Immunopharmacology and inflammation

Anti-inflammatory actions of Chemoattractant Receptor-homologousmolecule expressed on Th2 by the antagonist MK-7246 in a novel ratmodel of Alternaria alternata elicited pulmonary inflammation

Malgorzata A. Gil a, Michael Caniga a, Janice D. Woodhouse a, Joseph Eckman a,Hyun-Hee Lee b, Michael Salmon b, John Naber c, Valerie T. Hamilton d, Raquel S. Sevilla e,Kimberly Bettano f, Joel Klappenbach f, Lily Moy a, Craig C. Correll b, Francois G. Gervais f,Phieng Siliphaivanh g, Weisheng Zhang e, Jie Zhang-Hoover a,Robbie L. McLeod a, Milenko Cicmil a,n

a Pharmacology, Merck Research Laboratories, Boston, MA 02115, USAb Biology Discovery, Merck Research Laboratories, Boston, MA 02115, USAc Discovery Pharmaceutical Sciences, Merck Research Laboratories, Boston, MA 02115 , USAd Safety Assessment and Laboratory Animal Sciences, Merck Research Laboratories, West Point, PA 19486, USAe Imaging, Merck Research Laboratories, Boston, MA 02115, USAf Target & Pathway Biology, Merck Research Laboratories, Boston, MA 02115, USAg Discovery Chemistry, Merck Research Laboratories, Boston, MA 02115, USA

a r t i c l e i n f o

Article history:Received 20 May 2014Received in revised form11 September 2014Accepted 15 September 2014Available online 23 September 2014

Keywords:Alternaria alternataCRTH2MK-7246Brown Norway Rat

a b s t r a c t

Alternaria alternata is a fungal allergen linked to the development of severe asthma in humans. In view ofthe clinical relationship between A. alternata and asthma, we sought to investigate the allergic activity ofthis antigen after direct application to the lungs of Brown Norway rats.

Here we demonstrate that a single intratracheal instillation of A. alternata induces dose and timedependent eosinophil influx, edema and Type 2 helper cell cytokine production in the lungs of BN rats.

We established the temporal profile of eosinophilic infiltration and cytokine production, such asInterleukin-5 and Interleukin-13, following A. alternata challenge. These responses were comparable toOvalbumin induced models of asthma and resulted in peak inflammatory responses 48 h following asingle challenge, eliminating the need for multiple sensitizations and challenges. The initial perivascularand peribronchiolar inflammation preceded alveolar inflammation, progressing to a more sub-acuteinflammatory response with notable epithelial cell hypertrophy. To limit the effects of an A. alternatainflammatory response, MK-7246 was utilized as it is an antagonist for Chemoattractant Receptor-homologous molecule expressed in Th2 cells. In a dose-dependent manner, MK-7246 decreasedeosinophil influx and Th2 cytokine production following the A. alternata challenge. Furthermore,therapeutic administration of corticosteroids resulted in a dose-dependent decrease in eosinophil influxand Th2 cytokine production.

Reproducible asthma-related outcomes and amenability to pharmacological intervention by mechan-isms relevant to asthma demonstrate that an A. alternata induced pulmonary inflammation in BN rats isa valuable preclinical pharmacodynamic in vivo model for evaluating the pharmacological inhibitors ofallergic pulmonary inflammation.& 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND

license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

Contents lists available at ScienceDirect

journal homepage: www.elsevier.com/locate/ejphar

European Journal of Pharmacology

http://dx.doi.org/10.1016/j.ejphar.2014.09.0210014-2999/& 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

Abbreviations: A. alternata, Alternaria alternata; OVA, Ovalbumin; Th2, Type 2 helper cells; CRTH2, Chemoattractant Receptor-homologous molecule expressed on Th2 cells;MK-7246, {(7R)-7-[[(4-fluorophenyl)sulfonyl](methyl)amino]-6,7,8,9-tetrahydropyrido[1,2-a]indol-10-yl}acetic acid; ST2 (IL-1R1), interleukin-1 receptor family member;ILC2, innate lymphoid cells type 2; IL-4, interleukin-4; IL-5, interleukin-5; IL-25, interleukin-25; IL-18, interleukin-18; IL-6, interleukin-6; IL-1 β, interleukin-1 β; PAR-2,protease-activated receptor 2; BN, Brown Norway; BAL fluid, bronchoalveolar lavage fluid; PBS, phosphate buffered saline; PGD2, prostaglandin D2; MIP-1α, macrophageinflammatory Ppotein-1; RANTES, regulated on activation, normal T cell expressed and secreted; MCP-1, monocyte chemotactic protein-1; AFA, adaptive focused acoustics;TSLP, thymic stromal lymphopoietin

n Corresponding author. Tel.: þ1 617 992 2395.E-mail address: milenko.cicmil@merck.com (M. Cicmil).

