Neutrophils are a major source of the epithelial barrier disrupting cytokine oncostatin M in patients with mucosal airways disease

Accepted Manuscript Neutrophils are a major source of the epithelial barrier disrupting cytokine Oncostatin M in mucosal airways disease Kathryn L. Pothoven, PhD, James E. Norton, MSc, Lydia A. Suh, BSc, Roderick G. Carter, BSc, Kathleen E. Harris, BSc, Assel Biyasheva, PhD, Kevin Welch, MD, Stephanie Shintani-Smith, MD, David B. Conley, MD, Mark C. Liu, MD, Atsushi Kato, PhD, Pedro C. Avila, MD, Qutayba Hamid, MD, PhD, Leslie C. Grammer, III, MD, Anju T. Peters, MD, Robert C. Kern, MD, Bruce K. Tan, MD, Robert P. Schleimer, PhD PII:

S0091-6749(16)31462-2

DOI:

10.1016/j.jaci.2016.10.039

Reference:

YMAI 12512

To appear in:

Journal of Allergy and Clinical Immunology

Received Date: 27 June 2016 Revised Date:

13 October 2016

Accepted Date: 19 October 2016

Please cite this article as: Pothoven KL, Norton JE, Suh LA, Carter RG, Harris KE, Biyasheva A, Welch K, Shintani-Smith S, Conley DB, Liu MC, Kato A, Avila PC, Hamid Q, Grammer III LC, Peters AT, Kern RC, Tan BK, Schleimer RP, Neutrophils are a major source of the epithelial barrier disrupting cytokine Oncostatin M in mucosal airways disease, Journal of Allergy and Clinical Immunology (2017), doi: 10.1016/j.jaci.2016.10.039. 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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Neutrophils are a major source of the epithelial barrier disrupting cytokine Oncostatin M

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in mucosal airways disease

3 4 Kathryn L. Pothoven1 PhD, James E. Norton1 MSc, Lydia A. Suh1 BSc, Roderick G.

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Carter1 BSc, Kathleen E. Harris1 BSc, Assel Biyasheva1 PhD, Kevin Welch2 MD,

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Stephanie Shintani-Smith2 MD, David B. Conley2 MD, Mark C. Liu3 MD, Atsushi Kato1

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PhD, Pedro C. Avila1 MD, Qutayba Hamid4 MD, PhD, Leslie C. Grammer III1 MD, Anju T.

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Peters1 MD, Robert C. Kern2 MD, Bruce K. Tan2 MD, Robert P. Schleimer1,2 PhD

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Feinberg School of Medicine, Chicago, Ill

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Chicago, Ill

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Johns Hopkins Asthma and Allergy Center, Baltimore, Md

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Research Institute, Montreal, Quebec, Canada

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Divisions of Allergy and Clinical Immunology, Pulmonary and Critical Care Medicine,

Meakins-Christie Laboratories of McGill University and McGill University Health Center

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Department of Otolaryngology, Northwestern University Feinberg School of Medicine,

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Division of Allergy-Immunology, Department of Medicine, Northwestern University

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This study was supported in part by Grants R37HL068546, R01HL078860,

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U19AI106683 and T32AI007476-16 from the NIH and by the

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Ernest S. Bazley Foundation

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Capsule Summary:

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In this study, we show that neutrophils are a major source of OSM, the epithelial barrier

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disrupter, in chronic rhinosinusitis (CRS). GM-CSF, a known stimulus of OSM

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production, was also elevated in nasal polyps and was detected at levels sufficient to

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induce in vitro OSM production in peripheral blood neutrophils. OSM-producing

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neutrophils were found in tissues from patients with severe asthma. Neutrophils may be

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an important source of OSM resulting in impaired epithelial barrier function.

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34 Clinical implications:

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Neutrophils are a major source of OSM producing cells in CRS, and severe asthma. GM-

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CSF was elevated in CRS, at levels sufficient to induce neutrophil derived OSM

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production. Therapeutic targeting of OSM, neutrophils or GM-CSF may be beneficial in

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mucosal disease through the restoration of epithelial barrier function.

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40 Keywords:

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Oncostatin M, Epithelial barrier, Neutrophils, Chronic rhinosinusitis, Atopic asthma,

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Granulocyte-Monocyte Colony Stimulating Factor

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Abbreviations:

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ARG1: Arginase 1

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BAL: Bronchoalveolar lavage

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CRS: Chronic rhinosinusitis

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CRSsNP: CRS without nasal polyps

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CRSwNP: CRS with nasal polyps

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ECP: Eosinophil cationic protein

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FEV1: Forced Expiratory Volume in 1 Second

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FSTL1: Follistatin-like 1

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GM-CSF: Granulocyte-Macrophage colony stimulating factor

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HKSA: Heat killed staphylococcus aureus

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NHBE: Normal human bronchial epithelial cells

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MMR: Macrophage mannose receptor

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PMN: Polymorphonuclear leukocyte/neutrophil

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OSM: Oncostatin M

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UT: Uncinate tissue

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Abstract:

62 Background: We have previously shown that Oncostatin M (OSM) is elevated in nasal

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polyps of chronic rhinosinusitis (CRS) patients, as well as in bronchoalveolar lavage

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(BAL) fluids after segmental allergen challenge in allergic asthmatics. We also showed in

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vitro that physiological levels of OSM impair barrier function in differentiated airway

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epithelium.

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Objective: We sought to determine which hematopoietic or resident cell type(s) were the

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source of the OSM expressed in mucosal airways disease.

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Methods: Paraffin-embedded NP sections were stained with fluorescence-labeled

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specific antibodies against OSM, GM-CSF and hematopoietic cell specific markers. Live

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cells were isolated from NP and matched blood samples for flow cytometric analysis.

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Neutrophils were isolated from whole blood, cultured with the known OSM inducers GM-

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CSF and FSTL1, and levels of OSM were measured in the supernatants. Bronchial

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biopsy sections from controls, moderate asthmatics and severe asthmatics were stained

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for OSM and neutrophil elastase.

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Results: OSM staining was observed in NP, showed co-localization with neutrophil

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elastase (n=10), and did not co-localize with markers for eosinophils, macrophages, T

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cells or B cells (n=3-5). Flow cytometric analysis of NP (n=9) showed that 5.1±2% of

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CD45+ cells were OSM+, and of the OSM+ cells, 56±7% were CD16+Siglec8-, indicating

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neutrophil lineage. Only .6±.4% of CD45+ events from matched blood samples (n=5)

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were OSM+, suggesting that elevated OSM in CRS was locally stimulated and produced.

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A majority of OSM+ neutrophils expressed Arginase 1 (72.5±12%), suggesting a N2

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phenotype. GM-CSF was elevated in nasal polyp tissue compared to control, and was

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sufficient to induce OSM production (p<.001) in peripheral blood neutrophils in vitro.

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OSM+ neutrophils were also observed at elevated levels in biopsies from patients with

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severe asthma. Additionally, OSM protein was elevated in induced sputum from

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asthmatic patients compared to controls (p<.05).

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Conclusions: Neutrophils are a major source of OSM producing cells in CRS and severe

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asthma.

