mTOR- and HIF-1α-mediated aerobic glycolysis as metabolic basis for trained immunity

Abstract

Epigenetic reprogramming of myeloid cells, also known as trained immunity, confers nonspecific protection from secondary infections. Using histone modification profiles of human monocytes trained with the Candida albicans cell wall constituent β-glucan, together with a genome-wide transcriptome, we identified the induced expression of genes involved in glucose metabolism. Trained monocytes display high glucose consumption, high lactate production, and a high ratio of nicotinamide adenine dinucleotide (NAD(+)) to its reduced form (NADH), reflecting a shift in metabolism with an increase in glycolysis dependent on the activation of mammalian target of rapamycin (mTOR) through a dectin-1-Akt-HIF-1α (hypoxia-inducible factor-1α) pathway. Inhibition of Akt, mTOR, or HIF-1α blocked monocyte induction of trained immunity, whereas the adenosine monophosphate-activated protein kinase activator metformin inhibited the innate immune response to fungal infection. Mice with a myeloid cell-specific defect in HIF-1α were unable to mount trained immunity against bacterial sepsis. Our results indicate that induction of aerobic glycolysis through an Akt-mTOR-HIF-1α pathway represents the metabolic basis of trained immunity.

Figure 1. Monocyte Trained Immunity

(A) Schematic of in vitro trained immunity experimental setup. (B) TNFα levels after 7 days in β-glucan treated cells. (C) Genome-wide H3K4me3 (blue) and H3K27me3 (purple) epigenetic modification analysis after 7 days post β-glucan treatment. (D) The epigenetic modifications in the promoter regions of the genes involved in glycolysis and mTOR (left panels) pathways and the schematic representation of the upregulated enzymes (red) in the glycolysis pathway. (E) Representative screenshots of the H3K4me3 (blue) and H3K27Ac (purple) modification in the promoter region of pyruvate kinase (PKM) and hexokinase. (F)The heat-map analysis of the total RNA expression determined by RNA sequencing between β-glucan treated group and control group was presented. Genes in the glycolysis pathway that are upregulated by the β–glucan training are highlighted in the box. Three mice per group were used for the RNA sequencing.

Figure 2. Post β-glucan Treatment Physiology

(A) The representative oxygen consumption rate of RPMI and β-glucan trained monocytes determined by high-resolution respirometry (Oxygraph) (B) Baseline (upper panel) and maximum oxygen consumption rate (lower panel)of untrained (open bar) and β-glucan-trained (closed bar) monocytes determined by respirometry and normalized to the leak oxygen consumption. Kinetic changes of (C) glucose consumption and (D) lactate production from day 1, day 3 and day 7 of the RPMI and β-glucan trained monocytes. (E) The kinetics of NAD+/NADH determined at day 1, day 3 and day 7.

Figure 3. mTOR Signaling in β-glucan Treated Monocytes

(A) Schematic representation of the upregulated enzymes (red) in mTOR signaling pathway. (B) Screenshot of the H3K4me3 (blue) and H3K27Ac (purple) modification in the promoter region of EIF4eBP, the main target of mTOR. (C) Western blot from cell lysate harvest at day 7 for endogenous phospho-mTOR, total mTOR, phospho-AMPK, AMPK and actin. p-mTOR/mTOR ratio is shown as a bar chart. (D) The endogenous p-mTOR status of Dectin-1 deficient patients and healthy control by Western blot from cell lysate harvest at day 7. p-mTOR/mTOR ratio is shown as a bar chart. The relative cytokine production was determined from the cells incubated with (E) rapamycin (mTOR inhibitor), (F) AICAR (AMPK inhibitor) and ascorbate (HIF1α inhibitor) in a dose-dependent manner.

Figure 4. AKT/mTOR/HIF1α Pathway downstream of β-glucan Stimulation

(A) AKT phosphorylation induced by β-glucan was determined in a kinetic manner by Western blot in monocytes in the presence or absence of wortmannin. (B) AKT phosphorylation and p-AKT/AKT ratio induced by β-glucan from the dectin-1 were determined by Western blot. The effect of PI3 kinase inhibitors on (C) AKT and mTOR phosphorylation were determined by Western blot or (D) by cytokine production upon LPS restimulation. (E) The relative cytokine production was determined from the cells incubated with metformin (AMPK inhibitor) in a dose-dependent manner. (F) Survival of wild type C57BL/6J mice infected with live C. albicans and AMPK-mTOR pathway training, Metformin or PBS were given from one day before the first non-lethal dose live C. albicans challenge till 3 days post challenge on a daily basis. (G) HIF-KO alveolar macrophages have increased metabolic activity. *Inset shows 20hr time point absorbance values. (F) Survival curve of wild type or mHIF-1α KO mice primed with β-glucan and challenge with a lethal dose of Staphylococcus aureus infection.

Figure 5

Model of metabolic activation of trained monocytes, characterized by shift towards increased aerobic glycolysis and decreased oxidative phosphorylation. The potential role of rapamycin and metformin in inhibition of trained immunity is also depicted. The metabolic differences between naïve monocytes and trained monocytes is summarized in the panel.