Tissue factors are disease determinants in allergy
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UNIVERSITY MEDICAL CENTER UTRECHT DEPARTMENT OF DERMATOLOGY AND ALLERGOLOGY AND DEPARTMENT OF IMMUNOLOGY Tissue factors are disease determinants in allergy Jessica Wijngaarden 3734730 Infection and Immunity Supervised by Dr Edward Knol December 2012 Abstract Over 40% of the western population is atopic however, only a limited percentage develops allergic disease. Many chromosomal loci containing immune related genes have been associated with allergic disease, but immune dysregulation alone cannot explain disease development. In recent years the contribution of tissue restricted factors to development of allergic disease has become a research focus. The skin and the airway epithelium are highly structured barriers as the first line of defense against allergen entry and sensitization. Keratinocytes and epithelial cells are also now recognized to play a driving role in allergic inflammation. Epidermal barrier breakdown due to genetic polymorphisms and/or environmental factors can lead to the development of atopic dermatitis (AD). Genetic polymorphisms in filaggrin, a key component of the stratum corenum and lipid lamellae barriers of the skin, are associated with a higher risk of AD. Dysregulation of desquamation due to Kallikrein-related peptidase-7, Lymphoepithelial Kazal-type-related inhibitor and cystatin A polymorphisms and breakdown of the tight junction barrier due to claudin-1 polymorphisms are also associated with AD. Airway epithelial barrier integrity is essential in protection against asthma development. This barrier depends on cell-cell adhesion, primarily through tight and adherens junctions. Many adhesive proteins including claudin-1, occludin, zonula occludens-1, e-cadherin, α-catenin and protocadherin-1 have been associated with asthma development. Airway remodelling, a common feature of asthma, was believed to be caused by chronic airway inflammation in the asthmatic lung; however there are recent indications that it also has a genetic component. ADAM33 and claudin-1 have been associated with airway remodelling. With the evidence that tissue dysregulation is at the base of allergic disease, therapeutic approaches have shifted to address the underlying tissue irregularities rather than simply suppressing inflammation. This shift opens the door for development of preventative therapies. -2- Index Abstract .................................................................................................................................................. 2 Introduction ............................................................................................................................................ 4 Allergy and Atopy ....................................................................................................................................... 4 Tissue Role in Allergy .................................................................................................................................. 6 Role of the Skin in Allergy .............................................................................................................................. 7 Atopic Dermatitis ................................................................................................................................... 9 Introduction ................................................................................................................................................ 9 Tissue Factors ........................................................................................................................................... 11 Filaggrin ........................................................................................................................................... 11 Kallikrein-related peptidases........................................................................................................... 13 Lymphoepithelial Kazal-type-related inhibitor ............................................................................... 13 Cystatin A ........................................................................................................................................ 14 Claudin-1 ......................................................................................................................................... 15 Role of the Lung Epithelium in Allergy ........................................................................................................ 17 Asthma .................................................................................................................................................. 19 Introduction .............................................................................................................................................. 19 Tissue Factors ........................................................................................................................................... 20 ADAM33 .......................................................................................................................................... 20 Tight and Adherens Junctions ......................................................................................................... 22 Claudin-1............................................................................................................................ 22 Occludin ............................................................................................................................. 22 Zonula Occludens-1 ........................................................................................................... 23 E-cadherin .......................................................................................................................... 23 α-catenin............................................................................................................................ 24 Protocadherin-1 .............................................................................................................................. 24 Filaggrin ........................................................................................................................................... 25 Serine protease inhibitor, Kazal-type, 5 ....................................................................................... 25 Current Therapies and Future Perspective.......................................................................................... 26 Atopic Dermatitis ...................................................................................................................................... 26 Asthma ...................................................................................................................................................... 26 Conclusions ........................................................................................................................................... 28 References ............................................................................................................................................ 29 -3- Introduction Allergy and Atopy Over the past 50 years the prevalence of allergic diseases in the developed world has significantly risen.1-3 The hygiene hypothesis is commonly used to explain this phenomenon. Infants naturally have a Th2 dominated immune response. As they develop and encounter microbial antigens, their immune systems switch to a Th1 dominated response. The hygiene hypothesis states that the lack of microbial encounters in the developed world results in a failure to induce a Th1 regulated immune system and allows for the development of Th2 dominated allergic diseases. This is supported by decreased incidence of allergic disease in children who grow up on a farm or with older siblings and presumably encounter more microbes.1,3 The classical view of allergic disease is based on immune dysregulation and atopy: a predisposition to produce IgE antibodies in response to innocuous environmental stimuli.1,3 Allergic diseases progress in three stages: (1) allergen sensitization followed by recurrent cycles of (2) acute phase and (3) late/chronic phase responses upon allergen re-exposure. In atopic individuals, initial allergen encounters lead to a sensitization response which primes the immune system to over-react to subsequent encounters. The allergen is also processed by antigen presenting cells (APCs) and presented on MHC-II molecules to induce a Th2 response. Polarization to a Th2 response requires an initial Th2 environment, particularly IL-4; the source of this initial IL-4 is unknown, though it is postulated to be produced either by naive CD4+ T cells in the absence of a strong Th1 inducer or by infiltrating basophils.2 Th2 cell interactions with B cells through MHC-II, co-stimulatory molecules such as CD40/CD40L and through secreted IL-4 and IL-13 drive the B cell isotype switch to produce one of the primary hallmarks of allergic disease: IgE antibodies.1,4 IgE binds to the high affinity FcєRI on mast cells, arming them to respond to subsequent allergen encounters. IgE also binds to FcєRI on APCs where it functions to optimize antigen presentation.1 The acute phase response begins within minutes of antigen re-encounter due to crosslinking of mast cell bound IgE which triggers the release of pre-formed granules containing histamine, cysteinyl leukotrines, proteases such as tryptase, and lipid mediators such as PGD2.1,4 IgE cross linking also stimulates mast cell production of Th2 cytokines and various chemokines to recruit Th2 cells, basophils and eosinophils: the mediators of the late phase response.1,3 Tissue resident APCs take up, process and present allergens on MHC-II to memory Th2 cells activating them to secrete a myriad of Th2 cytokines.5 Together these initiate the inflammatory immune response and recruit basophils, eosinophils and more Th2 cells.1,3 The late phase response, which typically peaks within 6-9 hours of allergen exposure, is characterized by an influx of immune effector cells, primarily basophils, eosinophils, CD8+ T cells and CD4+ Th2 cells.1,3 Similar to mast cells, basophils are activated by cross-linking of surface bound IgE to release pre-formed granules containing various inflammatory mediators, lipid mediators, proteases, cytokines and chemokines. Basophil activation also triggers continued production of inflammatory cytokines and chemokines.3 Eosinophils contain pre-formed granules which, upon activation, release toxic proteins, inflammatory mediators and activators of basophils, mast cells, neutrophils and eosinophils. Some of the eosinophil produced factors may also play a role in tissue repair.6 Th2 cells can be activated at the site of allergen entry by tissue resident APCs or by activated APCs in the lymph nodes. They are then recruited to the site of allergic inflammation by mast cell and basophil secreted chemokines. Activated Th2 cells produce IL-3, IL-4, IL-5, IL-9, IL-13 and GM-CSF, which perpetuate the inflammatory conditions2 (see Table 1 for a description of the soluble factors in allergic disease). Many atopic children initially present with atopic dermatitis and develop other atopic diseases such as asthma and allergic rhinitis as they age. This progression of atopic disease is referred to as the atopic