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Introduction:

114 Epithelial barrier dysfunction has been shown to be important in the pathogenesis of

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many diseases including asthma, atopic dermatitis, chronic rhinosinusitis (CRS), and

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eosinophilic esophagitis (EoE) 1, 2, 3, 4, 5, 6, 7. Our laboratory has previously shown that the

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cytokine Oncostatin M (OSM) was elevated in nasal polyps of chronic rhinosinusitis

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(CRS) patients, in esophageal biopsies from eosinophilic esophagitis (EoE) patients, and

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in BAL fluids after allergen challenge in allergic asthmatic patients 8. Physiological levels

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of OSM were sufficient to induce barrier dysfunction in ex vivo cultured airway epithelium

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as measured by decreased epithelial resistance, increased epithelial permeability, and

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loss of tight junction structure. Additionally, levels of OSM in both nasal tissue from CRS

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patients and bronchoalveolar lavage fluid (BAL) from allergen-challenged allergic

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asthma patients correlated with markers of epithelial leak, suggesting that OSM may

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mediate barrier dysfunction in vivo in mucosal disease 8. We hypothesized that OSM-

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induced loss of barrier function could allow the entry of various allergens, pathogens and

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environmental factors into the tissue where they could elicit a chronic immune response.

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It is important now to identify the cell type responsible for OSM production in mucosal

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disease in order to understand the mechanism of barrier disruption in eosinophilic

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mucosal disease.

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Neutrophil-derived OSM has been shown to contribute to the pathogenesis of many

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conditions including acute lung injury, asthma, breast cancer, and rheumatoid arthritis 9,

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complement factor 5a, and thrombin have all been shown to induce OSM expression in

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various cell types 13, 14, 15, 16, 17, only GM-CSF was sufficient to induce OSM in neutrophils

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11, 18

. Although osteopontin, prostaglandin E2 (PGE2), follistatin-like 1 (FSTL1),

. Elbjeirami et al. showed that OSM was induced during neutrophil transendothelial

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migration in response to endothelial production of GM-CSF 18. Queen et al. showed that

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co-culture of neutrophils with the breast cancer cell lines MDA-MB-231 and T-47D

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induced OSM production that was lost when GM-CSF was blocked 11. Additionally, Miller

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et al, have shown that follistatin like-1 (FSTL1) was necessary for OSM induction in a

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mouse model of chronic asthma 16. Both GM-CSF and FSTL1 have been shown to be

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elevated in airways disease 19, 20, 21, 22, and we hypothesized that one or both of these

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cytokines may induce neutrophil derived OSM in CRS.

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Recent studies have described subtypes of polarized neutrophils, the classical,

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proinflammatory N1 neutrophil and the anti-inflammatory, or tumorigenic N2 neutrophil

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M1 and M2 macrophage counterparts 23. N2 neutrophils specifically have been shown to

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promote tumor growth and metastasis, as well as play an important role in repair

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processes and the resolution of inflammation 24, 25, 26. N1 and N2 neutrophil polarization

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has been best studied in mice, and N2 neutrophils have been shown to express the

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macrophage mannose receptor (MMR), arginase 1 (ARG1), chitinase-like 3 (Ym1), IL-10

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and TGFβ, which are also characteristic markers of M2 macrophages. Both M2

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macrophages and N2 neutrophils have been associated with tissue repair mechanisms

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cytokines and chemokines such as TNF, IL-1β, CCL3, CCL5, IL-6 and IL-12 25. Although

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progress is being made, N2 neutrophils in humans are much less extensively studied,

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and the markers used to define N2 neutrophils in mice may not all be relevant as

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markers for human N2 neutrophils 27. Because N2 neutrophils and OSM have been

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shown to be important for repair processes, we wanted to test the hypothesis that N2

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neutrophils were responsible for OSM production in CRS. In this manuscript we show

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that neutrophils are a major source of OSM producing cells in nasal polyps and

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. These N1 and N2 neutrophils have been shown to be very similar in function to their

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. Compared to N2, N1 neutrophils have been shown to express more proinflammatory

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neutrophils also express OSM in severe asthma. We also show that the OSM inducer,

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granulocyte-macrophage colony-stimulating factor (GM-CSF), was elevated in nasal

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polyps at levels sufficient to induce OSM production in cultured neutrophils.

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Methods:

192 Patients and tissue sample collection:

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Control subjects and patients with CRS were recruited from the Allergy-Immunology and

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Otolaryngology clinics of the Northwestern Medicine and the Northwestern Sinus Center

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at Northwestern Medicine. Nasal polyps and peripheral blood were obtained during

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routine functional endoscopic sinus surgery from patients with CRS, as defined by the

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American Academy of Otolaryngology—Head and Neck Surgery Chronic Rhinosinusitis

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Task Force 28. Patients with established immunodeficiency, pregnancy, coagulation

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disorder, or cystic fibrosis were excluded from this study. Characteristics of subjects in

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the study are shown in table E1. Control subjects did not have a history of sinonasal

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inflammation, and tissue was collected during endoscopic skull-base tumor excisions,

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and benign skull-base procedures including cerebrospinal fluid leak, encephalocele, and

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petrous apex cholesterol granuloma. The tumors excised include: pituitary adenoma (5),

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chordoma (4), meningioma (2), esthesioneuroblastoma (1), clival metastatic carcinoma

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(1), inverted Schneiderian papilloma (1) and osteoma (1). Asthma and atopic status

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were determined by the surgeon treating the patient. Segmental allergen challenge and

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bronchoalveolar lavage (BAL) were performed in a patient cohort of allergic asthmatics,

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as previously described 29, 30. Bronchial biopsies were obtained from asthmatics during

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bronchoscopy at the Meakins-Christie Laboratories at McGill University Montreal,

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Quebec with the approval of the respective Institutional Review Boards, which has been

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previously described 31. Induced sputum was obtained in the Allergy-Immunology clinic

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of Northwestern Medicine as previously described 32, 33.

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Immunofluorescent staining:

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Nasal polyp specimens were processed for histology as previously described 34.

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Sections were blocked with PBS containing 5% goat serum (Vector Laboratories,

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Burlingame, Calif) and .5% Triton X-100 (Sigma Aldrich, St Louis, MO) for 1 hour at

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room temperature (RT), then incubated with either rabbit anti-OSM (N-1, 1:400, Santa

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Cruz Biotechnology, Dallas, TX), mouse anti-neutrophil elastase (NP57, 1:100, Dako

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North America, Carpinteria, CA), mouse anti-CD68 (PG-M1, 1:50, Thermofisher), mouse

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anti-ECP (EG2, 1:1000, Diagnostics Development, Uppsala, Sweden), mouse anti-

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tryptase (AA1, 1:10000, Thermofisher), mouse anti-CD3 (SP7, 1:1, Thermofisher)

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mouse anti-CD20 (L26, 1:250, Thermofisher) or rat anti-GM-CSF (BVD2-21C11, 1:100,

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Abcam, Cambridge, MA) for 1 hour at RT. The sections were then incubated with

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secondary antibody for 1 hour at RT with Alexa Fluor 488 goat anti-rabbit antibody,

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Alexa Fluor goat anti-mouse 568, Alexa Fluor goat anti-rat 568, Alexa Fluor goat anti

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rabbit 647, or Alexa Fluor goat anti-mouse 647(Thermofisher). Slides were mounted with

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slowfade gold antifade reagent with DAPI counter stain (Thermofisher). To control for

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background staining, additional slides were stained with the secondary antibody only.

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Imaging was performed using a Nikon A1R confocal microscope using the 20x objective.

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Images were processed using ImageJ software.

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Flow Cytometric Analysis:

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Live cells were isolated from nasal polyp tissues obtained during endoscopic sinus

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surgery. Nasal polyps were incubated overnight on an oscillator in 1mg/mL collagenase I

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(Worthington Biochemical Corporation, Lakewood, NJ) and 30ug/mL DNase

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(Worthington Biochemical Corporation) in RPMI at 4 degrees in a gentleMACS tube.

