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Bile acid homeostasis in gastrointestinal and metabolic complications of cystic fibrosis

  • Ivo P. van de Peppel
    Correspondence
    Corresponding authors at: Section of Molecular Metabolism and Nutrition, Department of Pediatrics, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9713, GZ, Groningen, the Netherlands.
    Affiliations
    Pediatric Gastroenterology and Hepatology, University of Groningen, University Medical Center Groningen, the Netherlands

    Section of Molecular Metabolism and Nutrition, Department of Pediatrics, University of Groningen, University Medical Center Groningen, the Netherlands
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  • Frank A.J.A. Bodewes
    Affiliations
    Pediatric Gastroenterology and Hepatology, University of Groningen, University Medical Center Groningen, the Netherlands
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  • Henkjan J. Verkade
    Affiliations
    Pediatric Gastroenterology and Hepatology, University of Groningen, University Medical Center Groningen, the Netherlands

    Section of Molecular Metabolism and Nutrition, Department of Pediatrics, University of Groningen, University Medical Center Groningen, the Netherlands
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  • Johan W. Jonker
    Correspondence
    Corresponding authors at: Section of Molecular Metabolism and Nutrition, Department of Pediatrics, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9713, GZ, Groningen, the Netherlands.
    Affiliations
    Section of Molecular Metabolism and Nutrition, Department of Pediatrics, University of Groningen, University Medical Center Groningen, the Netherlands
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Open ArchivePublished:September 07, 2018DOI:https://doi.org/10.1016/j.jcf.2018.08.009

      Abstract

      With the improved treatment of the pulmonary complications of cystic fibrosis (CF), gastrointestinal problems have become more important in the morbidity in CF. A hallmark of the gastrointestinal phenotype of CF, apart from pancreatic insufficiency, is a disruption of bile acid homeostasis. Bile acid homeostasis is important for many gastrointestinal processes including fat absorption, inflammation, microbial composition, as well as regulation of whole body energy metabolism. This review describes the impairment of bile acid homeostasis in CF, its possible consequences for gastrointestinal and metabolic complications and its potential as a target for therapy.

      Keywords

      1. Introduction

      Cystic fibrosis (CF), caused by a mutation in the gene encoding the CF transmembrane conductance regulator (CFTR), results in production of abnormally thick, viscous mucus in various organ systems [
      • Rowe S.M.
      • Miller S.
      • Sorscher E.J.
      Cystic Fibrosis.
      ]. Aside from pulmonary problems, CF patients often suffer from GI disorders, hepatobiliary problems and cystic fibrosis related diabetes (CFRD). A specific GI feature common among CF patients is impaired bile acid (BA) homeostasis manifesting via BA malabsorption and subsequent increased fecal excretion [
      • O'Brien S.
      • Mulcahy H.
      • Fenlon H.
      • O’Broin A.
      • Casey M.
      • Burke A.
      • et al.
      Intestinal bile acid malabsorption in cystic fibrosis.
      ]. Recent studies show the interrelation of BA homeostasis with various other intestinal, hepatic and metabolic parameters. In this review we discuss the role of impaired BA homeostasis in CF, explaining its potential role in other GI and metabolic complications and as a therapeutic target.

