Immunity to Fungi

Abstract

The global increase in fungal disease burden, the emergence of novel pathogenic fungi, and the lack of fungal vaccines have focused intense interest in elucidating immune defense mechanisms against fungi. Recent studies in animal models and in humans identify an integrated role for C-type lectin and Toll-like receptor signaling in activating innate and adaptive responses that control medically relevant fungi. Beyond the critical role of phagocytes in host defense, the generation and balance of specific T helper subsets contributes to sterilizing immunity. These advances form a basis for the development of fungal vaccines and immune-based therapeutic adjuncts.

Introduction

Humans encounter, ingest, and inhale fungi daily. Although only several hundred of the estimated 10⁵ fungal species cause human disease, the incidence of clinically relevant fungal infections has risen substantially in the past 3–4 decades. This increase is largely due to the AIDS pandemic and the advent of transplantation, chemotherapy, immunosuppression, and vascular access in modern medicine. In sub-Saharan Africa, cryptococcal meningitis exceeds well-known infections such as tuberculosis as a cause of HIV-associated deaths [1]. The Pacific Northwest Cryptococcus gattii outbreak [2], and the worldwide amphibian (chytridiomycosis; Batrachochytrium dendrobatidis) and North American bat extinction (white nose syndrome; Geomyces destructans) highlight emerging fungal pathogens as agents of human and animal disease [3,4].

Fungi are eukaryotic, saprophytic organisms with a rigid cell wall. Yeasts form round, oval, or spherical cells that usually divide asexually by budding. Molds form asexual spores (conidia) that are dispersed in the environment and germinate into tubular filaments, termed hyphae. Dimorphic fungi (e.g. Blastomyces dermatitidis, Coccidioides posadasii, Histoplasma capsulatum, and Paracoccidioides brasiliensis) can exist as yeast cells (in human tissues) or as hyphae (in soil) with environmental conditions (e.g. temperature) guiding the transition between morphologic states.

Fungi cause a broad range of diseases with syndromes that involve superficial, tissue-invasive, and allergenic manifestations (Fig. 1). Route of acquisition, tissue tropism, and nutritional requirements all contribute to fungal pathogenesis. Deep-seated mycoses develop from inhalation of infectious propagules into the airways of susceptible hosts (e.g. Aspergillus fumigatus, Cryptococcus neoformans, Pneumocystis jiroveci) or penetration of mucosal breaches by commensal organisms such as Candida albicans. Disruption of host microbial communities (e.g. vulvovaginal candidiasis ensuing antibiotic therapy) or metabolic disorders (e.g. mucormycosis in diabetic ketoacidosis; [5••]) can predispose to superficial and invasive disease.

Figure 1.

Medically relevant fungi, syndromes, and common sites of disease.

The asterisks (*) indicate examples of organ involvement following fungal dissemination from the primary site of infection. Disseminated fungal disease occurs typically in severely immune compromised patients, e.g. disseminated cryptococcosis in patients with CD4 T cell dysfunction.

This review focuses on emerging concepts in fungal recognition and effector mechanisms, the generation of protective T cell responses, and current efforts to develop vaccine-based therapies for vulnerable patient groups. A more in-depth analysis of innate and adaptive immunity to human fungal pathogens and host evasion strategies is discussed elsewhere [6–8].

Fungal innate immune activation

Fungi bind antibodies, complement, surfactant, and soluble pattern recognition receptors at portals of entry. For example, the soluble collectin pentraxin-3 binds to inhaled A. fumigatus conidia, triggering complement deposition and CD32-dependent uptake by neutrophils [9]. Opsonization by these mechanisms promotes fungal uptake and innate immune activation by a wide range of phagocytic and signaling receptors [6].

