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Abstract Background To protect the gastrointestinal tract from infection, intestinal epithelial cells (IECs) lining the mucosa possess cytosolic complexes called inflammasomes which detect pathogenic insult and rapidly initiate inflammation to stop infectious spread. Active inflammasomes mediate crucial effector functions such as epithelial cell extrusion, pyroptotic cell death, and the release of interleukin (IL)-18 and prostaglandin E2 (PGE2). Recent literature has shown that IL-18 and PGE2 mediate intestinal repair following epithelial damage from DSS-induced colitis. Exogenous administration of IL-18 in inflammasome deficient mice improves survival and reduces pathology. PGE2 activates intestinal cellular reprogramming to drive intestinal wound healing. Whether inflammasome derived IL-18 and PGE2 mediate epithelial repair during enteric infection is unclear. Aims Using murine small intestinal organoids (mSIOs), we will investigate how IL-18 and PGE2 production by intestinal inflammasomes directly impacts IECs at both transcriptional and physiological levels. We hypothesize that inflammasome derived IL-18 and PGE2 will act on the intestinal epithelium resulting in transcriptional reprogramming that promotes tissue regeneration and barrier reinforcement. Methods WT and Nlrc4-/- mSIOs were stimulated with flagellin or FlaTox to activate the NAIP-NLRC4 inflammasome. The transcriptomic landscape was assessed with bulk-RNA sequencing and inflammasome activation was assessed by Western blot. IL-18 and PGE2 dependent gene targets were identified using Il-18-/- and indomethacin treated mSIOs by RT-qPCR. Results Bulk-RNA sequencing revealed an inflammasome activation dependent transcriptional signature in murine IECs upon NAIP-NLRC4 inflammasome activation. GO term analysis showed these genes to be involved in actin cytoskeleton rearrangement, epidermal growth factor signaling, and regulation of cell-cell adhesion. The expression of PGE2 gene targets were significantly reduced by indomethacin treatment while IL-18 gene targets remained unaffected in Il-18-/- IECs. FlaTox induced the same transcriptional changes but substantially amplified their expression. FlaTox but not flagellin led to a significant drop in Lgr5 (stem cells) and Lyz1 (Paneth cell) expression and an increase in Ly6a, a marker of fetal-like stem cells. Conclusions This data demonstrates that inflammasome signaling causes transcriptional reprogramming of IECs that activates pathways involved in maintaining epithelial barrier integrity. While IL-18 had no effects on gene expression at 4 hours, PGE2 paracrine signaling contributed to the expression of wound healing genes. Stronger inflammasome mediated damage with FlaTox amplified the repair response at the transcript level and we observed transient reprogramming of IECs into a fetal-like state suggesting active tissue regeneration. Funding Agencies CCC, CIHREPIC: Emerging & Pandemic Infections Consortium
Ranger et al. (Wed,) studied this question.