1. Volume 140, Issue 1, Pages 275-285.e3 (January 2011)
Transient Receptor Potential Vanilloid 4 Activated Inflammatory Signals by Intestinal Epithelial Cells and Colitis in Mice 
Emilie D'Aldebert, Nicolas Cenac, Perrine Rousset, Laurence Martin, Corinne Rolland, Kevin Chapman, Janick Selves, Laurent Alric, Jean–Pierre Vinel, Nathalie Vergnolle 
Gastroenterology 
Volume 140, Issue 1, Pages e3 (January 2011)
DOI: /j.gastro
Copyright © 2011 AGA Institute Terms and Conditions

2. Figure 1
TRPV4 expression in human intestinal epithelial cells. TRPV4 expression was studied by immunohistochemistry. Human colonic resections from control (non-IBD) patients (upper panels) or patients with IBD (middle panels for tissues from patients with ulcerative colitis [UC] and lower panels for tissues from patients with Crohn's disease [CD]) were stained with (A) anti-TRPV4 antibody (green) and anti–cytokeratin 18 antibody (red), (B) anti-TRPV4 antibody (green) and anti-S100 antibody (red), and (C) anti-TRPV4 antibody (green) and anti-CD45 (red). Cytokeratin 18 was used as an epithelial cell marker, S100 as a glial cell marker, and CD45 as an immune cell marker. Arrows in B and C show colocalization of cellular marker and TRPV4. Selected images are representative of n = 5 patients in each group.
Gastroenterology  , e3DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

3. Figure 2
Basolateral expression of TRPV4 in Caco-2 cells. TRPV4 localization was studied by immunohistochemistry and confocal microscopy. Caco-2 cells were stained with anti-TRPV4 antibody (green), anti-occludin (red), and nucleus with TOPRO-3 (blue). Occludin was used as a tight junction cell marker. (A) Apical confocal z plane. (B) Nucleus confocal z plane. (C) Basolateral confocal z plane. a, apical side; bl, basolateral side.
Gastroenterology  , e3DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

4. Figure 3
Up-regulation of TRPV4 in colon of DSS-treated mice. (A) mRNA was extracted from colonic tissue of control mice (white bar) or DSS-treated (3%) mice (black bar). TRPV4 mRNA detection was performed by real-time quantitative reverse-transcription polymerase chain reaction. Data are expressed as fold increases over control normalized by HPRT. Data represent means ± SEM. **P < .01, DSS vs control by a Mann–Whitney test. (B) TRPV4 expression was studied by immunohistochemistry and confocal microscopy. Mouse colonic slides from control tissues or DSS (3%) tissues were stained with anti-TRPV4 antibody (green) and anti–cytokeratin 18 antibody (red) (first and second row of panels) or (C) slides were stained with anti-TRPV4 antibody (green) and anti-CD45 antibody (red) (third and fourth row of panels). Original magnification 40×. Cytokeratin 18 was used as an epithelial cell marker and CD45 as a leukocyte marker.
Gastroenterology  , e3DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

5. Figure 4
TRPV4 activation in Caco-2 and T84 cells. (A and C) Caco-2 cells and (B) T84 cells were stimulated with 1, 10, 50, or 100 μmol/L 4αPDD or vehicle (0.6% dimethyl sulfoxide). (A) Stimulation was performed with (+ calcium) or without (− calcium) calcium in extracellular milieu. (C) Caco-2 cells pretreated with siRNA TRPV4 (light gray bar), control siRNA (white bar) or ruthenium red 10 μmol/L (medium gray bar), or 50 μmol/L (dark gray bar) were stimulated with 4αPDD (100 μmol/L). Intracellular calcium mobilization (nmol/L) was measured with the probe fluo-3-AM immediately after stimulation. Cellular response to 100 μmol/L 4αPDD was considered as 100% response (measured on n = 12 experiments); in the other conditions of incubation, cellular response was expressed as percent of inhibition of this 100% response to 4αPDD. Data represent means ± SEM. *P < .05, **P < .01, ***P < .005 treated vs vehicle tested by one-way analysis of variance.
Gastroenterology  , e3DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

6. Figure 5
TRPV4 agonist induced chemokine released by Caco-2 and T84 cells. (A, C, and D) Caco-2 cells and (B) T84 cells were treated with 100 μmol/L 4αPDD or with vehicle solution containing 0.6% dimethyl sulfoxide, and time-dependent chemokine/cytokine release was measured (A and B). (C) Dose-dependent release of IL-8 protein (black bars) and IL-8 mRNA expression (white bars) in response to 4αPDD. (D) IL-8 protein release in response to 10 (white bars) or 50 (black bars) μmol/L 4αPDD in Caco-2 cells incubated with TRPV4-directed siRNA (T) or control siRNA (C), the TRPV4 antagonist ruthenium red (10 μmol/L), the NFAT inhibitor VIVIT (5 or 10 μmol/L), a nuclear factor κB inhibitor (5 or 10 μmol/L), or the AP-1 inhibitor (SR11302; 10 μmol/L). IL-8 release in response to 4αPDD in the absence of any compound was considered as the 100% response (measured on n = 8 experiments); then, in the presence of those different compounds, IL-8 release was expressed as percent of inhibition of this 100% response to 4αPDD (D). Data represent means ± SEM. *P < .05, **P < .01, ***P < .005 treated vs corresponding control, tested by one-way analysis of variance.
Gastroenterology  , e3DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

