Investigating the role of CFTR in human and mouse myometrium.

Background: Abnormal cystic fibrosis transmembrane conductance regulator (CFTR) function in cystic fibrosis (CF) has been linked to airway smooth muscle abnormalities including bronchial hyperresponsiveness. However, a role for CFTR in other types of smooth muscle, including myometrium, remains largely unexplored. As CF life expectancy and the number of pregnancies increases, there is a need for an understanding of the potential role of CFTR in myometrial function. Methods: We investigated the role of CFTR in human and mouse myometrium. We used immunofluorescence to identify CFTR expression, and carried out contractility studies on spontaneously contracting term pregnant and non-pregnant mouse myometrium and term pregnant human myometrial biopsies from caesarean sections. Results: CFTR was found to be expressed in term pregnant mouse myometrium. Inhibition of CFTR, with the selective inhibitor CFTRinh-172, significantly reduced contractility in pregnant mouse and human myometrium in a concentration-dependent manner (44.89 ± 11.02 term pregnant mouse, 9.23 ± 4.75 term-pregnant human; maximal effect at 60 µM expressed as a percentage of the pre-treatment control period). However, there was no effect of CFTRinh-172 in non-pregnant myometrium. Conclusion: These results demonstrate decreased myometrial function when CFTR is inhibited, which may have implications on pregnancy and labour outcome and therapeutic decisions for labour in CF patients.

NF-κB2 signalling in enteroids modulates enterocyte responses to secreted factors from bone marrow-derived dendritic cells.

Alternative pathway NF-κB signalling regulates susceptibility towards developing inflammatory bowel disease (IBD), colitis-associated cancer and sepsis-associated intestinal epithelial cell apoptosis and shedding. However, the cell populations responsible for the perturbed alternative pathway NF-κB signalling in intestinal mucosal pathology remain unclear. In order to investigate the contribution of the epithelial compartment, we have tested whether NF-κB2 regulated transcription in intestinal epithelial cells controls the intestinal epithelial response to cytokines that are known to disrupt intestinal barrier permeability. Enteroids were generated from the proximal, middle and distal regions of small intestine (SI) from C57BL/6J wild-type mice and displayed region-specific morphology that was maintained during sub-culture. Enteroids treated with 100 ng/mL TNF were compared with corresponding regions of SI from C57BL/6J mice treated systemically with 0.33 mg/kg TNF for 1.5 h. TNF-induced apoptosis in all regions of the intestine in vitro and in vivo but resulted in Paneth cell degranulation only in proximal tissue-derived SI and enteroids. TNF also resulted in increased enteroid sphericity (quantified as circularity from two-dimensional bright field images). This response was dose and time-dependent and correlated with active caspase-3 immunopositivity. Proximal tissue-derived enteroids generated from Nfκb2-/- mice showed a significantly blunted circularity response following the addition of TNF, IFNγ, lipopolysaccharide (LPS) activated C57BL/6J-derived bone marrow-derived dendritic cells (BMDC) and secreted factors from LPS-activated BMDCs. However, Nfκb1-/- mouse-derived enteroids showed no significant changes in response to these stimuli. In conclusion, the selection of SI region is important when designing enteroid studies as region-specific identity and response to stimuli such as TNF are maintained in culture. Intestinal epithelial cells are at least partially responsible for regulating their own fate by modulating NF-κB2 signalling in response to stimuli known to be involved in multiple intestinal and systemic diseases. Future studies are warranted to investigate the therapeutic potential of intestinal epithelial NF-κB2 inhibition.