Innate and Adaptive Immune Responses to Fungi in the Airway

Abstract

Fungi are ubiquitous outdoors and indoors. Exposure and/or sensitization to fungi is strongly associated with development of asthma and allergic airway diseases. Furthermore, global climate change will likely increase the prevalence of fungi and enhance their antigenicity. Major progress has been made during the past several years regarding our understanding of antifungal immunity. Fungi contain cell wall molecules, such as β-glucan and chitin, and secrete biologically active proteases and glycosidases. Airway epithelial cells and innate immune cells, such as dendritic cells, are equipped with cell surface molecules that react to these fungal products, resulting in production of cytokines and pro-inflammatory mediators. As a result, the adaptive arm of antifungal immunity, including Th1-, Th2-, and Th17-type CD4⁺ T cells, is established, reinforcing protection against fungal infection and, by the same token, causing detrimental immunopathology in certain individuals. We are only in the beginning stages of understanding the complex biology of fungi and detailed mechanisms of how they activate the immune response that may protect or drive diseases in humans. Here we describe our current understanding with an emphasis on airway allergic immune responses. The gaps in our knowledge and desirable future directions are also discussed.

INTRODUCTION

Fungi are ubiquitous organisms that make their homes both indoors and outdoors.¹ Humans are constantly exposed to environmental fungal spores, often at levels 1000-fold higher than levels of grass and tree pollens.² Virtually everyone in the U.S. is exposed to Alternaria antigens at home.³ Molecular evidence suggests that animals and fungi have co-evolved since diverging from plants more than 1 billion years ago.⁴ Since then, the immune system has been crucial in establishing a close relationship between the host and fungi by ‘keeping the peace’ at barrier surfaces.⁵ In immune-compromised hosts, fungi can colonize and infect the lungs and other organs, causing increases in morbidity and mortality. Alternatively, fungi and their products can cause exaggerated immune responses and pathological changes in organs in certain individuals.

While numerous environmental factors are associated with asthma and allergic diseases, allergen exposure likely plays a key role in triggering and exacerbating asthma and allergy symptoms.⁶ In particular, exposure to airborne allergens derived from animals, arthropods, and molds is considered to be an important risk factor.^7–9 In humans, an association between fungal exposure and/or sensitization, in particular to Alternaria and Aspergillus, and asthma is recognized in various countries.^{2, 10} For example, Alternaria-sensitivity is closely linked with the development of allergic asthma.^11–13 Severe asthma and life-threatening acute exacerbations of asthma have also been associated with increased airborne exposure to Alternaria^14,15 Increased spore counts during crop harvest and after thunderstorms may contribute to asthma exacerbations as well.¹⁶ Furthermore, water damage and other environmental impacts lead to increased growth of fungi, such as Stachybotrys chartarum, compromising air quality and promoting airway inflammation.¹⁷ In experimental animals, the highly potent actions of Alternaria and Aspergillus in stimulating innate and adaptive type 2 immunity have been recognized by a number of investigators.^18–25

In the coming years, global climate change may increase the incidence of fungal sensitization and allergic airway diseases because increased fungal colonization of plants occurs at higher CO₂ levels.²⁶ Indeed, Alternaria alternata grown on plants in a high CO₂ environment showed increased spore production and higher antigen content per spore.²⁷ Higher air temperature was correlated with more days that Cladosporium spore counts exceeded the allergenic threshold.²⁸ The likelihood of an increasing impact of fungi on human health provides an incentive to learn more about the mechanisms of infection and immune response to fungi.

Alterations in the bacterial microbiome have a dramatic impact on host immunity and contribute to a number of immune-mediated diseases.²⁹ Fungi are naturally present in the mammalian intestine,³⁰ and recent sequencing technologies have expanded our understanding of fungal communities at barrier surfaces.³¹ For example, intestinal fungal dysbiosis has been shown to influence gastrointestinal diseases, such as inflammatory bowel disease (IBD)³² as well as allergic inflammation of the lungs.³³ Furthermore, a large number of fungal species have been found in sputum specimens from patients with asthma.³⁴

Thus, there is clearly a need to understand the host’s innate and adaptive immune responses to fungi and their implications in pathophysiology of human diseases. In this review article, we will summarize current information in the field, identify the gaps in our knowledge, and discuss the need for future clinical and basic science studies. While a variety of topics could be covered, we will focus our discussion on asthma and allergic immune responses that are specifically relevant to the JOURNAL. Readers are encouraged to refer to recent excellent reviews^31,35,36 and other articles in this issue for other specific topics.

FUNGI AND THEIR PRODUCTS

Fungal biology

Understanding key biologic aspects of fungi is necessary to understand the host’s immune responses to these organisms. Fungi are a kingdom of eukaryotes, and over 100,000 species have been identified to date; this likely accounts for 5% of the predicted fungal species.¹⁶ Almost 98% of known fungal species belong to one of two phyla, the Ascomycota and the Basidiomycota.³⁷ The genera most commonly associated with the development of allergy in humans are Alternaria, Cladosporium, Penicillium, and Aspergillus³ Fungi are heterotrophs, thus cannot make their own nutrients and must depend on other organisms to provide food.³⁹ While most animals ingest food and then digest it internally, fungi secrete enzymes that digest their food extracellularly before it can be taken up as nutrients.

Fungi produce spores, or conidia, that can remain dormant until they are ready to germinate, at which time they swell and send out hyphae.⁴⁰ Dormant fungal spores likely evade the immune system by production of a protective hydrophobin layer.⁴¹ Fungal growth depends on temperature. Mesophilic fungi, such as Alternaria and Cladosporium, grow best in moderate temperatures, typically between 20–30 °C. Because they are unable to grow at body temperature, mesophilic fungi rarely cause human infections; nonetheless, their airborne fragments and secreted products can cause respiratory allergies. In contrast, thermotolerant fungi, such as Candida, Penicillium and Aspergillus, are capable of growing at human body temperature (37 °C), and they serve both as a part of the microbiome and as a cause of pathogenic infections.³⁷ Indeed, Aspergillus is the most prominent fungus to infect the lungs.⁴² The ability of fungi to colonize and germinate in the respiratory mucosa makes them unique compared to other respiratory allergens (e.g. house dust mite, grass and tree pollens) that do not grow within the host. Most fungal allergens are released during⁴³ and after¹⁶ spore germination, suggesting the importance of keeping the fungal life cycle in consideration.

