<i>Nfkbiz</i>^−/− mouse T cells fail to generate Th17 cells <i>in vitro</i>.

<p>(A–D) Expression of IL-17A protein or <i>Il-17a</i> mRNA (A, B) and of the Th17-related mRNAs <i>Ccr6</i> and <i>Il-17f</i> (C, D) in CD4⁺ T cells from <i>Nfkbiz</i>^+/+ and <i>Nfkbiz</i>^−/− mice, cultured for 48 h under Th0 or Th17 conditions. (E) Diagram of <i>Il-17a</i> and <i>Il-17f</i> gene conservation. Red-arrows indicate the <i>Il-17a</i> promoter, the <i>Il-17f</i> promoter, and the CNS 1, CNS 2, and CNS 3 regions. (F) ChIP analysis of H3K27Ac. Cells were cultured under Th0 or Th17 conditions for 48 h. Data shown are from one experiment that was representative at three independent experiments. (A–D) Data shown represent mean ± S.E. (n = 3). Paired data were evaluated using the Student's t test. *<i>p</i><0.05, **<i>p</i><0.01.</p

Characteristics of immune homeostasis in <i>Nfkbiz</i>^−/− mice.

<p>(A, B) Naive CD4⁺ cells in the spleen and lymph nodes (LNs) of 8–12 week old <i>Nfkbid</i>^+/+ and <i>Nfkbiz</i>^−/− mice. (C, D) Flow cytometric analysis of IFN-γ- and IL-17-producing CD4⁺ cells isolated from the spleen (C) and LNs (D) of <i>Nfkbid</i>^+/+ and <i>Nfkbiz</i>^−/− mice at 8–12 weeks of age. Paired data were evaluated using the Student's t test. **p<0.01.</p

<i>Nfkbiz</i>^−/− T cells show decreased RORγt expression.

<p>RORγt expression in CD4⁺ T cells from <i>Nfkbiz</i>^+/+ and <i>Nfkbiz</i>^−/− mice, cultured for 72 h under Th0 or Th17 conditions. Data are representative of three independent experiments.</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-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

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

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

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

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

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