Introduction

The role of commensal fungi (referred to as the mycobiome, reviewed in 1-3 in educating the immune system in health and disease has been increasingly appreciated (reviewed in 4). Such a long‐established commensalism indicates that commensal fungi are an integral part of the eukaryotic host. To date, the human fungal community has been shown to be comprised of Candida and Malassezia on mucosal surfaces, skin, and oral cavity, Pneumocystis jirovecii, a lung commensal, and consensus members of the basal human salivary mycobiome including Candida/Pichia, Cladosporium/Davidiella, Alternaria/Lewia, Aspergillus/Emericella/Eurotium, Fusarium/Gibberella, Cryptococcus/Filobasidiella, and Aureobasidium 1, 5-7. Given that different body sites harbor specific fungal populations, unique mycobiome patterns are associated with various diseases. By interfacing with the microbiome, as well as with its host, the mycobiome plays an important role in health and disease, most likely by regulating immune reactivity at mucosal surfaces where an integration of physical and metabolic factors is of fundamental importance (reviewed in 1, 8, 9). Indeed, owing to their capacity to activate immune responses, commensal fungi have been shown to influence immune reactions at local 10-12 and distal sites 13, 14, such as in colitis and allergy in the airways. Conversely, owing to microbial dysbiosis or defects in the innate or adaptive immune systems, commensal fungi may shift from commensalism to parasitism, and they may cause severe fungal infections and diseases, including those associated with gut inflammation 15 or chemotherapy‐related damage 16.

Fungal infections in the metagenomic era Infections by opportunistic fungi have long been considered as resulting from a nonspecific pathogenic mechanism that renders a ‘‘weakened’’ host more susceptible to infectious noxae. However, a better understanding of the importance of the mycobiota in the host's immune and metabolic activities 4 has revealed an unexpected level of intricacy in the interaction between fungi and their hosts. Compelling evidence points to a significant role for the mycobiome in immune regulation, inflammation, and allergy 1 and in shaping the mutually related patterns of disease resistance (i.e. the ability to eradicate the microbes, irrespective of any danger associated with overreacting responses) and disease tolerance (i.e. the ability to reduce the detrimental effects of the microbes on host fitness, regardless of its eradication) 17. Fungal commensals coexist in a complex milieu of bacteria within the human body. Therefore, it is not surprising that bacterial–fungal interactions are highly dynamic and relevant to human diseases (reviewed in 18). Indeed, the complex interactions, as revealed by metagenomics, between fungal and bacterial commensals in the gut and skin — either directly or through the participation of the host immune system — all impact on the pathophysiology of a number of inflammatory disease that, in turn, lead to secondary fungal infections 19. The occurrence of intractable candidiasis in association with antibiotic‐induced dysbiosis and its exacerbation by antibiotics have long been known 20. In patients with enteric infections, the high level of fungal colonization in the gut has been linked to a decreased colonization resistance by the gut bifidobacteria and to the subsequent presence of Escherichia coli and nonfermenting Gram‐negative bacteria 21. In the skin of patients with chronic mucocutaneous candidiasis and hyper‐IgE syndrome, the increased risk for fungal diseases has been attributed to changes in the skin microbiome, which in turn suppress the cytokine response against fungal and microbial pathogens 22. Compelling evidence indicates that a variety of superficial and invasive fungal infections are caused by specific single‐gene inborn errors of immunity 23. Thus, strategically targeting microbial dysbiosis through the rational deployment of antibiotics, probiotics, prebiotics, and metabiotics (the last designating structural components of probiotic microorganisms and/or their metabolites and/or signaling molecules with a defined chemical structure 24), could provide the basis to develop new antifungal therapies in support of strategies that target the immune deficiencies.