Highly conserved fungal cell wall components and nucleic acids trigger innate immune activation in macrophages, dendritic cells, and neutrophils, as well as in epithelial cells [6]. The latter is exemplified by a MAP kinase response that discriminates between C. albicans yeast cell and filamentous growth [10•]. Deciphering the role of specific pattern recognition receptors in these interactions is complex since fungal cell wall composition changes during yeast cell and filamentous growth as well as after cell division (e.g. C. albicans bud scars), morphotype switching, and antifungal drug exposure [11]. In some fungi immune-activating polysaccharides are concealed beneath immunologically inert polymers such α-glucan (H. capsulatum) and hydrophobins (A. fumigatus) [6].

Toll-like receptors

The ligation of fungal cell wall polysaccharides and nucleic acids [12–14] activates murine Toll-like receptor (TLR; TLR1-4, 6, 7, and 9) signaling (Fig. 2). Although deficiency of the TLR adaptor protein MyD88 renders mice susceptible to C. neoformans and C. albicans and delays A. fumigatus clearance [6], fungal infections are not a prominent clinical phenotype in humans with MyD88 deficiency [15], suggesting that MyD88-dependent signals are not essential to prevent the development of symptomatic fungal infections in pediatric patients. However, TLR1 polymorphisms predispose to candidemia [16] and TLR4 polymorphisms to invasive aspergillosis in allogeneic hematopoietic stem cell transplant (HCT) patients [17], consistent with the notion that TLR-dependent signals contribute to optimal host defense against fungal pathogens in humans. The biological role of other receptors, such as the mannose receptor, DC-SIGN, CD36, complement receptor 3, and Galectin-3, implicated in fungal recognition and immune activation is discussed in detail elsewhere [6–8].

Figure 2.

Pattern recognition receptors and signaling pathways contributing to innate and adaptive antifungal responses. The recognition of fungal cell wall components by CLRs induces Syk-dependent and –independent signaling events that converge in the activation of NFAT, NF-κB and processing enzymes and result in the production of cytokines including those that drive T cell differentiation. The depicted signaling pathways are all induced by Dectin-1, but whether in particular the inflammasome and RAF signaling are also activated by Dectin-2 and Mincle, and whether Mincle can trigger antifungal T cell differentiation remains to be demonstrated. Phospholipomannans and O-linked mannans are recognized by TLRs at the plasma membrane, whereas fungal nucleic acids are sensed by endosomal TLRs and induce NF-κB-, MAPK- and IRF-dependent cytokine production. Numbers refer to human genetic polymorphisms listed in Table I. Th17 activation in response to CLR signaling is drawn out of scale to illustrate the polymorphisms that affect Th17 differentiation and function.

C-type lectin receptors

Three immunoreceptor tyrosine-based activation motif (ITAM)-dependent C-type lectin receptors (CLRs; Dectin-1, Dectin-2, and Mincle) activate innate and adaptive antifungal responses (Fig. 2). Dectin-2 and Mincle lack an intracellular ITAM motif but pair with FcRγ, an ITAM-containing adaptor protein. Particulate β-(1,3)-glucan activates Dectin-1 signaling through a mechanism that excludes the phosphatases CD45 and CD148 from the phagocytic cup, facilitating phosphorylation of its intracellular ITAM-like motif and activation of spleen tyrosine kinase (Syk) [18••]. Dectin-1/Syk signaling induces the assembly of the CARD9/BCL10/MALT1 (Caspase recruitment domain-containing protein 9/ B cell leukemia/lymphoma 10/Mucosa-associated lymphoid tissue lymphoma translocation protein 1) complex via PKCδ [19] and subsequent activation of the canonical NK-κB pathway via the kinase TAK1, resulting in TNF, IL-6, IL-23, and IL-1β synthesis C. albicans and particulate β-(1,3)-glucans can trigger Syk- and phospholipase Cγ2 (PLCγ2)-dependent nuclear factor of activated T cells (NFAT) activation via Dectin-1 or Dectin-2, resulting in the production of IL-2, IL-10, lipid mediators, and Egr transcription factors [6]. Dectin-1-dependent phagocytosis by macrophages and non-canonical NK-κB activation via Raf-1 [20] illustrate Syk-independent functions of the receptor.

