Anti-inflammatory actions of Chemoattractant Receptor-homologous molecule expressed on Th2 by the antagonist MK-7246 in a novel rat model of Alternaria alternata elicited pulmonary inflammation

European Journal of Pharmacology 743 (2014) 106–116

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Immunopharmacology and inflammation

Anti-inflammatory actions of Chemoattractant Receptor-homologous molecule expressed on Th2 by the antagonist MK-7246 in a novel rat model 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, USA Biology Discovery, Merck Research Laboratories, Boston, MA 02115, USA c Discovery Pharmaceutical Sciences, Merck Research Laboratories, Boston, MA 02115 , USA d Safety Assessment and Laboratory Animal Sciences, Merck Research Laboratories, West Point, PA 19486, USA e Imaging, Merck Research Laboratories, Boston, MA 02115, USA f Target & Pathway Biology, Merck Research Laboratories, Boston, MA 02115, USA g Discovery Chemistry, Merck Research Laboratories, Boston, MA 02115, USA b

art ic l e i nf o

a b s t r a c t

Article history: Received 20 May 2014 Received in revised form 11 September 2014 Accepted 15 September 2014 Available online 23 September 2014

Alternaria alternata is a fungal allergen linked to the development of severe asthma in humans. In view of the clinical relationship between A. alternata and asthma, we sought to investigate the allergic activity of this 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 time dependent 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 as Interleukin-5 and Interleukin-13, following A. alternata challenge. These responses were comparable to Ovalbumin induced models of asthma and resulted in peak inflammatory responses 48 h following a single challenge, eliminating the need for multiple sensitizations and challenges. The initial perivascular and peribronchiolar inflammation preceded alveolar inflammation, progressing to a more sub-acute inflammatory response with notable epithelial cell hypertrophy. To limit the effects of an A. alternata inflammatory response, MK-7246 was utilized as it is an antagonist for Chemoattractant Receptorhomologous molecule expressed in Th2 cells. In a dose-dependent manner, MK-7246 decreased eosinophil influx and Th2 cytokine production following the A. alternata challenge. Furthermore, therapeutic administration of corticosteroids resulted in a dose-dependent decrease in eosinophil influx and Th2 cytokine production. Reproducible asthma-related outcomes and amenability to pharmacological intervention by mechanisms relevant to asthma demonstrate that an A. alternata induced pulmonary inflammation in BN rats is a valuable preclinical pharmacodynamic in vivo model for evaluating the pharmacological inhibitors of allergic 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/).

Keywords: Alternaria alternata CRTH2 MK-7246 Brown Norway Rat

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, macrophage inflammatory 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.: þ 1 617 992 2395. E-mail address: [email protected] (M. Cicmil). http://dx.doi.org/10.1016/j.ejphar.2014.09.021 0014-2999/& 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/).