Immunity and tolerance to fungi Immunity to fungi is a dynamic interplay between multiple components of the immune system. A regulated immune response that recognizes and controls fungal growth is required to limit tissue damage and restore immune homeostasis while preserving the balance of the local microbiota 25. Resistance is meant to reduce, if not eradicate, the pathogen burden, through innate and adaptive immune mechanisms. In contrast, tolerance aims at mitigating the substantial cost imposed by resistance on the host's fitness. Among the plethora of tolerance mechanisms, in the case of chronic fungal infections dominated by nonresolving, persisting inflammation, IL‐10 produced by regulatory T (Treg) cells may act as a homeostatic host‐driven response that, by controlling the quality and magnitude of innate and adaptive responses, keeps inflammation under control. Treg cells with anti‐inflammatory activity have been described in fungal infections of both mice and humans (reviewed in 4). It is not surprising that many of the strategies that mammalian hosts have developed to coexist peacefully with their microbiota can be hijacked or manipulated by commensal fungi, to ensure their own survival. These hijacking mechanisms include escape from immune recognition by disparate stealth mechanisms 26, 27 as well as regulation of inflammation through the production of a number of mediators, such as resolvins, lipoxins, and prostaglandins 28 which limit tissue damage. Manipulation of the tolerogenic network of the host by the mycobiome is one additional mechanism to ensure fungal survival (reviewed in 4). In the case of Candida albicans, besides commensalism, the induction of tolerance by the fungus also results in amelioration of gut inflammation through the induction of IL‐10‐producing Treg cells (Fig. 1) 10. In healthy skin, the fungus Malassezia has been shown to downregulate inflammation via TGF‐β and IL‐10, thus establishing itself as a commensal 29. Thus, similar to gut symbionts, the mycobiome may contribute to the balance of inflammation and tolerance at local and distal sites (Fig. 1). Indeed, a healthy gut microbiome contains classes of microbes that enhance metabolism, resilience to infection, and inflammation, resistance to cancer or autoimmunity, and other functions via secretion of factors that modulate intestinal permeability and motility, epithelial cell function, innate, and adaptive immunity 30. Examples of bacterial taxa that have been shown to affect immune homeostasis include bacteroides, bifidobacterium, clostridia, segmented filamentous bacteria (SFB), and lactobacilli (Fig. 1). Figure 1 Open in figure viewer PowerPoint Examples of how microbiota shape host mucosal and systemic immunity. Lactobacilli in the gut have been shown to activate ILC3s to produce IL‐22 and thereby drive mucosal protection via AMP production. B. fragilis, Clostridia, and C. albicans have been shown to induce Treg cells, which in turn promote immune homeostasis locally and tolerance systemically. C. albicans, segmented filamentous bacteria and B. fragilis have also been shown to promote Th17 and Th1 cells, which drive inflammation locally and may contribute to autoimmunity. ILC3: Innate lymphoid cell 3; AMP: antimicrobial peptides; Treg cells: regulatory T cells. However, considering that inflammation may promote fungal colonization 31 and fungal colonization promotes further inflammation 32, these effects may create a vicious circle by which the mycobiome, by triggering inflammation, Th17 responses and molecular mimicry, is also associated with several diseases of the gastrointestinal tract 11, 33 as well as with tumor progression 34. In a number of cutaneous diseases, such as atopic dermatitis and psoriasis, an altered skin barrier may enhance fungal sensitization and infectivity, thus promoting disease exacerbation 35. Accordingly, Candida‐specific Th9 cells, which have both autocrine and paracrine proinflammatory capacity, have been found in abundance in psoriatic lesions 12 and basic and clinical evidence support a role for Malassezia yeasts in psoriasis exacerbations and skin carcinogenesis 36.

