Small animals models for drug discovery

Current and Future Models for Understanding Airway Diseases

Pulmonary Pharmacology & Therapeutics 24 (2011) 513e524

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Pulmonary Pharmacology & Therapeutics journal homepage: www.elsevier.com/locate/ypupt

Small animals models for drug discoveryq James G. Martin*, Mauro Novali Meakins Christie Laboratories, McGill University, 3626 St. Urbain St., Montreal, Quebec, Canada H2X 2P2

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 December 2010 Received in revised form 28 April 2011 Accepted 5 May 2011

There has been an explosion of studies of animal models of asthma in the past 20 years. The elucidation of fundamental immunological mechanisms underlying the development of allergy and the complex cytokine and chemokines networks underlying the responses have been substantially unraveled. Translation of findings to human asthma have been slow and hindered by the varied phenotypes that human asthma represents. New areas for expansion of modeling include virally mediated airway inflammation, oxidant stress, and the interactions of stimuli triggering innate immune and adaptive immune responses. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Asthma Inflammation Cytokines Chemokines Growth factors Remodeling Airway hyperresponsiveness Therapy

1. Introduction Small animal models have displaced larger animals for the study of potential pathophysiological mechanisms of asthma. The limitations imposed by size on the assessment of respiratory system mechanics and airway responsiveness to methacholine in small animals have been largely overcome by the availability of commercially available small animal ventilators which provide detailed information concerning the lung compartments contributing to the altered mechanical properties [1]. The large array of reagents for mice and to a lesser extent for rats has also facilitated the task of pathway exploration. The ability to probe the role of particular gene products through genetic manipulation of the mouse has perhaps been the single most cogent factor in promoting their use. Despite the advantages associated with the use of small animals to model asthma many investigators have lost sight of the fact that these animals may be a facsimile of asthma but only that. The increasingly liberal use of terms such as “murine asthma” may serve to enhance the chances of publication but obscures the fact that important differences between models and the true disease entity may be present. In addition to species differences in the simulated aspects of asthma, significant problems in the prediction

q Supported by the Canadian Institutes of Health Research. * Corresponding author. Tel.: þ514 398 3864x00137; fax: þ514 398 7483. E-mail address: [email protected] (J.G. Martin). 1094-5539/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.pupt.2011.05.002

of drug targets may have been the result of a narrow view of asthma pathogenesis. Indeed the limited success of animal models in the development of new modalities of treatment suggests as much. 2. What do we model in animals Asthma is a chronic inflammatory disorder of the airways that involves a variety of inflammatory and structural cells that interact to cause airway inflammation. Clinical consequences of airway inflammation include airway hyperresponsiveness, which in turn predisposes to recurrent episodes of wheezing, breathlessness, chest tightness, and coughing [2]. These episodes are usually associated with widespread, but variable, airflow obstruction within the lung which is often either spontaneously reversible or requires treatment. An additional element that is increasingly incorporated into the definition of asthma is airway remodeling. Airway inflammation that is typically eosinophil-rich and associated with T cell activation and airway hyperresponsiveness triggered by allergen sensitization and challenge have been adopted as the usual surrogates for asthma in animal models. Typical early and late responses to allergen challenge have also been documented in a variety of animal models [3e7] and remain a frequent experimental model in human subjects [8,9]. Other features of asthma are either not present or have not been looked at in any detail in small animals. An additional limitation of the small animal models is that they do not have a described clinical condition analogous to human

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asthma; only horses and cats have been described to develop an illness comparable to human allergic asthma [10,11]. Small animal models of asthma have been almost exclusively allergen-driven, although more recently the effects of respiratory viral infections have received more attention [12]. 2.1. Methodological considerations The tools available to measure lung mechanics and responsiveness to methacholine have evolved substantially in the past 15 years. There are several excellent reviews on the topic [13,14]. Noninvasive measurement are available to accommodate the need for repeated measurements although there is often only an indirect relationship of the outcomes to changes in respiratory mechanics [15]. These include barometric plethysmography (Penh system) that detects changes in breathing pattern and double chamber plethysmography that detects changes in mid-expiratory flow. This latter approach has been compared to concurrent estimates of lung resistance and elastance by measuring pleural pressure change with a catheter tip transducer system and has been shown to allow airway responses to be followed, albeit with less sensitivity to change [16]. Both of these non-invasive techniques are potentially confounded by upper airway reactions to allergen or methacholine. It has also been possible to repeatedly oro-tracheally intubate mice and measure respiratory mechanics [17] but few investigators have used this technique. Small animal ventilators equipped with software to model the respiratory system using the so-called constant phase model provide the greatest precision and also provide information about the site within the airway tree that has altered properties [18]. Other methodological considerations pertinent to these murine models is that they are generally acute and involve high level exposures for short periods. Variability in strains and sensitizationchallenge protocols used make short term asthma models difficult to compare. Sensitization protocols also vary with both systemic and intra-pulmonary routes employed. Antigen is administered via intranasal or intratracheal instillation but in high doses that activates acute inflammatory pathways that may differ from the acute on chronic inflammation that is typical of human asthma. Inflammation is also predominantly perivascular and peribronchial and the subepithelial compartrment is virtually non-existent in the murine. On occasion parenchymal inflammation and an associated pulmonary fibrosis is described, which is not typical of human asthma [19]. 2.2. Mechanisms of allergic sensitization Small animal models have contributed enormously to our understanding of the processes that lead to sensitization to antigens and to allergic inflammation. The success of the dissection of the immunological pathways involved is almost certainly related to the degree to which immunity has been conserved in mammals. The initial identification of CD4þ T cells subsets leading to distinct forms of inflammation, now termed Th1 and Th2 is attributable to research in the mouse [20]. The functional distinctions among T cell subsets have stood the test of time and have been expanded to include Th17, Th22 and Th9 cells [21e23]. The presence of such subsets has been established in human subjects although mixed T cell populations are well known to occur. The basis for most murine models of asthma is the creation of a systemic Th2 allergen-specific immune response prior to a pulmonary challenge with an antigen. The most frequently used model is based on administration of chicken egg ovalbumin (OVA) to systemically prime the mice towards developing OVA-specific immune responses followed by an aerosol challenge. This model

