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MUC5AC Upregulation by Bile Salts: an in Vitro Study

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MUC5AC Upregulation by Bile Salts: an in Vitro Study Central Annals of Otolaryngology and Rhinology Short Communication *Corresponding author M S Ali, Department of Otolaryngology, Institute for Cell and Molecular Biosciences, Newcastle University, UK, MUC5AC Upregulation by Bile Salts: Email: Submitted: 28 November 2016 An In vitro Study Accepted: 16 December 2016 Published: 19 December 2016 1,2 1 1 Mahmoud El-Sayed Ali *, Shruti Parikh , and Jeffrey P Pearson ISSN: 2379-948X 1 Institute for Cell and Molecular Biosciences, Newcastle University, UK Copyright 2 Department of Otolaryngology, Mansoura University Hospital, Egypt © 2016 El-Sayed Ali et al. OPEN ACCESS Abstract Introduction: Bile acids, as a constituent of refluxed and aspirated duodenal juice, could Keywords alter upper and lower airway major mucins such as MUC5AC in the airway mucosa. Human • Bile salts goblet cell line derived from the human colon carcinoma cells HT29-MTX has been found to • Duodenal reflux express MUC5AC as the main mucin and could be employed to study this hypothesis. • Hypersecretion Methods: The cell line HT29-MTX was challenged by various concentrations of a physiologic combination of bile salts. We modified an ELISA technique to measure the expressed MUC5AC within the culture media. Results: The Cultured HT29-MTX cells viability was maintained when challenged with bile salts up to 20 µmol/L. MUC5AC mucin production was upregulated by bile salts in a dose dependant manner within the first 24 hours. Discussion: Amongst the other constituents of duodenal reflux that reaches the upper and lower airways, bile salts could represent an important element of airway mucin alteration by upregulating MUC5AC production. This effect could be direct due to stimulated mucin release from cell stores or indirect via the release of pro-inflammatory cytokines which stimulate mucin biosynthesis and release. Conclusion: Bile salts challenge of the HT29-MTX cell line resulted in MUC5AC upregulation and this effect could be extrapolated to the airway goblet cells. INTRODUCTION cells HT29, adapted to increasing concentrations of methotrexate® (MTX), was cultured according to a previously described protocol Gastric and duodenal reflux is a common disease and the [6]. Cell viability was measured with the CellTiter-Blue Cell refluxate entering the upper aerodigestive, then the lower Viability Assay (Promega, USA) in a 96-well plate (Maxisorp, airways could alter the mucosal secretory elements to chemical NUNC). injury of reflux. Bile salts and trypsin/chymotrypsin are the main components of duodenal reflux. Bile salt aspiration has previously Using 24 well plates, cells were seeded at ~50000/well and been documented in paediatric and adult populations [1, 2]. As challenged with a physiological mixture of bile salts (50% cholic mucus hypersecretion is a key element in airway inflammation acid, 30 % chenodeoxycholic acid, 15 % deoxycholic acid and 5 and epithelial damage [3, 4], there is a potential of aspirated bile % lithocholic acid) [7] at concentrations of 3, 6, 9, 12, 15, 18, 20 salts to alter mucin expression by the airway mucosa. Previous µmol/L. We used negative controls containing media free from studies have also shown that bile salts can up-regulate mucin bile salts and blank controls by omitting the primary antibody. At genes e.g. the MUC4 gene in oesophageal cancer [5]. 24, 48 and 72 hours, 0.2ml media was collected and assayed for MUC5ACELISA for using the a measurement developed ELISA. of MUC5AC mucin We, therefore, assumed that bile salts could also alter mucin expression in the airways. This study investigates the effect of a mixture composed of various bile salts in proportions similar to Mucin was isolated from the non mucin components in the those present in human bile on mucus production using a goblet culture media of the HT29-MTX cells by Caesium Chloride (CsCl) cell model of MUC5AC, one of the major mucins expressed in equilibrium density gradient centrifugation [8]. Centrifuged upperMETHODS and lower airways. samples were fractionated and assayednd for mucin glycoprotein Goblet cell culture (HT29-MTX) by periodic acid Schiff assay and mucin rich fractions were pooled together and subjected to a 2 CsCl centrifugation for mucin purification. Baseline mucin produced was used as a A human goblet cell line derived from human colon carcinoma standard. The ELISA was carried out using a mouse monoclonal Cite this article: El-Sayed Ali M, Parikh S, Pearson JP (2016) MUC5AC Upregulation by Bile Salts: An In vitro Study. Ann Otolaryngol Rhinol 3(12): 1154. El-Sayed Ali et al. (2016) Email: Central primary antibody at 1:200 (abcam, UK) followed by anti-mouse and as we found that this cell line remained viable to bile salt secondary (peroxidise conjugated) at 1:5000 and casein blocking challenges up to a concentration of 20µmol/L, we think that these