Metabolic regulation of tolerance at the host–mycobiome interface via tryptophan A central role in immune tolerance to commensal fungi is played by tryptophan (trp), an essential, diet‐derived amino acid that is the rarest among those humans who consume from external sources. In addition to protein synthesis, trp is used in a variety of processes, including the production of biogenic amines such as serotonin, melatonin, tryptamine, and trp degradation yields byproducts collectively called kynurenines. Most of the free trp in mammals is oxidized along the kynurenine pathway 37. The first and rate‐limiting step in this series of reactions is catalyzed by three enzymes, indoleamine 2,3‐dioxygenase 1 (IDO1), tryptophan 2,3‐dioxygenase (TDO or TDO2) and a relatively newly identified enzyme, IDO2 38, 39. IDO1 has been conserved through the past 600 million years of evolution 40. Initially recognized via its role in infection because of its biostatic activity (‘‘tryptophan starvation’’ of intracellular parasites) 41, IDO1, which is expressed by a variety of immune and nonimmune cells 42, acts locally to modulate trp levels in a range of settings, including cancer, chronic infections, allergy, and autoimmunity 43. Consistent with the notion that increased amino acid catabolism inhibits immunity and promotes tolerance in a variety of inflammatory settings 43, 44, IDO1 is now widely recognized as a suppressor of inflammation and a regulator of mammalian immune homeostasis (reviewed in 40). Microbial stimuli, including fungi, induce IDO1 to dampen host immunity and promote pathogen persistence (reviewed in 40). Thus, in certain infectious settings, trp starvation may be a host strategy for restraining infection by natural trp auxotrophic microbes, whereas IDO1 may be utilized as an evasion mechanism for microbes that establish commensalism or chronic infection 40. However, the recent finding that Mycobacterium tuberculosis can synthesize trp under stress conditions, thus counteracting the starvation‐driven antibacterial mechanism, underscores the intricate behavior of mammals and microbes surrounding trp 45. In their capacity as inducers of tolerogenic dendritic cells (DCs) 42, 46, IDO1 and kynurenines reveal an unexpected potential in the control of inflammation, allergy, and Th‐driven inflammation to ubiquitous or commensal fungi through the activation of Treg cells 4, 47, 48 (Fig. 2). Although both Th1 and Th17 cells are instrumental in antifungal immunity via the production of cytokines that activate innate antifungal defenses 23, 49-51, downregulation of adaptive immunity is required to prevent pathogenic chronic inflammation 4. The Th17 pathway, in particular, which inhibits trp catabolism, may favor pathology, providing some evidence accommodating the apparently paradoxical association of chronic inflammation with opportunistic fungal diseases 52. In contrast, because IFN‐γ is a potent IDO1 activator, the IFN‐γ/IDO1 axis may accommodate fungal persistence in a host environment rich in IFN‐γ. As already mentioned, fungi can interfere with the host tolerogenic program dictated by IDO1 activity 4, likely contributing to either commensalism and/or fungal persistence. Of great interest, the occurrence of IDOs in fungi 53 points to the ability of commensal and noncommensal fungi to exert an unanticipated immunoregulatory function in a host‐autonomous fashion, and suggests that trp metabolites may participate in the interkingdom dialogue. Figure 2 Open in figure viewer PowerPoint Proposed model of the role of IDO1 and AhR at mucosal sites. The tryptophan metabolism pathway is exploited by the mammalian host and by commensals to activate antifungal resistance and immune tolerance. (A) In the gut, the indole derivative IAld, which is generated through conversion from dietary tryptophan by commensal lactobacilli, acts as endogenous ligand for the aryl hydrocarbon receptor (AhR), and thereby contributes to IL‐22 production by innate lymphoid cells 3 (ILC3). IL‐22 targets epithelial cells, leading to activation of signal transducer and activator of transcription 3 (STAT3) and, together with IL‐17A, to the production of antimicrobial peptides (AMP). (B) Fungus‐induced activation of tryptophan catabolism by indoleamine 2,3‐dioxygenase 1 (IDO1) expressed by tolerogenic dendritic cells and epithelial cells leads to the production of immunologically active L‐kynurenines (L‐Kyn), which induce the transcription of FOXP3 and suppress the transcription of RORγ‐t, thus promoting immune tolerance via Treg cells. Thus, consistent with their high level expression at mucosal sites, IDO1 and AhR act as dominant key players in antifungal immunity, regulating both the size and metabolic activity of gut microbiota and the quality of the adaptive immune response. FOXP3: forkhead box P3; RORgT: RAR‐related orphan receptor gamma; Treg cells: regulatory T cells.

