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Role of interleukin 33 in respiratory allergy and asthma Heidi Makrinioti, Marie Toussaint, David J Jackson, Ross P Walton, Sebastian L Johnston Lancet Respir Med 2014; 2: 226–37 Published Online January 23, 2014 http://dx.doi.org/10.1016/ S2213-2600(13)70261-3 Airway Disease Infection Section, National Heart and Lung Institute, Imperial College London, London, UK (H Makrinioti MD, M Toussaint PhD, D J Jackson MRCP, R P Walton PhD, Prof S L Johnston FRCP); Medical Research Council and Asthma UK Centre in Allergic Mechanisms of Asthma, London, UK; (H Makrinioti, M Toussaint, D J Jackson, R P Walton, S L Johnston); Centre for Respiratory Infection, Imperial College, London, UK (H Makrinioti, M Toussaint, D J Jackson, R P Walton, S L Johnston); and Imperial College Healthcare NHS Trust, London, UK (D J Jackson, S L Johnston) Correspondence to: Dr Heidi Makrinioti, Airway Disease Infection Section, National Heart and Lung Institute, Imperial College London, Norfolk Place, London W2 1PG, UK [email protected]
Since the discovery of interleukin 33 as the adopted ligand for the then orphan ST2 receptor, many studies have implicated this cytokine in the pathogenesis of respiratory allergy and asthma. Although some extracellular functions of interleukin 33 have been well defined, many aspects of the regulation and secretion of this cytokine need clarification. Interleukin 33 has been identified as a trigger of T-helper-type-2 cell differentiation, which by interacting with both the innate and the adaptive immune systems, can drive allergy and asthma pathogenesis. However, induction of interleukin 33 by both environmental and endogenous triggers implies a possible role during infection and tissue damage. Further understanding of the biology of interleukin 33 will clarify its possible role in future therapeutic interventions.
Introduction Interleukin 33, a member of the interleukin 1 cytokine family, was originally characterised as a protein expressed in lymph node-associated endothelial cells.1 The interleukin 33 receptor, termed IL1RL1 (ST2), was first identified as an interleukin 1 receptor-like molecule by Tominaga in 1989.2 The receptor has two main isoforms— one transmembrane form, ST2 (ST2L), and one soluble form, ST2 (sST2)—the expression of which is controlled by two distinct promoters. sST2 binds interleukin 33 and blocks its functional bioactivities that are initiated by its ligation to ST2L.3 Consistent with other interleukin 1-related cytokine receptor family members, the interleukin 33 receptor is a heterodimer, composed of ST2 and interleukin 1-receptor accessory protein (IL1RAP). Signalling via this receptor complex leads to activation of downstream signalling molecules, such as nuclear factor κB (NF-κB) and activating protein 1.4 Unlike other interleukin 1 family members, interleukin 33 has been associated with the promotion and amplification of both systemic and localised T helper (Th) 2 cell responses.5,6 The full length form, prointerleukin 33, is localised in the nucleus. The exact function of this interleukin in the nucleus is unclear; however, recent studies show that it binds to dimerised histone H2A and H2B at the surface of the nucleosome
Key messages • Interleukin 33 is an alarmin that is released in the lung mainly by epithelial cells and possibly also by macrophages after cell necrosis or cell activation • Interleukin 33 binds its receptor, ILRL1, and triggers allergic airway inflammation by interacting with both the innate and adaptive arms of the immune system • The pathogenic mechanisms that underlie the release of interleukin 33 by epithelial cells and the intracellular signals triggered by its interaction with its receptor need to be clarified further • Interleukin 33 has been associated with the incidence and pathogenesis of asthma and with induction of permanent structural changes in the airways • Data for the role of interleukin 33 during exacerbations of asthma (that are mainly virus-induced) are scarce; however, non-peer-reviewed evidence suggests that the role of interleukin 33 is probably important, and further research is urgently needed • Although no studies in man have targeted interleukin 33, the results from animal studies pave the way for trials in human beings, which are eagerly anticipated
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and might exhibit transcriptional repressor activity. Nuclear localisation of interleukin 33 is not cell specific and is probably fundamental to both the activity and function of the protein. Although interleukin 33 shares various characteristics with other interleukin 1 cytokine family members, the mechanisms regulating its extracellular migration from the nucleus seem to differ, and have not been completely clarified.4,7–9 The dominant hypothesis is that interleukin 33 is released after necrosis or cell damage, and acts as an alarmin to elicit a protective immune response. During necrosis, interleukin 33 remains in its active form; however, in apoptotic conditions, caspase 3 and caspase 7 cleave interleukin 33 rendering it inactive, suggesting a differential role on programmed cell death.7 However, the neutrophil derived serine proteases cathepsin G and elastase have also been reported to cleave interleukin 33 and amplify its bioactivity, thus suggesting that cleavage of interleukin 33 does not exclusively lead to its inactivation.10 Further work is clearly needed to better understand the effects of cleavage on interleukin 33 activity. The main sources of interleukin 33 protein are structural cells such as fibroblasts, epithelial cells, and endothelial cells. The ST2 receptor is expressed on various immune cells—namely T cells, B cells, eosinophils, mast cells, innate lymphoid cells, and dendritic cells.11 Interleukin 33 is thought to play a part in a range of diseases, such as infectious and autoimmune diseases, cardiovascular diseases, and neurological disorders.11 Additionally, increased evidence shows the ability of interleukin 33 to act as both a chemoattractant and an immunomodulator, and thus it is characterised as an alarmin because of its ability to activate cells of both the innate and the adaptive immune system.12–14 To date, research has shown that necrotic death and mechanical injury of epithelial cells can undoubtedly lead to interleukin 33 release.15 Furthermore, accumulating evidence from studies in mouse innate lymphoid cells infected with influenza suggests that interleukin 33 might be released as a result of viral infection.16 A pronounced role has been suggested for interleukin 33 in asthma pathogenesis. Asthma is a heterogeneous www.thelancet.com/respiratory Vol 2 March 2014
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inflammatory airway disease with a complex cause, stimulated by various triggers. Allergy can be placed centrally in the pathogenesis of asthma in most patients and the importance of the bronchial epithelium in the disease process is becoming clearer. The interleukin 33/ ST2 pathway has been suspected to mediate the pathogenesis of both asthma and allergy through diverse pathways.17,18 Epithelial cells are a primary source of interleukin 33, which suggests a mechanism whereby the respiratory tract has the potential, in response to a so-called danger signal, to drive immune responses other than the expected Th1 response. This signal could be either an infection (viral or bacterial), any other environmental signal (allergen or pollutant), or a combination of both. Secretion of interleukin 33 could perpetuate an unresolved airway inflammation, especially in patients with asthma.19 Therefore, the role of interleukin 33 in respiratory infection, allergy, and asthma pathogenesis is of particular interest, and a plethora of studies implicate interleukin 33 as an attractive therapeutic target. The increasing volume of published work in interleukin 33 leads to an increasing number of questions requiring answers. This Review aims to answer these questions and to discuss whether a clinical application of anti-interleukin 33 therapy might be useful for the future treatment of respiratory allergy or asthma.