march.7 Immune dysregulation is thought to be the driver behind the atopic march. In support of this, many immune related genes have been associated with atopy and -4- Table 1 Key soluble factors in allergic disease Factor Produced by Immunoglobulins IgE B cells (under Th2 env’t) Phase in allergic disease Function in allergic disease Produced during sensitization and initiates acute phase reaction Recognizes allergens and initiates immune response through mast cell and basophil activation and enhances antigen processing by APCs1,4,9 Critical inflammatory mediator in anaphylaxis, urticaria and rhinoconjunctivitis, but insignificant in asthma and atopic dermatitis. Can cause an increase in vasodilation to aid in effector cell recruitment.3,10 Th1,Th2 and Th17 attractant,5 causes vasodilation, increased vascular permeability and in asthma: induces smooth muscle contraction and increased mucous production1 Upregulates epithelial and endothelial cell adhesion molecule expression to attract basophils and eosinophils1,3 Mediators Histamine Mast cells and basophils Acute phase Leukotrines Mast cells and basophils Acute phase Tryptase Mast cells Acute phase Th2 cells Basophils, eosinophils and Th2 cells Late phase Required during sensitization, production initiated in acute phase. Primarily mediates late phase response Late phase Cytokines IL-3 IL-4 IL-5 Th2 cells IL-9 Th2 cells, mast cells, basophils and eosinophils Late phase IL-13 Mast cells, basophils, eosinophils and Th2 cells Production initiated in acute phase. Primarily mediates late phase response IL-25 Late phase GM-CSF Th2 cells, mast cells, basophils, eosinophils, epithelial cells and endothelial cells Stored intra-cellularly in epithelial cells and produced by mast cells Epithelial cells, fibroblasts, keratinocytes and mast cells Th2 cells Chemokines PGD2 Mast cells Acute phase Mast cells Fibroblasts, keratinocytes and airway epithelial cells Epithelial cells and mast cells Epithelial cells and mast cells Keratinocytes Acute phase Acute phase IL-33 TSLP CCL1 CCL5 (RANTES) CCL17 CCL22 CCL27 Late phase Late phase Late phase Involved in eosinophil survival1 Important in the initiation of the allergic Th2 response,10-11 drives isotype switch to IgE. Th2 survival factor2 and mast cell development factor1,11 In asthma: promotes mucous over-production1 Important eosinophil growth, differentiation and survival factor1 Important eosinophil development factor and mast cell development factor. In asthma: important in lung inflammation, promotes airway hyper responsiveness, and mucous over-production1-2,11 Drives isotype switch to IgE. Mast cell differentiation and maturation factor. Eosinophil maturation and survival factor, and involved in basophil recruitment.2 In asthma: promotes airway hyper responsiveness and mucous over-production1,11 Activates eosinophils and T cells.11 Activates mast cells (without triggering degranulation), basophils, eosinophils, Th2 cells and B cells. Also aids in skewing the T cell response to Th2.11 Important in activating DCs to polarize to Th2 response. Enhances mast cell IL-13 production, and Th2 IL-4 production. Also involved in eosinophil recruitment and basophil activation.11 Involved in eosinophil survival. In asthma: enhances antigen presentation and recruitment of macrophages1 Th2 chemo-attractant and activator, also involved in eosinophil and basophil recruitment and activation2,5 Th2 chemo-attractant5 Eosinophil and memory CD4+ T cell chemo-attractant12-14 Acute phase Th2 chemo-attractant2,5 Acute phase Th2 chemo-attractant2,5 Constitutively expressed Th1 and Th2 chemo-attractant15 -5- allergic diseases. One of the most consistent associations is with chromosomal locus 5q23-33, which contains the IL-4 cytokine gene cluster including IL3, IL4, IL5, IL9 and IL13.3,8 Tissue Role in Allergy Approximately 40% of the western population is atopic but only a fraction develops allergic disease.2 This, combined with evidence of non-atopic forms of allergic diseases points to something other than only immune dysregulation at the core of these disorders.16-17 The lack of complete responses to immune targeted therapies such as anti-IL-4, anti-IL-5, anti-IL-13 and anti-IgE antibody treatments, recombinant soluble IL-4 receptor, and chemokine receptor antagonists has shifted the focus from immune dysregulation to the role of the tissue in allergic disease.2,4,18 Recent evidence has shown that tissue restricted factors play a central role in initiation and perpetuation of allergic diseases. The skin and the lung epithelium provide the primary physical and chemical barriers against allergen entry and sensitization.17,19 Barrier disruptions allow allergen entry and interaction with immune effector cells initiating the allergic immune response. Allergen interaction with innate immune receptors on epidermal keratinocytes and lung epithelial cells propagates allergic inflammation.19-20 As discussed below, many epidermal and epithelial restricted genetic polymorphisms have been associated with the development and severity of atopic dermatitis and asthma respectively, reinforcing the importance of an intact barrier in the protection against allergic disease. -6- Role of the Skin in Allergy Skin structure The skin is a multi-level barrier against water loss and entry of unwanted or harmful matter, such as pathogens or allergens. The barrier includes the stratum corneum (SC), the lipid lamellae, the acid mantle and the tight junction barrier (see Figure 1).17,21 The stratum corneum, the outer most layer of the skin, acts as the air-liquid barrier. It is composed of corneocytes, which are dead, terminally differentiated keratinocytes. These corneocytes no longer contain intracellular organelles, a nucleus or cell membranes .21 Their keratin skeleton has been bundled together by filaggrin to flatten the cells into squames. Corneodesmosomes join the cells to form the SC barrier. Surrounding the squames is a cornified envelope composed of cross-linked structural proteins including loricrin, involucrin, filaggrin and small proline-rich proteins.17 The cornified envelope is linked to the surrounding lipid lamellae through the keratin matrix to further enhance the barrier.17, 21-22 The lipid lamellae is important in maintaining skin hydration and preventing the entry of soluble irritants. It is composed of ceramides, cholesterol, fatty acids and cholesterol esters which are secreted during differentiation from lamellar granules at the interface of the stratum granulosum (SG) and SC layers of the skin.17,22 Lamellar granules also secrete anti-microbial peptides such as LL-37 and β-defensin 2.23 The acidic pH in the upper layers of the skin, termed the acid mantle, is an important component of the skin’s barrier function. It is anti-microbial, helps maintain the lipid lamellae and regulates desquamation: the shedding of the uppermost corneocyte layers.17 Desquamation is tightly controlled by multiple pH-dependent KLK serine proteases, which breakdown the corneodesmosomes to release the corneocytes from the lipid lamellae, and by protease inhibitors which prevent excess barrier breakdown.17,22 The final line of defense in the skin is the tight junction barrier joining the cells of the SG.21,24 Keratinocytes Keratinocytes react to mechanical stress and environmental stimuli through surface toll-like receptors (TLRs) and NOD-like receptors (NLRs) to produce a number of cytokines and chemokines which, under certain conditions, can initiate an allergic immune response (see Table 2).25 Proteolytic allergens can cause cell damage which immediately triggers keratinocyte secretion of potent inflammatory cytokines IL-1, IL-18, TNF-α, and GM-CSF and upregulation of cell adhesion molecules to attract immune effector cells.20,25 One of the major keratinocyte derived factors contributing to the allergic response is TSLP. Allergen derived injury or allergen recognition by TLR2/6 and TLR5 induces TSLP secretion and its production can be enhanced by other TLR or NLR ligands, viral or microbial co-morbidities, smoke, exhaust or chemicals.24,26 TSLP primes skin DCs to activate Th2 cells and is therefore one of the main drivers of the Th2 allergic response.20,25-26 TSLP can also act synergistically with keratinocyte produced IL-1 and TNF-α to induce mast cell production of Th2 cytokines.20 In addition, keratinocytes and skin fibroblasts produce eotaxin, RANTES (CCL5) and CCL27 to recruit eosinophils to further enhance the allergic response.12,15 Following the induction of the allergic response, keratinocytes are involved in its maintenance. They express histamine receptors, which upon stimulation increase inflammatory cytokine production.20 The allergic inflammatory response negatively impacts keratinocyte barrier function and increases production of TSLP and T cell recruiting cytokines.20,26 IFN-γ (produced in the Th1 phase of atopic dermatitis) is a potent keratinocyte activator which induces cytokine and chemokine production, up-regulation of antigen presenting molecules, costimulatory molecules and adhesion molecules as well as the expression of Fas. Fas interacts with FasL on infiltrating T cells causing keratinocyte apoptosis and breakdown of the skin barrier.12,20 Th2 inflammatory cytokines IL4 and IL-13 inhibit keratinocyte anti-microbial peptide synthesis which further disrupts the skin -7- barrier.12,22 See Table 2 for a list of keratinocyte produced factors and their function in allergic disease. Table 2 Keratinocyte derived factors contributing to the allergic immune response Soluble factor Production stimulated by Function in allergic disease Cytokines IL-1(α and β) Typically stored intra-cellularly but Pro-inflammatory.25 Also promotes keratinocyte released upon mechanical trauma and differentiation and activates LCs27 20,27 barrier disruption IL-18 mechanical trauma and barrier Pro-inflammatory25 involved in Th1 cell proliferation and disruption25 differentiation27 TSLP IL-4, IL-13 and TNF-α. Barrier directs skin DCs to induce a Th2 response20,25-26 also acts disruption, TLR (TLR2/6 and TLR5) or synergistically with IL-1 and TNF-α to induce mast cell NLR ligands, viruses, microbes, production of Th2 cytokines.20 20,26 chemicals, exhaust, smoke TNF-α Mechanical trauma and skin barrier Increases adhesion molecule expression on endothelial disruption20 and IFN-y and IL-1.27 cells. Can induce keratinocyte apoptosis leading to barrier damage and increased immune response.27 GM-CSF Chemokines CXCL10 CCL5 (RANTES) CCL17 CCL22 CCL27 CXCR3 ligands Eotaxin Autocrine IL-1α, TNF-α and T cell produced IFN-γ and IL-4.12 Mechanical trauma and skin barrier disruption20 Important skin DC survival and maturation factor. Upregulates CD80 and CD86 expression on LCs and may aid in antigen presentation. Stimulates keratinocyte proliferation. Production is increased in atopic individuals.27 Th2 cytokines20 Allergen challenge13 and Th2 cytokines28 Th2 cytokines20 Th2 cytokines20 Constitutively expressed15 INF-y activation27 Th2 cytokines13 T cell recruitment20 Eosinophil recreuitment12-13 T cell recruitment20 T cell recruitment20 Binds CCR10 to recruit skin homing Th1 and Th2 cells15 attract activated T cells27 Eosinophil recruitment12 -8- Atopic Dermatitis Atopic dermatitis (AD) is a chronic inflammatory disease of the skin that typically appears early in childhood and affects 15-25% of children worldwide.7,24,29 