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1 Infection, Inflammation and Immunity Division, School of Medicine, Southampton University, Southampton General Hospital, Southampton, United Kingdom
Correspondence and requests for reprints should be addressed to Stephen T. Holgate, M.D., F.R.C.P., F.Med.Sci., Infection, Inflammation and Immunity Division, School of Medicine, Southampton University, Southampton General Hospital, Southampton SO166YD, UK. E-mail: sth{at}soton.ac.uk
ABSTRACT
Asthma is an inflammatory disorder of the airways dominated by a Th2-type pattern. Because of this, most research has focused on investigating the role of allergic pathways with the hope of discovering novel therapeutic targets. Unfortunately, this strategy (which has been extended to animal models) has failed to identify any therapeutic modalities other than anti-IgE and leukotriene modifiers directed to targets known about for many years. It seems that the problem lies in placing allergy at the center of disease pathogenesis, when in practice other environmental factors may be equally if not more important in the induction and then progression of asthma. An alternative view is that asthma is primarily a defect of epithelial barrier function that, as in atopic dermatitis, allows greater access of environmental allergens, microorganisms, and toxicants to the airway tissue. Evidence is provided to show that both the physical and functional barrier of the airway epithelium is defective in asthma with disrupted tight junctions, reduced antioxidant activity, and impaired innate immunity. This explains the remarkable susceptibility of asthmatic airways to respiratory viruses and the impact of air pollutants on asthma exacerbations. It also provides a mechanism for programming of dendritic cells to drive a Th2 response in the origins of asthma. Viewing asthma primarily as an epithelial disease with adoption of a chronic wound scenario also provides a route to airway wall remodeling and the varying asthma phenotypes over the life course.
Key Words: asthma • epithelium • allergens • rhinovirus • exacerbation
SHORTFALLS IN ASTHMA TREATMENTS
Although asthma has been considered as a single disease entity driven largely by the use of bronchodilators and corticosteroids for its treatment, sub-phenotypes are now recognized with differing immunology, pathology, clinical expression, response to treatments, and long-term outcomes (1). Most asthma exhibits a Th2-type inflammatory response with coordinated up-regulation of cytokines of the IL-4 gene cluster on chromosome 5q linked to atopy; however, overinterpretation of this immunologic pathway has led to the simplistic view that asthma results purely from allergen sensitization and exposure. Indeed, this concept has driven the great majority of animal "models" of the disease used by industry to identify novel therapeutic targets, but despite almost 50 years of intensive investment by the pharmaceutical industry, the only successful novel treatments to emerge are leukotriene modifiers and anti-human IgE monoclonal antibodies (Mab) (omalizumab), directed at targets identified many years ago (2). Atopy affects up to half of the adult population, yet the great majority do not develop asthma (3). Both in adults and in children the indistinguishable pathologic features of nonallergic and allergic asthma emphasizes that asthmatic inflammation and remodeling can occur independently of atopy (4). While allergen sensitization does contribute to asthma in many patients, attempts to prevent or reverse asthma using allergen reduction strategies (5) or allergen-specific immunotherapy (6) have overall proven disappointing in anything but the mildest disease, despite efficacy in allergic rhinitis (7).
In severe asthma, activated T cells are present in abundance, but despite initial promise, T cell–specific inhibitors (cyclosporine A, methotrexate, azathioprine, anti-CD4 or -CD25 blocking Mab) and blockade of Th2 cytokines (IL-4, -5, -9, and -13) have so far failed to translate into clinical use (2). Based on these concerns, the view that chronic airway inflammation and remodeling in asthma is caused primarily by allergen sensitization is being challenged.
