Clinical & Experimental Allergy, 43, 1302–1306
doi: 10.1111/cea.12204
© 2013 John Wiley & Sons Ltd
EDITORIAL
Clinical The role of tissue eosinophils in asthmatic airway & remodelling Experimental This editorial discusses the findings of the paper in this issue by S. J. Wilson et al. [7] pp. 1342–1350. Allergy A. Shikotra and S. Siddiqui Department of Infection, Immunity and Inflammation, Institute for Lung Health, University of Leicester, Leicester, UK
Asthma is an important cause of morbidity and mortality affecting 300 million people world-wide [1, 2]. Asthma is characterized by the presence of inflammation and structural changes within the airway wall often described as airway remodelling – these features were originally described in fatal asthmatics in the seminal report of Huber and Koessler published in 1922 [3]. Despite nearly a century of on-going asthma research, the pathogenesis of airway remodelling remains poorly understood. The structural and cellular changes which have been described repeatedly in studies of asthmatic airway remodelling include an increase in the numbers of tissue eosinophils (and in some cases neutrophils), thickening of the reticular basement membrane, increased airway smooth muscle mass, goblet cell hyperplasia and angiogenesis [4–6]. The limitation of these aforementioned studies is that the following: (i) many of them fail to consider the importance of colocalization of inflammatory/immune cells to key airway structural cells, (ii) most studies are cross-sectional, and some features of remodelling notably basement membrane thickening have been described in early life questioning the progressive nature of the remodelling process and (iii) few studies have evaluated the role of asthma therapy upon features of both structural and cellular remodelling. As a consequence, it is difficult to determine Correspondence: Dr Salman Siddiqui, Department of Infection, Immunity and Inflammation, Institute for Lung Health, University of Leicester Respiratory Research BRU, Glenfield Hospital, Groby Road, Leicester, LE3 9QP, UK. E-mail: [email protected] Cite this as: A. Shikotra and S. Siddiqui and Clinical & Experimental Allergy, 2013 (43) 1302–1306. This logo highlights the Editorial article on the cover and the first page of the article.
whether airway inflammatory cells drive structural remodelling in asthma. In this issue of Clinical & Experimental Allergy, Wilson et al. examine the relationship between airway remodelling and submucosal eosinophilia in a cohort of patients with mild steroid-na€ıve asthma and matched healthy subjects [7]. Using standard immunohistochemistry techniques, eosinophils were identified in the bronchial submucosa, and based upon the submucosal expression, the asthmatic subjects were subdivided into two groups: eosinophilshigh and eosinophilslow. The group subsequently assessed airway structural remodelling by assessing three key features: (i) epithelial damage, (ii) goblet cell hyperplasia and (iii) airway smooth muscle hypertrophy and cellular infiltration. The report has described a number of novel and interesting findings. Notably that patients with the tissue eosinophilshigh phenotype had evidence of increased epithelial damage in vivo (based upon epithelial EGF-R expression), increased Mucin-2 expression and increased numbers of both eosinophils and T cells within the airway smooth muscle (ASM) bundle itself when compared to eosinophilslow asthmatics and healthy controls. Interestingly, in this report, mast cell numbers in the ASM were only increased significantly in the eosinophilshigh asthma phenotype, and other features of structural remodelling such as increased ASM mass were similar in both tissue eosinophil phenotypes. Taken together, these observations suggest that tissue eosinophils may selectively modulate a number of aspects of structural airway remodelling in asthma and may be causally involved in perpetuating the aberrant cycle of dysregulated epithelial repair seen in asthma. The airway epithelium has emerged as a central orchestrator of asthmatic airway inflammation and remodelling [8]. The cycle of frequent damage and inadequate repair of the epithelium is thought to facilitate airway remodelling via epithelial–mesenchymal–
Tissue eosinophils in airway remodelling
inflammatory cell crosstalk [8]. Wilson et al. have quantified epithelial damage using epidermal growth factor receptor (EGF-R) protein expression. Puddicombe et al. previously demonstrated that the percentage of EGF-R-positive damaged epithelium increases as disease severity progresses despite the use of corticosteroids in asthma and that EGF-R immunoreactivity is evident throughout the epithelium in bronchial tissue. In the same study in vitro, EGF-R was not identified immediately after wounding but was identified subsequently at 3 and 9 hours postwounding [9]. These observations have also been reported in childhood asthma [10] and are associated with collagen deposition in the lamina reticularis. The use of EGF-R protein expression to quantify epithelial damage is novel as damaged epithelium is a common observation in bronchial biopsies and it is difficult to assess how much is artefact. Wilson et al. have differentiated between the two, using immunostaining. EGF-R-positive epithelium was defined as in vivo damaged epithelium, and EGR-R-negative epithelium were classed as artefact related possibly to sampling-induced damage. A number of previous reports have described increased numbers of eosinophils within the submucosa and airway epithelium itself in asthma [Reviewed in 11]. The authors extend these observations showing that in vivo epithelial damage was elevated only in the eosinophilshigh subgroup suggesting a role for eosinophils in epithelial damage. The notion that tissue eosinophils drive epithelial damage in asthma is supported by extensive literature linking eosinophilic granules and reactive oxygen species to epithelial damage in ex vivo models [12]. In addition, the damaged asthmatic epithelium is known to release a number of cytokines/chemokines that promote TH2 polarization including IL-13, IL-5, TARC, IL-33, RANTES, eotaxins and MCP-3. Therefore, it is plausible that a vicious cycle of epithelial damage and inflammation may promote tissue eosinophilia and further damage in asthma. Despite these observations, the clinical importance of tissue eosinophilia remains to be established. Tissue eosinophilia and associated basement membrane thickening have been reported in other diseases where airway hyper responsiveness is absent – such as nonasthmatic eosinophilic bronchitis [13]. In addition, targeting tissue eosinophilia with a selective monoclonal antibody to IL-5 over a 10 weeks period had little impact upon airway physiology despite a marked reduction in immunoreactive tenascin, collagen III and lumican present in the lamina reticularis [14]. These features were not evaluated in the current report. Therefore, it remains to be determined whether interactions between tissue eosinophils and epithelial cells drive clinically important outcomes in asthma with some reports suggesting a protective role for basement membrane remodelling in asthma [15].