      2. Gastrointestinal complications of cystic fibrosis

      A functional GI system is essential for maintaining adequate nutritional status and whole body homeostasis. Similar to pulmonary complications, viscous mucus, as a consequence of deficient surface fluid and bicarbonate flux is an important underlying factor in the GI phenotype of CF [
      • De Lisle R.C.
      • Borowitz D.
      The cystic fibrosis intestine.
      ]. Exocrine pancreatic insufficiency (EPI) is used as a marker for severity of the CF phenotype. However, other manifestations of the GI phenotype of CF are often highly variable and do not strongly correlated to allelic CFTR variation [
      • Haller W.
      • Ledder O.
      • Lewindon P.J.
      • Couper R.
      • Gaskin K.J.
      • Oliver M.
      Cystic fibrosis: an update for clinicians. Part 1: Nutrition and gastrointestinal complications.
      ]. Patients experience various symptoms including malabsorption, fatty stools (steatorrhea), abdominal pain, nausea, anorexia, bloating, gastro-esophageal reflux, constipation, distal intestinal obstruction syndrome (DIOS) and flatulence. Although most of the GI complications are interrelated, they can be subdivided in pancreatic, hepatobiliary and intestinal-luminal categories.
      A severe CFTR gene mutation in both alleles results in little or no CFTR Cl channel activity and destruction of the exocrine pancreas [
      • Ahmed N.
      • Corey M.
      • Forstner G.
      • Zielenski J.
      • Tsui L.-C.
      • Ellis L.
      • et al.
      Molecular consequences of cystic fibrosis transmembrane regulator (CFTR) gene mutations in the exocrine pancreas.
      ]. EPI is an early sign of CF and can present at birth or develop in the first months of life [
      • O'Sullivan B.P.
      • Baker D.
      • Leung K.G.
      • Reed G.
      • Baker S.S.
      • Borowitz D.
      Evolution of pancreatic function during the first year in infants with cystic fibrosis.
      ]. Ultimately, around 85% of CF patients develop EPI and these patients are prone to nutritional deficiencies, severe malnutrition and growth retardation [
      • Gaskin K.J.
      Nutritional care in children with cystic fibrosis: are our patients becoming better?.
      ]. Fortunately, EPI can be successfully treated with pancreatic enzyme replacement therapy (PERT). However, even with optimal PERT, fat malabsorption and GI complaints are often not fully corrected [
      • Kalivianakis M.
      • Minich D.M.
      • Bijleveld C.M.A.
      • Van Aalderen W.M.C.
      • Stellaard F.
      • Laseur M.
      • et al.
      Fat malabsorption in cystic fibrosis patients receiving enzyme replacement therapy is due to impaired intestinal uptake of long-chain fatty acids.
      ,
      • Borowitz D.
      • Durie P.R.P.
      • Clarke L.L.
      • Werlin S.L.
      • Taylor C.J.
      • Semler J.
      • et al.
      Gastrointestinal outcomes and confounders in cystic fibrosis.
      ,
      • Bodewes F.A.J.A.
      • Verkade H.J.
      Persistent fat malabsorption in cystic fibrosis - lessons from patients and mice.
      ]. Mice with targeted mutations in the Cftr gene do not display EPI but nevertheless have a lower bodyweight upon ad libitum feeding, possibly due to bacterial overgrowth or from impaired epithelial absorption of nutrients [
      • Bijvelds M.J.C.
      • Bronsveld I.
      • Havinga R.
      • Sinaasappel M.
      • de Jonge H.R.
      • Verkade H.J.
      Fat absorption in cystic fibrosis mice is impeded by defective lipolysis and post-lipolytic events.
      ]. This suggests that, in addition to EPI, there are other changes in the intestinal tract in CF that have important effects on nutrient absorption and growth.
      CF patients can suffer from a multitude of hepatobiliary problems including gall stones, hepatitis, steatosis and cirrhosis. Hepatobiliary problems are common in pediatric CF patients with reported prevalence rates up to 25% [
      • Feranchak A.P.
      • Sokol R.J.
      Cholangiocyte biology and cystic fibrosis liver disease.
      ,
      • Boelle P.
      • Debray D.
      • Guillot L.
      • Clement A.
      • Corvol H.
      Cystic fibrosis liver disease: outcomes and risk factors in a large cohort of French patients.
      ]. Cystic fibrosis related liver disease (CFLD) was thought to develop mainly in early childhood. However, a recent follow-up study of a cohort of CF patients into adulthood incorporated novel markers into the CFLD diagnostic algorithm and suggests an additional wave of adult-onset CFLD with a median age of 37 years [
      • Koh C.
      • Sakiani S.
      • Surana P.
      • Zhao X.
      • Eccleston J.
      • Kleiner D.E.
      • et al.
      Adult onset cystic fibrosis liver disease: diagnosis and characterization of an underappreciated entity.
      ]. Another recent study that assessed a large retrospective cohort of French CF patients found that CFLD incidence increased by approximately 1% every year reaching 32.2% by the age of 25 [
      • Boelle P.
      • Debray D.
      • Guillot L.
      • Clement A.
      • Corvol H.
      Cystic fibrosis liver disease: outcomes and risk factors in a large cohort of French patients.
      ].
      In the liver, CFTR is exclusively expressed at the apical membrane of cholangiocytes lining the bile ducts [
      • Cohn J.A.
      • Strong T.V.
      • Picciotto M.R.
      • Nairn A.C.
      • Collins F.S.
      • Fitz J.G.
      Localization of the cystic fibrosis transmembrane conductance regulator in human bile duct epithelial cells.
      ]. CFLD is characterized by focal biliary cirrhosis which can lead to multilobular cirrhosis and portal hypertension in 1–10% of patients [
      • Moyer K.
      • Balistreri W.
      Hepatobiliary disease in patients with cystic fibrosis.
      ]. The pathophysiology of biliary cirrhosis has been hypothesized to be secondary to occlusion of small bile ducts and/or to increased bile toxicity. In CF mouse models, however, evidence to support the hypothesis that increased bile toxicity contributes to CFLD has not been reported [
      • Bodewes F.A.J.A.
      • Bijvelds M.J.
      • De Vries W.
      • Baller J.F.W.
      • Gouw A.S.H.
      • De Jonge H.R.
      • et al.
      Cholic acid induces a Cftr dependent biliary secretion and liver growth response in mice.
      ].
      Luminal GI complications are highly prevalent in CF. Approximately 15–20% of CF infants present with meconium ileus, an obstruction of the distal small intestine by dehydrated mucofeculent material [
      • Casaccia G.
      • Trucchi A.
      • Nahom A.
      • Aite L.
      • Lucidi V.
      • Giorlandino C.
      • et al.
      The impact of cystic fibrosis on neonatal intestinal obstruction: the need for prenatal/neonatal screening.
      ]. After the neonatal phase, acute fecal obstruction of the ileocecum known as DIOS can occur and incidence increases with age [
      • Van Der Doef H.P.J.
      • Kokke F.T.M.
      • Van Der Ent C.K.
      • Houwen R.H.J.
      Intestinal obstruction syndromes in cystic fibrosis: meconium ileus, distal intestinal obstruction syndrome, and constipation.
      ,
      • Munck A.
      • Alberti C.
      • Colombo C.
      • Kashirskaya N.
      • Ellemunter H.
      • Fotoulaki M.
      • et al.
      International prospective study of distal intestinal obstruction syndrome in cystic fibrosis: associated factors and outcome.
      ]. Nearly half of pediatric CF patients suffer from constipation and this is even more prevalent in adulthood [
      • van der Doef H.P.J.
      • Kokke F.T.M.
      • Beek F.J.A.
      • Woestenenk J.W.
      • Froeling S.P.
      • Houwen R.H.J.
      Constipation in pediatric Cystic Fibrosis patients: an underestimated medical condition.
      ]. Another common luminal GI feature of CF is a change in intestinal microbiota characterized by small intestinal bacterial overgrowth (SIBO) and colonic dysbiosis [
      • Garg M.
      • Ooi C.Y.
      The ENIGMATIC GUT IN CYSTIC FIBROSIS: linking inflammation, dysbiosis, and the increased risk of malignancy.
      ]. Important contributing factors include delayed intestinal transit time, luminal hyperacidity due to decreased bicarbonate secretion by pancreas and intestinal epithelium, frequent antibiotic use and inspissated mucus. Intestinal microbial composition is important for immune function and various metabolic processes in the body [
      • Nicholson J.K.
      • Holmes E.
      • Wilson I.D.
      Gut microorganisms, mammalian metabolism and personalized health care.
      ]. Its disruption in CF is therefore likely to contribute to various aspects of the phenotype [
      • Li L.
      • Somerset S.
      The clinical significance of the gut microbiota in cystic fibrosis and the potential for dietary therapies.
      ].
      Along with the increased life expectancy the CF population has been shown to become exposed to an increased risk of malignant tumors especially of the small intestine and colon [
      • Garg M.
      • Ooi C.Y.
      The ENIGMATIC GUT IN CYSTIC FIBROSIS: linking inflammation, dysbiosis, and the increased risk of malignancy.
      ,
      • Maisonneuve P.
      • Marshall B.C.
      • Knapp E.A.
      • Lowenfels A.B.
      Cancer risk in cystic fibrosis: a 20-year nationwide study from the United States.
      ,
      • Neglia J.P.
      • Fitzsimmons S.C.
      • Maisonneuve P.
      • Schöni M.H.
      • Schöni-Affolter F.
      • Corey M.
      • et al.
      The risk of cancer among patients with cystic fibrosis.
      ], possibly due to an increased proliferation rate of epithelial cells and disruption of anti-apoptotic pathways [
      • Ooi C.Y.
      • Durie P.R.
      Cystic fibrosis from the gastroenterologist's perspective.
      ]. Additionally, a recent study has shown a direct role of CFTR as a tumor suppressor gene in intestinal cancer [
      • Than B.L.N.
      • Linnekamp J.F.
      • Starr T.K.
      • Largaespada D.A.
      • Rod A.
      • Zhang Y.
      • et al.
      CFTR is a tumor suppressor gene in murine and human intestinal cancer.
      ]. After lung transplantation the risk for malignancies in CF patients is even further increased due to the use of immunosuppressant drugs [
      • Maisonneuve P.
      • Marshall B.C.
      • Knapp E.A.
      • Lowenfels A.B.
      Cancer risk in cystic fibrosis: a 20-year nationwide study from the United States.
      ,
      • Fink A.K.
      • Yanik E.L.
      • Marshall B.C.
      • Wilschanski M.
      • Lynch C.F.
      • Austin A.A.
      • et al.
      Cancer risk among lung transplant recipients with cystic fibrosis.
      ].

      3. Impaired bile acid homeostasis and farnesoid X receptor signaling in cystic fibrosis

      One of the hallmarks of the GI complications in CF patients as well as in murine CF models is an up to 3-fold increase in fecal bile acid (BA) excretion [
      • O'Brien S.
      • Mulcahy H.
      • Fenlon H.
      • O’Broin A.
      • Casey M.
      • Burke A.
      • et al.
      Intestinal bile acid malabsorption in cystic fibrosis.
      ,
      • Strandvik B.
      • Einarsson K.
      • Lindblad A.
      • Angelin B.
      Bile acid kinetics and biliary lipid composition in cystic fibrosis.
      ,
      • Bijvelds M.J.C.
      • Jorna H.
      • Verkade H.J.
      • Bot A.G.M.
      • Hofmann F.
      • Agellon L.B.
      • et al.
      Activation of CFTR by ASBT-mediated bile salt absorption.
      ,
      • Bodewes F.A.J.A.
      • van der Wulp M.Y.M.
      • Beharry S.
      • Doktorova M.
      • Havinga R.
      • Boverhof R.
      • et al.
      Altered intestinal bile salt biotransformation in a cystic fibrosis (Cftr−/−) mouse model with hepato-biliary pathology.
      ]. This increase is independent of exocrine pancreatic insufficiency and fat malabsorption. In the physiological situation the enterohepatic circulation of BAs is a tightly regulated system in which ~95% of total BAs are reabsorbed and the remaining ~5% is excreted via the feces (Fig. 1).
      Fig. 1
      Fig. 1Schematic representation of the enterohepatic circulation of bile acids.
      Bile acids (BAs) are synthesized and conjugated in the liver after which they are actively secreted via the bile duct into the duodenum. In the ileum BAs are reabsorbed by the enterocytes through the apical sodium-dependent bile acid transporter (ASBT). In the enterocyte BAs activate the nuclear farnesoid X receptor (FXR) which leads to subsequent release of fibroblast growth factor 19 (FGF19 or 15 in mice). FGF15/19 travels to the liver where it binds to fibroblast growth factor receptor 4 (FGFR4) β-Klotho complex and inhibits cholesterol 7α-hydroxylase (CYP7A1), the rate controlling enzyme of BA synthesis. Reabsorbed BAs can also travel directly to the liver and inhibit CYP7A1 by activating hepatic FXR. Microbiota in the intestine can further biotransform BAs to deconjugated and secondary BA species. After reabsorption, the remaining 5% of BAs (in the physiological situation) is excreted in feces.
      Reabsorption mainly takes place by active transport of conjugated BAs into the ileal enterocyte by the apical sodium-dependent bile acid transporter (ASBT, SLC10A2) [
      • Dawson P.A.
      • Lan T.
      • Rao A.
      Bile acid transporters.
      ]. In the ileal enterocyte BAs activate the farnesoid X receptor (FXR), a ligand-activated transcription factor of the family of nuclear receptors, which leads to increased expression and subsequent release of fibroblast growth factor 19 (FGF19, Fgf15 in mice) in the circulation [
      • Inagaki T.
      • Choi M.
      • Moschetta A.
      • Peng L.
      • Cummins C.L.
      • McDonald J.G.
      • et al.
      Fibroblast growth factor 15 functions as an enterohepatic signal to regulate bile acid homeostasis.
      ]. In the liver FGF19 can bind to and activate the FGF receptor 4 (FGFR4)/β-Klotho complex which in turn exerts negative feedback on the rate controlling enzyme of BA synthesis, cholesterol 7α-hydroxylase (CYP7A1). Reabsorbed BAs can also cause negative feedback by directly activating hepatic FXR. However, organ specific Fxr knockout studies in mice indicated a much more prominent role for the FXR-FGF15/19 axis in CYP7A1 repression [
      • Kim I.
      • Ahn S.-H.
      • Inagaki T.
      • Choi M.
      • Ito S.
      • Guo G.L.
      • et al.
      Differential regulation of bile acid homeostasis by the farnesoid X receptor in liver and intestine.
      ].
      The importance of FXR-FGF15/19 signaling in BA homeostasis is illustrated by various BA malabsorption syndromes. Clinically, BA malabsorption causes diarrhea due to high BA concentrations in the colon that lead to secretion of water and electrolytes and stimulation of propulsive contractions [
      • Camilleri M.
      Advances in understanding of bile acid diarrhea.
      ]. Patients with primary BA diarrhea, a condition in which BA malabsorption occurs in the absence of ileal or other obvious GI disease, display lower levels of FGF19 and higher levels of 7α-hydroxy-4-cholesten-3-one (C4), a surrogate marker for BA synthesis [
      • Pattni S.S.
      • Brydon W.G.
      • Dew T.
      • Walters J.R.F.
      Fibroblast growth factor 19 and 7α-hydroxy-4-cholesten-3-one in the diagnosis of patients with possible bile acid diarrhea.
      ,
      • Vijayvargiya P.
      • Camilleri M.
      • Carlson P.
      • Lueke A.
      • O'Neill J.
      • Burton D.
      • et al.
      Performance characteristics of serum C4 and FGF19 measurements to exclude the diagnosis of bile acid diarrhoea in IBS-diarrhoea and functional diarrhoea.
      ]. Additionally, in a murine model of BA malabsorption it has been shown that Fxr activation or Fgf15 administration reduces fecal BA excretion [
      • Jung D.
      • Inagaki T.
      • Gerard R.D.
      • Dawson P.A.
      • Kliewer S.A.
      • Mangelsdorf D.J.
      • et al.
      FXR agonists and FGF15 reduce fecal bile acid excretion in a mouse model of bile acid malabsorption.
      ].
      Even though CF patients display BA malabsorption, (BA) diarrhea is rare and constipation due to inspissated mucus and delayed intestinal transit is more common (further explained in section 6). However, BA malabsorption in CF patients does result in impaired FXR-FGF19 signaling. Our group recently demonstrated that plasma FGF19 levels are lower and C4 levels higher in CF patients with a G551D gating-mutation as compared to healthy controls [
      • Bodewes F.A.J.A.
      • Doktorova M.
      • van de Peppel I.P.
      • van der Ley C.
      • Jonker J.W.
      • Verkade H.J.
      Ivacaftor restores the enterohepatic feedback regulation of the bile acid homeostasis in patients with a Cftr G551D mutation.
      ]. Conversely, improving CTFR function by treatment with the CFTR potentiator ivacaftor, altered the levels of these parameters towards normality, supporting a role for CFTR involvement in BA homeostasis. These results also indicate the possibility of using plasma C4 and FGF19 as surrogate biomarkers for CFTR function in the GI tract in CF. This is important as currently GI markers in CF are limited and/or difficult to obtain [
      • Bodewes F.A.J.A.
      • Verkade H.J.
      • Taminiau J.A.J.M.
      • Borowitz D.
      • Wilschanski M.
      Cystic fibrosis and the role of gastrointestinal outcome measures in the new era of therapeutic CFTR modulation.
      ].
      BA homeostasis has been more extensively studied in CF mouse models than in CF patients. Debray et al. showed that Cftr−/− mice have lower ileal expression levels of Fgf15 compared to wild type controls, while having similar fecal BA excretion rates [
      • Debray D.
      • Rainteau D.
      • Barbu V.
      • Rouahi M.
      • El Mourabit H.
      • Lerondel S.
      • et al.
      Defects in gallbladder emptying and bile acid homeostasis in mice with cystic fibrosis transmembrane conductance regulator deficiencies.
      ]. Interestingly, most other studies in CF mouse models showed an about 3-fold increase in fecal BA excretion, similar to what has been observed in CF patients, but did not directly assess FXR-FGF15/19 signaling [
      • Bijvelds M.J.C.
      • Jorna H.
      • Verkade H.J.
      • Bot A.G.M.
      • Hofmann F.
      • Agellon L.B.
      • et al.
      Activation of CFTR by ASBT-mediated bile salt absorption.
      ,
      • Bodewes F.A.J.A.
      • van der Wulp M.Y.M.
      • Beharry S.
      • Doktorova M.
      • Havinga R.
      • Boverhof R.
      • et al.
      Altered intestinal bile salt biotransformation in a cystic fibrosis (Cftr−/−) mouse model with hepato-biliary pathology.
      ]. This discrepancy in fecal BA excretion might be explained by genotypic and dietary differences between studies and requires further investigation [
      • Bijvelds M.J.C.
      • De Jonge H.R.
      • Verkade H.J.
      Bile acid handling in cystic fibrosis: Marked phenotypic differences between mouse models.
      ]. Indeed, unpublished data from our group confirms lower ileal expression levels of Fgf15 in combination with increased hepatic Cyp7a1 levels in Cftr−/− mice, supporting the disruption of FXR-FGF15/19 signaling. The exact mechanism underlying the impaired FXR-FGF15/19 signaling in CF, however, remains unknown.