European Journal of Pharmacology 743 (2014) 106–116

1. Introduction

Asthma is a heterogeneous respiratory disease with a complexpathophysiology. It continues to be a significant societal health burden,utilizes a large amount of health resources, and diminishes quality oflife (Adcock et al., 2008; Bel, 2013). Particularly noteworthy are a smallpercentage of patients with severe symptoms leading to significantairway obstruction, which require frequent hospital admissions due toexacerbated asthma symptoms and are refractory to standard of caredrugs. While these refractory asthmatics represent only a smallpercentage of asthmatic patients, the medical costs for treatingrefractory asthmatics represents about 50% of the total healthcare costfor asthma (Jang, 2012). The disease is characterized by a persistentTh2 inflammation, airflow obstruction, bronchoconstriction and air-way hyperresponsiveness (Howarth, 1995). While “asthma triggers”are loosely defined as substances or events that precipitate an acuteonset or worsening of asthma symptoms (Vernon et al., 2012), thetemporal understanding of the pathways by which these triggers driverespiratory pathophysiology are not fully understood. Nonetheless, it iswell accepted that exposing asthmatics to aeroallergens, viruses, andrespiratory irritants leads to an aggravation of the disease (Kim andGern, 2012). In recent years, fungi sensitization has emerged as animportant potential contributor to allergic lower airway disease(Knutsen et al., 2012). Indeed, patients with allergic asthma oftenhave detectable levels of serum IgE antibodies to fungi (Crameri et al.,2009). Schwartz et al. (1978) was the first to describe a relationshipbetween Aspergillus skin test reactivity and the severity of airwayobstruction in asthmatic patients. Subsequently, links between sensi-tization of various fungi (e.g. Alternaria, Aspergillus, Aureobasidium,Cladosporium, Helminthosporium and Trichophyton) to respiratorypathology were suggested (Peat et al., 1993; Neukirch et al., 1999; Fairset al., 2010; Agarwal, 2011). For instance, Arbes et al. (2007) found thatA. alternata is independently associated with asthma cases attributableto atopy. Further support for a relationship between A. alternata andasthma includes the following observations: (1) Childhood sensitiza-tion to A. alternata correlates with persistent adult asthma (Stern, et al.,2008); (2) sensitization to A. alternata is associated with fatal episodesof asthma (Arbes et al., 2007; Knutsen et al., 2010), (3) when delivereddirectly to the airways, A. alternata spores induce brochoconstriction inpeople with mild asthma (Licorish et al., 1985) and (4) environmentalfluctuations in A. alternata spore counts appear to associate withsymptom severity, as observed in the cases of “thunderstorm asthma”,where there is a significant rise in this airborne antigen (Pulimoodet al., 2007; Nasser and Pulimood, 2009).

The mechanism(s) and pathways by which A. alternata initiatesrespiratory inflammation in sensitized patients are not fullyunderstood. However, it is known that certain key immune cellssuch as lung epithelial cells, dendritic cells, and eosinophils likelyplay a pivotal role in orchestrating a Th2-mediated inflammationprecipitated by lung exposure to A. alternata antigens. Murai et al.(2012) demonstrated that A. alternata extracts in cultured humanbronchial epithelial cells induce the rapid release of Interleukin-18(IL-18) which can directly promote Th2 differentiation of naïveCD4þ T-cells through a NF-κB dependent pathway (Murai et al.,2012). Another route through which A. alternata and other fungimay induce Th2 inflammation is by the activation of a protease-activated receptor, particularly Protease Activated Receptor-2(Matsuwaki et al., 2011). In view of the clinical relationshipbetween A. alternata and asthma, we sought to characterize theactivity of this antigen after direct application to the lungs of BNrats. In this study, we describe a novel Th2-mediated model ofpulmonary inflammation due to the acute intratracheal instillationof A. alternata in Brown Norway rats. In addition, we conductedpharmacological validation of this model using clinically relevantdrugs, such as glucocorticoids and a CRTH2 antagonist, MK-7246(Gervais et al., 2010).

2. Materials and methods

2.1. Animal care and use

All studies were performed in accordance with the NationalInstitute of Health Guide to the Care and Use of Laboratory Animalsand the Animal Welfare Act, in Association with the Assessmentand Laboratory Animal Care Program. All experiments wereapproved by Merck Institutional Animal Care and Use Committee.

2.2. Characterization of A. alternata evoked pulmonaryinflammation responses in brown Norway rats

Male Brown Norway rats (225–275 g) were purchased fromCharles River Laboratories (Kingston, NY) and housed in an animalroom at a temperature of 25 1C (75 1C), relative humidity:30–70%, a daily light–dark cycle (07:00 to 19:00 light). Chow andwater were supplied ad libitum and the animals were studiedbetween 80 and 85 days of age. Experimental rats were intra-tracheally challenged with A. alternata extracts (Greer Labs, Lenoir,NC) in 100 μl sterile phosphate buffered saline (PBS), using a Penn-Century microsprayer device (Penn-Century, Philadelphia, PA).A series of experiments were conducted to examine: (1) theimpact of A. alternata on respiratory inflammation, (i.e. total BALfluid cells, eosinophils and neutrophils) after topical administra-tion to the lungs (n¼8 animals per treatment group); (2) thetemporal relationship between Th2 cytokine secretion and inflam-matory cellular influx into the BAL fluid after a single intratrachealA. alternata challenge (n¼8 animals per treatment group); (3) his-tological and CT scan observations after an A. alternata challenge(n¼6 animals per treatment group); and (4) pharmacologicalintervention of A. alternata responses by classical glucocorticoids(i.e. budesonide) and by MK-7246, a selective orally active CRTH2antagonist (n¼8 animals per treatment group).

