Promotion of Intestinal Epithelial Cell Turnover by Commensal Bacteria: Role of Short-Chain Fatty Acids

<div><p>The life span of intestinal epithelial cells (IECs) is short (3–5 days), and its regulation is thought to be important for homeostasis of the intestinal epithelium. We have now investigated the role of commensal bacteria in regulation of IEC turnover in the small intestine. The proliferative activity of IECs in intestinal crypts as well as the migration of these cells along the crypt-villus axis were markedly attenuated both in germ-free mice and in specific pathogen–free (SPF) mice treated with a mixture of antibiotics, with antibiotics selective for Gram-positive bacteria being most effective in this regard. Oral administration of chloroform-treated feces of SPF mice to germ-free mice resulted in a marked increase in IEC turnover, suggesting that spore-forming Gram-positive bacteria contribute to this effect. Oral administration of short-chain fatty acids (SCFAs) as bacterial fermentation products also restored the turnover of IECs in antibiotic-treated SPF mice as well as promoted the development of intestinal organoids in vitro. Antibiotic treatment reduced the phosphorylation levels of ERK, ribosomal protein S6, and STAT3 in IECs of SPF mice. Our results thus suggest that Gram-positive commensal bacteria are a major determinant of IEC turnover, and that their stimulatory effect is mediated by SCFAs.</p></div

Promotion of the proliferative activity and turnover of IECs in GF mice by chloroform-resistant commensal bacteria.

<p>(<b>A</b>) Chloroform-treated feces from untreated SPF mice or chloroform-treated PBS as a control were administered orally to 8-week-old GF mice. The resulting exGF and control mice, respectively, were housed separately for 3 weeks in isolators, after which frozen sections of the ileum from both groups of mice (11-week-old) as well as from age-matched SPF mice were immunostained with antibodies to Ki67 (red) and to β-catenin (green). Representative images are shown in the left panels. Scale bar, 20 μm. The number of Ki67-positive cells per crypt was also determined from such sections (right panel). Data are means ± SE for 30 crypts from one mouse and are representative of four mice. ***<i>P</i> < 0.001 (ANOVA and Tukey’s test). (<b>B</b>) Lysates of IECs isolated from mice (11-week-old) treated as in (A) were subjected to immunoblot analysis with antibodies to cyclin D1 and to β-tubulin (left panels). The cyclin D1/β-tubulin band intensity ratio was also determined from such blots and expressed relative to the value for GF mice (right panel). Data are means ± SE from three separate experiments. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001 (ANOVA and Tukey’s test). (<b>C</b>) Frozen sections of the ileum were prepared from SPF, GF, and exGF mice (11-week-old) at 2 days after BrdU injection and were immunostained with mAbs to BrdU (red) and to β-catenin (green). Representative images are shown in the left panels. Scale bar, 100 μm. The migration distance for BrdU-positive cells was also determined from such sections (right panel). Data are means ± SE for 30 villi from one mouse and are representative of three mice. ***<i>P</i> < 0.001 (ANOVA and Tukey’s test). (<b>D</b>) PCR analysis of 16S rRNA genes of total bacteria, <i>C</i>. <i>leptum</i>, <i>C</i>. <i>coccoides</i>, segmented filamentous bacteria (SFB), or <i>B</i>. <i>fragilis</i> in fecal pellets from GF, exGF, or SPF mice (11-week-old).</p

Numbers of different types of IECs in antibiotic-treated mice.

<p>(<b>A</b>) SPF mice (4-week-old) were provided with drinking water supplemented (or not) with an antibiotic cocktail (Abx: ampicillin, vancomycin, metronidazole, neomycin) for 4 weeks, after which paraffin-embedded sections of the ileum from mice (8-week-old) were prepared and subjected to in situ hybridization analysis of Olfm4 mRNA. Representative images are shown in the left panels. Scale bar, 50 μm. The number of Olfm4 mRNA–positive cells per crypt was also determined from such sections (right panel). Data are means ± SE for 25 crypts from one mouse and are representative of three mice. (<b>B</b>) Frozen sections prepared from the ileum of mice (8-week-old) treated as in (A) were immunostained with antibodies to Muc2 (red) and to β-catenin (green). Representative images are shown in the left panels. Scale bar, 100 μm. The numbers of β-catenin–positive absorptive enterocytes and Muc2-positive goblet cells per villus were also determined from such sections. Data are means ± SE for 30 villi from one mouse and are representative of seven mice. *<i>P</i> < 0.05 (Student’s <i>t</i> test). (<b>C</b>) Frozen sections prepared from the ileum of mice (8-week-old) treated as in (A) were immunostained with antibodies to lysozyme (red) and to β-catenin (green). Representative images are shown in the left panels. Scale bar, 100 μm. The number of lysozyme-positive Paneth cells per crypt was also determined from such sections (right panel). Data are means ± SE for 30 crypts from one mouse and are representative of seven mice. *<i>P</i> < 0.05 (Student’s <i>t</i> test).</p

Importance of MEK-ERK signaling in the SCFA-dependent development of intestinal organoids.

