Rob J.W. Arts

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

Introduction Trained immunity refers to the memory characteristics of the innate immune system. Memory traits of innate immunity have been reported in plants and invertebrates, as well as in mice lacking functional T and B cells that are protected against secondary infections after exposure to certain infections or vaccinations. The underlying mechanism of trained immunity is represented by epigenetic programming through histone modifications, leading to stronger gene transcription upon restimulation. However, the specific cellular processes that mediate trained immunity in monocytes or macrophages are poorly understood. Aerobic glycolysis as metabolic basis for trained immunity. In naïve macrophages during aerobic conditions, glucose metabolism is mainly geared toward oxidative phosphorylation providing adenosine triphosphate (ATP) as the energy source. In contrast, long-term functional reprogramming during trained immunity requires a metabolic shift toward aerobic glycolysis and is induced through a dec tin-1–Akt–mTOR–HIF-1α pathway. Methods We studied a model of trained immunity, induced by the β-glucan component of Candida albicans, that was previously shown to induce nonspecific protection against both infections and malignancies. Genome-wide transcriptome and histone modification profiles were performed and pathway analysis was applied to identify the cellular processes induced during monocyte training. Biological validations were performed in human primary monocytes and in two experimental models in vivo. Results In addition to immune signaling pathways, glycolysis genes were strongly upregulated in terms of histone modification profiling, and this was validated by RNA sequencing of cells from β-glucan–treated mice. The biochemical characterizations of the β-glucan–trained monocytes revealed elevated aerobic glycolysis with reduced basal respiration rate, increased glucose consumption and lactate production, and higher intracellular ratio of nicotinamide adenine dinucleotide (NAD+) to its reduced form (NADH). The dectin-1–Akt–mTOR–HIF-1α pathway (mTOR, mammalian target of rapamycin; HIF-1α, hypoxia-inducible factor–1α) was responsible for the metabolic shift induced by β-glucan. Trained immunity was completely abrogated in monocytes from dectin-1–deficient patients. Blocking of the mTOR–HIF-1α pathway by chemical inhibitors inhibited trained immunity. Mice receiving metformin, an adenosine monophosphate–activated protein kinase (AMPK) activator that subsequently inhibits mTOR, lost the trained immunity–induced protection against lethal C. albicans infection. The role of the mTOR–HIF-1α pathway for β-glucan–induced innate immune memory was further validated in myeloid-specific HIF-1α knockout (mHIF-1α KO) mice that, unlike wild-type mice, were not protected against Staphylococcus aureus sepsis. Discussion The shift of central glucose metabolism from oxidative phosphorylation to aerobic glycolysis (the “Warburg effect”) meets the spiked need for energy and biological building blocks for rapid proliferation during carcinogenesis or clonal expansion in activated lymphocytes. We found that an elevated glycolysis is the metabolic basis for trained immunity as well, providing the energy and metabolic substrates for the increased activation of trained immune cells. The identification of glycolysis as a fundamental process in trained immunity further highlights a key regulatory role for metabolism in innate host defense and defines a potential therapeutic target in both infectious and inflammatory diseases. A BLUEPRINT of immune cell development To determine the epigenetic mechanisms that direct blood cells to develop into the many components of our immune system, the BLUEPRINT consortium examined the regulation of DNA and RNA transcription to dissect the molecular traits that govern blood cell differentiation. By inducing immune responses, Saeed et al. document the epigenetic changes in the genome that underlie immune cell differentiation. Cheng et al. demonstrate that trained monocytes are highly dependent on the breakdown of sugars in the presence of oxygen, which allows cells to produce the energy needed to mount an immune response. Chen et al. examine RNA transcripts and find that specific cell lineages use RNA transcripts of different length and composition (isoforms) to form proteins. Together, the studies reveal how epigenetic effects can drive the development of blood cells involved in the immune system. Science, this issue 10.1126/science.1251086, 10.1126/science.1250684, 10.1126/science.1251033 Epigenetic profiling identifies the cellular metabolic substrate of innate immune memory. 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.

