Cystic fibrosis (CF) is a fatal genetic disease associated with widespread exocrine gland dysfunction. Studies have suggested activating effects of resveratrol, a naturally-occurring polyphenol compound with antioxidant and anti-inflammatory properties, on CF transmembrane conductance regulator (CFTR) protein function. We assayed, in F508del-CFTR homozygous (CF) and in wild-type mice, the effect of resveratrol on salivary secretion in basal conditions, in response to inhibition by atropine (basal β-adrenergic-dependent component) and to stimulation by isoprenaline (CFTR-dependent component). Both components of the salivary secretion were smaller in CF mice than in controls. Two hours after intraperitoneal administration of resveratrol (50 mg/kg) dissolved in DMSO, the compound was detected in salivary glands. As in both CF and in wild-type mice, DMSO alone increased the response to isoprenaline in males but not in females, the effect of resveratrol was only measured in females. In wild-type mice, isoprenaline increased secretion by more than half. In CF mice, resveratrol rescued the response to isoprenaline, eliciting a 2.5-fold increase of β-adrenergic-stimulated secretion. We conclude that the salivary secretion assay is suitable to test DMSO-soluble CFTR modulators in female mice. We show that resveratrol applied in vivo to mice reaches salivary glands and increases β-adrenergic secretion. Immunolabelling of CFTR in human bronchial epithelial cells suggests that the effect is associated with increased CFTR protein expression. Our data support the view that resveratrol is beneficial for treating CF. The salivary secretion assay has a potential application to test efficacy of novel CF therapies.
This study was designed to test the hypothesis that resveratrol stimulates in vivo salivary secretory responses in the presence of the F508del-CFTR protein. We showed in controlled experiments that detectable levels of resveratrol are achieved in salivary glands two hours after a single intraperitoneal injection of the compound and that it partially restores the refractory β-adrenergic response of salivary glands in CF female mice. The effect seemed to be associated with increased CFTR expression. The data show that the salivary secretion assay is suitable for testing the efficacy of DMSO-soluble compounds for CF therapies.
In 2005, Best and Quinton developed a salivary secretion assay in mouse ( Best and Quinton, 2005 ). They showed that salivary glands of mice knocked out for the CFTR protein (cftr −/− ) failed to respond to the β-adrenergic agonist isoprenaline, while the cholinergic induced secretion remained intact. Another in vivo study showed that CFTR potentiators (MPB-07, genistein) increased salivary secretion in wild-type but not in cftr −/− mice, highlighting the potentiality of this method to test the preclinical efficacy of CFTR modulators ( Noel et al., 2008 ). The fact that only water and ethanol soluble compounds were used in this study ( Noel et al., 2008 ) is quite important: in drug discovery programs, compounds are primarily synthesized to optimize their pharmacological activity, often resulting in highly lipophilic compounds with poor water solubility. Most of them require hydrophobic solvents, such as DMSO which can act as a chemical chaperone for CFTR ( Planas et al., 2012 ). However, possible unwanted effects of DMSO on the salivary secretion assay have not been evaluated.
Cystic Fibrosis (CF), a complex disease associated with widespread exocrine gland dysfunction, is caused by loss of function of the CF transmembrane conductance regulator (CFTR) chloride channel. Almost 2000 mutations have been described, but ∼70% of patients carry at least one F508del-CFTR mutation. The deletion of the phenylalanine at position 508 of CFTR impairs the folding of the protein which is retained in the endoplasmic reticulum and undergoes early degradation by the proteasome. The loss of CFTR contribution to ion homeostasis drives an impaired mucociliary clearance and promotes cycles of chronic inflammation and bacterial infection. Therefore, CF patients would strongly benefit from pharmacological compounds combining the ability to correct both the deficient ion transport and the deregulated inflammatory responses.
