Expressions of Tight Junction Proteins Occludin and Claudin-1 Are under the Circadian Control in the Mouse Large Intestine: Implications in Intestinal Permeability and Susceptibility to Colitis

<div><p>Background & Aims</p><p>The circadian clock drives daily rhythms in behavior and physiology. A recent study suggests that intestinal permeability is also under control of the circadian clock. However, the precise mechanisms remain largely unknown. Because intestinal permeability depends on tight junction (TJ) that regulates the epithelial paracellular pathway, this study investigated whether the circadian clock regulates the expression levels of TJ proteins in the intestine.</p><p>Methods</p><p>The expression levels of TJ proteins in the large intestinal epithelium and colonic permeability were analyzed every 4, 6, or 12 hours between wild-type mice and mice with a mutation of a key clock gene Period2 (Per2; mPer2^m/m). In addition, the susceptibility to dextran sodium sulfate (DSS)-induced colitis was compared between wild-type mice and mPer2^m/m mice.</p><p>Results</p><p>The mRNA and protein expression levels of Occludin and Claudin-1 exhibited daily variations in the colonic epithelium in wild-type mice, whereas they were constitutively high in mPer2^m/m mice. Colonic permeability in wild-type mice exhibited daily variations, which was inversely associated with the expression levels of Occludin and Claudin-1 proteins, whereas it was constitutively low in mPer2^m/m mice. mPer2^m/m mice were more resistant to the colonic injury induced by DSS than wild-type mice.</p><p>Conclusions</p><p>Occludin and Claudin-1 expressions in the large intestine are under the circadian control, which is associated with temporal regulation of colonic permeability and also susceptibility to colitis.</p></div

Clock and Bmal1 bind to the E-box element in the promoter regions of Occludin and Claudin-1 genes and affect their transcriptional responses.

<p>(<b>A</b>) CHiP assays for recruitments of Clock and Bmal1 to E-box elements in Occludin and Claudin-1 promoter region using colonic IECs obtained at the indicated time points from wild-type mice and mPer2^m/m mice (n = 3 per group). Similar results were obtained in two independent experiments. (<b>B</b>) CHiP assays for recruitments of CLOCK and BMAL1 to E-box elements in Occludin and Claudin-1 promoter region using HCT116 cells (n = 3 per group). (<b>C</b>) Relative luciferase activity in HCT116 cells transfected with Occludin and Claudin-1 promoter reporter plasmids together with Clock and Bmal1 expression vectors or a control empty vector (n = 3 per group). *p<0.05.</p

Occludin and Claudin-1 expressions exhibit a time of day-dependent variation in colonic epithelium, relying on normal Per2 activity.

<p>(<b>A</b>) Real-time PCR analysis for Occludin, Zo-1, Claudin-1, -2, -3, -4, -7, and JAM mRNAs expression in the colon tissue samples obtained from the wild-type mice and mPer2^m/m mice at the indicated time points (n = 8 per group). (<b>B</b>) Representative pictures of Western blot for Occludin, Claudin-1 and β-actin expression in colonic epithelial cell samples from wild-type and mPer2^m/m mice obtained at the indicated time points (n = 3 per group). (<b>C</b>) The quantitative analysis of (<b>B</b>) (n = 3 per group). *p<0.05.</p

Colonic permeability is under the circadian control.

<p>(<b>A</b>) Permeability to evans blue <i>in vivo</i> at the indicated time points from wild-type mice and mPer2^m/m mice (n = 5 per group). (<b>B</b>) Permeability to FITC-dextran 4000 <i>ex vivo</i> obtained at the indicated time points from wild-type mice and mPer2^m/m mice (n = 6 per group). (<b>C</b>) Permeability to HRP <i>ex vivo</i> obtained at the indicated time points from wild-type mice and mPer2^m/m mice (n = 8 per group). *p<0.05.</p

Resveratrol inhibits OVA plus CT-induced Th1/2 cell differentiation <i>in vitro</i>.

<p>DO11.10 mice-derived splenocytes were un-stimulated or stimulated with 300 µg/ml OVA plus 12 pM CT and were cultured in the presence or absence of 10 or 30 µM resveratrol for the indicated times. Then the analysis described below was performed. A. After 3 day-culture, RNA samples were extracted from the cells and the cDNA samples were synthesized using reverse transcriptase system. Quantitative real-time PCR analysis was then done for T-bet, GATA3, and Foxp3 mRNAs. Relative expression levels are shown (n = 3 per group). B. After 3 day-culture, the supernatants were collected and IFN-ã, IL-4, and IL-13 concentrations were measured by ELISA (n = 9 per group). C. After 3 day-culture, the cells were subjected to a WST assay for evaluation of the cell viability (n = 4 per group). Values represent the mean ± SD. *P<0.05 in comparison to the corresponding controls. (ND: non-detected).</p

Resveratrol inhibits CT-induced cAMP elevation in BMDCs.

