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International audience; In this review we discuss the possibility that the phenomenon of microglial priming can be explained by the mechanisms that underlie trained immunity. The latter involves the enhancement of inflammatory responses by epigenetic mechanisms that are mobilized after first exposure to an inflammatory stimulus. These mechanisms include long-lasting histone modifications, including H3K4me1 deposition at latent enhancer regions. Although such changes may be beneficial in peripheral infectious disease, in the context of microglial priming they may drive increased microglia reactivity that is damaging in diseases of brain aging.

Summary A growing number of studies show that innate immune cells can undergo functional reprogramming, facilitating a faster and enhanced response to heterologous secondary stimuli. This concept has been termed “trained immunity.” We outline here a protocol to recapitulate this in vitro using adherent monocytes from consecutive isolation of peripheral blood mononuclear cells. The induction of trained immunity and the associated functional reprogramming of monocytes is described in detail using β-glucan (from Candida albicans) and Bacillus Calmette-Guérin as examples. For complete details on the use and execution of this protocol, please refer to Repnik et al. (2003) and Bekkering et al. (2016)., Graphical abstract, Highlights • Isolation of PBMCs and monocytes using discontinuous density gradients • In vitro induction of trained immunity in adherent monocytes • Induction of trained immunity is assessed by cytokine production levels • Generally applicable to test multiple stimuli and pharmacological compounds, A growing number of studies show that innate immune cells can undergo functional reprogramming, facilitating a faster and enhanced response to heterologous secondary stimuli. This concept has been termed “trained immunity.” We outline here a protocol to recapitulate this in vitro using adherent monocytes from consecutive isolation of peripheral blood mononuclear cells. The induction of trained immunity and the associated functional reprogramming of monocytes is described in detail using β-glucan (from Candida albicans) and Bacillus Calmette-Guérin as examples.

International audience; Exposure of mononuclear phagocytes to β-glucan, a naturally occurring polysaccharide, contributes to the induction of innate immune memory, which is associated with long-term epigenetic, metabolic, and functional reprogramming. Although previous studies have shown that innate immune memory induced by β-glucan confers protection against secondary infections, its impact on autoinflammatory diseases, associated with inflammasome activation and IL-1β secretion, remains poorly understood. In particular, whether β-glucan-induced long-term reprogramming affects inflammasome activation in human macrophages in the context of these diseases has not been explored. We found that NLRP3 inflammasome-mediated caspase-1 activation and subsequent IL-1β production were reduced in β-glucan-reprogrammed macrophages. β-Glucan acted upstream of the NLRP3 inflammasome by preventing potassium (K+) efflux, mitochondrial ROS (mtROS) generation, and, ultimately, apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization and speck formation. Importantly, β-glucan-induced memory in macrophages resulted in a remarkable attenuation of IL-1β secretion and caspase-1 activation in patients with an NLRP3-associated autoinflammatory disease, cryopyrin-associated periodic syndromes (CAPS). Our findings demonstrate that β-glucan-induced innate immune memory represses IL-1β-mediated inflammation and support its potential clinical use in NLRP3-driven diseases.

International audience; Seminal work by Louis Pasteur revealed the contribution of fungi - yeasts and microsporidia to agro-industry and disease in animals, respectively. More than 150 years later, the impact of fungi on human health and beyond is an ever-increasing issue, although often underestimated. Recent studies estimate that fungal infections, especially those caused by Candida, Cryptococcus and Aspergillus species, kill more than one million people annually. Indeed, these neglected infections are in general very difficult to cure and the associated mortality remains very high even when antifungal treatments exist. The development of new antifungals and diagnostic tools that are both necessary to fight fungal diseases efficiently, requires greater insights in the biology of the fungal pathogens of humans in the context of the infection, on their epidemiology, and on their role in the human mycobiota. We also need a better understanding of the host immune responses to fungal pathogens as well as the genetic basis for the increased sensitivity of some individuals to fungal infections. Here, we highlight some recent progress made in these different areas of research, in particular based on work conducted in our own laboratories. These progresses should lay the ground for better management of fungal infections, as they provide opportunities for better diagnostic, vaccination, the development of classical antifungals but also strategies for targeting virulence factors or the host. (C) Springer Nature Limited 2019 reproduced with permission of SNCSC.

