Introduction Genome wide association studies and functional experiments in inflammatory bowel disease (IBD) have delineated the importance of autophagy in IBD pathogenesis. We aimed to determine the effect of commonly utilised IBD drugs on autophagy induction and the pathways involved in vitro .
Methods Cells naturally expressing (HCT116) and not expressing (HEK293) NOD2, both stably expressing green fluorescent protein-labelled light chain 3 (LC3), were treated with varying concentrations of 6-thioguanine, azathioprine, methotrexate or infliximab at different time points; rapamycin, serum-starvation and bafilomycin A1 served as positive controls. Cells were also treated with ERK (U0126) and autophagy (3-methyladenine) inhibitors where appropriate. For immunofluorescent microscopy images were captured using an Axioskop 2 fluorescent microscope and ImageJ software used to identify cells with >5 punctate foci indicating autophagy induction. For western blot analysis cell lysates were immunoblotted with antibodies to LC3, p62, phospho-rpS6 or total rpS6. All statistical analyses were performed using GraphPad Prism.
Results All four drugs induced significant autophagy in HCT116 cells, with only azathioprine inducing autophagy robustly in both cell lines. Azathioprine induced autophagy in a dose-dependent manner in HEK293 cells with significant autophagy induction at all concentrations (30–90 μM) in HCT116 cells. HCT116 cells treated with 6-thioguanine, azathioprine and methotrexate showed strong LC3-I to LC3-II conversion and a reduction in p62, with 6-thioguanine and azathioprine showing loss of phospho-S6K suggesting autophagy induction through the mTORC1 pathway. Use of U0126 and 3-methyladenine in HCT116 cells treated with azathioprine demonstrated that azathioprine may exert its autophagic effect via mTORC1 through the class I PI3K/Akt pathway.
Conclusion Common IBD drugs effect autophagy induction in vitro suggesting that manipulation of the autophagy pathway may be partly involved in the mechanism of action of many of these drugs, most convincingly azathioprine. Further work is now required to replicate these findings and further delineate the pathways in vivo .
Disclosure of Interest None Declared.