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Cells were then dissociated using a gentleMACS cell dissociator (Miltenyi Biotec, San

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Diego, CA) and then passed through a 70µm cell strainer (BD Biosciences, San Jose,

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CA). Live blood cells were isolated from the buffy coat of centrifuged peripheral blood to

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ensure that the granulocyte population would be present for analysis. For flow cytometry,

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cells were first stained using the LIVE/DEAD Aqua dead cell staining kit (Thermofisher,

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Waltham, MA), to facilitate gating only on live cells. Cells were then blocked using 10µL

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of Fc blocking reagent for 15min (Miltenyi Biotec, San Diego, CA) and then cells were

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stained at RT for 15min with one of 3 panels of antibodies listed in supplemental table

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E2. Following cell surface staining, cells were then fixed and permeablized using the BD

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cytofix/cytoperm kit for intracellular staining following manufacturer’s protocols (BD

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Biosciences, San Jose, CA), and cells were stained intracellularly for 30min at 4°C. Cells

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were analyzed on an LSRII flow cytometer with FACSDiva software. All analysis and

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compensation were done using FlowJo software (Treestar, Ashland, OR). All flow

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cytometric studies were prepared using the appropriate single stained control beads (BD

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Biosciences, San Jose, CA and eBioscience, San Diego, CA) and fluorescence minus

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one (FMO) controls.

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Neutrophil isolation and cell culture:

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Neutrophils were isolated from peripheral blood using an EasySep direct human

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neutrophil isolation kit (Stemcell, Vancouver, BC). Cells were then counted and cultured

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at 107 cells/mL in RPMI supplemented with 5% fetal bovine serum and 1%

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penicillin/streptomycin. Neutrophil cultures were treated with GM-CSF (1-25 ng/mL, R&D

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Systems, Minneapolis, MN), FSTL1 (100-1000 ng/mL, R&D Systems), Interferon-γ

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(IFNγ) (10 ng/mL, R&D Systems), lipopolysaccharide (LPS) (1µg/mL, Enzo Life

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Sciences, Farmingdale, NY), Interleukin 4 (IL-4) (20 ng/mL, R&D Systems), Interleukin

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13 (IL-13) (20 ng/mL, R&D Systems), Interleukin 25 (IL-25) (10-100ng/mL, R&D

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Systems), Interleukin 33 (IL-33) (10-100 ng/mL, R&D Systems), thymic stromal

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lymphopoietin (TSLP) (10-100 ng/mL, R&D Systems) or Leukotriene C4 (LTC4) (10-6-10-7

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Cayman Chemical, Ann Arbor, MI) for 20 hours, cell culture supernatants were collected

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for protein analysis and cell lysates were collected to isolate RNA. Plates were coated

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with E-selectin, ICAM (50ng/well, Peprotech, Rocky Hill, NJ) or bovine serum albumin

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(BSA) (50ng/well, Sigma Aldrich) in pH 8 tris buffered saline (TBS) overnight at 4

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degrees. Neutrophils were seeded into the wells and the culture supernatants were

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harvested at 20 hours.

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275 NHBE cell culture and injury:

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Normal human bronchial epithelial (NHBE) cells were cultured at air-liquid interface

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(ALI), as previously described 8. As a model of injury, fully differentiated NHBE were

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treated apically with .5mL of either PBS, or PBS containing 5x108 particles/mL of heat

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killed staphylococcus aureus (HKSA). Following culture for 48h, cell supernatants were

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collected for protein analysis.

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Protein quantification:

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Oncostatin M protein was detected in neutrophil cell culture supernatants using a

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DuoSet ELISA kit (R&D Systems, Minneapolis, MN). FSTL1 and GM-CSF protein were

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detected in neutrophil cell culture supernatants and FSTL1 was detected in sinus tissue

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extracts using DuoSet ELISA kits (R&D Systems). GM-CSF protein was detected in

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sinus tissue lysates using a Human Magnetic Protein Luminex Assay (R&D Systems).

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OSM protein was detected in sinus tissue lysates as previously described 8. Neutrophil

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Elastase protein (Hycult Biotech, Plymouth Meeting, PA) and Eosinophilic Cationic

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Protein (MBL International, Woburn, MA) were detected using ELISA, and sinus tissue

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extracts were prepared as previously described 35.

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295 Quantitative RT-PCR and microarray analysis:

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RNA isolation from tissue samples was prepared as previously described 34. Single-

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strand cDNA was synthesized from 0.5µg of total RNA with SuperScript II reverse

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transcriptase and random primers (Invitrogen, Carlsbad, Calif). Quantitative real-time

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RT-PCR was performed using an Applied Biosystems 7500 Sequence Detection System

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(Applied Biosystems, Foster City, Calif) in 20µL reactions (10µL of 2x TaqMan Master

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mix [Applied Biosystems], 400nmol/L of each primer and 200nmol/L of TaqMan probe

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plus 10µg of cDNA). Primer and probe sets for OSM (Hs00968300_g1) ARG1 and

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MRC1 (Hs00267207_m1) were purchased from Applied Biosystems. Primers

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(Forward:GAAGGTGAAGGTCGGAGTC, Reverse: GAAGATGGTGATGGGATTTC) and

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probe (6-VIC-CAAGCTTCCCGTTCTCAGCC-MGB) for GAPDH were synthesized by

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IDT technologies (IDT Technologies, Coralville, IA) and used to detect the housekeeping

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gene in all RT-PCR studies. Microarray analysis was performed, as previously described

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. All microarray data have been deposited to gene expression omnibus: GSE36830.

310 Statistical Analysis:

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All data were analyzed using GraphPad Prism 6 software (GraphPad Software, La Jolla,

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Calif). All data are reported as mean ± SEM. Differences between groups were analyzed

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using a non-parametric Kruskal-Wallis ANOVA or Mann-Whitney U test. Correlations

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were assessed using the Spearman correlation. Significance: *p<0.05, **p<0.01,

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***p<0.001, ****p<.0001.

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Results:

323 Neutrophils are a major source of OSM in Nasal Polyps:

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To determine which cell type was making OSM in CRS, we first obtained sections from

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nasal polyps and used immunofluorescence and specific antibodies to stain for OSM

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and markers of various cell types including CD3, CD20, tryptase, elastase, ECP, and

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CD68. We did not observe co-localization of OSM with markers for macrophages (Figure

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1A-B) or eosinophils (Figure 1C-D), while we observed some co-localization with a minor

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population of mast cells (Figure 1E-F). We also did not observe co-localization of OSM

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with T cells or B cells (data not shown). Elastase positive cells made up the majority of

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the OSM positive cells, suggesting that neutrophils are an important source of OSM

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(Figure 1G-H). Control images with no primary antibody for CD68, ECP, tryptase, and

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elastase staining strategies are shown in Figure E1A-D. Further analysis of our

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previously published microarray database (GEO: GSE36830) showed that OSM mRNA

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expression correlated with the granulocyte marker CD16 (FCGR3) (r = .61, p<.01; Figure

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E1E), and the neutrophil specific protease, cathepsin G (CTSG) (r = .56, p<.01; Figure

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E1F) in nasal polyps and uncinate tissue from control, CRSsNP and CRSwNP patients,

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suggesting a potential relationship between OSM expression and neutrophils

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FCGR3 is not neutrophil specific, FCGR3 and CTSG correlated with each other in the

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database (r =.56, p<.01; Figure E1G), suggesting that neutrophils are responsible for

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much of the FCGR3 expression in sinonasal tissue. We also observed a positive

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correlation between elastase and OSM using lysates from both nasal polyps, and

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uncinates from control, CRSsNP, and CRSwNP patients (r = .65, p<.01; Figure E1H).

