TRPA1 and Substance P Mediate Colitis in Mice

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TRPA1 and Substance P Mediate Colitis in Mice MATTHIAS A. ENGEL,*,‡ ANDREAS LEFFLER,§,储 FLORIAN NIEDERMIRTL,§ ALEXANDRU BABES,¶ KATHARINA ZIMMERMANN,* MILOŠ R. FILIPOVIC´,# IWONA IZYDORCZYK,* MIRJAM EBERHARDT,* TATJANA I. KICHKO,* SONJA M. MUELLER–TRIBBENSEE,* MOHAMMAD KHALIL,* NORBERT SIKLOSI,* CARLA NAU,§ IVANA IVANOVIC´–BURMAZOVIC´,# WINFRIED L. NEUHUBER,** CHRISTOPH BECKER,‡ MARKUS F. NEURATH,‡ and PETER W. REEH* *Institute of Physiology and Pathophysiology, ‡First Department of Medicine, §Department of Anesthesiology, #Department of Chemistry and Pharmacy, **Institute of Anatomy I, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.储Department of Anesthesiology and Intensive Care Medicine, Hannover Medical School, Hannover, Germany; and ¶Department of Animal Physiology and Biophysics, Faculty of Biology, University of Bucharest, Bucharest, Romania

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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 calciumimaging, 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-hydroxytrans-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-isopropylphenyl)-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. Keywords: Inflammatory Bowel Diseases; Nervous; Immune System; 4-HNE.

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nimal models of intestinal inflammation are essential to gain a better understanding of the pathogenesis of human inflammatory bowel diseases. Intracolonic instil-

lation of 2,4,6-trinitrobenzene-sulfonic-acid (TNBS) or peroral administration of dextran-sulfate-sodium-salt (DSS) are used most commonly for the induction of intestinal inflammation in rodents.1 TNBS induces an acute and chronic form of colitis, resembling Crohn’s disease, by haptenizing colonic autologous or microbiotic proteins and rendering them immunogenic to the host immune system, whereas DSS induces primary chronic colitis through direct toxic effects on colonic epithelial cells resembling human ulcerative colitis.1– 4 The autoinflammatory reaction in both models is not well understood, however, several reports have suggested that primary sensory neurons, definitely those expressing the capsaicin receptor transient receptor potential vanilloid subtype 1 (TRPV1), are crucial for the induction and propagation of the aberrant immune response in colitis.5–11 Activation of these sensory neurons is accompanied by the release of the neuropeptides calcitonin gene related peptide (CGRP) and substance P (SP), which may function as mediators at the interface between the nervous and immune systems. CGRP and SP induce neurogenic inflammation indicated by vasodilatation, plasma extravasation, and leukocyte migration, and they also have been reported to activate immune cells.12–16 However, with regard to colitis, opposite effects of the neuropeptides, largely derived from pharmacologic experiments, have been published on proinflammatory and anti-inflammatory roles of SP and CGRP, respectively, and there are controversial reports about the receptor channels that control their release from sensory neurons.5–11,17–23 The other irritant receptor transient receptor potential of the ankyrin type-1 (TRPA1) is expressed in a subset of Abbreviations used in this paper: AITC, allyl isothiocyanate; CGRP, calcitonin gene-related peptide; Cys, cysteine; DiI, 1,1=-dioctadecyl3,3,3=,3=-tetramethylindocarbocyanine perchlorate; DRG, dorsal root ganglion; DSS, dextran-sulfate-sodium-salt; HC-030031, 2-(1, 3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7Hpurin-7-yl)-N-(4-isopropylphenyl)-acetamide; IP, intraperitoneally; hTRPA1-3C, human TRPA1C621S/C641S/C665S; MPN, myenteric plexus neuron; RTX, resiniferatoxin; SP, substance P; TNBS, 2,4,6-trinitrobenzene-sulfonic-acid; TRPA1, transient receptor potential of the ankyrin type-1; TRPV, transient receptor potential vanilloid subtype; WT, wild-type. © 2011 by the AGA Institute 0016-5085/$36.00 doi:10.1053/j.gastro.2011.07.002

TRPV1-positive sensory neurons,24 and is ascribed a key role in visceral mechanosensation and inflammatory hyperalgesia.25,26 TRPA1 has emerged as the principle detector of numerous, in particular electrophilic, compounds that form adducts with thiols and primary amines and activate TRPA1 by reversible covalent modification of cysteine (Cys) residues within the cytoplasmic N-terminus of the channel.27,28 In analytic chemistry TNBS is used to quantify protein solutions because of its ability to bind to primary amines.29 Trinitrophenol, a compound structurally related to TNBS, activates recombinant TRPA1 in vitro.30 The TRPA1 agonists allyl isothiocyanate (AITC) and formalin enemas have been shown to induce colitis in rodents.31,32 Thus, we hypothesized that TNBS also may activate TRPA1 in sensory neurons with subsequent neuropeptide release, and this may be crucial for inducing TNBS colitis. Moreover, because TRPA1 also is activated by endogenous inflammatory mediators33,34 and by oxidative stress metabolites,35 we further investigated whether the activation of TRPA1/neuropeptide release could play a role in maintenance of the inflammatory process in chronic colitis. We established the novel role of TRPA1 in TNBS and DSS colitis and identified the dominant proinflammatory role of SP over the protective effects of CGRP.

