Abstract

Common airborne allergens (pollen, animal dander and those from fungi and insects) are the main triggers of type I allergic disorder in the respiratory system and are associated with allergic rhinitis, allergic asthma, as well as immunoglobulin E (IgE)-mediated allergic bronchopulmonary aspergillosis. These allergens promote IgE crosslinking, vasodilation, infiltration of inflammatory cells, mucosal barrier dysfunction, extracellular matrix deposition and smooth muscle spasm, which collectively cause remodelling of the airways. Fungus and insect (house dust mite and cockroaches) indoor allergens are particularly rich in proteases. Indeed, more than 40 different types of aeroallergen proteases, which have both IgE-neutralising and tissue-destructive activities, have been documented in the Allergen Nomenclature database. Of all the inhaled protease allergens, 85% are classed as serine protease activities and include trypsin-like, chymotrypsin-like and collagenolytic serine proteases. In this article, we review and compare the allergenicity and proteolytic effect of allergen serine proteases as listed in the Allergen Nomenclature and MEROPS databases and highlight their contribution to allergic sensitisation, disruption of the epithelial barrier and activation of innate immunity in allergic airways disease. The utility of small-molecule inhibitors of allergen serine proteases as a potential treatment strategy for allergic airways disease will also be discussed.

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This review highlights the contribution of common airborne indoor allergen serine proteases to epithelial damage, sensitisation and pro-inflammatory processes in allergic airway disease. https://bit.ly/3IoVFSB

Introduction

Sensitisation to airborne allergens is the main risk factor for allergic respiratory disease, including allergic asthma, allergic rhinitis (AR), chronic rhinosinusitis (CRS) and immunoglobulin E (IgE)-mediated components of allergic bronchopulmonary aspergillosis (ABPA), the prevalence of which has increased globally over the last number of decades [1]. In 2019, asthma was reported to affect an estimated 262 million people worldwide and caused 455 000 deaths [2]. AR, characterised by anterior or posterior rhinorrhoea, sneezing, nasal blockage and/or itching of the nose, as well as a number of non-nasal symptoms such as allergic rhinoconjunctivitis, post-nasal drip and cough, affects an estimated 25% of children and 40% of adults globally [3]. CRS, a complex, heterogeneous inflammatory disease of the sinonasal mucosa, has a prevalence of 10.9% in the EU and 12.3% in the USA across all age groups [4]. ABPA is a fungal infection of the lung that affects severe asthmatics, but also individuals with cystic fibrosis (CF). It is characterised by evidence of allergy to the antigens of Aspergillus (Asp.) species upon its colonisation of the airways. The most common airborne fungus is Asp. fumigatus [5]. Although these conditions may exist independently from one another, many asthmatics (40–90%) will also show symptoms of AR, CRS or ABPA [6]. Such comorbidity is frequently associated with more frequent asthmatic attacks, leading to a greater number of emergency room visits as well as increased asthma-related hospitalisations and medical costs [7]. Avoidance of asthma triggers such as airborne allergens, in addition to adequate inhaled medication, is therefore critically important in the management of asthma symptoms.

A hallmark of allergic airways disease is the presence of raised IgE against common aeroallergens. Exposure to an allergen induces the generation of a specific IgE that binds to the high-affinity IgE receptor (FcεRI) on mast cells and basophils. A second exposure thus initiates an IgE-mediated hypersensitivity. The antigen binds to the IgE–FcεRI complex that is already present on the surfaces of mast cells and basophils. The crosslinking of the antigen-triggered bound IgE–FcεRI complex results in degranulation of mast cells and increased secretion of histamine, leukotrienes, heparin, proteases and other inflammatory mediators. This leads to elevated vascular permeability, peripheral vasodilation and smooth muscle contraction [8]. Allergens with proteolytic activity not only cause atopic effects, but also damage epithelial barrier integrity enabling allergens to directly interact with the immune system. This promotes allergic inflammation and subsequent secondary allergen sensitisation and other infection [9].

Herein, specific allergen serine proteases were identified from the Allergen Nomenclature (as listed by the World Health Organization and International Union of Immunological Societies (WHO/IUIS) Allergen Nomenclature Sub-Committee) and MEROPs (peptidases) databases, which allowed a search to be performed on PubMed to ascertain their association with inflammatory or cellular changes in relevant cell and animal models or their clinical relevance to the progression of allergic asthma, AR and CRS. In particular, we highlight the allergenicity and proteolytic effect of indoor serine protease allergens from fungi (to include moulds and yeast) and arthropods (mites and cockroaches) and their contribution to allergic sensitisation, disruption of the epithelial membrane barrier and activation of innate immunity in allergic airways disease. The utility of small-molecule inhibitors of the allergen serine proteases as a potential treatment strategy for allergic airways disease is also discussed.

