Indoor Allergens and Allergic Respiratory Disease

Abstract

Purpose of review

The purpose of this review is to evaluate the most recent findings on indoor allergens and their impact on allergic diseases.

Recent findings

Indoor allergens are present inside buildings (home, work environment, school), and given the chronic nature of the exposures, indoor allergies tend to be associated with the development of asthma. The most common indoor allergens are derived from dust mites, cockroaches, mammals (including wild rodents and pets), and fungi. The advent of molecular biology and proteomics has led to the identification, cloning, and expression of new indoor allergens, which have facilitated research to elucidate their role in allergic diseases. This review is an update on new allergens and their molecular features, together with the most recent reports on their avoidance for allergy prevention and their use for diagnosis and treatment.

Summary

Research progress on indoor allergens will result in the development of new diagnostic tools and design of coherent strategies for immunotherapy.

Introduction

Sensitization and exposure to indoor allergens is a risk factor for allergic respiratory diseases, including rhinitis and asthma [1, 2]. In this report, an overview of the most important indoor allergens is presented, together with an update on the most current cutting edge research on their molecular structure and function (Table 1) [3•] and approaches to assess and treat indoor allergies.

Table 1.

Most relevant indoor allergens

Source	Group	Allergen	Protein family
Mite	1	Der p 1, Der f 1	Cysteine protease
	2	Der p 2, Der f 2	Immunoglobulin-like
	5	Der p 5	Structural protein
	7	Der p 7, Der f 7	LPS-binding protein
	10	Der p 10, Der f 10	Tropomyosin
	11	Der p 11, Der f 11	Paramyosin
	23	Der p 23	Chitin-binding protein homologue
Cockroach	1	Bla g 1, Per a 1	Microvilli protein homologue
	2	Bla g 2, Per a 2	Inactive aspartic protease
	4	Bla g 4	Lipocalin
	5	Bla g 5	Glutathione S-transferase
	9	Per a 9	Arginine kinase
	11	Bla g 11, Per a 11	α-Amylase
Cat	1	Fel d 1	Uteroglobulin
	4	Fel d 4	Lipocalin
Dog	1	Can f 1	Lipocalin
	2	Can f 2	Lipocalin
	5	Can f 5	Arginine esterase
Rodents	1	Mus m 1, Rat n 1	Lipocalin
Fungi	1	Asp f 1	Mitogillin
	1	Alt a 1	Unknown
	6	Cla h 6	Enolase
	8	Cla h 8	Mannitol dehydrogenase
	13	Pen ch 13	Serine protease
	18	Pen ch 18	Serine protease

Arthropods

Mites

House dust mites are an important cause of allergies worldwide, associated with diseases such as allergic rhinitis, atopic dermatitis, and asthma [1]. Mite allergens are classified into 33 groups, listed in the systematic Allergen Nomenclature Database maintained by the World Health Organization and International Union of Immunological Societies (WHO/IUIS) (www.allergen.org). More than 95 % of the allergen accumulating in mite cultures is associated with fecal particles, where Der p 1, the first allergen to be isolated, was estimated to be present at 10 mg/ml [4, 5]. These particles, from 10 to 40 μm of diameter, become airborne upon disturbance. Although inhalation is the most common mechanism of exposure, ingestion of foods made with mite-contaminated wheat flour has also been reported as a source of allergic reactions, including anaphylaxis (“the pancake syndrome”) [6]. The two most common species of dust mites are Dermatophagoides pteronyssinus and Dermatophagoides farinae. Additional mite species are Dermatophagoides microceras, Euroglyphus maynei, and Blomia tropicalis, as well as storage mites Glycyphagus domesticus, Lepidoglyphus destructor, Acarus siro, and Tyrophagus putrescentiae.

Groups 1 and 2

The group 1 and 2 allergens cause sensitization in >80 % of mite-allergic patients. These potent allergens account for 50–60 % of anti-house dust mite IgE antibodies in allergic subjects [7]. The group 1 allergens, Der p 1 and Der f 1, are cysteine proteases. The structures of natural Der p 1 and Der f 1 were determined, alone or in complex with fragments of monoclonal antibodies (mAb) that inhibit IgE antibody binding [8, 9, 10••] (Fig. 1). The proteolytic activity of group 1 has been reported to contribute to allergenicity by cleaving molecules involved in the immune response (i.e., CD23, CD25) and increasing membrane permeability [11]. Group 3, 6, and 9 allergens are serine proteases and may have similar effects.