Inhalation of fungal spores, hyphae, fragments or particles of secreted products is likely involved in the induction and/or exacerbation of asthma and allergic diseases.⁴⁰ Up to 50,000 spores/m³ are detected at peak times of late summer and early fall;¹⁶ theoretically, humans can be exposed to ~1 million spores per day. Fungal spores are highly variable in size, ranging from <2 μm to 250 μm in diameter. Spores larger than 10 μm, such as Alternaria spores, may deposit in the upper airways (where the temperature is also lower than 37 °C) while Aspergillus and Penicillium spores that are <10 μm can easily reach the lower airways.¹⁶ One should note that fungal fragments and submicron particles of fungal organisms may also serve as a aeroallergen source.⁴⁴ An extensive list of fungal allergens is available in a recent review article.³⁶

Fungal products

Biological molecules that are produced by fungi can be associated with their cell wall or secreted extracellularly. Major fungal products include chitin,⁴⁵ β-glucan,⁴⁶ proteases,⁴⁷ and glycosidases⁴⁸ (Figure 1). Chitin is intimately intertwined with β-glucans, galactomannans, and mannoproteins to form the structural foundation of the hyphal cell wall.³⁵ Chitin and β-glucans from Aspergillus induce eosinophilic inflammation in mouse lungs.⁴⁹ Fungal proteases and glycosidases are presumed to degrade polymers and are used by fungi to capture nutrients from plant and mammalian hosts. The potential impact of these hydrolyases is likely significant because fungi contribute the majority of protease activity in house dust samples.⁵⁰ Certain fungal allergens are also proteases themselves, including Asp f 5, f 6 and f 11 from Aspergillus.² Indeed, the protease activities produced by fungi were sufficient^18,51 and necessary⁵⁰ for induction of the robust allergic immune response induced by Aspergillus in the airway.

Figure 1.

Fungal products and their receptors. Fungi and their products are recognized by several families of PRRs, including Toll-like receptors (TLRs) and C-type lectin receptors [(CLRs; Dectin-1 and DC-specific ICAM3-grabbing non-integrin (DC-SIGN)] and the mannose receptor (MR). Fungi and spores may also be recognized by the receptor for advanced glycation end-products (RAGE) and ficolins, respectively. Fungus-derived proteases are recognized directly by protease-activated receptor 2 (PAR2) or indirectly through production of endogenous ligands, such as fibrinogen-cleavage products (FCPs), which activate TLR4

IMMUNE RECEPTORS THAT RECOGNIZE FUNGAL MOLECULES

In general, allergens can be recognized by the cellular immune system via three major mechanisms: 1) engagement of pattern recognition receptors (PRRs), 2) molecular mimicry of Toll-like receptor (TLR) signaling complex molecules, and 3) recognition of the allergen’s biological activities, such as proteases.^{52, 53} Recognition of fungi and fungal allergens likely occurs by the first and third mechanisms. Several families of PRRs have been shown to be involved in interactions with fungi.

Toll-like receptors

Toll-like receptors (TLRs) are a family of PRRs that respond to pathogen-associated molecular patterns (PAMPs) arising from a variety of pathogens, including bacteria, viruses and fungi. To date, 12 TLRs have been identified in humans.⁵⁴ TLRs may act as surface receptors (TLR1, 2, 4, 5, 6 and 10) or as intracellular receptors (TLR3, 7, 8, and 9) while TLR4 may also be intracellular and TLR3 and TLR9 may also be extracellular.⁵⁵ Fungal products that interact with TLRs include zymosan (i.e. a glucan), O-linked mannans, phospholipomannans, unmethylated CpG-rich DNA and double-stranded RNA.^{36, 56} The TLR1/TLR2 heterodimer recognizes β-glucan from fungal conidia and hyphae.⁵⁷ TLR2 also interacts with phospholipomannans from Candida albicans and unidentified ligands from Aspergillus fumigatus.⁵⁷ Activation of TLR4 by C. albicans and Cryptococcus neoformans occurs via O-linked mannans.⁵⁷ Intracellular TLRs, such as TLR3 and TLR9, are activated by dsRNA from conidia and unmethylated CpG from fungal DNA,⁵⁷ respectively. The immunological outcomes of activation of these TLRs could be either pro- or anti-inflammatory, depending on the TLRs or target cell types.⁵⁷ For example, activation of TLR4 in conjunction with Dectin-1 and the mannose receptor (MR) in DCs induces a pro-inflammatory Th17-type response.⁵⁸ In contrast, engagement of TLR2 elicits a combined Th2-type and regulatory T (Treg) cell response, resulting in persistent fungal infection but diminished immunopathology.⁵⁹

C-type lectin receptors

C-type lectin receptors (CLRs) are a class of receptors that generally recognize carbohydrates, lipids and proteins.³⁶ CLRs that recognize fungal products include Dectin-1 and Dectin-2, Dendritic Cell-Specific intercellular adhesion molecule-3-Grabbing Nonintegrin (DC-SIGN), and the mannose receptor (MR).⁶⁰ Dectin-1 binds β-glucan while Dectin-2, DC-SIGN and MR recognize mannose-based molecules.⁶⁰ Activation of these receptors triggers proinflammatory immune responses, including phagocytosis and production of cytokines and lipid mediators.⁶⁰ For example, Dectin-1 activation by β-glucan induces production of IL-12 and IL-4/IL-13 by macrophages⁶¹ and rat mast cells,⁶² respectively.