Materials and Methods 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 isolated mouse colon was prepared ex vivo as described previously.38,39 Details of the commercial CGRP and SP assay kit (Bertin Pharma, Montigny le Bretonneux, France) and its validation have been described previously.12 Neuropeptide release measurements from isolated dorsal root ganglion (DRG) neurons and colonic myenteric plexus neurons (MPNs) were performed as previously described (Supplementary Material and Methods).40

Primary Culture of Mouse DRGs and MPNs Mouse thoracolumbosacral DRG neurons were cultured as described previously,41 using nerve growth factor (100 ng/mL mouse NGF 2.5 S; Alomone Labs, Jerusalem, Israel). Mouse colon from newborn C57BL/6 mice (4 weeks of age) were used to isolate MPNs as described previously (Supplementary Material and Methods).42

Ratiometric [Ca2ⴙ]i Measurements Ratiometric [Ca2⫹]i measurements on cultured DRG neurons using Fura-2 (5 ␮mol/L) were essentially performed as

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recently published.43 See the Supplementary Material and Methods for analysis and protocol details.

Patch Clamp Recordings Whole-cell recordings in voltage-clamp mode were performed on mouse DRG neurons and transfected HEK-293t cells as recently published.41 Cells were held at ⫺60 mV at room temperature and only one cell was used for recording from each dish.

Depletion of Sensory Nerves by Resiniferatoxin Treatment with resiniferatoxin (RTX), an ultrapotent TRPV1 agonist, was performed according to an established protocol of cumulative subcutaneous injections (Supplementary Material and Methods).44

Induction of Colitis TNBS colitis. TNBS colitis was induced by instillation of a single enema of 200 mmol/L TNBS (Sigma-Aldrich, Taufkirchen, Germany) in 50% ethanol and water in TRPA1⫺/⫺ and wild-type (WT) littermates as previously described.45 A high concentration of TNBS was used because C57BL/6 strain is less susceptible to TNBS colitis46 and high TNBS concentrations induce colitis for several weeks.2 Mice were killed and examined 3 days (acute) or 3 weeks (chronic) after TNBS administration. Treatment with the TRPA1 antagonist 2-(1,3-dimethyl-2,6dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopro-pylphenyl)acetamide (HC-030031) (300 mg/kg, IP, once daily; Tocris, Bristol, UK) was performed for the first 3 days or throughout the last chronic week. DSS colitis. DSS colitis was induced by adding 2% DSS (molecular weight, 36,000 –50,000) (MP Biomedicals, Illkirch, France) to the drinking water as described previously.1 Treatment with HC (300 mg/kg, IP, once daily) was performed throughout the DSS period, starting 1 hour before DSS administration. Mouse body weight was monitored daily. To assess colitis severity, a combined sum score was used that recently was introduced and proven sensitive to experimental therapy (Supplementary Tables 1–3).45

HNE-Adduct Enzyme-Linked Immunosorbent Assay Measurements Levels of colonic 4-HNE– histidine protein adducts (pg/mL supernatant) were measured by enzyme-linked immunosorbent assay using a commercial kit (Cell Biolaboratories, San Diego, CA) (Supplementary Material and Methods).

Results 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 mechanism for calcium influx-dependent vesicular exocytosis.47 We used gene knockout mice to establish a

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molecular target for TNBS action. TNBS-induced CGRP release was abrogated in the colon of TRPA1⫺/⫺ but not TRPV1⫺/⫺ mice (Figure 1D). TRPA1-mediated TNBS effect was confirmed pharmacologically by co-application of the selective TRPA1 antagonist HC (50 ␮mol/L), which abolished TNBS-induced colonic CGRP release (Figure 1E). To characterize the specific sensory neurons activated by TNBS, ratiometric [Ca2⫹]i measurements in cultured DRG neurons were performed (Figure 2). We labeled colon-specific sensory neurons using intracolonic injections of the retrograde tracer DiI (Figure 2A). Representative intracellular calcium transients in response to consecutive administrations of TNBS (1 mmol/L), AITC (100 ␮mol/ L), capsaicin (300 nmol/L), and KCl (60 mmol/L) are shown in Figure 2B, obtained from colonic DRG neurons of TRPA1⫹/⫹ (n ⫽ 103) and TRPA1⫺/⫺ (n ⫽ 134) mice. Forty of 103 (39%) colonic DRG neurons from TRPA1⫹/⫹ mice were TNBS (1 mmol/L)-sensitive, whereas all of these neurons responded when exposed to AITC (100 ␮mol/L) and capsaicin (300 nmol/L) (Figure 2C, left). Complete overlap of AITC- and TNBS-positive cells was absent because the effective concentration of TNBS was lower than that of AITC, as shown in the concentration-response curves of the neuropeptide release (Figure 1A and B). Application of TNBS at 10 mmol/L resulted in an almost complete overlap of AITC (100 ␮mol/L) and TNBS (10 mmol/L) responsiveness (n ⫽ 157) (Figure 2C, right). Less than 1% of colonic DRGs from TRPA1⫺/⫺ mice showed small TNBS (at 1 mmol/L [n ⫽ 134] and 10 mmol/L [n ⫽ 182]) or AITC responses, and 56% were capsaicin-positive (data not shown). An alternative sequence of capsaicin