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347 Flow cytometric analysis of OSM producing cells in nasal polyps:

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To further validate the observation that neutrophils are a major source of OSM

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producers in nasal polyps, we utilized flow cytometry. Live cells were isolated from nasal

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polyps, and stained with CD45, OSM, CD16 and Siglec-8. Within the CD45 gate,

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5.1±2% of the cells were OSM+, and the OSM+ cells were determined using the

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appropriate FMO control. Of the OSM+ cells, 29.8±8% were excluded during analysis of

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the data because the wavelengths of the fluorophores used to stain for CD16-APCH7

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siglec 8-Alexa 647 were near each other and the rigorous compensation algorithm that

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we used excluded them to ensure the accuracy of the data. The majority of the cells that

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were excluded were single positive for either CD16 or siglec 8, suggesting they were

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granulocytes. Within the OSM+ gate, we observed two main populations; the major

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population was CD16+Siglec-8- (80.4±5%), which were neutrophils, and the minor

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population was CD16+Siglec-8+ (14.1±5%), which were either mast cells, basophils, or

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eosinophils. We did observe a small CD16-Siglec-8- population (5.5±3%) that was

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present in less than half of the polyps we analyzed (Figure 2A-C). Additionally,

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neutrophils were isolated from peripheral blood and nasal polyp tissue of five matched

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CRSwNP patients, and OSM expression in blood neutrophils was only minimally

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observed in one patient (.6±.4%), indicating that in vivo OSM induction was likely a

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locally induced event (Figure 2D). The full gating strategy for this antibody panel,

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including the OSM FMO control is available in Figure E2.

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OSM was expressed in a minor population of mast cells:

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To further identify the cells within the minor CD16+Siglec8+ population of OSM

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producers, we stained live cells isolated from nasal polyps with CD45, OSM, CD16,

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FcεRI, c-kit, and either EMR-1 or Siglec8. In the CD45+ gate, 4.6±2% events were

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OSM+. Within the OSM+, 15.6±10% were FcεRI+c-kit+, indicating mast cells, 3.7±1%

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were FcεRI+c-kit-, indicating basophils, and of the FcεRI-c-kit- cells, only 2.3±1% were

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emr1+ or siglec8+, which would indicate eosinophils, leaving the remaining 73.3±11% of

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the cells neutrophils (Figure 3). The full gating scheme for this antibody panel is

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available in Figure E3, and the sample chosen as the representative example was

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selected to best show the gating strategy for basophils and mast cells.

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GM-CSF was elevated in nasal polyps, and induces neutrophil derived OSM production

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in vitro:

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Since GM-CSF and FSTL-1 have been shown to be important inducers of OSM, our next

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aim was to determine whether GM-CSF and/or FSTL1 may be playing a role in in vivo

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induction of neutrophil derived OSM. We first wanted to determine whether GM-CSF or

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FSTL1 were sufficient to induce OSM in neutrophils. We treated neutrophils isolated

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from whole blood in vitro for 20 hours with GM-CSF and/or FSTL1 11, 16, 18. GM-CSF

387

alone induced OSM protein release into cell culture supernatants at 1, 5, and 25 ng/mL,

388

whereas FSTL1 alone at 100 or 1000ng/mL did not induce OSM in purified neutrophils;

389

the combination of 1 or 25 ng/mL GM-CSF with 1000ng/mL FSTL1 was not more active

390

than GM-CSF alone (Figure 4A). This data was analyzed using both mean and median

391

and the statistics were the same; the data represented in Figure 4A shows the mean

392

values. Analysis of OSM mRNA following culture showed that both GM-CSF- and

393

FSTL1-treated neutrophils expressed OSM transcripts, suggesting that neutrophils are

394

capable of de novo production of OSM, although levels of OSM were not elevated

395

compared to control. (Figure E4A). We next wanted to determine whether other type 2

396

mediators were sufficient to induce neutrophil derived OSM. Neutrophils were either left

397

unstimulated, or stimulated with GM-CSF (25ng/mL), IL-25 (10-100 ng/mL), IL-33 (10-

398

100 ng/mL), TSLP (10-100 ng/mL) or leukotriene C4 (10-6 – 10-7 M) Figure E4B). Only

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GM-CSF was sufficient to induce OSM in neutrophils. We also wanted to determine

400

whether neutrophil ligation with E-selectin and ICAM would induce neutrophil derived

401

OSM. We coated plates with E-selectin, ICAM, and BSA as a control, and then cultured

402

neutrophils for 20 hours in the coated wells. We did not observe any induction of OSM

403

with E-selectin or ICAM ligation (Figure E4C).

404

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OSM has been shown to be important for the early stages of epithelial repair, specifically

406

by inducing basal cell proliferation and migration 38. We next hypothesized that injured

407

epithelium may upregulate GM-CSF to induce OSM production from infiltrating

408

neutrophils, which would then promote the early stages of epithelial repair. To determine

409

whether GM-CSF could potentially be produced from injured epithelium, we injured

410

normal human bronchial epithelial (NHBE) cells that were grown at air-liquid interface,

411

and when they were fully differentiated they were treated with heat killed Staphlococcus

412

aureus (HKSA) as described 39. Exposure for 48 hr to HKSA at a concentration of 5 x 108

413

particles/mL induced GM-CSF secretion into cell culture supernatants (Figure 4B).

414

Although HKSA can activate epithelium by various pathways, these data support the

415

concept that injured epithelium is capable of producing GM-CSF, which has the potential

416

to induce OSM expression in neutrophils that migrate to the site of injury. Since GM-CSF

417

was sufficient to induce neutrophil-derived OSM secretion, we next hypothesized that

418

GM-CSF would be elevated in nasal polyps compared to control UT. To do this, we

419

measured GM-CSF and FSTL1 protein expression in nasal polyps and UT from CRS

420

patients and controls. GM-CSF was elevated in nasal polyps compared to control UT

421

while FSTL1 was not elevated, suggesting that GM-CSF could be responsible for

422

induction of neutrophil OSM expression in nasal polyps (Figure 4C, D).

423

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To determine the cell types responsible for GM-CSF production in nasal polyps, we

425

stained nasal polyp sections (n=4) for OSM, neutrophil elastase, and GM-CSF (Figure

426

4E and Figure E4D-E). Surprisingly, the neutrophils that expressed OSM also expressed

427

GM-CSF. GM-CSF has been shown to protect neutrophils against apoptosis 40,

428

suggesting that neutrophils may have an autocrine pathway by which GM-CSF

429

expression prolongs their own survival and promotes expression of OSM. We did not

430

observe GM-CSF expression in the epithelium in nasal polyps.

