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Expression of surfactant protein D in airways of asthmatics and interleukin-13 modulation of surfactant… Xu, Jie; Singhera, Gurpreet K; Dorscheid, Delbert R Feb 15, 2015

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RESEARCH Open AccessExpression of surfactant protein D in airways ofasthmatics and interleukin-13 modulation ofuman models of airwayimplications on the increased susceptibility to infections and altered inflammatory response in asthmatic patients.Xu et al. Respiratory Research  (2015) 16:26 DOI 10.1186/s12931-015-0177-7Center for Heart Lung Innovation, University of British Columbia and St.Paul’s Hospital, Vancouver, British ColumbiaFuture functional studies on the role of SP-D in asthma can provide better insight into defects in the structure andregulation of SP-D.Keywords: Surfactant protein D, Asthma, Airway epithelium, Air-liquid-interface, Interleukin-13* Correspondence: del.dorscheid@hli.ubc.caConclusions: SP-D is expressed differently in airways of astepitheliumJie Xu, Gurpreet K Singhera and Delbert R Dorscheid*AbstractBackground: Surfactant protein D (SP-D), a pattern recognition molecule, has been shown to play roles in hostdefense such as opsonisation, aggregation of pathogens, and modulation of the inflammatory response. In light ofinfection-induced exacerbations and damage to the airway epithelium from inflammation, these functions of SP-Dmake it relevant in the development and pathogenesis of asthma.Methods: Expression of SP-D was examined in human airway sections and primary airway epithelial cells (AEC)grown in air-liquid interface (ALI) cultures and comparisons were made between those from asthmatic andnon-asthmatic donors. ALI cultures of AEC from non-asthmatic donors were examined for SP-D, Mucin 5AC, andcytokeratin-5 expression at different stages of differentiation. Interleukin-13 (IL-13) treatment of airway epitheliumand its effect on SP-D expression was studied using ALI and monolayer cultures of primary AEC from non-asthmaticand asthmatic donors.Results: Airway epithelium of asthmatics, compared to that of non-asthmatics, expressed increased levels of SP-D asdemonstrated in airway tissue sections (fraction of epithelium 0.66 ± 0.026 vs. 0.50 ± 0.043, p = 0.004) and ALI cultures(fraction of epithelium 0.50 ± 0.08 vs. 0.25 ± 0.07). SP-D expression decreased as ALI cultures differentiated from 7 daysto 21 days (fraction of epithelium 0.62 ± 0.04 to 0.23 ± 0.03, p = 0.004). Treatment with IL-13 decreased SP-D expressionin both ALI cultures (fraction of epithelium 0.21 ± 0.06 vs. 0.62 ± 0.04, p = 0.0005) and monolayer cultures (proteinexpression fold change 0.62 ± 0.05) of non-asthmatic AEC; however, IL-13 had no significant effect on SP-D expressionin monolayer cultures of asthmatic AEC. Experiments with non-asthmatic monolayer cultures indicate IL-13 exert itseffect on SP-D through the IL-13 receptor alpha1 and transcription factor STAT6.hmatics relative to that of non-asthmatics. This can havesurfactant protein D in h© 2015 Xu et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly credited. The Creative Commons Public DomainDedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,unless otherwise stated.Xu et al. Respiratory Research  (2015) 16:26 Page 2 of 12IntroductionAsthma is a chronic inflammatory disorder of the airwayswhich affects people of all ages. It has increased in preva-lence over the past 30 years and currently affects 235 millionpeople worldwide [1]. Clinically, asthma is characterized byrecurrent and reversible episodes of wheezing, chest tight-ness, breathlessness, and coughing. Structural and functionalchanges associated with asthma include bronchoconstric-tion, airway inflammation, epithelial goblet cell hyperplasia,bronchial smooth muscle hypertrophy, and proliferation ofairway blood vessels and nerves [2]. Higher proportion ofbasal cells as opposed to differentiated cells has also beenfound in airways of asthmatics [3].Asthmatic patients may have increased susceptibility toviral and bacterial infections in the airways. Respiratoryviral infections have detrimental effects on patients withestablished asthma; they have been associated with almost80% of asthma exacerbations [4]. Respiratory syncytialvirus (RSV) is the most common cause of acute lower re-spiratory infection in infants and children worldwide [5].Several studies have demonstrated an association of severeRSV infection in infancy with subsequent development ofrecurrent wheezing and asthma in children [6-9]. Asthmahas traditionally been closely associated with the adaptiveimmune system; however, more recent studies have indi-cated that the innate immune response is also importantin its development and progression [10]. Therefore, therole of the innate immune system in host defence againstinhaled pathogens and subsequent inflammatory responseis of particular interest.Pulmonary surfactant was originally identified as a lipo-protein complex which reduces surface tension in thealveoli. More recently, the surfactant proteins A and D(SP-A, SP-D), were found to be involved in host defensemechanisms [11]. SP-A and SP-D, belong to the collectinfamily of proteins and are pattern recognition moleculeswhich bind a broad spectrum of pathogens including vi-ruses, bacteria, and fungi [11]. Its roles in host defense in-clude