Based on the data above, we hypothesized that histamine-induced TRPA1 and TRPV4 sensitization, in parallel to TRPV1, could be involved in VHS in patients with IBS, thereby explaining the previously reported beneficial effect of the H 1 R antagonist ebastine in IBS ( 44 ). To test this, we compared the response of IBS and HS rectal submucosal neurons to TRPA1 and TRPV4 agonists. Moreover, we assessed the ability of histamine and IBS biopsy supernatants to sensitize these TRP channels in primary cultured murine dorsal root ganglion (DRG) neurons.

Altered TRP channel function can be induced by several proinflammatory factors, including mast cell mediators that play an important role in IBS ( 3 , 5 , 20 ). Previously, we ( 4 , 44 ) showed that sensitization of TRPV1 in rectal submucosal neurons of patients with IBS was produced by the mast cell mediator histamine via activation of histamine 1 receptor (H 1 R). Furthermore, treatment of patients with IBS with the H 1 R antagonist ebastine resulted in significant improvement of abdominal pain ( 44 ), suggesting that H 1 R-mediated sensitization of TRPV1 and possibly other TRP channels underlies increased abdominal pain in IBS. In line with this, histamine can also sensitize TRPV4 both in vitro and in vivo, resulting in VHS in mice ( 12 ). Although submucosal neurons are not involved in visceral pain perception, it should be emphasized that visceral afferent sensory neurons reside in the same environment and thus will be exposed to the same environmental triggers. Therefore, evaluation of submucosal neurons in biopsies indirectly supports a role for neuronal sensitization in IBS. To date, human data supporting a role for histamine-driven TRPA1 and TRPV4 potentiation in patients with IBS is, however, lacking.

Similar to TRPV1, evidence is accumulating supporting an important role for TRPV4 in visceral pain. TRPV4 can be activated by mechanical force, osmotic pressure, or innocuous temperature (27–34°C). Intracolonic infusion of supernatants from IBS biopsies, but not from healthy subjects (HS), induced VHS in mice, whereas knockdown of TRPV4 inhibited this hypersensitivity ( 13 ). Furthermore, human serosal nociceptor mechanosensitivity is attenuated by application of the TRPV4 antagonist HC067047, further underscoring the potential role of TRPV4 in visceral pain perception ( 27 ). Along the same line, TRPA1 is suggested to play a role in visceral pain in preclinical models ( 7 , 10 , 16 ). TRPA1 is activated by cold, pungent compounds, such as allyl isothiocyanate, and mechanical distention. Intracolonic administration of allyl isothiocyanate in rodents results in an increased visceromotor response, which is absent in Trpa1 knockout mice ( 7 , 10 , 16 ). Taken together, these data demonstrate that TRPA1 and TRPV4 are involved in VHS.

Upregulation and/or sensitization of nociceptors, in particular of transient receptor potential (TRP) channels, are recognized to play a major role in somatic pain ( 29 , 31 ). For example, potentiation of TRP vanilloid 1 (TRPV1), TRP vanilloid 4 (TRPV4), and TRP ankyrin 1 (TRPA1) induces mechanical and thermal hyperalgesia in mice treated with the chemotherapeutic drug paclitaxel ( 15 ). Furthermore, TRPV4 sensitization and upregulation in trigeminal sensory neurons was described in an inflammatory model of temporomandibular joint pain ( 14 ). In parallel to their role in somatic pain perception, altered TRP channel function is also recognized as an important mechanism underlying aberrant visceral pain ( 20 ). Especially TRPV1, the capsaicin receptor, has repeatedly been shown to be involved in VHS. TRPV1 expression is increased in preclinical models of VHS ( 1 , 32 ), whereas sensitivity to colorectal distention is decreased by TRPV1 antagonists ( 39 , 42 ) and reduced in Trpv1 knockout mice ( 23 ).

Irritable bowel syndrome (IBS) is the most frequently diagnosed disorder by gastroenterologists worldwide, affecting >10% of the western population ( 19 ). IBS is a functional gastrointestinal disorder characterized by recurrent abdominal pain or discomfort associated with altered defecation pattern in the absence of an organic cause ( 33 ). Visceral hypersensitivity (VHS) or aberrant pain perception is present in ≤60% of the patients and represents one of the hallmarks of IBS ( 9 , 25 , 30 ). The underlying pathophysiological mechanism of VHS is, however, not fully understood.

Statistical analyses of the peak F340/F380 ratios for the Ca 2+ -imaging experiments were performed after correction for the individual baseline Ca 2+ , using GraphPad Prism. Values are expressed as means ± SE from n cells. When deviations from normality were observed by the use of Shapiro-Wilk normality test, medians and interquartile values were presented. Statistical comparisons were performed by a Wilcoxon signed-rank test (for 2 groups) or Mann-Whitney U test as appropriate or ANOVA (for >2 groups). Categorical data were analyzed by Fisher exact test.

