The potential for repositioning antithyroid agents as antiasthma drugs

Accepted Manuscript The potential for repositioning anti-thyroid agents as anti-asthma drugs Shoichi Suzuki, PhD, Masahiro Ogawa, PhD, Shoichiro Ohta, MD, PhD, Kazuhiko Arima, MD, PhD, Satoshi Nunomura, PhD, Yasuhiro Nanri, PhD, Yasutaka Mitamura, MD, Tomohito Yoshihara, MD, Yutaka Nakamura, MD, PhD, Kohei Yamauchi, MD, PhD, Kazuyuki Chibana, MD, PhD, Yoshiki Ishii, MD, PhD, James J. Lee, PhD, Yasuaki Aratani, PhD, Shigeru Kakuta, PhD, Sachiko Kubo, PhD, Yoichiro Iwakura, PhD, Hiroki Yoshida, MD, PhD, Kenji Izuhara, MD, PhD

PII:

S0091-6749(16)30446-8

DOI:

10.1016/j.jaci.2016.04.047

Reference:

YMAI 12173

To appear in:

Journal of Allergy and Clinical Immunology

Received Date: 15 July 2015 Revised Date:

12 April 2016

Accepted Date: 20 April 2016

Please cite this article as: Suzuki S, Ogawa M, Ohta S, Arima K, Nunomura S, Nanri Y, Mitamura Y, Yoshihara T, Nakamura Y, Yamauchi K, Chibana K, Ishii Y, Lee JJ, Aratani Y, Kakuta S, Kubo S, Iwakura Y, Yoshida H, Izuhara K, The potential for repositioning anti-thyroid agents as anti-asthma drugs, Journal of Allergy and Clinical Immunology (2016), doi: 10.1016/j.jaci.2016.04.047. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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The potential for repositioning anti-thyroid agents as

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anti-asthma drugs

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Shoichi Suzuki PhD,a Masahiro Ogawa PhD,a Shoichiro Ohta MD, PhD,b

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Kazuhiko Arima MD, PhD,a Satoshi Nunomura PhD,a Yasuhiro Nanri PhD,a

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Yasutaka Mitamura MD,a Tomohito Yoshihara MD,a Yutaka Nakamura MD,

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PhD,c Kohei Yamauchi MD, PhD,c Kazuyuki Chibana MD, PhD,d Yoshiki Ishii MD,

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PhD,d James J. Lee PhD,e Yasuaki Aratani PhD,f Shigeru Kakuta PhD,g, h

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Sachiko Kubo PhD,h, i Yoichiro Iwakura PhD, h, i Hiroki Yoshida MD, PhD,j and

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Kenji Izuhara MD, PhDa

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From aDivision of Medical Biochemistry, jDivision of Immunology, Department of

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Biomolecular Sciences, bDepartment of Laboratory Medicine, Saga Medical School,

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Saga, Japan; cDivision of Pulmonary Medicine, Allergy, and Rheumatology, Department

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of Internal Medicine, Iwate Medical University School of Medicine, Morioka, Japan;

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d

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University School of Medicine, Mibu, Japan; eDivision of Pulmonary Medicine, Mayo

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Clinic Arizona, Scottsdale, AZ, USA; fGraduate School of Nanobioscience, Yokohama

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City University, Yokohama, Japan; gDepartment of Biomedical Science, Graduate

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School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan;

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Science, The University of Tokyo, Tokyo, Japan; iResearch Institute for Biomedical

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Sciences, Tokyo University of Science, Noda, Japan

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Department of Pulmonary Medicine and Clinical Immunology, Dokkyo Medical

Center for Experimental Medicine and Systems Biology, The Institute of Medical

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Corresponding author: Kenji Izuhara MD, PhD, Division of Medical Biochemistry,

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Department of Biomolecular Sciences, Saga Medical School, 5-1-1, Nabeshima, Saga,

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849-8501, Japan. TEL: +81-952-34-2261, FAX: +81-952-34-2058, E-mail:

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[email protected].

