Crohn's disease adherent-invasive Escherichia coli colonize and induce strong gut inflammation in transgenic mice expressing human CEACAM

Abnormal expression of CEACAM6 is observed at the apical surface of the ileal epithelium in Crohn's disease (CD) patients, and CD ileal lesions are colonized by pathogenic adherent-invasive Escherichia coli (AIEC). We investigated the ability of AIEC reference strain LF82 to colonize the intestinal mucosa and to induce inflammation in CEABAC10 transgenic mice expressing human CEACAMs. AIEC LF82 virulent bacteria, but not nonpathogenic E. coli K-12, were able to persist in the gut of CEABAC10 transgenic mice and to induce severe colitis with reduced survival rate, marked weight loss, increased rectal bleeding, presence of erosive lesions, mucosal inflammation, and increased proinflammatory cytokine expression. The colitis depended on type 1 pili expression by AIEC bacteria and on intestinal CEACAM expression because no sign of colitis was observed in transgenic mice infected with type 1 pili–negative LF82-ΔfimH isogenic mutant or in wild-type mice infected with AIEC LF82 bacteria. These findings strongly support the hypothesis that in CD patients having an abnormal intestinal expression of CEACAM6, AIEC bacteria via type 1 pili expression can colonize the intestinal mucosa and induce gut inflammation. Thus, targeting AIEC adhesion to gut mucosa represents a new strategy for clinicians to prevent and/or to treat ileal CD

Involvement of the Wnt/β-catenin signaling pathway in the cellular and molecular mechanisms of fibrosis in endometriosis.

BACKGROUND:During the development and progression of endometriotic lesions, excess fibrosis may lead to scarring, chronic pain, and altered tissue function. However, the cellular and molecular mechanisms of fibrosis in endometriosis remain to be clarified. OBJECTIVES:The objective of the present study was to investigate whether the Wnt/β-catenin signaling pathway was involved in regulating the cellular and molecular mechanisms of fibrosis in endometriosis in vitro and to evaluate whether fibrosis could be prevented by targeting the Wnt/β-catenin pathway in a xenograft model of endometriosis in immunodeficient nude mice. METHODS:Seventy patients (40 with and 30 without endometriosis) with normal menstrual cycles were recruited. In vitro effects of small-molecule antagonists of the Tcf/β-catenin complex (PKF 115-584 and CGP049090) on fibrotic markers (alpha smooth muscle actin, type I collagen, connective tissue growth factor, fibronectin) and collagen gel contraction were evaluated in endometrial and endometriotic stromal cells from patients with endometriosis. In vitro effects of activation of the Wnt/β-catenin signaling pathway by treatment with recombinant Wnt3a on profibrotic responses were evaluated in endometrial stromal cells of patients without endometriosis. The effects of CGP049090 treatment on the fibrosis of endometriotic implants were evaluated in a xenograft model of endometriosis in immunodeficient nude mice. RESULTS:Treatment with PKF 115-584 and CGP049090 significantly decreased the expression of alpha smooth muscle actin, type I collagen, connective tissue growth factor and fibronectin mRNAs in both endometriotic and endometrial stromal cells with or without transforming growth factor-β1 stimulation. Both endometriotic and endometrial stromal cell-mediated contraction of collagen gels was significantly decreased by treatment with PKF 115-584 and CGP049090 as compared to that of untreated cells. The animal experiments showed that CGP049090 prevented the progression of fibrosis and reversed established fibrosis in endometriosis. CONCLUSION:Aberrant activation of the Wnt/β-catenin pathway may be involved in mediating fibrogenesis in endometriosis

Effects of β-catenin siRNA on fibrotic markers in endometrial and endometriotic stromal cells from patients with endometriosis.

<div><p>Effects of β-catenin siRNA on the mRNA expression of <i>αSMA</i>, <i>Col-I</i>, <i>CTGF</i>, and <i>FN</i> in endometriotic (A) and endometrial (B) stromal cells with or without TGF-β1 stimulation.</p> <p>C: control siRNA-transfected cells; ß: ß-catenin siRNA-transfected cells.</p> <p>*: p<.05 versus control (C) cells without TGF-β1 stimulation.</p> <p>**: p<.05 versus control (C) cells with TGF-β1 stimulation.</p> <p>Numerical values are presented as the mean + SEM. Expression levels of <i>αSMA</i>, <i>Col-I</i>, <i>CTGF</i>, and <i>FN</i> mRNA are given relative to the expression level of the reference gene, <i>GAPDH</i>.</p> <p>Endometriotic stromal cells (n=10), endometrial stromal cells (n=10).</p></div

In Vitro Effects of a Small-Molecule Antagonist of the Tcf/ß-Catenin Complex on Endometrial and Endometriotic Cells of Patients with Endometriosis