has been criticized as not using a pertinent aeroallergen and other allergens such as cockroach [24], ragweed [25] and dust mite [26] are increasing in popularity. However, none of the major surrogates for asthma have been shown to be affected differently when animals are sensitized and challenged with different allergens although major differences in gene expression have been reported. Inhaled allergen is taken up by dendritic cells that are numerous under the epithelium and these immature dendritic cells migrate to local lymph nodes [27] where they provide the opportunity for stochastic interactions with circulating T cells [28]. The cells mature en route and are efficient antigen presenting cells by the time they reach the lymph node. When the sampling of the dendritic cell surface by T cells results in a match between the MHC class II molecule on the dendritic cell surface bearing the antigenic peptide and the T cell receptor clonal expansion of the CD4þ T cell results. The T cells will then egress the lymph node and re-circulate before homing the mucosal surface of the airways. The factors that lead to a Th2 differentiation are several and include IgE on the dendritic cell surface [29], the secretion of thymic stromal lymphopoietin by the epithelium [30] or by basophils in the lymph node [31], through the activation of TLR2 by eosinophil derived neurotoxin [32] or other ligands [33] and the secretion of cysteinyl-leukotrienes by dendritic cells [34]. The synthesis of cysteinyl-leukotrienes by murine dendritic cells, triggered by antigens such as Aspergillus fumigatus and house dust mite, is dependent on dectin-2, a lectin receptor [35]. Semi-mature dendritic cells also reside in the airway mucosa and may interact closely with T cells in this location [36] (Fig. 1). When antigen is inhaled the rapidity of T cell-dependent airway responses suggests that dendritic cell-T cell interactions within the airway wall may short-circuit the usual antigen presenting mechanisms [37] that require migration of the dendritic cells to lymph nodes and local antigen presentation may lead to T cell reactions. 2.3. Early and late allergic responses Allergen challenge evokes early and late responses in rats that exhibit similar timing and biochemical mediators as do human airway responses [4]. Both are cysteinyl-leukotriene dependent [38] although the early response is serotonin and not histamine driven. Detailed kinetics of the production of cysteinyl-leukotrienes has demonstrated two peaks of leukotriene synthesis corresponding to the early and late responses [39]. The source of the leukotrienes responsible for the late response has never been determined although it has generally been assumed that eosinophils are the source. Cysteinyl-leukotrienes are not produced by rodent eosinophils [40] so an alternative cellular source is required. Perhaps as mentioned above dendritic cells may contribute to this second burst of cysteinyl leukotriene synthesis. The measurement of late responses, although a popular model for screening drug effects in human allergic subjects, is not common in small animals. Extensive studies have been performed on the rat using prolonged intubation and anesthesia. The initial promise of non-invasive respiratory function measurements for murine models has not been borne out by experience [15] and so following late responses in the mouse after allergen challenge is not popular. 2.4. Airway hyperresponsiveness Investigators have substituted airway hyperresponsiveness (AHR) to methacholine as a principal asthma surrogate in small animals. It should be kept in mind that this is not the same as the late allergic response although they have been correlated in human subjects [41]. The inhibition of one does not necessarily imply the inhibition of the other. Furthermore the mechanism of airway

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Fig. 1. Current and future targets for asthma based on adaptive immune mechanisms. CysLtr1: cysteinyl leukotriene receptor 1; TLR: toll like receptor; IL: interleukin.

hyperresponsiveness continues to elude investigators. However AHR is a complex process resulting from airway smooth muscle contraction, but is affected by parenchymal attachments that transmit the mechanical impedance exerted by the lung elastic recoil, mediators released by chronic inflammation and remodeling. Heterogeneous bronchoconstriction and airway closure may also affect AHR. Loss of airway surfactant may also be of importance in small animals since surface forces may promote airway closure in small airways [42]. Different mouse strains show different types of AHR: A/J mice show intrinsic AHR, possibly resulting from altered smooth muscle properties, while Balb/C mice show AHR after antigen sensitization and challenge, with inflammation, airway wall thickening and airway closure [43,44]. 2.5. Airway remodeling The most recent aspect of allergic asthma to be modeled in animals is the structural changes in the airway tissues that result from multiple allergen exposures. Although changes in the airway tissues have been recognized for more than a 100 years and careful morphometric studies were published as early as the mid-sixties [45,46] there has been a flurry of activity in this area in the past 10 years. Following epidemiological data linking sensitization and prolonged exposure to allergen to the development of asthma, investigators pursued the induction of airway hyperresponsiveness and its link to airway remodeling by repeatedly exposing sensitized rats [47e49] and, subsequently, mice to repeated aerosols of allergen [50,51]. Repeated high levels of exposure have generally been used with a view to the study of airway remodeling [50]. Airway smooth muscle hyperplastic growth, goblet cell differentiation, subepithelial fibrosis and proteoglycan deposition have all have been described following repeated allergen challenges