Release of interleukin 33 by respiratory epithelium during allergic respiratory response Airway epithelial cells form the interface between the host and the environment, and are the first line of defence against inhaled microorganisms and allergens.20 In this exposed position, epithelial cells express a broad array of pattern recognition receptors (PRRs) that detect environmental stimuli, responding to pathogenassociated molecular patterns (PAMPs) present on microbes or to danger-associated molecular patterns (DAMPs) released after tissue damage, cell death, or cellular stress. Activation of these PRRs on epithelial cells leads to the release of soluble mediators, including cytokines and chemokines, which can alert and activate the immune system to any potential threat.20 Among the cytokines released by the airway epithelium, interleukin 33 is highly secreted in response to allergen, with human epithelial cells of the bronchus and small airways showing constitutive expression of interleukin 33 mRNA.21,22 This expression is increased in bronchial epithelial cells of patients with asthma compared with healthy individuals.23 In resting conditions, epithelial cells store interleukin 33 in the nucleus.14 However, during injury or necrosis caused by exogenous triggers (such as virus, smoke, airborne allergens), or endogenous triggers (such as ATP, DNA, HMGB1), interleukin 33 is released by damaged cells. Additionally, these triggers might also directly activate PRRs, including Toll-like receptors (TLRs) on epithelium cells, resulting in the release of biologically www.thelancet.com/respiratory Vol 2 March 2014
active full-length interleukin 33.24 Recent studies in mouse models of allergic asthma induced by house dust mites—one of the most common allergens in human beings—have shown the importance of TLR4 signalling in the induction of interleukin 33 release by airway epithelial cells. Willart and colleagues21 showed that administration of low dose house dust mites during allergic sensitisation induced interleukin 1α release via TLR4 ligation and signalling on bronchial epithelial cells. This release occurred through PRR detection of the major allergen Derp2 and the endotoxin contained in the faecal pellets of the mite. Subsequently released interleukin 1α acts in an autocrine manner on bronchial epithelial cells, stabilising interleukin 1R1 surface expression, inducing the release of interleukin 33 and perpetuating a Th2 immune response against the allergen. In accordance with this, Collison and colleagues25 confirmed the importance of TLR4 activation by endogenous triggers for interleukin 33 release by the epithelium. After house dust mite exposure in mice, E3 ubiquitin ligase midline 1, which is known to induce interleukin 33 expression, was upregulated in bronchial epithelial cells in a TLR4dependent manner.25 An alternative pathway for release of interleukin 33 by airway epithelial cells that is independent of conventional TLR activation has been suggested. Exposure of airway epithelial cells to the fungus Alternaria alternata in vitro induces the rapid extracellular release of biologically active interleukin 33. The proposed mechanism is via the allergen inducing an acute extracellular accumulation of ATP, which signals in an autocrine way via P2 purinergic receptors, and leads to subsequent interleukin 33 extracellular secretion.26 Because most studies show that the lung epithelium is a major source of interleukin 33, topical administration of anti-interleukin 33/ST2 therapy could be considered as a future treatment strategy, thus reducing the risk of systemic side-effects. The appendix p 1 summarises studies investigating interleukin 33 production by airway epithelial cells.
See Online for appendix
Macrophages as both target and source of interleukin 33 Macrophages are specialised phagocytic cells found in various tissues and organs, which play a central part in homoeostasis, tissue remodelling, and host defence. Depending on the microenvironment, macrophages can be polarised into various different phenotypes and are classified as classically activated macrophages or alternatively activated macrophages. Alternatively activated macrophages promote and regulate type 2 immune responses and can be characterised by expression of mannose receptor and arginase 1 upregulation.27 Mouse macrophages have been shown to constitutively express ST2 mRNA and proteins.28 Yang and colleagues29 showed that mouse bone marrow-derived macrophages stimulated in vitro with interleukin 33 227
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substantially increased the expression of interleukin 13 in a time-dependent and concentration-dependent manner. Production of interleukin 13 from macrophages activates macrophages through an alternative autocrine pathway.29 Kurowska-Stolarska and colleagues30 reported that alternatively activated macrophages play a crucial part in type 2 immunity; mice deficient in ST2 had an impaired type 2 immune response, which is associated with a decrease in differentiation of alternatively activated macrophages. These investigators confirmed that interleukin 33 changes the quiescent phenotype of alveolar macrophages towards an alternatively activated macrophages phenotype in an interleukin 13-dependent manner during interleukin 33-induced eosinophilia in the lung. Moreover, depletion of alveolar macrophages reduced interleukin 33-induced airway inflammation.30 Additionally, the cell lineage and maturation markers, CD11b and CD86, and the ST2 receptor are upregulated by interleukin 33 in bone marrow-derived macrophages.29 Tjota and colleagues31 reported that allergen-specific IgG can enhance secondary Th-2 mediated immune responses in the lungs via an interleukin 33-dependent mechanism. Finally, treatment of primary mouse macrophages and alveolar macrophages with TLR3 (polyinosinic:polycytidylic acid) and TLR4 (lipopolysaccharide) agonists greatly increased expression of both interleukin 33 mRNA and protein.32 These findings suggest that viral infection could amplify type 2 immune responses through the production of interleukin 33 by alveolar macrophages during asthma exacerbations. Thus, macrophages are capable of both producing and responding to interleukin 33, and inhibition of interleukin 33/ST2 downregulates macrophage mediated type 2 inflammation. However, further work is needed to investigate the role of interleukin 33/ST2 in macrophage activation during stable, moderate, and severe asthma and in virus-induced asthma exacerbations, which might answer the question of whether the use of anti-interleukin 33/ST2 treatment could be a possible symptomatic therapy. The appendix pp 2 outlines studies investigating the relation between interleukin 33 and airway inflammation via macrophage interactions.
Interactions between interleukin 33/ST2 and immune cells in asthma Mast cells Mast cells are present in all tissues and are especially numerous at sites exposed to external environments, such as the epithelium. On activation, mast cells rapidly produce and secrete a range of soluble mediators, providing a rapid response to tissue damage or cell injury, initiating and augmenting the ensuing inflammatory response. Mast cells express the high affinity IgE receptor (FCεRI), and induce IgE-mediated immune responses. Several studies have shown that mast cells play an important part in asthma development, but also 228
that they are highly responsive to interleukin 33 that is released from necrotic structural cells.33 Both mouse and human mast cells constitutively express functional interleukin 33 receptors (ST2).34 In human mast cells, the transcription factor GATA2 is a positive regulator of ST2 expression.35 Interleukin 33 directly stimulates primary human mast cells to produce several proinflammatory cytokines (interleukin 1β, interleukin 6, interleukin 8, interleukin 13, and tumour necrosis factor α), chemokines (CCL1 and CXCL8), and growth factors (granulocyte-macrophage colony-stimulating factor) through activation of the NF-κB pathway.36 Although interleukin 33 does not trigger human mast cell degranulation, interleukin 33 might augment mast cell degranulation in sensitised individuals with high concentrations of IgE.37,38 Interleukin 33 prolongs survival of cultured mast cells derived from human cord blood by promoting cell adhesion.39 Interleukin 33 can also act in concert with thymic stromal lymphopoietin, a potent mast cell activator, to accelerate in-vivo maturation of human CD34+ mast cell precusors and induce secretion of Th2 cytokines and Th2-attracting chemokines.36 Together, these results suggest that interleukin 33 could play an important part in mast cell-mediated allergic lung inflammation. Junttila and colleagues40 have shown that mouse mast cells require two separate cytokine signals— interleukin 33 and interleukin 3—for optimum production of interleukin 13. The requirement of two separate signals could represent a protective mechanism to control non-adapted interleukin 13 release by mast cells. Finally, Hsu and colleagues41,42 have shown that mouse mast cells express low concentrations of the interleukin 33 gene and protein constitutively; however, expression of interleukin 33 was induced by IgEmediated activation. The same investigators also showed that expression of interleukin 33 by mast cells was dependent on calcium activation via IgE/FcεRI, but not ST2 activation.41,42 This endogenous production of interleukin 33 could be an important mechanism through which mast cells affect the recruitment and activation of inflammatory cells into sites of IgEdependent activation. Interleukin 33 clearly has the capacity to directly stimulate mast cells to produce various cytokines and chemokines, but degranulation of mast cells requires the presence of IgE as well. These findings suggest that interleukin 33 mediates asthma exacerbation via mast cells mainly in predisposed atopic individuals. The appendix pp 3 summarises studies relating interleukin 33 to airway inflammation through mast cell interactions.
Basophils In allergic inflammation, basophils are recruited from the blood to peripheral organs where they play a pivotal part in allergic inflammation by rapidly releasing chemical mediators, such as interleukins 4, 5, 8, and 13, in addition to sustained production of Th2 cytokines, including www.thelancet.com/respiratory Vol 2 March 2014
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interleukin 4. Unlike mast cells, human and mouse basophils express few ST2 on the cell surface.43 This could be explained by the high level of GATA1 expression in basophils, a negative regulator for ST2 transcription.35 In human basophils, surface expression of ST2 can be enhanced by stimulation with interleukin 3, a growth and differentiation factor for human basophils.18,43 Moreover, interleukin 33 alone is able to increase the production of interleukin 4 from human basophils,18,43 along with interleukin 5, interleukin 8, and interleukin 13.18 In addition to augmenting IgE crosslinking, interleukin 33 enhances human basophil degranulation and eotaxin mediated migration without affecting cell surface CCR3 expression.43 Interleukin 33 can further synergise with interleukin 3 to promote increased interleukin 4 production and CD11b expression by human basophils.43 However, interleukin 33 does not directly induce degranulation or function as a basophil chemoattractant, but instead exerts a priming effect on mouse basophils.43 Finally, in mice, interleukin 33 alone can directly activate mouse basophils, but by contrast with human basophils, it induces their preferential secretion of interleukin 6 (as opposed to interleukin 4) to indirectly promote basophil expansion in vivo through initiating an autocrine feedback loop via GM-CSF and interleukin 3 by the basophils.44 Thus interleukin 33 is an important regulator of basophil function and could be considered as a key cytokine in the pathogenesis of Th2-dominant inflammation. The appendix p 4 shows the outcomes of studies relating interleukin 33 to airway inflammation through basophil interactions.