Approximately 40-50% of childhood cases resolve with age but the remaining cases persist into adulthood.7 AD is characterized by dry, itchy, erythematous lesional patches of skin concentrated on the face, neck and extensor/flexural surfaces.7,17,29 In early childhood AD presents with increased edema leading to an oozing and crusting appearance. After about one year the skin dries out and presents as more typical AD.7 AD progresses from an acute, recurrent lesional-phase disease to chronic lesional disease. During the acute phase encountered allergens are captured by Langerhans cell (LC) bound IgE. LC bound IgE enhances allergen uptake and presentation to tissue resident memory Th2 cells activating them to secrete Th2 cytokines and initiate allergic inflammation.28 Allergens can also trigger keratinocyte production of inflammatory cytokines and chemokines.12,20 The Th2 allergic environment leads to an upregulation of cell adhesion molecules, inflammatory cytokines and chemokines which perpetuates recruitment of inflammatory cells such as basophils, eosinophils and Th2 cells. Infiltrating basophils and eosinophils also contribute to the inflammatory environment. Infiltrating T cells are activated by tissue resident APCs which drive a Th2 response leading to increased secretion of IL-4, IL-5 and IL-13.12,20,29 Initially, AD is a Th2 mediated disease; however, during the chronic phase it switches to a Th1 mediated disease. This switch is mediated by IL-12 which drives T cell production of IFN-γ leading to a Th1 dominated response. Although the source of IL-12 is unknown, it may be produced by keratinocytes, Th2 stimulated eosinophils, or inflammatory dendritic epidermal cells.6,20 The increased IFN-γ in the chronic phase induces keratinocyte expression of Fas which interacts with FasL on the infiltrating T cells, triggering keratinocyte apoptosis. This initiates increased production of Th1 chemokines resulting in increased keratinocyte apoptosis, perpetual skin barrier damage and chronic Th1inflammation.12,20 Macrophages and eosinophils continue to infiltrate the lesional tissue and maintain the inflammatory environment.12 Lesional tissue in chronic AD undergoes remodelling in which the skin thickens and becomes lichenified.7,20 In acute and chronic AD the non-lesional skin is also affected; it is characterized by mild hyperplasia and T cell infiltration as well as increased numbers of tissue resident mast cells.12,20 Similar to other allergic diseases, AD is highly heritable. Genome wide scans have identified many chromosomal regions linked to disease risk; however, untangling the specific underlying genes is a challenge.24,29 More than 100 candidate gene studies have reported associations with AD. However, these studies are often under powered and determining true associations is difficult. Many immune related genes, including IL4, IL4RA, IL5, IL13, RANTES, IL18, CD14, have been repeatedly associated with AD, indicating the importance of a misdirected immune response in allergic disease.24 Environmental factors also play an important role in AD. Proteolytic allergens can breakdown the skin barrier and trigger an inflammatory response through keratinocyte innate PRRs.25,30 Bacterial and viral infections complicate AD. Over 90% of AD patients are infected with Staphylococcus aureus, compared to only 5-30% of non-AD patients. This is likely due to genetic and environmental breakdown of the skin’s anti-microbial barrier.24 S. aureus toxins can act as superantigens which potently activate tissue resident mast cells and T cells, further perpetuating the inflammatory environment and epidermal barrier breakdown.7,12,24,29,31 The course and severity of AD is determined by a combination of underlying genetic factors, the immune response mounted against a specific set of allergens, and various environmental factors; however, the tissue specific genetic factors are pivotal in the initiation of disease. AD skin is intrinsically defective as a physical and chemical barrier against allergen entry which allows allergen sensitization and perpetuation of the allergic inflammatory response.24 The following is a summary of some of the common tissue factors that have been associated with AD in recent years (see Figure 1). -9- A SC Barrier FLG KLK7 SPINK5 CSTA SC SG TJ Barrier CLDN1 Stratum spinoza Stratum basale Basement membrane Dermis NMFs Filaggrin degradation Filaggrin monomers B Corneodesmosome Desquamation SC pH (approx) pH mantle FLG KLK LEKTI SG TJ Barrier Lipid Lamellae FLG Figure 1 Schematic representation of the skin barrier and the tissue factors associated with atopic dermatitis. (A) Genetic polymorphisms in genes involved at each layer of the skin have been associated to AD. These polymorphisms result in barrier dysfunction. (B) The epidermis is a highly stratified, multi-layer barrier. (i) The tight junction barrier is located at the SG-SC interface. (ii) Lamellar granules are secreted outside of the TJ barrier and play a role in the SC barrier. (iii) SG1 cells lose their TJs as they undergo (iv) terminal differentiation into corneocytes. (v) The mature corneocytes are surrounded by a cornified envelope and the lipid lamellae (both part of the SC barrier). (vi) Corneodesmosomes anchor the corneocytes together adding to the barrier strength. (vii) pH differences in the upper SC control the activation of KLKs and inhibition of LEKTI allowing controlled barrier breakdown in the form of desquamation. Adapted from Kubo et al (2012)21 - 10 - Filaggrin Filaggrin is a multi-functional barrier protein found in the skin, oral and nasal mucosa and in the conjunctiva.32-33 FLG is initially translated to profilaggrin, a large highly-phosphorylated polypeptide composed of an N-terminal domain, 10-12 filaggrin monomer repeats and a C-terminal domain.34-35 Profilaggrin localizes to the keratohyalin granules in the stratum granulosum keratinocytes.32,34 During keratinocyte differentiation, secreted profilaggrin is dephosphorylated and broken down by extracellular serine proteases. This process is highly regulated by pH and the presence of protease inhibitors.35 The N-terminal domain likely contributes to keratinocytes differentiation; it contains an S100-like calcium binding domain, which may be involved in calcium dependent steps of terminal differentiation or profilaggrin processing, and a nuclear localization domain, which may be involved in keratinocytes enucleation.34 The function of the C-terminal domain is still unknown, however it seems to be necessary for profilaggrin processing to filaggrin.34-36 The filaggrin monomers are essential to the epidermal barrier. They bind to constituents of the keratin cytoskeleton and other filament proteins to form tight bundles during terminal differentiation of keratinocytes into corneocytes. Filaggrin is also an important part of the cornified cell envelope which acts as a barrier to water loss by cross-linking the corneocytes. Excess filaggrin in the skin is degraded into its constituent amino acids. The most abundant breakdown products, histidine, argenine and glutamine, make up the majority of the natural moisturizing factor (NMF) which helps maintain skin hydration and may help regulate the pH balance.34-35 Histidine is further metabolized to urocanic acid (UCA) and glutamine is further metabolized to pyrrolidone-5-carboxylic acid (PCA), both of which are also important parts of the NMF and help maintain the pH gradient in the epidermis.34,37 There is some evidence that filaggrin is also involved in protection against UVmediated damage.34 Despite the importance of filaggrin, it has a relatively short half-life of six hours.35 Filaggrin polymorphisms Homozygous or compound heterozygous mutations in the FLG gene have been causally linked to ichthyosis vulgaris (IV).34,38 Since IV has similar symptoms and often co-presents with AD, the R501X (a non-sense mutation) and 2282del4 (a 4 base pair deletion causing frameshift) null mutations were also investigated for a role in AD.32 Using the initial IV family-based study patients, Palmer et al (2006)32 found an association between the two FLG mutations and AD. They repeated these results with a cohort study of Irish pediatric AD patients, a case-control cohort study of Scottish school children and a longitudinal study of Danish children with AD.32 These results were subsequently confirmed in a German family-based study by Weidinger et al (2006)39 and have since been confirmed in over 20 case-control studies and 8 family based studies.33-34,36,40-41 To date, over 40 null mutations in the FLG gene have been identified in European and Asian populations (see Figure 2). The expression patterns of the mutations vary/segregate across ethnic groups.36 Approximately 9-10% of the European population carry one of the two most common FLG null-mutations, whereas 25-50% of AD patients have FLG mutations.32,36,42 As such, FLG mutations account for approximately 15% of the population attributable risk of AD development.43 R501X and 2282del4 null-mutations are semi-dominant, with homozygous or compound heterozygous mutations correlating to the highest risk of developing IV, AD or other skin diseases.32,44 FLG null mutations in AD lead to a number of barrier defects including impaired corneocyte differentiation due to impaired filament/keratin aggregation. Cell-cell adhesion is also affected by FLG mutations which result in decreased corneodesmosin, an important component of corneodesmosomes and decreased numbers of tight junctions. FLG deficiencies also lead to the development of a defective lipid lamellae, and lack of NMF leading to water loss and increased epidermal pH.37,42 Increased SC pH can lead to a blockade of lamellar body secretion, increased - 11 - desquamation due to activation of proteases and deactivation of protease inhibitors, and increased bacterial colonization.17,22,42 Together, these barrier function deficiencies likely allow for increased allergen sensitization and contribute to the inflammatory response. This is supported by the association of FLG mutations with increased IgE levels.36,39 FLG mutations have also been weakly associated with AD severity, and AD patients with FLG mutations tend to have increased skin infections with herpes virus, and greater risk of other allergies and asthma than those without mutations.41-42 A B Normal skin barrier Filaggrin staining in normal skin Ichthyosis vulgaris and atopic dermatitis Filaggrin granules Defective skin barrier No filaggrin granules Figure 2 FLG in atopic dermatitis (A) Schematic representation of the FLG gene and mutations associated with AD in European and Asian populations. Mutations in red are recurrent in the population and mutations in black are rare or family-specific. (B) Filaggrin immunostaining in healthy skin shows filaggrin concentration in the upper SC and in lamellar granules (left panel). Filaggrin immunostaining of patient skin with a homozygous FLG loss-of-function mutation and suffering from IV and AD shows a complete lack of filaggrin in the SC and the lamellar granules (right panel). Adapted from Irvine et al (2011)42 and Irvine and Mclean (2006)45 Other factors influencing filaggrin expression levels Although FLG mutations are the leading predictive factor for the development of AD, only 40-60% of FLG mutation carriers develop AD.35,42 Factors other than genetic polymorphisms also