Role of the xenobiotic receptor in inflammatory bowel disease

Abstract

Background: Gene-environment interplay modulates inflammatory bowel diseases (IBD). Dioxin-like compounds can activate the aryl hydrocarbon receptor (AhR) and alter macrophage function as well as T-cell polarization. We hypothesized that attenuation of the AhR signaling pathway will ameliorate colitis in a murine model of IBD.

Methods: Dextran sulfate sodium (DSS) colitis was induced in C57BL/6 AhR null mice (AhR(-/-) ), heterozygous mice (AhR(-/+) ), and their wildtype (WT) littermates. Clinical and morphopathological parameters were used to compare the groups.

Patients: AhR pathway activation was analyzed in biopsy specimens from 25 IBD patients and 15 healthy controls.

Results: AhR(-/-) mice died before the end of the treatment. However, AhR(-/+) mice exhibited decreased disease activity compared to WT mice. The AhR(-/+) mice expressed less proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α) (6.1- versus 15.7-fold increase) and IL17 (23.7- versus 67.9-fold increase) and increased antiinflammatory IL-10 (2.3-fold increase) compared with the AhR(+/+) mice in the colon. Colonic macrophage infiltration was attenuated in the AhR(-/+) group. AhR and its downstream targets were significantly upregulated in IBD patients versus control (CYP1A1 -19.9, and IL8- 10-fold increase).

Conclusions: Attenuation of the AhR receptor expression resulted in a protective effect during DSS-induced colitis, while the absence of AhR exacerbated the disease. Abnormal AhR pathway activation in the intestinal mucosa of IBD patients may promote chronic inflammation. Modulation of AhR signaling pathway via the diet, cessation of smoking, or administration of AhR antagonists could be viable strategies for the treatment of IBD.

Figure 1. The severity of DSS-induced colitis is attenuated in AhR ^−/+ mice.

Mice were administered 3.5% DSS dissolved in water for 7 days followed by 3 days of water. Control mice received water alone for 10 days. [A] Kaplan-Meier plot showing premature lethality of male AhR ^−/− when compared with WT and AhR ^−/+ littermates [B] AhR mRNA gene expression in the colonic tissue [C] Body weight [D] Hematocrit [E] Colon length [F] Histopathological grading of colonic inflammation at the end of the experiment. Data are expressed as mean ± SE, n=10/group.

Figure 2. Decreased histological severity in AhR ^−/+ mice during DSS-induced colitis.

[A] H&E-staining of colons from WT [upper panel] and AhR ^−/+ mice [lower panel]. [B] Immunohistochemistry for epithelial cell proliferative index [PCNA] in sections of the colonic tissue of WT [upper panel] and AhR ^−/+ mice [lower panel].

Figure 3. The expression of pro-inflammatory cytokines and macrophage marker are reduced inAhR ^−/+ mice during colitis

[A] TNFα gene expression, [B] TNFα protein level, [C] F4/80 gene expression [D] MCP1 gene expression, [E] IL10 gene expression and [F] IL10 protein level in colonic tissue. Data are expressed as mean ± SE, n=10/group

Figure 4. Differential expression of master regulators for Treg and Th17 cells in the colon of WT and AhR ^−/+ mice

Increased expression of [A] FoxP3 mRNA gene expression and attenuated gene expression of [B] RORγ and [C] IL17 with a low level of [D] IL17 protein expression in AhR ^−/+ mice treated with DSS. Data are expressed as mean ± SE, n=10/group.

Figure 5. Proinflammatory adipokines are decreased in AhR ^−/+ mice treated with DSS

The pro-inflammatory adipokines [A] Angiotensinogen [B] ACE, [C] AT1a receptor, [D] Osteopontin gene expression were significantly lower in AhR heterozygous mice during colitis. Data are expressed as mean ± SE, n=10/group. [E] Fluorescence immunohistochemistry for angiotensin II [green] and AT1_aR [red] in AhR ^−/+ and WT mice treated with DSS. [a], [b] show dual staining.

Figure 6. Adiponectin is negatively regulated during colitis only in WT mice and not in the AhR ^−/+ mice

[A] Adiponectin gene expression in WT mice treated with DSS was significantly lower compared to AhR ^−/+ mice. [B] Decreased T- Cadherin receptor expression in WT mice with colitis. [C] and [D] Decreased adiponectin receptors in the context of low adiponectin expression may further impair this adipokine signaling pathway. [E] Increased gene expression of chaperone protein ERP₄₄ relative to its partner, endoplasmic reticulum oxidoreductin [ERO] can block the secretion of adiponectin in WT mice with colitis. Data are expressed as mean ± SE, n=10/group.

Figure 7. Reduced macrophage recruitment during DSS colitis in the AhR ^−/+ mice

Fluorescent immunohistochemistry showing macrophage-restricted protein - F4/80 expression in colonic tissue of AhR^−/+ mice with colitis. [A] Macrophage-restricted protein-F4/80 [green] in control groups, [B] Macrophage-restricted protein-F4/80 [green] in DSS-treated mice groups.

Figure 8. AhR activation in patients with IBD

(A) CYP1A1 mRNA gene expression level is increased in IBD patients compared with control. (B) IL8 mRNA gene expression increases during the inflammatory process in IBD patients. Control n=15, IBD n=25. Data are expressed as median. (C, D) Fluorescent Immunohistochemistry staining of the AhR in a control patient (C) versus a patient with Crohn’s disease (D). Representative images are shown of biopsies obtained from a control subject (C) and from a patient with Crohn’s disease (D). Red staining indicates binding of the antibody to human AhR. All samples were counterstained with DAPI (blue) to visualize nuclei. Magnification is 20X. E - Enterocytes and LP- Lamina Propria.