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Acute cigarette smoke exposure activates apoptotic and inflammatory programs but a second stimulus is… Murray, Lynne A; Dunmore, Rebecca; Camelo, Ana; Da Silva, Carla A; Gustavsson, Malin J; Habiel, David M; Hackett, Tillie L; Hogaboam, Cory M; Sleeman, Matthew A; Knight, Darryl A May 3, 2017

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RESEARCH Open AccessAcute cigarette smoke exposure activatesapoptotic and inflammatory programs buta second stimulus is required to induceepithelial to mesenchymal transition inCOPD epitheliumLynne A. Murray1*, Rebecca Dunmore1, Ana Camelo1, Carla A. Da Silva2, Malin J. Gustavsson2, David M. Habiel4,Tillie L Hackett3,5, Cory M. Hogaboam4, Matthew A. Sleeman1 and Darryl A. Knight3,5,6,7AbstractBackground: Smoking and aberrant epithelial responses are risk factors for lung cancer as well as chronic obstructivepulmonary disease and idiopathic pulmonary fibrosis. In these conditions, disease progression is associated withepithelial damage and fragility, airway remodelling and sub-epithelial fibrosis. The aim of this study was toassess the acute effects of cigarette smoke on epithelial cell phenotype and pro-fibrotic responses invitro and in vivo.Results: Apoptosis was significantly greater in unstimulated cells from COPD patients compared to control, butproliferation and CXCL8 release were not different. Cigarette smoke dose-dependently induced apoptosis, proliferationand CXCL8 release with normal epithelial cells being more responsive than COPD patient derived cells. Cigarette smokedid not induce epithelial-mesenchymal transition. In vivo, cigarette smoke exposure promoted epithelial apoptosis andproliferation. Moreover, mimicking a virus-induced exacerbation by exposing to mice to poly I:C, exaggerated theinflammatory responses, whereas expression of remodelling genes was similar in both.Conclusions: Collectively, these data indicate that cigarette smoke promotes epithelial cell activation and hyperplasia, buta secondary stimulus is required for the remodelling phenotype associated with COPD.Keywords: TGFβ1, Poly I:C, Remodelling, ApoptosisBackgroundChronic obstructive pulmonary disease (COPD) is achronic lung disease commonly associated with inhalationof cigarette smoke (CS). While the pathology of COPD isgenerally considered to be destructive in nature, epithelialremodeling and sub-epithelial fibrosis of the small airwaysis now recognized as a key histopathological feature of thedisease [1, 2]. While CS directly activates and damages theepithelium, viral infections, which occur frequently insmokers with and without COPD also influences epithelialphenotype and function [3]. Moreover, viral-induced exac-erbations contribute significantly to disease progression,accelerated decline of lung function and disease morbidityand mortality [3]. Unfortunately, the mechanisms thatdrive changes to the epithelium in COPD following expos-ure to CSE and respiratory viruses remains largelyunexplored.What is known, suggests that damage to the epitheliumtriggers a temporal cascade of inflammatory and cellsignaling events that under normal circumstances leads toinflammation, resolution of inflammation and repair.However, under conditions of chronic inflammation, theepithelium fails to repair effectively with structural andfunctional changes including goblet cell hyperplasia,* Correspondence: murrayl@medimmune.com1Respiratory, Inflammation and Autoimmunity, MedImmune Ltd, Granta Park,Cambridge CB21 6GH, United KingdomFull list of author information is available at the end of the article© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Murray et al. Respiratory Research  (2017) 18:82 DOI 10.1186/s12931-017-0565-2squamous cell metaplasia, induction of pro-inflammatorychemokines [4], matrix metalloproteinases [5] and epithe-lial cell apoptosis and proliferation [6]. The mechanismsby which these changes occur are unclear, although endo-plasmic reticulum (ER) stress and oxidative damage havebeen proposed as initiators of epithelial cell apoptosis inCOPD [7–11].One aberrant epithelial response that has been associatedwith abnormal repair and fibrosis is epithelial to mesenchy-mal transition (EMT). This phenomenon, commonlyobserved in cancer and a component of normal organ de-velopment, occurs when epithelial cells transform intohighly motile mesenchymal