affect filaggrin expression levels. The number of filaggrin monomer repeats in the FLG gene naturally varies from 10 to 12. An increased number of filaggrin repeats has been associated with a decreased risk of dry skin and AD.32,34,46 Filaggrin expression can also be influenced by the inflammatory environment. IL-4, IL-13, IL22, and IL-25 have been shown to decrease FLG expression.34,42 IL-22 may also down-regulate the profilaggrin processing enzyme cathepsin D and other Th2 cytokines can down-regulate caspase 14, which deiminates the filaggrin monomers before they can be degraded to individual amino acids and - 12 - metabolized to the NMF components.34,47 Disease severity has also been found to affect NMF levels, indicating that disease severity and the inflammatory environment play a role in FLG expression.37 In addition, FLG has been shown to interact with an eczema risk variant on the 11q13.5 chromosomal region and with polymorphisms on the IL-10 and IL-13 genes.34 These interactions highlight the complex role of FLG in the skin barrier and that a combination of mutations in interacting genes, in combination with environmental factors, may be responsible for the resulting phenotype. Kallikrein-related peptidases Kallikrein-related peptidases (KLKs) are a family of 15 serine proteases encoded by genes on chromosome 19q13.4.22,48-50 KLKs are synthesized as inactive pre-pro-enzymes with a 15-30 aminoacid pre-sequence on the N-terminal end that is cleaved off before secretion into the SC.17,49-50 At neutral pH, as in the lower SC, pro-KLK5 zymogens can self activate through trypsin digestion to remove the pro-peptide and initiate an activation cascade of other KLKs.17,50 KLK5, KLK7 and KLK14 are most abundant and the only KLKs that are found in their active form in the SC.49-50 KLKs primarily function in desquamation (see Figure 1).17,22,49,51 They initiate corneodesmosome degradation through the cleavage of key constituent proteins desmoglein-1 (DSG-1) by KLK5 and KLK14, and corneodesmosin (Cdsn) and desmocollin-1 by KLK7.17,22,48 KLKs also affect the skin barrier through degradation of lipid processing enzymes that work at the SG-SC interface to create the lipid lamellae from the lamellar granule secretions.22,51-52 KLK5 and KLK14 can signal through PAR2 to downregulate lamellar granule secretion,22,53 and increase inflammatory cytokine secretion, particularly TSLP.17,26,48-49,51,53 In addition, KLKs are involved in profilaggrin processing both directly and through activation of ELA-2, another important profilaggrin processing enzyme.48,51 Kallikrein-related peptidase-7 polymorphisms AD patients exhibit abnormally high levels of KLK7 and increased activity of KLK5, KLK7 and KLK14 in lesional skin.50 In an initial study, Vasilopoulos et al (2004)54 screened for KLK7 polymorphisms and their role in AD. They found a 4 base-pair insertion (AACC) in the 3’ UTR which, they hypothesize, increases KLK7 mRNA stability thereby increasing KLK7 activity. In their study, the two-insertion repeat allele (AACCAACC) was significantly associated with AD.54 However, these results have not been replicated. In a large study Weidinger et al (2008)55 found no association between KLK7 3’UTR insertions and AD. They conclude that although the 3’ UTR insertion variant of KLK7 does not increase the risk of AD development, the KLKs are important skin barrier regulatory enzymes and other undiscovered polymorphisms may play a role.55 Lymphoepithelial Kazal-type-related inhibitor Lymphoepithelial Kazal-type-related inhibitor (LEKTI) is a serine-protease inhibitor encoded by serine protease inhibitor, Kazal-type, 5 (SPINK5) on chromosome 5q31-33 at the distal end of the IL-4 cytokine gene cluster.14,52,56-58 LEKTI is composed of an initial peptide sequence and 15 inhibitory domains.49,58 Variable transcriptional processing results in three LEKTI variants differing at the Cterminal end. LEKTI-FL is the most abundant and contains all 15 domains, LEKTI-Sh is shorter, containing only the first 13 domains and LEKTI-L is elongated by a 30 amino-acid insertion between domains 13 (D13) and D14.49,51 D2 and D15 contain true Kazal motifs (6 cysteine residues) whereas the other domains contain Kazal-like motifs (4 cysteine residues). These cysteine residues, which form two or three disulfide bonds forcing a loop structure within the domain to mimic serine protease substrates, are responsible for the inhibitory action.49 In the skin LEKTI is packaged in lamellar granules, where it is processed into polypeptides by furin cleavage of the argenine or lysine rich linker sequences and secreted at the SG-SC interface.48 Each of the three LEKTI variants undergo - 13 - similar processing to produce D1, D5, D6, D7, D6-D9, D7-D9, D8-D9, D10-15, and D10-D13 polypeptides.49-50,58 Although LEKTI can control filaggrin expression through inhibition of profilaggrin proteolytic enzymes,51 it primarily functions as a KLK serine protease inhibitor regulating desquamation in the SC.49-50 LEKTI secretion from lamellar granules occurs before KLK secretion to prevent protease activity in the lower SC.48-49,59 The secreted domain fragments exhibit variable patterns of inhibition on the different KLKs. All domains except D1 inhibit KLK5 with varying strength; D5, D6, D8-11 and D9-15 inhibit KLK5, KLK7 and KLK14. D6-9 and D9-12 inhibit KLK5 and KLK14 and D10-D15 fragments primarily inhibit KLK7.49-50 LEKTI function is regulated by the pH gradient in the SC. At the SG-SC interface the pH is near neutral allowing optimal LEKTI function and inhibition of KLK activity. The progressive pH decrease in the outer SC layers inhibits LEKTI function to allow KLK activity and desquamation in the outermost SC.17,21,50 Serine protease inhibitor, Kazal-type, 5 polymorphisms Autosomal recessive SPINK5 loss-of-function mutations result in the rare skin disease Netherton syndrome.22,57 Netherton patients completely lack LEKTI resulting in unrestricted desquamation and severe barrier defects.52 Since Netherton syndrome patients always also present with AD-like symptoms, SPINK5 mutations were investigated for an association to AD.53 In two independent panels of British families Walley et al (2001)58 found a maternally derived association of the E420K polymorphism to AD, atopy, and elevated IgE. These results were further confirmed by Kato et al (2003)60 who found an association between seven SPINK5 polymorphisms, including E420K, and AD. Nishio et al (2003)61 also found an association of the E420K polymorphism to AD. In a large study of German children, the E420K polymorphism was associated with AD when presenting with asthma but not to AD alone.62 A second study of a German population and a French study also failed to find an association between AD and the E420K polymorphism.52 Since then, however, there have been more studies linking the polymorphism to AD: Weidinger et al (2008)55 found an association of maternally inherited E420K polymorphisms to AD and a Chinese study by Zhao et al (2012)52 found an association of E420K polymorphisms to AD.52,55 Although the E420K polymorphism has been repeatedly associated with AD, until recently its function was entirely unknown.52,63 It lies near the furin cleavage site within the D6-D7 linker region causing preferential cleavage. This altered fragmentation pattern results in a complete lack of the D6-D9 fragment, the most potent inhibitor of KLK5 activity.51 Fortugno et al (2012)51 found that a 420KK genotype increases KLK5 and KLK7 activity resulting in increased DSG1 degradation and decreased barrier function. They also found a correlation between increased TSLP levels and the 420KK variant, indicating that an imbalance between serine proteases and their inhibitors leads to increased PAR-2 activation and TSLP secretion driving a Th2 response.51 These results show for the first time how the 420K LEKTI polymorphism may be involved in the drive towards AD. LEKTI 420KK variants have also been associated with increased ELA-2 activity, weaker inhibition of profilaggrin proteolytic enzymes and decreased profilaggrin and filaggrin monomer expression.51 Increased SC pH due to decreased filaggrin levels leads to KLK activation, decreased skin barrier and initiation of the inflammatory cascade through PAR-2.22 Due to the functional links between KLKs, LEKTI and filaggrin, and the potential association of each of these to AD, Weidinger et al (2008)55 investigated the interaction between KLK7, SPINK5 and FLG polymorphisms. However, they found no effect of KLK7 polymorphisms on AD risk and no interaction of KLK7 or SPINK5 polymorphisms with FLG polymorphisms.55 Cystatin A CSTA, located on the AD susceptibility locus of chromosome 3q21, encodes the cysteine protease inhibitor cystatin A.17 Cystatin A is expressed in the SC and in sweat. In the SC it functions - 14 - as a cathepsin inhibitor. It inhibits the cysteine proteases cathepsins B, H and L which are involved in corneodesmosomal degradation during desquamation.64 In sweat, cystatin A forms a protective barrier against exogenous proteases. It is a potent inhibitor of common house dust mite allergens, Der p 1 and Der f 1, and S. aureus derived proteases.64-65 It is estimated that 50% of IgE antibodies in allergic diseases are against Der p 1 and Der f 1.65 These allergens are not only able to breakdown the corneodesmosomal barrier, but they can also activate keratinocytes to produce IL-8 and GM-CSF potentially leading to enhanced allergen sensitization.65-66 Cystatin A is an important inhibitor of endogenous and exogenous cysteine proteases to prevent epidermal barrier breakdown and allergen sensitization. Cystatin A polymorphisms Seguchi et al (1996)67 reported decreased cystatin A levels in lesional skin of AD patients, but no reduction in non-lesional AD skin. Recently, Vasilopoulos et al (2007)64 analyzed three CSTA single nucleotide polymorphisms (SNPs): T-190C, T+162C and C+344T. They found a significant association between the +344C variant and AD. Interestingly the rare +344T allele seemed to be protective against AD development. They performed functional analysis on the +344C variant and found that +344C mRNA has a much higher degradation rate, resulting in lower cystatin A levels.64 Together these data indicate that CSTA polymorphisms may predispose to AD and that the AD phenotype may regulate cystatin A levels. Although there have not been many studies on cystatin A, it appears to be an important barrier protein involved in regulating desquamation and preventing exogenous protease damage and allergen sensitization. Claudin-1 Tight junctions are important connective structures between cells. They act as a barrier against unwanted material and control trans-epithelial water loss (TEWL) and trans-epithelial electric resistance (TEER).68-69 Tight junctions are composed of two types of proteins: trans-membrane proteins which anchor neighboring cells and scaffold proteins which anchor the trans-membrane proteins to the intracellular matrix.68 Occludins and claudins are the most abundant and conserved tight junction trans-membrane proteins (see Figure 3 for TJ structure).30 Claudin-1 is encoded by CLDN1 on chromosome 3q28-q29.68 It has four trans-membrane domains and two extracellular loops that interact with the neighbouring cell.69 Claudin-1 is known to increase barrier function in tight junctions, but beyond this little is known of