Keywords

These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

I. Introduction: Dynamics of the Host–Fungus Interaction

The past two decades have seen an unprecedented number of fungal diseases (Fisher et al. 2012). The fungal kingdom is characterized by enormous biodiversity, with over 70,000 known species and an estimated 1.5 million species, 150–400 of which have already been associated with human and/or animal disease. In general, pathogenic fungi (e.g., Aspergillus fumigatus, Cryptococcus neoformans, Pneumocystis jirovecii and the thermally dimorphic Histoplasma capsulatum, Paracoccidioides brasiliensis, Coccidioides immitis and posadasii, Blastomyces dermatitidis, and Sporothrix schenckii) are distinguished from commensals (e.g., Malassezia spp. and Candida albicans) by their strategies for survival and replication within a host, or for transmission from a host, that eventually lead to cellular and tissue damage (Romani 2011). Pathogenicity tactics can vary a great deal and often define unique signatures for specific fungal species, as these involve particular mechanisms for gaining access to the host, adhering to and colonizing a niche, evading immune defenses, and multiplying (Rappleye and Goldman 2008). Indeed, disease onset is often critically dependent on the ability of fungi to reversibly switch morphotypes in infection, a trait that, on the other hand, has forced the host immune system to continuously evolve its repertoire of cross-regulatory and overlapping antifungal responses at different body sites (Romani 2011). Thus, in the context of a dynamic host–fungus interaction, the strategies used by the host to limit fungal infectivity are necessarily assorted in order to cope with the multitude of fungal survival strategies; in retaliation, fungi have developed their own elaborate tactics to evade or modulate host defenses and to survive.

Given the advances in medical care witnessed in the last few decades, specifically regarding transplantation and cancer treatment, the number of immunocompromised patients has risen, resulting in an increased incidence of fungal diseases (Kontoyiannis et al. 2010; Pappas et al. 2010). For this reason, opportunistic fungi, such as Candida and Aspergillus spp. have become major concerns in clinical care of immunocompromised patients. Despite the ability of fungi to survive and persevere within the human host, the truth is that fungal diseases in immunocompetent hosts are fairly uncommon, indicating that fungi have evolved particular adaptation mechanisms that allow them to persist relatively unnoticed by the host’s immune system (Cooney and Klein 2008). This “peaceful” coexistence may digress into overt disease under conditions of immune deregulation, which may modify the environmental conditions perceived by the fungus. Fungi are very proficient at sensing their surroundings and in responding to cues that promote their survival in changing environments. One such example is the sensing of mammalian interleukin (IL)-17A, which triggers virulence by increasing fungal adhesion and filamentous growth, leading to enhanced biofilm formation and resistance to local antifungal defenses (Zelante et al. 2012). Besides the immunologically dynamic context, fungi have often to sustain extreme environmental abiotic stress conditions, the adaptation to which relies on profound metabolic changes (Grahl et al. 2011).

Thus, in the absence of a relatively inert immune response, fungal adaptation to hostile surroundings may trigger complex genomic microevolution (Odds and Jacobsen 2008; Magditch et al. 2012) and structural and metabolic adjustments that, by “awakening” host immunity, may paradoxically contribute to disease. Although such adaptation mechanisms may improve fungal fitness, they can nonetheless provide important insights into potential therapeutic targets (Richie et al. 2009).

Given that existing antifungal therapy is often toxic and ineffective, there is currently a pressing demand for the development of antifungal vaccination strategies (Cassone and Casadevall 2012; Iannitti et al. 2012). For this purpose, a clear understanding of the mechanisms of adaptive immunity is ultimately required to foster the development of vaccines or strategies aiming at modulating the host’s immune response. The permanent interaction between the host and fungi and the commensal relationship with some of them may pose significant challenges in eliciting durable protective immunity, owing to repeated exposure or sensitivity to fungal antigens. A balanced host–fungus relationship and the concomitant fine tuning of pro- and anti-inflammatory signaling to allow host survival irrespective of pathogen elimination is a prerequisite for coexistence and requires the concerted actions of both innate and adaptive immune systems (Romani 2011). Therefore, generation of antifungal immunity presents a challenge that relies on a precarious equilibrium between pathogen clearance and tissue damage restriction, while preserving the host microbiota ecology.

This chapter focuses on new findings on adaptive immunity to the major medically important fungi and emphasizes how dendritic cells (DCs), through the discrimination of fungal molecular patterns, prime responses that nurture and shape the differentiation of T cell subsets. Also discussed is the contribution of T cell subsets to resistance and tolerance mechanisms of antifungal immune protection and how these mechanisms can be exploited for effective antifungal vaccine design.

II. Resistance and Tolerance Mechanisms in Antifungal Protection

The immune system protects from infections primarily by detecting and eliminating invading pathogens through a variety of host resistance effector mechanisms (Romani 2011; Medzhitov et al. 2012). Resistance is meant to reduce pathogen burden during infection through innate and adaptive immune mechanisms, whereas tolerance mitigates the substantial cost to host fitness of resistance. Even in the absence of overt tissue damage, resistance mechanisms commonly occur at a cost to normal tissue function, thus causing immunopathology. This means that the optimal immune response is determined by the balance between efficient pathogen clearance and an acceptable level of immunopathology.