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

1. Introduction

2. Materials and methods

Asthma is a heterogeneous respiratory disease with a complex pathophysiology. It continues to be a significant societal health burden, utilizes a large amount of health resources, and diminishes quality of life (Adcock et al., 2008; Bel, 2013). Particularly noteworthy are a small percentage of patients with severe symptoms leading to significant airway obstruction, which require frequent hospital admissions due to exacerbated asthma symptoms and are refractory to standard of care drugs. While these refractory asthmatics represent only a small percentage of asthmatic patients, the medical costs for treating refractory asthmatics represents about 50% of the total healthcare cost for asthma (Jang, 2012). The disease is characterized by a persistent Th2 inflammation, airflow obstruction, bronchoconstriction and airway hyperresponsiveness (Howarth, 1995). While “asthma triggers” are loosely defined as substances or events that precipitate an acute onset or worsening of asthma symptoms (Vernon et al., 2012), the temporal understanding of the pathways by which these triggers drive respiratory pathophysiology are not fully understood. Nonetheless, it is well accepted that exposing asthmatics to aeroallergens, viruses, and respiratory irritants leads to an aggravation of the disease (Kim and Gern, 2012). In recent years, fungi sensitization has emerged as an important potential contributor to allergic lower airway disease (Knutsen et al., 2012). Indeed, patients with allergic asthma often have detectable levels of serum IgE antibodies to fungi (Crameri et al., 2009). Schwartz et al. (1978) was the first to describe a relationship between Aspergillus skin test reactivity and the severity of airway obstruction in asthmatic patients. Subsequently, links between sensitization of various fungi (e.g. Alternaria, Aspergillus, Aureobasidium, Cladosporium, Helminthosporium and Trichophyton) to respiratory pathology were suggested (Peat et al., 1993; Neukirch et al., 1999; Fairs et al., 2010; Agarwal, 2011). For instance, Arbes et al. (2007) found that A. alternata is independently associated with asthma cases attributable to atopy. Further support for a relationship between A. alternata and asthma includes the following observations: (1) Childhood sensitization to A. alternata correlates with persistent adult asthma (Stern, et al., 2008); (2) sensitization to A. alternata is associated with fatal episodes of asthma (Arbes et al., 2007; Knutsen et al., 2010), (3) when delivered directly to the airways, A. alternata spores induce brochoconstriction in people with mild asthma (Licorish et al., 1985) and (4) environmental fluctuations in A. alternata spore counts appear to associate with symptom severity, as observed in the cases of “thunderstorm asthma”, where there is a significant rise in this airborne antigen (Pulimood et al., 2007; Nasser and Pulimood, 2009). The mechanism(s) and pathways by which A. alternata initiates respiratory inflammation in sensitized patients are not fully understood. However, it is known that certain key immune cells such as lung epithelial cells, dendritic cells, and eosinophils likely play a pivotal role in orchestrating a Th2-mediated inflammation precipitated by lung exposure to A. alternata antigens. Murai et al. (2012) demonstrated that A. alternata extracts in cultured human bronchial epithelial cells induce the rapid release of Interleukin-18 (IL-18) which can directly promote Th2 differentiation of naïve CD4 þ T-cells through a NF-κB dependent pathway (Murai et al., 2012). Another route through which A. alternata and other fungi may induce Th2 inflammation is by the activation of a proteaseactivated receptor, particularly Protease Activated Receptor-2 (Matsuwaki et al., 2011). In view of the clinical relationship between A. alternata and asthma, we sought to characterize the activity of this antigen after direct application to the lungs of BN rats. In this study, we describe a novel Th2-mediated model of pulmonary inflammation due to the acute intratracheal instillation of A. alternata in Brown Norway rats. In addition, we conducted pharmacological validation of this model using clinically relevant drugs, such as glucocorticoids and a CRTH2 antagonist, MK-7246 (Gervais et al., 2010).

2.1. Animal care and use

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All studies were performed in accordance with the National Institute of Health Guide to the Care and Use of Laboratory Animals and the Animal Welfare Act, in Association with the Assessment and Laboratory Animal Care Program. All experiments were approved by Merck Institutional Animal Care and Use Committee. 2.2. Characterization of A. alternata evoked pulmonary inflammation responses in brown Norway rats Male Brown Norway rats (225–275 g) were purchased from Charles River Laboratories (Kingston, NY) and housed in an animal room at a temperature of 25 1C (75 1C), relative humidity: 30–70%, a daily light–dark cycle (07:00 to 19:00 light). Chow and water were supplied ad libitum and the animals were studied between 80 and 85 days of age. Experimental rats were intratracheally challenged with A. alternata extracts (Greer Labs, Lenoir, NC) in 100 μl sterile phosphate buffered saline (PBS), using a PennCentury microsprayer device (Penn-Century, Philadelphia, PA). A series of experiments were conducted to examine: (1) the impact of A. alternata on respiratory inflammation, (i.e. total BAL fluid cells, eosinophils and neutrophils) after topical administration to the lungs (n ¼8 animals per treatment group); (2) the temporal relationship between Th2 cytokine secretion and inflammatory cellular influx into the BAL fluid after a single intratracheal A. alternata challenge (n ¼8 animals per treatment group); (3) histological and CT scan observations after an A. alternata challenge (n ¼6 animals per treatment group); and (4) pharmacological intervention of A. alternata responses by classical glucocorticoids (i.e. budesonide) and by MK-7246, a selective orally active CRTH2 antagonist (n ¼8 animals per treatment group). 2.3. Impact of A. alternata on respiratory inflammation and the temporal relationship of Th2 cytokine secretions To determine if rats that were given increasing doses (500– 2000 μg) of intratracheal A. alternata induced a more severe inflammatory response, BAL fluid cell composition was analyzed at 24 and 48 h post-provocation. BAL fluid collection and processing were carried out using the methods previously described (Lieber et al., 2013). Briefly, after an overdose (150–200 mg/kg via intratperitoneal injection) of pentobarbital, the trachea was catheterized, BAL fluid was obtained by the intratracheal insertion of a catheter, followed by two lavages with 3 ml 0.9% sodium chloride injection, USP (Hospira, Lake Forest, IL). One aliquot ( 2 ml), was analyzed for total and differential cell counts performed using an Advia 120 Automated Hematology Analyzer (Siemens, Washington DC). For some studies, a small BAL fluid aliquot (  0.5 ml) was collected for cytokine analysis. Specifically, BAL fluid samples were centrifuged at 1500g, 4 1C for 10 min to eliminate cellular debris and supernatant was aliquoted and frozen at  80 1C for further analysis. Once defrosted, a Luminex-Milliplex MAP magnetic beadbased Rat 22-plex immunoassay (Millipore, Billerica, MA) analysis of BAL fluid supernatants was performed following manufacturer's directions. We also utilized a Meso Scale Discovery (MSD, Rockville, MD) Rat 7plex ultra-sensitive kit for a panel of certain cytokines. ELISA for IL-13 (Invitrogen, Grand Island, NY) was performed on the BAL fluid supernatant according to the manufacturer's instructions and all ELISA plates were read with a BioRad Model 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 determined. Additional characterization of the effects of locally applied

