Table 1.
Human and mouse assays used in qPCR detection.
Table 2.
Mouse genes detected by the Quantigene 2.0 Plex RNA Assay in distal colon homogenates of DSS-treated WT and TRPA1 KO mice.
Figure 1.
Transient Receptor Potential Ankyrin 1 (Trpa1) and Vanilloid 1 (Trpv1) gene expressions determined by (A, C) qPCR and (B, D) RNA assay in the distal colon samples of intact mice (water-drinking, non-inflamed), as well as after 3, 7 and 10 days of dextran-sulphate (DSS) administration (inflamed; n = 3-5/group).
Panels E and F demonstrate TRPA1 and TRPV1 expression in human colon biopsies (non-inflamed n = 5, tumor n = 8, active inflammatory bowel diseases/IBD+ n = 5, inactive phase of colitis/IBD- n = 5). Columns represent means±SEM, *p<0.05 vs. respective non-inflamed control (Mann-Whitney U test for the murine samples, Kruskal-Wallis and Dunn's post test for the human samples).
Figure 2.
Representative immunohistochemical pictures of (A) TRPA1 and (B) TRPV1 labelling of intact, non-inflamed distal colon sections of C57Bl/6 mice drinking water or inflamed tissues of mice receiving dextran-sulphate (DSS) for 7 days.
Insert: KP1 (anti-CD68) antibody-labelled macrophages. Asterix: colon lumen. Arrows: a: weak immunopositivity on mucosal epithelial cells; b: remarkable immunopositivity around mucosal nerves and blood vessels; c: myenteric plexus nerve fibers; d: myenteric plexus ganglia; e: submucous plexus. g: infiltrating immune cells; h: marked immunopositivity on inflammatory leukocytes. i: interstitial plasma cells in the submucosa.
Figure 3.
Representative photomicrographs of (A, B, C) TRPA1 and (D, E, F) TRPV1 immunohistochemically labeled sections of colon biopsies from control patients with non-inflamed colon, colitis ulcerosa and active Crohn's disease.
Arrowhead: crypt epithelial cells. Arrows: a: granulated gland cells corresponding to neuroendocrine cells; b: Paneth cells. c: interstitial macrophages. d: infiltrating plasma cells and macrophages.
Figure 4.
Disease Activity Index of mice calculated daily on the basis of body weight, stool consistency and fecal blood content (see Table 1) (A) shown every day and (B) as areas under curve during the total 10-day-experiment.
Wildtype (WT) and TRPA1 knock-out (KO) mice were orally administered 2% dextran-sulfate (DSS) and compared to intact, water-consuming control animals. Data points represent means±SEM (n = 14-15). In panel A **p<0.01 and *p<0.05, KO DSS vs. WT DSS (two-way ANOVA with Bonferroni's post-test); effect of genotype (column factor) p<0.0001; in panel B *p<0.05 (Mann-Whitney U test).
Figure 5.
Representative light micrographs of distal colon samples of (A) water-treated non-inflamed wildtype (WT) and TRPA1-deficient (Trpa1 KO) mice, as well as after 10 days of dextran-sulphate (DSS) drinking.
Sections are stained with haematoxylin-eosin. Arrows show a) intact crypts, b) damaged crypt, c) mucosal neutrophil infiltration, d) submucosal neutrophil infiltration, asterisk shows the colon lumen. Magnification: 100×; inserts: 400×. Panel (B) demonstrates the box plots of the semiquantitative histopathological scores of distal colon sections of water-treated non-inflamed wildtype (WT) and TRPA1-deficient (Trpa1 KO) mice, as well as after 3, 7 and 10 days of dextran-sulphate (DSS).
Figure 6.
Gene expression profiles of inflammatory cytokines, such as (A) TNF-alpha, (B) BLC, (C) M-CSF, (D) IL-1rn in the distal colon homogenates of water-treated non-inflamed wildtype (WT) and TRPA1-deficient (Trpa1 KO) mice, as well as after 3, 7 and 10 days of dextran-sulphate (DSS) determined by quantitative PCR.
Columns represent means±SEM. *p<0.05, **p<0.01 vs. respective water-receiving control; #p<0.05, #p<0.01 WT vs. KO (n = 3-5/group, Mann-Whitney U test).
Figure 7.
Protein expression profiles of inflammatory cytokines, such as (A) IL-1b, (B) MCP-1, (B) RANTES, and (C) MIG in the distal colon homogenates of water-treated non-inflamed wildtype (WT) and TRPA1-deficient (Trpa1 KO) mice, as well as after 3, 7 and 10 days of dextran-sulphate (DSS) determined by multiplex bead array.
Columns represent means±SEM. *p<0.05, **p<0.01 vs. respective water-receiving control; #p<0.05, #p<0.01 WT vs. KO (n = 3-5/group, Mann-Whitney U test).
Figure 8.
Gene expression profiles of (A) TRPV1 receptor, sensory neuropeptides, such as (B) PACAP, (C) VIP, and their receptors (D) PAC1R, (E) VIPR1, (F) VIPR2, as well as another sensory neuropeptide (G) somatostatin and its receptors (H) SSTR1, (I) SSTR4, and tachykinins (J) TAC 1, (K) TAC 3, as well as their receptor NK1.
Gene expression was determined in the distal colon homogenates of water-treated non-inflamed wildtype (WT) and TRPA1-deficient (Trpa1 KO) mice, as well as after 3, 7 and 10 days of dextran-sulphate (DSS) determined by multiplex bead array. Columns represent means±SEM. *p<0.05, **p<0.01 vs. respective water-receiving control; #p<0.05, #p<0.01 WT vs. KO (n = 3-5/group, Mann-Whitney U test).