Dectin-1, Dectin-2, and Mincle contribute to murine host defense against systemic candidiasis [21–23], aspergillosis [24] and pneumocystosis [25]. While individual CLRs are dispensable, deficiencies in PKCδ [19] PLCγ2 [26], and CARD9 [27] result in rapid lethality in systemic candidiasis. The association of mucosal candidiasis and aspergillosis (in the context of allogeneic HCT) and defined allelic variations in Dectin-1 and CARD9 (Table I) further illustrates the critical role of the CLR/Syk/CARD9 pathway in antifungal defense.

Table I.

Genetic Polymorphisms associated with Spontaneous Fungal Disease Development

	Gene	Mutation1	Associated Disease	Ref.
1	Dectin-12	Y238X (AR) biological activity	mild CMC3 (vaginal candidiasis, onychomycosis) susceptibility to aspergillosis (2 of 3 studies) and Candida colonization in HCT recipients	[85] [86–88]
2	CARD9	Q295X (AR) lack of protein expression	CMC3 (thrush, vaginal candidiasis), dermatophytosis	[89]
3	gp91phox p22phox p67phox p47phox	X-linked (gp91phox) or AR (other subunits) mutations NADPH oxidase function	CGD bacterial and fungal infections, particularly aspergillosis (A. nidulans is a significant pathogen)	[90]
4	IL-12Rβ1	various mutations (AR)	mild CMC (thrush, in 25% of cases)	[91]
5	STAT3	mutations in DNA-binding or SH2 domain (AD); DNA binding activity (DN)	Hyper IgE syndrome (HIES) CMC (thrush, vaginal candidiasis, onychomycosis, dermatophytosis), aspergillosis, recurrent Staphylococcus aureus infections	[92,93]
6	STAT1	mutations in coiled-coil domain4 (AD) gain of function mutation	CMC	[94,95]
7	IL-17F	S65L (AD with incomplete penetrance) deficient receptor binding	CMC	[96]
8	IL-17RA	Q284X (AR) lack of protein expression	C. albicans dermatitis, S. aureus dermatitis	[96]
9	AIRE	various (~ 60 known, AR) IL-17A, IL-17F, and IL-22 autoantibodies	APS-I / APECED5: autoimmune APS-I / APECED5: autoimmune endocrinopathies, hypoparathyroidism, adrenal insufficiency, CMC	[97,98]

¹first line: mutation (AR = autosomal recessive, AD = autosomal dominant); second line: effect on protein function

²The Dectin-1 Y238X allele is a common polymorphism in specific European and African populations.

³CMC = chronic mucocutaneous candidiasis

⁴the coiled-coil domain plays a key role in unphosphorylated STAT1 dimerization and STAT1 nuclear dephosphorylation

⁵APS-I = autoimmune polyendocrine syndrome type I; APECED = autoimmune polyendocrinopathy candidiasis ectodermal dystrophy

Simultaneous CLR and TLR activation by fungal antigens augments NF-κB responses in macrophages and dendritic cells [6]. Fonsecaea pedrosoi, the most common cause of chromoblastomycosis, triggers Mincle/FcRγ/Syk-dependent cytokine production that is inadequate to eradicate the organism, resulting in chronic infection [28••]. Administration of the TLR7 agonist imiquinod enhances cytokine production by F. pedrosoi-stimulated dendritic cells in vitro and enhances fungal clearance in a TNF-dependent manner in vivo, providing a novel therapeutic strategy for this disfiguring soft tissue infection.