enhancement of phagocytosis through opsonisation,aggregation of pathogens, and regulation of inflammatorymediators [11]. SP-A and SP-D are constitutive mediatorsof antigen clearance which can interact with cellular com-ponents of both innate and adaptive immune system onthe mucosal surface [12].Interleukin-13 (IL-13) is a cytokine secreted by manycell types including TH2 cells, macrophages, and activatedmast cells [13-15]. Several animal models have indicatedthat IL-13 mediates features of asthma such as airwayhyper-reactivity, mucus cell hyperplasia, and sub-epithelialairway fibrosis independent of other cytokines [13,16,17].The role of IL-13 in mediating phenotypes of asthma wasalso supported by reports of increased IL-13 in lungs ofasthmatic patients [18]. IL-13 has two receptors: IL-13receptor alpha 1 (IL-13Rα1) and IL-13 receptor alpha 2(IL-13Rα2). IL-13Rα1 forms a heterodimeric receptorcomplex with IL-4Rα subunit. IL-13 signalling viaIL-13Rα1 activates the Janus kinase (JAK)-signal trans-ducer and activator of transcription (STAT) pathway,specifically the transcription factor STAT6. IL-13Rα2is known as a high-affinity receptor and can signal viathe transcription factor, activator protein 1[19].The present study aims to characterize the expression ofSP-D in airways of asthmatics, with the hypothesis that itwould differ from that of non-asthmatics. The effects ofdifferentiation and IL-13 exposure on the expression ofSP-D were investigated using air-liquid interface (ALI) cul-tures of primary airway epithelial cells (AEC) from non-asthmatic donors and monolayer cultures of AEC fromnon-asthmatic and asthmatic donors.Material and methodsReagentsMouse anti-human SP-D antibody used for immunohisto-chemistry (IHC) and Western blot analysis (catalog no.HYB-245-01-02) was purchased from Thermo Scientific(Rockford, IL). Mouse anti-β-actin monoclonal antibodyconjugated to horseradish peroxidase (sc-47778) was pur-chased from Santa Cruz Biotechnology (Santa Cruz, CA).Mouse anti-Mucin 5AC (MUC5AC) antibody (AB24071)and rabbit anti-cytokeratin 5 (CK-5, AB24647) antibodywere purchased from Abcam Inc. (Toronto, ON) for IHC.Recombinant human IL-13 (213-IL), goat anti-humanIL-13Rα1 (AF-152) and IL-13Rα2 (AF-146) neutralizingantibodies and normal goat IgG (AB-108-C) were pur-chased from R&D Systems (Minneapolis, MN). Mouseanti-phosphorylated STAT6 (p-STAT6) antibody (611566)and mouse anti-STAT6 antibody (611290) used for Westernblotting were purchased from BD Biosciences (Mississauga,ON). Trizol reagent (15596018) was purchased fromLife Technologies (Burlington, ON).Cell cultureHuman lungs of de-identified asthmatic and non-asthmatic donors, deemed unsuitable for transplant-ation and donated to medical research, were obtainedfrom the International Institute for the Advancementof Medicine (Edison, NJ) for primary cell isolation asper approval by the Research Ethics Board (REB) ofUniversity of British Columbia/Providence Healthcare(REB# H00-50110). Airway epithelial cells (AEC) wereisolated by protease digestion as described by Gray andcolleagues [20], cultured in bronchial epithelial growthmedium (Lonza, Mississauga, ON, CC-3170), incubated at37°C in 5% CO2, and grown in 12-well plates. Treatment ofprimary AEC in monolayer occurred between passages twoand three. Donor-matched air-liquid interface (ALI) epithe-lial cultures were generated using cells at passages one ortwo in PneumaCult medium (Stemcell Technologies,Xu et al. Respiratory Research  (2015) 16:26 Page 3 of 12Vancouver, BC, 05001). ALI epithelial cultures were grownfor 7, 14, or 21 days to represent various stages ofdifferentiation.Stimulation of cell culturePrimary AEC from non-asthmatic donors were grown to80% confluence and treated for 24 hours with IL-13 (10ng/mL) alone or pre-incubated with either goat IgG isotypecontrol, neutralizing antibodies for IL-13Rα1 (20 μg/mL)or IL-13Rα2 (2 μg/mL) for one hour prior to the additionof IL-13. Primary AEC from asthmatic donors were simi-larly grown to 80% confluence and treated for 24 hourswith IL-13 (10 ng/mL).Pseudostratified ALI cultures of AEC isolated fromnon-asthmatic donors were grown for 28 days to studythe effects of IL-13 treatment. Starting at days 14 and21, IL-13 was added on alternate days for the durationof two weeks and one week respectively.Immunohistochemical analysisHuman airway tissue from asthmatic and non-asthmaticdonors and ALI cultures grown from non-asthmatic AEC(untreated and IL-13 treated) were fixed in 10% formalin,embedded in paraffin, and sectioned into 4 μm slices. Sec-tions were deparaffinized in CitriSolv (Fisher Scientific,Toronto, ON, 22-143-975) and rehydrated. Antigen re-trieval was performed by autoclaving in Citra solution atpH 6.0 (Life Technologies, Burlington, ON) for 22 mi-nutes. All sections were blocked consecutively using Back-ground Sniper (Biocare Medical, Markham, ON, B5966L)and Dual Endogenous Block (Dako, Burlington, ON,K5361). Sections were stained with anti-SP-D (0.25 μg/mL), MUC5AC (1 μg/mL), or CK-5 (1 μg/mL) antibodiesusing biotin-free MACH 3 AP-Polymer Detection kit (Bio-care Medical, Markham, ON, M3U532 for mouse andM3R533 for rabbit primary antibodies) containing alkalinephosphatase with Warp Red Chromogen (Biocare Med-ical, Markham, ON, WR806) as substrate. Matched iso-type control antibodies were used as negative controls forIHC