Activity and source of indoor serine protease allergens

Common airborne allergens, such as fungi, house dust mites (HDMs) and cockroaches, are rich in serine protease activities that are highly involved in the development of allergic inflammation and barrier dysfunction in the airways. Serine proteases are characterised by the presence of a primary serine residue in the catalytic triad of their active site, which acts as a nucleophile to catalyse the hydrolysis of peptide bonds [10]. There are more than 40 types of aeroallergen serine proteases documented in the Allergen Nomenclature database created by the WHO/IUIS Allergen Nomenclature Sub-Committee, including subtilisin-like, elastinolytic, trypsin-like, chymotrypsin-like and collagenolytic serine proteases [11]. Table 1 summarises the source, biochemical classification, size, IgE binding ability and cross-reactivity of all the allergen serine proteases documented in the peptidase (MEROPS) and Allergen Nomenclature databases.

TABLE 1

Serine proteases associated with airborne allergens as documented in the peptidase (MEROPS) and Allergen Nomenclature databases

Fungi

Fungi predominantly contain elastinolytic and subtilisin-like serine proteases. The S1 specificity pocket of elastinolytic serine proteases are gated with two bulkier valine residues, which only allows them to cleave peptides containing amino acids with small side chains (e.g. alanine, serine, leucine and valine) in the complementary P1 position. Subtilisin-like serine proteases are an example of convergent evolution and share a similar active site (His-Asp-Ser) with chymotrypsin, i.e. the catalytic triad that also enables the formation of the oxyanion hole required for substrate hydrolysis. A difference, however, is that the oxyanion hole in the active site of subtilisin-like serine proteases is not formed by the peptide backbone but is assembled by the amine group from the side chain of an asparagine residue. Some of the elastinolytic serine proteases not only contains elastase activities, but also show homology to subtilisin-like protease, especially Asp fl 13, Asp f 13, Asp o 13 and Asp v 1 found in Asp. species.

Insects (arthropods)

Within the animal kingdom, arthropods (mite and cockroach) shed allergenic proteins in their egg casings, skin casts, whole bodies, secretions and faecal materials which can then become airborne. These proteins are rich in trypsin-like (TLP) and chymotrypsin-like serine proteases (CTLP) (table 1). TLPs in contrast to elastinolytic proteases, cleave long and positively charged peptide bonds (e.g. arginine and lysine) while CTLPs cleave peptide residues with aromatic or long, nonpolar side chains (e.g. phenylalanine or leucine). Der p 1, a cysteine protease, is the most abundant protease found in mite faecal pellets and is a major inducer of the IgE allergic response. Der p 1 activity is also pivotal to the activation of several HDM serine proteases, Der p 3, 6 and 9, that further contribute to allergenicity [111]. The impact of Der p 1 in allergic airway disease has been extensively reviewed by other researchers [112, 113].

Per a 10 is an abundant TLP found in the American cockroach (Periplaneta (Per.) americana) that has been found to alter immune signalling in the airways [114]. Bla g 2 is a potent allergen isolated from the German cockroach (Blattella (Bla.) germanica) that upon further characterisation was determined to be an inactive aspartic protease (therefore excluded from table 1). Pro-inflammatory effects of Bla. germanica extract on airway epithelial cells have, however, been attributed to three biochemically distinct serine TLP activities (designated E1, E2 and E3, but not listed in the WHO/IUIS Allergen Nomenclature) with a 57–71% sequence identity to Per a 10 and able to trigger both Ca2+ and mitogen-activated protein kinase signalling via activation of protease-activated receptor (PAR)-2 [93].

Mammals

Only a very small number of serine proteases are derived from the dander of mammals, such as dogs (Can f 5) and cats (Fel d 1). Can f 5 is a kallikrein expressed in the prostate and hence present only in the urine of male dogs with levels reduced upon neutering; Can f 5 has cross-reactivity with human prostate-specific antigen. It may also be present in extracts of dog hair and dander [31]. Studies have shown that the 18-kDa form of Fel d 1 is capable of degrading gelatin (denatured collagen), can cleave the A chain of plasma fibronectin and may be inhibited by the serine protease inhibitors aprotinin and N-p-tosyl-L-lysine chloromethyl ketone (TLCK). It does, however, lack a serine peptidase catalytic triad, which suggests that the inhibitory action of aprotinin and TLCK may be due to noncatalytic site interactions [115].