Inflammation is an essential process required for immune resistance, particularly at mucosal tissues, during the transition from the rapid innate to the slower adaptive response. However, the downside of this powerful mechanism of protection against fungi is the collateral damage to the host. These side effects may be more devastating than infection itself. Thus, the ability to tolerate a pathogen’s presence is a distinct host defense strategy that may have evolved to favor protective mechanisms without pathogen killing (Romani 2011; Medzhitov et al. 2012). A plethora of tolerance mechanisms, although not as well known as resistance mechanisms, protect the host from immune- or pathogen-induced damage (Cobbold et al. 2010; Saraiva and O’Garra 2010). Therefore, the term “tolerance” is semantically used here to refer to the multitude of anti-inflammatory mechanisms, including immunological tolerance (i.e., unresponsiveness to self-antigens). At this stage, however, whether “unwanted” immune responses against “self ” environmental antigens and commensal microorganisms occur is not clearly defined, although there is evidence that fungal sensitization contributes to auto-reactivity against self-antigens due to shared epitopes with homologous fungal allergens (Zeller et al. 2008).

T helper (Th)1 and Th17 cells, which provide antifungal resistance, and T regulatory (Treg) cells, which limit the inflammation-associated deleterious effects, crucially contribute to the activation and preservation of these two disparate antifungal mechanisms. Combined deficiency of the Th1 and Th17 pathways predisposes to fungal diseases (van de Veerdonk et al. 2011), thus emphasizing the important role played by both pathways in protection against fungi (Moraes-Vasconcelos et al. 2005; Romani 2011; Hardison and Brown 2012). Thus, the Th1/Th17 pathways and Treg cells, capable of fine-tuning protective antimicrobial immunity to lessen harmful immune pathology, are key components of the current view of immunity to fungi. The enzyme indoleamine 2,3-dioxygenase 1 (IDO1) and its downstream catabolites sustain this delicate balance by providing the host with adequate protective immune mechanisms without necessarily eliminating the pathogen or causing undesirable tissue damage (Zelante et al. 2009). As a result of their ability to induce differentiation of Treg cells and inhibit Th17 cells, IDO1 is critical to cell lineage commitment in experimental fungal infections and contributes to the overall outcome of inflammation, allergy, and Th17-driven inflammation in these infections. Under these circumstances, the Th17 pathway, by inhibiting tryptophan catabolism, may instead favor pathology and provides evidence accommodating the apparently paradoxical association of chronic inflammation with fungal disease (Romani et al. 2008b).

III. Fungi and Inflammation: Evolving Concepts

As in autoimmunity and chronic inflammation, an imbalance between pro- and anti-inflammatory signals may prevent successful host–fungal interaction, thus leading to infection and disease (Romani and Puccetti 2007). Indeed, despite the occurrence of severe fungal infections in immunocompromised patients, clinical evidence indicates that fungal disease also occurs in the setting of a heightened inflammatory response, in which immunity occurs at the expense of host damage and pathogen eradication (Perfect 2012). Although inflammation is an essential component of the protective response, fungi have evolved ways to exploit and subvert it, thereby affecting their ability to persist in the host and pathogenicity (Romani and Puccetti 2008). A hyperinflammatory response does, in fact, enhance the virulence of some fungi. This is well illustrated by the commensal lifestyle of Malassezia spp. in normal skin, possibly due to the downregulation of inflammation via tumor growth factor-β1 (TGF-β1) and IL-10 (Ashbee 2006). In contrast, in atopic dermatitis and psoriasis, the skin barrier enhances release of allergens and molecules involved in hyperproliferation, cell migration, and disease exacerbation. Additional fungal diseases are also important examples of such dichotomy. For example, in chronic mucocutaneous candidiasis (CMC), C. albicans yeasts persist in recurring lesions of the skin, nails, and mucous membranes (Lilic 2002). Although CMC has occasionally been associated with autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy (a condition of dysfunctional T cell activity), evidence has highlighted the contribution of deregulated inflammation and immune responses to disease pathogenesis (Liu et al. 2011; Puel et al. 2011; van de Veerdonk et al. 2011). As already mentioned, fungal sensing of IL-17A is a newly described mechanism by which host inflammation may favor fungal infectivity and promote the transition from fungal commensalism to infection (Zelante et al. 2012). Thus, commensals or ubiquitous fungi have evolved a contingency-based system during co-evolution to guarantee their persistence in an inflammatory host environment.

The main implication of these findings is that, at least in specific clinical settings, it is a heightened inflammatory response that probably compromises a patient’s ability to eradicate infection and not an “intrinsic” susceptibility to infection that determines a state of chronic or intractable diseases (Romani and Puccetti 2007). The conceptual principle highlighting a truly bipolar nature of the inflammatory process in infection is best exemplified by the occurrence of severe fungal infections in patients with chronic granulomatous disease (Romani et al. 2008a) or with immune reconstitution syndrome, an entity characterized by localized and systemic inflammatory reactions and worsening disease in opportunistic and non-opportunistic infections that are associated with immunological recovery (Gupta and Singh 2011; Perfect 2012). Additionally, a high incidence of fungal infections and sensitization to Aspergillus spp. has been described in the hyper-IgE syndrome, in which increased levels of pro-inflammatory gene transcripts have been found (Antachopoulos et al. 2007; Holland et al. 2007). These observations suggest that an inflammatory loop hampering a patient’s capacity to counter infection seems to be at work, at least in specific clinical settings. The manipulation of this loop may offer strategies to control or prevent exacerbations of these diseases.

