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Ectopic Respiratory Epithelial Cell Differentiation in Bronchiolised Interstitial lung disease Ectopic respiratory epithelial cell differentiation in Thorax: first published as 10.1136/thx.2010.151555 on 21 March 2011. Downloaded from bronchiolised distal airspaces in idiopathic pulmonary fibrosis Laurent Plantier,1 Bruno Crestani,2,3 Susan E Wert,1 Monique Dehoux,3,4 Barbara Zweytick,5 Andreas Guenther,6,7 Jeffrey A Whitsett1 See Editorial, p 647 ABSTRACT < Additional figures and tables Background Bronchiolisation of distal airspaces is an Key messages are published online only. To unexplained feature of idiopathic pulmonary fibrosis (IPF). view these files please visit the The authors sought to identify mechanisms driving the What is the key question? journal online (http://thorax.bmj. differentiation of mucus cells during the bronchiolisation < Abnormal differentiation of the respiratory com). process. 1 epithelium is a feature of idiopathic pulmonary Section of Neonatology, Methods Pathways governing airway mucus cell fibrosis (IPF). In this work, pathways governing Perinatal and Pulmonary Biology, differentiation include SRY (sex determining region Y)- Perinatal Institute, Cincinnati mucus cell differentiation were investigated in box 2 (SOX2), Notch, forkhead box A3(FOXA3)/SAM Children’s Hospital Medical lung tissues from patients with IPF and other pointed domain containing ETS transcription factor Center, Cincinnati, Ohio, USA chronic pulmonary disorders. 2Assistance Publique-Hoˆpitaux (SPDEF), epidermal growth factor (EGF) and the EGF- de Paris, Hoˆpital Bichat, Service related neuregulins NRG1a and NRG1b. Immunostaining What is the bottom line? de Pneumologie A, Paris, France < 3Inserm U 700, Universite´ Paris for components of those pathways and mucins were Mucus cells lining abnormal airways from Diderot-Paris 7, UFR de performed on lung tissue obtained from patients with IPF patients with IPF expressed MUC5B and SOX2 Me´decine-site Bichat, Paris, (n¼20), chronic obstructive pulmonary disease (n¼13), but lacked SAM pointed domain containing ETS France ¼ 4 idiopathic pulmonary artery hypertension (n 5) and from transcription factor (SPDEF) and forkhead box Assistance Publique-Hoˆpitaux organ donors (n¼6). NRG1a and NRG1b were quantified a de Paris, Hoˆpital Bichat, Service A3(FOXA3). Neuregulin1 , which drives mucus de Biochimie A, Paris, France in bronchoalveolar lavage fluid (BALF) of patients with cell differentiation in vitro, was expressed in 5Department of Thoracic early IPF (n¼20), controls (n¼9), and patients with other normal airway submucosal glands and in lungs Surgery, Vienna General interstitial pneumonias (n¼13). from patients with IPF. Hospital, Vienna, Austria Results In IPF, the bronchiolised and enlarged distal 6University of Giessen Lung airspaces stained for SOX2 are consistent with epithelial Why read on? Centre, Department of Internal < differentiation characteristic of conducting airway Activation of abnormal respiratory epithelial Medicine, Giessen, Germany http://thorax.bmj.com/ 7Lung Clinic epithelium. IPF mucus cells expressed MUC5B but low differentiation programs may contribute to the Waldhof-Elgershausen, levels of MUC5AC and MUC2, a profile typical of expression of MUC5B and bronchiolisation of Greifenstein, Germany submucosal glands. Singularly, SPDEF, a transcription the distal lung, a salient feature of IPF. < In contrast to findings in chronic obstructive Correspondence to factor associated with mucus metaplasia, was rarely Jeffrey A Whitsett, Division of detected in mucus cells in IPF. The Notch target, HES1, pulmonary disease and T helper 2-driven mucus Pulmonary Biology, Cincinnati was present in mucus cells from all groups. NRG1a was metaplasia in which SPDEF, FOXA3, and Children’s Hospital Medical detected in serous cells within normal submucosal MUC5AC are expressed at high levels, MUC5B Center, Perinatal Institute, was the predominant mucin and neither SPDEF glands and in epithelial cells lining honeycombing areas on October 2, 2021 by guest. Protected copyright. MLC7029, 3333 Burnet Avenue, nor FOXA3 were detected in the abnormal Cincinnati, OH 45229-3039, in IPF, and was not detected in other patients. NRG1a USA; [email protected] concentrations were elevated in BALF from patients with mucus cells characteristic of the IPF lesions. early IPF. Received 21 September 2010 Conclusion Expression of SOX2 and MUC5B and lack of Accepted 10 February 2011 Published Online First SPDEF in atypically differentiated cells of bronchiolised supported by experimental models in which 2 21 March 2011 distal airspaces are consistent with abnormal epithelial cell injury or direct activation of programming of airway epithelial cells in IPF. NRG1a may remodelling processes leads to pulmonary fibrosis.3 contribute to