Leptospira interrogans are pathogenic spirochetes responsible for leptospirosis, a worldwide reemerging zoonosis. Many Leptospira serovars have been described, and prophylaxis using inactivated bacteria provides only short-term serovar-specific protection. Therefore, alternative approaches to limit severe leptospirosis in humans and morbidity in cattle would be welcome. Innate immune cells, including macrophages, play a key role in fighting infection and pathogen clearance. Recently, it has been shown that functional reprograming of innate immune cells through the activation of pattern recognition receptors leads to enhanced nonspecific antimicrobial responses upon a subsequent microbial encounter. This mechanism is known as trained immunity or innate immune memory. We have previously shown that oral treatment with Lactobacillus plantarum confers a beneficial effect against acute leptospirosis. Here, using a macrophage depletion protocol and live imaging in mice, we established the role of peritoneal macrophages in limiting the initial dissemination of leptospires. We further showed that intraperitoneal priming of mice with CL429, a TLR2 and NOD2 agonist known to mimic the modulatory effect of Lactobacillus, alleviated acute leptospiral infection. The CL429 treatment was characterized as a training effect since i.) it was linked to peritoneal macrophages that produced ex vivo more pro-inflammatory cytokines and chemokines against 3 different pathogenic serovars of Leptospira, independently of the presence of B and T cells, ii.) it had systemic effects on splenic cells and bone marrow derived macrophages, and iii.) it was sustained for 3 months. Importantly, trained macrophages produced more nitric oxide, a potent antimicrobial compound, which has not been previously linked to trained immunity. Accordingly, trained macrophages better restrict leptospiral survival. Finally, we could use CL429 to train ex vivo human monocytes that produced more cytokines upon leptospiral stimulation. In conclusion, host-directed treatment using a TLR2/NOD2 agonist could be envisioned as a novel prophylactic strategy against acute leptospirosis., Author summary Leptospirosis is a worldwide zoonotic neglected disease caused by pathogenic Leptospira. In addition to cattle and pets, it affects one million people per year and causes 60000 deaths. Antibiotics are efficient when given early after infection, but because the symptoms are not specific, leptospirosis is difficult to diagnose in a timely fashion. There are ~300 serovars of pathogenic Leptospira, which complicates the development of a universal vaccine against leptospirosis. Therefore, prophylactic treatments would be welcome. Recently, a new kind of immune memory known as “innate immune memory” or “trained immunity” has been described. It is not linked to adaptive immunity but only to innate immune cells, such as macrophages. Initial stimulation by a bacterial or fungal component can “train” macrophages to respond better but nonspecifically to a second encounter with a microbe. In this work, we show that trained immunity can be induced in mice to alleviate the symptoms of experimental acute leptospirosis. Cells from treated mice stimulated ex vivo by different serovars of pathogenic leptospires secreted more antimicrobial compounds and showed enhanced leptospire killing. Moreover, this treatment was able to boost human monocytes. This work presents a host-directed therapeutic approach that could be used as a novel prophylactic strategy against leptospirosis.

International audience; The binary classification of mammalian immune memory is now obsolete. Innate immune cells carry memory characteristics. The overall capacity of innate immune cells to remember and alter their responses is referred as innate immune memory and the induction of a non-specific memory resulting in an enhanced immune status is termed "trained immunity". Historically, trained immunity was first described as triggered by the human fungal pathogen Candida albicans. Since, numerous studies have accumulated and deciphered the main characteristics of trained immunity mediated by fungi and fungal components. This review aims at presenting the newly described aspect of memory in innate immunity with an emphasis on the historically fungal mediated one, covering the known molecular mechanisms associated with training. In addition, the review uncovers the numerous non-specific effect that β-glucans trigger in the context of infectious diseases and septicaemia, inflammatory diseases and cancer.

Contains fulltext : 202899.pdf (Publisher’s version ) (Open Access) Sodium butyrate is well-known for its immune-modulatory properties. Studies until now only focused on the in vitro effects of butyrate or assessed local effects in the gut upon butyrate administration. In this trial, we studied the systemic anti-inflammatory effects induced by sodium butyrate supplementation in humans. Nine healthy (Lean) and ten obese (metabolic syndrome group, MetSyn) males were given 4 grams sodium butyrate daily for 4 weeks. PBMCs were isolated before and after supplementation for direct stimulation experiments and induction of trained immunity by oxidized low-density lipoprotein (oxLDL), beta-glucan, or Bacillus Calmette-Guerin vaccine (BCG). Butyrate supplementation moderately affected some of the cytokine responses in the MetSyn group. In the direct stimulation setup, effects of butyrate supplementation were limited. Interestingly, butyrate supplementation decreased oxLDL-induced trained immunity in the MetSyn group for LPS-induced IL-6 responses and Pam3CSK4-induced TNF-alpha responses. Induction of trained immunity by beta-glucan was decreased by butyrate in the MetSyn group for Pam3CSK4-induced IL-10 production. In this study, while having only limited effects on the direct stimulation of cytokine production, butyrate supplementation significantly affected trained immunity in monocytes of obese individuals with metabolic complications. Therefore, oral butyrate supplementation may be beneficial in reducing the overall inflammatory status of circulating monocytes in patients with metabolic syndrome.

International audience

International audience; Recognition of the fungal cell wall carbohydrate β‐glucan by the host receptor Dectin‐1 elicits broad immunomodulatory responses, such as phagocytosis and activation of oxidative burst. These responses are essential for engulfing and killing fungal pathogens. Phagocytic monocytes are key mediators of these early host inflammatory responses to infection. Remarkably, whether phagocytosis of fungal β‐glucan leads to an inflammatory response in human monocytes remains to be established. Here, we show that phagocytosis of heat‐killed Candida albicans is essential to trigger inflammation and cytokine release. By contrast, inhibition of actin‐dependent phagocytosis of particulate (1‐3,1‐6)‐β‐glucan induces a strong inflammatory signature. Sustained monocyte activation, induced by fungal β‐glucan particles upon actin cytoskeleton disruption, relies on Dectin‐1 and results in the classical caspase‐1 inflammasome formation through NLRP3, generation of an oxidative burst, NF‐κB activation, and increased inflammatory cytokine release. PI3K and NADPH oxidase were crucial for both cytokine secretion and ROS generation, whereas Syk signaling mediated only cytokine production. Our results highlight the mechanism by which phagocytosis tightly controls the activation of phagocytes by fungal pathogens and strongly suggest that actin cytoskeleton dynamics are an essential determinant of the host's susceptibility or resistance to invasive fungal infections.

International audience; β-glucan, the most abundant fungal cell wall polysaccharide, has gained much attention from the scientific community in the last few decades for its fascinating but not yet fully understood immunobiology. Study of this molecule has been motivated by its importance as a pathogen-associated molecular pattern upon fungal infection as well as by its promising clinical utility as biological response modifier for the treatment of cancer and infectious diseases. Its immune effect is attributed to the ability to bind to different receptors expressed on the cell surface of phagocytic and cytotoxic innate immune cells, including monocytes, macrophages, neutrophils, and natural killer cells. The characteristics of the immune responses generated depend on the cell types and receptors involved. Size and biochemical composition of β-glucans isolated from different sources affect their immunomodulatory properties. The variety of studies using crude extracts of fungal cell wall rather than purified β-glucans renders data difficult to interpret. A better understanding of the mechanisms of purified fungal β-glucan recognition, downstream signaling pathways, and subsequent immune regulation activated, is, therefore, essential not only to develop new antifungal therapy but also to evaluate β-glucan as a putative anti-infective and antitumor mediator. Here, we briefly review the complexity of interactions between fungal β-glucans and mononuclear phagocytes during fungal infections. Furthermore, we discuss and present available studies suggesting how different fungal β-glucans exhibit antitumor and antimicrobial activities by modulating the biologic responses of mononuclear phagocytes, which make them potential candidates as therapeutic agents.

Item does not contain fulltext The Drosophila Toll-signaling pathway controls the systemic antifungal host response. Gram-negative binding protein 3 (GNBP3), a member of the beta-glucan recognition protein family senses fungal infections and activates this pathway. A second detection system perceives the activity of proteolytic fungal virulence factors and redundantly activates Toll. GNBP3(hades) mutant flies succumb more rapidly to Candida albicans and to entomopathogenic fungal infections than WT flies, despite normal triggering of the Toll pathway via the virulence detection system. These observations suggest that GNBP3 triggers antifungal defenses that are not dependent on activation of the Toll pathway. Here, we show that GNBP3 agglutinates fungal cells. Furthermore, it can activate melanization in a Toll-independent manner. Melanization is likely to be an essential defense against some fungal infections given that the entomopathogenic fungus Beauveria bassiana inhibits the activity of the main melanization enzymes, the phenol oxidases. Finally, we show that GNBP3 assembles "attack complexes", which comprise phenoloxidase and the necrotic serpin. We propose that Drosophila GNBP3 targets fungi immediately at the inception of the infection by bringing effector molecules in direct contact with the invading microorganisms. 01 mei 2010

Neutrophils and monocytes provide a first line of defense against infections as part of the innate immune system. Here we report the integrated analysis of transcriptomic and epigenetic landscapes for circulating monocytes and neutrophils with the aim to enable downstream interpretation and functional validation of key regulatory elements in health and disease. We collected RNA-seq data, ChIP-seq of six histone modifications and of DNA methylation by bisulfite sequencing at base pair resolution from up to 6 individuals per cell type. Chromatin segmentation analyses suggested that monocytes have a higher number of cell-specific enhancer regions (4-fold) compared to neutrophils. This highly plastic epigenome is likely indicative of the greater differentiation potential of monocytes into macrophages, dendritic cells and osteoclasts. In contrast, most of the neutrophil-specific features tend to be characterized by repressed chromatin, reflective of their status as terminally differentiated cells. Enhancers were the regions where most of differences in DNA methylation between cells were observed, with monocyte-specific enhancers being generally hypomethylated. Monocytes show a substantially higher gene expression levels than neutrophils, in line with epigenomic analysis revealing that gene more active elements in monocytes. Our analyses suggest that the overexpression of c-Myc in monocytes and its binding to monocyte-specific enhancers could be an important contributor to these differences. Altogether, our study provides a comprehensive epigenetic chart of chromatin states in primary human neutrophils and monocytes, thus providing a valuable resource for studying the regulation of the human innate immune system.

Little is known regarding the role of NK cells during primary and secondary disseminated Candida albicans infection. We assessed the role of NK cells for host defense against candidiasis in immunocompetent, as well as immunodeficient, hosts. Surprisingly, depletion of NK cells in immunocompetent WT mice did not increase susceptibility to systemic candidiasis, suggesting that NK cells are redundant for antifungal defense in otherwise immunocompetent hosts. NK-cell-depleted mice were found to be protected as a consequence of attenuation of systemic inflammation. In contrast, the absence of NK cells in T/B/NK-cell-deficient NSG (NOD SCID gamma) mice led to an increased susceptibility to both primary and secondary systemic C. albicans infections compared with T/B-cell-deficient SCID mice. In conclusion, this study demonstrates that NK cells are an essential and nonredundant component of anti-C. albicans host defense in immunosuppressed hosts with defective T/B-lymphocyte immunity, while contributing to hyperinflammation in immunocompetent hosts. The discovery of the importance of NK cells in hosts with severe defects of adaptive immunity might have important consequences for the design of adjunctive immunotherapeutic approaches in systemic C. albicans infections targeting NK-cell function.

Upon priming with Candida albicans or with the fungal cell wall component β-glucan, monocytes respond with an increased cytokine production upon restimulation, a phenomenon termed “trained immunity.” In contrast, the prestimulation of monocytes with lipopolysaccharide has long been known to induce tolerance. Because the vast majority of commensal microorganisms belong to bacterial or viral phyla, we sought to systematically investigate the functional reprogramming of monocytes induced by the stimulation of pattern recognition receptors (PRRs) with various bacterial or viral ligands. Monocytes were functionally programmed for either enhanced (training) or decreased (tolerance) cytokine production, depending on the type and concentration of ligand they encountered. The functional reprogramming of monocytes was also associated with cell shape, granulocity, and cell surface marker modifications. The training effect required p38- and Jun N-terminal protein kinase (JNK)-mediated mitogen-activated protein kinase (MAPK) signaling, with specific signaling patterns directing the functional fate of the cell. The long-term effects on the function of monocytes were mediated by epigenetic events, with both histone methylation and acetylation inhibitors blocking the training effects. In conclusion, our experiments identify the ability of monocytes to acquire adaptive characteristics after prior activation with a wide variety of ligands. Trained immunity and tolerance are two distinct and opposing functional programs induced by the specific microbial ligands engaging the monocytes.

Contains fulltext : 138552.pdf (Publisher’s version ) (Open Access) We have recently shown that BCG (Bacillus Calmette-Guerin) vaccination in healthy volunteers induces epigenetic reprogramming of monocytes, leading to increased cytokine production in response to nonrelated pathogens for up to 3 months after vaccination. This phenomenon was named 'trained immunity'. In the present study we assessed whether BCG was able to induce long-lasting effects on both trained immunity and heterologous T helper 1 (Th1) and Th17 immune responses 1 year after vaccination. The production of TNFalpha and IL-1beta to mycobacteria or unrelated pathogens was higher after 2 weeks and 3 months postvaccination, but these effects were less pronounced 1 year after vaccination. However, monocytes recovered 1 year after vaccination had an increased expression of pattern recognition receptors such as CD14, Toll-like receptor 4 (TLR4) and mannose receptor, and this correlated with an increase in proinflammatory cytokine production after stimulation with the TLR4 ligand lipopolysaccharide. The heterologous production of Th1 (IFN-gamma) and Th17 (IL-17 and IL-22) immune responses to nonmycobacterial stimulation remained strongly elevated even 1 year after BCG vaccination. In conclusion, BCG induces sustained changes in the immune system associated with a nonspecific response to infections both at the level of innate trained immunity and at the level of heterologous Th1/Th17 responses. (c) 2013 S. Karger AG, Basel.

We have morphologically characterized Candida tropicalis isolates resistant to amphotericin B (AmB). These isolates present an enlarged cell wall compared to isolates of regular susceptibility. This correlated with higher levels of β-1,3-glucan in the cell wall but not with detectable changes in chitin content. In line with this, AmB-resistant strains showed reduced susceptibility to Congo red. Moreover, mitogen-activated protein kinases (MAPKs) involved in cell integrity were already activated during regular growth in these strains. Finally, we investigated the response elicited by human blood cells and found that AmB-resistant strains induced a stronger proinflammatory response than susceptible strains. In agreement, AmB-resistant strains also induced stronger melanization of Galleria mellonella larvae, indicating that the effect of alterations of the cell wall on the immune response is conserved in different types of hosts. Our results suggest that resistance to AmB is associated with pleiotropic mechanisms that might have important consequences, not only for the efficacy of the treatment but also for the immune response elicited by the host.

This research was originally published in the Journal of Biological Chemistry. Lisa Rizzetto, Daniela C. Ifrim, Silvia Moretti, Noemi Tocci, Shih-Chin Cheng, Jessica Quintin, Giorgia Renga, Vasilis Oikonomou, Carlotta De Filippo, Tobias Weil, Bastiaan A. Blok, Marcello S. Lenucci, Manuel A. S. Santos, Luigina Romani, Mihai G. Netea, and Duccio Cavalieri. Fungal Chitin Induces Trained Immunity in Human Monocytes during Cross-talk of the Host with Saccharomyces cerevisiae. J. Biol. Chem. 2016 291: 7961-7972 © the American Society for Biochemistry and Molecular Biology; International audience; The immune system is essential to maintain the mutualistic homeostatic interaction between the host and its micro- and mycobiota. Living as a commensal,Saccharomyces cerevisiaecould potentially shape the immune response in a significant way. We observed thatS. cerevisiaecells induce trained immunity in monocytes in a strain-dependent manner through enhanced TNFα and IL-6 production upon secondary stimulation with TLR ligands, as well as bacterial and fungal commensals. Differential chitin content accounts for the differences in training properties observed among strains, driving induction of trained immunity by increasing cytokine production and direct antimicrobial activity bothin vitroandin vivo These chitin-induced protective properties are intimately associated with its internalization, identifying a critical role of phagosome acidification to facilitate microbial digestion. This study reveals how commensal and passenger microorganisms could be important in promoting health and preventing mucosal diseases by modulating host defense toward pathogens and thus influencing the host microbiota-immune system interactions.

International audience; Innate and adaptive immunity have evolved as sophisticated mechanisms of host defence against invading pathogens. Classically the properties attributed to innate immunity are its rapid pleiotropic response, and to adaptive immunity its specificity and ability to retain a long-term memory of past infections. It is now clear that innate immunity also contributes to raising a memory response upon pathogenic assault. In this review we will discuss the interaction between bacterial, viral, fungal and parasitic molecular patterns and innate immune cells in which a memory response is imposed, or has the potential to be imposed.

Item does not contain fulltext Immunological memory in vertebrates is often exclusively attributed to T and B cell function. Recently it was proposed that the enhanced and sustained innate immune responses following initial infectious exposure may also afford protection against reinfection. Testing this concept of "trained immunity," we show that mice lacking functional T and B lymphocytes are protected against reinfection with Candida albicans in a monocyte-dependent manner. C. albicans and fungal cell wall beta-glucans induced functional reprogramming of monocytes, leading to enhanced cytokine production in vivo and in vitro. The training required the beta-glucan receptor dectin-1 and the noncanonical Raf-1 pathway. Monocyte training by beta-glucans was associated with stable changes in histone trimethylation at H3K4, which suggests the involvement of epigenetic mechanisms in this phenomenon. The functional reprogramming of monocytes, reminiscent of similar NK cell properties, supports the concept of "trained immunity" and may be employed for the design of improved vaccination strategies.