Inflammasome function during fungal infection

Production of IL-1β is critical for host defense in murine models of candidiasis [6]. The production and release of IL-1β requires two independent signals: one regulating the transcription and translation of pro-IL-1β, the other inducing its proteolytic cleavage into active IL-1β. Fungi trigger both steps of IL-1β synthesis by CLR-dependent caspase activation via the assembly of inflammasomes with distinct subunit composition. The NOD-like receptor NLRP3 (Nucleotide oligomerization domain-like receptor family, pyrin domain containing 3) and the adaptor protein ASC (Apoptosis-associated speck-like protein containing a CARD) form the scaffold of the NLRP3 inflammasome for caspase-1-activation. NLRP3- and ASC-deficient mice lack caspase-1 activation and IL-1β secretion and display increased mortality in response to C. albicans infection [29–32]. In humans, allelic variations in NLRP3 have been associated with recurrent vulvovaginal candidiasis [33].

The NLRC4 (NLR family CARD domain-containing protein 4) inflammasome is protective in systemic candidiasis and its function may be particularly important in the stromal compartment [34]. However, alternative IL-1β processing mechanisms have been reported. Caspase-1 may exist in an active form in circulating monocytes and induce IL-1β maturation independent of additional activation signals [35]. In neutrophils, caspase-1-independent IL-1β maturation is also mediated by serine proteinases, including proteinase 3, elastase and cathepsin G. Finally, C. albicans or A. fumigatus-induced Dectin-1/Syk signaling can recruit caspase-8 into a complex with CARD9, BCL10, and MALT1 to generate IL-1β [36•].

Innate Fungal Killing Mechanisms

Beyond the established roles of mononuclear phagocytes, neutrophils, and NK cells in antifungal defense [6], recent studies identify a role for plasmacytoid dendritic cells in aspergillosis [37] and invariant NKT (iNKT) cells in cryptococcosis [38] and aspergillosis [39]. iNKT cells accumulate and produce interferon-γ (IFN-γ) in fungus-infected airways [39]. In vitro, a wide range of fungi stimulate dendritic cells to activate iNKTs in a Dectin-1- and CD1d-dependent fashion, with no apparent requirement for fungal lipid antigen presentation [39].

NADPH oxidase is a conserved antifungal effector mechanism that underlies Dectin-1-dependent [24] and -independent activation, as demonstrated by β2-integrin (CD18)-, phosphoinositide-3-kinase-β- and -δ-, and Syk-dependent ROS activation by killed A. fumigatus hyphae [40]. Control of neutrophil NADPH oxidase activity by Vav guanine nucleotide exchange factors appears to be a critical defense mechanism against systemic candidiasis [41]. Loss of NADPH oxidase activity results in chronic granulomatous disease (CGD) in humans and GCD patients have a 25–40% lifetime risk of developing aspergillosis (Table I). Beyond the production of fungicidal ROS, loss of NADPH oxidase-dependent indoleamine-2,3-dioxygenase activity results in dysregulated IL-17 production and lethal immunopathology in a murine model of CGD [42]. Restoration of NADPH oxidase activity by gene therapy can resolve treatment-refractory aspergillosis [43] and correlates with the ability of neutrophils to form extracellular traps by releasing decondensed chromatin fibers decorated with antifungal proteins, e.g. calprotectin. These neutrophil extracellular traps ensnare C. albicans [44] and A. fumigatus hyphae [45], though their role in fungal killing remains controversial [46].

The phosphatase calcineurin B, the target of cyclosporine A and FK506, controls neutrophil killing of C. albicans without perturbing phagocytosis or ROS production [47•]. Neutrophil serine protease activity plays a protective role in murine systemic candidiasis [48], but appears to be dispensable for host defense following pulmonary A. fumigatus challenge [49]. Iron sequestration exerts fungistatic effects, exemplified by CGD neutrophil lactoferrin release to limit A. fumigatus hyphal growth [6]. H. capsulatum exerts control over macrophage phagolysosomal fusion to create an obligate intracellular niche for survival. In turn, macrophages sequester zinc, however the precise mechanism of this antifungal strategy has not been elucidated [50].