Fig. 1.

X-ray crystal structures of Bla g 1 (Protein Data Bank ID code 4JRB), Der p 1 (3RVW), Mus m 1 (1MUP), and Fel d 1 (2EJN)

Group 2 mite allergens have immunoglobulin-like folds, which bind lipids in the internal cavity. Der p 2 activates the innate immune system through Toll-like receptors (TLR-4) by mimicking the action of human MD-2 (myeloid differentiation antigen-like lipid-binding protein), a structural homologue that loads lipopolysaccharide (LPS) onto these receptors [12]. A TLR4-associated phospholipase D1 activation has recently been reported to be crucial for Der f 2-induced IL-13 production [13].

Groups 4, 5, 7, and 21

Allergens belonging to groups 4, 5, 7, and 21 together account for 30 % of house dust mite IgE antibodies, and each of them binds IgE in approximately 50 % of mite-allergic subjects [7]. Allergens from group 4 are α-amylases. Groups 5 and 21 contain structurally related proteins with a three-helical bundle [14]. Allergens from group 7 have a similar structure to LPS-binding proteins that interact with Toll-like receptors upon binding of LPS and other bacterially derived lipid ligands [15].

Group 8 allergens are glutathione S-transferases (GST), and group 10 allergens are tropomyosins. The degree of homology of these mite allergens with allergens from other species is an important determinant of allergenic cross-reactivity or lack thereof [16•]. Recently, a tropomyosin was reported from Chortoglyphus arcuatus, a storage mite to whom some patients are mono-sensitized in the northwest of Spain [17]. A mixture of purified mite allergens from groups 1, 2, 5, 7, 8, and 10 bound, on average, 76 % of mite-specific IgE antibodies [18]. Group 11 allergens are paramyosins, and Der p 11 is a new marker allergen for house dust mite-allergic patients suffering from atopic dermatitis [19••].

Der p 23 has been described as a new major allergen with a high prevalence of IgE reactivity (74 %) [20, 21]. However, this allergen accounts for a small percentage of the IgE response to mite allergens, which is dominated by Der p 1 and Der p 2 [22]. Interestingly, RNA expression level of Der p 23 is the lowest of the major allergens. The allergen is a small, globular protein stabilized by two disulphide bonds [22]. Der p 23 is homologous to chitin-binding proteins, but recent studies have shown that the allergen does not bind chitin and must have a different function [22, 23].

In 2015, genomic-transcriptomic and/or proteomic approaches have been used to identify up to 33 mite allergen groups [24, 25••].

Cockroaches

The first report on positive skin test responses to cockroach allergen dates back to 1964 [26]. A strong association between cockroach allergy, allergic rhinitis, and asthma has been demonstrated [27–29]. Inner-city asthma studies in the USA have shown that exposure and sensitization to cockroach allergens are associated with increased asthma morbidity in children [28, 29]. The two most common species are German and American cockroach (Blattella germanica and Periplaneta americana, respectively). The WHO/IUIS Allergen Nomenclature database currently lists 12 groups of cockroach allergens.

Groups 1 and 2

The molecular structure of group 1 cockroach allergens (Bla g 1 and Per a 1) consists of tandem repeats of ~100 amino acids. The determination of the three-dimensional structure of these allergens, challenging due to protein fragmentation, has recently been achieved for Bla g 1 thanks to the expression of its basic structural unit. The Bla g 1 unit comprises two consecutive repeats of six helices each, which encapsulate a large hydrophobic cavity that contains lipids (Fig. 1). This structure allowed the definition of 1 unit of Bla g 1 as 104 ng of allergen, which facilitates allergen standardization [30].

