Pseudomonas aeruginosa Reduces VX-809 Stimulated F508del-CFTR Chloride Secretion by Airway Epithelial Cells

Background P. aeruginosa is an opportunistic pathogen that chronically infects the lungs of 85% of adult patients with Cystic Fibrosis (CF). Previously, we demonstrated that P. aeruginosa reduced wt-CFTR Cl secretion by airway epithelial cells. Recently, a new investigational drug VX-809 has been shown to increase F508del-CFTR Cl secretion in human bronchial epithelial (HBE) cells, and, in combination with VX-770, to increase FEV1 (forced expiratory volume in 1 second) by an average of 3-5% in CF patients homozygous for the F508del-CFTR mutation. We propose that P. aeruginosa infection of CF lungs reduces VX-809 + VX-770- stimulated F508del-CFTR Cl secretion, and thereby reduces the clinical efficacy of VX-809 + VX-770. Methods and Results F508del-CFBE cells and primary cultures of CF-HBE cells (F508del/F508del) were exposed to VX-809 alone or a combination of VX-809 + VX-770 for 48 hours and the effect of P. aeruginosa on F508del-CFTR Cl secretion was measured in Ussing chambers. The effect of VX-809 on F508del-CFTR abundance was measured by cell surface biotinylation and western blot analysis. PAO1, PA14, PAK and 6 clinical isolates of P. aeruginosa (3 mucoid and 3 non-mucoid) significantly reduced drug stimulated F508del-CFTR Cl secretion, and plasma membrane F508del-CFTR. Conclusion The observation that P. aeruginosa reduces VX-809 and VX-809 + VX-770 stimulated F508del CFTR Cl secretion may explain, in part, why VX-809 + VX-770 has modest efficacy in clinical trials.


Introduction
CFTR is a cyclic-AMP regulated Cl channel localized to the apical plasma membrane of epithelial cells in the lungs [1][2][3][4]. Cl secretion via wt-CFTR is the major driving force for the production of a thin layer of liquid overlying the lung epithelium, which is essential for effective mucociliary transport that mechanically clears debris and pathogens from the airways and, thereby, serves a vital role in innate immunity [4][5][6]. Mutations in the CFTR gene cause Cystic Fibrosis (CF), an autosomal recessive genetic disease that causes progressive loss of lung function and death in the 3 rd decade of life due to a decrease in airway surface liquid and reduced mucociliary transport, leading to chronic bacterial lung infections [1][2][3]6]. The F508del mutation in CFTR increases its degradation in the endoplasmic reticulum, dramatically reducing establish confluent, polarized monolayers, 0.5× 10 6 CFBE cells were seeded onto 24-mm Transwell permeable supports or 12mm Snapwell permeable supports (0.4μm pore size, Corning, Corning, NY) coated with Vitrogen plating medium containing human fibronectin (10 μg/ ml, Collaborative Biomedical Products, Bedford, MA), PureCol (1%, Advanced BioMatrix, San Diego, CA), and bovine serum albumin (10 μg/ml, Invitrogen) and grown in an air-liquid interface culture at 37°C for 6-9 days, as described [18,19]. To establish confluent, polarized monolayers of CF-HBE cells, 1 × 10 6 cells were seeded onto 24-mm Transwell permeable supports or 250,000 cells were seeded onto 12mm Snapwell permeable supports (0.4-μm pore size, Corning, Corning, NY) coated with 50μg/ml Collagen type IV (Sigma) and grown in an air-liquid interface culture at 37°C for 3-4 weeks, as described [15]. Cells grown on Transwell filters were used for biochemical studies and cytokine analysis, and cells grown on Snapwell filters were used in Ussing chamber experiments.

Bacterial strains and growth conditions
In these studies, we used P. aeruginosa strains PAO1, PA14 and PAK, and six clinical isolates of P. aeruginosa (three mucoid: SMC1585, SMC5450, SMC5451 and three non mucoid: SMC1587, SMC1595, SMC1596) isolated from the sputa of six independent CF patients at the Dartmouth-Hitchcock Medical Center (Hanover, NH, USA). In addition, studies were conducted with Staphylococcus newman and Streptococcus salavari. All P. aeruginosa strains and Streptococcus salavari were grown and maintained in LB medium (Lysogeny Broth, LB) at 37°C [20]. Staphylococcus newman was grown in THY broth with Oxyrase. For co-culture studies, P. aeruginosa, Staphylococcus newman or Streptococcus salavari were harvested from overnight cultures, washed twice in CFBE cell-growth medium, and then suspended in cell-growth medium without antibiotics or phenol red. The cell suspensions were added in 300 μl of cell growth medium to the apical face of CFBE or CF-HBE monolayers for 6 hours. For control monolayers the same volume of fluid, without bacteria, was added to the apical face of CFBE and CF-HBE cells. None of the P. aeruginosa isolates or Staphylococcus newman and Streptococcus salavari had any effect on LDH release by CFBE cells over the course of the experiment (n = 3/group), indicating that the bacteria studied had no effect on epithelial cell viability.