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OSM producing neutrophils may have a non-classical phenotype:

434

We next tested whether OSM-producing neutrophils were phenotypically classical,

435

inflammatory N1 neutrophils or tissue repair-associated N2 neutrophils. We utilized flow

436

cytometry, and stained cells isolated from nasal polyps for CD45, OSM, CD16, IL-5R,

437

myeloperoxidase (MPO) and Arg1 (Figure E5A). No flow cytometry antibodies were

438

available for elastase, so we used anti-MPO instead. Of cells within the CD45+ gate,

439

3.7±1% were OSM+, and of the OSM+ cells, 56.0±8% were CD16+IL-5R- neutrophils. In

440

the neutrophil gate, we found two populations; MPOhiArg1+ (72.5±12%) and MPOloArg1-

441

(22.1±8%) (Figure 5A-B). The expression of Arg1 suggests that the majority of

442

neutrophils taken from polyp tissue in vivo were not classical N1 neutrophils. As

443

discussed above, GM-CSF and FSTL1 are two potential inducers of OSM in vivo. To

444

determine whether GM-CSF induced neutrophils to assume a non-classical phenotype,

445

we compared neutrophils treated in vitro with GM-CSF to neutrophils that were polarized

446

either toward an N1 phenotype using LPS and IFNγ or toward an N2 phenotype using

447

IL-4 and IL-13. As expected we saw increased levels of OSM protein released into the

448

culture supernatants in response to GM-CSF, but we did not see any increased OSM in

449

the supernatants in either the N1 or N2 polarizing conditions (Figure 5C). To determine

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whether neutrophil phenotypes influence OSM expression, we polarized neutrophils to

451

N1 or N2 conditions, or left them unstimulated for 24 hours. Following polarization, the

452

neutrophils were either left unstimulated or stimulated with GM-CSF. Unactivated, N1

453

and N2 neutrophils had no difference in expression of OSM, suggesting that all

454

neutrophils are capable of OSM expression (data not shown). To further define the

455

phenotype of our GM-CSF and FSTL1 treated neutrophils, we analyzed mRNA

456

expression of the type 2 markers mannose receptor (MMR) and arginase-1 (Arg1).

457

Neither GM-CSF or FSTL1 or their combination caused any induction of Arg1

458

(FigureE5B). However, GM-CSF and FSTL1 were both sufficient to induce elevated

459

mRNA for another N2 marker, MRC1, suggesting that GM-CSF and FSTL1 may induce

460

neutrophils to assume elements of an N2-like phenotypic state (Figure 5D).

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Neutrophil expression of OSM in asthmatic patients:

463

To determine whether neutrophils were also a source of OSM in asthma, we stained

464

bronchial biopsies from control patients (n=4) as well as those with moderate (n=5) and

465

severe (n=6) asthma for OSM and neutrophil elastase (Figure 6A). We counted

466

neutrophils and OSM+ cells in each specimen, and the tabulated data are represented in

467

Table E3. We did not observe any OSM+ cells in the control biopsies, but we detected

468

OSM+ cells in 60% of biopsies from moderate asthmatics and 100% of biopsies from

469

severe asthmatics (Figure E6A). Of the neutrophils counted in each specimen, 0±0%

470

were OSM positive in control biopsies, 14.0±9.8% were positive in moderate asthmatic

471

biopsies, and 30.1±10.2% were OSM positive in severe asthmatic biopsies (Figure E6B).

472

Additionally, of the OSM+ cells counted, 0±0% were neutrophils in control biopsies,

473

35.0±21.8% were neutrophils in moderate asthmatic biopsies, and 52.1±15.9% were

474

neutrophils in severe asthmatic biopsies (Figure 6B). Neutrophil counts were normalized

475

per mm of basement membrane within the biopsy section, and no differences in the total

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neutrophil counts/mm basement membrane were observed between controls and

477

asthmatics, suggesting an increased state of activation of neutrophils rather than

478

increased neutrophil numbers (Figure E6C).

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479 To further test whether OSM is produced in the airways of patients with asthma, we

481

evaluated a set of induced sputum samples from controls and asthmatic subjects. We

482

observed elevated levels of OSM in induced sputum from asthmatic patients compared

483

to controls (Figure 6C). Finally, we previously reported that segmental allergen

484

challenge in allergic asthmatic patients leads to increased levels of OSM protein in BAL

485

fluids 8. We reevaluated the previous data set to test the role of neutrophils in the

486

response and found that OSM levels correlated with total neutrophil counts, total

487

lymphocyte counts, and total cell counts in the BAL (Figure E6D-E, H), but not with total

488

eosinophil counts or total macrophage counts (Figure E6F-G). Additionally, we

489

previously reported that a concentration of 50pg/mL of GM-CSF was measured in the

490

BAL in a separate cohort of allergen challenged allergic asthmatics, suggesting that GM-

491

CSF may also induce neutrophil-derived OSM production in asthmatic patients 20.

492

Together, these data suggest that OSM is produced by neutrophils in both sinus and

493

lung tissue undergoing allergic inflammation and may play a role in epithelial barrier

494

dysfunction.

496 497 498 499 500 501

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502 Discussion:

504

We have previously showed that OSM was elevated in nasal polyp tissue from CRS

505

patients, and that physiological levels of OSM are sufficient to induce profound loss of

506

barrier function in differentiated bronchial and nasal epithelial cells cultured at air-liquid

507

interface 8. We hypothesized that elevated OSM in CRS may mediate epithelial barrier

508

dysfunction in vivo. Nasal polyps have been shown to express robust type 2

509

inflammation 41, 42, 43, and we originally speculated that the OSM producers in nasal

510

polyps were likely to be eosinophils, M2 macrophages, or mast cells. In the present

511

manuscript, we found rather that neutrophils are a major population of cells expressing

512

OSM in CRS tissues (Figure 1-3). We also identified a minor population of OSM+ cells

513

that were siglec 8+, and these cells were found to be mast cells (Figure 2-3). Neutrophils

514

are classically thought to be a first line of defense in response to tissue injury 44, and

515

neutrophil-derived OSM has been shown to be important in the early stages of epithelial

516

repair when basal cells proliferate and cover a wound 45. Neutrophils are known to be

517

increased in CRSsNP, CRSwNP and in certain forms of asthma, and production of OSM

518

by neutrophils could participate in both ongoing epithelial repair as well as promote the

519

epithelial barrier dysfunction that is observed in these diseases. While we did not

520

investigate whether neutrophils were responsible for OSM production in CRSsNP, we

521

suspect that neutrophil-derived OSM may also play a role in barrier dysfunction in this

522

non-polypoid form of CRS. It is possible that under normal circumstances, neutrophil-

523

derived OSM production is transient, allowing basal cells to reinitiate contact inhibition,

524

stop proliferating, and then differentiate back into mature epithelium. However, chronic

525

expression of OSM by neutrophils in mucosal disease may prolong or even perpetuate

526

the state of the epithelial repair process (Figure 7). OSM has been shown to induce

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527

epithelial-mesenchymal transition (EMT) 46, which antagonizes epithelial differentiation,

528

and expression of EMT biomarkers is well established in both CRS and asthma 47.

529 Neutrophils are not classically associated with type 2 immune responses, and are

531

generally considered to be a type 1 immune response effector cell. However,

532

neutrophilic subsets of CRS and asthma have been described. While nasal polyps in

533

Western countries tend to be highly eosinophilic, a significant proportion of nasal polyps

534

from patients in Eastern Asian countries are neutrophilic 214, 215, 216. In unpublished

535

studies not shown, we found that nasal polyps, that are highly eosinophilic in our

536

Chicago cohort, nonetheless have significant levels of neutrophils and neutrophil

537

elastase. Increased neutrophilia within nasal polyps has been linked to decreased

538

responsiveness to corticosteroid treatment 217. Severe asthma is a subtype of asthma

539

that is often characterized by neutrophilia 218, 219. It is thought that neutrophilia in asthma

540

associates with more severe disease, again, because neutrophils are not particularly

541

responsive to corticosteroid treatment 220. However, little is known about the role of

542

neutrophils in eosinophilic mucosal disease outside of these known neutrophilic

543

subtypes of CRS and asthma. Our data suggest that neutrophils may be capable of

544

playing a pathogenic role in eosinophilic mucosal disease through the induction of

545

epithelial barrier dysfunction mediated by the concurrent production of OSM and GM-

546

CSF.

