Breast Milk-Transforming Growth Factor-β2 Specifically Attenuates IL-1β-Induced Inflammatory Responses in the Immature Human Intestine via an SMAD6- and ERK-Dependent Mechanism

Abstract

Background: Breast milk is known to protect the infant against infectious and immuno-inflammatory diseases, but the mechanisms of this protection are poorly understood. Objectives: We hypothesized that transforming growth factor-β₂ (TGF-β₂), an immunoregulatory cytokine abundant in breast milk, may have a direct anti-inflammatory effect on immature human intestinal epithelial cells (IECs). Methods: Human fetal ileal organ culture, primary human fetal IECs, and the human fetal small intestinal epithelial cell line H4 were stimulated with interleukin 1β (IL-1β) with or without TGF-β₂. Pro-inflammatory cytokine secretion and mRNA expression were measured by ELISA and quantitative real-time polymerase chain reaction, respectively. Alterations in ERK signalling were detected from IECs by immunoblotting and in fetal ileal tissue culture by immunohistochemistry. SMAD6 knockdown was performed by transfecting the cells with SMAD6 siRNA. Results: TGF-β₂ significantly attenuated IL-1β-induced pro-inflammatory cytokine production in fetal intestinal organ culture and the cell culture models. In addition, TGF-β₂ reduced the IL-1β-induced IL-8 and IL-6 mRNA response in H4 cells. TGF-β₂ markedly inhibited IL-1β-induced phosphorylation of ERK, which was necessary for the cytokine response. The inhibitory effect of TGF-β₂ on IL-1β-induced cytokine production was completely abrogated by SMAD6 siRNA knockdown. Conclusions: TGF-β₂ attenuates IL-1β-induced pro-inflammatory cytokine production in immature human IECs by inhibiting ERK signalling. The anti-inflammatory effect of TGF-β₂ is dependent on SMAD6. Breast milk TGF-β₂ may provide the neonate with important immunoregulatory support. TGF-β₂ might provide a novel means to improve intestinal immunophysiology in premature neonates.