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

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

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

2.6. Histopathological analysis

Intratracheal budesonide was dosed 1 h prior to and 23 h post the A. alternata intratracheal dose while oral budesonide (3 mg kg  1) was administered 2 h before and 22 h post the A. alternata extract instillation. An intratracheally dosed budesonide was prepared using the Covaris formulation as described (Kakumanu and Schroeder, 2012). Covaris AFA is an in-situ method which allows the construction 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 h before and 23 h post an A. alternata extract instillation in order to examine the effect of the CRTH2 antagonist on A. alternata elicited pulmonary inflammatory responses. Budesonide dosed orally was used as a positive control in both experiments. The animals were lightly anesthetized with 3% isoflurane (supplemented with 100% oxygen), either 2 h following an oral dosing or 1 h following an intratracheal dosing. The animals were also secured on a rodent work stand to facilitate the localization of the larynx and tracheal openings. The microsprayer needle was inserted into the trachea and 0.1 ml of 10,000 μg ml  1 (total of 1000 μg) A. alternata extract was administered using a microsprayer. The animals were observed until they recovered from anesthesia and then returned to their cages and allowed food and water ad libitum. The animals were euthanized with an overdose of pentobarbital 48 h after an A. alternata intratracheal instillation and BAL fluid was collected. 2.5. Micro-CT imaging acquisition, image reconstruction, and analysis CT scans were performed on rats anesthetized with 3% isoflurane (supplemented with 100% oxygen), using a GE eXplore Locus Ultra Pre-Clinical CT scanner (GE Healthcare, Cleveland, OH, USA). Rats were imaged one at a time. The entire chest area was scanned at once, with 1000 projections collected in one full rotation of the gantry in approximately 16 s. The X-ray tube settings were 80 kV and 70 mA and the isotropic resolution was 185 μm. Projection data was reconstructed using the graphics processing unit (GPU)-based AxRecon reconstruction platform (Acceleware, Inc., Ontario, Canada). The three dimensions of the image data set (x, y, and z), were manually adjusted to 600  500  800 slices at 100 μm cubic voxel dimensions for reconstruction and images were scaled to Hounsfield units. The reconstructed data was then cropped into the easier-to-manage Analyze format using a MATLAB (MathWorks, Inc., Natick, MA) application that was developed to automate this conversion process. An image analysis method we used was based on a method published by Haines et al. (2009) that relies on thresh-holding and region-grow algorithms to segment separate anatomical regions of interest 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 a mouse model. After the functional volume was recorded, the total chest space was selected using semi-automated contouring and manual selection, eliminating the heart and liver. Vasculature found in the lung was included in this total lung measurement, since edema and vascular tissues have similar grayscale values and cannot be differentiated based on threshold. Once the total chest space was determined for each animal, the functional chest space was subtracted from that volume, leaving the intentionally selected vasculature, as well as edema. The final readout, (Edema volume with vasculature¼ Total chest volume functional lung volume), was the

