Impact of global Fxr deficiency on experimental acute pancreatitis and genetic variation in the FXR locus in human acute pancreatitis

Abstract

Background: Infectious complications often occur in acute pancreatitis, related to impaired intestinal barrier function, with prolonged disease course and even mortality as a result. The bile salt nuclear receptor farnesoid X receptor (FXR), which is expressed in the ileum, liver and other organs including the pancreas, exhibits anti-inflammatory effects by inhibiting NF-κB activation and is implicated in maintaining intestinal barrier integrity and preventing bacterial overgrowth and translocation. Here we explore, with the aid of complementary animal and human experiments, the potential role of FXR in acute pancreatitis.

Methods: Experimental acute pancreatitis was induced using the CCK-analogue cerulein in wild-type and Fxr-/- mice. Severity of acute pancreatitis was assessed using histology and a semi-quantitative scoring system. Ileal permeability was analyzed in vitro by Ussing chambers and an in vivo permeability assay. Gene expression of Fxr and Fxr target genes was studied by quantitative RT-PCR. Serum FGF19 levels were determined by ELISA in acute pancreatitis patients and healthy volunteers. A genetic association study in 387 acute pancreatitis patients and 853 controls was performed using 9 tagging single nucleotide polymorphisms (SNPs) covering the complete FXR gene and two additional functional SNPs.

Results: In wild-type mice with acute pancreatitis, ileal transepithelial resistance was reduced and ileal mRNA expression of Fxr target genes Fgf15, SHP, and IBABP was decreased. Nevertheless, Fxr-/- mice did not exhibit a more severe acute pancreatitis than wild-type mice. In patients with acute pancreatitis, FGF19 levels were lower than in controls. However, there were no associations of FXR SNPs or haplotypes with susceptibility to acute pancreatitis, or its course, outcome or etiology.

Conclusion: We found no evidence for a major role of FXR in acute human or murine pancreatitis. The observed altered Fxr activity during the course of disease may be a secondary phenomenon.

Figure 1

A – Representative pancreatic histology of wild-type mice from the control group (a, b) and mice with early (c, d) and late (e, f) acute pancreatitis (H&E staining, 20x and 100x magnifications consecutively). Control mice have normal pancreatic morphology, whereas mice of the early pancreatitis group exhibit edema, influx of neutrophils and necrosis. Mice of the late pancreatitis group have no edema or necrosis, but show influx of lymphocytes and fibroblasts. B – Transepithelial electrical resistance of the ileum measured by Ussing chamber experiments. The resistance of the ileum was lower in the early pancreatitis group in comparison to both controls and the late pancreatitis group. C – Ileal mRNA expression of Fxr and FXR target-genes Asbt, Shp, Fgf15, and Ibabp, and Villin in wild-type mice of the control group, and the early and late pancreatitis groups. Expression of Fxr, Asbt and Villin did not differ between experimental groups. Expression of the other Fxr target genes was lower in early acute pancreatitis, but not in late pancreatitis. D – Ileal mRNA expression of genes implicated in intestinal barrier function, iNos, Ang1, Car12, IL18. Expression of Ang1 was lowered in the early pancreatitis group, the other genes remained similar in the different experimental conditions. E – Hepatic expression of Fxr, Shp, iNos and Ang1. Hepatic Ang1 was increased in early pancreatitis, whereas the expression of the other genes was lowered in the early acute pancreatitis group. Expression levels were normalized to cyclophilin expression. Bars indicate means and SEM, * p<0.05, ** p<0.01, *** p<0.001.

Figure 2

A - Representative pancreatic histology following induction of acute pancreatitis (H&E staining, 20x and 100x consecutive magnifications for each experimental group): wild-type control (a,b); wild-type acute pancreatitis (c,d); Fxr^-/- control (e,f); Fxr^-/- acute pancreatitis (g,h). B – Semi-quantitative composite pancreatitis severity score of histopathological examination of pancreas samples from wild-type and Fxr^-/- mice with and without acute pancreatitis. Absence of Fxr does not result in more severe acute pancreatitis. C – Ileal mRNA expression of Fgf15 in wild-type and Fxr^-/- mice with and without early acute pancreatitis. Fgf15 expression was decreased in wild-type mice with acute pancreatitis. Bars indicate mean and SEM.

Figure 3. FGF19 plasma levels in patients with predicted severe acute pancreatitis during continuous enteral nutrition.

For comparison, FGF19 plasma levels of healthy controls in the fasting state and after a single bolus of fat are also shown. The postprandial levels represent the average of plasma FGF19 levels at 2, 3, 4 and 6 hours after fat ingestion. There appears to be a blunted FGF19 release in the pancreatitis group. Bars indicate mean and SD.