The ability of airborne serine protease allergens to compromise the epithelial barrier, create a pro-inflammatory environment and to induce airway remodelling, through cleavage of tight junction (TJ) and extracellular matrix (ECM) proteins and regulation of airway epithelial, immune and smooth muscle cells, is summarised in figure 1 and will be discussed in detail in the various sections below.

FIGURE 1

Allergenic and proteolytic activities of serine proteases from airborne allergens induce airway remodelling and promote a pro-inflammatory environment through regulation of airway epithelial, immune and smooth muscle cells, cleavage of tight junctions (TJs) and degradation of extracellular matrix (ECM) proteins. Initial damage of TJ proteins of airway epithelial cells is caused by allergen proteolytic activities triggering various interactions with immune cells. The synergistic pro-inflammatory effects of epithelial and dendritic cells (DCs) promote formation of a type 2 environment, as characterised by increased expression of interleukin (IL)-4, IL-5, IL-9, IL-13 and IL-23 by Th2 cells, accompanied by Th17 inflammatory responses which contributes to disruption of airway epithelium. This involves airway dehydration, excess mucus production, goblet cell hyperplasia and deciliation. An imbalance of protease and anti-protease activities in the ECM is characterised by increased elastase, tryptase and matrix metalloproteinase (MMP)-9 expression accompanied by reduced α1 antitrypsin (A1AT) and serine protease inhibitor Kazal-type 5 (SPINK5) expression. The alteration of ECM components also includes elevated growth factors (transforming growth factor-β (TGF-β), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF) and fibroblast growth factor (FGF)), therefore contributing to epithelial–mesenchymal transition and airway smooth muscle (ASM) hypertrophy and hyperplasia. CXCL1: chemokine ligand 1; CCL20: chemokine ligand 20; COX-2: cyclooxygenase-2; DAMPs: damage-associated molecular patterns; GM-CSF: granulocyte macrophage–colony-stimulating factor; IgE: immunoglobulin E; ILC2: type 2 innate lymphoid cell; PGE-2: prostaglandin E2; Tfh: T-follicular helper cells; Th2: T-helper 2 cells; Th17: T-helper 17 cells; TNF-α: tumour necrosis factor-α; TSLP: thymic stromal lymphopoietin; UA: uric acid. Figure created with BioRender.com.

Allergen serine proteases and airway epithelial barrier dysfunctionDisruption of TJs and effects on epithelial permeability

The airway epithelium acts as the first line of defence for the lungs and provides a physical, chemical and immunologic barrier in response to invasion of external environmental factors. Although repeated exposure to potentially harmful external factors can damage the pseudostratified structure of the epithelium, a slow but continuous process of renewal and repair helps to maintain airway homeostasis [116]. The pseudostratified epithelium is formed by different types of airway epithelial cells, including suprabasal, club, goblet, deuterosomal and ciliated cells, with rare cell types such as ionocytes, pulmonary neuroendocrine cells, tuft cells and “hillock” cells also present [117].

Mucociliary clearance mechanisms

An appropriate balance of mucosecretory and ciliated cells is vital for the effectiveness of mucociliary clearance (MCC) and underlies the physical innate defence mechanisms of the airways. The cilia are bathed by an airway surface liquid (ASL) comprising two layers, an upper mucus layer and a lower periciliary layer (PCL), and serves to protect the barrier function of the airway epithelium and the lower airways from harmful environmental substances. Mucin glycoproteins within the mucus layer physically trap inhaled particles while the aqueous PCL, whose volume is regulated by a balance of Na+ and Cl− transport, provides airways hydration and lubrication to promote the upward movement of the mucus layer via synchronised ciliary beating [118]. A serine protease from HDMs (trypsin-like and chymotrypsin-like protease) has been reported to increase apical Cl− secretion through calcium-activated chloride channels and CF transmembrane conductance regulator (CFTR), which is associated with the watery rhinorrhoea commonly seen in allergic airway diseases [119]. The activation of PAR-2 by proteases can also lead to an increase in cilia beating [120]. The impact of other types of allergen serine proteases, such as subtilisin-like and elastinolytic serine proteases on MCC, however, remains unknown. Bacterial elastase-like protease activities from Pseudomonas aeruginosa have been shown to disrupt coordinated secretion involving CFTR, PAR-2 and vasoactive intestinal peptide receptor in air–liquid interface (ALI) culture of Calu-3 cells, so it is possible that the elastase-like allergen serine proteases also contribute to the reduction of fluid secretion in serous cells.