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Funding

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This work was funded in part by Grants-in-Aid for Scientific Research from the Japan

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Society for the Promotion of Science (S.S., #24591467, K.I., #25293224) and by the

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Grant for Joint Research Project of the Institute of Medical Science, the University of

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Tokyo (Shigeru Kakuta, Sachiko Kubo, Yoichiro Iwakura, and H.Y., #201).

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Word count: 1043 words

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Number of figures and table: 2 figures

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Keywords: asthma, anti-thyroid agent, allergic inflammation, drug repositioning,

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peroxidase, mouse model

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Abbreviations used

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OSCN-: hypothiocyanite

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MPO: myeloperoxidase

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EPX: eosinophil peroxidase

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LPO: lactoperoxidase

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BALF: bronchoalveolar lavage fluids

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ICS: inhaled corticosteroids 2

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methimazole: 2-mercapto-1-methylimidazole

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PTU: 6-propyl-2-thiouracil

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OVA: ovalbumin

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AHR: airway hyperresponsiveness

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CAPSULE SUMMARY

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The deleterious effects of peroxidases in asthma remain undetermined. Peroxidase

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inhibitors widely used as anti-thyroid agents, inhibited allergic airway inflammation in

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the model mice, which suggests that anti-thyroid agents can be repositioned as

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anti-asthma drugs.

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To the Editor:

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Bronchial asthma is a common disease in which type 2 immunity is dominant.1 Based

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on this immunological background, various anti-asthma drugs targeting type 2 immune

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mediators, such as IL-4, IL-5, IL-13, and CRTH2, are now under development.2

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However, to develop novel drugs, particularly biologics, huge investments of time and

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money are required and safety risks are involved. Drug repositioning, which is the

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process of finding new therapeutic indications for existing drugs, is highly desirable as

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an alternative strategy.3,4 However, to our knowledge, there has been no instance of drug

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repositioning in bronchial asthma.

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Airway peroxidase functions as a potent defense system against microbes by producing biocidal compounds, including hypothiocyanite (OSCN-), together with

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hydrogen peroxide generated by Duox1 and Duox2, which are members of the

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Nox/Duox family.5,6 Three heme peroxidases, myeloperoxidase (MPO), eosinophil

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peroxidase (EPX), and lactoperoxidase (LPO) (expressed at neutrophils, eosinophils,

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and epithelial cells, respectively), are involved in this system in the lung tissues. In

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contrast, the deleterious effects of the airway peroxidase system in bronchial asthma

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remain undetermined. To define the pathological roles of peroxidase in bronchial

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asthma, we applied heme peroxidase inhibitors widely used as anti-thyroid agents to a

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mouse model of allergic airway inflammation. We found that these agents efficiently

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inhibited allergic airway inflammation. These results suggest that anti-thyroid agents

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can be repositioned as anti-asthma drugs.

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We first examined the expression of three airway peroxidase genes in a mouse

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model of allergic airway inflammation. The pulmonary expression of all three heme

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peroxidases (Mpo, Epx, and Lpo) was significantly upregulated in response to an 5

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allergen challenge (Fig 1, A). The peroxidase activities were also enhanced in the

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bronchoalveolar lavage fluids (BALFs) of allergen-challenged mice (Fig 1, B). We then

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collected bronchial biopsy samples from 10 asthma patients (Table E1) to determine

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whether or not the expression of the heme peroxidases was also enhanced in the

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bronchial tissues of asthma patients. The peroxidase activities and the expression levels

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of LPO were not statistically enhanced. No expression of EPX or MPO was detected or

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was invariant between the patients and normal donors in contrast to the mouse analyses

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(Fig 1, C, data not shown). However, some patients showed distinctly high peroxidase

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activities and LPO expression. This may be due to the background (steps 2–4 according

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to the GINA2014 criteria), effects of the treatment for the patients (under good control

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of inhaled corticosteroids [ICSs]), and/or heterogeneity among asthma patients

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regarding airway peroxidase expression. These results suggest the involvement of the

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airway peroxidase system at least in the mouse model of allergic airway inflammation

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and possibly in some asthma patients.

