Apical-Out Enteroids as an Innovative Model for Necrotizing Enterocolitis

Abstract

Introduction: Necrotizing enterocolitis (NEC) is a gastrointestinal disease of premature neonates. We previously validated a NEC enteroid model derived from human infant intestinal tissue. Typical enteroid configuration is basolateral-out (BO) without direct access to the luminal (apical) surface. Apical access is necessary to allow physiologic comparison of pathogen interaction with the intestinal epithelial barrier. We hypothesize that apical-out (AO) enteroids will provide a relevant NEC model to study this relationship.

Methods: Following the institutional review board approval (#11610-11611), neonatal intestinal tissue was collected from surgical specimens. Stem cells were collected; enteroids were generated and grown to maturity in BO conformation then everted to AO. Enteroids were untreated or treated for 24 h with 100 μg/mL lipopolysaccharide and hypoxia. Protein and gene expression were analyzed for inflammatory markers, tight junction (TJ) proteins and permeability characteristic of NEC.

Results: Apical TJ protein zonula occludens-1 and basolateral protein β-catenin immunofluorescence confirmed AO configuration. Treated AO enteroids had significantly increased messenger RNA (P = 0.001) and protein levels (P < 0.0001) of tumor necrosis factor-α compared to controls. Corrected total cell fluorescence of toll-like receptor 4 was significantly increased in treated AO enteroids compared to control (P = 0.002). Occludin was found to have significantly decreased messenger RNA in treated AO enteroids (P = 0.003). Expression of other TJ proteins claudins-1, -4 and zonula occludens-1 was significantly decreased in treated AO enteroids (P < 0.05).

Conclusions: AO enteroids present an innovative model for NEC with increased inflammation and gut barrier restructuring. This model allows for a biologically relevant investigation of the interaction between the pathogen and the intestinal epithelial barrier in NEC.

Keywords: Apical-out; Enteroids; Model; Necrotizing enterocolitis.