Stavely, Rhian (2018) Mechanisms of Mesenchymal Stem Cell Therapy for Enteric Neuropathy Associated with Colitis. PhD thesis, Victoria University.

Abstract

Due to the limited efficacy and high toxicity of current treatments, the development of novel therapies is crucial for inflammatory bowel disease (IBD). Mesenchymal stem cell (MSC) therapies have demonstrated positive outcomes in IBD patients that are refractory to conventional treatment options and produce fewer side-effects. To develop and optimise MSC therapies, their mechanism of action must be fully elucidated. This thesis aims to explore the mechanisms of MSC treatments in experimental intestinal inflammation with a focus on damage to the enteric nervous system (ENS). The guinea-pig model of TNBS-induced colitis was utilised in proof of principle experiments to evaluate the neuroprotective potential of MSCs. Treatments with MSCs attenuated acute inflammation as well as neuronal and nerve fibre loss. MSCs suppressed leukocyte infiltration to the myenteric plexus (plexitis) and the production of superoxide by myenteric neurons. Furthermore, MSCs derived from the bone-marrow (BM-MSCs) were more efficacious than those isolated from adipose tissue (AT-MSCs) in ameliorating damage to the ENS. For the first time, the effects of MSC treatments were explored in a model of spontaneous chronic colitis (Winnie mice). Using high-throughput RNA sequencing, Winnie mice were determined to closely replicate the transcriptome of human IBD with a high degree of accuracy not observed previously in models of chemically-induced colitis. Treatments with BM-MSCs decreased the disease activity of colitis and reduced leukocyte infiltration to the mucosa in Winnie mice. BM-MSCs were determined to reduce the expression of many proinflammatory factors in Winnie mice that contribute to IBD in human patients. The concordance of inflammatory gene expression in Winnie mice was highly representative of IBD. Thus, this model and in vitro organotypic cultures of longitudinal muscle-myenteric plexus were used to elucidate mechanisms of inflammation-associated enteric neuropathy. The expression of many genes associated with the ENS and neurotransmission pathways were normalised by BM-MSC treatments in Winnie mice. BM-MSCs restored neuronal density and attenuated plexitis in Winnie mice to near control levels. This correlated with a reduction in the disease activity of colitis and may have contributed to their ability to normalise many neuronal and synapse-associated genes. In Winnie mice, myenteric neurons were sensitive to oxidative stress with a strong accumulation of oxidised DNA/RNA adducts and superoxide generation from the mitochondria. This was attenuated to control levels by BM-MSCs. In in vitro studies, oxidative stimuli caused neuronal loss which was inhibited by BM-MSCs in a paracrine manner and was mediated, at least in part, by superoxide dismutase 1. BM-MSC treatments also upregulated several genes associated with metabolism and antioxidant defences in Winnie mice that may contribute to the resolution of oxidative injury. In vitro experiments provided evidence that cytoplasmic translocation of the damage associated molecular pattern, high-mobility group box 1 (HMGB1) protein, is induced by oxidative stress in myenteric neurons. HMGB1 was determined to be translocated in myenteric neurons of Winnie mice which correlated with neuronal loss. Treatment with BM-MSCs inhibited HMGB1 translocation in myenteric neurons of Winnie mice in vivo and organotypic cultures in vitro. Pharmacological inhibition of HMGB1 attenuated neuronal loss in Winnie mice without reducing plexitis and mitochondrial superoxide production. This suggests that BM-MSC treatments increased neuronal density by attenuating plexitis and oxidative stress which is upstream of HMGB1 translocation and myenteric neuronal death. In these studies, we have defined potent neuroprotective properties elicited by BM-MSCs which ameliorate damage to the myenteric ganglia in experimental colitis. The use of high-throughput transcriptome sequencing illustrates the complex alterations to the nervous system in chronic experimental colitis and IBD patients. The results of this thesis may be utilised as a reference to provide future direction in the fields of MSC therapies and ENS pathophysiology in intestinal inflammation.

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