SCFAs (short-chain fatty acids), fermentation products of bacteria, influence epithelial-specific gene expression. We hypothesize that SCFAs affect goblet-cell-specific mucin MUC2 expression and thereby alter epithelial protection. In the present study, our aim was to investigate the mechanisms that regulate butyrate-mediated effects on MUC2 synthesis. Human goblet cell-like LS174T cells were treated with SCFAs, after which MUC2 mRNA levels and stability, and MUC2 protein expression were analysed. SCFA-responsive regions and cis-elements within the MUC2 promoter were identified by transfection and gel-shift assays. The effects of butyrate on histone H3/H4 status at the MUC2 promoter were established by chromatin immunoprecipitation. Butyrate (at 1 mM), as well as propionate, induced an increase in MUC2 mRNA levels. MUC2 mRNA levels returned to basal levels after incubation with 5-15 mM butyrate. Interestingly, this decrease was not due to loss of RNA stability. In contrast, at concentrations of 5-15 mM propionate, MUC2 mRNA levels remained increased. Promoter-regulation studies revealed an active butyrate-responsive region at -947/-371 within the MUC2 promoter. In this region we identified an active AP1 (c-Fos/c-Jun) cis-element at -818/-808 that mediates butyrate-induced activation of the promoter. Finally, MUC2 regulation by butyrate at 10-15 mM was associated with increased acetylation of histone H3 and H4 and methylation of H3 at the MUC2 promoter. In conclusion, 1 mM butyrate and 1-15 mM propionate increase MUC2 expression. The effects of butyrate on MUC2 mRNA are mediated via AP-1 and acetylation/methylation of histones at the MUC2 promoter.

Paneth cell dysfunction has been suggested in necrotizing enterocolitis (NEC). The aim of this study was to i) study Paneth cell presence, protein expression, and developmental changes in preterm infants with NEC and ii) determine Paneth cell products and antimicrobial capacity in ileostomy outflow fluid. Intestinal tissue from NEC patients (n = 55), preterm control infants (n = 22), and term controls (n = 7) was obtained during surgical resection and at stoma closure after recovery. Paneth cell abundance and protein expression were analyzed by immunohistochemistry. RNA levels of Paneth cell proteins were determined by real-time quantitative RT-PCR. In ileostomy outflow fluid, Paneth cell products were quantified, and antimicrobial activity was measured in vitro. In acute NEC, Paneth cell abundance in small intestinal tissue was not significantly different from preterm controls. After recovery from NEC, Paneth cell hyperplasia was observed in the small intestine concomitant with elevated human alpha-defensin 5 mRNA levels. In the colon, metaplastic Paneth cells were observed. Ileostomy fluid contained Paneth cell proteins and inhibited bacterial growth. In conjunction, these data suggest an important role of Paneth cells and their products in various phases of NEC.

Background Mucin Muc2 is the structural component of the intestinal mucus layer. Absence of Muc2 leads to loss of this layer allowing direct bacterial-epithelial interactions. We hypothesized that absence of the mucus layer leads to increased expression of innate defense peptides. Specifically, we aimed to study the consequence of Muc2 deficiency (Muc2 −/− ) on the expression of regenerating islet-derived protein 3 beta (Reg3β), regenerating islet-derived protein 3 gamma (Reg3γ), and angiogenin-4 (Ang4) in the intestine shortly before and after weaning. Methods Intestinal tissues of Muc2 −/− and wild-type (WT) mice were collected at postnatal day 14 (P14, i.e. pre-weaning) and P28 (i.e. post-weaning). Reg3β, Reg3γ, and Ang4 expression was studied by quantitative real-time PCR, Western-blot, in situ hybridization, and immunohistochemistry. Results Reg3β and Reg3γ were expressed by diverging epithelial cell types; namely enterocytes, Paneth cells, and goblet cells. Additionally, Ang4 expression was confined to Paneth cells and goblet cells. Expression of Reg3β , Reg3γ , and Ang4 differed between WT and Muc2 −/− mice before and after weaning. Interestingly, absence of Muc2 strongly increased Reg3β and Reg3γ expression in the small intestine and colon. Finally, morphological signs of colitis were only observed in the distal colon of Muc2 −/− mice at P28, where and when expression levels of Reg3β , Reg3γ , and Ang4 were the lowest. Conclusions Expression of Reg3 proteins and Ang4 by goblet cells point to an important role for goblet cells in innate defense. Absence of Muc2 results in up-regulation of Reg3β and Reg3γ expression, suggesting altered bacterial-epithelial signaling and an innate defense response in Muc2 −/− mice. The inverse correlation between colitis development and Reg3β , Reg3γ , and Ang4 expression levels might point toward a role for these innate defense peptides in regulating intestinal inflammation.

Muc2-deficiency leads to an active inflammatory response in 2- and 4-week-old Muc2(-/-) mice as demonstrated by the altered expression in immune response related genes. In addition, 4-week-old Muc2(-/-) mice also showed a decrease in epithelial barrier function and an increase in epithelial proliferation as indicated by, respectively, the altered expression in tight junction-related genes and upregulation of genes stimulating cell growth. Remarkably, upregulation of genes stimulating cell growth correlated with increased crypt length and increased epithelial proliferation in 4-week-old Muc2(-/-) mice. Together, these data demonstrate that there are distinct phases in colitis development in 2-4-week-old Muc2(-/-) mice.

Short chain fatty acids (SCFAs), fermentation products of bacteria, influence epithelial-specific gene expression. We hypothesize that SCFAs affect goblet cell-specific mucin MUC2 expression and thereby alter epithelial protection. Our aim was to study the mechanisms that regulate butyratemediated effects on MUC2 synthesis. Human goblet cell-like LS174T cells were treated with SCFAs, after which MUC2 mRNA levels and stability and MUC2 protein expression were analyzed. SCFAresponsive regions and cis-elements within the MUC2 promoter were identified by transfection and gel shift assays. Effects of butyrate on histone H3/H4 status at the MUC2 promoter were established by chromatin immunoprecipitation. One mM butyrate as well as propionate induced an increase in MUC2 mRNA levels. MUC2 mRNA levels returned to basal levels after incubation with 5-15 mM of butyrate. Interestingly, this decrease was not due to loss of RNA stability. In contrast, at concentrations of 5-15 mM of propionate MUC2 mRNA levels remained increased. Promoter regulation studies revealed an active butyrate-responsive region at -947/-371 within the MUC2 promoter. In this region we identified an active AP1 (c-Fos/c-Jun) cis-element at -818/-808 that mediates butyrate-induced activation of the promoter. Finally, MUC2 regulation by butyrate at 10-15 mM was associated with increased acetylation of histone H3 and H4 and methylation of H3 at the MUC2 promoter. In conclusion, 1 mM of butyrate and 1-15 mM of propionate increase MUC2 expression. The effects of butyrate on MUC2 mRNA are mediated via AP-1 and acetylation/methylation of histones at the MUC2 promoter.