2.3. Impact of A. alternata on respiratory inflammation and thetemporal relationship of Th2 cytokine secretions

To determine if rats that were given increasing doses (500–2000 μg) of intratracheal A. alternata induced a more severeinflammatory response, BAL fluid cell composition was analyzedat 24 and 48 h post-provocation. BAL fluid collection and proces-sing were carried out using the methods previously described(Lieber et al., 2013). Briefly, after an overdose (150–200 mg/kg viaintratperitoneal injection) of pentobarbital, the trachea was cathe-terized, BAL fluid was obtained by the intratracheal insertion of acatheter, followed by two lavages with 3 ml 0.9% sodium chlorideinjection, USP (Hospira, Lake Forest, IL). One aliquot (�2 ml), wasanalyzed for total and differential cell counts performed using anAdvia 120 Automated Hematology Analyzer (Siemens, WashingtonDC). For some studies, a small BAL fluid aliquot (�0.5 ml) wascollected for cytokine analysis. Specifically, BAL fluid samples werecentrifuged at 1500g, 4 1C for 10 min to eliminate cellular debrisand supernatant was aliquoted and frozen at �80 1C for furtheranalysis. Once defrosted, a Luminex-Milliplex MAP magnetic bead-based Rat 22-plex immunoassay (Millipore, Billerica, MA) analysisof BAL fluid supernatants was performed following manufacturer'sdirections. We also utilized a Meso Scale Discovery (MSD, Rock-ville, MD) Rat 7plex ultra-sensitive kit for a panel of certaincytokines. ELISA for IL-13 (Invitrogen, Grand Island, NY) wasperformed on the BAL fluid supernatant according to the manu-facturer's instructions and all ELISA plates were read with a BioRadModel 680 microplate reader.

In separate studies, the time related (i.e. 24, 48, 72 and 96 h)effects of A. alternata on BAL fluid cells and cytokines were deter-mined. Additional characterization of the effects of locally applied

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A. alternata to the airways was studied, utilizing histological andimaging methodologies (see below).

2.4. Pharmacological actions of glucocorticoids and MK-7246 onpulmonary inflammation responses in Brown Norway rats

Intratracheal budesonide was dosed 1 h prior to and 23 h post theA. alternata intratracheal dose while oral budesonide (3 mg kg�1)was administered 2 h before and 22 h post the A. alternata extractinstillation. An intratracheally dosed budesonide was prepared usingthe Covaris formulation as described (Kakumanu and Schroeder,2012). Covaris AFA is an in-situ method which allows the construc-tion of tightly distributed suspensions, achieving micron sized(1–10 μm) suspensions using Covaris Focused-ultrasonicator E220.MK-7246 (3, 10, 30 and 100 mg kg�1) was administered orally 1 hbefore and 23 h post an A. alternata extract instillation in order toexamine the effect of the CRTH2 antagonist on A. alternata elicitedpulmonary inflammatory responses. Budesonide dosed orally wasused as a positive control in both experiments. The animals werelightly anesthetized with 3% isoflurane (supplemented with 100%oxygen), either 2 h following an oral dosing or 1 h following anintratracheal dosing. The animals were also secured on a rodent workstand to facilitate the localization of the larynx and tracheal openings.The microsprayer needle was inserted into the trachea and 0.1 ml of10,000 μg ml�1 (total of 1000 μg) A. alternata extract was adminis-tered using a microsprayer. The animals were observed until theyrecovered from anesthesia and then returned to their cages andallowed food and water ad libitum. The animals were euthanizedwith an overdose of pentobarbital 48 h after an A. alternata intra-tracheal instillation and BAL fluid was collected.

2.5. Micro-CT imaging acquisition, image reconstruction, andanalysis

CT scans were performed on rats anesthetized with 3% isoflur-ane (supplemented with 100% oxygen), using a GE eXplore LocusUltra Pre-Clinical CT scanner (GE Healthcare, Cleveland, OH, USA).Rats were imaged one at a time. The entire chest area was scannedat once, with 1000 projections collected in one full rotation of thegantry in approximately 16 s. The X-ray tube settings were 80 kVand 70 mA and the isotropic resolution was 185 μm.

Projection data was reconstructed using the graphics proces-sing unit (GPU)-based AxRecon reconstruction platform (Accele-ware, Inc., Ontario, Canada). The three dimensions of the imagedata set (x, y, and z), were manually adjusted to 600�500�800slices at 100 μm cubic voxel dimensions for reconstruction andimages were scaled to Hounsfield units. The reconstructed datawas then cropped into the easier-to-manage Analyze format usinga MATLAB (MathWorks, Inc., Natick, MA) application that wasdeveloped to automate this conversion process.

An image analysis method we used was based on a methodpublished by Haines et al. (2009) that relies on thresh-holding andregion-grow algorithms to segment separate anatomical regions ofinterest using the Amira 5.4.2 software (Mercury Computer Systems,Inc., Chelmsford, MA). We adjusted the functional lung volume from�1000 to �300 since the current studies utilized a rat instead of amouse model. After the functional volume was recorded, the totalchest space was selected using semi-automated contouring andmanual selection, eliminating the heart and liver. Vasculature foundin the lung was included in this total lung measurement, sinceedema and vascular tissues have similar grayscale values and cannotbe differentiated based on threshold. Once the total chest space wasdetermined for each animal, the functional chest space was sub-tracted from that volume, leaving the intentionally selected vascu-lature, as well as edema. The final readout, (Edema volume withvasculature¼Total chest volume� functional lung volume), was the

edema plus the vascular tissue, which should remain relativelyconstant given the weeklong timeframe of this type of study.

