Complex Regulatory Role of the TRPA1 Receptor in Acute and Chronic Airway Inflammation Mouse Models

The Transient Receptor Potential Ankyrin 1 (TRPA1) cation channel expressed on capsaicin-sensitive afferents, immune and endothelial cells is activated by inflammatory mediators and exogenous irritants, e.g., endotoxins, nicotine, crotonaldehyde and acrolein. We investigated its involvement in acute and chronic pulmonary inflammation using Trpa1 gene-deleted (Trpa1-/-) mice. Acute pneumonitis was evoked by intranasal Escherichia coli endotoxin (lipopolysaccharide: LPS) administration, chronic bronchitis by daily cigarette smoke exposure (CSE) for 4 months. Frequency, peak inspiratory/expiratory flows, minute ventilation determined by unrestrained whole-body plethysmography were significantly greater, while tidal volume, inspiratory/expiratory/relaxation times were smaller in Trpa1-/- mice. LPS-induced bronchial hyperreactivity, myeloperoxidase activity, frequency-decrease were significantly greater in Trpa1-/- mice. CSE significantly decreased tidal volume, minute ventilation, peak inspiratory/expiratory flows in wildtypes, but not in Trpa1-/- mice. CSE remarkably increased the mean linear intercept (histopathology), as an emphysema indicator after 2 months in wildtypes, but only after 4 months in Trpa1-/- mice. Semiquantitative histopathological scores were not different between strains in either models. TRPA1 has a complex role in basal airway function regulation and inflammatory mechanisms. It protects against LPS-induced acute pneumonitis and hyperresponsiveness, but is required for CSE-evoked emphysema and respiratory deterioration. Further research is needed to determine TRPA1 as a potential pharmacological target in the lung

Upregulation of the Transient Receptor Potential Ankyrin 1 Ion Channel in the Inflamed Human and Mouse Colon and Its Protective Roles

<div><p>Transient Receptor Potential Ankyrin 1 (TRPA1) channels are localized on sensory nerves and several non-neural cells, but data on their functional significance are contradictory. We analysed the presence and alterations of TRPA1 in comparison with TRP Vanilloid 1 (TRPV1) at mRNA and protein levels in human and mouse intact and inflamed colons. The role of TRPA1 in a colitis model was investigated using gene-deficient mice. TRPA1 and TRPV1 expressions were investigated in human colon biopsies of healthy subjects and patients with inflammatory bowel diseases (IBD: ulcerative colitis, Crohn's disease) with quantitative PCR and immunohistochemistry. Mouse colitis was induced by oral 2% dextran-sulphate (DSS) for 10 days. For investigating the functions of TRPA1, Disease Activity Index (weight loss, stool consistency, blood content) was determined in C57BL/6-based Trpa1-deficient (knockout: KO) and wildtype (WT) mice. Sensory neuropeptides, their receptors, and inflammatory cytokines/chemokines were determined with qPCR or Luminex. In human and mouse colons TRPA1 and TRPV1 are located on epithelial cells, macrophages, enteric ganglia. Significant upregulation of TRPA1 mRNA was detected in inflamed samples. In Trpa1 KO mice, Disease Activity Index was significantly higher compared to WTs. It could be explained by the greater levels of substance P, neurokinins A and B, neurokinin 1 receptor, pituitary adenylate-cyclase activating polypeptide, vasoactive intestinal polypeptide, and also interleukin-1beta, macrophage chemoattractant protein-1, monokine induced by gamma interferon-1, tumor necrosis factor-alpha and B-lymphocyte chemoattractant in the distal colon. TRPA1 is upregulated in colitis and its activation exerts protective roles by decreasing the expressions of several proinflammatory neuropeptides, cytokines and chemokines.</p></div

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.

<p>Insert: KP1 (anti-CD68) antibody-labelled macrophages. Asterix: colon lumen. Arrows: <i>a:</i> weak immunopositivity on mucosal epithelial cells; <i>b:</i> remarkable immunopositivity around mucosal nerves and blood vessels; <i>c:</i> myenteric plexus nerve fibers; <i>d:</i> myenteric plexus ganglia; <i>e</i>: submucous plexus. <i>g:</i> infiltrating immune cells; <i>h:</i> marked immunopositivity on inflammatory leukocytes. <i>i:</i> interstitial plasma cells in the submucosa.</p

Mouse genes detected by the Quantigene 2.0 Plex RNA Assay in distal colon homogenates of DSS-treated WT and TRPA1 KO mice.

<p>Mouse genes detected by the Quantigene 2.0 Plex RNA Assay in distal colon homogenates of DSS-treated WT and TRPA1 KO mice.</p

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.

<p>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).</p

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.

<p>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).</p

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).

<p>Panels <b>E</b> and <b>F</b> 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).</p