IV. Activation of Antifungal Immunity

A. Dendritic Cells

Immunity to fungi is a dynamic interplay between every arm of the immune system. Innate immune mechanisms are used by the host to respond to a range of fungal pathogens in an acute and conserved fashion (as discussed in chapter “Receptor-Ligand Interactions in Fungal Infections”). The induction of innate immunity shapes the development of the adaptive immune response. As sentinels of the immune system, DCs are responsible for sampling antigenic material in the environment, shaping T cell responses through secretion of cytokines, and priming T cells via antigen presentation (Steinman 2012). Priming of T cells by DCs is mediated by pathogen-associated antigens on major histocompatibility complex class I (MHC-I) or MHC class II (MHC-II) molecules for priming of CD8+ or CD4+ T cells, respectively. After priming of naïve T cells, the response is generally described as Th1, Th2, Th17, or Treg (described in detail in a later section B) based on the pattern of cytokine production. Thus, the ability to control the fate of the immune response makes DCs both central to balancing antifungal immunity and a prime target for vaccination strategies.

For years, DC biologists have oscillated between two apparently opposing concepts: functional specialization of DC subsets (division of labor) and plasticity (multitasking). More recently, a third hypothesis is gathering support: crosstalk between functionally distinct DC subsets. This reveals a previously unappreciated hierarchy of organization within the DC system, and provides a conceptual framework for understanding how cooperation between functionally distinct, yet plastic, DC subsets can shape adaptive immunity and immunological memory (Pulendran et al. 2008).

1. The Role of DCs in Antifungal Immunity

DCs are uniquely proficient at decoding the fungus-associated antigens and translating them into qualitatively different adaptive T cell immune responses (Romani et al. 2002; Romani 2011; Roy and Klein 2012; Wuthrich et al. 2012). DCs have the exceptional competence to initiate distinct adaptive antifungal immune responses as a result of cooperation between subsets (Romani and Puccetti 2006; Romani et al. 2008a) and activation of distinct intracellular signaling pathways (Bonifazi et al. 2009, 2010). The functional plasticity of DCs is mostly defined by the discriminative recognition of different fungal species and morphotypes by the full range of pattern recognition receptors (PRRs). As a matter of fact, whole−genome transcriptional analysis of fungus-pulsed DCs revealed the presence of a specific transcriptional program governing fungal recognition (Rizzetto and Cavalieri 2010). Among DC subsets, plasmacytoid (p)DCs have a prominent role in fungal infections. Not only are pDCs rapidly recruited and activated in response to fungi at mucosal sites, such as the lung (Bonifazi et al. 2010; Ramirez-Ortiz et al. 2011; Carvalho et al. 2012; De Luca et al. 2012) and gut (De Luca et al. 2007; Bonifazi et al. 2009), but infusion of pDCs in bone marrow-transplanted mice has been found to trigger Th1/Treg cell priming, eventually leading to fungal growth restriction, limited inflammatory pathology and, interestingly, transplantation tolerance (Romani et al. 2006). More recently, Toll-like receptor (TLR) 3-dependent recognition of fungal RNA by CD8+ DCs induced potent cytotoxic CD8+ T cell responses to A. fumigatus, both in mice and humans (Carvalho et al. 2012). TLR3 deficiency renders mice highly susceptible to aspergillosis, and a TLR3 mutation affecting the ability of CD8+ DCs to cross-present antigens renders hematopoietic stem cell-transplanted patients more susceptible to invasive aspergillosis.

The activation of distinct signaling pathways in DCs translates recognition of fungi into distinct inflammatory and adaptive immune responses (Bonifazi et al. 2009, 2010). The screening of signaling pathways in DCs through a systems biology approach was exploited for the development of therapeutics to attenuate inflammation in experimental fungal infections and diseases. In vivo targeting inflammatory [phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR)] or anti-inflammatory [signal transducer and activator of transcription (STAT3)/IDO1] DC pathways by intranasally delivered small interfering RNA (siRNA) modified resistance and tolerance to infection. Thus, the screening of signaling pathways in DCs through a systems biology approach may be exploited for the development of siRNA therapeutics to attenuate inflammation in respiratory fungal infections and diseases (Bonifazi et al. 2010).

DCs are now being exploited to improve vaccine efficacy (Steinman 2008). The potential use of tolerogenic pDCs as negative cellular vaccines to induce experimental transplantation tolerance has been suggested (Turnquist and Thomson 2008).

Over recent years, experimental models have shown that it is possible to exploit the mechanisms that normally maintain immune homeostasis and tolerance to self-antigens to induce tolerance to allo-antigens (Waldmann and Cobbold 2004; Martinic and von Herrath 2006). Like natural tolerance, transplantation tolerance is achieved through control of T cell reactivity by central and peripheral mechanisms of tolerance.

Fungus-pulsed DCs or RNA-transfected DCs acted as potent fungal vaccines in experimental hematopoietic stem cell transplantation (Bozza et al. 2003), a model in which autologous reconstitution of host stem cells is greatly reduced due to the benefit of a long-term, donor-type chimerism in more than 95% of the mice and low incidence of graft rejection. Protection was associated with myeloid and T cell recovery, the activation of CD4 + Th1 lymphocytes, and the concomitant IL-10-driven Treg cells. Thus, tolerogenic DCs proved to be pivotal in the generation of some form of dominant regulation that ultimately controlled inflammation, pathogen immunity, and tolerance in transplant recipients eventually leading to prevention of graft-versus-host reaction and reduction of aspergillosis incidence rates. These results, along with the finding that fungus-pulsed DCs could reverse T cell anergy of patients with fungal diseases, further supports the utility of targeting DCs for antifungal vaccination strategies (Bozza et al. 2004).

