Phagocytosis depends on TRPV2-mediated calcium influx and requires TRPV2 in lipids rafts: alteration in macrophages from patients with cystic fibrosis.

Whereas many phagocytosis steps involve ionic fluxes, the underlying ion channels remain poorly defined. As reported in mice, the calcium conducting TRPV2 channel impacts the phagocytic process. Macrophage phagocytosis is critical for defense against pathogens. In cystic fibrosis (CF), macrophages have lost their capacity to act as suppressor cells and thus play a significant role in the initiating stages leading to chronic inflammation/infection. In a previous study, we demonstrated that impaired function of CF macrophages is due to a deficient phagocytosis. The aim of the present study was to investigate TRPV2 role in the phagocytosis capacity of healthy primary human macrophage by studying its activity, its membrane localization and its recruitment in lipid rafts. In primary human macrophages, we showed that P. aeruginosa recruits TRPV2 channels at the cell surface and induced a calcium influx required for bacterial phagocytosis. We presently demonstrate that to be functional and play a role in phagocytosis, TRPV2 might require a preferential localization in lipid rafts. Furthermore, CF macrophage displays a perturbed calcium homeostasis due to a defect in TRPV2. In this context, deregulated TRPV2-signaling in CF macrophages could explain their defective phagocytosis capacity that contribute to the maintenance of chronic infection

Correction of the Delta Phe508 Cystic Fibrosis Transmembrane Conductance Regulator Trafficking Defect by the Bioavailable Compound Glafenine

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes a cAMP-activated anion channel expressed in epithelial cells. The most common mutation Delta Phe508 leads to protein misfolding, retention by the endoplasmic reticulum, and degradation. One promising therapeutic approach is to identify drugs that have been developed for other indications but that also correct the CFTR trafficking defect, thereby exploiting their known safety and bioavailability in humans and reducing the time required for clinical development. We have screened approved, marketed, and off-patent drugs with known safety and bioavailability using a Delta Phe508-CFTR trafficking assay. Among the confirmed hits was glafenine, an anthranilic acid derivative with analgesic properties. Its ability to correct the misprocessing of CFTR was confirmed by in vitro and in vivo studies using a concentration that is achieved clinically in plasma (10 mu M). Glafenine increased the surface expression of Delta Phe508-CFTR in baby hamster kidney (BHK) cells to similar to 40% of that observed for wild-type CFTR, comparable with the known CFTR corrector 4-cyclohexyloxy-2-{1-[4-(4-methoxybenzensulfonyl)-piperazin-1-yl]-ethyl} -quinazoline (VRT-325). Partial correction was confirmed by the appearance of mature CFTR in Western blots and by two assays of halide permeability in unpolarized BHK and human embryonic kidney cells. Incubating polarized CFBE41o(-) monolayers and intestines isolated from Delta Phe508-CFTR mice (treated ex vivo) with glafenine increased the short-circuit current (I-sc) response to forskolin + genistein, and this effect was abolished by 10 mu M CFTRinh 172. In vivo treatment with glafenine also partially restored total salivary secretion. We conclude that the discovery of glafenine as a CFTR corrector validates the approach of investigating existing drugs for the treatment of CF, although localized delivery or further medicinal chemistry may be needed to reduce side effects

Mitochondrial Alterations and Oxidative Stress in Cystic Fibrosis

Cystic fibrosis (CF) is the most frequent autosomal recessive disease and is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Since the discovery of the deletion in the phenylalanine 508 site (ΔF508) of the CFTR gene, the study of its function as chloride channel occupied most investigations. Now, we know that CFTR is also involved in the GSH and HCO3− transport, and its function could regulate the mitochondrial function and ROS production. In this way, the notion of the CFTR as a simple chloride channel has begun to change toward a more complex function as molecular hub that integrates different cellular signals. There is a growing body of evidence that shows mitochondrial dysfunctions and increased oxidative stress in CF. Here, we review the mitochondrial defects induced by the altered function of the CFTR in CF, focusing on oxidative stress and inflammation as targets for therapy.Fil: Valdivieso, Ángel Gabriel. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentin