Fungal mediated innate immune memory, what have we learned?

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 ß-glucans trigger in the context of infectious diseases and septicaemia, inflammatory diseases and cancer.

Innate immune memory through TLR2 and NOD2 contributes to the control of Leptospira interrogans infection.

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.

Innate immune memory in mammals.

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.

Studying fungal pathogens of humans and fungal infections: fungal diversity and diversity of approaches.

Seminal work by Louis Pasteur revealed the contribution of fungi-yeasts and microsporidia to agroindustry 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 progress 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.

Impaired phagocytosis directs human monocyte activation in response to fungal derived ß-glucan particles.

Recognition of the fungal cell wall carbohydrate ß-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 ß-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)-ß-glucan induces a strong inflammatory signature. Sustained monocyte activation, induced by fungal ß-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.

Fungal Chitin Induces Trained Immunity in Human Monocytes during Cross-talk of the Host with Saccharomyces cerevisiae.

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.

Cell Wall Changes in Amphotericin B-Resistant Strains from Candida tropicalis and Relationship with the Immune Responses Elicited by the Host.

We have morphologically characterizedCandida tropicalisisolates resistant to amphotericin B (AmB). These isolates present an enlarged cell wall compared to isolates of regular susceptibility. This correlated with higher levels of ß-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 ofGalleria mellonellalarvae, 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.

Autophagy controls BCG-induced trained immunity and the response to intravesical BCG therapy for bladder cancer.

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 ß-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.

Systematic phenotyping of a large-scale Candida glabrata deletion collection reveals novel antifungal tolerance genes.

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.

Differential role of NK cells against Candida albicans infection in immunocompetent or immunocompromised mice.

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.

Oxidized low-density lipoprotein induces long-term proinflammatory cytokine production and foam cell formation via epigenetic reprogramming of monocytes.

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.

The Drosophila Toll pathway controls but does not clear Candida glabrata infections.

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.

Candida albicans primes TLR cytokine responses through a Dectin-1/Raf-1-mediated pathway.

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 ß-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 ß-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.

Bacille Calmette-Guerin 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-1ß, 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.

Role of Dectin-2 for host defense against systemic infection with Candida glabrata.

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.

BCG-induced trained immunity in NK cells: Role for non-specific protection to infection.

Adaptive features of innate immunity, also termed 'trained immunity', have recently been shown to characterize monocytes of BCG vaccinated healthy volunteers. Trained immunity leads to increased cytokine production in response to non-related pathogens via epigenetic reprogramming of monocytes. Recently, memory-like properties were also observed in NK cells during viral infections, but it is unknown if memory properties of NK cells contribute to trained immunity due to BCG vaccination. BCG vaccination of healthy volunteers increased proinflammatory cytokine production following ex vivo stimulation of NK cells with mycobacteria and other unrelated pathogens up until at least three months after vaccination. In addition, in a murine model of disseminated candidiasis, BCG vaccination led to an increased survival in SCID mice, which was partially dependent on NK cells. These findings suggest that NK cells may contribute to the non-specific (heterologous) beneficial effects of BCG vaccination.

The Drosophila PRR GNBP3 assembles effector complexes involved in antifungal defenses independently of its Toll-pathway activation function.

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.

In vitro induction of trained immunity in adherent human monocytes.

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 ß-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).

The N-terminal domain of Drosophila Gram-negative binding protein 3 (GNBP3) defines a novel family of fungal pattern recognition receptors.

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.