1303
An alternative mechanism linking tissue eosinophilia to asthma morbidity is mucous hyper secretion that may arise due to hyperplasia of goblet cells and glands. Previous reports have identified increased goblets cells in mild asthma [16]. Furthermore, MUC5AC and 5B alongside eosinophilic granule proteins are known to increase the viscosity and tenacity of airway mucous [17, 18]. Interestingly, in the current report, only MUC2 protein expression was increased in eosinophilshigh asthma in contrast to the MUC5AC and MUC5B. These observations are difficult to fully interpret as (i) MUC2 comprises at most only 2.5% of the weight of gel forming mucins, (ii) glandular expression of mucins was not evaluated and is likely to be an important source and (iii) post-translational modifications of the major mucins MUC5AC and MUC5B that alter their glycosylation and charge status may significant influence the viscosity of mucous. Further studies are required to determine the role of eosinophilic tissue inflammation in modulating altered mucin protein expression in airway glands and mucous rheology in asthma. One of the key features of airway remodelling is an increase in ASM mass. The mechanisms underlying ASM remodelling are likely to involve ASM hyperplasia, hypertrophy and deposition of extracellular matrix (ECM) proteins [19, 20]. Interestingly, in the current report by Wilson et al., increased ASM mass was found to be independent of tissue eosinophilia suggesting that other processes may drive this phenomenon. It is plausible that the phenotype of increased ASM mass in asthma may therefore not respond to current antieosinophilic therapies. Alternative approaches to targeting this phenotype include bronchial thermoplasty which has been shown to improve patient reported outcome measures in asthma [21–23]. Future studies are required to evaluate whether ASM mass can be utilized to further stratify the response to thermoplasty. There is increasing recognition that inflammatory cells not only infiltrate the submucosa but also microlocalize to the structural components of the airway [11]. Research in this area has primarily focused on the airway smooth muscle bundle, and the infiltration of mast cells within the ASM bundle has been well documented [4, 13]. Similarly, Wilson et al. in this issue describe mast cell infiltration in the context of mild asthma and the eosinophilshigh phenotype. In addition, this is one of the very few studies that have observed T-cell infiltration into the smooth muscle [24]. The finding that eosinophils colocalize to the ASM is novel. Our group has not consistently observed these cells within the ASM bundle itself, and it is possible that some of these cells were within the perimeter of the ASM bundle or juxtaposed between ASM cells. In either case, the findings are important as we have previously shown that mast cells within the perimeter of the ASM bundle [25]
© 2013 John Wiley & Sons Ltd, Clinical & Experimental Allergy, 43 : 1302–1306
1304 A. Shikotra & S. Siddiqui can increase the expression of contractile proteins in vivo, and it is possible that eosinophilic proteins may facilitate similar functional changes within ASM cells. Future studies should evaluate the functional effects of eosinophils upon asthmatic ASM cells in coculture models and the ability of ASM chemokines to facilitate migration and adhesion of eosinophils. In addition, the findings suggest that a complex interplay between T cells, eosinophils and mast cells may be responsible for the observed association between AHR and infiltration of mast cells within the ASM previously reported. The importance of the localization of inflammatory cells to the structural cells is not only limited to the ASM. As discussed, epithelial damage is a key component of airway remodelling. Eosinophils adhere to epithelial cells through CD18 dependent mechanism regulated by the local cytokine environment [26, 27]. This adhesion leads to degranulation and together with T cells promotes epithelial apoptosis which may be a contributing factor to the epithelial damage [28]. Furthermore, in vitro eosinophil-derived major basic protein, eosinophil peroxidase and eosinophilic cationic protein are cytotoxic to the epithelium [29, 30]. It would have been particularly interesting to evaluate whether eosinophils colocalized to the epithelium were associated with locally up-regulated EGF-R expression in the current report, and future studies evaluating the mecha-
