P0204 Impact of crypt niche factors and oxygen gradient on intestinal epithelial differentiation and inflammatory response
Authors: Dotti I , Rodríguez-Camejo C , Esteller M , Robbins L M , Ricart E , Salas A 
Published: January 2025

Abstract

Background
The gastrointestinal mucosa, with its rich vascularization juxtaposed to the anaerobic lumen, creates a steep physiological oxygen gradient along the intestinal crypt. This gradient, together with soluble stromal factors, is critical for maintaining barrier homeostasis. In inflammatory bowel disease (IBD), active inflammation disturbs stem cell niche environment and oxygen perfusion 1. However, the impact of niche factors and oxygen gradients on epithelial behaviour in health and IBD remains poorly understood. Here, we aim to elucidate how crypt niche factors and oxygen tension influence epithelial differentiation, barrier permeability and response to pro-inflammatory stimuli.

Methods
We used air-liquid interface (ALI) cultures derived from human intestinal organoids as a physiologically relevant crypt model 2. Differentiation was monitored across four culture conditions with progressively reduced stem cell niche factors (including Wnt3A and Rspondin), and upon exposure to IBD-associated cytokines IFN-ɣ, IL22 and IL17A. Submerged cultures were used to model low oxygen tension during differentiation. Epithelial differentiation kinetics and response to inflammatory stimuli were assessed using transcriptional analysis, trans-epithelial resistance measurement and morphological evaluation.

Results
Transition of the epithelial cultures to ALI conditions resulted in transient over-expression of stem/transit-amplifying (TA) markers (LGR5, MKI67), with expression levels correlating to stem cell niche factor availability (Fig. 1A). Culture conditions significantly influenced the differentiation kinetics, with progressive up-regulation of differentiation markers (AQP8, ZG16, CHGA) under decreasing stem cell niche factor availability (Fig. 1B). These factors also affected epithelial permeability (Fig. 1C) and morphology (Fig. 1D). In submerged cultures, differentiation and polarization were entirely inhibited, while stem/TA marker expression showed only partial perturbations (Fig. 1E). Oxygen tension variations further modulated epithelial responses to IFN-ɣ, IL22 and IL17A. Notably, submerged cultures exhibited enhanced expression of IFN-ɣ- and IL22-specific targets (CXCL10 and OLFM4, respectively) (Fig. 1F), despite the limited effect of cytokine stimulation on barrier integrity (Fig. 1G).

Conclusion
In conclusion, our findings indicate that crypt niche factors and oxygen gradients distinctly regulate epithelial behaviour, with the differentiated compartment being particularly sensitive to oxygen availability. These results suggest that diminished oxygen tension, as commonly observed in active IBD, may exacerbate epithelial barrier dysfunction and amplify the epithelial responses to pro-inflammatory stimuli.

References
1. Taylor CT, Colgan SP. Hypoxia and gastrointestinal disease. J Mol Med. 2007;85(12):1295-1300.
2. Wang Y, Chiang IL, Ohara TE, et al. Long-Term Culture Captures Injury-Repair Cycles of Colonic Stem Cells. Cell. 2019;179(5):1144-1159.e15.