All statistical analyses were performed using GraphPad Prism 7.04. Continuous data were summarized by their mean and standard deviation. When deviations from normality were observed by the use of Shapiro-Wilk normality test, medians and interquartile values were presented. Comparisons between groups were made using a t -test or Wilcoxon rank-sum test, as appropriate. Statistical significance is assumed when P ≤ 0.05 after Bonferroni correction for multiple testing.

Translocation of TRPA1 or TRPV4 to the membrane of the cell was measured with ImageJ as a ratio of the TRPA1 or TRPV4 fluorescence intensity (expressed as mean gray value) in the membrane area on the TRPA1 or TRPV4 fluorescence intensity in the cytoplasm, both normalized to the background. Total TRPA1 and TRPV4 was quantified combining the TRPA1 or TRPV4 fluorescence intensity in the membrane and in the cytoplasm. Areas of interest were defined using phase-contrast images of the cells.

To evaluate the translocation of TRPA1 and TRPV4 to the membrane on stimulation, cultured DRG neurons were incubated with 10–100 µM histamine or vehicle for 10 min or overnight (for TRPA1 and TRPV4, respectively). Cells were then fixed in 4% paraformaldehyde for 15 min before permeabilization in 0.1% Triton X-100 for 10 min. After blocking in 5% donkey serum for 3 h, cells were stained with rabbit anti-TRPA1 (1:200; Alomone, Jerusalem, Israel) or rabbit anti-TRPV4 (1:200; Alomone) overnight. After washing, cells were incubated with goat anti-rabbit Cy3 (1:500; Jackson ImmunoResearch, West Grove, PA) for 2 h and 4′,6′-diamidino-2-phenylindole for 15 min. Confocal images were taken with a Zeiss LSM510 confocal microscope in the Cell Imaging Core, University of Leuven.

RNA was extracted from mouse DRG, which were overnight incubated with histamine (10–100 µM), and from 5- to 10-mg human rectal biopsies, which were stored in RNAlater (Qiagen Benelux, Venlo, The Netherlands), using RNeasy Mini Kit (Qiagen, Hilden, Germany). cDNA of 2-µg total RNA was synthesized using qScript cDNA SuperMix (Quanta Biosciences, Gaithersburg, MD) according to the manufacturer’s instructions. Real-time quantitative PCR was performed for quantification of neuronal TRPV4 and TRPA1 mRNA expression FastStart Essential DNA Green Master (Roche, Mannheim, Germany) relative to the housekeeping gene β-actin ( ACTB ; primer sequences are listed in Table 1 ). Wells of an AmpliStar 96-well LightCycler 480 QPCR Plate (Westburg, Leusden, The Netherlands) were loaded with 2.5 µl of each cDNA sample together with 5 µl of FastStart Essential DNA Green Master (Roche), 0.2 µl of oligonucleotides (10 µM), and 2.3 µl of RNase-free water (Applied Biosystems, Halle, Belgium). Gene expression was normalized to the endogenous reference gene β-actin, and the relative gene expression was calculated by the comparative cycle threshold method ( 26 ).

The intracellular Ca 2+ measurements were performed using a monochromator-based imaging system consisting of either a Polychrome IV monochromator (TILL Photonics, Martinsried, Germany) and a Roper Scientific (Tucson, AZ) charge-coupled device camera connected to a Zeiss (Oberkochen, Germany) Axiovert 200 M inverted microscope or an Olympus (Tokyo, Japan) Cell^M system. The fluorescence intensity was measured during excitation at 340 and 380 nm, and the ratio of the fluorescence intensity at both excitation wavelengths (F340/F380) was monitored. Intracellular Ca 2+ concentrations were determined as previously described ( 41 ). Experiments were performed using standard Krebs solution (containing in mM: 120.9 NaCl, 5.9 KCl, 1.2 MgCl 2 , 2.5 CaCl 2 , 11.5 glucose, 14.4 NaHCO 3 , and 1.2 NaH 2 PO 4 ). To identify neurons in the DRG cultures, we applied a Krebs-based solution in which the KCl concentration was increased to 45 mM by iso-osmotic substitution of NaCl. The baseline was monitored for 120 s, and the chamber was thereafter superfused with the TRP agonists.

Finally, rectal biopsies from HS and patients with IBS were overnight incubated in RPMI (Lonza, Verviers, Belgium) supplemented with fetal calf serum (10%; PAN-Biotech, Aidenbach, Germany), penicillin-streptomycin (1%; Lonza), and amphotericin B/gentamicin (0.2%; Invitrogen, Gent, Belgium) at 37°C, 5% CO 2 . Twenty-four hours later, supernatants were collected and stored in −80°C until murine DRG neurons were incubated overnight with 142 µl of these supernatants derived from either HS or patients with IBS in the presence or absence of histamine (10 µM for TRPA1 activation or 100 µM for TRPV4 activation) with or without pyrilamine (1 µM). Thereafter, we evaluated the responses of these cells to CA (10 µM) and GSK1016790A (1 µM in the presence of 1 µM SB-366791).