[52e54]. Features such as ASM growth and goblet cell differentiation in the BN rat require a few challenges only but are reversible changes [55]. Two weeks of alternate day exposure to antigen evoke an increase in fibronectin that is not reversible by steroid treatment although it is at least partially preventable when steroids are administered concurrently [56]. Remodeling in the mouse requires large numbers of challenges and several months of challenges have been used to induce long-lasting changes. In murine models of chronic antigen exposure it has been possible to obtain features of remodeling, but AHR does not invariably persist after the resolution of the acute inflammatory response. In a chronic challenge model proposed by Leigh et al remodeling and AHR persisted up to 8 weeks after the last challenge. Interestingly in this model the increase in extracellular matrix became significant after 4 weeks, when the inflammatory response was no longer evident [50]. In a similar long term model IL-13 was shown to be important for subepithelial matrix deposition and goblet cell differentiation [57]. Lack of IL-4 and IL-13 in mice appears to result in an increase in airway smooth muscle mass constitutively but allergen challenge does not further significantly increase its mass [58]. Persistent AHR is dependent on these cytokines. A chronic fungal model has also been established in mice and has features that are reminiscent of allergic bronchopulmonary aspergillosis and include aspects of airway remodeling such as goblet cell hyperplasia and subepithelial fibrosis and also demonstrates a prolonged AHR [59]. The roles of chemokines and TLRs have been thoroughly explored in this model. There is increasing recognition of the contribution of oxidative stress to the inflammatory response following allergen challenge. Many aeroallergens have intrinsic oxidant properties [60] and reactive oxygen species are generated by cells such as dendritic cells following allergen exposure [61]. There is also evidence of the

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induction of an asthma-like illness following substantial exposures to highly irritant or oxidant substances [62e64]. Models of irritant induced asthma have been developed using chlorine and ozone exposures [65,66]. Other stimuli may lead to activation of other T cell subsets such as the invariant natural killer cells and lead to forms of asthma that are also not dependent on adaptive immune mechanisms [67,68]. However there is a need to integrate the inflammatory mechanisms following airway injury with models based on adaptive immunity. 3. Drug targets suggested by animal models The study of the mechanisms of allergic sensitization, the consequences of allergen challenge for airway function and the complex cytokine and chemokine networks have identified a large number of biologically plausible targets for drug development (Fig. 1). The inhibition of allergen-induced airway hyperresponsiveness and eosinophilic airway inflammation by anticytokine treatments may be effective in the model systems but the findings may not translate well to a clinical context in human asthmatic subjects. The predictive value of the asthma surrogates employed in small animal models has not been clearly established. Indeed the modeling of clinical asthma with its diverse causes and triggers may not be achieved by use of allergen provocation models even in human subjects. 3.1. Targeting inflammatory cells Depletion and adoptive transfer experiments have been used to demonstrate the dependence of airway inflammation and airway dysfunction on various T cell subsets [37,69,70] (Table 1). The central place of the CD4þ T cell in allergic airway disease has been firmly established but important negative modulatory effects of CD8þ T cells and gamma delta T cell receptor bearing cells have also been shown, indicating a complex interplay of the different T cell subsets [68,71,72]. An added degree of complexity has been introduced by the identification and characterization of regulatory T cells. Adoptive transfer of naturally occurring Tregs suppresses AHR and airway eosinophilia following allergen challenge [73]. The absence of FoxP3, the transcription factor for Tregs is associated with severe inflammation in chronically challenged animals [74]. The kinetics of the appearance of Tregs after allergen challenge is more consistent with a role for these cells in the resolution of inflammation rather than in determining the intensity of the response to an acute challenge. Given the importance of CD4þ T cells in allergic disease it is perhaps surprising that CD4þ T celldeficient states such as human immunodeficiency virus infection causing lymphopenia have not been associated with a reduction in

airway hyperresponsiveness and asthma, suggesting preferential preservation of Th2 related processes [75]. Likewise the administration of potent lymphocytotoxic drugs such as rapamycin to transplant recipients is also associated with Th2 biased immunity. Trials of cyclosporine in humans have been unimpressive [76]. Corticosteroids have been shown to be efficacious in preventing cysteinyl-leukotriene synthesis and late allergic airway responses in the rat [77] and allergen-induced AHR in the mouse [78,79]. In this respect their therapeutic effects are similar to those in human subjects, similarly challenged [80]. Corticosteroids administered to animals prior to repeated allergen challenges also prevent airway remodeling [81]. These data lend support to the validity of the models. Therapies such as corticosteroids are administered to humans after the development of disease and there are scant data on their ability to reverse established changes. The administration of corticosteroids as therapy for asthma has served to validate models but their application in medicine antedated their study in current model systems. However the models have shed light on some of the mechanisms pertinent to the beneficial effects of steroids such as suppression of adhesion molecule expression and inhibition of cytokine, chemokine and mediator release. Although a minority of patients has refractory asthma these persons pose a significant clinical problem. Since they are, by definition, on steroids either by the inhaled or oral route without satisfactory control there is a need for better understanding of inflammatory processes in asthma that are relatively steroid unresponsive if we are to be able to resolve the issue of cure of asthma rather than palliation. However recent data on the role of Th17 cells in airway disease have demonstrated that following adoptive transfer of ovalbumin-specific Th17 cells ovalbumin induces neutrophilic inflammation and AHR that are steroid insensitive [82]. This is an advance on the previous descriptions of processes that were quite steroid-sensitive and opens up new areas for exploration with potentially high translational relevance. Sensitization to ovalbumin by the airways, when coupled with lipopolysaccharide, leads to weaker Th2 responses than intraperitoneal route of sensitization employed in mouse models but evokes a strong Th17 response and a neutrophil-dependent AHR [83]. Such models may provide information pertinent to alternative mechanisms of AHR, steroid insensitive processes and perhaps other asthma phenotypes. 3.2. Targeting adhesion molecules The recognition of the key role of adhesion molecules in allowing the egress of inflammatory cells from the circulation and their migration within the tissues has led to the study of these molecules as possible targets. In the late allergic airway response