its expression patterns and function in humans.68 Tight junctions were thought to be primarily involved in simple epithelial barriers; however, Furuse et al (2002)70 demonstrated the functional importance of tight junctions and claudin-1 in stratified epithelia through the generation of CLDN1-/- mice. These mice had wrinkled skin and a severely damaged epidermal barrier and died within one day after birth.70 Claudin-1 polymorphisms De Benadetto et al (2011)68,71 have found an approximate 50% decrease of claudin-1 mRNA and protein levels in non-lesional AD skin. They also found functional defects in claudin-1 deficient lesional and non-lesional AD skin. Interestingly, in their system IL-4 and IL-13 caused claudin-1 upregulation. Together these data point to an intrinsic claudin-1 deficiency in AD, rather than a disease-driven deficiency. Analysis of 24 CLDN1 SNPs common to both an African-American and European-American population revealed several significant associations in the African-American population. SNP rs9290927 was associated with increased risk of AD. Two SNPs (rs893051 in intron 1 and rs9290929 in the promoter) were associated with increased disease severity and, interestingly, SNP rs17501010 was associated with decreased AD risk. They were only able to find modest associations in the European-American population.68 - 15 - In a second study, De Bernadetto et al (2011)71 investigated the potential association of CLDN1 polymorphisms and herpes simplex virus-1 (HSV-1) infections. HSV-1 infections in patients with AD can lead to eczema herpaticum (EH), a rare and severe widespread skin infection. Previous studies have shown that susceptibility of keratinocytes to HSV-1 infection is inversely correlated to the amount of cell-cell contact. They found that claudin-1 silencing by siRNA significantly increased HSV-1 infection levels. They subsequently reanalyzed the African-American and European-American populations from their previous study for CLDN1 SNPs associated to EH. Initially, they were only able to find modest associations; however, when they excluded patients with FLG mutations, they found a significant association of two CLDN1 SNPs (rs3774032 and rs3732923) to EH in the EuropeanAmerican population. After the exclusion of FLG polymorphisms the African-American population was too small to analyze.71 Although both of these studies are quite small and require replication, they indicate the importance of tight junction integrity in resistance to development of AD and prevention of viral exacerbations. Adhesion genes associated to AD CLDN1 Adhesion genes associated to asthma CLDN1 OCLN ZO1 PCHD1 CDH1 CTNNA1? CTNNA Figure 3 Tight junction, adherens junction and desmosome structure and genes associated with adhesion abnormalities and atopic diseases. Adapted from Nawijn et al (2011)72 - 16 - Role of the Lung Epithelium in Allergy Airway epithelium structure The airway epithelium is the largest surface that is in continual contact with the environment and serves as the first line of defense against inhaled particles.19,73 The airway is equipped with a mucous barrier, a physical barrier and a catabolic barrier to prevent entry of harmful substances. The epithelial cell layer is composed of ciliated columnar cells, mucous secreting goblet cells, and surfactant secreting Clara cells. The mucous traps inhaled particles, allergens and microbes and the ciliated cells act as a conveyor belt to sweep away unwanted materials. The physical barrier is formed by tight junctions joining the apical ends of the epithelial cells, to tightly bind the cells and prevent soluble factors from passing between them.13,19,73-74 Other cell-cell and cell-extracellular matrix interactions, such as adherens junctions, desmosomes and hemidesmosomes, further strengthen the physical barrier of the airway epithelium (see Figure 3).19,73-74 The basement membrane lies directly under the epithelial cell layer and acts as a barrier between the epithelium and the underlying mesenchyme.75 It is composed of collagen, laminin, fibronectin, proteoglycans and other extracellular matrix (ECM) proteins.75-77 Fibroblasts in the lamina propria and airway smooth muscle cells produce the extracellular matrix proteins found in the basement membrane.76 See Figure 4 for airway barrier structure. Finally, the epithelial cells form a catabolic barrier through the expression of surface peptidases to degrade damage-inducing peptides, and the secretion of protease inhibitors to limit the effects of allergen or pathogen derived proteases.13 To protect against reactive oxygen species (ROS)-induced tissue damage from biological or chemical insults, the epithelia contains many anti-oxidant enzymes including superoxide dismutase and glutathione peroxidase.19,73 Epithelial cells also produce antimicrobial peptides such as defensins, cathelicidins, and collectins which are toxic to invading pathogens and recruit immune cells.78 Epithelial cells Epithelial cells (ECs) play an important role in the initiation of airway allergic inflammation. They sample the environment through various pattern recognition receptors (PRRs), including TLRs, NLRs, C-type lectins and protease activated receptors (PARs).79 Many allergens interact with TLRs and PARs activating the ECs to produce cytokines, such as IL-25, IL-33 and TSLP, and chemokines, such as CCL17 and CCL22, that drive the allergic Th2 inflammatory response (see Table 3 for a more extensive list).2,13,19,79-80 IL-25, IL-33 and TSLP potently promote a Th2 immune response by inducing Th2 cell, mast cell and basophil production of IL-4 and/or IL-13. They also promote eosinophil survival and inflammatory cytokine production. TSLP also primes DCs to initiate a Th2 response.78 Inhaled TLR ligands can act as adjuvants further activating the ECs to produce Th2 inducing cytokines and chemokines.2 Epithelial cells are also involved in the perpetuation of airway allergic inflammation. IL-4 and IL-13 stimulated ECs increase production of GM-CSF, CCL11 and CCL17 to increase recruitment of eosinophils and Th2 cells to the airways. In addition, IL-4 and IL-13 disrupt the epithelial barrier which activates the EC immune response and maintains allergic inflammation.79 IL-25, IL-33 and TSLP may mediate secretion of IL-13 and TGF-β resulting in increased fibroblast production of extracellular matrix proteins. This contributes to basement membrane thickening, one of the key features of airway remodelling.78 EC derived GM-CSF may also play a role in airway remodelling.73 - 17 - Table 3 Epithelial cell derived factors contributing to the allergic immune response Soluble factor Production stimulated by Function in allergic disease Cytokines IL-6 Pro-inflammatory cytokine Stimulates Th2 response, IgA mucosal production and B cell stimulation, fungal or dust mite differentiation13 proteases, or respiratory viruses13 IL-11 Pro-inflammatory cytokine Involved in B cell activation (dependent on T cells). Increased stimulation or respiratory levels observed in severe asthma patients13 13 viruses IL-16 T cell and eosinophil attractant13 2 IL-25 TLR stimulation enhances Th2 memory response, interacts with mast cells, basophils, eosinophils and endothelial cells to enhance the inflammatory response and important in DC activation2,79 2 IL-33 TLR stimulation induces Th2 proliferation and cytokine production interacts with mast cells, basophils, eosinophils and endothelial cells to enhance the inflammatory response and activates DCs to induce Th2 response2,79 GM-CSF Pro-inflammatory cytokines, TLR activates neutrophils, eosinophils and macrophages. stimulation2 eosinophil survival factor13 Important in DC activation and promotes Th2 response2,79 TSLP TLR stimulation2 primes DCs to induce Th2 response13,79 interacts with mast cells, basophils, eosinophils and endothelial cells to enhance the inflammatory response.2 important in DC activation. stimulates bronchial EC proliferation and IL-13 production. Important in basophil growth and differentiation. May also be involved in epithelial repair mechanisms 79 Chemokines CXC chemokines mast cell recruitment13 CCL11, CCL24, CCL26 (eotaxins 1-3) CCL5 (RANTES) CCL17 CCL22 CCL20 CCL28 IL-4 and IL-1313 IL-4 and allergens, TLR stimulation2 and epithelial damage73 TLR stimulation2 and epithelial damage73 inflammatory cytokines and ambient particles13 inflammatory cytokines13 Bind to CCR3 on eosinophils and basophils13 eosinophil attractant13 Th2 recruitment13,19 Th2 recuitment19 recruitment of immature DCs13 recruitment of eosinophils and T cells13 - 18 - Asthma Asthma is a chronic inflammatory disease of the conducting airways that affects 5-10% of the developed world.2,16 Allergic asthma typically begins in early childhood with allergen-triggered episodes of airway hyper-responsiveness and inflammation. As it progresses to a chronic state it spreads to the distal airways and is associated with airway remodelling.19,81 Asthma is characterized by symptom free periods and periods of exacerbation caused by viral infections or allergen exposure.82 During periods of exacerbations patients experience coughing, wheezing, chest tightness and difficulty breathing.79 Asthma is caused by the interaction of genetic and environmental factors. It has been estimated that 36-79% of asthma is heritable. Over 120 genes have been associated with asthma but without a clear inheritance pattern. Chromosomal regions 5q23-31, 5p15 and 12q1424.2, which contain immune related genes such as IL3, IL4, IL5, IL9, IL13, IFNγ and FcєRIβ are consistently associated with asthma.83 Early life viral infections also seem to play a driving role in asthma development. Persistent viral infections in the lower respiratory tract, particularly with rhinovirus (HRV) or respiratory syncytial virus (RSV), are associated with an increased risk of asthma development.19,84 In contrast, early life microbial exposure may play a protective role.2 Recent studies also indicate that early life changes in lung or gut microbiome compositions can increase risk of asthma development.85-86 The asthmatic lung is characterized by infiltration of mast cells, eosinophils and Th2 cells in the epithelium and the lamina propria.3,19,79,81 Allergen recognition by IgE sensitized mast cells and basophils triggers degranulation releasing histamine, leukotrines, inflammatory cytokines and chemokines. This triggers acute bronchial smooth muscle contractions and recruits inflammatory immune cells.19,87 The infiltrating eosinophils and Th2 cells perpetuate the bronchial contractions and inflamed conditions through the secretion of lipid, peptide and protein mediators and Th2 cytokines such as IL-4, IL-5, and IL-13.13,81,87 Although most asthma is primarily Th2 mediated, in some patients the primary infiltrates are neutrophils. High INF-γ responses are also observed during acute asthma attacks in some patients.16,79,87 These, and similar observations, have led to the view of asthma as a range of diseases with distinct phenotypes.16 Bronchial hyperresponsiveness (BHR) and airway remodelling are key features of asthma. BHR is characterized by airway obstruction in response to a non-allergic physical or chemical stimulus. This may be due to underlying airway Th2 or eosinophilic inflammation.79,88 Airway remodelling is characterized by thickened basal lamina, increased smooth muscle around the airway walls, goblet cell