Targeted CFTR gene disruption with zinc-finger nucleases in human intestinal epithelial cells induces oxidative stress and inflammation

• We used the zinc finger nuclease method to produce a total ablation of cystic fibrosis transmembrane conductance regulator (CFTR) gene expression in intestinal Caco-2 cells.
• CFTR deletion resulted in a spontaneously weakened antioxidant defense in association with lipid peroxidation and inflammation.
• The exposure of CFTR-silenced intestinal cells to iron/ascorbate and LPS revealed their enhanced susceptibility to oxidative stress and inflammation.

Cystic fibrosis (CF) is a multisystemic pathology caused by mutations of the CF transmembrane conductance regulator (CFTR) gene.ObjectivesAs the intestine harbors the greatest number of CFTR transcripts after birth and since CFTR plays a role in glutathione transport, we hypothesized that CFTR deletion might produce oxidative stress (OxS) and inflammation in CF intestinal epithelial cell.MethodsCFTR gene was abrogated in Caco-2/15 enterocytes through the zinc-finger nuclease system. Their oxidative and inflammatory characteristics were appreciated under basal conditions and after the treatment with the pro-oxidant iron-ascorbate (Fe/Asc) complex and pro-inflammatory lipopolysaccharide (LPS).ResultsIntestinal epithelial cells with CFTR knockout spontaneously exhibited an increased lipid peroxidation level, reflected by malondialdehyde overproduction and reduced antioxidant defense characterized by low enzymatic activities of glutathione peroxidase and catalase. CFTR silencing also resulted in elevated protein expression of pro-inflammatory tumor necrosis Factor-α, interleukin-6, cyclooxygenase-2, and the transcription factor nuclear factor-κB. Moreover, exaggerated OxS and inflammation processes occurred in CFTR−/− cells in response to the addition of Fe/Asc and LPS, respectively.ConclusionsIntestinal Caco-2/15 cells with CFTR deletion, display innate oxidative and inflammatory features while being more sensitive to pro-oxidant and pro-inflammatory stimuli. These two pathophysiological processes could be implicated in CF-related intestinal disorders.

Figure optionsDownload high-quality image (127 K)Download as PowerPoint slide