      In wild type mice it has been shown that uptake of BAs by Asbt activates Cftr [
      • Bijvelds M.J.C.
      • Jorna H.
      • Verkade H.J.
      • Bot A.G.M.
      • Hofmann F.
      • Agellon L.B.
      • et al.
      Activation of CFTR by ASBT-mediated bile salt absorption.
      ]. Altered functionality or expression of ASBT might also be involved in BA malabsorption in CF. However, results on ileal Asbt expression in murine CF models have been conflicting, [
      • Bijvelds M.J.C.
      • Jorna H.
      • Verkade H.J.
      • Bot A.G.M.
      • Hofmann F.
      • Agellon L.B.
      • et al.
      Activation of CFTR by ASBT-mediated bile salt absorption.
      ,
      • Debray D.
      • Rainteau D.
      • Barbu V.
      • Rouahi M.
      • El Mourabit H.
      • Lerondel S.
      • et al.
      Defects in gallbladder emptying and bile acid homeostasis in mice with cystic fibrosis transmembrane conductance regulator deficiencies.
      ,
      • Stelzner M.
      • Somasundaram S.
      • Lee S.P.
      • Kuver R.
      Ileal mucosal bile acid absorption is increased in Cftr knockout mice.
      ], likely due to differences in experimental setups. Additionally, gene expression might not adequately reflect protein expression or activity. When looking at protein abundance, Debray et al. [
      • Debray D.
      • Rainteau D.
      • Barbu V.
      • Rouahi M.
      • El Mourabit H.
      • Lerondel S.
      • et al.
      Defects in gallbladder emptying and bile acid homeostasis in mice with cystic fibrosis transmembrane conductance regulator deficiencies.
      ] found decreased expression of Asbt by western blot in Cftr−/− mice, while Bijvelds et al. [
      • Bijvelds M.J.C.
      • Jorna H.
      • Verkade H.J.
      • Bot A.G.M.
      • Hofmann F.
      • Agellon L.B.
      • et al.
      Activation of CFTR by ASBT-mediated bile salt absorption.
      ] showed a robust Asbt immunohistochemistry staining pattern and intensity in both Cftr−/− and F508del-Cftr mice indistinguishable from WT mice. The regulation of ASBT expression is complex and can be influenced by many factors including intestinal BA concentrations and microbial composition [
      • Dawson P.A.
      • Lan T.
      • Rao A.
      Bile acid transporters.
      ]. ASBT expression is under a negative feedback regulation by intestinal BA concentrations in mice and humans [
      • Neimark E.
      • Chen F.
      • Li X.
      • Shneider B.L.
      Bile acid-induced negative feedback regulation of the human ileal bile acid transporter.
      ,
      • Chen F.
      • Ma L.
      • Dawson P.A.
      • Sinal C.J.
      • Sehayek E.
      • Gonzalez F.J.
      • et al.
      Liver receptor homologue-1 mediates species- and cell line-specific bile acid-dependent negative feedback regulation of the apical sodium-dependent bile acid transporter.
      ]. The proposed mechanism of this feedback regulation is that BAs activate FXR in the enterocyte which leads to subsequent small heterodimer partner (SHP) and liver receptor homologue-1 (LRH-1) activation which downregulate ASBT expression. This hypothesis is supported by the fact that BA depletion by feeding mice a BA binding resin increased Asbt expression [
      • Torchia E.C.
      • Cheema S.K.
      • Agellon L.B.
      Coordinate regulation of bile acid biosynthetic and recovery pathways.
      ]. However, Debray et al. showed, besides decreased Asbt expression in Cftr−/− mice, a decrease in expression of ileal Fxr target genes, Fgf15 and Shp, arguing against suppression of ASBT by FXR [
      • Debray D.
      • Rainteau D.
      • Barbu V.
      • Rouahi M.
      • El Mourabit H.
      • Lerondel S.
      • et al.
      Defects in gallbladder emptying and bile acid homeostasis in mice with cystic fibrosis transmembrane conductance regulator deficiencies.
      ]. Conversely, while decreased intestinal BA concentrations seemed to induce ASBT expression, Stravitz et al. showed an induction of Asbt gene and protein expression in rats by feeding cholic acid or perfusing the intestine with taurocholic acid [
      • Stravitz R.T.
      • Sanyal A.J.
      • Pandak W.M.
      • Vlahcevic Z.R.
      • Beets J.W.
      • Dawson P.A.
      Induction of sodium-dependent bile acid transporter messenger RNA, protein, and activity in rat ileum by cholic acid.
      ]. Intestinal dysbiosis or SIBO could contribute to altered ASBT expression in CF. Germfree or antibiotic treated mice generally show higher expression levels of Asbt and a decrease in fecal BA excretion [
      • Sayin S.I.
      • Wahlström A.
      • Felin J.
      • Jäntti S.
      • Marschall H.U.
      • Bamberg K.
      • et al.
      Gut microbiota regulates bile acid metabolism by reducing the levels of tauro-beta-muricholic acid, a naturally occurring FXR antagonist.
      ,
      • Out C.
      • Patankar J.V.
      • Doktorova M.
      • Boesjes M.
      • Bos T.
      • De Boer S.
      • et al.
      Gut microbiota inhibit Asbt-dependent intestinal bile acid reabsorption via Gata4.
      ,
      • Miyata M.
      • Takamatsu Y.
      • Kuribayashi H.
      • Yamazoe Y.
      Administration of ampicillin elevates hepatic primary bile acid synthesis through suppression of ileal fibroblast growth factor 15 expression.
      ]. Higher expression levels of Asbt were also observed more proximal in the intestine when mice were treated with antibiotics, regulated through reduced expression of the transcription factor Gata4 [
      • Out C.
      • Patankar J.V.
      • Doktorova M.
      • Boesjes M.
      • Bos T.
      • De Boer S.
      • et al.
      Gut microbiota inhibit Asbt-dependent intestinal bile acid reabsorption via Gata4.
      ].
      Next to their indirect effect on ASBT expression, microbiota are also directly involved in BA homeostasis and FXR-FGF15/19 signaling. Different species of intestinal microbiota have the ability to biotransform BAs mainly by deconjugation and subsequent de-hydroxylation, the latter resulting in more hydrophobic secondary BAs. The various BA species have different properties regarding absorption and receptor activation. Many species of microbiota express bile salt hydrolase (BSH) activity which results in deconjugation of part of the luminal BAs, thereby making them unable to be reabsorbed by ASBT but, due to decreased polarity (more hydrophobic), easier to be passively reabsorbed. Germ-free mice lack the microbial ability to deconjugate BAs, resulting in higher concentrations of tauro-β-muricholic acid, a conjugated BA that acts as an Fxr antagonist that can lower Fgf15 levels [
      • Sayin S.I.
      • Wahlström A.
      • Felin J.
      • Jäntti S.
      • Marschall H.U.
      • Bamberg K.
      • et al.
      Gut microbiota regulates bile acid metabolism by reducing the levels of tauro-beta-muricholic acid, a naturally occurring FXR antagonist.
      ].
      CF patients and murine CF models display intestinal dysbiosis and sometimes even bacterial overgrowth, potentially affecting BA handling. Cftr−/− mice generally show a decreased intestinal microbiota biodiversity with an increase in species associated with inflammation [
      • Lynch S.V.
      • Goldfarb K.C.
      • Wild Y.K.
      • Kong W.
      • De Lisle R.C.
      • Brodie E.L.
      Cystic fibrosis transmembrane conductance regulator knockout mice exhibit aberrant gastrointestinal microbiota.
      ]. Decreases within the Bacteroides and Firmicutes phyla were observed in Cftr−/− mice as well as CF patients [
      • Li L.
      • Somerset S.
      The clinical significance of the gut microbiota in cystic fibrosis and the potential for dietary therapies.
      ]. The Firmicutes phylum contains species known to be involved in de-hydroxylation of BAs [
      • Ridlon J.M.
      • Kang D.-J.
      • Hylemon P.B.
      Bile salt biotransformations by human intestinal bacteria.
      ]. A decrease in one of the most important secondary BAs, deoxycholic acid (DCA), has been observed in feces of Cftr−/− mice [
      • Bodewes F.A.J.A.
      • Bijvelds M.J.
      • De Vries W.
      • Baller J.F.W.
      • Gouw A.S.H.
      • De Jonge H.R.
      • et al.
      Cholic acid induces a Cftr dependent biliary secretion and liver growth response in mice.
      ,
      • Bodewes F.A.J.A.
      • van der Wulp M.Y.M.
      • Beharry S.
      • Doktorova M.
      • Havinga R.
      • Boverhof R.
      • et al.
      Altered intestinal bile salt biotransformation in a cystic fibrosis (Cftr−/−) mouse model with hepato-biliary pathology.
      ]. These observations suggest an important relation between intestinal dysbiosis in CF and BA handling. The exact role, however, of microbiota in BA homeostasis in CF remains to be elucidated.