hyperplasia and metaplasia and angiogenesis (see Figure 4).73,89 Increased fibroblast proliferation and differentiation into myofibroblasts results in increased deposition of ECM proteins including collagen, fibronectin, laminin and proteoglycan.27,77 Airway smooth muscle (ASM) undergoes hyperplasia and hypertrophy contributing to airway constriction and breathing difficulties.76,79 ASM is also known to produce ECM proteins contributing to basal laminar thickening.76 Increased production of highly viscous mucous due to goblet cell hyperplasia and metaplasia also contributes to breathing difficulties.19,79 Angiogenesis allows for increased infiltration of immune effector cells and perpetuation of the allergic inflammatory response.90 The cause of airway remodelling is unknown, however both Th2 cytokines and genetic factors have been implicated.76,89-90 An emerging theory is that an aberrant epithelial repair response leads to continual activation of the epithelial mesenchymal trophic unit (EMTU) resulting in airway remodelling.89 The EMTU is defined as the bidirectional communication between the epithelium and the underlying mesenchyme resulting in cytokine and growth factor production.73 In recent years the suspected origins of asthma have been re-evaluated. Atopy-driven chronic inflammation was thought to be the driving force behind airway remodelling. However, airway remodelling is also found in young children and in the relative absence of inflammation.13,73 In light of this, the contribution of the epithelium to both the development and perpetuation of asthma is a major research focus.2,19 The epithelium acts as the primary barrier against allergen entry. Intrinsic barrier dysfunction can increase the risk of allergen sensitization and asthma development.13,19 Recently tissue restricted genes have been associated with increased asthma risk. - 19 - As discussed below, many of these genes are involved in the maintenance of the epithelial barrier and some may play a role in airway remodelling (see Figure 4). Healthy airway wall TJ and AJ barrier CLDN1? OCLN ZO1 CDH1 CTNNA1? CTNNA Asthmatic airway wall Legend Barrier breakdown Th2 inflammation Airway remodelling Lymphocyte Macrophage Eosinophil Neutrophil Epithelial cell Other genes associated with asthma PCHD1 FLG? SPINK5? Goblet cell Smooth muscle cell (myo)fibroblast Blood vessel Airway remodelling ADAM33 CLDN1 Extracellular matrix Basement membrane Tight junction Figure 4 Schematic representation of the airway epithelial structure in healthy tissue and asthmatic tissue. Genetic polymorphisms have been associated with the epithelial barrier breakdown and airway remodelling in asthma. Adapted from Rydell-Törmänen et al (2012)76 ADAM protein family The ADAM (a disintegrin and metalloprotease) family is a sub-group of the zinc-dependent metalloprotease superfamily.91 ADAMs are large membrane bound proteases with eight domains: an N-terminus signal sequence, a pro-domain, a metalloprotease domain, a disintegrin-like domain, a cysteine rich domain, an EGF-like domain, a trans-membrane domain and a cytoplasmic domain.91-93 They are unique because of the presence of both a disintegrin domain and a metalloprotease domain. The disintegrin-like domain contains a 14 amino acid RGD motif in the disintegrin loop that is necessary for integrin binding to facilitate cell-cell or cell-matrix interactions.92,94 The metalloprotease domain contains a histidine-rich zinc-binding consensus sequence. Loss of the histidine residues in this consensus sequence results in an inability to bind zinc and a loss of proteolytic function.94-95 The metalloprotease domain is thought to be involved in extracellular matrix breakdown and in the shedding of membrane bound cytokines, growth factors and receptors.92 ADAMs are expressed in a variety of tissues and are thought to play a role in multiple processes including cell adhesion, proliferation, differentiation, signalling, apoptosis and the inflammatory response. 83,92,94 There are six ADAM subfamilies each with distinct characteristics.94 - 20 - ADAM33 polymorphisms ADAM33 belongs to subfamily E which also includes ADAM8, 12, 15, and 19.94-95 All the members of this subfamily have active metalloprotease domain.91 ADAM33 is located on chromosome 20p13 and exists in multiple splice variants, differing in the number of domains expressed.93-94,96 The function of ADAM33 is largely unknown. Van Eerdewegh et al (2002)91 were the first to associate ADAM33 with asthma. Genome wide scans of 460 Caucasian families identified an association between the 20p13 gene region and asthma. Analysis of 135 SNPs in 23 genes in this region identified a significant association of ADAM33 to asthma and bronchial hyperresponsiveness. Van Eerdewegh et al (2002)91 found six SNPs that were significantly associated with asthma in a US population and seven SNPs that were significantly associated with asthma in a UK population, some of which were in linkage disequilibrium with SNPs in adjacent genes.91 Howard et al (2003)97 confirmed the association of ADAM33 to asthma in four populations: African-American, US white, US Hispanic and Dutch white. They typed eight SNPs in the 3’ region and found a significant association of at least one SNP in each population.97 Lind et al (2003)98 studied six individual SNPs and combinations thereof in US Puerto Rican and Mexican populations; however they found no association of any individual SNP or combination of SNPs with asthma risk in either population.98 Raby et al (2004)99 tested 17 SNPs (9 of which were found to be significant by Van Eerdewegh et al (2002)91) in North American children. They were not able to find an association of any SNP to asthma and only found a slight association of one haplotype to asthma.99 Since then the association of ADAM33 with asthma has not gotten clearer. Positive associations have been found in German, Chinese, Thai and Indian populations, however other studies failed to find associations in Chinese, Australian, Colombian, and Indian populations.83,100-101 The lack of consistent SNP associations across multiple populations may indicate that the major causative SNP in ADAM33 has not yet been found, or that the true association of chromosome 20p13 is caused by a different gene, which may be in linkage disequillbrium with ADAM33.97 Interestingly, a study by Holgate et al (2006)102 found an association of three ADAM33 SNPs with early life lung function and asthma severity in later life indicating that ADAM33 may not directly affect asthma risk but rather may be associated to decreased lung function which predisposes to asthma development.102 In the lungs ADAM33 is primarily found in smooth muscle cells, myofibroblasts and fibroblasts.83,91,95 There are some reports of ADAM33 expression in lung epithelial cells, however as ADAM33 has been found to be silenced by DNA methylation in epithelial cells of both asthma patients and healthy controls it is considered functionally absent.96,103-104 Due to its localization, ADAM33 is also postulated to be involved in airway remodelling.83,91,95 As ADAM33 has a functional metalloprotease domain it likely functions as a sheddase, releasing cytokines, growth factors and receptors from the cell surface. The release of these factors may increase proliferation and differentiation of fibroblasts leading to increased deposition of ECM components and may influence smooth muscle growth leading to the smooth muscle thickening seen in asthma patients.83,95,102 Lee et al (2006)105 identified a soluble variant of ADAM33 (sADAM33) in the bronchoalveolar lavage fluid of asthma patients which correlated with decreased lung function. Puxeddu et al (2008)106 discovered that sADAM33 is produced from ectodomain shedding of membrane bound ADAM33. As such, it contains the metalloprotease domain but has lost the cytoplasmic domain. This allows sADAM33 to access proteolytic targets the membrane bound form was excluded from, potentially increasing its function. They were able to confirm that sADAM33 promotes angiogensis, likely through increased shedding of angiogenic factors in the ECM. Many of the ADAM33 SNPs associated with asthma are in the transmembrane or cytoplasmic domains. These SNPs may play a role in sADAM33 shedding or localization.106 This study shows, for the first time, a function of ADAM33 and proposes a potential effect of ADAM33 polymorphisms. - 21 - Tight and Adherens Junctions The airway epithelial barrier is primarily maintained through tight junctions (TJs), adherens junctions (AJs) and desmosomes which tightly bind neighbouring cells but still allow water and solute passage between them. Tight junctions are primarily composed of trans-membrane proteins, including claudins, occludins and junctional adhesion molecules (JAM), and scaffold proteins, the zonula occludens (ZO-1, -2, -3) which link the trans-membrane proteins to the actin cytoskeleton.72,107 Adherens junctions are primarily composed of e-cadherin which is bound to the keratin and actin cytoskeleton by p120 catenin, α-catenin, β-catenin and γ-catenin.72,108 Desmosomes are primarily composed of desmoleins and desmocollins.77 See Figure 3 for a schematic representation of these adhesion complexes. Recently asthma has been recognized as a disease of compromised epithelial barrier. Tight junctions are intrinsically deficient in asthmatic tissue and many of these junctional proteins have been associated with a risk of developing asthma (see Figure 3 and Figure 5).73 Figure 5 Tight junction distribution is abnormal in asthmatic epithelium. TJ component ZO-1 is stained green. Left panel represents healthy bronchial tissue. Right panel represents asthmatic bronchial tissue. Adapted from Holgate (2007)73 Claudin-1 Claudin-1 is a trans-membrane protein which, in epithelial cell tight junctions, regulates plasma membrane permeability.72 Interestingly, claudin-1 is also expressed in the nucleus and cytoplasm of ASM cells where it seems to play a role in cell proliferation. Fuijta et al (2011)69 found that over-expression of claudin-1 in ASM cells results in increased cell proliferation and production of VEGF, an important angiogenic factor. IL-1β and TNF-α seemed to upregulate claudin-1 expression in ASM whereas Th2 cytokines IL-4 and IL-13 down-regulated expression. They found increased claudin-1 expression in asthma patients ASM cells. These results indicate that claudin-1 may also play an important role in airway remodelling.69 Future testing is needed to determine if genetic polymorphisms are responsible for the increased expression of claudin-1 in asthmatic ASM. It would also be interesting to examine if CLDN1 is increased in the TJs of these patients and the effects of potential CLDN1 polymorphisms on TJ integrity. Occludin Occludin is a trans-membrane TJ protein that is involved in de novo TJ assembly.72 Xiao et al (2011)107 found decreased protein levels of occludin in asthma patients. The mRNA levels, however, were normal suggesting epigenetic down-regulation. They were able to determine that occludin down-regulation was not caused by the Th2 cytokine milieu found in asthma.107 Ahdieh et al (2001)109 however, found that IL-4 and IL-13 induced decreased expression of occludin resulting in decreased epithelial barrier function. These studies indicate the potential role of both genetic (or epigenetic) polymorphisms and local environment in