buffer and antibody diluent. cells could be used as a model for airway goblet cells.. MUC5AC mucin concentration was measured in duplicate The MUC5AC mucin expression by the HT-29 cell line using the locally developed indirect ELISA and absorbance was response to bile salt challenge developed and reached its measured at 405 nm with a plate reader (Bio-Tek EL808). nearly the same level. Therefore, for further bile salt stimulation Statistical analysis experimentsmaximum within at these the firstconcentrations 24 hours and it would continued be importantafter that toat analyse the media at multiple time points up to 24 hours to obtain We used One-Way ANOVA (Repeated Measures ANOVA, more clear information as regard to the temporal up regulation Dunn’s Multiple Comparison post test) and non-parametric of MUC5AC production. The increase in MUC5AC production was unpaired tests (Spearman’s correlation test) with Prism close to linear up to 20 µmol/L bile salts and the viability of the (GraphPad Software Inc., La Jolla, California, USA) with a HT-29 cells was maintained at 100% in those conditions. The concentration of bile salts could therefore be increased above the significantRESULTS p-value of ≤0.05. concentrations would compromise the viability of the HT29-MTX The cultured cells remained viable through all the experiment 20 µmol/L concentration used in this study to find out if higher conditions and there was no evidence of cell death at any of the production by the viable HT29-MTX cells becomes maximal. challenge conditions. cells and to find out if and at what bile salt concentration MUC5AC MUC5AC up regulation by bile salts could be achieved by a Basal MUC5AC mucin secretion in the controls at all time direct effect of bile salts on the mucin expression at the mRNA points was 40 µg/ml. MUC5AC production was stimulated by bile levels or by the release of MUC5AC from cell stores. It could also salts (Figure) in a concentration dependant manner. Maximal MUC5AC secretion was noted at 20 µmol/L bile salts and the level cytokines, such as tumour necrosis factor alpha, Interleukin (IL)- remained the same at 24h [50 ± 0.9 µg/ml (mean ± SD)], 48h [50 6be and an IL-17indirect [12, effect 13]. throughPrevious thework release in our of lab pro-inflammatory has shown that ± 0.5 µg/ml (mean ± SD)] and 72h [50 ± 0.5 µg/ml (mean ± SD)] MUC5AC expression was upregulated by IL-8 [14] and bacterial above the baseline levels (Figure 1). lipopolysaccharide and IL-8 [15]. DISCUSSION This study suggests that bile acid aspiration among the other This study investigated the effect of bile salts in proportions similar to those in human bile on goblet cell production of constituents of duodenal reflux could be involved in the airway MUC5AC. Previous work carried out in our lab [9] has shown has previously been reported in both the paediatric and adult inflammation and epithelial damage. As bile salt aspiration that exposure of primary bronchial epithelial cells to bile salt challenges resulted in cell injury and death. Previous studies to lung injury in upper and lower airway diseases. populations, bile acid reflux should be considered as a contributor showed that HT-29 cell line express mainly MUC5AC [10, 11] CONCLUSION MUC5AC was upregulated by bile salts and the maximum bile salt challenge. production of MUC5AC was achieved within the first 24 hours of REFERENCES 1. Blondeau K, Dupont LJ, Mertens V, Verleden G, Malfroot A, Vandenplas Y, et al. Gastro-oesophageal reflux and aspiration of gastric contents in 2. adultBrodzicki patients J, withTrawinska-Bartnicka cystic fibrosis. Gut. 2008;M, Korzon 57: 1049-1055. M. Frequency, consequences and pharmacological treatment of gastroesophageal 3. refluxHamid in Q, children Springall with DR, cystic Riveros-Moreno fibrosis. Med Sci V, Monit. Chanez 2002; P, Howarth 8: 529-537. P, Redington A, Bousquet J, Godard P, Holgate S, Polak JM. Induction of 4. nitricMills PR,oxide Davies synthase RJ, Devalia in asthma. JL. Airway Lancet. epithelial 1993; 342: cells, 1510-1513. cytokines, and 5. pollutants.Mariette C, AmPerrais J Respir M, LeteurtreCrit Care Med.E, Jonckheere 1999;160: N, 38-43. Hémon B, Pigny P, et al. Transcriptional regulation of human mucin MUC4 by bile acids Figure 1 MUC5AC production by HT29-MTX goblet cells at 24, 48 in oesophageal cancer cells is promoter-dependent and involves and 72 hours of stimulation with bile salts measured with by indirect ELISA. The basal level of secretion at 0µmol/l bile acids was 40ug/ 6. activation of the phosp. Biochem J. 2004; 377: 701-708. ml and this was subtracted from the levels produced by bile acid to Methotrexate of Ht-29 Human Colon-Carcinoma Cells Is Associated stimulation. withLesuffleur Their T, Ability Barbat to A, DifferentiateDussaulx E, Zweibaum into Columnar A. Growth Absorptive Adaptation and Ann Otolaryngol Rhinol 3(12): 1154 (2016) 2/3 El-Sayed Ali et al. (2016) Email: Central 6725. 7. Mucus-SecretingDewar P, King R, JohnstonCells. Cancer D. Bile Res.
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