Background: Human milk (HM) is the ideal infant nutrition and reduces death and disease. HM contains thousands of bioactives, including extracellular vesicles (HMEVs). HMEVs are lipid bilayer-encased nanoparticles released from mammary epithelial cells that carry biological cargo to the infant. Pre-clinical models suggest that HMEVs may enhance barrier function and limit inflammation; however, if HMEVs are present in the neonatal intestine and how they interact with intestinal epithelial cells (IECs) is less defined. This limits the ability to leverage HMEVs as additives to infant nutrition or as therapeutics. This study aims to develop a neonatal enteroid model and EV isolation pipeline to study HMEV–IEC interactions with the ultimate goal of therapy development.
Hypothesis: HMEVs survive in vivo neonatal digestion to be taken up by infant IECs.
Methods: All studies are IRB-approved. Neonatal intestinal contents (digesta) were collected after gastric feeding from post-pyloric tubes. HMEVs were isolated by density-gradient ultracentrifugation and validated by electron microscopy, nanoparticle tracking analysis (NTA), and western blotting. HMEV sub-populations were examined by super-resolution microscopy. Uptake was tested using apical-out neonatal human enteroids isolated from a 2-month-old patient and validated by qPCR for proliferation and differentiation markers (mean ± SEM). Enteroid uptake was assessed with CMTPX dye-labeled-HMEVs, dynamin inhibitor, and confocal microscopy.
Results: Our isolation pipeline reliably isolates HMEVs with classic EV morphology and enriched in extracellular vesicle markers CD81/CD9/CD63, but depleted of b-casein and lactalbumin. NTA data indicates that only 17.8 ± 2.9% (p>0.0001) of input HMEVs survive to reach the intestine. Mammary gland-derived protein BTN1A1 is present in digested HMEVs. Apical-out human enteroids represent a less proliferative and more differentiated epithelium, e.g. down-regulated KI67 (0.05 ± 0.03-fold relative to basal-out, p<0.0001, n=4-6) and elevated CHGA (14.22 ± 9.30-fold relative to basal-out, p=0.0072, n=4-6) as measured by qRT-PCR and normalized to housekeeping gene RPLP0. Neonatal enteroids take up digested HMEVs largely via dynamin-mediated endocytosis, as measured by CMTPX dye intensity normalized to enteroid area using Fiji (2.05 x 10⁵ ± 2.95 x 10⁴vs 5.38 x 10⁴± 6.08 x 10³, p<0.0001, n=62-115 enteroids, HMEV-treatment alone and HMEVs + dynamin inhibitor, respectively).
Conclusion: Our data suggest that HMEVs survive to the neonatal intestine and can be absorbed by neonatal enteroids largely via dynamin-mediated endocytosis. These data are an important first step to leveraging HMEVs and their cargo to treat neonatal intestinal inflammation.
Funding: NIH K01DK129401, R01HD097367, USDA NIFA, Collins Medical Trust, Medical Research Foundation, OHSU Exploratory Research Seed Grant

Madeline Kuhn

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