Other PRRs

The receptor for advanced glycation end products (RAGE) is a transmembrane receptor that belongs to the immunoglobulin superfamily and recognizes PAMPs as well as a variety of glycated molecules. It still needs to be determined whether fungal products are directly recognized by RAGE or whether RAGE interacts with TLRs to mediate immune responses to fungi.⁶³ Nonetheless, in mice exposed to Alternaria, deletion of RAGE diminished production of IL-33 and protected the animals from developing airway inflammation.⁶⁴ Furthermore, increased levels of RAGE have been positively correlated with asthma.³⁶ Ficolins are soluble lectins that can bind fungal conidia and activate the lectin pathway of complement. Clearance of A. fumigatus was inhibited in ficolin-deficient mice.⁶⁵

Recognition of proteases

As described above, fungi produce a large amount of proteolytic and glycolytic enzymes. Pro-inflammatory activities of these fungal products have been recognized for a number of years.⁶⁶ Porter et al compared the extent of allergic airway inflammation in mice intranasally infected with wild-type (WT) or protease-deficient Aspergillus niger conidia.⁵⁰ While WT conidia induced robust Th2 cytokine production, eosinophilia, and airway hyperreactivity (AHR), these pathological changes decreased in protease-deficient conidia. Because other components, such as amounts of β-glucan, were similar between these strains, their observations point to the biological significance of fungal proteases in triggering allergic airway inflammation.

Several potential mechanisms can be considered as to the effects of fungal proteases. First, fungal proteases can disrupt airway epithelial tight junctions, leading to increased recognition of fungal proteins by immune cells resident within mucosal tissues.³⁷ Second, fungal proteases can be recognized directly by cell surface receptors of innate immune cells. Protease-activated receptors (PARs) comprise a family of four 7-transmembrane G-protein coupled receptors.⁶⁷ Within the structure of a PAR are both ligand and ligand-binding domain. Upon protease cleavage, the ligand is freed to interact with the ligand-binding domain to initiate signaling that drives Ca²⁺ flux and transcription of genes for inflammatory mediators. Indeed, Alternaria extract activates human eosinophils and airway epithelial cells through PAR2, resulting in extracellular release of granule proteins⁶⁸ and production and secretion of thymic stromal lymphopoietin (TSLP) and IL-33,^{19, 69} respectively. Furthermore, activation of epithelial cell PAR2 by Aspergillus proteases suppresses CXCL10 production by airway epithelial cells,⁷⁰ suggesting immunoregulatory roles for epithelial PAR2. Nonetheless, there are some controversies as to whether the increase in intracellular calcium concentration ([Ca²⁺]i) in airway epithelial cells that are exposed to Alternaria proteases is dependent on⁷¹ or independent of⁷² PAR2. Finally, fungal proteases promote immune responses indirectly by facilitating production of molecule(s) within the host, which in turn activate immune cells. For example, intranasal administration of proteases derived from Aspergillus oryzae to mouse airways induced allergic airway inflammation via cleavage of the clotting factor fibrinogen and generation of fibrinogen cleavage product(s) that activates TLR4.⁷³ Therefore, further studies are necessary to identify key fungal proteases and glycosidases and elucidate the cellular and molecular mechanisms involved in their activation of immune cells.

INNATE IMMUNE RESPONSES

Exposure to microbes, such as bacteria and viruses, induces innate immune responses in the host followed by development of adaptive immune responses. Exposure to fungi and their products also provokes analogous immunological pathways (Figure 2).

Figure 2.

Innate and adaptive immune responses to fungi. The immune responses to fungi consist of several layers of adaptive and innate immunity. Epithelial tight junctions and mucus secreted by goblet cells deter and physically remove fungi. Fungal exposure to epithelial cells causes production of cytokines, such as IL-25, IL-33, and TSLP, which activate group 2 innate lymphoid cells (ILC2s) and initiate Th2-type differentiation of naive CD4+ T cells. Dendritic cells (DCs) and their cytokines, such as IL-6, IL-23 and IL-12, also drive proliferation and differentiation of CD4+ T cells. Th1 cells activate macrophages via secretion of IFN-γ. Th17 cells produce IL-17 and IL-22, which mediate CXCL8-dependent neutrophil recruitment and production of antimicrobial peptides by epithelial cells, respectively. ILC2s and Th2 cells produce IL-5 and IL-13, which mediate eosinophilic inflammation, goblet cell hyperplasia and airway remodeling. The effector cells, including macrophages, neutrophils and eosinophils, use distinct strategies to provide antifungal immunity. AAM, alternatively-activated macrophages; Eos, eosinophils; ET, extracellular DNA trap; ΜΦ, macrophages; Neut, neutrophils; Treg, regulatory T; TSLP, thymic stromal lymphopoietin.

Epithelial barrier and epithelial cells

The first line of immune response against fungi is the epithelial barrier that provides protection by excluding the microbes and removing them physically.⁷⁴ Epithelial cells form tight junctions that prevent invasion of environmental pathogens into the host. Mucus provides a layer of protection, in which cilia and mucus work together to physically remove microbes.⁷⁵

Epithelial cells express PRRs and respond to microbes by producing antimicrobial peptides, cytokines and other pro-inflammatory molecules.⁷⁶ In particular, a triad of cytokines that promote type 2 immune responses, specifically IL-25, IL-33 and TSLP, is produced and released by airway epithelial cells in response to various environmental factors and microbial stimuli.⁷⁷ They are also produced by airway epithelial cells in response to Alternaria and/or proteases in vitro ^69,78,79 Notably, IL-33, which was pre-formed and stored in nuclei, was released quickly into the airway lumen when animals were exposed to Alternaria extract.^19,22,78,80 Other common aeroallergens did not have such properties,^19,78 suggesting a unique biological activity of the fungus Alternaria.