Induction of Adaptive Immunity

TLR and CLR signaling induces MHC class II, co-stimulatory molecule, and cytokine expression by antigen-presenting cells that dictate T cell differentiation (Fig. 2). In the lung, inflammatory monocytes and CCR2⁺Ly6C^hi monocyte-derived DCs are essential for fungal antigen transport to draining lymph nodes and facilitate Aspergillus-, Histoplasma-, and Blastomyces-specific CD4⁺ T cell priming and expansion [51,52•]. Following subcutaneous administration, multiple skin-resident and lymph node dendritic cell subsets cooperate to transport vaccine yeast and to prime antigen-specific CD4 T cell responses [53].

Th1 and Th17 cells are the principal T helper subsets that contribute to protective immunity to several pathogenic fungi. Below, we discuss the signaling pathways and other factors that influence T cell differentiation and mechanisms of protection. The recent development of A. fumigatus- and B. dermatitidis-specific T cell receptor (TCR) transgenic mice facilitates studies on antigen-specific CD4 T cell differentiation and memory development during fungal infections [54,55]. Interestingly, B. dermatitidis-specific 1807 TCR transgenic cells recognize a protective antigen that is shared among the major systemic dimorphic fungi including H. capsulatum, Coccidioides posadasii and Paracoccidioides brasiliensis [54].

IL-17 contributes to host resistance in oropharyngeal, cutaneous, and systemic infection with C. albicans [23,56,57], and pulmonary infection with H. capsulatum [58], A. fumigatus [24], and P. carinii [59]. In fungal infection models, IL-17 can arise from myeloid sources [60], γδ T cells [42] [61], or CD4 T cells that have differentiated into Th17 effector cells [62••]. Thus, the role of IL-17 and Th17 cells in antifungal host defense are not synonymous in all instances. The recent discovery of human polymorphisms in genes that regulate Th17 differentiation (IL-12Rβ1, STAT3, STAT1) and IL-17 function (IL-17F, IL-17RA, AIRE) in individuals suffering from mucocutaneous candidiasis underlines the importance of IL-17-dependent immunity (Table I).

In contrast to the studies cited above, IL-17 and Th17 cells have been suggested to be pathogenic in a model of gastric candidiasis and in pulmonary A. fumigatus infection [63]. In other experimental systems, deleterious effects of Th17 cells are kept in check by regulatory T (Treg) cells. In a murine model of pulmonary P. brasiliensis infection, TLR2 promoted the induction of Treg cells that limited Th17 cell development and tissue pathology [64]. Similarly, the chemokine receptor CCR5 regulates the balance between Tregs and Th17 cells in H. capsulatum infection [65]. In contrast, Tregs enhance Th17 responses and fungal clearance in a murine model of oral candidiasis [66].

TLR signaling mediates the development of antifungal Th1 cells. MyD88-dependent signals are dispensible for CD4⁺ T cell priming and trafficking to infected airways during respiratory fungal infection, but mediate the stepwise differentiation of A. fumigatus-specific CD4 T cells into IFN-γ-producing airway effector cells [55]. Vaccine-induced Th17 cells mediate resistance to B. dermatitidis, H. capsulatum and C. posadasii [62••,67]. C albicans, A. fumigatus and curdlan, a β-(1,3)-glucan agonist, can induce the development of Th17 cells via Dectin-1/Syk/CARD9-dependent signals [68,69]. In A. fumigatus-infected mice, Dectin-1 enhances Th17 differentiation by decreasing IL-12 and IFN-γ production in innate cells and T-bet expression in A. fumigatus-specific CD4⁺ T cells [69]. In contrast, development of vaccine-induced Blastomyces-specific Th17 cells depends on MyD88, but not Dectin-1 [62••], while Dectin-2 is also implicated in Th17 cell development during candidiasis [23,70].