Lungs from 6 rats/group were obtained at the scheduled terminations of 6, 24, 48, or 72 h following the challenge, along with lungs from naïve animals not administered A. alternata antigen or lavaged, but housed concurrently with the treated rats. Lungs from all dose groups except the naïve group were lavaged and infused with 10% neutral buffered formalin. Lungs from naïve rats were only infused with 10% neutral buffered formalin. After approximately 24 h, lung sections from all lobes were processed to demonstrate maximum longitudinal sectioning of the right caudal lobe and representative sections of the remaining right and left lung lobes along with major bronchi and trachea. Sections were processed and embedded in paraffin. Embedded tissues were cut into 4–6 μm sections and stained with hematoxylin and eosin (H&E). A histologic evaluation was performed on stained tissue sections from multiple lobes and all the rats. The histology score for each rat was based on alveolar damage, including alveolar leukocyte infiltration and edema, epithelial cell hypertrophy, along with perivascular and peribroncheolar cuffing and an estimate of the area involved with disease. Grading was based on a severity score scale of 0–5: 0 (no observable pathology), 1 (minimal or very slight), 2 (mild or slight), 3 (moderate), 4 (marked), or 5 (severe). The evaluation also included scoring any pre-existing chronic background inflammation present in the Brown Norway rat model (Noritake et al., 2007) and noted here in all the rats, including the naïve rats, which was separately graded to avoid influencing the evaluation of damage due to the A. alternata antigen. 2.7. Statistical analysis Statistical analysis was performed using Prism Software (GraphPad Software, Inc., La Jolla, CA). All measures were analyzed via oneway or two-way ANOVA, with post-hoc Bonferroni analyses performed 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% Tween 80 (for intratracheal dosing) or 0.5% methylcelluloseþ 0.24% sodium dodecyl 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 influx in 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 and dose-dependent increases in the number of total cells were observed 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 eosinophils in the A. alternata treated rats, with maximal numbers at 48 h with 492  103 7108 cells ml  1 compared to 21  103 74 cells ml  1 in the phosphate buffered saline (PBS) treated group (Po0.05) (Fig. 1B). A dose related increase in neutrophils levels was also observed reaching a peak levels at 24 h and were 5 fold less than the eosinophils, reaching a maximum of 80  103 720 cells ml  1

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for the 1000 μg dose compared to 25  103 77 cells ml  1 for the PBS treated group (Fig. 1C). Furthermore, we evaluated a more detailed temporal profile of inflammatory cell influx in the lungs of BN rats up to 96 h post A. alternata challenge in order to determine the optimal time-point for future experiments. As observed previously, the total cells (Fig. 2A) and eosinophils (Fig. 2B) in the lungs continued to increase following an A. alternata challenge, peaking at 48–72 h and remaining elevated for up to 96 h. By contrast, the neutrophils levels reached their maximum levels 24 h after an A. alternata challenge and rapidly declined in numbers reaching almost PBS control levels by 72 h (Fig. 2C). 3.2. Cytokine production in A. alternata induced lung inflammation in brown Norway rats Since the production and release of Th2 cytokines such as Interleukin-5 (IL-5) and IL-13 contribute to eosinophil influx and mucus secretion in humans as well as animal models of asthma, we evaluated the levels of these cytokines in BAL fluid recovered from BN rats (n¼8 per group) 24, 48, 72 and 96 h following a single intratracheal instillation of A. alternata. The levels of critical Th2 cytokines, such as IL-5 and IL-13, were significantly elevated in the BAL fluid 24 h, reaching levels of 19547658 pg ml  1 for IL-5 and 4107156 pg ml  1 for IL-13 in comparison to the PBS control 2577 pg ml  1 and 2075 pg ml  1 (respectively) (Po0.05). Elevated levels continued at 48 h following an A. alternata challenge, reaching levels of 14357370 pg ml  1 for IL-5 and 8067232 pg ml  1 for IL-13, in comparison to the PBS control 2679 pg ml  1 and 1476 pg ml  1 (respectively) (Po0.05). Both, IL-5 and IL-13 returned to baseline levels at 96 h (Fig. 3). A. alternata induced allergic responses develop within a short timeframe, without the need for prior sensitization or multiple 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 allergic inflammation-relevant cytokines and chemokines in the BAL fluid of BN rats 6, 24 and 48 h following an A. alternata challenge. Early response cytokines, such as interleukin-6 (IL-6) and interleukin-1 β (IL-1β) were significantly increased at 6 h following the challenge (Table 1), while IL-5, IL-13, Macrophage Inflammatory Protein-1α (MIP-1α), Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES), and monocyte chemotactic protein-1 (MCP-1) were significantly increased at later time points (Fig. 3, Table 1). 3.3. Histopathology in A. alternata induced lung inflammation in Brown Norway rats To determine A. alternata induced histopathological changes in the lungs of BN rats, we evaluated the temporal profile of lung pathology following an A. alternata challenge (Fig. 4). Evaluation of the lungs from rats (n ¼6 per group) dosed with A. alternata antigen demonstrated a pronounced and persistent alveolar inflammatory cell response and an increase in the perivascular and peribronchiolar inflammatory response (Fig. 4B and D), as compared to PBS-dosed rats (Fig. 4A and C), at all the time-points investigated. All lung lobes evaluated were also compared against lungs from naïve rats, in order to account for any background inflammatory changes. The lungs of rats challenged with PBS demonstrated very slight alveolar inflammatory changes, consisting of infiltration of neutrophils and eosinophils, primarily during the earliest time-points. This was resolved by 72 h post-dose, when the changes were considered comparable to naïve rats, indicating reversal of the minimal changes. By comparison, inflammation in the alveolar spaces of A. alternata antigen-dosed rats was more severe, as compared to the PBS-dosed