<div><p>Background</p><p>Our previous studies suggested that aberrant activation of Wnt/ß-catenin signaling might be involved in the pathophysiology of endometriosis. We hypothesized that inhibition of Wnt/ß-catenin signaling might result in inhibition of cell proliferation, migration, and/or invasion of endometrial and endometriotic epithelial and stromal cells of patients with endometriosis.</p><p>Objectives</p><p>The aim of the present study was to evaluate the effects of a small-molecule antagonist of the Tcf/ß-catenin complex (PKF 115–584) on cell proliferation, migration, and invasion of endometrial and endometriotic epithelial and stromal cells.</p><p>Methods</p><p>One hundred twenty-six patients (78 with and 48 without endometriosis) with normal menstrual cycles were recruited. In vitro effects of PKF 115–584 on cell proliferation, migration, and invasion and on the Tcf/ß-catenin target genes were evaluated in endometrial epithelial and stromal cells of patients with and without endometriosis, and in endometrial and endometriotic epithelial and stromal cells of the same patients.</p><p>Results</p><p>The inhibitory effects of PKF 115–584 on cell migration and invasion in endometrial epithelial and stromal cells of patients with endometriosis prepared from the menstrual phase were significantly higher than those of patients without endometriosis. Levels of total and active forms of MMP-9 were significantly higher in epithelial and stromal cells prepared from menstrual endometrium in patients with endometriosis compared to patients without endometriosis. Treatment with PKF 115–584 inhibited MMP-9 activity to undetectable levels in both menstrual endometrial epithelial and stromal cells of patients with endometriosis. The number of invasive cells was significantly higher in epithelial and stromal cells of endometriotic tissue compared with matched eutopic endometrium of the same patients. Treatment with PKF 115–584 decreased the number of invasive endometriotic epithelial cells by 73% and stromal cells by 75%.</p><p>Conclusions</p><p>The present findings demonstrated that cellular mechanisms known to be involved in endometriotic lesion development are inhibited by targeting the Wnt/β-catenin pathway.</p></div

Effects of small-molecule antagonists of the Tcf/β-catenin complex (PKF 115-584 and CGP049090) on stromal cell-mediated collagen gel contraction.

<div><p>A–F: Collagen gel contraction at 0, 4, 8, 12, and 24 h in matched endometrial and endometriotic stromal cells from the same patient (A), *: p<.05: EES:deep endometriosis versus EES: ovarian endometriosis, endometrial stromal cells of patients with and without endometriosis (B), *: p<.05: versus EuEs: patients with endometriosis, treated and vehicle-treated endometrial stromal cells of patients with (C), *: p<.05: versus treated EuEs, and without endometriosis (D), *: p<.05: versus treated EuEs, treated and vehicle-treated endometriotic stromal cells (E, F), *: p<.05: versus treated EES, and G, H: Representative photomicrographs of contracted gels taken at 24 h in endometriotic stromal cells with and without treatment.</p> <p>Numerical values are presented as the mean + SEM.</p> <p>EES: endometriotic stromal cells; EuES: endometrial stromal cells.</p> <p>Endo (-): endometrium of patients without endometriosis.</p> <p>Endo (+): Endometrium of patients with endometriosis.</p> <p>EuES: ovarian endometriosis (n=10); EES: ovarian endometriosis (n=10).</p> <p>EuES: deep endometriosis (n=10); EES: deep endometriosis (n=10).</p> <p>EuES: patients with endometriosis (n=20); EuES: patients without endometriosis (n=10).</p></div

Effects of small-molecule antagonists of the Tcf/β-catenin complex (PKF 115-584 and CGP049090) on fibrotic markers in endometrial and endometriotic stromal cells from patients with endometriosis.

<div><p>A, B: Effects of small-molecule antagonists of the Tcf/β-catenin complex (PKF 115-584 and CGP049090) on the mRNA expression of <i>αSMA</i>, <i>Col-I</i>, <i>CTGF</i>, and <i>FN</i> in endometriotic (A) (n=10) and endometrial (B) (n=10) stromal cells with or without TGF-β1 stimulation. *: p<.05 versus vehicle-treated controls without TGF-β1 stimulation. **: p<.05 versus vehicle-treated controls with TGF-β1 stimulation.</p> <p>C: Representative photomicrographs of endometriotic stromal cells after treatment with vehicle or CGP049090 (6.25 µM) for 24 h and immunostained for αSMA (green) and nuclei (blue). Scale bar, 100 μm.</p> <p>D: Percentage of αSMA-positive endometriotic stromal cells (n=10) after treatment with vehicle, PKF 115-584 (6.25 µM), or CGP049090 (6.25 µM) for 24 h. *: p<.05 versus vehicle-treated controls.</p> <p>Numerical values are presented as the mean + SEM. Expression levels of <i>αSMA</i>, <i>Col-I</i>, <i>CTGF</i>, and <i>FN</i> mRNA are given relative to the expression level of the reference gene, <i>GAPDH</i>.</p></div

Effects of PKF 115–584 on cell proliferation.

