Sinupret Activates CFTR and TMEM16A-Dependent Transepithelial Chloride Transport and Improves Indicators of Mucociliary Clearance

Introduction We have previously demonstrated that Sinupret, an established treatment prescribed widely in Europe for respiratory ailments including rhinosinusitis, promotes transepithelial chloride (Cl−) secretion in vitro and in vivo. The present study was designed to evaluate other indicators of mucociliary clearance (MCC) including ciliary beat frequency (CBF) and airway surface liquid (ASL) depth, but also investigate the mechanisms that underlie activity of this bioflavonoid. Methods Primary murine nasal septal epithelial (MNSE) [wild type (WT) and transgenic CFTR−/−], human sinonasal epithelial (HSNE), WT CFTR-expressing CFBE and TMEM16A-expressing HEK cultures were utilized for the present experiments. CBF and ASL depth measurements were performed. Mechanisms underlying transepithelial Cl− transport were determined using pharmacologic manipulation in Ussing chambers, Fura-2 intracellular calcium [Ca2+]i imaging, cAMP signaling, regulatory domain (R-D) phosphorylation of CFTR, and excised inside out and whole cell patch clamp analysis. Results Sinupret-mediated Cl− secretion [ΔISC(µA/cm2)] was pronounced in WT MNSE (20.7+/−0.9 vs. 5.6+/−0.9(control), p<0.05), CFTR−/− MNSE (10.1+/−1.0 vs. 0.9+/−0.3(control), p<0.05) and HSNE (20.7+/−0.3 vs. 6.4+/−0.9(control), p<0.05). The formulation activated Ca2+ signaling and TMEM16A channels, but also increased CFTR channel open probability (Po) without stimulating PKA-dependent pathways responsible for phosphorylation of the CFTR R-domain and resultant Cl− secretion. Sinupret also enhanced CBF and ASL depth. Conclusion Sinupret stimulates CBF, promotes transepithelial Cl− secretion, and increases ASL depth in a manner likely to enhance MCC. Our findings suggest that direct stimulation of CFTR, together with activation of Ca2+-dependent TMEM16A secretion account for the majority of anion transport attributable to Sinupret. These studies provide further rationale for using robust Cl− secretagogue based therapies as an emerging treatment modality for common respiratory diseases of MCC including acute and chronic bronchitis and CRS.


Introduction
Dysfunctional mucociliary clearance (MCC) is a common pathophysiologic process that underlies inflammation and infection in many airway diseases, including chronic rhinosinusitis (CRS) [1,2]. Transport of the mucus layer within the airway surface liquid (ASL) covering respiratory epithelia is influenced by the transepithelial movement of ions, such as Cl 2 , and the rate at which cilia beat. There is recent growing interest in the use of Cl 2 secretagogues for treatment of diseases caused by inadequate MCC, including COPD, asthma, and CRS. A primary Cl 2 channel in respiratory epithelia responsible for proper MCC is the cystic fibrosis transmembrane conductance regulator (CFTR), which is dysfunctional or absent in cystic fibrosis (CF) -resulting in a significant reduction in the rate of MCC. Ongoing drug discovery efforts designed to target CFTR (via high throughput compound library screening) are under development for CF and other lower airway diseases [3]. Enhancing MCC also represents an important therapeutic strategy for patients with active sinus disease (by accelerating clearance of bacterial pathogens), and as a preventative measure among individuals prone to developing recurrent infections (by minimizing the accumulation of inhaled particles, including allergens, pollutants, and debris) [4][5][6]. CRS is a prevalent respiratory illness affecting 16% of the U.S. population and incurs an estimated aggregated cost of 8.6 billion dollars annually in healthcare expenditures [7,8,9].
Sinupret is a phytopharmaceutical widely utilized for a variety of respiratory ailments including rhinosinusitis and bronchitis. The compound is a mixture derived from parts of five plants (elder flowers, primrose flowers with calyx, common sorrel herb, European vervain herb, and gentian root), and has been shown efficacious in reducing acute symptoms and signs of sinusitis [10]. The mechanisms underlying therapeutic benefit of the compound are presently unknown, but are thought to be derived, at least in part, from flavonoid components (flavonol glycosides and free aglycones) of the formulation. Numerous additional molecules have been identified in Sinupret (e.g., iridoid glycosides, caffeoyl derivatives, phenolic compounds, triterpenes, plastocyanin, and phenylpropanoids), and many other components have yet to be characterized [11].
Several flavonoids have been demonstrated to strongly activate CFTR-mediated Cl 2 currents [12][13][14]. Our previous studies have established that Sinupret robustly activates transepithelial Cl 2 secretion in mice in vitro and in airway epithelial cells in vivo. The purpose of the present study was to determine whether this bioflavonoid therapeutic activates ciliary beat frequency (CBF) and enhances ASL, and to investigate the underlying mechanism responsible for the Cl 2 secretagogue activity of Sinupret.