To isolate the β-adrenergic-sensitive fraction of the basal salivary secretion, the predominant cholinergic component of the salivary secretion was initially blocked with atropine ( Fig. 1 B). The average atropine-insensitive component of saliva secretion was significantly reduced by at least 50% in the CF group compared to the wild-type and the heterozygous groups. In heterozygous female mice, values significantly different from CF (P<0.05) but not from wild-type mice were observed ( Fig. 1 B). Integrity of the β-adrenergic-induced component was then tested by assessing the magnitude of the response to the β-agonist isoprenaline ( Fig. 1 C). In wild-type mice, a three- to four-fold increase in saliva secretion rate was observed after isoprenaline stimulation. This response was completely abolished in CF females as the median (25–75% interquartile range) obtained after β-adrenergic stimulation [3.2 (2.6–5.4) µg.min −1 .g −1 ; Fig. 1 C] did not significantly differ from the corresponding atropine-insensitive value [2.4 (1.7–4.9) µg.min −1 .g −1 ; Fig. 1 B]. However, a significant sensitivity to isoprenaline was detected in CF males: the average rate of isoprenaline-induced response in CF males was increased 4-fold after stimulation (P<0.05, compare Fig. 1 B and 1 C). However, beta-adrenergic-stimulated saliva secretion rates in CF males represented a small fraction (∼20%) of the corresponding values observed in wild-type mice ( Fig. 1 C). These data confirm that salivary secretion is altered in CF mice: the predominant atropine-sensitive component is intact and apparently not influenced by sex, while the remaining atropine-insensitive fraction is reduced and refractory to β-agonist stimulation, particularly in females.
The average rate of basal salivary secretion tested in the absence of any agonist or inhibitor did not differ among the different genotypes, and similar rates were found in females and in males ( Fig. 1 A). In CF males, the average rate of basal secretion was significantly reduced to two thirds of the value found in wild-type males. The possible influence of body weight, significantly larger in male mice than in female ( Table 1 ), was normalized by correcting rates of salivary secretion for animal body weight.
No adverse events occurred in any experimental groups. As lower rates of salivary secretion in female mice than in male have previously been reported in C57BL/6 ( Best and Quinton, 2005 ) and in other backgrounds ( Droebner and Sandner, 2013 ), we first analyzed the influence of genotype and sex on the different components of the salivary secretion in FVB/129 mice. Thus, between-group comparisons were performed with data obtained from the basal salivary secretion, the atropine-insensitive component and the isoprenaline-induced component.
Prior to evaluating the effect of resveratrol on salivary secretion in CF mice, control experiments were performed to test the effect of DMSO alone. Basal salivary secretion, atropine-insensitive secretion and isoprenaline-induced secretion were evaluated two hours after intraperitoneal injection of DMSO:NaCl (1:1) in CF and in wild-type mice of both sexes. Median rates of basal salivary secretion (tested in the absence of any agonist or inhibitor) and of atropine-insensitive secretion (tested under cholinergic blockade with atropine) were not influenced by DMSO (data not shown). However, distinct sex-dependent effects of DMSO were observed on the median rate of isoprenaline-stimulated salivary secretion ( Table 2 ). The vehicle did not modify isoprenaline-stimulated salivary secretion in females. However, in males, a significant response to isoprenaline was observed two hours after injection of the vehicle.
In DMSO-controlled experiments, we tested the effect of resveratrol on salivary secretion in CF and wild-type mice. Because the response to isoprenaline was largely refractory and no effect of DMSO was observed in CF females, only female mice were used to evaluate the effect of resveratrol. In wild-type mice, the effect of resveratrol on the rate of isoprenaline-induced saliva secretion was increased by more than half compared to that measured in the DMSO controlled group ( Fig. 2 A). In CF mice, resveratrol partially restored the response to isoprenaline, eliciting a 2.5-fold increase of the rate of β-adrenergic-stimulated salivary secretion ( Fig. 2 B).