      4. Bile acid homeostasis and its relation to metabolic function in cystic fibrosis

      BAs are primarily known as detergents with their main function to aid in digestion and absorption of fat and fat soluble vitamins. However, recent studies show that BAs also act as ligands for receptors resulting in the release of hormones that affect other metabolic processes in the body such as glucose metabolism [
      • Kuipers F.
      • Bloks V.W.
      • Groen A.K.
      Beyond intestinal soap--bile acids in metabolic control.
      ]. CF patients often display metabolic abnormalities, such as hyperglycemia, hypertriglyceridemia or steatosis, and these are more common with increasing age [
      • Georgiopoulou V.V.
      • Denker A.
      • Bishop K.L.
      • Brown J.M.
      • Hirsh B.
      • Wolfenden L.
      • et al.
      Metabolic abnormalities in adults with cystic fibrosis.
      ]. Table 1 summarizes several of the metabolic effects of BA homeostasis and metabolic abnormalities in CF patients. Malnutrition is generally regarded as an important problem in CF management. However, due to better nutritional and supportive therapy, the prevalence of malnutrition in CF has declined and some CF patients even develop overweight or obesity [
      • Hanna R.M.
      • Weiner D.J.
      Overweight and obesity in patients with cystic fibrosis: a center-based analysis.
      ]. A recent study showed that some CF patients display “normal weight obesity”, defined by a normal body mass index (BMI) but increased fat percentage that was associated with decreased pulmonary function [
      • Alvarez J.A.
      • Ziegler T.R.
      • Millson E.C.
      • Stecenko A.A.
      Body composition and lung function in cystic fibrosis and their association with adiposity and normal-weight obesity.
      ].
      Table 1Summary of the metabolic effects of bile acid homeostasis and the phenotype in CF as discussed in Section 4. BA: bile acid; CF: cystic fibrosis; TGR5: G-protein coupled bile acid receptor 1; FGF19: fibroblast growth factor 19; GLP-1: glucagon-like peptide-1; CFRD: cystic fibrosis related diabetes.
      Effects of BA homeostasisResultFindings in CF patients
      Lipid metabolismCatabolism of cholesterol for BA synthesisLower plasma cholesterolOften low plasma cholesterol [
      • Georgiopoulou V.V.
      • Denker A.
      • Bishop K.L.
      • Brown J.M.
      • Hirsh B.
      • Wolfenden L.
      • et al.
      Metabolic abnormalities in adults with cystic fibrosis.
      ,
      • Figueroa V.
      • Milla C.
      • Parks E.J.
      • Schwarzenberg S.J.
      • Moran A.
      Abnormal lipid concentrations in cystic fibrosis.
      ]