Primary Cell Culture
Institutional Review Board and Institutional Animal Care and Use Committee approval were obtained prior to initiating these studies. MNSE and HSNE cells were harvested, grown on Costar 6.5-mm diameter permeable filter supports (Corning Life Sciences, Lowell, MA), and submerged in culture medium as previously described [15][16][17][18][19][20][21][22]. Media was removed from the monolayers on day 4 after the epithelium reached confluence, and cells fed via the basal chamber. Transgenic CFTR 2/2 MNSE cultures were also investigated to test CFTR-dependent effects and evaluate contributions from the apical calcium-activated Cl 2 channel, TMEM16A. Differentiation and ciliogenesis occurred in all cultures within 10 to 14 days. Cultures were used for experiments when fully differentiated with widespread ciliogenesis.
A total of 420 MNSE and 48 HSNE cell monolayers were used to complete these experiments. Cultures with transepithelial resistances (R t ) .300 V*cm 2 were used to obtain short-circuit current measurements, monitor ciliary beating by inverted microscopy, and measure ASL depth. A minimum of 5 wells were tested per condition.

Using Chamber Analysis
Transwell inserts (Costar) were mounted in Ussing chambers to investigate pharmacologic manipulation of vectorial ion transport as previously described [15,17]. Monolayers were evaluated under short circuit current conditions following fluid resistance compensation using automatic voltage clamping (VCC 600; Physiologic Instruments, San Diego, CA). Serosal bath solutions contained (in mM): 120 NaCl, 25 NaHCO 3 , 3.3 KH 2 PO 4 , 0.8 K 2 HPO 4 , 1.2 MgCl 2 , 1.2 CaCl 2 , and 10 glucose. Baths for transwell filters were warmed to 37uC and gassed continuously with a 95%O 2 -5%CO 2 mixture that provides a pH of 7.4 under conditions studied here. Chemicals were obtained from Sigma (St. Louis, MO) except for Sinupret which was purchased online (Bionorica, Neumarkt, Germany). Ussing chamber experiments were performed with a nominal mucosal Cl 2 in which 6 mM replaced NaCl in the above solution. The following pharmacologic agents were used: amiloride (100 mM) -blocks epithelial Na + channels, as a means to isolate changes in short-circuit current (DI SC ) secondary to effects on Cl 2 channel activity [23]; forskolin (20 mM) -activates CFTR by elevating intracellular cAMP, which results in protein kinase A (PKA)-dependent phosphorylation of the CFTR regulatory domain (R-D); tannic acid (100 mM) -an inhibitor of calciumactivated Cl 2 channels (recently identified as TMEM16A [24]); INH-172 (10 mM)-a CFTR inhibitor that permits determination of CFTR-dependent contributions to I SC [17]; and Sinupret (2.5 mg/ml). Each solution was produced as 1,0006 stock and studied at 16 in the Ussing chamber or by CBF analysis. Stock solutions of Sinupret tablets were generated by dissolving in dimethyl sulfoxide (DMSO) followed by removal of insoluble matter by filtration (i.e., concentrations described here do not account for removal of insoluble particulates) [15]. Maximal solubility and activity of the drug was at 2.5 mg/ml with an EC 50 of 200 mg/ml. DMSO control solutions were tested for comparison in all cases. The I SC was continuously assessed at one current measurement per second. By convention, a positive deflection in I SC under conditions tested here was defined as the net movement of anions from the serosal to mucosal direction.

