Abstract
The overall aim of this thesis was to investigate whether myeloperoxidase and its oxidants are involved in the pathology inflammatory bowel disease (IBD). Myeloperoxidase is a major antimicrobial enzyme of neutrophils that produces the extremely reactive oxidant hypochlorous acid (HOCl), otherwise known as chlorine bleach. HOCl kills bacteria, but excessive HOCl might also drive inflammation and damage the host. HOCl reacts with the amino acid taurine to form taurine chloramine (Tau‑Cl), a weak oxidant. Additionally, HOCl oxidises glutathione, a physiological antioxidant, to form glutathione disulfide (GSSG) and the irreversible product glutathione sulfonamide (GSA). Calprotectin, the major cytosolic protein of neutrophils, is formed of two subunits, S100A8 (A8) and S100A9 (A9). HOCl cross-links calprotectin by forming either A8-A9 or A9-A9 with reversible disulfide bonds.
IBD is an umbrella term for two main diseases, Crohn’s disease and ulcerative colitis. These diseases are defined by excessive inflammation in the gastrointestinal tract. The presence of neutrophils in inflammation is a noted feature of IBD lesions. This is the basis for the use of faecal calprotectin as the current clinical biomarker for IBD severity. However, the role of myeloperoxidase and its oxidants in IBD has received little attention.
Faecal myeloperoxidase protein concentration and enzymatic activity were evaluated as biomarkers of IBD severity using an ELISA. Faecal samples were analysed from a cohort of individuals (n = 175) with colonoscopically assessed IBD activity. Faecal myeloperoxidase protein (r = 0.62, p < 0.0001) and activity (r = 0.64, p < 0.0001) were good biomarkers of IBD severity, performing as good as faecal calprotectin. Elevated faecal myeloperoxidase was associated with increased risk of a complicated IBD course. An assay was developed to extract myeloperoxidase from faeces to quickly measure its enzymatic activity, without the use of antibodies for purification. This assay could be developed into a cheap and rapid point-of-care test for inflammation in IBD.
Calprotectin cross-links were detected by western blotting of faecal samples from individuals with active IBD. To understand how calprotectin might be affected by myeloperoxidase-produced oxidants, the interaction between Tau-Cl and calprotectin was examined in detail. Tau-Cl reacted with calprotectin to form both the A8-A9 and A9-A9 cross-links, which were reducible by glutathione. Competition kinetics showed that formation of the A9-A9 cross-link by Tau-Cl was exceptionally fast. This result suggests that the A9 cysteine exists in part as a low pKa thiolate that is more reactive with Tau-Cl than a usual cysteine thiol.
Finally, the presence of GSA in the urine was evaluated as a biomarker of myeloperoxidase activity in IBD using liquid chromatography with tandem mass spectrometry. Urinary GSA strongly correlated with ulcerative colitis severity (r = 0.57, p < 0.0001), but the correlations with disease severity in Crohn’s disease were weaker. This result supports the theory that myeloperoxidase produces HOCl in ulcerative colitis lesions, possibly exasperating this disease. In summary, this thesis provides compelling evidence that myeloperoxidase is active in IBD. It gives strong rationale for conducting further research into how myeloperoxidase and oxidants contribute to the pathology of IBD.