cells. EMT has been shown tocontribute to airway disease and fibrosis in several organs,including the lung [12, 13]. However, the underlying mech-anisms and the functional consequences of EMT in theairways from COPD patients remain unclear [14].In this study we investigated the epithelial phenotypein COPD and responses to CS extract (CSE) on normaland COPD bronchial epithelial cell function in vitro. Weassessed the impact of CSE on apoptosis, proliferationand chemokine production. We show that in the shortterm, cigarette smoke does not modulate EMT, but ra-ther induces epithelial cell proliferation, apoptosis andCXCL8/IL8 production. Exposing mice to CSE com-bined with poly I:C challenge revealed that CSE inducesapoptosis and inflammation in the lung, but a secondarystimulus of poly I:C amplifies fibrotic changes. Thesedata suggest that CS alone is insufficient to induce thechronic airway remodeling that is observed in COPDand that an extra insult, such as viral infection isrequired.MethodsHuman COPD cohortThis study was approved by the Research Ethics Boardof the University of British Columbia, Canada. Humanlung tissue from patients with COPD (n = 37) and controls(n = 9) was obtained from patients undergoing surgeryand who gave informed consent. All COPD patients wereGOLD II/III, the majority were male and ex-smokersundergoing surgery for lung cancer. Control lung tissuewas from otherwise healthy, non-smoking subjects whoselungs were not used for transplant from the IIAM aspreviously described (Hackett et al., [12]). We attemptedto age match as best as possible, but the normal donorswere significantly younger than the COPD cohort.Epithelial cell cultureNormal human bronchial epithelial (NHBE, donor105116, donor 7 F4334 or 84749) cells and COPD humanbronchial epithelial cells (COPD-AEC donor OF3207,Lonza, UK) were maintained in BEGM™ (bronchial epithe-lial cell growth medium) plus BulletKit™ (Lonza). Both celltypes were cultured at 37 °C in the presence of 5% CO2.Primary bronchial epithelial cells were also isolated frompatients and cultured as previously described [12]. In all invitro epithelial cell experiments, control and COPD epi-thelial cells were used before passage 4. Cells were seededfor each experiment so they would be between 70 and80% confluent at the start of each experiment. In addition,cells were deprived of bovine pituitary extract (BPE),hydrocortisone, human epidermal growth factor (EGF),epinephrine, transferrin and triiodothyronine for 16 hprior to stimulations. Insulin, retinoic acid and GA-1000remained throughout the experiment.Induction of EMTMonolayer cultures were grown to 60–70% confluenceon 6-well tissue culture plates (BD Falcon, NJ), beforebeing quiesced and exposed to TGFβ1 (10 ng/ml) for48 h. Samples were then assayed for mesenchymal(EDA-Fn) and epithelial (E-Cadherin) markers usingquantitative polymerase chain reaction and immunoblot.Cigarette smoke extract (CSE) productionCigarette smoke extract (CSE) 100% stock was generatedfrom mainstream cigarette smoke by the combustion of1 1R3F reference cigarette (Tobacco Health Research,University of Kentucky, Kentucky, USA) with the filterremoved, using a peristaltic pump, through 25 mls ofculture medium without FBS. The CSE was then sterilefiltered through a 0.22 μM filter, and added to cellcultures at the required concentration.Immunohistochemical staining of human airway sectionsTissue sections were deparaffinized and rehydrated, andput through antigen retrieval by autoclaving (15 min,120 C, 30 psi) for 20 min in citrate target retrieval solu-tion (Dako, Mississauga, ON, Canada). Subsequently, en-dogenous peroxidase was quenched with 3% H2O2 andnon-specific interactions blocked for 30 min with 10%goat serum. Antibodies directed against α-smoothmuscle actin (ab5694, Abcam, Cambridge, MA), andvimentin (AF2105, R&D Systems) were added overnightat 4 °C in 25% goat serum. Sections were then incubatedwith either biotinylated goat anti-mouse or goatanti-rabbit secondary antibody (1:100, Vector Labs,Burlingame, CA) for 60 min followed by a 10-min treat-ment with Streptavidin-HRP (Dako). The antigen ofinterest was visualized by 3,3-diaminobenzidine (Dako)and counterstained with Harris Hematoxylin Solution(Sigma, Oakville, ON, Canada). Sections were then dehy-drated and mounted with Cytoseal 60 (Richard-AllanScientific, Kalamazoo, MI). Antibody dilutions and allwashes