Abstract
Necrotizing enterocolitis (NEC) is a severe inflammatory disease of the distal small intestine that affects 7-17% of all infants and affects preterm infants most severely due to their immature gastrointestinal tracts. The pathogenesis of NEC is not well understood, and there is no treatment for it other than surgery in the most severe cases; however, human milk (HM) has been found to reduce the risk of NEC. Hundreds of human milk (HM) peptides are released by digestion and are present in the distal small intestine, where they may exert bioactivity to reduce inflammation through the toll-like receptor 4 (TLR4) and myeloid differentiation factor 2 (MD2) inflammatory pathway. We hypothesize that HM peptides inhibit LPS binding to sensing cell surface receptors, TLR4 and MD2, that trigger the inflammatory nuclear factor kappa B (NF-κB) pathway. The secondary aim was to identify and verify HM peptides that interact with these cell surface receptors. Ten HM peptides were selected for synthesis based on correlation with lipopolysaccharide (LPS)-induced inflammatory responses in intestinal epithelial cells from previous cell culture experiments and predicted binding scores from structure-based molecular modeling techniques. Surface plasmon resonance (SPR) was first used to measure direct binding between HM peptides and TLR4, MD2 or LPS. Of the ten peptides, five were found to bind directly to MD2 and three bound directly to TLR4. Five of the ten peptides exhibited high binding affinity as demonstrated by equilibrium dissociation constants in µM or nM. Eight of the ten peptides bound with LPS. Binding HM peptides from SPR experiments were then used to treat human intestinal epithelial cells (HIEC-6) before either stimulating with LPS or left unstimulated. Inflammatory cytokines were measured using enzyme linked immunosorbent assays (ELISAs). Three peptides bound to all three targets (TLR4, MD2, and LPS), whereas two peptides bound to MD2 alone and five peptides bound to LPS alone. Two peptides with observed potential were selected to test at higher dosages and showed a dose response inflammation suppression in HIEC-6 cells stimulated with LPS, one of which significantly reduced inflammation compared to the LPS-control (p<0.001). Selected HM peptides were able to bind with TLR4, MD2, and LPS; and among them, some reduced inflammation in vitro. This study provides additional knowledge of how human milk may provide protection to infants via interfering with LPS-induced inflammation via cell surface receptors, which may subsequently reduce risk of NEC in preterm infants.