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(300 nmol/L), AITC (100 ␮mol/L), TNBS (10 mmol/L), and KCl (60 mmol/L) resulted in a similar pattern of calcium transients and responsive cell fractions in colonic DRGs from TRPA1 WT mice (n ⫽ 114) (Supplementary Figure 1A and B). When using the conventional whole-cell patch-clamp technique, we found TNBS-induced currents in AITC-sensitive TRPA1⫹/⫹ DRG neurons that were blocked by HC (Figure 3A). The effect of TNBS on recombinant rat TRPA1 was investigated in HEK-293t cells to verify the direct activation of TRPA1 by TNBS. TNBS activated large inward currents in rTRPA1-expressing HEK-293t cells, blocked by HC (100 ␮mol/L) (Figure 3B). Other TRPA1 agonists, such as AITC and acrolein, activate TRPA1 by forming covalent bonds with particular intracellular Cys residues (Michael addition).27,28 Because the morphology of TNBS-induced currents in rTRPA1 resembled those induced by AITC (ie, slowly activating and inactivating currents), we examined the effects of TNBS on the acrolein-insensitive mutant human TRPA1– C621S/C641S/C665S (hTRPA1–3C) lacking the decisive Cys residues.27 TNBS (1 mmol/L) induced significantly smaller inward currents in hTRPA1–3C (209 ⫾ 114 pA; n ⫽ 8) than those in WT hTRPA1 (1911 ⫾ 365 pA; n ⫽ 6) (Figure 3C; P ⬍ .001, unpaired Student t test), whereas the noncovalently binding TRPA1 agonist carvacrol induced inward currents of similar size in hTRPA1–WT and hTRPA1–3C (data not shown). The additional substitution of lysine by arginine at position 708 leads to complete insensitivity of this mutant to AITC.27 Therefore, we also examined the effects of TNBS (1 mmol/L) on the hTRPA1-C621S/C641S/C665S/K708R mutant (hTRPA1–

Figure 1. TNBS and AITC induced neuropeptide release from isolated mouse colon. (A and B) TNBS and AITC induced CGRP/SP release from the colons of WT mice in a concentration-dependent manner. (C) CGRP release by TNBS (1 mmol/L) was dependent on extracellular calcium ions (**P ⬍ .01, Mann–Whitney U test) and (D) was lacking in colon preparations from TRPA1⫺/⫺ (**P ⬍ .01) but not TRPV1⫺/⫺ mice. (E) In the presence of the TRPA1 antagonist HC (50 ␮mol/L), TNBS-induced colonic CGRP release was abolished (*P ⬍ .05). DSS (2%) did not induce colonic CGRP release. Baseline release: (A) CGRP, 115 ⫾ 6 pg/mL; SP, 69 ⫾ 6 pg/mL; (B) CGRP, 112 ⫾ 11 pg/mL; (C) CGRP, 81 ⫾ 7 pg/mL; (D) CGRP TRPA1⫹/⫹, 108 ⫾ 7 pg/mL; TRPA1⫺/⫺, 122 ⫾ 24 pg/mL; and TRPV1⫺/⫺, 117 ⫾ 30 pg/mL; and (E) CGRP, 115 ⫾ 12 pg/mL.

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Figure 2. TNBS activates irritant-sensing colonic DRG neurons during calcium-imaging. (A) Bright-field (left), DiI-fluorescence (middle), and merged (right) images of cultured DRG neurons. Colon-specific neurons are shown in red in the merged image. (B) Representative experiment in a single colon-specific DRG neuron showing calcium transients induced by TNBS (1 mmol/L) and AITC (100 ␮mol/L) in a capsaicin (Caps 300 nmol/L)sensitive neuron from TRPA1⫹/⫹ (left) and TRPA1⫺/⫺ (right) mice. (C) Fractions of cells responding to TNBS (1 mmol/L, left; 10 mmol/L, right), AITC, capsaicin, and KCl among colon-specific DRG neurons from TRPA1⫹/⫹ mice. KCl-positive (whole pie), capsaicin-responsive (white segment), AITC (light gray), and TNBS-positive (dark gray) cell fractions. In TRPA1⫺/⫺ mice less than 1% of colonic DRGs showed small TNBS (at 1 and 10 mmol/L) or AITC responses, whereas 56% were capsaicin-positive.

3CK). TNBS did not induce any inward currents in this mutant whereas 1 mmol/L 2-aminoethoxydiphenyl-borate induced normal current responses, suggesting normal protein expression (Figure 3C). Thus, it is likely that TNBS activates TRPA1 by post-translational modification of Cys and lysine residues in the cytoplasmatic N-terminal region of the channel. Considering that several members of the TRP-receptor family are expressed in sensory neurons, we also studied the effects of TNBS on recombinant TRPV1 (rTRPV1), rTRPV2, rTRPV4, and r-transient receptor potential ion channel of the melastatin type 8 expressed in HEK-293t cells to examine specificity. We did not observe any TNBS-induced currents (1–10 mmol/L) in nontransfected HEK-293t cells (data not shown) or in cells expressing rTRPV1, rTRPV2, rTRPV4, or r-transient receptor potential ion channel of the melastatin type 8 (Figure 3D). The other colitogenic agent DSS (2%; molecular weight, 36,000 –50,000) also was assessed for its ability to activate peptidergic sensory neurons but it did not

induce CGRP release from isolated mice colons (Figure 1E) and failed to induce calcium transients in cultured lumbosacral DRG neurons (Supplementary Figure 1C).

