Protective effects of transforming growth factor β2 in intestinal epithelial cells by regulation of proteins associated with stress and endotoxin responses.

Duc Ninh Nguyen,Pingping Jiang,Dereck E W Chatterton,Susanne Jacobsen,Emøke Bendixen,Per T Sangild,Jörn-Hendrik Weitkamp

doi:10.1371/journal.pone.0117608

Duc Ninh Nguyen, Pingping Jiang + Show 5 more

Open Access

https://doi.org/10.1371/journal.pone.0117608

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Abstract

Transforming growth factor (TGF)-β2 is an important anti-inflammatory protein in milk and colostrum. TGF-β2 supplementation appears to reduce gut inflammatory diseases in early life, such as necrotizing enterocolitis (NEC) in young mice. 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 IECs PsIc1 were treated with TGF-β2 and/or lipopolysaccharide (LPS), and changes in the cellular proteome were subsequently analyzed using two-dimensional gel electrophoresis-MS and LC-MS-based proteomics. TGF-β2 alone induced the differential expression of 13 proteins and the majority of the identified proteins were associated with stress responses, TGF-β and Toll-like receptor 4 signaling cascades. In particular, a series of heat shock proteins had similar differential trends as previously shown in the intestine of NEC-resistant preterm pigs and young mice. Furthermore, LC-MS-based proteomics and Western blot analyses revealed 20 differentially expressed proteins following treatment with TGF-β2 in LPS-challenged IECs. Thirteen of these proteins were associated with stress response pathways, among which five proteins were altered by LPS and restored by TGF-β2, whereas six were differentially expressed only by TGF-β2 in LPS-challenged IECs. Based on previously reported biological functions, these patterns indicate the anti-stress and anti-inflammatory effects of TGF-β2 in IECs. We conclude that TGF-β2 of dietary or endogenous origin may regulate the IEC responses against LPS stimuli, thereby supporting cellular homeostasis and innate immunity in response to bacterial colonization, and the first enteral feeding in early life.

Highlights

Transforming growth factor β2 (TGF-β2) is an important growth factor present in human and bovine milk (0.1–5.3 and 13–1150 ng/mL, respectively)
TGF-β2 is involved in different processes including cell proliferation and differentiation, and protects the immature intestine against inflammation [3,6]
Little is known about the molecular mechanisms underlying the effects of TGF-β2 in intestinal epithelial cells (IECs), which may help to explain how TGF-β2 improves intestinal development and protects the intestine against stress such as enteral feeding and endotoxin challenge in newborns

Summary

Introduction

Transforming growth factor β2 (TGF-β2) is an important growth factor present in human and bovine milk (0.1–5.3 and 13–1150 ng/mL, respectively). In a murine model, the activation of Toll-like receptor 4 (TLR-4) in intestinal epithelial cells (IECs) by lipopolysaccharide (LPS) derived from Gram-negative bacteria plays a central role in NEC progression through the inhibition of IEC migration and proliferation, thereby leading to IEC apoptosis and NEC development [8] These observations suggest that the synergy of TGF-β2 and LPS may play important roles in modulating intestinal diseases such as NEC. TGF-β2 has already been shown to decrease inflammatory cytokine secretion in both PsIc1 cells [15] and other types of IECs [16] It remains unknown whether TGF-β2 protects IECs by inducing differential protein expression in the IEC proteome, and whether these trends are similar to that found previously with the pattern of HSP regulation in the intestine of preterm pigs. The effects of TGF-β2 on the proteome of naïve and inflamed IECs induced by LPS were profiled using gel-based and LC-MS-based proteomics in PsIc1 cells

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