bronchiolisation of the distal lung seen in IPF. Knowledge about the mechanisms influencing the differentiation and function of the respiratory epithelium in IPF may provide opportunities to diagnose, prevent or treat this incurable disorder. INTRODUCTION Histopathological studies of IPF lungs reveal the Idiopathic pulmonary fibrosis (IPF) is a lethal typical ‘usual interstitial pneumonia’ pattern, with disease affecting 6.8e16.3 per 100 000 individuals epithelial hyperplasia and apoptosis overlaying annually in the USA.1 While the mechanisms areas of scarring with fibroblast foci. In a focal resulting in the loss of lung structure and function manner, the alveolar architecture is destroyed, but in this disorder remain poorly understood, there is regular lung structure may be found in adjacent increasing evidence that chronic injury to the regions. Bronchiolar abnormalities, including bron- respiratory epithelium activates aberrant repair chiolisation of enlarged alveolar ducts, cysts and processes, resulting in replacement of peripheral alveoli, are commonly seen, resulting in honey- lung tissue with fibrotic scars and cysts lined by combing when dilated airspaces communicate with abnormal epithelial cells. These concepts are proximal airways. Such bronchiolar changes are Thorax 2011;66:651e657. doi:10.1136/thx.2010.151555 651 Interstitial lung disease a salient feature of the disease.4 5 Recent data support the (pH 6) at 958C, sections were blocked with non-specific sera and concept that bronchiolar abnormalities in IPF may be caused by primary antibodies (online table S2) were incubated overnight at Thorax: first published as 10.1136/thx.2010.151555 on 21 March 2011. Downloaded from cell autonomous changes in epithelial cell differentiation. 48C. Non-immune immunoglobins were used as negative Nuclear localisation of ß-catenin, indicating activation of the controls. The HES120 and SPDEF21 antibodies were gifts from Dr Wnt pathway, has been reported in bronchiolar-like epithelia Stanger (University of Pennsylvania) and Dr Watson (University lining the conducting airways in patients with IPF.6 of South Carolina). Biotinylated secondary antibodies and An intriguing feature of the bronchiolar-like epithelium lining biotin-conjugated horseradish peroxidase complexes (both honeycombing regions in the IPF lung is the presence of mucin- from Vector Laboratories, Burlingame, California, USA) were laden cells, as mucus cells are normally absent from the distal sequentially applied and staining was developed with nickel- lung.7 Moreover, while inflammation seems to be the main force diaminobenzidine (Sigma, St Louis, Missouri, USA). Staining by inducing mucus cell differentiation in diseases such as asthma, the NRG1a (RB277) antibody was completely blocked when this cystic fibrosis, and chronic obstructive pulmonary disease antibody was pre-incubated with recombinant human NRG1a (COPD), inflammation is usually mild in IPF.5 These discrep- EGF-like domain (296-HR, R&D Systems, Minneapolis, Minne- ancies led us to hypothesise that the mechanisms governing sota, USA). Sections were counter-stained with Alcian Blue and mucus cell metaplasia in IPF may be distinct, and that their Nuclear Fast Red (Poly Scientific, Bay Shore, New York, USA) identification may provide insight into processes regulating cell and examined at 3100 magnification. Mucus cells were identi- fate and airway epithelial function in IPF. Mucus cells in the fied by Alcian Blue staining of the cytoplasm. The number of respiratory tract are thought to originate from the reversible cells expressing MUC2, MUC5B, MUC5AC, FOXA3, SPDEF and differentiation of basal, secretory or ciliated epithelial cells. HES1 was counted on each whole section and was expressed as During development, activation of the Wnt/ß-catenin8 and a percentage of the total number of mucus cells on the section. Notch9 pathways increases mucus cell numbers in the airway. Postnatally, mucus cell differentiation of SOX2-expressing Dual labelling to identify NRG1a and p63 airway epithelial cells10 is strongly driven by the epidermal After NRG1a staining, sections were incubated with a mouse growth factor receptor (EGFR or ErbB1) and T helper 2 (Th2) monoclonal antibody recognising p63 (sc-56188, Santa Cruz cytokines, which activate the transcription factors forkhead box Biotechnology, Santa Cruz, California, USA), exposed to an A3 (FOXA3) and SAM pointed domain containing ETS tran- alkaline phosphatase-conjugated goat anti-mouse antibody scription factor (SPDEF) in association with downregulation of (31320, Thermo Scientific, Waltham, Massachusetts, USA), FOXA2.11 12 Recently, activation of other members of the ErbB developed with Fast Red TR/Naphthol AS-MX (Sigma), and family by neuregulin-1 (NRG1) splice variants NRG1a and counterstained with
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