Chronic IL 9 and IL-13 Exposure Leads to an Altered Differentiation of Ciliated Cells in a Well-Differentiated Paediatric Bronchial Epithelial

Asthma is a chronic inflammatory disease characterised by airways remodelling. In mouse models IL-9 and IL-13 have been implicated in airways remodelling including mucus hypersecretion and goblet cell hyperplasia. Their role, especially that of IL-9, has been much less studied in authentic human ex vivo models of the bronchial epithelium from normal and asthmatic children. We assessed the effects of IL-9, IL-13 and an IL-9/IL-13 combination, during differentiation of bronchial epithelial cells from normal (n = 6) and asthmatic (n = 8) children. Cultures were analysed for morphological markers and factors associated with altered differentiation (MUC5AC, SPDEF and MMP-7). IL-9, IL-9/IL-13 combination and IL-13 stimulated bronchial epithelial cells from normal children had fewer ciliated cells [14.8% (SD 8.9), p = 0.048, 12.4 (SD 6.1), p = 0.016 and 7.3% (SD 6.6), p = 0.031] respectively compared with unstimulated [(21.4% (SD 9.6)]. IL-9 stimulation had no effect on goblet cell number in either group whereas IL-9/IL-13 combination and IL-13 significantly increased goblet cell number [24.8% (SD 8.8), p = 0.02), 32.9% (SD 8.6), p = 0.007] compared with unstimulated normal bronchial cells [(18.6% (SD 6.2)]. All stimulations increased MUC5AC mRNA in bronchial epithelial cells from normal children and increased MUC5AC mucin secretion. MMP-7 localisation was dysregulated in normal bronchial epithelium stimulated with Th2 cytokines which resembled the unstimulated bronchial epithelium of asthmatic children. All stimulations resulted in a significant reduction in transepithelial electrical resistance values over time suggesting a role in altered tight junction formation. We conclude that IL-9 does not increase goblet cell numbers in bronchial epithelial cell cultures from normal or asthmatic children. IL-9 and IL-13 alone and in combination, reduce ciliated cell numbers and transepithelial electrical resistance during differentiation of normal epithelium, which clinically could inhibit mucociliary clearance and drive an altered repair mechanism. This suggests an alternative role for IL-9 in airways remodelling and reaffirms IL-9 as a potential therapeutic target. Citation: Parker JC, Thavagnanam S, Skibinski G, Lyons J, Bell J, et al. (2013) Chronic IL9 and IL-13 Exposure Leads to an Altered Differentiation of Ciliated Cells in a Well-Differentiated Paediatric Bronchial Epithelial Cell Model. PLoS ONE 8(5): e61023. doi:10.1371/journal.pone. 0061023 Editor: Malcolm Gracie Semple, University of Liverpool, United Kingdom Received October 4, 2012; Accepted March 5, 2013; Published May 9, 2013 Copyright: 2013 Parker et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Northern Ireland Chest Heart and Stroke Association (grant number 200546), The Royal Belfast Hospital for Sick Children and The Irish Thoracic Society Boehringer Ingleheim Research Fellowship. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The following authors have declared that no competing interests exist: Jeremy Parker, Surendran Thavagnanam, Grzegorz Skibinski, Jennifer Bell, Jeremy Lyons. The authors have read the journal’s policy and have the following conflicts: Liam Heaney has received grant funding from Medimmune, Novartis UK, Genentech Inc, and Glaxo Smith Kline, has taken part in Advisory Boards and given lectures at meetings supported by Glaxo Smith Kline, Merck Sharpe & Dohme, Nycomed, Novartis and Astra Zeneca. He has received support funding to attend International Respiratory meetings (Astra Zeneca, Chiesi, Novartis and Glaxo Smith Kline) and has taken part in asthma clinical trials (GSK and Genentech) for which his Institution was remunerated. None of these activities have any direct relationship to the content of this manuscript. Michael Shields has no direct competing interests relating to this study but does declare that he has delivered lectures at educational events and received honoraria from GlaxoSmithKline, AstraZeneca, Merck Sharp & Dohme, Novartis and Alk Abello. In addition, he has received educational grant support to attend the British Thoracic and European Respiratory Societies annual meetings from Novartis and GlaxoSmithKline. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: l.heaney@qub.ac. uk . These authors contributed equally to this work. " These authors also contributed equally to this work.