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548

We present circumstantial evidence that the neutrophils responsible for OSM production

549

are of the N2 phenotype. Recent studies have shown that neutrophils can be polarized

550

into either classical N1 neutrophils, which are thought to have a more inflammatory

551

phenotype, and N2 neutrophils, which promote tissue repair and tumorigenesis and are

552

thought to have an anti-inflammatory or regulatory phenotype 23, 24, 25, 26. We showed that

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the majority of OSM+ neutrophils from nasal polyps expressed the type 2 marker Arg1

554

(Figure 5), indicating a phenotype resembling N2 neutrophils more than N1 neutrophils.

555

We also showed that both GM-CSF and FSTL1 induced in vitro-stimulated neutrophils to

556

express elevated MMR compared to unstimulated neutrophils and N1 polarized

557

neutrophils, also indicating that OSM producing neutrophils could be skewed toward the

558

N2 phenotype (Figure 5). Since both GM-CSF and FSTL1 have been shown to be

559

important inducers of OSM 11, 16, 18, we tested their ability to induce OSM in vitro in

560

cultured neutrophils; of these two stimuli, only GM-CSF induced OSM production.

561

However, since we did not see any induction of Arg1 in response to GM-CSF, another

562

factor may be necessary for full induction of the N2 phenotype. These studies implicate

563

GM-CSF as a potential inducer of OSM in neutrophils in vivo. Since circulating

564

neutrophils do not express OSM in patients, this induction likely takes place within nasal

565

polyp or lung tissue where these inducers can be found.

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Interestingly, immunofluorescent staining showed that GM-CSF localized to OSM-

568

producing neutrophils in nasal polyps, suggesting that OSM producing neutrophils may

569

induce their own OSM through autocrine production of GM-CSF (Figure 4E). There is

570

precedent for such a concept, as Wardlaw and colleagues showed that eosinophils

571

produce GM-CSF to prolong their own survival 48. In addition, Durand et al, have shown

572

that autoantibody ligation of FcγRIIIb (CD16) receptors on the surface of neutrophils

573

protects against apoptosis through the induction of autocrine GM-CSF 40. We have

574

previously shown elevated levels of autoantibodies in nasal polyps, suggesting that

575

elevated autoantibodies in nasal polyps may trigger GM-CSF expression in the

576

infiltrating neutrophils by a similar mechanism 49. Whether production of OSM by

577

neutrophils via an autocrine GM-CSF dependent pathway occurs in CRSwNP and

578

asthma is worthy of further investigation.

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579 Importantly, we have also shown that OSM was elevated in induced sputum from

581

asthmatics compared to controls and that OSM+ cells were present in bronchial biopsies

582

from 100% of severe asthma patients, 60% of moderate asthma patients and none of

583

the healthy controls. These results suggest that the expression of OSM and its

584

association with barrier disruption applies to CRSwNP, asthma and, as shown in our

585

previous study, eosinophilic esophagitis. In the present study, 52% of the OSM+ cells

586

were neutrophils in severe asthmatics and 35% of the neutrophils were OSM+ in

587

moderate asthmatics, suggesting that neutrophil derived OSM plays a role in the

588

pathogenesis of asthma. OSM has previously been implicated in the induction of fibrosis

589

in a mouse model of asthma, though it was derived from macrophages 16, 50.

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590

Given that GM-CSF may promote prolonged survival in neutrophils as well as induce

592

OSM, therapeutic targeting of GM-CSF could potentially be beneficial in the treatment of

593

CRS and possibly other mucosal diseases like asthma and eosinophilic esophagitis.

594

Inhibition of GM-CSF could block neutrophil-derived OSM production, allowing

595

epithelium to enter the late phase of repair and differentiation, potentially resolving

596

chronic inflammation resulting from barrier dysfunction. Two strategies have been used

597

to inhibit GM-CSF in human trials, monoclonal antibodies against GM-CSF, and a

598

monoclonal antibody against GM-CSFRα. Clinical trials have been conducted with

599

monoclonal antibodies against GM-CSF (MOR103, KB003, and namilumab) in

600

rheumatoid arthritis, plaque psoriasis, and severe asthma. A phase II trial was

601

conducted using KB003 in asthmatic patients that were poorly controlled with long-acting

602

bronchodilators and either inhaled or oral corticosteroids. They did not observe improved

603

forced expiratory volume in 1 second (FEV1) in the entire patient cohort. However, when

604

they separated patients based on markers of poorly controlled asthma, peripheral blood

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605

eosinophilia, low baseline FEV1, and high bronchodilator reversibility, they did detect an

606

improvement in FEV1 in response to treatment with KB003 51.

607 In summary, we have shown that neutrophils are a major source of OSM producing cells

609

in nasal polyps using both immunofluorescence and flow cytometric analyses. We have

610

also shown that GM-CSF is elevated in nasal polyps, and that physiological levels of

611

GM-CSF are sufficient to induce OSM production in cultured neutrophils. Interestingly,

612

GM-CSF is expressed by many of the same neutrophils that produce OSM in nasal

613

polyps, suggesting the presence of an autocrine activation. OSM-producing neutrophils

614

phenotypically resemble N2 neutrophils based on limited biomarker evaluation, but

615

further characterization of these cells will be required. OSM+ cells are also present in

616

biopsies from severe asthmatics, and approximately half of the OSM producing cells are

617

neutrophils, suggesting that neutrophil-derived OSM may also play a role in asthma.

618

Taken together, these studies further implicate OSM in barrier dysfunction in CRSwNP

619

as well as in asthma, and suggest that the OSM found in these diseases is produced by

620

neutrophils. Therapeutic intervention targeting either GM-CSF or OSM may thus be

621

beneficial in the treatment of CRS and asthma.

624 625 626 627 628 629 630

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Acknowledgements:

632

The authors wish to thank Dr. Bruce Bochner for the very kind gift of anti-siglec 8

633

antibody and would like to thank Ms. Jacqi Shaffer for the outstanding medical

634

illustration.

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635

This study was supported in part by Grants R37HL068546, R01HL078860, the Chronic

637

Rhinosinusitis Integrative Studies Program (CRISP - U19AI106683), and T32AI007476-

638

16 from the NIH, as well as by the Ernest S. Bazley Foundation.

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Imaging work was performed at the Northwestern University Center for Advanced

641

Microscopy generously supported by NCI CCSG P30 CA060553 awarded to the Robert

642

H. Lurie Comprehensive Cancer Center.

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Homma, T. et al. Involvement of Toll-like receptor 2 and epidermal growth factor receptor signaling in epithelial expression of airway remodeling factors. American journal of respiratory cell and molecular biology 52, 471481 (2015).

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Durand, V., Renaudineau, Y., Pers, J.O., Youinou, P. & Jamin, C. Cross-linking of human FcgammaRIIIb induces the production of granulocyte colonystimulating factor and granulocyte-macrophage colony-stimulating factor by polymorphonuclear neutrophils. Journal of immunology 167, 3996-4007 (2001).

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Akdis, C.A. et al. Endotypes and phenotypes of chronic rhinosinusitis: a PRACTALL document of the European Academy of Allergy and Clinical Immunology and the American Academy of Allergy, Asthma & Immunology. The Journal of allergy and clinical immunology 131, 1479-1490 (2013).

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Van Zele, T. et al. Differentiation of chronic sinus diseases by measurement of inflammatory mediators. Allergy 61, 1280-1289 (2006).

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Stevens, W.W. et al. Cytokines in Chronic Rhinosinusitis. Role in Eosinophilia and Aspirin-exacerbated Respiratory Disease. American journal of respiratory and critical care medicine 192, 682-694 (2015).

44.