were in TRIS-buffered saline solution.Murray et al. Respiratory Research  (2017) 18:82 Page 2 of 12Gene expression analysesFor gene expression analysis from lung tissue, total RNAwas obtained using TRIzol reagents (Invitrogen, Paisley,UK) according to manufacturer’s instructions. RNA wasreverse transcribed into cDNA using the high capacitycDNA kit (Applied Biosystems) and gene expression levelswere determined using qRT-PCR using an ABI Prism7900HT sequence detector (Applied Biosystems).Transcript levels of genes of interest were normalised to18s and fold change was to matched animal controls ornon-COPD lung using 2-ΔΔCt.Nu-PAGE and Western blottingNHBE cells were seeded at 12 × 104 cells/ml and cul-tured for 24 h in complete medium. Prior to stimulationcells were cultured overnight in basal medium supple-mented with only retinoic acid, insulin and antibiotics.TGFβ1 (10 ng/ml, R&D Systems, Abingdon, UK), CSE(5% or 10%), or both combined were added to the cells(media alone was used as a control) and left to incubatefor a further 48 h. Cells were scraped and lysed in RIPAbuffer (Sigma-Aldrich, Poole, UK) containing proteaseinhibitors (Roche Diagnostics, East Sussex, UK). Cellsuspensions were cleared by centrifugation at 8000 x gfor 10 min at 4 °C. 15 μg of protein was loaded onto aBis-Tris (4–12%) Nu-PAGE gel (Invitrogen, Paisley UK)followed by electrophoresis and then transferred toPVDF membranes using the iBlot dry blotting system(Invitrogen, Paisley, UK). Membranes were blocked inOdyssey blocking buffer (LI-COR) for 2 h and thenprobed with E-cadherin (clone 67A4 mIgG1, Santa Cruz,Heidelberg, Germany), fibronectin (clone IST-9 mIgG1,Santa Cruz, Heidelberg, Germany) or beta tubulin load-ing control (LI-COR) diluted in Odyssey blocking buffer(LI-COR) supplemented with 0.02% Tween-20 overnightat 4 °C. Membranes were washed 4 times in PBS-T for5 min each and were then probed with anti-mouse oranti-rabbit IRDye 800cw conjugated secondary antibody(LI-COR) in Odyssey blocking buffer supplemented with0.02% Tween-20. Membranes were washed as beforeand left to dry before scanning on the Odyssey ClxInfrared Imaging System (LI-COR). Western blots wereanalysed by densitometry using loading control fornormalisation and the fold change in expression wasdetermined compared to untreated controls. Experimentswere performed on 3 NHBE donors.CXCL8 ELISACXCL8 was measured from the supernatants of epithe-lial cells using a duoset ELISA (R&D Systems) accordingto manufacturer’s guidelines. Experiments were donethree times using triplicate wells each time.Apoptosis assayNHBE and COPD-AEC were plated at 1x105cells/ml andcultured for 24 h to reach around 80–90% confluency.BEGM media with 5, 10 or 20% CSE was added to cellsfor 6 and 24 h. Caspase-3/-7 activity was determined fromtreated epithelial cells using the Caspase-Glo® -3/-7 Assay(Promega) according to manufacturer’s guidelines.Experiments were done three times using triplicate wellseach time.Lactate Dehydrogenase (LDH) assayTo determine the levels of released LDH in cell-free su-pernatants, the CytoTox 96 Non-Radioactive CytotoxicityAssay (Promega) was used to measure levels in the super-natants of epithelial cells according to manufacturer’sguidelines. Measurements were performed on three indi-vidual experiments using triplicate wells each time.Proliferation assayNHBE and COPD-AEC cells were plated at 1x105cells/mland cultured to 80–90% confluency. At this point, cellswere exposed to CSE media at a concentration of 5, 10and 20% in low serum conditions. Cells were stimulatedwith media alone, CSE media, full serum complete mediaor media with 30 ng/ml of PDGF (R&D systems, UK) as apositive control for proliferation for 72 h. At this pointcells were pulsed with 30 μl/ml of [3H] thymidine for 6 hand proliferation measured in a topcount liquid scintilla-tion counter (Perkin-Elmer). Results were expressed ascounts per minute. Experiments were done three timesusing triplicate wells each time.MiceSix-week-old C57Bl/6 mice were purchased from TaconicEurope (Denmark). Experiments were conducted in ac-cordance with standards established by the Council ofEurope, AstraZeneca Global R&D Standards for animalcare, European Union Directive 86/609, and Swedishlegislation. The studies were approved by the regionalEthics Board, Malmö/Lund, Sweden (reference numberM230/09).Animal exposure to cigarette smoke and poly I:CThe