TRPA1 Is Crucial for TNBS and DSS Colitis The discovery that TNBS specifically functions as a TRPA1 agonist and induces massive neuropeptide release raised the question of whether these effects were significant for inducing colitis. The TNBS enema induced severe acute colitis in WT mice congenic with the C57BL/6J strain and also in their littermates deficient of TRPV1 (Figure 4A and B). However, null TRPA1 mutants were protected and did not become ill after TNBS administration. We also conducted a pharmacologic trial in WT mice because global knockouts can undergo developmental or transcriptional alterations other than those intended. HC was injected (300 mg/kg; IP) before and 2 days after the TNBS enema and prevented colitis in WT mice. If the animals lived after acute TNBS colitis, they remained ill

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Figure 3. TNBS activates TRPA1 in patch-clamp experiments. (A) Representative current trace of a TNBS-induced inward current in a DRG neuron (Vm, ⫺60 mV) which was reversibly blocked by the TRPA1 antagonist HC (100 ␮mol/L). (B) Representative current traces of TNBS and AITC induced inward currents in rTRPA1-expressing HEK-293t cells; cells were held at ⫺60 mV and TNBS (1 mmol/L) or AITC (100 ␮mol/L) was applied. HC (100 ␮mol/L) also blocked TNBS-induced inward currents in these cells. Comparison of mean inward currents. (C) TNBS (1 mmol/L) induced inward currents in HEK293t cells expressing hTRPA1-WT, hTRPA13C, or hTRPA1-3CK. Mean inward currents in HEK293t cells expressing WT or mutant hTRPA1 (each experiment, n ⫽ 5 or 8 cells). (D) Representative current traces of rTRPV1, rTRPV2, rTRPV4, and transient receptor potential ion channel of the melastatin type 8 r(TRPM8) expressed in HEK293t cells. Each TRP subtype was probed with TNBS (1 mmol/L); protein expression was controlled by administration of established agonists (capsaicin, 2-aminoethoxydiphenyl-borate [2-APB], 4-alpha-phorbol-12,13-didecanoate [4␣-PDD], or menthol).

and progressively lost weight, even after the chemical initiator and TRPA1 agonist TNBS was long eliminated. Nevertheless, HC administrated (once daily, IP) during the third week of chronic colitis was beneficial for improving the disease scores, leading to a remarkable gain in body weight (Figure 4C–F). This surprising therapeutic effect suggested a sustained activation of TRPA1 and consequent neurosecretion that contributed to the disease. This finding provided us with a reason to study another colitis model using DSS in the drinking water, a chemical inducer that does not activate TRPA1 directly (see earlier). WT mice showed a delayed (5 days) but progressive weight loss that was much less expressed and

progressive in TRPA1 knockout and WT littermates treated with HC, indicating a moderate contrast to severe DSS colitis (Figure 4C–F).

TRPA1 Is Activated by Inflammatory Products of Lipid Peroxidation and Sensitized in Colitis The striking therapeutic effect of the TRPA1 block in both chronic models of colitis suggested that TRPA1 is activated or sensitized by chemicals generated during the peculiar metabolism of inflammation. Apart from the classic mediator bradykinin, which sensitizes TRPA1,33 products of membrane phospholipid peroxidation, indi-

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Figure 4. TNBS and DSS colitis scores and representative histologic slides of WT, TRPA1⫺/⫺, and TRPV1⫺/⫺ mice. Asterisks indicate significance with regard to disease scores of WT mice with colitis (**P ⬍ .01, Mann–Whitney U test). (A) Acute TNBS colitis scores (3 days after enema) for the different genotypes and drug-treated mice. (B) H&E-stained histologic slides from distal colon sections showing severe acute colitis in TRPA1⫹/⫹ and TRPV1⫺/⫺ mice and normal tissue in TRPA1⫺/⫺ and HC-treated TRPA1⫹/⫹ mice. (C) Chronic TNBS colitis scores (21 days after enema) in TRPA1⫹/⫹ with and without treatment with the TRPA1 block and DSS colitis scores in TRPA1⫹/⫹ mice compared with those TRPA1⫺/⫺ and TRPA1⫹/⫹ mice treated with HC from days 0 –10. (D, upper panel) Body weights of TRPA1⫹/⫹ mice with chronic TNBS colitis decreased for 3 weeks after the TNBS enema. Treatment with HC during the last week led to considerable body weight gain. Arrows indicate HC treatment duration. The therapeutic effect of HC was significant from day 17 (*P ⬍ .05, **P ⬍ .01). Each point represents the mean body weight of 8 mice ⫾ standard error of the mean. Statistical comparisons were made using analysis of variance followed by the Fisher LSD test. (D, lower panel) Body weight losses in DSS colitis in TRPA1⫹/⫹ mice compared with those in TRPA1⫺/⫺and TRPA1⫹/⫹ mice treated with HC. Representative histologic slides of TRPA1⫹/⫹ with or without HC treatment in (E) chronic TNBS and (F) both TRPA1 genotypes with DSS colitis. See Supplementary Material and Methods for the colitis scoring system and mortality rates.

