Short-chain fatty acid butyrate, a breast milk metabolite, enhances immature intestinal barrier function genes in response to inflammation in vitro and in vivo

Abstract

Infants born under 1,500 g have an increased incidence of necrotizing enterocolitis in the ileum and the colon, which is a life-threatening intestinal necrosis. This is in part due to excessive inflammation in the immature intestine to colonizing bacteria because of an immature innate immune response. Breastmilk complex carbohydrates create metabolites of colonizing bacteria in the form of short-chain fatty acids (SCFAs). We studied the effect of breastmilk metabolites, SCFAs, on immature intestine with regard to anti-inflammatory effects. This showed that acetate, propionate, and butyrate were all anti-inflammatory to an IL-1β inflammatory stimulus. In this study, to further define the mechanism of anti-inflammation, we created transcription profiles of RNA from immature human enterocytes after exposure to butyrate with and without an IL-1β inflammatory stimulus. We demonstrated that butyrate stimulates an increase in tight-junction and mucus genes and if we inhibit these genes, the anti-inflammatory effect is partially lost. SCFAs, products of microbial metabolism of complex carbohydrates of breastmilk oligosaccharides, have been found with this study to induce an anti-IL-1β response that is associated with an upregulation of tight junctions and mucus genes in epithelial cells (H4 cells). These studies suggest that breastmilk in conjunction with probiotics can reduce excessive inflammation with metabolites that are anti-inflammatory and stimulate an increase in the mucosal barrier.NEW & NOTEWORTHY This study extends previous observations to define the anti-inflammatory properties of butyrate, a short-chain fatty acid produced by the metabolism of breastmilk oligosaccharides by colonizing bacteria. Using transcription profiling of immature enterocyte genes, after exposure to butyrate and an IL-1β stimulus, we showed that tight-junction genes and mucus genes were increased, which contributed to the anti-inflammatory effect.

Keywords: butyrate; immature intestinal inflammation; mucus genes; tight junction genes.

Graphical abstract

Figure 1.

Differentially expressed genes (DEGs) were identified in H4 cells after exposure to butyrate and IL-1β. H4 cells were pretreated with 20 mM of butyrate for 30 min before IL-1β stimulation (1 ng/ml for 4 h). A: histogram showing the number of DEGs relative to the control. B: Venn diagram showing the genes, treated with butyrate or IL-1 β (with or without butyrate) were significantly regulated (P < 0.05) by comparison with the control. CTL, control group; Buty, individual butyrate group; Buty+ IL-1β, combination of butyrate and IL-1β group.

Figure 2.

Comparison of gene expression ratios that were obtained by RNA-seq and quantitative (q)RT-PCR. The horizontal striped bars represent RNA-seq data, and the diagonal striped bars represent the qRT-PCR data. Results are expressed of 3 independent experiments, each performed in triplicate.

Figure 3.

Effect of butyrate and IL-1β on tight junction and mucin-related genes in H4 cells. H4 cells were pretreated with 20 mM of butyrate for 30 min before IL-1β stimulation (1 ng/ml for 4 h). mRNA expression of Occludin (Ocln; A), Claudin4 (Cldn4; B), Cldn11 (C), Cldn15 (D), and mucin20 (Muc20) were determined by real-time qRT-PCR. Data are represented as means ± SE of three independent experiments. One-way ANOVA and Tukey post hoc tests were used for statistic, n = 3. Differences were considered significant at *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4.

Induction of mucin and tight junction related genes mRNA expression in mouse ileum induced by IL-1β pretreated with butyrate. Ileum tissue collected from control, and mice administered with butyrate were incubated for 24 h with or without IL-1β. mRNA levels of Muc1 (A), Muc2 (B), Muc4 (C), Muc19 (D), Tjp1 (E), Occln (F), Cldn 1(G), Cldn3 (H), and Cldn 4 (I) were determined by quantitative RT-PCR. Data are represented as means ± SE of 2 independent experiments; n = 6 for each experiment. One-way ANOVA and Tukey post hoc tests were used for statistic. Differences were considered significant at *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5.

Butyrate reduced the macrophage inflammatory protein2 (MIP2) secretion in neonatal mouse ileum and colon in response to IL-1β stimulation. Ileum (A) and colon (B) tissue collected from mouse fed with PBS (control) or butyrate were incubated with or without IL-1β for 24 h. The secretion of MIP2 into the supernatants was assayed by ELISA. Data are represented as means ± SE from 2 independent experiments; n = 6 for each experiment. One-way ANOVA and Tukey post hoc tests were used for statistic. Differences were considered significant at *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6.

Mucin inhibitor partially inhibited the anti-inflammatory effects of butyrate in H4 cells. H4 cells were pretreated with the mucin inhibitor Talniflumate before butyrate treatment then exposed to human IL-1β. Supernatants were collected and stored for IL8 ELISA analysis. *P < 0.05 and ***P < 0.001.