Broad defects in the energy metabolism of leukocytes underlie immunoparalysis in sepsis

The acute phase of sepsis is characterized by a strong inflammatory reaction. At later stages in some patients, immunoparalysis may be encountered, which is associated with a poor outcome. By transcriptional and metabolic profiling of human patients with sepsis, we found that a shift from oxidative phosphorylation to aerobic glycolysis was an important component of initial activation of host defense. Blocking metabolic pathways with metformin diminished cytokine production and increased mortality in systemic fungal infection in mice. In contrast, in leukocytes rendered tolerant by exposure to lipopolysaccharide or after isolation from patients with sepsis and immunoparalysis, a generalized metabolic defect at the level of both glycolysis and oxidative metabolism was apparent, which was restored after recovery of the patients. Finally, the immunometabolic defects in humans were partially restored by therapy with recombinant interferon-γ, which suggested that metabolic processes might represent a therapeutic target in sepsis.

TREM-1: intracellular signaling pathways and interaction with pattern recognition receptors

TREM‐1 is an important signaling receptor expressed on neutrophils and monocytes that plays an important role in systemic infections. Here, we review the intracellular signaling pathways that mediate the immunological effects of TREM‐1. Because of the absence of signaling motifs, TREM‐1 constitutively associates with DAP12 for induction of intracellular signals. After phosphorylation of DAP12, production of chemokines and cytokines is induced. Moreover, TREM‐1 also modulates signaling pathways induced by known classes of PRRs, such as TLRs and NLRs. The exact mechanisms through which TREM‐1 influences TLR and NLR pathways are still largely elusive.

Glutaminolysis and Fumarate Accumulation Integrate Immunometabolic and Epigenetic Programs in Trained Immunity

Induction of trained immunity (innate immune memory) is mediated by activation of immune and metabolic pathways that result in epigenetic rewiring of cellular functional programs. Through network-level integration of transcriptomics and metabolomics data, we identify glycolysis, glutaminolysis, and the cholesterol synthesis pathway as indispensable for the induction of trained immunity by β-glucan in monocytes. Accumulation of fumarate, due to glutamine replenishment of the TCA cycle, integrates immune and metabolic circuits to induce monocyte epigenetic reprogramming by inhibiting KDM5 histone demethylases. Furthermore, fumarate itself induced an epigenetic program similar to β-glucan-induced trained immunity. In line with this, inhibition of glutaminolysis and cholesterol synthesis in mice reduced the induction of trained immunity by β-glucan. Identification of the metabolic pathways leading to induction of trained immunity contributes to our understanding of innate immune memory and opens new therapeutic avenues.

Trained innate immunity as underlying mechanism for the long‐term, nonspecific effects of vaccines

An increasing body of evidence shows that the innate immune system has adaptive characteristics that involve a heterologous memory of past insults. Both experimental models and proof‐of‐principle clinical trials show that innate immune cells, such as monocytes, macrophages, and NK cells, can provide protection against certain infections in vaccination models independently of lymphocytes. This process is regulated through epigenetic reprogramming of innate immune cells and has been termed “trained immunity.” It has been hypothesized that induction of trained immunity is responsible for the protective, nonspecific effects induced by vaccines, such as BCG, measles vaccination, and other whole‐microorganism vaccines. In this review, we will present the mechanisms of trained immunity responsible for the long‐lasting effects of vaccines on the innate immune system.

Immunometabolic Pathways in BCG-Induced Trained Immunity

Summary The protective effects of the tuberculosis vaccine Bacillus Calmette-Guerin (BCG) on unrelated infections are thought to be mediated by long-term metabolic changes and chromatin remodeling through histone modifications in innate immune cells such as monocytes, a process termed trained immunity. Here, we show that BCG induction of trained immunity in monocytes is accompanied by a strong increase in glycolysis and, to a lesser extent, glutamine metabolism, both in an in-vitro model and after vaccination of mice and humans. Pharmacological and genetic modulation of rate-limiting glycolysis enzymes inhibits trained immunity, changes that are reflected by the effects on the histone marks (H3K4me3 and H3K9me3) underlying BCG-induced trained immunity. These data demonstrate that a shift of the glucose metabolism toward glycolysis is crucial for the induction of the histone modifications and functional changes underlying BCG-induced trained immunity. The identification of these pathways may be a first step toward vaccines that combine immunological and metabolic stimulation.

β-Glucan Reverses the Epigenetic State of LPS-Induced Immunological Tolerance.

