The Role of Estrogen Signaling in a Mouse Model of Inflammatory Bowel Disease: A Helicobacter Hepaticus Model

<div><p>The pathogenesis of inflammatory bowel diseases (IBD), Crohn's disease and ulcerative colitis, is due in part to interactions between the immune system, genetics, the environment, and endogenous microbiota. Gonadal sex hormones (GSH), such as estrogen, are thought to be involved in the development of IBD as variations in disease severity occur during pregnancy, menopause, or oral contraceptives use. In certain strains of mice, infection with <i>Helicobacter hepaticus</i> triggers IBD-like mucosal inflammation that is more severe in female mice than in males, suggesting a role for GSH in this model. To determine the role of estrogen signaling in microbiota-induced intestinal inflammation, estrogen receptor (ER) α and β knock-out (KO) mice, ER agonists, and adoptive transfers were utilized. We demonstrate that, when signaling is limited to ERβ on a non-CD4⁺ cell subset, disease is less severe and this correlates with decreased expression of pro-inflammatory mediators.</p></div

Treatment with an ERβ agonist decreases disease severity and the expression of pro-inflammatory cytokines.

<p>Female A/J mice were ovariectomized, administered continuous-release pellets containing a placebo, ERα agonist (PPT) or ERβ agonist (DPN), inoculated with <i>H. hepaticus</i>, and necropsied three months later. (A) Cecal lesion scores of mice treated with a placebo (circles), PPT (squares) or DPN (triangles) pellet (n = 19, 22, and 21 respectively). Points represent the lesion scores of individual mice. Data analyzed by one-way ANOVA with a Student-Newman-Keuls post-hoc test; data presented as group means ± s.d (B) Frequency of mice with severe disease (lesion score ≥6). Data analyzed with chi-squared analysis. (C) Real-time quantitative PCR measurements of cecal mRNA expression levels in mice treated with an ERα agonist (squares), ERβ agonist (triangles), or placebo control (circles) (n = 8, 9, and 9 respectively). The mRNA expression levels of all cytokines are normalized to HPRT expression. Data analyzed with an exact Kruskal-Wallis test followed by Dunn's post-hoc comparisons; each point represents the expression level for an individual mouse and horizontal lines represent medians. For all data analysis p≤0.05 considered significant and indicated by horizontal capped bars.</p

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<p>Genotyping PCR Primers, Products, & Annealing Temperatures.</p

Estrogen treatment decreases disease severity and the expression of pro-inflammatory cytokines and chemokines.

<p>(A) Representative histologic images of ceca from ovariectomized A/J mice sacrificed 90 days after <i>Helicobacter hepaticus</i> inoculation and implantation with a subcutaneous pellet containing either (top) 17β-estradiol (1.5 mg/pellet) or (bottom) placebo. (B) Cecal lesion scores of placebo (circles) and estradiol (squares) treated mice (n = 8, 8 respectively). Each point represents the lesion score for an individual mouse. Lesion score data analyzed with an exact Wilcoxon-Mann-Whitney test; horizontal lines represent medians. (C) Real-time quantitative PCR measurements of cecal cytokine and chemokine mRNA expression levels in mice receiving placebo (circle) or estrogen (square) treatment (n = 8). mRNA expression was normalized to the expression of the housekeeping gene HPRT. All mRNA expression data with an exact Wilcoxon Rank Sum Test; each point represents the expression level for an individual mouse and horizontal lines represent medians. For all data analysis p≤0.05 considered significant and indicated by horizontal capped bars.</p

Lack of ER signaling in CD4⁺ cellular populations does not alter cecal inflammation or cytokine and chemokine expression.

<p>(A) Cecal lesion scores in female <i>H. hepaticus-</i>inoculated <i>RAG2</i>^−/− mice adoptively transferred <i>ERα</i>^−/− (squares) or <i>ERβ</i>^−/− (triangles) CD4⁺ lymphocytes (n = 8, 9 respectively). As a control group, <i>RAG</i>^−/− female mice were adoptively transferred wild type CD4⁺ lymphocytes (n = 20)(circles). Points represent the lesion scores of individual mice in each experimental group and horizontal bars indicate median lesion scores; horizontal lines represent medians. (B) Real-time quantitative PCR measurements of cecal cytokine and chemokine mRNA expression levels in <i>RAG2</i>^−/− mice adoptively transferred CD4⁺ lymphocytes deficient for ERα or ERβ expression. The mRNA expression levels of all cytokines were normalized to HPRT mRNA expression. Each point represents the normalized expression of an individual mouse (symbols the same as lesion score data). All data analyzed by an exact Kruskal-Wallis test and horizontal lines represent medians. For all data analysis p≤0.05 considered significant.</p

Absence of ERα expression decreases disease severity and mRNA expression of CXCL9 and IL-10.

<p>A/J female mice deficient for either ERα or ERβ expression and heterozygous/wild type littermate controls were inoculated with <i>H. hepaticus</i> and evaluated 3 months post-inoculation. (A) Cecal lesion scores of inoculated ERα (squares), ERβ (triangles) and control (circles) mice (n = 14, 9, and 27 respectively); each point represents the lesion score for an individual mouse. Lesion score data analyzed with an exact Kruskal-Wallis test followed by Dunn's post-hoc comparisons. Horizontal bars represent medians. (B) Frequency of mice with severe cecal inflammation (lesion score ≥6) within each genotype group. Data analyzed by exact Chi-squared test of independence. (C) Real-time quantitative PCR measurements of cecal cytokine and chemokine mRNA expression levels in mice deficient for ERα (squares) or ERβ (triangles) and littermate controls (circles). The mRNA expression of all cytokines were normalized to HPRT. Data analyzed with an exact Kruskal-Wallis test followed by Dunn's post-hoc comparisons; each point represents the expression level for an individual mouse and horizontal lines represent medians. For all data analysis p≤0.05 considered significant and indicated by horizontal capped bars.</p

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<p>Real time PCR Primers and Annealing Temperatures.</p

ERβ signaling on non-CD4⁺ cellular populations reduces cecal inflammation and IFN-γ expression.

<p>(A) Cecal lesion scores in female <i>H. hepaticus-</i>inoculated <i>ERα</i>^−/−<i>RAG2</i>^−/− (squares) or <i>ERβ</i>^−/−<i>RAG2</i>^−/− (triangles) female mice adoptively transferred wild type CD4⁺ lymphocytes (n = 11, 13 respectively). As a control group (circles), <i>RAG</i>^−/− female mice were adoptively transferred wild type CD4⁺ lymphocytes (n = 20). Points represent the lesion scores of individual mice in each experimental group and horizontal lines represent median lesion scores. Lesion score data analyzed with an exact Kruskal-Wallis test followed by Dunn's post-hoc comparisons. (B) Real-time quantitative PCR measurements of cecal cytokine and chemokine mRNA expression levels in <i>RAG2</i>^−/− mice deficient for either ERα or ERβ and adoptively transferred wild type CD4⁺ lymphocytes. All cytokine mRNA expression levels were normalized to <i>HPRT</i> expression. Data analyzed by an exact Kruskal-Wallis test followed by Dunn's post-hoc comparisons; each point represents the normalized expression of an individual mouse and horizontal lines represent medians. For all data analysis p≤0.05 considered significant and indicated by horizontal capped bars.</p