to demonstrate antibody specificity. Hematoxylin wasused for counter-staining of the nuclei. Colour segmenta-tion via ImagePro Plus (Media Cybernetics, Silver Spring,MD) was performed for quantification whereby area posi-tive was normalized to total epithelial area.Western blot analysisTotal cell lysates collected from primary human AECmonolayers and ALI cultures were used for Western blotanalysis according to standard techniques [21]. Twenty mi-crograms of reduced protein samples were electrophoresedin a 4-20% gradient SDS-PAGE gel and then transferred toa nitrocellulose membrane. The membrane was blockedfor 1 hour at room temperature in TBST (10 mmol/L Tris-HCl, 150 mmol/L NaCl, and 0.1% Tween-20) containing5% skimmed milk for non-phosphorylated proteins and 5%BSA for phosphorylated proteins. The membrane was thenincubated overnight at 4°C in respective primary anti-bodies in TBST containing 2.5% skimmed milk for non-phosphorylated protein or 1% BSA for phosphorylatedprotein. Primary antibody concentrations used were: anti-SP-D (1:1000) and anti-p-STAT6 (1:2000) followed by re-probing with anti-β-actin (1:2500) and STAT6 (1:1000) tonormalize for SP-D and p-STAT6 respectively. The mem-branes were washed three times with TBST, followedby incubation with a 1:2000 dilution of horseradishperoxidase-labeled goat anti-mouse IgG (BD Biosci-ences Canada, Mississauga, ON). Samples were detectedwith enhanced chemiluminescence Super Signal WestFemto (Pierce, Cheshire, United Kingdom). Densitometrywas performed via ImageJ (National Institutes of Health,Bethesda, MD) for quantification. In experiments withmultiple treatments, densitometry values for SP-D werenormalized first to β-actin and then to untreated controlto obtain fold change protein expression of SP-D.Quantitative real time PCRTotal RNA was isolated using Trizol reagent accordingto manufacturer’s protocol, and 0.6 μg of total RNA wasconverted to complementary DNA (cDNA) through re-verse transcription (RT) using qScript cDNA SuperMixfrom Quanta Biosciences (Gaithersburg, MD). RT reac-tion was incubated at 25°C for 5 minutes, 42°C for 30minutes, and 85°C for 5 minutes. Quantitative real-time PCR (qPCR) was performed on ABI ViiA 7 (LifeTechnologies, Burlington, ON) with 20 μL reactionscontaining 1 μL of cDNA, 5 μL of TaqMan master mix(Life Technologies, Burlington, ON), and 0.5 μl of SP-D TaqMan assay probe (Hs00358340_m1 SFTPD) orhypoxanthine phosphoribosyltransferase (HPRT) Taq-Man assay probe (Hs99999909_m1HPRT1) in 384-wellplates. qPCR conditions were one cycle of 50°C for 2minutes, one enzyme activation cycle of 95°C for 10minutes, and 40 cycles of amplification. Each amplifi-cation cycle consists of denaturation at 95°C for 15seconds, followed by simultaneous annealing and ex-tension at 60°C for 1 minute. Results were analyzed withViiA 7 software using the comparative quantificationmethod where CT values of SP-D were normalized toCT values of HPRT followed by normalization tountreated control samples to obtain ΔΔCT = [(CTSP-D –CTHPRT)Treated – (CTSP-D – CTHPRT)Untreated]. Expres-sion fold change was defined as 2-ΔΔCT.Statistical analysisGraphPad Prism 5 (GraphPad Software, San Diego, CA)was used for all statistical analyses. Colour segmentationdata from human airway sections and baseline ALI sec-tions between asthmatic and non-asthmatic donors wereanalyzed using a two-tailed, unpaired t test while datafrom IL-13 treated ALI sections was analyzed using two-tailed, paired t test. Data from IL-13 treated asthmaticand non-asthmatic monolayer cultures was also analyzedusing a two-tailed, paired t-test against the respectivecontrols. Data from ALI cultures that differentiated from7 to 21 days was analyzed by one-way ANOVA followedby Bonferroni’s Multiple Comparison Test and post-testfor linear trend where appropriate. Densitometry datafrom IL-13 treated non-asthmatic monolayer cultures(with and without neutralizing antibodies) was analyzedby one-way ANOVA with Bonferroni’s Multiple Compari-son Test. Values are presented as mean ± SEM. Statisticalsignificance is defined as p-value < 0.05.ResultsSP-D expression in human airway sectionsLung tissue sections obtained from 11 asthmatic and 11non-asthmatic donors were used to characterize the ex-pression of SP-D in human airways. Immunohistochem-istry was performed to study the distribution andrelative quantity of SP-D expression. Airways of bothasthmatic and non-asthmatic donors expressed SP-Din the cytoplasm of epithelial cells (Figure 1A). Whenpositive staining was expressed as a fraction of totalepithelial area, asthmatic airways demonstrated higherSP-D expression (0.66 ± 0.026) compared to non-asthmatic airways (0.50 ± 0.043) (n = 11, p = 0.004,Figure 1B). The presence of SP-D was confirmed inhuman airways and further, its expression was in-creased in airways of asthmatics. The non-uniform in-tensity indicates that SP-D may be expressed more incertain subtypes of airway epithelial cells, specificallyundifferentiated basal cells.SP-D expression in human air-liquid interface culturesWell-differentiated twenty-one day ALI cultures of pri-mary asthmatic AEC demonstrated higher levels of SP-Dexpression compared to that of non-asthmatics as de-tected by immunohistochemistry (fraction of total epithe-lial area: 0.50 ± 0.08 vs. 0.25 ± 0.07, n = 5, p = 0.04,Figure 2A, B). Western blotting also