cating oxidative stress in inflamed tissue, function as endogenous TRPA1 agonists. Typical factors are unsaturated aldehydes such as acrolein, 4-HNE, and 4-oxo-2nonenal, which are found in inflammatory exudates in micromolar to millimolar concentrations.48,49 Indeed, all 3, most effectively 4-oxo-2-nonenal, induced substantial neuropeptide release from the isolated colons of WT but not TRPA1⫺/⫺ mice (Figure 5A). We analyzed 4-HNE concentrations, a key molecule mediating many of the cytotoxic effects of reactive oxygen species, in healthy and

inflamed colons and found a distinct increase in 4-HNE concentrations in both forms of colitis (Figure 5B). We were interested in determining whether acute and chronic colitis can induce and maintain TRPA1 sensitization and concomitant neuropeptide release. The inflamed colons were isolated from WT mice 3 days after the TNBS enema and 12 days after DSS administration. After cleaning and equilibrating the preparation, the basal neuropeptide release levels were not increased significantly (Figure 5C and D). The newly identified selective agonist TNBS (1

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Figure 5. (A) 4-HNE-, 4-oxo-2-nonenal (4-ONE)–, and acrolein-induced neuropeptide release from isolated mouse colon (each n ⫽ 4). All 3 compounds induced CGRP and SP release in healthy WT but not TRPA1⫺/⫺ mice (*P ⬍ .05, Mann–Whitney U test). Baseline release: WT CGRP, 101 ⫾ 10 pg/mL; SP, 56 ⫾ 6 pg/mL; TRPA1⫺/⫺, CGRP 102 ⫾ 9 pg/mL; SP, 69 ⫾ 6 pg/mL. (B) 4-HNE concentration in inflamed colons from both DSS- and TNBS-treated mice increased compared with that in healthy tissue (*P ⬍ .05; **P ⬍ .01). (C) TNBS-induced CGRP and SP release from isolated colon preparations from WTs with colitis. Values are normalized to baseline neurosecretion (each n ⫽ 8). On day 3 of acute TNBS colitis, TNBS (1 mmol/L)-induced colonic CGRP and SP release increased compared with that in mice receiving a vehicle enema. The response was abolished when HC (50 ␮mol/L) was co-applied with the TNBS stimulus. (D) On day 12, mice with DSS colitis also showed a drastic increase in stimulated neuropeptide release, which was prevented by the TRPA1 block HC. *Significant increase in colonic neuropeptide release compared with controls. ⫹Abolished stimulated neuropeptide release from the inflamed colon by HC compared with controls. (ⴱ,⫹P⬍.05). Baseline release: (C) CGRP, 101 ⫾ 8 pg/mL; SP, 79 ⫾ 8 pg/mL; (D) CGRP, 119 ⫾ 7 pg/mL; SP, 72 ⫾ 10 pg/mL. (E) Chemical structure of a stable sulfinic acid transformation of the Cys-SH group by TNBS, as shown by mass spectrometry, which may contribute to sustained TRPA1 sensitization (see also Supplementary Figure 2).

mmol/L) was used to stimulate TRPA1, and approximately double the amount of CGRP/SP release was achieved from inflamed colons compared with normal colons (Figure 5C and D). HC completely blocked neuropeptide release, in agreement with the earlier-described therapeutic effects. For the colitogenic agent TNBS an alternative method of keeping TRPA1 in a sensitized state can be postulated based on chemical experiments (Figure 5E, Supplementary Figure 2). A model peptide, glutathione in 1 mmol/L aqueous solution, containing a reactive Cys-SH residue such as TRPA1, was mixed with 34.5 mmol/L TNBS at 37°C for 2 hours and then analyzed by electrospray ionization time-of-flight spectroscopy followed by mass spectrometry. A typical peak indicating reversible cova-

lent substitution of the SH residue by TNB (Michael adduct) in a fraction of the glutathione molecules was detected. However, the second peak detected corresponded with a sulfinic acid transformation of the Cys-SH group. Such an oxidation product is stable and could contribute to sustaining the activation/sensitization of the receptor channel if applied to the Cys residues in TRPA1.

Dominant Proinflammatory Role of SP Over the Protective Effects of CGRP The released neuropeptides are candidate mediators for linking the sensory neurons to the immune system and generating inflammation.17–23 We used null mu-

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surpassed by the knockout of CGRP (Figure 6C–E). In contrast, CGRP mutants treated with HC were protected against DSS colitis and SP⫺/⫺ mice were protected against both TNBS and DSS colitis (Figure 6). Interestingly, CGRP/SP double-knockout mice also completely were protected against the TNBS colitis (Figure 6A and B), indicating that the loss of the CGRP protective effect did not reduce the beneficial effect of the absence of SP. In

