Translocation of Crohn's disease Escherichia coli across M-cells: contrasting effects of soluble plant fibres and emulsifiers

Abstract

Background: Crohn's disease is common in developed nations where the typical diet is low in fibre and high in processed food. Primary lesions overlie Peyer's patches and colonic lymphoid follicles where bacterial invasion through M-cells occurs. We have assessed the effect of soluble non-starch polysaccharide (NSP) and food emulsifiers on translocation of Escherichia coli across M-cells.

Methods: To assess effects of soluble plant fibres and food emulsifiers on translocation of mucosa-associated E coli isolates from Crohn's disease patients and from non-Crohn's controls, we used M-cell monolayers, generated by co-culture of Caco2-cl1 and Raji B cells, and human Peyer's patches mounted in Ussing chambers.

Results: E coli translocation increased across M-cells compared to parent Caco2-cl1 monocultures; 15.8-fold (IQR 6.2-32.0) for Crohn's disease E coli (N=8) and 6.7-fold (IQR 3.7-21.0) for control isolates (N=5). Electron microscopy confirmed E coli within M-cells. Plantain and broccoli NSP markedly reduced E coli translocation across M-cells at 5 mg/ml (range 45.3-82.6% inhibition, p<0.01); apple and leek NSP had no significant effect. Polysorbate-80, 0.01% vol/vol, increased E coli translocation through Caco2-cl1 monolayers 59-fold (p<0.05) and, at higher concentrations, increased translocation across M-cells. Similarly, E coli translocation across human Peyer's patches was reduced 45±7% by soluble plantain NSP (5 mg/ml) and increased 2-fold by polysorbate-80 (0.1% vol/vol).

Conclusions: Translocation of E coli across M-cells is reduced by soluble plant fibres, particularly plantain and broccoli, but increased by the emulsifier Polysorbate-80. These effects occur at relevant concentrations and may contribute to the impact of dietary factors on Crohn's disease pathogenesis.

Figure 1

Co-culture model of M-cells. (A) Low power and (B) high power TEM images of representative control Caco2-cl1 monolayers, showing closely packed uniform microvilli on the apical aspect of the cell. (C) Low power and (D and E) high power images of in vitro derived M-cells. The M-cells possess an apical surface characteristically devoid of microvilli (indicated by black arrows). In images (A) and (C), the Millicell-PCF filter support can be seen on the lower portion of the image, as can the 3 μm pores within the filter (indicated by white arrows). Bar either 10 μm (A and C) or 1 μm (B, D and E). (F). TEER over time in Caco2-cl1 and M-cell monolayers. Monolayer TEER steadily increases during culture, indicating formation of fully confluent, differentiated monolayers (TEER values in excess of 300 Ω cm²). Monolayer incubation with Raji B cells in the lower chamber occurred on day 16 (dashed line) and continued for the following 5 days, leading to the formation of M-cells, while control Caco2-cl1 monolayers formed in the absence of Raji B cells. The TEER of the control Caco2-cl1 monolayers continued to increase over the following 5 days of incubation (day 16 to 21; solid boxes), as did the TEER of the M-cell monolayers (open boxes), although to a lesser extent (n=35 culture wells for each group). (G) Salmonella typhimurium and (H) Shigella sonnei show significantly greater translocation through M-cells than through Caco2-cl1 monolayers (N=3). **, p<0.01 and ***, p<0.001; Mann–Whitney U.

Figure 2

Translocation of human mucosal E coli isolates across M-cells. (A) Translocation across M-cell monolayers was first quantifiable within 15 min post-infection, while translocation across Caco2-cl1 monolayers could not be detected until at least 30 min post-infection. Data presented are for adherent, invasive Crohn's disease E coli HM605. (*, p<0.05 and ***, p<0.001; ANOVA; N=3). (B) Translocation is measured as CFU expressed relative to M-cell translocation of E coli K12 (N=8, with minimum n=8 replicates for each E coli). Crohn's disease E coli isolates were translocated through M-cell monolayers more readily than through Caco2-cl1 monolayers. Adherent, invasive control patient (irritable bowel syndrome/sporadic polyposis) E coli isolates also translocated through M-cell monolayers more readily, with the exception of isolate HM463. Neither E coli K12 nor E coli XL-1blue showed significantly better translocation through M-cells than through parent Caco2-cl1 cells. Translocation of the probiotic E coli Nissle 1917 was also very low through M-cells, and reduced compared to its translocation through Caco2-cl1 cells. *, p<0.05; **, p<0.01; and ***, p<0.001; ANOVA. (C) TEM showing the presence of intracellular E coli HM605 within M-cells (white arrows). Intracellular E coli HM605 were not seen within Caco2-cl1 monolayers. Bar=0.5 μm.

Figure 3

Plantain NSP blocks translocation of E coli across M-cells in vitro. (A) E coli HM605 translocation through M-cells is inhibited by the presence of plantain NSP at 5 mg/ml and 50 mg/ml (N=6). (B) Broccoli NSP inhibits bacterial translocation across M-cells at 0.5, 5 and 50 mg/ml (N=3). (C) Neither leek NSP nor (D) apple NSP inhibited HM605 translocation across M-cells (both N=2, each at least n=5 replicates). Translocation is measured as CFU expressed relative to M-cell translocation in the absence of fibres. (E) Plantain NSP (5 mg/ml) inhibits translocation across M-cells for a wide range of E coli (N=3). Translocation is measured as CFU expressed relative to M-cell translocation of E coli K12. For all, *, p<0.05; **, p<0.01; ***, p<0.001; ANOVA.

Figure 4

Plantain NSP blocks translocation of E coli across the human intestinal epithelium in Ussing chambers. Histology of (A) an ileal lymphoid follicle (LF) and overlying follicule associated epithelium (FAE) and of (B) villus epithelium (VE) following Ussing chamber experiments. ×20 magnification. (C and D) EGFP-expressing E coli HM615 translocation through both FAE (N=7) and VE (N=9) and is inhibited by the presence of plantain NSP. **p<0.01; ***, p<0.001; ANOVA. (E) Overnight culture of Ussing chamber serosal medium following 2 h translocation of EGFP-expressing E coli HM615 across isolated human epithelium, in the presence and absence of plantain NSP.

Figure 5

Dietary emulsifier polysorbate-80 increases translocation of E coli across the intestinal epithelium. Polysorbate-80 increased translocation of Crohn's disease E coli across both (A) M-cells and (B) human follicle-associated epithelium (FAE). Similarly, polysorbate-80 enhanced translocation of E coli across (C) parent Caco2-cl1 cells and (D) human villus epithelium (VE). *, p<0.05; **, p<0.01; ANOVA (N=4–8). Crohn's E coli HM605 and EGFP-expressing HM615 were used for the in vitro and ex vivo epithelium translocation studies respectively.