Eosinophils Eosinophils are proinflammatory granulocytes that play a central part in parasite immunity, but they are also implicated in the pathogenesis of bronchial asthma.45 Eosinophils primarily reside in tissues at mucosal surfaces but are also found circulating in the blood. As with mast cells and basophils, both ST2 mRNA and protein have been shown to be constitutively expressed by human and mouse eosinophils.46,47 Notably, interleukin 33 has been shown, independently of interleukin 3, interleukin 5, and GM-CSF, to potently activate human eosinophils, inducing production and release of the inflammatory mediators, such as CCL2/MCP-1 and CXCL8/interleukin 8, as a result of ST2 activation.47 In addition to these activities, interleukin 33 prolongs human eosinophil survival and adhesion, degranulation and production of superoxide, augments expression of CD11b, and modulates responsiveness to Siglec-8mediated apoptosis.46–48 Moreover, Dyer and colleagues49 have shown that systemic administration of interleukin 33 in mice induces profound interleukin 5-dependent eosinophilia; however, in this study, eosinophil haemopoiesis was interleukin 33 independent. Eosinophils are the main cell type that is increased in numbers in the inflamed airways of patients with www.thelancet.com/respiratory Vol 2 March 2014
allergic asthma. Although eosinophilic-mediated asthma has been regarded as the predominant asthma phenotype, research shows that there are patients with asthmas with non-eosinophilic-mediated inflammation, and that interleukin 4, interleukin 5, and interleukin 13 are not consistently elevated in the airways of all patients with asthma.50 Evidence showing that interleukin 33 has the potential to directly activate eosinophils suggests this molecule could be a therapeutic target for eosinophil-mediated asthma, but further research is awaited to show whether interleukin 33 could also be regarded as important in asthma exacerbations. The appendix pp 5 summarises studies relating interleukin 33 to airway inflammation through eosinophil interactions.
Innate lymphoid cells The recent discovery and subsequent phenotypic characterisation of a novel population of innate lymphoid cells has led to intense investigation into their mechanisms of activation and effector function. A population of innate lymphoid cells able to produce large amounts of the Th2 cytokines interleukin 5 and interleukin 13 has been described; these cells were initially termed lung natural helper cells or nuocytes, but the more standardised nomenclature is now type 2 innate lymphoid cells.51–53 Initially identified by their importance in driving helminth expulsion, these type 2 innate lymphoid cells respond to interleukin 33 by producing large quantities of interleukin 5 and interleukin 13.52 GATA3, a Th2 regulator, is a transcription factor involved in controlling type 2 innate lymphoid cell differentiation, maintenance, and function.54 In-vivo mouse studies of airway administration of glycolipid antigen have shown that interleukin 33 release by alveolar macrophages and dendritic cells leads to activation of type 2 innate lymphoid cells and subsequent interleukin 13 and interleukin 5 production.55 This process of interleukin 33-mediated interleukin 13 secretion by type 2 innate lymphoid cells is sufficient for the induction of airway hyperreactivity in the absence of an adaptive immune response.55 Using a purified system of mouse type 2 innate lymphoid cells, Halim and colleagues51 identified a crucial requirement for costimulation with other cytokines for activation of these cells and effector Th2 cytokine production. Combined administration of interleukin 33 with interleukin 2, interleukin 7, or thymic stromal lymphopoietin resulted in greatly enhanced interleukin 5 and interleukin 13 production by type 2 innate lymphoid cells.51 Moreover, in-vitro stimulation of lung explants from Rag1–/– mice (deficient for T cells and B cells) with papain (a protease allergen homologous to house dust mite-derived Der P1 that can induce airway inflammation in vivo) leads to an increase of interleukin 5 and interleukin 13 production by type 2 innate lymphoid cells. Additionally, Th2 229
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cytokine production by type 2 innate lymphoid cells stimulated with papain is strongly reduced when interleukin 33 signalling is neutralised with an antiinterleukin-33 antibody, whereas depletion of type 2 innate lymphoid cells in mice attenuates airway eosinophilia after papain inhalation.51 In two models of aero-allergen induced allergic asthma (house dust mites and ovalbumin) type 2 innate lymphoid cells are important producers of Th2 cytokines (interleukin 13 and interleukin 5) in both the airways and the lung tissue identified as crucial for airway hyperreactivity development during influenza A infection in mice.56 Here, influenza infection results in increased production of interleukin 33 by alveolar macrophages, which in turn activates type 2 innate lymphoid cells to produce interleukin 13.16 To facilitate worm expulsion, type 2 innate lymphoid cells are key secretors of interleukin 5 and interleukin 13, but not interleukin 4, in response to interleukin 33 and interleukin 25 released during helminth infection in the gut.52 During an ovalbumin model of allergic lung inflammation, numbers of type 2 innate lymphoid cells increased greatly in the airways and lung after ovalbumin challenge.57 After in-vivo stimulation with interleukin 33, type 2 innate lymphoid cells exhibit increased interleukin 5 and interleukin 13 cytokine production, and contribute to airway hyperreactivity development.57 Finally, Bartemes and colleagues58 have shown that type 2 innate lymphoid cells isolated from mouse lung produce large amounts of interleukin 5 and interleukin 13 when cultured with interleukin 33 in vitro. However, Rag–/– mice exposed to the fungus A alternata showed markedly increased interleukin 33 protein in the airways within 1 h of exposure. As a result of interleukin 33 release, type 2 innate lymphoid cells mediated downstream type-2 cytokine driven allergic airway inflammation in the lungs of these mice.58 In human beings, a role has been suggested for nasal epithelial cell-derived interleukin 33 and an interleukin 33-responsive type 2 innate lymphoid cell population in the pathology of chronic rhinosinusitis.59,60 Although these data imply a possible role for interleukin 33 and type 2 innate lymphoid cells interaction in asthma, little is still known in human disease. The identification and phenotypic and functional analysis of type 2 innate lymphoid cells in mice provides an exciting novel mechanism for innate development of type 2 immunity in the lung and induction of allergic airway disease; however, further evidence is needed to more precisely define their role, and little is known regarding the role played by these cells in human disease. There are no data on the possible role of an interleukin 33/type 2 innate lymphoid cells axis in virusinduced asthma exacerbations. The appendix p 6 shows the outcomes of studies relating interleukin 33 to airway inflammation through type 2 innate lymphoid cell interactions. 230
Dendritic cells Dendritic cells reside in the submucosa in close contact with the airway epithelium. Here, dendritic cells extend processes into the airway lumen sampling the inhaled milieu, whereby they capture antigen and present it to naive lymphocytes in draining lymph nodes. In this capacity, they play an essential part in initiating adaptive immune responses. Recent published work has presented dendritic cells as master regulators of the interleukin 33 pathway. ST2 is expressed on dendritic cells and T cells at both the gene and protein level.61,62 Lambrecht and colleagues63 showed that dendritic cell-induced Th2 development in the airways could be inhibited by blocking the interaction of ST2 with its, at that time unknown, ligand. Receptor blockade with antibodies at the time of intratracheal adoptive transfer of dendritic cells (during sensitisation) resulted in complete suppression of sensitisation to inhaled allergen, showing that ST2 presence and functionality on dendritic cells is crucial for the activation of immunogenic responses. Further, Besnard and colleagues64 showed that interleukin 33 is able to drive bone marrow-derived dendritic cells maturation in vitro through the upregulation of expression of costimulatory molecules CD40, CD80, and OX40L, however, no effect on MHC class II molecule or CD86 expression was observed. These findings were confirmed by Chu and colleagues65 who showed that blockade of OX40L significantly reduces allergic responses to both house dust mites and peanuts, and although induction of OX40L on dendritic cells in vitro was not interleukin 33 dependent, interleukin 33 signalling alone was sufficient for intact allergic immunity. By contrast, Rank and colleagues66 showed that interleukin 33 increased the expression of MHC class II and CD86 on the surface of dendritic cells, but not that of CD40 or CD80. Interleukin 33-mediated dendritic cell–T cell activation and subsequent Th2 polarisation has been reported in several studies, with ST2-deficiency in mice shown to attenuate allergic inflammation in ovalbumin driven airway inflammation models.61,62 Th2 polarisation downstream of interleukin 33 signalling on dendritic cells has been claimed to be non-canonical because Rank and colleagues66 showed that, on coculture of dendritic cells and CD4-positive T cells, production of interleukin 5 and interleukin 13 is enhanced, but interleukin 4 is unchanged. A novel in-vivo allergy model to cypress pollen has shown the capacity for the natural allergen to modulate the biological activity of dendritic cells by directly enhancing ST2 expression, and thus, increasing responsiveness to interleukin 33, with subsequently enhanced Th2 polarisation.8 Additionally, data from Su and colleagues67 show that dendritic cells produce interleukin 33 via a TLR/NF-κB signalling pathway on microbial stimulation, and furthermore suggest that local allergic inflammatory responses might be amplified by dendritic cell-produced interleukin 33 through potential autocrine regulation. Here, dendritic cells expressing the www.thelancet.com/respiratory Vol 2 March 2014
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receptor for interleukin 33 respond on ligation, proliferate, mature, and activate T cells to differentiate towards a Th2 phenotype. All these processes are controlled by an autocrine mechanism of interleukin 33 production and response. Overall, all seven studies8,61,63–67 describe that dendritic cells express the receptor for interleukin 33 and on ligation, they proliferate, mature, and are able to trigger activated T cells to secrete increased amounts of interleukin 5 and interleukin 13. This role is very important for the induction of allergic airway inflammation (figure 1). The ST2 receptor mediates these processes, because using ST2-deficient mice or blocking the ST2 receptor in dendritic cells seems crucial for the decrease in lung eosinophilia and airway inflammation. The appendix p 7 shows the outcomes of studies relating interleukin 33 to airway inflammation through dendritic cell interactions.