Table 1 Cells as targets. AHR: airway hyperresponsiveness; mAB: monoclonal antibody; VLA-4: very late antigen; BALF: bronchoalveolar lavage fluid; FeNO: Fraction of Exhaled Nitric Oxide. Target

Animal model

Human asthma

CD4þ

Adoptive transfer of CD4þ T cells induces AHR and eosinophilia. Administration of anti-CD4 mAb before challenge prevented AHR and eosinophil infiltration. Antigen primed CD8þ ab T cells can transfer allergen sensitivity to naive rats and can mediate the development of late responses after allergen challenge. Adoptive transfer of ovalbumin-specific Th17 cells induces neutrophilic inflammation and AHR after ovalbumin challenge. In sensitized mice, depletion of gd T cells before airway challenge leads to an increase in AHR. Anti VLA-4 antibody significantly affects lung mechanics and eosinophilic count in BALF.

T cells are increased in asthmatic airways in proportion to disease severity. CD4þ T cell deficiency states have not been associated with a reduction in airways hyperresponsiveness and asthma. Lymphocytotoxic drugs are associated with Th2 biased immunity.

CD8þ

Th17

gd T cell VLA-4

No effect on AHR and FeNO

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the adhesion molecule, very late antigen 4 (VLA-4), an integrin involved in leukocyteeendothelial interaction has been shown to be involved through administration of a neutralizing antibody to ovalbumin-sensitized and challenged rats [84,85] (Table 1). This particular molecule was selected for targeting because of its expression by T cells and eosinophils [86]. The expression of its counter receptor VCAM-1 is also upregulated in human asthmatic subjects and correlated with IL-4 expression in the airways [87]. In OVA sensitized mice, anti VLA-4 administration before challenges significantly affected lung mechanics and the eosinophil count in bronchoalveolar lavage fluid [88]. VLA-4 antagonists failed to achieve significant results in a clinical trial on patients with mild intermittent asthma, suggesting that this molecule may not play a pivotal role in human allergen-induced AHR and inflammation [89]. There has been a decline in interest in this therapeutic area for asthma, perhaps for lack of success in the development of compounds with efficacy or perhaps because of potential side effects. 3.3. Targeting specific mediators A large body of data has been gathered on the pathobiological role of cysteinyl-leukotrienes in asthma through models (Table 2). Rat models have been used to show that cys-LTs are important mediators of early and late airway narrowing after allergen [39,90]. AHR in the mouse after allergen challenge can also be inhibited by cys-LTr1 antagonists [91], although administration of a cys-LT biosynthesis inhibitor failed to inhibit allergen-induced AHR [92]. Cys-LTs administered to the concomitantly challenged mouse augment eosinophilic inflammation and AHR [91]. Airway remodeling in the rat subjected to repeated allergen challenge is also cys-LT-dependent [93,94]. Mouse models have confirmed the dependence of remodeling on cys-LTs and have suggested that reversal of remodeling might be facilitated by a cysteinyl-leukotriene antagonist such as montelukast [95]. Recent data suggest that cys-LTs act mediate airway inflammation and airway remodeling through transactivation of the epidermal growth factor receptor [52,96]. Despite the work on remodeling in murine models no human trials of leukotriene receptor antagonists or 5-lipoxygenase inhibitors have been performed on human asthmatics addressing the issue of remodeling. Montelukast has

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been shown to reduce myofibroblast numbers in the airways in allergen challenged human subjects [97]. However whether reversal of established remodeling is a therapeutic possibility is not known and has been scarcely addressed even in animals. Despite the impressive role of cysteinyl-leukotrienes in murine models of asthma leukotriene modifiers have had limited success in the clinic. Recent publications demonstrating a constitutive ligand independent inhibitory effect of the receptor GPR17 on the response of the cysteinyl-leukotriene 1 receptor response to leukotriene D4 [98,99] suggests that further investigation of this pathway may provide insights into this issue in human asthma. 3.4. Targeting cytokines/chemokines Most of the work performed on the immunopathology of asthma in murine models has resulted in the identification of suitable targets for treatment by biologicals such as proinflammatory cytokines (Table 2). Monoclonal antibody neutralization of IL-4 during sensitization to allergen inhibits murine “asthma” indicating an importance of Th2 mediated mechanisms in the generation of allergic airway responses [100]. A soluble IL-4Ra has been developed to block the effect of IL-4. In an in vivo animal model soluble IL-4Ra blocks antigen induced IgE production and inhibit allergen-induced airway eosinophil infiltration, vascular cell adhesion molecule-1 (VCAM-1) expression and mucus hypersecretion [101] The administration of the IL-4 soluble receptor to human subjects has had a modest effect on chronic persistent asthma [102]. The link between airway eosinophilia and the symptoms and control of human asthma has led to an intense investigation of the factors controlling the recruitment and survival of eosinophils from the bone marrow to the lungs. The role of eosinophils in asthma continues to be debated and the pathobiological effects they may have are still not entirely clear. The eosinophil does not appear to be activated in the mouse in the same fashion as in human disease. However when eotaxin and IL-5 are co-expressed in the mouse airway pathology with the important features required of an asthma model are present [103]. Mice lacking eosinophils do not develop allergen-induced AHR or mucus cell differentiation [103]. Since the most important cytokines involved in eosinophilic inflammation is IL-5 the eosinophil has also been targeted through

