Indole-containing capsules show preventative effect on colitis development in SPF mice.

<p>SPF mice were treated with indole- (n = 7) or MCT- (n = 7) containing capsules for 1 week, and then challenged by 5% DSS for 6 days. Body weight changes relative to the value prior to colitis induction are shown. Data are representative of two independent experiments and mean ± S.E.M of 7 mice at each time point is shown. *P<0.05. MCT, Medium-Chain Triglycerides.</p

Commensal Bacteria-Dependent Indole Production Enhances Epithelial Barrier Function in the Colon

<div><p>Microbiota have been shown to have a great influence on functions of intestinal epithelial cells (ECs). The role of indole as a quorum-sensing (QS) molecule mediating intercellular signals in bacteria has been well appreciated. However, it remains unknown whether indole has beneficial effects on maintaining intestinal barriers <i>in vivo</i>. In this study, we analyzed the effect of indole on ECs using a germ free (GF) mouse model. GF mice showed decreased expression of junctional complex molecules in colonic ECs. The feces of specific pathogen-free (SPF) mice contained a high amount of indole; however the amount was significantly decreased in the feces of GF mice by 27-fold. Oral administration of indole-containing capsules resulted in increased expression of both tight junction (TJ)- and adherens junction (AJ)-associated molecules in colonic ECs in GF mice. In accordance with the increased expression of these junctional complex molecules, GF mice given indole-containing capsules showed higher resistance to dextran sodium sulfate (DSS)-induced colitis. A similar protective effect of indole on DSS-induced epithelial damage was also observed in mice bred in SPF conditions. These findings highlight the beneficial role of indole in establishing an epithelial barrier <i>in vivo</i>.</p></div

Indole and indole metabolites are absent in GF mice.

<p>(A, B) Feces and serum were collected from either SPF (n = 3) or GF (n = 3) mice. The concentration of indole in the feces was measured by HPLC-FL, and the serum concentration of indoxyl sulfate was measured by LC-MS/MS. Data are representative of two independent experiments and show mean values ± S.D. of 3 mice. *P<0.05. SPF, specific pathogen free; GF, germ free.</p

Indole-containing capsules promote epithelial barrier function in GF mice.

<p>(A) Feces were collected from SPF mice, and GF mice treated with indole- or MCT- containing capsules. Three mice was analysed in each group. The concentration of indole in the feces was measured by HPLC-FL. Data show mean values ± S.D. of 3 samples. *P<0.05. n.s., not significant. SPF, specific pathogen free; GF, germ free; MCT, Medium-Chain Triglycerides. (B) Real-time quantitative RT-PCR analysis of mRNA expression of <i>Cldn7, Ocln, Tjp1, Ctnnb1</i>, and <i>Cdh1</i> in colonic epithelial cells of GF mice treated with indole- (n = 4) or MCT- (n = 4) containing capsules. Values were normalized to the expression of <i>Gapdh</i>. Data are representative of two independent experiments and show mean values ± S.D. of 4 samples performed in duplicate. *P<0.05. (C) Colonic tissues of GF mice treated with indole- or MCT- containing capsules were stained with anti-occludin antibody. Sections were analyzed using a confocal microscope. Bars, 20 µm. Data are representative of two independent experiments. (D) After oral administration with either indole- (n = 6) or MCT- (n = 6) containing capsules for 2 weeks, GF mice were treated by 4% DSS in drinking water for 3 days. Survival rate of the mice in each group is shown. Data are representative of two independent experiments. MCT, Medium-Chain Triglycerides.</p

Epithelial barrier functions is impaired in GF mice.

<p>(A) Real-time quantitative RT-PCR analysis of mRNA expression of <i>Cldn7, Ocln, Tjp1, Ctnnb1, Cdh1</i> in colonic ECs in SPF (n = 4) or GF (n = 4) mice. Values were normalized to that of <i>Gapdh</i>. Data are representative of two independent experiments and show mean values ± S.D. of 4 samples performed in duplicate. *P<0.05. (B) Mouse colonic tissue was stained with anti-occludin antibody. Sections were analyzed using a confocal microscope. Bars, 50 µm. Data are representative of two independent experiments. (C) SPF (n = 8) or GF (n = 8) mice were administered 4% DSS by drinking water for 3 days. Survival rates of the indicated mice are shown. Body weight changes relative to the value prior to colitis induction are shown. Data are mean ± S.E.M of 8 mice at each time point. SPF, specific pathogen free; GF, germ free.</p