TJs and the epithelial barrier

The barrier function of the epithelium is maintained by TJs between cells that are assembled by the scaffolding zonula occludens (ZO) family of proteins on the apical epithelium. TJs maintain the polarity of the airway epithelium, act as a bridge for cell–cell connections and communication, and play a critical role in limiting paracellular transport. The family of integral membrane proteins, including claudins and junctional adhesion molecules (JAMs), as well as occludins, tricellulin and marvel D3, is involved in the regulation of paracellular permeability. A charge-selective small-pore pathway is constructed by claudins to control ion conductance, while JAMs are involved in the regulation of macromolecule permeability [121]. At the basolateral membranes of epithelial cells, major intercellular adhesive junctions are formed by desmosomes creating a supracellular scaffold that enable the cell–cell contacts essential to tissue architecture [122]. Desmosomes are formed by specialised members of the cadherin family and associated cytoskeletal-linking proteins. They provide mechanical resilience to tissues and anchor intermediate filaments to the plasma membrane.

The effect of allergen serine proteases on epithelial permeability

Nearly all allergen serine proteases have been confirmed to have damaging proteolytic effects on TJs that result in the integrity of the epithelial barrier being compromised (figure 2). This includes fungal proteases, such as Alternaria (Alt.) alternata [123] and Asp. fumigatus serine proteases [124], Penicillium (Pen.) chrysogenum Pen ch 13 [125, 126], as well as HDM serine proteases [127, 128]. Serine proteases (groups 3, 6, 9) from HDM faecal pellets can cleave TJ proteins (occludins, ZO-1 and, to a lesser extent, E-cadherin) and concentrate desmosomal puncta (breaches), leading to high levels of epithelial permeability [127]. A study comparing the effect of HDM cysteine (Der p 1) and serine (Der p 6) proteases on ALI-polarised cultures of the lung epithelial cell line BEAS-2B demonstrated that the CTLP Der p 6 led to an increase in epithelial permeability [128]. In addition, degradation of occludin, ZO-1 and E-cadherin in Pen c 13 sensitised mice led to a progressive increase in epithelial permeability [126]. An increased production of mitochondrial reactive oxygen species (ROS) at the time of exposure to Asp. proteases also contributed to the epithelial cell barrier function being damaged, leading to pathological changes [129].

FIGURE 2

Allergen serine proteases impair airway epithelial barrier function. Allergen serine proteases from house dust mites, as well as Penicillium and Aspergillus species can cleave tight junction (TJ) proteins (zonula occludens-1 (ZO-1), junctional adhesion molecules (JAMs), occludin, claudin) and adherens junctions (AJs) (e.g. E-cadherin) and can create puncta in desmosomes. Production of mitochondrial reactive oxygen species (ROS) in response to exposure to Aspergillus proteases can also contribute to the epithelial cell barrier function being damaged, leading to pathological changes. Figure created with BioRender.com.

Stimulation of a pro-inflammatory response by allergen proteasesAlarmin and type 2 inflammatory response

Allergen serine proteases stimulate the production of epithelial and immune cell-derived inflammatory mediators which contribute to type 2 inflammation (figure 1). In particular, epithelial “alarmin” cytokines such as interleukin (IL)-33 and thymic stromal lymphopoietin (TSLP), released in response to tissue injury, play an important role in type 2 asthma. IL-33, which is a cytokine belonging to the IL-1 family, mainly targets IL-1-like receptor (ST2) on immune cells, especially tissue-resident mast cells, type 2 innate lymphoid cells (ILC2s) and tissue regulatory T-cells that express high levels of ST2 [130, 131]. Serum levels of IL-33 have been reported to be higher in patients with allergic and eosinophilic asthma phenotypes compared to nonallergic and noneosinophilic phenotypes [132]. In addition, allergen inhalation triggers upregulation of ST2 expression in eosinophils from blood and sputum derived from allergic asthma patients [133]. Exposure to Alt. alternata serine proteases led to the rapid release of IL-33 in lung epithelial cells from Alt. alternata challenged mice and simultaneously, also stimulated neutrophilia, eosinophilia and recruitment of tissue macrophages, as well as elevated IL-13 expression but had no effect on IL-5 levels [134].

