P159 Inhibition of Caspase-1 signalling subtly affect the gut microbiota and reduces Adherent-invasive Escherichia coli (AIEC)-induced inflammasome activation

Abstract

Abstract Background Inflammatory bowel disease (IBD), encompassing Crohn’s Disease and ulcerative colitis, is a chronic gastrointestinal disorder with increasing incidence worldwide. Microbial factors such as Adherent-invasive Escherichia coli (AIEC) have been suggested to play a significant role in the pathogenesis of IBD. The inflammasome, a multi-protein complex, senses environmental signals, including bacteria, resulting in the activation of caspase-1 leading to the subsequent maturation and secretion of IL-1β. In this study we examined the impact of AIEC on inflammasome activation and on the inhibition of Caspase-1 signaling in AIEC-colonised mice and on intestinal epithelial cells (IECs). Methods Human IECs (CaCO2Bee1 cells) were cultured for 3hr with AIEC (HM605 and its invasiveness mutant), washed with antibiotics and further cultured in full cell media for 13hrs, followed by analysis on cytokine and inflammasome gene expression and activation by qRT-PCR, ELISA, imaging and flow cytometry. To inhibit caspase-1 activation, cells were pre-treated for 1 hr with the caspase-1 inhibitor zVAD-fmk. To establish AIEC colonisation, C57BL/6 and IL-10-/- mice were treated with streptomycin for 24hrs followed by oral gavage of AIEC-HM605 (10-8 cfu/mL) and faecal and tissue samples were collected at 6 days post AIEC-infection. Caspase-1 inhibitor was orally gavaged every in mice. Faecal samples were prepared for 16S rRNA gene sequencing and colon tissue was analysed for cytokines and inflammasome markers by qRT-PCR and ELISA. Results IECs cultured with AIEC and AIEC-colonised C57BL/6 and IL-10-/- mice resulted in an increased expression and activation of both canonical (Caspase-1) and non-canonical inflammasome (caspase 4). The AIEC-induced inflammasome response was dependent on the invasiveness of AIEC. Treatment with a caspase-1 inhibitor reduced the AIEC-induced inflammasome markers in IECs and in C57BL/6 mice. No major changes in alpha or beta-diversity was detected in AIEC-colonised mice or upon Caspase-1 inhibition. AIEC-colonisation resulted in an increased abundance of Bacteroides and Alistipides and reduced abundance of Lachnospiraceae, alterations reversed by Caspase-1 inhibition. Conclusion We show that the invasion of AIEC in IECs and colonisation of murine intestine results in activation of caspase-1 inflammasome. AIEC colonisation provoke subtle microbiota alterations which are reversed by caspase-1 inhibition. In summary, our data supports a role of AIEC in IBD pathogenesis through the activation of caspase-1 inflammasome.