Tiotropium inhibits mucin production stimulated by neutrophil elastase but not by IL-13

Tiotropium, a muscarinic antagonist, is approved for the treatment of chronic obstructive pulmonary disease and poorly controlled asthma. Because mucus hypersecretion is characteristic of both of these diseases, and muscarinic agonists stimulate mucus secretion, we hypothesized that tiotropium would attenuate airway MUC5AC expression. We grew normal human bronchial epithelial (NHBE) cells to a goblet cell phenotype with 1 or 5 ng/mL of IL-13 and exposed these cells to 10 nM tiotropium or excipient for the full 14 days. Normally differentiated NHBE cells (without IL-13) were exposed to neutrophil elastase (NE) 1×10-7 or 5×10-7 M for 1 hour. MUC5AC was measured by quantitative PCR and ELISA. Acetylcholine production by the epithelium was evaluated by quantitative PCR and by choline/acetylcholine quantification. Tiotropium had no effect on IL-13-stimulated MUC5AC, but attenuated MUC5AC stimulated by NE (p=0.007 at 5×10-7 M). IL-13 increased CarAT mRNA (p<0.001 at 5 ng/mL) and acetylcholine concentration in the medium (p=0.018 at 5 ng/mL), while NE had no effect. Tiotropium had no direct effect on IL-13 or NE-induced CarAT or acetylcholine concentration. Tiotropium decreased MUC5AC stimulated by NE, but had no effect on MUC5AC stimulated by IL-13. These results may be due to IL-13, but not NE, increasing cetylcholine production. INTRODUCTION Tiotropium is a long-acting muscarinic antagonist bronchodilator which has been primarily used to treat chronic obstructive pulmonary disease (COPD), and has been approved for treating poorly controlled asthma [1]. Poorly controlled asthma is a heterogeneous disease with endotypes usually classified on the basis of biomarkers including eNO, periostin, and airway cell predominance (ie eosinophilic, neutrophilic, mixed cellularity) [2]. Severe asthma is often associated with mucous cell hyperplasia and mucus hypersecretion. Acting by different pathways, both interleukin (IL)-13, a TH2 cytokine, and neutrophil elastase (NE), which is increased in COPD and neutrophil dominant asthma, can cause mucus secretion [3, 4].It is known that acetylcholine is primarily released from parasympathetic nerve endings and induces airway smooth muscle contraction. More recent studies have demonstrated that acetylcholine is also released from non-neuronal cells (e.g. epithelial cells or inflammatory cells) and can also influence airway inflammation and remodeling through muscarinic receptors expressed on these non-neuronal cells [5, 6]. However, there are few studies focusing on the role of non-neuronal acetylcholine effects in the airway. To assess the impact of non-neuronal acetylcholine on mucus secretion from airway epithelial cells, we used a differentiated airway epithelial cell culture system that lacks nerve fibers.Tiotropium inhibits acetylcholine from binding to the muscarinic 3 receptor (M3R) by competitively blocking the binding site [7]. While there is clinical evidence that tiotropium reduces sputum production in patients with COPD [8, 9], its specific effects on IL-13 and NE induced mucus secretion have not been evaluated. We hypothesized that tiotropium would decrease mucin (MUC5AC) expression, and potentially prevent IL-13 stimulated goblet cell metaplasia in cultured human airway cells. The following reagents were purchased: recombinant human (rh) IL-13 (R&D Systems, Minneapolis, MN, USA); human NE (Innovative Research, MI, USA); DMEM, Ham’s F12 medium (Gibco, Grand Island, NY, USA); human bronchial epithelial (HBE) cell growth medium, SingleQuotR® kit and Hanks’ balanced salt solution (HBSS) (Lonza Walkersville Inc., Walkersville, MD, USA). Tiotropium was supplied by Boehringer Ingelheim (Rhein, Germany).The cultivation of HBE cells (Lonza Walkersville Inc.) and differentiation atair-liquid interface (ALI) have been previously reported (see the online supplement) [10, 11]. Cell viability was evaluated using WST-8 assay (Cell Counting Kit-8, Dojindo, Kumamoto, Japan).HBE cells were grown for 14 days at ALI with 0, 1, 5 ng/mL of IL-13 or IL-13 with 10 nM tiotropium by exposure from the basolateral side [12]. The medium was changed every 48 hours. At day 14, supernatants, medium