Crohn's disease: an in vitro analysis of T lymphocyte function and response to commensal microbes

Inflammatory bowel disease is a chronic, relapsing inflammation of the intestine. Approximately 0.5% of the western world is estimated to suffer from the disease. Crohn’s disease, a type of inflammatory bowel disease, consists of a patchy inflammation, that can occur throughout the entire gastrointestinal tract. Due to the heterogeneous nature of Crohn’s disease, little is known about the mechanisms of disease pathogenesis. Compared to healthy individuals, patients with Crohn’s disease often have elevated levels of inflammatory T lymphocyte subsets, their associated effector cytokines, and higher intestinal permeability. Crohn’s disease has been associated with genetic risk factors, sedentary lifestyles, and a loss of tolerance to the commensal members of the microbiome. In this research, T lymphocytes from non-IBD and Crohn’s disease patient peripheral blood mononuclear cells (PBMCs) were analysed for comparison. Flow cytometry was used to fluorescently label cells and analytes of interest. Visual analytic tools, such as CytoAnalytics’ EarlyBird and viSNE were used to visualise patient variation within, and between groups. It was hypothesised that Crohn’s disease T lymphocytes would have a bias for inflammation-inducing T lymphocyte subsets, and that these cells would have a higher capacity for inflammation, than non-IBD controls. Indeed, inflammatory T lymphocytes were present at higher frequencies in Crohn’s disease patient blood samples. Furthermore, Crohn’s disease patient PBMCs had a higher capacity for proliferation in response to T lymphocyte stimulation. In responses to the commensal bacterium, Faecalibacterium prausnitzii, Crohn’s disease patient PBMC Tregs increased in frequency, whereas CD4+ inflammatory T lymphocyte subsets maintained their frequency. In contrast, non-IBD PBMC Tregs were not influenced by co-culture with F. prausnitzii, and CD4+ inflammatory T lymphocyte subsets decreased in the presence of F. prausnitzii. Together, these data suggest that Crohn’s disease patient PBMCs respond abnormally to commensal bacteria, and this could play a role in disease pathogenesis. Intestinal organoids are derived from patient colonic biopsy stem cells. Organoids provide a unique in vitro model for the observation of intestinal interactions. In this thesis, a 2D intestinal monolayer was developed from the culture of 3D intestinal organoids. 2D monolayers emulate epithelial intestinal barrier cellular organisation. A difficult question to answer in Crohn’s disease research is whether intestinal permeability and inflammation is a cause or consequence of disease. As such, patient organoids and bacteria were integrated into the model stepwise, to analyse the influence each component has on the system, in the absence, or presence, on an active immune presence. Using this model, it was found that monolayers derived from Crohn’s disease patients were more permeable than non-IBD controls. Furthermore, Crohn’s disease patient monolayers had deleterious interactions in co-culture with F. prausnitzii, resulting in reduced epithelial integrity; in contrast to non-IBD patient monolayers which were unaffected. The addition of matched PBMCs into Crohn’s disease patient monolayers exacerbated epithelial degradation in the presence of F. prausnitzii. Taken together, it was found that Crohn’s disease patient PBMCs had greater inflammatory capacity than non-IBD control PBMCs. These data suggest that intestinal cells in patients with Crohn’s disease may have deleterious interactions with commensal bacteria, which could result in increased intestinal permeability. Furthermore, Crohn’s disease patient Treg responses to commensal bacteria suggest that intestinal areas of immune suppression may promote bacteria success, and potential dysbiosis. Translocation of the luminal microbiota into the lamina propria, combined with excessive T lymphocyte inflammatory capacity, could be an initial driving factor for Crohn’s disease pathogenesis. Data in this thesis provide valuable insight into the initiation of epithelial degradation in patients with Crohn’s disease. These data suggest that epithelial degradation may occur in Crohn’s disease patients in response to commensal bacteria, even in the absence of an immune cell influence. With further development, the intestinal organoid monolayer model may provide insight into the mechanisms of epithelial degradation in individual patients. Further, the monolayer model may also provide a tool to optimise individual patient treatments, in vitro

