Shaoyong Yu

TRPA1 in mast cell activation-induced long-lasting mechanical hypersensitivity of vagal afferent C-fibers in guinea pig esophagus

Sensitization of esophageal sensory afferents by inflammatory mediators plays an important role in esophageal nociception. We have shown esophageal mast cell activation induces long-lasting mechanical hypersensitivity in vagal nodose C-fibers. However, the roles of mast cell mediators and downstream ion channels in this process are unclear. Mast cell tryptase via protease-activated receptor 2 (PAR2)-mediated pathways sensitizes sensory nerves and induces hyperalgesia. Transient receptor potential A1 (TRPA1) plays an important role in mechanosensory transduction and nociception. Here we tested the hypothesis that mast cell activation via a PAR2-dependent mechanism sensitizes TRPA1 to induce mechanical hypersensitivity in esophageal vagal C-fibers. The expression profiles of PAR2 and TRPA1 in vagal nodose ganglia were determined by immunostaining, Western blot, and RT-PCR. Extracellular recordings from esophageal nodose neurons were performed in ex vivo guinea pig esophageal-vagal preparations. Action potentials evoked by esophageal distention and chemical perfusion were compared. Both PAR2 and TRPA1 expressions were identified in vagal nodose neurons by immunostaining, Western blot, and RT-PCR. Ninety-one percent of TRPA1-positive neurons were of small and medium diameters, and 80% coexpressed PAR2. Esophageal mast cell activation significantly enhanced the response of nodose C-fibers to esophageal distension (mechanical hypersensitivity). This was mimicked by PAR2-activating peptide, which sustained for 90 min after wash, but not by PAR2 reverse peptide. TRPA1 inhibitor HC-030031 pretreatment significantly inhibited mechanical hypersensitivity induced by either mast cell activation or PAR2 agonist. Collectively, our data provide new evidence that sensitizing TRPA1 via a PAR2-dependent mechanism plays an important role in mast cell activation-induced mechanical hypersensitivity of vagal nodose C-fibers in guinea pig esophagus.

Antigen inhalation induces mast cells and eosinophils infiltration in the guinea pig esophageal epithelium involving histamine-mediated pathway.

Antigen challenge in sensitized guinea pig esophagus in vitro induces mast cell degranulation and histamine release. This study tests the hypothesis that antigen inhalation in vivo induces infiltration of the esophageal epithelium by mast cells and eosinophils via a histamine pathway. Actively sensitized guinea pigs were exposed to inhaled 0.1% ovalbumin. One or 24 h after inhalation exposure, the esophagus was processed for immunofluorescent staining of mast cell tryptase and eosinophil major basic protein (MBP). Additional animals were pretreated with thioperamide, a histamine H4/H3 receptor antagonist. Total tryptase- and MBP-labeled cells and percent of positive cells in the epithelial layer were counted. The total number of mast cells was unchanged after inhalation challenge, but the percentage in the epithelium increased 1 h after challenge. The total number of eosinophils increased 1 h after challenge, and the percentage migrating to the epithelium increased by 24 h after challenge. Mast cell migration into the mucosal epithelium preceded that of eosinophils. Thioperamide inhibited mast cell and eosinophil migration. In conclusion, antigen inhalation in sensitized animals induces mast cells and eosinophils to infiltrate in the esophageal epithelium via histamine-mediated mechanism.

5‐Hydroxytryptamine selectively activates the vagal nodose C‐fibre subtype in the guinea‐pig oesophagus

