Optimising the induction of inflammation within preterm infant-derived intestinal epithelial organoids.

Preterm infants born <32 weeks gestation have abnormal microbial colonisation and dysregulated inflammation within the gut. Preterm infant-derived intestinal organoids (PIOs) represent a valuable model for investigating gut microbiome-host interactions and inflammatory responses. We optimised an inflammation model in PIO monolayers incubated within an anaerobic co-culture system that recreates the physiological oxygen gradient of the intestinal epithelium. We trialled multiple stimuli, including live and heat-killed pathobiont consortia, lipopolysaccharide (LPS) and flagellin. We found that a combination of apical LPS and basolateral flagellin, incubated for 3 h, elicited the most robust response. This was characterised by enhanced pro-inflammatory cytokine secretion, the potential for chemokine-driven immune recruitment, TNFα and IL17C pathway signalling, shifts from NF-κB to AP-1-mediated responses, and signs of tissue remodelling. This provides a framework for appropriate study design to disentangle the impacts of microbiome-host interactions in health and disease using intestinal organoids.