Bla g 2 is a globular protein that belongs to the family of aspartic proteases, but amino acid substitutions in the catalytic site render it inactive [31]. The antigenic surface of Bla g 2 has been analyzed by determining the structure of the allergen in complex with fragments (Fab or Fab′) of mAb that interfere with IgE antibody binding and by site-directed mutagenesis of residues involved in the epitopes [32–34, 35•]. These studies revealed IgE antibody binding sites and mechanisms of allergen-antibody interaction.

Group 3 allergens are homologous to arylphorins and insect hemocyanins. Different isoallergens and variants have been reported for Per a 3 with a wide range of skin test reactivities (26–95 %) [36]. Therefore, the relevance of this allergen remains controversial.

Group 4 allergens are lipocalins, with a similar molecular structure to mammalian lipocalin allergens, e.g., from cow, dog, cat, horse, rat, and mouse (Fig. 1). The molecular structure of Bla g 4 consists of an eight-stranded β-barrel and a C-terminal α-helix [37]. Most lipocalins share a low degree of amino acid identity (~20 %), and no significant cross-reactivity among them is expected.

Group 5 allergens are GST, thought to be involved in detoxification of toxic compounds. Bla g 5, together with Bla g 2, is one of the most important cockroach allergens in USA patients [38]. Recently, the X-ray crystal structures of Bla g 5 and the homologous allergens Der p 8 and Blo t 8 from mites and Asc s 13 from the nematode Ascaris suum were determined. These GST allergens showed a significant lack of cross-reactivity in a US population, suggesting that each individual allergen would be required for molecular diagnostic purposes [16•].

Groups 6, 7, and 8 are structural molecules including troponin C (group 6), tropomyosin (group 7), and myosin light chain. Tropomyosins are ubiquitous inhalant and food panallergens. In Brazil, cockroach tropomyosin is an important allergen, showing potential cross-reactivity with mite and shrimp tropomyosins [27]. Allergens from groups 6 and 8 are minor allergens and regulatory proteins involved in muscle contraction, which undergo structural changes upon calcium binding to EF-hand motifs [39].

Groups 9, 10, 11, and 12 are enzymes: arginine kinases, serine proteases, α-amylases, and chitinases, respectively, and mostly reported for P. americana, except for Bla g 11 [40–43]. A Per a 9-homolog from German cockroach and Bla g 11 have recently been reported to be immunodominant, together with Bla g 5, for T cell responses in asthmatic subjects [44]. The prevalence of IgE to these allergens was relatively high in the Asian countries where they were identified and needs to be evaluated in other parts of the world.

Mammalian Allergens

Animal allergens are primarily produced in the liver or secretory glands and are present on animal skin and in body fluids, such as urine, saliva, and blood. With the exception of the main cat allergen Fel d 1, most major animal allergens belong to the lipocalin family of proteins [45]. The proteins adhere to fur and can thus be efficiently distributed in the environment where they accumulate on fabrics, carpets, upholstery, and mattresses. Numerous studies have confirmed that the distribution of animal allergens in the environment is ubiquitous. Thus, allergy to animal proteins is considered a significant public health problem [46].

Cat (Felis domesticus)

Fel d 1 is a 38 kD tetrameric glycoprotein with a structure similar to that of uteroglobulin [47] (Fig. 1). IgE from over 90 % of cat-sensitized individuals reacts with this major cat allergen [48]. Fel d 1 is produced in sebaceous, anal, and salivary glands and transferred to the fur by grooming [49, 50]. While airborne Fel d 1 is mostly associated with larger particles (>9 μm), about 23 % of airborne Fel d 1 is carried on small particles (<4.7 μm diameter) that stay suspended in the air for several days, favoring distribution of the allergen in the environment [51]. In fact, the quantity of cat allergen in schools is directly correlated to the number of children in the class who live with cats in their homes [52]. Other relevant cat allergens include the cross-reactive serum albumin Fel d 2 and the lipocalins Fel d 4 and Fel d 7, which react with 15–40, 63, and 38 % of IgE from cat-allergic patients, respectively [53–55].