<p>(<b>A</b>) Mouse intestinal organoids were stimulated (or not) with 0.5 mM acetate, 0.5 mM propionate, 0.5 mM butyrate, or SCFAs (mixture of 0.5 mM acetate, 0.5 mM propionate, and 0.5 mM butyrate) for 20 min, after which they were lysed and subjected to immunoblot analysis of phosphorylated and total forms of ERK. (<b>B</b> and <b>C</b>) Mouse intestinal organoids were incubated in the absence (–) or presence of SCFAs (mixture of 0.5 mM acetate, 0.5 mM propionate, and 0.5 mM butyrate) with or without MEK inhibitor (10 μM U0126) for 4 days. Organoid area (B) and the number of buds per organoid (C) determined for cultures. Data are means ± SE from three separate experiments, with 30 organoids being examined for each condition in each experiment. **<i>P</i> < 0.01 (ANOVA and Tukey’s test).</p

Promotion of the proliferative activity and turnover of IECs by SCFAs.

<p>(<b>A</b>) SPF mice (4-week-old) were provided with drinking water supplemented (or not) with vancomycin (VCM) for 2 weeks and then with vancomycin alone or together with an SCFA cocktail (62.5 mM acetate, 25.9 mM propionate, 40 mM butyrate) for an additional 2 weeks. The mice (8-week-old) were then injected with BrdU, and frozen sections of the ileum prepared at 2 h after the injection were immunostained with mAbs to BrdU (red) and to β-catenin (green). Representative images are shown in the left panels. Scale bar, 100 μm. The number of BrdU-positive cells per crypt was also determined from such sections (right panel). Data are means ± SE for 30 crypts from one mouse and are representative of three mice. ***<i>P</i> < 0.001 (ANOVA and Tukey’s test). (<b>B</b>) Frozen sections of the ileum from mice (8-week-old) treated as in (A) were also prepared at 2 days after BrdU injection and subjected to immunostaining as in (A). Representative images are shown in the left panels. Scale bar, 100 μm. The migration distance for BrdU-positive cells was also determined from such sections (right panel). Data are means ± SE for 30 villi from one mouse and are representative of three mice. ***<i>P</i> < 0.001 (ANOVA and Tukey’s test).</p

Reduced proliferative activity and turnover of IECs in antibiotic-treated SPF mice.

<p>(<b>A</b>) SPF mice (4-week-old) were provided with drinking water supplemented (or not) with an antibiotic cocktail (Abx: ampicillin, vancomycin, metronidazole, neomycin) for 4 weeks, after which frozen sections of the ileum from mice (8-week-old) were prepared at 2 h or 2, 3, or 5 days after BrdU injection and were immunostained with mAbs to BrdU (red) and to β-catenin (green). The boxed areas in the left panels for the sections prepared at 2 h after BrdU injection are shown at higher magnification in the right panels. Scale bars, 20 μm (higher magnification) or 100 μm (lower magnification). (<b>B</b>) The number of BrdU-positive cells per crypt at 2 h after BrdU injection was determined from sections similar to those in (A). Data are means ± SE for 30 crypts from one mouse and are representative of four mice. ***<i>P</i> < 0.001 (Student's <i>t</i> test). (<b>C</b>) The migration distance for BrdU-positive cells at 2 days after BrdU injection was determined from sections similar to those in (A). Data are means ± SE for 30 villi from one mouse and are representative of four mice. ***<i>P</i> < 0.001 (Student's <i>t</i> test). (<b>D</b>) Frozen sections prepared from the ileum of mice (8-week-old) treated (or not) with antibiotics as in (A) were immunostained with antibodies to Ki67 (red) and to β-catenin (green). Representative images are shown in the left panels. Scale bar, 20 μm. The number of Ki67-positive cells per crypt in such sections was also determined (right panel). Data are means ± SE for 30 crypts from one mouse and are representative of three mice. ***<i>P</i> < 0.001 (Student's <i>t</i> test). (<b>E</b>) IECs isolated from mice (8-week-old) treated (or not) with antibiotics as in (A) were lysed and subjected to immunoblot analysis with antibodies to cyclin D1 and to β-tubulin (loading control). Representative blots are shown in the left panels. The cyclin D1/β-tubulin band intensity ratio for such blots was also determined and is expressed relative to the value for control mice (right panel). Data are means ± SE from three individual experiments. ***<i>P</i> < 0.001 (Student’s <i>t</i> test).</p

Reduced phosphorylation levels of ERK, S6, and STAT3 in the small intestine of antibiotic-treated SPF mice.

<p>(<b>A</b>) Mice (4-week-old) were provided with drinking water supplemented (or not) with an antibiotic cocktail (Abx: ampicillin, vancomycin, metronidazole, neomycin) for 4 weeks, after which the abundance of mRNAs for c-Myc, Axin2, Hes1, and Areg in freshly isolated IECs from mice (8-week-old) was determined by quantitative RT-PCR analysis. The amount of each mRNA was normalized by that of GAPDH mRNA and is expressed relative to the corresponding value for control mice. Data are means ± SE from four mice. (<b>B</b>–<b>F</b>) Lysates of IECs prepared from mice (8-week-old) treated as in (A) were subjected to immunoblot analysis with antibodies to total or phosphorylated (p) forms of ERK (B), AKT (C), S6 (D), STAT3 (E), or c-Src (F). Representative blots and densitometric analysis of the ratio of the band intensity for the phosphorylated protein to that for the total protein are shown, with the quantitative data being expressed relative to the corresponding value for control mice and presented as means ± SE from three separate experiments. *<i>P</i> < 0.05 (Student’s <i>t</i> test).</p