Student Travel Award Recipient – TGF-β2 Protects Intestinal Epithelial Cells by Regulation of Proteins Associated with Stress and Endotoxin Responses

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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