and cell lysates were collected for ELISA, choline/acetylcholine quantification assay, RNA for RT-PCR and Western blotting (Figure 1). In addition, to compare with a short exposure to NE, the ciliated cells, which were grown for 14 days without IL-13, were exposed to IL-13 for 1 hour.The cells were grown for 14 days at ALI with 0, or 10 nM tiotropium in the basolateral medium; changed every 48 hours. Neutrophils exist both in airway and in submucosa in neutrophil predominant asthma [13]. Therefore, HBE cells were exposed from the apical and basal side to 1 X 10-7 or 5 X 10-7 M of NE for 1 hour at day 14 as previously reported [4, 14]. Cell lysates were collected for RNA for RT-PCR and Western blotting (Figure 1).Total RNA was extracted from the cells after IL-13 or NE exposure and prepared for real-time PCR as described in the online supplement. In addition to MUC5AC m RNA, we measured expressions of carnitine acetyltransferase (CarAT) an enzyme that promotes acetylcholine production and acetylcholine esterase (AchE) that catalyzes the breakdown of acetylcholine, and the muscarinic receptors expressed in the airway (M1, M2, and M3) [15]. The following primers were used [11, 16].MUC5AC forward: 5’-TACTCCACAGACTGCACCAACTG-3’ MUC5AC reverse: 5’-CGTGTATTGCTTCCCGTCAA-3’ CarAT forward: 5’-AAGAAGCTGCGGTTCAACAT-3’CarAT reverse: 5’-GGGCTTAGCTTCTCCGACTT-3’ AchE forward: 5’-CCTCCTTGGACGTGTACGAT-3’ AchE reverse: 5’-CTGATCCAGGAGACCCACAT-3’Muscarinic 1 receptor forward: 5’-CCGCTACTTCTCCGTGACTC-3’ Muscarinic 1 receptor reverse: 5’-GTGCTCGGTTCTCTGTCTCC-3’ Muscarinic 2 receptor forward: 5’-TACGGCTATTGCAGCCTTCT-3’ Muscarinic 2 receptor reverse: 5’-GCAACAGGCTCCTTCTTGTC-3’ Muscarinic 3 receptor forward: 5’-GGTCATACCGTCTGGCAAGT-3’ Apically secreted MUC5AC protein was measured using an ELISA [11, 17].ELISA for MUC5AC protein in cell supernatants is described in the online supplement. Data are expressed as the percentage above the PBS control.Acetylcholine production was measured in cell culture medium by a colorimetric method using a commercial kit (BioVision Research Products, Milpitas, CA, USA). The kit detects choline before and after adding acetylcholine esterase to the reaction. This converts acetylcholine into choline which is detected by a colorimetric assay and concentration is calculated against the standard curve. Acetylcholine was measured as difference in choline before and after adding choline esterase. The absorbance was measured at 570 nm using the ELx808 Ultra Microplate Reader (Bio Tek Instruments, Inc., Winooski, VT, USA). Results are expressed as µ M.We measured M3R protein by immunoblotting. Extraction of cell lysates and western blot analysis were performed as detailed in the online supplement [11].The cells on porous filters were fixed in 10% formalin neutral buffer, embedded in paraffin, and cut into 8 mm slices. To examine morphology, haematoxylin and eosin staining and periodic acid-Schiff (PAS) staining were performed [10, 11]. Cell morphology was assessed by microscopy (CKX41; Olympus, Tokyo, Japan) and photographed using a digital camera system (AxioCam ICc 1; Carl Zeiss, Oberkochen, Germany).M3 receptor (M3R) expression was also evaluated by immunohistochemistry. Sections were stained with anti-MUC5AC antibody, or anti-M3R antibody as the first antibody. EnVisionTM 1 Dual Link System-HRP (Dako, North America Inc., Carpinteria, CA) was added as the second antibody. Antigen-antibody complexes were visualized using the Liquid DAB 1 Substrate Chromogen System (Dako) [11].Data are expressed as mean values ±SEM. Comparisons between the two groups were made by unpaired, two-tailed Student’s t-test. Multiple comparisons were made by two-way analysis of variance (ANOVA). A post hoc analysis for multiplicity was performed by using the Bonferroni method. P value less than 0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism 5 (La Jolla, CA, USA) and PASW Statistics for Windows v.21.0 (SPSS Inc., Chicago, IL, USA). RESULTS A 14-day exposure to IL-13 increased MUC5AC mRNA expression (1.01±0.10 in control vs 130.4±32.38 at 5 ng/mL of IL-13, p=0.013) and protein production (1.00±0.02 in control vs 10.01±1.19 at 5 ng/mL of IL-13, p<0.013), and this induced histologically confirmed goblet