2.1 Mycobiota in health

Whereas the impact of the bacterial microbiota on host physiology is relatively well described, much less is known about the interactions between the mycobiota and the host and the ensuing effects on the host. Foster et al. have argued that hosts are under strong selective pressure to control and mold their microbiota towards the retention of beneficial species (Foster, Schluter, Coyte, & Rakoff‐Nahoum, 2017). By this reasoning, one would predict that the mycobiota has also undergone selection by the host to provide advantages to the latter. Accordingly, a limited number of studies—to be detailed in the following sections—have demonstrated a role for symbiont fungi in mediating tissue homeostasis.

2.1.1 Symbiont fungi calibrate host immunological responses The ability of symbiont bacteria and viruses to calibrate the baseline immunological responsiveness of the host is well‐delineated, and we refer the reader to several excellent reviews for more information on the specific mechanisms involved (Cho & Blaser, 2012; Chung & Kasper, 2010; Jarchum & Pamer, 2011; Virgin, 2014). Furthermore, recent work by the Human Functional Genome Project (Netea et al., 2016) has identified associations between certain symbiont bacterial species and anti‐microbial cytokine responses of peripheral blood mononuclear cells (PBMCs) in healthy people, in support of a role for the microbiota in modifying systemic immune responses in humans (Schirmer et al., 2016). Evidence for an analogous immunomodulatory role for symbiont fungi (discussed in Hall & Noverr (2017), Rizzetto, De Filippo, & Cavalieri (2014)) has been limited largely to in vitro studies (Rizzetto et al., 2010, 2016) and in vivo infection or disease models in rodents (Iliev et al., 2012; Murdock et al., 2011; Wheeler et al., 2016). However, a recent study by Jiang et al. (2017) indicates that the mycobiota in and of itself can indeed substitute for symbiont bacteria and viruses in modulating homeostatic immunological functions both systemically and in mucosal tissue in mice. Antibiotics‐treated mice developed more fulminant dextran sodium sulfate (DSS)‐induced colitis and generated reduced levels of protective CD8+ T cells when infected with influenza A virus; oral inoculation of antibiotics‐treated mice with two common members of the gut mycobiota, Saccharomyces cerevisiae and Candida albicans, sufficed to mitigate both aforementioned defects (Jiang et al., 2017). The protective impact of the two fungal species could be recapitulated by the inoculation of mannans, an abundant component of fungal cell walls. The pathways underlying these broad effects of symbiont microbes on host physiology remain ambiguous, though several possibilities exist, some of which are discussed in the following sections. For instance, the phenotype and differentiation of the monocyte‐macrophage compartment is strongly influenced by the intestinal microbiota in mice (Chang, Hao, Offermanns, & Medzhitov, 2014; Ganal et al., 2012; Khosravi et al., 2014; Trompette et al., 2018), and human monocytes stimulated in vitro with chitin or β‐glucan, which represent other fungal cell wall determinants, evince sustained changes in their potential for secreting pro‐inflammatory cytokines (Quintin et al., 2012; Rizzetto et al., 2016). There, however, exists considerable variation in the capacity of fungi (and fungal cell wall components) to modulate immunocyte activity at the strain and species level (Rizzetto et al., 2010, 2013, 2016; Wagener, MacCallum, Brown, & Gow, 2017). It is therefore likely that diverse symbiotic fungal species can tune homeostatic immune responses via differential host sensing of fungal determinants such as mannans, with the net effect dependent on the composition of the mycobiota in each individual (Rizzetto et al., 2014).

2.1.2 Symbiont fungi promote development of peripheral lymphoid organs Germ‐free mice present with underdeveloped secondary lymphoid organs (SLOs), including a reduced volume and cellularity in both gut‐proximal and gut‐distal lymph nodes. This defect in the formation of lymphoid structures impairs the ability of the host to mount protective adaptive immune responses to infections (van de Pavert et al., 2014). Interestingly, Shi and coworkers identified symbiont fungi but not bacteria as primary drivers of the maturation of SLOs in mice (Z. Zhang et al., 2016). Development of SLOs requires a wave of migration of dendritic cells (DCs) expressing the retinol dehydrogenase enzyme, RALDH, from the intestines into peripheral lymph nodes in neonatal mice. The arrival of these RALDH+ DCs promotes a switch in the local expression of adhesion molecules from MAdCAM‐1 to PNAd, which in turn binds l‐selectin on naïve T cells and mediates their recruitment into and recirculation throughout the SLO network in the body. Treatment of mice with antifungals but not antibiotics diminishes the migration of RALDH+ DCs into SLOs, while inoculation of neonates with a single species of the murine indigenous mycobiota, Candida tropicalis, augmented the numbers of RALDH+ DCs in lymph nodes. However, the mechanisms by which symbiotic fungi promote the trafficking of RALDH+ DCs into SLOs remain unknown, nor is it apparent if the effect of C. tropicalis on SLO development is common to other members of the mycobiota.