Serine proteases from HDM have been reported to increase epithelial IL-33, TSLP and IL-25 secretion, resulting in activation of ILC2s, which produce IL-5, IL-9 and IL-13, in addition to IL-5 from eosinophils [135, 136]. Specifically, trypsin-like serine proteases have been identified to be the main allergen component of HDMs leading to the increased secretion of IL-33 and TSLP from asthma human primary bronchial epithelial cells (hPBECs) [137]. Upon exposure of epithelial cells to allergen proteases (fungi, cockroaches and mites), caspase-8 is activated via a novel signalling hub, the caspase-8 activating ripoptosome, a protein complex which consists of receptor-interacting serine/threonine-protein kinase 1 (RIPK1), Fas-associated via death domain and pro-caspase-8 [138, 139]. The liberated active caspase-8 then facilitates activation of caspases-3 and -7 (cleavage site at residues D175 and D178), which, in turn, directly promotes potentiation and maturation of IL-33 that is co-released with histone [138]. Secretion of IL-33 is then completed by neo-form murine amino-terminal p40 fragment gasdermin D, which promotes pore formation in the cell membrane [140]. The RIPK1–caspase 8 ripoptosome complex is, therefore, an allergen-sensing platform able to detect a diverse range of allergic stimuli. However, the actual signalling pathways, such as the role of PARs (see the section entitled “PARs”) and canonical pattern recognition receptors (PRRs) (see the section entitled “PRRs”), may lead to its activation have yet to be established [138]. Full-length IL-33 precursor (30 kDa) can also be directly processed by allergen serine proteases from HDMs, Alt. alternata, Asp. fumigatus, Asp. oryzae and cockroaches to generate mature IL-33 forms (18–21 kDa) [141]. Of note, mature IL-33 forms can have biological activity 10–30 times higher than that of their precursor and are thus capable of driving a cytokine storm [142]. The role of allergen proteases in the processing and maturation of IL-33 is summarised in figure 3.

FIGURE 3

Allergen serine proteases play a role in the maturation and secretion of interleukin (IL)-33 contributing to the type 2 inflammatory response. Allergen proteases can either directly process the full-length IL-33 (30 kDa) to mature IL-33 forms (18–21 kDa) or IL-33 can be potentiated and matured via the caspase-8 activating ripoptosome (a protein complex consisting of receptor-interacting serine/threonine-protein kinase 1 (RIPK1), Fas-associated via death domain (FADD) and pro-caspase-8). Ripoptosome activation is a pre-determined series of molecular events that starts with rapid RIPK1 phosphorylation and pro-caspase-8 activation, followed by degradation of RIPK1 and activation of caspases-3 and -7. Stress granule (SG) assembly permits the transportation of full-length (FL)-IL-33 from the nuclear compartment to the cytoplasm. The active caspases-3 and -7 then convert intracellular precursor IL-33 moieties with histone bonded into active mature (m)-IL-33. The involvement of protease activated receptors (PARs) and pattern recognition receptors (PRRs) in the initial signalling of this process remains unclear. Simultaneously, mature caspase-8 converts GSDMD-NT (consisting of an N-terminal domain (NTD) containing 242 amino acids) into neo-form murine amino-terminal p40 fragment gasdermin D (GSDMD), which leads to membrane pore formation to facilitate the release of m-IL-33. FL-IL-33 consists of three parts. The residues of amino acids from 1 to 65 are the nuclear domain, 65–112 are the protease sensor domain, which allows cleavage by endo-/exogenous proteases, and 112–270 are the IL-1-like domain containing the caspase cleavage sites at D175 and D178. Active IL-33 can then activate immune cells expressing IL-1-like (ST2) receptors (i.e. type 2 innate lymphoid cells (ILC2), mast cells, eosinophils, T-helper 2 (Th2) cells and dendritic cells (DCs)) to propagate a type 2 inflammatory response. Figure created with BioRender.com.

Other alarmins that are increased upon the exposure of human bronchial epithelial cells (HBECs) to serine proteases from HDMs, Artemisia vulgaris, Betula pendula and Alt. alternata, include ATP and uric acid (UA) [143]. Moreover, another study suggested that Alt. alternata and HDM serine proteases may possibly intensify the type 2 immune response through triggering DNA release from HBECs. The underlying mechanism involves the induction of ROS production by Alt. alternata and the subsequent apical release of nuclear and mitochondrial DNA. These DNAs serve as alarmins, which induce Ca2+ uptake via ATP release and subsequent activation of P2X purinergic receptors. The increase of intracellular Ca2+ concentration also elevates expression of the proprotein convertase furin, which promotes the maturation of the nuclear DNA fragmentation modulator, caspase-3 [144].