cell metaplasia. Tiotropium had no significant effect on MUC5AC mRNA expression or protein production. Goblet cell differentiation was not affected by tiotropium (Figure 2). A 1-hour exposure to IL-13 did not change MUC5AC expression in differentiated ciliated cells (Supplemental figure 1)A 14-day exposure to IL-13 significantly increased CarAT mRNA expression (1.00±0.06 in control vs 4.82±1.63 at 5 ng/mL of IL-13, p<0.001) and acetylcholine in the medium (0.69±0.47 µ M in control vs 2.02±0.65 µ M at 5 ng/mL of IL-13, p=0.018), but it did not change AChE mRNA expression (1.01±0.12 µM in control vs 1.11±0.44 µ M at 5 ng/mL of IL-13, p=0.999) (Figure 3). Tiotropium itself, did not affect CarAT or AChE mRNA expression, or acetylcholine concentration. We did not detect ChAT mRNA expression in our experimental system, which is consistent with a previous report [16].Unexpectedly, a 14-day exposure to IL-13 significantly decreased M3R mRNA expression (1.01±0.05 in control vs 0.47±0.05 at 5 ng/mL of IL-13, p<0.001) and protein production (1.00±0.01 in control vs 0.37±0.06 at 5 ng/mL of IL-13, p<0.001) (Figure 4A and B). M1R mRNA expression was increased after 14-days of IL-13 (1.18±0.30 in control vs 14.84±3.55 at 5 ng/mL of IL-13, p=0.024), but M2R mRNA expression was unchanged (Supplemental Figure 2). Tiotropium did not affect muscarinic receptor expression withIL-13 exposure. Immunohistochemistry showed that apical surface expression of M3R in IL-13 transformed goblet cells was less than in ciliated cells (Figure 4C).Tiotropium significantly decreased MUC5AC mRNA expression stimulated by 1-hour of NE exposure (1.28±0.07 in control vs 0.66±0.11, p=0.002 in 1X10-7 M of NE, 1.28±0.08 in control vs 0.76±0.13, p=0.007 in 5X10-7 M of NE) as shown in Figure 5A. Cellular viability was not affected by tiotropium or NE at these concentrations or exposure durations. Because the NE stimulation was only one hour, we did not measure MUC5AC protein secretion. Exposing HBE cells to NE for greater than1 hour caused cell death (data not shown).NE did not change CarAT mRNA expression (1.00±0.09 in control vs 1.36±0.56 at 5X10-7 M of NE, p=0.612), acetylcholine in medium (0.82±0.46 in control vs 0.89±0.62 at 5X10-7 M of NE, p=1.000), or AChE mRNA expression (1.00±0.05 in control vs 1.26±0.32 at 5X10-7 M of NE, p=0.624) (Figure 5B, C, and D). Tiotropium did not affect mRNA expression or acetylcholine concentration.NE unexpectedly increased M3R mRNA expression (1.01±0.07 in control vs 1.40±0.08 at 5X10-7 M of NE, p=0.002), however there was no significant increase in M3R protein (Figure 6). Tiotropium had no effect on M3R mRNA expression. NE had no significant effect on M1R or M2R mRNA expression (Supplemental figure 3). DISCUSSION It is reported that anticholinergics, like tiotropium, decrease mucus secretion in COPD [8, 9]. NE and IL-13 are two major drivers of mucus hypersecretion in COPD and severe asthma but these work through different mechanism [14, 18, 19]. We hypothesized that tiotropium would decrease MUC5AC mRNA and protein stimulated by IL-13 and/or NE and might prevent IL-13 induced goblet cell metaplasia. However, while tiotropium did decrease MUC5AC stimulated by NE, it had no effect on IL-13 induced mucin secretion or goblet cell metaplasia. Non-neuronal acetylcholine secreted by airway epithelium cells binds to M3R and induces inflammation and mucin production [6, 7, 20], and increased M3R expression appears to enhance these responses. It is reported that a murine knock-out of M3R, but not M1R or M2R, prevented both the inflammatory response induced by cigarette smoke and allergen-induced goblet cell metaplasia [6, 21]. We therefore investigated the effect of IL-13, NE, or tiotropium on acetylcholine production and on muscarinic receptor expression.We found that IL-13 increased the airway epithelial production of non-neuronal acetylcholine while NE had no effect on acetylcholine production. However, IL-13 exposure unexpectedly decreased M3R expression whilst NE increased M3R expression. The unanticipated finding that IL-13 decreases M3R may be due to negative feedback regulation to prevent an increased amount of acetylcholine from binding to M3R. In contrast, we found that IL-13 increased M1R