2. Metabolic Regulation of DC Plasticity in Response to Fungi

A wealth of evidence indicates that acquisition of an immunogenic or tolerogenic phenotype is a trait of a specific subset or lineage of DCs, but that it is an environmentally acquired feature. In this regard, the tryptophan metabolic pathway pivotally contributes to DC regulation, such that tolerance and Treg induction can be mediated by IDO1-expressing DCs (Orabona et al. 2004) (see below). In response to fungi, IDO1 expression was found to confer tolerogenic properties to DCs (Zelante et al. 2009) such that C. albicans-pulsed, IDO1-expressing gut DCs ameliorated experimental colitis (Bonifazi et al. 2009). By subverting the morphotype-specific program of activation of DCs, environmental factors and fungi themselves qualitatively affect DC functioning and Th/Treg cell selection in vivo, ultimately impacting on fungal virulence. Thus, the current view accommodates the concept of virulence as an important component of fungal fitness within the plasticity of immune responses orchestrated by DCs. Indeed, impaired DC maturation and function have been associated with disease in patients with CMC (Ryan et al. 2008).

B. T Cell Responses in Fungal Infection

Studies on nonmammalian hosts have provided means to examine the molecular elements of fungal virulence and host innate immunity (Fuchs and Mylonakis 2006; Mylonakis et al. 2007; Peleg et al. 2008). In higher organisms, however, innate sensing mechanisms are capable of distinguishing different fungal morphotypes and are hard-wired to activate distinct adaptive immune responses with protective and nonprotective functions against the different fungal species. It has been suggested that a memory-based immune mechanism may have evolved in vertebrates to accommodate colonization by symbiotic microbes while retaining the capacity to oppose their infectivity (McFall-Ngai 2007). This suggests that the adaptive immune system has co-evolved with ubiquitous or commensal fungi, with a price to be paid for this permissiveness. Stimulation of antigen-presenting macrophages, DCs, and, more recently, epithelial cells (ECs) leads to activation and recruitment of lymphocytes and the development of Th cell−specific antifungal responses. There is extensive plasticity in T cell responses to fungi (Fig. 6.1). The heterogeneity of the CD4+ and CD8+ T cell repertoire may account for the multiplicity and redundancy of effector mechanisms through which T lymphocytes participate in the control of fungal infections. Once committed, T cells express effector functions largely, but not exclusively, through release of cytokines, most notably interferon (IFN)-γ, tumor necrosis factor (TNF)-α and IL-17/IL-22, which are instrumental in mobilizing and activating antifungal effectors, thus providing prompt and effective control of infectivity once the fungus has established itself in tissues or spread to internal organs (Romani 2011).

Fig. 6.1
figure 00061

T cell activation in fungal infection. Dendritic cells (DCs) are at the crossroads of antifungal immunity and tolerance because both inflammatory and tolerogenic DCs may arise from their common immature progenitors. Following fungal recognition by specific pattern recognition receptors, inflammatory DCs may trigger the expression of subset-associated transcription factors and differentiation of naïve CD4+ T cells into T helper 1 (Th1), Th2, or Th17 cells, which may induce cytokine secretion and activation of specific antifungal immunity programs. On the other hand, plasmacytoid DCs may activate CD8+ cytotoxic lymphocytes (CTLs) that, by displaying direct fungicidal activity, also contribute to antifungal immunity. In addition, indoleamine 2,3-dioxygenase 1 (IDO1)-competent tolerogenic DCs produce kynurenines (Kyn) and cytokines that contribute to the expansion of interleukin (IL)-10-secreting T regulatory (Treg) cells, crucial for the maintenance of tolerance to the fungus. MR mannose receptor, IFN interferon, TLR Toll-like receptor, T-bet T-box expressed in T cells, Gata3 GATA-binding protein 3, Rorc retinoic acid receptor (RAR)-related orphan receptor C

Full size image

1. Th1 Cells

Generation of a dominant Th1 response driven by IL-12 is essentially required for the expression of protective immunity to fungi and is compulsory for the design of effective antifungal vaccines (Spellberg et al. 2008). Through the release of the signature cytokine IFN-γ and by helping the production of opsonizing antibodies, induction of Th1 cells is instrumental for the optimal activation of phagocytes at sites of infection. Of interest, Th1 cell-based cross-protection for different fungi has been recently demonstrated to be achievable using a single immunogenic epitope from the A. fumigatus cell wall, thus providing attractive opportunities for immunotherapeutic strategies (Stuehler et al. 2011). The failure to deliver activating signals to effector phagocytes may predispose patients to overwhelming infections, limit the therapeutic efficacy of antifungals and antibodies, and favor persistency and/or commensalism (Romani 2011).

As noted above, Th cell skewing is determined by the way that DCs respond to the combination of fungal-derived TLR and C-type lectin receptor (CLR) signaling. Interestingly, dectin-1-mediated signals can alter the Th1 profile of CD4+ T cell responses to fungal infection by decreasing the production of IL-12 and IFN-γ in innate cells and consequent expression of T-box expressed in T cells (T-bet) in A. fumigatus-specific CD4+ T cells, thus enabling Th17 differentiation (Rivera et al. 2011).

Further supporting the pivotal role of Th1 cell activation, immunological studies on patients with polar forms of paracoccidioidomycosis demonstrate an association between Th1-biased reactivity and asymptomatic and mild forms of infection, as opposed to the correlation of Th2 cell responses with severe disease and poor prognosis. Thus, the finding that estradiol favors Th1-type immunity may explain why paracoccidioidomycosis is much more frequent in men than in women (Pinzan et al. 2010).