Abstract  The afferent neurons innervating the oesophagus originate from two embryonic sources: neurons located in vagal nodose ganglia originate from embryonic placodes and neurons located in vagal jugular and spinal dorsal root ganglia (DRG) originate from the neural crest. Here, we address the hypothesis that 5‐hydroxytryptamine (5‐HT) differentially stimulates afferent nerve subtypes in the oesophagus. Extracellular recordings of single unit activity originating from nerve terminals were made in the isolated innervated guinea‐pig oesophagus. Whole cell patch clamp recordings (35 °C) were made from the primary afferent neurons retrogradely labelled from the oesophagus. 5‐Hydroxytryptamine (10 μmol L−1) activated vagal nodose C‐fibres (70%) in the oesophagus but failed to activate overtly vagal jugular nerve fibres and oesophagus‐specific spinal DRG neurons. The response to 5‐HT in nodose C‐fibre nerve terminals was mimicked by the selective 5‐HT3 receptor agonist 2‐methyl‐5‐HT (10 μmol L−1) and nearly abolished by the 5‐HT3 receptor antagonists ondansetron (10 μmol L−1) and Y‐25130 (10 μmol L−1). In patch clamp studies, 2‐methyl‐5‐HT (10 μmol L−1) activated a proportion of isolated oesophagus‐specific nodose capsaicin‐sensitive neurons (putative cell bodies of nodose C‐fibres). We conclude that the responsiveness to 5‐HT discriminates placode‐derived (vagal nodose) C‐fibres from the neural crest‐derived (vagal jugular and spinal DRG) afferent nerves in the oesophagus. The response to 5‐HT in nodose C‐fibres is mediated by the 5‐HT3 receptor in their neuronal membrane.

Prostaglandin D2 receptor d‐type prostanoid receptor 2 mediates eosinophil trafficking into the esophagus

Eosinophilic esophagitis is characterized by eosinophil-predominant inflammation in the esophagus. How eosinophils migrate and infiltrate into the esophagus, however, is less clear. Our previous study demonstrated that mast cell activation led to eosinophil infiltration in the esophagus. Prostaglandin D2 (PGD2) is an important mediator released from activated mast cells. The present study aims to determine whether PGD2 induces eosinophil infiltration into the esophagus via a d-type prostanoid receptor 2 (DP2) receptor-dependent mechanism. Using an in vivo guinea pig model, PGD2, d-type prostanoid receptor 1 (DP1) agonist, or DP2 agonist were injected into the esophagus. Esophageal tissues were removed 2 hours after injections and proceeded to either hematoxylin-eosin (HE) staining or immunofluorescent staining of eosinophil major basic protein (MBP) to compare each treatment-induced eosinophil infiltration in the esophagus. In a separate study, ovalbumin (OVA)-sensitized guinea pigs were pretreated with either DP2 or DP1 antagonists, followed by inhalation of OVA to induce mast cell activation. Esophageal tissues were then processed for immunofluorescent staining of MBP. PGD2 injection in the esophagus led to an increase of eosinophil infiltration in esophageal epithelium at the injection site as revealed by HE staining. Increased infiltration of eosinophils was further confirmed by the increased presence of MBP-labeled immunopositive (MBP-LI) cells in esophageal epithelium. Injection with DP2 agonist 15(R)-PGD2, but not DP1 agonist BW 245C, mimicked the PGD2-induced response. In OVA-sensitized animals, antigen inhalation increased MBP-LI cells in esophageal epithelium. Pretreatment with DP2 antagonist BAY-u3405, but not DP1 antagonist BW 868C, inhibited the antigen inhalation-induced increase of MBP-LI cells in esophageal epithelium. These data support the hypothesis that PGD2 induces eosinophil trafficking into the esophageal epithelium via a DP2-mediated pathway, suggesting a role of DP2 antagonist in the prevention of eosinophilic esophagitis.

ERK1/2 signaling pathway in mast cell activation-induced sensitization of esophageal nodose C-fiber neurons

Sensitization of esophageal nociceptive afferents by inflammatory mediators plays an important role in esophageal inflammatory nociception. Our previous studies demonstrated that esophageal mast cell activation increases the excitability of esophageal nodose C-fibers. But the intracellular mechanism of this sensitization process is still less clear. We hypothesize that extracellular signal-regulated kinases 1 and 2 (ERK1/2) signaling pathway plays an important role in mast cell activation-induced sensitization of esophageal nodose C-fiber neurons. Mast cell activation and in vivo esophageal distension-induced phosphorylations of ERK1/2 were studied by immuno-staining and Western blot in esophageal nodose neurons. Extracellular recordings were performed from nodose neurons using ex vivo esophageal-vagal preparations with intact nerve endings in the esophagus. Nerve excitabilities were compared by action potentials evoked by esophageal distensions before and after mast cell activations with/without pretreatment of mitogen-activated protein kinases (MAPK)/ERK kinase inhibitor U0126. The expressions of phospho-ERK1/2 (p-ERK1/2) in the same nodose ganglia were then studied by Western blot. Mast cell activation enhances in vivo esophageal distension-induced phosphorylation of ERK1/2 in nodose neurons. This can be prevented by pretreatment with mast cell stabilizer cromolyn. In ex vivo esophageal-vagal preparations, both mast cell activation and proteinase-activated receptor 2 (PAR2)-activating peptide perfusion increases esophageal distension-induced mechano-excitability of esophageal nodose C-fibers and phosphorylation of ERK1/2 in nodose neurons. Pretreatment with MAPK/ERK kinase inhibitor U0126 prevents these potentiation effects. Collectively, our data demonstrated that mast cell activation enhances esophageal distension-induced mechano-excitability and phosphorylation of ERK1/2 in esophageal nodose C-fiber neurons. This reveals a new intracellular pathway of esophageal peripheral sensitization and inflammatory nociception.

