Leo A. B. Joosten

Toll-like receptor 2 controls expansion and function of regulatory T cells

Tregs play a central role in the suppression of immune reactions and prevention of autoimmune responses harmful to the host. During acute infection, however, Tregs might hinder effector T cell activity directed toward the elimination of the pathogenic challenge. Pathogen recognition receptors from the TLR family expressed by innate immune cells are crucial for the generation of effective immunity. We have recently shown the CD4CD25 Treg subset in TLR2 mice to be significantly reduced in number compared with WT littermate control mice, indicating a link between Tregs and TLR2. Here, we report that the TLR2 ligand Pam3Cys, but not LPS (TLR4) or CpG (TLR9), directly acts on purified Tregs in a MyD88-dependent fashion. Moreover, when combined with TCR stimulation, TLR2 triggering augmented Treg proliferation in vitro and in vivo and resulted in a temporal loss of the suppressive Treg phenotype in vitro by directly affecting Tregs. Importantly, WT Tregs adoptively transferred into TLR2 mice were neutralized by systemic administration of TLR2 ligand during the acute phase of a Candida albicans infection, resulting in a 100-fold reduced C. albicans outgrowth. This demonstrates that in vivo TLR2 also controls the function of Tregs and establishes a direct link between TLRs and the control of immune responses through Tregs.

Human Dectin-1 Deficiency and Mucocutaneous Fungal Infections

Mucocutaneous fungal infections are typically found in patients who have no known immune defects. We describe a family in which four women who were affected by either recurrent vulvovaginal candidiasis or onychomycosis had the early-stop-codon mutation Tyr238X in the beta-glucan receptor dectin-1. The mutated form of dectin-1 was poorly expressed, did not mediate beta-glucan binding, and led to defective production of cytokines (interleukin-17, tumor necrosis factor, and interleukin-6) after stimulation with beta-glucan or Candida albicans. In contrast, fungal phagocytosis and fungal killing were normal in the patients, explaining why dectin-1 deficiency was not associated with invasive fungal infections and highlighting the specific role of dectin-1 in human mucosal antifungal defense.

Differential requirement for the activation of the inflammasome for processing and release of IL-1beta in monocytes and macrophages.

The processing of pro-interleukin-1beta depends on activation of caspase-1. Controversy has arisen whether Toll-like receptor (TLR) ligands alone can activate caspase-1 for release of interleukin-1beta (IL-1beta). Here we demonstrate that human blood monocytes release processed IL-1beta after a one-time stimulation with either TLR2 or TLR4 ligands, resulting from constitutively activated caspase-1 and release of endogenous adenosine triphosphate. The constitutive activation of caspase-1 depends on the inflammasome components, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and NALP3, but in monocytes caspase-1 activation is uncoupled from pathogen-associated molecular pattern recognition. In contrast, macrophages are unable to process and release IL-1beta solely by TLR ligands and require a second adenosine triphosphate stimulation. We conclude that IL-1beta production is differentially regulated in monocytes and macrophages, and this reflects their separate functions in host defense and inflammation.

Dense genotyping identifies and localizes multiple common and rare variant association signals in celiac disease.

Using variants from the 1000 Genomes Project pilot European CEU dataset and data from additional resequencing studies, we densely genotyped 183 non-HLA risk loci previously associated with immune-mediated diseases in 12,041 individuals with celiac disease (cases) and 12,228 controls. We identified 13 new celiac disease risk loci reaching genome-wide significance, bringing the number of known loci (including the HLA locus) to 40. We found multiple independent association signals at over one-third of these loci, a finding that is attributable to a combination of common, low-frequency and rare genetic variants. Compared to previously available data such as those from HapMap3, our dense genotyping in a large sample collection provided a higher resolution of the pattern of linkage disequilibrium and suggested localization of many signals to finer scale regions. In particular, 29 of the 54 fine-mapped signals seemed to be localized to single genes and, in some instances, to gene regulatory elements. Altogether, we define the complex genetic architecture of the risk regions of and refine the risk signals for celiac disease, providing the next step toward uncovering the causal mechanisms of the disease.

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.