2. Th2 Cells

Progressive fungal infections, especially those acquired through the respiratory tract, are eventually associated with a shift in dominance from Th1 to Th2. IL-4 and IL-13 provide the most potent proximal signals for commitment of naïve T cells to the Th2 cell lineage. Despite often occurring coincidentally with vigorous Th1 responses, Th2 reactivity results in a net effect of poor control of the fungal burden because it induces alternatively activated macrophages that are permissive for intracellular fungal growth and may be impervious to signaling by IFN-γ (Voelz et al. 2009). In addition, Th2 reactivity alters pulmonary physiology such that airway resistance increases, thus compounding the severity of infection and contributing to fungus-associated allergic responses (Jain et al. 2009). Accordingly, attenuating Th2 responses to A. fumigatus in cystic fibrosis patients with allergic bronchopulmonary aspergillosis (Kreindler et al. 2010) or limiting IL-4 production in experimental models of histoplasmosis (Szymczak and Deepe 2009) restored antifungal resistance.

For the vast majority of fungi, Th2 responses are associated with pathogenic allergic responses. The contradiction is pneumocystosis, in which a Th2 cell-dependent humoral immunity affords some degree of protection (Rapaka et al. 2010). In addition, alternatively activated macrophages driven by IL-13 and amplified by IL-33 displayed enhanced fungicidal activity regardless of the antibody-dependent effect, a finding that further supports a contribution of Th2 cells to fungal clearance (Bhatia et al. 2011).

3. Th17 Cells

Over the past several years, the Th1/Th2 dichotomy has been replaced by the belief that the “fates” for developing CD4+ T cells and associated cytokines are more flexible than formerly anticipated (Zhou et al. 2009). Th17 cells are a separate lineage of effector Th cells contributing to immune pathogenesis previously attributed to the Th1 lineage (Kaufmann and Kuchroo 2009). Th17 cells have an important function in the host response against extracellular pathogens, but they are also associated with the pathogenesis of many autoimmune and allergic disorders. It is now well accepted that Th17 cell activation occurs in fungal infections (Hernandez-Santos and Gaffen 2012), mainly through the spleen tyrosine kinase/caspase recruitment domain-containing protein 9 (Syk/CARD9), myeloid differentiation primary response protein 88 (MyD88), and mannose receptor (MR), signaling pathways in DCs and macrophages leading to the production of unique cytokines such as IL-17, IL-17F, and IL-22. This signaling is inhibited by Raf-1 kinase and TIR domain–containing adapter–inducing interferon-beta (TRIF)/type I IFN pathways, indicating that the molecular pathways defining activation or inhibition of Th17 cells are present downstream of both CLRs and TLRs. Indeed, the central role of Th17 cells in antifungal immunity is supported by studies reporting Mendelian susceptibility to fungal infections of individuals with inborn errors of dectin-1, CARD9, STAT1, STAT3 and, specifically, IL-17 immunity (Ferwerda et al. 2009; Glocker et al. 2009; Puel et al. 2011; van de Veerdonk et al. 2011) (Table 6.1). In experimental fungal infections, however, IL-17-dependent immunity has been reported to be either essential (Huang et al. 2004; Saijo et al. 2007; Conti et al. 2009; Wuthrich et al. 2011) or not (Lin et al. 2009; De Luca et al. 2010b; Hardison et al. 2010). This suggests that the protective or detrimental effects of this pathway may depend on the stage and site of infection, probably influenced by environmental stimuli that induce cells to produce cytokines of the IL-17 family, including IL-22 (see below). In this regard, recent evidence has demonstrated that Th17 cells can be segregated into pathogenic or nonpathogenic cells, depending on whether they produce granulocyte macrophage colony stimulating factor (GM-CSF) or IL-10, respectively (Codarri et al. 2011; El-Behi et al. 2011). These Th17 phenotypes are still to be described in the context of fungal diseases (Wuthrich et al. 2012).

Table 6.1. Mendelian susceptibility to fungal diseases
Full size table

Th17 cells are present in the fungus-specific T cell memory repertoire in humans (Acosta-Rodriguez et al. 2007; Bozza et al. 2009) and mediate some (Wuthrich et al. 2011), but not all (De Luca et al. 2012), vaccine-induced protection in mice. Interestingly, as in mice (De Luca et al. 2012), human host defense against A. fumigatus relies on Th1 rather than Th17 cell responses (Chai et al. 2010), and CMC patients (with or without autosomal dominant hyper-IgE syndrome) have defective Th17 and Th1 cell responses (Ryan et al. 2008; van de Veerdonk et al. 2011). This could be explained by the notion that Th17 cells, although found early during the initiation of an immune response, are involved in a broad range of both Th1- and Th2-type responses.

Indeed, a role for Th17 cells in supporting Th1 cell responses has been shown in experimental mucosal candidiasis (Conti et al. 2009; De Luca et al. 2010b). In addition, in experimental aspergillosis, enhanced Th2 cell responses and fungal allergy are observed in the absence of IL-17A receptor signaling (our unpublished observations). Thus, these findings point to an important regulatory function of the Th17 cell pathway in promoting Th1-type and restraining Th2-type immunity.

It is intriguing that Th17 cell responses are dampened by C. albicans (Cheng et al. 2010) and that failure to do so eventually results in chronic inflammation and failure to resolve the infection (Zelante et al. 2007; Loures et al. 2009). In this regard, it is fascinating that fungi are able to sense mammalian IL-17 in their surrounding environment and turn on molecular programs that result in enhanced virulence and survival aptitude (Zelante et al. 2012).

