I K-12 may influence the differentiation of specific cell lineages with a shift towards the goblet cell lineage. Nevertheless, the interplay of underlying mechanisms and the exact consequences of the effects on the differentiation markers need further study. Previously, the induction of HBD2 by E. coli Nissle 1917 was demonstrated to be dependent on flagellin [34]. Since Hes1, Hath1 and Muc1 were also regulated by E. coli Nissle 1917, we analyzed the role of flagellin with respect to these three factors. In contrast to E. coli Nissle 1917 wild type, Hes1 and Hath1 mRNA was not downregulated by the flagellin mutant strains EcNDfliA, EcNDfliC and EcNDflgE. Accordingly, Muc1 expression was enhanced in E. coli Nissle 1917 wild type, but not in EcNDfliA, EcNDfliC and EcNDflgE. This implies that Hes1, Hath1 and Muc1 are regulated by E. coli Nissle 1917 flagellin, similar to HBD2. To elucidate the effect of the intestinal microflora in vivo, we analysed the expression of mHes1, Math1 and mKLF4 in the colon of germ free mice compared to SPF and conventionalized mice. Similar to the cell culture data, we observed a significantly lower Math1 and mKLF4 mRNA and protein expression incolonized mice compared to germ free mice, whereas mHes1 expression was reduced on mRNA but not on protein level. This difference in mHes1 expression could be a result of posttranscriptional regulation mechanisms which need further investigations. Several arguments underline that intestinal bacteria play a crucial role in IBD pathogenesis: Inflammation in IBD is located in areas with a high density of bacteria (mostly colon and/or terminal ileum) [45]; germ free mice do not develop colitis [46]; exposure of fecal stream to the terminal ileum worsen inflammation [47]; antimicrobial peptides are insufficiently expressed in CD, and mutations of human receptors recognizing luminal bacteria, such as NOD2 [48,49] and TLR dysfunction [50,51] are linked to a higher risk of IBD development. Moreover, the intestinal microflora is altered in IBD as compared to healthy controls. Numerous studies described changes in the composition of the microflora between CD, UC and healthy patients [52?4], and mucosa-associated and even intracellular bacteria were found in both types of IBD [17,18]. Recent studies showed UC to be associated with goblet cell [21] and ileal CD with Paneth cell differentiation defects [20]. In addition, mice with an epithelialspecific defect leading to reduced Hes1 expression were recently shown to spontaneously develop colitis [55]. Considering these observations, our data suggest that in addition to the genetic predisposition, the luminal microbiota may directly affect epithelial differentiation and its defensive role. There are also reasons to suggest bacteria to be involved in colon cancer pathogenesis: intestinal cancer is mostly found in the colon, the GW0742 Hexokinase II Inhibitor II, 3-BP segment with the highest number of bacteria [56], some bacteria can induce malignancies, e.g. H. pylori and gastric neoplasia [57,58], and, moreover, patients with colon cancer have adherent bacteria [27] as well as more circulating antibodies against specific bacteria (e.g. S. gallolyticus) compared to healthy controls [59]. On the other hand, several studies reported that probiotics, such as L. acidophilus NCFM, suppress carcinogenesis [60,61]. In most colorectal cancers Notch signaling was found to be activated [62,63], whereas Hath1 and KLF4 were decreased [64?7]. It may be speculated that the downregulatio.I K-12 may influence the differentiation of specific cell lineages with a shift towards the goblet cell lineage. Nevertheless, the interplay of underlying mechanisms and the exact consequences of the effects on the differentiation markers need further study. Previously, the induction of HBD2 by E. coli Nissle 1917 was demonstrated to be dependent on flagellin [34]. Since Hes1, Hath1 and Muc1 were also regulated by E. coli Nissle 1917, we analyzed the role of flagellin with respect to these three factors. In contrast to E. coli Nissle 1917 wild type, Hes1 and Hath1 mRNA was not downregulated by the flagellin mutant strains EcNDfliA, EcNDfliC and EcNDflgE. Accordingly, Muc1 expression was enhanced in E. coli Nissle 1917 wild type, but not in EcNDfliA, EcNDfliC and EcNDflgE. This implies that Hes1, Hath1 and Muc1 are regulated by E. coli Nissle 1917 flagellin, similar to HBD2. To elucidate the effect of the intestinal microflora in vivo, we analysed the expression of mHes1, Math1 and mKLF4 in the colon of germ free mice compared to SPF and conventionalized mice. Similar to the cell culture data, we observed a significantly lower Math1 and mKLF4 mRNA and protein expression incolonized mice compared to germ free mice, whereas mHes1 expression was reduced on mRNA but not on protein level. This difference in mHes1 expression could be a result of posttranscriptional regulation mechanisms which need further investigations. Several arguments underline that intestinal bacteria play a crucial role in IBD pathogenesis: Inflammation in IBD is located in areas with a high density of bacteria (mostly colon and/or terminal ileum) [45]; germ free mice do not develop colitis [46]; exposure of fecal stream to the terminal ileum worsen inflammation [47]; antimicrobial peptides are insufficiently expressed in CD, and mutations of human receptors recognizing luminal bacteria, such as NOD2 [48,49] and TLR dysfunction [50,51] are linked to a higher risk of IBD development. Moreover, the intestinal microflora is altered in IBD as compared to healthy controls. Numerous studies described changes in the composition of the microflora between CD, UC and healthy patients [52?4], and mucosa-associated and even intracellular bacteria were found in both types of IBD [17,18]. Recent studies showed UC to be associated with goblet cell [21] and ileal CD with Paneth cell differentiation defects [20]. In addition, mice with an epithelialspecific defect leading to reduced Hes1 expression were recently shown to spontaneously develop colitis [55]. Considering these observations, our data suggest that in addition to the genetic predisposition, the luminal microbiota may directly affect epithelial differentiation and its defensive role. There are also reasons to suggest bacteria to be involved in colon cancer pathogenesis: intestinal cancer is mostly found in the colon, the segment with the highest number of bacteria [56], some bacteria can induce malignancies, e.g. H. pylori and gastric neoplasia [57,58], and, moreover, patients with colon cancer have adherent bacteria [27] as well as more circulating antibodies against specific bacteria (e.g. S. gallolyticus) compared to healthy controls [59]. On the other hand, several studies reported that probiotics, such as L. acidophilus NCFM, suppress carcinogenesis [60,61]. In most colorectal cancers Notch signaling was found to be activated [62,63], whereas Hath1 and KLF4 were decreased [64?7]. It may be speculated that the downregulatio.