Dog (Canis familiaris)

Four of six currently identified dog allergens, Can f 1, Can f 2, Can f 4, and Can f 6, are lipocalins [56]. About ~70 % of dog-allergic individuals have IgE antibodies specific to the major dog allergen Can f 1 [57, 58]. Can f 1 is detectable not only in all homes with a dog, but also in one third of homes without a dog [59, 60••]. The size distribution of particles associated with Can f 1 is similar to that of Fel d 1 [60••]. A wide variability in Can f 1 levels can be found between dog breeds, but there is no evidence for a hypoallergenic breed [61, 62]. Can f 5 is also considered a major allergen, with up to 70 % of dog-allergic patients having Can f 5-specific IgE. Interestingly, 38 % of dog-allergic patients were monosensitized to Can f 5 [63]. Other relevant dog allergens are the lipocalins Can f 2 and Can f 4 and the cross-reactive dog serum albumin Can f 3, which react with 20–30, 15–50, and 81 % of IgE from dog-allergic patients, respectively [64–67].

Rodents (Mus musculus, Rattus norvegicus)

Allergy to mice and rats is an important occupational health problem. The prevalence of rodent allergy among technicians, animal care takers, physicians, and scientists working in pharmaceutical industry, university laboratories, and animal facilities ranges from 11 to 44 % [68]. Besides exposure in occupational settings, rodent exposure also occurs in domestic environments as was shown in inner-city children with asthma in the USA, where mouse and rat sensitization rates were 11–47 and 21 %, respectively [69, 70•, 71]. In contrast, a recent study from Europe has reported very low sensitization prevalence for mouse and rat (1.5 and 0.5 %, respectively) in urban atopic populations without occupational exposures [72]. Mouse sensitization has also been associated with allergic rhinitis in urban children in the USA with comorbid asthma [73]. Urine is the main source of allergenic proteins in both mice and rats, and the major allergens Mus m 1 from mouse and Rat n 1 from rat are lipocalins. Mus m 1 is carried on small particles that stay airborne for a long time, favoring the distribution of the allergen within the facility and even outside the facility into the homes of laboratory animal workers [74, 75]. Indeed, children of parents who are occupationally exposed to rodents have a higher prevalence of sensitization to mouse, rat, and hamster compared to children of non-exposed parents [76].

Fungi

From more than 15 genera of fungi measurable in inner-city homes, Cladosporium, Penicillium, Aspergillus, and Alternaria species were the most commonly detected [77]. Alternaria and Cladosporium species also produce important outdoor allergens, and sensitization and exposure to species of these genera is associated with the development of asthma and rhinitis, as well as epidemics of asthma exacerbations, some of which are life threatening [78].

Alternaria alternata

The prevalence of sensitization to Alternaria is approximately 5 % and is strongly associated with asthma and allergic rhinitis [79]. Alt a 1 is the most important Alternaria allergen with a seroprevalence of over 90 % among Alternaria-sensitized individuals [80]. The structure of this dimeric allergen has been recently determined [81]. Other relevant Alternaria alternata allergens include Alt a 2, a 25 kD aldehyde dehydrogenase and major allergen, as well as Alt a 5, an enolase, which is recognized by approximately 20 to 50 % of Alternaria-sensitized individuals [82, 83].

Cladosporium herbarum

Similar to Alternaria, C. herbarum is frequently found in indoor and outdoor air and is a major source of fungal inhalant allergens [84]. While Alternaria alternata is a major allergen in humid climates, Cladosporium is the leading allergenic mold in cooler climates [85]. No dominant Cladosporium allergen had been found until the identification of Cla h 8, a NADP-dependent mannitol dehydrogenase. Cla h 8 is recognized by IgE antibodies of 57 % of all Cladosporium-allergic patients [86]. In addition, Cla h 6 (enolase) is recognized by ~50 % of sera from Cladosporium-sensitized patients [87].

Aspergillus fumigatus

Aspergillus fumigatus is a thermo-tolerant fungus with worldwide distribution. A. fumigatus is the principal etiologic agent of allergic bronchopulmonary aspergillosis (ABPA) and is also associated with asthma [88]. Both ABPA and allergic asthma are characterized by hypersensitivity and presence of A. fumigatus-specific IgE, but the sensitization patterns to individual allergens differ [89]. The major allergen, Asp f 1, is an 18 kD ribotoxin that is recognized by 85 % of Aspergillus-sensitized patients [90]. Besides Asp f 1, several other Aspergillus allergens (Asp f 2, Asp f 3, Asp f 4, Asp f 5, Asp f 9, Asp f 11, Asp f 15, and Asp f 18) are associated with a high prevalence of reactivity among Aspergillus-sensitized patients [78].