<p>A. Cultured BMDCs were un-stimulated or stimulated with 12 pM CT in the presence or absence of 10 or 30 µM resveratrol for 1 hour or 10 nM PGE2 (as a positive control) and intracellular cAMP levels were measured using a cAMP EIA kit. Relative changes in intracellular cAMP levels are indicated. Values represent the mean ± SD (n = 3 per group). B. Cultured BMDCs were un-stimulated (no treat) or stimulated with 300 µg/ml OVA plus 12 pM CT and were cultured in the presence or absence of 10 or 30 µM resveratrol for 3 days. The cells were then stained with APC-conjugated anti-CD11c and FITC-conjugated anti-CD80 or CD86 antibody and were subjected to FACS analysis. A representative histogram regarding surface CD80 and CD86 expression levels on CD11c gated cells is shown. Numbers indicate the mean fluorescence intensity (MFI) of CD80 and CD86-stained cells. The filled histogram indicates un-stimulated (no treat) BMDCs for comparison. Representative results from 3 independent experiments with similar results are shown.</p

Dietary resveratrol protects mice against a lethal dose of CT.

<p>Survival of 8-week-old C57BL/6 mice orally challenged with CT at a dose of 150 µg monitored until day 10 after challenge (n = 5 per group). Representative results from 2 independent experiments with similar results are shown. (S: standard diet-fed mice, S/R: standard diet plus resveratrol-fed mice).</p

Resveratrol inhibits CT-driven mucosal sensitization to OVA in mice.

<p>Mice were fed the standard diet or standard diet plus resveratrol (22.4 mg/kg diet, 0.01% resveratrol) for 5 weeks (day 0–35). The mice were orally administered 50 mg of OVA with 10 µg of CT 1 week after the start of resveratrol feeding and 4 times a week for 4 weeks and sacrificed at the 5 weeks (on day 35) for the analysis below. A. Experimental protocol. B. Serum samples were collected on day 35 from the standard diet-fed and standard diet plus resveratrol-fed mice with or without OVA plus CT sensitization. OVA-specific serum IgE concentrations were measured by ELISA (n = 7 per group). C. The standard diet-fed and standard diet plus resveratrol-fed mice sensitized with OVA plus CT were challenged intraperitoneally with OVA on day 35 and rectal temperatures were measured with a digital thermometer at 0, 5, 30, and 60 minutes after the challenge (n = 10 per group). D. Splenocytes (SPL) and mesenteric lymph node (MLN) cells were obtained on day 35 from the standard diet-fed and standard diet plus resveratrol-fed mice with or without OVA plus CT sensitization and the cells were re-stimulated <i>in vitro</i> with OVA for 3 days (n = 5 per group). The culture supernatants were then collected and IL-13 and IFN-ã concentrations were measured by ELISA. (S: standard diet-fed mice, S/R: standard diet plus resveratrol-fed mice) Values represent the mean ± SD. *P<0.05 in comparison to the corresponding controls. Representative results from 2 independent experiments with same results are shown.</p

Resveratrol inhibits early T cell activation and co-stimulatory molecule expression in APCs <i>in vitro</i>.

<p>DO11.10 mice-derived splenocytes were un-stimulated (no treat) or stimulated with 300 µg/ml OVA plus 12 pM CT and were cultured in the presence or absence of 10 or 30 µM resveratrol for 1 day. Then the analysis described below was performed. A. After 1 day-culture, whole splenocytes were stained with PE-conjugated anti-KJ1–26 and APC-conjugated anti-CD25 antibody. Representative FACS plots are shown. Numbers indicate the proportion of the KJ1–26⁺ CD25⁺ cells. B. Quantitative analysis of A (n = 3 per group). C. After 1 day-culture, the supernatants were collected and IL-2 concentrations were measured by ELISA (n = 9 per group). D. After 1 day-culture, RNA samples were extracted from the splenocytes and the cDNA samples were synthesized using reverse transcriptase system. Quantitative real-time PCR analysis was then performed to assess CD80 and CD86 mRNA levels. Relative expression levels are shown (n = 3 per group). Values represent the mean ± SD. *P<0.05 in comparison to the corresponding controls. (ND: non-detected).</p