      BA synthesis is elevated [
      • Bodewes F.A.J.A.
      • Doktorova M.
      • van de Peppel I.P.
      • van der Ley C.
      • Jonker J.W.
      • Verkade H.J.
      Ivacaftor restores the enterohepatic feedback regulation of the bile acid homeostasis in patients with a Cftr G551D mutation.
      ]
      Glucose metabolismIntestinal bile acids activate TGR5 signaling and GLP-1 releaseGLP-1 improves post-prandial insulin secretionReduced GLP-1 release in CF patients [
      • Hillman M.
      • Eriksson L.
      • Mared L.
      • Helgesson K.
      • Landin-Olsson M.
      Reduced levels of active GLP-1 in patients with cystic fibrosis with and without diabetes mellitus.
      ,
      • Perano S.J.
      • Couper J.J.
      • Horowitz M.
      • Martin A.J.
      • Kritas S.
      • Sullivan T.
      • et al.
      Pancreatic enzyme supplementation improves the incretin hormone response and attenuates postprandial glycemia in adolescents with cystic fibrosis: a randomized crossover trial.
      ,
      • Kuo P.
      • Stevens J.E.
      • Russo A.
      • Maddox A.
      • Wishart J.M.
      • Jones K.L.
      • et al.
      Gastric emptying, incretin hormone secretion, and postprandial glycemia in cystic fibrosis - effects of pancreatic enzyme supplementation.
      ]
      Intestinal bile acids activate FXR to release FGF19FGF19 inhibits bile acid synthesis and improves insulin sensitivityLower FGF19 due to reduced BA absorption [
      • Bodewes F.A.J.A.
      • Doktorova M.
      • van de Peppel I.P.
      • van der Ley C.
      • Jonker J.W.
      • Verkade H.J.
      Ivacaftor restores the enterohepatic feedback regulation of the bile acid homeostasis in patients with a Cftr G551D mutation.
      ]
      BA homeostasis is also highly involved in lipid and cholesterol metabolism [
      • Kuipers F.
      • Bloks V.W.
      • Groen A.K.
      Beyond intestinal soap--bile acids in metabolic control.
      ]. Conversion of cholesterol to BAs and subsequent fecal excretion is one of the main routes of cholesterol disposal. Most CF patients display serum low-density lipoprotein cholesterol (LDL-C) levels that are lower compared to control subjects of similar age [
      • Georgiopoulou V.V.
      • Denker A.
      • Bishop K.L.
      • Brown J.M.
      • Hirsh B.
      • Wolfenden L.
      • et al.
      Metabolic abnormalities in adults with cystic fibrosis.
      ,
      • Figueroa V.
      • Milla C.
      • Parks E.J.
      • Schwarzenberg S.J.
      • Moran A.
      Abnormal lipid concentrations in cystic fibrosis.
      ]. However, some CF patients display elevated LDL-C and triglyceride levels which are associated with older age and other metabolic abnormalities such as a high BMI and lower insulin sensitivity [
      • Ishimo M.-C.
      • Belson L.
      • Ziai S.
      • Levy E.
      • Berthiaume Y.
      • Coderre L.
      • et al.
      Hypertriglyceridemia is associated with insulin levels in adult cystic fibrosis patients.
      ,
      • Coderre L.
      • Fadainia C.
      • Belson L.
      • Belisle V.
      • Ziai S.
      • Maillhot G.
      • et al.
      LDL-cholesterol and insulin are independently associated with body mass index in adult cystic fibrosis patients.
      ,
      • Rhodes B.
      • Nash E.F.
      • Tullis E.
      • Pencharz P.B.
      • Brotherwood M.
      • Dupuis A.
      • et al.
      Prevalence of dyslipidemia in adults with cystic fibrosis.
      ]. With increasing age the incidence of CF related diabetes (CFRD) is also rising. The pathophysiology of CFRD is not completely understood but its incidence is correlated with EPI and fibrosis [
      • Kelly A.
      • Moran A.
      Update on cystic fibrosis-related diabetes.
      ]. A combination of partial insulin deficiency and episodes of insulin resistance are often present. Interestingly, CF patients display lower levels of glucagon like peptide 1 (GLP-1), an incretin hormone regulating postprandial insulin secretion, that is improved by pancreatic enzyme replacement therapy [
      • Hillman M.
      • Eriksson L.
      • Mared L.
      • Helgesson K.
      • Landin-Olsson M.
      Reduced levels of active GLP-1 in patients with cystic fibrosis with and without diabetes mellitus.
      ,
      • Perano S.J.
      • Couper J.J.
      • Horowitz M.
      • Martin A.J.
      • Kritas S.
      • Sullivan T.
      • et al.
      Pancreatic enzyme supplementation improves the incretin hormone response and attenuates postprandial glycemia in adolescents with cystic fibrosis: a randomized crossover trial.
      ,
      • Kuo P.
      • Stevens J.E.
      • Russo A.
      • Maddox A.
      • Wishart J.M.
      • Jones K.L.
      • et al.
      Gastric emptying, incretin hormone secretion, and postprandial glycemia in cystic fibrosis - effects of pancreatic enzyme supplementation.
      ]. Intestinal activation of the G-protein coupled bile acid receptor 1 (GPBAR1, GPCR19 also known as TGR5) by BAs enhances the secretion of GLP-1 [
      • Thomas C.
      • Gioiello A.
      • Noriega L.
      • Strehle A.
      • Oury J.
      • Rizzo G.
      • et al.
      TGR5-Mediated Bile Acid Sensing Controls Glucose Homeostasis.
      ]. Secondary BAs lithocholic acid (LCA) and DCA are the most potent naturally occurring TGR5 agonists. As these BA species are lower in murine CF models, it is tempting to speculate that TGR5 activation is reduced in CF due to impaired BA homeostasis. Unfortunately, however, no studies directly addressing this relationship have been performed.
      In addition to the role of TGR5, the FXR-FGF15/19 axis has been implicated in other aspects of glucose metabolism and metabolic disorders. In humans, both obesity and type 2 diabetes mellitus (T2DM) are associated with lower plasma FGF19 levels [
      • Roesch S.L.
      • Styer A.M.
      • Wood G.C.
      • Kosak Z.
      • Seiler J.
      • Benotti P.
      • et al.
      Perturbations of fibroblast growth factors 19 and 21 in type 2 diabetes.
      ,
      • Gallego-Escuredo J.M.
      • Gómez-Ambrosi J.
      • Catalan V.
      • Domingo P.
      • Giralt M.
      • Frühbeck G.
      • et al.
      Opposite alterations in FGF21 and FGF19 levels and disturbed expression of the receptor machinery for endocrine FGFs in obese patients.
      ]. A derivative of the naturally occurring FXR agonist CDCA, 6α-ethyl-CDCA (obeticholic acid, OCA), was recently approved by the Food and Drug Administration (FDA) for the treatment of primary biliary cholangitis (PBC), and is in clinical trials for treatment of non-alcoholic steatohepatitis. Administration of OCA to patients with non-alcoholic fatty liver disease and T2DM increased plasma FGF19 levels, decreased liver enzymes (alanine aminotransferase and γ-glutamyltransferase) and improved insulin sensitivity [
      • Mudaliar S.
      • Henry R.R.
      • Sanyal A.J.
      • Morrow L.
      • Marschall H.U.
      • Kipnes M.
      • et al.
      Efficacy and safety of the farnesoid x receptor agonist Obeticholic acid in patients with type 2 diabetes and nonalcoholic fatty liver disease.
      ]. In a phase 3 clinical trial, OCA ameliorated histology scores of non-alcoholic steatohepatitis patients [
      • Neuschwander-Tetri B.A.
      • Loomba R.
      • Sanyal A.J.
      • Lavine J.E.
      • Van Natta M.L.
      • Abdelmalek M.F.
      • et al.
      Farnesoid X nuclear receptor ligand obeticholic acid for non-cirrhotic, non-alcoholic steatohepatitis (FLINT): a multicentre, randomised, placebo-controlled trial.
      ]. Animal studies, however, have not generated unambiguous results regarding the effects of the FXR-FGF15/19 axis on metabolism. Transgenic mice with hepatic overexpression of FGF19 display an increased metabolic rate and decreased adiposity [
      • Tomlinson E.
      • Fu L.
      • John L.
      • Hultgren B.
      • Huang X.
      • Renz M.
      • et al.
      Transgenic mice expressing human fibroblast growth factor-19 increased metabolic rate and decreased adiposity.
      ]. Kir et al. found that administration of FGF19 to WT mice improved glucose metabolism by inducing hepatic glycogen and protein synthesis [
      • Kir S.
      • Beddow S.A.
      • Samuel V.T.
      • Miller P.
      • Previs S.F.
      • Suino-Powell K.
      • et al.
      FGF19 as a postprandial, insulin-independent activator of hepatic protein and glycogen synthesis.
      ]. Conversely, Fgf15−/− mice showed glucose intolerance and a reduced hepatic glycogen content. Administration of FGF19 was even found to reverse diabetes mellitus in ob/ob mice [
      • Fu L.
      • John L.M.
      • Adams S.H.
      • Yu X.X.
      • Tomlinson E.
      • Renz M.
      • et al.
      Fibroblast growth factor 19 increases metabolic rate and reverses dietary and leptin-deficient diabetes.
      ]. A direct involvement of Fxr-Fgf15 signaling was demonstrated by intestinal inactivation of Fxr or reducing intestinal Fxr signaling through remodeling the intestinal microbial profile by the anti-oxidant tempol, both of which reduced diet induced obesity and hepatic triglyceride accumulation in mice [
      • Li F.
      • Jiang C.
      • Krausz K.W.
      • Li Y.
      • Albert I.
      • Hao H.
      • et al.
      Microbiome remodelling leads to inhibition of intestinal farnesoid X receptor signalling and decreased obesity.
      ,
      • Jiang C.
      • Xie C.
      • Li F.
      Intestinal farnesoid X receptor signaling promotes nonalcoholic fatty liver disease.
      ]. The metabolic improvements by tempol were explained by reduced species of Lactobacillus and their BSH activity resulting in increased levels of tauro-β-muricholic acid, a naturally occurring FXR antagonist. This claim was further supported by a study in which administration of glyco-β-muricholic acid (Gly-MCA), a selective FXR inhibitor, reduced obesity and improved related metabolic abnormalities in mice [
      • Jiang C.
      • Xie C.
      • Lv Y.
      • Li J.
      • Krausz K.W.
      • Shi J.
      • et al.
      Intestine-selective farnesoid X receptor inhibition improves obesity-related metabolic dysfunction.
      ]. On the other hand, intestine-specific FXR activation using the intestinally restricted FXR agonist fexaramine, reduced diet-induced obesity, insulin resistance and steatosis in mice fed a HFD [
      • Fang S.
      • Suh J.M.
      • Reilly S.M.
      • Yu E.
      • Osborn O.
      • Lackey D.
      • et al.
      Intestinal FXR agonism promotes adipose tissue browning and reduces obesity and insulin resistance.
      ]. However, a recent study explained the beneficial effects of fexaramine by an indirect effect on the microbiota and activation of TGR5 via pronounced alterations in BA composition [
      • Pathak P.
      • Xie C.
      • Nichols R.G.
      • Ferrell J.M.
      • Boehme S.
      • Krausz K.W.
      • et al.
      Intestine farnesoid X receptor agonist and the gut microbiota activate G-protein bile acid receptor-1 signaling to improve metabolism.
      ].
      As the pathology of CFRD is thought to be mainly due to (relative) insulin deficiency rather than insulin resistance as in T2DM, the effects of modulating the FXR-FGF15/19 axis in CFRD could be less pronounced. On the other hand, CF is associated with direct impairment of BA homeostasis and FXR-FGF15/19 signaling. It will therefore be interesting to evaluate strategies to improve FXR-FGF15/19 signaling in CF, especially in the prevention of metabolic complications such as CFRD and hepatic steatosis.