Item does not contain fulltext 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-Guerin (BCG) vaccination in healthy volunteers led not only to a four- to sevenfold increase in the production of IFN-gamma, but also to a twofold enhanced release of monocyte-derived cytokines, such as TNF and IL-1beta, 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.

Studying infectious diseases requires suitable hosts for experimental in vivo infections. Recent years have seen the advent of many alternatives to murine infection models. However, the use of non-mammalian models is still controversial because it is often unclear how well findings from these systems predict virulence potential in humans or other mammals. Here, we compare the commonly used models, fruit fly and mouse (representing invertebrate and mammalian hosts), for their similarities and degree of correlation upon infection with a library of mutants of an important fungal pathogen, the yeast Candida glabrata. Using two indices, for fly survival time and for mouse fungal burden in specific organs, we show a good agreement between the models. We provide a suitable predictive model for estimating the virulence potential of C. glabrata mutants in the mouse from fly survival data. As examples, we found cell wall integrity mutants attenuated in flies, and mutants of a MAP kinase pathway had defective virulence in flies and reduced relative pathogen fitness in mice. In addition, mutants with strongly reduced in vitro growth generally, but not always, had reduced virulence in flies. Overall, we demonstrate that surveying Drosophila survival after infection is a suitable model to predict the outcome of murine infections, especially for severely attenuated C. glabrata mutants. Pre-screening of mutants in an invertebrate Drosophila model can, thus, provide a good estimate of the probability of finding a strain with reduced microbial burden in the mouse host., Summary: Can the fitness of deletion mutants in a murine model be predicted by their virulence in Drosophila melanogaster? For a fungal pathogen, the answer is, mostly, yes.

Contains fulltext : 154948.pdf (Publisher’s version ) (Closed access) Patients with signal transducer and activator of transcription-1 (STAT1)-dependent chronic mucocutaneous candidiasis (CMC) and patients with STAT3-dependent hyper-immunoglobulin (Ig)E syndrome (HIES) display defects in T helper type 17 (Th17) cytokine production capacity. Despite this similar immune defect in Th17 function, they show important differences in the type of infections to which they are susceptible. Recently, our group reported differential regulation of STAT-1 and STAT-3 transcription factors during epigenetic reprogramming of trained immunity, an important host defence mechanism based on innate immune memory. We therefore hypothesized that STAT1 and STAT3 defects have different effects on trained immunity, and this may partly explain the differences between CMC and HIES regarding the susceptibility to infections. Indeed, while trained immunity was normally induced in cells isolated from patients with HIES, the induction of innate training was defective in CMC patients. This defect was specific for training with Candida albicans, the main pathogen encountered in CMC, and it involved a type II interferon-dependent mechanism. These findings describe the role of STAT-1 for the induction of trained immunity, and may contribute to the understanding of the differences in susceptibility to infection between CMC and HIES patients. This study could also provide directions for personalized immunotherapy in patients suffering from these immunodeficiencies.

International audience; Drosophila melanogaster is a robust model to investigate many biological problems. It is however prone to some infections, which may endanger fly stocks if left unchecked for. One such infection is caused by an obligate fungal intracellular parasite, Tubulinosema ratisbonensis, which can be found in laboratory stocks. Here, we identify and briefly characterize a T. ratisbonensis strain that was infesting our Drosophila cultures and that required intensive measures to contain and eradicate the infection. We describe the phenotypes of infested stocks. We also report PCR-based techniques that allow the detection of infested stocks with a high sensitivity. We have developed a high-throughput qPCR assay that allows the efficient parallel screening of a large number of potentially-infested stocks. We also have investigated several prophylactic measures to prevent the further contamination of stocks, namely UV-exposure, ethanol treatment, bleaching, and desiccation. Bleaching was found to kill all spores. Other treatments were less effective but were found to be sufficient to prevent further contamination of noninfested stocks. Two treatments were efficacious in curing infested stocks (1) bleaching of eggs and subsequent raising of the larvae in clean vials; (2) fumagillin treatment. These cures only work on stocks that have not become too weak to withstand the procedures.

Contains fulltext : 96277.pdf (Publisher’s version ) (Open Access) BACKGROUND: Two NF-kappaB signaling pathways, Toll and immune deficiency (imd), are required for survival to bacterial infections in Drosophila. In response to septic injury, these pathways mediate rapid transcriptional activation of distinct sets of effector molecules, including antimicrobial peptides, which are important components of a humoral defense response. However, it is less clear to what extent macrophage-like hemocytes contribute to host defense. METHODOLOGY/PRINCIPAL FINDINGS: In order to dissect the relative importance of humoral and cellular defenses after septic injury with three different gram-positive bacteria (Micrococcus luteus, Enterococcus faecalis, Staphylococcus aureus), we used latex bead pre-injection to ablate macrophage function in flies wildtype or mutant for various Toll and imd pathway components. We found that in all three infection models a compromised phagocytic system impaired fly survival--independently of concomitant Toll or imd pathway activation. Our data failed to confirm a role of the PGRP-SA and GNBP1 Pattern Recognition Receptors for phagocytosis of S. aureus. The Drosophila scavenger receptor Eater mediates the phagocytosis by hemocytes or S2 cells of E. faecalis and S. aureus, but not of M. luteus. In the case of M. luteus and E. faecalis, but not S. aureus, decreased survival due to defective phagocytosis could be compensated for by genetically enhancing the humoral immune response. CONCLUSIONS/SIGNIFICANCE: Our results underscore the fundamental importance of both cellular and humoral mechanisms in Drosophila immunity and shed light on the balance between these two arms of host defense depending on the invading pathogen.