2.6. Histopathological analysis

Lungs from 6 rats/group were obtained at the scheduledterminations of 6, 24, 48, or 72 h following the challenge, alongwith lungs from naïve animals not administered A. alternataantigen or lavaged, but housed concurrently with the treated rats.Lungs from all dose groups except the naïve group were lavagedand infused with 10% neutral buffered formalin. Lungs from naïverats were only infused with 10% neutral buffered formalin. Afterapproximately 24 h, lung sections from all lobes were processed todemonstrate maximum longitudinal sectioning of the right caudallobe and representative sections of the remaining right and leftlung lobes along with major bronchi and trachea. Sections wereprocessed and embedded in paraffin. Embedded tissues were cutinto 4–6 μm sections and stained with hematoxylin and eosin(H&E). A histologic evaluation was performed on stained tissuesections from multiple lobes and all the rats. The histology scorefor each rat was based on alveolar damage, including alveolarleukocyte infiltration and edema, epithelial cell hypertrophy, alongwith perivascular and peribroncheolar cuffing and an estimate ofthe area involved with disease. Grading was based on a severityscore scale of 0–5: 0 (no observable pathology), 1 (minimal or veryslight), 2 (mild or slight), 3 (moderate), 4 (marked), or 5 (severe).The evaluation also included scoring any pre-existing chronicbackground inflammation present in the Brown Norway rat model(Noritake et al., 2007) and noted here in all the rats, including thenaïve rats, which was separately graded to avoid influencing theevaluation of damage due to the A. alternata antigen.

2.7. Statistical analysis

Statistical analysis was performed using Prism Software (Graph-Pad Software, Inc., La Jolla, CA). All measures were analyzed via one-way or two-way ANOVA, with post-hoc Bonferroni analyses per-formed on measures that showed significant (Po0.05) overall effect.

2.8. Drugs

Budesonide was purchased from Sigma Chemical Co. (St. Louis,MO, USA). MK-7246 was synthesized at Merck Research Laboratories.Budesonide was dissolved in physiological (0.9%) salineþ0.5% Tween80 (for intratracheal dosing) or 0.5% methylcelluloseþ0.24% sodiumdodecyl sulfate (for oral dosing). MK-7246 was dissolved in 0.5%methylcelluloseþ0.24% sodium dodecyl sulfate.

3. Results

3.1. A. alternata induces dose and time-dependent eosinophil influxin the lungs of brown Norway rats

We first determined the cellular profile of BAL fluid in BN rats,following a single intratracheal challenge of A. alternata. Time anddose-dependent increases in the number of total cells wereobserved in the BAL fluid recovered from BN rats (n¼8 per group)following the intratracheal challenge with A. alternata (Fig. 1A).There was a dose-dependent increase in the number of eosinophilsin the A. alternata treated rats, with maximal numbers at 48 h with492�1037108 cells ml�1 compared to 21�10374 cells ml�1 inthe phosphate buffered saline (PBS) treated group (Po0.05)(Fig. 1B). A dose related increase in neutrophils levels was alsoobserved reaching a peak levels at 24 h and were 5 fold less thanthe eosinophils, reaching a maximum of 80�103720 cells ml�1

M.A. Gil et al. / European Journal of Pharmacology 743 (2014) 106–116108

for the 1000 μg dose compared to 25�10377 cells ml�1 for thePBS treated group (Fig. 1C). Furthermore, we evaluated a moredetailed temporal profile of inflammatory cell influx in the lungs ofBN rats up to 96 h post A. alternata challenge in order to determinethe optimal time-point for future experiments. As observed pre-viously, the total cells (Fig. 2A) and eosinophils (Fig. 2B) in the lungscontinued to increase following an A. alternata challenge, peaking at48–72 h and remaining elevated for up to 96 h. By contrast, theneutrophils levels reached their maximum levels 24 h after anA. alternata challenge and rapidly declined in numbers reachingalmost PBS control levels by 72 h (Fig. 2C).

3.2. Cytokine production in A. alternata induced lung inflammationin brown Norway rats

Since the production and release of Th2 cytokines such asInterleukin-5 (IL-5) and IL-13 contribute to eosinophil influx andmucus secretion in humans as well as animal models of asthma, weevaluated the levels of these cytokines in BAL fluid recovered from BNrats (n¼8 per group) 24, 48, 72 and 96 h following a singleintratracheal instillation of A. alternata. The levels of critical Th2cytokines, such as IL-5 and IL-13, were significantly elevated in theBAL fluid 24 h, reaching levels of 19547658 pgml�1 for IL-5 and4107156 pg ml�1 for IL-13 in comparison to the PBS control2577 pgml�1 and 2075 pgml�1 (respectively) (Po0.05). Elevatedlevels continued at 48 h following an A. alternata challenge, reachinglevels of 14357370 pgml�1 for IL-5 and 8067232 pgml�1 for IL-13,in comparison to the PBS control 2679 pgml�1 and 1476 pgml�1

(respectively) (Po0.05). Both, IL-5 and IL-13 returned to baselinelevels at 96 h (Fig. 3). A. alternata induced allergic responses developwithin a short timeframe, without the need for prior sensitization ormultiple challenges, a variety of early and late response cytokines may

be involved in the initiation of the inflammatory cascade. Therefore,we tested the levels of a panel of early response cytokines and allergicinflammation-relevant cytokines and chemokines in the BAL fluid ofBN rats 6, 24 and 48 h following an A. alternata challenge. Earlyresponse cytokines, such as interleukin-6 (IL-6) and interleukin-1β (IL-1β) were significantly increased at 6 h following the challe-nge (Table 1), while IL-5, IL-13, Macrophage Inflammatory Protein-1α(MIP-1α), Regulated on Activation, Normal T Cell Expressed and Secr-eted (RANTES), and monocyte chemotactic protein-1 (MCP-1) weresignificantly increased at later time points (Fig. 3, Table 1).

