Loes E M Kistemaker1, Pieter S Hiemstra2, I Sophie T Bos1, Susanne Bouwman1, Maarten van den Berge3, Machteld N Hylkema4, Herman Meurs1, Huib A M Kerstjens3, Reinoud Gosens1. 1. Department of Molecular Pharmacology, University of Groningen, Groningen, The Netherlands GRIAC Research Institute, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands. 2. Department of Pulmonology, Leiden University Medical Center, Leiden, The Netherlands. 3. GRIAC Research Institute, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Department of Pulmonary Diseases, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands. 4. GRIAC Research Institute, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Department of Pathology and Medical Biology, University Medical Center Groningen, Groningen, The Netherlands.
Abstract
BACKGROUND: It has been shown that acetylcholine is both a neurotransmitter and acts as a local mediator, produced by airway cells including epithelial cells. In vivo studies have demonstrated an indirect role for acetylcholine in epithelial cell differentiation. Here, we aimed to investigate direct effects of endogenous non-neuronal acetylcholine on epithelial cell differentiation. METHODS: Human airway epithelial cells from healthy donors were cultured at an air-liquid interface (ALI). Cells were exposed to the muscarinic antagonist tiotropium (10 nM), interleukin (IL)-13 (1, 2 and 5 ng/mL), or a combination of IL-13 and tiotropium, during or after differentiation at the ALI. RESULTS: Human airway epithelial cells expressed all components of the non-neuronal cholinergic system, suggesting acetylcholine production. Tiotropium had no effects on epithelial cell differentiation after air exposure. Differentiation into goblet cells was barely induced after air exposure. Therefore, IL-13 (1 ng/mL) was used to induce goblet cell metaplasia. IL-13 induced MUC5AC-positive cells (5-fold) and goblet cells (14-fold), as assessed by histochemistry, and MUC5AC gene expression (105-fold). These effects were partly prevented by tiotropium (47-92%). Goblet cell metaplasia was induced by IL-13 in a dose-dependent manner, which was inhibited by tiotropium. In addition, tiotropium reversed goblet cell metaplasia induced by 2 weeks of IL-13 exposure. IL-13 decreased forkhead box protein A2 (FoxA2) expression (1.6-fold) and increased FoxA3 (3.6-fold) and SAM-pointed domain-containing ETS transcription factor (SPDEF) (5.2-fold) expression. Tiotropium prevented the effects on FoxA2 and FoxA3, but not on SPDEF. CONCLUSIONS: We demonstrate that tiotropium has no effects on epithelial cell differentiation after air exposure, but inhibits and reverses IL-13-induced goblet cell metaplasia, possibly via FoxA2 and FoxA3. This indicates that non-neuronal acetylcholine contributes to goblet cell differentiation by a direct effect on epithelial cells. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.
BACKGROUND: It has been shown that acetylcholine is both a neurotransmitter and acts as a local mediator, produced by airway cells including epithelial cells. In vivo studies have demonstrated an indirect role for acetylcholine in epithelial cell differentiation. Here, we aimed to investigate direct effects of endogenous non-neuronal acetylcholine on epithelial cell differentiation. METHODS:Human airway epithelial cells from healthy donors were cultured at an air-liquid interface (ALI). Cells were exposed to the muscarinic antagonist tiotropium (10 nM), interleukin (IL)-13 (1, 2 and 5 ng/mL), or a combination of IL-13 and tiotropium, during or after differentiation at the ALI. RESULTS:Human airway epithelial cells expressed all components of the non-neuronal cholinergic system, suggesting acetylcholine production. Tiotropium had no effects on epithelial cell differentiation after air exposure. Differentiation into goblet cells was barely induced after air exposure. Therefore, IL-13 (1 ng/mL) was used to induce goblet cell metaplasia. IL-13 induced MUC5AC-positive cells (5-fold) and goblet cells (14-fold), as assessed by histochemistry, and MUC5AC gene expression (105-fold). These effects were partly prevented by tiotropium (47-92%). Goblet cell metaplasia was induced by IL-13 in a dose-dependent manner, which was inhibited by tiotropium. In addition, tiotropium reversed goblet cell metaplasia induced by 2 weeks of IL-13 exposure. IL-13 decreased forkhead box protein A2 (FoxA2) expression (1.6-fold) and increased FoxA3 (3.6-fold) and SAM-pointed domain-containing ETS transcription factor (SPDEF) (5.2-fold) expression. Tiotropium prevented the effects on FoxA2 and FoxA3, but not on SPDEF. CONCLUSIONS: We demonstrate that tiotropium has no effects on epithelial cell differentiation after air exposure, but inhibits and reverses IL-13-induced goblet cell metaplasia, possibly via FoxA2 and FoxA3. This indicates that non-neuronal acetylcholine contributes to goblet cell differentiation by a direct effect on epithelial cells. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.
Authors: Siti Farah Rahmawati; Rémon Vos; I Sophie T Bos; Huib A M Kerstjens; Loes E M Kistemaker; Reinoud Gosens Journal: Sci Rep Date: 2022-06-30 Impact factor: 4.996
Authors: Tinne C J Mertens; Anne M van der Does; Loes E Kistemaker; Dennis K Ninaber; Christian Taube; Pieter S Hiemstra Journal: Physiol Rep Date: 2017-07-11