Mechanisms to underpin novel colonic therapies for colitis: thioguanine and microbiota

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Haywood, Alison

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Crane, Denis

Florin, Timothy

Oancea, Iulia

Ó Cuív, Páraic

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Inflammatory bowel diseases (IBD), Crohn’s and ulcerative colitis are the chronic inflammatory conditions affecting the gastrointestinal tract. Modulation of the host’s innate inflammatory and adaptive immunity remains the standard of treatment for acute IBD. Thiopurine pro-drugs azathioprine and mercaptopurine (MP) have been the mainstay of oral treatments employed by gastroenterologists worldwide to treat moderate to severe IBD, however they have numerous drawbacks. Thioguanine (TG), another thiopurine pro-drug, has major advantages over the widely used MP and azathioprine, but it has been frequently implicated with the serious side effect of hepatic nodular regenerative hyperplasia. Our lab has shown that this is dose related arising from high concentrations of thioguanosine triphosphate (TGTP) or related metabolites in the liver sinusoids. It is believed that thiopurine pro-drugs have an absolute requirement for the host’s purine salvage pathway for conversion to the main active drug, TGTP. Hypoxanthine guanine phosphoribosyl transferase (HPRT) enzyme is essential for metabolism of TG into its active thioguanosine nucleotides (TGNs) (TGTP being most active), thought to be responsible for improving colitis via effects on immune cells. Our lab recently published in Gut a novel observation which showed that TG could remarkably improve murine chronic colitis (induced) in the absence of Hprt. This led us to explore the role of effect of TG on gut microbiota, which also possess purine salvage enzymes, including HPRT. We investigated if gut bacteria could contribute to conversion of thiopurine pro-drugs TG and MP to their active TGN metabolites. A rapid high-pressure liquid chromatography-ultraviolet (HPLC-UV) method was developed to measure bacterial thiopurine nucleoside and thiopurine base as indirect measure of TGNs (difficult to measure) in bacteria. We showed that representative gut bacteria could convert TG and MP to TGNs and significantly less TGNs were produced from MP (likely because conversion was rate limited by inosine monophosphate dehydrogenase). In order to explore whether thiopurines especially TG could ameliorate spontaneous colitis in a murine model that lacked host Hprt we have generated HaW mice by crossing Hprt-/- and Winnie (spontaneous chronic colitis mouse model). Using HaW mice model, we showed that short term oral intervention with TG or a much higher dose of MP could ameliorate spontaneous colitis in HaW while avoiding systemic immunosuppression. We hypothesised that the amelioration of colitis in HaW mice involved bacterial HPRT metabolism. We could show that intra-rectal administration of TG but not MP (at similar dose) could ameliorate locally distal colitis in HaW mice. We further investigated the role of bacterial HPRT in metabolising thiopurine pro-drugs TG and MP to their active TGNs by generating an E. coli BL21 derived strain that carries an inactivating group II intron insertion in HPRT. Surprisingly, we found that E. coli BL21hprt could convert TG to TGNs. The amount of generated TGNs with TG incubation was five times higher than the E. coli BL21, but bacterial viability was affected after 8 hrs. We suspect existence of other yet to be discovered purine enzymes. Further research could provide novel insights into highly conserved purine pathways. Manipulating the gut microbiota with antibiotics, prebiotic/probiotic treatments or faecal microbiota transplant has been a burgeoning interest in recent times. In this thesis I have further explored the role of microbiome in IBD by manipulating the microbiome in Winnie and C57Bl/6 WT mice using a cocktail of five antibiotics (AB5). I showed that short term intervention (10 days) of AB5 successfully ameliorated spontaneous colitis in Winnie mice. AB5 also resulted in gigantic mucus filled caecum (a germ free-like intestinal phenotype) by the end of the treatment in both Winnie and WT mice. There was also a reduction in the mRNA expression of endoplasmic reticular stress, oxidative stress and pro-inflammatory cytokines markers in Winnie treated with AB5. Sequencing analysis of caecal mucosal microbiome revealed significant shifts with AB5 treatment to what was taxonomically a ‘dysbiosis’. AB5 treatment did not reduce total microbial load. Changes were qualitatively similar with caecal content and caecal mucosa. Pilot in vitro experiments with faecal waters from Winnie and WT +/- AB5 treatment using NF-B (central to the IBD associated inflammatory response) reporter cell line revealed that the ‘dysbiotic’ shifts observed with AB5 treatment were less pro-inflammatory compared to untreated Winnie faecal waters. I found that the positive clinical response induced with AB5 treatment could sustain for the tested 8 weeks observation period after treatment withdrawal. The AB5 induced germ free-like effect was lost at the end of 4 weeks observation period. To examine if that alteration in colonic microbial composition could directly influence inflammation, we induced colitis in WT mice using 1% dextran sulphate sodium (DSS) and co-treated the WT DSS mice with gavages of anaerobic caecal contents collected from earlier experiments with WT, Winnie and Winne AB5-treated mice. Caecal contents from WT and Winnie AB5 trended to improve DSS colitis in a WT mice and Winnie caecal content gavage aggravated it. Further investigation is required to better understand the mechanisms of how microbiome manipulation (AB5 or caecal gavage) can affect a reduction in chronic inflammation. The overall outcomes of my thesis provide insights in better understanding the mechanisms to design and develop novel colonic therapies for IBD which are fast acting, safe, economic and efficacious.

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Thesis (PhD Doctorate)

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Doctor of Philosophy (PhD)


School of Pharmacy and Pharmac

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