Intestinal Alkaline Phosphatase Prevents the Systemic Inflammatory Response Associated With Necrotizing Enterocolitis

Abstract

Introduction: Necrotizing enterocolitis (NEC) is the most common surgical emergency in neonates with an incidence of 0.5-2.4 cases per 1000 live births. the mortality rate is between 10-50% and has not improved significantly in the past two decades. Neonates affected by NEC develop a septic injury that is associated with an increased risk of chronic lung disease and neurological impairment due to intraventricular bleeding. Intestinal alkaline phosphatase (IAP) is an endogenous protein that has been shown to inactivate the endotoxin lipopolysaccharide (LPS), and has recently been used successfully as an adjunct to treat sepsis in adult patients. Our hypothesis is that systemic, exogenous IAP will mitigate the inflammatory response as measured by serum levels of pro-inflammatory cytokines in a rat model of NEC. Methods: Newborn Sprague Dawley rats were divided into groups. Control pups were breast-fed. in the remaining pups, NEC was induced by feeding formula containing LPS and exposure to intermittent hypoxia. NEC pups were also given intra-peritoneal injections of 4 or 40 glyceine units (U) of IAP or placebo twice daily. Rats were sacrificed on days 1-3. Intestine was harvested to grade intestinal injury and serum was collected for cytokine analysis using a bioplex assay as well as measurement of alkaline phosphatase (AP) activity. Results: Systemic IAP administration had no effect on increasing intestinal AP activity or preventing NEC as measured by histology. However, systemic IAP administration significantly increased serum AP activity in a dose and time dependent fashion. the serum AP activity for the 40 U IAP treated groups on days of life 1-3 were 0.285, 0.344, and 0.496 U/mL respectively, while the controls and NEC groups had serum activities ranging from 0.007-0.0489 U/mL on days 1-3 (p<0.05 for all). We also found that the pro-inflammatory cytokines (measured in pg/mL) TNF-a, IL-6, and IL-1b were significantly increased in NEC rats as compared to controls on days 2 and 3 (TNF-a: 1390 vs. 25 on DOL 2 and 2896 vs. 1332 on DOL 3; IL-1b: 3295 vs. 66 DOL 2 and 7010 vs. 2394 on DOL3; IL-6: 1718 vs. 21 on DOL2; (p<0.05 for all). Importantly, we found that treatment with 40 U of systemic IAP not only increased serum AP activity, but also decreased these pro-inflammatory cytokines back to near-control levels. (TNF-a: 26 on DOL 2 and 1018 on DOL3; IL-1b 183 on DOL 2 and 1339 on DOL 3; IL-6: 381 on DOL 3; (p<0.05 for all). Figure 1 shows serum TNF-a levels in control, NEC, and 40 U IAP treated pups. Conclusions: Systemic IAP administration appears effective in preventing the systemic inflammatory response associated with NEC, and may prove to be a valuable adjunctive treatment for NEC.