Cystic Fibrosis–Related Liver Diseases: New Paradigm for Treatment Based on Pathophysiology

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Abbreviations

CF

cystic fibrosis

CFLD

cystic fibrosis–associated liver disease

CFTR

cystic fibrosis transmembrane conductance regulator

KO

knockout

LPS

lipopolysaccharide

NF‐κB

nuclear factor‐κB

PPAR‐γ

peroxisome proliferator‐activated receptor gamma

TLR

Toll‐like receptor

UDCA

ursodeoxycholic acid

Cystic fibrosis (CF), the most common life‐threatening autosomal recessive disease in Caucasian populations, is caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR), a cyclic adenosine monophosphate–dependent Cl⁻ channel expressed in secretory epithelial cells of various organs.

Almost 2000 different CFTR mutations have been identified and are grouped into five classes based on their functional defect and decreased severity (class I: deficient synthesis; class II: altered CFTR processing; class III: defective gating; class IV: decreased Cl⁻ ion conductance; class V: reduced protein level). Deletion of a phenylalanine residue at position 508 (F508del, class II) is the most common mutation being present in 80% to 90% of patients with CF.1

CF is a complex multisystem disease that affects the epithelia of the respiratory tract, exocrine pancreas, intestine, hepatobiliary system, and sweat glands.2 CFTR is expressed on the apical membrane of the biliary epithelium where it regulates the alkalinity and fluidity of the bile; when CFTR is defective, bile secretion, hydration, and alkalinity are impaired.3 Progressive clinical liver disease occurs in about 10% of the patients but represents the third leading cause of death in CF. Cystic fibrosis–associated liver disease (CFLD) is a chronic inflammatory sclerosing cholangiopathy that may evolve into sclerosing cholangitis and eventually in multilobular cirrhosis, portal hypertension, and advanced liver disease and hepatic decompensation. Cirrhosis and portal hypertension aggravate the respiratory function of these patients and increase the risk for early mortality.4

Current Treatment of CFLD

A cure for CFLD is not yet available. Patients are usually treated with ursodeoxycholic acid (UDCA). UDCA has been shown to modify the bile acid pool and to improve liver biochemistry and some histological parameters in patients with CF; however, its efficacy is still unclear because of the lack of long‐term studies.5

Supportive treatments have significantly increased the prognosis of patients with CF.4 Current efforts in CF research aim at repairing the basic defect, using improvement in pulmonary function as an endpoint. The US Food and Drug Administration has recently approved small molecules that are able to modulate the channel function. Ivacaftor (VX770), a corrector of the gating defect of CFTR, was approved for patients with G551D, a class III mutation, and has shown promising improvements in outcomes, such as sweat chloride concentration, forced expiratory volume in 1 second, body mass index, and exacerbation rate. Lumacaftor (VX‐809), a channel corrector that allows the ΔF508 CFTR to bypass proteomic degradation and increases trafficking of the protein to the plasma membrane, has been very recently approved in combination with ivacaftor (Orkambi) in patients homozygous for F508Δ.6 Unfortunately, the efficacy is not impressive mainly because of the instability and reduced half‐life of the rescued CFTR at the plasma membrane. This is due in part to the inflammatory status of the airways epithelia and in part to more profound targeted defects.7 Thus far, no data have been reported on the effects of these molecules in patients with CFLD. However, recent discoveries in the field of CFLD pathophysiology may open new therapeutic paradigms.

Pathogenesis of CFLD and New Prospective for Therapy

Liver disease in CF has been classically considered a consequence of ductal cholestasis, caused by the defective function of CFTR, with changes in bile flow and composition and retention of hydrophobic bile acids and toxins that damage the biliary epithelium. The injured epithelium then secretes inflammatory mediators that recruit inflammatory and mesenchymal cells into the portal space.3

However, whereas all patients with CF present a biliary secretory defect, clinical data show that severe CFLD occurs in a low percentage of patients with CF. The association with possible modifier genes such as Serpin 1 and alpha‐1‐antitrypsin does not explain the phenotypic variability, suggesting that other insults/factors, in addition to the genetic mutation, are influencing the development and progression of the disease in the liver.