The molecular mechanisms that mediate IL-33 release are an active area of current research. IL-33 was initially described as a damage-associated molecular pattern (DAMP) that is released from damaged or necrotic cells.⁸¹ However, recent studies suggest that cells may actively respond to fungal exposure. For example, Alternaria-induced IL-33 release from normal human bronchial epithelial (NHBE) cells involves rapid extracellular accumulation of ATP, followed by autocrine and paracrine activation of P2 purinergic receptors and increases in [Ca²⁺]_i. The administration of Ca²⁺ chelators as well as pharmacologic or genetic inhibition of P2 purinergic receptors inhibited Alternaria-induced IL-33 release.^{22, 78} In addition, in airway epithelial cells exposed to Alternaria, oxidative stress pathways were activated and played a pivotal role in initiation of IL-33 release.^{22, 82} The unfolded protein response (UPR) may also play a role in epithelial response to Alternaria as mice deficient in ORMDL3 did not develop airway inflammation.⁸³ Interestingly, an agonist for glucagon-like peptide-1 (GLP-1) inhibited Alternaria-induced expression of dual oxidase 1 (DUOX1) and IL-33 release in vivo in mice.⁸⁰ Collectively, these findings suggest that oxidative stress, purinergic receptors, and intracellular calcium are likely to be key players in the signaling cascade involved in IL-33 release from airway epithelial cells and that it can be controlled by pharmacologic agents, such as GLP-1 agonists.

Group 2 innate lymphoid cells

In the early 2000s, group 2 innate lymphoid cells (ILC2s) were first described in mice infected with the parasite Nippostrongylus brasiliensis or those exposed to the fungus Aspergillus, as non-B/non-T cells that secrete IL-5 and IL-13 in response to IL-25.^{84, 85} ILC2s were later isolated and characterized as a population of innate immune cells by several investigators.^86–88

When naive mice were exposed to Alternaria extract, the levels of Th2 cytokines in the airway, including IL-5 and IL-13, increased as early as 6 h after the exposure.⁷⁸ Subsequent studies established that the IL-33-activated lung ILC2 population drives this rapid type 2 cytokine response.^{20, 21} Intranasal administration of Alternaria also induced rapid production of cysteinyl leukotrienes (CysLTs) that can activate ILC2s through CysLT1R, suggesting that an IL-33-independent pathway for fungus-induced ILC2 activation also exists.⁸⁹

Accumulating evidence points to a critical role for ILC2s in fungus-mediated immunopathology in the airway. For example, Alternaria exposure increased peribronchial infiltration of inflammatory cells and promoted epithelial hyperplasia, mimicking the observations in human asthma.^{20, 90} IL-7R-deficient mice that lack ILC2s and adaptive immune cells (e.g. T cells) were protected from developing these pathologic changes even when they were exposed to Alternaria. Conversely, adoptive transfer of ILC2s to these mice restored lung pathology,^{20, 90} suggesting that ILC2s are necessary and sufficient to mediate fungus-induced airway inflammation. Alternaria-induced activation of ILC2s further enhanced lung inflammation in mice that had been sensitized to an irrelevant allergen.⁹¹ Nevertheless, in mice exposed to Alternaria, ILC2s also produced amphiregulin that may play a role in epithelial repair,²¹ suggesting that ILC2s may provide tissue protection during allergic immune responses to fungi. Further studies will be necessary to elucidate contributions of ILC2s to allergic airway inflammation and pathological changes in animal models and in humans.

Dendritic cells

Dendritic cells (DCs) take up antigen at local tissue sites, undergo maturation via upregulation of costimulatory molecules, and migrate to lymph nodes (LNs), where they promote proliferation and differentiation of naive T cells. These DCs likely play a pivotal role in antifungal immunity. For example, pulmonary infections of C. neoformans and A. fumigatus induce predominantly Th1-type responses that are orchestrated by monocyte-derived DCs.^{92, 93} In mice depleted of these monocyte-derived DCs, Th2 or Th17 responses were generated, suggesting a key role for DCs in controlling T cell responses. DCs recognize fungal mannans directly through Dectin-2, DC-SIGN, and MR, triggering production of pro-inflammatory and immunoregulatory cytokines, including IL-1, IL-2, IL-6, IL-10 and IL-23.⁹⁴ Importantly, DCs distinguish between conidia and hyphae of fungi. For example, C. albicans conidia induce IL-12 production by DCs, resulting in a Th1-type immune response while hyphae induce IL-4 and 10, leading to a Th2-type response.⁹⁵ Thus, the life cycle of fungi likely affects the characteristics of the DC response, resulting in differential immunological outcomes.

In allergic immune responses, fungi can serve as an adjuvant to promote immune responses to fungal components as well as to bystander antigens, and DCs likely play a key role in recognition of the adjuvant activities of fungi. For example, airway exposure to innocuous allergens, such as OVA, is generally tolerogenic.⁹⁶ When these antigens were co-administered into the airway with fungal products, a strong Th2 response developed.⁹⁷ Bone marrow (BM)-derived DCs cultured with A. alternata extract upregulated expression of co-stimulatory molecules and increased production of IL-6, but not IL-12; when transferred to naive mice, these Alternaria-stimulated DCs promoted development of Th2-type CD4⁺ T cells and IgE antibody production.⁹⁷ In addition, in a mouse model of protease-induced allergic immune responses, DCs were activated by ILC2-derived IL-13 to mediate Th2-type memory T cells.⁹⁸ Altogether, DCs may be activated directly by fungal antigens or collaborate with other innate immune cells, such as ILC2s, to mediate innate immune responses to fungi and regulate adaptive immunity.

Neutrophils and eosinophils

Neutrophils and eosinophils are considered to be effector immune cells that mediate antifungal immunity. These granulocytes likely accomplish the task by both distinct and overlapping mechanisms. Neutrophils employ several mechanisms to control fungi, including production and/or extracellular release of granule proteins, antimicrobial peptides (AMPs), and reactive oxygen species (ROS) as well as formation of neutrophil extracellular DNA traps (NETs).⁹⁹ The smaller conidia are generally phagocytized by neutrophils while larger hyphae trigger NETs.¹⁰⁰ IgG opsonization of fungi can further enhance neutrophil phagocytosis and promote antibody-directed cell toxicity.¹⁰¹

Eosinophils are not professional phagocytes; therefore, they respond to fungi in different ways than neutrophils. For example, human eosinophils released granule proteins extracellularly in response to Alternaria conidia and hyphae in vitro; the process was dependent on eosinophil adhesion to fungi via eosinophil CD11b and fungal β-glucans.¹⁰² Human eosinophils also responded to Alternaria proteases through PAR2, resulting in extracellular release of granule contents.^{68, 103} Furthermore, murine BM-derived eosinophils killed A. fumigatus by a contactindependent mechanism in vitro¹⁰⁴ and eosinophils released their extracellular DNA traps (ETs), targeting fungal hyphae.¹⁰⁵ Eosinophil ETs may contribute to the formation of the viscous and sticky mucus seen in patients with allergic bronchopulmonary aspergillosis (ABPA) and other related disorders.¹⁰⁵ Further information will be necessary to better understand the roles of neutrophils and eosinophils in antifungal immunity in humans as well as the pathological outcomes of patients with fungus-associated diseases.