nisms that underpin EGF-R induction in the context of local eosinophilia may identify novel asthma targets. So what is the meaning of tissue eosinophilia in asthma? Airway eosinophila is a key feature of asthma. Bronchial biopsies, induced sputum and bronchiolar lavage (BAL) fluid demonstrate elevated eosinophil numbers in steroid-na€ıve asthmatics [31–33]. Sputum eosinophil counts serve as valuable diagnostic marker or steroid responsive airways disease in asthma, and targeting sputum eosinophils with monoclonal antibodies directed at IL-5 has been shown to reduce exacerbations in phase 2 and phase 3 studies [34, 35]. Despite these observations, sputum IL-5 and submucosal eosinophilia have been associated with obesity in severe asthma but not a sputum eosinophilia in a recent study [36]. This would suggest that more detailed studies evaluating the concurrent role of factors that promote tissue retention of eosinophils or luminal trafficking of eosinophilic inflammation are required to fully determine the clinical importance of a tissue eosinophilia. Furthermore, submucosal eosinophils are not completely attenuated following anti-IL-5 treatment [37], which may be related to the loss of IL-5 receptors from eosinophils as they enter the airway or (and more likely) that other inflammatory processes are important in mediating a tissue eosinophilia in asthma. A variety of innate and adaptive immune pathways may act syn-
Epithelial damage IL-33 IL-25
ASM contraction
TSLP Injury
EGF-R
IL-13 IL-4 MBP ECP
TGF-β IL-4 IL-13
Eosinophils TGF-β IL-4
ECM Production EMT?
Mesenchymal cell proliferation
Mast cell Activation and degranulation
Fig. 1. Schematic representation of factors influencing eosinophilic remodelling. Upon injury, the epithelium is damaged causing the release of cytokines (IL-33, IL-25, TSLP) which either directly or indirectly through TH2 cytokine-secreting cells (innate lymphoid cells type 2, TH2 cells, mast cells*) modulate development, recruitment and survival of eosinophils. Subsequently, tissue eosinophils contribute to remodelling through release of cytotoxic cellular contents directly facilitating epithelial damage through epithelial detachment and TGF-b production. The release of TGF-b by the epithelium and eosinophils contribute to extracellular matrix production (ECM) and potentially epithelial–mesenchymal transition (EMT) as well as mesenchymal cell proliferation. Tissue eosinophils also contribute to mast cell activation and airway smooth muscle contraction perpetuating tissue remodelling. © 2013 John Wiley & Sons Ltd, Clinical & Experimental Allergy, 43 : 1302–1306
Tissue eosinophils in airway remodelling
ergistically or independently to promote the eosinophilic tissue phenotype including classical TH2 pathways driven by archetypal effector cytokines IL-5 [38], IL-4 [39]/IL-13 [40] or TH2 amplification pathways such as epithelial cell-derived IL-25, IL-33 that may support the maintenance of adaptive TH2 cells in vivo directly or indirectly through eosinophils [41, 42, 47] (Fig. 1). IL-13 plays a key role in epithelial repair which is mediated by EGFR activation [44], and activation of type 2 innate lymphoid cells by IL-25 results in release of IL-5 [45, 46]. This may in turn perpetuate and amplify eosinophil survival and activation. More recently, blockade of IL-25 in animal models of allergic asthma has been shown to attenuate tissue eosinophilia alongside a reduction in innate epithelial-derived cytokines IL-33 and TSLP, collagen deposition, ASM hyperplasia, neovascularization and AHR suggesting an important role for this molecule in airway remodelling [47].
References 1 Asher MI, Montefort S, Bjorksten B et al. ISAAC Phase Three Study Group. Worldwide time trends in the prevalence of symptoms of asthma, allergic rhinoconjunctivitis, and eczema in childhood: ISAAC Phases One and Three repeat multicountry crosssectional surveys. Lancet 2006; 368: 733–43. 2 Masoli M, Fabian D, Holt S, Beasley R. Global Initiative for Asthma (GINA) Program. The global burden of asthma: executive summary of the GINA Dissemination Committee report. Allergy 2004; 59:469–78. 3 Huber HL, Koessler KK. The pathology of bronchial asthma. Arch Intern Med 1922;30:689–760. 4 Siddiqui S, Mistry V, Doe C et al. Airway hyperresponsiveness is dissociated from airway wall structural remodeling. J Allergy Clin Immunol 2008;122:335, 341.e3. 5 Benayoun L, Druilhe A, Dombret M-, Aubier M, Pretolani M. Airway structural alterations selectively associated with severe asthma. Am J Respir Crit Care Med 2003; 167:1360–8. 6 Wenzel SE, Schwartz LB, Langmack EL et al. Evidence that severe asthma can be divided pathologically into two inflammatory subtypes with distinct physiologic and clinical characteristics. Am J Respir Crit Care Med 1999; 160:1001–8.
