Fungal Recognition and Host Defense Mechanisms

ABSTRACT

Fungi have emerged as premier opportunistic microbes of the 21st century, having a considerable impact on human morbidity and mortality. The huge increase in incidence of these diseases is largely due to the HIV pandemic and use of immunosuppressive therapies, underscoring the importance of the immune system in defense against fungi. This article will address how the mammalian immune system recognizes and mounts a defense against medically relevant fungal species.

CONCEPTUAL FRAMEWORK FOR IMMUNOLOGY

The immune system has over millennia evolved strategies to discriminate between what is “self” and what is foreign (nonself) as well as “normal self” and “injured or altered self,” with the ultimate purpose being to defend and repair self. First, to fully appreciate the framework that governs most of our current understanding of how the immune system operates, it is imperative to be cognizant of some of the immunological models that have laid the foundation on which we stand.

Self-Nonself Discrimination Models

The 1960 Nobel Prize in Physiology or Medicine was awarded to F. MacFarlane Burnet and Peter Medawar for the discovery of immunological tolerance. Burnet suggested that each B lymphocyte, an immune cell responsible for the production of antibodies (or immunoglobulins), expresses numerous clones of cell surface immunoglobulin receptors specific for a foreign body (antiforeign body) and that when antibody receptor recognizes its cognate antigen, an immune response is initiated (1). This idea was solidified by Billingham, Brent, and Medawar’s findings, which demonstrated that adult mice “tolerate” skin grafts if they are transplanted at birth; that is, the immune system can learn to tolerate the foreign antigens until defensive intolerance is acquired (2).

The self-nonself discrimination model became the modus operandi of the immune system until it was modified in 1969 by Bretscher and Cohn, who postulated that a quiescent B lymphocyte requires interaction with a “helper” cell (now known as helper T lymphocyte) specific for the same antigen to induce an activation signal, whereas inactivation ensues when antigen is recognized in the absence of the helper cell (3). In 1975, Lafferty and Cunningham introduced a requirement for a costimulation signal and proposed that recognition of antigen by the helper T cell (which provides signal 1) together with a “stimulator cell” (signal 2) (known now as antigen-presenting cell [APC]) induced activation of the immune system (4, 5). Over a decade later, Jenkins and Schwartz provided experimental evidence for the requirement of costimulation for activation of resting lymphocytes (6). However, the prerequisite of costimulation introduced a conundrum because this meant that immune activation hinges on APCs that capture and present both self and nonself antigens; therefore, immunity could not be solely targeted against the invading foreigners.

Noninfectious Self and Infectious Nonself Model and the Inception of Pattern Recognition Receptors

Charles Janeway provided an elegant solution to the costimulation conundrum mediated by APCs. In his 1989 article Janeway suggested that the immune system has evolved specifically to recognize and respond to infectious organisms and that this involves recognition not only of antigenic determinants but also of certain characteristics or patterns common in infectious organisms but absent from the host, and he coined the term “pattern recognition receptor” (PRR) (7). He also pointed out that these germline-encoded receptors would act early during an immune response and imagined that they also play a role in shaping the adaptive T and B cell responses. Another piece of the puzzle was added by Polly Matzinger, who in 1994 proposed that resting APCs are activated by stimuli associated with host-cell damage, which she termed “danger signals” (8). She posited that by definition, pathogens damage the host and that recognition of damage could be instrumental in validating initiation of an immune response. A summary of the history of the immune models discussed above is depicted in Fig. 1.

FIGURE 1.

More than half a century of immunogical theories.

PRRs INVOLVED IN SENSING FUNGI

Several classes of PRRs have been discovered since their initial identification, providing experimental support for Janeway’s theory. It is noteworthy that pioneering work emanating from the use of fungi and their antigenic determinants has contributed significantly to our understanding of the initiation of an immune response (for a detailed timeline, see reference 9). Here, we will discuss PRRs that have been demonstrated to sense the presence of fungi and activate antifungal host immune responses as well as the implications of defective receptor/signaling networks on susceptibility to fungal infections.

Due to the ubiquitous presence of fungi in the environment, humans are constantly exposed to them; in fact, it has been proposed that we have coevolved with commensals such as Candida spp. and Malassezia (10). Despite this, fungi are associated with various diseases ranging from asthma to mucocutaneous infections to severe life-threatening systemic infections in patients with a compromised immune system (e.g., organ tissue transplant patients and HIV-infected individuals).