Table 2 Cytokines/mediators as targets. Cys-LTs: cysteinyl-leukotrienes; CysLTr1: cysteinyl leukotriene receptor 1; IL: interleukin; AHR: airway hyperresponsiveness; FeNO: Fraction of Exhaled Nitric Oxide; IL-4Ra: interleukin 4 receptor a; VCAM-1: vascular cellular adesion molecule-1; TNF-a: tumor necrosis factor a. Target

Animal model

Human studies

Cys-LTs

Cys-LTr1 antagonists inhibit AHR, reduce eosinophilia, smooth muscle hyperplasia and subepithelial fibrosis.

IL-4

Soluble IL-4Ra blocks antigen induced IgE production, inhibit airway eosinophil infiltration, VCAM-1 expression and mucus hypersecretion. Anti IL-5 monoclonal antibodies effective against eosinophilia and antigen induced AHR.

Cys-LTr1 antagonists improve lung function. Reduce myofibroblasts number in airway of allergen challenged subjects. Soluble IL-4Ra has modest effect on chronic persistant asthma.

IL-5

IL-13

Anti IL-13 antibodies reduce inflammation and AHR.

TNF-a

TNF-a blockade reduced airway eosinophilia, mucus cells metaplasia and late phase response.

IL-9

Anti IL-9 antibodies reduce allergen-driven eosinophilia, IgE levels, epithelial damage. Anti IL-12 antibodies reduce Th2 response, eosinophilia and blood IgE.

IL-12

Anti IL-5 monoclonal antibodies reduce blood and sputum eosinophils and exacerbations in refractory eosinophilic asthma. Anti IL-13 antibodies have no effect on allergen-induced AHR. TNF-a blockade improve clinical control and AHR in patients with severe refractory asthma. No data Anti IL-12 antibodies reduce blood and sputum eosinophilia in patients with mild allergic asthma. No effect on AHR. High prevalence of adverse events.

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this molecule. Inhibition of IL-5 using monoclonal antibodies demonstrated efficacy against eosinophilia and antigen induced airway hyperresponsiveness in both acute and chronic mice asthma models [104]. There have been notable exceptions to these findings [100,105] and generally differences have been found when neutralization of IL-5 preceded the phase of sensitization to allergen. Neutralization of IL-5 at the time of and immediately before allergen challenge has led to reduced eosinophilia but has not altered AHR [78]. It appears that eosinophils are involved in the development of Th2 responses and are not necessarily direct mediators of AHR [106]. If this is correct then it is perhaps not surprising that anti IL-5 treatment has not produced an inhibition of late allergic responses and AHR in human subjects [107]. IL-5 has been shown in human subjects to be an important mediator of blood and sputum eosinophilia and the administration of IL-5 antibody has demonstrated a therapeutic effect in a subgroup of patients with refractory eosinophilic asthma, reducing levels of blood and sputum eosinophils, allowing a reduction of prednisone and reducing exacerbations [108,109]. Attempts to alter airway inflammation using an antisense (AS) phosphorothioate oligodeoxynucleotide (ODN) that inhibited transcription of the common bc of IL-3, IL-5, and GM-CSF receptors were successful in reducing allergen induced eosinophilia and AHR to LTD4 in ovalbumin-sensitized and challenged BN rats [110]. The potential utility of a topical antisense ODN approach targeting bc for the treatment of asthma led to the development of a compound (TPI ASM8) that contained two modified phosphorothioate antisense ODNs designed to inhibit human CCR3 and the common beta chain (bc). TPI ASM8 inhibited sputum eosinophilia. Curiously the drug treatment inhibited the early and not the late asthmatic response in human subjects [8]. Gene based strategies for allergic airway diseases largely developed through murine models have been extensively reviewed recently and show considerable promise [111]. There has been considerable experimental evidence supporting the role of IL-13 in asthma models [112e114]. This Th2 cytokine is of particular interest because of its broader role with effects on both immune cells and on structural cells such as epithelium, fibroblasts and smooth muscle [115e117]. IL-13 is closely related to IL-4, they both share a surface receptor IL-4Ra. This cytokine is critical to the synthesis of IgE is involved in eosinophil recruitment and promotes Th2 differentiation. IL-13 upregulates mucus genes in epithelial cells [118], promotes transforming growth factor-b production by epithelial cells that promotes fibroblast differentiation towards the myofibroblast [119]. IL-13 inhibition in a murine model reduces inflammation and AHR [114] whereas absence of the IL-13Ra2 receptor, an endogenous inhibitor of IL-13, augments inflammatory responses in the mouse OVA model [120]. Exogenous IL-13 induces AHR within a few hours before there is much evidence of inflammation and indeed mediates AHR induced by allergen challenge independently of eosinophilia [113]. Enhancement of ASM contractility through both calcium-dependent and calcium independent mechanisms may account for this finding [105,121,122]. Despite the substantial evidence of the role of IL-13 in allergeninduced AHR in murine models, clinical studies have failed to show a comparable effect of anti IL-13 therapies on allergen induced AHR in human atopic subjects [123] despite an effect on the late airway response [9,123]. Targeting IL4Ra1 receptor with a recombinant protein (Pitrakinra) that is an IL-4 mutein, which binds to the IL-4Ra subunit and prevents the inflammation induced by IL-4 and IL-13, is being explored. Two formulations of this compound, for inhalation and subcutaneous administration, are currently in Phase IIa trials showing promising results on late phase asthmatic responses [9]. Allergen induced AHR is also inhibited in a primate model [124].

