L. W. Hansen1,2, W. Yang1,2, A. Khader1, J. M. Prince1,2, J. M. Nicastro1, G. F. Coppa1, P. Wang1,2 1Hofstra North Shore-LIJ School Of Medicine,Surgery,Manhasset, NY, USA 2The Feinstein Institute For Medical Research,Center For Translational Research,Manhasset, NY, USA
Introduction: Polymicrobial sepsis in neonates remains a major cause of morbidity and mortality. A breakdown in intestinal integrity contributes to the severity of this disease. Providing breast milk to premature infants can reduce the risk of necrotizing enterocolitis (NEC). Milk fat globule–EGF factor 8 (MFG-E8) is a secretory glycoprotein and is abundant in mammalian breast milk. MFG-E8 has anti-inflammatory activity and plays a major role in the maintenance of intestinal epithelial homeostasis. Thus, we hypothesize that MFG-E8 will reduce mortality in neonatal sepsis by protecting their intestinal integrity.
Methods: Newborn wild-type (WT) C57BL/6 and MFG-E8 knockout (KO) mice stayed with their mothers throughout the experiments. At age 5-7 days (3-4 g), the neonatal mice were made septic by intraperitoneal injection of cecal slurry (CS; 0.75 mg CS/g body weight) and monitored for a 7-day survival study. The CS was prepared from a mixture of cecal contents from 7 adult male WT mice (8-10 weeks old). To study intestinal injury, the neonatal mice were injected with 0.9 mg CS/g body weight (n=6-8 per group). At 10 h after injection, the small intestine was collected for analysis by ELISA, Western blot, myeloperoxidase activity, and endotoxin assay.
Results: The 7-day survival of septic WT neonates was 73%, while all the septic MFG-E8 KO neonates died within 2 days (Figure, P < 0.001). Protein levels of proinflammatory cytokines IL-6 (730.2 ± 105.9 pg/mg) and IL-1β (447.0 ± 45.6 pg/mg) in the intestine of septic MFG-E8 KO mice were increased 1.9- and 2.0-fold, respectively, compared to intestinal IL-6 (375.8 ± 50.9 pg/mg) and IL-1β (224.2 ± 47.9 pg/mg) in septic WT mice. The myeloperoxidase activity, a marker of neutrophil infiltration, in the intestine of septic MFG-E8 KO neonates was 3.8–fold higher than that in septic WT neonates (P < 0.05). There was a slight increase in serum endotoxin levels in septic MFG-E8 KO mice compared to septic WT mice (37.4 ± 9 vs. 25.8 ± 7 EU/ml). Additionally, septic MFG-E8 KO neonates had a reduced induction of phosphorylated AKT, which is an indication of activation of the cell survival pathway, compared to septic WT neonates.
Conclusion: Breast milk protein MFG-E8 can protect neonatal mice from polymicrobial sepsis by suppressing intestinal inflammation and promoting the cell survival pathway. Supplementation of MFG-E8 for critically ill infants may represent a viable therapeutic strategy.
