Persistent Proclivity to a Proinflammatory State in a Human Enteroid Model of Necrotizing Enterocolitis

Abstract

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.

Keywords: cytokines; enteroids; hyperinflammatory; necrotizing enterocolitis (NEC); premature.

FIG. 1.

Expression of tumor necrosis factor-α (TNF-α) in age-matched enteroids seen on reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). Enteroids derived from tissue with necrotizing enterocolitis (NEC) that underwent treatment had an increased level of TNF-α compared with the control (RT-qPCR, p = 0.0084; ELISA, p < 0.0001). Enteroids derived from tissue with NEC that underwent treatment had an elevated level of TNF-α compared with enteroids derived from control tissue that underwent treatment (RT-qPCR, p = 0.0042; ELISA, p = 0.0003). There was not an increase in the level of TNF-α seen when comparing control enteroids that underwent treatment with those that did not. *Denotes significance; ns = non-significant.

FIG. 2.

Interleukin-8 (IL-8) and interleukin-1β (IL-1β) expression in age-matched enteroids seen on reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). Necrotizing enterocolitis (NEC)-derived enteroids that underwent treatment demonstrated a higher level of IL-8 and IL1-β when compared to NEC-derived enteroids that did not undergo treatment (IL-8 RT-qPCR, p = 0.003; IL-8 ELISA, p = 0.0002; IL1-β, p = 0.024). Necrotizing enterocolitis-derived enteroids that underwent treatment again demonstrated an elevated level of IL-8 and IL1-β when compared with non-NEC–derived enteroids that underwent treatment (IL-8 RT-qPCR, p = 0.024; IL1-β, p = 0.0351) with a positive trend increase in IL-8 seen on ELISA. *Denotes significance; ns = nonsignificant

FIG. 3.

Tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8) expression in non-age–matched enteroids seen on enzyme-linked immunosorbent assay (ELISA). Necrotizing enterocolitis (NEC)-derived enteroids that underwent treatment demonstrate higher levels of TNF-α when compared to untreated NEC-derived enteroids (TNF-α, p = 0.0051; IL-8, p = 0.0015). There is a demonstrated positive trend when comparing NEC-derived enteroids that underwent treatment to non-NEC–derived enteroids that underwent treatment in both TNF-α and IL-8. *Denotes significance; ns = nonsignificant

FIG. 4.

Tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8) expression in fully recovered necrotizing enterocolitis (NEC) enteroids on reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). Enteroids from patients that have fully recovered from NEC demonstrate elevated levels of inflammatory markers TNF-α and IL-8 when compared with untreated recovered-NEC–derived enteroids (TNF-α RT-qPCR, p = 0.0010; IL-8 ELISA, p < 0.0001) and when compared with treated non-NEC–derived enteroids (TNF-α RT-qPCR, p = 0.0222; IL-8 ELISA, p = 0.003). Enteroids from patients with active NEC and recovered from NEC that underwent treatment demonstrate elevated levels of TNF-α when compared with active NEC recovered NEC enteroids that did not undergo treatment (RT-qPCR p < 0.0001 and p = 0.0002, respectively). There is no significant difference in TNF-α levels when comparing treated active NEC enteroids with treated fully recovered enteroids. *Denotes significance; ns = nonsignificant.