P3.A019: Characterization of the Extracellular Vesicles in Human Milk and Infant Formulas

Background: Breast milk (BM) is known to protect against inflammatory injury and is an effective strategy to prevent necrotizing enterocolitis (NEC) in premature babies. Extracellular Vesicles (EVs) are cell-derived vesicles released in response to various stimuli and are an important regulator of intercellular communication. EVs’ cargo contains microRNAs (miRNAs), small noncoding RNAs involved in various biological processes through regulation of posttranscriptional gene expression.

Methods: Characterization of milk EVs was compared in Term (collected week 1), Preterm BM samples ( < 32 weeks gestation, collected week 1 and 4), donor milk, and various formulas (Enfamil NeuroPro, Similac Special Care 24) using differential ultracentrifugation method. EV size and quantities were determined using NanoSight. To explore their biological effects, EVs were co-cultured with THP-1 immune cells exposed to lipopolysaccharide (LPS), and the supernatants were analyzed for cytokines production. Furthermore, we analyzed the miRNA composition of the milk EVs, specifically miR-146a, using RT-PCR. Finally, we evaluated the ability of Caco-2 intestinal cell line to uptake milk EVs using confocal microscopy.

Results: Our study demonstrated that EVs were present in all samples, with BM having higher levels than formulas. However, Enfamil NeuroPro showed significantly higher EV content than Similac Special Care 24 (Figure 1). Human milk and formula-derived EVs influenced cytokine production by THP-1 immune cells, indicating biological activity. Human milk contained more miR-146a per EV than formulas, and only the EV miR-146a mimic reduced TNF-α gene expression, indicating anti-inflammatory properties (Figure 2). Both human milk and formula-derived EVs were taken up by Caco-2 intestinal cell line, suggesting their potential role in intestinal health and development.

Conclusion: Our research sheds light on the presence and biological significance of EVs in human milk and infant formulas. The results highlight the EVs impact on immune function and their potential role in protecting the intestinal lining from uncontrolled pro-inflammatory responses, such as those seen in NEC. Clinicians should advocate for the use of mother's milk, utilizing donor breast milk or formula as alternatives only when necessary.