Activation of pruritogenic TGR5, MrgprA3, and MrgprC11 on colon-innervating afferents induces visceral hypersensitivity

Itch induces scratching that removes irritants from the skin, whereas pain initiates withdrawal or avoidance of tissue damage. While pain arises from both the skin and viscera, we investigated whether pruritogenic irritant mechanisms also function within visceral pathways. We show that subsets of colon-innervating sensory neurons in mice express, either individually or in combination, the pruritogenic receptors Tgr5 and the Mas-gene–related GPCRs Mrgpra3 and Mrgprc11. Agonists of these receptors activated subsets of colonic sensory neurons and evoked colonic afferent mechanical hypersensitivity via a TRPA1-dependent mechanism. In vivo intracolonic administration of individual TGR5, MrgprA3, or MrgprC11 agonists induced pronounced visceral hypersensitivity to colorectal distension. Coadministration of these agonists as an “itch cocktail” augmented hypersensitivity to colorectal distension and changed mouse behavior. These irritant mechanisms were maintained and enhanced in a model of chronic visceral hypersensitivity relevant to irritable bowel syndrome. Neurons from human dorsal root ganglia also expressed TGR5, as well as the human ortholog MrgprX1, and showed increased responsiveness to pruritogenic agonists in pathological states. These data support the existence of an irritant-sensing system in the colon that is a visceral representation of the itch pathways found in skin, thereby contributing to sensory disturbances accompanying common intestinal disorders.


Supplementary Figure 1: Functional co-expression of pruritogenic G protein-coupled receptors (GPCRs) and TRP channels using Ca 2+ imaging in mouse colon-innervating DRG neurons.
All data represent the percentage of DiI positive (colon-innervating) DRG neurons from individual coverslips For analysis, the raw EMG signal was integrated and the VMR was expressed as the AUC of the EMG signal during distension, corrected for the AUC during baseline.

Supplementary Figure 9: Application of TGR5 agonists and MRGPR agonists activates colonic afferents.
Representative examples showing that A) oleanoic acid (OA; 100µM) induces action potential firing in a subset of nociceptors from CVH mice. This was not observed with OA application to nociceptors from control mice (not shown). B) CCDC (100µM) application evoked responses in a small subset of colonic nociceptors from healthy mice with enhanced responses to CCDC in nociceptors from C) CVH and D) TGR5 over-expressing  Figure 11H, and Figure 11I.

Ex vivo single fibre colonic nociceptor recordings and responses to pruritogens
Tissues for ex vivo colonic nociceptor recordings were from healthy or CVH (6-12,16) C57BL/6J mice or Trpa1 -/-(1,2), Tgr5 -/- (3,4), Mrgpr cluster -/- (5) or Tgr5-Tg (3,4) mice. On the day of experimentation, mice were humanely killed by CO2 inhalation and the colon/rectum (5-6 cm) and attached splanchnic nerves were removed and afferent recordings from splanchnic nerves were performed as described previously (11,17,18 CaCl2, 11.1 D-glucose), and bubbled with carbogen (95% O2, 5% CO2) at a temperature of 34°C. All solutions contained the L-type calcium channel antagonist nifedipine (1µM) to suppress smooth muscle activity and the prostaglandin synthesis inhibitor indomethacin (3µM) to block endogenous prostaglandin production. The nerve bundle was extended into a paraffin-filled recording compartment in which finely dissected strands were laid onto a mirror, and single fibres placed on the platinum recording electrode. Action potentials, generated by mechanical stimuli to the receptive field, were recorded by a differential amplifier, filtered and sampled (20 kHz) using a 1401 interface (Cambridge Electronic Design, Cambridge, UK) and stored on a PC for off-line analysis. Recordings were made using standard protocols (8,9,17). Receptive fields were identified by systematically stroking the mucosal surface of the colon with a stiff brush to activate all subtypes of mechanoreceptors. Categorization of afferent properties was in accordance with our previously published classification system (2,(6)(7)(8)(9)(10)(11)17,19). Once identified, receptive fields were tested with three distinct mechanical stimuli to enable classification: static probing with calibrated von Frey hairs (vfh) (2 g force; applied 3 times for a period of 3 seconds), mucosal stroking with calibrated vfh (10 mg force; applied 10 times) or circular stretch (5 g; applied for a period of 1 min). We recorded from serosal afferents, also termed vascular afferents (20), from the splanchnic pathway. These colonic afferents have high-mechanical activation thresholds and respond to noxious distension (40 mmHg), stretch (≥7 g) or vfh filaments (2 g) but not to fine mucosal stroking (10 mg vfh) (9,10,17,21). They express and respond to agonists of algesic channels and receptors including TRPV1 (18)  to Microsoft Excel® and relative abundance to endogenous control genes estimated using delta Cq method, as described previously (2,22,28).