The mechanisms that link inflammation to chronic infection may lie in an inability to control inflammation following IL-17A-dependent neutrophil mobilization, thus preventing optimal protection and favoring fungal persistence. Thus, the Th17 cell pathway could be involved in the immunopathogenesis of chronic fungal disease, in which persistent fungal antigens may promote immune deregulation, as demonstrated in patients with autoimmune polyendocrine syndrome type 1 and in the absence of autoimmune regulator (AIRE), in which excessive Th17-type responses to fungi have been observed (Ahlgren et al. 2011).

As noted above, Th17 cells can concomitantly synthesize IL-22, a member of the IL-10 family of cytokines, which has been shown to play a more important role than IL-17 in host defense in the lung and gut (Zenewicz and Flavell 2008). Our recent findings suggest that the IL-23/IL-22/defensins pathway is crucially involved in the control of fungal growth at mucosal and nonmucosal sites in both candidiasis and aspergillosis, particularly in conditions of Th1 deficiency. Interestingly, memory IL-22+ CD4+ cells specific for C. albicans are present in humans (Liu et al. 2009) and are defective in patients with CMC (Eyerich et al. 2008). Thus, further tweaking the Th17 model, Th17 cells may exert their protective role in fungal infections through IL-22. Indeed, IL-22 has recently been demonstrated to be required for the control of C. albicans growth at mucosal sites in the absence of Th1 and Th17 cells (De Luca et al. 2010b). Specifically, IL-22 produced by NKp46+ innate lymphoid cells expressing the aryl hydrocarbon receptor directly targeted intestinal ECs and induced STAT3 phosphorylation and release of S100 calcium binding protein A8 (S100A8) and S100A9, peptides known to have antifungal activity and anti-inflammatory effects. Thus, in the relative absence of protective Th1/Treg, IL-22 + Th17 cells may fulfill the role of a protective response that exploits primitive effector defense mechanisms of antifungal resistance, as demonstrated also for experimental bacterial diseases (Aujla et al. 2008). Consistent with this role for IL-22, patients with autosomal dominant hyper-IgE syndrome, owing to dominant-negative mutations of STAT3, have a defective Th17 cell response to C. albicans (Milner et al. 2008). Accordingly, C. albicans-specific IL-22+ CD4+ memory T cells are present in healthy individuals (Liu et al. 2009) but are lacking in CMC patients (Eyerich et al. 2010), an observation pointing to IL-22 production in the mucosa as a primitive mechanism of resistance against fungi under conditions of limited inflammation. Of interest, dectin-1-mediated production of IL-22 in the lung has also been demonstrated to contribute to early innate immune resistance to A. fumigatus (Gessner et al. 2012), although it paradoxically promoted lung inflammation and immunopathology during persistent fungal exposure in an allergy model (Lilly et al. 2012). These seemingly discrepant findings further add to the complexity of IL-22 function in antifungal mucosal immunity and point to the existence of regulatory events leading to its production that depend on the stage and site of infection.

4. Treg Cells

During infection, the immune response must eliminate the fungus while limiting infection-associated costs to host fitness and restoring a homeostatic environment. Treg cells, by means of their anti-inflammatory activity, play a central role in this process. In experimental fungal infections, inflammatory immunity and immune tolerance in the respiratory or gastrointestinal mucosa have been shown to be controlled by the coordinated activation of different Treg cell subsets. Because Treg cell responses may handicap the efficacy of protective immunity, Treg cell activity decreases host tissue damage but may conversely promote fungal persistence (Romani and Puccetti 2006) and, eventually, immunosuppression (Ferreira et al. 2010). Some cells with this function, such as CD4+ Foxp3+ natural Tregs (nTregs), exist regardless of the presence of infectious stimuli, whereas others may be induced as a consequence of infection (iTregs) or in conditions of impaired co-stimulatory signaling and in the presence of deactivating cytokines and drugs. This scenario is crucially exemplified in experimental aspergillosis, in which inflammation was controlled at an early stage by nTregs suppressing neutrophils whereas, later tolerogenic iTregs inhibited Th2 cells and prevented fungal allergy (Montagnoli et al. 2006).

As already discussed, a reciprocal relationship has been described between the development of Foxp3+ Treg and effector Th17 cells, so that naïve T cell activation in the presence of innate stimuli redirects iTreg generation to Th17 generation. In this regard, CD4+ CD25+ Foxp3+ Treg cells have been recently found to promote IL-17 upregulation and contribute to suppression of mucosal candidiasis in vivo (Pandiyan et al. 2011). Thus, by controlling the quality and magnitude of effector innate and adaptive responses, the spectrum of Treg cell skills may go from protective tolerance and immune homeostasis to dominant effector activities. Furthermore, this suggests that this degree of interaction between fungi and the host immune system determines whether a fungus is perceived as commensal or pathogen, and that this definition may evolve constantly.

