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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.

Epigenetic programming of monocyte-to-macrophage differentiation and trained innate immunity

Introduction Monocytes circulate in the bloodstream for up to 3 to 5 days. Concomitantly, immunological imprinting of either tolerance (immunosuppression) or trained immunity (innate immune memory) determines the functional fate of monocytes and monocyte-derived macrophages, as observed after infection or vaccination. The epigenome, DNase I accessibility, and transcriptome were characterized in purified human circulating monocytes, in vitro differentiated naïve, tolerized (immunosuppression), and trained macrophages (innate immune memory). This allowed the identification of pathways functionally implicated in innate immune memory. This epigenetic signature of human monocyte-to-macrophage differentiation and monocyte training generates hypotheses to understand and manipulate medically relevant immune conditions. Methods Purified circulating monocytes from healthy volunteers were differentiated under the homeostatic macrophage colony-stimulating factor concentrations present in human serum. During the first 24 hours, trained immunity was induced by β-glucan (BG) priming, and postsepsis immunoparalysis was mimicked by exposure to lipopolysaccharide (LPS), generating endotoxin-induced tolerance. Epigenomic profiling of the histone marks H3K4me1, H3K4me3, and H3K27ac, DNase I accessibility, and RNA sequencing were performed at both the start of the experiment (ex vivo monocytes) and at the end of the 6 days of in vitro culture (macrophages). Results Compared with monocytes (Mo), naïve macrophages (Mf ) display a remodeled metabolic enzyme repertoire and attenuated innate inflammatory pathways, most likely necessary to generate functional tissue macrophages. Epigenetic profiling uncovered about 8000 dynamic regions associated with about 11,000 DNase I hypersensitive sites. Changes in histone acetylation identified most dynamic events. Furthermore, these regions of differential histone marks displayed some degree of DNase I accessibility that was already present in monocytes. H3K4me1 mark increased in parallel with de novo H3K27ac deposition at distal regulatory regions; H3K4me1 mark remained even after the loss of H3K27ac, marking decommissioned regulatory elements. β-glucan priming specifically induced about 3000 distal regulatory elements, whereas LPS tolerization induced H3K27ac at about 500 distal regulatory regions. At the transcriptional level, we identified coregulated gene modules during monocyte-to-macrophage differentiation, as well as discordant modules between trained and tolerized cells. These indicate that training likely involves an increased expression of modules expressed in naïve macrophages, including genes that code for metabolic enzymes. On the other hand, endotoxin tolerance involves gene modules that are more active in monocytes than in naïve macrophages. About 12% of known human transcription factors display variation in expression during macrophage differentiation, training, and tolerance. We also observed transcription factor motifs in DNase I hypersensitive sites at condition-specific dynamic epigenomic regions, implying that specific transcription factors are required for trained and tolerized macrophage epigenetic and transcriptional programs. Finally, our analyses and functional validation indicate that the inhibition of cyclic adenosine monophosphate generation blocked trained immunity in vitro and during an in vivo model of lethal Candida albicans infection, abolishing the protective effects of trained immunity. Discussion We documented the importance of epigenetic regulation of the immunological pathways underlying monocyte-to-macrophage differentiation and trained immunity. These dynamic epigenetic elements may inform on potential pharmacological targets that modulate innate immunity. Altogether, we uncovered the epigenetic and transcriptional programs of monocyte differentiation to macrophages that distinguish tolerant and trained macrophage phenotypes, providing a resource to further understand and manipulate immune-mediated responses. 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 Genome-wide approaches analyze human monocyte differentiation in vitro into functional macrophages. Monocyte differentiation into macrophages represents a cornerstone process for host defense. Concomitantly, immunological imprinting of either tolerance or trained immunity determines the functional fate of macrophages and susceptibility to secondary infections. We characterized the transcriptomes and epigenomes in four primary cell types: monocytes and in vitro–differentiated naïve, tolerized, and trained macrophages. Inflammatory and metabolic pathways were modulated in macrophages, including decreased inflammasome activation, and we identified pathways functionally implicated in trained immunity. β-glucan training elicits an exclusive epigenetic signature, revealing a complex network of enhancers and promoters. Analysis of transcription factor motifs in deoxyribonuclease I hypersensitive sites at cell-type–specific epigenetic loci unveiled differentiation and treatment-specific repertoires. Altogether, we provide a resource to understand the epigenetic changes that underlie innate immunity in humans.