current protocol was adapted from a previouslydescribed model by Kang et al. [16]. Briefly, mice wereexposed to cigarette smoke using a whole-body smokeexposure system (SIU-48, Promech Lab AB, Vintrie,Sweden). Mice were exposed to 10 1R3F referencecigarettes with filters removed for a period of approximately50 min, twice daily, for 18 consecutive days, control animals(i.e., sham-exposed animals) were exposed to room air only.Prior to exposure mice were acclimatized over a 3 dayperiod. On days 8, 11, 15 and 18 of cigarette smoke expos-ure, mice were lightly anesthetized and 50 μl aliquots ofMurray et al. Respiratory Research  (2017) 18:82 Page 3 of 12poly (I:C) (50 μg per animal, Sigma Aldrich, Sweden) or itsvehicle control were administered via nasal aspiration.Animals were euthanized at day 19 of the protocol andoutcome measures assessed.Evaluation of airway inflammation and remodelling inmiceTissue samples and bronchoalveolar lavage (BAL) fluidwere collected for assessment. For histologic evaluations,the left lung was inflated to 25 cm with 4% buffered for-maldehyde (Histolab Products AB, Sweden). Images oflung sections were captured at x20 final magnificationon a microscope using a Scanscope (Aperio, CA, USA).In addition, RNA was extracted from tissue for gene ex-pression analysis (see previous section). Total cellularitywas determined from BAL fluid and specific blood cellcounts were determined for macrophages, neutrophils,lymphocytes and eosinophils.Immunohistochemical stainingImmunohistochemical staining for Hematoxylin andeosin (H&E), proliferating cell nuclear antigen (PCNA);Novocastra Laboratories Ltd., Newcastle upon Tyne,UK,) and caspase-3 (rabbit anti-human/mouse caspase-3active, AF835; R&D Systems, UK) were performed. Anti-bodies were detected with EnVision™ kit (DAKO UKLtd, Cambridgeshire) and DAB as chromogen, accordingto the manufacturer’s instructions.Statistical analysisNormal distribution was assumed. Data were expressedas means ± SEM, and assessed for significance byStudent's t test or ANOVA as appropriate.ResultsCigarette smoke induces apoptosis, proliferation andchemokine generation in normal and COPD lungepithelial cellsTo determine the effect of CSE on apoptosis, airway epi-thelial cells from normal (NHBE) and COPD (COPD-AEC) lung were treated with increasing concentrationsof CSE for 6 h or 24 h and caspase 3/7 activity was mea-sured. There was no increase in caspase activity at 6 h(data not shown), however CSE induced an increase incaspase 3/7 activity at 24 h, with caspase activity max-imal at a concentration of 5% CSE (Fig. 1a). Baselinecaspase 3/7 activity was significantly higher in COPD-AEC compared to NHBE and CSE had only a modeststimulating effect, suggesting that endogenous caspaseactivity was already near maximum at rest (Fig. 1a). Theconcentrations of CSE used did not affect epithelial cellviability as observed morphologically (Additional file 1:Figure S1) or when assessing lactate dehydrogenaserelease (Additional file 1: Figure S2).Next we assessed the impact of CSE on epithelial cellproliferation and chemokine production. CSE increasedproliferation of NHBE and COPD-AEC at 5% CSE and10% CSE (Fig. 1b). We also measured the effects of CSEon the release of CXCL8, a chemokine known to be in-duced by CS [15], and found that CSE induced CXCL8release in a concentration dependent manner from bothNHBE and COPD-AEC, although surprisingly IL-8 re-lease from COPD-AEC was blunted in response to CSEwhen compared to the NHBE (Fig. 1c).Acute cigarette smoke exposure does not affect epithelialto mesenchymal transitionWe next assessed the effects of CSE on markers ofEMT. Epithelial cells from non-diseased donors werestimulated for 48 h with CSE in the presence or absenceof 10 ng/ml TGFβ1 and EMT markers such as e-cadherin and the EDA splice variant of fibronectin weremeasured. As shown in Fig. 2, TGFβ1 reduced E-cadherin expression and concomitantly increasedexpression of EDA-Fn suggesting an EMT response. Incontrast, addition of CSE at either 5 or 20% did notdirectly induce EMT nor modulate TGFβ1-inducedEMT (Fig. 2a-c).Decreased epithelial markers and elevated mesenchymalmarkers in COPDTo then determine if any quantitative differences in epi-thelial and mesenchymal markers exist in the COPDlung, diseased and healthy lung biopsy tissue wasassessed for the gene expression of epithelial and mesen-chymal specific