expression. It is possible that M1R might increase mucin production because this receptor plays a modulatory role in electrolyte and water secretion [22]. On the other hand, NE increased M3R mRNA expression but did not appear to increase acetylcholine synthesis. In this scenario, tiotropium might bind to the increased M3R, increasing signaling and thus decreasing NE-induced MUC5AC expression. Because prolonged exposure to NE is toxic to cells, we limited exposure time to 1 hour. Although IL-13 is not toxic to cells in the concentrations we studied, in order to compare with a 1-hour exposure to NE we also exposed differentiated airway cells, to IL-13 for just 1 hour.Given these data, we speculate that the increase in acetylcholine with IL-13 that was not attenuated by tiotropium could lead to competitive receptor binding, abrogating any effect that tiotropium might have on IL-13 driven mucus secretion. However, NE did not affect acetylcholine leaving less competition with tiotropium for receptor binding and allowing M3 antagonism to decrease mucin production. Acetylcholine has been shown to be a local signaling molecule synthesized in bronchial epithelial cells [23, 24] and this non-neuronal acetylcholine can stimulate the production of MUC5AC mucin protein [24].We extend these findings by demonstrating that IL-13 induces acetylcholine production and attenuates the inhibitory effect of tiotropium on mucin expression. As an anti-muscarinic drug, tiotropium is able to regulate non-neuronal acetylcholine induced inflammation and mucin production, but this effect appears to be specific for NE-induced stimulation.Our finding that tiotropium decreases NE-induced MUC5AC expression is consistent with studies using animal models of COPD [4, 25, 26]. Some in vivo studies using asthma models also demonstrated that tiotropium significantly decreased MUC5AC expression [27, 28], while in our study, tiotropium had no effect on IL-13 induced MUC5AC expression. Ovalbumin or house dust mite are frequently to develop animal models of allergic asthma. However, these allergens can also induce neutrophil inflammation [29]. Tiotropium appears to predominantly affect the neutrophilic component of this response. Asthma is a heterogeneous disease with endotypes usually classified on the basis of biomarkers such as predominance of different inflammatory cells (ie eosinophilic, neutrophilic, or mixed cellularity) [2]. Studies show that tiotropium is effective therapy in most, but not all patients with asthma [30].IL-13 is involved in the regulation of IgE synthesis, eosinophil migration, and mucus hypersecretion [31, 32]. We have previously shown that dexamethasone, effective corticosteroid therapy for TH2 dominant asthma, has no effect on IL-13 induced airway mucus secretion or goblet cell differentiation [10]. Albano and colleagues showed thatIL-13 can desensitize β2-adrenergic receptors on HBE cells [33]. Here we report that IL-13 can decrease the effect of a muscarinic receptor antagonist by increasing acetylcholine production; a novel and probably an important effect in some patients with asthma and mucus hypersecretion. Kistemaker and colleagues reported that tiotropium partially blocked goblet cell metaplasia induced with exposure to 1 ng/mL of IL-13, but tiotropium did not decrease MUC5AC gene expression [16]. This effect was only seen in some cell lines (R. Gosens, personal communication). We confirmed that tiotropium had no effect of MUC5AC that had been stimulated by or 5 ng/mL of IL-13.Tiotropium is an effective bronchodilator in asthma and COPD and this is likely to be independent of the effect of tiotropium on mucin secretion, an important cause of morbidity in severe asthma and COPD. The results reported here suggest that the inhibitionof mucus secretion by anticholinergics would be greatest in neutrophil dominant inflammation.Treatment of refractory asthma is challenging. We have now shown that IL-13 induced mucin production is not only corticosteroid resistant, but also tiotropium resistant. However, NE induced mucin production may be decreased by tiotropium. We speculate that combination therapy using tiotropium along with an Alvelestat anti-IL-13 antibody [34] may be complementary in treating severe asthma.