ADAPTIVE IMMUNE RESPONSES TO FUNGI

Adaptive immune cells, such as T cells and B cells, express genetically rearranged surface receptors that are exquisitely specific for a particular antigen. Innate and adaptive immune responses are inextricably intertwined, and a successful adaptive immune response is a coordinated effort requiring activation of tissue resident cells, antigen-presenting cells and antigen-specific T cells and B cells.

Th1/Th17 cell response

Successful clearance of fungal organisms by the host and recovery of tissue homeostasis involves a balance of Th1- and Th17-type responses and Treg cells. For example, activation of CLRs in vivo leads to the development of Th1- and Th17-type CD4⁺ T cells.⁶⁰ The Th1 response provides protective immunity against fungi through enhancing the functions of phagocytic cells via production of IFNγ and promoting B-cell production of opsonizing antifungal antibodies.¹⁰⁶ Th17 responses are also critical because they activate tissue cells, such as epithelial cells and fibroblasts, resulting in production of chemokines that recruit phagocytes to the site of immune response.¹⁰¹ In patients with primary aspergillosis, Aspergillus-specific T cells from the lungs displayed a Th17-type phenotype while circulating specific T cells showed a Th1-type profile.¹⁰⁷ Furthermore, airway exposure to fungal β-glucan enhanced allergen-driven Th2 type immune responses, resulting in a mixed Th17 and Th2 response that is resistant to glucocorticoid treatment.¹⁰⁸

Protective Th1- and Th17-type antifungal immunity is both promoted and downregulated by innate immunity. For example, commensal gut microbiota influence CD4⁺ T cell polarization to fungal infection. In mice infected with Aspergillus, depletion of gut microbiota by the antibiotic vancomycin suppressed Th17 cell development.¹⁰⁹ In pulmonary Paracoccidioides brasiliensis infection, mice deficient in TLR4 developed more Treg cells and showed diminished T cell activation and impaired clearance of fungal organisms.¹¹⁰ In addition, as described above, the same fungi may produce different types of adaptive immunity depending on the context. For example, in Aspergillus infection, factors from dormant conidia promoted Th1-type response while those from hyphae or swollen (i.e. germinating) spores favored Th2-type immunity.⁹⁵ Such differential T cell responses to fungi are likely regulated by innate immunity because, in macrophages, Aspergillus conidia mediated production of pro-inflammatory cytokines via TLR4 activation while hyphae mediated IL-10 production via TLR2.¹¹¹ Furthermore, in the absence of TLR2, mice infected with P. brasiliensis showed decreases in Treg cells and enhanced Th17 cells, resulting in efficient resolution of infection but increased pathologic outcomes.⁵⁹ Thus, Th1- and Th17-type immune responses to fungi are generally protective, but they need to be fine-tuned to provide effective immunity while minimizing damage to the host.

Th2 cell response

Th2-type immunity to fungi is generally characterized by an inability to clear fungal pathogens accompanied by detrimental allergic inflammation, including tissue eosinophilia, goblet cell hyperplasia and airway remodeling.¹¹² Furthermore, unlike classical macrophage activation induced by Th1 cell-derived cytokines, IL-4 and IL-13 induce an alternate form of macrophage activation (AAM) that is less efficient at controlling fungal growth.¹⁰¹

Major progress has been made during the past several years to understand the mechanisms involved in the Th2-type response to fungi. Generally, epithelial cells are triggered by exposure to allergens to release cytokines, such as IL-25, IL-33, GM-CSF and TSLP,¹¹³ which promote development and/or differentiation of Th2-type CD4⁺ T cells. These cytokines may not be required for priming of naive CD4⁺ T cells in draining lymph nodes (dLNs), but are necessary for effector functions of primed Th2-type CD4⁺ T cells at the site of inflammation.¹¹⁴ Additional factors, such as prostaglandins, may also be involved in regulation of type 2 immunity to fungi because PGE₂-deficient mice showed reduced levels of IL-33 as well as diminished eosinophilic inflammation in mice exposed to Alternaria.²⁴ Furthermore, IL-33 impaired clearance of Aspergillus by suppressing PGE₂ expression, resulting in diminished IL-17A and IL-22 production.¹¹⁵

To mimic recurrent and chronic exposure to fungi in humans, several mouse models exposed naive animals to fungal extracts, sometimes in combination with other allergens. For example, airway exposure of mice to combined extracts of house dust mite (HDM), ragweed, and Aspergillus broke tolerance and established chronic features of asthma, including eosinophilic inflammation, mast cell and smooth muscle hyperplasia, mucous production and airway remodeling.¹¹⁶ Interestingly, the pathological changes persisted for several weeks even after the cessation of allergen exposure, and antibodies to IL-5 or IL-13 showed minimal effects, suggesting the presence of the pathway independent of Th2 cells. In another study, chronic and multiple exposures of naive mice to Alternaria, Aspergillus and HDM extracts induced a robust increase in airway eosinophils, tissue remodeling and elevated plasma concentrations of allergen-specific IgE and IgG1 antibodies that also lasted for a prolonged period.¹¹⁷ Notably, a marked increase in the lung levels of IL-33 was observed, and the pathological changes were nearly abolished in mice deficient in the IL-33 receptor ST2. Consistent with this, IL-33 that is produced in mice exposed to Alternaria dysregulated tolerogenic Treg cells and drove them to a pro-inflammatory phenotype.¹¹⁸ Thus, animals that are exposed to fungal products for a prolonged period appear to provide robust models that mimic human asthma. While the IL-33 pathway may play a major role in the model, further studies are necessary to elucidate the mechanisms.

