(P30-013-23) Interactions Between Digestion-Released Human Milk Peptides and Cell Surface Receptors Targeting Inflammation

Objectives: Necrotizing enterocolitis (NEC) is a severe inflammatory disease of the distal small intestine in preterm infants. 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 NEC risk. Hundreds of HM peptides survive digestion and are present in the distal intestine where NEC occurs, and the peptides may retain their bioactivity. Cell surface receptors, TLR4 and MD2, have been found to be increased in NEC. We hypothesized that HM peptides following digestion will reduce risk for NEC by binding to cell surface receptors, TLR4 and MD2, thus preventing inflammation.

Methods: A total of 103 digestion-released HM peptides were found to be correlated with LPS-induced inflammatory responses in intestinal epithelial cells from our previous experiments. HM peptide-receptor binding was predicted using structure-based molecular modeling techniques. Ten candidate peptides were selected for synthesis (SP1-SP10) based on the predicted binding scores and the cellular inflammation data. Surface plasmon resonance (SPR) was used to examine direct binding between individual peptides and TLR4 or MD2.

Results: Of the ten peptides, five were found to bind directly to MD2 and three to TLR4. Five of the ten peptides exhibited high binding affinity as demonstrated by the dissociation constant (K_D): SP5, SP6, SP7, and SP9 bound with MD2, and SP2, SP5, and SP6 bound with TLR4. Out of the five peptides, two were predicted by modeling (SP2 and SP7), whereas all five peptides were correlated with high cellular inflammation data.

Conclusions: We demonstrated that select HM peptides can bind with cell surface receptors and may prevent inflammation by blocking LPS binding, thus reducing risk for NEC. While only two of the computer modeling predicted peptides were successful in receptor binding, the modeling technique is meant to be “trained” to improve prediction. Mechanistically, LPS binds to MD2 before complexing with TLR4 to initiate downstream activation of NF-κB leading to an inflammatory response. In this experiment, more peptides bound, and at higher affinity, to MD2 compared to TLR4, which may have physiologic implications for preventing excessive inflammation.

Funding Sources: University of Idaho Institute for Modeling Collaboration and Innovation (IMCI) Internal Grant