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We applied the heme peroxidase inhibitors to examine the role of the airway peroxidase system in allergic airway inflammation. The approach we took to understand

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the role of this production system during allergic inflammatory responses involved

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inhibiting all peroxidase-mediated events in the lung because no inhibitors specific for

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LPO, MPO, or EPX have been developed. We examined the effects of

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2-mercapto-1-methylimidazole (methimazole) and 6-propyl-2-thiouracil (PTU), which

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are agents that inhibit all peroxidases and are widely used as anti-thyroid agents

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targeting thyroid peroxidase. For the experiments of methimazole or PTU ingestion, 0.2

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mg/mL of methimazole (Wako Pure Chemical Industries, Osaka, Japan) or 0.5 mg/mL

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of PTU (Sigma-Aldrich, St. Louis, Mo) in drinking water was administered orally every

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day for the indicated times. We adopted two protocols of methimazole administration, a

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long and a short administration strategy (Met-L and Met-S, respectively) (Fig 2, A).

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Methimazole was administered orally every day from the start of sensitization (day 0) in

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the long administration and from two days before the start of the allergen airway

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challenge (day 20) in the short administration.

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The parameters of ovalbumin (OVA)-induced airway inflammation (i.e., airway hyperresponsiveness [AHR], infiltration of inflammatory cells in BALF, and

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histological changes) were completely inhibited in the long administration and less so

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(yet significantly) in the short administration (Fig 2, B–D). The use of another

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peroxidase-inhibiting anti-thyroid agent, PTU, showed effects similar to but smaller

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than those of methimazole. Nonetheless, these effects showed a statistically significant

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improvement of AHR at the dosages used in this experiment (0.2 mg/mL of

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methimazole vs. 0.5 mg/mL of PTU) (Fig E1). These results strongly suggest that heme

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peroxidase activities are critical for the setting of allergic airway inflammation in the

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model mice.

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The Met-L decreased free T4 in serum by inhibiting thyroid peroxidase (1.32 ±

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0.14 ng/dL vs. 2.0 ± 0.27 ng/dL, P < 0.001) (Fig E2). We complemented the thyroid

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function by administering thyroxine to exclude the possibility that the inhibition of

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airway inflammation by methimazole was due to impaired thyroid function; however,

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administering thyroxine had no effect on airway responsiveness (Fig E3).

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Next, we used mice deficient in each of the three peroxidases (Mpo, Epx, and

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Lpo) to determine which peroxidase dominantly contributes to the setting of allergic

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airway inflammation. Since Lpo-deficient mice were not available, we generated

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Lpo-deficient animals on a BALB/c background in preparation for these studies (Fig 7

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E4). Epx- and Lpo-deficient mice showed a nominal but not statistically significant

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decrease of AHR compared to their control littermates (Fig E5, A). The Mpo-deficient

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mice showed no change of AHR. Furthermore, infiltration of eosinophils and T cells

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was decreased in the BALF of the Lpo-deficient mice, whereas there was no change in

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the infiltration in the Mpo- or Epx-deficient mice (Fig E5, B). These results suggest that

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the contributions of the three peroxidases are redundant in the setting of allergic airway

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inflammation. However, Lpo appears to be dominant among the peroxidases,.

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Our results may help explain the findings of several reports showing the beneficial effects of the accidental administration of anti-thyroid agents to asthma

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patients,7, 8 although there is a conflicting report.9 It is of note that in most patients,

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bronchial asthma was exacerbated by discontinuing or tapering off of anti-thyroid

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agents.7, 8 Currently, it remains unknown how heme peroxidase plays a role in the

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pathogenesis of allergic airway inflammation. We found that OSCN−, a product of

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airway peroxidase, activates NF-κB in airway epithelial cells (unpublished data), which

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partly explains the role of peroxidase in bronchial asthma. Drug repositioning is now

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expected as an alternative to drug discovery and development. The use of anti-thyroid

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agents could be the first example of drug repositioning in bronchial asthma.