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tant mouse strains and generated a double-knockout line for CGRP and SP to study both mechanistically different colitis models (Figure 6). In comparison with WT, CGRP⫺/⫺ mice showed a particularly severe course of the acute TNBS colitis (Figure 6A and B). Furthermore, rapid, massive weight loss in the DSS model made the phenotypical difference rather than the disease scores, which were so high in WT mice that they could hardly be

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Figure 6. Acute TNBS colitis (A) sum scores and (B) representative histologic slides from WT, CGRP⫺/⫺, SP⫺/⫺, and double-null mutant mice; the latter 2 strains were completely protected. (C and D) After 8 days of DSS colitis, WT and CGRP⫺/⫺ mice were seriously ill, whereas CGRP⫺/⫺ mice treated with HC and SP⫺/⫺ mice were completely protected, (E) which is reflected in reduced body weight loss, whereas the aggravation in CGRP⫺/⫺ mice became evident as progressive wasting from day 2. Asterisks in the bar graphs indicate the significance of colitis scores for ill WT mice (*P ⬍ .05, **P ⬍ .01, Mann–Whitney U test). Statistical comparisons of the body weight course were made by analysis of variance followed by the Fisher LSD test (*P ⬍ .05, **P ⬍ .01).

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Figure 7. (A) Representative photomicrographs of whole-mount preparations from the mouse distal colon at the level of the submucosal plexus. In WT mice intrinsic ganglionic neurons were surrounded by CGRP-positive (red) and CGRP/TRPV1 co-expressing (yellow) fiber baskets, however, they were neither CGRP nor TRPV1 immunoreactive. RTX-pretreated mice showed a complete loss of TRPV1 (green)-expressing sensory neurons. However, some intrinsic enteric CGRP immunoreactive fibers were still observed. (B) Capsaicin (30 nmol/L) and TNBS (1 mmol/L) failed to induce CGRP (left) and SP (right) release from the colons of RTX-pretreated mice but did so in controls (Con; each n ⫽ 8, *P ⬍ .05, Mann–Whitney U test). The baseline release of CGRP was 84 ⫾ 8 pg/mL and that of SP was 55 ⫾ 9 pg/mL. (C–E) RTX-pretreated WT mice were protected from TNBS and DSS colitis. (C, lower panel) The latter was reflected in the body weights during the 8-day period of DSS colitis (analysis of variance followed by the Fisher LSD test, *P ⬍ .05, **P ⬍ .01). In both colitis models, RTX-pretreated WT mice had preserved healthy mucosal architecture. (F) Chemically induced CGRP release from DRG neurons and MPNs. TNBS (1 mmol/L), AITC (100 ␮mol/L), and capsaicin (30 nmol/L) (each n ⫽ 8) induced profound CGRP release from cultured lumbosacral DRG neurons, which was not evident in MPNs (**P ⬍ .01). However, nonspecific depolarization with KCl (60 mmol/L) (n ⫽ 24) induced CGRP release from both classes of neurons.

other words, CGRP was not required for colon protection if SP could not exert its deleterious effects.

Extrinsic Sensory Neurons Mediate TNBSInduced Neuropeptide Release, TNBS, and DSS Colitis Intrinsic enteric neurons are insensitive to capsaicin stimulation.50,51 Therefore, the established technique of chemically induced sustained functional denervation in vivo by RTX,44 an ultrapotent TRPV1 agonist, eliminates the extrinsic primary afferent population of sensory nerves rather than intrinsic enteric neurons. Indeed, immunohistochemical staining of the colons of RTX-pretreated mice showed effective ablation of TRPV1/CGRP co-expressing nerve fibers (Figure 7A), which were associated with a complete lack of capsaicin-induced colonic neuropeptide release (Figure 7B). The finding that TNBSinduced neuropeptide release also was abolished (Figure 7B) was in agreement with TRPA1 co-expression in a subset of TRPV1-positive sensory neurons.24 TNBS and

DSS colitis were abolished and DSS-induced weight loss was prevented in RTX-pretreated mice (Figure 7C–E), suggesting that the TRPV1/TRPA1- and CGRP/SP-positive subpopulation of extrinsic primary afferent neurons mediates the in vivo proinflammatory effects. Finally, we compared stimulated neuropeptide release between isolated colonic MPNs and DRG neurons. TNBS (1 mmol/ L), AITC (100 ␮mol/L), and capsaicin (30 nmol/L) induced robust CGRP release from DRG neurons, which was lacking in MPNs, confirming that activation of TRPV1 and TRPA1 is crucial for neuropeptide release from extrinsic sensory neurons (Figure 7F).

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. Because DSS does not directly activate TRPA1 and because stimulated neuropeptide release is enhanced in the inflamed colon, we propose that endogenous mediators of inflammation and oxidative stress such as 4-HNE and acrolein sensitize TRPA1 to induce sustained SP release, which leads to aberrant or exaggerated immune responses by as yet unknown neuroimmune interactions.