Ussing chamber analysis of F508del-CFTR Cl secretion
Ussing chamber measurements of F508del-CFTR Cl secretion were performed as described [16,17]. Briefly, CFBE and CF-HBE cells grown on Snapwell permeable supports were mounted in an Ussing chamber (Physiologic Instruments, San Diego, CA) and short circuit current (I sc ) was measured by voltage-clamping the transepithelial voltage across the monolayers to 0 mV with a voltage clamp (Physiologic Instruments). Amiloride (50 μM) was added to the apical bath solution (5 ml total volume in the apical and basolateral bath solutions) to inhibit I sc attributed to sodium reabsorption, and subsequently I sc was stimulated with forskolin (10 μm), followed by VX-770 (5 μm) to stimulate CFTR mediated I sc , and thiazolidinone (CFTR inh -172, 20 μm; EMD Millipore, Billerica, MA) to inhibit CFTR-mediated I sc . Data are expressed as CFTR inh -172 inhibited I sc in μA/cm 2 . Data collection and analysis were done with the Acquire & Analyze Data Acquisition System (Physiologic Instruments). To examine the effect of P. aeruginosa on F508del-CFTR Cl secretion P. aeruginosa (see above for strains) was added to the apical side of CFBE and CF-HBE cell monolayers, which have a layer of mucus overlying cells, at a multiplicity of infection (MOI) of 30:1 for 6 hours in the absence of antibiotics, and then I sc was measured as described above. Ussing chamber studies were also conducted to examine the effect of Staphylococcus newman and Streptococcus salavari on VX-809 stimulated F508del-CFTR Cl secretion by CFBE cells, as described above for P. aeruginosa.

Biochemical determination of apical membrane CFTR and transferrin receptor
Briefly, the biochemical determination of F508del-CFTR in the apical plasma membrane of cells was performed by domain selective cell surface biotinylation using EZ-Link Sulfo-NHS-LC-Biotin (Pierce) at 4°C, as described previously in detail [18,23]. PAO1 was added to the apical side of CFBE monolayers at a multiplicity of infection (MOI) of 30:1 for 6 hours in the absence of antibiotics, and, subsequently, biotinylated proteins were isolated by streptavidin-agarose beads, eluted into SDS sample buffer, and separated by 7.5% SDS-PAGE. The blots were probed for F508del-CFTR, Transferrin receptor (TfR) and for Na + -K + -ATPase as a loading control. Western blot analysis of F508del-CFTR, TfR and Na + -K + -ATPase were conducted by methods described previously [16,21]. Cytokines IL-6 and IL-8 produced by polarized monolayers of CFBE and CF-HBE cells were measured using the Bio-Rad Bio-Plex cytokine arrays (Hercules, CA) as described [19,24]. CFBE and CF-HBE cells were grown on Transwell filters as described above, and P. aeruginosa was added to the apical side of monolayers at a multiplicity of infection (MOI) of 30:1 for 1 h in the absence of antibiotics, and subsequently P. aeruginosa was removed by replacing the apical medium with MEM. Five hours after P. aeruginosa was washed from monolayers the apical and basolateral media were removed for analysis of cytokines.

Data analysis and statistics
Graphpad Prism version 5.0 for Macintosh (Graphpad, San Diego, CA) was used to perform a statistical analysis of the data. Means were compared using a t-test or ANOVA followed by Tukeys test, as appropriate. P<0.05 was significant, and all data are expressed as the mean ± SEM.