STAT1 Mutations in Autosomal Dominant Chronic Mucocutaneous Candidiasis

BACKGROUND Chronic mucocutaneous candidiasis (CMC) is characterized by susceptibility to candida infection of skin, nails, and mucous membranes. Patients with recessive CMC and autoimmunity have mutations in the autoimmune regulator AIRE. The cause of autosomal dominant CMC is unknown. METHODS We evaluated 14 patients from five families with autosomal dominant CMC. We incubated their peripheral-blood mononuclear cells with different combinations of stimuli to test the integrity of pathways that mediate immunity, which led to the selection of 100 genes that were most likely to contain the genetic defect. We used an array-based sequence-capture assay, followed by next-generation sequencing, to identify mutations. RESULTS The mononuclear cells from the affected patients were characterized by poor production of interferon-γ, interleukin-17, and interleukin-22, suggesting that the defect lay within the interleukin-12 receptor and interleukin-23 receptor signaling pathways. We identified heterozygous missense mutations in the DNA sequence encoding the coiled-coil (CC) domain of signal transducer and activator of transcription 1 (STAT1) in the patients. These mutations lead to defective responses in type 1 and type 17 helper T cells (Th1 and Th17). The interferon-γ receptor pathway was intact in these patients. CONCLUSIONS Mutations in the CC domain of STAT1 underlie autosomal dominant CMC and lead to defective Th1 and Th17 responses, which may explain the increased susceptibility to fungal infection. (Funded by the Netherlands Organization for Scientific Research and others.).

Bacille Calmette-Guérin induces NOD2-dependent nonspecific protection from reinfection via epigenetic reprogramming of monocytes

Adaptive features of innate immunity, recently described as “trained immunity,” have been documented in plants, invertebrate animals, and mice, but not yet in humans. Here we show that bacille Calmette-Guérin (BCG) vaccination in healthy volunteers led not only to a four- to sevenfold increase in the production of IFN-γ, but also to a twofold enhanced release of monocyte-derived cytokines, such as TNF and IL-1β, in response to unrelated bacterial and fungal pathogens. The enhanced function of circulating monocytes persisted for at least 3 mo after vaccination and was accompanied by increased expression of activation markers such as CD11b and Toll-like receptor 4. These training effects were induced through the NOD2 receptor and mediated by increased histone 3 lysine 4 trimethylation. In experimental studies, BCG vaccination induced T- and B-lymphocyte–independent protection of severe combined immunodeficiency SCID mice from disseminated candidiasis (100% survival in BCG-vaccinated mice vs. 30% in control mice). In conclusion, BCG induces trained immunity and nonspecific protection from infections through epigenetic reprogramming of innate immune cells.

Stimulation of TLR2 and TLR4 differentially skews the balance of T cells in a mouse model of arthritis

TLRs may contribute to the progression of rheumatoid arthritis through recognition of microbial or host-derived ligands found in arthritic joints. Here, we show that TLR2 and TLR4, but not TLR9, are involved in the pathogenesis of autoimmune arthritis and play distinct roles in the regulation of T cells and cytokines. We investigated the involvement of TLR2, TLR4, and TLR9 in the progression of arthritis using IL-1 receptor antagonist-knockout (IL1rn-/-) mice, which spontaneously develop an autoimmune T cell-mediated arthritis. Spontaneous onset of arthritis was dependent on TLR activation by microbial flora, as germ-free mice did not develop arthritis. Clinical and histopathological evaluation of IL1rn-/-Tlr2-/- mice revealed more severe arthritis, characterized by reduced suppressive function of Tregs and substantially increased IFN-gamma production by T cells. IL1rn-/-Tlr4-/- mice were, in contrast, protected against severe arthritis and had markedly lower numbers of Th17 cells and a reduced capacity to produce IL-17. A lack of Tlr9 did not affect the progression of arthritis. While any therapeutic intervention targeting TLR2 still seems complicated, the strict position of TLR4 upstream of a number of pathogenic cytokines including IL-17 provides an interesting potential therapeutic target for rheumatoid arthritis.

Inflammasome is a central player in the induction of obesity and insulin resistance

Inflammation plays a key role in the pathogenesis of obesity. Chronic overfeeding leads to macrophage infiltration in the adipose tissue, resulting in proinflammatory cytokine production. Both microbial and endogenous danger signals trigger assembly of the intracellular innate immune sensor Nlrp3, resulting in caspase-1 activation and production of proinflammatory cytokines IL-1β and IL-18. Here, we showed that mice deficient in Nlrp3, apoptosis-associated speck-like protein, and caspase-1 were resistant to the development of high-fat diet-induced obesity, which correlated with protection from obesity-induced insulin resistance. Furthermore, hepatic triglyceride content, adipocyte size, and macrophage infiltration in adipose tissue were all reduced in mice deficient in inflammasome components. Monocyte chemoattractant protein (MCP)-1 is a key molecule that mediates macrophage infiltration. Indeed, defective inflammasome activation was associated with reduced MCP-1 production in adipose tissue. Furthermore, plasma leptin and resistin that affect energy use and insulin sensitivity were also changed by inflammasome-deficiency. Detailed metabolic and molecular phenotyping demonstrated that the inflammasome controls energy expenditure and adipogenic gene expression during chronic overfeeding. These findings reveal a critical function of the inflammasome in obesity and insulin resistance, and suggest inhibition of the inflammasome as a potential therapeutic strategy.