Origin of Colonic Neuropeptide Release CGRP and SP are co-expressed in extrinsic primary afferents and intrinsic enteric neurons.13,50 –54 However, only the minor ␤ isoform of CGRP is present in intrinsic enteric nerves, which are insensitive to capsaicin treatment.50,51,55–57 Immunocytochemical reports have suggested that TRPV1 is expressed in intrinsic myenteric neuron cell bodies of the guinea pig ileum and colon as well as the rat and pig ileum.58 – 60 In contrast, intense TRPV1 immunoreactivity has been observed only in nerve fibers surrounding the myenteric neurons but not in cell bodies within myenteric ganglia of the rat small intestine.61,62 Moreover, only capsaicin-sensitive extrinsic sensory fibers are activated or sensitized by noxious conditions such as inflammation and tissue acidosis.55–57 Our data derived from cultured DRG and MPNs and RTXpretreated, sensory nerve-depleted mice support the concept that TRPV1/TRPA1 and CGRP/SP are functionally co-expressed only in extrinsic sensory neurons of the colon.

Endogenous TRPA1 Sensitization TRPA1-receptor expression in lumbosacral DRG neurons is up-regulated in experimental colitis.63 In addition, our data show functional sensitization of TRPA1 in colonic nerve endings during TNBS and DSS colitis, 4-HNE concentrations were increased in both forms of colitis, and all 3 unsaturated aldehydes induced colonic neuropeptide release in a TRPA1-dependent way, suggesting a likely role in the maintenance of colonic inflammation through ongoing TRPA1 activation and increased SP release.

TRPA1-Mediated Neuropeptide Release With Regard to Inflammation Caceres et al64 recently showed that activating TRPA1 mediates inflammatory leukocyte infiltration and enhances pulmonary mucus production and airway hyperreactivity supposedly through neuropeptide release in a murine model of allergic asthma. They induced a Th2 lymphocyte-driven allergic response using the ovalbumin mouse model of asthma.64 Similarly, we suggest a crucial role for TRPA1 activation and sensitization with concomitant neuropeptide release in 2 different colitis models. In fact, we propose a more general proinflammatory role for

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TRPA1 sensitization/activation because we refer to the mainly Th1-driven TNBS colitis and to DSS colitis, which depends on the innate rather than the adaptive immune system.1–3,65 Moreover, our data indicate a therapeutic benefit for the pharmacologic TRPA1 block even in the chronic stage of colitis, which probably occurs by attenuating chronically increased SP release that may be owing to the demonstrated sensitization of TRPA1 in sensory neurons. Our data from RTX-pretreated mice suggest that it is extrinsic sensory rather than intrinsic enteric neurons that promote inflammation in TNBS and DSS colitis.

Role of Sensory Neurons and Vagal Innervation in Colitis Extrinsic primary afferents co-expressing TRPV1, TRPA1-receptor channels, and the neuropeptides may reach the colon from the spinal dorsal root ganglia joining the splanchnic nerves and visceral blood vessels as well as through the vagus nerves. Vagal nerve stimulation is reported to attenuate the production of proinflammatory cytokines and inhibit the inflammatory processes in various experimental models through acetylcholine release from parasympathetic efferents that influence immune cell functions via the ␣7 nicotinic acetylcholine receptor.66 In addition, CGRP has been suggested to exert a protective effect in rat TNBS colitis depending on the integrity of the vagus nerves.20,67 Abundant TRPA1 and TRPV1 co-expression was shown in vagal afferent nerve fibers and nodose ganglion neurons.68 Thus, TRPA1-mediated effects on our colitis models could derive from vagal as well as spinal sensory neurons. However, surgical denervation around the inferior mesenteric artery affecting mainly spinal afferents also attenuated rat TNBS colitis.69 In accord with our RTX results, systemic capsaicin-induced desensitization ameliorated rat DSS and rat/rabbit TNBS colitis, but these experiments cannot differentiate between vagal and spinal neurons.5,8,9 DSS colitis in rats and mice also was attenuated, if the capsaicin receptor was pharmacologically inhibited by topical, intracolonic, administration of capsazepine,5,7 however, at drug concentrations that act partially agonistic on TRPV1.70 This may actually have led to a desensitization of TRPV1-expressing sensory nerve fibers, thus, explaining protection from colitis through depletion of sensory neurons/neuropeptides rather than TRPV1 antagonism. Very recently, a study suggested a deleterious role of TRPV1 in DSS colitis, however, protection of TRPV1⫺/⫺ mice receiving 2% DSS for 7 days was observed only in the distal, not proximal, parts of the colon. WT controls were only mildly affected, which prevented a comparison, and when treated with 5% DSS both genotypes developed severe colitis to the same degree.10 In opposition, Massa et al11 reported increased colitis susceptibility in TRPV1-deficient mice challenged with dinitrobenzene sulfonic acid (DNBS). Although the latter study was lacking a detailed disease activity score/histologic analysis, the measurement of myeloperoxidase activity and the macroscopic analysis suggested that TRPV1⫺/⫺

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mice were not protected. Thus, DNBS, which is structurally most similar to TNBS, actually may have activated TRPA1 and induced colitis. In the present study acute TNBS colitis did not depend on TRPV1 expression. Rather, TRPA1 activation by TNBS releasing massive amounts of SP appeared to initiate acute TNBS colitis. In chronic TNBS colitis, the TRPA1 block was less effective, and primary chronic DSS colitis was ameliorated only in TRPA1 null mutants, which leaves room for other SP release mechanisms besides TRPA1 activation to contribute to the chronic state of colitis.

