Early pathogenesis of cystic fibrosis gallbladder disease in a porcine model

Abstract

Hepatobiliary disease causes significant morbidity in people with cystic fibrosis (CF), yet this problem remains understudied. We previously found that newborn CF pigs have microgallbladders with significant luminal obstruction in the absence of infection and consistent inflammation. In this study, we sought to better understand the early pathogenesis of CF pig gallbladder disease. We hypothesized that loss of CFTR would impair gallbladder epithelium anion/liquid secretion and increase mucin production. CFTR was expressed apically in non-CF pig gallbladder epithelium but was absent in CF. CF pig gallbladders lacked cAMP-stimulated anion transport. Using a novel gallbladder epithelial organoid model, we found that Cl − or HCO 3 − was sufficient for non-CF organoid swelling. This response was absent for non-CF organoids in Cl − /HCO 3 − -free conditions and in CF. Single-cell RNA sequencing revealed a single epithelial cell type in non-CF gallbladders that co-expressed CFTR, MUC5AC, and MUC5B. Despite CF gallbladders having increased luminal MUC5AC and MUC5B accumulation, there was no significant difference in the epithelial expression of gel-forming mucins between non-CF and CF pig gallbladders. In conclusion, these data suggest that loss of CFTR-mediated anion transport and fluid secretion contribute to microgallbladder development and luminal mucus accumulation in CF.

Early intrahepatic duct defects in a cystic fibrosis porcine model

“…Our studies also found that CF intrahepatic biliary organoids had defective fluid secretion. This is consistent with our studies in the CF pig gallbladder (Zarei et al, 2020). The exact function of biliary fluid secretion, specifically pertaining to CFTR, is still not clear, but likely contributes to decreased bile flow in CF pigs (Uc et al, 2012).…”

“…Organoids were kept in suspension at 4ºC while they were permeabilized in 0.3% Triton for 1 h and blocked in Superblock (Thermo Fisher) with 4% normal goat serum for 48 h. Organoids were then incubated with primary antibodies overnight: mouse anti‐CFTR (clone 769) (1:100 dilution, University of North Carolina—Chapel Hill and the Cystic Fibrosis Foundation Therapeutics) and rabbit anti‐Na + /K + ‐ATPase (clone EP1845Y) (1:100 dilution, Abcam). The anti‐CFTR antibody was validated based on cellular localization, absent signal in CFTR‐deficient tissue, and previous studies (Zarei et al, 2020). The anti‐Na + /K + ‐ATPase was validated based on cellular localization and previous studies (Nieto‐Torres et al, 2014).…”

“…CFTR immunohistochemistry was performed with mouse anti-CFTR 769 (1:1200, 60 min; CFF/UNC), generously supplied by Dr. John Riordan, University of North Carolina-Chapel Hill and the Cystic Fibrosis Foundation Therapeutics. The anti-CFTR antibody was validated based on cellular localization, absent signal in CFTR-deficient tissue, and previous studies (Zarei et al, 2020). Secondary Ab (1:200, 30 min; Vector Biotinylated Anti-Mouse IgG) was then applied followed by Vector ABC Reagent (30 min, Standard VECTASTAIN ® Elite ® ABC Kit, Vector Laboratories, Inc.) and chromogen (room temperature, DAB plus for 5 min followed by DAB Enhancer for 3 min).…”

“…The exact pathogenesis contributing to these changes is not clear. In newborn CF pigs, we previously found reduced bile flow into the small intestine and that the CF pig gallbladder was obstructed by mucus without signs of infection, inflammation, or changes in mucin expression (Uc et al, 2012; Zarei et al, 2020). In the extrahepatic tract, we also showed that CF pig gallbladder organoids lacked cAMP‐mediated fluid secretion (Zarei et al, 2020).…”

“…In newborn CF pigs, we previously found reduced bile flow into the small intestine and that the CF pig gallbladder was obstructed by mucus without signs of infection, inflammation, or changes in mucin expression (Uc et al, 2012;Zarei et al, 2020). In the extrahepatic tract, we also showed that CF pig gallbladder organoids lacked cAMPmediated fluid secretion (Zarei et al, 2020). Our rationale for this study was to further examine the newborn CF pig liver and assess if fluid secretion defects and mucus changes contribute to early CF disease in the intrahepatic tract.…”