Other pro-inflammatory mediators

Aeroallergen serine proteases also promote secretion of other inflammatory mediators from airway epithelial cells. Alt. alternata serine proteases induced IL-8 and tumour necrosis factor-α (TNF-α) production from 16HBE cells, although reduced IL-8 production was observed in asthma ALI primary HBECs in response to Alt. alternata compared to healthy donors [123]. Protease activities of Pen ch 13 decreased cluster of differentiation (CD) 44 expression and increased expression of inflammatory mediators such as IL-8, prostaglandin-E2, cyclo-oxygenase-2 and transforming growth factor-β1 from HBEC lines (A549 and 16HBE14o-) and primary HBECs [125]. Additionally, Pen ch 13 has also been reported to stimulate histamine release from basophils of asthmatic patients [23] and IL-8 from HBECs [89]. Serine proteases from German cockroach induced elevation of chemokine (C-C motif) ligand 20 (CCL20) and granulocyte-macrophage colony-stimulating factor (GM-CSF) generation in the airways leading to the deployment and/or differentiation of myeloid dendritic cells (DCs) [145].

In terms of HDM allergens, the CTLP Der p 6 induces IL-6 production but decreases IL-8 and GM-CSF production in airway epithelial cells [128]. A similar effect on IL-6 production was observed with the collagenolytic serine TLP Der p 9. Der p 9 and Der p 3 also contributing to GM-CSF and eotaxin release from HBECs [66]. Treatment of primary HBECs and the epithelial cell line BEAS-2B by Der p 1 and Der p 9 induced both concentration- and time-dependent increases in the release of GM-CSF, IL-6 and IL-8 [146]. Der f 3 facilitates IL-6, IL-8, GM-CSF, IL-1β, chemokine ligand 1 and CCL20 production from HBECs, possibly contributing to the development of the T-helper (Th)-17 and Th2/Th17 mixed asthma endotypes [56]. An in vivo study further confirmed that HDM proteases trigger the generation of a chemokine, cytokine and oxidant response from airway epithelial cells, which activates IL-17A/IL-17R signalling, resulting in an increase in neutrophilic inflammation [147].

Allergen serine proteases as Th2 adjuvantsDendritic and Th cell recruitment

An increase in epithelial permeability caused by active allergen serine proteases (see the section entitled “TJs and the epithelial barrier”) permits their access to the sub-epithelial layer stimulating DC recruitment and allergic sensitisation. Fungal serine protease Asp f 13 (also called alkaline protease 1) can disrupt the TJs of bronchiolar epithelial club cells upon stimulation of mechanosensor transient receptor potential (TRPV) 4 channels, followed by elevation of intracellular Ca2+ [148]. Subsequently, Ca2+-regulated calcineurin can activate nuclear factor of activated T-cells, thus contributing to the production of an allergic, inflammation-driven cytokine and chemokine storm through employment of DCs and Th2 cells [148, 149]. DCs also trigger generation of T-follicular helper (Tfh) 1 and 2 cells and polarised Th2 cells.

Allergen serine proteases drive a Th2-adjuvant occurrence and altered airway remodelling

Th2-cells generate cytokines that are known to be involved in airway remodelling in asthma. Additionally, IL-4 secreted by Tfh cells stimulates IgM+ naive B-cells to change to IgG+ memory B-cells or IgE+ memory B-cells via class switch recombination (CSR). Basophils, mast cells and ILC2s also generate Th2 cytokines and contribute to the development of type 2 high airway diseases [150, 151].

Th2 adjuvant occurrence

Due to the abundance of proteolytic and sensitised activities in allergens, a growing number of studies recognise their ability to facilitate the Th2 adjuvant occurrence of the airway allergic inflammatory response. Alt. alternata serine proteases induce cell infiltration in murine airways [152]. Per a 10 from Per. americana induces DCs to a Th2-biased polarisation characterised by increased CD86 and TNF superfamily member 4 (also known as OX40 L) expression and reduced CD40 expression, which promotes a decrease of IL-12 and an elevation of IL-23, IL-13 and IL-4 expression in a murine lung model [15, 153–155]. Elevation of IgE and IgG1 in serum and an increased level of IL-4 and IL-5 in bronchoalveolar lavage fluid (BALF) and splenocyte culture supernatant was observed from mice challenged with Cur l 1 from C. lunata compared to an inactive Cur l 1 group [156]. Likewise, Pen c 13 induced elevated expression of Pen c 13-specific IgE and IgG1 in mice and increased IL-4, IL-5 and IL-13 production in Pen c 13 treated splenocytes [126].