7. Figure 6
TRPV4 agonist in the colon caused colonic inflammation. Mice were killed 3, 6, or 24 hours after intracolonic administration of 200 μg 4αPDD in 40% ethanol (black bars) or vehicle solution (PBS plus 40% ethanol) (white bars). In these colonic tissues, (A) macroscopic damage score (arbitrary unit) and (B) microscopic damage score (arbitrary unit) were evaluated. (C) Intestinal tissue samples stained with H&E from vehicle, treated mice (3 hours) (left panel) or 4αPDD-treated mice (3 hours) (right panel) were observed. White arrows represent submucosal edema and cellular infiltration. (D) MPO activity (unit MPO/mg tissue) was measured in these colonic tissues. (E) Three hours after intracolonic administration of 200 μg 4αPDD in 40% ethanol plus PBS (black bar) or vehicle solution containing 40% ethanol plus PBS (white bar), passage of labeled 51Cr-EDTA from the lumen to the blood was assessed. Data represent means (cpm/mL) ± SEM. ***P < .005 TRPV4 agonist treated vs vehicle treated, using a t test. (F) Mice were killed 3, 6, or 24 hours (white bars, gray bars, and black bars, respectively) after intracolonic administration of 200 μg 4αPDD in 40% ethanol plus PBS or vehicle solution containing 40% ethanol plus PBS, and chemokine or cytokine (IL-6, KC, MCP-1, and RANTES) release in colonic tissues was measured. Data are expressed as the ratio of chemokines or cytokines measured in 4αPDD-treated groups vs chemokines or cytokines measured in vehicle-treated group. Data represent means ± SEM. *P < .05, **P < .01, ***P < .005 TRPV4 agonist treated vs vehicle treated and tested using a one-way analysis of variance.
Gastroenterology  , e3DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

8. Figure 7
Repeated administration of TRPV4 agonist caused chronic colonic inflammation. Changes in (A) diarrhea, (B) colon thickness, (C) macroscopic damage score, and (D) MPO activity were observed in naïve mice (dark gray bars or black circles) or in mice that have received repeated administration (every day for 7 days) of 4αPDD (200 μg/mice, black bars or black triangles) or its vehicle (light gray bars or white triangles) or DSS treatment (3% in drinking water, white bars or white squares). Data represent means ± SEM. *P < .05, **P < .01, ***P < .005 treated vs corresponding control, tested using a one-way analysis of variance.
Gastroenterology  , e3DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

9. Supplementary Figure 1
4αPDD-induced calcium flux in HEK-FLPTREX-TRPV4 cells. HEK cells expressing TRPV4 were provided by Dr Nigel Bunnett and Dr Graham Cottrell. HEK cells were transiently transfected with TRPV4 using Lipofectamine 2000 (Invitrogen) and designated HEK-TRPV4 cells. A tetracycline-inducible system was used to generate stable cell lines expressing TRPV4, because continuous overexpression of this channel was toxic to the cells. HEK-FLPTREX cells were stably transfected with pcDNA5/FRT/TO plus hTRPV4 (designated HEK-FLPTREX-TRPV4 cells) and grown in Dulbecco's modified Eagle medium containing 10% tetracycline-free fetal bovine serum, hygromycin (50 μg/mL−1), and blasticidin (5 μg/mL−1). Calcium flux studies on these cells were performed as described in the report. To induce TRPV4 expression, tetracycline (0.1 μg/mL−1) was added to the medium 16 hours before use. Treatment of cells with 4αPDD (100 μmol/L) increased intracellular calcium concentration in TRPV4-transfected but not in nontransfected HEK cells. Dimethyl sulfoxide has no effect on calcium influx in both cell types. **P < .01 treated vs corresponding control, tested using a one-way analysis of variance.
Gastroenterology  , e3DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

10. Supplementary Figure 2
4αPDD induced an increase in IL-8 mRNA in Caco-2 cells. Total mRNA was extracted from Caco-2 cells using the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. As described in the report, after reverse transcription, quantitative real-time polymerase chain reaction was performed using the LightCycler 480 SYBR Green I Master (Roche). The primers used in real-time polymerase chain reaction were the following: forward 5′-TGGGAGGCCATCACATTGT-3′ and reverse 5′-TCCAG CAGGTCAGCAAAGAA-3′ for human HPRT (Invitrogen), and forward 5′-GCCTTCCTGATTTCTGCAGCT-3′ and reverse 5′-TGCACTGACATCTAAGTTCT TTAGCAC-3′ for human IL-8 (Invitrogen). Quantitative increase of IL-8 mRNA induced by 4αPDD (50 μmol/L) was decreased by treatment of Caco-2 cells with AP-1 (SR11302; 5 and 10 μmol/L) or nuclear factor κB (5 and 10 μmol/L) inhibitor, as well as ruthenium red (10 μmol/L). NFAT inhibitor (VIVIT; 10 μmol/L) had no effect on IL-8 mRNA expression in response to 4αPDD (50 μmol/L). These results are in complete accord with what was observed at the protein level (Figure 5D). *P < .05, **P < .01 treated vs corresponding control, tested using a one-way analysis of variance.
Gastroenterology  , e3DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

11. Supplementary Figure 3
Capsaicin treatment had no effect on 4αPDD-induced inflammation. Capsaicin (50 mg/kg subcutaneously) or its vehicle (80% saline, 10% ethanol, 10% Tween 80) was administered 7 days before intracolonic administration of 4αPDD (200 μg/mouse), and the efficacy of capsaicin pretreatment to ablate sensory nerves was tested just before the animals were killed, as previously described.1 MPO activity and damage score were quantified as described in the report. No difference was observed between the capsaicin-treated group and its vehicle. *P < .05, ***P < .005 treated vs corresponding control, tested using a one-way analysis of variance.
Gastroenterology  , e3DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions