LRH-1-mediated glucocorticoid synthesis in enterocytes protects against inflammatory bowel disease

Abstract

Liver receptor homolog-1 (LRH-1) is a nuclear receptor involved in intestinal lipid homeostasis and cell proliferation. Here we show that haploinsufficiency of LRH-1 predisposes mice to the development of intestinal inflammation. Besides the increased inflammatory response, LRH-1 heterozygous mice exposed to 2,4,6-trinitrobenzene sulfonic acid show lower local corticosterone production as a result of an impaired intestinal expression of the enzymes CYP11A1 and CYP11B1, which control the local synthesis of corticosterone in the intestine. Local glucocorticoid production is strictly enterocyte-dependent because it is robustly reduced in epithelium-specific LRH-1-deficient mice. Consistent with these findings, colon biopsies of patients with Crohn's disease and ulcerative colitis show reduced expression of LRH-1 and genes involved in the production of glucocorticoids. Hence, LRH-1 regulates intestinal immunity in response to immunological stress by triggering local glucocorticoid production. These findings underscore the importance of LRH-1 in the control of intestinal inflammation and the pathogenesis of inflammatory bowel disease.

Fig. 1.

Lrh-1^+/− mice are more susceptible to TNBS- or DSS-induced colitis than their wild-type littermates. (A) Representative macroscopic view of the colon of Lrh-1^+/+ or Lrh-1^+/− mice 48 h after vehicle (Ctrl) or TNBS administration. (B) Representative H&E staining of colon sections of Lrh-1^+/+ or Lrh-1^+/− mice 48 h after vehicle (Ctrl) or TNBS administration and 12 days after DSS administration. TNBS induces thickening of the colon wall, with predominant inflammatory infiltrate (open arrowheads) present in the lamina propria (lp) and submucosa (sm) of TNBS-treated Lrh-1^+/− mice. Compared with Lrh-1^+/+ mice, necrosis (filled arrowheads) is more severe in Lrh-1^+/− mice, with necrotic regions detected in the colonic epithelium (ep). After DSS administration, colons of Lrh-1^+/+ mice were characterized by a mild inflammatory infiltrate (open arrowheads) and advanced crypt regeneration. In Lrh-1^+/− mice, a massive infiltrate is associated with an important crypt loss and poor epithelium regeneration. (C) Macroscopic and histologic assessment of the colon of Lrh-1^+/+ (n = 21) or Lrh-1^+/− (n = 17) mice 48 h after TNBS administration, using the Wallace and Ameho scores, respectively. Quantification of myeloperoxidase levels was performed by immunoblotting. (D) Assessment of colonic weight/length ratio and histological score of Lrh-1^+/+ (n = 7) and Lrh-1^+/− (n = 7) mice after DSS-induced colitis.

Fig. 2.

Exaggerated inflammatory response in Lrh-1^+/− mice is closely associated with decreased intestinal corticosterone production. (A and B) Effect of LRH-1 haplodeficiency on TNF-α, IL-1β, and IL-6 gene expression in Lrh-1^+/+ (filled bars) or Lrh-1^+/− (open bars) mice after TNBS- or DSS-induced colitis. LRH-1, TNF-α, IL-1β, and IL-6 mRNA levels in the colon of Lrh-1^+/+ or Lrh-1^+/− mice challenged with vehicle or TNBS for 48 h (A) or 12 days after vehicle or DSS administration (B). +, P < 0.05 and ++, P < 0.01 depict significant differences between untreated and treated groups from the same genotype; *, P < 0.05 and **, P < 0.01 depict significant differences between genotypes receiving the same treatment. (C and D) mRNA levels of steroidogenic enzymes CYP11A1 and CYP11B1 (C) and in situ-produced glucocorticoids (D) in colon biopsies of Lrh-1^+/+ (filled bars) or Lrh-1^+/− (open bars) mice killed 24 h after vehicle or TNBS instillation. *, P < 0.05; **, P < 0.01.

Fig. 3.