Microbiota regulation of resistance to fungi through the AhR/IL‐22 axis The AhR is a cytosolic, ligand‐operated transcription factor that is involved in many biological processes, including development, cellular differentiation and proliferation, xenobiotic metabolism, and the immune response (reviewed in 54). The evolutionary conservation of this transcription factor 55 along with its widespread expression in the vertebrate immune system highlight its important physiological functions. Although the AhR was initially proposed to affect Treg‐ and Th17‐cell development 56, IL‐22 production by innate lymphoid cells (ILCs) is even more specifically dependent on AhR activation. Indeed, AhR is crucial for the maintenance and function of group 3 innate lymphoid cells (ILC3), which produce IL‐22 in the gut 57, 58 and skin 59. In addition to its main function in tissue repair and host defense at the epithelial barrier 60, IL‐22 has recently been found to affect the homeostatic balance between immunity and microbiota in the gut, by regulating microbial composition 61-63. For instance, IL‐22 negatively regulates Th17 cells in the gut by inhibiting the expansion of commensal SFB, known to promote Th17‐cell activation 64. IL‐22 has also been shown to be exploited by pathogens such as Salmonella enterica Typhimurium to suppress the growth of indigenous competitors, such as Enterobacteriaceae 61. Thus IL‐22 has a unique challenge at the host–microbiota interface, being positioned to exert beneficial tolerance to commensals by controlling both the tolerant state of the host and its microbial composition. A steady state dialogue between the host and its microbiota is therefore required for adjusting intestinal immune homeostasis to the microbiota status. Decoding this dialogue may guide the development of rational therapies for dysbiosis‐dependent inflammatory diseases and infections. Consistent with the highly conserved nature of the AhR signaling pathway, a variety of endogenous ligands have been shown to interact with the receptor 65. Among the trp metabolites generated by mammalian IDO1 in the kynurenine pathway, kynurenic acid, xanthurenic acid, cinnabarinic acid and l‐kynurenine, an amino acid itself, have all been shown to act as direct ligands of AhR, with the capacity to stimulate AhR‐dependent gene expression at physiologically attainable concentrations 66-70. In addition, a variety of indole derivatives, which are generated through the catabolism of dietary trp by tryptophanase in commensal intestinal bacteria 71, act as endogenous ligands for AhR 72. Evidence has shown that AhR is also involved in the (patho)physiology of skin, including regulation of skin pigmentation, photocarcinogenesis, and skin inflammation (73, and reviewed in 74). Of interest, the ability of Malassezia‐derived indoles to activate AhR correlates with local immunoregulation in the skin through inhibition of DC responses to Toll‐like receptor ligands 75, 76. In general, metabolomics, a field of study in systems biology which involves the identification and quantification of metabolites present in a biological system 77, have revealed that gut bacteria impact the host's metabolism and immunity through a variety of chemically different metabolites, including amino acid metabolites 71. In particular, a dietary lack of trp has been shown to impair intestinal immunity — associated with a deficiency in angiotensin I converting enzyme 2 — and to alter the gut microbial community 78, suggesting that mucosal homeostasis is a multifactorial phenomenon of which trp metabolism is an important regulatory component. We have recently identified a microbial trp metabolic pathway that has evolved to preserve immune physiology at mucosal surfaces while inducing anticandidal resistance via the recruitment of ILC3 63. Metabolomics have revealed that bioactive indoles of microbial origin with AhR agonist activity are present in mice with candidiasis, particularly under conditions of unrestricted trp availability, and that these microbially produced indoles can successfully control C. albicans growth and infectivity at mucosal surfaces 79. Although indoles could directly impair the virulence of C. albicans 80, the IL‐22–upregulated mucosal response by indoles allows for the survival of mixed microbial communities, providing colonization resistance to C. albicans. IL‐22 has demonstrated its pivotal role in fungal infections 81. Not only are naturally occurring IL‐22+ cells highly enriched at mucosal sites, where continuous exposure to fungi occurs, but C. albicans‐specific IL‐22+CD4+ memory T cells are also present in human peripheral cells 82 and are low in number in patients with chronic mucocutaneous candidiasis 83. In recurrent vulvovaginal candidiasis, functional genetic variants in IL‐22 have been found to be associated with heightened resistance to infection, and they correlated with increased local expression of IL‐22 84. Thus, IL‐22+ cells, employing ancient effector mechanisms of immunity, may represent a primitive mechanism of resistance to fungi, driven by the microbiota, under conditions of limited inflammation. The fact that IL‐22 production is driven by commensals 63, 85 may provide mechanistic insights into how antibiotic‐related dysbiosis and cancer therapy may predispose to candidiasis.