Role of interleukin 33 in adaptive immune responses T helper cells have a pivotal role in directing immune responses to eliminate viral and bacterial pathogens via Th1 responses, but also mediate allergic airway inflammation Th2 responses. The elicited response is dependent on the pathogen-antigen interaction, interaction with APCs, and the microenvironment, including soluble mediators, in which the T cells are activated. Interleukin 33 has been shown to affect Th cell responses via either direct or indirect interaction with T cells and to furthermore intensify allergic inflammatory responses.68 T helper cells have been shown to express the ST2 receptor, but its regulation is not yet fully clear. Early data showed that expression of ST2 and secretion of Th2 cytokines is not dependent on the presence of interleukin 4.69 Numerous mouse studies have shown that Th2 cells
selectively express ST2, with interleukin 4 and interferon γ differentially modulating receptor expression.24 Naive CD4+ T cells do not express ST2, suggesting a requirement for antigen or mitogen stimulation in the presence of interleukin 4 for its expression, although a certain redundancy exists regarding the necessity of interleukin 4.69,70 These observations have been confirmed in exvivo human culture systems showing increased expression of ST2 on Th2 cells suggesting ST2 as a selective marker for Th2 cells.5,71 However, despite evidence relating ST2 expression with Th2 cells, in other research interleukin 33 acted as an amplifier, rather than a polariser, of T cell responses because recombinant human interleukin 33 induced the inflammatory potential of both Th1 and Th2 polarised cells in vitro, whereas it repressed interleukin 10 secretion by Th1 cells.5 The reported mechanism of interleukin 33-mediated Th2 polarisation via dendritic cell–T cell interactions, as shown in a number of allergic inflammatory models, could be dependent on the form of interleukin 33 expressed.72,73 Caspase-cleaved mouse interleukin 33 is unable to induce Th2 differentiation in vivo, but mediates infiltration of the lung with inflammatory cells in an ST2-independent way, whereas uncleaved interleukin 33 triggers secretion of Th2 cytokines in vivo by interacting with the ST2 receptor.74 The mechanism by which interleukin 33 induces Th2 differentiation in vivo has not been thoroughly clarified. Trefoil factor 2 and FOXa2 have both been implicated in the interleukin 33-Th2 pathway,75 and Wills-Karp and colleagues76 suggested that trefoil factor 2 drives Th2 differentiation in vivo via an interleukin 33-dependent pathway. Although the classic route of Th2 cell differentiation involves STAT6 and results in GATA3 induction,
Airway lumen Allergen Draining lymph node Damage Mature dendritic cell
Epithelial cells Lung tissue Maturation and migration of dendritic cell Immature dendritic cell
↑expression of MHC II, CD86, CD40, CD80, OX40L Naive T cell
Th2 differentiation Interleukin 33 Allergen Pattern recognition receptors ST2 receptor
Migrati
on to th
MHC class II T-cell receptor
e lung ti
ssue
Specific Th2 cell induction
Figure 1: Overview of the role of interleukin 33 in asthma Interleukin 33 released by damaged or stimulated airway epithelial cells attracts, activates, and polarises dendritic cells. Interleukin 33-activated dendritic cells migrate to the draining lymph nodes and drive Th2 responses. Th2=T-helper-type-2 cell. CD86=cluster of differentiation 86. CD40=cluster of differentiation 40. CD80=cluster of differentiation 80. OX40L=ligand for cluster of differentiation 134.
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interleukin 33 was shown to follow a so-called noncanonical pathway that might be mediated by STAT5 activation.77–79 In published studies, interleukin 33 triggers airway inflammation by inducing interleukin 5 and interleukin 13 secretion, but not interleukin 4.79 T cell responses other than Th2 responses have also been reported after interleukin 33 stimulation in vivo in a mouse model of allergic asthma. In eosinophil-deficient mice, on interleukin 33 stimulation, mast cells were shown to drive Th17 responses.80 Interleukin 33 was also shown to be important not only for the establishment of the allergic inflammatory response, but also for its maintenance. On injection of an antibody blocking the ST2 receptor during ovalbumin challenge in mice, lung expression of interleukin 4 was reduced and persistence of the airway hyperresponsiveness was abrogated.19 All the 25 studies analysed show that interleukin 33 is able to induce T cell proliferation and Th2 cell differentiation, thus placing the interleukin 33–Th2 pathway in the centre of asthma pathogenesis. In vivo, on interleukin-33 stimulation, mouse T cells produce increased interleukin 5 and interleukin 13 that induce interleukin 33-dependent eosinophilic airway inflammation, airway hyperresponsiveness, and airway obstruction. These findings show the necessity for novel clinical trials that will investigate the role of interleukin 33 as a possible central mediator of asthma pathogenesis in humans. Understanding the possible role of interleukin 33 in promoting Th1 or Th17 Number of patients and demographic
differentiation clearly requires further work because existing evidence is not yet conclusive. The appendix pp 8 shows the outcomes of studies relating interleukin 33 to airway inflammation through T cell interactions.
