Gastroenterology

Gastroenterology

Volume 141, Issue 4, October 2011, Pages 1346-1358
Gastroenterology

Original Research
Basic and Translational—Alimentary Tract
TRPA1 and Substance P Mediate Colitis in Mice

https://doi.org/10.1053/j.gastro.2011.07.002Get rights and content

Background & Aims

The neuropeptides calcitonin gene-related peptide (CGRP) and substance P, and calcium channels, which control their release from extrinsic sensory neurons, have important roles in experimental colitis. We investigated the mechanisms of colitis in 2 different models, the involvement of the irritant receptor transient receptor potential of the ankyrin type-1 (TRPA1), and the effects of CGRP and substance P.

Methods

We used calcium-imaging, patch-clamp, and neuropeptide-release assays to evaluate the effects of 2,4,6-trinitrobenzene-sulfonic-acid (TNBS) and dextran-sulfate-sodium-salt on neurons. Colitis was induced in wild-type, knockout, and desensitized mice.

Results

TNBS induced TRPA1-dependent release of colonic substance P and CGRP, influx of Ca2+, and sustained ionic inward currents in colonic sensory neurons and transfected HEK293t cells. Analysis of mutant forms of TRPA1 revealed that TNBS bound covalently to cysteine (and lysine) residues in the cytoplasmic N-terminus. A stable sulfinic acid transformation of the cysteine-SH group, shown by mass spectrometry, might contribute to sustained sensitization of TRPA1. Mice with colitis had increased colonic neuropeptide release, mediated by TRPA1. Endogenous products of inflammatory lipid peroxidation also induced TRPA1-dependent release of colonic neuropeptides; levels of 4-hydroxy-trans-2-nonenal increased in each model of colitis. Colitis induction by TNBS or dextran-sulfate-sodium-salt was inhibited or reduced in TRPA1−/− mice and by 2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopro-pylphenyl)-acetamide, a pharmacologic inhibitor of TRPA1. Substance P had a proinflammatory effect that was dominant over CGRP, based on studies of knockout mice. Ablation of extrinsic sensory neurons prevented or attenuated TNBS-induced release of neuropeptides and both forms of colitis.

Conclusions

Neuroimmune interactions control intestinal inflammation. Activation and sensitization of TRPA1 and release of substance P induce and maintain colitis in mice.

Section snippets

Animals

Mice were killed in a 100% CO2 atmosphere (approved by the Animal Protection Authority, District Government Mittelfranken, Ansbach, Germany). Initial breeding pairs of TRPV1+/– and TRPA1+/– mice were donated by Dr Davis (Glaxo Smith Kline, Harlow, UK)36 and Drs Kwan and Corey (Harvard, Boston, MA),37 respectively, and CGRP+/− and SP+/− mice were donated by Dr Zimmer (University Bonn, Bonn, Germany). For details of mouse breeding and genotyping see the Supplementary Material and Methods.

Release of CGRP and SP

The

TNBS Activates TRPA1 in Sensory Neurons

Whether TNBS directly activates colonic sensory nerves and functions through the irritant receptor TRPA1 to release neuropeptides was investigated using an established ex vivo mouse colon preparation.38 TNBS induced CGRP and SP release in a concentration-dependent manner, similar to the established TRPA1 agonist AITC-induced CGRP release (Figure 1A and B). TNBS-induced (1 mmol/L) CGRP release was abolished in a calcium-free extracellular solution (Figure 1C), indicating a physiological

Discussion

We identified a TRPA1- and SP-dependent sensory neuronal mechanism of TNBS and DSS inducing colitis in mice. TRPA1 receptor and SP expression in extrinsic primary afferent neurons was required for the development of TNBS colitis, and a pharmacologic block of TRPA1 improved chronic colitis. In addition, primary chronic DSS colitis was ameliorated in TRPA1−/− and in WT littermates treated with the selective TRPA1 antagonist HC-030031, it was abrogated in SP−/− but aggravated in CGRP−/− mice.

Conclusions

Along with our results, evidence is growing for the etiologic relevance of neuroimmune interactions in colitis. The present study expands the established concept of hapten-induced TNBS and cytotoxic DSS colitis by sensory neuronal factors that finally lead to a loss in immune tolerance. Sensitization of the TRPA1 receptor by endogenous mediators of inflammation and metabolites of oxidative stress, which lead to increased SP release, may play a decisive role in the pathogenesis of human

Acknowledgments

The authors thank A. Kuhn and S. Haux-Oertel for technical assistance, J. Schramm and R. Fischer for excellent maintenance of animal breeding, and D. Metzner for graphic assistance (Institute of Physiology and Pathophysiology). The authors also thank K. Loeschner, H. Symowski, and A. Hecht for preparing the histologic slides (Institute of Anatomy I).

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    Conflicts of interest The authors disclose no conflicts.

    Funding Peter Reeh and Matthias Engel were supported by the Federal Ministry of Education and Res. (BMBF0315449C); Matthias Engel received additional support from the Marohn-Stiftung of the Friedrich-Alexander-Universität Erlangen-Nürnberg; Alexandru Babes was supported by grant PN2 Idei 164/2007 from the Romanian Government and by a visiting scientist grant from the Humboldt Foundation; and the chemical analytic work by Miloš Filipović and Ivana Ivanović–Burmazović was supported by the Deutsche Forschungsgemeinschaft.

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