Opposite Role of SP and CGRP in Colitis

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Previous reports on pharmacologic block of the SP high-affinity receptor neurokinin1 using SR14033 and CP-96345 showed protective effects in rat TNBS and DSS colitis.17,18,21 In contrast, another neurokinin1 antagonist was ineffective in rat TNBS colitis.19,22 An opposite, protective, role of CGRP was suggested by several pharmacologic studies on rat TNBS colitis.20,22,71 In addition, knockdown by double-stranded RNA against the calcitonin receptor-like receptor for CGRP resulted in a significantly greater degree of edema and necrosis than in sham-treated rats with TNBS colitis.72 We confirmed opposite effects of CGRP and SP in both TNBS and DSS colitis, after studying neuropeptide gene-targeted mice in colitis models. Beyond that, the failure to induce colitis in CGRP/SP double-knockout mice indicates that the loss of the protective CGRP effect does not promote colitis if SP is absent. The absence of DSS and TNBS colitis in SP⫺/⫺ and in RTX-pretreated mice emphasizes the abundant proinflammatory role of SP released from extrinsic sensory neurons.

Neuroimmune Interactions We did not analyze the direct effects of CGRP and SP on immune cells. Numerous previous reports have shown that neurosecretion of CGRP and SP contributes to different inflammatory processes through various direct effects on dendritic, mast, T, and B cells.14 –16,73,74 Gad et al23 showed that a pharmacologic block of the NK-1 receptor for SP or capsaicin-induced ablation of extrinsic sensory neurons protects mice with severe combined immunodeficiency from disease in a purely immunogenic T-cell transfer model of chronic colitis. However, it appears that the molecular mechanisms of how CGRP and SP directly affect the immune system still remain unclear, particularly with regard to colitis.

TRPA1 and Gut Motility Most recently, TRPA1 immunoreactivity was detected in inhibitory motoneurons, descending interneurons, cholinergic and intrinsic sensory neurons of the mouse cecum and colon. The study showed no significant differences in gastric emptying and small intestinal and colonic transit between TRPA1⫹/⫹ and TRPA1⫺/⫺ mice, which could have had an impact on our experiments. However, administration of the TRPA1 agonist AITC is

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reported to slow colonic transit in TRPA1⫹/⫹ but not TRPA1⫺/⫺.75 Thus, also the TRPA1 agonist TNBS could have been egested faster from the colon of TRPA1⫺/⫺ and HC-030031–treated than untreated WT mice, causing less damage, which would suggest an unspecific beneficial effect of TRPA1 deletion and block. Against this interpretation can be argued that chronic TNBS colitis, when TNBS is long expulsed, also was improved by HC treatment as well as DSS colitis that is not induced by a TRPA1 agonist.75 In addition, SP, if at all, shows propulsive effects in the colon, but nonetheless, SP knockouts were protected from TNBS as well as DSS colitis in our experiments.76 –78

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 inflammatory bowel diseases.

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GASTROENTEROLOGY Vol. 141, No. 4 70. Pethö G, Izydorczyk I, Reeh PW. Effects of TRPV1 receptor antagonists on stimulated iCGRP release from isolated skin of rats and TRPV1 mutant mice. Pain 2004;109:284 –290. 71. Evangelista S, Tramontana M. Involvement of calcitonin generelated peptide in rat experimental colitis. J Physiol Paris 1993; 87:277–280. 72. Clifton MS, Hoy JJ, Chang J, et al. Role of calcitonin receptor-like receptor in colonic motility and inflammation. Am J Physiol Gastrointest Liver Physiol 2007;293:36 – 44. 73. Peters EM, Ericson ME, Hosoi J, et al. Neuropeptide control mechanisms in cutaneous biology: physiological and clinical significance. J Invest Dermatol 2006;126:1937–1947. 74. Niizeki H, Alard P, Streilein JW. Calcitonin gene-related peptide is necessary for ultraviolet B-impaired induction of contact hypersensitivity. J Immunol 1997;159:5183–5186. 75. Poole DP, Pelayo JC, Cattaruzza F, et al. Transient receptor potential ankyrin 1 is expressed by inhibitory motoneurons of the mouse intestine. Gastroenterology 2011;141:565–575.e4. 76. Holzer P, Holzer-Petsche U. Tachykinin receptors in the gut: physiological and pathological implications. Curr Opin Pharmacol 2001;1:583–590. 77. King SK, Sutcliffe JR, Ong SY, et al. Substance P and vasoactive intestinal peptide are reduced in right transverse colon in pediatric slow-transit constipation. Neurogastroenterol Motil 2010;22: 883– 892. 78. Tzavella K, Riepl RL, Klauser AG, et al. Decreased substance P levels in rectal biopsies from patients with slow transit constipation. Eur J Gastroenterol Hepatol 1996;8:1207–1211. Received December 27, 2010. Accepted July 6, 2011. Reprint requests Address requests for reprints to: Peter W. Reeh, MD, PhD, Institute of Physiology and Pathophysiology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Universitätsstr. 17, D-91054 Erlangen, Germany. e-mail: [email protected]; fax: (49) 9131-85-22497. 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). 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 FriedrichAlexander-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 Milo
Filipovic´ and Ivana Ivanovic´–Burmazovic´ was supported by the Deutsche Forschungsgemeinschaft.