Engelhardt, E. et al. Chemokines IL-8, GROalpha, MCP-1, IP-10, and Mig are sequentially and differentially expressed during phase-specific infiltration of

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leukocyte subsets in human wound healing. The American journal of pathology 153, 1849-1860 (1998). Goren, I. et al. Oncostatin M expression is functionally connected to neutrophils in the early inflammatory phase of skin repair: implications for normal and diabetes-impaired wounds. The Journal of investigative dermatology 126, 628-637 (2006).

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Nightingale, J. et al. Oncostatin M, a cytokine released by activated mononuclear cells, induces epithelial cell-myofibroblast transdifferentiation via Jak/Stat pathway activation. Journal of the American Society of Nephrology : JASN 15, 21-32 (2004).

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Schleimer, R.P. Immunopathogenesis of Chronic Rhinosinusitis and Nasal Polyposis. Ann Rev Path (2016).

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Anwar, A.R., Moqbel, R., Walsh, G.M., Kay, A.B. & Wardlaw, A.J. Adhesion to fibronectin prolongs eosinophil survival. The Journal of experimental medicine 177, 839-843 (1993).

49.

Tan, B.K. et al. Evidence for intranasal antinuclear autoantibodies in patients with chronic rhinosinusitis with nasal polyps. The Journal of allergy and clinical immunology 128, 1198-1206 e1191 (2011).

50.

Scaffidi, A.K. et al. Oncostatin M stimulates proliferation, induces collagen production and inhibits apoptosis of human lung fibroblasts. British journal of pharmacology 136, 793-801 (2002).

51.

Molfino, N.A. et al. Phase 2, randomised placebo-controlled trial to evaluate the efficacy and safety of an anti-GM-CSF antibody (KB003) in patients with inadequately controlled asthma. BMJ Open 6, e007709 (2016).

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Figure 1: Oncostatin M was expressed in neutrophils.

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Nasal polyp sections were stained for OSM in green and various cell type specific

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markers as indicated in red; two representative examples from separate patients are

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shown for each staining strategy. OSM did not co-localize with macrophages (A, B)

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(n=3) or eosinophils (C,D) (n=5) and had minimal co-localization with mast cells (E,F)

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(n=6). OSM did co-localize with neutrophils (G,H) (n=10).

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Figure 2: Flow cytometric analysis showed that neutrophils are a major source of

901

OSM producing cells in nasal polyps.

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(A) Within the CD45 gate, 5.1± 1.7% of cells were OSM+. (B) Within the OSM+ gate,

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80.4±5% of cells were neutrophils (CD16+Sig8-) and 14.1±5% of cells were either

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eosinophils, mast cells or basophils (CD16+Sig8+). (C) Quantification of the percentage

905

of OSM+ cells within the CD45+ gate, and the percentage of neutrophils (CD16+Sig8-),

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and CD16+Sig8+ cells within the OSM+ gate, n=9. (D) In matched blood and nasal

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polyps, .60± .36% of CD45+ cells in the blood were OSM+ while 6.3± 2.9% of CD45+

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cells in nasal polyps were OSM+ (n=5, p< .05, Mann-Whitney U test), suggesting that

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OSM was locally induced, n=5.

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Figure 3: Flow cytometric analysis showed that mast cells were a minor source of

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OSM producing cells in nasal polyps.

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(A) Representative flow cytometric plot of the OSM+ gate. (B) Representative flow

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cytometric plots of the c-kit-FcεRI- gate. (C) Quantification of the relative representation

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of different cell types among the OSM+ cells. Mast cells (MC) were 15.7±9.6%,

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basophils (Baso) were 3.7±1.5%, eosinophils (Eo) were 2.3±.9% and neutrophils (PMN)

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were 73.3±10.6% within the OSM+ gate, n=4. OSM+ cells were 4.6±1.9% of the CD45+

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cells.

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Figure 4: GM-CSF was elevated in nasal polyps and induced neutrophil derived

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OSM.

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(A) Blood neutrophils were stimulated with the indicated concentrations of GM-CSF,

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FSTL1 or both and levels of OSM protein in the culture supernatant were measured;

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(n=7-13, * p< .05, * p< .001, Kruskal-Wallis test). (B) Fully differentiated NHBE cells

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were either unstimulated, or stimulated with HKSA, and levels of GM-CSF protein in the

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cell culture supernatants were measured. HKSA treated NHBE released more GM-CSF,

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21.3± 6.9pg/mL, than control NHBE, .87±.87 (n=4, p< .05, Mann-Whitney U test). (C)

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GM-CSF protein, 2.6±.93pg/mg total protein, was elevated in nasal polyps compared to

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control UT, .68±.42pg/mg total protein, (n=16-24, p< 05, Mann-Whitney U test). (D)

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FSTL1 protein, .60±.47ng/mL, was elevated in nasal polyps compared to control UT,

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.80±.33, (n=15-23, p=.31, Mann-Whitney U test). (E) OSM (alexa 488), GM-CSF (alexa

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568) and neutrophil elastase (alexa 647) colocalized in nasal polyps (n=4).

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Figure 5: OSM producing neutrophils did not express a classical N1 phenotype.

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(A) OSM+ neutrophils contained a distinct population of Arg1+MPOhi cells, representative

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data. (B) 3.7±1.3% of cells in the CD45 gate were OSM+; within the OSM+ gate, 56.0±

936

8.2% of the cells were CD16+IL-5R- neutrophils. Within the neutrophil gate 72.56± 11.9%

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of the cells were Arg1+MPOhi, and 22.1± 8.0% were Arg1-MPOlo, n=5. (C) Blood

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neutrophils were either left untreated, or treated under N1 polarizing conditions

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(LPS/IFNγ), N2 polarizing conditions (IL-4/IL-13) or with GM-CSF. GM-CSF treated

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neutrophils secreted elevated levels of OSM into the cell culture supernatants, while N1

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and N2 polarizing conditions did not induce OSM, (n=4-7, p< .01, Kruskal-Wallis test).

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(D) Levels of MRC1 mRNA were elevated in neutrophils stimulated with GM-CSF either

943

alone or together with FSTL1, (n=3-7, p< .05, Kruskal-Wallis test).

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Figure 6: OSM was elevated in asthmatic patients

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(A) Bronchial biopsies from control patients, moderate asthmatics and severe asthmatics

947

were stained for OSM in green and neutrophil elastase in red, n=4-6. (B) Counts were

948

obtained of neutrophils and OSM+ cells; none of the control biopsies had any OSM+

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cells, and of the OSM+ cells 35± 21.8% were neutrophils in moderate asthmatics, and

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52.1± 15.9% were neutrophils in severe asthmatics. (C) Levels of OSM in the sputum of

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asthmatic patients, 26.9± 9.5pg were elevated compared to control patients 3.9± 2.6pg,

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(n=11-12, p< .05, Mann-Whitney U test).

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Figure 7: Proposed mechanism of OSM mediated barrier dysfunction in mucosal

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disease

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Under normal circumstances, when epithelium is injured, neutrophils are recruited to the

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site of injury and potentially, transiently make OSM to promote the early stages of repair.

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Due to transient OSM expression, once the epithelial cells become contact inhibited they

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are able to enter the later stages of repair and redifferentiate back into functional

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epithelium. However, under pathogenic conditions we hypothesize that neutrophils are

961

recruited to the injury site, and once at the site of injury the neutrophils are converted

962

into an alternative phenotype, potentially N2, that makes both OSM and GM-CSF. The

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GM-CSF alone is sufficient to induce production of OSM in neutrophils, and will also

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contribute to long-term survival of OSM producing neutrophils that may prevent late

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stage repair, causing a long-term state of barrier dysfunction.