Retrograde tracing to identify DRG neurons innervating the colon
Mice (C57BL/6) were anesthetized with 5% isoflurane. The descending colon was exposed through a midline incision and the overlying viscera were kept moist with saline-soaked gauze.
Dicarbocyanine dye,1,1-dioctadecyl-3,3,3,3-tetramethlindocarbocyanine methanesulfonate (DiI, 2% in ethanol; Invitrogen, Carlsbad, CA) was injected into in the wall of the colon (3-4 sites, 2 μL per site) using a Hamilton microliter syringe and a 32-gauge needle. To prevent leakage to other organs, the needle was held in place for 30 seconds after the injection, and any excess dye was carefully removed.
The abdominal wall and skin were closed, and animals were allowed to recover. After 7-10 days, animals were killed and DRG (T10-L1) were removed. Alternatively, cholera toxin subunit B conjugated to AlexaFluor 488 (CTB-488); Invitrogen, Carlsbad, CA) was injected at three sites sub-serosally within the wall of the distal colon of healthy control or CVH mice mice instead of DiI (2,7,9,13). After 4 days, animals were humanely killed by CO2 inhalation for subsequent thoracolumbar (TL: T10-L1) DRG removal and dissociation.

Single cell RT-PCR of pruritogenic targets within colon-innervating DRG neurons from healthy control and CVH mice
After retrograde tracing, DRG were collected from thoracolumbar regions from healthy and CVH mice and were dissociated as previously described (7,29). Dissociated neurons were plated onto poly D-lysine-(0.1 mg.ml -1 ) and laminin-(0.004 mg.ml -1 ) coated cover slips and were incubated in

Visualisation of pERK activated neurons within the dorsal horn of the spinal cord following intra-colonic application of pruritogens and noxious colorectal distension (CRD).
Healthy control or CVH C57BL/6J mice or Trpa1 -/-(1,2), Tgr5 -/- (3,4) or Tgr5-Tg (3,4) mice were fasted overnight with free access to water and a 5% glucose solution, ensuring faecal pellets were absent from the colorectum for colorectal distension (CRD). In the first series of experiments mice were briefly anaesthetized with isoflurane anaesthetic and a 100 µl enema of CCDC (100 µM) or saline administered intra-colonically via a catheter. Subsequently, a 4 cm balloon catheter was inserted into the perianal canal and secured to the tail so that the start of the balloon sat 0.5 cm from the anal opening and the tube secured to the tail (7-9). The balloon catheter was attached to a sphygmomanometer pressure gauge and a 20ml syringe via a three-way stopcock. Mice were removed from the isoflurane chamber and upon regaining consciousness the balloon was distended for 10 seconds to a pressure of 40 mmHg, applied via the syringe. This pressure was released, and the balloon deflated (0 mmHg) for 5 seconds. This process was repeated 5 times, as per our previous studies (7)(8)(9)13 hours, before block freezing in 100% OCT. Frozen sections (12μm) were cut using a cryostat and placed onto gelatin-coated slides. Immunohistochemistry for pERK was performed in a paired fashion, with tissue from healthy and CVH mice exposed to pruritogenic compound or saline run simultaneously.

In vivo visceromotor responses (VMR) to colorectal distension (CRD) following intra-colonic administration of pruritogens
Noxious distension of the colorectum triggers the VMR, a nociceptive brainstem reflex consisting of the contraction of the abdominal muscles (30). Using abdominal electromyography (EMG), this technique allows assessment of visceral sensitivity in vivo in fully awake animals (6,12,23,31). At least three days prior to the VMR and under isoflurane anaesthesia, the bare endings of two Teflon-

In vivo assessment of animal behaviour
Behavioural testing was conducted on male healthy control mice (aged 12-14 weeks) with an average body weight of ~28 g (24 -32g) on the experimental day. Mice were transferred in their home cage to a temperature-controlled test room (24 ± 1 • C) and allowed to acclimatize for at least 10 min prior to testing. All experiments were performed during the light phase at the same time each day

Quantitative RT-PCR analysis of pruritogenic ion channels and receptors in human colonic biopsies and human DRG
For human colonic biopsy studies, RNA was extracted from each biopsy using a PureLink RNA Mini kit (Invitrogen, Sydney, Australia, cat# 12183025) including an on-column DNAse treatment (LifeTechnologies, Sydney, Australia cat#12185-010) according to manufacturer's instructions. RNA was stored at -80°C in aliquots of 1-3 µL. Quantity and purity were checked using a NanoDrop spectrometer.
RNA quality was assessed using a 2100 Bioanalyzer (Agilent) and all samples ranged from a RIN of 6 -9.
None of the measured samples showed a DNA peak in the profile.
For whole human DRG studies (7)

Single-cell PCR analysis of pruritogenic ion channels and receptors within individual human DRG neurons using RT-PCR
For single cell studies human DRG were dissociated, plated and single neurons picked using precision pulled and fire polished glass capillaries and a high-fidelity micromanipulator (7) Images were acquired at 0.2Hz for 3 minutes with the following protocol: 1 minute of baseline, 1 minute of agonist application and 1 minute of washout. Agonists were applied in the following sequence and concentrations: 100 µM CCDC (BioVision), 1 µM Chloroquine (Sigma), 2 µM BAM8-22 (Tocris) and 2 µM NPFF (Tocris). An unrecorded 5 minutes washout period was applied between each agonist application. As positive controls, 100 nM Capsaicin (Sigma) and 50 µM AITC (Sigma) were applied for 20 seconds each at the end of the experiment, with a 20-minute washout interval between each application.
In order to mimic a pathological state, we incubated neurons with an "inflammatory soup"

Study approval
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