demonstrated higherSP-D expression in ALI cultures derived from asthmaticAEC (1.02 ± 0.15, n = 3) compared to those from non-asthmatic AEC (0.27 ± 0.11, n = 7, p = 0.005, Figure 2C).These findings are in concordance with those of airwaytissue sections and suggest that ALI epithelial cultures area viable model for studying the expression of SP-D.. Se oXu et al. Respiratory Research  (2015) 16:26 Page 4 of 12Figure 1 Surfactant protein D expression in human airway sectionsand asthmatic donors via immunohistochemistry, where pink was indicativusing colour segmentation in ImagePro Plus. Expression of SP-D in the airwaynon-asthmatic donors (n = 11, t-test p = 0.004) [B].P-D expression was examined in sections of airways from non-asthmaticf positivity [A]. Fraction of total epithelial area positive was quantifiedepithelia of asthmatic donors was significantly higher compared toonA].aatein.05Xu et al. Respiratory Research  (2015) 16:26 Page 5 of 12Figure 2 Surfactant protein D expression ALI cultures. SP-D expressidonors via immunohistochemistry, where pink was indicative of positivity [segmentation in ImagePro Plus. Expression of SP-D in the ALIs of asthmAEC (n = 5, t-test p = 0.04) [B]. SP-D protein expression in total cell lyswas quantified by densitometry using ImageJ and normalized to β-actof SP-D compared to that of non-asthmatic AEC (n = 3-7, t-test p = 0.0SP-D expression and differentiation of ALI culturesALI cultures grown from non-asthmatic AEC were ex-posed to air and allowed to differentiate for 7, 14, and 21days. The fraction of epithelium stained positive for SP-Dsignificantly decreased from 0.62 ± 0.04 at 7 days to 0.47 ±0.09 at 14 days and to 0.23 ± 0.03 at 21 days, as detectedby immunohistochemistry (n = 5, p = 0.004, Figure 3A, B).Similarly, the fold change of SP-D protein expressionrelative to day 0 decreased from 1.68 ± 0.33 at 7 daysto 1.06 ± 0.21 at 14 days and to 0.74 ± 0.18 at 21 daysas detected by Western blotting (n = 3, Figure 3C).While the decrease in Western blotting was not signifi-cant, a linear trend with slope -0.5 was detected over thetwo-week course from day 7 to day 21 (p = 0.04). Addingto the finding of non-uniform staining in the airways, thissuggests that undifferentiated cells have higher expressionof SP-D.MUC5AC, a mucin expressed in airway goblet cells, isused here as a marker for goblet cell differentiation. Im-munohistochemistry of ALI cultures grown from non-asthmatic AEC demonstrated an increased expression ofMUC5AC from day 7 through 21 (fraction of total epithe-lial area: 0.08 ± 0.02 7 days; 0.20 ± 0.06 14 days; 0.29 ±0.05 21 days; n = 4, p = 0.007, Figure 3A and D). This indi-cated that differentiation into mucin-secreting goblet cellsoccurred in the ALI model throughout 21 days of culture.CK-5, a marker for basal epithelial cells [3], is used todemonstrate the extent of differentiation in ALI cultureswas examined in ALI sections grown from non-asthmatic and asthmaticFraction of total epithelial area positive was quantified using colourtic AEC was significantly higher compared to that of non-asthmatics of ALI cultures was analyzed by Western blot. Expression of SP-DALIs of asthmatic AEC demonstrated significantly higher expression) [C].grown from non-asthmatic AEC. Immunohistochemistryon these cultures demonstrated significantly reduced ex-pression of CK-5 (fraction of total epithelial area: 0.59 ±0.04 7 days; 0.36 ± 0.07 14 days; 0.27 ± 0.05 21 days; n = 4,p = 0.002, Figure 3A and E). The decrease in CK-5 expres-sion coordinate with decreased SP-D expression duringdifferentiation suggests that the same CK-5 positive basalcells could potentially also be the main contributor of SP-D production in the airway epithelium.IL-13 effect on SP-D expressionTwo-week IL-13 treatment of ALI cultures grown fromnon-asthmatic AEC induced characteristic changes includ-ing altered epithelial thickness and cell type distribution(Figure 4A). With respect to SP-D expression, IL-13 in-duced a significant reduction in the fraction of epitheliumthat stained positive for SP-D (0.21 ± 0.06) compared tountreated (0.62 ± 0.04) as shown by immunohistochemistry(n = 4, p = 0.0005, Figure 4B). In a time-course experiment,IL-13 treatment of ALI cultures resulted in a fold reductionin SP-D protein expression relative to untreated cultures asdemonstrated by Western blotting (0.65 ± 0.15 at 24 hours,0.47 ± 0.10 at 7 days, 0.32 ± 0.06 at 14 days; n = 4-6,Figure 4C). A significant reduction was also found in SP-Dprotein expression at day 14 compared to day 7 (p < 0.05).Due to concurrent changes in cell growth, cell type, andepithelial structure, it is unclear whether this reduction is aXu et al. Respiratory Research  (2015) 16:26 Page 6 of 12direct effect of IL-13 or an indirect consequence of IL-13-induced changes in differentiation.IL-13 regulation of SP-D in monolayer cultureTo study SP-D regulation in response to IL-13, monolayercultures grown from non-asthmatic AEC were treatedwith IL-13 (10 ng/mL) for 24 hours. When normalized toFigure 3 Surfactant protein D, Mucin 5AC and cytokeratin-5 expressioand CK-5 expression were examined in ALI sections grown for 7, 14, and 21 dwas indicative of positivity [A]. Fraction of total epithelial area positive was quin the airway epithelia decreased significantly as the cultures differentiated froexpression in 7, 14, and 21-day ALI cultures