<p>A, B: Basal cell proliferation in non-treated endometrial epithelial (A) and stromal (B) cells of patients with and without endometriosis. C, D: Percent inhibition of cell proliferation in endometrial epithelial (C) and stromal (D) cells of patients with and without endometriosis treated with PKF 115–584. Results are presented as the mean+SEM. Basal cell proliferation is presented as OD. Percent inhibition of cell proliferation is calculated as percent of vehicle control. M: menstrual phase, P: proliferative phase, ES: early secretory phase, MS: mid- secretory phase, LS: late secretory phase. Endo (+): Endometrium of patients with endometriosis (M: n = 6, P: n = 20, ES: n = 7, MS: n = 15, LS: n = 6). Endo (–): endometrium of patients without endometriosis (M: n = 4, P: n = 11, ES: n = 8, MS: n = 8, LS: n = 4). a: p<.05 versus patients without endometriosis.</p

Effects of Wnt3a treatment on profibrotic responses in endometrial stromal cells of patients without endometriosis.

<div><p>A: Axin2 mRNA expression in endometrial stromal cells treated with vehicle or Wnt3a (150 ng/mL) for 0, 4, 8, 12, 24, 48, or 72 h (n=5). *: p<.05 versus vehicle-treated controls.</p> <p>B: Effects of β-catenin siRNA on the mRNA expression of <i>αSMA</i>, <i>Col-I</i>, <i>CTGF</i>, and <i>FN</i> in endometrial stromal cells treated with vehicle, Wnt3a (150 ng/mL), or TGF-β1 (5 ng/mL) for 24 h (n=10). C: control siRNA-transfected cells; ß: ß-catenin siRNA-transfected cells.</p> <p>*: p<.05 versus control (C) cells without stimulation.</p> <p>**: p<.05 versus control (C) cells with Wnt3a stimulation.</p> <p>***: p<.05 versus control (C) cells with TGF-β1 stimulation.</p> <p>C: Cell proliferation of endometrial stromal cells treated with vehicle or Wnt3a (150 ng/mL) for 72 h (n=5). *: p<.05 versus vehicle-treated controls.</p> <p>D: Cell migration of endometrial stromal cells treated with vehicle or Wnt3a (150 ng/mL) for 72 h (n=5). *: p<.05 versus vehicle-treated controls.</p> <p>E: Collagen gel contraction of stromal cells treated for 24 h with vehicle or Wnt3a (150 ng/mL) (n=5), and representative photomicrographs of contracted gels taken at 24 h in stromal cells treated with vehicle or Wnt3a (150 ng/mL). *:p<.05 versus vehicle-treated controls.</p> <p>F: Percentage of αSMA-positive cells treated for 24 h with vehicle, Wnt3a (150 ng/mL), or TGF-β1 (5 ng/mL) (n=5). *:p<.05 versus vehicle-treated controls.</p> <p>G: Representative photomicrographs of endometrial stromal cells from the same patient after treatment with vehicle, Wnt3a (150 ng/mL), or TGF-β1 (5 ng/mL) for 24 h and stained for f-actin (red) or αSMA (green) and nuclei (blue). Scale bar, 100 μm.</p> <p>Numerical values are presented as the mean + SEM. Expression levels of axin2, <i>αSMA</i>, <i>Col-I</i>, <i>CTGF</i>, and <i>FN</i> mRNAs are given relative to the expression of the reference gene, <i>GAPDH</i>.</p></div

Effects of PKF 115–584 on cell migration and invasion.

<p><b>A, B</b>: Number of migrated cells/mm² in non-treated and PKF 115–584–treated epithelial (A) and stromal (B) cells of endometriotic tissue and matched eutopic endometrium of the same patient. <b>C, D</b>: Representative photomicrographs of migration of non-treated and PKF 115–584–treated epithelial (C) and stromal (D) cells of endometriotic tissue and matched eutopic endometrium of a same patient (magnification x100). <b>E, F</b>: Number of invasive cells/mm² in non-treated and PKF 115–584–treated epithelial (E) and stromal (F) cells of endometriotic tissue and matched eutopic endometrium of the same patients. <b>G, H</b>: Representative photomicrographs of invasion of non-treated and PKF 115–584–treated epithelial (G) and stromal (H) cells of endometriotic tissue and matched eutopic endometrium of a same patient (magnification x100). Results are presented as the mean+SEM. Epithelial cells of endometriotic tissue and matched eutopic endometrium of the same patients (n = 16). Stromal cells of endometriotic tissue and matched eutopic endometrium (n = 16). a: p<.05 versus matched eutopic endometrium of the same patients.</p

Representative photomicrographs of cell migration and invasion.

<p><b>A, B</b>: Representative photomicrographs of migration of non-treated and PKF 115–584–treated menstrual endometrial epithelial (A) and stromal (B) cells of patients with and without endometriosis (magnification x100). <b>C, D</b>: Representative photomicrographs of invasion of non-treated and PKF 115–584–treated menstrual endometrial epithelial (C) and stromal (D) cells of patients with and without endometriosis (magnification x100). Endo (+): Endometrium of patients with endometriosis prepared from the menstrual phase. Endo (–): endometrium of patients without endometriosis prepared from the menstrual phase.</p