P076 Role of gut microbiota in mediating the effects of thiopurines

Background A general consensus exists that patients with inflammatory bowel disease (IBD) present compositional changes in the gut microbiota (dysbiosis), including an increase in the abundance of Enterobacteriaceae. Thiopurine drugs are commonly used in the maintenance of remission in IBD. In this context, the purpose of the project is to explore the role of candidate bacterial strains in mediating the effects of thiopurines in vitro. Methods Azathioprine (AZA), mercaptopurine (MP) and thioguanine (TG) (400 µM) were incubated in minimal salts medium (M9) in presence or not of E. coli, S. enterica and K. pneumoniae and of their growth phase broths (GPB) for 4 h at 37°C. The viability of NALM6 (B cells) and JURKAT (T cells) exposed to serial dilution of drugs (ranging from 0.2 to 15 μM of AZA, from 0.3 to 20 μM of MP, from 0.08 to 5 μM of TG) previously incubated or not with bacteria and with their GPB was determined by the MTT assay. Absorbance peaks of thiopurines were analysed by UV spectrophotometry. Statistical significance was assessed by two-way ANOVA and Bonferroni’s post-test for MTT tests and by one-way ANOVA for UV spectra. Results In NALM6 cells, the cytotoxic effects of 15 μM of AZA, 2.5 μM of MP and 1.25 μM of TG decreased significantly (p < 0.001) after incubation with K. pneumoniae (respectively 45 ± 2.9%; 34 ± 2.5%% and 21 ± 0.6%) and its GPB (respectively 41 ± 7.7%; 41 ± 5.1% and 27 ± 3.5%) compared with the drugs not previously exposed (respectively 76 ± 2.3%; 69 ± 1.7% and 43 ± 3.8%). In JURKAT cells, the cytotoxic effects of 15 μM of AZA, 2.5 μM of MP and 1.25 μM of TG decreased significantly (p < 0.001) after incubation with K. pneumoniae (respectively 46 ± 2.8%; 38 ± 1.29% and 19 ± 3.3%) and its GPB (respectively 49 ± 9.4%; 38 ± 1.5% and 26 ± 1.5%) in comparison with the drugs not exposed (respectively 75 ± 4.0%; 50 ± 3.5% and 54 ± 4.0%). E. coli and S. enterica did not affect the cytotoxicity of the thiopurines. UV analysis evidenced a reduction of absorbance peaks of AZA (21 ± 0.05%), MP (32 ± 0.015%) and TG (30 ± 0.03%) after incubation with K. pneumoniae but not with its growth phase broth (GPB). Conclusion The activity of thiopurines decreased after incubation with both K. pneumoniae and its GPB. UV analysis suggested that the lower cytotoxicity of thiopurines exposed to the bacterial strain is due to the reduction of the concentration of the drugs exposed to K. pneumoniae. Moreover, the reduction of drug availability after the exposure to GPB could be explained with a possible interaction between thiopurines and capsular polysaccharides released by the bacteria.