Detection of Regulatory Domain (R-D) Phosphorylation and cAMP levels
Cellular cAMP was measured using an ELISA-based detection kit (Cayman Chemicals, Ann Arbor, MI) as previously described [25]. To evaluate R-D phosphorylation, polyclonal NIH-3T3 cells expressing the HA-tagged R-domain were treated with Sinupret solution for 20 minutes, and compared to forskolin (20 uM) 65 minutes (as a positive control) or DMSO alone (negative vehicle control). Following lysis, equal amounts of protein (50 mg) from total cell lysate were electrophoresed through a 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), and immunoblotted with antibody to the HA tag (Covance, Cumberland, VA). Phosphorylation of the R-domain was visualized as a 2-4 kD shift in migration, as previously described [25].

Fura-2 Imaging
Changes in cytosolic calcium [Ca 2+ ] i were measured with dual excitation wavelength fluorescence microscopy after cells were loaded with the Ca 2+ -sensitive dye Fura-2 [26]. HSNE cells were perfused with solutions containing either Sinupret 2.5 mg/ml, DMSO control, or 140 mM UTP (Sigma) as a positive control. Alteration in fluorescence intensity was monitored by a Photon Technology International (Birmingham, NJ) dual wavelength spectrofluorometer (excitation wavelength: 340/380 nm and emission wavelength: 510 nm). The intensity of fluorescence was calculated automatically. The Rmax and Rmin values were determined by the addition of 3 mM Ca 2+ and 5 mM EGTA plus 5 mM ionomycin, respectively.

Whole Cell Patch Clamp Analysis
Mouse TMEM16A subcloned in frame within the pEGFP-N1 vector was kindly provided by Professor Lily Y. Jan (University of California, San Francisco). The TMEM16A-GFP construct was as described previously by Schroeder et al. [27] HEK 293 cells were transfected using Lipofectamine 2000 Transfection reagent (Life technologies, Cat. No:11668) according to the manufacturer's instructions. Stably TMEM16A transfected HEK 293 cells were maintained in DMEM supplemented with 10% FBS and 0.4 mg/ ml G418 at 37uC in 5% CO 2 and passaged every 2-5 d. Cells were split and seeded on glass coverslips one day before whole-cell patch clamp analysis. Each cover slip with cells was transferred to an experimental chamber mounted on the stage of an Olympus microscope. TMEM16A currents were recorded in whole cell mode of the patch clamp technique using an Axopatch 2000B amplifier interfaced to a computer through DIGITA 1200 A. The data was recorded and analyzed using Pclamp software (Molecular Devices). During the experiments, cells were perfused with an external solution of the following ionic composition (in mM): 145 CsCl, 2 MgCl 2 , 2 Cacl 2 , 5.5 Glucose, 10 HEPES pH 7.4 (1 N NaOH). The pipette resistance used for whole recording ranged from 3 to 5 GV when filled with the following intrapipette solution (in mM): 135 CsCl, 10 KCl, 2 MgCl 2 , 0.1 EGTA, 5.5 Glucose, 10 HEPES, pH 7.2 (1N KOH). All experiments were performed at room temperature. Cells were continuously perfused with the external solution until formation of the recording whole cell configuration. Activators and inhibitors of TMEM16A were applied through the same perfusion system. The rate of perfusion was adjusted to 1 ml/min.