5. Indoleamine 2,3-Dioxygenase 1 is a Critical Regulator of Tolerance to Fungi

IDO1 is an IFN-γ-inducible intracellular enzyme that catalyzes the catabolism of tryptophan (Puccetti and Grohmann 2007; Mellor and Munn 2008). The effects of IDO1 activity are tryptophan deficiency, excess tryptophan breakdown products (kynurenines), and consumption of reactive oxygen species. IDO1 and kynurenines serve many roles in fungal infections. A number of studies have established that the proper control of infection and associated inflammatory reactions require IDO1 induction and consequent production of tryptophan metabolites with immunoregulatory activities, contributing to the maintenance of the Treg/Th17 balance (Romani et al. 2008b). As already mentioned, IDO1-expressing DCs are regarded as regulatory DCs specialized in antigen-specific deletional tolerance or induction of CD4+ CD25+ Treg cells. These findings disclose a mutual interaction between DCs and Treg cells for the preservation of immunological tolerance. Indeed, IDO1 blockade greatly exacerbated experimental fungal infections and the associated inflammatory pathology, and swept away resistance to re-infection, as a result of deregulated innate and adaptive immune responses caused by the impaired activation and functioning of suppressor CD4+ CD25+ Treg cells producing IL-10 (Romani and Puccetti 2006). IDO1 expression is paradoxically upregulated in patients with allergy or autoimmune inflammation, a finding pointing to the occurrence of a homeostatic mechanism to halt ongoing inflammation (Grohmann et al. 2007). During experimental fungal allergy, modulation of tryptophan catabolism via the glucocorticoid-induced tumor necrosis factor receptor (GITR) and its ligand, GITRL, inhibited Th2 cell responses and allergy and induced the expression of Foxp3+ Treg cells through IDO1-dependent mechanisms (Grohmann et al. 2007), a finding pointing to the potential relevance of IDO1 in the anti-inflammatory action of corticosteroids. As already mentioned, a reciprocal antagonistic relationship exists between IDO1 and the Th17 pathway, with IDO1 restraining Th17 responses and IL-17A inhibiting IDO1 (Zelante et al. 2007).

Recent evidence indicates that the non-hematopoietic compartment also contributes to tolerance to fungi via IDO1 (Cunha et al. 2010; de Luca et al. 2010a). ECs are key players in tolerance to respiratory pathogens via an IFN-γ–IDO1 axis culminating in the inhibition of Th17 cell responses (Desvignes and Ernst 2009; de Luca et al. 2010a). IDO1 overexpression in airway ECs was found to restrain CD4+ T cell activation to A. fumigatus, an activity that was nevertheless dispensable in the presence of IDO1-expressing tolerogenic DCs. However, IDO1 induction in ECs could compensate for the lack of IDO1 on hematopoietic cells (Paveglio et al. 2011). The expression of IDO1 on ECs occurred through the TLR3/TRIF-dependent pathway, a finding consistent with the abundant expression of TLR3 both intracellularly and on the cell surface of ECs. The failure to activate IDO1 probably accounted for the lack of tolerance to the fungus observed in experimental stem cell transplantation in conditions in which either the recipient or the donor (or even more when both) were TRIF- or TLR3-deficient (de Luca et al. 2010a).

Overall, these data shed light on pathways of immune resistance and tolerance to the fungus that probably take place in a hematopoietic stem cell transplantation setting. It appears that protective tolerance to the fungus is achieved through a TLR3/TRIF-dependent pathway activating Th1/Treg cells via IDO1 expressed on both the hematopoietic and non-hematopoietic compartments. In contrast, the MyD88 pathway provides antifungal resistance, i.e., the ability to restrict fungal growth through defensins and, probably, other effector mechanisms (de Luca et al. 2010a). However, the ability of mice to clear the fungus in the relative absence of the MyD88 pathway (Bretz et al. 2008) clearly indicates redundancies and hierarchy in antifungal mechanisms of resistance. Ultimately, the finding that both C. albicans (De Luca et al. 2007) and A. fumigatus (de Luca et al. 2010a), two major human fungal pathogens, exploit the TRIF/IDO1-dependent pathway at the interface with the mammalian hosts indicates that the exploitation of tolerance mechanisms is an advantageous option.

V. Immune Memory and Antifungal Vaccines

A successful vaccination relies on the elicitation of pathogen-specific immune memory that mediates long-term protection from infection or disease. Given the plethora of fungal ligands present at the cell surface, as well as those that become available to immune sensing upon processing of the fungus by phagocytic cells, it is clear that vaccine-induced protection to attenuated fungal strains occurs through distinct PRRs and downstream signaling adapters (Wuthrich et al. 2011; De Luca et al. 2012). For instance, Th17-induced acquisition of vaccine immunity to live attenuated strains of B. dermatitidis, H. capsulatum , and C. posadasii was found to require MyD88 signaling (Wuthrich et al. 2011 ), whereas Th1-induced protection to A. fumigatus relied on TRIF (De Luca et al. 2012 ). Of interest, vaccination with purified A. fumigatus antigens was found to be dependent on the MyD88 pathway in the presence of the appropriate adjuvant (Carvalho et al. 2012; De Luca et al. 2012), a finding pointing to the crucial role of adjuvants in promoting T cell differentiation along specific effector pathways. Thus, fungal innate sensing is one critical step in mounting immune responses, eventually defining appropriate effector responses to maximize protection (Levitz and Golenbock 2012).

Although CD4+ Th1 cells have been historically considered the cornerstone of cell-mediated defense against intracellular fungi (Cassone and Casadevall 2012; Iannitti et al. 2012), CD8+ T cells have also earned a place in this category (Cutler et al. 2007). Indeed, in a mouse model of vaccination against blastomycosis, both the numbers and function of protective antifungal memory CD8+ T cells were maintained, even in the absence of CD4+ T cell help (Nanjappa et al. 2012b). Under these circumstances, a distinct lineage of CD8+ T cells able to produce IL-17 (Tc17 cells) has been found to be nonredundant for vaccine immunity to fungal infection by mediating protection in a neutrophil-dependent manner (Nanjappa et al. 2012a).

