Chronic obstructive pulmonary disease ORIGINAL ARTICLE Thorax: first published as 10.1136/thoraxjnl-2017-210593 on 10 July 2018. Downloaded from NOTCH3 contributes to rhinovirus-induced goblet cell hyperplasia in COPD airway epithelial cells Yaxun Jing,1 Joao Antonio Gimenes,2 Rahul Mishra,1 Duc Pham,1 Adam T Comstock,1 Daohai Yu,3 Umadevi Sajjan1,2,4 ► Additional material is ABSTRact published online only. To view Rationale Goblet cell hyperplasia (GCH) is one of Key messages please visit the journal online (http:// dx. doi. org/ 10. 1136/ the cardinal features of chronic obstructive pulmonary thoraxjnl- 2017- 210593). disease (COPD) and contributes to airways obstruction. What is the key question? Rhinovirus (RV), which causes acute exacerbations in ► Does rhinovirus infection induce goblet cell 1Department of Pediatrics patients with COPD, also causes prolonged airways hyperplasia in chronic obstructive pulmonary and Communicable Diseases, obstruction. Previously, we showed that RV enhances disease (COPD) and what is the underlying University of Michigan, Ann mechanism? Arbor, Michigan, USA mucin gene expression and increases goblet cell number 2 Department of Thoracic Surgery in a COPD mouse model. This study examines whether What is the bottom line? and Medicine, Temple University, RV causes sustained GCH in relevant models of COPD. ► Rhinovirus, which is associated with acute Philadelphia, Pennsylvania, USA Methods Mucociliary-differentiated COPD and normal 3Department of Clinical exacerbations in COPD, can promote goblet airway epithelial cell cultures and mice with normal Sciences, Temple University, cell hyperplasia, one of the features of airway or COPD phenotype were infected with RV or sham Philadelphia, Pennsylvania, USA epithelial remodelling in COPD. 4Department of Physiology, and examined for GCH by immunofluorescence and/ Temple University, Philadelphia, or mucin gene expression. In some experiments, RV- Why read on? Pennsylvania, USA infected COPD cells and mice with COPD phenotype ► We demonstrated that rhinovirus induces were treated with γ-secretase inhibitor or interleukin-13 goblet cell hyperplasia in in vitro and in Correspondence to Professor Umadevi Sajjan, neutralising antibody and assessed for GCH. To vivo models of COPD through NOTCH Departments of Physiology, and determine the contribution of NOTCH1/3 in RV-induced signalling independent of well-characterised Thoracic Medicine and Surgery, GCH, COPD cells transduced with NOTCH1/3 shRNA interleukin-13 pathway. Center for Inflammation, were used. Translational and Clinical Lung Results RV-infected COPD, but not normal cell cultures, Research, Lewis Katz Medical School, Temple University, showed sustained GCH and increased mucin genes and this may be a result of abnormal responses to Philadelphia PA 19140, USA; expression. Microarray analysis indicated increased infections or other noxious agents. http://thorax.bmj.com/ uma. sajjan@ temple. edu expression of NOTCH1, NOTCH3 and HEY1 only in RV- Rhinovirus (RV), which causes self-limiting upper infected COPD cells. Blocking NOTCH3, but not NOTCH1, airway infections in healthy subjects, is associated Received 31 May 2017 with acute exacerbations in COPD. Patients with Revised 15 May 2018 attenuated RV-induced GCH in vitro. Inhibition of NOTCH Accepted 11 June 2018 signalling by γ-secretase inhibitor, but not neutralising mild COPD experimentally infected with RV show antibody to IL-13, abrogated RV-induced GCH and mucin more severe and prolonged lower airway symptoms gene expression. including airflow obstruction.1 We demonstrated Conclusions RV induces sustained GCH via NOTCH3 that RV enhances mucin gene expression and particularly in COPD cells or mice with COPD phenotype. GCH within 4 days postinfection in two different on September 23, 2021 by guest. Protected copyright. This may be one of the mechanisms that may contribute models of COPD.2 3 Therefore, it is plausible that to RV-induced prolonged airways obstruction in COPD. RV may induce or perpetuate GCH in patients with COPD, thus causing excessive mucus production and airways obstruction. However, mechanisms by which RV induces sustained GCH in COPD are not INTRODUCTION well understood. Mucus overlaying the airway epithelium traps the Most studies conducted on respiratory virus-in- inhaled pathogens and other noxious agents and duced GCH have focused on models of allergic the coordinated beating of cilia clear mucus along asthma or virus infection model that induces with the trapped inhaled agents. Thus, both cilia type II responses. Although viral infection causes and mucus contribute to primary defence mech- airway obstruction in both patients with asthma © Author(s) (or their anism of airway epithelium. However, in chronic and