Fungal vaccination strategies

Despite a growing medical need, no fungal vaccines are commercially available. Virulence-attenuated mutants represent one of the best ways to induce sterilizing immunity and achieve maximal protection, as evidenced in experimental models of blastomycosis, histoplasmosis and coccidiodomycosis [54,71,72]. In addition, vaccination with attenuated mutants induces antifungal memory CD8⁺ T cells that are maintained without numeric or functional loss for at least 6 months [73]. Although these vaccine strains are not likely to be safe in immunocompromised individuals, the knowledge gained from experimental vaccine models is crucial for the rational design of human vaccine candidates. Experimental vaccines against candidiasis, aspergillosis, and the endemic mycosis [62,67,74,75] owe their protection chiefly to Th17 and Th1 cells, a novel paradigm for human vaccines.

Two subunit vaccines designed to protect against C. albicans infections are in Phase 1 clinical trials. The rAls3p-N vaccine (NovaDigm Therapeutics, USA) contains the N terminal portion of the adhesin agglutinin-like sequence (Als) 3 and stimulates protective immunity to C. albicans and S. aureus infection by eliciting Th17 and Th1 T cells that enhance microbial killing independent of B cell function [74]. The antigen specificity of the protective Als3p-N-specific T cells may reside within a naturally processed and presented epitope that is reactive to multiple, clinically relevant Candida species [76•]. Another T cell epitope from the A. fumigatus cell wall glucanase Crf1 cross-protects against infection with A. fumigatus and C. albicans [77].

Naturally acquired infections with fungi and virulence-attenuated live vaccines do not induce protective isotype-switched antibodies, but conserved natural IgM antibodies are beneficial during pneumocystosis [78]. Protective monoclonal antibodies (mAbs) recognize the cryptococcal capsule, β-glucan on C. albicans and A. fumigatus, and the secreted virulence factor aspartyl proteinase 2 (Sap2) from C. albicans. Neutralizing antibodies to Sap2 provide the functional basis of the second fungal vaccine currently in a Phase 1 clinical trial. The virosome-formulated vaccine induces Sap-neutralizing antibodies (PEV7 by Pevion Biotech, Switzerland) that confer protection against candidal vaginitis [79]. A conjugate vaccine that elicits β-glucan antibodies protects in experimental models of aspergillosis, candidiasis, and cryptococcosis is in preclinical development [80,81].

In summary, fungal vaccines confer protection by T cells and antibodies. T cell based vaccines mediate acquired resistance by the production of inflammatory cytokines (e.g. IFN-γ, TNF, IL-17 and IL-22) that regulate the influx and killing by phagocytes and the synthesis of epithelial-cell derived antimicrobial proteins (e.g. cathelicidins and histatins) [8,82]. Antibodies contribute to protection by neutralizing virulence factors (e.g. adhesins), inhibiting fungal growth, and by stimulating direct killing, opsonophagocytosis, and complement activation. Thus, strategies that harness both arms of the adaptive antifungal immune response may yield the most rational and successful vaccine candidates [80,81,83,84].

Conclusions

In the past several years, researchers have made significant progress in understanding innate and adaptive immunity to medically important fungi. Important milestones include the dissection of fungal recognition and killing pathways in animal models and the discovery of the genetic causes of human syndromes characterized by fungal disease. These lines of investigation identify a key role for CLR signaling and genes related to IL-17-mediated immunity in regulating tissue inflammation and fungal clearance. Integrated CLR/TLR signals underlie host control of chromoblastomycosis and can be elicited by administering a TLR agonist, establishing a novel treatment paradigm for fungal disease. The identification of new vaccine antigens and their underlying mechanisms of protection in preclinical models of disease are essential steps in the development of candidate human vaccines. Greater understanding of the molecular and cellular basis of antifungal immunity will undoubtedly provide opportunities for novel therapeutic strategies and applications.

Highlights.

Genetic defects predispose to fungal infections and illustrate key defense mechanisms

C type lectin receptor signaling pathways play a central role in immunity to fungi

Toll-like receptor agonism can resolve fungal infections

IL-17 is a key mediator of antifungal immunity, particularly at mucosal surfaces

Experimental fungal vaccines elicit protective immunity by Th1 and Th17 cells