RESUMO
Background: Necrotizing enterocolitis (NEC) is the leading gastrointestinal cause of death of premature neonates. NEC is associated with prematurity, a hyperinflammatory response, and dysregulation of intestinal barrier function. We hypothesize that patients with NEC will have, and continue to have after recovery, an increased hyperinflammatory intestinal response compared to those patients without NEC. Methods: Neonates with NEC, those that have recovered from NEC, and those without NEC undergoing intestinal resections had specimens collected and snap frozen or generated into enteroids. The enteroids were treated with 100ug/mL lipopolysaccharide (LPS) and subjected to 24 hr of hypoxia together, then compared with untreated controls. Expression of Tumor Necrosis Factor (TNF-α) and interleukin 8 (IL-8) were evaluated via RT-qPCR and ELISA to measure inflammatory response. ANOVA determined statistical significance (p<0.05). Results: There was no difference in inflammatory markers in recovered NEC tissue compared to non-NEC tissue on RTqPCR (p=0.701 TNF-α and 0.861 IL-8). However, recovered NEC enteroids demonstrate elevated levels of inflammatory markers after treatment compared to non-NEC enteroids after treatment on RTqPCR (p=0.0485 TNF-α, p=0.0057 IL-8) and ELISA (p=0.0354 TNF-α, p=0.0011 IL-8). Recovered NEC enteroids that underwent treatment demonstrated increased inflammatory markers compared to recovered NEC enteroids without treatment on RTqPCR (p=0.0045 TNF-α, p=0.0002 IL-8) and ELISA (p=0.034 TNF-α, p=0.0002 IL-8) suggesting a heightened inflammatory response to a second hit. Conclusion: Intestinal tissue resected from neonates with NEC has an elevated hyperinflammatory response compared to neonates recovered from NEC and neonates without NEC. Enteroids generated from patients that have recovered from NEC have a heightened inflammatory response in response to NEC inducing stimuli compared to controls. This tendency towards an increased hyperinflammatory state may be correlated with an infant's proclivity to develop NEC and demonstrates the significance of a second hit on this tissue creating a heightened inflammatory response. This could be correlated with the impact and trajectory of an illness post recovery from NEC.
RESUMO
Background: Necrotizing enterocolitis (NEC) is a devastating disease of premature neonates with substantial morbidity and mortality. Necrotizing enterocolitis is associated with prematurity, a hyperinflammatory response, and dysregulation of intestinal barrier function. We hypothesize that patients with NEC will have an increased hyperinflammatory intestinal response compared with those without NEC. Patients and Methods: Enteroids were generated from intestinal tissue from neonates undergoing resection. They were treated with 100 mcg/mL lipopolysaccharide (LPS), subjected to 24 hours of hypoxia inducing experimental NEC, then compared with untreated controls. Expression of tumor necrosis factor (TNF-α) and interleukin 8 (IL-8) were evaluated via reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) to measure inflammatory response. Analysis of variance (ANOVA) determined statistical significance (p < 0.05). Results: Treated NEC-derived enteroids expressed significantly higher levels of IL-8 (RT-qPCR, p = 0.003; ELISA, p = 0.0002) compared with untreated NEC-derived enteroids with an increase in inflammatory marker concentration in those with a greater degree of prematurity (ELISA, p = 0.0015). A higher level of IL-8 was seen in NEC-derived enteroids compared with control after treatment (RT-qPCR, p = 0.024). Tumor necrosis factor-α levels were elevated in treated NEC-derived enteroids compared with untreated NEC-derived enteroids (RT-qPCR, p = 0.006; ELISA, p = 0.002) and compared with treated non-NEC-derived enteroids (RT-qPCR, p = 0.025; ELISA, p < 0.0001). Conclusions: Enteroids generated from neonates with NEC have an elevated hyperinflammatory response in response to NEC-inducing stimuli compared with controls. Enteroids generated from neonates with NEC with a greater degree of prematurity have a larger increase in inflammatory markers. This tendency toward a hyperinflammatory state may be correlated with an infant's proclivity to develop NEC and further demonstrates the hyperinflammatory state of prematurity.
