Human milk metabolites modulate gut barrier and immunity-related genes in an <i>in vitro</i> multicellular model of intestinal epithelium

Human milk (HM) is a complex food that meets nutritional newborn needs. The role of its bioactive components, particularly metabolites, in neonatal development remains poorly understood. This study focused on evaluating the effects of HM short chain fatty acids (SCFA), polyamines, tryptophan derivatives, gamma-aminobutyric acid (GABA), serotonin and lactate on several neonatal gut functions. The effects of these metabolites, at HM concentration, were analyzed individually or in mixture (MTB mix), on an <i>in vitro</i> multicellular model of intestinal epithelium, including Caco-2 (enterocytes), HT29-MTX (goblet cells), NCI-H716 (enteroendocrine cells) and M cells. Transcriptomic semi-screening revealed the impact of these metabolites, especially combined as a mixture, on various intestinal functions. MTB mix upregulated <i>CLDN3</i> and <i>CLDN4</i> while downregulating <i>CLDN1</i> and this was associated with a higher transepithelial resistance, highlighting its potential role in strengthening the intestinal epithelial barrier (IEB). MTB mix also reduced the expression of genes involved in mucus formation (<i>MUC1</i>, <i>TFF3</i>). Besides, MTB mix decreased immune-related gene expression (<i>CXCL8</i>, <i>MYD88</i>, <i>GPX2</i>), suggesting an immunomodulatory effect. Lastly, MTB mix decreased nutrient transporter and enzyme gene expression (<i>SLC2A1</i>, <i>SLC15A1</i>, <i>LCT</i>), suggesting that the mixture modulates digestive function. SCFA, especially butyrate, drove most of these effects, with a contribution from polyamines also, especially on IEB. Individually, GABA had a significant impact on all the examined functions, although these effects were absent with the MTB mix. Overall, this study highlights the ability of HM metabolites to modulate IEB and some genes related to the immune, digestive and endocrine functions <i>in vitro</i>, with some cumulative or attenuated effects when taken altogether <i>vs.</i> individually, emphasizing the importance of studying them as a mixture.