Tight Junction Changes in Neonatal Enteroid Model of Necrotizing Enterocolitis

Author(s):
Guillermo Ares; Christie Buonpane; Carrie Yuan; Douglas Wood; Catherine Hunter

Background:

Necrotizing enterocolitis (NEC) is a severe and often fatal gastrointestinal disorder of the newborn. Improved survival in early premature infants due to advances in neonatal care has led to an increase in the incidence of NEC globally. Despite decades of research, our understanding of the pathogenesis NEC is incomplete. Enteroids are 3-dimensional structures that allow for the study of the intestinal epithelium and the manipulation of such, serving as a human tissue model of NEC. Tight junction (TJ) proteins are pivotal in regulating intestinal permeability.

Hypothesis:

We hypothesize that strengthening TJ proteins, such as Claudin 4 (C4), will be underexpressed, and pore-forming proteins, like Claudin 2 (C2), will be increased in an enteroid model of NEC.

Methods:

Human intestinal stem cells were harvested from bowel resection samples obtained from neonates with NEC vs other conditions (control). The isolated stem cells were grown in a basement membrane matrix with clean media vs media with lipopolysaccharide (LPS) to induce NEC. Additionally, we compared findings to an established rat model of NEC, where premature rat pups were fed clean formula (control) vs formula containing bacteria to induce NEC. Human tissue, enteroids, and rat bowels were collected and analyzed by RT-PCR, western blot, and mean immunofluorescent intensity (MFI). Differences were compared with student’s T-test for significance.

Results:

C4 gene expression was downregulated in enteroids with experimental NEC and in humans with NEC (p<0.005). Likewise, C4 protein expression was decreased in enteroids and rats with experimental NEC (p=0.025). Conversely, C2 gene expression and protein were increased in enteroids and rats with experimental NEC vs control (p=0.02). Enteroids, rats, and humans with NEC had increased immunofluorescence of C2 (MFI 714±27 vs 528±29, p<0.0001). Not only was the MFI different in NEC, but there were also congruent changes in the subcellular localization of C2 and C4.

Conclusions:

Human NEC is associated with changes in TJ protein structure that may be responsible for the changes in intestinal permeability seen in these infants. Neonatal human enteroids are a novel model in the study of NEC that allow for the manipulation of study parameters in human tissue. Further research into the pathophysiology and therapeutic targets against NEC using this new model is warranted.