Human milk extracellular vesicles protect protein cargo from human digestion and are taken up by neonatal human enteroids
Authors: Zhang Yang , Kuhn Madeline , Yung Claire , Kim Bum-Jin , Armstrong Randall , Dallas Dave , Scottoline Brian , Andres Sarah 
Published: May 2023

Background: Human milk (HM) is the ideal infant nutrition and reduces infant death and disease. For example, HM is the best-known preventative for the deadly neonatal intestinal inflammatory disease, necrotizing enterocolitis (NEC), for which there is no cure. How HM reduces NEC risk is relatively unknown. HM contains thousands of molecular components, including extracellular vesicles (EVs). EVs are lipid bilayer-encased particles released from cells that carry biological cargo. EVs are putative regulators of intestinal function and HM EVs offer a mechanism for the transfer of proteins from the mom to the infant’s gut. It is unknown if milk EVs or protein cargo survive digestion. This limits the ability to leverage HM EV proteins as treatments for NEC, additives to infant nutrition, or therapeutics for other diseases. The objective of this proposal is to examine if HM EV protein cargo survives in vivo human digestion and if surviving cargo confers anti-inflammatory benefits in neonatal human enteroids. Hypothesis: HM EVs transport key protein cargo to intercellular targets and protect against intestinal inflammation. Methods: All studies were conducted under approved IRB protocols. Neonatal intestinal contents (digesta) were collected after gastric feeding from naso- or orojejunal sampling tubes. EVs were isolated from HM and digesta by density-gradient ultracentrifugation. EVs were validated by electron microscopy, nanoparticle tracking analysis, and western blot. EV protein cargo from n=3 paired HM and digesta samples were profiled by a C18-UPLC with Orbitrap mass spectrometry. Apical-out neonatal human enteroids were validated by qPCR for proliferation and differentiation markers (mean ± SEM). Enteroid EV uptake was assessed with CMPTX dye-labeled EVs. Effects of digesta EVs on inflammation were assessed by qPCR in LPS-treated enteroids. Results: EVs are enriched for markers CD63, CD81, CD9, and TSG101 and de-enriched for casein, milk fat globules. Only 4.68% ± 0.02 (p=0.0012 vs HM) of EV cargo proteins from HM reach the human intestine, but nearly half 64.08% ± 0.02 (p=0.007 vs HM) of the protein diversity is preserved. Human mammary-derived protein BTN1A1 is present in digesta EVs. Apical-out enteroids gene expression is consistent with a more differentiated epithelium, e.g. elevated ChgA (7.60 ± 4.04 vs 1.37 ± 0.39, p=0.0026) and down-regulated Ki67 (0.13 ± 0.04 vs 1.00 ± 0.04, p<0.0001) Enteroids take up milk EVs. Filtered (0.22μm) neonatal digesta (contains EVs) attenuates LPS-induced TNFα gene expression. Conclusion: These novel findings demonstrate that a mostly pure population of EVs can be isolated from a 1mL starting volume. A subset of HM EVs reaches the neonatal human intestine where they can be taken up by the epithelium and may act to reduce inflammation. These data demonstrate the importance of examining EV cargo that survives to the human intestine when investigating potential HM-mediated mechanisms of disease prevention. NIH K01DK129401, USDA NIFA, Collins Medical Trust, Medical Research Foundation, OHSU Exploratory Research Seed Grant This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.