CEACAM6 acts as a receptor for adherent-invasive E. coli, supporting ileal mucosa colonization in Crohn disease

Abstract

The ileal mucosa of Crohn disease (CD) patients is abnormally colonized by adherent-invasive E. coli (AIEC) that are able to adhere to and invade intestinal epithelial cells. Here, we show that CD-associated AIEC strains adhere to the brush border of primary ileal enterocytes isolated from CD patients but not controls without inflammatory bowel disease. AIEC adhesion is dependent on type 1 pili expression on the bacterial surface and on carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) expression on the apical surface of ileal epithelial cells. We report also that CEACAM6 acts as a receptor for AIEC adhesion and is abnormally expressed by ileal epithelial cells in CD patients. In addition, our in vitro studies show that there is increased CEACAM6 expression in cultured intestinal epithelial cells after IFN-gamma or TNF-alpha stimulation and after infection with AIEC bacteria, indicating that AIEC can promote its own colonization in CD patients.

Figure 1. Adhesion of CD-associated AIEC strain LF82 to the brush border of ileal enterocytes.

(A) Phase contrast microscopy of the adhesion of strain LF82 to the brush border of ileal enterocytes isolated from a CD patient and a control. Original magnification, ×1,000. (B) Indices of adhesion of AIEC strain LF82 to the brush border of ileal enterocytes isolated from 15 CD patients and 9 controls. The number of bacteria adherent to the brush border of 30–50 enterocytes was determined. Adhesion index was expressed as the mean number of bacteria adherent to the brush border of 1 enterocyte. Two separate experiments were performed in duplicate.

Figure 2. Type 1 pili–mediated adhesion to the brush border of ileal enterocytes.

(A) Adhesion of the LF82-derived type 1 pili–negative mutant 52D11 (fimA::Tn5phoA) to the brush border of enterocytes isolated from CD patients and controls. (B) Adhesion of AIEC LF82 to the brush border of enterocytes isolated from CD patients and controls in the absence or presence of 2% d-mannose. (C) Adhesion of LF82 bacteria expressing type 1 pili variant and of nonpathogenic E. coli K-12 transformed with pPil38 to induce the expression of K-12 type 1 pili (left panel) or of LF82 bacteria expressing either LF82 type 1 pili variant or K-12 type 1 pili (middle panel) to the brush border of enterocytes isolated from CD patients. Western blot analysis using anti–type 1 pili antibodies performed on whole bacterial extracts showed similar amounts of type 1 pili expressed by the various strains (right panel). (D) Expression of a highly mannosylated molecule on the brush border of ileal enterocytes isolated from CD patients. Phase contrast microscopy and fluorescence labeling using ConA-FITC of ileal enterocytes from a CD patient and a control. Original magnification, ×400.

Figure 3. CEACAM6 expression supports AIEC adhesion.

(A) Western blot analysis of whole protein extracts from ileal biopsies taken in both involved and uninvolved areas of 9 CD patients and from 9 ileal biopsies from controls using anti-CEACAM5, anti-CEACAM6, and anti–β-actin antibodies. (B) Western blot analysis of whole protein extracts from colonic biopsies from 3 controls, 3 UC patients, and 3 CD patients using anti-CEACAM6 and anti–β-actin antibodies. (C) Adhesion indices of AIEC strain LF82 to the brush border of enterocytes from CD patients in the presence of monoclonal antibodies raised against CEACAM1, CEACAM5, CEACAM6, THP, and CD48. (D) Adhesion indices of AIEC strain LF82 to the brush border of enterocytes from 4 CD patients in the presence of anti-CEACAM6 monoclonal antibody. (E) Confocal microscopic analysis of HeLa cells transfected with human CEACAM6 cDNA cloned and infected with AIEC LF82 harboring a GFP construct. Original magnification, ×1,000. CEACAM6 expression was detected using anti-CEACAM6 monoclonal antibody and a Texas red–conjugated anti-mouse IgG. Arrows show clear colocalization (yellow staining) between CEACAM6 and bacteria.

Figure 4. Tissue expression of CEACAM6.

Immunohistochemistry staining of ileal sections from CD patients (A) and controls (B) using anti-CEACAM6 monoclonal antibody clone 9A6 or isotype control. Original magnification, ×100. (C) Analysis of CEACAM6 expression in 20 CD patients and 20 controls monitored from negative (–) to strongly positive (+++) staining as described in Methods.

Figure 5. CEACAM6 expression and LF82 adhesion ability with various intestinal epithelial cells.

(A) Western blot analysis using monoclonal antibodies CEACAM6 clone 9A6 and anti–β-actin. Ten micrograms of total protein from different intestinal epithelial cell lines were loaded onto 4%–12% Tris-glycine gel. (B) Confocal microscopic analysis of differentiated Caco-2 cells infected with GFP-expressing LF82 bacteria. Original magnification, ×400. CEACAM6 was detected using anti-CEACAM6 monoclonal antibody clone 9A6 and a Texas red–conjugated anti-mouse IgG (top panel). Arrows show colocalization (yellow) between CEACAM6 and bacteria. A 3D reconstruction (bottom panel) showed apical expression of CEACAM6 (red) and adherent bacteria (green). (C) Western blot analysis showing expression levels of CEACAM6, CEACAM5, and CEACAM1 by Caco-2 cells after 48 hours of stimulation with IFN-γ or TNF-α or after a 3-hour infection period with AIEC LF82 bacteria at an MOI of 10. As loading control, a labeling was performed using anti–β-actin polyclonal antibodies (D) Adhesion ability of AIEC LF82 bacteria was quantified after a 3-hour infection period at an MOI of 10 in Caco-2 intestinal epithelial cells after 1 and 2 days of IFN-γ stimulation. For RNA silencing, IFN-γ–stimulated Caco-2 cells were transfected with 10 ng of siRNA-blocking CEACAM6 (CEACAM6 siRNA) or 10 ng of nonworking siRNA (control siRNA). Expression of CEACAM6 was analyzed by Western blot analysis using anti-CEACAM6 monoclonal antibody clone 9A6 or anti–β-actin polyclonal antibodies. *P < 0.05 compared with nonstimulated Caco-2 cells; ^#P < 0.05 compared with Caco-2 cells stimulated for 2 days with IFN-γ and untransfected or transfected with control siRNA.