Human milk-derived MANF, as an immuno‐nutritional factor, maintains the intestinal epithelial barrier and protects against necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a leading cause of death in preterm infants. Compared to formula milk, breastfeeding protects against NEC. However, the composition of breast milk is quite complicated, and many immunological compositions remain unknown. In this study, we aimed to investigate the concentration of a secreted protein, Mesencephalic astrocyte-derived neurotrophic factor (MANF), in breastmilk and evaluate its immune-regulatory function in protecting the intestinal epithelial barrier. Our data indicated that MANF was secreted in human milk but could not be detected in infant formulas. More importantly, the amount of MANF in colostrum was higher than that in mature milk. We also clarified that MANF was mainly expressed in intestinal macrophages and was capable of inducing apoptosis and decreasing the inflammation of pro-inflammatory macrophages in both NEC intestinal tissues and BMDMs. Mechanismly, MANF protein significantly inhibited the apoptosis of intestinal epithelial cells and protected epithelial tight junctions through downregulation of the NF-κB pathway in pro-inflammatory macrophages. These results reveal the crucial function of human milk-derived MANF in intestinal macrophages, which contributes to downregulating the intestinal inflammatory response and protecting the homeostasis of intestinal epithelial cells. Our study not only demonstrates a potential mechanism underlying breastfeeding protective effects in NEC but also, more importantly, enables clinical translation, facilitating new strategies for the development of nutritional interventions in the prevention of NEC.