As described above, ILC2s provide rapid and innate immune responses to fungi. Notably, recent findings suggest that ILC2s in conjunction with CD4+ T cells or ILC2s alone contributed to persistent immune responses to fungi. For example, in mice exposed to fungal antigens for a prolonged period (e.g. 4 weeks), the number of ILC2s increased by >2-fold, and both the proportion and absolute number of IL-5- or IL-13-producing ILC2s and CD4⁺ T cells increased by approximately 4-fold, suggesting both ILC2s and Th2-type CD4⁺ T cells may provide antifungal immunity.¹¹⁷ Furthermore, intranasal administration of protease stimulated both ILC2s and Th2 cells, causing allergic airway inflammation and elevated levels of IgE antibodies;^{98, 119} this process was severely impaired in ILC2-deficient mice. Moreover, ILC2s that are responsive to TSLP contributed to airway inflammation and AHR in mice that had been exposed to multiple allergens, including Aspergillus¹²⁰ In a similar model, IL-33-responsive ILC2s played a role in the persistence of airway remodeling and AHR.¹²¹ Importantly, epithelial IL-33 activated ILC2s to produce IL-13, which in turn promoted production of IL-33 and ST2 expression by airway epithelial cells.¹²¹ Thus, during a chronic phase of fungus-induced airway inflammation, tripartite interactions among airway epithelial cells, Th2 cells, and ILC2s may form a positive feedback loop, resulting in continued and persistent immunopathology.

CLINIAL IMPLICATIONS

Detailed information on the clinical aspects of fungus-associated airway diseases is provided in recent excellent review articles.^{16, 37} Here we will highlight some of the wide-ranging implications of fungi in airway diseases, such as asthma and chronic rhinosinusitis (CRS). As discussed in the Introduction, epidemiological studies suggest that asthma is induced or exacerbated by exposure to fungi and/or by increased immune reactivity to fungal organisms. Fungi likely contribute to asthma via two distinct mechanisms. In the case of thermotolerant fungi, such as Aspergillus, fungal colonization may provide an on-going source of fungal allergens, leading to persistent immune response, airway inflammation and tissue damage. Alternatively, temperature sensitive fungi, such as Alternaria, may cause disease exacerbations that are tied closely to increased exposure to fungal spores or fragments and to fungal products (e.g. proteases).

IgE antibodies to fungi are associated with increased asthma severity; individuals with severe asthma are often sensitized to multiple fungal species.¹⁶ Furthermore, greater lung colonization with A. fumigatus is observed in asthma patients than healthy individuals.¹²² The combination of severe asthma and fungal sensitivity as documented by skin prick test (SPT) or serum IgE test is now recognized as a phenotype, known as severe asthma with fungal sensitization (SAFS).^{2, 123} Individuals with SAFS experience more bronchiectasis and fixed airflow obstruction than other asthma patients with comparable severity, suggesting more advanced disease pathology.¹⁶ In another phenotype of asthma, fungi colonize the airway. For example, in allergic bronchopulmonary mycosis (ABPM), fungal colonization of the asthmatic airway leads to IgE sensitization to fungi, recurrent and transient radiographic infiltrates, peripheral and pulmonary eosinophilia and bronchiectasis.¹²⁴ Initially called allergic bronchopulmonary aspergillosis due to increased serum levels of Aspergillus-specific IgE and IgG, this syndrome was subsequently named ABPM when other fungi were found to be involved. While the etiology of ABPM is not fully understood, poor airway clearance, airway remodeling, and mucus overproduction may allow the spores of thermotolerant fungi, such as A. fumigatus, A, flavus, A. niger, and Bipolaris spp, to remain in the airways long enough to germinate, produce hyphae and release their biological products (e.g. proteases).³⁷

In the upper airway, CRS may be co-morbid with eosinophilic mucin (thick mucus with degranulating eosinophils and fungal hyphae) in the sinus cavity and IgE antibodies to fungi,¹²⁵ a condition known as allergic fungal rhinosinusitis (AFRS). AFRS occurs in approximately 5–10% of individuals with CRS⁴²; ~50% of individuals with AFRS also have asthma.¹⁶ AFRS is associated with various fungi, including Bipolaris, Curvularia, Cladosporium, Alternaria, and Aspergillus,¹²⁵ and is more severe than other forms of CRS.³⁷ Thus, both upper and lower airway subtypes of asthma and CRS clearly involve increased immune sensitivity to fungi or colonization of respiratory mucosa with fungal organisms. However, a fundamental question still remaining is the proportion of patients with asthma and CRS whose diseases are impacted by fungi themselves or by immune responses to them.

This question is complicated by difficulties in establishing diagnostic criteria that detect involvement of fungi, both in terms of pathophysiology and diagnostic tools. For example, SAFS may include both individuals whose sensitization to fungi does not contribute to the pathophysiology of their diseases and individuals with sensitization to fungi that colonize the lungs and cause damage to their airways. Fungal sensitization may occur even in individuals without allergic symptoms or evidence of airway inflammation.³⁷ Furthermore, the detection of fungal organisms in biological specimens (e.g. sputum) is complicated by species-associated variations in optimal culture conditions, poor recovery of organisms or spores, and possible contamination from environmental fungi. Assessment of fungal sensitivity is not straightforward either, with SPT and serum IgE tests often giving conflicting results.³⁷ Fungal extracts are not standardized and are highly variable due to strain differences, batch-to-batch variations, diverse source materials (e.g. spore, hyphae, secreted proteins) and enzymatic degradation.¹²⁶

Progress has been made in addressing these current shortcomings. For example, fungi were detected in >60 % of patients with CRS with nasal polyps (CRSwNP) when sterile sinus lavage fluids were collected during endoscopic surgery, even in those not diagnosed with AFRS.¹²⁷ Fungi were also detected in ~ 20 % of control subjects. Measurement of an in vitro cellular immune response to fungi by an IL-4 production assay identified sensitivity to fungi in up to 60 % of patients with CRSwNP,¹²⁷ suggesting that prevalence of both fungal organisms and fungal sensitivity in Th2-type chronic airway diseases may be higher than previously anticipated. These data indicate the need for further refinement of tools for evaluating the involvement of fungi in allergic and chronic airway diseases, as well as studies to elucidate the immunological mechanisms by which fungi promote or modulate immunopathology.