In order to correspond to the delay of in vivo salivary tests (pharmacodynamics effects), pharmacokinetic studies were performed two hours after intraperitoneal administration of resveratrol. Plasma and salivary gland concentrations are given in Table 3 . Concentration values at the limit of detection (0.5 ng/ml) were found in plasma samples from 3 out of 4 mice. In salivary glands, the median concentration was about 30 times larger than in plasma. Typical ion chromatograms of sample extracts obtained from a blank sample, an internal standard or from a sample spiked at the 1 ng/ml are shown in supplementary material Fig. S1 . Chromatographs of a blood sample and of a salivary gland sample at 26.6 ng/ml and 27.4 ng/mg resveratrol respectively are also shown ( supplementary material Fig. S1 ).
Quantification of CFTR protein by immunofluorescence in human bronchial epithelial cells. (A) Representative immunohistochemical labeling of CFTR (green) and zonula occludens (ZO-1, red) 24 h after incubation with DMSO (left) or 100 µM resveratrol (right) in wild-type 16HBE14o− (WT, top) or in CFBE41o− (CF, bottom) cultures. Nuclei (blue) stained by DAPI. Bar: 20 µm. (B) Relative position corresponding to intensity of the CFTR signal quantified by morphometric analysis of cross-sectional profiles in individual (n=8 cells per condition, randomly selected). (C) Average area under the fluorescence curves (±s.e.m.) obtained in B normalized to that measured in DMSO-treated wild-type cells and expressed as a measure of total CFTR protein expression (*P<0.05, Student t-test). Data from a representative experiment selected from a series of 3 experiments with similar results. Resv, Resveratrol.
Quantification of CFTR protein by immunofluorescence in human bronchial epithelial cells. (A) Representative immunohistochemical labeling of CFTR (green) and zonula occludens (ZO-1, red) 24 h after incubation with DMSO (left) or 100 µM resveratrol (right) in wild-type 16HBE14o− (WT, top) or in CFBE41o− (CF, bottom) cultures. Nuclei (blue) stained by DAPI. Bar: 20 µm. (B) Relative position corresponding to intensity of the CFTR signal quantified by morphometric analysis of cross-sectional profiles in individual (n=8 cells per condition, randomly selected). (C) Average area under the fluorescence curves (±s.e.m.) obtained in B normalized to that measured in DMSO-treated wild-type cells and expressed as a measure of total CFTR protein expression (*P<0.05, Student t-test). Data from a representative experiment selected from a series of 3 experiments with similar results. Resv, Resveratrol.
To assess the potential mechanism involved in the activating effect of resveratrol on CFTR-dependent salivary secretion, we examined the cellular expression of CFTR mRNA and protein in human immortalized CF and wild-type bronchial cells after incubation with the compound. No significant changes in CFTR mRNA expression were found in 16HBE14o− ( Fig. 3 A) or CFBE41o− cells ( Fig. 3 B) after 2 h or 24 h exposure to resveratrol. This finding suggests that resveratrol does not act by altering mRNA. We then tested the effect of the compound on the protein expression by immunohistochemical analysis. In DMSO-treated wild-type and CF cells examined in confluent HBE cells cultures, the fluorescence labelling was limited to a perinuclear ring, hardly any signal being observed in more peripheral areas of the cytoplasm. By contrast, in resveratrol-treated wild-type confluent cultures, a perinuclear ring was less clearly visible, and the fluorescence signal tended to spread out, leading to a diffuse labelling throughout the whole cytoplasm ( Fig. 4 , compare panels A and B). Likewise, in CF cells treated with resveratrol, the same differences were observed in the localization of the perinuclear versus peripheral labelling, but in addition, the intensity of the whole fluorescence signal was increased ( Fig. 4 A, compare panels C and D). To improve data analysis, we performed morphometric studies of the images. The immunohistochemical results were quantified using the subcellular method described by Pasyk et al. ( Pasyk et al., 2015 ). Briefly, cross-sectional scans of pixel intensities of individual cells were measured at their largest diameter and analyzed using AxioVision software (version 4.9.1). The mean CFTR-related intensity across each cell (n=8 cells per condition, randomly selected) was measured as representative of CFTR distribution. This allowed confirming that CFTR overall expression is significantly decreased in CF cells. Indeed, CF cells showed a decreased normalized area under the curve (see Fig. 4 C). After incubation in the presence of resveratrol, CF cells showed increased CFTR-related global (peripheral and perinuclear) fluorescence intensity. It did not significantly affect the global intensity of the signal in wild-type cells. In addition, redistribution of the fluorescence intensity signal towards cell membranes was observed even in wild-type cells. Redistribution of CFTR with a larger intensity of the signal close to membrane areas colocalized with zonula occludens ZO-1, used as dual labelling. As conclusions, in CF cells, resveratrol increased both the intensity of the whole fluorescence signal and the modified the localization of the perinuclear versus peripheral CFTR labelling. These findings suggest that the increased rate of isoprenaline-induced salivary secretion in CF salivary glands after resveratrol treatment could be associated with an increased CFTR protein expression.
