The dietary fibre rhamnogalacturonan improves intestinal epithelial barrier function in a microbiota-independent manner.

BACKGROUND AND PURPOSE: Dietary fibre comprises a complex group of polysaccharides that are indigestible but are fermented by gut microbiota, promoting beneficial effects to the intestinal mucosa indirectly through the production of short chain fatty acids. We found that a polysaccharide, rhamnogalacturonan (RGal), from the plant Acmella oleracea, has direct effects on intestinal epithelial barrier function. Our objective was to determine the mechanism whereby RGal enhances epithelial barrier function. EXPERIMENTAL APPROACH: Monolayers of colonic epithelial cell lines (Caco-2, T84) and of human primary cells from organoids were mounted in Ussing chambers to assess barrier function. The cellular mechanism of RGal effects on barrier function was determined using inhibitors of TLR-4 and PKC isoforms. KEY RESULTS: Apically applied RGal (1000 µg ml-1 ) significantly enhanced barrier function as shown by increased transepithelial electrical resistance (TER) and reduced fluorescein isothiocyanate (FITC)-dextran flux in Caco-2, T84 and human primary cell monolayers, and accelerated tight junction reassembly in Caco-2 cells in a calcium switch assay. RGal also reversed the barrier-damaging effects of inflammatory cytokines on FITC-dextran flux and preserved the tight junction distribution of occludin. RGal activated TLR4 in TLR4-expressing HEK reporter cells, an effect that was inhibited by the TLR4 inhibitor, C34. The effect of RGal was also dependent on PKC, specifically the isoforms PKCδ and PKCζ. CONCLUSION AND IMPLICATIONS: RGal enhances intestinal epithelial barrier function through activation of TLR4 and PKC signalling pathways. Elucidation of RGal mechanisms of action could lead to new, dietary approaches to enhance mucosal healing in inflammatory bowel diseases.

N-Terminomics/TAILS Profiling of Proteases and Their Substrates in Ulcerative Colitis.

Dysregulated protease activity is often implicated in the initiation of inflammation and immune cell recruitment in gastrointestinal inflammatory diseases. Using N-terminomics/TAILS (terminal amine isotopic labeling of substrates), we compared proteases, along with their substrates and inhibitors, between colonic mucosal biopsies of healthy patients and those with ulcerative colitis (UC). Among the 1642 N-termini enriched using TAILS, increased endogenous processing of proteins was identified in UC compared to healthy patients. Changes in the reactome pathways for proteins associated with metabolism, adherens junction proteins (E-cadherin, liver-intestinal cadherin, catenin alpha-1, and catenin delta-1), and neutrophil degranulation were identified between the two groups. Increased neutrophil infiltration and distinct proteases observed in ulcerative colitis may result in extensive break down, altered processing, or increased remodeling of adherens junctions and other cellular functions. Analysis of the preferred proteolytic cleavage sites indicated that the majority of proteolytic activity and processing comes from host proteases, but that key microbial proteases may also play a role in maintaining homeostasis. Thus, the identification of distinct proteases and processing of their substrates improves the understanding of dysregulated proteolysis in normal intestinal physiology and ulcerative colitis.

Inhibition of Intestinal Epithelial Wound Healing through Protease-Activated Receptor-2 Activation in Caco2 Cells.

The mechanisms of epithelial wound healing are not completely understood, especially in the context of proteases and their receptors. It was recently shown that activation of protease-activated receptor-2 (PAR2) on intestinal epithelial cells induced the expression of cyclooxygenase-2 (COX-2), which has protective functions in the gastrointestinal tract. It was hypothesized that PAR2-induced COX-2 could enhance wound healing in intestinal epithelial cells. Caco2 cells were used to model epithelial wound healing of circular wounds. Cellular proliferation was studied with a 5-ethynyl-2'-deoxyuridine assay, and migration was studied during wound healing in the absence of proliferation. Immunofluorescence was used to visualize E-cadherin and F-actin, and the cellular transcription profile during wound healing and PAR2 activation was explored with RNA sequencing. PAR2 activation inhibited Caco2 wound healing by reducing cell migration, independently of COX-2 activity. Interestingly, even though migration was reduced, proliferation was increased. When the actin dynamics and cell-cell junctions were investigated, PAR2 activation was found to induce actin cabling and prevent the internalization of E-cadherin. To further investigate the effect of PAR2 on transcriptionally dependent wound healing, RNA sequencing was performed. This analysis revealed that PAR2 activation, in the absence of wounding, induced a similar transcriptional profile compared with wounding alone. These findings represent a novel effect of PAR2 activation on the mechanisms of epithelial cell wound healing that could influence the resolution of intestinal inflammation.

A simple, cost-effective method for generating murine colonic 3D enteroids and 2D monolayers for studies of primary epithelial cell function.

Cancer cell lines have been the mainstay of intestinal epithelial experimentation for decades, due primarily to their immortality and ease of culture. However, because of the inherent biological abnormalities of cancer cell lines, many cellular biologists are currently transitioning away from these models and toward more representative primary cells. This has been particularly challenging, but recent advances in the generation of intestinal organoids have brought the routine use of primary cells within reach of most epithelial biologists. Nevertheless, even with the proliferation of publications that use primary intestinal epithelial cells, there is still a considerable amount of trial and error required for laboratories to establish a consistent and reliable method to culture three-dimensional (3D) intestinal organoids and primary epithelial monolayers. We aim to minimize the time other laboratories spend troubleshooting the technique and present a standard method for culturing primary epithelial cells. Therefore, we have described our optimized, high-yield, cost-effective protocol to grow 3D murine colonoids for more than 20 passages and our detailed methods to culture these cells as confluent monolayers for at least 14 days, enabling a wide variety of potential future experiments. By supporting and expanding on the current literature of primary epithelial culture optimization and detailed use in experiments, we hope to help enable the widespread adoption of these innovative methods and allow consistency of results obtained across laboratories and institutions.NEW & NOTEWORTHY Primary intestinal epithelial monolayers are notoriously difficult to maintain culture, even with the recent advances in the field. We describe, in detail, the protocols required to maintain three-dimensional cultures of murine colonoids and passage these primary epithelial cells to confluent monolayers in a standardized, high-yield and cost-effective manner.