CONCLUSIONS

Fungi, like helminth parasites, are complex organisms and produce a wide variety of molecules depending on their life cycles. Mammalian immune responses to fungi are also complex, as demonstrated by protective Th1/Th17-type responses to cell wall β-glucan and pathologic Th2-type responses to secreted proteases. Furthermore, fungi provide not only allergens but also potent “adjuvants”, and they promote or modulate immune reactions to bystander allergens. We recognize, however, several major gaps in our current knowledge. First, our knowledge regarding fungal products and their “receptor(s)” in immune cells is still limited. For example, while fungal proteases likely play a key role in mediating type 2 immunity, we still do not know which specific proteases from each fungal species are involved. Furthermore, while certain fungal products, such as chitin, have been used successfully to dissect the mechanisms of type 2 immunity,¹²⁸ our understanding of how they are recognized by “receptor(s)” in the mammalian immune system is limited. Questions of whether and how other fungal biological molecules (e.g. glycosidases) contribute to immune responses remain unanswered. Second, as described in Figure 2, a variety of cell types and their products, in particular cytokines, are involved in the host’s immune responses to fungi. While DCs and conventional CD4⁺ T cells have been studied extensively, recent studies suggest potential roles for tissue cells (e.g. airway epithelial cells) and ILCs. The hierarchy and importance of these cell types needs to be elucidated. Is there a specific cell type that plays a “central” role in regulating antifungal immunity? Alternatively, do mammalian immune cells form a network in which several cell types contribute together? How do these cell types or networks adjust to the changing life stages and products of fungal organisms? Third, clinically, the diagnosis and treatment of fungal sensitivity has been a challenge. Fungal sensitization often occurs without IgE-mediated type 1 hypersensitivity responses, and results are inconsistent between SPT and serum IgE measurements.³⁷ As discussed earlier, fungal extracts are highly variable.³⁹ New diagnostic approaches, such as those monitoring the CD4⁺ T cell response to fungi, may improve detection.¹²⁷ Furthermore, better treatment options for patients with hypersensitivities to fungi are needed. Avoidance is impractical due to the ubiquitous nature of these organisms. The efficacy of anti-fungal immunotherapy has been controversial.¹²⁹ Would conventional glucocorticoids or anti-fungal agents work? In this regard, it is imperative to develop better diagnostic tools to identify specific cohorts of patients with fungal involvement and to develop safe and efficacious treatment for them. Newer agents with a safer pharmacologic profile and novel therapeutic routes and regimens may also need to be considered. Finally, as described in recent studies,³⁰ fungi are likely a part of mammalian microbiome. A major question still remains regarding the overall significance of fungal exposure and colonization to human disease and health at the population level. As asthma is associated with polymorphisms in certain immune molecules,¹³⁰ it will be important to identify genetic variants that are associated with increased infection/colonization with fungal organisms or increased immune reactivity to them. Furthermore, as global climate change is imminent, it is imperative to expand our body of knowledge regarding how fungi interact with our immune systems and how immune responses will mediate and modulate disease processes in various organs, such as respiratory, gastrointestinal and cutaneous immune systems. Future efforts that involve collaborations among clinicians, immunologists, and fungal experts will be necessary to address these questions.

What do we know?

• Fungal allergen exposure is closely linked to asthma development, and fungal sensitivity is associated with more severe asthma.

• Fungi are unique among airway allergens as they can cause both acute and chronic responses in the airway by inhalation and colonization.

• Fungi express pathogen-associated molecular patterns and secrete proteases, glycosidases and other enzymes; these molecules may promote type 2 immune responses in animals and humans.

• Various pattern recognition receptors expressed by immune cells are activated by fungal products.

• Immune responses to fungi are mediated by a network of innate and adaptive immune cells, including but not limited to airway epithelial cells, ILC2s, DCs, and CD4⁺ T cells.

What is still unknown?

• Which specific fungal components and secreted products are potent triggers of immune responses?

• Which specific cell types and which specific cellular receptors are involved in responding to the fungal products?

• How can we identify patients who are immunologically sensitive to fungi and their products?

• How can we treat patients with increased fungal sensitivity safely and effectively?

• Which genetic signatures are associated with fungus-mediated diseases?

• What role do fungi play in the human microbiome and in regulation of the immune system?

KEY MESSAGES.

• Fungi express and/or release biologically-active molecules depending on their life cycles, which are recognized by various cell surface molecules on immune cells.

• Exposure to fungi activates airway epithelial cells and innate immune cells, such as DCs and ILC2s.

• The adaptive immune responses to fungi consist of Th1-, Th2-, or Th17-type CD4⁺ T cells that may vary depending on the types of fungi and/or molecules they produce.

• Further studies are necessary to better understand the impact of exposure to and colonization with fungi in human health and disease.

ABBREVIATIONS

AAM

alternatively-activated macrophages

ABPA

allergic bronchopulmonary aspergillosis

ABPM

allergic bronchopulmonary mycosis

AFRS

allergic fungal rhinosinusitis

AHR

airway hyperreactivity

AMPs

antimicrobial peptides

BM

bone marrow

CLRs

C-type lectin receptors

CRS

chronic rhinosinusitis

CRSwNP, CRS

with nasal polyps

CysLTs

cysteinyl leukotrienes

DAMP

damage-associated molecular pattern

DCs

dendritic cells

DC-SIGN

dendritic cell-specific intercellular adhesion molecule-3 grabbing Nonintegrin; dLNs, draining lymph nodes

DUOX1

dual oxidase 1

ET

extracellular DNA traps

GLP-1

glucagonlike peptide-1

HDM

house dust mite

IBD

inflammatory bowel disease

ILC2s

group 2 innate lymphoid cells

LN

lymph node; MR, mannose receptor

NETs

neutrophil extracellular DNA traps

NHBE

normal human bronchial epithelial

PAMP

pathogen-associated molecular pattern

PAR

protease-activated receptors

PRR

pattern recognition receptors

RAGE

receptor for advanced glycation end products

ROS

reactive oxygen species

SAFS

severe asthma with fungal sensitization

SPT

skin prick test; TLR, Toll-like receptors

TSLP

thymic stromal lymphopoietin

UPR

unfolded protein response

WT

wild type

Glossary: JACI 18–00321 Innate and Adaptive Immune Responses to Fungi

B-glucan

β-D-glucose polysaccharides found in the cell walls of bacteria, fungi, yeasts, algae, lichens, and plants, such as oats and barley.