DRG neurons were exposed to 10 µM CA before and after acute application (10 min) of histamine (10 µM). These experiments were repeated in Hrh1 knockout mice or in the presence of the H 1 R antagonist pyrilamine (1 µM) in wild-type mice. TRPA1-expressing neurons were identified by application of 300 µM CA at the end of the protocol. The response of DRG neurons to GSK1016790A (1 µM) was determined before and after acute (10 min) incubation with 10 µM histamine. In other experiments, DRG neurons were incubated overnight with vehicle (Krebs) or 10/100 µM histamine and exposed to 1 µM GSK1016790A. These experiments were performed in the presence of the TRPV1 antagonist SB-366791 or in cells isolated from Trpv1 knockout mice, since high doses of GSK1016790A can activate TRPV1. In accordance with TRPA1, these experiments were repeated in Hrh1 knockout mice or in the presence of pyrilamine (1 µM) in wild-type mice.

The lumbosacral (L5–S2) DRG from three to four adult mice were bilaterally excised under a dissection microscope. The ganglia were washed in 10% fetal calf serum Neurobasal-A Medium (basal medium) and then incubated (95% air-5% CO 2 ) at 37°C in a mix of 1 mg/ml collagenase (GIBCO, Gent, Belgium) and 2.5 mg/ml dispase (GIBCO) for 45 min. Digested ganglia were gently washed twice with basal medium and mechanically dissociated in B-27-supplemented (2%) Neurobasal-A Medium (Invitrogen, Gent, Belgium) containing 2 ng/ml glial cell line-derived neurotrophic factor (Invitrogen, Gent, Belgium), 10 ng/ml neurotrophin 4 (PeproTech, London, United Kingdom), 100 µg/ml penicillin-streptomycin (Invitrogen, Gent, Belgium), and GlutaMAX (complete medium; Invitrogen, Gent, Belgium). Neurons were seeded on poly-l-ornithine or poly-d-lysine/laminin-coated glass coverslips and cultured for 12–18 h at 37°C. Cultured DRG neurons were subsequently loaded with 2 μM fura 2-AM for 20 min at 37°C.

All animal experiments were carried out in accordance with the European Community Council guidelines and were approved by the local ethics committee of the Katholieke Universiteit Leuven (KU Leuven; ECD P157/2014). Ten- to twelve-week-old male mice were used in all experiments. C57BL/6 mice were purchased from Janvier, and Hrh1 knockout mice were purchased from Oriental Bioservice (Kyoto, Japan). In addition, Trpv1 knockout mice were obtained from Jackson ( https://jaxmice.jax.org/strain/003770.html ). Trpv1 / Trpv4 double-knockout mice were obtained from an in-house breeding program. All knockout mice were backcrossed ≥10 times in the C57BL/6 background. Mice were housed under identical conditions, with a maximum of 4 animals per cage on a 14:10-h light-dark cycle and with food and water ad libitum.

The responses of HS and IBS submucosal neurons to the TRPA1 agonist cinnamaldehyde (CA; 10 nM and 1 µM; Sigma-Aldrich, Diegem, Belgium) and to the TRPV4 agonist GSK1016790A (0.1 and 1 nM; Sigma-Aldrich) were compared. The perfusion rate was 1 ml/min for 5 s. In addition, we evaluated the effects of 10-min and overnight preincubation with 10–100 µM histamine (Sigma-Aldrich) on the responses of HS submucosal neurons to CA (10 nM) and GSK1016790A (0.1 nM). Finally, we also evaluated the TRPA1- and TRPV4-mediated responses after preincubation with 10 µM histamine combined with 1 µM pyrilamine (Sigma-Aldrich) in HS submucosal neurons.

Fig. 1. Transient receptor potential (TRP) ankyrin 1 (TRPA1) and TRP vanilloid 4 (TRPV4) on human submucosal neurons of patients with irritable bowel syndrome (IBS) is more sensitive compared with healthy subjects. Shown are representative images of a ganglion loaded with fluo 4 ( A , left ) and HuCD immunostaining ( A , right ) and traces of neurons responding to high K + ( B ). Red and blue squares and arrows in A correspond to the respective response represented by a red and blue line in B . C : representative traces of the intracellular Ca 2+ response of human submucosal neurons in biopsies of healthy subjects (HS; blue) and patients with IBS (red) to acute application of cinnamaldehyde (CA; 10 nM) and data showing the amplitude of the Ca 2+ flux and the number of responding neurons (in percentage) to CA in patients with IBS ( n = 14) and HS ( n = 10). D : representative traces of the intracellular Ca 2+ response of human submucosal neurons in biopsies of HS (blue) and patients with IBS (red) to acute application of GSK1016790A (GSK; 0.1 nM) and data showing the amplitude of the Ca 2+ flux and the number of responding neurons to GSK in patients with IBS ( n = 7) and HS ( n = 10). Data are presented as medians ± interquartile range ( left ) and means ± SD ( right ). ** P < 0.01, *** P < 0.001, unpaired t -test ( left ) and Fisher exact test ( right ). F 0 , baseline fluorescence.

