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

Abstract

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.

Variations in the Composition of Human Milk Oligosaccharides Correlates with Effects on Both the Intestinal Epithelial Barrier and Host Inflammation: A Pilot Study

“…HMOs are reported to exert a number of protective effects in the intestinal tract, including the stimulation of the growth of commensal bacteria [ 24 , 25 ], inhibition of the growth of enteric pathogens and elaborated toxins [ 13 , 26 ], promotion of goblet cell-derived mucin production and secretion [ 27 ], modulation of both innate and adaptive immunity [ 28 ] and maintenance of the integrity of the epithelial cell barrier [ 17 , 19 ]. Traditionally, the structure and specificity of responses of HMOs are associated with effects on microbial functions related to their role either as prebiotics or anti-adhesive molecules [ 3 ].…”

“…These observations confirm previous studies, where it has been postulated that epithelial barrier protection is a core function, which is common in various HMOs. For instance, Natividad et al showed, using a mixture of fucosylated, sialylated and acetylated synthetic HMOs at physiologic concentrations, an increase in TER in polarized epithelial (Caco-2 and HT-29) cells, whereas exposure to any one oligosaccharide alone did not result in an increase in TER [ 17 ]. HMOs may also act to promote gut homeostasis via the induction of mucin production [ 19 ] and enhanced glycocalyx expression [ 39 ].…”

“…To date, most studies have evaluated the effects of a single HMO without considering the functional relevance of inter-individual variations in the composition of HMOs. Using RNA sequencing, we recently demonstrated that five synthetic HMOs each induced a unique set of host cell transcriptomic changes with little overlap between individual structures [ 17 ]. In the present study, we performed in vitro functional comparisons of HMOs extracted from the mature human milk of four individual mothers.…”

Human Milk Oligosaccharides: A Critical Review on Structure, Preparation, Their Potential as a Food Bioactive Component, and Future Perspectives

“…They found that individual HMOs had different degrees of protection against NEC. However, their mechanisms were different, indicating that the protective effect of HMOs on NCE was related to their structures . In addition to intestinal inflammation-related diseases, studies have found that HMO can delay and inhibit the development of type I diabetes in non-obese diabetic mice and can also improve psoriasis in mice induced by imiquimod, a chronic immune-mediated inflammatory skin disease …”

“…Wu et al explored the intestinal epithelial transcriptome of five HMOs (2′-FL, 3-FL, LNnT, LNT, and 6′-SL). They found that different HMOs can protect against intestinal barrier damage and inflammation, but each HMO regulates a unique signal pathway . In addition, HMOs have also been proven to enhance gut barrier function by stimulating the development of glycocalyx on intestinal epithelial cells. , Therefore, HMOs may prevent the development of diabetes by maintaining the integrity of the gut barrier.…”