It has been shown that liver inflammation in susceptible individuals can be caused by activation of innate immune responses to bacterial products, coming from the enteric microflora. Once in the liver, gut‐derived endotoxins or pathogen‐associated molecular pattern is normally cleared by the hepatocytes that take up endotoxins and discharge them partly unmodified into the bile via vesicular transport. The biliary epithelium expresses several toll‐like receptors (TLRs), including TLR4, TLR2, and TLR5. In normal conditions, the biliary epithelium is highly tolerant toward TLR ligands due to a process of “endotoxin tolerance” that prevents an excessive inflammatory response after lipopolysaccharide (LPS) stimulation.8

In mouse models of CF, induction of colitis with dextran sodium sulfate, a protocol known to cause increased intestinal permeability and portal release of bacterial products, triggers biliary damage and inflammation in Cftr knockout (Cftr‐KO) mice, but not in wild type mice exposed to the same treatment.9 The biliary damage is characterized by the expansion of the ductular reactive component and portal inflammation, with extensive infiltration of neutrophils. Interestingly, recovery of bile secretion by administration of choleretic bile acids did not limit the liver damage and inflammation. Instead, hepatic inflammation was reduced by bowel decontamination using a combination of oral antibiotics. In addition, cholangiocytes isolated from Cftr‐KO mice showed significantly higher nuclear factor‐κB (NF‐κB) activity and secreted a larger amount of NF‐κB‐dependent inflammatory cytokines when exposed to the TLR4‐ligand LPS.9 By investigating the mechanism that links CFTR with TLR4,10 we have found that, independent of its channel function, CFTR controls TLR signaling and inflammation in secretory epithelia by increasing the activation of Src, a non‐receptor tyrosine kinase. When CFTR is absent from the membrane, uncontrolled kinase activation enhances the response of TLR4 to endotoxins. Moreover, increased Src kinase activation destabilizes the structure of cell–cell junctions and the architecture of the cortical cytoskeleton, thus contributing to the pathogenesis of CFLD. Inhibition of Src in vivo improves the inflammatory phenotype in Cftr‐KO mice exposed to gut‐derived endotoxins, confirming that Src has a pathogenetic role in CF and is a possible target for treatment.

Thus, CFTR deficiency in cholangiocytes alters the normal endotoxin tolerance of the epithelium that responds to gut‐derived endotoxins with a stronger inflammatory reaction. These findings challenge the current paradigm of the pathogenesis of CFLD and suggest that an effective therapeutic approach should also target the TLR4/NF‐κB‐dependent inflammatory pathway. Decreased inflammation and Src activation might, very likely, also improve the efficacy of newly approved combination treatment.

Nuclear receptors, a superfamily of ligand‐dependent transcription factors, might represent a novel strategy to control inflammation in the CF biliary epithelium because among other cell function they are able to modulate inflammation by controlling TLR‐dependent signaling pathways. In a recent study,11 we have shown that the expression of the nuclear receptor peroxisome proliferator‐activated receptor gamma (PPAR‐γ) is upregulated in CFTR‐defective cholangiocytes, and stimulation of PPAR‐γ by pioglitazone and rosiglitazone negatively regulates NF‐kB‐dependent innate immune responses in CF biliary epithelium by stimulating the NF‐kB regulator IkBα.

Conclusion

In conclusion, these new findings indicate that the effective treatment of CFLD should include a combination of synergistic approaches: repairing the basic defect and decreasing inflammation. In addition to the classic anti‐inflammatory agents, many new drugs able to target the signaling mechanisms described earlier are in development.

Figure 1.

Current and new proposed treatment approaches for CFLD based on the pathophysiology of the disease. CFTR is expressed on the apical membrane of cholangiocytes where together with the anion exchanger isoform 2 (AE‐2) they regulate the alkalinity and fluidity of the bile in response to cyclic adenosine monophosphate. The most common mutation among patients with CF is the deletion of a phenylalanine residue at position 508 (ΔF508‐CFTR) that affects CFTR function by preventing its trafficking from the endoplasmic reticulum (ER) to the plasma membrane, causing a gating defect and decreasing its half‐life when rescued. In ΔF508‐CFTR cholangiocytes, bile secretion is impaired and bile composition is altered, resulting in the accumulation of endotoxins and toxic bile acids that damages the epithelium and causes an aberrant TLR4/NF‐kB innate immune response and production of inflammatory cytokines. Current treatment is limited to the administration of UDCA, a hydrophobic bile acid that by modifying the bile acid pool decreases its toxicity and slows down the epithelial damage. However, based on new insights on the pathophysiology of CFLD, a novel therapeutic approach should target inflammation (i.e., nuclear receptor PPAR‐γ agonists) and correct the basic CFTR defect (correctors and potentiators).