Human Milk Oligosaccharides Protect against Necrotizing Enterocolitis by Activating Intestinal Cell Differentiation

Abstract

Scope: Necrotizing enterocolitis (NEC) is a devastating gastrointestinal emergency and currently the leading cause of mortality in preterm infants. Recent studies show that human milk oligosaccharides (HMOs) reduce the frequency and incidence of NEC; however, the molecular mechanisms for their protection are largely unexplored. Methods and results: To address this gap, a genome-wide profiling of the intestinal epithelial transcriptome in response to HMOs using RNA-sequencing is performed. It is found that HMOs alter the host transcriptome in 225 unique target genes pertaining to cell proliferation and differentiation, including upregulation of stem cell differentiation marker HMGCS2. To validate these results, differentiation in Caco-2Bbe1 (Caco-2) intestinal cells is verified by Alcian Blue staining and transepithelial electrical resistance (TER) recordings. Furthermore, an in vivo model of NEC is also employed whereby neonatal pups are gavage fed HMOs. Interestingly, HMOs-fed pups show enhanced cell MUC2 differentiation and HMGCS2 expression. Conclusions: These findings demonstrate HMOs protect against NEC in part by altering the differentiation of the crypt-villus axis. In addition, this study suggests that pooled HMOs directly induce a series of biological processes, which provide mechanistic insights to how HMOs protect the host intestine.

Enteral Feeding Interventions in the Prevention of Necrotizing Enterocolitis: A Systematic Review of Experimental and Clinical Studies

“…NEC induced increase in paracellular translocation of FITC-labelled dextran was reduced by enteral HMO administration in a murine NEC model [38]. In addition, HMO administration normalizes the number of goblet cells in the intestinal villi (mucin 2 (Muc2) positive cells) that is decreased by NEC protocol exposure [38,86] and tended to increase the mRNA expression of Muc2 and trefoil factor 3 (TFF3) in NEC protocol exposed mice [38]. Interestingly, the effect of enteral HMO treatment on goblet cell numbers was abolished in the presence of an inhibitor of the ER chaperone protein PDI, suggesting a mechanism behind the protective effects of HMO administration could be induction of the unfolded protein response (UPR) [38].…”

“…Fish oil (rich in n-3 PUFA) [82,83] MPL [84] MFGM [48] Very low fat diet [85] Reduced long chain triacylglycerol diet (considered pre-digested) [85] Pomegranate seed oil [47] Carbohydrate/sugar-based interventions HMO [38,42,49,[86][87][88][89][90] Neutral HMO (no sialic acids) [42] −2 HMO (two sialic acids) [42] DSLNT (HMO) [42] DSLNnT (synthetic disialyl glycan) [89,90] DS'LNnT (synthetic disialyl glycan) [89,90] 2 -FL [87,91,92] 6 -SL [92] 2 -FL and 6 -SL [42,92] Sialylated HMO [93] Sialylated GOS [87] GD3 [50] Hyaluronan 35 kD [94] Protein/amino acid-based interventions L-Glutamine/glutamine [84,[95][96][97] Arginine [98,99] L-Carnitine [99] N-Acetylcysteine [85] Lactadherin [51] OPN [100] Lactoferrin [101] IAP [102,103] [104] HB-EGF [41,[58]…”

“…HMO have been shown to promote intestinal proliferation and reduce apoptosis in the context of NEC. In a mouse NEC model, orogastric administration of HMO restored the amount of cells positive for the proliferation marker Ki67 in the ileum [49,86,88], whereas this effect was not seen with supplementation with infant formula oligosaccharides [49]. In addition, loss of Sox9-positive stem cells was prevented by HMO treatment, but not by infant formula oligosaccharides [49].…”

“…In a preterm pig model of NEC, treatment with a mixture of four HMO did not change small intestinal mRNA expression of proliferating cell nuclear antigen (PCNA) [134]. Enteral administration of HMO reduced apoptosis (TUNEL) [86] and decreased ileal cleaved caspase-3 and hypoxia-inducible factor 1α (HIF1α) protein levels [88] in a murine NEC model. Both in a pig and murine NEC model, enteral administration of 2 -FL, 6 -SL and a combination of the two reduced intestinal epithelial apoptosis [92].…”

“…PUFA Endotoxemia (plasma) ↓ [45] DHA Ileal effective diffusivity amine modified particles ↓ [131] Ileal effective diffusivity carboxyl modified particles ↓ [131] Ileal linear movements E. coli through intestinal mucus ↓ [131] Ileal movement speed E. coli through intestinal mucus ↓ [131] Ileal sialic acid content mucus ↓ [131] Ileal mucus structure (confocal imaging/SEM) = [cells ↑[38,86] Ileal Muc2 (mRNA) ↑ (trend)[38] Ileal TFF3 (mRNA) ↑ (trend)[38] Mixture of four HMOsSmall intestinal bacterial adhesion and tissue invasion = [134]Small intestinal Muc1 (mRNA) =[134] Small intestinal Muc2 (mRNA) =[134] …”