal1^+/+ and Bmal1^-/- DASA/LPS bands is relative to their LPS bands. These bands are indicated by * symbols. *p < 0.05, **p < 0.01 ***p < 0.001, ****p < 0.0001.

Figure 5

Schematic of immunometabolic changes in macrophages with deletion of Bmal1. Glucose metabolism is increased in macrophages with deletion of Bmal1 which potentiates expression of the pro-inflammatory cytokine IL-1β. In the absence of Bmal1 in macrophages, increased expression of the glucose transporter GLUT1 leads to increased glucose uptake and higher glycolytic pathway activity. Increased dimerization of the glycolytic enzyme PKM2 facilitates its translocation to the nucleus where it phosphorylates STAT3 to drive IL-1β expression. In the mitochondria, flux of pyruvate through pyruvate dehydrogenase (PDH) and oxygen consumption is increased alongside increased Krebs cycle flux which fuels accumulation of the intermediate succinate. Activity of the electron transport chain complex succinate dehydrogenase (SDH) is also increased in Bmal1 ^-/- macrophages which produces heightened levels of ROS which stabilizes HIF-1α to also promote IL-1β expression. Therefore, BMAL1 is regulating glucose metabolism in macrophages to impact upon the expression of IL-1β.