A Novel Human Epithelial Enteroid Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is a devastating disease of newborn infants. It is characterized by multiple pathophysiologic alterations in the human ...

Northwestern University

Necrotizing enterocolitis (NEC) is a serious intestinal disease that occurs in newborn infants. It is associated with major morbidity and affects 5% of all infants admitted to neonatal intensive care units. Probiotics have variable efficacy in preventing necrotizing enterocolitis. Tight junctions (TJ) are protein complexes that maintain epithelial barrier integrity. We hypothesized that the probiotics  and  strengthen intestinal barrier function, promote TJ integrity, and protect against experimental NEC. Both an  and an  experimental model of NEC were studied. Cultured human intestinal Caco-2 cells were pretreated with  and  probiotics. TJ were then disrupted by EGTA calcium switch or LPS to mimic NEC . Trans-epithelial resistance (TER) and flux of fluorescein isothiocynate dextran was measured. TJ structure was evaluated by ZO-1 immunofluorescence.  effects of ingested probiotics on intestinal injury and ZO-1 expression were assessed in a rat model of NEC infected with  (CS). Caco-2 cells treated with individual probiotics demonstrated higher TER and lower permeability compared to untreated cells (p<0.0001). ZO-1 immunofluorescence confirmed TJ stability in treated cells. Rat pups fed probiotics alone had more intestinal injury compared with controls (p=0.0106). Probiotics were protective against injury when given in combination with CS, with no difference in intestinal injury compared to controls (p=0.21). Increased permeability was observed in the probiotic and CS groups (p=0.03, p=0.05), but not in the probiotic plus CS group (p=0.79).  sp. strengthened intestinal barrier function and preserved TJ integrity in an  experimental model of NEC. , probiotic bacteria were not beneficial when given alone, but were protective in the presence of CS in a rat model of NEC.

Probiotic  Species Strengthen Intestinal Barrier Function and Tight Junction Integrity in Experimental Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC) is a deadly disease that occurs in 5-10% of neonates. Although NEC has been extensively studied, no single therapeutic target has been identified. Rho kinase (ROCK) is a serine/threonine kinase that affects multiple cellular processes, including tight junction (TJ) function, cellular permeability, and apoptosis. We hypothesized that ROCK inhibition would decrease cellular permeability, stabilize TJ proteins (occludin), and decrease the severity of NEC. To test this hypothesis, human colon epithelial cells (Caco-2) and human endothelial cells were studied. Cells were treated with lipopolysaccharide to simulate an in vitro model of NEC. The effect of ROCK inhibition was measured by transepithelial membrane resistance (TEER) and cellular permeability to FITC-dextran. The effects of ROCK inhibition in vivo were analyzed in the rat pup model of NEC. NEC was induced by feeding formula supplemented with Cronobacter sakazakii with or without gavaged ROCK inhibitor. Rat intestines were scored based on histological degree of injury. RNA and protein assays for occludin protein were performed for all models of NEC. Treatment with ROCK inhibitor significantly decreased cellular permeability in Caco-2 cells and increased TEER. Intestinal injury scoring revealed decreased scores in ROCK inhibitor-treated pups compared with NEC only. Both cell and rat pup models demonstrated an upregulation of occludin expression in the ROCK inhibitor-treated groups. Therefore, we conclude that ROCK inhibition protects against experimental NEC by strengthening barrier function via upregulation of occludin. These data suggest that ROCK may be a potential therapeutic target for patients with NEC. NEW & NOTEWORTHY These studies are the first to demonstrate an upregulation of occludin tight junction protein in response to Rho kinase (ROCK) inhibition. Furthermore, we have demonstrated that ROCK inhibition in experimental models of necrotizing enterocolitis (NEC) is protective against NEC in both in vitro and in vivo models of disease.

Rho kinase inhibition maintains intestinal and vascular barrier function by upregulation of occludin in experimental necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal emergency of neonates. Epithelial tight junction (TJ) proteins, such as claudins, are essential for regulation and function of the intestinal barrier. Rho kinase (ROCK) affects cellular permeability and TJ regulation. We hypothesized that TJ protein changes would correlate with increased permeability in experimental NEC, and ROCK inhibitors would be protective against NEC by regulation of key claudin proteins. We tested this hypothesis using an in vivo rat pup model, an in vitro model of experimental NEC, and human intestinal samples from patients with and without NEC. Experimental NEC was induced in rats via hypoxia and bacteria-containing formula, and in Caco-2 cells by media inoculated with LPS. The expression of claudins was measured by gene and protein analysis. Experimental NEC in rat pups and Caco-2 cells had increased permeability compared to controls. Gene and protein expression of claudin 2 was increased in experimental NEC. Sub-cellular fractionation localized increased claudin 2 protein to the cytoskeleton. ROCK inhibition was associated with normalization of these alterations and decreased severity of experimental NEC. Co-immunoprecipitation of caveolin-1 with claudin 2 suggests that caveolin-1 may act as a shuttle for the internalization of claudin 2 seen in experimental NEC. In conclusion, NEC is associated with intestinal permeability and increased expression of claudin 2, increased binding of caveolin-1 and claudin 2, and increased trafficking of claudin 2 to the cytoskeleton.

Douglas Wood

Department of Pediatrics

Northwestern University

A Novel Human Epithelial Enteroid Model of Necrotizing Enterocolitis

Douglas Wood

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Pediatrics

Northwestern University

A Novel Human Epithelial Enteroid Model of Necrotizing Enterocolitis

Department of Pediatrics