Role of interleukin 33 on clinical outcomes Globally, asthma is one of the most common chronic diseases in children and adults. Although respiratory allergy and asthma are generally regarded as Th2mediated diseases, these associations have been challenged and therapies designed to suppress Th2 function are not effective for all patients. Interleukin 33 is a novel cytokine that has been strongly linked to the pathogenesis of both respiratory allergy and asthma and several human studies have confirmed this association.81,82 Genetic polymorphisms close to the IL33 gene have been associated with the incidence of nasal polyposis and with increased blood eosinophil numbers.6 Interleukin 33 protein was significantly increased in the nasal secretions of patients with Japanese cedar pollinosis at peak season, suggesting a possible relation with exacerbation of allergic rhinitis.83 Genome-wide association studies (GWAS) of asthma have consistently shown that polymorphisms in IL33 and IL1RL1 (ST2) genes are strongly related to increased susceptibility to asthma development,84–87 but not to decreased lung function as assessed by spirometry in patients with asthma.84 In a Dutch GWAS of asthmatic families, polymorphisms in adjacently located genes (IL33,
Variables measured
Outcome
p value
Soluble IL1R1 levels in serum and asthma prevalence (questionnaire); blood eosinophil counts at 4 years and 8 years of age and asthma prevalence (questionnaire)
Polymorphisms in ILRL1 gene associated with increased asthma prevalence at 8 years old; Polymorphisms in ILRL1 gene associated with soluble IL1RL1 concentrations and eosinophils counts
Role of interleukin 33 in respiratory allergy and asthma Heidi Makrinioti, Marie Toussaint, David J Jackson, Ross P Walton, Sebastian L Johnston Lancet Respir Med 2014; 2: 226–37 Published Online January 23, 2014 http://dx.doi.org/10.1016/ S2213-2600(13)70261-3 Airway Disease Infection Section, National Heart and Lung Institute, Imperial College London, London, UK (H Makrinioti MD, M Toussaint PhD, D J Jackson MRCP, R P Walton PhD, Prof S L Johnston FRCP); Medical Research Council and Asthma UK Centre in Allergic Mechanisms of Asthma, London, UK; (H Makrinioti, M Toussaint, D J Jackson, R P Walton, S L Johnston); Centre for Respiratory Infection, Imperial College, London, UK (H Makrinioti, M Toussaint, D J Jackson, R P Walton, S L Johnston); and Imperial College Healthcare NHS Trust, London, UK (D J Jackson, S L Johnston) Correspondence to: Dr Heidi Makrinioti, Airway Disease Infection Section, National Heart and Lung Institute, Imperial College London, Norfolk Place, London W2 1PG, UK [email protected]
Since the discovery of interleukin 33 as the adopted ligand for the then orphan ST2 receptor, many studies have implicated this cytokine in the pathogenesis of respiratory allergy and asthma. Although some extracellular functions of interleukin 33 have been well defined, many aspects of the regulation and secretion of this cytokine need clarification. Interleukin 33 has been identified as a trigger of T-helper-type-2 cell differentiation, which by interacting with both the innate and the adaptive immune systems, can drive allergy and asthma pathogenesis. However, induction of interleukin 33 by both environmental and endogenous triggers implies a possible role during infection and tissue damage. Further understanding of the biology of interleukin 33 will clarify its possible role in future therapeutic interventions.
Introduction Interleukin 33, a member of the interleukin 1 cytokine family, was originally characterised as a protein expressed in lymph node-associated endothelial cells.1 The interleukin 33 receptor, termed IL1RL1 (ST2), was first identified as an interleukin 1 receptor-like molecule by Tominaga in 1989.2 The receptor has two main isoforms— one transmembrane form, ST2 (ST2L), and one soluble form, ST2 (sST2)—the expression of which is controlled by two distinct promoters. sST2 binds interleukin 33 and blocks its functional bioactivities that are initiated by its ligation to ST2L.3 Consistent with other interleukin 1-related cytokine receptor family members, the interleukin 33 receptor is a heterodimer, composed of ST2 and interleukin 1-receptor accessory protein (IL1RAP). Signalling via this receptor complex leads to activation of downstream signalling molecules, such as nuclear factor κB (NF-κB) and activating protein 1.4 Unlike other interleukin 1 family members, interleukin 33 has been associated with the promotion and amplification of both systemic and localised T helper (Th) 2 cell responses.5,6 The full length form, prointerleukin 33, is localised in the nucleus. The exact function of this interleukin in the nucleus is unclear; however, recent studies show that it binds to dimerised histone H2A and H2B at the surface of the nucleosome
Key messages • Interleukin 33 is an alarmin that is released in the lung mainly by epithelial cells and possibly also by macrophages after cell necrosis or cell activation • Interleukin 33 binds its receptor, ILRL1, and triggers allergic airway inflammation by interacting with both the innate and adaptive arms of the immune system • The pathogenic mechanisms that underlie the release of interleukin 33 by epithelial cells and the intracellular signals triggered by its interaction with its receptor need to be clarified further • Interleukin 33 has been associated with the incidence and pathogenesis of asthma and with induction of permanent structural changes in the airways • Data for the role of interleukin 33 during exacerbations of asthma (that are mainly virus-induced) are scarce; however, non-peer-reviewed evidence suggests that the role of interleukin 33 is probably important, and further research is urgently needed • Although no studies in man have targeted interleukin 33, the results from animal studies pave the way for trials in human beings, which are eagerly anticipated
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and might exhibit transcriptional repressor activity. Nuclear localisation of interleukin 33 is not cell specific and is probably fundamental to both the activity and function of the protein. Although interleukin 33 shares various characteristics with other interleukin 1 cytokine family members, the mechanisms regulating its extracellular migration from the nucleus seem to differ, and have not been completely clarified.4,7–9 The dominant hypothesis is that interleukin 33 is released after necrosis or cell damage, and acts as an alarmin to elicit a protective immune response. During necrosis, interleukin 33 remains in its active form; however, in apoptotic conditions, caspase 3 and caspase 7 cleave interleukin 33 rendering it inactive, suggesting a differential role on programmed cell death.7 However, the neutrophil derived serine proteases cathepsin G and elastase have also been reported to cleave interleukin 33 and amplify its bioactivity, thus suggesting that cleavage of interleukin 33 does not exclusively lead to its inactivation.10 Further work is clearly needed to better understand the effects of cleavage on interleukin 33 activity. The main sources of interleukin 33 protein are structural cells such as fibroblasts, epithelial cells, and endothelial cells. The ST2 receptor is expressed on various immune cells—namely T cells, B cells, eosinophils, mast cells, innate lymphoid cells, and dendritic cells.11 Interleukin 33 is thought to play a part in a range of diseases, such as infectious and autoimmune diseases, cardiovascular diseases, and neurological disorders.11 Additionally, increased evidence shows the ability of interleukin 33 to act as both a chemoattractant and an immunomodulator, and thus it is characterised as an alarmin because of its ability to activate cells of both the innate and the adaptive immune system.12–14 To date, research has shown that necrotic death and mechanical injury of epithelial cells can undoubtedly lead to interleukin 33 release.15 Furthermore, accumulating evidence from studies in mouse innate lymphoid cells infected with influenza suggests that interleukin 33 might be released as a result of viral infection.16 A pronounced role has been suggested for interleukin 33 in asthma pathogenesis. Asthma is a heterogeneous www.thelancet.com/respiratory Vol 2 March 2014
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inflammatory airway disease with a complex cause, stimulated by various triggers. Allergy can be placed centrally in the pathogenesis of asthma in most patients and the importance of the bronchial epithelium in the disease process is becoming clearer. The interleukin 33/ ST2 pathway has been suspected to mediate the pathogenesis of both asthma and allergy through diverse pathways.17,18 Epithelial cells are a primary source of interleukin 33, which suggests a mechanism whereby the respiratory tract has the potential, in response to a so-called danger signal, to drive immune responses other than the expected Th1 response. This signal could be either an infection (viral or bacterial), any other environmental signal (allergen or pollutant), or a combination of both. Secretion of interleukin 33 could perpetuate an unresolved airway inflammation, especially in patients with asthma.19 Therefore, the role of interleukin 33 in respiratory infection, allergy, and asthma pathogenesis is of particular interest, and a plethora of studies implicate interleukin 33 as an attractive therapeutic target. The increasing volume of published work in interleukin 33 leads to an increasing number of questions requiring answers. This Review aims to answer these questions and to discuss whether a clinical application of anti-interleukin 33 therapy might be useful for the future treatment of respiratory allergy or asthma.
Release of interleukin 33 by respiratory epithelium during allergic respiratory response Airway epithelial cells form the interface between the host and the environment, and are the first line of defence against inhaled microorganisms and allergens.20 In this exposed position, epithelial cells express a broad array of pattern recognition receptors (PRRs) that detect environmental stimuli, responding to pathogenassociated molecular patterns (PAMPs) present on microbes or to danger-associated molecular patterns (DAMPs) released after tissue damage, cell death, or cellular stress. Activation of these PRRs on epithelial cells leads to the release of soluble mediators, including cytokines and chemokines, which can alert and activate the immune system to any potential threat.20 Among the cytokines released by the airway epithelium, interleukin 33 is highly secreted in response to allergen, with human epithelial cells of the bronchus and small airways showing constitutive expression of interleukin 33 mRNA.21,22 This expression is increased in bronchial epithelial cells of patients with asthma compared with healthy individuals.23 In resting conditions, epithelial cells store interleukin 33 in the nucleus.14 However, during injury or necrosis caused by exogenous triggers (such as virus, smoke, airborne allergens), or endogenous triggers (such as ATP, DNA, HMGB1), interleukin 33 is released by damaged cells. Additionally, these triggers might also directly activate PRRs, including Toll-like receptors (TLRs) on epithelium cells, resulting in the release of biologically www.thelancet.com/respiratory Vol 2 March 2014
active full-length interleukin 33.24 Recent studies in mouse models of allergic asthma induced by house dust mites—one of the most common allergens in human beings—have shown the importance of TLR4 signalling in the induction of interleukin 33 release by airway epithelial cells. Willart and colleagues21 showed that administration of low dose house dust mites during allergic sensitisation induced interleukin 1α release via TLR4 ligation and signalling on bronchial epithelial cells. This release occurred through PRR detection of the major allergen Derp2 and the endotoxin contained in the faecal pellets of the mite. Subsequently released interleukin 1α acts in an autocrine manner on bronchial epithelial cells, stabilising interleukin 1R1 surface expression, inducing the release of interleukin 33 and perpetuating a Th2 immune response against the allergen. In accordance with this, Collison and colleagues25 confirmed the importance of TLR4 activation by endogenous triggers for interleukin 33 release by the epithelium. After house dust mite exposure in mice, E3 ubiquitin ligase midline 1, which is known to induce interleukin 33 expression, was upregulated in bronchial epithelial cells in a TLR4dependent manner.25 An alternative pathway for release of interleukin 33 by airway epithelial cells that is independent of conventional TLR activation has been suggested. Exposure of airway epithelial cells to the fungus Alternaria alternata in vitro induces the rapid extracellular release of biologically active interleukin 33. The proposed mechanism is via the allergen inducing an acute extracellular accumulation of ATP, which signals in an autocrine way via P2 purinergic receptors, and leads to subsequent interleukin 33 extracellular secretion.26 Because most studies show that the lung epithelium is a major source of interleukin 33, topical administration of anti-interleukin 33/ST2 therapy could be considered as a future treatment strategy, thus reducing the risk of systemic side-effects. The appendix p 1 summarises studies investigating interleukin 33 production by airway epithelial cells.
See Online for appendix
Macrophages as both target and source of interleukin 33 Macrophages are specialised phagocytic cells found in various tissues and organs, which play a central part in homoeostasis, tissue remodelling, and host defence. Depending on the microenvironment, macrophages can be polarised into various different phenotypes and are classified as classically activated macrophages or alternatively activated macrophages. Alternatively activated macrophages promote and regulate type 2 immune responses and can be characterised by expression of mannose receptor and arginase 1 upregulation.27 Mouse macrophages have been shown to constitutively express ST2 mRNA and proteins.28 Yang and colleagues29 showed that mouse bone marrow-derived macrophages stimulated in vitro with interleukin 33 227
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substantially increased the expression of interleukin 13 in a time-dependent and concentration-dependent manner. Production of interleukin 13 from macrophages activates macrophages through an alternative autocrine pathway.29 Kurowska-Stolarska and colleagues30 reported that alternatively activated macrophages play a crucial part in type 2 immunity; mice deficient in ST2 had an impaired type 2 immune response, which is associated with a decrease in differentiation of alternatively activated macrophages. These investigators confirmed that interleukin 33 changes the quiescent phenotype of alveolar macrophages towards an alternatively activated macrophages phenotype in an interleukin 13-dependent manner during interleukin 33-induced eosinophilia in the lung. Moreover, depletion of alveolar macrophages reduced interleukin 33-induced airway inflammation.30 Additionally, the cell lineage and maturation markers, CD11b and CD86, and the ST2 receptor are upregulated by interleukin 33 in bone marrow-derived macrophages.29 Tjota and colleagues31 reported that allergen-specific IgG can enhance secondary Th-2 mediated immune responses in the lungs via an interleukin 33-dependent mechanism. Finally, treatment of primary mouse macrophages and alveolar macrophages with TLR3 (polyinosinic:polycytidylic acid) and TLR4 (lipopolysaccharide) agonists greatly increased expression of both interleukin 33 mRNA and protein.32 These findings suggest that viral infection could amplify type 2 immune responses through the production of interleukin 33 by alveolar macrophages during asthma exacerbations. Thus, macrophages are capable of both producing and responding to interleukin 33, and inhibition of interleukin 33/ST2 downregulates macrophage mediated type 2 inflammation. However, further work is needed to investigate the role of interleukin 33/ST2 in macrophage activation during stable, moderate, and severe asthma and in virus-induced asthma exacerbations, which might answer the question of whether the use of anti-interleukin 33/ST2 treatment could be a possible symptomatic therapy. The appendix pp 2 outlines studies investigating the relation between interleukin 33 and airway inflammation via macrophage interactions.
Interactions between interleukin 33/ST2 and immune cells in asthma Mast cells Mast cells are present in all tissues and are especially numerous at sites exposed to external environments, such as the epithelium. On activation, mast cells rapidly produce and secrete a range of soluble mediators, providing a rapid response to tissue damage or cell injury, initiating and augmenting the ensuing inflammatory response. Mast cells express the high affinity IgE receptor (FCεRI), and induce IgE-mediated immune responses. Several studies have shown that mast cells play an important part in asthma development, but also 228
that they are highly responsive to interleukin 33 that is released from necrotic structural cells.33 Both mouse and human mast cells constitutively express functional interleukin 33 receptors (ST2).34 In human mast cells, the transcription factor GATA2 is a positive regulator of ST2 expression.35 Interleukin 33 directly stimulates primary human mast cells to produce several proinflammatory cytokines (interleukin 1β, interleukin 6, interleukin 8, interleukin 13, and tumour necrosis factor α), chemokines (CCL1 and CXCL8), and growth factors (granulocyte-macrophage colony-stimulating factor) through activation of the NF-κB pathway.36 Although interleukin 33 does not trigger human mast cell degranulation, interleukin 33 might augment mast cell degranulation in sensitised individuals with high concentrations of IgE.37,38 Interleukin 33 prolongs survival of cultured mast cells derived from human cord blood by promoting cell adhesion.39 Interleukin 33 can also act in concert with thymic stromal lymphopoietin, a potent mast cell activator, to accelerate in-vivo maturation of human CD34+ mast cell precusors and induce secretion of Th2 cytokines and Th2-attracting chemokines.36 Together, these results suggest that interleukin 33 could play an important part in mast cell-mediated allergic lung inflammation. Junttila and colleagues40 have shown that mouse mast cells require two separate cytokine signals— interleukin 33 and interleukin 3—for optimum production of interleukin 13. The requirement of two separate signals could represent a protective mechanism to control non-adapted interleukin 13 release by mast cells. Finally, Hsu and colleagues41,42 have shown that mouse mast cells express low concentrations of the interleukin 33 gene and protein constitutively; however, expression of interleukin 33 was induced by IgEmediated activation. The same investigators also showed that expression of interleukin 33 by mast cells was dependent on calcium activation via IgE/FcεRI, but not ST2 activation.41,42 This endogenous production of interleukin 33 could be an important mechanism through which mast cells affect the recruitment and activation of inflammatory cells into sites of IgEdependent activation. Interleukin 33 clearly has the capacity to directly stimulate mast cells to produce various cytokines and chemokines, but degranulation of mast cells requires the presence of IgE as well. These findings suggest that interleukin 33 mediates asthma exacerbation via mast cells mainly in predisposed atopic individuals. The appendix pp 3 summarises studies relating interleukin 33 to airway inflammation through mast cell interactions.
Basophils In allergic inflammation, basophils are recruited from the blood to peripheral organs where they play a pivotal part in allergic inflammation by rapidly releasing chemical mediators, such as interleukins 4, 5, 8, and 13, in addition to sustained production of Th2 cytokines, including www.thelancet.com/respiratory Vol 2 March 2014
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interleukin 4. Unlike mast cells, human and mouse basophils express few ST2 on the cell surface.43 This could be explained by the high level of GATA1 expression in basophils, a negative regulator for ST2 transcription.35 In human basophils, surface expression of ST2 can be enhanced by stimulation with interleukin 3, a growth and differentiation factor for human basophils.18,43 Moreover, interleukin 33 alone is able to increase the production of interleukin 4 from human basophils,18,43 along with interleukin 5, interleukin 8, and interleukin 13.18 In addition to augmenting IgE crosslinking, interleukin 33 enhances human basophil degranulation and eotaxin mediated migration without affecting cell surface CCR3 expression.43 Interleukin 33 can further synergise with interleukin 3 to promote increased interleukin 4 production and CD11b expression by human basophils.43 However, interleukin 33 does not directly induce degranulation or function as a basophil chemoattractant, but instead exerts a priming effect on mouse basophils.43 Finally, in mice, interleukin 33 alone can directly activate mouse basophils, but by contrast with human basophils, it induces their preferential secretion of interleukin 6 (as opposed to interleukin 4) to indirectly promote basophil expansion in vivo through initiating an autocrine feedback loop via GM-CSF and interleukin 3 by the basophils.44 Thus interleukin 33 is an important regulator of basophil function and could be considered as a key cytokine in the pathogenesis of Th2-dominant inflammation. The appendix p 4 shows the outcomes of studies relating interleukin 33 to airway inflammation through basophil interactions.