COPD, the underlying mechanisms may differ employer(s)) 2018. No inflammatory airway disease including chronic because of different type of responses. In allergic commercial re-use. See rights and permissions. Published obstructive pulmonary disease (COPD), there is asthma, viral infection predominantly induces type by BMJ. excessive mucus production, which may subse- 2 cytokines, such as interleukin (IL)-13, IL-4 and quently cause airways obstruction and disease IL-5.4 IL-13 has been shown to play a critical role in To cite: Jing Y, Gimenes JA, exacerbation. Excessive production is a result of inducing GCH via activation of STAT6, SPDEF and Mishra R, et al. Thorax Epub 5–7 ahead of print: [please increased synthesis and secretion of mucins and is FOXA3. Therefore, most of the published studies include Day Month Year]. often associated with increase in number of goblet on allergic asthma have focused on delineating the doi:10.1136/ cells (goblet cell hyperplasia or GCH). Patients with molecular mechanisms by which respiratory viruses thoraxjnl-2017-210593 COPD show GCH in both small and large airways, induce IL-13.8–11 In COPD, however, viral infection Jing Y, et al. Thorax 2018;0:1–15. doi:10.1136/thoraxjnl-2017-210593 1 Chronic obstructive pulmonary disease is associated predominantly with CXCL-8, CXCL-10, TNF-α, Institutional Review Board, University of Michigan. Age and Thorax: first published as 10.1136/thoraxjnl-2017-210593 on 10 July 2018. Downloaded from IFN-γ and IL-6.12 Therefore, the role of IL-13 in virus-induced clinical status of COPD subjects and healthy donors are provided GCH in COPD is not clear. in online supplementary table 1. Airway basal cells at passage Another mechanism by which respiratory viruses may one were cultured at air/liquid interface as described previously induce GCH is via activation of epidermal growth factor to promote mucociliary differentiation.15 27 For details, please receptor (EGFR). Activation of EGFR has been shown to see the supplemental file. The sample size for COPD and normal induce GCH independently or in concert with IL-13. For cell cultures was determined based on our previous study,28 in instance, persistent activation of EGFR in mucociliary-differ- which we examined the goblet cell number. With the calculated entiated cell cultures by treatment with reactive oxygen species effect size of 2.87, α=0.05 and power of 0.95, we determined has been demonstrated to reduce number of ciliated cells and that we will require cells from six normal subjects and six subjects increase goblet cells.13 Chronic EGFR activation by Sendai virus with COPD. Therefore, we used cells from 8 to 9 patients in our induces GCH in well-differentiated airway epithelial cells by initial experiments to determine the difference in responses to promoting survival and transdifferentiation of ciliated to goblet RV between normal and COPD cell cultures. The sample size cells in concert with IL-13.14 Recently, we demonstrated that required for detecting significant differences between sham and RV promotes goblet cell differentiation in a model of injured RV infection in COPD cultures was determined based on the but not in normal airway epithelium via persistent activation of results of initial experiments (figure 2D), in which we quantified EGFR.15 Since EGFR activity is already enhanced in COPD,16 17 GCH induced by RV. With the effect size of 3.89, α=0.05 and RV may further activate EGFR to drive GCH independently or power of 0.95, we determined that we will require cells from in concert with IL-13. three normal subjects and three subjects with COPD. Therefore, In recent years, NOTCH signalling has emerged as one of the we used cells from 3– to 6 subjects to determine the mechanism key players in regulating goblet cell differentiation in conductive by which RV induces GCH in COPD cells. airways. NOTCH signalling pathway is evolutionarily conserved, and therefore all mammals express NOTCH receptors and Rhinovirus ligands. There are four NOTCH receptors (NOTCH1–4) and RV16 and RV1B purchased from ATCC were propagated in H1 five NOTCH ligands (delta-like ligand (DLL) 1, 2, 3, 4, and HeLa cells, partially purified, and viral titre was determined by jagged (JAG) 1 and 2). Human bronchial epithelial cells express plaque assay as described.29 Less than 100 kDa fraction from all four NOTCH receptors and all NOTCH ligands except for purified RV preparation was used as sham control. All in vitro DLL3.18 Overexpression of active domain of either NOTCH experiments were conducted with major group RV RV16 and in 1 or 3, but not 2 or 4, skewed differentiation of cells towards vivo studies with minor group virus RV1B, because major group secretory cells with a parallel decrease in ciliated cells. Similarly, viruses