Duc Ninh Nguyen, Department of Food Science, University of Copenhagen, Denmark
Duc Ninh Nguyen1, Pingping Jiang2, Susanne Jacobsen3, Per T. Sangild2, Stine B. Bering2, Emøke Bendixen4, Dereck E.W. Chatterton1,2
1 Department of Food Science, University of Copenhagen, Denmark
2 Department of Nutrition, Exercise and Sports, University of Copenhagen, Denmark
3 Department of Systems Biology, Technical University of Denmark, Denmark
4 Department of Molecular Biology and Genetics, Aarhus University, Denmark
Transforming growth factor (TGF)-β2, an important anti-inflammatory protein in milk, possesses protective effects against intestinal inflammation by limiting the production of inflammatory cytokines. TGF-β2-fortified infant formula has shown potential in reducing the incidence of necrotizing enterocolitis (NEC) and associated inflammation in preterm neonates. However, the molecular mechanisms by which TGF-β2 protects immature intestinal epithelial cells (IECs) remain to be more clearly elucidated before interventions in infants can be considered. Porcine intestinal epithelial cells (IECs) PsIc1 were treated with TGF-β2 (3 ng/mL, close to the concentration in breast milk) alone and in the presence of lipopolysaccharide (LPS). The changes of the cellular proteome were analyzed using two-dimensional gel electrophoresis (2DE) and LC-MS-based proteomics. TGF-β2 alone induced differential expression of 13 proteins, and the majority of the identified proteins were associated with stress response, TGF-β and toll-like receptor 4 (TLR4) signaling (heat shock protein A5 (HSPA5), HSPA8, HSPA9, HSP60, HSP90B1 and RACK1). Furthermore, by LC-MS-based proteomics and Western blot, 20 differentiated proteins were identified following treatment with TGF-β2 in LPS-challenged IECs. Thirteen of these proteins were associated with stress response pathways. Among them, five proteins (GRP58, PDI, cyclophilin A, TIMP3, and PKC-α) were altered by LPS and restored by TGF-β2, while six proteins (HSPA8, HSP90, apoptosis inducing factor, annexin II, calreticulin and tropomyosin α3) were differentiated only by TGF-β2 in LPS-challenged IECs. In addition, the protein-protein interaction network of all identified proteins analyzed by STRING showed all HSPs and stress-associated proteins interacting together in central clusters. As TLR4 signaling, and TLR4 and HSP interaction are indicated to be involved in inflammation and NEC pathogenesis, our data suggest that TGF-β2 of dietary or endogenous origin may modulate the cellular responses against LPS-induced stress and thereby support cellular homeostasis, and the innate immunity in response to bacterial colonization and the first enteral feeds in early life. These mechanisms may play a role in the protection against NEC induced by feeding mother’s milk to immune-compromised preterm infants. Further investigation is important to conclude the protective effects of TGF-β2 used as the supplement in preterm infant formula.
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