Single Channel Studies
Conventional inside out patch clamp recordings were performed using CFBE cells expressing WT CFTR. Recording pipettes were constructed from borosilicate glass capillaries (Warner Instruments, Hamden CT) using a Narishige PP83 microelectrode puller and fire polished with a PP90 microforge (Narishige, Tokyo). The pipettes were partially filled with a solution containing (in mmol/l) 150 CsCl, 1 CaCl 2 , 1 MgCl 2 and 10 HEPES (pH 7.2) and with tip resistances of 6-8 MV. Experiments were performed at room temperature (20-22uC). Single channel currents were obtained using an Axopatch 200B patch clamp amplifier (Axon Instruments (AI), Molecular Devices, USA) with voltage commands and data acquisition controlled by Clampex software (pClamp 10, Axon Instruments) and digitized (Digidata 1440A interface, AI) at a sampling frequency of 1 kHz. CFBE cells were seeded on glass coverslips and mounted in a flow through chamber. To obtain seals, bath solutions contained (in mmol/l) 140 NaCl, 4.0 KCl, 1.8 CaCl 2 , 1.0 MgCl 2 , 10 glucose, 10 HEPES, pH 7.4. For inside out patches, bath solution contained (in mmol/l) 150 CsCl, 5 EDTA and 10 HEPES pH 7.4 designed to prevent calcium-activated currents. Sinupret was added to the CsCl bath solution and used at a final concentration of 2.5 mg/ml from a 250 mg/ml stock dissolved in DMSO. Vehicle controls were included in all studies [DMSO (0.01%)]. As an additional control, patches were exposed to ATP (1 mM/PKA 40 units/ml) after Sinupret following perfusion if channel activity was not observed. Confirmation that stimulated currents were due to CFTR was achieved using the inhibitor CFTR(inh)-172 at 10 mM. Single channel recordings were analyzed using pClamp 10 software (AI).

Ciliary Beat Frequency Analysis
Images were visualized using a 206 objective on an inverted scope (Fisher Scientific, Pittsburgh, Pa.). Data were captured using a Model A602f-2 Basler area scan high-speed monochromatic digital video camera (Basler AG, Ahrensburg, Germany) at a sampling rate of 100 frames per second. Image analysis was performed with the Sisson-Ammons Video Analysis (SAVA) system, version 2.1.6 [28]. For each experiment, a large area of beating cilia within air-liquid interface cultures was identified with the inverted microscope. Images were analyzed with virtual instrumentation software designed specifically for measuring CBF. A baseline recording of CBF was conducted for each cell monolayer prior to applying an apical test solution. All experiments were compared to the corresponding control using vehicle alone (apical fluid addition, by itself, can increase CBF). Each recording was normalized to fold-change over baseline (Hertz preto post-treatment). All studies were performed at ambient temperature (23uC).

Statistical analysis
Statistical analyses were performed using unpaired t tests or analysis of variance with Tukey-Kramer post hoc analysis as appropriate +/2 standard deviation. A p value,0.05 was considered statistically significant.

