Anti-Infectives Restore ORKAMBI® Rescue of F508del-CFTR Function in Human Bronchial Epithelial Cells Infected with Clinical Strains of P. aeruginosa

Abstract

Chronic infection and inflammation are the primary causes of declining lung function in Cystic Fibrosis (CF) patients. ORKAMBI® (Lumacaftor-Ivacaftor) is an approved combination therapy for Cystic Fibrosis (CF) patients bearing the most common mutation, F508del, in the cystic fibrosis conductance regulator (CFTR) protein. It has been previously shown that ORKAMBI®-mediated rescue of CFTR is reduced by a pre-existing Pseudomonas aeruginosa infection. Here, we show that the infection of F508del-CFTR human bronchial epithelial (HBE) cells with lab strain and four different clinical strains of P. aeruginosa, isolated from the lung sputum of CF patients, decreases CFTR function in a strain-specific manner by 48 to 88%. The treatment of infected cells with antibiotic tobramycin or cationic antimicrobial peptide 6K-F17 was found to decrease clinical strain bacterial growth on HBE cells and restore ORKAMBI®-mediated rescue of F508del-CFTR function. Further, 6K-F17 was found to downregulate the expression of pro-inflammatory cytokines, interleukin (IL)-8, IL-6, and tumor necrosis factor-α in infected HBE cells. The results provide strong evidence for a combination therapy approach involving CFTR modulators and anti-infectives (i.e., tobramycin and/or 6K-F17) to improve their overall efficacy in CF patients.

The CFTR Mutation c.3453G > C (D1152H) Confers an Anion Selectivity Defect in Primary Airway Tissue that Can be Rescued by Ivacaftor

“…Then, CFTR inhibitor (CFTRinh-172, 10 µM) was added to deactivate CFTR. The peak changes in fluorescence to CFTR agonists were normalized relative to fluorescence immediately before agonist (forskolin) addition [ 24 , 28 , 29 ].…”

Photochemically Activated Notch Signaling Hydrogel Preferentially Differentiates Human Derived Hepatoblasts to Cholangiocytes

“…Immunoblotting: WT and KO-CFTR 16HBE14o-cells were grown as previously described. [70] Following 7 d of culture on Jag1-HA versus HA hydrogels, the cells were lysed in a modified radioimmunoprecipitation assay (RIPA) buffer (50 × 10 −3 m Tris-HCl, 150 × 10 −3 m NaCl, 1 × 10 −3 m EDTA, pH 7.4, 0.2% SDS, and 0.1% triton X-100) containing a protease inhibitor cocktail (Roche, Mannheim, Germany) for 10 min. [71] Soluble fractions were analyzed by SDS-PAGE on 6% tris-glycine gels (Life Technologies).…”

Novel Anti-Inflammatory Approaches for Cystic Fibrosis Lung Disease: Identification of Molecular Targets and Design of Innovative Therapies

“…We can imagine that the improvement of various dysregulated parameters will have long-term effects on the inflammation present in CF patients, even if indirectly. A recent article has highlighted that by Tobramycin or the AMP, 6K-F17 could restore the effects of Orkambi on p.Phe508del-CFTR protein, suggesting a significant role of infection in the CF pathology ( Laselva et al., 2020 ). Furthermore, using this approach, they have demonstrated that the active AMP can down-regulate the expression of pro-inflammatory cytokines like IL-8, IL-6, and TNF-α in p.Phe508del-CFTR human BECs ( Laselva et al., 2020 ).…”