CFTR Knockdown induces proinflammatory changes in intestinal epithelial cells

Abstract

BackgroundHyperinflammation is a hallmark feature of cystic fibrosis (CF) airways. However, inflammation has also been documented systemically and, more recently, in extrapulmonary CF-affected tissues such as the pancreas and intestine. The pathogenesis of CF-related inflammation and more specifically the role of the cystic fibrosis transmembrane conductance regulator (CFTR) in that respect are not entirely understood. We have tested the hypothesis that genetic depletion of CFTR will affect the inflammatory status of human intestinal epithelial cell lines.MethodsCFTR expression was genetically depleted from Caco-2/15 and HT-29 cells using short hairpin RNA interference (shRNAi). Inflammatory conditions were induced by the addition of human recombinant tumor necrosis factor (TNF) or Interleukin-1β (IL-1β) for various periods of time. Gene expression, mRNA stability and secreted levels of interleukin (IL)-6, −8 and 10 were assessed. Analysis of pro- and anti-inflammatory signaling pathways including mitogen-activated protein kinases (p38, ERK 1/2 and JNK), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα), and nuclear factor-kappa B (NF-κB) was also performed. Eosinophils were counted in the jejunal mucosa of Cftr−/− and Cftr+/+ mice.ResultsCFTR gene and protein knockdown caused a significant increase in basal secretion of IL-8 as well as in IL-1β-induced secretion of IL-6 and −8. Release of the anti-inflammatory cytokine, IL-10, remained unaffected by CFTR depletion. The enhanced secretion of IL-8 stems in part from increased IL8 mRNA levels and greater activation of ERK1/2 MAPK, IκBα and NF-κB in the CFTR knockdown cells. By contrast, phosphorylation levels of p38 and JNK MAPK did not differ between control and knockdown cells. We also found a higher number of infiltrating eosinophils in the jejunal mucosa of Cftr −/− females, but not males, compared to Cftr +/+ mice, thus providing in vivo support to our in vitro findings.ConclusionCollectively, these data underscore the role played by CFTR in regulating the intestinal inflammatory responses. Such findings lend support to the theory that CFTR exerts functions that may go beyond its role as a chloride channel whereby its disruption may prevent cells to optimally respond to exogenous or endogenous challenges. These observations are of particular interest to CF patients who were found to display alterations in their intestinal microbiota, thus predisposing them to pathogens that may elicit exaggerated inflammatory responses.Electronic supplementary materialThe online version of this article (doi:10.1186/s12950-015-0107-y) contains supplementary material, which is available to authorized users.

Inactivation of CFTR by CRISPR/Cas9 alters transcriptional regulation of inflammatory pathways and other networks

“…These result bear similarity to a report showing in- creased CXCL8 (IL8) expression in response to siRNA interference of CFTR in Caco-2 cells. However, that report found differential expression even without exogenous stimulation [ 9 ], a significant difference from the work shown here. Whether the siRNA treatment itself acted as an inflammatory stimulus is unclear, as those were acute experiments, while our cultures were carried without exposure to any exogenous stimuli for baseline expression measurements.…”

“…To study the effects of CFTR's absence on other genes and cellular processes such as inflammation, we used the CRISPR/Cas9 genome editing technology to generate subclonal cell lines from single cells of Caco-2 [ 13 ], a colonic adenocarcinoma-derived line that expresses high levels of CFTR [ 8 ]. Caco-2 cells have been reported to differ in cytokine induction upon siRNA-mediated knockdown of CFTR [ 9 ] and thus were used here to investigate cytokine regulation in models with the CFTR locus rendered inactive, akin to CF patients' cells.…”

Increased cytokines in cystic fibrosis patients’ upper airways during a new P. aeruginosa colonization

“…Moreover, we described an elevated protease‐antiprotease ratio, in particular neutrophil elastase (NE) versus secretory leukocyte protease inhibitor (SLPI), in NL of our CF patient cohort, indicating insufficient control of an exuberant and persistent neutrophil‐dominated immune response that does not manage to clear the bacterial infections. Recent data lend support to the theory that inflammation in CF is an inherent effect of dysfunctional CFTR . Inflammation in turn has been recognized as a priming factor for infection with pathogens such as PsA, which is associated with increased mortality and morbidity in patients with CF.…”

ABCC7/CFTR Expression is Down-regulated in Patients with Active Ulcerative Colitis

“…Similar ndings by Defu Li et al who found increase in the NF-ĸB activity, IL-8 and IL-6 production in human bronchial epithelial cells (16HBE) that had a down-regulation of ABCC7/CFTR 18 as well as in patients with human lung adenocarcinoma cell lines 25 . In other cell lines from colorectal carcinoma, Crites K et al reported that a down-regulation of ABCC7/CFTR increased the production of IL-6, IL-1β and IL-8 in CACO2 and HT29 cells due to the activation of ERK1/2, MAPK, IΚBα and NF-ĸB pathways 26 . In a knock-out animal model of ABCC7/CFTR, they found that the down regulation of CFTR produced in ammation through the increased of NF-ĸB, TNFα and IL-6 in the mouse intestine, CACO2 cells and colangiocytes stimulated by lipopolysaccharides from Escherichia coli 2326,27 .…”

“…with activation of NF-ĸB and subsequent production of pro-in ammatory cytokines such as IL-6 and IL-8. 23,26,27,33,34 Microbiota dysbiosis is associated with reduction of short-chain fatty acids, an antiin ammatory molecules that regulates the immune response in the intestine, this fatty acids ameliorates the intestinal in ammation in IBD in animal model. 35 But, is possible that down-regulation of ABCC7/CFTR contribute to IL17 up-regulation in patients with UC allowing the persistent activity as we observed in the active group in our study.…”