TNF-a is involved in a variety of chronic inflammatory diseases and therapeutics targeting this cytokine have been a major advance in the treatment of rheumatoid arthritis, Cröhns disease and psoriasis. Different properties of this cytokine suggest its potential importance in the pathogenesis of refractory asthma such as neutrophil recruitment, induction of steroid resistance, and promotion of remodeling through TGF-b. The role of TNF-a has been supported by observations in a murine model. TNF-a blockade reduced eosinophilia, goblet cell differentiation and the late airway response determined from changes in enhanced pause (Penh) through a mechanism that involved cytosolic phopholipase A2 [125,126]. In a clinical trial involving patients with severe refractory asthma the upregulation of TNF-a axis has been observed, and in these patients administration of soluble TNF receptor improved clinical control, lung function and AHR [127]. A larger clinical trial which failed to reveal any benefit of treatment and there was an unacceptably high rate of adverse effects of antibody treatment against TNF-a [128]. The beneficial effect of TNFa suppression may be confined to patients with refractory asthma and highlights the need for careful patient selection in the application of this treatment. In vitro and in vivo studies have demonstrated the role of IL-9 mediating asthmatic allergic inflammation. This cytokine potentiates the Th2 response, amplifies mast cell mediator release and increases serum IgE. Transgenic IL-9 over-expressing mice have increased lung inflammation, increase numbers of mast cells in the airways, increases in IgE levels and AHR [129]. The effects of IL-9 over-expression were however mediated through IL-13 production by the epithelium [130]. Anti IL-9 antibody reduces allergendriven eosinophilia, IgE levels, AHR and epithelial damage in the mouse [131]. Similar results were not observed in IL-9 knockout mice and the authors concluded that IL-4 and IL-13 could compensate for the prolonged IL-9 deficiency [132]. Anti IL-9 antibody also reduces indices of remodeling in a chronically allergen exposed mouse model [133]. Expression of IL-9 mRNA is increased in asthmatic patients’ airways and correlates with loss of FEV1 and methacholine responsiveness [134]. Two phase I trials have shown an acceptable safety profile supporting further investigation of anti IL-9 in human disease [135]. Stem cell factor (SCF) has interest for asthma pathogenesis because of its effects on mast cells and eosinophils. Neutralization of SCF reduced peribronchial fibrosis, eosinophilic inflammation and AHR in a cockroach model of murine asthma, and concomitant reductions in the chemokines CCL6 and CCL17, and potential downstream effects on T cells recruitment [24]. Experiments on a murine model with prolonged Aspergillus infection and changes reminiscent of allergic bronchopulmonary aspergillosis have revealed substantive roles for certain chemokines and chemokine receptors in the asthmatic features seen in this model. CCR1 deficient mice had reduced Th2 cytokines and fewer goblet cells and subepithelial fibrosis [136]. CCR5 deficient mutants had less subepithelial fibrosis, goblet cell differentiation and AHR, particularly at late time points (21 and 40 days) after Aspergillus administration [137]. RANTES was shown to be responsible for the findings by immuno-neutralization. Eotaxin, in contrast was involved only in the immediate AHR and eosinophilia that followed administration of Aspergillus [138]. Clinical trials of chemokine receptor antagonists may reveal subsets of patients with asthma that are responsive to these agents. CCR4 that appears to mediate the chemoattraction of Th2 cells in response to CCL17 synthesis in the airways in human allergic asthma [139] also appears to have promise in a murine model. Its inhibition in a humanized severe combined immunodeficient mouse model driven by house dust mite allergen leads to an inhibition of airway eosinophilia, IgE, goblet cell hyperplasia and AHR [140].

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3.5. Strategies based on immune-biasing Murine models have also shown clearly the inhibitory potential of Th1 cytokines for Th2 responses. IL-12 production by dendritic cells is key in directing Th1 cell differentiation [112]. IFN-g is also inhibitory of IgE synthesis and deficiency of IFN-g or its transcription factor Tbet leads to exaggerated Th2 responses [141]. Ovalbumin challenged mice show inhibition of AHR and airway eosinophilia by IL-12 administration [142,143]. Peripheral blood mononuclear cells from asthmatics showed that IL-12 reduces IL-5 production and upregulates IFN-g production [144]. Attempts to suppress the Th2 response through the administration of IFN-g and IL-12 have not been very successful in clinical studies and, in the case of IL-12, associated with a high prevalence of adverse events [145]. Subcutaneous administration of human recombinant IL-12 in patients with mild allergic asthma reduced blood and sputum eosinophilia but had no effect on AHR to histamine and late asthmatic response after inhalational allergen challenge [146]. Perhaps IL-12 administered during sensitization might inhibit Th2 shift but is difficult to test. An additional problem is that exogenous cytokine may target many cells when a precise delivery to the desired site of action is necessary. An alternative treatment option for allergic asthma could be the use of unmethylated cytosine-guanine dinucleotides (CpG) to skew the immune response away from Th2 to Th1. Epidemiological studies prompted the hygiene hypothesis in asthma pathogenesis, motivating attention to bacterial products and TLRs as a means to suppress allergic airway inflammation. CpG motifs are presents in bacterial DNA but not in vertebrate DNA and synthetic oligodeoxynucleotides containing CpG motifs (CpG ODN) have immunostimulatory activity through TLR-9 [147]. In mouse models systemic administration of CpG DNA causes Th1 rather than a Th2 immune

