TGF-ß2 induces maturation of immature human intestinal epithelial cells and inhibits inflammatory cytokine responses induced via the NF-κB pathway.

OBJECTIVES: Breast milk transforming growth factor (TGF)-ß2 is associated with healthy immune maturation and reduced risk of immune-mediated disease in infants. We sought to investigate whether conditioning with TGF-ß2 may result in a more mature immune responder phenotype in immature human intestinal epithelial cells (IECs). METHODS: Primary human fetal IECs (hFIECs) and the human fetal small intestinal epithelial cell line (H4 cells) were conditioned with breast milk levels of TGF-ß2, and an inflammatory response was subsequently induced. Inflammatory cytokine secretion and mRNA expression were measured by enzyme-linked immunosorbent assay and quantitative real-time polymerase chain reaction, respectively. Alterations in activation of inflammatory signaling pathways were detected from IECs by immunoblotting and immunofluorescence. The effects of TGF-ß2 conditioning on gene expression patterns in hFIECs were assessed by cDNA microarray analysis and quantitative PCR. RESULTS: Conditioning with TGF-ß2 significantly attenuated subsequent interleukin (IL)-1ß-, TNF-α-, and poly I:C-induced IL-8 and IL-6 responses in immature human IECs. Conditioning with TGF-ß2 inhibited IL-1ß-induced IκB-α degradation and NF-κB p65 nuclear translocation, which may partially result from TGF-ß2-induced changes in the expression of genes in the NF-κB signaling pathway detected by cDNA microarray and qPCR. CONCLUSIONS: Conditioning with TGF-ß2 attenuates the subsequent inflammatory cytokine response in immature human IECs by inhibiting signaling in the NF-κB pathway. The immunomodulatory potential of breast milk may in part be mediated by TGF-ß2, which may provide a novel means of supporting intestinal immune maturation in neonates.

Breast milk-transforming growth factor-ß2 specifically attenuates IL-1ß-induced inflammatory responses in the immature human intestine via an SMAD6- and ERK-dependent mechanism.

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-ß2 (TGF-ß2), 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 1ß (IL-1ß) with or without TGF-ß2. 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-ß2 significantly attenuated IL-1ß-induced pro-inflammatory cytokine production in fetal intestinal organ culture and the cell culture models. In addition, TGF-ß2 reduced the IL-1ß-induced IL-8 and IL-6 mRNA response in H4 cells. TGF-ß2 markedly inhibited IL-1ß-induced phosphorylation of ERK, which was necessary for the cytokine response. The inhibitory effect of TGF-ß2 on IL-1ß-induced cytokine production was completely abrogated by SMAD6 siRNA knockdown. CONCLUSIONS: TGF-ß2 attenuates IL-1ß-induced pro-inflammatory cytokine production in immature human IECs by inhibiting ERK signalling. The anti-inflammatory effect of TGF-ß2 is dependent on SMAD6. Breast milk TGF-ß2 may provide the neonate with important immunoregulatory support. TGF-ß2 might provide a novel means to improve intestinal immunophysiology in premature neonates.

Human milk adiponectin is associated with infant growth in two independent cohorts.

BACKGROUND: Adiponectin, a circulating adipocyte protein, is associated with lower obesity. We have previously shown that adiponectin is present in human milk. This study determined whether higher milk adiponectin is associated with infant growth and investigated milk adiponectin's oligomeric form. DESIGN AND METHODS: This is a study of two parallel longitudinal cohorts of breastfed infants born between 1998 and 2005. Forty-five mother-infant pairs from Cincinnati, OH and 277 mother-infant pairs from Mexico City, Mexico were analyzed. All participants were healthy, term infants breastfed at least 1 month who completed 6 months of follow-up. Monthly milk samples (n = 1,379) up to 6 months were assayed for adiponectin by radioimmunoassay. Infant weight-for-age, length-for-age, and weight-for-length Z-scores up to 6 months of age were calculated using World Health Organization standards. Repeated-measures analysis was conducted. The structural form of human milk adiponectin was assessed by western blot. RESULTS: In the population studies, initial milk adiponectin was 24.0 +/- 8.6 microg/L and did not differ by cohort. Over the first 6 months, higher milk adiponectin was associated with lower infant weight-for-age Z-score (-0.20 +/- 0.04, p < 0.0001) and weight-for-length Z-score (-0.29 +/- 0.08, p = 0.0002) but not length-for-age Z-score, adjusted for covariates, with no difference by cohort. By western blot, human milk adiponectin was predominantly in the biologically active high-molecular-weight form. CONCLUSIONS: Our data suggest milk adiponectin may play a role in the early growth and development of breastfed infants.

A novel technique for differentiation of proteins in the development of acid gel structure from control and heat treated milk using confocal scanning laser microscopy.

The incorporation of caseins and whey proteins into acid gels produced from unheated and heat treated skimmed milk was studied by confocal scanning laser microscopy (CSLM) using fluorescent labelled proteins. Bovine casein micelles were labelled using Alexa Fluor 594, while whey proteins were labelled using Alexa Fluor 488. Samples of the labelled protein solutions were introduced into aliquots of pasteurised skim milk, and skim milk heated to 90 degrees C for 2 min and 95 degrees C for 8 min. The milk was acidified at 40 degrees C to a final pH of 4.4 using 20 g glucono-delta-lactone/l (GDL). The formation of gels was observed with CSLM at two wavelengths (488 nm and 594 nm), and also by visual and rheological methods. In the control milk, as pH decreased distinct casein aggregates appeared, and as further pH reduction occurred, the whey proteins could be seen to coat the casein aggregates. With the heated milks, the gel structure was formed of continuous strands consisting of both casein and whey protein. The formation of the gel network was correlated with an increase in the elastic modulus for all three treatments, in relation to the severity of heat treatment. This model system allows the separate observation of the caseins and whey proteins, and the study of the interactions between the two protein fractions during the formation of the acid gel structure, on a real-time basis. The system could therefore be a valuable tool in the study of structure formation in yoghurt and other dairy protein systems.