cell markers. As shown in Fig. 3a-d, ex-pression of the epithelial specific markers keratin-18 andkeratin-19 were downregulated in COPD lung tissue,whilst the expression of mesenchymal markers collagen1a2 and vimentin were highly upregulated compared tocontrol lung tissue (Fig. 3a-d). To further support thesefindings and localise the increase in mesenchymalmarkers, immunohistochemical staining identifiedalpha-smooth muscle actin (αSMA) and vimentin stain-ing within the epithelial layer itself (Fig. 3e).Cigarette smoke induces epithelial cell apoptosis in vivobut fibrotic changes only occur after viral exacerbationWe next used an in vivo mouse model of virus-induced COPD exacerbations using cigarette smokewith poly I:C [16]. Exposure to CSE or poly I:C chal-lenges significantly induced an inflammatory response,as observed by increased infiltration of macrophages,neutrophils and eosinophils in the bronchoalveolarlavage (BAL) of animals compared to animals exposedto air alone. In addition, further enhancement of theinflammatory effect was observed when CSE and polyI:C were used in combination (Fig. 4a-d). HistologicalMurray et al. Respiratory Research  (2017) 18:82 Page 4 of 12analysis of lung sections showed PCNA staining inareas of inflammation in the lungs of animals treatedwith CSE or poly I:C and this was further enhancedin mononuclear cells and type II pneumocytes whenCSE and poly I:C were administered in combination(Fig. 4e). Minimal caspase-3 staining was observed incontrol animals, while a marginal staining in the peri-vascular inflammatory cuffs was observed in the polyI:C group. Exposure to CSE significantly increasedcaspase-3 staining, primarily in the areas of alveolarinflammation (Fig. 4e). Addition of poly I:C had noeffect on the CSE induce caspase-3 expression. Inaddition to increased expression of caspase 3 and -7(Fig. 5a and b), we also observed increases in otherapoptotic markers such as fas (Fig. 5c) and gsk3b(Fig. 5d) with CSE alone or in combination with polyI:C. In contrast, we saw an increase in the epithelialmarker E-cadherin (Fig. 5e), the mesenchymalmarkers tgfb1 (Fig. 5f ), aSMA (Fig. 5g), collagen 1a1(Fig. 5h) and fibronectin (Fig. 5i), but the most robustand elevated responses were observed in the animalstreated with CSE and poly I:C. This suggests that anadditional stimulus, beyond CSE, is required for ab-normal repair and fibrosis to occur.DiscussionGiven its prime location, it is perhaps not unexpectedthat the epithelium of people who smoke, and par-ticularly those with COPD would be abnormal.Indeed, chronic inflammation and remodeling of themucosa are typical features of COPD. We were inter-ested in looking at the effects of CSE on the epithe-lium and found that using primary epithelial cellsfrom patients with COPD and healthy controls: (1)COPD-AEC have increased basal levels of apoptosisand that exposure to CSE increases caspase activity inhealthy and COPD-AEC; (2) CSE increases prolifera-tion and IL-8 release in a concentration-dependentmanner although intriguingly, IL-8 release by COPD-AEC was significantly lower than normal-AEC. (3)We then used a mouse model of CSE and poly I:C tomimic viral exacerbations and show that while polyI:C clearly induces lung inflammation in its own rightand enhances CSE-induced inflammation, it has noeffect on caspase-3/-7 activity either alone or in com-bination with CSE; (4) finally we show that short-term CSE challenge has no effect on markers of EMTeither in vitro or in vivo. Our data suggests thatcigarette smoke induces a marked apoptotic burdenFig. 1 Cigarette smoked induces apoptosis, proliferation and IL-8 production in normal and COPD epithelial cells. NHBEs (normal humanbronchial epithelial cells) or COPD-AECs (COPD diseased human bronchial epithelial cells; Lonza) were treated with increasing concentrations ofCSE (2–20% as indicated) and after 24 h CSE promoted apoptosis was increased as assessed by elevated caspase 3/7 activity (a), and cellproliferation (b) as measured using 3H-incorporation, (n = 3). CXCL8/IL8 was induced in a dose-dependent manner by CSE treatment (c), asmeasured by ELISA in the supernatants following stimulation for 24 h (n = 2 donors, each repeated n = 3 times).Murray et al. Respiratory Research  (2017) 18:82 Page 5 of 12on epithelial cells, but at the concentrations andtimes used does not induce EMT either in vitro or invivo.One of the strengths of our study was the use of pri-mary AEC from COPD patients where we show for thefirst time that basal apoptotic load is significantly higherin cultured AEC obtained from these patients than AECobtained from healthy donors. While caspase activity in-creased in AEC from normal donors after CSE exposure,we saw no statistically significant effect in COPD-AEC.However, basal caspase activity in COPD-AEC was thesame as the maximal CSE-induced activity in normal-AEC, suggesting activity of caspase -3/7 is constitutivelyhigh in COPD-AEC. In contrast, Chiappara andFig. 