Quantitative Characterization of the Neuropeptide Level Changes in Dorsal Horn and Dorsal Root Ganglia Regions of the Murine Itch Models

Emily G Tillmaand, Krishna D B Anapindi, Eduardo De La Toba, Changxiong J Guo, Jessica Krebs, Ashley E Lenhart, Qin Liu, Jonathan V Sweedler

Abstract

Chronic itch can be extremely devastating and, in many cases, difficult to treat. One challenge in treating itch disorders is the limited understanding of the multitude of chemical players involved in the communication of itch sensation from the peripheral to central nervous system. Neuropeptides are intercellular signaling molecules that are known to be involved in the transmission of itch signals from primary afferent neurons, which detect itch in the skin, to higher-order circuits in the spinal cord and brain. To investigate the role neuropeptides play in transmitting itch signals, we generated two mouse models of chronic itch-Acetone-Ether-Water (AEW, dry skin) and calcipotriol (MC903, atopic dermatitis). For peptide identification and quantitation, we analyzed the peptide content of dorsal root ganglia (DRG) and dorsal horn (DH) tissues from chronically itchy mice using liquid chromatography coupled to tandem mass spectrometry. De novo-assisted database searching facilitated the identification and quantitation of 335 peptides for DH MC903, 318 for DH AEW, 266 for DRG MC903, and 271 for DRG AEW. Of these quantifiable peptides, we detected 30 that were differentially regulated in the tested models, after accounting for multiple testing correction (q<0.1). These include several peptide candidates derived from neuropeptide precursors, such as proSAAS, protachykinin-1, proenkephalin and calcitonin gene-related peptide, some of them previously linked to itch. The peptides identified in this study may help elucidate our understanding about these debilitating disorders. Data are available via ProteomeXchange with identifier PXD015949.

Calcium imaging of primary canine sensory neurons: Smalldiameter neurons responsive to pruritogens and algogens

Joy Rachel C. Ganchingco| Tomoki Fukuyama | Jeffrey A. Yoder | Wolfgang Bäumer

Abstract

Calcium imaging of primary canine sensory neurons: Smalldiameter neurons responsive to pruritogens and algogensAbstract Introduction: Rodent primary sensory neurons are commonly used for studying itch and pain neurophysiology, but translation from rodents to larger mammals and humans is not direct and requires further validation to make correlations. Methods: This study developed a primary canine sensory neuron culture from dorsal root ganglia (DRG) excised from cadaver dogs. Additionally, the canine DRG cell cultures developed were used for single‐cell ratiometric calcium imaging, with the activation of neurons to the following pruritogenic and algogenic substances: histamine, chloroquine, canine protease‐activated receptor 2 (PAR2) activating peptide (SLIGKT), compound 48/80, 5‐hydroxytryptamine receptor agonist (5‐HT), bovine adrenal medulla peptide (BAM8‐22), substance P, allyl isothiocyanate (AITC), and capsaicin. Results: This study demonstrates a simple dissection and rapid processing of DRG collected from canine cadavers used to create viable primary sensory neuron cultures to measure responses to pruritogens and algogens. Conclusion: Ratiometric calcium imaging demonstrated that small‐diameter canine sensory neurons can be activated by multiple stimuli, and a single neuron can react to both a pruritogenic stimulation and an algogenic stimulation.

K E Y W O R D S dorsal laminectomy dissection, dorsal root ganglia cell culture, fura‐2AM, ratiometric calcium imagingCalcium imaging of primary canine sensory neurons: Smalldiameter neurons responsive to pruritogens and algogens

Activation of mas-related G-protein-coupled receptors by the house dust mite cysteine protease Der p1 provides a new mechanism linking allergy and inflammation.

Reddy VB¹, Lerner EA².

Author information

Abstract

Cysteine and serine proteases function via protease-activated and mas-related G-protein-coupled receptors (Mrgprs) to contribute to allergy and inflammation. Der p1 is a cysteine protease and major allergen from the house dust mite and is associated with allergic rhinitis and allergic asthma. Der p1 activates protease-activated receptor 2 and induces the release of the pro-inflammatory cytokine IL-6 from cells. However, the possibility that Der p1 acts on Mrgprs has not been considered. We report here that ratiometric calcium imaging reveals that Der p1 activates the human receptor MRGPRX1 and the mouse homolog MrgprC11, implicated previously in itch. Der p1 cleavage of N-terminal receptor peptides followed by site-directed mutagenesis of the cleavage sites links receptor activation to specific amino acid residues. Der p1 also induced the release of IL-6 from heterologous cells expressing MRGPRX1. In summary, activation of Mrgprs by the allergen Der p1 may contribute to inflammation.

