NF-\u03baB2 signalling in enteroids modulates enterocyte responses to secreted factors from bone marrow-derived dendritic cells

Lauren G Jones,Carrie A Duckworth,Jorge H Caamaño,D Mark Pritchard,Andra Vaida,Jonathan M Williams,Louise M Thompson,Fabio Miyajima,Michael D Burkitt,Felix I Ikuomola,Barry J Campbell

doi:10.1038/s41419-019-2129-5

Abstract

Alternative pathway NF-κB signalling regulates susceptibility towards developing inflammatory bowel disease (IBD), colitis-associated cancer and sepsis-associated intestinal epithelial cell apoptosis and shedding. However, the cell populations responsible for the perturbed alternative pathway NF-κB signalling in intestinal mucosal pathology remain unclear. In order to investigate the contribution of the epithelial compartment, we have tested whether NF-κB2 regulated transcription in intestinal epithelial cells controls the intestinal epithelial response to cytokines that are known to disrupt intestinal barrier permeability. Enteroids were generated from the proximal, middle and distal regions of small intestine (SI) from C57BL/6J wild-type mice and displayed region-specific morphology that was maintained during sub-culture. Enteroids treated with 100 ng/mL TNF were compared with corresponding regions of SI from C57BL/6J mice treated systemically with 0.33 mg/kg TNF for 1.5 h. TNF-induced apoptosis in all regions of the intestine in vitro and in vivo but resulted in Paneth cell degranulation only in proximal tissue-derived SI and enteroids. TNF also resulted in increased enteroid sphericity (quantified as circularity from two-dimensional bright field images). This response was dose and time-dependent and correlated with active caspase-3 immunopositivity. Proximal tissue-derived enteroids generated from Nfκb2−/− mice showed a significantly blunted circularity response following the addition of TNF, IFNγ, lipopolysaccharide (LPS) activated C57BL/6J-derived bone marrow-derived dendritic cells (BMDC) and secreted factors from LPS-activated BMDCs. However, Nfκb1−/− mouse-derived enteroids showed no significant changes in response to these stimuli. In conclusion, the selection of SI region is important when designing enteroid studies as region-specific identity and response to stimuli such as TNF are maintained in culture. Intestinal epithelial cells are at least partially responsible for regulating their own fate by modulating NF-κB2 signalling in response to stimuli known to be involved in multiple intestinal and systemic diseases. Future studies are warranted to investigate the therapeutic potential of intestinal epithelial NF-κB2 inhibition.

Highlights

We have recently identified components of the alternative NFκB signalling pathway that are important in modulating the susceptibility to inflammatory bowel disease (IBD), colitis-associated cancer and intestinal epithelial apoptosis and cell shedding in mice
As we have shown that Tumour necrosis factor (TNF) causes apoptosis and Paneth cell depletion at the dose and timepoints tested in mice in vivo and in enteroids, we wanted to determine whether this treatment had any impact on intestinal proliferation
We have previously shown that Nfκb2−/− mice are resistant to TNF-induced villus tip apoptosis and cell shedding compared with their wild-type C57BL/6J counterparts[17]

Summary

Introduction

The intestine is lined by a single layer of columnar epithelial cells that function to maintain a barrier betweenOfficial journal of the Cell Death Differentiation AssociationJones et al Cell Death and Disease (2019)10:896 recently shown that defective intestinal barrier function caused by cell shedding in the terminal ileum predicts relapse of inflammatory bowel disease (IBD) in humans[4].Defects in intestinal barrier function may be involved in the pathogenesis of systemic conditions, such as metabolic endotoxaemia and sepsis[5,6].Several pro-inflammatory cytokines are present at increased concentrations in the circulation, intestinal lumen and lamina propria of the intestine during active IBD, infectious diarrhoea, coeliac disease and sepsis[7,8,9,10,11,12].Tumour necrosis factor (TNF) is a well characterised cytokine that is produced in these conditions and is a key mediator of mucosal pathology[3,13]. The intestine is lined by a single layer of columnar epithelial cells that function to maintain a barrier between. Jones et al Cell Death and Disease (2019)10:896 recently shown that defective intestinal barrier function caused by cell shedding in the terminal ileum predicts relapse of inflammatory bowel disease (IBD) in humans[4]. Defects in intestinal barrier function may be involved in the pathogenesis of systemic conditions, such as metabolic endotoxaemia and sepsis[5,6]. Several pro-inflammatory cytokines are present at increased concentrations in the circulation, intestinal lumen and lamina propria of the intestine during active IBD, infectious diarrhoea, coeliac disease and sepsis[7,8,9,10,11,12]. Anti-TNF therapies are widely used clinically to ameliorate active Crohn’s disease[14]

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Conclusion