Abstract
Crohn’s disease (CD) is a chronic inflammatory bowel disease with many contributing factors: the immune response, genetic susceptibility, environmental factors, intestinal epithelium, and the host microbiome. Current treatments aim to control excessive inflammation but are not always effective. Human intestinal organoids can be grown from patient intestinal biopsy stem cells and can be used in in vitro experimental models to account for patient heterogeneity when investigating the pathogenesis of CD. I developed a two-dimensional (2D) intestinal organoid monolayer transwell model that corresponds to the lumen and lamina propria of the human gut. The monolayer model was used to manipulate apical (lumen) and basal (lamina propria) compartments with the addition of commensal bacteria and patient-matched peripheral blood mononuclear cells (PBMCs), respectively, and to study the contribution of the immune response on the epithelial integrity.
Using these 2D human intestinal organoids, I modelled the gut immune responses in people with CD compared to healthy controls (HC), as well as the effect of bacteria and immune cells from the same patient on the epithelial integrity of the monolayers. Addition of PBMCs and heat killed bacteria to CD monolayers resulted in reduced epithelial resistance compared to monolayers derived from HC donors.
I developed a model of inflammation using HC monolayers and polyclonally active immune cells. The degree of immune cell activation was correlated with reduced epithelial integrity. Epithelial integrity was restored with the addition of anti-tumour necrosis factor (TNF) antibody in all HC derived monolayers and some CD derived monolayers (responders) and this response was associated with reduced abundance of CD4 and CD8 T cells and reduced inflammatory cytokine (TNF and interferon (IFN)-γ) production.
CD is associated with microbial dysbiosis and probiotic use has been an attractive therapeutic option to alter the microbiota in patients with CD. In this thesis, I developed a co-culture of human organoid monolayers, autologous immune cells and live probiotic bacteria. The influence of live probiotic bacteria on cytokine production, PBMCs phenotypes and epithelial integrity in the inflammation model was investigated. Addition of either the live probiotics Escherichia coli Nissle 1917 (EcN) or Vivomixx restored epithelial integrity in only some HC and CD derived monolayers, and affected the expression and localisation of tight junction proteins. Epithelial protection mediated by addition of probiotics was associated with reduced inflammatory immune cell phenotypes and reduced TNF and IFN-g cytokine production.
Taken together, activated immune cells caused inflammatory mediated epithelial damage to human organoid monolayers, and epithelial protection by anti-TNF or probiotic bacteria was associated with reduced abundance of T cells populations and decreased concentrations of TNF and IFN-γ, suggesting that epithelial damage in CD is the result of these inflammatory cytokines. Investigating the interaction of gut bacteria, immune cells and the intestinal epithelial cells will help in understanding the pathogenesis of CD. This organoid model, which is derived from individual patient tissues, accounts for patient heterogeneity and leads towards a personalised medicine approach.