The anti-tuberculosis-vaccine Bacillus Calmette-Guérin (BCG) is the most widely used vaccine in the world. In addition to its effects against tuberculosis, BCG vaccination also induces non-specific beneficial effects against certain forms of malignancy and against infections with unrelated pathogens. It has been recently proposed that the non-specific effects of BCG are mediated through epigenetic reprogramming of monocytes, a process called trained immunity. In the present study we demonstrate that autophagy contributes to trained immunity induced by BCG. Pharmacologic inhibition of autophagy blocked trained immunity induced in vitro by stimuli such as β–glucans or BCG. Single nucleotide polymorphisms (SNPs) in the autophagy genes ATG2B (rs3759601) and ATG5 (rs2245214) influenced both the in vitro and in vivo training effect of BCG upon restimulation with unrelated bacterial or fungal stimuli. Furthermore, pharmacologic or genetic inhibition of autophagy blocked epigenetic reprogramming of monocytes at the level of H3K4 trimethylation. Finally, we demonstrate that rs3759601 in ATG2B correlates with progression and recurrence of bladder cancer after BCG intravesical instillation therapy. These findings identify a key role of autophagy for the nonspecific protective effects of BCG., Author Summary Next to its effects against tuberculosis, BCG vaccination also induces non-specific beneficial effects on immune cells to increase their ability to control unrelated pathogens. It has been recently proposed that the non-specific effects of BCG are mediated through epigenetic reprogramming of monocytes, a process called trained immunity. Little is known regarding the intracellular events controlling its induction. In this study we identified autophagy as a key player in trained immunity. Pharmacological inhibition of autophagy as well as polymorphisms in autophagy-related genes blocked BCG-induced trained immunity. Furthermore, BCG vaccine is also used to treat bladder cancer. Genetic polymorphisms in autophagy-related genes correlated with progression and recurrence of bladder cancer after treatment with BCG therapy. These findings open new possibilities for improvement of future BCG-based vaccines to be used against infections and malignancies.

Contains fulltext : 133902.pdf (Publisher’s version ) (Open Access) RATIONALE: To prevent or combat infection, increasing the effectiveness of the immune response is highly desirable, especially in case of compromised immune system function. However, immunostimulatory therapies are scarce, expensive, and often have unwanted side-effects. beta-glucans have been shown to exert immunostimulatory effects in vitro and in vivo in experimental animal models. Oral beta-glucan is inexpensive and well-tolerated, and therefore may represent a promising immunostimulatory compound for human use. METHODS: We performed a randomized open-label intervention pilot-study in 15 healthy male volunteers. Subjects were randomized to either the beta -glucan (n = 10) or the control group (n = 5). Subjects in the beta-glucan group ingested beta-glucan 1000 mg once daily for 7 days. Blood was sampled at various time-points to determine beta-glucan serum levels, perform ex vivo stimulation of leukocytes, and analyze microbicidal activity. RESULTS: beta-glucan was barely detectable in serum of volunteers at all time-points. Furthermore, neither cytokine production nor microbicidal activity of leukocytes were affected by orally administered beta-glucan. CONCLUSION: The present study does not support the use of oral beta-glucan to enhance innate immune responses in humans. TRIAL REGISTRATION: ClinicalTrials.gov NCT01727895.

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

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

Contains fulltext : 154433.pdf (Publisher’s version ) (Closed access) A large variety of fungi are present in the environment, among which a proportion colonizes the human body, usually without causing any harm. However, depending on the host immune status, commensals can become opportunistic pathogens that induce diseases ranging from superficial non-harmful infection to life-threatening systemic disease. The interplay between the host and the fungal commensal flora is being orchestrated by an efficient recognition of the microorganisms, which in turn ensures a proper balance between tolerance of the normal fungal flora and induction of immune defense mechanisms when invasion occurs. Pattern recognition receptors (PRRs) play a significant role in maintaining this balance due to their capacity to sense fungi and induce host responses such as the induction of proinflammatory cytokines involved in the activation of innate and adaptive immune responses. In the present review, we will discuss the most recent findings regarding the recognition of Candida albicans and Aspergillus fumigatus and the different types of immune cells that play a role in antifungal host defense.

Objective— Although the role of monocytes in the pathogenesis of atherosclerosis is well established, the persistent vascular inflammation remains largely unexplained. Recently, our group reported that stimulation of monocytes with various microbial products can induce a long-lasting proinflammatory phenotype via epigenetic reprogramming, a process termed trained immunity. We now hypothesize that oxidized low-density lipoprotein (oxLDL) also induces a long-lasting proinflammatory phenotype in monocytes, which accelerates atherosclerosis by proinflammatory cytokine production and foam cell formation. Approach and Results— Isolated human monocytes were exposed for 24 hours to medium or oxLDL. After washing and resting for 6 days, the cells were exposed to toll-like receptor 2 and 4 agonists. Pre-exposure to oxLDL increased mRNA expression and protein formation on toll-like receptor 2 and 4 stimulation of several proatherogenic proteins, including interleukin-6, interleukin-18, interleukin-8, tumor necrosis factor-α, monocyte chemoattractant protein 1, and matrix metalloproteinase 2 and 9. In addition, foam cell formation was enhanced after oxLDL exposure, which was associated with an upregulation of scavenger receptors CD36 and scavenger receptor-A and downregulation of ATP-binding cassette transporters, ABCA1 and ABCG1. Chromatin immunoprecipitation performed 6 days after oxLDL stimulation demonstrated increased trimethylation of lysine 4 at histone 3 in promoter regions of tnfα , il-6 , il-18 , mcp-1 , mmp2 , mmp9 , cd36 , and sr-a. Finally, pretreatment of the monocytes with the histone methyltransferase inhibitor methylthioadenosine completely prevented the oxLDL-induced long-lasting proinflammatory phenotype. Conclusions— Brief exposure of monocytes to a low concentration of oxLDL induces a long-lasting proatherogenic macrophage phenotype via epigenetic histone modifications, characterized by increased proinflammatory cytokine production and foam cell formation.