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Therefore, establishing the expression of proremodelling cytokines that may bridge innate and adaptive immunity in vivo (IL-25 & IL-33), and their relationship to the pathological remodelling phenotype is required to assess how modulation of these pathways may attenuate eosinophilic remodelling. Future remodelling studies evaluating tissue eosinophila in the context of inhibition of these cytokines are required to fully identify the role of tissue eosinophils in asthma. It would not be unreasonable for remodelling end-points to be included as secondary exploratory outcome measures in these studies. Until then, the field of airway remodelling has progressed only incrementally since the seminal observations of Huber and Koessler [3]. Conflicts of interest: The authors declare no conflict of interest.
7 Wilson SJ, Rigden HM, Ward AJ, Laviolette M, Jarjour NN, Djukanovic R. The relationship between eosinophilia and airway remodelling in mild asthma. Clin Exp Allergy 2013; 43: 1342–50. 8 Holgate ST. The sentinel role of the airway epithelium in asthma pathogenesis. Immunol Rev 2011; 242:205– 19. 9 Holgate ST, Lackie PM, Howarth PH et al. Invited lecture: activation of the epithelial mesenchymal trophic unit in the pathogenesis of asthma. Int Arch Allergy Immunol 2001; 124:253–8. 10 Puddicombe SM, Polosa R, Richter A et al. Involvement of the epidermal growth factor receptor in epithelial repair in asthma. FASEB J 2000; 14:1362–74. 11 Fedorov IA, Wilson SJ, Davies DE, Holgate ST. Epithelial stress and structural remodelling in childhood asthma. Thorax 2005; 60:389–94. 12 Siddiqui S, Hollins F, Saha S, Brightling CE. Inflammatory cell microlocalisation and airway dysfunction: cause and effect? Eur Respir J 2007; 30:1043–56. 13 Blanchard C, Rothenberg ME. Biology of the eosinophil. Adv Immunol 2009; 101:81–121. 14 Brightling CE, Bradding P, Symon FA, Holgate ST, Wardlaw AJ, Pavord ID. Mast-cell infiltration of airway smooth muscle in asthma. N Engl J Med 2002; 346:1699–705.
© 2013 John Wiley & Sons Ltd, Clinical & Experimental Allergy, 43 : 1302–1306
15 Flood-Page P, Menzies-Gow A, Phipps S et al. Anti-IL-5 treatment reduces deposition of ECM proteins in the bronchial subepithelial basement membrane of mild atopic asthmatics. J Clin Invest 2003; 112:1029–36. 16 Milanese M, Crimi E, Scordamaglia A et al. On the functional consequences of bronchial basement membrane thickening. J Appl Physiol 2001; 91:1035–40. 17 Ordonez CL, Khashayar R, Wong HH et al. Mild and moderate asthma is associated with airway goblet cell hyperplasia and abnormalities in mucin gene expression. Am J Respir Crit Care Med 2001; 163:517–23. 18 Kirkham S, Sheehan JK, Knight D, Richardson PS, Thornton DJ. Heterogeneity of airways mucus: variations in the amounts and glycoforms of the major oligomeric mucins MUC5AC and MUC5B. Biochem J 2002; 361:537–46. 19 Voynow JA, Rubin BK. Mucins, mucus, and sputum. Chest 2009; 135:505–12. 20 Woodruff PG, Dolganov GM, Ferrando RE et al. Hyperplasia of smooth muscle in mild to moderate asthma without changes in cell size or gene expression. Am J Respir Crit Care Med 2004; 169:1001–6. 21 Yick CY, Ferreira DS, Annoni R et al. Extracellular matrix in airway smooth muscle is associated with dynamics of airway function in asthma. Allergy 2012; 67:552–9.