Submucosal plexuses were loaded with 1 µM fluo 4-AM (Molecular Probes, Invitrogen, Merelbeke, Belgium) to perform intracellular Ca 2+ imaging as previously described ( 17 , 44 ). Next, the recording chamber was mounted onto an upright Zeiss Examiner microscope equipped with a ×20 (numerical aperture 1) water dipping lens and coupled to a monochromator (Polychrome V) and cooled charge-coupled device camera (Imago QE) both from TILL Photonics (Gräfelfing, Germany). A gravity-fed perfusion system ensured continuous and constant perfusion (1 ml/min) of the preparation with 95% oxygen-5% carbon dioxide-gassed Krebs solution (at room temperature), and excess solution was removed via a peristaltic suction pump, which kept the experimental volume constant (3 ml). Fluo 4 was excited at 475 nm, and its fluorescence emission was collected at 525/50 nm. Images were acquired at 2 Hz and collected by TILLvisION software (TILL Photonics, Oberhausen, Germany). Data were analyzed by custom-written macros in Igor Pro (WaveMetrics, Lake Oswego, OR). Neurons within ganglia were selected based on fluo 4 signal and morphology (big round shape, surrounded by glial cells, and enclosed within nerve fibers). These were only included in the analysis when a sharp Ca 2+ response was displayed after perfusion with high-K + concentration (75 nM; Fig. 1, A and B ). Thus any other intraganglionic nonneuronal cell was excluded. Moreover, neurons over- or underlapped by a nerve fiber or blood vessels were excluded to avoid nonspecific Ca 2+ . Regions of interest were drawn over each neuron, fluorescence intensity was normalized to the basal fluorescence at the onset of the recording for each region of interest, and peaks were analyzed. Background autofluorescence and the bleaching thereof were corrected using a Runge-Kutta iterative deconvolution algorithm assuming monoexponential fluorescence decay. Fluorescence intensities were normalized and expressed as a ΔF/F 0 ratio (F 0 = baseline fluorescence) and percentage of responsive neurons.

During the rectal proctoscopy, biopsies were taken by experienced endoscopists using standard biopsy forceps (single-use biopsy forceps without pin; Onis, Lasne, Belgium). After collection, biopsies were immediately immersed in ice-cold (4°C) Krebs solution (in mM: 120.9 NaCl, 5.9 KCl, 1.2 MgCl 2 , 2.5 CaCl 2 , 11.5 glucose, 14.4 NaHCO 3 , and 1.2 NaH 2 PO 4 ) previously oxygenated (95% oxygen-5% carbon dioxide) and kept on ice for transport. Biopsies were subsequently carefully stretched and pinned flat in a Sylgard-lined petri dish and dissected under a stereomicroscope while continuously perfused with oxygenated (95% oxygen-5% carbon dioxide) ice-cold Krebs solution. The inner submucous layer was carefully removed from the mucosa using watchmaker’s forceps. Then, the tissue was gently stretched and pinned flat in a special recording chamber (own design) in which in- and outflow volume could be tightly controlled.

HS [ n = 38, median age = 24 yr, interquartile range (IQR) = 23–47, 21 women] were recruited by public advertisement, were free of abdominal symptoms, had no history of gastrointestinal disease and no previous gastrointestinal surgery, and were not on gastrointestinal medication. Patients with IBS ( n = 39, median age = 31 yr, IQR = 24–53, 30 women) were recruited from the outpatient clinic of the University Hospital Leuven and had to fulfill the Rome III criteria for IBS. All participants were invited to undergo a proctoscopy to collect rectal biopsies. Not every subject participated to all subprotocols described below. Ethical committee of the University Hospitals Leuven approved the protocols (ref. S55484). Informed consent was obtained from all participants.

Next, we investigated whether, similar to our results in human submucosal neurons, H 1 R is involved in in the histamine-mediated sensitization of TRPA1 and TRPV4 in murine DRG neurons. Therefore, we first tested the effect of the H 1 R antagonist pyrilamine on sensitization of TRPA1 and TRPV4 by IBS supernatants in DRG neurons. Pyrilamine (1 µM) prevented TRPA1 and TRPV4 sensitization in DRG neurons pretreated with IBS supernatants ( Fig. 7 B ). Similarly, the sensitizing effect of overnight incubation of DRG neurons with HS supernatants supplemented with histamine (TRPA1, 10 µM; TRPV4, 100 µM) was blocked by pyrilamine (1 µM) for both TRP channels ( Fig. 7 A ). In addition, pyrilamine (1 µM) significantly reduced the response to CA and GSK1016790A in DRG neurons pretreated with histamine for 10 min (TRPA1, 10 µM; TRPV4, 100 µM; Figs. 5 and 6 ), and histamine preincubation did not induce TRPA1 or TRPV4 sensitization in DRG neurons lacking H 1 R ( Figs. 5 and 6 ), confirming the key role of H 1 R in this process.