Results
Sinupret increases transepithelial Cl 2 transport through CFTR and calcium-activated pathways in Ussing chamber analysis One of the goals of this study was to test whether Sinupret stimulated CFTR or calcium-activated Cl 2 (TMEM16A) channels on the apical cell surface. The primary method to evaluate anion transport involved pharmacological manipulation of cell cultures mounted in Ussing chambers followed by measurements of shortcircuit current (DI SC ). After blocking epithelial sodium channels with amiloride, Sinupret-dependent DI SC (in mA/cm 2 ) was tested in wild type (WT) MNSE ( Figure 1A), CFTR 2/2 MNSE ( Figure 1B), and HSNE cultures ( Figure 1C). Summary data is presented in Figure 1D. Sinupret significantly increased DI SC when compared to vehicle control in WT MNSE (20.7+/20.9 vs. 5.6+/20.9, p,0.05). In addition, Sinupret significantly stimulated Cl 2 secretion in HSNE cultures when compared to DMSO control (DI SC , 20.7+/20.3 vs. 6.4+/20.9, p,0.05) establishing that the therapeutic is a strong Cl 2 secretagogue in human sinus and nasal epithelium. The formulation also amplified DI SC in CFTR 2/2 MNSE cultures (DI SC , 10.1+/21.0 vs. 0.9+/20.3, p, 0.05), albeit at levels approximately 50% of WT. These data suggest stimulation of transepithelial Cl 2 secretion is partially mediated through channels other than CFTR.
Another means to investigate overall contribution of CFTR channel activation to the Sinupret -mediated DI SC is by blocking CFTR with the specific inhibitor INH-172 or pre-treating the cells with tannic acid (to eliminate calcium-activated I SC ). We observed  (Figure 2B and 2C). In epithelial monolayers from CFTR 2/2 mice, pre-incubation with tannic acid abolished the Sinupret-dependent I SC (Figure 2A). These findings suggest that the two major components of DI SC stimulated by the formulation are attributable to CFTR-mediated and calciumactivated Cl 2 secretion.
Sinupret activates Ca 2+ -mediated, but not cAMP/PKAdependent cell signaling pathways CFTR-mediated anion secretion is regulated by cAMP/PKAdependent second messenger signaling, which results in phosphorylation of the regulatory (R)-domain. TMEM16A-dependent transport relies on fluxes in intracellular Ca 2+ . In order to determine whether Sinupret works via these second messenger pathways, intracellular Ca 2+ , cAMP, and a gel shift assay used to detect R-domain phosphorylation were utilized. Sinupret applied to MNSE cultures at 0.1, 1.0, and 2.5 mg/ml did not increase cAMP (in pmol/ml) in contrast to that seen with the positive control 20 mM forskolin ( Figure 3A). Additionally, Sinupret did not confer phosphorylation of the CFTR R-domain ( Figure 3B). We performed Fura-2 Ca 2+ imaging to assess whether the formulation stimulates transport via an increase in [Ca 2+ ] i [24]. An increase in [Ca 2+ ] i (in nM) was observed by this assay following treatment with 2.5 mg/ml Sinupret (262.8+/214.4 vs. 48.3+/2 28.5, p,0.05). The Sinupret-induced [Ca 2+] i rise was approximately 70% of the UTP-induced [Ca 2+ ] i rise, which served as the positive control (377.8+/270.3). UTP response following Sinupret (300.7+/284.1) was not significantly different than UTP alone (Figure 4).

Sinupret activates TMEM16A and increases CFTR channel open time as judged by whole cell and single channel patch clamp analysis
To determine whether TMEM16A was definitively activated by Sinupret, HEK cells stably transfected with TMEM16A were subjected to whole cell patch clamp analysis ( Figure 5). Perfusion with Sinupret induced activation of a Cl 2 current that was inhibited by tannic acid. DMSO controls had no effect on TMEM16A activity at the cell surface, while positive controls had UTP-dependent activation that was prominently blocked by tannic acid. Application of 2.5 mg/ml Sinupret resulted in activation of a small conductance Cl 2 channel consistent with CFTR (with the anticipated single channel conductance and INH-172 sensitivity) in 4 out of 14 inside-out patches from CFBE cells. In 4 experiments, ATP/PKA was added to the bath following Sinupret exposure but with no channel activation. For patches in which Sinupret activated CFTR, NPO increased from 0.7760.26 to 2.2160.98-a 2.9 fold increase in activity (NPO/n: 0.1860.06 to 0.4360.15, where n = number of discernable channel levels). Thus, in the absence of other stimulatory molecules, Sinupret increased CFTR channel Po. This is consistent with our finding of CFTR activation in an intact cell preparation (Figure 1) in which Sinupret increased CFTR channel activity in the absence of in vitro stimulation by PKA (Figure 3).

Sinupret enhances ASL depth
It is well-established that stimulation of transepithelial Cl 2 secretion leads to airway surface hydration and promotes ASL depth. In order to evaluate effects of the formulation on ASL, MNSE cultures were exposed to Sinupret and stained to visualize overlying surface liquid. Sinupret significantly increased ASL depth in these cultures (in mm: 9.1460.42 vs. 5.2560.38, control, *p,0.05) indicating a vigorous treatment effect (Figure 7).