Type 2 environment and altered airway remodelling

The type 2 environment with elevation of IL-4 and IL-13 contributes to alteration of airway epithelial remodelling, including goblet cell metaplasia and deciliation [150, 157], and drives excessive goblet cell differentiation due to disruption of the mucin (MUC) 5AC and MUC5B balance in the airway, a common feature found in asthma, AR and CRS. Overproduction of MUC5AC induces increased ASL viscosity and diminished ciliary beating that alongside airway dehydration disrupts MCC [116]. The increased expression of MUC5AC has also been observed in an HBEC line (NCI-H292) treated with Asp. fumigatus, an effect that was diminished by serine protease inhibitors [158].

Allergen serine proteases contribute to dysregulated airways remodelling in allergic asthmaAllergen proteases can alter ECM components to promote airway remodellingECM structures in healthy airways

In healthy tissues, a thin layer of ECM called the basement membrane (BM) is located on the basal side of the epithelium and is responsible for separating the epithelium from underlying connective tissue to maintain the stability of the overall airway structure. Below the BM, a fibril-like network of ECM, which acts as indispensable scaffold for maintaining tissue integrity, surrounds the cells and connects them within a biological three-dimensional structure in the airways smooth muscle (ASM) layer [159]. The upper airway is typically resistant to environmental insults and can mount an inflammatory response in concert with effective airway injury repair processes [160]. The BM and ECM are composed of a variety of matrix proteins including collagen I, III and V, elastins, proteoglycans, fibronectin, biglycan, lumican, versican, decorin, and tenascin [161].

Structural changes in asthmatic airways

Structural changes in the airways have long been recognised as the main feature of asthma and are also observed in other upper airway diseases. Key features observed in allergen-associated airway diseases including AR, CRS with nasal polyps (CRSwNP) and allergic asthma include epithelial shedding, excessive goblet cell differentiation, BM thickening in both the upper and lower airways, and an active epithelial–mesenchymal trophic unit within the epithelial layer. Mucus gland hypertrophy and ECM deposition in the submucosal layer may also be observed in CRSwNP and allergic asthma, while these features can remain normal in AR [160]. Asthma is also characterised by ASM hypertrophy, which is a more severe phenotype of airway remodelling.

Airways remodelling in asthmatic airways

Host matrix metalloproteinases (MMPs) (a group of collagenases and gelatinases) are the major proteases responsible for the normal remodelling of the ECM to achieve effective organisational and morphological changes. Damage of the epithelial barrier by allergen serine proteases during allergen sensitisation can stimulate an increased expression of adhesion molecules and growth factors to promote tissue repair. The deposition of ECM proteins is, therefore, a sign of epithelial restoration.

Elevation of MMP-9 (gelatinase B) levels have, however, been observed in BALF from patients with severe asthma [162]. Allergen serine proteases from Asp. fumigatus, Pen. chrysogenum and Asp. versicolor can induce the release of pro-MMP-9 from human leukaemia monocytic cells (THP-1) [163] and HBECs [164]. The inactive form of pro-MMP-9 generated by eosinophils, neutrophils, alveolar macrophages and airway epithelial cells can then be cleaved by endogenous or exogenous proteases to generate active MMP-9 [165]. For instance, serine proteases from cockroach frass and HDMs can cleave pro-MMP-9 in a dose- and time-dependent manner that is abrogated by pre-treating frass with an inhibitor of serine, but not cysteine protease activity, and can neutralise the endogenous, highly protective, neutrophil elastase (NE) inhibitor α1-antitrypsin (AAT; also known as α1-proteinase inhibitor or SERPIN-A1) [166]. The much higher serine protease activities in cockroaches are also reflected in a heightened ability to activate endogenous pro-MMP-9, compared to HDMs [164]. Allergen proteases, therefore, contribute to the negative skewing of the protease–antiprotease balance observed in allergic asthma that increases the risk of irreversible lung tissue damage [164].

