P040 Caspase-8 blockade attenuates NSAID-induced colitis and cell death via NLRP3 inflammasome and NF-κB activation

Abstract

Abstract Background Non-steroidal anti-inflammatory drugs (NSAIDs) are believed to exacerbate symptoms and inflammation in patients with inflammatory bowel disease (IBD). Recent studies indicate the gut microbiota may play a role in NSAID-promoted inflammation. However, mechanism(s) underpinning NSAID-induced inflammation are not fully understood. In this study, using the piroxicam-accelerated model of colitis (PAC) in IL-10-/- mice, we examined the mechanism(s) regulating NSAID-induced colitis. Methods To establish whether microbiota plays a role in PAC, IL-10-/- mice were treated with streptomycin for 24hrs followed by feeding with 100 ppm piroxicam in their food for 9 days. In the inhibitor study, mice were injected intra-peritoneally with the caspase-8 inhibitor (Z-IETD-FMK, 10mg/kg) every other day starting before piroxicam-feeding. Colon tissue was collected on day 14 for analysis on inflammation and cell death markers by qRT-PCR, MesoScaleDiscovery (MSD)-assay, western blot, and histology. Faecal samples were collected at different time-points for 16S rRNA gene sequencing. Results A significantly higher expression of colonic inflammatory markers and a reduced expression of epithelial barrier proteins and elongated crypts were found in PAC-mice, which was independent of microbiota. Piroxicam feeding did not cause major changes in fecal microbiota diversity or composition on day 14. An activation of members of the NLRP3 inflammasome family including caspase-1 and IL-1b, Caspase-8, and the pro-apoptotic markers -cleaved caspase 3 and PARP- were associated with piroxicam-induced colitis. Inhibition of caspase-8 ameliorated NSAID-provoked colitis and apoptosis by preventing mitochondrial damage, and downregulating NF-κB and NLRP3 activation as well as reducing cleaved caspase-3 and PARP levels. Histology analysis revealed an improvement in colonic epithelial morphology associated with shorter crypts and reduced immune cell infiltration to the lamina propria. Conclusion Our data provides evidence on how NSAIDs can promote intestinal inflammation by activating caspase-8, NLRP3 inflammasome and NF-κB, resulting in induction of inflammation and apoptosis. Targeting Caspase-8 could be a potential treatment strategy for patients with NSAID-worsened symptoms and inflammation