were detected by Western blottinwas observed (n = 3, p = 0.04) [C]. Expression of MUC5AC in the airway epdemonstrated by immunohistochemistry (n = 4, one-way ANOVA p = 0.007) [the cultures grew from day 7 to 21 (n = 4, one-way ANOVA p = 0.002) [E].untreated control, treatment with IL-13, either alone orafter pre-incubation with IgG isotype control for 24hours induced a reduction in SP-D mRNA (0.62 ± 0.05and 0.60 ± 0.03 respectively, n = 3, Figure 5A). Cells pre-incubated with IL-13Rα1 neutralizing antibody prior toIL-13 treatment demonstrated a mitigation of the IL-13 ef-fect on SP-D mRNA expression (0.99 ± 0.11). In contrast,n in ALI cultures at different differentiation stages. SP-D, MUC5AC,ays from non-asthmatic donors via immunohistochemistry, where pinkantified using colour segmentation in ImagePro Plus. Expression of SP-Dm day (d) 7 to 21 (n = 5, one-way ANOVA p = 0.004) [B]. SP-D proteing, quantified by densitometry relative to cultures at 0 days. A linear trendithelia increased significantly as the cultures grew from day 7 to 21 asD]. Expression of CK-5 in the airway epithelia decreased significantly asLItchquL-1ternf SPXu et al. Respiratory Research  (2015) 16:26 Page 7 of 12Figure 4 Surfactant protein D (SP-D) expression in non-asthmatic AIL-13-treated (10 ng/ml, 14-day duration) ALI sections grown from mawas indicative of positivity [A]. Fraction of total epithelial area positive wasin the airway epithelia decreased significantly in the cultures treated with Itreated with IL-13 for 24 hours, 7 days, and 14 days were detected by Wescontrol to obtain fold changes. All three durations resulted in reductions ocells pre-incubated with IL-13Rα2 neutralizing antibodydemonstrated no significant difference in SP-D mRNA ex-pression compared to cells pre-incubated with IgG isotypecontrol (0.54 ± 0.07).Total protein lysates collected from parallel experi-ments were analysed by Western blotting. Relative tountreated control, treatment with IL-13, either alone orafter pre-incubation with IgG isotype control for 24hours induced a reduction in SP-D protein (0.51 ± 0.08and 0.65 ± 0.07 respectively, n = 6, Figure 5B). Pre-incubation with IL-13Rα1 neutralizing antibody prior toIL-13 demonstrated a similar mitigation of the IL-13 effecton SP-D protein expression (0.85 ± 0.13). Pre-incubationwith IL-13Rα2 neutralizing antibody produced no signifi-cant difference in SP-D protein expression compared toeither IL-13 treatment alone or IL-13 treatment with IgGpre-incubation (0.60 ± 0.06).Activation of the transcription factor STAT6 was ana-lyzed in matched protein lysates coordinate with SP-Dexpression to examine downstream activity of IL-13Rα1.The ratio of phosphorylated STAT6 to total STAT6 as amarker of activation was low in untreated control (0.04 ±0.01), markedly increased in IL-13 treated cells (1.57 ± 0.27),in IgG pre-incubated cells (1.59 ± 0.28), and in IL-13Rα2neutralizing antibody pre-incubated cells (1.25 ± 0.17)(n = 5, p = 0.005, Figure 5C). In contrast, pre-incubationwith IL-13Rα1 neutralizing antibody resulted in a markedreduction in the ratio (0.52 ± 0.08).cultures treated with IL-13. SP-D expression was examined ined non-asthmatic donors via immunohistochemistry, where pinkantified using colour segmentation in ImagePro Plus. Expression of SP-D3 (n = 4, t-test p = 0.0005) [B]. SP-D protein expression in ALI culturesblotting, quantified by densitometry, and normalized to untreated-D expression (n = 6, one-way ANOVA p = 0.02) [C].STAT6 activation, studied in ALI cultures exposed to IL-13 for 24 hours, one week, and two weeks, was quantifiedby the phosphorylated STAT6 to total STAT6 ratio. Thisratio was low in untreated ALI control (0.07 ± 0.03) andincreased in IL-13 treatment groups (n = 3, Figure 5D).The highest ratio was observed at 24 hours (1.76 ± 0.27)which progressively decreased at 7 days (1.27 ± 0.32) andat 14 days (1.15 ± 0.21). This suggests that the cells contin-ued to respond to IL-13 through IL-13Rα1 and the STAT6pathway during the two weeks of exposure.The effect of IL-13 on SP-D expression in asthmaticAEC was studied in monolayer cultures and comparedwith non-asthmatic AEC. Total protein lysates of untreatedand IL-13-treated (10 ng/ml) asthmatic and non-asthmaticAEC were collected at 24 hours post-treatment and subse-quently analysed by Western blotting. No significant differ-ence in SP-D expression was detected in asthmatic AEC ofthe IL-13 treated group relative to untreated control cells(n = 4), whereas as significant reduction in SP-D expres-sion was demonstrated in IL-13 treated (0.31 ± 0.06)non-asthmatic AEC relative to untreated control cells(0.62 ± 0.11) (n = 5, p = 0.03, Figure 5E).DiscussionIn this study, we compared the expression of SP-D inairways of asthmatics to non-asthmatics and began toexplore what may regulate this expression. The role ofSP-D as a pattern recognition molecule in the innateFigure 5 (See legend on next page.)Xu et al. Respiratory Research  (2015) 16:26 Page 8 of 12IL-ertopsd gw-trA[Bcu.00ificD pnoXu et al. Respiratory Research  (2015) 16:26 Page 9 of 12immune system makes it relevant to asthma as multiplestudies have demonstrated that asthma can occur subse-quent to and be exacerbated by respiratory infections[7-9,22,23]. More recent studies have included the innateimmune system, acknowledging that it may play import-ant roles in the