The Human Milk-derived Peptide Drives Rapid Regulation of Macrophage Inflammation Responses in the Neonatal Intestine.

Background & Aims: The interactions between human milk and the regulation of innate immune homeostasis in newborns, and their impact on intestinal health, are not fully understood. This study aimed to explore the role of peptides in human milk extracellular vesicles (EVs) in this process.

Methods: A comprehensive screening of peptides within human milk EVs was performed, leading to the identification of a beta-casein-derived peptide (CASB). The effects of CASB on intestinal injury were evaluated in a rat necrotizing enterocolitis (NEC) model. Immunofluorescence analysis was used to determine its distribution, and its impact on NF-κB signaling and inflammation was studied in bone marrow-derived macrophages (BMDMs) and intestinal macrophages. Protein-protein interaction (PPI) analysis, single-cell RNA-seq (scRNA-seq), and co-immunoprecipitation (co-IP) experiments were conducted to explore the mechanism underlying CASB function.

Results: CASB significantly mitigated intestinal injury in the rat NEC model. Immunofluorescence analysis revealed that CASB could target intestinal macrophages and rapidly inhibited NF-κB signaling and reduced inflammation. ScRNA-seq analyses indicated a strong association between FHL2 and NEC development, and co-IP confirmed the interaction between CASB and FHL2. CASB disrupted the FHL2/TRAF6 complex, reducing TRAF6 protein levels. Mutation of key amino acids in CASB disrupted its interaction with FHL2 and abolished its ability to inhibit NF-κB signaling, which also prevented its protective effect in vivo. RNA-seq of intestinal tissue further highlighted the impact of CASB on the NF-κB signaling pathway.

Conclusions: Our study identifies CASB, a novel peptide in human milk EVs, that rapidly regulates macrophage inflammatory responses and protects against NEC-induced intestinal injury. These findings provide new insights into the role of human milk in modulating the infant immune system and intestinal health.