      5. Bile acid homeostasis and its role in cystic fibrosis liver disease

      Cystic fibrosis liver disease (CFLD) is a severe complication of CF and an independent risk factor for mortality [
      • Rowland M.
      • Gallagher C.G.
      • O'Laoide R.
      • Canny G.
      • Broderick A.
      • Hayes R.
      • et al.
      Outcome in cystic fibrosis liver disease.
      ,
      • Colombo C.
      Liver disease in cystic fibrosis.
      ]. According to the 2016 CF patient registry report, CFLD accounted for 2.7% of mortality in CF patients [

      Cystic Fibrosis Foundation. Annual Data Report 2016 Cystic Fibrosis Foundation Patient Registry 2017.

      ]. CFLD is an umbrella term used to describe various types of liver dysfunction in CF of which the pathophysiology is often not completely understood [
      • Van De Peppel I.P.
      • Bertolini A.
      • Jonker J.W.
      • Bodewes F.A.J.A.
      • Verkade H.J.
      Diagnosis, follow-up and treatment of cystic fibrosis-related liver disease.
      ]. In the liver, CFTR is expressed exclusively at the apical membrane of cholangiocytes lining the bile ducts [
      • Cohn J.A.
      • Strong T.V.
      • Picciotto M.R.
      • Nairn A.C.
      • Collins F.S.
      • Fitz J.G.
      Localization of the cystic fibrosis transmembrane conductance regulator in human bile duct epithelial cells.
      ,
      • Feranchak A.P.
      CFTR: Actin(g) as a master regulator of cholangiocyte function.
      ]. One proposed mechanism for the development of CFLD is that loss of CFTR function leads to obstruction of the bile ducts by thickening of the mucus, eventually resulting in obstructive biliary cirrhosis. Murine CF models generally do not display CFLD except upon ageing [
      • Durie P.R.
      • Kent G.
      • Phillips M.J.
      • Ackerley C.A.
      Characteristic multiorgan pathology of cystic fibrosis in a long-living cystic fibrosis transmembrane regulator knockout murine model.
      ,
      • Wilke M.
      • Buijs-Offerman R.M.
      • Aarbiou J.
      • Colledge W.H.
      • Sheppard D.N.
      • Touqui L.
      • et al.
      Mouse models of cystic fibrosis: Phenotypic analysis and research applications.
      ]. The absence of CFLD in murine CF models might be partially explained by lower BA toxicity due to differences in biliary BA composition between mice and humans. Mice generally have a higher concentration of hydrophilic BAs which is regarded as less cytotoxic since hydrophilic BAs have a lower detergent capacity than hydrophobic BAs [
      • van Nieuwerk C.M.J.
      • Groen A.K.
      • Ottenhoff R.
      • Van Wijland M.
      • Weerman M.A.V.D.B.
      • Tytgati G.N.J.
      • et al.
      The role of bile salt composition in liver pathology of mdr2 (−/−) mice: differences between males and females.
      ]. High concentrations of hydrophobic BAs, mainly DCA, have been associated with increased risk of cholesterol gallstone disease, colon cancer and liver cancer [
      • Ridlon J.M.
      • Kang D.-J.
      • Hylemon P.B.
      Bile salt biotransformations by human intestinal bacteria.
      ,
      • Ridlon J.M.
      • Kang D.-J.
      • Hylemon P.B.
      • Bajaj J.S.
      Bile acids and the Gut microbiome.
      ].
      FXR-FGF15/19 signaling and hepatic FXR activation have been consistently shown to affect liver regeneration and proliferation [
      • Naugler W.E.
      Bile acid flux is necessary for normal liver regeneration.
      ,
      • Zhang L.
      • Wang Y.D.
      • Chen W.D.
      • Wang X.
      • Lou G.
      • Liu N.
      • et al.
      Promotion of liver regeneration/repair by farnesoid X receptor in both liver and intestine in mice.
      ,
      • Uriarte I.
      • Fernandez-Barrena M.G.
      • Monte M.J.
      • Latasa M.U.
      • Chang H.C.Y.
      • Carotti S.
      • et al.
      Identification of fibroblast growth factor 15 as a novel mediator of liver regeneration and its application in the prevention of post-resection liver failure in mice.
      ,
      • Huang W.
      • Ma K.
      • Zhang J.
      • Qatanani M.
      • Cuvillier J.
      • Liu J.
      • et al.
      Nuclear receptor-dependent bile acid signaling is required for normal liver regeneration.
      ]. This regenerative/proliferative response, measured by hepatic staining of the proliferation marker Ki67, is absent in Cftr−/− mice upon feeding cholic acid (CA), a strong FXR agonist [
      • Bodewes F.A.J.A.
      • Bijvelds M.J.
      • De Vries W.
      • Baller J.F.W.
      • Gouw A.S.H.
      • De Jonge H.R.
      • et al.
      Cholic acid induces a Cftr dependent biliary secretion and liver growth response in mice.
      ]. This suggests that a CF liver might have reduced regenerative ability due to BA malabsorption. Interestingly, in humans the presence of CFLD is associated with normalization of fecal BA excretion which could be due to impaired production as declining liver function occurs but this has not been further investigated [
      • O'Brien S.
      • Mulcahy H.
      • Fenlon H.
      • O’Broin A.
      • Casey M.
      • Burke A.
      • et al.
      Intestinal bile acid malabsorption in cystic fibrosis.
      ].
      Currently, ursodeoxycholic acid (UDCA) is the only recommended and widely used drug in the treatment of CFLD. However, the clinical efficacy of UDCA is controversial. The most recent Cochrane review only identified a small number of trials assessing the effectiveness of UDCA [
      • Cheng K.
      • Ashby D.
      • Smyth R.L.
      Ursodeoxycholic acid for cystic fibrosis-related liver disease.
      ]. The authors concluded that there is ‘currently insufficient evidence to justify its routine use in cystic fibrosis’. UDCA treatment is often started early in life to prevent severe CFLD and related complications. This was also challenged by the results of a recent study, showing that treatment with UDCA started earlier in life had no effect on development of severe CFLD [
      • Boelle P.
      • Debray D.
      • Guillot L.
      • Clement A.
      • Corvol H.
      Cystic fibrosis liver disease: outcomes and risk factors in a large cohort of French patients.
      ].
      The effects of UDCA on BA homeostasis and FXR signaling have not been fully elucidated. UDCA increases hepatocellular and cholangiocellular secretion thereby increasing bile flow and reducing biliary toxicity [
      • Beuers U.
      • Trauner M.
      • Jansen P.
      • Poupon R.
      New paradigms in the treatment of hepatic cholestasis: from UDCA to FXR, PXR and beyond.
      ]. UDCA is also suggested to decrease hepatic steatosis in mice [
      • Quintero P.
      • Pizarro M.
      • Solís N.
      • Arab J.P.
      • Padilla O.
      • Riquelme A.
      • et al.
      Bile acid supplementation improves established liver steatosis in obese mice independently of glucagon-like peptide-1 secretion.
      ,
      • Tsuchida T.
      • Shiraishi M.
      • Ohta T.
      • Sakai K.
      • Ishii S.
      Ursodeoxycholic acid improves insulin sensitivity and hepatic steatosis by inducing the excretion of hepatic lipids in high-fat diet-fed KK-A y mice.
      ,
      • Fujita K.
      • Iguchi Y.
      • Une M.
      • Watanabe S.
      Ursodeoxycholic acid suppresses lipogenesis in mouse liver: possible role of the decrease in β-muricholic acid, a farnesoid X receptor antagonist.
      ]. In obese subjects UDCA was reported to lower FGF19 and subsequently increase hepatic bile acid synthesis [
      • Mueller M.
      • Thorell A.
      • Claudel T.
      • Jha P.
      • Koefeler H.
      • Lackner C.
      • et al.
      Ursodeoxycholic acid exerts farnesoid X receptor-antagonistic effects on bile acid and lipid metabolism in morbid obesity.
      ]. The authors explained this as UDCA having FXR antagonistic effects which was supported by showing decreased FXR activation in an avidin biotin complex DNA-assay. However, in vitro assays suggest UDCA has neither FXR agonistic nor antagonistic properties [
      • Fujita K.
      • Iguchi Y.
      • Une M.
      • Watanabe S.
      Ursodeoxycholic acid suppresses lipogenesis in mouse liver: possible role of the decrease in β-muricholic acid, a farnesoid X receptor antagonist.
      ,
      • Zhang Y.
      • Lacerte C.
      • Kansra S.
      • Jackson J.P.
      • Brouwer K.R.
      • Edwards J.E.
      Comparative potency of obeticholic acid and natural bile acids on FXR in hepatic and intestinal in vitro cell models.
      ]. UDCA is readily absorbed and constitutes a predominant part of the BA pool in UDCA treated patients (from 40% in PBC treated patients [
      • Dilger K.
      • Hohenester S.
      • Winkler-Budenhofer U.
      • Bastiaansen B.A.J.
      • Schaap F.G.
      • Rust C.
      • et al.
      Effect of ursodeoxycholic acid on bile acid profiles and intestinal detoxification machinery in primary biliary cirrhosis and health.
      ] to almost 90% in obese patients [
      • Mueller M.
      • Thorell A.
      • Claudel T.
      • Jha P.
      • Koefeler H.
      • Lackner C.
      • et al.
      Ursodeoxycholic acid exerts farnesoid X receptor-antagonistic effects on bile acid and lipid metabolism in morbid obesity.
      ]), which is likely to contribute to the effects on the FXR-FGF15/19 axis. Fujita et al. showed a clear decrease in relative and absolute levels of muricholic acid levels in mouse livers after UDCA treatment, arguing the beneficial effects on hepatic steatosis (at least in mice) might be due to a reduction of the FXR antagonistic muricholic acid species [
      • Fujita K.
      • Iguchi Y.
      • Une M.
      • Watanabe S.
      Ursodeoxycholic acid suppresses lipogenesis in mouse liver: possible role of the decrease in β-muricholic acid, a farnesoid X receptor antagonist.
      ].
      FXR agonism has been shown to protect against hepatoxicity in a rat model of intrahepatic cholestasis [
      • Liu Y.
      • Binz J.
      • Numerick M.J.
      • Dennis S.
      • Luo G.
      • Desai B.
      • et al.
      Hepatoprotection by the farnesoid X receptor agonist GW4064 in rat models of intra- and extrahepatic cholestasis.
      ]. In that study a systemic FXR agonist (i.e. GW4064) was used and effects could therefore be at least partly due to hepatic FXR activation. Direct evidence of the benefits of intestinal FXR-FGF15/19 signaling has been shown by Modica et al. [
      • Modica S.
      • Petruzzelli M.
      • Bellafante E.
      • Murzilli S.
      • Salvatore L.
      • Celli N.
      • et al.
      Selective activation of nuclear bile acid receptor FXR in the intestine protects mice against cholestasis.
      ] who demonstrated that transgenic overexpression of intestinal FXR or administration of FGF19 in mice protects against liver damage in three different models of cholestasis. The beneficial effects were attributed to a reduced BA pool size and more hydrophilic (i.e. less cytotoxic) biliary BA composition. However, FGF19 is a growth factor and is also associated with the induction of liver proliferation and growth of cancer cells [
      • Lin B.C.
      • Desnoyers L.R.
      FGF19 and cancer.
      ]. To overcome the potential tumorigenic effects of FGF19, a modified variant of FGF19 (M70) has been produced, with reduced tumorigenicitiy but retained benefits in cholestatic liver disease in mice [
      • Luo J.
      • Ko B.
      • Elliott M.
      • Zhou M.
      • Lindhout D.A.
      • Phung V.
      • et al.
      Liver Disease: a nontumorigenic variant of FGF19 treats cholestatic liver diseases.
      ,
      • Zhou M.
      • Learned R.M.
      • Rossi S.J.
      • Depaoli A.M.
      • Tian H.
      • Ling L.
      Engineered fibroblast growth factor 19 reduces liver injury and resolves sclerosing cholangitis in Mdr2-deficient mice.
      ].
      These results make intestinal FXR an interesting target in developing treatment and prevention strategies for CFLD. Unfortunately, CF mice have not been an ideal model for CFLD. Recently, however, other potentially more useful CF animal models have been developed, including the CF pig which already shows signs of CFLD at birth [
      • Olivier A.K.
      • Gibson-Corley K.N.
      • Meyerholz D.K.
      Animal models of cystic fibrosis: gastrointestinal, pancreatic, and hepatobiliary disease and pathophysiology.
      ,
      • Lavelle G.M.
      • White M.M.
      • Browne N.
      • Mcelvaney N.G.
      • Reeves E.P.
      Animal models of cystic fibrosis pathology: phenotypic parallels and divergences.
      ]. CF pigs could therefore be interesting to study CFLD [
      • Rogers C.S.
      • Stoltz D.A.
      • Meyerholz D.K.
      • Ostedgaard L.S.
      • Rokhlina T.
      • Taft P.J.
      • et al.
      Disruption of the CFTR gene produces a model of cystic fibrosis in newborn pigs.
      ,
      • Meyerholz D.K.
      • Stoltz D.A.
      • Pezzulo A.A.
      • Welsh M.J.
      Pathology of gastrointestinal organs in a porcine model of cystic fibrosis.
      ].