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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-13 concentrations in the BAL fluid after instillation of a single dose of A. alternata. Each point represents the Mean 7 S.E.M. (n¼ 8 per treatment group). *Po 0.05 compared to control animals receiving intratracheal PBS treatment.

Table 1 Temporal profile of various cytokines concentration following a single i.t. instillation of A.alternata. Time post A. alternata challenge (h) Cytokine (pg ml  1) MIP1α RANTES MCP-1 IL-6 IL-1b a

6 250 7 44 97 3 1845 7 525 487a 7 43 49a 7 14

24 261a 7 32 21a 7 3 10222 7 1947 1547 11 237 4

48 651a 795 42a 76 46,741a 715,667 198 7 36 21 7 5

P o0.05 compared to control animals receiving i.t. PBS treatment.

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

(PMNC). These cells were mainly eosinophils within alveolar spaces that are associated with edema and fibrin in addition to the prominent perivascular and peribronchiolar inflammatory responses. By 48 h, the lung lobes of A. alternata challenged rats contained coalescing areas of inflammation (Fig. 4B and D). By 72 h, the affected alveolar spaces were lined with hypertrophied epithelium, with significantly widened septa by macrophages and fibroblasts and alveolar spaces filled by mononuclear cells, occasional multinucleated cells and rare PMNC. 3.4. Micro-CT imaging as a tool to monitor A. alternata induced edema in the lung To further investigate the pathophysiology of an A. alternata challenge, we conducted micro-CT scanning in BN rats (n¼6 per group) at 0, 24, and 48 h post A. alternata challenges. The total edema, plus vasculature (E&V) volume, increased from 369757 to

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Fig. 4. Histo-pathological evaluation of BN rats lung tissue following intratracheal instillation of A. alternata. Sections of lungs from rats (n¼ 6 per treatment group) following a 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 cell infiltration within alveolar spaces of rats treated with A. alternata shown at low (Panel B) and higher magnification (Panel D). Rats treated with PBS demonstrated minimal to no alveolar inflammatory cell infiltrates by 48 h as shown at low magnification (Panel A) and higher magnification inset (Panel C).

56075 mm3 by the 24 h time-point for the vehicle treated PBS challenged group (Fig. 5). By 48 h post-PBS instillation, the E&V increased to 6427118 mm3. The vehicle treated A. alternata group had a baseline of 394735 mm3, which increased to 16547149 mm3 by 48 h and resulted in a 5 fold increase from the baseline to 21037208 mm3 at 48 h (Fig. 5). A statistically significant (Po0.05) increase in E&V was observed in A. alternata challenged groups compared to PBS group at 24 h with further increase over a period of 48 h. The slight increase in E&V of edema after the PBS challenge is most likely due to the reabsorption of the instilled fluid in the lung. Taken together, these data demonstrates that a single dose of A. alternata induces pulmonary edema, cellular infiltration in the lungs and the production of asthma-relevant cytokines and chemokines.

3.5. Effect of prophylactic and therapeutic corticosteroid treatment on A. alternata induced lung inflammation in Brown Norway rats To examine the prophylactic effect of corticosteroids on A. alternata elicited pulmonary inflammatory responses, we examined the effect of intratracheally administered Budesonide (0.01, 0.1 and 1 mg kg  1) in A. alternata challenged BN rats (n¼8 per group). Intratracheally dosed Budesonide led to a dose-dependent decrease in the number of eosinophils, which was maximal at 1 mg kg  1 dose with complete inhibition, compared to vehicle treated group at 48 h following the intratracheal challenge with A. alternata (Po0.05) (Fig. 6). Oral budesonide was administered to BN rats 2 h prior to and 22 h post the A. alternata challenge and used as a positive control. As expected, orally administered budesonide significantly reduced A. alternata induced eosinophil influx in the lungs of BN rats. Similarly, intratracheally dosed mometasone produced a dose dependent attenuation in the number of eosinophils in the BAL fluid recovered from BN rats 48 h following the intratracheal challenge with A. alternata (not shown). These results demonstrate that intratracheal and the systemic administration