3.3. Histopathology in A. alternata induced lung inflammation inBrown Norway rats

To determine A. alternata induced histopathological changes inthe lungs of BN rats, we evaluated the temporal profile of lungpathology following an A. alternata challenge (Fig. 4).

Evaluation of the lungs from rats (n¼6 per group) dosed withA. alternata antigen demonstrated a pronounced and persistentalveolar inflammatory cell response and an increase in theperivascular and peribronchiolar inflammatory response (Fig. 4Band D), as compared to PBS-dosed rats (Fig. 4A and C), at all thetime-points investigated. All lung lobes evaluated were alsocompared against lungs from naïve rats, in order to account forany background inflammatory changes. The lungs of rats chal-lenged with PBS demonstrated very slight alveolar inflammatorychanges, consisting of infiltration of neutrophils and eosinophils,primarily during the earliest time-points. This was resolved by72 h post-dose, when the changes were considered comparable tonaïve rats, indicating reversal of the minimal changes.

By comparison, inflammation in the alveolar spaces of A. alternataantigen-dosed rats was more severe, as compared to the PBS-dosed

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Fig. 1. Effect of intratracheal dosing of A. alternata on BAL fluid cells in the Brown Norway rat. Figure displays the effect of A. alternata dose–response curve (500–2000 μg,intratracheal) on total cell counts (Panel A), eosinophils (Panel B) and neutrophils (Panel C) 24 and 48 h following direct instillation to the lungs. Each bar represents theMean7S.E.M. (n¼8 per treatment group). *Po0.05 compared to control animals receiving intratracheal PBS treatment.

M.A. Gil et al. / European Journal of Pharmacology 743 (2014) 106–116 109

rats at all time-points. A notable perivascular and peribronchiolarinflammation preceded alveolar inflammation 6 h following an A.alternata challenge. At 24 h and 48 h following an A. alternatachallenge, there was a notable increase in polymorphonuclear cells

(PMNC). These cells were mainly eosinophils within alveolar spacesthat are associated with edema and fibrin in addition to theprominent perivascular and peribronchiolar inflammatory responses.By 48 h, the lung lobes of A. alternata challenged rats containedcoalescing areas of inflammation (Fig. 4B and D). By 72 h, the affectedalveolar spaces were lined with hypertrophied epithelium, withsignificantly widened septa by macrophages and fibroblasts andalveolar spaces filled by mononuclear cells, occasional multinu-cleated cells and rare PMNC.

3.4. Micro-CT imaging as a tool to monitor A. alternata inducededema in the lung

To further investigate the pathophysiology of an A. alternatachallenge, we conducted micro-CT scanning in BN rats (n¼6 pergroup) at 0, 24, and 48 h post A. alternata challenges. The totaledema, plus vasculature (E&V) volume, increased from 369757 to

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Fig. 3. Time related effects of intratracheal A. alternata on BAL fluid cytokines in the Brown Norway rat. Panels A and B illustrate the temporal profile of IL-5 and IL-13concentrations in the BAL fluid after instillation of a single dose of A. alternata. Each point represents the Mean7S.E.M. (n¼8 per treatment group). *Po0.05 compared tocontrol animals receiving intratracheal PBS treatment.

Table 1Temporal profile of various cytokines concentration following a single i.t. instilla-tion of A.alternata.

Time post A. alternata challenge (h)

Cytokine (pg ml�1) 6 24 48MIP1α 250744 261a732 651a795RANTES 973 21a73 42a76MCP-1 18457525 1022271947 46,741a715,667IL-6 487a743 154711 198736IL-1b 49a714 2374 2175

a Po0.05 compared to control animals receiving i.t. PBS treatment.

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56075 mm3 by the 24 h time-point for the vehicle treated PBSchallenged group (Fig. 5). By 48 h post-PBS instillation, the E&Vincreased to 6427118 mm3. The vehicle treated A. alternata grouphad a baseline of 394735 mm3, which increased to 16547149mm3

by 48 h and resulted in a 5 fold increase from the baseline to21037208 mm3 at 48 h (Fig. 5). A statistically significant (Po0.05)increase in E&V was observed in A. alternata challenged groupscompared to PBS group at 24 h with further increase over a periodof 48 h. The slight increase in E&V of edema after the PBS challengeis most likely due to the reabsorption of the instilled fluid in thelung. Taken together, these data demonstrates that a single doseof A. alternata induces pulmonary edema, cellular infiltration inthe lungs and the production of asthma-relevant cytokines andchemokines.

3.5. Effect of prophylactic and therapeutic corticosteroid treatmenton A. alternata induced lung inflammation in Brown Norway rats

To examine the prophylactic effect of corticosteroids on A. alternataelicited pulmonary inflammatory responses, we examined the effect ofintratracheally administered Budesonide (0.01, 0.1 and 1mg kg�1) inA. alternata challenged BN rats (n¼8 per group). Intratracheally dosedBudesonide led to a dose-dependent decrease in the number ofeosinophils, which was maximal at 1 mg kg�1 dose with completeinhibition, compared to vehicle treated group at 48 h following theintratracheal challenge with A. alternata (Po0.05) (Fig. 6). Oralbudesonide was administered to BN rats 2 h prior to and 22 h postthe A. alternata challenge and used as a positive control. As expected,orally administered budesonide significantly reduced A. alternatainduced eosinophil influx in the lungs of BN rats. Similarly, intratra-cheally dosed mometasone produced a dose dependent attenuation inthe number of eosinophils in the BAL fluid recovered from BN rats48 h following the intratracheal challenge with A. alternata (notshown). These results demonstrate that intratracheal and the systemicadministration

of corticosteroids can suppress A. alternata induced allergic lunginflammation in BN rats.