1306 A. Shikotra & S. Siddiqui 22 Cox G, Thomson NC, Rubin AS et al. AIR Trial Study Group. Asthma control during the year after bronchial thermoplasty. N Engl J Med 2007; 356:1327–37. 23 Castro M, Rubin AS, Laviolette M et al. AIR2 Trial Study Group. Effectiveness and safety of bronchial thermoplasty in the treatment of severe asthma: a multicenter, randomized, double-blind, sham-controlled clinical trial. Am J Respir Crit Care Med 2010; 181:116– 24. 24 Cox G, Thomson NC, Rubin AS et al. Asthma control during the year after bronchial thermoplasty. N Engl J Med 2007; 356:1327–37. 25 Begueret H, Berger P, Vernejoux JM, Dubuisson L, Marthan R, Tunon-deLara JM. Inflammation of bronchial smooth muscle in allergic asthma. Thorax 2007; 62:8–15. 26 Woodman L, Siddiqui S, Cruse G et al. Mast cells promote airway smooth muscle cell differentiation via autocrine up-regulation of TGF-beta 1. J Immunol 2008; 181:5001–7. 27 Sato M, Takizawa H, Kohyama T et al. Eosinophil adhesion to human bronchial epithelial cells: regulation by cytokines. Int Arch Allergy Immunol 1997; 113:203–5. 28 Godding V, Stark JM, Sedgwick JB, Busse WW. Adhesion of activated eosinophils to respiratory epithelial cells is enhanced by tumor necrosis factoralpha and interleukin-1 beta. Am J Respir Cell Mol Biol 1995; 13:555–62. 29 Trautmann A, Schmid-Grendelmeier P, Kruger K et al. T cells and eosinophils cooperate in the induction of bronchial epithelial cell apoptosis in asthma. J Allergy Clin Immunol 2002; 109:329– 37. 30 Ayars GH, Altman LC, McManus MM et al. Injurious effect of the eosinophil peroxide-hydrogen peroxide-
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halide system and major basic protein on human nasal epithelium in vitro. Am Rev Respir Dis 1989; 140:125– 31. Motojima S, Frigas E, Loegering DA, Gleich GJ. Toxicity of eosinophil cationic proteins for guinea pig tracheal epithelium in vitro. Am Rev Respir Dis 1989; 139:801–5. Frigas E, Loegering DA, Solley GO, Farrow GM, Gleich GJ. Elevated levels of the eosinophil granule major basic protein in the sputum of patients with bronchial asthma. Mayo Clin Proc 1981; 56:345–53. Broide DH, Gleich GJ, Cuomo AJ et al. Evidence of ongoing mast cell and eosinophil degranulation in symptomatic asthma airway. J Allergy Clin Immunol 1991; 88:637–48. Bradding P, Roberts JA, Britten KM et al. Interleukin-4, -5, and -6 and tumor necrosis factor-alpha in normal and asthmatic airways: evidence for the human mast cell as a source of these cytokines. Am J Respir Cell Mol Biol 1994; 10:471–80. Haldar P, Brightling CE, Hargadon B et al. Mepolizumab and exacerbations of refractory eosinophilic asthma. N Engl J Med 2009; 360:973–84. Pavord ID, Korn S, Howarth P et al. Mepolizumab for severe eosinophilic asthma (DREAM): a multicentre, double-blind, placebo-controlled trial. Lancet 2012; 380:651–9. Desai D, Newby C, Symon FA et al. Elevated sputum interleukin-5 and submucosal eosinophilia in obese severe asthmatics. Am J Respir Crit Care Med 2013; 188:657–63. Flood-Page PT, Menzies-Gow AN, Kay AB, Robinson DS. Eosinophil’s role remains uncertain as anti-interleukin-5 only partially depletes numbers in asthmatic airway. Am J Respir Crit Care Med 2003; 167:199–204.
39 Brightling CE, Symon FA, Birring SS, Bradding P, Pavord ID, Wardlaw AJ. TH2 cytokine expression in bronchoalveolar lavage fluid T lymphocytes and bronchial submucosa is a feature of asthma and eosinophilic bronchitis. J Allergy Clin Immunol 2002; 110:899– 905. 40 Siddiqui S, Mistry V, Doe C, Stinson S, Foster M, Brightling C. Airway wall expression of OX40/OX40L and interleukin-4 in asthma. Chest 2010; 137:797–804. 41 Saha SK, Berry MA, Parker D et al. Increased sputum and bronchial biopsy IL-13 expression in severe asthma. J Allergy Clin Immunol 2008; 121:685– 91. 42 Wang YH, Angkasekwinai P, Lu N et al. IL-25 augments type 2 immune responses by enhancing the expansion and functions of TSLP-DC-activated Th2 memory cells. J Exp Med 2007; 204:1837–47. 43 Gregory LG, Jones CP, Walker SA et al. IL-25 drives remodelling in allergic airways disease induced by house dust mite. Thorax 2013; 68:82–90. 44 Lloyd CM. IL-33 family members and asthma - bridging innate and adaptive immune responses. Curr Opin Immunol 2010; 22:800–6. 45 Allahverdian S, Harada N, Singhera GK, Knight DA, Dorscheid DR. Secretion of IL-13 by airway epithelial cells enhances epithelial repair via HB-EGF. Am J Respir Cell Mol Biol 2008; 38:153–60. 46 Neill DR, Wong SH, Bellosi A et al. Nuocytes represent a new innate effector leukocyte that mediates type-2 immunity. Nature 2010; 464:1367–70. 47 Moro K, Yamada T, Tanabe M et al. Innate production of T(H)2 cytokines by adipose tissue-associated c-Kit(+) Sca-1(+) lymphoid cells. Nature 2010; 463:540–4.