Polymyxin B and polymyxin E induce anaphylactoid response through T mediation of Mas-related G protein–coupled receptor X2

Yingzhuan Zhan, Nan Ma, Rui Liu, Nan Wang, Tao Zhang, Langchong He* School of Pharmacy, Health Science Center, Xi’an Jiaotong University, Xi’an, Shaanxi Province, 710061, PR China

Keywords: Polymyxin B, Polymixin E, Anaphylactoid reaction, MRGPRX2

ABSTRACT

Polymyxin B (PMB) and polymyxin E (PME) are cyclic, peptide antibiotics which derived from various species of Paenibacillus (Bacillus) polymyxa. They are decapeptide antibiotics with an antimicrobial spectrum that includes Gram-negative bacteria, and reused as therapeutic agents due to the emergence of multidrug-resistant (MDR) Gram-positive bacteria. PMB or PME-induced anaphylactoid reactions in the clinic have been documented. However, the mechanism underlying anaphylactoid reaction induced by polymyxin has not yet been reported. Here, we report that human Mas-related G protein-coupled receptor X2 (MRGPRX2) and its mouse homologue Mas-related G protein-coupled receptor B2 (MrgprB2) are the receptors mediating the anaphylactoid response provoked by PMB and PME. We firstly investigated the anaphylactoid reactions induced by PMB and PME in LAD2 cells in vitro and in vivo, and found that treatment with PMB and PME led to significant release of mast cell granules such as histamine and β-hexosaminidase, secretion of pro-inflammatory cytokines, such as TNF-α and PGD2, and provocation of calcium flux in LAD2 cells. Furthermore, treatment with PMB and PME led to reduced release of β-hexosaminidase in MRGPRX2 knockdown-LAD2 cells, and obvious increased calcium release in MRGPRX2 overexpressing HEK293 cells, which suggested that MRGPRX2 are involved in mast cell activation provoked by PMB or PME. In vivo, MRGPRB2 knockout mice exhibited lower pseudo-allergic reactions than wild type mice. Activation of MrgprB2 also triggers increased capillary permeability and paw swelling. Our results elucidated the role of MRGPRX2 in PMB and PME-induced anaphylactoid response and suggested that MRGPRX2 as a potential therapeutic target to control the anaphylactoid reactions which triggered by PMB or PME.

MRGPRX4 is a bile acid receptor for human cholestatic itch

Huasheng Yu,^1,2,3Tianjun Zhao,^1,2,3Simin Liu,¹Qinxue Wu,⁴Omar Johnson,⁴Zhaofa Wu,^1,2Zihao Zhuang,¹Yaocheng Shi,⁵Luxin Peng,⁵Renxi He,^1,2Yong Yang,⁶Jianjun Sun,⁷Xiaoqun Wang,⁸Haifeng Xu,⁹Zheng Zeng,¹⁰Peng Zou,⁵Xiaoguang Lei,^3,5Wenqin Luo,⁴ and  Yulong Li^1,2,3,11

Abstract

Patients with liver diseases often suffer from chronic itch, yet the pruritogen(s) and receptor(s) remain largely elusive. Here, we identify bile acids as natural ligands for MRGPRX4. MRGPRX4 is expressed in human dorsal root ganglion (hDRG) neurons and co-expresses with itch receptor HRH1. Bile acids elicited Ca²⁺ responses in cultured hDRG neurons, and bile acids or a MRGPRX4 specific agonist induced itch in human subjects. However, a specific agonist for another bile acid receptor TGR5 failed to induce itch in human subjects and we find that human TGR5 is not expressed in hDRG neurons. Finally, we show positive correlation between cholestatic itch and plasma bile acids level in itchy patients and the elevated bile acids is sufficient to activate MRGPRX4. Taken together, our data strongly suggest that MRGPRX4 is a novel bile acid receptor that likely underlies cholestatic itch in human, providing a promising new drug target for anti-itch therapies.

Research organism: Human

Supplementary Figures. https://elifesciences.org/articles/48431/figures#fig8s1

Unexpected Ca²⁺-mobilization of oxaliplatin via H1 histamine receptors.

Potenzieri A¹, Riva B¹, Genazzani AA².