Since prophylactically administered corticosteroids suppressedA. alternata induced pulmonary inflammation, we sought toinvestigate if equivalent therapeutic dosing (after an A. alternatachallenge) of corticosteroids could also exert a similar effect. InFig. 7, we show that orally dosed budesonide (3 mg kg�1) at 2, 6 or23 h post an intratracheal challenge with A. alternata decreasedthe number of eosinophils and cytokines, such as IL-5 and IL-13levels, in a time dependent manner 48 h after the A. alternatachallenge. Budesonide dosed 1 h prior to A. alternata challengewas used as a positive control. Eosinophil numbers (Fig. 7A)following A. alternata challenge were significantly decreased inthe BAL fluid at the earlier therapeutic budesonide dosing time-points (2 and 6 h post-challenge) with 9872% and 7179% ofinhibition respectively. In comparison to the later (23 h postchallenge) therapeutic dosing time-point inhibition, which inhib-ited only 31712% of eosinophils at the 48 h time point. (Po0.05)(Fig. 7).

IL-5 and IL-13 cytokine levels (Fig. 7 B and C) followingA. alternata challenge were significantly decreased in the BAL fluidat the earlier therapeutic budesonide dosing time-points (2 and6 h post-challenge), with 9873% and 10372% for IL-5 and10371% and 10571% for IL-13 of inhibition respectively. Incomparison to the later (23 h post-challenge), therapeutic dosingtime- point inhibition, which produced only 31712% of eosino-phils inhibition at the 48 h time point. (Po0.05) (Fig. 7a). Inaddition, therapeutic dosing produced 81710% and 9074% of IL-5 and IL-13 cytokine levels inhibition at the 48 h time point.(Po0.05) (Fig. 7 B and C).

3.6. Effect of a CRTH2 antagonist on A. alternata elicited pulmonaryinflammation

In addition to corticosteroids, we also investigated whether theinhibition of a clinically-relevant mechanism of allergic lung

Fig. 4. Histo-pathological evaluation of BN rats lung tissue following intratracheal instillation of A. alternata. Sections of lungs from rats (n¼6 per treatment group) followinga single instillation of either PBS (Panels A and C) or A. alternata (Panels B and D), and euthanized after 48 h. At 48 h, there is multifocal polomorphonuclear inflammatory cellinfiltrationwithin alveolar spaces of rats treated with A. alternata shown at low (Panel B) and higher magnification (Panel D). Rats treated with PBS demonstrated minimal tono alveolar inflammatory cell infiltrates by 48 h as shown at low magnification (Panel A) and higher magnification inset (Panel C).

M.A. Gil et al. / European Journal of Pharmacology 743 (2014) 106–116 111

inflammation such as CRTH2 would lead to a suppression ofinflammatory responses in A. alternata challenged Brown Norwayrats (n¼8 per group). Mast cell derived production of Prostaglan-din D2 (PGD2) is believed to be a prime mediator of allergicinflammation. Indeed, PGD2 is known to drive Th2 inflammationthrough its receptor, CRTH2, which is expressed on Th2 cells,eosinophils, basophils and innate lymphoid cells type 2 (ILC2).Activation of CRTH2 has been shown to activate these cellularsubtypes and drive chemotaxis, as well as cytokine production,

including IL-4, IL-5 and IL-13. Since CRTH2 plays an important rolein the early aspects of the allergic inflammation cascade (Kostenisand Ulven, 2006), we examined the effect of the CRTH2 antagoniston A. alternata elicited pulmonary inflammatory responses. Weorally administered the CRTH2 inhibitor MK-7246, 1 h before and23 h post-intratracheal instillation of the A. alternata. The MK-7246 produced a dose dependent decrease in the number ofeosinophils with a maximal inhibition of 7475% in the100 mg kg�1 group (Po0.05) (Fig. 8 a), IL-5 (80712%) and IL-13

Hours post-challenge

Volu

me

mm

3

0

1000

2000

3000

0 24 48

*

*Veh/ A. alternataVeh/PBS

Fig. 5. CT images of the lungs from Brown Norway rats 24 and 48 h following intratracheal instillation of A. alternata (n¼6 per treatment group). Upper panel (A) isdisplaying representative images from PBS treated animals serving as a negative control group. In the lower panel (A) images are representative of animal lungs challengedwith A. alternata. Baseline measurements were taken 24 h prior to PBS or A. alternata instillation. A summary plot of the effects of PBS or A. alternata where each pointrepresents the Mean7S.E.M. (n¼6 per treatment group) is shown in panel (B). *Po0.05 compared to control animals receiving intratracheal PBS treatment.