© 2013 John Wiley & Sons Ltd, Clinical & Experimental Allergy, 43 : 1302–1306
doi: 10.1111/cea.12204
© 2013 John Wiley & Sons Ltd
EDITORIAL
Clinical The role of tissue eosinophils in asthmatic airway & remodelling Experimental This editorial discusses the findings of the paper in this issue by S. J. Wilson et al. [7] pp. 1342–1350. Allergy A. Shikotra and S. Siddiqui Department of Infection, Immunity and Inflammation, Institute for Lung Health, University of Leicester, Leicester, UK
Asthma is an important cause of morbidity and mortality affecting 300 million people world-wide [1, 2]. Asthma is characterized by the presence of inflammation and structural changes within the airway wall often described as airway remodelling – these features were originally described in fatal asthmatics in the seminal report of Huber and Koessler published in 1922 [3]. Despite nearly a century of on-going asthma research, the pathogenesis of airway remodelling remains poorly understood. The structural and cellular changes which have been described repeatedly in studies of asthmatic airway remodelling include an increase in the numbers of tissue eosinophils (and in some cases neutrophils), thickening of the reticular basement membrane, increased airway smooth muscle mass, goblet cell hyperplasia and angiogenesis [4–6]. The limitation of these aforementioned studies is that the following: (i) many of them fail to consider the importance of colocalization of inflammatory/immune cells to key airway structural cells, (ii) most studies are cross-sectional, and some features of remodelling notably basement membrane thickening have been described in early life questioning the progressive nature of the remodelling process and (iii) few studies have evaluated the role of asthma therapy upon features of both structural and cellular remodelling. As a consequence, it is difficult to determine Correspondence: Dr Salman Siddiqui, Department of Infection, Immunity and Inflammation, Institute for Lung Health, University of Leicester Respiratory Research BRU, Glenfield Hospital, Groby Road, Leicester, LE3 9QP, UK. E-mail: [email protected] Cite this as: A. Shikotra and S. Siddiqui and Clinical & Experimental Allergy, 2013 (43) 1302–1306. This logo highlights the Editorial article on the cover and the first page of the article.
whether airway inflammatory cells drive structural remodelling in asthma. In this issue of Clinical & Experimental Allergy, Wilson et al. examine the relationship between airway remodelling and submucosal eosinophilia in a cohort of patients with mild steroid-na€ıve asthma and matched healthy subjects [7]. Using standard immunohistochemistry techniques, eosinophils were identified in the bronchial submucosa, and based upon the submucosal expression, the asthmatic subjects were subdivided into two groups: eosinophilshigh and eosinophilslow. The group subsequently assessed airway structural remodelling by assessing three key features: (i) epithelial damage, (ii) goblet cell hyperplasia and (iii) airway smooth muscle hypertrophy and cellular infiltration. The report has described a number of novel and interesting findings. Notably that patients with the tissue eosinophilshigh phenotype had evidence of increased epithelial damage in vivo (based upon epithelial EGF-R expression), increased Mucin-2 expression and increased numbers of both eosinophils and T cells within the airway smooth muscle (ASM) bundle itself when compared to eosinophilslow asthmatics and healthy controls. Interestingly, in this report, mast cell numbers in the ASM were only increased significantly in the eosinophilshigh asthma phenotype, and other features of structural remodelling such as increased ASM mass were similar in both tissue eosinophil phenotypes. Taken together, these observations suggest that tissue eosinophils may selectively modulate a number of aspects of structural airway remodelling in asthma and may be causally involved in perpetuating the aberrant cycle of dysregulated epithelial repair seen in asthma. The airway epithelium has emerged as a central orchestrator of asthmatic airway inflammation and remodelling [8]. The cycle of frequent damage and inadequate repair of the epithelium is thought to facilitate airway remodelling via epithelial–mesenchymal–
Tissue eosinophils in airway remodelling
inflammatory cell crosstalk [8]. Wilson et al. have quantified epithelial damage using epidermal growth factor receptor (EGF-R) protein expression. Puddicombe et al. previously demonstrated that the percentage of EGF-R-positive damaged epithelium increases as disease severity progresses despite the use of corticosteroids in asthma and that EGF-R immunoreactivity is evident throughout the epithelium in bronchial tissue. In the same study in vitro, EGF-R was not identified immediately after wounding but was identified subsequently at 3 and 9 hours postwounding [9]. These observations have also been reported in childhood asthma [10] and are associated with collagen deposition in the lamina reticularis. The use of EGF-R protein expression to quantify epithelial damage is novel as damaged epithelium is a common observation in bronchial biopsies and it is difficult to assess how much is artefact. Wilson et al. have differentiated between the two, using immunostaining. EGF-R-positive epithelium was defined as in vivo damaged epithelium, and EGR-R-negative epithelium were classed as