Fungi are eukaryotes and thus are similar to mammalian cells; however, they possess a cell wall, which is absent in mammalian cells. The cell wall is thought to provide several advantages to fungi including protection against environmental stresses and structural rigidity for invading ecological niches (11). For many fungal species, the molecular details of the structural composition and organization of cell wall components have not been fully elucidated. However, for most of the medically relevant fungi, polysaccharides account for more than 90% of the wall content, with the central core comprising branched β-1,3 and β-1,6 glucan that associate with chitin via a β-1,4 linkage (11). The core structure is decorated by unique extensions depending on the pathogen; for instance, O- or N-linked mannans and mannoproteins are found in Candida yeasts, while glucuronoxylomannan and galactoxylomannan coat Cryptococcus (12). Because of their exclusivity, the components of the fungal cell wall contribute a large reservoir of Janeway’s “patterns,” which are now commonly termed “pathogen-associated molecular patterns” (PAMPs). Chitin, mannans, and β-glucan are the three major PAMPs present in all fungi that cause human disease, with the possible exception of Pneumocystis jirovecii which lacks chitin (13, 14). Thus, the framework for induction of an immune response to most fungi follows similar sets of rules and involves recognition of PAMPs by several classes of germ-line-encoded PRRs expressed by innate immune cells, including C-type lectin receptors (CLRs), Toll-like receptors (TLRs), nucleotide-oligomerization domain (NOD)-like receptors (NLRs), and retinoic acid-inducible gene 1 (RIG-I)-like receptors (RLRs), discussed below.

TLRs and Fungal Sensing

By the mid-1980s, the nuclear factor-κB (NF-κB) was known to mediate upregulation of many genes involved in immunity and stress in mammalian cells (15). In Drosophila melanogaster, the NF-κB homologue, Dorsal, was shown by Nüsslein-Volhard and colleagues to drive expression of genes involved in development (16). Furthermore, activation of Toll by its ligand Spätzle was found to be the critical receptor involved in triggering the signaling cascade resulting in activation and nuclear translocation of Dorsal (17). Notably, work emanating from the Hoffman laboratory also demonstrated the expression and nuclear translocation of Dorsal upon microbial challenge (18). Importantly, this group demonstrated that the Spätzle-Toll-Dorsal signaling axis mediated expression of the antifungal peptide drosomycin, with Toll mutant flies succumbing to infection with Aspergillus fumigatus (19). Of interest to note is that Drosophila possesses only an innate immune system, while mammals also have an adaptive immune system. Medzhitov and Janeway subsequently demonstrated that a human cDNA clone of Toll homologue (now known as TLR4) also activated NF-κB and induced expression of costimulatory molecule B7.1 required for activating the adaptive immune responses (20). Seminal findings from Buetler’s laboratory using mice with genetic mutations that abolished recognition of lipopolysaccharide, a major PAMP conserved in Gram-negative bacteria, established that TLR4 is the innate immune sensor of infection critical for host immunity (21).

Since the discovery of TLR4, over 12 mammalian TLRs have been described and their ligands and functions in immunity extensively studied (22). Similar to Drosophila Toll, the extracellular domain of TLRs contains leucine-rich repeat motifs and the intracellular domain structure that is homologous to the interleukin-1 (IL-1) receptor and pathogen resistance proteins found in plants—hence the name TIR (Toll/IL-1 receptor/resistance domain) (23, 24). TLRs transmit signals by interacting with several TIR-containing proteins such as myeloid differentiation primary response (MyD88), TIR domain-containing adaptor protein inducing interferon-β (IFN-β) (TRIF), and TRIF-related adaptor molecule (Fig. 2). With the exception of TLR3 and to some degree TLR4, all TLRs couple the MyD88 adaptor, which interacts with the IL-1 and IL-4 receptor-associated kinase (IRAK1/4) via its N-terminus death domain, resulting in downstream activation of several transcription factors including NF-κB, activation protein 1, interferon regulatory factor (IRF) 3, IRF5, IRF7, and mitogen-activated protein kinases that induce expression of inflammatory mediators (25).

FIGURE 2.