of corticosteroids can suppress A. alternata induced allergic lung inflammation in BN rats. Since prophylactically administered corticosteroids suppressed A. alternata induced pulmonary inflammation, we sought to investigate if equivalent therapeutic dosing (after an A. alternata challenge) of corticosteroids could also exert a similar effect. In Fig. 7, we show that orally dosed budesonide (3 mg kg  1) at 2, 6 or 23 h post an intratracheal challenge with A. alternata decreased the number of eosinophils and cytokines, such as IL-5 and IL-13 levels, in a time dependent manner 48 h after the A. alternata challenge. Budesonide dosed 1 h prior to A. alternata challenge was used as a positive control. Eosinophil numbers (Fig. 7A) following A. alternata challenge were significantly decreased in the BAL fluid at the earlier therapeutic budesonide dosing timepoints (2 and 6 h post-challenge) with 98 72% and 71 79% of inhibition respectively. In comparison to the later (23 h post challenge) therapeutic dosing time-point inhibition, which inhibited only 31 712% of eosinophils at the 48 h time point. (P o0.05) (Fig. 7). IL-5 and IL-13 cytokine levels (Fig. 7 B and C) following A. alternata challenge were significantly decreased in the BAL fluid at the earlier therapeutic budesonide dosing time-points (2 and 6 h post-challenge), with 9873% and 10372% for IL-5 and 103 71% and 105 71% for IL-13 of inhibition respectively. In comparison to the later (23 h post-challenge), therapeutic dosing time- point inhibition, which produced only 31 712% of eosinophils inhibition at the 48 h time point. (Po 0.05) (Fig. 7a). In addition, therapeutic dosing produced 81 710% and 90 74% of IL5 and IL-13 cytokine levels inhibition at the 48 h time point. (P o0.05) (Fig. 7 B and C). 3.6. Effect of a CRTH2 antagonist on A. alternata elicited pulmonary inflammation In addition to corticosteroids, we also investigated whether the inhibition of a clinically-relevant mechanism of allergic lung

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Fig. 5. CT images of the lungs from Brown Norway rats 24 and 48 h following intratracheal instillation of A. alternata (n¼ 6 per treatment group). Upper panel (A) is displaying representative images from PBS treated animals serving as a negative control group. In the lower panel (A) images are representative of animal lungs challenged with 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 point represents the Mean 7 S.E.M. (n¼6 per treatment group) is shown in panel (B). *Po 0.05 compared to control animals receiving intratracheal PBS treatment.

inflammation such as CRTH2 would lead to a suppression of inflammatory responses in A. alternata challenged Brown Norway rats (n¼ 8 per group). Mast cell derived production of Prostaglandin D2 (PGD2) is believed to be a prime mediator of allergic inflammation. Indeed, PGD2 is known to drive Th2 inflammation through 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 cellular subtypes and drive chemotaxis, as well as cytokine production,

including IL-4, IL-5 and IL-13. Since CRTH2 plays an important role in the early aspects of the allergic inflammation cascade (Kostenis and Ulven, 2006), we examined the effect of the CRTH2 antagonist on A. alternata elicited pulmonary inflammatory responses. We orally administered the CRTH2 inhibitor MK-7246, 1 h before and 23 h post-intratracheal instillation of the A. alternata. The MK7246 produced a dose dependent decrease in the number of eosinophils with a maximal inhibition of 74 75% in the 100 mg kg  1 group (Po 0.05) (Fig. 8 a), IL-5 (80 7 12%) and IL-13

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(76 714%) cytokines levels (P o0.05) (Fig. 8 B and C). The results reported show BAL fluid recovered from BN rats 48 h following the intratracheal. challenge with A. alternata. Orally dosed budesonide was used as a positive control producing significant inhibition of eosinophilic infiltration as well as both IL-5 and IL-13 cytokine levels. These results demonstrate that A. alternata induced eosinophil influx and inflammatory lung responses in BN rats may

Fig. 6. Dose dependent effects of Budesonide in Brown Norway rats challenged with A. alternata. Figure displays BAL fluid eosinophils influx 48 h after instillation of a single dose of A. alternata. Each bar represents the Mean 7 S.E.M. (n ¼8 per treatment group). *Po 0.05 compared to control A. alternata challenged animals receiving intratracheal vehicle treatment (0.9% PBSþ 0.5% Tween 80).

