Structure–Function Relationships of Human Milk Oligosaccharides on the Intestinal Epithelial Transcriptome in Caco‐2 Cells and a Murine Model of Necrotizing Enterocolitis

Scope Necrotizing enterocolitis (NEC) is a devastating gastrointestinal emergency affecting preterm infants. Breastmilk protects against NEC, partly due to human milk oligosaccharides (HMOs). HMO compositions are highly diverse, and it is unclear if anti‐NEC properties are specific to carbohydrate motifs. Here, this study compares intestinal epithelial transcriptomes of five synthetic HMOs (sHMOs) and examines structure–function relationships of HMOs on intestinal signaling. Methods and Results This study interrogates the transcriptome of Caco‐2Bbe1 cells in response to five synthetic HMOs (sHMOs) using RNA sequencing: 2′‐fucosyllactose (2′‐FL), 3‐fucosyllactose (3FL), 6′‐siallyllactose (6′‐SL), lacto‐N‐tetraose (LNT), lacto‐N‐neotetraose (LNnT). Protection against intestinal barrier dysfunction and inflammation occurred in an HMO‐dependent manner. Each sHMO exerts a unique set of host transcriptome changes and modulated unique signaling pathways. There is clustering between HMOs bearing similar side chains, with little overlap in gene regulation which is shared by all sHMOs. Interestingly, most sHMOs protect pups against NEC, exerting divergent mechanisms on intestinal cell morphology and inflammation. Conclusions These results demonstrate that while structurally distinct HMOs impact intestinal physiology, their mechanisms of action differ. This finding establishes the first structure–function relationship of HMOs in the context of intestinal cell signaling responses and offers a functional framework by which to screen and design HMO‐like compounds. Human milk oligosaccharides (HMOs) are known to protect intestinal epithelial barrier function and prevent the onset of necrotizing enterocolitis. However, it is unclear if these benefits are shared across individual HMOs with different structures. Here this study compares the intestinal epithelial transcriptomes across individual HMOs using RNA sequencing and demonstrates the first structure–function mapping of HMO‐mediated cell responses.