epithelial barrier disruption. - 22 - Zonula occludens-1 Zonula occludens-1 (ZO-1) acts primarily as a scaffold protein in TJs to secure claudins, occludins and JAMs to the intracellular actin cytoskeleton. It can, however, also act as a transcription factor when bound to ZO-1-associated nucleic acid binding protein (ZONAB).108 de Boer et al (2008)108 were the first to report significantly reduced levels of ZO-1 in asthmatic epithelia. This was confirmed by Xiao et al (2011)107 who also reported significant reductions in ZO-1 protein levels in asthmatic bronchial epithelium. Although they found no reductions in ZO-1 mRNA levels, indicating epigenetic or environmental control, cultures of asthmatic bronchial cells in the absence of Th2 cytokines did not revert back to wild type ZO-1 expression indicating intrinsic deficiencies.107 Ahdieh et al (2001)109 found that Th2 cytokines, IL-4 and IL-13, are also able to decrease expression of ZO-1. Intrinsic defects in ZO-1 and disease induced down-regulation may contribute to the disrupted epithelial barrier in asthma (see Figure 5). E-cadherin E-cadherin is the adhesion protein that has most consistently been associated with asthma. E-cadherin, encoded by CDH1 on chromosome 16q22.1, is a trans-membrane glycoprotein with extracellular domains that form Ca+2 dependent interactions with cadherins on neighboring cells. These interactions maintain a mechanical connection to surrounding cells, playing an important role in the AJ epithelial barrier. The intracellular tail is highly conserved and interacts with scaffold proteins to anchor e-cadherin to the cytoskeleton. In addition to its role in cell adhesion, e-cadherin can regulate cell proliferation and differentiation through interactions with ZONAB, and can negatively regulate multiple signalling cascades.72,110-111 As a CD103 ligand, E-cadherin may play a role in Treg retention in the lungs and may limit DC activation during bronchial lumen sampling.72 de Boer et al (2008)108 and Hackett et al (2011)112 reported significantly decreased levels of e-cadherin in asthma patient epithelia. Ierodiakonou et al (2011)111 repeated these results and investigated the role of CDH1 polymorphisms. They found five SNPs (rs8056633, rs16958383, rs2276330, rs3785078 and rs7203904) that were associated with significantly decreased e-cadherin expression levels in asthma patients without inhaled corticosteroid (ICS) use. Recent evidence shows potential of ICS to repair the epithelial barrier and increase e-cadherin expression, so Ierodiakonou et al (2011)111 also looked at CHD1 polymorphisms in asthma patients with ICS use. In these patients they found 7 SNPs associated with airway remodelling, 3 with CD8+ T cell counts, 2 with eosinophil counts and 7 with lung function decline. These results indicate that ICS use may alter the functional effects of CHD1 polymorphisms.111 E-cadherin expression levels are also influenced by environmental factors such as RSV infection and pro-inflammatory cytokines.77,112 Trautmann et al (2005)77 found that IFN-γ and TNF-α cause e-cadherin down-regulation. T cell produced IFN-γ sensitizes epithelial cells to eosinophil TNFα induced apoptosis, which may result in epithelial cell shedding, a characteristic feature of asthma. They also found significantly decreased e-cadherin levels in asthma patients with epithelial shedding indicating a potential role of e-cadherin loss in epithelial shedding.77 Heijink et al (2007)110 investigated the signalling role of e-cadherin. E-cadherin is known to reduce EGFR ligand binding and co-localize with EGFR to limit its movement in the plasma membrane. E-cadherin knock-down by siRNA resulted in increased EGFR phosphorylation and increased activation of downstream transcription factor inducers, ERK and p38. Increased EGFR signalling, due to decreased e-cadherin, resulted in increased production of TARC (CCL17), a Th2 recruiting chemokine and TSLP, a potent Th2 inducing cytokine.110 E-cadherin is a multi-functional protein that plays an important role in the development and pathogenesis of asthma. Future studies will continue to unravel the functional role of e-cadherin variants in allergic disease. - 23 - α-catenin α-catenin is a scaffolding protein which regulates AJ structure through binding e-cadherin to the actin cytoskeleton. de Boer et al (2008)108 found significantly reduced levels of α-catenin in the bronchial epithelia of asthma patients. The α-catenin gene, CTNNA1, is found on chromosome 5q31 near the cytokine cluster which is repeatedly associated with atopy and allergic diseases.108 Together these indicate a potential role for CTNNA1 in asthma AJ defects. Protocadherin-1 Protocadherin-1 is a member of the δ1-protocadherin subfamily.113 PCDH1 is located on chromosome 5q31.1, which has been previously associated with atopy, asthma and atopic dermatitis.114 PCDH1 has five exons, which can be differentially processed resulting in multiple protein variants. The two most common variants are a three exon form and a longer five exon form. Although the 3 exon variant of protocadherin-1 lacks the majority of the intracellular domain it expresses an additional sequence in exon 2.114-115 Protocadherin-1 is a membrane spanning protein whose extracellular domain contains seven cadherin repeats and intracellular domain contains three conserved motifs (CM1, CM2 and CM3), an RVTF consensus domain and a PDZ-domain binding site at the C-terminal end. The RVTF consensus motif can interact with protein phosphatase-1α (PP1α) which is a signalling molecule reportedly involved in lung morphogenesis.113-115 There have been reports that the CM2 and CM3 domains also interact with PP1α, but otherwise their function is unknown.114-115 PDZ-containing proteins generally bind to the cytoskeleton and function in cell signalling, either directly or as scaffold proteins.115 Protocadherin-1 is expressed in the brain, skin, lung and epithelial tissues in mice. In the lungs it is found in fibroblasts and terminally differentiated bronchial epithelial cells, where it primarily locates to the cell-cell boundaries.115-116 Although the exact function of protocadherin-1 is unknown, it is thought to be involved in cell adhesion and maintenance of the epithelial barrier. Due to its interactions with PP1α, primary expression in terminally differentiated epithelial cells, and possible interaction with SMAD3 it may also be involved in cell signalling and epithelial cell differentiation.113-115 Protocadherin-1 polymorphisms In an initial study Koppelman et al (2009)114 identified PCDH1 as a novel BHR susceptibility gene. They identified 22 polymorphisms in PCDH1, one of which (rs3797054, Ala750Ala) was significantly associated to BHR in three Dutch populations and one US population. Interestingly, this SNP does not result in an amino acid substitution. As it is located in the 3’ UTR of exon three it may influence mRNA stability or splicing. A three base-pair insertion/deletion polymorphism (IVS3-116) was also associated with BHR risk in two Dutch populations and two US populations. This polymorphism is also located in the 3’ UTR of exon three. Both rs3797054 and IVS3-116 were also significantly associated with asthma risk. The major allele of a polymorphism in the fifth cadherin repeat (rs3822357, Ala514Thr) was also associated with BHR in a US and a UK population.114 Toncheva et al (2012)116 attempted to replicate these results. As PCDH1 is located on chromosome 5q31, nearby a cytokine cluster previously linked to atopy, asthma and atopic dermatitis, they performed linkage disequilibrium (LD) analysis to confirm that PCDH1 is itself associated to BHR. There was no LD between PCDH1 and surrounding genes, indicating that it is indeed a susceptibility gene for BHR. However, they were unable to find a significant association of rs3797054, rs3822357 or IVS3-116 with BHR in their cross-sectional German population. They did find a protective association of rs7719391 to asthma. Upon further analysis, they found no polymorphic associations to atopic asthma and two SNPs (rs2974704 and rs11167761) that were significantly associated to non-atopic asthma.116 Their failure to replicate the results of Koppelman - 24 - et al (2012)114 may be due to different determinants of BHR or due to the small, underpowered study design.116 Although PCDH1 polymorphisms have been associated with BHR and asthma, the functional role of these variants is unknown. Koning et al (2012)117 have also found an association of the IVS3116 variant of PCDH1 with AD in two Dutch populations. Future studies will be needed to determine the function of the various polymorphic forms and splice variants in the bronchial epithelium and the epidermis. The association of PCDH1 polymorphisms to susceptibility risk of asthma and atopic dermatitis also needs to be confirmed in larger studies. Filaggrin Filaggrin is not expressed in the bronchial epithelium118 however, numerous studies have found an association between FLG mutations and asthma when it co-presents with AD.32-33,39,42,44 The common interpretation is that a decreased skin barrier leads to increased allergen sensitization encouraging the atopic march towards asthma.32,39,42 Rogers et al (2007 and 2008)44,119 however, disagree with this interpretation. Similar to Palmer et al (2006)32 they have found an association between FLG mutations and asthma with AD and no association of FLG mutations in asthma without AD. The lack of association between FLG polymorphisms and asthma alone has led them to conclude that FLG mutations do not predispose towards asthma at all, but rather that FLG mutations predispose to AD and AD predisposes to asthma as part of the atopic march.44,119 It is interesting to note that asthma patients with FLG polymorphisms have been found to have more severe disease independent of AD status.42,120 Although FLG mutations may not directly increase the risk of developing asthma, they seem to interact with other factors to increase disease severity or encourage the atopic march. Serine protease inhibitor, Kazal-type, 5 Asthma has previously been linked to the 5q31-33 chromosomal region where SPINK5 is located.121 In addition, LEKTI is an inhibitor of tryptase, an important asthma mediator.57 Due to this, SPINK5 has been investigated for a role in asthma development and some associations between asthma and SPINK5 polymorphisms have been reported. Walley et al (2001)58 found a weak association of the E420K polymorphism to asthma and Kabesch et al (2004)62 repeated this finding in a large German population-based study. However, in more cases the association is not found.61-62,121 Recently, Jongepier et al (2005)121 found no association of SPINK5 polymorphisms with asthma in two Dutch populations. They also found that SPINK5 is not expressed in the lungs. This, combined with the lack of asthma over-expression in Netherton syndrome patients, indicates that SPINK5 polymorphisms likely do not increase the risk of developing asthma.121 - 25 - Current Therapies and Future Perspective Disruption of tissue homeostasis, due to genetic and environmental factors, is now seen as fundamental in the initiation and perpetuation of allergic inflammation and disease. This paradigm shift has led to changes in therapeutic approaches and the development of novel, tissue-targeted treatments. Atopic Dermatitis Topical corticosteroids are the