E-NPP3 controls plasmacytoid dendritic cell numbers in the small intestine

<div><p>Extracellular adenosine 5’-triphosphate (ATP) performs multiple functions including activation and induction of apoptosis of many cell types. The ATP-hydrolyzing ectoenzyme ecto-nucleotide pyrophosphatase/phosphodiesterase 3 (E-NPP3) regulates ATP-dependent chronic allergic responses by mast cells and basophils. However, E-NPP3 is also highly expressed on epithelial cells of the small intestine. In this study, we showed that E-NPP3 controls plasmacytoid dendritic cell (pDC) numbers in the intestine through regulation of intestinal extracellular ATP. In <i>Enpp3</i>^-/- mice, ATP concentrations were increased in the intestinal lumen. pDC numbers were remarkably decreased in the small intestinal lamina propria and Peyer’s patches. Intestinal pDCs of <i>Enpp3</i>^-/- mice showed enhanced cell death as characterized by increases in annexin V binding and expression of cleaved caspase-3. pDCs were highly sensitive to ATP-induced cell death compared with conventional DCs. ATP-induced cell death was abrogated in <i>P2rx7</i>^-/- pDCs. Accordingly, the number of intestinal pDCs was restored in <i>Enpp3</i>^-/- <i>P2rx7</i>^-/- mice. These findings demonstrate that E-NPP3 regulates ATP concentration and thereby prevents the decrease of pDCs in the small intestine.</p></div

Decrease in the number of intestinal pDCs in <i>Enpp3</i>^<i>-/-</i> mice.

<p><b>(A</b>–<b>C)</b> Frequency and number of CD45⁺ PDCA-1⁺ CD11c^med pDCs (A, C) and CD45⁺ PDCA-1^- CD11c ^high cDCs (B, C) in the Peyer’s patches (PPs), small intestinal lamina propria (SILP), bone marrow (BM), and spleen (SPL) of wild-type and <i>Enpp3</i>^<i>-/-</i> mice. All data are mean values ± SD (n = 7 for PP and SILP, and n = 6 for SPL and BM). *<i>p</i> < 0.05, NS: not significant. <b>(D)</b> Frequency of CD45⁺ PDCA-1⁺ CD11c^med pDCs in the PPs and SILP of wild-type (n = 6), <i>Enpp3</i>^<i>-/-</i> (n = 8), <i>Kit</i>^{<i>W-sh/W-sh</i>} (n = 7), and <i>Enpp3</i>^<i>-/-</i> <i>Kit</i>^{<i>W-sh/W-sh</i>} (n = 6) mice. All data are mean values ± SD. *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001, NS: not significant. <b>(E, F)</b> Frequency of annexin V-positive (E) and active caspase-3-positive (F) cells gated on CD45⁺ PDCA-1⁺ CD11c^med pDCs from the PPs and SILP of wild-type and <i>Enpp3</i>^<i>-/-</i> mice. Representative histograms are shown (left) and the means ± SD of the percentages of positive cells (right) are shown (n = 6 in e, and n = 5 in f). *<i>p</i> < 0.05, **<i>p</i> < 0.01. <b>(G)</b> Frequency of PDCA-1⁺ CD11c^med pDCs in the PPs and SILP from antibiotic-treated wild-type (n = 11) and <i>Enpp3</i>^<i>-/-</i> (n = 12) mice as well as untreated wild-type (n = 10) and <i>Enpp3</i>^<i>-/-</i> (n = 10) mice. Data are the means ± SD of the percentages of pDCs. *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001, NS: not significant.</p

Expression of <i>Enpp3</i> in small intestinal epithelia.

