http://repub.eur.nl/res/aut/15348/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/33621/ 2011-10-01T00:00:00Z The mucin Muc2 is the structural component of the colonic mucus layer. Adult Muc2 knockout (Muc2-/-) mice suffer from severe colitis. We hypothesized that Muc2 deficiency induces inflammation before weaning of mother's milk [postnatal day (P) 14] with aggravation of colitis after weaning (P28). Muc2-/-and wild-type mice were killed at embryonic day 18.5 and P1.5, P7.5, P14, P21, and P28. Colonic morphology, influx of T cells, and goblet cell-specific protein expression was investigated by (immuno)histochemistry. Cytokine and Tolllike receptor (TLR) profiles in the colon were analyzed by quantitative RT-PCR. Muc2-/-mice showed an increased and persistent influx of Cd3ε-positive T cells in the colonic mucosa as of P1.5. This was accompanied by mucosal damage at P28 in the distal colon but not in the proximal colon. At P14, the proinflammatory immune response [i.e., increased interleukin (IL)-12 p35, IL-12 p40, and tumor necrosis factor-α, expression] in the distal colon of Muc2-/-mice presented with an immune suppressive response [i.e., increased Foxp3, transforming growth factor (TGF)-β1, IL-10, and Ebi3 expression]. In contrast, at P28, a proinflammatory response remained in the distal colon, whereas the immune suppressive response (i.e., Foxp3 and TGF-β1 expression) declined. The proximal colon of Muc2-/-mice did not show morphological damage and was dominated by an immune suppressive response at P14 and P28. Interestingly, changes in expression of TLRs and TLR-related molecules were observed in the distal colon at P14 and P28 and in the proximal colon only at P28. Colitis in Muc2-/-mice is limited before weaning by immune suppressive responses and exacerbates in the distal colon after weaning because of the decline in the immune suppressive response. http://repub.eur.nl/res/pub/25182/ 2009-06-01T00:00:00Z 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. http://repub.eur.nl/res/pub/28845/ 2008-06-24T00:00:00Z Expression of the mucin MUC2, the structural component of the colonic mucus layer, is lowered in ulcerative colitis. Furthermore, interleukin (IL)-10 knockout (IL-10-/-) mice develop colitis and have reduced Muc2 levels. Our aim was to obtain insight into the role of Muc2 and IL-10 in epithelial protection. Muc2-IL-10 double-knockout (Muc2/IL-10DKO) mice were characterized and compared to Muc2 knockout (Muc2-/-), IL-10-/-and wild-type (WT) mice. Clinical symptoms, intestinal morphology and differences in epithelial-specific protein levels were analyzed. In addition, levels of the pro-inflammatory cytokines in colonic tissue and serum were determined. IL-10-/-mice were indistinguishable from WT mice throughout this experiment and showed no clinical or histological signs of colitis. Muc2/IL-10DKOand Muc2-/-mice showed significant growth retardation and clinical signs of colitis at 4 and 5 weeks, respectively. Muc2/IL-10DKOmice had a high mortality rate (50% survival/5 weeks) compared to the other types of mice (100% survival). Microscopic analysis of the colon of Muc2/IL-10DKOmice showed mucosal thickening, increased proliferation, superficial erosions and a diminished Muc4 expression. Furthermore, pro-inflammatory cytokines were significantly upregulated, both in tissue (mRNA) and systemically in Muc2/IL-10DKOmice. In conclusion, Muc2/IL-10DKOmice develop colitis, which is more severe in every aspect compared to Muc2-/-and IL-10-/-mice. These data indicate that (i) in case of Muc2 deficiency, the anti-inflammatory cytokine IL-10 can control epithelial damage, though to a limited extent and (ii) the mucus layer is most likely a key factor determining colitis. http://repub.eur.nl/res/pub/28987/ 2008-05-16T00:00:00Z The mucin Muc2 is the main component of the intestinal mucus layer and thus plays important roles in intestinal protection. Therefore, it is important to understand its regulation during goblet cell differentiation. Foxa1 and Foxa2 forkhead box transcription factors (TFs) participate in transcriptional programs governing intestinal cell differentiation. Using immunohistochemistry, we showed a spatio-temporal pattern of expression of both TFs in developing and adult mouse intestine and their expression in Muc2-expressing intestinal cells. Down-regulation of Foxa1 and Foxa2 by RNA interference in cultured intestinal cells decreased Muc2 mRNA level by half, and abolished Muc2 protein expression. Chromatin immunoprecipitation and gel shift assays showed that these two TFs directly bind to the Muc2 promoter. Co-transfection experiments indicated that both TFs activate the Muc2 promoter and that mutations of three Foxa cis-elements inhibit Muc2 transactivation. In conclusion, this work identifies Foxa1 and Foxa2 as important regulators of Muc2 expression in the intestine. http://repub.eur.nl/res/pub/37114/ 2007-06-25T00:00:00Z Cyclooxygenases (COXs) play a significant role in many different viral infections with respect to replication and pathogenesis. Here we investigated the role of COXs in the mouse hepatitis coronavirus (MHV) infection cycle. Blocking COX activity by different inhibitors or by RNA interference affected MHV infection in different cells. The COX inhibitors reduced MHV infection at a post-binding step, but early in the replication cycle. Both viral RNA and viral protein synthesis were affected with subsequent loss of progeny virus production. Thus, COX activity appears to be required for efficient MHV replication, providing a potential target for anti-coronaviral therapy. http://repub.eur.nl/res/pub/13485/ 2004-09-01T00:00:00Z Elevated levels of prostaglandins (PGs), products of cyclooxygenases (COXs), are found in the plasma and stool of rotavirus-infected children. We sought to determine the role of COXs, PGs, and the signal transduction pathways involved in rotavirus infection to elucidate possible new targets for antiviral therapy. Human intestinal Caco-2 cells were infected with human rotavirus Wa or simian rotavirus SA-11. COX-2 mRNA expression and secreted PGE2 levels were determined at different time points postinfection, and the effect of COX inhibitors on rotavirus infection was studied by an immunofluorescence assay (IFA). To reveal the signal transduction pathways involved, the effect of MEK, protein kinase A (PKA), p38 mitogen-activated protein kinase (MAPK), and NF-kappaB inhibitors on rotavirus infection was analyzed. In infected Caco-2 cells, increased COX-2 mRNA expression and secreted PGE2 levels were detected. Indomethacin (inhibiting both COX-1 and COX-2) and specific COX-1 and COX-2 inhibitors reduced rotavirus infection by 85 and 50%, respectively, as measured by an IFA. Indomethacin reduced virus infection at a postbinding step early in the infection cycle, inhibiting virus protein synthesis. Indomethacin did not seem to affect viral RNA synthesis. Inhibitors of MEK, PKA, p38 MAPK, and NF-kappaB decreased rotavirus infection by at least 40%. PGE2 counteracted the effect of the COX and PKA inhibitors but not of the MEK, p38 MAPK, and NF-kappaB inhibitors. Conclusively, COXs and PGE2 are important mediators of rotavirus infection at a postbinding step. The ERK1/2 pathway mediated by PKA is involved in COX induction by rotavirus infection. MAPK and NF-kappaB pathways are involved in rotavirus infection but in a PGE2-independent manner. This report offers new perspectives in the search for therapeutic agents in treatment of severe rotavirus-mediated diarrhea in children.