VX-809 stimulates F508del-CFTR Cl secretion
VX-809 (3 μM for 48 hours) increased F508del-CFTR Cl secretion by CFBE cells (Fig 1, S1  Table). Three laboratory isolates of P. aeruginosa (PAO1, PA14 and PAK) reduced VX-809-stimulated F508del-CFTR Cl secretion by 48%, 38% and 43%, respectively, after 6 hours of co-incubation (See Fig 2, S2 Table for representative traces of F508del-CFTR Cl currents). Additional studies were conducted using recent clinical isolates of P. aeruginosa, including three non-mucoid and three mucoid strains obtained from CF patients at the Dartmouth Hitchcock Medical Center [20]. All of the clinical isolates caused a significant reduction in VX-809 stimulated F508del-CFTR Cl secretion in CFBE cells, with a range of 25% to 45% inhibition (Fig 1,  S1 Table). Additional studies were performed to determine if P. aeruginosa also impacted drug rescued F508del-CFTR Cl secretion in CF-HBE cells in primary culture. As seen in CFBE cells,   Table). Moreover, three non-mucoid and three mucoid strains also reduced VX-809 stimulated F508del-CFTR Cl secretion in CF-HBE cells, with a range of 46% to 59% inhibition (Fig 3, S3 Table).
Although P. aeruginosa reduced VX-809 stimulated F508del-CFTR Cl secretion in CFBE cells and in CF-HBE cells in primary culture, neither Staphylococcus newman nor Streptococcus salavari had any effect on VX-809 stimulated F508del-CFTR Cl secretion in CFBE cells (Fig 4, S4 Table).  Recently, as the studies in this report were being written for publication, two groups independently reported that co-administration of VX-770 with VX-809 for 48 hours decreased the ability of VX-809 to increase F508del-CFTR Cl currents in primary cultures of CF-HBE cells [22,23]. Thus, we conducted additional experiments with the co-administration of VX-809 + VX-770 (48 hours) to examine the effect of P. aeruginosa on F508del-CFTR Cl secretion.  Table).

P. aeruginosa reduces F508del-CFTR in the plasma membrane
Previous studies have shown that VX-809 reduces the degradation of F508del-CFTR in the proteasome, and increases plasma membrane F508del-CFTR [24]. Thus, studies were conducted to determine if P. aeruginosa reduces VX-809 stimulated F508del-CFTR Cl secretion by reducing F508del-CFTR in the apical cell membrane of CFBE cells. VX-809 increased the amount of F508del-CFTR in the apical membrane of CFBE cells as well as in cell lysates (Fig 6,  S1, S2 and S4 Figs). However, P. aeruginosa reversed the VX-809-stimulated increase in F508del-CFTR in both the plasma membrane and cell lysate (Fig 6, S1, S2 and S4 Figs). By contrast, neither VX-809 nor P. aeruginosa had any effect on the amount of transferrin receptor in the plasma membrane or in cell lysates of CFBE cells (Fig 7, S3 and S4 Figs). Thus, P. aeruginosa reduced VX-809 stimulated F508-CFTR Cl secretion, in part, by decreasing the amount of F508del-CFTR in the apical plasma membrane. VX-809 has no effect on IL-6 and IL-8 secretion In a previous study, we reported that neither VX-325 nor Corr4a, investigational compounds that increase F505del-CFTR Cl secretion, had any effect on cytokine secretion by CFBE cells treated with vehicle or P. aeruginosa [17]. Thus, studies were conducted to determine if VX-809 alone or VX-809 + VX-770 reduced cytokine secretion by CFBE and CF-HBE cells. Neither VX-809 alone nor VX-809 + VX-770 had a significant effect on constitutive IL-6 and IL-8 secretion, or on PAO1 simulated IL-6 and IL-8 secretion in CFBE or CF-HBE cells (Fig 8).