Single-molecule force spectroscopy reveals a highly compliant helical folding for the 30-nm chromatin fiber

The compaction of eukaryotic DNA into chromatin has been implicated in the regulation of all DNA processes. To unravel the higher-order folding of chromatin, we used magnetic tweezers and probed the mechanical properties of single 197-bp repeat length arrays of 25 nucleosomes. At forces up to 4 pN, the 30-nm fiber stretches like a Hookian spring, resulting in a three-fold extension. Together with a high nucleosome-nucleosome stacking energy, this points to a solenoid as the underlying topology of the 30-nm fiber. Unexpectedly, linker histones do not affect the length or stiffness of the fiber but stabilize its folding. Fibers with a nucleosome repeat length of 167 bp are stiffer, consistent with a two-start helical arrangement. The observed high compliance causes extensive thermal breathing, which forms a physical basis for the balance between DNA condensation and accessibility.

Catalytic activity of the yeast SWI/SNF complex on reconstituted nucleosome arrays

A novel, quantitative nucleosome array assay has been developed that couples the activity of a nucleosome ‘remodeling’ activity to restriction endonuclease activity. This assay has been used to determine the kinetic parameters of ATP‐dependent nucleosome disruption by the yeast SWI/SNF complex. Our results support a catalytic mode of action for SWI/SNF in the absence of nucleosome targeting. In this quantitative assay SWI/SNF and ATP lead to a 100‐fold increase in nucleosomal DNA accessibility, and initial rate measurements indicate that the complex can remodel one nucleosome every 4.5 min on an 11mer nucleosome array. In contrast to SWI/SNF action on mononucleosomes, we find that the SWI/SNF remodeling reaction on a nucleosome array is a highly reversible process. This result suggests that recovery from SWI/SNF action involves interactions among nucleosomes. The biophysical properties of model nucleosome arrays, coupled with the ease with which homogeneous arrays can be reconstituted and the DNA accessibility analyzed, makes the described array system generally applicable for functional analysis of other nucleosome remodeling enzymes, including histone acetyltransferases.

Phosphorylation of linker histones regulates ATP-dependent chromatin remodeling enzymes

Members of the ATP-dependent family of chromatin remodeling enzymes play key roles in the regulation of transcription, development, DNA repair and cell cycle control. We find that the remodeling activities of the ySWI/SNF, hSWI/SNF, xMi-2 and xACF complexes are nearly abolished by incorporation of linker histones into nucleosomal array substrates. Much of this inhibition is independent of linker histone-induced folding of the arrays. We also find that phosphorylation of the linker histone by Cdc2/Cyclin B kinase can rescue remodeling by ySWI/SNF. These results suggest that linker histones exert a global, genome-wide control over remodeling activities, implicating a new, obligatory coupling between linker histone kinases and ATP-dependent remodeling enzymes.

SWI-SNF-Mediated Nucleosome Remodeling: Role of Histone Octamer Mobility in the Persistence of the Remodeled State