Effect of synthetic cationic protein on mechanoexcitability of vagal afferent nerve subtypes in guinea pig esophagus

Eosinophilic esophagitis is characterized by increased infiltration and degranulation of eosinophils in the esophagus. Whether eosinophil-derived cationic proteins regulate esophageal sensory nerve function is still unknown. Using synthetic cationic protein to investigate such effect, we performed extracellular recordings from vagal nodose or jugular neurons in ex vivo esophageal-vagal preparations with intact nerve endings in the esophagus. Nerve excitabilities were determined by comparing action potentials evoked by esophageal distensions before and after perfusion of synthetic cationic protein poly-L-lysine (PLL) with or without pretreatment with poly-L-glutamic acid (PLGA), which neutralized cationic charges of PLL. Perfusion with PLL did not evoke action potentials in esophageal nodose C fibers but increased their responses to esophageal distension. This potentiation effect lasted for 30 min after washing out of PLL. Pretreatment with PLGA significantly inhibited PLL-induced mechanohyperexcitability of esophageal nodose C fibers. In esophageal nodose Aδ fibers, perfusion with PLL did not evoke action potentials. In contrast to nodose C fibers, both the spontaneous discharges and the responses to esophageal distension in nodose Aδ fibers were decreased by perfusion with PLL, which can be restored after washing out PLL for 30-60 min. Pretreatment with PLGA attenuated PLL-induced decrease in spontaneous discharge and mechanoexcitability of esophageal nodose Aδ fibers. In esophageal jugular C fibers, PLL neither evoked action potentials nor changed their responses to esophageal distension. Collectively, these data demonstrated that synthetic cationic protein did not evoke action potential discharges of esophageal vagal afferents but had distinctive sensitization effects on their responses to esophageal distension.

200 Allergen Challenge Sensitizes TRPA1 in Vagal Sensory Neurons and Afferent C-Fibers in Guinea Pig Esophagus

Introduction: Eosinophilic esophagitis (EoE) often presents with symptoms that are considered to relate to esophageal dysfunctions. Our previous study demonstrated that TRPA1 plays important role in tissue mast cell activation-induced increase in the excitability of esophageal vagal nodose C fibers. The present study aims to determine whether prolonged antigen exposure in vivo sensitizes TRPA1 in esophageal vagal afferents in a guinea pig model of EoE. Method: Antigen challenge-induced responses in esophageal mucosa were first studied by histological assessments and Ussing chamber methods. TRPA1 functions in vagal sensory neurons were then studied by calcium imaging and by whole-cell patch clamp recordings in DiI-labeled esophageal nodose and jugular neurons. Extracellular single-fiber recordings were performed in nodose and jugular C-fiber neurons using ex vivo esophagealvagal preparations with intact nerve endings in the esophagus. Results: (1) Prolonged antigen challenge (0.1% Ovalbumin, 30-second/day for 2-week) in antigen-sensitized guinea pigs increased inflammation scores from 2.0±0.47 (naive) to 4.33±0.27 (OVA-2w) in the esophagus (p<0.05). OVA challenge also increased the infiltrations of both mast cells (from 8.2±2.1 to 63.5±4.5 in mucosa, and from 14.0±2.4 to 43.1±4.5 in muscle layers, both p<0.05) and eosinophils (from 2.6±0.9 to 63.5±20.0 in mucosa, and from 0 to 5.2±2.55/ in muscle layers, both p<0.05) in the cross-sections of the esophagus. (2) Antigen challenge decreased the transepithelial resistance in esophageal epithelium from 564.7±63.4 V·cm2 to 356.0±45.5V·cm2 (p<0.05). (3) Antigen challenge increased TRPA1 agonist AITC-induced calcium influx in both nodose (from 43.5% to 66.7%) and jugular neurons (from 38.9% to 51.9%)(both p<0.05). (4) Antigen challenge increased current density elicited by TRPA1 agonist AITC in DiI-labeled esophageal nodose neurons (naive vs OVA-2w: 24.3±5.4 vs 59.7 ±4.7 pA/pF, p<0.05) and jugular neurons (naive vs OVA-2w: 31.5±5.3 vs 65.8 ±6.2 pA/pF, p<0.05). (5) In naive animals, intra-esophageal infusion of TRPA1 agonist AITC did not evoke action potential discharge in either esophageal nodose or jugular C fibers. After OVA challenge, intra-esophageal infusion of AITC was able to evoke action potential discharges in both nodose C fibers (baseline vs AITC: 0.75±0.25 Hz vs 4.63±1.03 Hz, p<0.01, n=8) and Jugular C fibers (baseline vs AITC: 0.88±0.25 Hz vs 2.5±0.38Hz, p<0.01, n=8). Conclusion: These results demonstrated that prolonged antigen challenge induced allergic inflammation, decreased epithelial barrier resistance, and sensitized TRPA1 in vagal nociceptive neurons and afferent C fibers in the esophagus. These changes enabled intra-esophageal noxious chemical to activate esophageal nociceptor. This novel finding may help to better understand esophageal dysfunction in EoE.

