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

Myosin Vb Is Required for Trafficking of the Cystic Fibrosis Transmembrane Conductance Regulator in Rab11a-specific Apical Recycling Endosomes in Polarized Human Airway Epithelial Cells

Cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl(-) secretion across fluid-transporting epithelia is regulated, in part, by modulating the number of CFTR Cl(-) channels in the plasma membrane by adjusting CFTR endocytosis and recycling. However, the mechanisms that regulate CFTR recycling in airway epithelial cells remain unknown, at least in part, because the recycling itineraries of CFTR in these cells are incompletely understood. In a previous study, we demonstrated that CFTR undergoes trafficking in Rab11a-specific apical recycling endosomes in human airway epithelial cells. Myosin Vb is a plus-end-directed, actin-based mechanoenzyme that facilitates protein trafficking in Rab11a-specific recycling vesicles in several cell model systems. There are no published studies examining the role of myosin Vb in airway epithelial cells. Thus, the goal of this study was to determine whether myosin Vb facilitates CFTR recycling in polarized human airway epithelial cells. Endogenous CFTR formed a complex with endogenous myosin Vb and Rab11a. Silencing myosin Vb by RNA-mediated interference decreased the expression of wild-type CFTR and DeltaF508-CFTR in the apical membrane and decreased CFTR-mediated Cl(-) secretion across polarized human airway epithelial cells. A recombinant tail domain fragment of myosin Vb attenuated the plasma membrane expression of CFTR by arresting CFTR recycling. The dominant-negative effect was dependent on the ability of the myosin Vb tail fragment to interact with Rab11a. Taken together, these data indicate that myosin Vb is required for CFTR recycling in Rab11a-specific apical recycling endosomes in polarized human airway epithelial cells

Serum and glucocorticoid-inducible kinase1 increases plasma membrane wt-CFTR in human airway epithelial cells by inhibiting its endocytic retrieval

Background: Chloride (Cl) secretion by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) located in the apical membrane of respiratory epithelial cells plays a critical role in maintenance of the airway surface liquid and mucociliary clearance of pathogens. Previously, we and others have shown that the serum and glucocorticoid-inducible kinase-1 (SGK1) increases wild type CFTR (wt-CFTR) mediated Cl transport in Xenopus oocytes by increasing the amount of wt-CFTR protein in the plasma membrane. However, the effect of SGK1 on the membrane abundance of wt-CFTR in airway epithelial cells has not been examined, and the mechanism whereby SGK1 increases membrane wt-CFTR has also not been examined. Thus, the goal of this study was to elucidate the mechanism whereby SGK1 regulates the membrane abundance of wt-CFTR in human airway epithelial cells. Methods and Results: We report that elevated levels of SGK1, induced by dexamethasone, increase plasma membrane abundance of wt-CFTR. Reduction of SGK1 expression by siRNA (siSGK1) and inhibition of SGK1 activity by the SGK inhibitor GSK 650394 abrogated the ability of dexamethasone to increase plasma membrane wt-CFTR. Overexpression of a constitutively active SGK1 (SGK1-S422D) increased plasma membrane abundance of wt-CFTR. To understand the mechanism whereby SGK1 increased plasma membrane wt-CFTR, we examined the effects of siSGK1 and SGK1-S442D on the endocytic retrieval of wt-CFTR. While siSGK1 increased wt-CFTR endocytosis, SGK1-S442D inhibited CFTR endocytosis. Neither siSGK1 nor SGK1-S442D altered the recycling of endocytosed wt-CFTR back to the plasma membrane. By contrast, SGK1 increased the endocytosis of the epidermal growth factor receptor (EGFR). Conclusion: This study demonstrates for the first time that SGK1 selectively increases wt-CFTR in the plasma membrane of human airway epithelia cells by inhibiting its endocytic retrieval from the membrane. © 2014 Bomberger et al

Monomethylarsonous Acid (MMAIII) Has an Adverse Effect on the Innate Immune Response of Human Bronchial Epithelial Cells to Pseudomonas Aeruginosa

Arsenic is the number one contaminant of concern with regard to human health according to the World Health Organization. Epidemiological studies on Asian and South American populations have linked arsenic exposure with an increased incidence of lung disease, including pneumonia, and chronic obstructive pulmonary disease, both of which are associated with bacterial infection. However, little is known about the effects of low dose arsenic exposure, or the contributions of organic arsenic to the innate immune response to bacterial infection. This study examined the effects on Pseudomonas aeruginosa (P. aeruginosa) induced cytokine secretion by human bronchial epithelial cells (HBEC) by inorganic sodium arsenite (iAsIII) and two major metabolites, monomethylarsonous acid (MMAIII) and dimethylarsenic acid (DMAV), at concentrations relevant to the U.S. population. Neither iAsIII nor DMAV altered P. aeruginosa induced cytokine secretion. By contrast, MMAIII increased P. aeruginosa induced secretion of IL-8, IL-6 and CXCL2. A combination of iAsIII, MMAIII and DMAV (10 pbb total) reduced IL-8 and CXCL1 secretion. These data demonstrate for the first time that exposure to MMAIII alone, and a combination of iAsIII, MMAIII and DMAV at levels relevant to the U.S. may have negative effects on the innate immune response of human bronchial epithelial cells to P. aeruginosa