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

4. Discussion Asthma continues to be a significant health concern (Polosa and Benfatto, 2009). New emerging treatments, including therapeutic antibodies to inflammatory cytokines (i.e. IL-5, IL-13, IL-4 and IL-17), allergen-specific immunotherapy, combination-inhaled agents (i.e. ICS plus LAMA and or LABA), or small molecule G-protein coupled receptors (i.e. CRTH2 antagonist), may ultimately enhance the armaments used to treat this heterogeneous disease. However, the effectiveness of these approaches is currently being determined in human asthma trials (Albertson et al., 2013). From a preclinical perspective, animal models have been historically useful in the progression of novel targets from the laboratory to the clinic although their human predictability remains subject of continuous debate (Canning, 2003). While mouse models of allergic airway diseases have proven to be invaluable for informing on various aspects and pathways related to respiratory biology and pathology, they are not necessarily the preferred species from a drug development perspective. The rat has historically been the rodent species of choice for the majority of nonclinical safety assessment and toxicology studies. There is a significant knowledge and many years of accumulated data regarding baseline clinical chemistry and hematology ranges, as well as database of compounds that have been evaluated in singleand repeat-dose rat toxicology studies. In addition, a recent report by Seok et al. (2013) suggested that genomic responses in mice

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 influx in 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 Mean 7S.E.M. (n¼ 8 per treatment group). *Po 0.05 compared to control A. alternata challenged animals receiving p.o. vehicle treatment (0.5% MC þ 0.24% SDS).

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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-7246 administration. Panels (B) and (C) show IL-5 and IL-13 concentrations in the BAL fluid following the MK-7246 treatment. Each bar represents the Mean 7 S.E.M. (n¼ 8 per treatment group). *Po 0.05 compared to control A. alternata challenged animals receiving p.o. vehicle treatment (0.5% MC þ 0.24% SDS).

poorly mimic human inflammatory diseases, thereby casting an additional shadow on the translatability of mouse models to human inflammatory diseases. In humans, mounting evidence suggests that atopy to mold allergens is related to asthma severity (Black et al. 2000; Zureik et al., 2002; Denning et al., 2006). Here, we describe the respiratory impact of a single intratracheal instillation of A. alternata in BN rats, with a goal of establishing a simple but clinically relevant rodent asthma mechanism model that may be used to probe pathway biology of novel pharmacological targets. The mucosal surface of airway epithelium is the first anatomical interface where environmental allergens evoke host defense mechanisms. Extracts from A. alternata spores have been shown to stimulate the release of various pro-inflammatory cytokines including Thymic stromal lymphopoietin (TSLP), granulocyte macrophage colonystimulating factor, and IL-33 from epithelial cells (Kouzaki et al., 2009, Kouzaki et al., 2011). These events have been reported to be dependent on Protease Activated Receptor-2 signaling and the ST2 receptor (Boitano et al., 2011). Chronic instillation of the A. alternata extract via the intranasal route in mice has been shown to release IL-33, IL-5 and IL-13, followed by recruitment of eosinophils in the BAL fluid, independent of T or B cells involvement (Doherty et al., 2012). These events have been described to be mediated by innate lymphoid cells (ILC) (Bartemes et al., 2012), which are capable of mediating asthma-like pathological environments, by secreting Th2 cytokines such as IL-5 and IL-13, prior to the initiation of adaptive immune responses. The ILCs are not only present in lungs, but in other organs such as intestines and have been 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 of these cells is to mediate Th2 type allergic inflammatory responses independent of adaptive immunity (Monticelli et al., 2012). In our study, we found that a single intratracheal administration of A. alternata extract can mediate allergic responses similar to some aspects of the human asthma phenotype. We established a temporal profile of differential cell recruitment and proinflammatory cytokines after a challenge with an A. alternata extract. The A. alternata challenge is characterized by the release of early response pro-inflammatory cytokines such as IL-6, IL-1b, MIP-1a and MCP-1. The A. alternata instillation triggers a small and transient neutrophilic response which is resolved and returns to baseline 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 around 48 h post-challenge and returned to basal levels within 72 h. In addition to inflammatory responses, intratracheal instillation of A. alternata leads to pathological changes, such as increased alveolar infiltrates of neutrophils and eosinophils associated with edema, as compared to rats dosed with PBS alone. The initial perivascular and peribronchiolar inflammation was followed by alveolar inflammation and cell accumulation, progressing to a more sub-acute inflammatory response with notable epithelial cell hypertrophy in affected areas at 72 h following an A. alternata challenge. The cellular infiltration upon an A. alternata challenge was further confirmed in our CT imaging experiments measuring the temporal profile of microvascular leakage, leading to lung edema. In this study we also report the effect of commonly used corticosteroids, such as budesonide on A. alternata challenged Brown Norway rats. Budesonide dosed via an intratracheal and oral route, 2 h prior to an A. alternata challenge, inhibited Th2