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Table E1: Patient Demographics

Age (y), median (range)

n = 33 (15F/18M) 45.5 (2273)

n = 24 (14F/10M)

CRSwNP n = 89 (36F/53M)

48 (19-75)

48 (18-69)

Y 5 1

N 15 32

U 13 0

Y 10 4

N 10 20

(p=.0142) (p=.2659)

1 3

30 18

2 1

1 0

23 24

Immunofluorescence Age (y), median (range) Neutrophil culture Age (y), median (range)

n = 13 (3F/10M) 37 (22-63) n = 20 (12F/8M) 58 (25-73)

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Protein lysates Age (y), median (range)

CRSwNP polyp

U 4 0

Y 53 43

N 29 45

U 7 1

0 0

18 10

68 78

2 1

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Chisquare (p<.0001) (p<.0001)

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Atopy Asthma Peri operative systemic steroids Antibiotics Methodology used Flow cytometry Age (y), median (range)

CRSsNP

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Total no. of subjects

Control

n = 6 (3F/3M) 53 (25-75)

n = 18 (11F/7M) 46 (19-67)

n=16(8F/8M) 44(18-69) n = 21 (8F/13M) 45 (25-66)

n = 8 (2F/6M) 36 (28-65) n = 20 (7F/13M) 48 (28-69)

n = 24 (11F/13M) 54 (25-69)

Dilution 1:10 1:40 1:40 1:10 A10 1:20 MHE-18 1:100 104D2 1:50 17022 1:10 HI30 1:40 3G8 1:40 26815 1:20 MPO455-8E6 1:20 658922 1:20

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Clone 17022 HI30 3G8

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Table E2: Flow Cytometry Antibodies Antibody Figure 2 and 3: anti-human OSM-PE anti-human CD45-V450 anti-human CD16-APC-H7 anti-human Siglec 8-Alexa 647 anti-human EMR1-Alexa 647 anti-human FCεR1-Pe-Cy7 anti-human c-kit-Alexa 488 Figure 5: anti-human OSM-PE anti-human CD45-V450 anti-human CD16-APC-H7 anti-human IL-5R-APC anti-human MPO-FITC anti-human Arg1-Alexa 700

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Manufacturer R&D systems, Minneapolis, MN BD Biosciences, San Jose, CA BD Biosciences, San Jose, CA Gift from Dr. Bruce Bochner AbD Serotec, Raleigh, NC BioLegend, San Diego, CA Biolegend, San Diego, CA R&D systems, Minneapolis, MN BD Biosciences, San Jose, CA BD Biosciences, San Jose, CA R&D systems, Minneapolis, MN ebioscience, San Diego, CA R&D systems, Minneapolis, MN

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5

2

7

1

10

9

6 6

0 4

2 6

2 2

39

19

25

9

0 20 7 8 15 23 11 22 0

%PMN PMN/mm %OSM+ that are basement OSM+ membrane PMN

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60

15

35.27

10

14.29

14.69

0 66.67

0 66.67

9.85 8.86

25

6

76

48.72

52.25

9

0

100

36

17.93

0

0

0

0

0

0

10

10

0

100

50

31.97

2 0 4 0 0 0 0

2 0 1 0 0 0 0

0 0 75 0 0 0 0

0 0 20 0 0 0 0

8.41 13.24 22.44 43.08 16.41 39.15 0

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Patient abb 0120908 Y abb 0151108 Y abb 0308207 Y abb0235206 Y abb 0120709 Y abb 0305807 Y Moderate abb asthma 0167606 N abb 0144408 Y abb 0240008 Y abb0200412 N abb0164406 Y Control abb349911 N abb0150705 N abb0160706 N abb0165706 N

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Patient groups Severe asthma

OSM+ nonTotal OSM+ OSM PMN PMN OSM+ PMN Y/N count count count count

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Table E3: Bronchial biopsy cell counts

0 0 3 0 0 0 0

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Figure E1: Control images from OSM staining with CD68 (A), ECP (B), tryptase (C), and elastase (D). (E) OSM expression levels correlated with the granulocyte marker FCGR3 and (F) with the

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neutrophil specific protease CTSG in the microarray dataset. (G) FCGR3 correlated with CTSG in the microarray. (H) OSM protein correlated with elastase protein in lysates from both nasal

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polyps and uncinates from CRS patients and controls.

Figure E2:

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Gating strategy for identification of OSM+ cells. Cells were first gated on forward and side scatter, and doublets (not shown) and dead cells were gated out. Cells were then gated on the CD45+ population, and then subsequently gated on the OSM+ population. The OSM+ population was then plotted based on CD16 and siglec 8 expression, and the CD16+Sig8- and CD16+Sig8+

Figure E3:

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populations were quantified.

Contribution of mast cells, eosinophils and basophils to the OSM+ cell population. Cells were

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first gated on forward and side scatter, and doublets (not shown) and dead cells were gated out. Cells were then gated on the CD45+ population, and then gated on the OSM+ population. The

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OSM+ population was then plotted on c-kit and FcεRI expression, and the mast cell, ckit+FcεRI+, and basophil c-kit-FcεRI+, populations were quantified. The c-kit-FcεRI- population was then plotted on CD16 and either EMR1 or Siglec 8 to discriminate neutrophils (CD16+EMR1/Siglec8-) from eosinophils, (CD16+EMR1/Siglec 8+).

Figure E4: (A) Blood neutrophils were isolated and either left unstimulated, or were stimulated with GMCSF, FSTL1 or both. There was a trend toward both GM-CSF and FSTL1 to elevate OSM

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mRNA compared to unstimulated controls. (B) Neutrophils were either left unstimulated or stimulated with 25ng/mL of GM-CSF or the indicated concentrations of IL-25, IL-33, TSLP or leukotriene C4 for 20h (n=5-6). Only GM-CSF was sufficient to induce OSM. (C) E-selectin and

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ICAM ligation did not induce neutrophil derived OSM (n=5-6). (D) OSM (alexa 488) and GMCSF (alexa 568) colocalized in nasal polyps, (n=4). (E) GM-CSF (alexa 568) and neutrophil

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elastase (alexa 647) colocalized in nasal polyps, (n=4).

Figure E5:

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Identification of putative N1 and N2 cells among the OSM+ neutrophils. (A) Cells were first gated on forward and side scatter, and doublets and dead cells were gated out. Cells were then gated on the CD45+ population, and then gated on the OSM+ population. The OSM+ population was then plotted on CD16 and IL-5R to discriminate neutrophils (CD16+IL-5R-) and eosinophils (CD16+IL-5R+). Cells were then gated on the neutrophil population and plotted on MPO and

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Arg1, and the Arg1+MPOhi, and Arg1+MPOlo populations were quantified. (B) Levels of ARG1 mRNA were not elevated in neutrophils stimulated with GM-CSF either alone or together with

Figure E6:

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FSTL1 compared to control.

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Bronchial biopsy sections from healthy controls and asthma patients were stained for OSM and neutrophil elastase (A): none of the control biopsies had any OSM+ cells, 60% of moderate asthmatic biopsies and 100% of severe asthmatic biopsies had OSM+ cells. (B) Of the neutrophils counted, 0±0% were OSM+ in controls, 14.0±9.8% were positive in moderate asthmatics, and 30.1±10.1% were positive in severe asthmatics. (C) No differences were seen in the number of neutrophils/mm of basement membrane. Levels of OSM in BAL from segmental allergen challenge of allergic asthmatics correlated with (D) total neutrophil count, (E)

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total lymphocyte count, and (H) total cell count, but not with (F) total eosinophil count or (G) total

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macrophage count.

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