CFTR inhibition mimics the cystic fibrosis inflammatory profile

Abstract

Primary airway epithelial cells grown in air-liquid interface differentiate into cultures that resemble native epithelium morphologically, express ion transport similar to those in vivo, and secrete cytokines in response to stimuli. Comparisons of cultures derived from normal and cystic fibrosis (CF) individuals are difficult to interpret due to genetic differences besides CFTR. The recently discovered CFTR inhibitor, CFTRinh-172, was used to create a CF model with its own control to test if loss of CFTR-Cl− conductance alone was sufficient to initiate the CF inflammatory response. Continuous inhibition of CFTR-Cl− conductance for 3–5 days resulted in significant increase in IL-8 secretion at basal ( P = 0.006) and in response to 109 Pseudomonas ( P = 0.0001), a fourfold decrease in Smad3 expression ( P = 0.02), a threefold increase in RhoA expression, and increased NF-κB nuclear translocation upon TNF-α/IL-1β stimulation ( P < 0.000001). CFTR inhibition by CFTRinh-172 over this period does not increase epithelial sodium channel activity, so lack of Cl− conductance alone can mimic the inflammatory CF phenotype. CFTRinh-172 does not affect IL-8, IL-6, or granulocyte/macrophage colony-stimulating factor secretion in two CF phenotype immortalized cell lines: 9/HTEo− pCEP-R and 16HBE14o− AS, or IL-8 secretion in primary CF cells, and inhibitor withdrawal abolishes the increased response, so CFTRinh-172 effects on cytokines are not direct. Five-day treatment with CFTRinh-172 does not affect cells deleteriously as evidenced by lactate dehydrogenase, trypan blue, ciliary activity, electron micrograph histology, and inhibition reversibility. Our results support the hypothesis that lack of CFTR activity is responsible for the onset of the inflammatory cascade in the CF lung.

SUMOylation of Tissue Transglutaminase as Link between Oxidative Stress and Inflammation

“…IB3-1 cell lines were incubated for 24 h with EUK-134 (50 g/ml; Alexis Biochemicals), TG inhibitor R283 (250 M), KCC009 (250 M), or cystamine (400 M; Sigma-Aldrich) followed or not by rosiglitazone for 6 h (10 M; Alexis Biochemicals). A549 and 16HBE cell lines were incubated for 1 h with H 2 O 2 (33) (2 mM; Sigma-Aldrich) and for 24 h with rotenone (1 M; Sigma-Aldrich) or buthionine sulfoximine (10 M; Sigma-Aldrich) in the presence or absence of EUK-134 as well as with CFTR-inh172 (20,22) (20 M;Calbiochem). A549 and 16HBE cell lines were also cultured under hypoxic conditions in a humidified hypoxia chamber (Cop Laboratories) for 1 h at 1% O 2 (5% CO 2 balance N 2 ), and temperature was maintained at 37°C (34).…”

Pulmonary vascular dysfunction in end-stage cystic fibrosis: role of NF-κB and endothelin-1

“…CF is characterised by an intense pulmonary inflammation, and NFkB upregulation is among its key markers. It is a major inflammatory transcription factor [29] partly responsible for the high levels of interleukin-8, among other inflammatory mediators [30,31]. In addition to the latter, NF-kB might also lead to the transcription of vasoactive and proliferative mediators, such as ET-1 for instance.…”

Hyperinflammation in airways of cystic fibrosis patients: what’s new?

“…Interestingly, we had shown that the de novo expression of IκBα protein could be obtained in CF human bronchial epithelial cells following treatment with the flavonoid genistein [25]. Perez and colleagues have also observed that cultured primary normal human bronchial epithelial cells have significantly increased basal secretion of proinflammatory cytokines, nuclear NF-κB translocation and stimulated secretion when these cells are first treated with a new class of specific inhibitor of CFTR chloride conductance, CFTR inh-172 [39]. These data support the hypothesis that lack of CFTR activity in airway epithelial cells is responsible for the onset of the inflammatory cascade in the CF lung.…”