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response to schistosome eggs, preventing eosinophilic inflammation in sensitized animals. Alterations in dendritic cell antigen presentation to Th2 cells and inhibition of IgE mediated Th2 cytokine release appear to explain the effects of these immune stimulatory sequences [148]. CpG in combination with ragweed antigen Amb a1 has been tested in allergic rhinitis and at the end of the ragweed season the subjects showed evidence of benefit [149]. However administration of immunostimulatory sequences to human allergic asthmatic subjects did not affect airway responses to allergen challenge despite induction of interferon-inducible genes [150]. Studies of a synthetic TLR7/8 agonist, S28463 (resiquimod, R-848) in the rat and mouse have shown marked antiinflammatory effects and prevention of airway remodeling in response to repeated allergen challenges [151,152]. It has also been possible to target TLR on IL-13 responsive cells with a bacterial product, Pseudomonas exotoxin, fused to IL-13 and to ameliorate features of a murine model of allergic bronchopulmonary aspergillosis [153]. The vast majority of studies in models study interventions prior to sensitization with allergen or challenge but models of reversal of established disease would be more relevant to clinical practice. In a recent study the intranasal administration of pneumococcal conjugate vaccine to OVA sensitized and challenged mice suppressed eosinophilic and Th2 mediated inflammation, AHR, circulating IgE and mucus secretion [154]. This treatment was effective in suppressing airway disease both when administrated during sensitization or after animals have received OVA challenges and were then re-challenged at a later time point, a so-called established airway disease model. The mechanism of action appeared to involve the induction of regulatory T cells. Similar actions seem to result from pneumococcal infection of mice or treatment with heat-killed bacteria [155].

Table 3 Other targets. AS ODN: antisense oligodeoxynucleotide; bc: Beta chain; GM-CSF: granulocyte macrophage colony stimulating factor; AHR: airway hyperresponsiveness; LTD4: Leukotriene D4; BN: Brown-Norway; CpG: cytosine-guanine dinucleotides; TLR: toll like receptor; MAPK: miogeno-activated protein kinase; ERK: extracellular signalregulated kinase; BALF: bronchoalveolar lavage fluid; OVA: ovalbumin; EGFR: epithelial growth factor receptor; S1P: sphingosine 1-phosphate; SphK: sphingosine kinase. Target

Animal Model

Human asthma

Antisense phosphorothioate oligodeoxynucleotide

AS phosphorothioate ODN that inhibit transcription of the common bc of IL-3, IL-5, and GM-CSF receptors were successful in reducing allergen-induced eosinophilia and AHR to LTD4 in ovalbuminsensitized and challenged BN rats In mouse models CpG DNA causes Th1 response to schistosome eggs, preventing eosinophilic inflammation in sensitized animals. A synthetic TLR7/8 agonist showed marked anti-inflammatory effects and prevented airway remodeling in response to repeated allergen challenges in rats. A MAPK/ERK kinase inhibitor was effective in reducing BALF inflammation, serum level of total and specific IgG and IgE, AHR in OVA challenged mice. Inhibition of p38 pathway reduces airway eosinophilia in OVA sensitized and OVA challenged mice. c-Jun N terminal kinase inhibition reduced AHR, smooth muscle proliferation, cellular infiltration and IgE production in mouse and rats models of asthma. Inhibition of EGFR receptor tyrosine kinase activation in the airways inhibits mucus synthesis, goblet cell hyperplasia and smooth muscle hyperplasia in the repeatedly allergen challenged rat. Nebulized administration of SphK inhibitors before OVA challenge decrease S1P levels in BALF, eosinophil infiltration, peroxidase activity and AHR. Similar treatment after OVA inhalation reduces AHR but has no effect on eosinophil infiltration

AS ODNs designed to inhibit human CCR3 and the common bc inhibited sputum eosinophilia. Inhibited early but not late asthmatic response.

Unmethylated cytosineguanine dinucleotides

MAPK/ERK kinase inhibitors

p38 c-Jun

EGFR

S1P

CpG in combination with an antigen benefited subjects with allergic rhinitis, but did not affect the airway response to allergen challenge.

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3.6. Targeting growth factors and signaling pathways Signaling pathways involved in immune responses have the potential to be useful downstream targets for therapy (Table 3). A limitation of such targets is the multiple roles that these molecules may have in cell functions, increasing the probability of off-target effects. The topic of kinase signaling pathways as targets in asthma has been reviewed recently [156]. A specific MAPK/ERK kinase inhibitor was effective in reducing BAL inflammation, serum levels of total and specific IgE and IgG1 and AHR in OVA challenged mice [157]. The inhibition of p38 pathway has also received attention and its inhibition reduces airway eosinophilia after OVA challenge in OVA sensitized mice and in guinea pigs after leukotriene D4 inhalation [158]. c-Jun N terminal kinase inhibition has been tested on mouse and rat models of asthma, reducing AHR, smooth muscle proliferation, cellular infiltration and IgE production [159,160]. Spleen tyrosine kinase (Syk) may also provide a target for the inhibition of allergen-driven airway inflammation that is mast cell driven [161,162]. The epidermal growth factor receptor is over-expressed in asthma [163] and is induced by allergen challenge in the rat [164]. Inhibition of EGFR receptor tyrosine kinase activation in the airways inhibits mucus synthesis, goblet cell hyperplasia and smooth muscle hyperplasia in the repeatedly allergen challenged rat [52]. Over-expression of platelet derived growth factor (PDGF) by adenoviral transduction in the mouse leads to airway smooth muscle growth, and PDGF is elevated in the BALF after prolonged allergen challenges [165]. No selective inhibitor of the tyrosine kinase is available to probe the role of PDGF at present. Overexpression of vascular endothelial growth factor in the mouse supports a role for this molecule in Th2 type inflammation and vascular and other remodeling [166]. Sphingolipids metabolites such as ceramide and sphingosine 1-phosphate (S1P) are important signaling molecules involved in