Signaling pathways involved in antifungal immunity.

Mice lacking MyD88 have been shown to be sensitive to infections with various pathogens, presumably, in addition to TLR unresponsiveness, defective immune activation due to a lack of IL-1R, IL-18R, and IL-33R signaling, which also utilize the TIR-MyD88 module (26–28). In experimental settings, MyD88-deficient mice displayed broad susceptibility to bacterial, viral, parasitic, and fungal pathogens, although only selected bacterial infections were observed in humans with deficiencies in MyD88 or IRAK4 (see reference 29 for a detailed review). Nonetheless, polymorphisms in TLR4 were found to be associated with pulmonary infections with Aspergillus and systemic candidiasis (30–32). Additionally, mutations in TLR3 resulting in reduced activation of NF-κB and decreased production of IFN-γ were associated with cutaneous candidiasis (33). Furthermore, defective TLR9 expression was implicated in allergic bronchopulmonary aspergillosis in humans, suggesting a possible accessory role of TLRs in antifungal mammalian immunity (32). Thus far, fungal PAMPs including α-1,4-glucan, glucuronoxylo/phospholipido-mannans, cytosolic nucleic acids, and O-linked/rhamnomannans have been reported to be recognized by TLRs (12) (Fig. 3).

FIGURE 3.

PRRs and the fungal components they recognize. (Adapted with modifications from reference 12).

Contribution of NLRs and RLRs

NLRs comprise a large family of cytoplasmic receptors made up of three characteristic domains (NH₂-terminal protein-protein interaction domain, a central NOD, and a C-terminal leucine-rich repeat) and can be grouped into subfamilies based on the nature of the NH₂-terminal domain structure (34). Of particular interest to antifungal immunity is NALP3—or NACHT (NAIP [neuronal apoptosis inhibitor], CIITA [MHC class II transcription activator], HET-E [heterokaryon incompatibility protein], and TP1 [telomerase-associated protein])–LRR (leucine-rich repeats)–PYD (pyrin domains)-containing protein 3—which activates a multiprotein complex known as the inflammasome by recruiting the adaptor molecule apoptosis-associated speck-like protein containing a caspase-associated recruitment domain (CARD) to the protein-protein interaction pyrin domain that subsequently activates caspase-1-mediated cleavage of the inflammatory cytokines, pro-IL-1β and pro-IL-18, to their mature forms (35–38) (Fig. 2). To this end, several studies have linked defective NALP3-mediated IL-1β release with susceptibility to several fungi including Candida albicans and A. fumigatus (39–42). Interestingly, the main component of Cryptococcus neoformans capsule, glucoronoxylomannan, was suggested to facilitate intracellular survival of this pathogen via inhibition of Syk-mediated NALP3 inflammasome activation (43).

RLRs are another important family of cytosolic sensors responsible for detecting double-stranded RNA and are distinct from TLR detection of endosome-associated RNA (22). Members of this family include RIG-I, melanoma differentiation-associated gene 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2). All these proteins have a central ATPase-containing DExD/H box helicase domain as a common feature (22). RIG-I and MDA5 also contain N-terminal CARD domains that facilitate downstream signaling via an adaptor molecule mitochondrial antiviral signal, resulting in activation of mitogen-activated protein kinase, NF-κB, type I IFNs (IFNα/β), and IFN-stimulated genes (44–46) (Fig. 2).

To date, MDA5 is the only member of the RLR family suggested to play a role in antifungal defense. In both mice and humans, altered expression or function of MDA5 correlated with chronic mucocutaneous candidiasis and was associated with susceptibility to systemic Candida infections, presumably due to reduced expression of type I IFNs (47). Although the C. albicans ligand(s) that mediates activation of MDA5 is currently unknown, there is a possibility that fungal components gain access to the cytosol and induce immune responses, but this warrants further investigation. The role of type I IFNs, IRFs, and IFN-stimulated genes in antifungal immunity also needs more clarity. For instance, a protective role of IFNs was reported in mice and humans infected with C. albicans (48, 49), whereas in a different study using Candida glabrata, these cytokines were associated with susceptibility to infection (50).

