The Differential Response of Human Epithelial Derived Colonic Organoids to TLR Agonists

Abstract

The intestinal epithelium expresses a range of pattern recognition receptors and is thought to contribute to the innate immune response in the intestine. However, as the cytokines produced by the epithelial cells are the same as those produced by the intestinal immune system it is difficult to define the epithelial response. Here we have used epithelial derived colonic organoids from healthy individuals to characterise the response of the colonic epithelium to toll like receptor (TLR) agonists. Colonoids were grown for 15 days from biopsies from the transverse colon of healthy control patients. The expression of TLRs in the organoids was compared with that of the native colonic epithelium with qPCR and Western blotting, and the response of the organoids to TLR agonists was quantified with qPCR. The response of SW480 cells to agonists was used as a positive control. n = number of patients from which organoids were grown. Two distinct types of organoids developed, colonospheres, which have a poorly developed epithelial layer consisting of thin fusiform cells, and colonoids, which have a well‐developed columnar epithelium. Both colonospheres and colonoids had transcript levels of TLR 1, 2, 4, 5 & 6 comparable to that seen in the native colonic epithelium. Furthermore, transcript for the accessory proteins associated with TLR4 ‐ CD14, lipopolysaccharide binding protein (LBP) and myeloid differentiation factor 2 (MD2) ‐ were also present in the organoids and the expression of TLR 4 & 5 protein in the organoids was comparable to that of the native epithelium (n=5). Despite this, treatment of the organoids with either the TLR 2 agonists [Pam 2CSK4 & Pam3CSK4 (200ng ml−1) or lipoteichoic acid (LTA, 10μg ml−1)] or the TLR4 agonist (lipopolysaccharide (LPS, 200ng ml−1)] failed to stimulate an increase in either IL‐8 or TNF‐α transcript at 3, 6 or 24 h in either colonospheres or colonoids (n=5). In contrast, flagellin (1 μg ml−1) stimulated an increase in both IL‐8 and TNF‐α at all time points (n=5). To further investigate the differential response to the TLR agonists, the effect of increasing concentrations of LPS and flagellin (1, 10, 100, 1000 ng ml−1) on IL‐8 and TNF‐α transcript levels in colonospheres and colonoids was investigated (n=5). Flagellin stimulated a dose dependent increase in both cytokines, whereas LPS failed to stimulate an increase in either IL‐8 or TNF‐α at any dose, although comparable measurements with LPS in SW480 cells resulted in a robust response. Furthermore, LPS (1 μg ml−1) did not stimulate an increase in transcript levels of a range of cytokines associated with intestinal epithelial cells (TSLP, IL33, IL‐1β, or APRIL), whereas flagellin (1 μg ml−1) significantly increased expression of these cytokines (n=5). These data indicate that colonic epithelial cells are unresponsive to common components of the commensal bacteria (LTA, LPS) but flagellin, a virulence factor associated with both gram positive and negative bacteria, induces a robust response. It remains to be determined what the roles of the TLR2 and TLR4 receptors are in the colonic epithelial cells.Support or Funding InformationSupported by a University of Otago Research Grant, a University of Otago postgraduate scholarship and grants from and the Department of Physiology and the Dean's Funds, Otago School of Biomedical Sciences and Dunedin School of Medicine.