P058 Bacterial activation of thioguanine results in lymphocyte independent improvement in murine colitis
I. Oancea1, 2, I. Das1, D. Aguirre de Cárcer3, R. Movva1, 4, V. Schreiber1, Y. Yang1, 5, M. Proctor1, R. Wang1, Y. Sheng1, M. Lobb1, P. Ó Cuiv6, J. A. Duley1, 4, J. Begun*2, 7, T. H. J. Florin1, 2
1University of Queensland, Mater Research Institute, Brisbane, Australia, 2University of Queensland, School of Medicine, Brisbane, Australia, 3CSIRO, Preventative Health National Research Flagship and Division of Livestock Industries, Brisbane, Australia, 4Griffith University, School of Pharmacy, Gold Coast, Australia, 5University of Queensland, School of Pharmacy, Brisbane, Australia, 6University of Queensland, Diamantina Institute, Brisbane, Australia, 7University of Queensland, Mater Research Institute, South Brisbane, Australia
Thiopurines are the most commonly prescribed oral immunomodulator drugs to treat IBD. They are pro-drugs. Their treatment effect is believed to require host conversion to active drug and drug loading of circulating lymphocytes. We examined the necessity for lymphocytes, host metabolism of one of the thiopurines, thioguanine (TG), and the microbiome, for the amelioration of colitis in spontaneous and induced animal colitis models.
C57Bl/6 mice with or without genetic alterations were gavaged daily for up to 28 days with TG (0.5–2.5 mg/kg/d), mercaptopurine(MP 2.5 mg/kg/d) or water (control). Disease activity (diarrhoea, rectal bleeding, and body weight) was scored daily. At sacrifice, blinded scoring of colonic histology, cytokine message, caecal luminal, and mucosal microbiomes were analysed. Western blots of TG treated cell lysates were used for autophagy determination.
TG but not MP (2.5 mg/kg/d) rapidly ameliorated spontaneous colitis in Winnie mice (p < 0.01). These mice have a point mutation in Muc2 secretory mucin, which leads to a Th17 dominant distal colitis. TG (0.5 mg/kg/d) also ameliorated dextran sodium sulphate-induced chronic colitis in wild-type mice (p < 0.01). Interestingly, Rag1-/-;Winnie mice, which lack T- and B-lymphocytes, develop spontaneous colitis, which was improved by TG therapy (p < 0.01). Disease activity (p < 0.001) and blinded histological colitis scores (p < 0.01) improved without immunosuppressive effects in the absence of host hypoxanthine(guanine) phosphoribosyltransferase (Hprt) mediated conversion of TG (0.5 mg/kg/d) to active drug. The caecal mucosa-associated microbiome shifted with TG treatment (DPCOA), but the genetic structure of the microbiome was not shifted in Winnie mice even at the highest TG dose. Colonic bacteria rapidly converted TG but not MP to the active metabolite, indicating that it is intestinal bacterial metabolism of the pro-drug that reduces inflammation in the mice lacking host Hprt. TG rapidly induced an Hprt-dependent autophagic flux in epithelial and macrophage cell lines and augmented intracellular bacterial killing in epithelial cells.
The treatment effects of TG are not necessarily dependent on the known actions of thiopurines to inhibit the antigen presenting cell-T lymphocyte synapse that leads to apoptosis of activated lymphocytes. Rapid local bacterial conversion of TG pro-drug to active metabolite augmented epithelial autophagy and intracellular bacterial killing and decreased intestinal inflammation and immune activation in our models. Preferential delivery of TG to sites of intestinal inflammation in IBD could decrease inflammation and immune activation while avoiding unwanted side effects because of portal and blood leukocyte metabolism.