<i>Pseudomonas aeruginosa</i> Reduces VX-809 Stimulated F508del-CFTR Chloride Secretion by Airway Epithelial Cells

<div><p>Background</p><p><i>P</i>. <i>aeruginosa</i> is an opportunistic pathogen that chronically infects the lungs of 85% of adult patients with Cystic Fibrosis (CF). Previously, we demonstrated that <i>P</i>. <i>aeruginosa</i> 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 <i>P</i>. <i>aeruginosa</i> 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.</p><p>Methods and Results</p><p>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 <i>P</i>. <i>aeruginosa</i> 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 <i>P</i>. <i>aeruginosa</i> (3 mucoid and 3 non-mucoid) significantly reduced drug stimulated F508del-CFTR Cl secretion, and plasma membrane F508del-CFTR.</p><p>Conclusion</p><p>The observation that <i>P</i>. <i>aeruginosa</i> 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.</p></div

Effect of <i>P</i>. <i>aeruginosa</i> on F508del-CFTR Cl secretion in CFBE cells (A) and CF-HBE cells (B) treated with VX-809 alone and VX-809 + VX-770.

<p>VX-770 (5 μM) did not significantly alter VX-809 (3 μM) stimulated Cl secretion in CFBE cells. Both PAO1 and PA14 reduced VX-809 + VX-770 stimulated Cl secretion in CFBE cells. By contrast VX-770 significantly reduced VX-809 stimulated Cl secretion in CF-HBE cells. As in CFBE cells both PAO1 and PA14 reduced the VX-809 + VX-770 stimulated Cl secretion. *P<0.05 versus control. **P<0.05 versus VX-809. ***P<0.05 versus VX-809 + VX-770. N = 3 to 6/treatment for CFBE cells. For CF-HBE cells, N = 3 donors, from each donor 3 to 7 monolayers of cells were studied/treatment.</p

Analysis of plasma membrane transferrin receptor in the presence of VX-809 alone and in combination with <i>P</i>. <i>aeruginosa</i>.

<p>(A) Representative western blot of apical plasma membrane transferrin receptor (TfR) in CFBE cells treated with vehicle (Control), VX-809 (3 μM), PA14 alone, or VX-809 (3 μM) + PA14. ns, not significantly different. N = 3/treatment. (B) Top: Representative western blot of cell lysate TfR in CFBE cells treated with vehicle (Control), VX-809 (3 μM), PA14 alone, or VX-809 (3 μM) + PA14. Bottom: Representative western blot of cell lysate Na⁺-K⁺-ATPase in CFBE cells treated with vehicle (Control), VX-809 (3 μM), PA14 alone, or VX-809 (3 μM) + PA14. N = 4/treatment. ns, not significantly different. All samples run on the same gel but cut for presentation.</p

A. Effects of VX-809 alone and in combination with <i>P</i>. <i>aeruginosa</i> on CFBE cells.

<p>VX-809 (3 μM, 48 hours) increased Cl secretion compared to vehicle treated control in CFBE cells. PA01, PA14, PAK and 6 clinical isolates of <i>P</i>. <i>aeruginosa</i> reduced VX-809-stimulated F508del-CFTR Cl secretion compared to VX-809 alone. *P<0.05 versus control. **P<0.05 versus VX-809. N = 3 to 46/treatment.</p

Analysis of apical CFTR in the presence of VX-809 alone and in combination with <i>P</i>. <i>aeruginosa</i>.

<p>(A) Western blot of apical membrane F508del-CFTR in CFBE cells treated with vehicle (Control), VX-809 (3 μM) alone, PA14 alone, or VX-809 (3 μM) + PA14. Na⁺-K⁺-ATPase is a loading control. *P<0.05 versus all other groups. N = 4/group. **P<0.05 versus VX-809 alone, ***P<0.05 versus Control. N = 4/group. (B) Top: Representative western blot of F508del-CFTR in cell lysates of CFBE cells treated with vehicle (Control), VX-809 (3 μM) alone, PA14 alone, or VX-809 (3 μM) + PA14. Bottom: Representative western blot of Na⁺-K⁺-ATPase (gel loading control) in cell lysates of CFBE cells treated with vehicle (Control), VX-809 (3 μM) alone, PA14 alone, or VX-809 (3 μM) + PA14 *P<0.05 versus all other groups. **P<0.05 versus VX-809 alone, ***P<0.05 versus Control. N = 4/group. All samples run on the same gel but cut for presentation. N = 4/treatment.</p