2 TGFβ1 but not CSE drives a mesenchymal phenotype in lung epithelial cells. Exposure of NHBE (a-c) or COPD-AEC (D-F) cells to TGF β1(10 ng/ml) decreased E-cadherin and increased EDA-Fn expression (A-C). Exposure to CSE (5%) did not impact on E-cadherin expression in thepresence or absence of TGF-β1 (a, b), but at a high concentration (20%) did reduce TGFβ1-induced EDA-Fn expression (a, c). COPD-AEC wereresistant to EMT. Exposure of AEC from COPD patients (Lonza) to TGFβ1 had minimal effect on E-Cadherin expression (d, e) although it did induceEDA-Fn expression (d, f). Exposure to CSE (5%) did not impact E-Cadherin (d, e) or EDA-Fn (d, f) expression in the presence or absence of TGF-β1.Representative western blots from NHBE donor cells (a) or COPD donor cells (d), shown alongside densitometry from 3 NHBE donors (b, c) or 1COPD donor repeated 3 times (e, f), protein levels normalised to β-tubulin loading controlMurray et al. Respiratory Research  (2017) 18:82 Page 6 of 12colleagues [17] showed that epithelial caspase-3 expres-sion was similar in patients with COPD and normal con-trols and that CSE did not influence apoptosis in atransformed human airway epithelial cell line (16HBE).We hypothesize that since Chiappara et al., examinedexpression but not activity of caspase-3 and used atransformed cell line which are generally resistant toapoptosis are possible reasons for these disparate find-ings. We also investigated caspase-3 expression as wellas caspase-3/7 activity in a mouse model of CSE andpoly I:C to mimic viral exacerbations. We show that polyI:C clearly induces lung inflammation and enhancesCSE-induced inflammation, that it increases caspase-3expression in areas of alveolar inflammation but has noeffect on lung tissue caspase-3/7 activity either alone orin combination with CSE. A similar model developed byKang et al [16] demonstrated that the combination ofCSE and poly I:C were associated with the induction oftype I interferon (IFN) and IL-18, followed by the induc-tion of IL-12/IL-23 p40 and IFN-γ, and the activation ofPKR (double-stranded RNA-dependent protein kinase)and eIF-2α (eukaryotic initiation factor-2α). Theseauthors also demonstrated that CSE enhanced the effectsof influenza, but no other agonists of innate immunity,suggesting that CSE selectively augments the airway andalveolar inflammatory and remodelling responses in-duced in the murine lung by poly I:C and viruses [16].Furthermore, Chiappara et al also showed that expres-sion of two different proliferation markers, PCNA andKi67 were differentially expressed in epithelium inCOPD sections; expression of PCNA was virtuallyabsent in COPD and smoking groups, whereas Ki67 ex-pression was significantly higher in the COPD/smokinggroups compared to control. Proliferation was notFig. 3 Epithelial and mesenchymal marker expression in COPD lung. Lung tissue samples were analysed for epithelial marker gene expression (a) krt18and (b) krt19 which were down regulated in patients with COPD whilst the mesenchymal markers (c) col1a1 and (d) vim were concomitantlyupregulated. Immunohistochemical staining of COPD lung sections identified positive staining (brown) for (e) alpha-smooth muscle actin and (f)vimentin, which appeared to be localised to the epithelial layer as indicated by the arrowsMurray et al. Respiratory Research  (2017) 18:82 Page 7 of 12Fig. 4 Cigarette smoke-induced lung inflammation is moderately enhanced by poly (I:C) in mice. C57Bl/6 mice were exposed to cigarette smoke (CSE),poly (I:C) (pI:C, 50 μg), a combination of both (CSE + pI:C) or their respective controls (Air). One day after the last poly (I:C) challenge (Day 19 of the protocol),the mice were killed, bronchoalveolar lavage (BAL) was undertaken, and lung sections were prepared. CSE induced an increase in BAL total cell