Fig. 8. A : representative images of murine dorsal root ganglion (DRG) neurons used to quantify expression/translocation of transient receptor potential (TRP) vanilloid 4 (TRPV4) on histamine stimulation. Left : merge bright-field + TRPV4 channel + 4′,6′-diamidino-2-phenylindole (DAPI) channel. Middle : bright-field. Right : TRPV4 channel. TRP ankyrin 1 (TRPA1; n = 19–21; B ) and TRPV4 ( n = 20–22; C ) quantification of channel translocation to the plasma membrane ( top ) and total expression in the cell ( bottom ) in cultured DRG neurons treated with histamine (TRPA1: 10 µM for 10 min; TRPV4: 100 µM overnight) are shown. Data are shown as means ± SE. * P < 0.05 with Mann-Whitney test or Student’s t -test (as appropriate). Ctrl, control; cyto, cytoplasm; mb, membrane; MGV, mean gray value.

To assess whether the increased TRPA1- and TRPV4-mediated Ca 2+ responses after histamine treatment in DRG neurons resulted from upregulation of Trpa1 and Trpv4 , we compared their mRNA expression levels in cells incubated for 10 min (10 µM, for TRPA1) or overnight (100 µM, for TRPV4) with histamine or vehicle. In line with the results obtained with rectal biopsies, no differences in mRNA expression could be detected (data not shown). Moreover, 10-min incubation with histamine did not increase translocation or the total amount of TRPA1 ( Fig. 8 ). On the other hand, overnight incubation with histamine increased TRPV4 protein expression and translocation to the membrane in DRG neurons ( Fig. 8 ), in line with previous studies ( 12 ).

Fig. 7. Sensitization of transient receptor potential (TRP) ankyrin 1 (TRPA1) and TRP vanilloid 4 (TRPV4) by irritable bowel syndrome (IBS) supernatants on dorsal root ganglion (DRG) neurons is mediated by histamine and the histamine 1 receptor (H 1 R). A : data showing the effect of overnight incubation with healthy subject (HS) supernatants ( n = 8) supplemented with histamine (+H; TRPA1, 10 µM; TRPV4, 100 µM; n = 8) or histamine and pyrilamine (+H+P; 1 µM; TRPA1, n = 5; TRPV4, n = 3) on the Ca 2+ response to cinnamaldehyde (CA; left ) and GSK1016790A ( right ). B : data showing the CA ( left ) and GSK1016790A ( right )-induced Ca 2+ response after overnight incubation with supernatant of cultured rectal biopsies of patients with IBS ( n = 8) in the presence or absence of pyrilamine (+P; 1 µM; n = 8). * P < 0.05, ** P < 0.01, Mann-Whitney U test (amplitudes).

Fig. 6. Transient receptor potential vanilloid 4 (TRPV4) is sensitized by histamine via histamine 1 receptor (H 1 R) in murine dorsal root ganglion (DRG) neurons. A : representative traces of the effect of histamine and pyrilamine on the Ca 2+ response of DRG neurons evoked by 1 µM GSK1016790A (GSK). Conditions vehicle, histamine, histamine + pyrilamine, and Hrh1 −/− were combined with the TRPV1 antagonist (SB-366791). Overnight (ON) incubation with 100 µM histamine potentiates the effect of GSK1016790A, an effect that is blocked in the presence of the H 1 R antagonist pyrilamine (1 µM). B and C : effect of histamine, pyrilamine, and DRG neurons lacking H 1 R ( Hrh1 −/− ), TRPV1 ( Trpv1 −/− ), and TRPV1V4 ( Trpv1 −/− v4 −/− ) on the amplitude of the Ca 2+ response ( B ) and the percentage of DRG neurons ( C ) responding to 1 µM GSK1016790A. Conditions vehicle, histamine, histamine + pyrilamine, and Hrh1 −/− were combined with the TRPV1 antagonist (SB-366791). Data are shown as means ± SE. ** P < 0.01 *** P < 0.001; 1-way ANOVA with Bonferroni multiple-comparison correction (Amplitudes) and Fisher exact test (percentage of responding neurons). Hist, histamine; Pyr, pyrilamine; Veh, vehicle.

