Project description:Background: Cystic fibrosis (CF) is caused by mutations in the CFTR gene that impair function of this cAMP-regulated Cl- channel. In the small intestine, loss of CFTR function creates a dehydrated, acidic luminal environment which is believed to cause an accumulation of mucus, a phenotype characteristic of CF. CF mice have an innate immune response and impaired intestinal transit as well. We investigated whether lubiprostone, which activates the CLC2 Cl- channel, would improve the CF intestinal phenotype. Methods: Cftrtm1UNC (CF) and wildtype (WT) littermate mice on the C57BL/6 background were used. Lubiprostone (10ug/kg-day) was administered by gavage for two weeks. Mucus accumulation was estimated from crypt lumen widths in Carnoy's-fixed, PAS/AB stained sections. Luminal bacterial load was measured by qPCR for the bacterial 16S gene. Gastric emptying and small intestinal transit were assessed by gavage of rhodamine dextran. Gene expression was evaluated by Affymetrix Mouse430 2.0 microarray. Results: Crypt width in control CF mice was 700% that of WT mice (P<0.001). Lubiprostone did not affect WT crypt width but, unexpectedly, increased CF crypt width 22% (P=0.001). Lubiprostone increased bacterial load in WT mice to 490% of WT control levels (P=0.008). Conversely, lubiprostone decreased bacterial overgrowth in CF mice by 60% (P=0.005). Lubiprostone significantly increased gastric emptying at 20 min postgavage in both WT (P<0.001; control=57±3%, treated=81±4%) and CF mice (P<0.001; control=48±4%, treated=75±5%). After lubiprostone small intestinal transit was significantly enhanced in WT mice (P=0.024) but the effect was not significant in CF mice (P=0.377). Among other innate immune markers, expression of mast cell genes was elevated ~20-fold in the control CF intestine and lubiprostone decreased expression to WT control levels. Conclusions: These results indicate that lubiprostone has some benefits for the CF intestinal phenotype, especially on bacterial overgrowth and the innate immune response. The unexpected observation of increased mucus accumulation in the crypts of lubiprostone-treated CF mice suggests the possibility that lubiprostone increases mucus secretion.

Project description:Background: Cystic fibrosis (CF) is caused by mutations in the CFTR gene that impair function of this cAMP-regulated Cl- channel. In the small intestine, loss of CFTR function creates a dehydrated, acidic luminal environment which is believed to cause an accumulation of mucus, a phenotype characteristic of CF. CF mice have an innate immune response and impaired intestinal transit as well. We investigated whether lubiprostone, which activates the CLC2 Cl- channel, would improve the CF intestinal phenotype. Methods: Cftrtm1UNC (CF) and wildtype (WT) littermate mice on the C57BL/6 background were used. Lubiprostone (10ug/kg-day) was administered by gavage for two weeks. Mucus accumulation was estimated from crypt lumen widths in Carnoy's-fixed, PAS/AB stained sections. Luminal bacterial load was measured by qPCR for the bacterial 16S gene. Gastric emptying and small intestinal transit were assessed by gavage of rhodamine dextran. Gene expression was evaluated by Affymetrix Mouse430 2.0 microarray. Results: Crypt width in control CF mice was 700% that of WT mice (P<0.001). Lubiprostone did not affect WT crypt width but, unexpectedly, increased CF crypt width 22% (P=0.001). Lubiprostone increased bacterial load in WT mice to 490% of WT control levels (P=0.008). Conversely, lubiprostone decreased bacterial overgrowth in CF mice by 60% (P=0.005). Lubiprostone significantly increased gastric emptying at 20 min postgavage in both WT (P<0.001; control=57±3%, treated=81±4%) and CF mice (P<0.001; control=48±4%, treated=75±5%). After lubiprostone small intestinal transit was significantly enhanced in WT mice (P=0.024) but the effect was not significant in CF mice (P=0.377). Among other innate immune markers, expression of mast cell genes was elevated ~20-fold in the control CF intestine and lubiprostone decreased expression to WT control levels. Conclusions: These results indicate that lubiprostone has some benefits for the CF intestinal phenotype, especially on bacterial overgrowth and the innate immune response. The unexpected observation of increased mucus accumulation in the crypts of lubiprostone-treated CF mice suggests the possibility that lubiprostone increases mucus secretion. For each group (control wild type, lubiprostone-treated wild type, contol Cftr null, and lubiprostone-treated cftr null), equal amounts of total RNA extracted from the entire small intestine were pooled for analysis.

Project description:Epithelial cells of the mammalian intestine are covered with a mucus layer that prevents direct contact with intestinal microbes but also constitutes a substrate for mucus-degrading bacteria. To study the effect of mucus degradation on the host response, germ-free mice were colonized with Akkermansia muciniphila. This anaerobic bacterium belonging to the Verrucomicrobia is specialized in the degradation of mucin, the glycoprotein present in mucus, and found in high numbers in the intestinal tract of human and other mammalian species. Efficient colonization of A. muciniphila was observed with highest numbers in the cecum, where most mucin is produced. In contrast, following colonization by Lactobacillus plantarum, a facultative anaerobe belonging to the Firmicutes that ferments carbohydrates, similar cell-numbers were found at all intestinal sites. Whereas A. muciniphila was located closely associated with the intestinal cells, L. plantarum was exclusively found in the lumen. The global transcriptional host response was determined in intestinal biopsies and revealed a consistent, site-specific, and unique modulation of about 750 genes in mice colonized by A. muciniphila and over 1500 genes after colonization by L. plantarum. Pathway reconstructions showed that colonization by A. muciniphila altered mucosal gene expression profiles toward increased expression of genes involved in immune responses and cell fate determination, while colonization by L. plantarum led to up-regulation of lipid metabolism. These indicate that the colonizers induce host responses that are specific per intestinal location. In conclusion, we propose that A. muciniphila modulates pathways involved in establishing homeostasis for basal metabolism and immune tolerance toward commensal microbiota. Keywords: Analysis of target gene regulation by using microarrays Adult germ-free female NMRI-KI mice (45 – 65 days) were used for bacterial mono-association. Two bacterial strains were used in this study, A. muciniphila MucT (ATTC BAA-835) and L. plantarum WCFS1 (NCIMB 8826). A. muciniphila was grown anaerobically in a basal mucin based medium and L. plantarum was grown anaerobically at 37°C in Man-Rogosa-Sharpe broth (MRS; Le Pont de Claix, France). After 7 days of colonization, mice were killed by cervical dislocation and terminal ileum, cecum and ascending colon specimens were sampled.

			Action	DRS
	ERR12188105_1.fastq.gz	Fastqsanger.gz
	ERR12188105_2.fastq.gz	Fastqsanger.gz
	ERR12188106_1.fastq.gz	Fastqsanger.gz
	ERR12188106_2.fastq.gz	Fastqsanger.gz
	ERR12188107_1.fastq.gz	Fastqsanger.gz

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