CLRs Are Central for Recognition of Fungi and Antifungal Host Defense

CLRs constitute a superfamily of more than 1,000 soluble and membrane-bound proteins classically characterized by the presence of at least one C-type lectin domain (CTLD) and can be clustered into at least 17 subgroups based on architecture of the CTLD (51). The EPN (Glu-Pro-Asn) motif within the CTLD specifies binding to mannose, N-acetylglucosamine, l-fucose, and glucose, whereas QPD (Gln-Pro-Asp) confers specificity to galactose and N-acetylgalactosamine (52). While calcium-dependent binding of carbohydrates is commonly associated with the function of the CTLD, many CTLDs are known to bind a vast array of noncarbohydrate ligands including proteins, lipids, and lipoproteins (51). Of note are membrane CLRs expressed on myeloid cells that function as PRRs and prime the immune system such as the dendritic cell-associated C-type lectin 1 (Dectin-1, also known as CLEC7A), discovered in 2001 by Brown and Gordon as the receptor for β-1,3-glucan (a PAMP expressed in many, if not all, medically important fungi) (53).

Dectin-1 binding of β-glucan results in phosphorylation of the integral immunoreceptor tyrosine-based-like motif embedded in the cytoplasmic tail providing docking sites for the Src homology 2 domain of spleen tyrosine kinase (Syk) (54). Syk subsequently activates the signaling scaffold CARD9-Bcl-10-Malt-1 through PKCδ and a recently described complex of CARD9-H-Ras-Ras-GRF-1, which promotes nuclear translocation of several transcription factors including NF-κB, nuclear factor and activator of transcription, IRF1, IRF5, and the activation of extracellular-regulated kinases pathway (55). In addition, unlike the Syk pathway, which activates both classical NF-κB (p65 and c-Rel) and noncanonical NF-κB (RelB), Dectin-1 also activates the Raf-1 kinase pathway that sequesters RelB by forming inactive p65/RelB dimers, thereby promoting Syk-mediated activation of p65 (56) (Fig. 2). Signaling via Dectin-1 regulates several processes including phagocytosis, autophagy, NETosis, respiratory burst, and gene expression of proinflammatory mediators including cytokines such as tumor necrosis factor (TNF), IL-23, IL-6, and chemokines (C-C motif) CCL2, CCL3, and (C-X-C motif) CXCL1 (57). Activation of the Dectin-1-Syk-CARD9 signaling pathway by fungi upregulates IL-1β and IL-18 transcripts and is implicated in “priming” of the NALP3/caspase 1 inflammasome, while reactive oxygen species, potassium efflux, and cathepsin B activate inflammasome activity (58, 59). More recently, the Dectin-1-Syk pathway was reported to activate a noncanonical inflammasome comprising MALT1-caspase 8 and apoptosis-associated speck-like protein containing a CARD domain that cleaved pro-IL-1β (60).

Several studies in mice have demonstrated a critical role of Dectin-1 in host defense against fungal infections. In mice lacking Dectin-1, a failure to sense the presence of fungi results in tissue overgrowth of C. albicans or A. fumigatus and is associated with an overall failure to induce appropriate inflammatory responses (such as IL-6 and granulocyte colony-stimulating factor [G-CSF]) that would normally result in the recruitment of monocytes and neutrophils to the site of infection (61–64). Importantly, individuals with polymorphisms in Dectin-1 that affect the ability of leukocytes to bind β-glucan display increased susceptibility to chronic mucocutaneous candidiasis, recurrent episodes of vulvovaginal candidiasis, onychomycosis, and intra-abdominal Candida infections and a reduced Th17 cell response (discussed below) (65).

CLRs such as Dectin-2, macrophage-inducible C-type lectin (Mincle), dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), and the mannose receptor have also been shown to elicit an immune response against fungi (66–68). Dectin-2 recognizes α-mannans from several fungi including C. albicans, Histoplasma capsulatum, and C. neoformans, as well as glycoproteins containing O-linked mannobiose-rich residues from Malassezia (69, 70). Signals transmitted via Dectin-2 activate the Syk-CARD9 pathway through association with an integral immunoreceptor tyrosine-based-bearing Fc receptor γ chain (FcRγ) adaptor molecule, which results in improved reactive oxygen species production by neutrophils, expression of IL-23p19, and IL-1β release by APCs (71, 72). Dectin-2-deficient mice display increased susceptibility to infection with C. albicans (Fig. 2) (73, 74). Similar to Dectin-2, Mincle recognition of glucosyl- and mannosylglycolipids from Malassezia and ill-defined ligands from C. albicans and Fonsecaea pedrosoi also activates the Syk-CARD9 pathway via the FcRγ adaptor (75). However, the role of Mincle in antifungal immunity may be context dependent. In a recent report, interaction of Mincle with the causative agent of chromoblastomycosis, Fonsecaea monophora, was shown to suppress IL-12p35 (an important cytokine required for T helper cell polarization) via Syk-CARD9 activation of E3 ubiquitin ligase Mdm2-dependent degradation of the transcription factor IRF-1 (76).