Lactobacilli produce indole‐3‐aldehyde (IAld) upon adaptation to trp All lactobacilli are phylogenetically closely related by their small genomes and common metabolic pathways for sugar fermentation and lactic acid production (reviewed in 86). The mammalian/human stomach favors colonization by acid‐resistant lactobacilli and is a normal habitat of various fungal taxa in rodents and humans, where yeasts are associated with the mucin layer covering the secreting epithelial cells 87, 88. Within the stomach, lactobacilli are known to promote resistance to colonization by fungi 14. Thus a functional interplay between C. albicans and lactobacilli does occur, a disturbance of which may affect microbial/C. albicans symbiosis 1. Although the mechanisms of protection against C. albicans in the stomach may include the production of several mediators and direct microbial antagonism by lactobacilli, we have recently shown a metabolic pathway of IL‐22 production via AhR stimulation by lactobacilli that is contingent upon the trp metabolic environment, which involves microbial nutritional adaptation, and is modulated by diet 63. Both Lactobacillus johnsonii 89 and Lactobacillus reuteri 90 are transcriptionally very active in the stomach, where distinct nutritional adaptations provide niche differentiation that allows cohabitation by the two strains in murine stomachs 91. Vancomycin‐resistant but ampicillin‐sensitive L. reuteri was found to expand under conditions of unrestricted availability of trp, such as in settings of IDO1 deficiency or on supplemental trp feeding 91. These findings indicate that the IDO1‐mediated restriction of local trp availability could affect the qualitative and quantitative nature of lactobacilli populations in vivo. Most importantly, the increased availability of intestinal trp not only selectively expanded specific lactobacilli populations, but could also promote alternate pathways of trp degradation by the lactobacilli population being expanded 63. Indeed, metabolic profiling revealed that, of the different putative metabolites, indole‐3‐aldehyde (IAld) was abundantly produced by L. reuteri in the gut in the presence of trp and was capable of activating ILC3s to produce IL‐22 via AhR (Fig. 2). A mutant L. reuteri strain, no longer able to produce IAld in the presence of trp, lost its capacity to induce IL‐22. Thus, in addition to contributing to immune tolerance (reviewed in 86), lactobacilli appear to stimulate gut ILC3s to produce IL‐22, an activity which occurs independently of gut Th17 cells (Fig. 2) 63. As a matter of fact, consistent with the ability of ILC3s to inhibit the expansion of SFB 64, SFB were not expanded under conditions of trp availability 63. The lactobacilli/IAld/AhR/IL‐22 axis was also shown to be involved in murine vaginal candidiasis, in which L. acidophilus, a constituent of the human vaginal microbiota which prevents vaginal candidiasis 92, was abundantly expanded in the presence of trp, and produced IAld that induced IL‐22 63. The very high diversity of Lactobacillus spp. in the human vagina may thus offer a plausible explanation as to why healthy women remain entirely asymptomatic despite being colonized by Candida spp. The ability of some lactobacilli to affect host antifungal reactivity may offer a possible explanation not only for the susceptibility to candidiasis in certain clinical settings but also for the strain‐specific effects observed after the administration of probiotic lactobacilli. Commensal lactobacilli are greatly reduced by stress 88, 93 and in the neonatal period 94, as well as in bacterial vaginitis, clinical conditions in which the empirical use of lactobacilli as probiotics to prevent infection has long been recommended yet never until now been mechanistically explained. Moreover, the species‐specific effects of lactobacilli on C. albicans colonization may offer a plausible explanation for the organ tropism of mucosal candidiasis, for the susceptibility to infection in specific clinical settings, as well as for the variable and inconsistent effects of probiotic administration in human candidiasis 20, 95. We also anticipate that responsiveness to the different probiotic lactobacilli species is not only determined by the characteristics of the consumed strains but also by microbial adaptation, and the metabolic and nutritional status of the host. Considering that probiotic L. reuteri of human origin also produces IAld when growing on trp 63, these findings imply that trp supplementation in the context of antibiotic coverage may optimize antifungal and probiotic therapy, and, likely, immune physiology.