Gene targeting and tamoxifen-induced deletion of the Lrh-1 gene in the intestinal epithelium. (A) Restriction maps of the Lrh-1 genomic locus, targeting vector, floxed allele +neo, and floxed allele −neo. The indicated probes (5′ probe and 3′ probe) were used to assess recombination events. Boxes represent the respective exons. (B) Southern blot analysis revealing homologous recombination in ES cells electroporated with targeting vector. DNA derived from ES cells was digested with XbaI or SacI. Hybridizing fragments of wild-type (+/+) and floxed (+/L2) alleles to the 5′ probe or the 3′ probe, and their respective sizes, are indicated. (C) Recombination of loxP sites as demonstrated with PCR analysis of genomic DNA from ES cells. Size of PCR products in floxed and wild type are indicated. (D–F) Conditional loss of LRH-1 in intestinal epithelium (Lrh-1^vil−/−) after tamoxifen-induced recombination in Lrh-1^L2/L2 mice mated to vil-Cre-ER^T2 transgenic mice (vil-Cre-ER^T2/Lrh-1^L2/L2). (D) Analysis of recombination of Lrh-1 locus by PCR on genomic DNA isolated from various tissues of Lrh-1^L2/L2 and vil-Cre-ER^T2/Lrh-1^L2/L2 mice treated with olive oil/ethanol (10:1) (Veh) or tamoxifen (Tam). (E) LRH-1 mRNA levels in the colon of Lrh-1^L2/L2 mice (filled bars, n = 4) and vil-Cre-ER^T2/Lrh-1^L2/L2 mice (open bars, n = 4) as determined by quantitative RT-PCR analysis. *, P < 0.05. (F) LRH-1 immunoblotting performed on protein extracts of nuclear membrane and chromatin fraction from colon of Lrh-1 ^L2/L2 and vil-Cre-ER^T2/Lrh-1 ^L2/L2 mice (LRH-1^vil−/−). Specific bands corresponding to long (LRH-1L) and short (LRH-1S) isoforms of mouse LRH-1 are indicated by arrowheads.

Fig. 4.

TNBS-induced intestinal glucocorticoid production is impaired in enterocyte-specific LRH-1-deficient mice and leads to severe colitis. (A) H&E sections from control (Lrh-1^L2/L2) or mutant (Lrh-1^vil−/−) colons 24 h after vehicle (Ctrl) or TNBS administration. Filled arrowheads depict high-degree of necrosis. (Magnification: ×50.) (B) Macroscopic and histological assessment of the colon from Lrh-1^L2/L2 or Lrh-1^vil−/− mice 24 h after TNBS administration. Quantification of myeloperoxidase levels was performed by immunoblotting. (C) LRH-1, TNF-α, IL-1β, and IL-6 mRNA levels in the colon of Lrh-1^L2/L2 (filled bars, n = 7) and Lrh-1^vil−/− (open bars, n = 7) mice challenged with vehicle or TNBS for 24 h. +, P < 0.05 and ++, P < 0.01 depict significant differences between untreated and treated groups from the same genotype; *, P < 0.05 and **, P < 0.01 depict significant differences between genotypes receiving the same treatment. (D and E) mRNA levels of the steroidogenic genes CYP11A1 and CYP11B1 (D) and corticosterone production (E) in the colon biopsies of Lrh-1^L2/L2 (filled bars, n = 7) and Lrh-1^vil−/− (open bars, n = 7) mice challenged with vehicle or TNBS for 24 h.

Fig. 5.

Inflammation in colons of IBD patients is inversely correlated with LRH-1 and downstream targets required for local glucocorticoid synthesis. (A and B) Gene expression profiles of inflammation markers TNF-α, IL-1β, and IL-6 (A) and LRH-1, CYP11A1, and CYP11B1 (B) mRNA in colon biopsies of control subjects (C) (n = 12) or in healthy (H) (n = 12) or inflamed (I) (n = 12) mucosa of patients with CD or UC. *, P < 0.05; **, P < 0.01. (C and D) Representative immunoblots of CD (n = 2) (C) and UC (n = 2) (D) patients showing that the isoforms hLRH-1a (61 kDa) and hLRH-1b (56 kDa) are specifically decreased in the inflamed (I) parts of the colon compared with the healthy (H) part of the colon. No difference between the healthy-looking part of the colon (H) and the colon of control subjects (C) could be observed. β-actin was used as an internal loading control.