pathogenesis of asthma, affecting hostsusceptibility to infections and immune response topathogens.We demonstrated an elevated expression of SP-D in air-ways of asthmatics compared to that of non-asthmatics inlung tissue sections and ALI cultures, a model for epithelialdifferentiation. The increased expression of SP-D couldarise from several possibilities: from structural differences,such as proliferation of a cellular subtype in the epithelium,to functional differences, such as over-production or lackof inhibition affecting gene expression of SP-D. Previously,Cheng et al. have shown elevated concentrations of SP-Din the bronchial alveolar lavage (BAL) of asthmatic patientscompared to non-asthmatic controls; while a higher aver-age concentration was found, the difference was not sig-nificant [24]. Koopmans et al. observed increased serumSP-D in allergic patients both at baseline and after allergen(See figure on previous page.)Figure 5 SP-D expression in monolayer epithelial cultures treated bytreated with IL-13 (10 ng/mL) alone and in cultures pre-treated with eith(20 μg/mL), or IL-13Rα2 neutralizing antibody (2 μg/mL) one hour priornormalizing SP-D mRNA to HPRT mRNA and normalizing treatment grouthe IgG pre-treated group and IL-13Rα1 neutralizing antibody pre-treatenon-asthmatic monolayer cultures under the same treatment conditionsbetween the IL-13 treated group and IL-13Rα1 neutralizing antibody preprotein expression was normalized to untreated control (n = 6, one-waynon-asthmatic monolayer cultures from the same experiment as panelincreased the phosphorylation of STAT6 compared to control while pre-inphosphorylation of STAT6 compared to IgG (n = 5, one-way ANOVA p = 0treated with IL-13 (10 ng/mL) over the course of two weeks show signp = 0.001) [D]. IL-13 treatment resulted in a significant decrease in SP-p = 0.03) whereas no significant change was detected in asthmatic mochallenge [25]. Mouse models of chronic inflammatoryconditions using P. Carinii-induced lung injury have found7.4 fold and 71 fold increases of SP-D in BAL and serumrespectively [26]. Murine models of acute allergic lung in-flammation have demonstrated disparity in SP-D levels inBAL of C57BL/6 mice compared to BALB/c mice, suggest-ing a genetic component to the baseline production ofSP-D in the lung [12].SP-D gene deficient mice were found to exhibit hyper-eosinophilia and increased levels of IL-5 and IL-13 uponAspergillus fumigatus allergen challenge, a response thatwas reversible by treating the mice with SP-D [27]. AsSP-D participates in host defense and modulates inflam-mation, an increase in SP-D levels could potentially bebeneficial if it plays a protective or even compensatoryrole in asthma and other chronic inflammatory conditions.While higher levels of SP-D in the airways of asthmaticsseem counterintuitive in the context of increased suscepti-bility to viral infection in asthma, this suggest that under-lying differences in the function of SP-D may exist inhumans between asthmatics and non-asthmatics. In astudy by Wang et al. purified SP-A and SP-D suppressedDermatophagonides pteronyssinus-stimulated lymphocyteproliferation in naïve mice, but this suppressive effect wasreduced in sensitized mice [28]. While the study was con-ducted in a murine model, this suggests that there may bedysregulated processes in the immunomodulatory roles ofSP-D in sensitized patients such as those with asthma.With regards to SP-D function, Kishore et al. have pro-posed that SP-A and SP-D in naïve lungs can help miti-gate potential damage from a low level of exogenousinsults; however, when overwhelmed by high levels of in-sults, these collectins assume a pro-inflammatory role tocomplement innate and adaptive immunity [29].Immunohistochemistry of ALI cultures demonstrateddecreasing levels of SP-D expression as they differentiatedover three weeks. Visual inspection led to the observationof SP-D in columnar cells and basal cells. Using MUC5ACas a marker for the presence of mucin-producing goblet13. SP-D mRNA was examined in non-asthmatic monolayer culturesgoat IgG isotype control (20 μg/mL), IL-13Rα1 neutralizing antibodyIL-13 exposure. To obtain fold changes, ΔΔCT values were obtained byto untreated control. A significant difference was observed betweenroup (n = 3, one-way ANOVA p = 0.01) [A]. SP-D protein levels inere detected via Western blotting. A significant difference was foundeated group. Quantification was performed by densitometry and SP-DNOVA p = 0.04) [B]. Phosphorylated-STAT6 (pSTAT6) expression in] was examined via Western blotting. IL-13 treatment significantlybation with IL-13Rα1 neutralizing antibody significantly reduced the5) [C]. pSTAT6 to STAT6 protein ratio in non-asthmatic ALI culturesant increases relative to untreated control (n = 3, one-way ANOVArotein expression in non-asthmatic monolayer culture (n = 5, t-testlayer culture (n = 4) [E].cells, little to no SP-D staining was observed in goblet cells.Previously, Madsen et al. have localized SP-D in humanlungs to alveolar type II cells, Clara cells, and on or withinalveolar macrophages [30]. Kim et al. found surfactant pro-teins expressed in the ciliated cells of the nasal epitheliumbut not the goblet cells of human nasal mucosa [31]. Herewe present novel data on characterization of SP-D withinthe airway epithelia of conducting bronchus. Our observa-tions are