Chitin-a

long-chain polysaccharide similar to cellulose, chitin is a primary component of cell walls in fungi, the exoskeletons of arthropods, insects, and the scales of fish and lissamphibians.

CpG sites

regions of DNA where a cytosine nucleotide occurs next to a guanine nucleotide separated by only 1 phosphate. Methylation of the cytosine within a gene can turn the gene off.

CXCL10 (C-X-C Motif Chemokine Ligand 10)

a chemokine that elicits its effects by binding to the cell surface chemokine receptor CXCR3 resulting in pleiotropic effects, including stimulation of monocytes, natural killer and T-cell migration, and modulation of adhesion molecule expression.

Dual oxidase 1 (DUOX1)

a member of the NADPH oxidase family. This protein generates hydrogen peroxide and thereby plays a role in the activity of thyroid peroxidase, lactoperoxidase, and in lactoperoxidase-mediated antimicrobial defense at mucosal surfaces.

Galactomannans

multifunctional macromolecular carbohydrates found in various albuminous or endospermic seeds.

Glucagon-like peptide-1 (GLP-1)

a 30-amino acid peptide hormone produced in the intestinal epithelial endocrine L-cells by differential processing of proglucagon, the gene which is expressed in these cells.

Glycosidases

(also called glycoside hydrolases or glycosyl hydrolases) an enzyme that catalyzes the hydrolysis of glycosidic bonds in complex sugars, thus degrading oligosaccharides and glycoconjugates

GM-CSF (Granulocyte-macrophage colony-stimulating factor)

also known as colony stimulating factor 2 (CSF2), is a monomeric glycoprotein secreted by macrophages, T cells, mast cells, NK cells, endothelial cells and fibroblasts that functions as a cytokine. GM-CSF functions as a white blood cell growth factor and stimulates stem cells to produce granulocytes (neutrophils, eosinophils, and basophils) and monocytes.

IFN-γ (interferon gamma)

a type II interferon, IFNγ is a cytokine that is required for innate and adaptive immunity against viral, bacterial and protozoal infections. IFNγ has been shown to be an important activator of macrophages and inducer of Class II major histocompatibility complex (MHC) molecule expression. IFNγ is produced predominantly by natural killer (NK) and natural killer T (NKT) cells as part of the innate immune response, and by CD4 Th1 and CD8 cytotoxic T lymphocyte (CTL) effector T cells once antigen-specific immunity develops.

IL-12

a cytokine produced by dendritic cells, macrophages, neutrophils, and human B-lymphoblastoid cells (NC-37) in response to antigenic stimulation and has been shown to be required for the differentiation of naive T cells into Th1 cells.

IL-25

a pro inflammatory cytokine that shares sequence similarity with IL-17 and has shown to favor the Th2-type immune response. IL-25 can induce NFкB activation and stimulate the production of IL-8.

IL-33

a member of the IL-1 family of cytokines that potently drives production of Th2-associated cytokines

Mannoproteins

antigenic proteins found in the yeast cell wall with many mannose groups attached.

ORMDL3

(Orosomucoid-like 3) a gene on chromosome 17q21 highly linked to asthma, has been shown to upregulate airway smooth muscle proliferation, contraction, and Ca²⁺ oscillations in asthma

Reactive oxygen species (ROS)

a natural byproduct of oxygen metabolism that has a critical role in cell signaling and homeostasis. However, during times of environmental stress, ROS levels can increase significantly and cause damage to cell structures, which is known as oxidative stress.

Toll Like Receptors (TLRs)

members of the toll-like receptor (TLR) family which play a fundamental role in pathogen recognition and activation of innate immunity.

TLR1/TLR2 heterodimer

a complex where TLR1 recognizes peptidoglycan and triacyl lipoproteins in combination with TLR2

TLR2

a membrane surface receptor that recognizes many bacterial, fungal, viral, and certain endogenous substances and plays a fundamental role in pathogen recognition and the activation of innate immunity.

TLR3

a membrane surface receptor that recognizes dsRNA associated with viral infection, and induces the activation of interferon regulatory factor 3 (IRF3), unlike all other toll-like receptors which activate NF-κB.

TLR4

a transmembrane protein which belongs to the pattern recognition receptor (PRR) family. Its activation leads to the activation of the innate immune system via an intracellular signaling pathway NF-κB and inflammatory cytokine production. TLR4 recognizes lipopolysaccharide (LPS), which is a component present in many Gram-negative bacteria and select Gram-positive bacteria. Its ligands also include several viral proteins, polysaccharide, and a variety of endogenous proteins.

TLR9

an important receptor expressed in dendritic cells, macrophages, natural killer cells, and other antigen presenting cells that preferentially binds bacterial and viral DNA, and triggers signaling cascades that lead to a pro-inflammatory cytokine response.

Thymic stromal lymphopoietin (TSLP)

A IL-2 family cytokine that stimulates the maturation of T cells through the activation of antigen presenting cells such as dendritic cells and macrophages. This cytokine is implicated in Th2-type immune response.

Unfolded protein response (UPR)

a signaling network that is activated by stresses that compromise the endoplasmic reticulum (ER) which impair maturation resulting the accumulation of mis-folded proteins to alleviate this stress and restore ER homeostasis, promoting cell survival and adaptation. Although under irresolvable ER stress conditions, the UPR promotes apoptosis.