426 Allergic Inflammation-Induced Structural and Functional Changes in Esophageal Epithelium in a Guinea Pig Model of Eosinophilic Esophagitis

Background: Neuro-immune interaction has evolved as an integral part in the pathophysiology of gut and crosstalk between nerves andmast cells is a typical example of such interactions. We have already reported that CGRP-immunoreactive intrinsic nerve fibers are specifically increased along with the development of food allergy in the colonic mucosa of our food allergy model mice. Furthermore, our previous studies have provided vivid evidences of mucosal mast cells (MMCs) juxtaposed with CGRP-positive enteric nerve fibers in the colonic mucosa of food allergy model mice. IgE-antigen stimulation has not only documented to cause degranulation in mast cells but also, recently, reported to cause activation of neurons in superior cervical ganglion. However, no study so far employed isolated enteric neurons and MMCs to elaborate this interaction which closely mimics to the pathophysiological state of the gut in the allergic condition. In the present study, we cultured both isolated enteric neurons and MMCs and examine their interaction. Material & methods: Enteric neurons were isolated from small intestine of BALB/c (4 to 6 weeks old) and examined by anti-β3tubulin antibody (anti-pan-neuronal marker antibody). Presence of high-affinity IgE receptors (FceRIs) on myentric neurons was examined in longitudinal muscle/myenteric plexus (LMMP) preparations by immunohistochemistry (IHC). Mucosal type bone marrow-derived mast cells (mBMMCs) were prepared from the femurs of BALB/c mice using four cytokines (SCF, IL-3, IL-9 and TGF-β1) and degranulation was performed by incubation with IgEantigen (IgE-DNP) to give mast cell juice (MCJ). Isolated enteric neurons were stimulated with either IgE-DNP or MCJ and analyzed by calcium imaging using fluo-8, a fluorescent calcium indicator. Finally, isolated enteric neurons were co-incubated with IgE-pretreated mBMMCs followed by stimulation with DNP. Only isolated enteric neurons were loaded with Fluo-8. Results: IHC studies revealed the presence of FceRI on the cell body of myentric neurons. Stimulation of isolated enteric neurons with IgE-DNP demonstrated an increase in intracellular calcium concentration ([Ca2+]i). Similarly, treatment of isolated enteric neurons with MCJ also led to an increase in [Ca2+]i. Furthermore, stimulation with DNP in an isolated enteric neuron/IgE-pretreated mBMMC co-culture condition produced an elevation of [Ca2+]i in isolated enteric neurons. Conclusion: We demonstrated here for the first time the activation of isolated enteric neurons by IgE-antigen, MCJ, and activated mBMMCs via calcium imaging. Therefore, we infer both direct and indirect involvement of crosstalk between enteric neurons and mucosal mast cells in allergic and inflammatory/ functional diseases of gut like food allergy or irritable bowel syndrome.