M.A. Gil et al. / European Journal of Pharmacology 743 (2014) 106–116112

(76714%) cytokines levels (Po0.05) (Fig. 8 B and C). The resultsreported show BAL fluid recovered from BN rats 48 h following theintratracheal. challenge with A. alternata. Orally dosed budesonidewas used as a positive control producing significant inhibition ofeosinophilic infiltration as well as both IL-5 and IL-13 cytokinelevels. These results demonstrate that A. alternata induced eosi-nophil influx and inflammatory lung responses in BN rats may

serve as a valuable pharmacodynamic (PD) model to interrogatepharmacological inhibition of asthma-relevant mechanisms.

4. Discussion

Asthma continues to be a significant health concern (Polosaand Benfatto, 2009). New emerging treatments, including ther-apeutic antibodies to inflammatory cytokines (i.e. IL-5, IL-13, IL-4and IL-17), allergen-specific immunotherapy, combination-inhaledagents (i.e. ICS plus LAMA and or LABA), or small moleculeG-protein coupled receptors (i.e. CRTH2 antagonist), may ulti-mately enhance the armaments used to treat this heterogeneousdisease. However, the effectiveness of these approaches is cur-rently being determined in human asthma trials (Albertson et al.,2013). From a preclinical perspective, animal models have beenhistorically useful in the progression of novel targets from thelaboratory to the clinic although their human predictabilityremains subject of continuous debate (Canning, 2003). Whilemouse models of allergic airway diseases have proven to beinvaluable for informing on various aspects and pathways relatedto respiratory biology and pathology, they are not necessarily thepreferred species from a drug development perspective. The rathas historically been the rodent species of choice for the majorityof nonclinical safety assessment and toxicology studies. There is asignificant knowledge and many years of accumulated dataregarding baseline clinical chemistry and hematology ranges, aswell as database of compounds that have been evaluated in single-and repeat-dose rat toxicology studies. In addition, a recent reportby Seok et al. (2013) suggested that genomic responses in mice

Fig. 6. Dose dependent effects of Budesonide in Brown Norway rats challengedwith A. alternata. Figure displays BAL fluid eosinophils influx 48 h after instillationof a single dose of A. alternata. Each bar represents the Mean7S.E.M. (n¼8 pertreatment group). *Po0.05 compared to control A. alternata challenged animalsreceiving intratracheal vehicle treatment (0.9% PBSþ0.5% Tween 80).

Fig. 7. Effect of a prophylactic or therapeutic Budesonide dosing in A. alternata challenged Brown Norway rats. Panel (A) illustrates the temporal profile of eosinophils influxin the lung. Panels (B) and (C) illustrate IL-5 and IL-13 concentrations in the BAL fluid after instillation of a single dose of A. alternata. Each bar represents the Mean7S.E.M.(n¼8 per treatment group). *Po0.05 compared to control A. alternata challenged animals receiving p.o. vehicle treatment (0.5% MCþ0.24% SDS).

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poorly mimic human inflammatory diseases, thereby casting anadditional shadow on the translatability of mouse models tohuman inflammatory diseases.

In humans, mounting evidence suggests that atopy to moldallergens is related to asthma severity (Black et al. 2000; Zureiket al., 2002; Denning et al., 2006). Here, we describe the respira-tory impact of a single intratracheal instillation of A. alternata inBN rats, with a goal of establishing a simple but clinically relevantrodent asthma mechanism model that may be used to probepathway biology of novel pharmacological targets. The mucosalsurface of airway epithelium is the first anatomical interfacewhere environmental allergens evoke host defense mechanisms.Extracts from A. alternata spores have been shown to stimulate therelease of various pro-inflammatory cytokines including Thymicstromal lymphopoietin (TSLP), granulocyte macrophage colony-stimulating factor, and IL-33 from epithelial cells (Kouzaki et al.,2009, Kouzaki et al., 2011). These events have been reported to bedependent on Protease Activated Receptor-2 signaling and theST2 receptor (Boitano et al., 2011). Chronic instillation of theA. alternata extract via the intranasal route in mice has beenshown to release IL-33, IL-5 and IL-13, followed by recruitment ofeosinophils in the BAL fluid, independent of T or B cells involve-ment (Doherty et al., 2012). These events have been described tobe mediated by innate lymphoid cells (ILC) (Bartemes et al., 2012),which are capable of mediating asthma-like pathological environ-ments, by secreting Th2 cytokines such as IL-5 and IL-13, prior tothe initiation of adaptive immune responses. The ILCs are not onlypresent in lungs, but in other organs such as intestines and havebeen shown to produce Th2 cytokine in response to Interleukin-25(IL-25) and IL-33 (Moro et al., 2010, Neill et al., 2010, Saenz, et al.,2010). Although the repertoire of cytokines released by these ILCs

in different organs can vary, it appears that the common feature ofthese cells is to mediate Th2 type allergic inflammatory responsesindependent of adaptive immunity (Monticelli et al., 2012).

In our study, we found that a single intratracheal administra-tion of A. alternata extract can mediate allergic responses similarto some aspects of the human asthma phenotype. We establisheda temporal profile of differential cell recruitment and pro-inflammatory cytokines after a challenge with an A. alternataextract. The A. alternata challenge is characterized by the releaseof early response pro-inflammatory cytokines such as IL-6, IL-1b,MIP-1a and MCP-1. The A. alternata instillation triggers a small andtransient neutrophilic response which is resolved and returns tobaseline within 24 h. This is followed by a release of IL-5 and IL-13,as detected in BAL fluid. Both cytokines reached their peak around48 h post-challenge and returned to basal levels within 72 h. Inaddition to inflammatory responses, intratracheal instillation ofA. alternata leads to pathological changes, such as increasedalveolar infiltrates of neutrophils and eosinophils associated withedema, as compared to rats dosed with PBS alone. The initialperivascular and peribronchiolar inflammation was followed byalveolar inflammation and cell accumulation, progressing to amore sub-acute inflammatory response with notable epithelialcell hypertrophy in affected areas at 72 h following an A. alternatachallenge. The cellular infiltration upon an A. alternata challengewas further confirmed in our CT imaging experiments measu-ring the temporal profile of microvascular leakage, leading tolung edema.