Smooth muscle constriction and airway hyperresponsiveness

Epithelial barrier dysfunction, alteration of ECM components and inflammatory cell infiltration contribute to a continuous cycle of remodelling within the asthmatic airway. This is also driven by raised levels of pro-inflammatory cytokines and chemokines, growth factors as well as contractile agonists (histamine, acetylcholine and substance P), which facilitate ASM cell hypertrophy and fibroblast hyperactivity. An increased deposition of ECM, epithelial and mucous cell hyperplasia, and hypersecretion is also observed. Abundance of Asp f 13 from Asp. fumigatus highly correlates with the severity of asthma, as it can penetrate the airway submucosa. Moreover, it was found that Asp f 13 can proteolytically disrupt ASM and ECM interactions by degrading collagen I and fibronectin [167]; the underlying mechanism associated with enhancement of G protein coupled receptor induced inositol 1,4,5-trisphosphate (InsP3) production. In addition, a small concentration of Asp f 13 (0.001 μg·mL−1) can cause calcium mobilisation in ASM cells, which contributes to airway bronchoconstriction, while Asp f 13 (3 μg·mL−1) can promote ASM cell detachment and elongation in a protease-dependent manner.

Asp f 13 may also directly interact with the ASM layer as a result of epithelial barrier disruption, which prompts airway hyperresponsiveness (AHR) and remodelling [168, 169]. In addition, mice sensitised with active Epi p 1, from the Epicoccum purpurascens dust mite species, showed elevation of Epi p 1-specific IgE and IgG in serum, higher levels of IL-4 and IL-13 in BALF, as well as goblet cell metaplasia, increased mucus production, enhanced leucocyte infiltration and airway narrowing, compared to the inactive Epi p 1 group [79].

Signalling pathways involved in mediating the allergen serine protease-induced responsePARsExpression and activation of PARs in the airways

PARs are the major G-coupled protein receptor involved in allergen serine protease-mediated allergic airway diseases. The PAR family consists of four subtypes including PAR-1, -2, -3 and -4 [170]. It has been reported that the PARs are expressed in epithelial, endothelial and smooth muscle cells, while PARs 1–3 are also expressed in fibroblasts. In immune cells, PAR-1 and -2 are expressed on mast cells, eosinophils, neutrophils, monocytes, macrophages, T- and B-cells, and DCs, while to date, PAR-3 expression has only been discovered in mast cells, eosinophils, monocytes, macrophages and DCs. Expression of PAR-4 is found on mast cells, monocytes, macrophages and T-cells [171]. Among these four subtypes, PAR-1, -3 and -4 can be activated by thrombin and PAR-2 by TLP and CTLP activities. Canonical protease activation of PARs is achieved by irreversible cleavage of the extracellular N-terminus, which exposes a new N-terminal peptide that acts as a tethered ligand to activate the transmembrane receptor domain, triggering G-protein and β-arrestin activation [170]. PARs regulate several cellular responses by mediating conversion of G-proteins from guanosine diphosphate to guanosine triphosphate (GTP), resulting in dissociation of GTP-bound activated Gα from Gβ/γ subunits and can activate multiple second messenger signalling cascades such as Ca2+, cAMP and diacyl glycerol [170].

Activation of PARs by allergen serine proteases

Activation of PARs by allergen serine proteases is involved in the regulation of MCC in the epithelial barrier, Th2 cell polarisation as well as ASM cell contraction. Figure 4 and table 2 further summarise the role of PARs in allergen serine protease-associated inflammation in allergic airway diseases. Based on these studies, it can be concluded that activation of PARs by allergen serine proteases leads to a pro-inflammatory immune response by promoting the release of alarmins such as IL-33 and TSLP, induction of cytokines, and chemokine expression such as IL-8, IL-6, GM-CSF, IL-1β and CCL20, as well as immune cell recruitment. Simultaneously, PAR activation by allergen proteases also increases total IgE levels and promotes Th2 polarisation. In addition, elevation of intracellular calcium by activation of PARs contributes to constriction of ASM and an increased secretion of pro-inflammatory mediators by airway epithelial cells. Of note, PAR-2 expression is elevated in asthmatic airway epithelial cells and correlates with asthma severity [188]. Moreover, increased expression of PAR-2 has been observed in monocytes derived from patients with AR or asthma, thereby contributing to TNF-α expression. Asthma and healthy hPBECs grown at the ALI also show different Ca2+ signalling in response to stimulation by HDM with the apical response of asthma airway cells thought to involve signalling via PAR-2, PAR-4 and TRPV1, transient receptor potential ankyrin 1 and TRPV4; the antagonist of these channels only had a small impact on basolateral HDM-induced Ca2+ response [137]. It is also possible that other transient receptor potential channels such as TRPV2 (localised on the basolateral side) may play a role in the regulation of basolateral HDM-induced Ca2+ mobilisation, thus further study is required.