SCOPE Necrotizing enterocolitis (NEC) is a leading cause of morbidity and death in preterm infants, occurring more often in formula-fed than breastfed infants. Studies in both rats and humans show that human milk oligosaccharides (HMOs) lower the incidence of NEC, but the mechanism underlying such protection is currently unclear. METHODS AND RESULTS By extracting HMOs from pooled human breastmilk, the impact of HMOs on the intestinal mucin levels in a murine model of NEC are investigated. To confirm the results, the findings are validated by exposing human intestinal epithelial cells and intestinal organoids to HMOs and evaluated for mucin expression. HMO-gavage to pups increases Muc2 levels and decreases intestinal permeability to macromolecular dextran. HMO-treated cells have increased Muc2 expression, decreased bacterial attachment and dextran permeability during challenge by enteric pathogens. To identify the mediators involved in HMO induction of mucins, it is demonstrated that HMOs directly induce the expression of chaperone proteins including protein disulfide isomerase (PDI). Suppression of PDI activity removes the protective effects of HMOs on barrier function in vitro as well as NEC protection in vivo. CONCLUSIONS Taken together, the results provide insights to the possible mechanisms by which HMOs protect the neonatal intestine through upregulation of mucins.

Inflammatory bowel disease (IBD) is characterized by severe and recurrent inflammation of the gastrointestinal tract, associated with altered patterns of cytokine synthesis, excessive reactive oxygen species (ROS) production, and high levels of the innate immune protein, lipocalin-2 (LCN-2), in the mucosa. The major source of ROS in intestinal epithelial cells is the NADPH oxidase NOX1, which consists of the transmembrane proteins, NOX1 and p22PHOX, and the cytosolic proteins, NOXO1, NOXA1, and Rac1. Here, we investigated whether NOX1 activation and ROS production induced by key inflammatory cytokines in IBD causally affects LCN-2 production in colonic epithelial cells. We found that the combination of TNFalpha and IL-17 induced a dramatic upregulation of NOXO1 expression that was dependent on the activation of p38MAPK and JNK1/2, and resulted into an increase of NOX1 activity and ROS production. NOX1-derived ROS drive the expression of LCN-2 by controlling the expression of IkappaBzeta, a master inducer of LCN-2. Furthermore, LCN-2 production and colon damage were decreased in NOX1-deficient mice during TNBS-induced colitis. Finally, analyses of biopsies from patients with Crohn's disease showed increased JNK1/2 activation, and NOXO1 and LCN-2 expression. Therefore, NOX1 might play a key role in mucosal immunity and inflammation by controlling LCN-2 expression.

Aquaporin (AQP) 3 expression is altered in inflammatory bowel diseases, although the exact mechanisms regulating AQP abundance are unclear. Although interferon gamma (IFNgamma) is centrally involved in intestinal inflammation, the effect of this cytokine on AQP3 expression remains unknown. HT-29 human colonic epithelial cells were treated with IFNgamma to assess AQP3 mRNA expression by real-time RT-PCR and functional protein expression through the uptake of radiolabelled glycerol. Transient knockdown of signal transducer and activator of transcription 1 (STAT1), STAT3, Sp1, and Sp3 were performed to determine the involvement of these transcription factors in the IFNgamma-induced signalling cascade. AQP3 promoter regions involved in the response to IFNgamma were assessed using a luciferase reporter system. Likewise, enteroids derived from human colonic biopsies were also treated with IFNgamma to assess for changes in AQP3 mRNA expression. IFNgamma decreased AQP3 mRNA expression in HT-29 cells in a time- and concentration-dependent manner and reduced functional AQP3 protein expression (decreased 3H-labelled glycerol uptake). IFNgamma also reduced AQP3 expression in enteroids derived from human colonic biopsies. Knockdown of STAT1 partially prevented the IFNgamma-induced downregulation of AQP3 expression, whereas STAT3 and Sp3 knockdowns resulted in increased baseline expression of AQP3 but did not alter IFNgamma-induced downregulation. Constitutive transcription of AQP3 is downregulated by IFNgamma as demonstrated using the luciferase reporter system, with Sp3 bound to the AQP3 promoter as shown by chromatin immunoprecipitation. AQP3 constitutive transcription in intestinal epithelial cells is downregulated by IFNgamma. This response requires STAT1 that is postulated to drive the downregulation of AQP3 expression through increased acetylation or decreased deacetylation the AQP3 promoter, ultimately resulting in decreased constitutive transcription of AQP3. KEY MESSAGES • IFNgamma suppresses the expression of AQP3 in intestinal epithelial cells. • Proximal AQP3 promoter elements are sufficient to drive constitutive expression and mediate the IFNgamma-induced downregulation of AQP3 mRNA expression. • IFNgamma-induced suppression of AQP3 is dependent upon STAT1 expression, but not STAT3, Sp1, or Sp3.

Due to their complex chemical and physical properties, the effects and mechanisms of action of natural sources of dietary fiber on the intestine are unclear. Pigs are commonly fed high-fiber diets to reduce production costs and non-starch polysaccharide (NSP)-degrading enzymes have been used to increase fiber digestibility. We evaluated the expression of mucin 2 (MUC2), presence of goblet cells, and ileal immune profile of pigs housed individually for 28 days and fed either a low fiber diet based on corn-soybean meal (CSB, n = 9), or two high fiber diets formulated adding 40{\%} corn distillers' dried grains with solubles (DDGS, n = 9) or 30{\%} wheat middlings (WM, n = 9) to CSB-based diet. Pigs were also fed those diets supplemented with a NSP enzymes mix (E) of xylanase, beta-glucanase, mannanase, and galactosidase (n = 8, 10, and 9 for CSB+E, DDGS+E and WM+E, respectively). Feeding DDGS and WM diets increased ileal MUC2 expression compared with CSB diet, and this effect was reversed by the addition of enzymes. There were no differences in abundance of goblet cells among treatments. In general, enzyme supplementation increased gene expression and concentrations of IL-1beta, and reduced the concentrations of IL-4, IL-17A and IL-11. The effects of diet-induced cytokines on modulating intestinal MUC2 were assessed in vitro by treating mouse and swine enteroids with 1 ng/ml of IL-4 and IL-1beta. In accordance with previous studies, treatment with Il-4 induced Muc2 and expansion of goblet cells in mouse enteroids. However, swine enteroids did not change MUC2 expression or number of goblet cells when treated with IL-4 or IL-1beta. Our results suggest that mucin and immune profile are regulated by diet in the swine intestine, but by mechanisms different to mouse, emphasizing the need for using appropriate models to study responses to dietary fiber in swine.