Epistem - Epithelial Response to Pro-inflammatory Cytokines

Intestinal epithelial barrier function is maintained by the tight regulation of several processes, such as cell proliferation, apoptosis and cell shedding rates.

Dysregulation of these processes results in the break-down of the intestinal barrier, which is believed to be a contributing factor to the development of multiple diseases, such as inflammatory bowel disease (IBD).

Pro-inflammatory cytokines are present at high concentration in the intestinal lumen and lamina propria during active IBD and can induce mucosal injury. Tumour necrosis factor (TNF), for example, can induce apoptosis and cell shedding from the small intestine villus tip within 1.5 hours of intraperitoneal administration in mice and is accompanied by diarrhoea.

The alternative NF-KB signalling pathway has been recently demonstrated to have a role in the susceptibility to IBD, colitis-associated cancer and intestinal epithelial apoptosis and cell shedding in mice. Indeed, Nfkb2^-/- mice were resistant to the induction of apoptosis and cell shedding by LPS or its downstream effector TNF.

Most of the studies characterising the alternative NF-KB signalling pathway, so far, have focused on the immune cell compartment, leaving its role in the epithelial cell response not understood in meaningful depth.

In a recent study, Jones et al. analysed the interaction between intestinal epithelium and immune cell derived cytokines by using the small intestinal organoid model. The authors hypothesised that Nfkb2 epithelial activation could sensitise the cells to the induction of apoptosis by pro-inflammatory cytokines and then explored whether Nfkb2 inhibition could be a potential therapeutic approach to improve inflammation-associated intestinal disease.

TNF induced organoids apoptosis and Paneth cells depletion without modulating cell proliferation.

Apoptosis and cell shedding into the lumen was positively correlated with an increase in the organoid circularity, due to the build-up of apoptotic cells into the lumen with no compensatory increase in cell proliferation.

Both wild-type and Nfkb1^-/- derived organoids were affected by TNF treatment while no induction of circularity was detected in organoids derived from Nfkb2^-/-, highlighting the importance of the alternative NF-KB signalling pathway in the response to cytokines.

Co-culture of LPS activated BMDCs with Nfkb2^-/- derived organoids did not induce an increase in circularity, whereas wild-type and Nfkb1^-/- organoids were affected. The effect on circularity was not due to TNF in this context, since it was produced at very low concentration, but high levels of other cytokines, such as IL6, were detected, suggesting their potential contribution to modulating epithelial cell dynamics.

Taken together, this data suggests that Nfkb2 signalling pathway may be targetable to improve intestinal inflammation.

Reference

Jones et al, NF-κB2 signalling in enteroids modulates enterocyte responses to secreted factors from bone marrow-derived dendritic cells. Cell Death and Disease (2019) 10:896

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GI Toxicity or Organoids?

Epistem offers validated small intestinal in vitro organoid models derived from variety of species, including human, canine and rodents. Our organoid cultures display cellular architecture that closely resembles that observed in vivo. Cultures mimic the stem cell niche allowing cell proliferation and differentiation to occur. The established organoid protocols allow MOA determination and provide in vitro to in vivo biological and PD linkages through following readouts:

Viability assays (visual or with MTS)
Histology/IHC or RNAscope to assess the effect on specific protein expression including LGR5
Gene expression (NGS, Microarray or qRT-PCR) to evaluate the effect of treatment on expression of signalling pathways genes
FACS analysis to identify specific cell populations affected
ELISA to identify and quantify cytokines of interest within organoid's spend media

Epistem's intestinal organoid models allow screening of multiple agents, with over 5000 screened to date including Wnt pathway regulators, cytokines, growth factor, chemotherapy drugs, steroids and lectins.