number(a), neutrophil (b), macrophage (c) and lymphocyte (d) number. e Histological assessments indicated an increase in interstitial inflammation (hematoxylin &eosin; H&E; top panel) and apoptosis (proliferating cell nuclear antigen or cyclin; PCNA; middle panel stains, caspase-3; casp3, bottom panel stains, x20 oforiginal magnification) following CSE and poly I:C challenge. Results are presented as the mean ± SEM of 6–8 animals per group. * p< 0.05, ** p< 0.01,*** p< 0.005Murray et al. Respiratory Research  (2017) 18:82 Page 8 of 12Fig. 5 (See legend on next page.)Murray et al. Respiratory Research  (2017) 18:82 Page 9 of 12quantified in vitro. Our data suggest that CSE induces aconcentration-dependent proliferative response in vitro,which was maximal at 10% and then sharply declining,presumably due to toxicity. CSE also promoted lung epi-thelial cell proliferation in vivo as observed by PCNAstaining. These results are supported by previous reportsthat showed smoking generates a dose-dependent pro-proliferative response in epithelial cells of smokers [18].Release of CXCL8 from normal-AEC dose dependentlyincreased with CSE exposure. Kode et al showed similareffects in primary cultures of small airway epithelial cells[19]. In agreement with previous studies [20], basalCXCL8 release was similar in normal and COPD-AEC.These findings differ from those of Schneider et al., whoobserved increased basal CXCL8 production in epithelialcells from COPD patients [21]. These disparate findingsmay be due the different culture procedures, asSchneider et al generated differentiated air-liquid inter-face cultures, whereas both Heijink and colleagues andthis study used submerged monolayer cultures. Likewise,disease severity may play a role. Although GOLD statuswas not reported, 8 of the COPD patients included inthe study by Schneider had severe emphysema. Never-theless, Schulz et al [22] were also not able to detect dif-ferences in baseline CXCL8 production in submergedcultured PBEC from currently smoking COPD patientsand smoking controls.Epithelial-mesenchymal transition (EMT) has beencategorized into three different types: Type I represent-ing a natural process such as occurs during embryogen-esis; Type II – leading to organ fibrosis and Type III,pro-angiogenesis and associated with cancer. In the con-text of COPD, Sohal and colleagues documented TypeIII EMT in the epithelium of large airways from patientswith COPD [23] and Nishioka et al., showed that epithe-lial cells from COPD patients have display a partial EMTphenotype under basal conditions [24–30]. Recent find-ings indicate that similar changes can also be observedin the small (<1 mm) airways of smokers. However, des-pite CSE-induced increases in expression of TGFβ1,GSK3β and col1α1, we saw no evidence of EMT, sinceexpression of αSMA and E-cadherin was not changed.Given this gene expression signature was derived fromwhole lung which may have masked epithelial specificresponses, we performed similar experiments using pri-mary cultures of human airway epithelial cells. We showthat following exposure to TGFβ1, epithelial cells in-crease expression of mesenchymal marker EDA-Fn,while down regulating expression of the epithelialmarker E-cadherin as expected. Exposure to CSE had noeffect on these markers when used alone and did notmodify the response to TGFβ1. Recent studies haveshown that cigarette smoke condensate (CSC) inducesan EMT-like process in BEAS-2B cells [31]. The reasonsfor these different findings may relate to the type of cellcultures or duration of exposure to CSE, since Veljkovicand colleagues exposed their cells for 30 days, whereaswe used 48 h.ConclusionsIn summary, the main findings of this study was that ex-posure to CSE in vitro and in vivo induces a profoundinflammatory and pro-apoptotic response of the epithe-lium under normal conditions, but these responses areblunted in COPD-AEC. Furthermore, CSE does not in-duce an EMT-like phenotype in vitro or in vivo, at leastat the time points measured. Our results highlight thecapacity of the epithelium to respond to environmentalinsult is compromised in COPD.Additional fileAdditional file 1: Figure S1. Cigarette smoke extract (CSE) atconcentrations of 50% or lower did not change normal epithelial cellmorphology. Figure S2. Cigarette smoke extract (CSE) and/or TGFβ1 didnot increase cell cytotoxicity in lung epithelial cells. (DOCX 903 