International audience; Although Candida glabrata is an important pathogenic Candida species, relatively little is known about its innate immune recognition. Here, we explore the potential role of Dectin-2 for host defense against C. glabrata. Dectin-2-deficient (Dectin-2(-/-)) mice were found to be more susceptible to C. glabrata infections, showing a defective fungal clearance in kidneys but not in the liver. The increased susceptibility to infection was accompanied by lower production of T helper 1 (Th1) and Th17-derived cytokines by splenocytes of Dectin-2(-/-) mice, while macrophage-derived cytokines were less affected. These defects were associated with a moderate yet significant decrease in phagocytosis of the fungus by the Dectin-2(-/-) macrophages and neutrophils. Neutrophils of Dectin-2(-/-) mice also displayed lower production of reactive oxygen species (ROS) upon challenge with opsonized C. glabrata or C. albicans. This study suggests that Dectin-2 is important in host defense against C. glabrata and provides new insights into the host defense mechanisms against this important fungal pathogen.

Item does not contain fulltext 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.

The opportunistic fungal pathogen Candida glabrata is a frequent cause of candidiasis, causing infections ranging from superficial to life-threatening disseminated disease. The inherent tolerance of C. glabrata to azole drugs makes this pathogen a serious clinical threat. To identify novel genes implicated in antifungal drug tolerance, we have constructed a large-scale C. glabrata deletion library consisting of 619 unique, individually bar-coded mutant strains, each lacking one specific gene, all together representing almost 12% of the genome. Functional analysis of this library in a series of phenotypic and fitness assays identified numerous genes required for growth of C. glabrata under normal or specific stress conditions, as well as a number of novel genes involved in tolerance to clinically important antifungal drugs such as azoles and echinocandins. We identified 38 deletion strains displaying strongly increased susceptibility to caspofungin, 28 of which encoding proteins that have not previously been linked to echinocandin tolerance. Our results demonstrate the potential of the C. glabrata mutant collection as a valuable resource in functional genomics studies of this important fungal pathogen of humans, and to facilitate the identification of putative novel antifungal drug target and virulence genes., Author Summary Clinical infections by the yeast-like pathogen Candida glabrata have been ever-increasing over the past years. Importantly, C. glabrata is one of the most prevalent causes of drug-refractory fungal infections in humans. We have generated a novel large-scale collection encompassing 619 bar-coded C. glabrata mutants, each lacking a single gene. Extensive profiling of phenotypes reveals a number of novel genes implicated in tolerance to antifungal drugs that interfere with proper cell wall function, as well as genes affecting fitness of C. glabrata both during normal growth and under environmental stress. This fungal deletion collection will be a valuable resource for the community to study mechanisms of virulence and antifungal drug tolerance in C. glabrata, which is particularly relevant in view of the increasing prevalence of infections caused by this important human fungal pathogen.

The immune system is essential to maintain homeostasis with resident microbial populations, ensuring that the symbiotic host–microbial relationship is maintained. In parallel, commensal microbes significantly shape mammalian immunity at the host mucosal surface, as well as systemically. Candida albicans is an opportunistic pathogen that lives as a commensal on skin and mucosa of healthy individuals. Little is known about its capacity to modulate responses toward other microorganisms, such as colonizing bacteria (e.g., intestinal microorganisms). The aim of this study was to assess the cytokine production of PBMCs induced by commensal bacteria when these cells were primed by C. albicans. We show that C. albicans and β-1,3-glucan induce priming of human primary mononuclear cells and this leads to enhanced cytokine production upon in vitro stimulation with TLR ligands and bacterial commensals. This priming requires the β-1,3-glucan receptor dectin-1 and the noncanonical Raf-1 pathway. In addition, although purified mannans cannot solely mediate the priming, the presence of mannosyl residues in the cell wall of C. albicans is nevertheless required. In conclusion, C. albicans is able to modify cytokine responses to TLR ligands and colonizing bacteria, which is likely to impact the inflammatory reaction during mucosal diseases.

The pathogenicity of Candida glabrata to patients remains poorly understood for lack of convenient animal models to screen large numbers of mutants for altered virulence. In this study, we explore the minihost model Drosophila melanogaster from the dual perspective of host and pathogen. As in vertebrates, wild-type flies contain C. glabrata systemic infections yet are unable to kill the injected yeasts. As for other fungal infections in Drosophila, the Toll pathway restrains C. glabrata proliferation. Persistent C. glabrata yeasts in wild-type flies do not appear to be able to take shelter in hemocytes from the action of the Toll pathway, the effectors of which remain to be identified. Toll pathway mutant flies succumb to injected C. glabrata. In this immunosuppressed background, cellular defenses provide a residual level of protection. Although both the Gram-negative binding protein 3 pattern recognition receptor and the Persephone protease-dependent detection pathway are required for Toll pathway activation by C. glabrata, only GNBP3, and not psh mutants, are susceptible to the infection. Both Candida albicans and C. glabrata are restrained by the Toll pathway, yet the comparative study of phenoloxidase activation reveals a differential activity of the Toll pathway against these two fungal pathogens. Finally, we establish that the high-osmolarity glycerol pathway and yapsins are required for virulence of C. glabrata in this model. Unexpectedly, yapsins do not appear to be required to counteract the cellular immune response but are needed for the colonization of the wild-type host.