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ACKNOWLEDGEMENTS

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We thank Dr. Dovie R. Wylie for the critical review of this manuscript and Prof.

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Hidenobu Soejima and Drs. Kenichi Nishioka and Hidetaka Watanabe for the

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instruction of Droplet Digital PCR. We also thank Maki Futamata, Tameko Takahashi,

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Chizuko Kondo, Kazuyo Yoshida, and Seiji Kawasaki for technical assistance.

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REFERENCES

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1. Peters MC, Mekonnen ZK, Yuan S, Bhakta NR, Woodruff PG, Fahy JV. Measures of

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gene expression in sputum cells can identify TH2-high and TH2-low subtypes of

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asthma. J Allergy Clin Immunol 2014;133:388-94.

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3. Tobinick EL. The value of drug repositioning in the current pharmaceutical market. Drug News Perspect 2009;22:119-25.

4. Li YY, Jones SJ. Drug repositioning for personalized medicine. Genome Med 2012;4:27.

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regarding periostin in bronchial asthma. Allergol Int 2015;64:S3-10.

5. Hawkins CL. The role of hypothiocyanous acid (HOSCN) in biological systems. Free Radic Res 2009;43:1147-58.

6. Barrett TJ, Hawkins CL. Hypothiocyanous acid: benign or deadly? Chem Res Toxicol 2012;25:263-73.

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2. Izuhara K, Matsumoto H, Ohta S, Ono J, Arima K, Ogawa M. Recent developments

7. Settipane GA, Schoenfeld E, Hamolsky MW. Asthma and hyperthyroidism. J Allergy Clin Immunol 1972;49:348-55.

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8. Nakazawa T, Kobayashi S. Influence of antithyroidal therapy on asthma symptoms in

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the patients with both bronchial asthma and hyperthyroidism. J Asthma

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1991;28:109-16.

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9. Grembiale RD, Naty S, Iorio C, Crispino N, Pelaia G, Tranfa CM. Bronchial asthma induced by an antithyroid drug. Chest 2001;119:1598-9.

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FIGURE LEGENDS

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FIG 1. Enhanced expression of airway peroxidase in the lung tissues of

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allergen-challenged mice and asthma patients

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A, Mpo, Epx, and Lpo expression (left) and immunostaining for Lpo (rigt) in the lung

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tissues of mice with or without OVA inhalation. The arrows indicate Lpo-positive cells.

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B, Heme peroxidase activities converted to EPX concentration in BALF of mice with or

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without OVA inhalation. C, Heme peroxidase activities converted to EPX concentration

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in BALF of asthma patients and LPO expression in the bronchial tissues of asthma

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patients. The averages of control samples are set as one-fold. *P<0.05, **P<0.01, and

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NS: not significant.

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FIG 2. Inhibitors against heme peroxidases improve airway allergic

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inflammation in mice

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A, The experimental protocol of the long and the short administrations of methimazole

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(Met-L and Met-S). B, Airway reactivity. Open circle: without OVA inhalation, closed

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circle: with OVA inhalation, closed square: Met-L, and closed triangle: Met-S. C, D,

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Analyses of BALF (C), and lung histology (D). *P<0.05, **P<0.01, #P<0.001, and NS:

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not significant (vs. with OVA inhalation).

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Heme peroxidase activity (×102 ng/mL)

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Copy number (×102/µg)

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Heme peroxidase activity (×103 ng/mL)

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Copy number (×103/µg)

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Lpo

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0 Control Asthma Suzuki et al. Figure 1

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Eosinophil

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Lung function BALF Histology Gene expression

Met-L Day 0

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4 8 Methacholine (mg/mL)

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Macrophage *

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Suzuki et al. Figure 2

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ONLINE REPOSITORY MATERIALS Induction of airway allergic inflammation in mice Seven- to ten-week-old female mice were used. Animal studies were undertaken

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following the guidelines for care and use of experimental animals of the Japanese

Association for Laboratory Animals Science (1987) and were approved by the Saga University Animal Care and Use Committee (Saga, Japan).

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The mouse model of airway allergic inflammation was generated as previously described1 with minor modifications. Mice were sensitized by intra-peritoneal injections

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of 50 µg ovalbumin (OVA, Sigma-Aldrich) and 1 mg alum (LSL, Tokyo, Japan) in 500 µL saline on days 0 and 12. Mice inhaled 1% OVA in saline on days 22, 26, and 30 or on days 22, 23, and 24, followed by assessment of airway responsiveness on the following day and of BALF, lung histology and gene expression two days later. In some

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experiments, 0.05 µg/mouse thyroxine (Sigma-Aldrich) was administered intra-peritoneally every day for the indicated times.

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Assessment of AHR

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Airway responsiveness to methacholine (Sigma-Aldrich) was assessed with a four-chamber whole-body plethysmograph from Buxco Electronics (Wilmington, NC) or flexiVentTM from SCIREQ (Montreal, Canada).

Digital PCR Droplet Digital PCR was performed on QX200 Droplet Digital PCR system (Bio-Rad, Hercules, CA) using ddPCR supermix for probes or ddPCR EVAGreen supermix. The sequences of primers are follows: Mpo, 5’-CCCTTCCTAAACTGAACCTGAC-3’ and

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5’-ATGGCCTCCGTCCTTCTC-3’; Epx, 5’-CTCCTGACTAACCGCTCTGC-3’ and 5’-TCACTTGACCGAGTGTCACC-3’. Predesigned TaqMan Gene Expression Assays (Life Technologies, Carlsbad, CA) for Lpo (Mm00475466_m1) and LPO

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(Hs00976400_m1) were used.

Analysis of BALF

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After mice were anesthetized, lungs were lavaged with 500 µL of BAL liquid (0.1%

BSA/50 µM EDTA/PBS), and BALF was collected. This procedure was repeated three

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times. BALF cell counts were determined with a particle counter (CDA500, Sysmex, Japan). Eosinophils, neutrophils, T cells, and macrophages were defined as siglec-F+ cells (BD Bioscience, San Diego, CA), Gr-1high cells (eBioscience, San Diego, CA),

Lung histology

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CD3+ cells (eBioscience), and strongly autofluorescent/bigger-size cells,2 respectively.

Paraffin-embedded lung sections were prepared and stained with hematoxylin and eosin

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or periodic acid-Schiff. For immunostaining, sections were incubated with anti-Lpo

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antibodies (Thermo Fisher Scientific, Waltham, MA) followed by visualization according to a standard procedure using 3,3’-diaminobenzidine (DAB).

Heme peroxidase activity One hundred µl of TMB solution (1-step Ultra TMB-ELISA, Thermo Fisher Scientific) was added to 100 µl of BALF in the presence or absence of 1mM sodium azide (Wako Pure Chemical Industries). The reaction was stopped by adding 50 µl of 2M sulfuric acid and the enzyme activity was evaluated by the absorbance at 450 nm. The

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peroxidase activity was converted to peroxidase concentrations using a standard curve generated with commercial human EPX (Lee BioSolutions, Maryland Heights, MO).

activity from total peroxidase activity.

Free T4

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Heme peroxidase activity was calculated by subtracting azide-insensitive peroxidase

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Free T4 was measured by Elecsys FT4II (Roche Diagnostics, Basel, Switzerland).

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Asthma patients

Ten asthma patients and ten healthy donors were recruited from the Iwate Medical University Hospital. The backgrounds of the asthma patients were described in Table E1. The asthma patients had no other medical disorders and were not current smokers; the

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ten control subjects were non-allergic and non-asthmatic patients. All the asthma patients were at step 2 to 4 according to the GINA2014 criteria and were well controlled with inhaled corticosteroids. Biopsy samples were taken from the bifurcations of the

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subsegmental bronchi under bronchoscopy. This study was approved by the Iwate Medical University Hospital Ethics Committee. Healthy donors had both normal

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spirometry and required a lung resection for treatment of lung carcinoma. They were not current smokers and had no history of asthma and allergy or systemic diseases.