THE EPITHELIAL–MESENCHYMAL TROPHIC UNIT
In 2000 we first proposed that allergic-type inflammation and aberrant epithelial injury/repair mechanisms were parallel phenomena leading to a range of different asthma subtypes (8). In asthma the epithelial–mesenchymal trophic unit (EMTU), which controls the local tissue microenvironment and maintains tissue homeostasis, becomes dysregulated (9). Thus, epithelial damage by environmental agents results in production of signals that act on the underlying mesenchyme to propagate and amplify inflammatory and remodeling responses in the submucosa. The finding that many novel asthma susceptibility genes identified as a result of hypothesis-independent approaches are expressed in the epithelium (ESE 1 and 3, DPP10, NPSR1, PCDH1, CHI3L1, GSTP1, GSDML, OPN3, and HLA-G) and mesenchyme (ADAM33, KCNMB1, MYLK, and C/EBP
) places the EMTU at the center of asthma pathogenesis (10, 11). The most frequent risk factors for developing, exacerbating and prolonging asthma act through the EMTU—namely biologically active allergens, air pollutants, irritants, environmental tobacco smoke (ETS), and respiratory viruses. These strengthen the case for a dynamic interaction between the epithelium and formed elements of the airways in the development of the different asthma subphenotypes (1).
Rather than being a primary cause of asthma, allergen sensitization may well be the consequence of a defective airway epithelium leading to inappropriate programming of mucosal dendritic cells (DCs) toward promoting a Th2 phenotype. In support of this, we and others have recently reported defective epithelial tight junction (TJ) formation both in asthmatic biopsies and in the epithelium differentiated at an air–liquid interface (ALI) in vitro in association with impaired barrier function (12). Similar mechanisms are now known to operate in predisposing to other allergic diseases such as atopic dermatitis, where polymorphisms in the filaggrin gene affect skin permeability (13), and in food allergy (14)—both leading to enhanced allergen sensitization.
Further evidence for defective epithelial repair in asthma comes from the observation of overexpression of the epidermal growth factor (EGF) receptor in proportion to disease severity (15), with receptor activation revealed by phosphorylation of the intracellular domain (16). These changes (in the presence of reduced evidence of epithelial cell cycling) point to a chronic wound scenario present, not only in adult asthma, but also in the airways of asthmatic children (17). Most recently, airway epithelial cells cultured from atopic children with asthma compared with those from atopic normal children have been shown to be inherently dysfunctional in their ability to repair after injury, a defect linked to marked up-regulation of plasminogen activator inhibitor-1 (18). Defective epithelial repair is a potent stimulus for the release of profibrogenic growth factors, as has recently been shown in a mouse model of naphthalene-induced epithelial injury (19).
Bronchial biopsies from very young children with early life virus–associated wheezing have revealed little abnormal pathology, but by the age of 3 years, epithelial injury and thickening of the lamina reticularis is evident, either in the absence or presence of Th2-type inflammation (20, 21). While thickening of the lamina reticularis is a highly characteristic feature of asthma, there remains debate over its significance to remodeling, since it does not relate to asthma duration (22), although it does increase with disease severity (23). This feature of the asthmatic airway is present both in asthma associated with atopy and in nonallergic asthma. Based upon its unique presence in the airways of individuals with asthma and also after lung transplantation (24); the deposition of new extracellular matrix in the lamina reticularis maybe another indicator of chronic epithelial injury.
In severe asthma, the extent of epithelial expression of EGF receptors closely correlates with immunoreactive CXCL8 (IL-8) (25). Activation of EGF receptors either directly by specific ligand or through transactivation in the presence of an oxidative stimulus leads to the secretion of proinflammatory cytokines such as CXCL8, a potent chemoattractant for neutrophils (Figure 1). The generation of reactive oxygen from epithelial damage as well as from inflammatory cells can also transactivate the EGF receptor in addition to its interaction with specific ligands (15, 16). Through such amplification mechanisms, delayed and incomplete epithelial repair both promotes ongoing chronic inflammation and initiates remodeling in an attempt to limit penetration of the airway by potentially damaging inhaled toxicants or microorganisms.