ABSTRACT SWI-SNF is an ATP-dependent chromatin remodeling complex that disrupts DNA-histone interactions. Several studies of SWI-SNF activity on mononucleosome substrates have suggested that remodeling leads to novel, accessible nucleosomes which persist in the absence of continuous ATP hydrolysis. In contrast, we have reported that SWI-SNF-dependent remodeling of nucleosomal arrays is rapidly reversed after removal of ATP. One possibility is that these contrasting results are due to the different assays used; alternatively, the lability of the SWI-SNF-remodeled state might be different on mononucleosomes versus nucleosomal arrays. To investigate these possibilities, we use a coupled SWI-SNF remodeling–restriction enzyme assay to directly compare the remodeling of mononucleosome and nucleosomal array substrates. We find that SWI-SNF action causes a mobilization of histone octamers for both the mononucleosome and nucleosomal array substrates, and these changes in nucleosome positioning persist in the absence of continued ATP hydrolysis or SWI-SNF binding. In the case of mononucleosomes, the histone octamers accumulate at the DNA ends even in the presence of continued ATP hydrolysis. On nucleosomal arrays, SWI-SNF and ATP lead to a more dynamic state where nucleosomes appear to be constantly redistributed and restriction enzyme sites throughout the array have increased accessibility. This random positioning of nucleosomes within the array persists after removal of ATP, but inactivation of SWI-SNF is accompanied by an increased occlusion of many restriction enzyme sites. Our results also indicate that remodeling of mononucleosomes or nucleosomal arrays does not lead to an accumulation of novel nucleosomes that maintain an accessible state in the absence of continuous ATP hydrolysis.

Recruitment of a chromatin remodelling complex by the Hog1 MAP kinase to stress genes

For efficient transcription, RNA PolII must overcome the presence of nucleosomes. The p38‐related MAPK Hog1 is an important regulator of transcription upon osmostress in yeast and thereby it is involved in initiation and elongation. However, the role of this protein kinase in elongation has remained unclear. Here, we show that during stress there is a dramatic change in the nucleosome organization of stress‐responsive loci that depends on Hog1 and the RSC chromatin remodelling complex. Upon stress, the MAPK Hog1 physically interacts with RSC to direct its association with the ORF of osmo‐responsive genes. In RSC mutants, PolII accumulates on stress promoters but not in coding regions. RSC mutants also display reduced stress gene expression and enhanced sensitivity to osmostress. Cell survival under acute osmostress might thus depend on a burst of transcription that in turn could occur only with efficient nucleosome eviction. Our results suggest that the selective targeting of the RSC complex by Hog1 provides the necessary mechanistic basis for this event.

Recruitment of chromatin remodeling machines

The assembly of eukaryotic DNA into folded nucleosomal arrays has drastic consequences for many nuclear processes that require access to the DNA sequence, including RNA transcription, DNA replication, recombination, and repair. Two types of highly conserved chromatin remodeling enzymes have been implicated as regulators of the repressive nature of chromatin structure: ATP‐dependent remodeling complexes and nuclear histone acetyltransferases (HATs). Recent studies indicate that both types of enzymes can be recruited to chromosomal loci through either physical interactions with transcriptional activators or via the global accessibility of chromatin during S phase of the cell cycle. Here we review these recent observations and discuss the implications for gene‐specific regulation by chromatin remodeling machines. J. Cell. Biochem. 78:179–185, 2000.

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.

Purification and biochemical properties of yeast SWI/SNF complex.

Publisher Summary This chapter discusses the purification method and biochemical properties of yeast Saccharomyces cerevisiae SWI/SNF complex. SWI/SNF complex is a 2-MDa multiprotein assembly containing 11 distinct polypeptides. Genetic and biochemical evidence indicate that this macromolecular machine participates in the induction of genes otherwise repressed at a transcriptional level via nucleosome-dependent mechanisms. In the context of nucleosome arrays, SWI/SNF also enhances nucleosomal DNA binding by Gal4 protein, which can result in the persistent disruption of a nucleosome. The stimulation of transcription-factor binding is not readily amenable to kinetic analysis as it depends on the detection of a reversible event. However, the restriction enzyme cleavage of DNA is more amenable because it involves an enzymatic covalent modification that is easy to detect. The chapter discusses various methods that are used to assemble radiolabeled nucleosome arrays and determine the degree of saturation of the reconstituted arrays.