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cytokines and eosinophilia recruitment in the BAL fluid in a dose dependent manner. Interestingly, the maximal dose of budesonide is partially effective if dosed 6 h after the challenge and completely ineffective if dosed 23 h post an A. alternata challenge. Thus, our results suggest that prophylactic budesonide intervention and the blockade of early events mediated by A. alternata is an essential driver/event in this model. Pharmacological intervention of corticosteroids is likely to occur at multiple stages during the A. alternata elicited signaling pathways. Specifically, corticosteroids can either directly or indirectly regulate the transcription (trans-repression) of various cytokines (i.e. IL-1, IL-2, IL-3, IL-4, IL5, 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 (vascular cell adhesion moleculeintracellular cell adhesion molecule 1), inflammatory enzymes, receptors and peptides (Barnes, 2006). Therefore, our results show that budesonide profoundly impacted IL-13 and IL-5 in the A. alternata model, which is consistent with the already described pharmacology of corticosteroids (Braun et al., 1997). In addition to evaluating the actions of corticosteroids in our A. alternata model, we also profiled the impact of the CRTH2 antagonist, MK-7246. The antagonism of CRTH2, while not being as efficacious as the corticosteroid, budesonide, in response to an A. alternata challenge, clearly impacted the eosinophil influx, as well as impacting overall IL-5 and IL-13 levels at 48 h. This is a novel finding, in that CRTH2 is believed to be involved in an adaptive immune response (Kostenis and Ulven, 2006), with significant effects in typical sensitized allergic models (Mauser et al., 2013). Recent investigations have expanded a role for CRTH2 outside the initial adaptive immune response and have identified and characterized CRTH2 on innate lymphoid cells as well (Mjösberg et al., 2011). In response to an allergen, epithelial derived cytokines (IL-25/IL-33/TSLP) promote a Th2 like response, by stimulating an innate lymphoid population, which in turn can produce the canonical IL-5 and IL-13 response (Licona- Limón et al., 2013). The type 2 innate lymphoid cells (ILC2) represent a critical nexus for expanding a Th2 repertoire in response to an epithelial insult by parasites or allergens (Neill et al., 2010). Human ILC2s expressing CRTH2 are responsive to both IL-25 and IL-33, resulting in production of IL-13 (Mjösberg et al., 2012). Additional studies directly demonstrated CRTH2 mediated induction of ILC2s migration and IL-4/IL-5/IL-13 production (Xue et al., 2013). In addition, Xue et al., 2013, reported that CRTH2 up-regulated the expression of receptor subunits for IL-33 and IL-25 (ST2 and Interleukin-17 Receptor A) in ILC2s. Furthermore, peripheral ILC2s demonstrated a 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 lymphoid cells, suggests an important role in modulating and controlling Th2 inflammation. Currently, CRTH2 antagonism is being investigated in clinical trials by several pharmaceutical companies for the treatment of allergic diseases such as asthma, allergic rhinitis and atopic dermatitis (Norman, 2014).

5. Summary In summary, since no single animal model exhibits all facets of asthma, animal models replicating various features of asthma are important tools in investigating the mechanisms of the disease pathogenesis and can provide an insight in identifying novel therapeutic targets. Modeling asthma has proven to be challenging, in part due to the clinical and pathophysiological complexity and heterogeneity of the human disease. In this study we established a novel pharmacodynamic model that recapitulates some cardinal features of asthma pathogenesis and can be used as a tool in drug

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discovery. Moreover, we demonstrated for the first time that the CRTH2 antagonist can reduce eosinophilia, most likely through inhibiting ILC2 mediated responses in the A. alternata model. Further studies are underway to establish the role of early cytokines releases, leading to heighten eosinophilic responses and underlying pathology after an A. alternata challenge.

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