      6. Modulating bile acid homeostasis to improve gastrointestinal outcomes in cystic fibrosis

      Considering the close relation between BA homeostasis and GI and metabolic function, it is interesting to speculate about the effect of modulating the factors involved. In the previous section the effects of altering FXR-FGF15/19 and TGR5 signaling to improve metabolism were considered. Modulating the FXR-FGF15/19 axis might also ameliorate GI outcomes. In turn, modulating certain GI factors could improve BA homeostasis and metabolic outcomes.
      CF patients often display SIBO or colonic dysbiosis. Not only do these conditions generate direct symptoms including abdominal discomfort, diarrhea and flatulence, they also increase the risk of developing metabolic abnormalities and liver disease [
      • Arslan N.
      Obesity, fatty liver disease and intestinal microbiota.
      ]. The relationship between BAs and intestinal microbiota is complex, with mutual interactions [
      • Ridlon J.M.
      • Kang D.-J.
      • Hylemon P.B.
      • Bajaj J.S.
      Bile acids and the Gut microbiome.
      ,
      • Wahlströ A.
      • Sayin S.I.
      • Marschall H.-U.
      • Bä Ckhed F.
      Intestinal crosstalk between bile acids and microbiota and its impact on host metabolism.
      ]. In the CF intestine, inspissated mucus accumulates, making it easier for harmful bacteria to thrive [
      • Ooi C.Y.
      • Durie P.R.
      Cystic fibrosis from the gastroenterologist's perspective.
      ]. Other factors contributing to an altered microbial profile include a low intestinal pH due to reduced bicarbonate secretion, a longer intestinal transit time and exposure to antibiotics that CF patients frequently receive for (suspected) pulmonary infections. Interestingly, bacterial overgrowth itself has also been suggested to contribute to mucus secretion. Antibiotic treatment aimed at eradication of bacterial overgrowth in Cftr−/− mice reduced mucus accumulation without a major effect on mucin gene expression, suggesting a more direct role for bacteria on mucus secretion by intestinal epithelium [
      • De Lisle R.C.
      • Roach E.A.
      • Norkina O.
      Eradication of small intestinal bacterial overgrowth in the cystic fibrosis mouse reduces mucus accumulation.
      ].
      Treatment with probiotics can also be used to alter the microbiota profile of CF patients. One study reports that administration of the probiotic Lactobacillus Reuteri improved digestive health and inflammation [
      • del Campo R.
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      • et al.
      Improvement of digestive health and reduction in proteobacterial populations in the gut microbiota of cystic fibrosis patients using a Lactobacillus reuteri probiotic preparation: a double blind prospective study.
      ]. The fecal microbial profile changed, showing a decrease in Proteobacteria and an increase of the Firmicutes phylum. As numerous species of the Firmicutes phylum are involved in BA biotransformation, one could speculate that this change affects BA homeostasis. Treatment with another probiotic, Lactobacillus GG, decreased fecal calprotectin, a marker for intestinal inflammation, and changed microbial composition partially towards that of healthy controls [
      • Bruzzese E.
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      ,
      • Bruzzese E.
      • Raia V.
      • Gaudiello G.
      • Polito G.
      • Buccigrossi V.
      • Formicola V.
      • et al.
      Intestinal inflammation is a frequent feature of cystic fibrosis and is reduced by probiotic administration.
      ]. However, in a large trial in CF children, one year of treatment with Lactobacillus GG versus placebo did not affect hospitalization, pulmonary outcomes or BMI [
      • Bruzzese E.
      • Raia V.
      • Ruberto E.
      • Scotto R.
      • Giannattasio A.
      • Bruzzese D.
      • et al.
      Lack of efficacy of Lactobacillus GG in reducing pulmonary exacerbations and hospital admissions in children with cystic fibrosis: a randomised placebo controlled trial.
      ]. Nevertheless, as probiotic use does seem to improve the GI phenotype of CF, it will be interesting to evaluate its effects on BA homeostasis and metabolic function.
      Another GI feature of CF patients is a delayed intestinal transit time, possibly leading to constipation or in severe cases distal intestinal obstruction syndrome (DIOS) [
      • Rovner A.J.
      • Schall J.I.
      • Mondick J.T.
      • Zhuang H.
      • Mascarenhas M.R.
      Delayed small bowel transit in children with cystic fibrosis and pancreatic insufficiency.
      ,
      • Hedsund C.
      • Gregersen T.
      • Joensson I.M.
      • Olesen H.V.
      • Krogh K.
      Gastrointestinal transit times and motility in patients with cystic fibrosis.
      ,
      • Gelfond D.
      • Ma C.
      • Semler J.
      • Borowitz D.
      Intestinal ph and gastrointestinal transit profiles in cystic fibrosis patients measured by wireless motility capsule.
      ]. Interestingly, Bijvelds et al. showed that active BA absorption in the ileum triggered CFTR activation and subsequent local salt and water excretion [
      • Bijvelds M.J.C.
      • Jorna H.
      • Verkade H.J.
      • Bot A.G.M.
      • Hofmann F.
      • Agellon L.B.
      • et al.
      Activation of CFTR by ASBT-mediated bile salt absorption.
      ]. It is therefore tempting to speculate that the absence of this postprandial ileal water release contributes to specific distal localization of obstruction occurring in CF patients. The inability to sufficiently hydrate intestinal content and mucus likely explains the fact that BA malabsorption does not lead to diarrhea in CF.
      As CF patients often suffer from constipation, laxatives are commonly prescribed. Laxative treatment shortens intestinal transit time and is able to alter microbiota and BA homeostasis. In rats, the commonly used laxative polyethylene glycol (PEG) decreased BA dehydroxylation, increasing the amount of primary BAs in the BA pool [
      • van der Wulp M.Y.M.
      • Cuperus F.J.C.
      • Stellaard F.
      • van Dijk T.H.
      • Dekker J.
      • Rings E.H.H.M.
      • et al.
      Laxative treatment with polyethylene glycol does not affect lipid absorption in rats.
      ]. Whole body Cftr knockout mice display a severe intestinal phenotype and need to be kept either on a liquid diet or a solid diet in combination with laxative. A study by De Lisle et al. compared the effects of either a solid diet with PEG or a liquid diet with or without n-acetylcysteine (NAC), a mucolytic agent, on various aspects of the intestinal phenotype [
      • De Lisle R.C.
      • Roach E.
      • Jansson K.
      Effects of laxative and N-acetylcysteine on mucus accumulation, bacterial load, transit, and inflammation in the cystic fibrosis mouse small intestine.
      ]. Laxative treatment had pronounced effects on the intestine and improved markers of intestinal inflammation and reduced bacterial overgrowth. In CF patients, laxative treatment was also found to be associated with a decrease in occurrence of SIBO [
      • Fridge J.L.
      • Conrad C.
      • Gerson L.
      • Castillo R.O.
      • Cox K.
      Risk factors for small bowel bacterial overgrowth in cystic fibrosis.
      ]. Considering these beneficial effects of laxative treatment on the CF intestine, it is tempting to speculate that laxative treatment could decrease fecal BA excretion.

      7. Summary

      The presently increased life span of the CF population changes the frequency and spectrum of symptoms regarding GI and metabolic function including intestinal dysbiosis, constipation, intestinal cancer, liver disease and diabetes. One part of the CF phenotype that recently received more attention is the GI tract, including impaired BA homeostasis, characterized by increased fecal BA excretion and reduced FXR-FGF15/19 signaling. Modulating BA homeostasis directly by altering FXR-FGF15/19 or TGR5 signaling or indirectly by improving intestinal transit or modifying intestinal microbiota are potential strategies to improve other GI and metabolic CF complications. Lastly, emerging research into BA homeostasis and the GI phenotype of CF could provide novel easily measurable surrogate biomarkers (e.g. C4, FGF19). Especially in the current era of new CFTR modulator therapies (e.g. ivacaftor and lumacaftor), the need for such biomarkers has increased.

      Acknowledgements

      None.

      Funding sources

      This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

      Declaration of interest

      None.

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