Summary Innate immune memory is the phenomenon whereby innate immune cells such as monocytes or macrophages undergo functional reprogramming after exposure to microbial components such as lipopolysaccharide (LPS). We apply an integrated epigenomic approach to characterize the molecular events involved in LPS-induced tolerance in a time-dependent manner. Mechanistically, LPS-treated monocytes fail to accumulate active histone marks at promoter and enhancers of genes in the lipid metabolism and phagocytic pathways. Transcriptional inactivity in response to a second LPS exposure in tolerized macrophages is accompanied by failure to deposit active histone marks at promoters of tolerized genes. In contrast, β-glucan partially reverses the LPS-induced tolerance in vitro. Importantly, ex vivo β-glucan treatment of monocytes from volunteers with experimental endotoxemia re-instates their capacity for cytokine production. Tolerance is reversed at the level of distal element histone modification and transcriptional reactivation of otherwise unresponsive genes.

TREM-1 interaction with the LPS/TLR4 receptor complex

Triggering receptor expressed on myeloid cells 1 (TREM-1) is an activating receptor expressed on neutrophils and monocytes that amplifies inflammation induced by stimulation of pattern-recognition receptors. In this study, several lines of evidence are presented that TREM-1 interacts with the toll-like receptor 4 (TLR4) receptor complex, or is a component of this complex. Blocking anti-TREM-1 antibodies specifically inhibited LPS-induced TNF-α production, while the alternative approach of blocking TLR4 by a specific inhibitor led to a down-regulation of the effects of TREM-1 cross-linking. These data are in line with the TLR4-TREM1 co-localization in human neutrophils and suggests that, at least some of the biological effects of TREM-1 may be due to its interaction with the TLR4/LPS-receptor complex.

Metabolic Induction of Trained Immunity through the Mevalonate Pathway

Innate immune cells can develop long-term memory after stimulation by microbial products during infections or vaccinations. Here, we report that metabolic signals can induce trained immunity. Pharmacological and genetic experiments reveal that activation of the cholesterol synthesis pathway, but not the synthesis of cholesterol itself, is essential for training of myeloid cells. Rather, the metabolite mevalonate is the mediator of training via activation of IGF1-R and mTOR and subsequent histone modifications in inflammatory pathways. Statins, which block mevalonate generation, prevent trained immunity induction. Furthermore, monocytes of patients with hyper immunoglobulin D syndrome (HIDS), who are mevalonate kinase deficient and accumulate mevalonate, have a constitutive trained immunity phenotype at both immunological and epigenetic levels, which could explain the attacks of sterile inflammation that these patients experience. Unraveling the role of mevalonate in trained immunity contributes to our understanding of the pathophysiology of HIDS and identifies novel therapeutic targets for clinical conditions with excessive activation of trained immunity.

An enigma: why vitamin A supplementation does not always reduce mortality even though vitamin A deficiency is associated with increased mortality

Background: Vitamin A deficiency (VAD) is associated with increased mortality. To prevent VAD, WHO recommends high-dose vitamin A supplementation (VAS) every 4–6 months for children aged between 6 months and 5 years of age in countries at risk of VAD. The policy is based on randomized clinical trials (RCTs) conducted in the late 1980s and early 1990s. Recent RCTs indicate that the policy may have ceased to be beneficial. In addition, RCTs attempting to extend the benefits to younger children have yielded conflicting results. Stratified analyses suggest that whereas some subgroups benefit more than expected from VAS, other subgroups may experience negative effects. Methods and Results: We reviewed the potential modifiers of the effect of VAS. The variable effect of VAS was not explained by underlying differences in VAD. Rather, the effect may depend on the sex of the child, the vaccine status and previous supplementation with vitamin A. Vitamin A is known to affect the Th1/Th2 balance and, in addition, recent evidence suggests that vitamin A may also induce epigenetic changes leading to down-regulation of the innate immune response. Thus VAS protects against VAD but has also important and long-lasting immunological effects, and the effect of providing VAS may vary depending on the state of the immune system. Conclusions: To design optimal VAS programmes which target those who benefit and avoid those harmed, more studies are needed. Work is ongoing to define whether neonatal VAS should be considered in subgroups. In the most recent RCT in older children, VAS doubled the mortality for males but halved mortality for females. Hence, we urgently need to re-assess the effect of VAS on older children in large-scale RCTs powered to study effect modification by sex and other potential effect modifiers, and with nested immunological studies.