thapsigargin and Inflammatory-Bowel-Diseases

The Unfolded Protein Response, a mechanism triggered by the cell in response to Endoplasmic reticulum stress, is linked to inflammatory responses. Our aim was to identify novel Unfolded Protein Response-mechanisms that might be involved in triggering or perpetuating the inflammatory response carried out by the Intestinal Epithelial Cells in the context of Inflammatory Bowel Disease.. We analyzed the transcriptional profile of human Intestinal Epithelial Cell lines treated with an Endoplasmic Reticulum stress inducer (thapsigargin) and/or proinflammatory stimuli. Several genes were further analyzed in colonic biopsies from Ulcerative Colitis patients and healthy controls. Lastly, we generated Caco-2 cells lacking HMGCS2 by CRISPR Cas-9 and analyzed the functional implications of its absence in Intestinal Epithelial Cells.. Exposure to a TLR ligand after thapsigargin treatment resulted in a powerful synergistic modulation of gene expression, which led us to identify new genes and pathways that could be involved in inflammatory responses linked to the Unfolded Protein Response. Key differentially expressed genes in the array also exhibited transcriptional alterations in colonic biopsies from active Ulcerative Colitis patients, including NKG2D ligands and the enzyme HMGCS2. Moreover, functional studies showed altered metabolic responses and epithelial barrier integrity in HMGCS2 deficient cell lines.. We have identified new genes and pathways that are regulated by the Unfolded Protein Response in the context of Inflammatory Bowel Disease including

Topics: Caco-2 Cells; Colitis, Ulcerative; Endoplasmic Reticulum Stress; Epithelial Cells; Humans; Hydroxymethylglutaryl-CoA Synthase; Inflammatory Bowel Diseases; Thapsigargin

Endoplasmic reticulum [ER] stress was shown to be pivotal in the pathogenesis of inflammatory bowel disease. Despite progress in inflammatory bowel disease [IBD] drug development, not more than one-third of patients achieve steroid-free remission and mucosal healing with current therapies. Furthermore, patient stratification tools for therapy selection are lacking. We aimed to identify and quantify epithelial ER stress in a patient-specific manner in an attempt towards personalised therapy.. A biopsy-derived intestinal epithelial cell culture system was developed and characterised. ER stress was induced by thapsigargin and quantified with a BiP enzyme-linked immunosorbent assay [ELISA] of cell lysates from 35 patients with known genotypes, who were grouped based on the number of IBD-associated ER stress and autophagy risk alleles.. The epithelial character of the cells was confirmed by E-cadherin, ZO-1, and MUC2 staining and CK-18, CK-20, and LGR5 gene expression. Patients with three risk alleles had higher median epithelial BiP-induction [vs untreated] levels compared with patients with one or two risk alleles [p = 0.026 and 0.043, respectively]. When autophagy risk alleles were included and patients were stratified in genetic risk quartiles, patients in Q2, Q3, and Q4 had significantly higher ER stress [BiP] when compared with Q1 [p = 0.034, 0.040, and 0.034, respectively].. We developed and validated an ex vivo intestinal epithelial cell culture system and showed that patients with more ER stress and autophagy risk alleles have augmented epithelial ER stress responses. We thus presented a personalised approach whereby patient-specific defects can be identified, which in turn could help in selecting tailored therapies.

Topics: Adult; Alleles; Autophagy; Autophagy-Related Protein-1 Homolog; Autophagy-Related Proteins; Biopsy; Cells, Cultured; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Enzyme Inhibitors; Epithelial Cells; Female; Genotype; GTP-Binding Proteins; Heat-Shock Proteins; Humans; Inflammatory Bowel Diseases; Intestinal Mucosa; Intracellular Signaling Peptides and Proteins; Keratin-18; Keratin-20; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Male; Membrane Proteins; Middle Aged; Patient Selection; Precision Medicine; Protein Tyrosine Phosphatases, Non-Receptor; Receptors, G-Protein-Coupled; RNA, Messenger; Thapsigargin; X-Box Binding Protein 1