Shih-chin Cheng

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.

The macrophage mannose receptor induces IL-17 in response to Candida albicans.

The cytokine IL-17 controls neutrophil-mediated inflammatory responses. The pattern recognition receptor(s) that induce Th17 responses during infection, in the absence of artificial mitogenic stimulation with anti-CD3/anti-CD28 antibodies, remain obscure. We investigated the innate immune receptors and pathogen-associated molecular patterns involved in triggering Th17 responses during pathogen-specific host defense. The prototypic fungal pathogen Candida albicans was found to induce IL-17 more potently than Gram-negative bacteria. Candida mannan, but not zymosan, beta-glucans, Toll-like receptor (TLR) agonists, or the NOD2 ligand MDP, induced IL-17 production in the absence of anti-CD3/anti-CD28 antibodies. Candida-induced IL-17 response was dependent on antigen-presenting cells and the macrophage mannose receptor (MR), demonstrating that Candida mannan is not simply a mitogenic stimulus. The TLR2/dectin-1 pathway, but not TLR4 or NOD2, amplified MR-induced IL-17 production. This study identifies the specific pattern recognition receptors that trigger the Th17 response induced by a human pathogen in the absence of mitogenic stimulation.

The dectin-1/inflammasome pathway is responsible for the induction of protective T-helper 17 responses that discriminate between yeasts and hyphae of Candida albicans

In the mucosa, the immune pathways discriminating between colonizing and invasive Candida, thus inducing tolerance or inflammation, are poorly understood. Th17 responses induced by Candida albicans hyphae are central for the activation of mucosal antifungal immunity. An essential step for the discrimination between yeasts and hyphae and induction of Th17 responses is the activation of the inflammasome by C. albicans hyphae and the subsequent release of active IL‐1β in macrophages. Inflammasome activation in macrophages results from differences in cell‐wall architecture between yeasts and hyphae and is partly mediated by the dectin‐1/Syk pathway. These results define the dectin‐1/inflammasome pathway as the mechanism that enables the host immune system to mount a protective Th17 response and distinguish between colonization and tissue invasion by C. albicans.

Human TLR10 is an anti-inflammatory pattern-recognition receptor

Significance We demonstrate the biological role of TLR10, the only member of the Toll-like receptor (TLR)-family so far without a known function. We show that TLR10 acts as an inhibitory receptor, with suppressive effects. Blocking TLR10 by specific antibodies significantly upregulated TLR2-mediated cytokine production. Additionally, we show that individuals carrying loss-of-function SNPs in TLR10 display upregulation of TLR2-mediated cytokine production. After challenging human TLR10 transgenic mice with TLR2 ligand pam3CSK4 (Pam3Cys), less inflammation could be observed when compared with wild-type mice. Taking these data together, we show that TLR10 is the only pattern-recognition receptor within the TLR family that is able to dampen TLR2 responses, thereby suppressing immune responses through production of IL-1Ra. Toll-like receptor (TLR)10 is the only pattern-recognition receptor without known ligand specificity and biological function. We demonstrate that TLR10 is a modulatory receptor with mainly inhibitory effects. Blocking TLR10 by antagonistic antibodies enhanced proinflammatory cytokine production, including IL-1β, specifically after exposure to TLR2 ligands. Blocking TLR10 after stimulation of peripheral blood mononuclear cells with pam3CSK4 (Pam3Cys) led to production of 2,065 ± 106 pg/mL IL-1β (mean ± SEM) in comparison with 1,043 ± 51 pg/mL IL-1β after addition of nonspecific IgG antibodies. Several mechanisms mediate the modulatory effects of TLR10: on the one hand, cotransfection in human cell lines showed that TLR10 acts as an inhibitory receptor when forming heterodimers with TLR2; on the other hand, cross-linking experiments showed specific induction of the anti-inflammatory cytokine IL-1 receptor antagonist (IL-1Ra, 16 ± 1.7 ng/mL, mean ± SEM). After cross-linking anti-TLR10 antibody, no production of IL-1β and other proinflammatory cytokines could be found. Furthermore, individuals bearing TLR10 polymorphisms displayed an increased capacity to produce IL-1β, TNF-α, and IL-6 upon ligation of TLR2, in a gene-dose–dependent manner. The modulatory effects of TLR10 are complex, involving at least several mechanisms: there is competition for ligands or for the formation of heterodimer receptors with TLR2, as well as PI3K/Akt-mediated induction of the anti-inflammatory cytokine IL-1Ra. Finally, transgenic mice expressing human TLR10 produced fewer cytokines when challenged with a TLR2 agonist. In conclusion, to our knowledge we demonstrate for the first time that TLR10 is a modulatory pattern-recognition receptor with mainly inhibitory properties.

Innate immune memory: towards a better understanding of host defense mechanisms

Innate immunity is classically defined as unable to build up immunological memory. Recently however, the assumption of the lack of immunological memory within innate immune responses has been reconsidered. Plants and invertebrates lacking adaptive immune system can be protected against secondary infections. It has been shown that mammals can build cross-protection to secondary infections independently of T-lymphocytes and B-lymphocytes. Moreover, recent studies have demonstrated that innate immune cells such as NK cells and monocytes can display adaptive characteristics, a novel concept for which the term trained immunity has been proposed. Several mechanisms are involved in mediating innate immune memory, among which epigenetic histone modifications and modulation of recognition receptors on the surface of innate immune cells are likely to play a central role.

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.

Functional genomics identifies type I interferon pathway as central for host defense against Candida albicans

Candida albicans is the most common human fungal pathogen causing mucosal and systemic infections. However, human antifungal immunity remains poorly defined. Here, by integrating transcriptional analysis and functional genomics, we identified Candida-specific host defense mechanisms in humans. Candida induced significant expression of genes from the type I interferon (IFN) pathway in human peripheral blood mononuclear cells. This unexpectedly prominent role of type I IFN pathway in anti-Candida host defense was supported by additional evidence. Polymorphisms in type I IFN genes modulated Candida-induced cytokine production and were correlated with susceptibility to systemic candidiasis. In in-vitro experiments, type I IFNs skewed Candida-induced inflammation from a Th17-response toward a Th1-response. Patients with chronic mucocutaneaous candidiasis displayed defective expression of genes in the type I IFN pathway. These findings indicate that the type I IFN pathway is a main signature of Candida-induced inflammation and plays a crucial role in anti-Candida host defense in humans.

Anti-Aspergillus human host defence relies on type 1 T helper (Th1), rather than type 17 T helper (Th17), cellular immunity.

Both interferon‐γ‐producing type 1 T helper (Th1)‐ and interleukin‐17 (IL‐17)‐producing Th17 cells have been proposed to be involved in anti‐fungal host defence. Although invasive aspergillosis is one of the most severe human fungal infections, little is known regarding the relative importance of the Th1 versus Th17 cellular immune pathways for the human anti‐Aspergillus host defence. Using human peripheral blood mononuclear cells and a system consisting of monocyte‐derived macrophages with lymphocytes, we found that Aspergillus fumigatus is a weak inducer of human IL‐17 but induces a strong Th1 response. These data were validated by the very low IL‐17 levels in bronchoalveolar lavage fluid and serum of patients with invasive aspergillosis. Surprisingly, live A. fumigatus reduced IL‐17 production induced by mitogenic stimuli. This effect was mediated through the propensity of A. fumigatus to metabolize tryptophan and release kynurenine, which modulates the inflammatory response through inhibition of IL‐17 production. In conclusion, A. fumigatus does not stimulate production of IL‐17 and human host defence against aspergillosis may not rely on potent Th17 responses.

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.