DC-SIGN and the mannose receptor recognize branched N-linked mannans and have been implicated in trafficking of mannosylated fungal antigens into the APC endocytic pathway, facilitating antigen processing and presentation to T helper cells (77, 78). In mice, a lack of mannose receptor was shown to result in susceptibility to infection with C. neoformans but not C. albicans (79, 80).

Although mouse models and in vitro studies with human leukocytes provide experimental support for a role in immunity of CLRs, mentioned above, to date, only polymorphisms in Dectin-1 and mutations in the signaling adaptor molecule CARD9 (which acts downstream of Syk-coupled CLRs) have been associated with susceptibility to fungal infections in humans. While polymorphisms in Dectin-1 were predominantly linked with superficial infections, CARD9 deficiency in both mice and humans was found to be critical for protection against systemic Candida and Exophiala infection, particularly of the central nervous system (49, 65, 81–83). CARD9-mediated protection against Candida spinal osteomyelitis and meningoencephalitis was shown to require neutrophil chemotaxis into the central nervous system (49, 65, 81, 84, 85).

EFFECTOR MECHANISMS DRIVING HOST PROTECTION FROM FUNGI

Epithelium

Apart from the obvious physical barrier function, the epithelium, through secretion of factors such as mucins and antimicrobial peptides, plays an important role in protection against microbial tissue invasion (86). Salivary mucins in host defense and disease prevention were shown to repress formation of C. albicans hyphae (a morphological form thought to be associated with host invasion) and displayed direct candidacidal activities in the oral cavity, respectively (87).

Although research into “self versus nonself” interactions has for the most part focused on pathogens, commensal microorganisms, including the mycobiota (fungal communities), colonize mucosal surfaces such as the gut, and their role in health and disease is slowly gaining attention (88). For instance, increased Candida gut colonization and differential fungal profiles were observed in patients with inflammatory bowel disease and in ulcerative colitis patients, respectively (89–91). Notably, in these studies, an absence of Dectin-1 signaling correlated with severe forms of ulcerative colitis in both mice and humans. Intriguingly, Dectin-1 and CARD9 have also been implicated in regulation of intestinal homeostasis by modulating gut microbiota. Dectin-1-mediated induction of antimicrobial peptides S100A8 and S100A9 paralleled decreases in Lactobacilli numbers; these are commensal gut bacteria best known for metabolizing tryptophan into aryl hydrocarbon receptor ligands and generation of regulatory helper CD4 T cells (discussed below) (92, 93). By contrast, gut microbiota from CARD9-deficient mice failed to metabolize tryptophan and consequently displayed decreased aryl hydrocarbon receptor ligands and IL-22 and increased sensitivity to colitis (94).

Phagocytes

Derived from the Greek term phagein, meaning “to eat,” phagocytes are critical for clearing foreign bodies and debris from dead or dying cells. Tissue-resident macrophages and neutrophils are key phagocytic cells that mediate host protection against fungi (95). Here, a few examples of the mechanisms deployed by phagocytic cells in antifungal immunity are described. Upon sensing fungi, macrophages recruit other immune cells to sites of infection through secretion of proinflammatory cytokines and chemokines, discussed above (95). In vivo depletion of macrophages or deficiency of CX₃C-chemokine receptor 1 (CX₃CR1), which is associated with poor survival of macrophages, was observed to result in unrestricted fungal growth in tissues and heightened mortality rates in mice systemically challenged with C. albicans (96). Importantly, patients with decreased expression of CX₃CR1 are highly susceptible to disseminated candidiasis (96). In the lung, alveolar macrophages kill inhaled A. fumigatus conidia and Pneumocystis carinii cysts through mechanisms involving reactive oxidant intermediates (97, 98). As immunologists increasingly define functional “subsets” of cells, specific populations of monocytes and macrophages that protect against fungi are being described. For instance, several studies have reported a protective role of CCR2⁺Ly6^hi inflammatory monocytes in systemic candidiasis (99, 100). In mice, depletion of this subset resulted in lethal invasive aspergillosis (101).