Eosinophils Eosinophils are proinflammatory granulocytes that play a central part in parasite immunity, but they are also implicated in the pathogenesis of bronchial asthma.45 Eosinophils primarily reside in tissues at mucosal surfaces but are also found circulating in the blood. As with mast cells and basophils, both ST2 mRNA and protein have been shown to be constitutively expressed by human and mouse eosinophils.46,47 Notably, interleukin 33 has been shown, independently of interleukin 3, interleukin 5, and GM-CSF, to potently activate human eosinophils, inducing production and release of the inflammatory mediators, such as CCL2/MCP-1 and CXCL8/interleukin 8, as a result of ST2 activation.47 In addition to these activities, interleukin 33 prolongs human eosinophil survival and adhesion, degranulation and production of superoxide, augments expression of CD11b, and modulates responsiveness to Siglec-8mediated apoptosis.46–48 Moreover, Dyer and colleagues49 have shown that systemic administration of interleukin 33 in mice induces profound interleukin 5-dependent eosinophilia; however, in this study, eosinophil haemopoiesis was interleukin 33 independent. Eosinophils are the main cell type that is increased in numbers in the inflamed airways of patients with www.thelancet.com/respiratory Vol 2 March 2014
allergic asthma. Although eosinophilic-mediated asthma has been regarded as the predominant asthma phenotype, research shows that there are patients with asthmas with non-eosinophilic-mediated inflammation, and that interleukin 4, interleukin 5, and interleukin 13 are not consistently elevated in the airways of all patients with asthma.50 Evidence showing that interleukin 33 has the potential to directly activate eosinophils suggests this molecule could be a therapeutic target for eosinophil-mediated asthma, but further research is awaited to show whether interleukin 33 could also be regarded as important in asthma exacerbations. The appendix pp 5 summarises studies relating interleukin 33 to airway inflammation through eosinophil interactions.
Innate lymphoid cells The recent discovery and subsequent phenotypic characterisation of a novel population of innate lymphoid cells has led to intense investigation into their mechanisms of activation and effector function. A population of innate lymphoid cells able to produce large amounts of the Th2 cytokines interleukin 5 and interleukin 13 has been described; these cells were initially termed lung natural helper cells or nuocytes, but the more standardised nomenclature is now type 2 innate lymphoid cells.51–53 Initially identified by their importance in driving helminth expulsion, these type 2 innate lymphoid cells respond to interleukin 33 by producing large quantities of interleukin 5 and interleukin 13.52 GATA3, a Th2 regulator, is a transcription factor involved in controlling type 2 innate lymphoid cell differentiation, maintenance, and function.54 In-vivo mouse studies of airway administration of glycolipid antigen have shown that interleukin 33 release by alveolar macrophages and dendritic cells leads to activation of type 2 innate lymphoid cells and subsequent interleukin 13 and interleukin 5 production.55 This process of interleukin 33-mediated interleukin 13 secretion by type 2 innate lymphoid cells is sufficient for the induction of airway hyperreactivity in the absence of an adaptive immune response.55 Using a purified system of mouse type 2 innate lymphoid cells, Halim and colleagues51 identified a crucial requirement for costimulation with other cytokines for activation of these cells and effector Th2 cytokine production. Combined administration of interleukin 33 with interleukin 2, interleukin 7, or thymic stromal lymphopoietin resulted in greatly enhanced interleukin 5 and interleukin 13 production by type 2 innate lymphoid cells.51 Moreover, in-vitro stimulation of lung explants from Rag1–/– mice (deficient for T cells and B cells) with papain (a protease allergen homologous to house dust mite-derived Der P1 that can induce airway inflammation in vivo) leads to an increase of interleukin 5 and interleukin 13 production by type 2 innate lymphoid cells. Additionally, Th2 229
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cytokine production by type 2 innate lymphoid cells stimulated with papain is strongly reduced when interleukin 33 signalling is neutralised with an antiinterleukin-33 antibody, whereas depletion of type 2 innate lymphoid cells in mice attenuates airway eosinophilia after papain inhalation.51 In two models of aero-allergen induced allergic asthma (house dust mites and ovalbumin) type 2 innate lymphoid cells are important producers of Th2 cytokines (interleukin 13 and interleukin 5) in both the airways and the lung tissue identified as crucial for airway hyperreactivity development during influenza A infection in mice.56 Here, influenza infection results in increased production of interleukin 33 by alveolar macrophages, which in turn activates type 2 innate lymphoid cells to produce interleukin 13.16 To facilitate worm expulsion, type 2 innate lymphoid cells are key secretors of interleukin 5 and interleukin 13, but not interleukin 4, in response to interleukin 33 and interleukin 25 released during helminth infection in the gut.52 During an ovalbumin model of allergic lung inflammation, numbers of type 2 innate lymphoid cells increased greatly in the airways and lung after ovalbumin challenge.57 After in-vivo stimulation with interleukin 33, type 2 innate lymphoid cells exhibit increased interleukin 5 and interleukin 13 cytokine production, and contribute to airway hyperreactivity development.57 Finally, Bartemes and colleagues58 have shown that type 2 innate lymphoid cells isolated from mouse lung produce large amounts of interleukin 5 and interleukin 13 when cultured with interleukin 33 in vitro. However, Rag–/– mice exposed to the fungus A alternata showed markedly increased interleukin 33 protein in the airways within 1 h of exposure. As a result of interleukin 33 release, type 2 innate lymphoid cells mediated downstream type-2 cytokine driven allergic airway inflammation in the lungs of these mice.58 In human beings, a role has been suggested for nasal epithelial cell-derived interleukin 33 and an interleukin 33-responsive type 2 innate lymphoid cell population in the pathology of chronic rhinosinusitis.59,60 Although these data imply a possible role for interleukin 33 and type 2 innate lymphoid cells interaction in asthma, little is still known in human disease. The identification and phenotypic and functional analysis of type 2 innate lymphoid cells in mice provides an exciting novel mechanism for innate development of type 2 immunity in the lung and induction of allergic airway disease; however, further evidence is needed to more precisely define their role, and little is known regarding the role played by these cells in human disease. There are no data on the possible role of an interleukin 33/type 2 innate lymphoid cells axis in virusinduced asthma exacerbations. The appendix p 6 shows the outcomes of studies relating interleukin 33 to airway inflammation through type 2 innate lymphoid cell interactions. 230
Dendritic cells Dendritic cells reside in the submucosa in close contact with the airway epithelium. Here, dendritic cells extend processes into the airway lumen sampling the inhaled milieu, whereby they capture antigen and present it to naive lymphocytes in draining lymph nodes. In this capacity, they play an essential part in initiating adaptive immune responses. Recent published work has presented dendritic cells as master regulators of the interleukin 33 pathway. ST2 is expressed on dendritic cells and T cells at both the gene and protein level.61,62 Lambrecht and colleagues63 showed that dendritic cell-induced Th2 development in the airways could be inhibited by blocking the interaction of ST2 with its, at that time unknown, ligand. Receptor blockade with antibodies at the time of intratracheal adoptive transfer of dendritic cells (during sensitisation) resulted in complete suppression of sensitisation to inhaled allergen, showing that ST2 presence and functionality on dendritic cells is crucial for the activation of immunogenic responses. Further, Besnard and colleagues64 showed that interleukin 33 is able to drive bone marrow-derived dendritic cells maturation in vitro through the upregulation of expression of costimulatory molecules CD40, CD80, and OX40L, however, no effect on MHC class II molecule or CD86 expression was observed. These findings were confirmed by Chu and colleagues65 who showed that blockade of OX40L significantly reduces allergic responses to both house dust mites and peanuts, and although induction of OX40L on dendritic cells in vitro was not interleukin 33 dependent, interleukin 33 signalling alone was sufficient for intact allergic immunity. By contrast, Rank and colleagues66 showed that interleukin 33 increased the expression of MHC class II and CD86 on the surface of dendritic cells, but not that of CD40 or CD80. Interleukin 33-mediated dendritic cell–T cell activation and subsequent Th2 polarisation has been reported in several studies, with ST2-deficiency in mice shown to attenuate allergic inflammation in ovalbumin driven airway inflammation models.61,62 Th2 polarisation downstream of interleukin 33 signalling on dendritic cells has been claimed to be non-canonical because Rank and colleagues66 showed that, on coculture of dendritic cells and CD4-positive T cells, production of interleukin 5 and interleukin 13 is enhanced, but interleukin 4 is unchanged. A novel in-vivo allergy model to cypress pollen has shown the capacity for the natural allergen to modulate the biological activity of dendritic cells by directly enhancing ST2 expression, and thus, increasing responsiveness to interleukin 33, with subsequently enhanced Th2 polarisation.8 Additionally, data from Su and colleagues67 show that dendritic cells produce interleukin 33 via a TLR/NF-κB signalling pathway on microbial stimulation, and furthermore suggest that local allergic inflammatory responses might be amplified by dendritic cell-produced interleukin 33 through potential autocrine regulation. Here, dendritic cells expressing the www.thelancet.com/respiratory Vol 2 March 2014
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receptor for interleukin 33 respond on ligation, proliferate, mature, and activate T cells to differentiate towards a Th2 phenotype. All these processes are controlled by an autocrine mechanism of interleukin 33 production and response. Overall, all seven studies8,61,63–67 describe that dendritic cells express the receptor for interleukin 33 and on ligation, they proliferate, mature, and are able to trigger activated T cells to secrete increased amounts of interleukin 5 and interleukin 13. This role is very important for the induction of allergic airway inflammation (figure 1). The ST2 receptor mediates these processes, because using ST2-deficient mice or blocking the ST2 receptor in dendritic cells seems crucial for the decrease in lung eosinophilia and airway inflammation. The appendix p 7 shows the outcomes of studies relating interleukin 33 to airway inflammation through dendritic cell interactions.