kb)AcknowledgmentsThe authors would like to thank Tom Kisby for technical assistance.FundingThis study was supported by the following funding: for CMH: R01HL123899;for DAK: NHMRC 1099575.Availability of data and materialsAll of the data presented in this study is reported; no additional databaseswere used or generated.Authors’ contributionsLM designed the studies, analysed the data, wrote the manuscript. RD andAC carried out the in vitro work and analysed in vivo samples. CDS and MGcarried out the in vivo studies. DMH conducted human gene analysis. TLH(See figure on previous page.)Fig. 5 Cigarette smoke alone induces apoptotic markers in the lungs of mice and increases some mesenchymal marker expression although thisis further enhanced by poly (I:C). C57Bl/6 mice were exposed to cigarette smoke (CSE), poly (I:C) (pI:C, 50 μg), a combination of both (CSE + pI:C)or saline controls. One day after the last poly (I:C) challenge, the mice were killed and lung tissue was taken for RNA extraction and genetranscript analysis. The apoptotic markers caspase 3 (a), caspase 7 (b), fas (c) and gsk3b (d) were increased by cigarette smoke alone and nofurther enhancement was observed in combination with poly (I:C). The epithelial marker e-cadherin (e) was slightly elevated in the presence ofcigarette smoke. tgfβ1 (f) expression was clearly upregulated following cigarette smoke exposure and further still when in combination with polyI:C. The mesenchymal markers alpha smooth muscle actin (g), collagen1a1 (h) and fibronectin (i) were also more robustly increased in thecombined poly I:C cigarette smoke group. * p < 0.05, ** p < 0.01, **** p < 0.001Murray et al. Respiratory Research  (2017) 18:82 Page 10 of 12conducted in vitro assessments. CMH conducted human sample analysesand designed studies. MAS participated in the coordination and helped draftthe manuscript. DAK designed the studies, analysed the data and wrote themanuscript. All authors read and approved the final manuscript.Competing interestsI declare that no authors have any competing interests.Consent for publicationAll authors consent to the publication of this article. There is no person-specific data in this manuscript therefore it is not applicable to seekadditional consent to publish beyond the author list. We declare that noneof the authors have any competing interests associated with the publicationof this manuscript.Ethics approval and consent for participateHuman lung tissue from patients with COPD (n = 37) and controls (n = 9)was obtained from patients undergoing surgery and who gave informedconsent. This study was approved by the Research Ethics Board of theUniversity of British Columbia, Canada. For the in vivo experiments, allstudies were conducted in accordance with standards established by theCouncil of Europe, AstraZeneca Global R&D Standards for animal care,European Union Directive 86/609, and Swedish legislation.Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims in publishedmaps and institutional affiliations.Author details1Respiratory, Inflammation and Autoimmunity, MedImmune Ltd, Granta Park,Cambridge CB21 6GH, United Kingdom. 2Respiratory, Inflammation andAutoimmunity innovative Medicines Unit, AstraZeneca R&D, Mölndal,Sweden. 3Department of Anesthesiology, Pharmacology and Therapeutics,University of British Columbia, Vancouver, Canada. 4Department of Medicine,Cedars-Sinai Medical Center, Los Angeles, CA, USA. 5James Hogg ResearchCentre, University of British Columbia, Vancouver, Canada. 6School ofBiomedical Sciences and Pharmacy, Newcastle, Australia. 7Priority ResearchCentre for Healthy Lungs, University of Newcastle and Hunter MedicalResearch Institute, New South Wales, Australia.Received: 28 June 2016 Accepted: 27 April 2017References1. 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ToxicolIn Vitro. 2011;25(2):446–53.•  We accept pre-submission inquiries •  Our selector tool helps you to find the most relevant journal•  We provide round the clock customer support •  Convenient online submission•  Thorough peer review•  Inclusion in PubMed and all major indexing services •  Maximum visibility for your researchSubmit your manuscript atwww.biomedcentral.com/submitSubmit your next manuscript to BioMed Central and we will help you at every step:Murray et al. Respiratory Research  (2017) 18:82 Page 12 of 12

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