In this study we also report the effect of commonly usedcorticosteroids, such as budesonide on A. alternata challengedBrown Norway rats. Budesonide dosed via an intratracheal andoral route, 2 h prior to an A. alternata challenge, inhibited Th2

Fig. 8. Effects of CRTh2 antagonist MK-7246 in A. alternata challenged Brown Norway rats. Panel (A) shows effect on BAL fluid eosinophils levels following MK-7246administration. Panels (B) and (C) show IL-5 and IL-13 concentrations in the BAL fluid following the MK-7246 treatment. Each bar represents the Mean7S.E.M. (n¼8 pertreatment group). *Po0.05 compared to control A. alternata challenged animals receiving p.o. vehicle treatment (0.5% MCþ0.24% SDS).

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cytokines and eosinophilia recruitment in the BAL fluid in a dosedependent manner. Interestingly, the maximal dose of budesonideis partially effective if dosed 6 h after the challenge and completelyineffective if dosed 23 h post an A. alternata challenge. Thus, ourresults suggest that prophylactic budesonide intervention and theblockade of early events mediated by A. alternata is an essen-tial driver/event in this model. Pharmacological intervention ofcorticosteroids is likely to occur at multiple stages during theA. alternata elicited signaling pathways. Specifically, corticoster-oids can either directly or indirectly regulate the transcription(trans-repression) of various cytokines (i.e. IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-11, IL-12, IL-13, IL-18, TNFα), chemokines (i.e. IL-8,RANTES, MIP-1α, MCP-1, eotaxin), adhesion molecules (vascularcell adhesion moleculeintracellular cell adhesion molecule 1),inflammatory enzymes, receptors and peptides (Barnes, 2006).Therefore, our results show that budesonide profoundly impactedIL-13 and IL-5 in the A. alternata model, which is consistent withthe already described pharmacology of corticosteroids (Braunet al., 1997). In addition to evaluating the actions of corticosteroidsin our A. alternata model, we also profiled the impact of the CRTH2antagonist, MK-7246. The antagonism of CRTH2, while not beingas efficacious as the corticosteroid, budesonide, in response to anA. alternata challenge, clearly impacted the eosinophil influx, aswell as impacting overall IL-5 and IL-13 levels at 48 h. This is anovel finding, in that CRTH2 is believed to be involved in anadaptive immune response (Kostenis and Ulven, 2006), withsignificant effects in typical sensitized allergic models (Mauseret al., 2013).

Recent investigations have expanded a role for CRTH2 outsidethe initial adaptive immune response and have identified andcharacterized CRTH2 on innate lymphoid cells as well (Mjösberget al., 2011). In response to an allergen, epithelial derived cytokines(IL-25/IL-33/TSLP) promote a Th2 like response, by stimulating aninnate lymphoid population, which in turn can produce thecanonical IL-5 and IL-13 response (Licona- Limón et al., 2013).The type 2 innate lymphoid cells (ILC2) represent a critical nexusfor expanding a Th2 repertoire in response to an epithelial insultby parasites or allergens (Neill et al., 2010). Human ILC2s expres-sing CRTH2 are responsive to both IL-25 and IL-33, resulting inproduction of IL-13 (Mjösberg et al., 2012). Additional studiesdirectly demonstrated CRTH2 mediated induction of ILC2s migra-tion and IL-4/IL-5/IL-13 production (Xue et al., 2013). In addition,Xue et al., 2013, reported that CRTH2 up-regulated the expressionof receptor subunits for IL-33 and IL-25 (ST2 and Interleukin-17Receptor A) in ILC2s. Furthermore, peripheral ILC2s demonstrateda synergistic production of IL-13 that required CRTH2 stimulation,along with the presence of IL-33 and IL-25 (Barnig et al., 2013).CRTH2 expression on both innate, as well as adaptive lymphoidcells, suggests an important role in modulating and controllingTh2 inflammation. Currently, CRTH2 antagonism is being investi-gated in clinical trials by several pharmaceutical companies for thetreatment of allergic diseases such as asthma, allergic rhinitis andatopic dermatitis (Norman, 2014).

5. Summary

In summary, since no single animal model exhibits all facets ofasthma, animal models replicating various features of asthma areimportant tools in investigating the mechanisms of the diseasepathogenesis and can provide an insight in identifying novel ther-apeutic targets. Modeling asthma has proven to be challenging, inpart due to the clinical and pathophysiological complexity andheterogeneity of the human disease. In this study we established anovel pharmacodynamic model that recapitulates some cardinalfeatures of asthma pathogenesis and can be used as a tool in drug

discovery. Moreover, we demonstrated for the first time that theCRTH2 antagonist can reduce eosinophilia, most likely throughinhibiting ILC2 mediated responses in the A. alternata model. Furtherstudies are underway to establish the role of early cytokines releases,leading to heighten eosinophilic responses and underlying pathologyafter an A. alternata challenge.

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