Objective Mercaptopurine (MP) and pro-drug azathioprine are ‘first-line’ oral therapies for maintaining remission in IBD. It is believed that their pharmacodynamic action is due to a slow cumulative decrease in activated lymphocytes homing to inflamed gut. We examined the role of host metabolism, lymphocytes and microbiome for the amelioration of colitis by the related thioguanine (TG).
Design C57Bl/6 mice with or without specific genes altered to elucidate mechanisms responsible for TG's actions were treated daily with oral or intrarectal TG, MP or water. Disease activity was scored daily. At sacrifice, colonic histology, cytokine message, caecal luminal and mucosal microbiomes were analysed.
Results Oral and intrarectal TG but not MP rapidly ameliorated spontaneous chronic colitis in Winnie mice (point mutation in Muc2 secretory mucin). TG ameliorated dextran sodium sulfate-induced chronic colitis in wild-type (WT) mice and in mice lacking T and B lymphocytes. Remarkably, colitis improved without immunosuppressive effects in the absence of host hypoxanthine (guanine) phosphoribosyltransferase (Hprt)-mediated conversion of TG to active drug, the thioguanine nucleotides (TGN). Colonic bacteria converted TG and less so MP to TGN, consistent with intestinal bacterial conversion of TG to so reduce inflammation in the mice lacking host Hprt. TG rapidly induced autophagic flux in epithelial, macrophage and WT but not Hprt−/− fibroblast cell lines and augmented epithelial intracellular bacterial killing.
Conclusions Treatment by TG is not necessarily dependent on the adaptive immune system. TG is a more efficacious treatment than MP in Winnie spontaneous colitis. Rapid local bacterial conversion of TG correlated with decreased intestinal inflammation and immune activation.
- IBD MODELS
- DRUG METABOLISM
- COLONIC BACTERIA
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Contributors IO had major roles in experimental design, data acquisition analysis and interpretation and writing of the manuscript; RM performed with direction from IO experiments in figures 3–6; ID was involved with acquisition and analysis of data in figures 1 and 2; DAdC produced the bacterial community profiles and performed the microbiome analyses and led interpretation in figure 4; VS and BH contributed to data acquisition and analysis for figure 6; YY performed with IO the experiments in figure 3; MP contributed to data acquisition in figures 2–7; RW contributed to data acquisition in figures 3, 5 and 6; YS contributed to data acquisition in figures 2–4; AP contributed along with IO to the organoid cultures and intrarectal experiments; RL instigated the organoid culture systems in our lab; ML contributed to method development, figure 5; PÓC provided oversight of the microbiology and contributed to the writing of the manuscript; JAD provided expertise throughout concerning thiopurine biochemistry measurement and intermediate metabolism; JB contributed to experimental design, method development and data analysis for figure 6 and contributed to manuscript writing; THJF conceived the overall project and experimental design, is responsible overall for the integrity of the data, data analysis and interpretation and the writing and final manuscript.
Funding THJF gratefully acknowledges funding from the Gutsy Charity (Incorporated Victoria, Australia). IO and THJF also received funding from NHMRC project grant 1064440. IO and JB have had UQ Reginald Ferguson Fellowships. JB and THJF receive funding from the Mater Foundation.
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.
Data sharing statement Provisional Patent filed 29 September 2015. PAT-02211-AU-01 “Novel Formulation and Treatment Methods”. Data and Materials availability: Accession number PRJEB10595 (deposited at European Nucleotide Archive).
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