Hepatocyte nuclear factor 4alpha in the intestinal epithelial cells protects against inflammatory bowel disease

Abstract

Background: Hepatocyte nuclear factor 4alpha (HNF4alpha; NR2A1) is an orphan member of the nuclear receptor superfamily expressed in liver and intestine. While HNF4alpha expression is critical for liver function, its role in the gut and in the pathogenesis of inflammatory bowel disease (IBD) is unknown.

Methods: Human intestinal biopsies from control and IBD patients were examined for expression of mRNAs encoding HNF4alpha and other nuclear receptors. An intestine-specific HNF4alpha null mouse line (Hnf4alpha(DeltaIEpC)) was generated using an Hnf4alpha-floxed allele and villin-Cre transgene. These mice and their control floxed counterparts (Hnf4alpha(F/F)), were subjected to a dextran sulfate sodium (DSS)-induced IBD colitis protocol and their clinical symptoms and gene expression patterns determined.

Results: In human intestinal biopsies, HNF4alpha was significantly decreased in intestinal tissues from Crohn's disease and ulcerative colitis patients. HNF4alpha expression was also suppressed in the intestine of DSS-treated mice. In Hnf4alpha(DeltaIEpC) mice, disruption of HNF4alpha expression was observed in the epithelial cells throughout the intestine. In the DSS-induced colitis model Hnf4alpha(DeltaIEpC) mice showed markedly more severe changes in clinical symptoms and pathologies associated with IBD including loss of body weight, colon length, and histological morphology as compared with Hnf4alpha(F/F) mice. Furthermore, the Hnf4alpha(DeltaIEpC) mice demonstrate a significant alteration of mucin-associated genes and increased intestinal permeability, which may play an important role in the increased susceptibility to acute colitis following an inflammatory insult.

Conclusions: While HNF4alpha does not have a major role in normal function of the intestine, it protects the gut against DSS-induced colitis.

Fig. 1

Nuclear receptor gene expression in intestinal samples from human IBD patients. The gene expression of hepatocyte nuclear factor 4α (HNF4α), farnesoid X receptor (FXR), liver X receptor (LXR), mineralocorticoid receptor (MR), peroxisome proliferator-activated receptor α, β, γ (PPARα, PPARβ, PPARγ), vitamin D receptor (VDR), and pregnane X receptor (PXR) were performed by qPCR to assess their gene expression in human IBD patient tissues. Mann-Whitney U test was performed to assess significant differences between normal and patients group. P values are expressed as *, P<0.05, ** P<0.01, and ***, P<0.001.

Fig. 2

HNF4α gene expression after DSS treatment. Expression of HNF4α mRNA, as measured by quantitative real-time PCR (qPCR), was decreased at day 7 after 2.5% DSS and 5% DSS treatment (A). Expression of HNF4α mRNA was decreased at day 1, 3, 5, and 7 after 2.5% DSS treatment (B). HNF4α protein, as measured by western blot analysis, was also decreased at day 1, 3, 5, and 7 after 2.5% DSS treatment and relative density to β-actin was decreased at day 1, 3, 5, and 7 after 2.5% DSS treatment (C). Both relative mRNA and protein levels of HNF4α were decreased after DSS treatment. Data represent the mean value ± standard deviations, ***, p<0.001 compared with control mice.

Fig. 3

Generation of the intestinal epithelial cells-specific Hnf4α-null, Hnf4α^ΔIEpC mice. Schematic of generation of intestinal specific Hnf4α-null mice (A) and their genotypes by PCR (B) using microsomal epoxide hydrolase (mEH) primers as a positive control for amplication. The loss of intestine-specific HNF4α gene were analyzed by northern blot analysis (C) and immunohistochemical analysis (D). Immunohistochemistry analysis with HNF4α antibody of Hnf4α^F/F (D; a, c, and e) and Hnf4α^ΔIEpC (D; b, d, and f) mice in duodenum (a and b), jejunum (c and d), ileum (e and f). Note the loss of HNF4α-positive cells in nucleus from Hnf4α^F/F mice. HNF4α related gene expression as assessed by northern blot analysis in whole intestine of Hnf4α^F/F and Hnf4α^ΔIEpC mice. Pooled total RNA (n=7 for each genotype) was isolated from liver, kidney, duodenum, jejunum, ileum, and colon (L, K, D, J, I, and C, respectively).

Fig. 4

Histological and immunohistochemical analysis of the intestine in Hnf4α^F/F and Hnf4α^ΔIEpC mice by H&E stained intestine sections (A), ultrastructure (B), PAS, and alcian blue stain(C). H&E stained intestine sections from Hnf4α^F/F (A; left panel; a, c, e, and g) and Hnf4α^ΔIEpC (A; right panel; b, d, f, and h) mice (original magnification X 200) and ultrastructure of the colon from Hnf4α^F/F (i) and Hnf4α^ΔIEpC (j) mice. N; nucleus, MG; mucous granules. Immunohistochemistry of HNF4α, and polysaccharide and acidic mucopolysaccharide stain in the colon of Hnf4α^F/F and Hnf4α^ΔIEpC mice (C). Immunohistochemistry against HNF4α antibody (k and l), PAS (m and n), and Alcian blue stain (o and p) from in the colon of Hnf4α^F/F (C; left panel) and Hnf4α^ΔIEpC (C; right panel) mice.

Fig. 5

Assessment of DSS-induced IBD in Hnf4α^F/F and Hnf4α^ΔIEpC mice. Lack of HNF4α in intestinal epithelial cells is associated with increased susceptibility to DSS-induced IBD. Daily changes in body weight following DSS treatment (A), loss of body weight at day 5 after DSS treatment (B), picture of the isolated colon from Hnf4α^F/F and Hnf4α^ΔIEpC mice with or without DSS treatment (C), and colon length, which was shortened by severe IBD (D). Data represent the mean value ± standard deviations of n=14 for each group, *, p<0.05, and ***, p<0.001 compared with DSS-treated Hnf4α^F/F mice.

Fig. 6

Histological assessment of 5-day DSS or control treatment in Hnf4α^F/F and Hnf4α^ΔIEpC mice. H&E stained colon sections from Hnf4α^F/F (A) and Hnf4α^ΔIEpC (B) mice following control treatment, Hnf4α^F/F (C) and Hnf4α^ΔIEpC (D) mice after 5-day DSS treatment. Histological score was analyzed from colonic H&E stained sections of Hnf4α^F/F and Hnf4α^ΔIEpC mice after 5-day DSS treatment (E). Data represent the mean value ± standard deviations, *, p<0.05 compared with DSS-treated Hnf4α^F/F mice.

Fig. 7

Cytokine gene expression from colon tissue following 5-day DSS or control treatment in Hnf4α^F/F and Hnf4α^ΔIEpC mice. IL-1β, IL-6, IL-10, TNFα, IFNγ, iNOS, CCR2, MCP-1, and ICAM-1 mRNA expressions were analyzed by qPCR. Expression was normalized to 36B4. Data represent the mean value ± standard deviations of n=8 for each group, *, p<0.05 compared with DSS-treated Hnf4α^F/F mice.

Fig. 8

In vivo intestinal permeability assay. Intestinal permeability using a FITC-labeled-dextran method were measured at 2, 4, and 8 hr of fluorescence units after FITC-dextran oral administration following 3-day DSS treatment between Hnf4α^F/F and Hnf4α^ΔIEpC mice. Data represent the mean value ± standard deviations, * p<0.05.