Fig. 5. Transient receptor potential ankyrin 1 (TRPA1) is sensitized by histamine via histamine 1 receptor (H 1 R) in murine dorsal root ganglion (DRG) neurons. A : representative traces of the effect of histamine and pyrilamine on the Ca 2+ responses of DRG neurons to 10 µM cinnamaldehyde (CA). Histamine (10 µM) potentiates the effect of CA, an effect that is completely abolished in the presence of the H 1 R antagonist pyrilamine (1 µM). B and C : effect of histamine, pyrilamine, and DRG neurons lacking H 1 R ( Hrh1 −/− ) on the amplitude of the Ca 2+ response ( B ) and the percentage of DRG neurons ( C ) responding to 10 µM CA. Data are shown as means ± SE. *** P < 0.001, 1-way ANOVA with Bonferroni multiple-comparison correction (amplitudes) and Fisher exact test (percentage of responding neurons). Hist, histamine; Pre, CA response before incubation; Pyr., pyrilamine; Veh, vehicle.

In line with the effect of histamine on human submucosal neurons, preincubation of murine DRG neurons for 10 min with histamine resulted in an increased Ca 2+ response and number of responding neurons to CA ( Fig. 5 ) compared with vehicle. This effect was not observed for GSK1016790A (data not shown). However, longer (overnight) incubation of DRG neurons with 100 µM, but not 10 µM (data not shown), histamine resulted in an increased response to GSK1016790A ( Fig. 6 ). To confirm that GSK1016790A (1 µM) does not activate TRPV1 ( 37 ), even at high doses (1 µM), the experiments were repeated in the presence of the TRPV1 antagonist SB-366791 (1 µM) and in cells isolated from Trpv1 knockout mice ( Fig. 6, B and C ). Moreover, sensitization of TRPA1 and TRPV4 was absent in Trpa1 −/− (data not shown) and double Trpv1 −/− Trpv4 −/− knockout mice, respectively ( Fig. 6, B and C ). In keeping with these findings, overnight incubation of DRG neurons with HS supernatants supplemented with histamine increased the Ca 2+ response to both TRP agonists ( Fig. 7 A ).

Fig. 4. Rectal biopsy supernatants from patients with irritable bowel syndrome (IBS) sensitize transient receptor potential (TRP) ankyrin 1 (TRPA1) on murine dorsal root ganglion (DRG) neurons. A : effect of overnight incubation of murine DRG neurons with supernatant of cultured rectal biopsies of patients with IBS ( n = 8) or healthy subjects (HS; n = 8) on the Ca 2+ response ( left ) and percentage of responding neurons ( right ) to cinnamaldehyde (10 µM). B : data showing the effect of overnight incubation with HS ( n = 8) or IBS ( n = 8) supernatants on the Ca 2+ response ( left ) and percentage of responding neurons ( right ) to GSK1016790A (1 µM). * P < 0.05, *** P < 0.001, Mann-Whitney U test (amplitudes) or Fisher exact test (percentage of responding neurons). Data are shown as medians ± interquartile range. TRPV4, TRP vanilloid 4.

Although we showed TRP channel sensitization of submucosal neurons in IBS, it should be emphasized that these neurons are not involved in visceral pain perception. However, visceral afferent sensory neurons reside in the same environment and thus will be exposed to the same environmental triggers. To test the hypothesis that bioactive mediators in the microenvironment may also affect visceral afferents, we assessed the effect of the supernatants of biopsies on isolated murine DRG neurons. Overnight incubation of DRG neurons with IBS supernatants of six out of eight patients significantly increased the Ca 2+ response to CA compared with HS ( Fig. 4 A ). Moreover, the number of neurons responding to CA was significantly increased by supernatants of seven out of eight patients compared with HS. Similarly, IBS supernatants of two of the eight patients with IBS significantly increased the Ca 2+ response to the TRPV4 agonist GSK1016790A, whereas the supernatants of three patients with IBS activated significantly more neurons compared with those of HS ( Fig. 4 B ).

Fig. 3. Histamine sensitizes transient receptor potential (TRP) ankyrin 1 (TRPA1) and TRP vanilloid 4 (TRPV4) through histamine 1 receptor (H 1 R) in human submucosal neurons. A : representative traces of submucosal neurons in biopsies of healthy subjects (HS) on application of cinnamaldehyde (CA; 10 nM) and GSK1016790A (GSK; 1 nM) before and after incubation with histamine (Hist.; 10 µM; TRPA1, n = 7; TRPV4, n = 7) in the presence or absence of the H 1 R antagonist pyrilamine (Pyril.; 1 µM; TRPA1, n = 6; TRPV4, n = 5). Data show the effect of histamine in the absence ( B ) or presence ( C ) of pyrilamine on the amplitude of the Ca 2+ response and the percentage of responding neurons. Data are shown as medians ± interquartile range (amplitude) and means ± SE (percentage of responding neurons). * P < 0.05, Wilcoxon signed-rank test (amplitudes) and Fisher exact test (percentage of responding neurons). F 0 , baseline fluorescence.

