Toll like receptor signalling in human colonic organoids

Three-dimensional primary cultures of colonic epithelium, or colonic organoids, are thought to be a suitable culture system to investigate the properties of human intestinal epithelium and its interaction with commensal microbiota. However, to be of use, the colonic organoids will need to express the pattern recognition receptors found in the native intestinal epithelium and respond to appropriate microbial associated molecular patterns (MAMPs). Therefore, we have investigated the expression of toll like receptors (TLRs) by colonic organoids and their response to TLR agonists from healthy individuals and how the response is altered in CD patients. Colonic organoids were grown from crypts isolated from transverse colonic biopsies from healthy and CD patients, maintained in Matrigel© overlaid with stem cell media. Ultrastructure of epithelial cells was determined by transmission electron microscopy. Expression of TLR1, 2, 4 & 5 transcript and protein in colonic organoids were determined by qPCR & immunoblotting and compared with the expression in isolated crypts. The colonic organoids from healthy and CD patients were exposed with TLR agonists [TLR2-Pam2CSK4 & Pam3CSK4 (200 ng ml-1) and lipoteichoic acid (LTA, 10 µg ml-1), TLR- lipopolysaccharide (LPS, 200 ng ml -1), and flagellin (1 µg ml-1)] for 3, 6 & 24 h after minus 15d of culture. Following this, dose response curve was measured for both LPS and flagellin in 15 d colonic organoids (1, 10, 100 & 1000 ng ml-1). IL-8 and TNF-α transcript expression was measured using qRT-PCR. IL-8 and TNF-α transcript was determined in colonic organoids grown ± SB#202190, p38 MAPKinase inhibitor, exposed to LPS (1 µg ml-1) for 6 h after minus 15 d culture. IFN-1β transcript was measured in colonic organoids exposed with LPS (1 µg ml-1) for 6h after minus 15d culture. IL-8 and TNF-α transcript was determined in colonic organoids with same patient and passage number when exposed to LPS (1 µg ml-1) for 6h minus 4 d and 6 h minus 15 d culture. Colonic organoids from healthy and CD patients were exposed with LPS and flagellin at (1 µg ml-1) for 6h. IL-8, IL-33, APRIL, TSLP, IL-1β and TNF-α transcript were measured using qRT-PCR. IL-8 and TNF-α transcript was measured by qPCR in colonic organoids from healthy patients exposed to commensal and pathogenic LPS (1 µg ml-1) & flagellin (1 µg ml-1) for 6h after minus 15 d culture. IL-8 and TNF-α transcript was measured by qPCR in colonic organoids from healthy patients grown in media containing ± (10% Fetal bovine serum) and exposed to LPS (1 µg ml-1) for 6 h after minus 15d culture. Colonic organoids from healthy patients were grown to day4 and exposed to LPS (1 µg ml-1) for 10 days. Ki67 was used to determine proliferation and differentiation by counting the number of goblet cells stained by alcian blue. Measurement of the size of colonic organoids was also determined from the same treatment. Two morphologically distinct types of organoids developed in cultures, colonospheres having thin, non-polar and colonoids having a well-developed columnar epithelium. In both colonospheres and colonoids TLR1, 2, 4, 5 and 6 were expressed at levels comparable to that in crypts from healthy individuals and TLR4 transcript expression was significantly upregulated in CD crypts and colonoids. Addition of the TLR4 agonist from the basal end does not increase IL-8 and TNF-α transcript expression but addition of TLR5 agonist cause a large significant increase in the transcript expression of IL-8, TNF-α, IL-33, TSLP, APRIL & IL-1β (P