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The chronic pruritic inflammatory skin disease, atopic dermatitis or eczema, is also associated with adaptive Th2 response to common environmental allergens or a mixed Th1/Th2 response, as seen in severe asthma. Atopic dermatitis is characterized by increased epithelial permeability, increased transepidermal water loss, increased penetration of exogenous substances (allergens, bacteria), and decreased subcutaneous hydration. Impaired or absent secretion of the filaggrin peptide consequent upon loss-of-function mutations of the FLG gene on chromosome 1q21 leads to a defect in forming the cornified envelope to maintain barrier function (13), filaggrin being among the last barrier function protein to be incorporated. FLG mutations are a powerful determinant of both early-onset and severe atopic dermatitis, and lead to enhanced allergen sensitization. It is probably through such a mechanism that a link between severe asthma and atopic dermatitis occurs. Indeed, there are remarkable parallels with defective barrier function in asthma. Different from the skin, it is the TJs in the airway epithelium that regulate the paracellular passage of inhaled materials. Both in biopsies and in differentiated cultured epithelial cells, TJ integrity is compromised with disordered assembly of the TJ components such as occluding and zonula occludens-1 (ZO-1) that stabilize barrier function through coupling to the perijunctional cytoskeleton (12, 26, 27). Environmental factors such as proteolytic dust mite and pollen allergens as well as virus infection enhance TJ disassembly (27). In contrast, high levels of exogenous EGF or keratinocyte growth factor are capable of restoring TJ integrity and re-establishing barrier function both at baseline and in response to environmental toxicants.
We suggest that damage to the epithelium results in augmented expression of EGF receptors, but on account of a reduced ability of signaling through these to repair the epithelium through "primary intention" in response to the levels of EGFR ligands available, the epithelium enters into "frustrated repair (or repair by secondary intention)" leading to enhanced profibrotic growth factor release and a tendency toward epithelial–mesenchymal transition. The defect in TJ assembly in asthma may simply be the result of impaired EGFR signaling and thus be another feature of the chronic wound process.
THE ROLE OF VIRUSES IN EXACERBATIONS
Respiratory viral infections are the major cause of asthma exacerbations, with human rhinoviruses (HRV) dominating (28). Accordingly, asthma exacerbations peak in the autumn and winter months, with much smaller summer peaks linked to pollenosis (29). The bronchial epithelium is the target for such virus infections using ICAM1 (major HRV subclass) or the VLDL (minor HRV subclass) receptor for gaining entry (30). We have shown that the epithelial cells of asthmatic airways are sensitive to the cytotoxic effects of HRV on account of reduced capacity to restrict viral replication and initiate epithelial cell apoptosis (31). This results in extensive cell death, viral shedding, and mediator release, features of an asthma exacerbation. Neutrophils as well as eosinophils are recruited, accounting for the partial response to corticosteroids (32).
In normal epithelial cells the double-stranded RNA of the virus is recognized by microsomal Toll-like receptor (TLR) 3 that, through activation of interferon regulatory factor (IRF) 3, induces the activation of the interferon (IFN)-β gene (33). Secreted IFN-β then activates its own receptor to initiate the complex cascade of downstream antiviral protective mechanisms. However, in asthma the early TLR3 signaling is defective, leading to a grossly inadequate IFN-β and IFN-
responses that account for the impaired viral clearance and epithelial damage in this disease (31, 34) (Figure 2). Since asthmatic epithelial cells produce IFN-β normally in response to polyIC, a surrogate of viral DS RNA, the most likely site of the impaired IFN response in asthmatic cells is somewhere in the first step of the antiviral response (Figure 3). Inhaled IFN-β is now being developed as a treatment for severe virus-induced asthma exacerbations.
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Most asthma has its origins early in life, suboptimal fetal growth, maternal micronutrient deficiencies, or smoking during pregnancy being associated with impaired infant lung function and later asthma. Both bronchial hyperresponsiveness (BHR) and persistent asthma also have a strong genetic basis independent of atopy. For example, polymorphism of the asthma susceptibility gene ADAM33 is associated with impaired lung function in infants, increased risk of RSV-induced bronchiolitis, and the later development of BHR (35, 36). Birth cohort studies have also revealed that severe asthma is predicted by impaired infant lung function and BHR; however, the role of allergy as the sole initiator of asthma is in doubt. In most children who develop asthma, atopy has little influence on the expression of the disease until 5 years of age, after which it predicts disease persistence (37), with those destined for severe disease acquiring IgE sensitization earlier (age 3–4 yr) in the presence of high aeroallergen exposure (38).