The importance of neutrophils is underscored by the fact that neutropenia is one of the major risk factors driving disseminated fungal infections in mouse models and in humans (95, 102, 103). Neutrophils accomplish their potent fungicidal activities through generation of reactive oxygen radicals and nonoxidative effector mechanisms including secretion of lysozyme, lactoferrin, elastase, β-defensin, gelatinases, and cathepsin G (104). Neutrophil extracellular traps, released during the last stages of neutrophil active cell death, known as NETosis, have been implicated as another strategy of antifungal defense (105–107). Neutrophil extracellular trap formation coincides with neutrophil degranulation and release of microbicidal factors such as cathelicidin (108). Proteinase 3-mediated cleavage of cathelicidin into LL-37 was shown to interfere with fungal cell membranes, suppress formation of biofilms and fungal attachment, induce reactive oxygen species production, enhance chemotaxis, and suppress neutrophil apoptosis (109–114).

Reinforcing Effectors: Cytokines

The basics of the immune process as we currently understand it were discussed in the preceding sections; that is, PAMPs are recognized by PRRs expressed by innate immunocytes, resulting in activation of the innate immune system. As described above, the bridge between innate and adaptive host immunity is mediated by professional APCs, dendritic cells, discovered in 1973 by Steinman and Cohn (115). Dendritic cells provide signal 1 (antigen-presentation) and signal 2 (costimulation) to naive T helper CD4 cells (bearing αβ T cell receptors) initiating “effector” antigen-specific helper CD4 T cell responses (116, 117). Other aspects including generation of cytotoxic CD8 T cells and B cell development into immunoglobulin-producing plasma cells are also activated but will be not be discussed here. In the last section, we discuss the nature of helper cells induced upon fungal recognition and the type of help they provide (Fig. 4).

FIGURE 4.

Schematic representation of the sequential inflammatory immune reaction involved in antifungal immune responses.

Cytokines produced by APCs in response to PAMPs provide signal 3i, which instructs polarity of T cells (Th) into functionally distinct helper subsets. Members of the IL-12 family of cytokines are known critical mediators of Th polarization, and these include IL-12 (IL-12p35:IL-12p40), IL-23 (IL-12p19:IL-12p40), IL-27 (IL-12p28:EBi3), and IL-35 (IL-12p35:EBi3) (118). Binding of these cytokines to their cell surface receptors activates the Janus kinase and signal transducer and activator of transcription (STAT) signaling pathways, trans-activating gene expression, and in the case of naive T cells, differentiation (119).

IL-12 and IL-27 secretion by innate immune cells as well as innate lymphocyte-derived IFN-γ skew naive cells toward a Th1 differentiation program driven by STAT1, STAT4, and T box transcription factor T-bet (120, 121). Classically, Th1 cells are defined by production of mainly IFN-γ but also produce other important proinflammatory cytokines such as TNF and GM-CSF. The role of Th1 cells in protective immunity against most fungal pathogens in both experimental mouse models and humans is well established. Several laboratories have shown that IFN-γ and TNF stimulate macrophages to release nitric oxide and reactive oxygen species, causing intracellular growth arrest of several fungal species such as H. capsulatum (122–124). An absence of Th1 immunity or expression of the effector cytokines as demonstrated in IL-12, IFN-γ, or TNF deficiency in both mice and humans results in increased susceptibility to a myriad of fungal infections, including C. neoformans. Patients on IFN-γ immunotherapy show augmented protection against aspergillosis, cryptococcosis, and coccidioidomycosis (125). GM-CSF, on the other hand, was shown to mediate antifungal responses in macrophages by a mechanism involving sequestering intracellular zinc (a micronutrient required by yeast cells), stimulating reactive oxygen species, as well as neutrophil recruitment (126, 127). In contrast, several laboratories reported that STAT1 (signal transducer of IL-27, type I IFNs, and IFN-γ) gain of function mutations in humans resulted in autosomal dominant chronic mucocutaneous candidiasis, potentially due to excess induction of type 1 IFNs which promote IL-27 and IL-10, resulting in inhibition IL-17-producing T cells (128, 129) (Table 1).

