Characterising and investigating the role of Granzyme A in inflammatory bowel disease

Abstract

Inflammatory bowel disease (IBD) defines two chronic inflammatory diseases of the gastrointestinal tract, ulcerative colitis and Crohn’s disease. The prevalence of these diseases is increasing in the western world, and there is currently no cure for either. Biological therapy is widely used to treat IBD; however, patients often lose response to these treatments. There is a need to stratify patients into further disease subsets, which require biomarkers that could act as read-outs of disease progression and severity.

Granzymes (A, B, H, K and M) are released by cytotoxic T cells and NK cells, with granzyme A and B the most abundant forms found in humans. Granzyme A has previously been reported to be elevated in tissue biopsies from IBD patients who responded well to anti-integrin therapy (etrolizumab) and subsequently achieved clinical remission, in comparison to those who did not respond. Granzyme A has also recently been reported to have a pro-inflammatory role in other inflammatory diseases including rheumatoid arthritis. Granzyme B, typically thought of as a cytotoxic protease, has been reported to be elevated in the serum of IBD patients.

Based on these data, in chapter 3, we sought to characterise the expression of granzymes A and B in IBD tissue samples and to determine whether the expression of granzymes A or B could be used as blood and/or stool-based readout of IBD activity. We also investigated the source of granzyme A in IBD tissue, using fresh tissue biopsies taken from patients undergoing research colonoscopies.

Granzymes typically must be activated before they exert their effects on target cells. We therefore hypothesised that activity of an enzyme would be a more informative biomarker than expression of the enzyme, as only when active are these granzymes known to have cytotoxic effects. To have a read-out of granzyme A activity, in Chapter 4, we sought to create substrate-based probes that would fluoresce when cleaved by active granzyme A but would remain intact in the presence of inactive granzyme A.

Lastly, in Chapter 5, we also sought to understand the pro-inflammatory role of granzyme A and identified that it could induce the secretion of pro-inflammatory cytokines from monocytes. We investigated the receptors responsible for the pro-inflammatory effects on monocytes and used an inhibitor to block the pro-inflammatory effects of granzyme A. Finally, we synthesised a new granzyme A inhibitor, which was shown to be efficacious in vitro. We performed preliminary in vivo experiments in a mouse model of colitis using a PAR-1 receptor inhibitor and the granzyme A inhibitor but observed no efficacy at the concentrations used.

Altogether, the work in this thesis characterised the expression of granzymes A and B in gut tissue from IBD patients and created new substrate-based probes for granzyme A that could act as a read-out of enzyme activity rather than expression. We also began to investigate how granzyme A could induce inflammation in an IBD setting and lastly created a granzyme A inhibitor to attenuate the pro-inflammatory effects of the enzyme.