Recently, our group ( 44 ) showed that sensitization of TRPV1 in IBS is mediated by the mast cell mediator histamine. To determine whether histamine is also involved in the sensitization of TRPA1 and TRPV4, the effect of histamine (10 µM) incubation was assessed on the response to CA (10 µM) and GSK1016790A (1 µM). Of note, rectal submucosal neurons of HS pretreated with 10 µM histamine showed increased amplitude of the Ca 2+ response and an increased percentage of neurons responding to CA (10 nM) and GSK1016790A (0.1 nM; Fig. 3, A and B ).

Then, to evaluate whether the increased response to TRPA1 and TRPV4 agonists resulted from upregulation of TRPA1 and TRPV4 , mRNA expression levels of both TRP channels were evaluated in 30 HS and 30 IBS biopsies. No differences in mRNA TRPA1 and TRPV4 expression could be detected ( Fig. 2 ). In addition, TRPA1 and TRPV4 mRNA expression was not different between IBS subtypes (data not shown). Furthermore, none of the patients with IBS with submucosal TRPA1 and/or TRPV4 sensitization ( n = 6) had increased TRPA1 and TRPV4 mRNA expression (data not shown). Taken together, these results suggest that the increased Ca 2+ response for these TRP channels is due to sensitization rather than to upregulation.

Rectal biopsies of patients with IBS and HS were collected to compare the response of submucosal neurons to the TRPA1 agonist CA (10 nM and 1 µM) and the TRPV4 agonist GSK1016790A (0.1 and 1 nM). Application of CA (10 nM and 1 µM) and GSK1016790A (0.1 and 1 nM) induced significantly higher Ca 2+ responses in submucosal neurons of patients with IBS compared with those of HS ( Fig. 1, C and D ). Furthermore, exposure to CA (10 nM and 1 µM) and GSK1016790A (0.1 and 1 nM) activated more submucosal neurons in patients with IBS than HS ( Fig. 1, C and D ).

DISCUSSION

In the present study, we provide the first evidence for TRPA1 and TRPV4 sensitization in the rectal submucosal plexus of patients with IBS, an effect mediated by the mast cell mediator histamine via activation of H 1 R. Moreover, histamine and IBS biopsy supernatants sensitized TRPA1 and TRPV4 on murine DRG neurons via H 1 R activation. These results indicate that not only TRPV1 (4, 44), but also TRPA1 and TRPV4 are involved in the pathophysiology of IBS, further underscoring the concept that histamine-mediated TRP channel sensitization is an important mechanism in IBS. Moreover, our data provide further evidence underscoring H 1 R antagonism (44) as a novel therapeutic approach for IBS.

Although the exact pathophysiological mechanisms in IBS are still incompletely understood, upregulation of TRP channel expression or altered TRP channel function have been shown to underlie aberrant visceral pain perception in preclinical models (10, 11, 20, 39, 40). Moreover, we (4, 40, 44) recently showed that TRPV1 sensitization plays an important role in VHS in patients with IBS. However, to date, data supporting the involvement of sensitization of other TRP channels in IBS is lacking. In the present study, we provide evidence for TRPA1 and TRPV4 sensitization in IBS. Application of the TRPA1 agonist CA and TRPV4 agonist GSK1016790A induced significantly higher Ca2+ responses in rectal submucosal neurons of patients with IBS compared with those of HS. To what extent these data support a role in abnormal pain perception in IBS can be questioned, especially as, to date, submucosal neurons have not been shown to be directly involved in visceral pain transmission. Nevertheless, our findings indicate that the gut microenvironment contains bioactive mediators that significantly affect neural signaling. Afferent nerve endings of nociceptive DRG neurons, transmitting pain signals to the spinal cord, reside in the same sensitizing microenvironment as submucosal neurons and thus may be similarly affected. We indeed recently demonstrated sensitization of murine colonic afferents to mechanical probing by IBS supernatant (4), suggesting pronociceptive changes in the gut microenvironment. Along the same line, intracolonic administration of diarrhea-predominant IBS biopsy supernatants induced VHS in mice through a TRPV4-dependent mechanism (13). Moreover, increased neuronal excitability of DRG neurons in response to IBS supernatant has been repeatedly reported (8, 13, 22, 38, 44). In the present study, we provide additional evidence that IBS supernatant contains mediators sensitizing not only TRPV1 (44), but also TRPA1 and TRPV4, clearly illustrating that pronociceptive mediators are released by biopsies collected from patients with IBS. We, therefore, propose that these mediators affect the excitability not only of submucosal neurons in IBS, but also of visceral afferents residing in the same microenvironment. To date, we have only access to human submucosal neurons to unravel the underlying mechanism in patients with IBS. Human nociceptive neurons including colonic/rectal afferent nerves and DRG from surgical resections can be collected from patients undergoing surgery; however, these patients do not suffer from IBS. Thus these tissues can merely be used to characterize human TRP channels and investigate TRP channel sensitization by inflammatory mediators, including histamine. Interestingly, a recent study showed decreased human serosal nociceptor mechanosensitivity after incubation with the TRPV4 antagonist HC067047, further underscoring the role of TRPV4 in human visceral pain perception (27). Taken together, we propose that sensitization of TRPV1, TRPV4, and TRPA1 represents one of the mechanisms contributing to aberrant pain signaling in IBS.