consistent with the previous studies with regardsto the lack of SP-D in goblet cells. Differences in SP-D ex-pression within specific cell types could arise from the dif-ferent regions of the respiratory tract studied.In human airway sections, the more intense SP-Dstaining in basal cells relative to the remainder of theepithelium suggest that basal cells either produced moreor retained more SP-D within their cytoplasm. Thisconcurs with the observation of decreasing SP-D levelsXu et al. Respiratory Research  (2015) 16:26 Page 10 of 12in the differentiating ALI cultures where the pluripotentbasal cell, visualized with CK-5 as a marker, becomes sig-nificantly less abundant over 21 days of culture. In light ofthese observations, the increased SP-D expression in air-ways of asthmatics could be due to a greater proportion ofundifferentiated cells. The increased number of gobletcells in asthma, which theoretically results in lower SP-Dexpression, may play a relatively minor role compared tothe undifferentiated cells.Treatment with IL-13 of non-asthmatic human airwayepithelial cells, grown in both monolayer and ALI culture,resulted in decreased SP-D expression. Previous studiesfrom our laboratory have demonstrated that IL-13 treat-ment of primary airway epithelial cells reduced spontan-eous apoptosis in culture [32], thereby apoptosis-inducedreduction of SP-D is unlikely. Studies to date have notcome to consensus on how IL-13 modulates SP-D expres-sion in the lungs. Haczku et al. found SP-D mRNA andprotein levels in the lungs of mice increased in responseto IL-4 and IL-13 treatment [33]. Ito and Mason foundthat IL-13 reduced mRNA and protein levels of SP-D incultured human alveolar type II cells [34]. We have dem-onstrated that IL-4 and IL-6 (Additional file 1; Additionalfile 2: Figure S1), in addition to IL-13, all decrease SP-Dexpression by non-asthmatic human AEC. This discrep-ancy in SP-D response to IL-4 and IL-13 may be attributedto differences in species; for instance, a non-specificSTAT-binding site is present in the SP-D promoter of ratsand mice but not that of humans [35]. In contrast to theeffect of IL-13 on non-asthmatic AEC, no significant dif-ference was detected in SP-D expression between IL-13treated and untreated asthmatic AEC. This suggests thatasthmatic AEC have an altered response to IL-13 withregards to SP-D expression. This, in turn, may contributeto the increased SP-D observed in airway epithelium ofasthmatics despite the elevated IL-13 levels in asthmaticairways as established by several groups [18,36-38].Potential pathways for IL-13 regulation of SP-D werestudied using monolayer cultures of human AEC. It hasbeen shown that monolayer cultures have the same IL-13 receptors as ALI cultures [39]. Neutralizing anti-bodies for IL-13Rα1 and IL-13Rα2 were used to blockthe two receptors of IL-13. Data from both mRNA andprotein demonstrate that IL-13 decreased the expres-sion of SP-D and suggest that such a reduction was me-diated through the IL-13Rα1 pathway as opposed toIL-13Rα2. We also demonstrated an increase in phos-phorylated STAT6, a transcription factor downstreamof IL-13Rα1, which is coordinate with the reduction inSP-D expression. Protein analysis of STAT6 phosphoryl-ation affirmed that IL-13 treatment activated the STAT6transcription factor and that this activation was to a largedegree blocked by the pre-incubation with IL-13Rα1 neu-tralizing antibody.Studies in monolayer cultures grown from non-asthmaticdonors with exposure to IL-4 also demonstrated SP-D re-duction and STAT6 phosphorylation. This supports the roleof the heterodimeric receptor, consisting of IL4Rα andIL-13Rα1, in modulating SP-D levels (Additional file 1;Additional file 2: Figure S1). Treatment of monolayercultures with IL-6 induced a decrease in SP-D as well(Additional file 2: Figure S1). While IL-6 signalsthrough STAT3 [40], it is still possible that the path-ways through which SP-D decreases in response toIL-13, IL-4, and IL-6 share common components.Study of STAT6 activation on IL-13 treated ALI cul-tures grown from non-asthmatics demonstrated a drasticinitial increase in the expression of phosphorylatedSTAT6 24 hours post-treatment followed by a gradualdecrease over the span of two weeks. The sustained de-crease in SP-D level over the same period suggests thatthe IL-13 effect on SP-D expression in non-asthmaticAEC is chronic and sustained.Airway epithelium differentiation and IL-13 signallingare both implicated in the structural and functionalchanges associated with asthma. Hackett et al. havedemonstrated that asthmatic patients have less differen-tiated airway epithelium [3]. This may be a consequenceof persistent injury to the epithelium and result in an al-tered proportion of cell types as well as their protein ex-pression profiles. IL-13 has been shown to mediatefeatures of asthma independent of other cytokines inanimal models [13,16,17]. While IL-13 is closely associ-ated with allergic asthma and adaptive immunity, it isalso valuable to know how it may affect molecules andpathways of innate immunity in asthma.We have demonstrated that airway epithelia of asth-matics express more SP-D whereas non-asthmatic differ-entiated ALI cultures have decreases in SP-D expressionin parallel with decreasing basal cells (via CK-5 marker)and increasing goblet cells (via MUC5AC marker). IL-13treatment, which