Developing pharmacological therapies to rescue mutated CFTR processing and function has been the goal of an increasing number of drug discovery programs over the past decade (reviewed in Becq et al., 2011). In vitro screening methods have led to the discovery of strong hits. However, these methods do not predict future in vivo efficacy or potential adverse effects in human. Animal testing is needed, and the mouse is the only model currently available for routine drug testing in CF. Like every animal model of human disease, the CF mouse model has been criticized, mainly based on the lack of spontaneous lung disease, most prominent in CF patients. However, it does recapitulate several phenotypical features of the human disease. Indeed, F508del-CFTR mice present typical transepithelial ion transport abnormalities (van Doorninck et al., 1995) as we (Lubamba et al., 2009,, 2008) and others (Droebner and Sandner, 2013; Saussereau et al., 2013) have demonstrated by measuring nasal potential difference. The failure of salivary glands to respond to β-adrenergic stimulation is an additional defect recapitulated in CF mice. Using normal and cftr−/− mice in a C57BL/6 background (CFTRtm1Unc), Best and Quinton have developed a rapid and straightforward method to quantify salivary secretion. Importantly, the method allows isolating the β-adrenergic component from the largely predominant cholinergic response (Best and Quinton, 2005), and thereby to test its integrity. The method was further used in wild-type mice to measure the effects of local injections of CFTR activators such as MPB-07 and genistein (Noel et al., 2008), highlighting its application to conduct preclinical in vivo studies on CFTR activity. Homozygous F508del-CFTR mice similar to those used in our work have already been studied to test the corrector effect of DMSO-soluble glafenine, an anthranilic acid derivative (Robert et al., 2010). Our current study provides additional control experiments; indeed we determined (1) the effects of the F508del-CFTR mutation relative to the FVB/129 wild-type mouse; (2) a possible influence of sex; and (3) the effect of the DMSO vehicle, all being crucial to validate this technique as a preclinical assay to study CFTR function in this CF mouse model.
In this study, we determined the salivary secretion rate in wild-type and F508del-CFTR homozygous and heterozygous mice. As originally reported for cftr−/− mice (Best and Quinton, 2005), atropine drastically inhibited secretion in all CFTR genotypes. The atropine-insensitive secretion was lower in CF than in wild-type mice of both sexes although it did not reach significance in male. Isoprenaline increased secretion rate in wild-type animals, but not significantly in CF mice.
An influence of sex, probably dependent on the genetic background, on the β-adrenergic component of secretion has been previously reported (Best and Quinton, 2005; Droebner and Sandner, 2013). Here we found that basal salivary secretion tended to be lower in CF male mice compared to wild-type animals, but the difference was not observed in female groups. In contrast with Best and Quinton's findings on the C57BL/6 background, our results do not suggest a major influence of sex on the different components of salivary secretion in FVB/129 wild-type animals. However, we show here that CF female mice seem to be completely refractory to isoprenaline stimulation, whereas a significant response was evident in male mice, though the response was dramatically reduced relative to wild-type animals (compare Fig. 1B and 1C). Our data are in line with those obtained in other secretory glands, such as sweat glands, that may also be influenced by sex as recently shown in CF patients (Quinton et al., 2012).