Environmental factors other than allergen exposure are emerging in the early life origins of asthma. In a longitudinal birth cohort study in the United States (COAST), repeated lower respiratory tract infections with HRV during the first 3 years of life increased the risk of developing asthma 26-fold by age 6 years, compared with 3-fold for allergen sensitization (39). The key role of early life virus infection also extends into adult asthma in the European Community Respiratory Health Survey (40). In a U.S. 95,000-infant cohort study, the timing of birth in relationship to the winter virus season conferred a 30% increased risk of developing asthma by age 6 years (41), while a Perth cohort has shown that respiratory virus infection (RV: 70% and RSV: 16%) positively interacts with atopy to promote later asthma at age 5 (42). Importantly, the target for these viruses, environmental tobacco smoke, and other pollutants is the airway epithelium. Understanding why the airway epithelium of these children is so susceptible to these stimuli and how it affects allergic sensitization provides a potential route to prevent asthma.
Antigen-presenting cells such as airway dendritic cells (DCs) play a critical role in initiating and regulating early inflammatory events. While in the first year of life infants do not typically exhibit airway DCs in the absence of inflammation, severe respiratory infection is associated with the appearance in infant airways of mature DCs (43). This raises the possibility that viral respiratory infection in early life may play a key role in the etiology of atopic asthma by recruiting appropriately primed antigen-presenting cells to the epithelium, possibly as a consequence of disruption of the epithelial barrier. This proposal is supported by showing impaired barrier function as a property of asthmatic epithelium that respiratory viruses disrupt epithelial TJs (44) and that in adult asthma airway epithelial cells make less IFN-β and more thymic stromal lymphopoetin (TSLP) in response to TLR3 agonists, suggesting a preferential drive toward a Th2 response. Based on these studies, we hypothesize that a structurally and functionally defective airway epithelium underlies abnormal responses to respiratory viruses and other components of the inhaled environment. This promotes a microenvironment that facilitates allergic sensitization, supports different types of inflammation, and predisposes the airways to development of asthma during childhood.
Using a co-culture system of airway epithelial cells and differentiating DCs in the presence of IL-4 and GM-CSF, Rate and colleagues have shown that resting airway epithelial cells direct adjacent DC differentiation in favor of a Th1-type profile and in doing so down-modulate the capacity for expression of pro-allergic Th2 immunity (45). Thus, in an individual with potentially defective epithelial cell production of IFN-β in response to respiratory viral infections (genetic or epigenetic in origin), DCs would be driven toward a Th2 phenotype. Such a mechanism would help explain the interaction between virus infection in early life and the later acquisition of atopy.
CONCLUSIONS
The airway epithelium is crucial to both the origins and progression of asthma well as playing a key role in asthma exacerbations. Viral infections especially with HRV seem especially important, but what it is about this virus type that is so "asthmagenic" is a mystery. However, the recent recognition of HRV C as a new subclass that has been closely linked to a high proportion of pediatric asthma (46) raises the profile of virus infection in asthma pathogenesis further and supports the view that in this disease the airway epithelium plays a crucial role in its origin and subsequent natural history.
FOOTNOTES
S.T.H. is a UK Medical Research Professor, and much of the authors' work cited in the review was funded by the MRC, Asthma UK, or the British Lung Foundation.
Conflict of Interest Statement: S.T.H. has received reimbursement for consultancies with Synairgen ($10,001–$50,000), and also sat on an advisory board for Synairgen ($5,001–$10,000). He also has stock ownership or options with Synairgen ($10,001–$50,000). G.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. H.S.A. has received reimbursement for consultancies with Rotta Laboratorium ($1,001–$5,000), and an honorarium for lectures with ALk Abello (up to $1,000). P.H.H. has received reimbursement for consultancies with Shionogi Pharamaceuticals ($1,001–$5,000), and serving on advisory boards for Wyeth Pharmaceuticals ($1,001–$5,000), Funxional Therapeutics ($1,001–$5,000), and Merck (up to $1,000); he has also received grants for research with Shionogi Pharmaceuticals ($50,001–$100,000), and Schering Plough ($50,001–$100,000). D.E.D. has received reimbursement for consultancies with Synairgen ($10,001–$50,000), and has received or is awaiting a pending patent with Synairgen. She also has stock ownership or options with Synairgen ($50,001–$100,000).
(Received in original form July 21, 2009; accepted in final form September 28, 2009)
REFERENCES
R, et al. The role of the epidermal growth factor receptor in sustaining neutrophil inflammation in severe asthma. Clin Exp Allergy 2003;33:233–240.[CrossRef][Medline]
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