is known to induce goblet cell hyperpla-sia, reduced SP-D expression in non-asthmatic airway epi-thelial cultures via IL-13Rα1 but had no effect on SP-Dexpression in asthmatic airway epithelial cultures. Thissuggests that in airways of asthmatics, increased SP-D pro-duction is predominantly contributed by a less differenti-ated phenotype, while the increased number of gobletcells is negligible in affecting the expression of SP-D.Additionally, IL-13 regulation of SP-D expression in asth-matic airways was different from non-asthmatic airways,thus contributing to the observed disparity in the SP-D ex-pression. Asthmatic patients being more susceptible to viralinfections despite their increased SP-D expression leadsone to question whether the SP-D molecules produced inthese patients are less effective in their host defence roles.Limitations of this study include the lack of functionaldata on SP-D. While SP-D expression levels were studiedmune response in the airways cannot be compared be-model exhibiting the pseudo-stratified airway epithelium.matics do not exhibit full functionality, hence cannot me-SP-A, and SP-D, in allergen-induced airway inflammation. Immunobiology.Xu et al. Respiratory Research  (2015) 16:26 Page 11 of 12diate inflammation or clear pathogens, and consequentlyproduction continues as the inflammation or infectionpersists. Another potential factor could be cell type anddifferentiation differences between the airway epitheliumof asthmatics and non-asthmatics. Lastly, altered regula-tory pathways through which SP-D expression is affected,such as IL-13, in the asthmatic airway epithelium may leadto airway remodelling differences.Additional filesAdditional file 1: This includes the data obtained on monolayercultures grown from non-asthmatic donors treated with IL-4, IL-6 orIL-13.Additional file 2: Figure S1. Graphical representation of SP-D levels inmonolayer cultures grown from non-asthmatic donors treated with eitherIL-4 and IL-6 or IL-13.Competing interestsIL-13 signalling was studied in non-asthmatic AEC atthe receptor level and any links, direct or indirect, be-tween this and SP-D gene expression remains to be found.Future studies on SP-D can add depth to the molecularmechanisms in both asthmatic and non-asthmatic airways.These can provide insight into the role SP-D plays inasthma and sources from which dysregulation or dysfunc-tion may arise with regards to innate immunity.ConclusionOverall, this study demonstrated a difference in SP-D ex-pression in airways of asthmatics relative to that of non-asthmatics. This can have implications on the increasedsusceptibility to infections and altered inflammatory re-sponse in asthmatic patients. There could be several fac-tors contributing to the increased SP-D levels observed inthe airway epithelium of asthmatics and further studiesare required to elucidate the underlying mechanisms. Up-regulation of SP-D expression in asthma could be in re-sponse to the chronic inflammatory milieu of the airways.It may also be that SP-D molecules produced by asth-tween people with and without asthma. Another limitationis related to the resolution of immunohistochemistry andthe inability to clearly separate differentiated and undiffer-entiated cells of the airway epithelium. Visual inspectioncould only offer qualitative observations on the localizationof SP-D. It is difficult to compare the contribution ofSP-D expression in each cell type of the airway or in ahere, its function in binding pathogens and modulating im-We would like to declare that there are no competing interests of personalor financial type with any person or organization regarding the work donefor this manuscript.2007;212(4–5):417–25.13. Wills-Karp M. Interleukin-13 in asthma pathogenesis. Immunol Rev.2004;202:175–90.14. Hancock A, Armstrong L, Gama R, Millar A. Production of interleukin 13 byalveolar macrophages from normal and fibrotic lung. Am J Respir Cell MolBiol. 1998;18(1):60–5.15. Burd PR, Thompson WC, Max EE, Mills FC. Activated mast cells produceinterleukin 13. J Exp Med. 1995;181(4):1373–80.Authors’ contributionsDRD conceived the idea of this project. JX, GKS and DRD participated inexperimental design, coordination and execution of the experiments andpreparation of the manuscript. JX and GKS participated in the in vitroexperimentation and sampling. JX contributed in acquisition of data (IHC),Western blotting, and PCR studies, analysis and interpretation of data. JX,GKS, DRD gave final approval for the submission.AcknowledgementsThis work was supported by the operating grants from Canadian Institutesof Health Research (CIHR, MOP-97755) (DRD) and AllerGen-National Centresof Excellence (AllerGen-NCE #09B9) (DRD). JX is a recipient of the AllerGenCAIDATI Award (2011) and UBC Faculty of Medicine Summer StudentResearch Program Award provided by CIHR Health Professional StudentResearch Award and Providence Health Care Research Institute (2011-2014).DRD received personal support awards from the Parker B Francis Fellowship inPulmonary Research, Michael Smith Foundation for Health Research (SeniorScholar) and the Canadian Institute of Health Research. The authors gratefullythank S. Jasemine Yang for intellectual input and critical reading of themanuscript.Received: 27 August 2014 Accepted: 20 January 2015References1. 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