<p><b>(A)</b> Quantitative RT-PCR analysis of <i>Enpp3</i> mRNA expression in the indicated tissues (n = 3). <b>(B)</b> Quantitative RT-PCR analysis of <i>Enpp3</i> mRNA expression in epithelial cells and lamina propria in four parts of the small intestine. A smaller number represents a more proximal portion of the intestine (n = 4). <b>(C)</b> Quantitative RT-PCR analysis of <i>Enpp3</i> mRNA expression in epithelial cells of the small intestine in specific-pathogen free (SPF) and germ-free (GF) mice (n = 3). <b>(D)</b> Immunohistochemical analysis of the small intestine. E-NPP3 (red) and DAPI (blue). Swiss roll frozen sections were stained with the anti-mouse E-NPP3 antibody. A smaller number represents more proximal portion of the intestine. Scale bars, 100 μm. <b>(E)</b> ATP concentrations in luminal contents of the small intestine of wild-type and <i>Enpp3</i>^<i>-/-</i> mice. All data are mean values ± SD (n = 6 per groups). *<i>p</i> < 0.05. <b>(F)</b> ATP concentration in the proximal portion of the small intestinal lumen from wild-type, <i>Enpp3</i>^<i>-/-</i>, <i>Kit</i>^{<i>W-sh/W-sh</i>} and <i>Enpp3</i>^<i>-/-</i> <i>Kit</i>^{<i>W-sh/W-sh</i>} mice. All data are mean values ± SD (n = 4 per groups). *<i>p</i> < 0.05, NS: not significant. <b>(G)</b> ATP concentration in the proximal portion of the small intestinal lumen from <i>Kit</i>^{<i>W-sh/W-sh</i>} and <i>Enpp3</i>^<i>-/-</i> <i>Kit</i>^{<i>W-sh/W-sh</i>} mice with or without adoptive transfer of wild-type or <i>Enpp3</i>^<i>-/-</i> bone marrow-derived mast cells. All data are mean values ± SD (n = 5 for mast cells transferred mice groups and <i>Kit</i>^{<i>W-sh/W-sh</i>} mice group, and n = 3 for non transferred <i>Enpp3</i>^<i>-/-</i> <i>Kit</i>^{<i>W-sh/W-sh</i>} mice group). *<i>p</i> < 0.05, NS: not significant.</p

P2X7-dependent induction of pDC apoptosis in vivo.

<p><b>(A, B)</b> Frequencies of annexin V-positive (A) and active caspase-3-positive (B) cells gated on CD45⁺ PDCA-1⁺ CD11c^med pDCs from the PPs and SILP of wild-type, <i>Enpp3</i>^<i>-/-</i>, <i>P2rx7</i>^<i>-/-</i>, and <i>Enpp3</i>^<i>-/-</i> <i>P2rx7</i>^<i>-/-</i> mice (n = 7 per groups in a, and n = 5 per groups in b). Representative histograms are shown (left) and the means ± SD of the percentages of annexin V-positive (A) and active caspase-3-positive cells (B) are shown (right). *<i>p</i> < 0.05, **<i>p</i> < 0.01 <b>(c)</b> Frequency and number of PDCA-1⁺ CD11c^med pDCs in the PPs and SILP from wild-type, <i>Enpp3</i>^<i>-/-</i>, <i>P2rx7</i>^<i>-/-</i>, and <i>Enpp3</i>^<i>-/-</i> <i>P2rx7</i>^<i>-/-</i> (n = 13 per groups) mice. Representative dot plots are shown (left) and the means ± SD of the percentages of pDCs are shown (right). *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001.</p

CREBH Determines the Severity of Sulpyrine-Induced Fatal Shock

<div><p>Although the pyrazolone derivative sulpyrine is widely used as an antipyretic analgesic drug, side effects, including fatal shock, have been reported. However, the molecular mechanism underlying such a severe side effect is largely unclear. Here, we report that the transcription factor CREBH that is highly expressed in the liver plays an important role in fatal shock induced by sulpyrine in mice. CREBH-deficient mice were resistant to experimental fatal sulpyrine shock. We found that sulpyrine-induced expression of cytochrome P450 2B (CYP2B) family genes, which are involved in sulpyrine metabolism, in the liver was severely impaired in CREBH-deficient mice. Moreover, introduction of CYP2B in CREBH-deficient liver restored susceptibility to sulpyrine. Furthermore, ectopic expression of CREBH up-regulated CYP2B10 promoter activity, and <em>in vivo</em> knockdown of CREBH in wild-type mice conferred a significant resistance to fatal sulpyrine shock. These data demonstrate that CREBH is a positive regulator of CYP2B in response to sulpyrine administration, which possibly results in fatal shock.</p> </div