Item does not contain fulltext NK cells have direct activity against fungal pathogens. Using an unbiased systematic approach, Li et al. (2013) find that NKp30 is a major NK cell receptor responsible for fungal recognition. Moreover, diminished NKp30 expression is associated with reduced antifungal activity in NK cells isolated from HIV-infected persons.

Contains fulltext : 96477.pdf (Publisher’s version ) (Closed access) Immune responses in vertebrates are classically divided into innate and adaptive, with only the latter being able to build up immunological memory. However, although lacking adaptive immune responses, plants and invertebrates are protected against reinfection with pathogens, and invertebrates even display transplant rejection. In mammals, past "forgotten" studies demonstrate cross-protection between infections independently of T and B cells, and more recently memory properties for NK cells and macrophages, prototypical cells of innate immunity, have been described. We now posit that mammalian innate immunity also exhibits an immunological memory of past insults, for which we propose the term "trained immunity." Understanding trained immunity will revolutionize our view of host defense and immunological memory, and could lead to defining a new class of vaccines and immunotherapies.

Item does not contain fulltext To gain an in-depth grasp of infectious processes one has to know the specific interactions between the virulence factors of the pathogen and the host defense mechanisms. A thorough understanding is crucial for identifying potential new drug targets and designing drugs against which the pathogens might not develop resistance easily. Model organisms are a useful tool for this endeavor, thanks to the power of their genetics. Drosophila melanogaster is widely used to study host-pathogen interactions. Its basal immune response is well understood and is briefly reviewed here. Considerations relevant to choosing an adequate infection model are discussed. This review then focuses mainly on infections with two categories of pathogens, the well-studied Gram-negative bacterium Pseudomonas aeruginosa and infections by fungi of medical interest. These examples provide an overview over the current knowledge on Drosophila-pathogen interactions and illustrate the approaches that can be used to study those interactions. We also discuss the usefulness and limits of Drosophila infection models for studying specific host-pathogen interactions and high-throughput drug screening. 01 juni 2011

International audience; Gram-negative binding protein 3 (GNBP3), a pattern recognition receptor that circulates in the hemolymph of Drosophila, is responsible for sensing fungal infection and triggering Toll pathway activation. Here, we report that GNBP3 N-terminal domain binds to fungi upon identifying long chains of beta-1,3-glucans in the fungal cell wall as a major ligand. Interestingly, this domain fails to interact strongly with short oligosaccharides. The crystal structure of GNBP3-Nter reveals an immunoglobulin-like fold in which the glucan binding site is masked by a loop that is highly conserved among glucan-binding proteins identified in several insect orders. Structure-based mutagenesis experiments reveal an essential role for this occluding loop in discriminating between short and long polysaccharides. The displacement of the occluding loop is necessary for binding and could explain the specificity of the interaction with long chain structured polysaccharides. This represents a novel mechanism for beta-glucan recognition.

International audience; Sex-based differences influence incidence and outcome of infectious disease. Women have a significantly greater incidence of urinary tract infection (UTI) than men, yet, conversely, male UTI is more persistent with greater associated morbidity. Mechanisms underlying these sex-based differences are unknown, in part due to a lack of experimental models. We optimized a model to transurethrally infect male mice and directly compared UTI in both sexes. Although both sexes were initially equally colonized by uropathogenic E. coli, only male and testosterone-treated female mice remained chronically infected for up to 4 weeks. Female mice had more robust innate responses, including higher IL-17 expression, and increased γδ T cells and group 3 innate lymphoid cells in the bladder following infection. Accordingly, neutralizing IL-17 abolished resolution in female mice, identifying a cytokine pathway necessary for bacterial clearance. Our findings support the concept that sex-based responses to UTI contribute to impaired innate immunity in males and provide a rationale for non-antibiotic-based immune targeting to improve the response to UTI.

International audience; Despite the fact that Candida albicans is an important human fungal pathogen and Dectin-2 is a major pattern recognition receptor for fungi, our knowledge regarding the role of Dectin-2 for the host defense against disseminated candidiasis is limited. Dectin-2 deficient (Dectin-2(-/-)) mice were more susceptible to systemic candidiasis, and the susceptibility was mirrored by an elevated fungal load in the kidneys that correlated with the presence of large inflammatory foci. Phagocytosis of Candida by the macrophages lacking the Dectin-2 receptor was moderately decreased, while production of most of the macrophage-derived cytokines from Dectin-2(-/-) mice with systemic candidiasis was decreased. No striking differences among several Candida mutants defective in mannans could be detected between naïve wild-type and Dectin-2(-/-) mice, apart from the β-mannan-deficient bmt1Δ/bmt2Δ/bmt5Δ triple mutant, suggesting that β-mannan may partially mask α-mannan detection, which is the major fungal structure recognized by Dectin-2. Deciphering the mechanisms responsible for host defense against the majority of C. albicans strains represents an important step in understanding the pathophysiology of systemic candidiasis, which might lead to the development of novel immunotherapeutic strategies.