Peroxidase-deficient mice Mpo- or Epx-deficient mice were prepared as previously described (Fig E6).3,4 Lpoflox/+ chimeric mice were obtained by injecting Lpotm1a(EUCOMM)Wtsi ES cells (JM8.N4, C57BL/6N background, European Mouse Mutant Cell Repository, Neuherberg,

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Germany) into blastocysts from ICR mice (Japan SLC, Hamamatsu, Japan). Lpoflox/+ mice were selected by mating Lpoflox/+ chimeric mice and C57BL/6 mice (Kyudo, Tosu, Japan). Then Lpo-deficient mice were generated by mating Lpoflox/+ mice and Cre

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transgenic mice (BALB/c-Tg(CMV-cre)1Cgn/J #3465, The Jackson Laboratory, Bar Harbor, ME, USA) and were backcrossed onto BALB/c mice (Japan SLC) for eight

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generations.

REFERENCES 1. Matsushita H, Ohta S, Shiraishi H, Suzuki S, Arima K, Toda S, et al. Endotoxin

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tolerance attenuates airway allergic inflammation in model mice by suppression of the T-cell stimulatory effect of dendritic cells. Int Immunol 2010;22:739-47. 2. van Rijt LS, Kuipers H, Vos N, Hijdra D, Hoogsteden HC, Lambrecht BN. A rapid flow cytometric method for determining the cellular composition of bronchoalveolar

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lavage fluid cells in mouse models of asthma. J Immunol Methods 2004;288:111-21. 3. Aratani Y, Koyama H, Nyui S, Suzuki K, Kura F, Maeda N. Severe impairment in early host defense against Candida albicans in mice deficient in myeloperoxidase.

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Infect Immun 1999;67:1828-36.

4. Denzler KL, Borchers MT, Crosby JR, Cieslewicz G, Hines EM, Justice JP, et al.

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Extensive eosinophil degranulation and peroxidase-mediated oxidation of airway proteins do not occur in a mouse ovalbumin-challenge model of pulmonary inflammation. J Immunol 2001;167:1672-82.

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Supplemental table 1 Clinical backgrounds, LPO expression, and heme peroxidase activity in asthma patients

44

2

35

M

134

3

36

M

52

4

38

M

34

5

40

M

34

6

36

F

30

7

65

M

39

8

48

M

25

9

39

M

62

10

40

F

46

FP 200 µg/day FP 500 µg/day FP 500 µg/day FP 400 µg/day FP 200 µg/day BUD 200 µg/day FP 200 µg/day FP 400 µg/day FP 100 µg/day BUD 400 µg/day

Eosinophil (/µL)

528

98.0

33

82

53.3

286

2657

96.8

1286

LPO mRNA (copy number/µg)

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M

FEV1 (%)

Heme peroxidase activity (ng/mL)

11600

809

4100

2792

659

130

367

63.0

296

640

8317

96.3

280

16400

567

82.8

341

76500

483

48.1

252

12300

392

293 572 909

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IgE (IU/mL)

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Treatment

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FeNO (ppb)

88

65.9

391

N.D.

550

134

104.8

293

N.D.

6083

190

59.1

179

960

967

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Sex

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Age (year)

FeNO, fractional exhaled nitric oxide; FP, fluticasone propionate; BUD, budesonide; FEV1, forced expiratory volume in 1 second; N.D., not determined.