TABLE 1.

Selected human genetic associations with fungal susceptibility discussed in this article

Gene	Immunological phenotype	Associated disease
Dectin-1	Dimunition of cytokine responses	Chronic mucocutaneous candidiasis, vulvovaginal candidiasis, onychomycosis, and intra-abdominal Candida
CARD9	Dimunition of cytokine responsesDefective neutrophil chemotaxis	Disseminated candidiasis
TLR1	Decreased IL-1β, IL-6, and IL-18	Candidemia
TLR2	Decreased IFN-γ and IL-8	Candidemia
TLR3	Reduced activation of NF-κB and decreased IFN-γ	Cutaneous candidiasis
NALP3	Decreased IL-1β?	Candidiasis-mediated vestibulitis
MDA5	Decreased type I IFNs	Systemic candidiasis
STAT1	Decreased IL-17 production, elevated cell response to IFNs, IFN-γ,s and IL-27Defective IL-12R and IL-23R signaling	Chronic mucocutaneous candidiasis, coccidioidomycosis, histoplasmosis
STAT3	Defective Th17 polarization, decreased IL-17/IL-22-expressing T cells, decreased IL-17-induced antimicrobial peptides	Chronic mucocutaneous candidiasis, coccidioidomycosis, histoplasmosis, cryptococcosis, aspergillosis, nail dermatophytosis
Autoimmune regulator	Neutralizing antibodies to IL-17 and IL-22	Chronic mucocutaneous candidiasis
IL-17/IL-17R	Lack of IL-17 cellular responses	Chronic mucocutaneous candidiasis
IL-12RA	Impaired IL-12 and IL-23-mediated expression of IFN-γ	Coccidioidomycosis, paracoccidioidomycosis, histoplasmosis, cryptococcosis

It is now well established that IL-17-producing T cells play a significant role in the clearance of fungi, particularly at the mucosae. This lineage differs from Th1 in that it requires STAT3 activation by IL-6 and IL-21 as well as transforming growth factor β induction of retinoid-related orphan receptor γt, while IL-23 is required for stabilization of the phenotype (130). Effector cytokines, particularly IL-17 and IL-22, were shown to promote the release of various antimicrobial peptides by keratinocytes and epithelial cells as well as strengthening of the mucosal barrier preventing disease dissemination (131–134). The importance of this lineage is made obvious by the heightened susceptibility to a wide range of fungal infections in individuals with loss of function mutations in STAT3 or defects in the IL-17 signaling axis (135–138). In addition, mutations in the autoimmune regulator gene that result in antibodies targeted at Th17 cytokines predispose to chronic and recurrent mucocutaneous candidiasis as noted in individuals with autoimmune polyendocrinopathy with candidiasis and ectodermal dystrophy (APECED) (125). Decreased antimicrobial peptide secretion as well as reduced neutrophil-attracting CXC chemokines due to a lack of Th17-mediated immune responses are thought to be key drivers of susceptibility to mucosal fungal infections (135, 138).

Polarization toward the Th2 subset requires IL-4-mediated STAT6 activation and GATA3 expression, which drive induction of the signature cytokines: IL-4, IL-5, and IL-13. Th2-cytokines are considered important for promoting alternatively activated macrophages reported to permit intracellular growth of fungi due to decreased nitric oxide expression (139). In mouse models, a pathogenic role of an elevated Th2 immune response is best exemplified by the observation of progressive lung infections with C. neoformans and H. capsulatum (69).

Regulatory T cells (T regs) are unique helper cells that play the essential role of restraining immune responses limiting collateral damage to the host. Mechanisms of action mediated by T regs include secretion of inhibitory cytokines such as IL-10, transforming growth factor β, IL-27, IL-35, contact-dependent mechanisms, and sequestration of IL-2 (125). Decreased T reg numbers as observed in TLR2- or CCR5-deficient mice are thought to drive efficient clearance of several fungal infections including C. albicans (140, 141). Interestingly, defective natural T reg development is associated with pathogenesis of chronic mucocutaneous candidiasis in APECED patients, highlighting the importance of the delicate balance between host-driven immune pathology and resolution of infection (142).