2.1.3 Symbiont fungi promote T cell responses Mucosal and systemic fungal infections typically provoke CD4+ T cell (Th17 and Th1) responses characterized by the production of the cytokines interleukin (IL)‐17A and interferon (IFN)‐γ, respectively (Kashem et al., 2015). An assortment of evidence supports a role for the mycobiota in promoting similar T cell responses at steady‐state. Germ‐free or antibiotics‐treated mice colonized with C. albicans developed robust colonic Th17 responses without any overt signs of intestinal inflammation (Atarashi et al., 2015; Leonardi et al., 2018), and antibiotics‐treated mice gavaged with C. albicans generated memory and effector T cells in the gut (Xin et al., 2014). In healthy people, memory T cells specific for C. albicans and Aspergillus fumigatus can be isolated from the peripheral blood (Bacher et al., 2014). Circulating A. fumigatus‐specific T cells are primarily Th1 cells (Chaudhary, Staab, & Marr, 2010; Hebart et al., 2002; Jolink et al., 2013), although the predominant T cell subset induced by the fungus likely varies considerably among individuals due to substantial intra‐strain variation in inducing Th1 versus Th17 populations (Rizzetto et al., 2013). On the other hand, C. albicans‐specific T cells in human blood typically secrete the cytokines IL‐17A, IL‐22, and IFN‐γ but not IL‐10, thereby presenting a mixed Th1‐Th17 phenotype (Zielinski et al., 2012). Healthy individuals also harbor a circulating subset of Th9 cells that secrete the cytokine IL‐9, and the majority of Th9 cells are specific for C. albicans and express the skin‐homing receptor, cutaneous leucocyte‐associated antigen (CLA) (Schlapbach et al., 2014). In contrast, C. albicans‐specific Th17 cells typically express the gut‐homing integrin α 4 β 7 (Schlapbach et al., 2014). Thus, C. albicans (and by extension, other symbiotic fungi) can drive diverse and functional T cell responses in a tissue‐contextual fashion. Since microbiota‐induced T cell subsets have been described to mediate heterologous protection against other pathogens at the mucosal surface (Ivanov et al., 2009; Naik et al., 2015), we hypothesize that mycobiota‐driven T cell subsets and other immunocyte populations can similarly confer the host with cross‐protection against other infections. A. fumigatus and C. albicans are pathobionts—symbionts that are ordinarily benign but that can turn pathogenic under certain circumstances—and it remains possible that the Th1 and Th17 populations elicited by the two fungal species represents a pro‐inflammatory response to subclinical infections rather than a homeostatic response. Interestingly, S. cerevisiae, another common member of the mycobiota that neither forms hyphae nor causes infections in people, can also induce both Th1 and Th17 subsets from human CD4+ T cells in vitro (Rizzetto et al., 2010). S. cerevisiae yeasts promote Th1 differentiation while the sporulated form favors Th17 induction. The divergent effects of S. cerevisiae yeasts and spores and C. albicans on T cell responses were traced to a differential impact of fungal mannans on DCs that in turn prime the T cells (Rizzetto et al., 2010, 2012). Hence both nonpathogenic and pathobiont members of the mycobiota can drive robust T cell responses via a diversity of mechanisms. Since the host immune system has to remain tolerant towards the mycobiota to maintain homeostasis, the inflammatory T cell subsets generated in response to symbiotic fungi are likely countervailed by regulatory immunocyte populations. Indeed, Foxp3+ regulatory T (Treg) cells specific to C. albicans and A. fumigatus have been detected in the peripheral blood of healthy individuals, whereby they outnumber their fungus‐specific effector T cell counterparts (Bacher et al., 2014). Whether fungus‐specific Treg cells are capable of dampening excessive inflammation in the context of nonfungal infections such as autoimmunity and bacterial infections remains unclear, though this possibility is implied by the precedent of bacteria‐specific Treg populations mitigating the severity of colitis and allergic diarrhea in mice (Atarashi et al., 2011, 2013).

2.1.4 Fungi train host immunity Recent studies have demonstrated that two common members of the intestinal mycobiota, C. albicans and S. cerevisiae, are capable of eliciting a form of innate immunological memory in myeloid cells, a phenomenon known as trained immunity (Netea, Quintin, & van der Meer, 2011). Trained immunity refers to the heightened immune response to a secondary infection that, unlike classical immunological memory, occurs independently of the adaptive immune system and can be directed against both the causative microbe of the primary infection and other microbes (Netea et al., 2011). Human monocytes pre‐exposed to C. albicans or S. cerevisiae produced elevated amounts of pro‐inflammatory cytokines, including IL‐6 and TNF‐α, upon subsequent stimulation with various infectious stimuli such as toll‐like receptor ligands (Quintin et al., 2012; Rizzetto et al., 2016). The authors then identified β‐1,3‐glucan and chitin as sufficient to recapitulate the training effects of C. albicans and S. cerevisiae on myeloid cells, respectively. Accordingly, mice treated with β‐1,3‐glucan or chitin were significantly protected from a subsequent systemic challenge with C. albicans compared to untreated controls (Quintin et al., 2012; Rizzetto et al., 2016). It is tempting to speculate that resident C. albicans and S. cerevisiae cells can train mucosal or circulating myeloid cells via similar mechanisms, thereby raising the baseline resistance of the host to other pathogens. An analogous effect has already been described for intestinal symbiotic bacteria, which enhance the antimicrobial activity of neutrophils systemically via shedding of peptidoglycan, a component of bacterial cell walls (Clarke et al., 2010). Mechanistically, trained immunity in myeloid cells by fungal β‐glucan requires substantial rewiring of cellular metabolism, including an increase in aerobic glycolysis and adaptations in cholesterol biosynthesis, which in turn promote epigenetic remodeling of genes involved in inflammation (Arts et al., 2016; Bekkering et al., 2018; Cheng et al., 2014; Mitroulis et al., 2018). Of note, S. cerevisiae strains exhibiting the highest chitin content and the strongest capacity for training monocytes were clinical isolates from human stools and not laboratory or environmental isolates (Rizzetto et al., 2016), suggesting host selection of fungal strains for their immunostimulatory properties.