Discussion
This study demonstrates that 3 well characterized laboratory strains and 6 clinical isolates of P. aeruginosa (3 mucoid and 3 non-mucoid) inhibit the VX-809 and VX-809 + VX-770 stimulated increase in F508del-CFTR Cl secretion in a cell line (CFBE) and in primary cultures of CF-HBE cells. The P. aeruginosa induced inhibition of VX-809 stimulated F508del-CFTR Cl secretion is similar to that reported previously for the effect of P. aeruginosa on wt-CFTR Cl secretion [14,16,27]. The observation that P. aeruginosa reduces VX-809 + VX-770 stimulated F508del-CFTR Cl secretion, coupled with the fact that~85% of adult CF patients are colonized with P. aeruginosa suggests that the modest effect of VX-809 + VX-770 on FEV1 in CF patients (3-5% increase) may be due in part to chronic infection with P. aeruginosa.
In addition, if P. aeruginosa inhibits VX-809 + VX-770 stimulated F508del-CFTR Cl secretion one would expect to see a disproportional improvement in FEV1 in subjects not infected with P. aeruginosa compared to those who are infected with P. aeruginosa. However, at the North American CF Conference in 2014 data were presented demonstrating that there was no significant difference between the effect of VX-809 + VX-770 on FEV1 in subjects positive or negative for P. aeruginosa [11]. Although this observation is at odds with our conclusion that infection with P. aeruginosa may reduce the efficacy of VX-809 + VX-770 in vivo, it is important to note that other bacteria in the CF lungs, including Acinetobacter nosocomialis and Acinetobacter baumannii secrete a virulence factor (aCif) that reduces CFTR abundance in CFBE cells [26], thus, it is possible that infection with other bacterial that are known to reduce CFTR abundance, in addition to P. aeruginosa, may influence the response to VX-809 + VX-770. Clearly, additional studies are required to determine if P. aeruginosa, and or infection with other bacteria such as Acinetobacter that reduces CFTR abundance, suppresses the efficacy of VX-809 + VX-770 in vivo.
In two recent studies, it was reported that VX-770 dramatically reduced the ability of VX-809 to increase F508del-CFTR Cl secretion [22,23]. This is at odds with clinical trials with VX-809 + VX-770 that demonstrated significant improvement in FEV1 and several other improvements in clinical outcomes in CF patients homozygous for the F508del-CFTR mutation [11], as well as the studies in this report in which the combination of VX-809 + VX770 (48 hours) significantly increased F508del-CFTR Cl secretion in CFBE cells and in CF-HBE cells (Figs 1-3 and 5, S1-S3 and S5 Tables). Several factors may contribute to the variable effects of VX-809 + VX-770 on F508del-CFTR Cl secretion by HBE cells, including the possible differential expression of modifier genes among donors studied that may affect export of F508del-CFTR from the endoplasmic reticulum, endocytic trafficking of F508del-CFTR and channel open probability [27,28]. While additional experiments are required to determine why these studies reach different conclusions regarding the effect of VX-770 on VX-809 stimulated F508del-CFTR Cl secretion, our data show that P. aeruginosa inhibits both the VX-809 and the VX-809 + VX-770 stimulated increase in F508del-CFTR Cl secretion in primary cultures of CF-HBE cells.
We have shown that several laboratory strains of P. aeruginosa (PAO1, PA14 and PAK) as well as 6 clinical isolates of P. aeruginosa reduce VX-809 (and VX-809 + VX-770) stimulated F508del-CFTR Cl secretion. Furthermore, preliminary studies presented by Guimbellot and colleagues at the 2014 North American Cystic Fibrosis Conference confirm our results that P. aeruginosa reduces VX-809 stimulated F508del-CFTR Cl secretion [29]. Interestingly, these authors demonstrated that other bacteria known to colonize the CF lung, including Haemophilus influenza and Staphylococcus aureus, had no effect on F508del-CFTR Cl secretion. Our results with Staphylococcus newman and Streptococcus salivari demonstrate that these bacteria also have no effect on VX-809 stimulated F508del-CFTR Cl secretion.
Finally, studies were also conducted to determine if VX-809 alone or VX-809 + VX-770 reduced P. aeruginosa induce cytokine production. A reduction of cytokine secretion would be beneficial in CF patients since the hyperinflammatory lung milieu that has been linked to worse clinical outcomes in CF [30]. However, neither VX-809 alone nor VX-809 + VX-770 reduced constitutive nor the P. aeruginosa stimulated increase in IL-6 and IL-8 secretion by CFBE or CF-HBE cells, a result consistent with a previous study in which we demonstrated that two other "correctors", VX-325 and Corr 4a, had no effect on the P. aeruginosa induced inflammatory response by CFBE cells [17]. Taken together these studies from our laboratory and from others demonstrate that although VX-325, VX-809 and Corr4a increase F508del-CFTR Cl secretion, these drugs do not modify the hyperinflammatory response of CF airway cells to P. aeruginosa infection. The most effective drugs for CF patients would ideally enhance F508del-CFTR Cl secretion and reduce the proinflamatory response to bacterial infection.

Conclusion
Because 85% of adult CF patients are chronically colonized by P. aeruginosa, and because P. aeruginosa inhibited VX-809 and VX-809 +VX-770 F508del-CFTR Cl secretion by CFBE and CF-HBE cells, the observation in this paper may explain, in part, why VX-809 + VX-770 has only modest clinical benefit.