P001 Chronic exposure to LPS but not MDP induces goblet cell development in human colonic enteroids
E. Rodrigues1, 2, L. Slobbe1, S. Gadeock1, A.G. Butt1, M. Schultz*2
1University of Otago, Department of Physiology, Otago School of Medical Sciences, Dunedin, New Zealand, 2University of Otago, Department of Medicine, Dunedin School of Medicine, Dunedin, New Zealand
Inflammatory Bowel Disease (IBD) is thought to be the result of an aberrant response of the intestinal immune system to the commensal microbiota in a genetically susceptible host. It is generally felt that disruption of the intestinal barrier contributes to the development of IBD. However, development of the intestinal barrier is dependent upon the commensal bacterial, although whether this is a result of direct interaction between the commensal bacterial and the epithelium or mediated by intestinal immune system is unknown. Here we have used primary cultures of human colonic intestinal epithelium (enteroids), to investigate the effect of microbial stimuli on the development of the intestinal epithelium via the modulation of intestinal stem cells, independent of immune input.
Enteroids were grown from crypts isolated from the transverse colon of healthy individuals and transferred to Matrigel and growth media for 15 days in the presence and absence of lipopolysaccharide (LPS, 20 ng ml-1), a TLR 4 agonist, or muramyl dipeptide (MDP, 20 ng ml-1), a NOD 2 agonist. Enteroid structure was assessed by light and transmission electron microscopy and gene expression by microarray, qPCR, Western blotting and immunohistochemistry (IHC).
At 15 days the enteroids consisted primarily of columnar epithelial cells resembling colonocytes with little evidence of goblet cells, which normally make up 15-20% of the colonic epithelium. Inclusion of LPS or MDP in the growth medium resulted in increased expression of transcript of genes associated with cell cycle, growth and differentiation. However, LPS also induced increased expression of genes associated with goblet cells (MUC2, TFF3, KLF4, Clca1, Zg16, Mpgc60, microtubule-associated protein and resistin-like beta protein) and this was associated with an increase in the number of goblet cells to 18.5 ± 5% (P<0.05, ANOVA with Dunnets test) of the total cells compared with 2%±2.1% in the MDP treated cells and 1.5 ± 1.4% in the untreated controls. Western blot confirmed an increased expression of MUC2, while IHC confirmed an increase in the number of MUC2 and TFF3 positive cells following treatment with LPS, suggesting a lineage change towards goblet cell development.
These data indicate that components of the commensal microbiota differentially induce changes in gene expression in intestinal epithelial cells independent of input from intestinal immune cells. This suggests a direct cross-talk between the intestinal stem cells and the microbiota occurs, which differs dependent on the receptor involved, and will influence epithelial homeostasis and barrier properties in the intestinal epithelium. Disruption of this cross talk may contribute to the development of IBD.