Mo2033 Allergic Inflammation-Induced Up-Regulation of Acid and TRPV1 Sensing in Vagal Afferent Neurons in a Guinea Pig Model of Eosinophilic Esophagitis

pathways downstream of BDNF in bladder afferent neuronal activation in trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats. The METHODS used include induction of colitis with intracolonic instillation of TNBS (1.5 mL/kg of 60 mg/mL solution in 50 % EtOH) and Fast Blue labeling of bladder afferent neurons in these animals. TrkB was examined by immunohistochemistry and polymerase chain reaction (PCR). The intracellular signaling molecules downstream of BDNF including phospholipase C (PLC)γ, Akt, extracellular signalregulated kinase (ERK), and cAMP response element-binding (CREB) were examined by immunohistochemistry in bladder afferent neurons. We also used single cell calcium imaging and cultural of DRG neuron and glia for ex vivo studies. Our RESULTS showed that TrkB was expressed in small to medium-sized DRG neurons; in comparison, TrkB was not expressed by DRG glial cells. This was consistent with that BDNF (50 ng/mL) stimulation increased intracellular calcium level in cultured small to medium-sized DRG neurons but not in DRG glial cells, and a selective TrkB agonist 7,8-Dihydroxyflavone (1 μM) had the same effect in these cells. At 7 days of colitis, the elevated level of TrkB in bladder afferent neurons may enhance the responsiveness of these neurons to BDNF. Among the intracellular signaling molecules downstream of BDNF/TrkB, the expression level of PLCγ (not Akt or ERK) was increased in specifically labeled bladder afferent neurons during colitis, suggesting an involvement of calcium pathways in colitis-induced bladder afferent activation. At 7 days of colitis, the phosphorylation (activation) level of CREB, a calcium-dependent transcription factor, was also increased in bladder afferent neurons. BDNF stimulation of DRG explants increased the level of phospho-CREB in DRG neurons. CONCLUSION: BDNF-mediated bladder overactivity during colitis involves activation of the PLCγ/calcium pathways in bladder afferent neurons.

Prostaglandin D2 receptord-type prostanoid receptor 2 mediates eosinophil trafficking into the esophagus: PGD2 in esophageal eosinophil trafficking

Eosinophilic esophagitis is characterized by eosinophil-predominant inflammation in the esophagus. How eosinophils migrate and infiltrate into the esophagus, however, is less clear. Our previous study demonstrated that mast cell activation led to eosinophil infiltration in the esophagus. Prostaglandin D2 (PGD2) is an important mediator released from activated mast cells. The present study aims to determine whether PGD2 induces eosinophil infiltration into the esophagus via a d-type prostanoid receptor 2 (DP2) receptor-dependent mechanism. Using an in vivo guinea pig model, PGD2, d-type prostanoid receptor 1 (DP1) agonist, or DP2 agonist were injected into the esophagus. Esophageal tissues were removed 2 hours after injections and proceeded to either hematoxylin-eosin (HE) staining or immunofluorescent staining of eosinophil major basic protein (MBP) to compare each treatment-induced eosinophil infiltration in the esophagus. In a separate study, ovalbumin (OVA)-sensitized guinea pigs were pretreated with either DP2 or DP1 antagonists, followed by inhalation of OVA to induce mast cell activation. Esophageal tissues were then processed for immunofluorescent staining of MBP. PGD2 injection in the esophagus led to an increase of eosinophil infiltration in esophageal epithelium at the injection site as revealed by HE staining. Increased infiltration of eosinophils was further confirmed by the increased presence of MBP-labeled immunopositive (MBP-LI) cells in esophageal epithelium. Injection with DP2 agonist 15(R)-PGD2, but not DP1 agonist BW 245C, mimicked the PGD2-induced response. In OVA-sensitized animals, antigen inhalation increased MBP-LI cells in esophageal epithelium. Pretreatment with DP2 antagonist BAY-u3405, but not DP1 antagonist BW 868C, inhibited the antigen inhalation-induced increase of MBP-LI cells in esophageal epithelium. These data support the hypothesis that PGD2 induces eosinophil trafficking into the esophageal epithelium via a DP2-mediated pathway, suggesting a role of DP2 antagonist in the prevention of eosinophilic esophagitis.