AIDS and susceptibility to fungal infections such as Cryptococcus, Candida, Pneumocystis, and Histoplasma have focused attention on the critical contribution of T cells to the host defense arsenal against fungal pathogens (143). Yet cells of the innate immune system such as natural killer cells, γδ T cells, and the recently described innate lymphoid cells also contribute to the effector cytokine pool, and the contribution of these cells in antifungal defense is under intense investigation (133). A case in point is work from LeinbundGut-Landmann’s laboratory demonstrating that antibody depletion of innate lymphoid cells in Rag-deficient mice (which lack mature classical T helper cells) resulted in attenuation of IL-17 and increased susceptibility to oropharyngeal candidiasis infection (144). Moreover, this group also reported that IL-17-mediated immune responses are critical for natural killer cell activation and secretion of GM-SCF, which is essential for antifungal immunity (145). Another study reported natural killer cell direct killing of C. neoformans and Cryptococcus gattii through mechanisms that involved perforin (146). The contribution of these cells to invasive fungal disease and their relevance in human disease are still under investigation.

IMMUNE-BASED THERAPIES AGAINST FUNGAL INFECTIONS

Vaccines

A major hurdle in this area has been largely impacted by the lack of understanding of the requirements for induction of protective immunity and defining surrogate markers for these responses. This is made apparent by the lack of any fungal human vaccine approved for clinical use to date. However, there are exciting developments including studies showing that mice immunized with β1-3-d-glucan-laminarin-diphtheria toxoid conjugate vaccine or antigens encapsulated with glucan particles induce a strong antibody response and a long-term antifungal T cell response that protects mice against several fungal species including Cryptococcus and Candida (147, 148). The use of a live vaccine strategy has also shown promising results in several settings. Examples such as immunization with a C. neoformans-expressing IFN-γ strain offer advantages for developing therapeutic vaccines that offer protection in conditions where CD4 helper T cells are deficient (149).

Adaptive Cell Therapies

Fungal-specific T cell transfer studies have been shown to improve disease outcomes in high-risk individuals such as hematopoietic transplant patients by providing enhanced control of Aspergillus antigenemia and reducing the mortality rate (150). More recently, T cells bearing modified chimeric antigen receptors have been investigated for potential use in fungal diseases (for details about chimeric antigen receptor technology see reference 151). In a proof-of-concept study, T cells bearing Dectin-1 (directing T cell specificity to fungal cell wall glucans) were shown to mediate protection against A. fumigatus (152).

As discussed above, neutropenia is associated with susceptibility to fungal infections, and approaches that reduce the duration of neutropenia in chemotherapy patients have obvious attractive therapeutic implications. Thus, using G-CSF and GM-CSF to increase the yield of granulocytes from donors is gaining a lot of interest in clinical settings where granulocyte transfusions are frequently used as supportive therapy (153). Another exciting approach is immune modulation via exogenous provision of costimulation to effectively activate the immune responses. This was demonstrated by a recent study showing that chronicity of chromoblastomycosis caused by F. pedrosoi was due to lack of costimulation with TLRs; thus, topical application of TLR ligands such as imiquimod (which signals via TLR7) resulted in an adequate inflammatory response that resolved the infection (154).

FUTURE PERSPECTIVES

In the 27 years since the inception of the PRR theory, much progress has been accomplished. We now know that there are other pathogen-sensing mechanisms exemplified by ideas such as the guard theory, which posits that the mammalian immune system detects cellular processes that are targeted by the pathogen’s virulence factors. Furthermore, identification of the cell biology components such as compartmentalization of sensing receptors into membrane-bound, cytosolic, or endosomal modules has emerged as an important framework for the activity of the innate immune system. Understanding how all of these pathways are integrated and controlled from different compartments in the context of fungal infections will be essential for advancing our understanding of antifungal immune responses and developing future immunotherapies. Moreover, additional efforts using a systems biology approach to better define immunological, cellular, and molecular pathways involved in antifungal responses at different anatomical sites will also expand our knowledge. Studies characterizing the complex interplay between host genetics, microbiota, and the virome in health and disease will help decipher an age-old question: what is the basis for mammalian protective immunity?