Penicillium

Penicillium species are prevalent indoor fungi that are associated with allergic disease in sensitized individuals. Penicillium citrinum and Penicillium chrysogenum are the most studied and the two most abundant species in the USA. The major allergens of P. chrysogenum include the serine proteases Pen ch 13 and Pen ch 18 with specific IgE reactivities of 88 and 82 %, respectively [91, 92]. Generally, IgE reactivity to allergens from P. citrinum was lower, and the highest reactivity of 46 % among Penicillium-sensitized asthmatic patients was reported for Pen c 3, an 18 kD membrane protein [93].

Cross-Reactive Indoor Allergens

The most relevant protein families involved in cross-reactivity with indoor allergens are tropomyosins and serum albumins. Tropomyosin is a highly conserved protein found in both muscle and non-muscle cells of all species of vertebrates and invertebrates. Allergenic tropomyosins are found in invertebrates such as crustaceans, arachnids (house dust mites), insects (cockroaches), and mollusks (squid). Immunological cross-reactivity has been demonstrated between crustaceans, cockroaches, and house dust mites, suggesting that tropomyosin is an important cross-sensitizing panallergen. More than 50 % of European house dust mite-allergic patients with IgE sensitization to tropomyosin (Der p 10) have clinically relevant cross-reactivity to eating seafood [94]. However, there is a lack of allergenic cross-reactivity between these tropomyosins and those from vertebrates such as bony fish, beef, pork, or chicken, which are considered nonallergenic [95].

Allergic sensitization to serum albumin can occur by inhalation as well as ingestion [96]. Serum albumins are found in dander and saliva and are important inhalant allergens of cat (Fel d 2) and dog (Can f 3). Chicken serum albumin (Gal d 5) is a major hen egg allergen that is associated with the bird-egg syndrome, a cross-reactivity between ingested egg allergens and inhaled feather and dander allergens [97]. Similarly, the cat-pork syndrome is based on cross-reactivity between Fel d 2 and pork serum albumin (Sus s 6). In this rare syndrome, patients develop an IgE antibody response specific for cat serum albumin Fel d 2 that cross-reacts with porcine albumin Sus s 6 and can lead to severe or even fatal allergic reactions on occasions when pork is consumed [98].

Indoor Allergens for Diagnosis

Commercial allergen extracts are standardized based on in-house assays and standards and are not comparable between different manufacturers [99]. Efforts to improve standardization and enable cross-product comparisons using individual recombinant allergens were initiated in 2001 with the CREATE project of the European Union [100]. Purified allergens from birch, timothy grass, olive pollen, and dust mites were compared with the natural counterparts, and allergen-specific candidate ELISA systems were investigated. Two allergens, rBet v 1 and rPhl p 5, were further characterized through the BSP090 Biological Standardization Programme (BSP) of the European Directorate for the Quality of Medicines and HealthCare (EDQM) [101]. These allergens have since been included in the European Pharmacopeia.

The selection of an optimal set of allergens for diagnosis needs to be evaluated for each source. Sensitization to Fel d 1 in childhood is a good predictor of cat allergy symptoms during adolescence [102]. However, other allergen sources such as cockroach do not have a dominant allergen. Early studies revealed that Bla g 1, Bla g 2, Bla g 4, and Bla g 5 identify ~95 % of cockroach-allergic patients in the USA [103]. As more allergens have been identified around the world, different patterns of IgE sensitization to cockroach have been found in other populations. IgE reactivity to cockroach tropomyosin was found to be dominant in Brazil [27]. Overall, a cocktail of five cockroach allergens from groups 1, 2, 4, 5, and 7 would allow to diagnose 50–65 % of patients worldwide [103].