standard treatment for acute lesions in atopic dermatitis. They interact with glucocorticoid receptors on immune cells, keratinocytes and fibroblasts to block the transcription of pro-inflammatory genes.122 Although topical corticosteroids are quite effective at reducing symptoms and disease severity they fail to address the underlying mechanism of disease and there is some evidence that they further disrupt the skin barrier and increase the risk of microbial infection.23 Topical emollients are also widely used to treat AD. They are creams or ointments that create a superficial barrier of non-physiologic lipids, such as petrolatum, lanolin mineral oil or silicon.122-123 Emollients have been quite successful in treating AD symptoms; however there is some evidence that the non-physiologic lipids may obstruct the underlying barrier defects rather than correct them.23 In healthy skin the lipid lamellae is composed of approximately 50% ceramides, 25% cholesterol and 10-20% fatty acids. In AD skin these three lipids are significantly reduced. Emollient therapies containing these physiologic lipids have recently become available. When topically applied, physiologic lipids are thought to penetrate the SC and get processed into lamellar bodies for secretion into the SC where they can restore the epidermal barrier.23,123There is some evidence that on their own or in the incorrect ratio they may have a negative effect on barrier repair.123 Ceramide dominant treatments, such as EpiCeram® which uses a 3:1:1 ratio of ceramides, cholesterol and fatty acids, seems to provide optimal barrier repair.23,123 CeraVe,® another ceramide based cream which is under development uses multilamellar vesicle emulsions to slowly release the lipids over a 24 hour period. This is promising as a once-daily barrier-repair therapy for AD.123 In addition, gene repair therapies may be available in the coming years. Ex-vivo lentiviral transduction of keratinocytes with SPINK5 has been shown to restore LEKTI production in human NS epidermal cells grafted onto a mouse model.124 As the available treatments for AD are very effective, not only at addressing the symptoms but also at repairing the underlying barrier defects, this type of treatment will likely only be used in cases of sever barrier breakdown, such as in NS. Future research should focus on identifying biomarkers of early disease to detect at-risk children. Prophylactic treatment of these children with barrier repairing creams could potentially prevent allergen sensitization and development of atopic dermatitis. There have been a few studies indicating that prophylactic emollient treatments in infants may reduce the incidence of atopic dermatitis and enhance skin barrier.122 Asthma Asthma is a very heterogeneous disease; therefore it is unlikely that a single treatment or therapy with be completely effective. However, with a clearer definition of asthma subtypes, targeted therapies could be quite successful.18,84 Many immune targeted therapies, with varying levels of success are under development. Anti-IgE therapy, which was expected to be curative for allergic diseases, has been disappointing. Omalizumab, an anti-IgE monoclonal antibody (mAb), has shown some benefit in patients with moderate to severe asthma in that it can reduce systemic IgE levels, FcєRI on mast cells and basophils and can reduce eosinophil levels in some patients. However, it likely fails to reduce site-specific IgE levels and has low cost-effectiveness.4,18 Many cytokine targeted therapies are also under development. In clinical trials some patients show improved - 26 - pulmonary function and reduced symptoms in response to anti-IL-12 mAbs. Multiple anti-IL-4 mAbs are also undergoing clinical trials; however, their success has been much lower than anticipated. This may be due to the ability of IL-13 to signal through the same receptor complex negating the effect of IL-4 reduction. Anti-IL-13 mAbs are also under development. A phase 2 clinical trial shows some promise in certain subgroups of asthma. Anti-IL-5 mAbs were expected to greatly reduce asthma symptoms; although they reduce eosinophil levels to some extent, they did not result in any significant lung function improvement. Blocking TSLP signalling with a soluble antagonist and small molecule targeting of chemokine receptors including CX3CR1 on CD4+ T cells and CCR3 on eosinophils have also shown some promise.2,18 Allergen specific immunotherapy (SIT) is the only curative treatment for asthma; however it is controversial due to the risk of anaphylaxis. In randomized control trials SIT has been fairly successful in reducing asthma symptoms and decreasing medication use, however there is a high risk of local and systemic side effects and this treatment does not seem to improve overall lung function.125 Currently the standard asthma treatment is inhaled corticosteroids (ICS) alone or combined with long-acting bronchodilators.79 Corticosteroids are immunosuppressive agents that target eosinophilic airway inflammation, while bronchodilators aim to reverse bronchoconstriction and relieve breathing difficulties.4,125 In most patients this treatment is very successful in suppressing asthma symptoms.125 Although the currently available asthma treatments are quite effective, they primarily target the symptoms or immune mediated mechanisms of the disease.79 Given the critical role of the epithelium in both the disease initiation and perpetuation, novel therapeutic design should focus on epithelial barrier repair and the prevention of disease. Xiao et al (2011)107 have shown that topically applied (inhaled) epidermal growth factor (EGF) can increase TJ numbers and improve the epithelial barrier function in asthmatic patients without causing uncontrolled cell proliferation or goblet cell hyperplasia. There is recent evidence that ICS may increase tight junction integrity and aid in barrier repair as well as suppress excess cell proliferation, potentially decreasing airway remodelling.126-127 Therapeutically targeting the epithelial may prevent inflammation, rather than simply suppressing it. Some studies of PRR modulation have shown promise. In mouse models TLR7/8 agonists significantly reduce airway inflammation and prevent airway remodelling. TLR7/8 stimulation resulted in decreased cytokine production, decreased inflammatory cell infiltration in the lungs, decreased smooth muscle proliferation and decreased goblet cell hyperplasia and metaplasia.128-129 Targeting the airway epithelium with a combination of TLR agonists and barrier repair factors may prevent allergen entry and the initiation of an allergic inflammatory response. Many asthma patients develop disease as part of the atopic march. These patients will have presented with atopic dermatitis before developing asthma. Early identification of children at-risk of developing asthma subsequent to AD could allow for administration of preventative treatments. This could include topical epidermal barrier repair treatment to prevent systemic allergen sensitization and prevention of respiratory viral infections to avoid compromising the airway epithelium. Many of the genetic loci associated with asthma are distinct from the loci associated with AD indicating that, while in some cases AD can increase the risk of asthma development in line with the atopic march, asthma is also a distinct disease.29 As such, potentially preventative interventions also need to be targeted at asthma directly. In most cases of childhood diagnosed asthma, wheezing precedes disease development.84 Viral infections are often the cause of this wheezing and can cause epithelial barrier damage increasing risk of asthma development. Vaccinating young children against RSV or HRV, the two most common respiratory viruses associated with asthma risk, may decrease asthma incidence. Anti-RSV antibodies are under development but not yet available for clinical testing in children.84 Edlmayr et al (2009)130 developed a novel vaccine for allergy and rhinovirus infection. They created a fusion protein of VP1, a major HRV surface protein, and a synthetic Phl p 1 protein of a major grass pollen allergen. Vaccination with this fusion protein resulted in a strong protective IgG antibody response against both the allergen and the virus.130 This study indicates the potential of prophylactic allergic vaccination. - 27 - Conclusions Allergic diseases arise from complex interactions between genetic predispositions and environmental factors. The classical view of allergic diseases is based primarily on immune dysregulation; however, this alone cannot fully explain their development. Over 40% of the western population is atopic however, only 7-10% percent of these develop asthma and only a small proportion develops atopic dermatitis (AD).73-74 The recent association of tissue restricted factors with asthma and AD indicates that local tissue factors play a critical role in determining the location of disease establishment.17,19 The primary tissue role is as a barrier against allergen entry and sensitization. Disruption of the barrier by physical damage or allergen activity can result in the production of Th2 cytokines and chemokines driving the allergic immune response.26,78 The skin and the airway epithelium are the primary sites susceptible to allergen entry. As such, in healthy individuals, they are highly structured barriers. The skin is a multi-layer barrier which, when functional, protects against water loss and invasion of unwanted or harmful environmental materials. The barrier consists of the stratum corneum with its associated structural and regulatory components, the lipid lamellae, the acid mantle and tight junctions.17,21 Intrinsic deficiencies at any of these levels predisposes towards allergen sensitization and the development of atopic dermatitis. Genetic polymorphisms in FLG, KLK7, SPINK5 and CSTA can result in a breakdown of the SC barrier.32,54,58,64 FLG polymorphisms can also affect the pH mantle and lipid lamellae barriers and CLDN1 polymorphisms can disrupt the TJ barrier.32,68 Although it can be difficult to unravel the exact contribution of these polymorphisms to disease development, they all indicate that the epidermal barrier is an essential protective force against allergic disease in the skin. The airway epithelium is a single layer barrier that depends on the adhesive forces between neighbouring cells to prevent allergen, microbial and chemical entry. Tight and adherens junctions are the primary enforcers of the epithelial barrier. Cell adhesion genes, CLDN1, OCLN, ZO-1, CDH1, PCDH1 and CTNNA1, have all been associated with increased risk of asthma highlighting their essential role in epithelial barrier integrity and the protective nature of the barrier against allergen sensitization and disease development.107-108,112 The association of genes functionally involved in airway remodelling in asthma, such as ADAM33 and CLDN1, indicate that tissue related factors are also involved in disease progression.69,91 A predisposition to epithelial barrier dysfunction and aberrant barrier repair may lead to allergen sensitization and development of an overactive immune response in asthma patients. The current understanding of tissue barrier dysregulation at the core of allergic diseases has opened the door to novel therapeutic options. 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