artefact related possibly to sampling-induced damage. A number of previous reports have described increased numbers of eosinophils within the submucosa and airway epithelium itself in asthma [Reviewed in 11]. The authors extend these observations showing that in vivo epithelial damage was elevated only in the eosinophilshigh subgroup suggesting a role for eosinophils in epithelial damage. The notion that tissue eosinophils drive epithelial damage in asthma is supported by extensive literature linking eosinophilic granules and reactive oxygen species to epithelial damage in ex vivo models [12]. In addition, the damaged asthmatic epithelium is known to release a number of cytokines/chemokines that promote TH2 polarization including IL-13, IL-5, TARC, IL-33, RANTES, eotaxins and MCP-3. Therefore, it is plausible that a vicious cycle of epithelial damage and inflammation may promote tissue eosinophilia and further damage in asthma. Despite these observations, the clinical importance of tissue eosinophilia remains to be established. Tissue eosinophilia and associated basement membrane thickening have been reported in other diseases where airway hyper responsiveness is absent – such as nonasthmatic eosinophilic bronchitis [13]. In addition, targeting tissue eosinophilia with a selective monoclonal antibody to IL-5 over a 10 weeks period had little impact upon airway physiology despite a marked reduction in immunoreactive tenascin, collagen III and lumican present in the lamina reticularis [14]. These features were not evaluated in the current report. Therefore, it remains to be determined whether interactions between tissue eosinophils and epithelial cells drive clinically important outcomes in asthma with some reports suggesting a protective role for basement membrane remodelling in asthma [15].
1303
An alternative mechanism linking tissue eosinophilia to asthma morbidity is mucous hyper secretion that may arise due to hyperplasia of goblet cells and glands. Previous reports have identified increased goblets cells in mild asthma [16]. Furthermore, MUC5AC and 5B alongside eosinophilic granule proteins are known to increase the viscosity and tenacity of airway mucous [17, 18]. Interestingly, in the current report, only MUC2 protein expression was increased in eosinophilshigh asthma in contrast to the MUC5AC and MUC5B. These observations are difficult to fully interpret as (i) MUC2 comprises at most only 2.5% of the weight of gel forming mucins, (ii) glandular expression of mucins was not evaluated and is likely to be an important source and (iii) post-translational modifications of the major mucins MUC5AC and MUC5B that alter their glycosylation and charge status may significant influence the viscosity of mucous. Further studies are required to determine the role of eosinophilic tissue inflammation in modulating altered mucin protein expression in airway glands and mucous rheology in asthma. One of the key features of airway remodelling is an increase in ASM mass. The mechanisms underlying ASM remodelling are likely to involve ASM hyperplasia, hypertrophy and deposition of extracellular matrix (ECM) proteins [19, 20]. Interestingly, in the current report by Wilson et al., increased ASM mass was found to be independent of tissue eosinophilia suggesting that other processes may drive this phenomenon. It is plausible that the phenotype of increased ASM mass in asthma may therefore not respond to current antieosinophilic therapies. Alternative approaches to targeting this phenotype include bronchial thermoplasty which has been shown to improve patient reported outcome measures in asthma [21–23]. Future studies are required to evaluate whether ASM mass can be utilized to further stratify the response to thermoplasty. There is increasing recognition that inflammatory cells not only infiltrate the submucosa but also microlocalize to the structural components of the airway [11]. Research in this area has primarily focused on the airway smooth muscle bundle, and the infiltration of mast cells within the ASM bundle has been well documented [4, 13]. Similarly, Wilson et al. in this issue describe mast cell infiltration in the context of mild asthma and the eosinophilshigh phenotype. In addition, this is one of the very few studies that have observed T-cell infiltration into the smooth muscle [24]. The finding that eosinophils colocalize to the ASM is novel. Our group has not consistently observed these cells within the ASM bundle itself, and it is possible that some of these cells were within the perimeter of the ASM bundle or juxtaposed between ASM cells. In either case, the findings are important as we have previously shown that mast cells within the perimeter of the ASM bundle [25]
© 2013 John Wiley & Sons Ltd, Clinical & Experimental Allergy, 43 : 1302–1306