For mites, Der p 1 and Der p 2 will diagnose most mite-allergic patients. Der p 23 has also been defined as a major allergen [20]. However, the contribution of Der p 23 to mite-specific IgE was small (4 %) compared to Der p 1 and Der p 2 combined (85 %) in a North American population [22]. Der p 1 and Der p 2 diagnosed 96 % of mite-allergic patients, and addition of Der p 23 did not show further improvement. Similarly, a European study reported an IgE prevalence of 89 % for Der p 1 and Der p 2 combined, and the addition of Der p 23 increased the percentage only by 3 % [104].

Allergen Exposure Assessment

Measurements of major allergens in dust and air samples have proved to be an effective approach to assess allergen exposure and to relate exposure to allergic sensitization. While in the past, these measurements were made by ELISA, they are increasingly being replaced by multiplex technology (MARIA) which enables 6–10 allergens to be measured in a single assay [105]. In keeping with the CREATE project, ELISA and MARIA use purified allergens as standards. For example, cockroach allergens are used to be measured in arbitrary units but are now measured in absolute units using purified allergen standards [30, 106]. The advantages of immunoassays are that they provide high throughput and are ideally suited to large cohorts involving hundreds or thousands of environmental samples. Alternatively, much progress has recently been made using mass spectrometry for allergen measurements, and this highly sensitive technology is increasingly being used in the pharmaceutical industry (reviewed in [107]). The joint task force of the AAAAI and ACAAI recently published several practice parameters on environmental exposure assessments as part of allergy practice [108–111]. The parameters provided systematic reviews of the categories of evidence linking allergen exposure and allergic symptoms. The practice parameters did not make specific recommendations regarding allergen exposure thresholds for health effects. Further work in this area is needed to improve indoor air quality in the homes of allergic patients.

Indoor Allergens and Therapy

Avoidance of indoor allergen exposure is an important factor that may ameliorate symptoms but is not always sufficient. Immunotherapy via either subcutaneous or sublingual routes has shown benefits in patients with allergic rhinitis and allergic asthma induced by house dust mites. However, there is a lack of consensus on basic treatment parameters (i.e., dose and duration) and a need for rigorous, long-term, double-blind, placebo-controlled randomized clinical trials for house dust mite allergies [112]. Four pilot studies of cockroach immunotherapy suggest that immunotherapy with cockroach allergen is more likely to be effective with SCIT than SLIT [113].

Modified Indoor Allergens for Immunotherapy

The availability of natural and recombinant purified allergens has led to the design of new immunotherapeutic molecules [114]. The rationale for using modified allergens for immunotherapy is to reduce side effects due to IgE cross-linking during the administration of increasing doses of allergen, while maintaining immunogenicity. Hypoallergenic chemically modified extracts (allergoids) are successfully used in Europe for rhinitis, asthma, and atopic dermatitis. These include carbamylated allergoids and depigmented-polymerized extracts [115–117]. However, the Federal Drug Administration has not approved their use in the USA, because these preparations lack structurally well-defined molecules and are difficult to standardize.

Alternative approaches to immunotherapy are under study, based on current knowledge of the molecular structure of allergens. One of them uses short T cell epitope synthetic peptides from the allergen sequence and has been extensively studied for cat allergy and less for mite [118, 119].

Additional approaches to immunotherapy have become possible with the advent of molecular biology to generate modified allergens expressed as recombinant proteins [114]. The design of protein modifications is based on structural features of the allergens and aims to reduce IgE antibody reactivity while preserving immunogenicity. For example, hybrid molecules were obtained by either combining two allergens, such as Der p 1 and Der p 2 [120, 121], or by combining two fragments in inverse order [122]. Allergens fused to viral domains or viral-like particles have shown immunomodulatory capacity [21]. Another strategy is to perform site-directed mutagenesis of known IgE epitopes, based on the X-ray crystal structures of the allergens alone or in complex with monoclonal antibodies that interfere with IgE antibody binding [9, 10••, 32, 34, 35•, 123, 124].

Conclusions

In the past 20 years, a broader knowledge of indoor allergens from mite, cockroach, cat, dog, rodents, and fungi has led to the development of new strategies for the diagnosis and treatment of allergic disease. These include the use of modified allergens with reduced IgE reactivity, allergen peptides, or fusion proteins with molecules that modulate immune responses.