Role of interleukin 33 in adaptive immune responses T helper cells have a pivotal role in directing immune responses to eliminate viral and bacterial pathogens via Th1 responses, but also mediate allergic airway inflammation Th2 responses. The elicited response is dependent on the pathogen-antigen interaction, interaction with APCs, and the microenvironment, including soluble mediators, in which the T cells are activated. Interleukin 33 has been shown to affect Th cell responses via either direct or indirect interaction with T cells and to furthermore intensify allergic inflammatory responses.68 T helper cells have been shown to express the ST2 receptor, but its regulation is not yet fully clear. Early data showed that expression of ST2 and secretion of Th2 cytokines is not dependent on the presence of interleukin 4.69 Numerous mouse studies have shown that Th2 cells
selectively express ST2, with interleukin 4 and interferon γ differentially modulating receptor expression.24 Naive CD4+ T cells do not express ST2, suggesting a requirement for antigen or mitogen stimulation in the presence of interleukin 4 for its expression, although a certain redundancy exists regarding the necessity of interleukin 4.69,70 These observations have been confirmed in exvivo human culture systems showing increased expression of ST2 on Th2 cells suggesting ST2 as a selective marker for Th2 cells.5,71 However, despite evidence relating ST2 expression with Th2 cells, in other research interleukin 33 acted as an amplifier, rather than a polariser, of T cell responses because recombinant human interleukin 33 induced the inflammatory potential of both Th1 and Th2 polarised cells in vitro, whereas it repressed interleukin 10 secretion by Th1 cells.5 The reported mechanism of interleukin 33-mediated Th2 polarisation via dendritic cell–T cell interactions, as shown in a number of allergic inflammatory models, could be dependent on the form of interleukin 33 expressed.72,73 Caspase-cleaved mouse interleukin 33 is unable to induce Th2 differentiation in vivo, but mediates infiltration of the lung with inflammatory cells in an ST2-independent way, whereas uncleaved interleukin 33 triggers secretion of Th2 cytokines in vivo by interacting with the ST2 receptor.74 The mechanism by which interleukin 33 induces Th2 differentiation in vivo has not been thoroughly clarified. Trefoil factor 2 and FOXa2 have both been implicated in the interleukin 33-Th2 pathway,75 and Wills-Karp and colleagues76 suggested that trefoil factor 2 drives Th2 differentiation in vivo via an interleukin 33-dependent pathway. Although the classic route of Th2 cell differentiation involves STAT6 and results in GATA3 induction,
Airway lumen Allergen Draining lymph node Damage Mature dendritic cell
Epithelial cells Lung tissue Maturation and migration of dendritic cell Immature dendritic cell
↑expression of MHC II, CD86, CD40, CD80, OX40L Naive T cell
Th2 differentiation Interleukin 33 Allergen Pattern recognition receptors ST2 receptor
Migrati
on to th
MHC class II T-cell receptor
e lung ti
ssue
Specific Th2 cell induction
Figure 1: Overview of the role of interleukin 33 in asthma Interleukin 33 released by damaged or stimulated airway epithelial cells attracts, activates, and polarises dendritic cells. Interleukin 33-activated dendritic cells migrate to the draining lymph nodes and drive Th2 responses. Th2=T-helper-type-2 cell. CD86=cluster of differentiation 86. CD40=cluster of differentiation 40. CD80=cluster of differentiation 80. OX40L=ligand for cluster of differentiation 134.
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interleukin 33 was shown to follow a so-called noncanonical pathway that might be mediated by STAT5 activation.77–79 In published studies, interleukin 33 triggers airway inflammation by inducing interleukin 5 and interleukin 13 secretion, but not interleukin 4.79 T cell responses other than Th2 responses have also been reported after interleukin 33 stimulation in vivo in a mouse model of allergic asthma. In eosinophil-deficient mice, on interleukin 33 stimulation, mast cells were shown to drive Th17 responses.80 Interleukin 33 was also shown to be important not only for the establishment of the allergic inflammatory response, but also for its maintenance. On injection of an antibody blocking the ST2 receptor during ovalbumin challenge in mice, lung expression of interleukin 4 was reduced and persistence of the airway hyperresponsiveness was abrogated.19 All the 25 studies analysed show that interleukin 33 is able to induce T cell proliferation and Th2 cell differentiation, thus placing the interleukin 33–Th2 pathway in the centre of asthma pathogenesis. In vivo, on interleukin-33 stimulation, mouse T cells produce increased interleukin 5 and interleukin 13 that induce interleukin 33-dependent eosinophilic airway inflammation, airway hyperresponsiveness, and airway obstruction. These findings show the necessity for novel clinical trials that will investigate the role of interleukin 33 as a possible central mediator of asthma pathogenesis in humans. Understanding the possible role of interleukin 33 in promoting Th1 or Th17 Number of patients and demographic
differentiation clearly requires further work because existing evidence is not yet conclusive. The appendix pp 8 shows the outcomes of studies relating interleukin 33 to airway inflammation through T cell interactions.
Role of interleukin 33 on clinical outcomes Globally, asthma is one of the most common chronic diseases in children and adults. Although respiratory allergy and asthma are generally regarded as Th2mediated diseases, these associations have been challenged and therapies designed to suppress Th2 function are not effective for all patients. Interleukin 33 is a novel cytokine that has been strongly linked to the pathogenesis of both respiratory allergy and asthma and several human studies have confirmed this association.81,82 Genetic polymorphisms close to the IL33 gene have been associated with the incidence of nasal polyposis and with increased blood eosinophil numbers.6 Interleukin 33 protein was significantly increased in the nasal secretions of patients with Japanese cedar pollinosis at peak season, suggesting a possible relation with exacerbation of allergic rhinitis.83 Genome-wide association studies (GWAS) of asthma have consistently shown that polymorphisms in IL33 and IL1RL1 (ST2) genes are strongly related to increased susceptibility to asthma development,84–87 but not to decreased lung function as assessed by spirometry in patients with asthma.84 In a Dutch GWAS of asthmatic families, polymorphisms in adjacently located genes (IL33,
Variables measured
Outcome
p value
Soluble IL1R1 levels in serum and asthma prevalence (questionnaire); blood eosinophil counts at 4 years and 8 years of age and asthma prevalence (questionnaire)
Polymorphisms in ILRL1 gene associated with increased asthma prevalence at 8 years old; Polymorphisms in ILRL1 gene associated with soluble IL1RL1 concentrations and eosinophils counts