cell differentiation, proliferation, migration and apoptosis. S1P levels are regulated via sphingosine kinase stimulation (SphK) and are increased in BALF from allergen challenged asthmatic subjects. S1P stimulates human ASM cell proliferation in vitro, suggesting a potential role in remodeling [167]. SphK activity is increased in the OVA sensitized and challenged mouse. Nebulized administration of SphK inhibitors before OVA challenge decrease S1P levels in BALF, eosinophil infiltration, peroxidase activity and AHR. Similar treatment after OVA inhalation reduces AHR but has no effect on eosinophil infiltration [168]. S1P mediates the egress of T cells from the lymph node [169] and stable analogs of S1P such as FTY 720 may have the potential to provide a measure of useful immunosuppression in asthma. However, unwanted side effects may prevent development of this target for asthma. 3.7. Other strategies Animal models have been valuable not only for allergen induced asthma, but also to understand the role of oxidative stress in asthma pathogenesis. Mice exposed to different concentrations of irritants such as ozone or chlorine have developed a pathology that seems to model irritant induced asthma [65,66,170]. Oxidative stress is a key component in these forms of irritant induced asthma and the effectiveness of anti-oxidant treatment has been demonstrated in these animal models [65,171]. Invariant NKT cells mediate ozone-induced AHR in the mouse by mechanisms that involve IL-17 [66]. In clinical studies an inverse correlation between circulating anti-oxidant levels and asthma control has been shown and there is increasing interest in oxidant stress associated with allergic asthma [172] and exercise induced asthma [173]. Countering oxidant stress may ameliorate allergen-driven responses in mouse models. Overexpressing of thioredoxin, en endogenous anti-oxidant protein, reduces AHR, downstream of Th1 or Th2 cytokines [174]. Administration of thioredoxin also reduces airway inflammation,

Fig. 2. Other current and future targets. CysLtr1: cysteinyl leukotriene receptor 1; CysLTs: cysteinyl-leukotrienes; TLR: toll like receptor; ROS: reactive oxygen species; EGFR: epithelial growth factor receptor; HB-EGF: eparin binding epithelial growth factor; TGF-b: transforming growth factor beta; iNKT: invariant natural killer T cell; IL: interleukin.

J.G. Martin, M. Novali / Pulmonary Pharmacology & Therapeutics 24 (2011) 513e524

remodeling and AHR in the context of a more chronic model [174]. Products of oxidation may signal through transient receptor potential channels promoting inflammatory responses [175] (Fig. 2). Inhibitors of these channels may serve a useful function in preventing neurogenic inflammation in airway disease. Neurokinins are among the downstream mediators of neurogenic inflammation and may prove useful if and when effective antagonists are available for human subjects. Murine models support an important role for neurokinins [176] but quantitative species differences may lessen their importance in human. 4. What should we model in the future The success of animal models in predicting useful targets for asthma has been quite limited. Some of these limitations have been reviewed elsewhere [177]. However the limitations seem less related to the species differences in biological processes than to the failure to choose appropriate models for asthma. Much of the symptomatology of asthma does not appear to be allergen-driven although most models have relied on the similarity between the inflammation evoked by allergen in animals and human asthma. There seems to have been a longstanding, if somewhat implicit, view that allergen-induced late responses may be equivalent to spontaneous asthma. Viral infections frequently trigger significant asthma attacks and other stimuli which trigger bronchoconstriction such as strong odors and perfumes likely do so through activation of transient receptor potential channels, causing neurogenic inflammation [178]. The innate immune system is activated by a variety of signals, including ligands derived from tissue damage [179]. Many irritants cause oxidative stress and epithelial responses to these irritants need to be characterized. Models based on other stimuli need to be employed in exploring new targets. In testing the potential for drug efficacy in human asthma other models than allergen challenge with late responses and AHR should be considered. 5. Conclusions Using animal models, the dissection of the pathways of inflammation pertinent to human asthma has been possible. The translational potential of this research has only just begun. The limitations of model systems need to be kept in mind and greater focus on realistic models of human asthma is required. There is a substantial lag-time between the identification of a potential target and the application of the knowledge for therapeutic purposes. The complexity and multiple phenotypes contained in the term asthma are adding to the difficulty in advancing in the field. Failure of therapies in clinical trials may in some cases have resulted from inadequacy of the model system but may also reflect the failure to select the appropriate phenotype for study. The increasing focus on innate immune mechanisms of airway inflammation may prove to be fruitful in identifying further targets for testing. References [1] Schuessler TF, Bates JH. A computer-controlled research ventilator for small animals: design and evaluation. IEEE Trans Biomed Eng 1995;42:860e6. [2] Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease (COPD) and asthma. This official statement of the American Thoracic Society was adopted by the ATS Board of Directors. Am Rev Respir Dis November 1986;1987(136):225e44. [3] Shampain MP, Behrens BL, Larsen GL, Henson PM. An animal model of late pulmonary responses to alternaria challenge. Am Rev Respir Dis 1982;126: 493e8. [4] Eidelman DH, Bellofiore S, Martin JG. Late airway responses to antigen challenge in sensitized inbred rats. Am Rev Respir Dis 1988;137:1033e7.

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