Edinburgh Napier University

  Crohn’s disease (CD), one of the main forms of Inflammatory Bowel Disease (IBD), is a complex disorder characterised by chronic inflammation of the gastrointestinal (GI) tract. The aetiology of CD involves genetics and environmental factors that trigger an abnormal immune response to intestinal bacteria. Genome-wide association studies have strongly linked genes involved in autophagy, such as ATG16L1, to CD. Autophagy is a cellular degradation process that clears intracellular bacteria and regulates inflammatory responses. Recent studies suggest that enhancing autophagy in CD patients may be therapeutically beneficial. The aim of this study was to characterise the mechanism of action of a panel of commonly used IBD drugs in the context of autophagy and autophagy-related pathways, such as the unfolded protein response (UPR) and apoptosis.

Modulation of autophagy was assessed in vitro, and in peripheral blood mononuclear cells (PBMCs) and GI biopsies from paediatric IBD patients. Several complimentary techniques to monitor the autophagy marker LC3 and master regulator of autophagy, mechanistic target of rapamycin (mTORC1), were used. Varying stages of apoptosis were assessed using a range of techniques and activity of UPR mediators was measured using RT-qPCR and western immunoblotting. The clearance of CD-associated adherent-invasive E. coli (AIEC) was assessed using gentamicin protection assays, and pro-inflammatory cytokines were monitored by RT-qPCR.

Our results reveal that the immunosuppressant drug azathioprine is a strong inducer of autophagy and this response was independent of apoptosis. Azathioprine induced autophagy via inhibition of mTORC1 and up-regulation of the UPR. Azathioprine also enhanced the clearance of intracellular AIEC and dampened pro-inflammatory cytokine responses. Furthermore, azathioprine induced autophagy in paediatric patient samples, and this response was more pronounced in patients harbouring the CD-associated ATG16L1 variant. A better understanding of IBD drug mechanism of action can contribute to patient stratification for the development of a more personalised therapeutic approach.