Crohn's disease: an in vitro analysis of T lymphocyte function and response to commensal microbes

Inflammatory bowel disease is a chronic, relapsing inflammation of the intestine. Approximately 0.5% of the western world is estimated to suffer from the disease. Crohn’s disease, a type of inflammatory bowel disease, consists of a patchy inflammation, that can occur throughout the entire gastrointestinal tract. Due to the heterogeneous nature of Crohn’s disease, little is known about the mechanisms of disease pathogenesis. Compared to healthy individuals, patients with Crohn’s disease often have elevated levels of inflammatory T lymphocyte subsets, their associated effector cytokines, and higher intestinal permeability. Crohn’s disease has been associated with genetic risk factors, sedentary lifestyles, and a loss of tolerance to the commensal members of the microbiome. In this research, T lymphocytes from non-IBD and Crohn’s disease patient peripheral blood mononuclear cells (PBMCs) were analysed for comparison. Flow cytometry was used to fluorescently label cells and analytes of interest. Visual analytic tools, such as CytoAnalytics’ EarlyBird and viSNE were used to visualise patient variation within, and between groups. It was hypothesised that Crohn’s disease T lymphocytes would have a bias for inflammation-inducing T lymphocyte subsets, and that these cells would have a higher capacity for inflammation, than non-IBD controls. Indeed, inflammatory T lymphocytes were present at higher frequencies in Crohn’s disease patient blood samples. Furthermore, Crohn’s disease patient PBMCs had a higher capacity for proliferation in response to T lymphocyte stimulation. In responses to the commensal bacterium, Faecalibacterium prausnitzii, Crohn’s disease patient PBMC Tregs increased in frequency, whereas CD4+ inflammatory T lymphocyte subsets maintained their frequency. In contrast, non-IBD PBMC Tregs were not influenced by co-culture with F. prausnitzii, and CD4+ inflammatory T lymphocyte subsets decreased in the presence of F. prausnitzii. Together, these data suggest that Crohn’s disease patient PBMCs respond abnormally to commensal bacteria, and this could play a role in disease pathogenesis. Intestinal organoids are derived from patient colonic biopsy stem cells. Organoids provide a unique in vitro model for the observation of intestinal interactions. In this thesis, a 2D intestinal monolayer was developed from the culture of 3D intestinal organoids. 2D monolayers emulate epithelial intestinal barrier cellular organisation. A difficult question to answer in Crohn’s disease research is whether intestinal permeability and inflammation is a cause or consequence of disease. As such, patient organoids and bacteria were integrated into the model stepwise, to analyse the influence each component has on the system, in the absence, or presence, on an active immune presence. Using this model, it was found that monolayers derived from Crohn’s disease patients were more permeable than non-IBD controls. Furthermore, Crohn’s disease patient monolayers had deleterious interactions in co-culture with F. prausnitzii, resulting in reduced epithelial integrity; in contrast to non-IBD patient monolayers which were unaffected. The addition of matched PBMCs into Crohn’s disease patient monolayers exacerbated epithelial degradation in the presence of F. prausnitzii. Taken together, it was found that Crohn’s disease patient PBMCs had greater inflammatory capacity than non-IBD control PBMCs. These data suggest that intestinal cells in patients with Crohn’s disease may have deleterious interactions with commensal bacteria, which could result in increased intestinal permeability. Furthermore, Crohn’s disease patient Treg responses to commensal bacteria suggest that intestinal areas of immune suppression may promote bacteria success, and potential dysbiosis. Translocation of the luminal microbiota into the lamina propria, combined with excessive T lymphocyte inflammatory capacity, could be an initial driving factor for Crohn’s disease pathogenesis. Data in this thesis provide valuable insight into the initiation of epithelial degradation in patients with Crohn’s disease. These data suggest that epithelial degradation may occur in Crohn’s disease patients in response to commensal bacteria, even in the absence of an immune cell influence. With further development, the intestinal organoid monolayer model may provide insight into the mechanisms of epithelial degradation in individual patients. Further, the monolayer model may also provide a tool to optimise individual patient treatments, in vitro