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OVA ip

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OVA inhalation

8 22 23 24 2526 Lung function BALF

Eosinophil

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OVA−

OVA+

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PTU

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1 0

0

**

* * ** **

0

2

4 8 Methacholine (mg/ml)

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Neutrophil

0.8 0.6 0.4

0

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Met

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Macrophage

25

×104 cells/BALF

3

26

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PTU Day 20 Met Day 20

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Met

PTU Suzuki et al. Figure E1

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1.5

*

AC C

Free T4 (ng/dL)

2.5

Control

Met(−)

Met-L

Met-S

Suzuki et al. Figure E2

A

OVA ip

Day 0

OVA inhalation

12

22

31

RI PT M AN U

16 12

TE D

8

* **

EP

4 0

31

SC

20

0

AC C

Respiratory resistance (cmH2O.s/ml)

Met-L + thyroxine Day 0

15 30 Methacholine (mg/mL)

60

3.0 2.5 Free T4 (ng/dL)

C

30 31

Lung function

ACCEPTED MANUSCRIPT

Met-L Day 0

B

26

2.0

**

1.5

#

1.0 0.5 0

Met(−)

Met-L

Met-L + thyroxine Suzuki et al. Figure E3

ACCEPTED MANUSCRIPT

FRT

loxP

Exon 2

FRT loxP

LacZ

neo

Exon 3

Exon 4

Lpo -3’arm

Lpo-5’arm Cre recombinase LacZ

Exon 4

SC

Exon 2

LAR3

M AN U

Lpo-5’arm

loxP

RI PT

A

B Lpo+/−

Lpo−/−

AC C

EP

Lpo+/+

TE D

Lpo-5’arm : CAACCTGGAGAGAGCAAGCCGAGC Lpo -3’arm : CACTCCGACCCCGGCAGCCATTAG LAR3: CAACGGGTTCTTCTGTTAGTCC

Lpo-5’ arm/Lpo-3’ arm (543 bp) Lpo-5’ arm/LAR3 (278 bp)

Suzuki et al. Figure E4

NS

NS

6 4 NS

2 0

0

15

30

60

80

8 6

NS

4

NS NS

2 0

0

15

Methacholine (mg/mL)

0

×104 cells/BALF

10

5

WT

KO

3

4

NS

NS

2

0

0

15

OVA−

WT

T cell

NS

2 1

OVA−

KO

*

NS

30

60

80

Methacholine (mg/mL)

8

WT

Macrophage NS

6 4 2 0

0

0 OVA−

80

M AN U

10

4

NS

NS

20

60

Mpo

Neutrophil

15

×104 cells/BALF

×104 cells/BALF

Eosinophil

30

30

Methacholine (mg/mL)

B 40

*

6

×104 cells/BALF

NS

SC

8

10

Lpo

8

RI PT

10

Respiratory resistance (cmH2O.s/mL)

ACCEPTED MANUSCRIPT Epx 12

Mpo

12

Respiratory resistance (cmH2O.s/mL)

Respiratory resistance (cmH2O.s/mL)

A

OVA−

KO

WT

KO

WT

Eosinophil

**

4

WT

KO

12

×104 cells/BALF

8

OVA−

OVA−

WT

OVA−

6 3

KO

WT

Macrophage

T cell

* 2

WT

KO

NS

4 2

16

4

OVA−

6

0

KO

6

0

×104 cells/BALF

2

8

NS

WT

NS

4

Lpo

Neutrophil

OVA−

6

0

KO

9

0

×104 cells/BALF

1

0

KO

12

8

×104 cells/BALF

OVA−

T cell

8

×104 cells/BALF

2

EP

3

0

Neutrophil

NS

×104 cells/BALF

6

16

3

NS

9

0

×104 cells/BALF

Eosinophil

AC C

×104 cells/BALF

12

TE D

Epx

OVA−

WT

KO

Macrophage

12 8

NS

4 0

OVA−

WT

KO Suzuki et al. Figure E5

ACCEPTED MANUSCRIPT

Mpo+/+ Mpo+/− Mpo−/−

WT allele (608 bp) KO allele (361 bp)

AC C

EP

TE D

KO allele (991 bp) WT allele (687 bp)

M AN U

SC

RI PT

Epx+/+ Epx+/− Epx−/−

Suzuki et al. Figure E6