Interestingly, we also found an influence of sex on the effect of DMSO, an epithelial differentiating agent that has been suspected to have a corrector effect on F508del-CFTR maturation defect (Bebök et al., 1998). Accordingly, it has been shown in a porcine kidney cell line, LLC-PK1, that insertion of functional F508del-CFTR to the plasma membrane was significantly increased four days after treatment with DMSO (Bebök et al., 1998). In female mice of both genotypes, DMSO did not significantly alter the response to isoprenaline. Similar observation was made in wild-type male mice. However, in males, DMSO induced a significant increase in isoprenaline-induced salivary secretion in CF male mice. Therefore, we conclude that the method is suitable for testing DMSO-soluble CFTR activators or potentiators administrated by intraperitoneal injection. However, because of a large effect of DMSO in male mice, only female animals were used to evaluate the effects of resveratrol in this study.
In plants, resveratrol exists as the trans- and the cis- isomers. Trans-resveratrol is the primary form found in high abundance in the skin of grapes as well as in many other fruits and vegetables such as berries and peanuts. Trans-resveratrol has the largest biological activity and has been extensively investigated. Dozens of investigators have shown that resveratrol can prevent or slowdown the progression of a variety of diseases in animal models and humans. Although its effects on the lifespan of many model organisms remain controversial (Kasiotis et al., 2013), anticancer (Bhat and Pezzuto, 2002), anti-inflammatory (Donnelly et al., 2004) and beneficial cardiovascular (Wu and Hsieh, 2011) effects have been reported. The distribution of polyphenols in organ tissues seems to vary depending on the mode of administration (Planas et al., 2012). In our studies, resveratrol was administrated by intraperitoneal injection; we observed that two hours after administration, very low circulating levels were detected with higher levels being found in the salivary glands. The dose we tested corresponded to the minimal dose that has been shown to be active on CFTR function. We performed the salivary secretion assay two hours after treatment based on previous observation (Hamdaoui et al., 2011) that the effect of resveratrol on F508del-CFTR expression in CF pancreatic cell line CFPAC-1 is stronger after two hours of exposure.
The mechanism of action of resveratrol on CFTR activity is largely unknown and it seems that several pathways could be responsible for the in vivo effects. For example, it has been shown that resveratrol displays proteasome- (Qureshi et al., 2012) or cAMP-dependent phosphodiesterases (PDE) (Park et al., 2012) inhibiting effects. In the context of CFTR, proteasome inhibitors have been recently shown to rescue F508del-CFTR function in mouse intestinal tissues (Wilke et al., 2012) and we have demonstrated that inhibitors of PDE5 can act as correctors and potentiators of CFTR mutant proteins (Lubamba et al., 2008). Moreover, resveratrol has also been shown to modulate various epigenetic processes including DNA methylation, histone modification, chromatin remodeling, proteostasis balance and microRNA (miRNA) regulation (reviewed in Wang et al., 2013). Several lines of evidence have indicated that CFTR mutations may alter major signaling pathways through altered expression of miRNAs (Xu et al., 2011) or by modulation of proteostasis network (Balch et al., 2011). As we evaluated effects of resveratrol two hours after injection, it is likely that they are due to acute potentiator effect (as resveratrol induced salivary secretion in wild-type animals as well) and corrector effect rather than epigenetic mechanisms. Our data showed an increased CFTR immunolabelling in human CF and wild-type bronchial epithelial cell lines, suggesting that the increased rate of isoprenaline-induced secretion in salivary glands could be associated with an increased CFTR protein expression. In addition, the redistribution of the fluorescence intensity signal towards cell membranes in both CF and wild-type cells suggests that resveratrol could also act as a corrector. However, longer exposure could alter various epigenetic pathways and be an additional therapeutic benefit for CF.
In conclusion, we showed here that resveratrol partially rescues the refractory CFTR-dependent β-adrenergic response of saliva secretion in female CF mice. Our data indicate that the salivary secretion assay has a potential application to test efficacy of novel CF therapies.
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