Discussion
Sinupret has been utilized extensively throughout Europe for more than 70 years as treatment of airway diseases associated with inadequate MCC, and has an excellent safety profile. The medication is registered as a regulated phyto-pharmaceutical in Germany [29], and has been available in the United States for 9 years where it is considered an herbal supplement (not regulated by the Food and Drug Administration) [11]. The drug has been extensively investigated in a variety of scientific studies and clinical settings. Prophylactic administration to mice led to increased resistance against respiratory tract infection following intranasal application of Sendai virus (Parainfluenza viridae) [30]. Mucus clearance properties have been observed in rabbit tracheas in vivo [31]. Several clinical trials have tested the effect of Sinupret either alone or in combination with antibiotics for rhinosinusitis. Significant improvement in radiologic outcomes and headache were demonstrated in a randomized, placebo-controlled trial [32]. When used as an adjunctive treatment with antibiotics and/or nasal decongestants in this setting, two clinical studies demonstrated benefit versus placebo [33].
In the present experiments, we establish that Sinupretstimulates Cl 2 secretion in part by activating apical CFTR channels. This finding is supported by: 1) a significant decrease in DI SC with chemical inhibition (INH-172) in WT airway epithelial   monolayers, 2) stimulation of I SC despite pretreatment with tannic acid in WT MNSE, 3) blunted Cl 2 secretion in CFTR 2/2 MNSE lacking CFTR, and 4) excised inside-out patch clamp analysis demonstrating increase in CFTR channel Po. Overall Cl 2 transport remained significantly greater than controls despite absence of CFTR channel function (Figure 1), suggesting either increased Cl 2 gradient (secretory driving force) due to activation of basolateral K + channels (NKCC1) that promote Cl 2 transport through TMEM16A, enhanced intracellular Ca 2+ , and/or increased gating of TMEM16A was responsible for the residual transepithelial Cl 2 secretion in the absence of CFTR. Notably, Cl 2 -dependent I SC was abrogated following addition of the TMEM16A blocker tannic acid in CFTR 2/2 cell monolayers, indicating that non-CFTR activity was responsible for these findings. It has been reported previously that the absence of CFTR in murine airways may confer enhanced expression or activity of other Cl 2 secretory pathways, including TMEM16A [34]. Therefore, the Cl 2 secretory mechanisms available for stimulation in CFTR 2/2 mice may differ from those in WT. Nevertheless, Fura2-imaging suggests intracellular Ca 2+ release contributes to TMEM16A activation in this setting, while activation of current in whole cell patch clamp analysis in TMEM16A transfected cells confirms these channels are stimulated by Sinupret. Therefore, Cl 2 transport is clearly mediated via effects on both apical CFTR and TMEM16A.
Two key components of MCC include ciliary beat and the composition of the ASL. Because Sinupret stimulates intracellular Ca 2+ signaling cascades known to stimulate both TMEM16Amediated Cl 2 transport and CBF in mammalian respiratory epithelial cells [16,35], it is not surprising that an increase in CBF as well as ASL depth was noted in this study. Stimulation of CFTR-dependent pathways through increased channel Po (as opposed to cAMP/PKA-mediated phosphorylation of the Rdomain) may also contribute [36,37] and are likely related to the flavonoid components of the formulation [4,6]. Activation of mechanisms such as these would improve MCC and probably account for underlying health benefits of the drug.

Conclusions
Sinupret likely derives its clinical benefit, at least in part, through stimulating transepithelial Cl 2 secretion, CBF, and MCC. Augmenting fluid and electrolyte secretion represents a means of improving mucus clearance in individuals affected by acute or chronic rhinosinusitis, specifically in the setting of WT CFTR. The present studies also point to molecular pathways that might be targeted as a means to improve therapy of MCC in diseases such as CRS and COPD in the future.