OC-043 Nfkb1 deficiency alters susceptibility to helicobacter spp. induced il-1Β secretion in bone marrow derived dendritic cells
Authors: JM Tang, DM Pritchard, CA Duckworth, J Caamano, MD Burkitt
Journal: Gut (2015)

<h3>Introduction</h3> Deletion of specific NF-κB subunits in mice alters the outcome of <i>Helicobacter felis</i>infection. <i>Nfkb1<sup>-/-</sup></i>mice developed more severe gastric atrophy than wild-type (WT) mice 6 weeks after <i>H. felis</i>infection, whereas <i>Nfkb2<sup>-/-</sup></i>mice were protected from this pathology. The mechanisms underlying these outcomes remain unclear, but gastric <i>Il1b</i>transcript abundance was increased in <i>H. felis</i>infected <i>Nfkb1<sup>-/-</sup></i>mice relative to WT, and polymorphisms at the IL1B locus have also been associated with gastric cancer in humans. <i>Il1b</i>transcription is regulated by classical pathway NF-κB signalling. IL-1β secretion also requires the formation of inflammasome complexes, which form following intracellular pathogen recognition, and lead to the autocatalysis of pro-caspase 1 and subsequent cleavage of IL-1β. We hypothesised that inflammasome signalling was altered in mice with abrogated NF-κB signalling and that this <i>influenced H. felis</i>induced pathology. We therefore investigated inflammasome mediated signalling in bone marrow derived dendritic cells (BMDCs) from mice lacking specific NF-κB subunits. <h3>Method</h3> Cells were harvested from C57BL/6, <i>Nfkb1<sup>-/-</sup>,</i><i>Nfkb2<sup>-/-</sup></i>and <i>c-Rel<sup>-/-</sup></i>bone marrow. Dendritic cells were differentiation using 20 ng/ml GM-CSF for 7 days. BMDCs and WT derived gastric epithelial organoids were primed with 20 ng/ml LPS and exposed to 300 μg/ml silica, 5 mM ATP, <i>H. pylori</i>(ATCC 53726) or <i>H. felis</i>(ATCC 49179) (MOI 1:100) with or without a pan-caspase inhibitor (Z-VAD-fmk), an inhibitor of NADPH oxidase (APDC) or 50 mM KCl. Secreted IL-1β and TNF concentrations were measured by ELISA. <h3>Results</h3> LPS alone induced TNF, but not Il-1β, secretion in all genotypes of BMDCs. Both silica and ATP induced IL-1β secretion in WT BMDCs pre-stimulated with LPS (750 ± 65 and 530 ± 77 pg/ml). BMDCs derived from NF-κB deficient mice secreted similar amounts of Il-1β in response to these stimuli. Inflammasome inhibitors returned IL-1β secretion to unstimulated levels. Gastric epithelial organoid cultures treated with LPS and silica did not secrete either IL-1β or TNF. Following exposure to <i>H. felis</i>or <i>H. pylori</i>, <i>Nfkb1<sup>-/-</sup></i>BMDCs, but not other genotypes, exhibited a 2.6 fold increase in IL-1β secretion compared to untreated cells or cells stimulated with LPS. This was abrogated by Z-VAD-fmk. <h3>Conclusion</h3> These data identify potent inflammasome activation in NF-κB deficient BMDCs. <i>H. pylori</i>and <i>H. felis</i>also weakly stimulated inflammasome activation in <i>NFkb1-/-</i>BMDCs. This mechanism may contribute to the more severe gastric phenotype that is observed in <i>NFkb1<sup>-/-</sup></i>mice <i>in vivo</i>following <i>H. felis</i>infection. Further studies are required to identify how NF-κB1 deletion influences inflammasome formation, and whether altered IL-1β transcription is involved. <h3>Disclosure of interest</h3> None Declared.