1304 A. Shikotra & S. Siddiqui can increase the expression of contractile proteins in vivo, and it is possible that eosinophilic proteins may facilitate similar functional changes within ASM cells. Future studies should evaluate the functional effects of eosinophils upon asthmatic ASM cells in coculture models and the ability of ASM chemokines to facilitate migration and adhesion of eosinophils. In addition, the findings suggest that a complex interplay between T cells, eosinophils and mast cells may be responsible for the observed association between AHR and infiltration of mast cells within the ASM previously reported. The importance of the localization of inflammatory cells to the structural cells is not only limited to the ASM. As discussed, epithelial damage is a key component of airway remodelling. Eosinophils adhere to epithelial cells through CD18 dependent mechanism regulated by the local cytokine environment [26, 27]. This adhesion leads to degranulation and together with T cells promotes epithelial apoptosis which may be a contributing factor to the epithelial damage [28]. Furthermore, in vitro eosinophil-derived major basic protein, eosinophil peroxidase and eosinophilic cationic protein are cytotoxic to the epithelium [29, 30]. It would have been particularly interesting to evaluate whether eosinophils colocalized to the epithelium were associated with locally up-regulated EGF-R expression in the current report, and future studies evaluating the mecha-
nisms that underpin EGF-R induction in the context of local eosinophilia may identify novel asthma targets. So what is the meaning of tissue eosinophilia in asthma? Airway eosinophila is a key feature of asthma. Bronchial biopsies, induced sputum and bronchiolar lavage (BAL) fluid demonstrate elevated eosinophil numbers in steroid-na€ıve asthmatics [31–33]. Sputum eosinophil counts serve as valuable diagnostic marker or steroid responsive airways disease in asthma, and targeting sputum eosinophils with monoclonal antibodies directed at IL-5 has been shown to reduce exacerbations in phase 2 and phase 3 studies [34, 35]. Despite these observations, sputum IL-5 and submucosal eosinophilia have been associated with obesity in severe asthma but not a sputum eosinophilia in a recent study [36]. This would suggest that more detailed studies evaluating the concurrent role of factors that promote tissue retention of eosinophils or luminal trafficking of eosinophilic inflammation are required to fully determine the clinical importance of a tissue eosinophilia. Furthermore, submucosal eosinophils are not completely attenuated following anti-IL-5 treatment [37], which may be related to the loss of IL-5 receptors from eosinophils as they enter the airway or (and more likely) that other inflammatory processes are important in mediating a tissue eosinophilia in asthma. A variety of innate and adaptive immune pathways may act syn-
Epithelial damage IL-33 IL-25
ASM contraction
TSLP Injury
EGF-R
IL-13 IL-4 MBP ECP
TGF-β IL-4 IL-13
Eosinophils TGF-β IL-4
ECM Production EMT?
Mesenchymal cell proliferation
Mast cell Activation and degranulation
Fig. 1. Schematic representation of factors influencing eosinophilic remodelling. Upon injury, the epithelium is damaged causing the release of cytokines (IL-33, IL-25, TSLP) which either directly or indirectly through TH2 cytokine-secreting cells (innate lymphoid cells type 2, TH2 cells, mast cells*) modulate development, recruitment and survival of eosinophils. Subsequently, tissue eosinophils contribute to remodelling through release of cytotoxic cellular contents directly facilitating epithelial damage through epithelial detachment and TGF-b production. The release of TGF-b by the epithelium and eosinophils contribute to extracellular matrix production (ECM) and potentially epithelial–mesenchymal transition (EMT) as well as mesenchymal cell proliferation. Tissue eosinophils also contribute to mast cell activation and airway smooth muscle contraction perpetuating tissue remodelling. © 2013 John Wiley & Sons Ltd, Clinical & Experimental Allergy, 43 : 1302–1306
Tissue eosinophils in airway remodelling
ergistically or independently to promote the eosinophilic tissue phenotype including classical TH2 pathways driven by archetypal effector cytokines IL-5 [38], IL-4 [39]/IL-13 [40] or TH2 amplification pathways such as epithelial cell-derived IL-25, IL-33 that may support the maintenance of adaptive TH2 cells in vivo directly or indirectly through eosinophils [41, 42, 47] (Fig. 1). IL-13 plays a key role in epithelial repair which is mediated by EGFR activation [44], and activation of type 2 innate lymphoid cells by IL-25 results in release of IL-5 [45, 46]. This may in turn perpetuate and amplify eosinophil survival and activation. More recently, blockade of IL-25 in animal models of allergic asthma has been shown to attenuate tissue eosinophilia alongside a reduction in innate epithelial-derived cytokines IL-33 and TSLP, collagen deposition, ASM hyperplasia, neovascularization and AHR suggesting an important role for this molecule in airway remodelling [47].
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Therefore, establishing the expression of proremodelling cytokines that may bridge innate and adaptive immunity in vivo (IL-25 & IL-33), and their relationship to the pathological remodelling phenotype is required to assess how modulation of these pathways may attenuate eosinophilic remodelling. Future remodelling studies evaluating tissue eosinophila in the context of inhibition of these cytokines are required to fully identify the role of tissue eosinophils in asthma. It would not be unreasonable for remodelling end-points to be included as secondary exploratory outcome measures in these studies. Until then, the field of airway remodelling has progressed only incrementally since the seminal observations of Huber and Koessler [3]. Conflicts of interest: The authors declare no conflict of interest.
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