A plethora of proinflammatory mediators induce modulation of TRP channels on peripheral sensory nerve endings leading to increased pain perception (31), but there is increasing evidence that histamine could be particularly important. Recently, we (44) showed that treatment of patients with IBS with H 1 R antagonist ebastine improved abdominal pain, possibly by blocking histamine-mediated sensitization of TRPV1. Here, we show that histamine is also involved in TRPV4 and TRPA1 sensitization. The observed increase in TRPA1 and TRPV4 Ca2+ responses reported here can result from increased synthesis of the TRP channels, translocation of more receptors to the cell membrane, or phosphorylation and subsequent sensitization. It should be emphasized that not only neurons express TRP channels, but also we failed to show an increase in TRPA1 and TRPV4 mRNA levels in mucosal biopsies from IBS compared with HS. Moreover, mRNA expression levels of TRPA1 and TRPV4 were not altered in murine DRG neurons incubated with histamine. In contrast, using immunohistochemistry, we were able to demonstrate that histamine promoted translocation of TRPV4 to the cell membrane of murine DRG neurons, as previously demonstrated (12). Cenac et al. (12) further showed that TRPV4 plasma membrane relocation is mediated via a specific MAPKK pathway. Of interest, we did not observe translocation of TRPA1 in response to histamine, indicating that sensitization of TRPA1 most likely explains TRPA1 potentiation. Sensitization of TRP channels via coupling with G protein-coupled receptors such as histamine receptors (3, 6, 43) has been repeatedly demonstrated in sensory neurons, a mechanism mediated by stimulation of the phospholipase C/protein kinase C signaling pathway with phosphorylation of the TRP channels (12, 24, 34, 35). Of interest, the increased TRPV4 Ca2+ response induced by histamine is also dependent on this pathway (12), suggesting that increased TRPV4 signaling might result from both receptor relocation and sensitization.

Of interest, we observed that sensitization of TRPV4 requires prolonged incubation with histamine. Indeed, sensitization of TRPA1 and TRPV1 (44) in murine DRG neurons was already induced with 10 µM histamine after 10 min, whereas only overnight incubation with 100 µM histamine sensitized TRPV4. In line with our results, Cenac et al. (12) showed TRPV4 sensitization in DRG neurons but only with higher (50 and 100 µM) concentrations of histamine. Moreover, potentiation of TRPV4 on murine DRG neurons by other inflammatory mediators was different compared with TRPV1 and TRPA1 sensitization. For example, TRPV4 potentiation by serotonin required a higher dose (12) compared with serotonin-induced TRPV1 sensitization on DRG neurons (36). On the other hand, TRPV4 sensitization via protease-activated receptor 2 was induced after a longer incubation period of protease-activated receptor 2 agonists (21) compared with TRPV1 (2) and TRPA1 (18). These results suggest that sensitization of TRPV4 requires a longer incubation period and/or a higher concentration of mediators such as histamine compared with TRPV1 (44) and TRPA1 and might explain why only two out of eight IBS supernatants were able to sensitize TRPV4. Further investigation to explain the differences in TRPV1, TRPA1, and TRPV4 sensitization is, however, warranted.

Taken together, our data indicate that the intestinal microenvironment in IBS contains histamine and/or histamine metabolites, which, in parallel to TRPV1 (4, 44), sensitize TRPA1 and TRPV4 via H 1 R activation, contributing to VHS in IBS. These data further underscore H 1 R antagonism as potential treatment for IBS. Stratifying patients with IBS for a specific treatment is of particular importance in IBS as the patient population is very heterogeneous and includes patients with different underlying mechanisms. This most likely explains why not all IBS supernatants were able to sensitize TRP channels and why not all patients with IBS respond to H 1 R antagonism (44). Therefore, identifying an indicator that can predict the therapeutic response to H 1 R antagonism would represent a major step forward. Interestingly, a recent clinical trial showed that urinary histamine concentrations could predict the therapeutic response to low fermentable oligosaccharides, disaccharides, and monosaccharides and polyols diet in patients with IBS (28). Together with our results, measuring concentrations of histamine and/or its metabolites in patient samples could be helpful in the future to predict whether this patient would respond to H 1 R antagonism counteracting TRP channel sensitization.