The persistence of immunological memory and how it relates to vaccination strategies is a question of central importance. Memory T cells are derived from normal T cells that have learned how to overcome a pathogen by “remembering” the strategy used to defeat previous infections (Sallusto et al. 2010). In addition to central memory T cells present in secondary lymphoid organs, which scrutinize the presence of remote pathogens via DCs, effector memory T cells reside in peripheral non-lymphoid tissues such as the skin and mucosa. The latter are heterogeneous in terms of homing receptor expression and effector function and comprise the Th1, Th2, Th17, and Treg cells and cytotoxic T lymphocytes. Memory CD8+ cytotoxic T cells are also induced in fungal infections (Nanjappa et al. 2012b) and exhibit a pleiotropic activity by mediating protection via production of IFN-γ and cytolytic activity against fungus-laden cells or the fungus itself (Carvalho et al. 2012; De Luca et al. 2012). As such, CD8+ T cells, especially if long-lasting, are regarded as ideal candidates for expansion at mucosal surfaces by vaccination strategies.

The nature of the antifungal vaccine, the route of antigen delivery, and the mode of antigen routing and presentation are important for determining the success of a fungal vaccine. Indeed, recent evidence has highlighted striking differences in antigen presentation pathways in DCs leading to the activation of CD4+ or CD8+ T cells (De Luca et al. 2012). Memory CD4+ T cells were activated by purified antigens from A. fumigatus that were routed to the endosome/lysosome-dependent MHC-II presentation pathway via MyD88 with the involvement of distinct upstream PRRs. En route to lysosomes, purified antigens were also targeted to the mildly acidic stable early endosomal compartment where uptake by MR led to presentation on MHC-I molecules and Th1 polarization. Consistently, mannosylation has been reported to significantly enhance antifungal CD8+ T cell priming (Luong et al. 2007). In contrast to soluble antigens, phagocytosed cells or particulate antigens activated CD8-dependent memory through a pathway relying on TLR3/TRIF signaling (De Luca et al. 2012). Similar to the situation for C. neoformans (Wozniak and Levitz 2008), phagocytosed cells were routed to the late endosome/lysosome compartment and also to the rat sarcoma (RAS)-related protein (Rab)14+ compartment, which is known to limit routing of antigens from early endosomes to the acidic lysosomal environment, thus limiting antigen degradation and favoring cross-presentation (Saveanu et al. 2009). The escape of fungal cells from the endosomal/lysosomal compartment to the Rab14+ compartment occurs through TLR3-dependent autophagy. Accordingly, CD8+ T cell memory to conidia was abrogated when autophagy was defective (e.g., in the absence of TLR3) or under conditions of defective endosomal alkalinization (e.g., in NADPH deficiency) (Savina et al. 2006) (Fig. 6.2). In these conditions, long-lasting antifungal protection and disease control was successfully achieved upon vaccination with purified fungal antigens that activate CD4+ T cells. Thus, CD8+ T cells can provide antifungal memory in CD4+ T cell deficiency and vice versa. These data highlight how understanding memory at a basic level, including information obtained from suitable animal models, may be exploited to personalize vaccination strategies against fungal diseases. Refinement of these approaches could lead to antifungal vaccines and adjuvants tailored to the different target populations, administered either alone or in combination with immunomodulators targeting antigen trafficking and presentation pathways. In this regard, promoting autophagy restores defective CD8+ T cell memory (De Luca et al. 2012), a finding that broadens the role of autophagy in adaptive immunity to include response to vaccines.

Fig. 6.2.
figure 00062

En route to antifungal vaccines. Antifungal vaccines undergo specific antigen presentation pathways leading to the activation of CD4+ and CD8+ T cells. Shown are results from co-localization studies using live conidia of A. fumigatus or protective and nonprotective soluble antigens (Ag). Memory, major histocompatibility complex (MHC) class II-restricted CD4+ T cells were activated by live conidia and purified Ags that were routed to the endosome/lysosome-dependent MHC class II presentation pathway. En route to lysosomes, purified fungal Ags also targeted to the mildly acidic stable early endosomal compartment and to MHC class II+ organelles. In contrast, phagocytosed cells activated MHC class I-restricted CD8+ T cells upon routing to the Rab14+ compartment, known to favor cross-presentation, and to MHC class I+ organelles. Of interest, the escape of fungal cells from endosomal/lysosomal degradation to the Rab14+ compartment occurred through activation of autophagy. MR mannose receptor, LAMP1 lysosomal-associated membrane protein 1, LC3 microtubule-associated protein 1A/1B-light chain 3, Rab14 rat sarcoma (RAS)–related protein (Rab) 14

Full size image

VI. Final Remarks

The control of inflammation leading to tolerance, the molecular bases of immune regulation and deregulation, and the way in which commensal but opportunistic fungal pathogens can switch from a “friendly” affinity with the host to a pathological relationship by evading or subverting host inflammation, are challenging issues in the field of medical mycology and infection-related immunological disorders. A related question is how and whether the fungal microbiota contributes to the regulation of inflammation in health and disease. By the use of multidisciplinary approaches based on whole-genome immunogenetics, cutting-edge “omics” techniques, advanced bioinformatics, and systems biology applied to immune profiling, it will be possible to challenge existing paradigms in the fields of fungal immunopathology, thereby leading to the discovery of “commensal signatures” for the fungal biota and the development of therapeutic approaches for mucosal and systemic fungal diseases.