Abstract

Oxaliplatin is a widely used chemotherapeutic drug and represents the cornerstone of colorectal cancer therapy, in combination with 5-fluorouracil and folinic acid. As with many chemotherapeutic agents, its use is associated with a number of side effects, ranging from hypersensitivity reactions to haematological dyscrasias. Oxaliplatin also induces acute and chronic peripheral neuropathy. While it is likely that the haematological side effects are associated with its anti-proliferative effects and with the ability to form DNA adducts, the molecular mechanisms underlying peripheral neuropathy and hypersensitivity reactions are poorly understood, and therefore the choice of adequate supportive therapies is largely empirical. Here we show that an acute low dose oxaliplatin application on DRG neurons is able to induce an increase in intracellular calcium that is dependent on the Histamine 1 receptor (H1). Oxaliplatin-induced intracellular calcium rises are blocked by two selective H1 antagonist, as well as by U73122, a PLC inhibitor, and by 2-APB, a non-specific IP₃ receptor blocker. Moreover, expression of the H1 receptor on HEK293 t cells unmasks an oxaliplatin-induced Ca²⁺-rise. Last, activation of H1 via either histamine or oxaliplatin activates TRPV1 receptors, a mechanism that has been associated with itch. These data, together with literature data that has shown that anti-histamine agents reduce the incidence of oxaliplatin-induced hypersensitivity, may provide a molecular mechanism of this side effect in oncological patients.

3’-O-Methylorobol Inhibits the Voltage-Gated Sodium Channel Nav1.7 with Anti-Itch Efficacy in A Histamine-Dependent Itch Mouse Model

Fan Zhang †, Ying Wu †, Shuwen Xue, Shuangyan Wang, Chunlei Zhang * and Zhengyu Cao *

State Key Laboratory of Natural Medicines and Jiangsu Provincial Key Laboratory for TCM Evaluation and Translational Development, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 211198, China; zhangfan20111112@126.com (F.Z.); wingwycpu@126.com (Y.W.); xsw19850856166@163.com (S.X.); shuangyanwcpu@163.com (S.W.)

* Correspondence: zhangchunlei11@sina.com (C.Z.); zycao1999@hotmail.com (Z.C.); Tel.: +86-25-8618-5955 (C.Z.); +86-25-8618-5955 (Z.C.)

† These authors contributed equally to this work.
Received: 15 November 2019; Accepted: 29 November 2019; Published: 1 December 2019

􏰀􏰁􏰂􏰀􏰃 􏰅􏰆􏰇

􏰈􏰉􏰊􏰋􏰌􏰂􏰍

Abstract: An itch is a clinical complication that affects millions of patients. However, few treatment options are available. The voltage-gated sodium channel Nav1.7 is predominantly expressed in peripheral sensory neurons and is responsible for the rising phase of action potentials, thereby mediating nociceptive conduction. A gain-of-function mutation of Nav1.7 results in the hyperexcitability of sensory neurons and causes the inherited paroxysmal itch. Conversely, a monoclonal antibody that selectively inhibits Nav1.7 is able to effectively suppress the histamine-dependent itch in mice. Therefore, Nav1.7 inhibitors may possess the potential to relieve the itch. In the present study, using whole-cell voltage-clamp recordings, we demonstrated that 3’-O-methylorobol inhibited Na+ currents in Nav1.7-CHO cells and tetrodotoxin-sensitive Na+ currents in mouse dorsal root ganglion (DRG) neurons with IC50 (half-maximal inhibitory concentration) values of 3.46 and 6.60 μM, respectively. 3’-O-methylorobol also suppressed the tetrodotoxin-resistant Na+ currents in DRG neurons, though with reduced potency (~43% inhibition at 30 μM). 3’-O-methylorobol (10 μM) affected the Nav1.7 by shifting the half-maximal voltage (V1/2) of activation to a depolarizing direction by ~6.76 mV, and it shifted the V1/2 of inactivation to a hyperpolarizing direction by ~16.79 mV. An analysis of 3’-O-methylorobol activity toward an array of itch targets revealed that 3’-O-methylorobol was without effect on histamine H1 receptor, TRPV1, TRPV3, TRPV4, TRPC4 and TRPM8. The intrathecal administration of 3’-O-methylorobol significantly attenuated compound 48/80-induced histamine-dependent spontaneous scratching bouts and the expression level of c-fos in the nuclei of spinal dorsal horn neurons with a comparable efficacy to that of cyproheptadine. Our data illustrated the therapeutic potential for 3’-O-methylorobol for histamine-dependent itching, and the small molecule inhibition of Nav1.7 may represent a useful strategy to develop novel therapeutics for itching.

Keywords: Nav1.7; 3’-O-methylorobol; gating modifier; itch relief; lead compound

Brain allopregnanolone induces marked scratching behaviour in diet-induced atopic dermatitis mouse model

Abstract

Allopregnanolone (ALLO) is a neurosteroid produced in the brain, but so far, no study has explored its link with itching. Herein, we used a diet-induced atopic dermatitis mouse model to examine whether exogenously administered and endogenously produced ALLO contribute to inducing scratching. Systemic administration of ALLO elicited robust scratching in the atopic dermatitis model, while it did not affect spontaneous and pruritogen-induced scratching in normal mice. ALLO caused scratching when administered intracisternally, but not when administered intrathecally or intradermally, suggesting the involvement of supraspinal mechanisms. Pharmacological analyses suggested that both γ-aminobutyric acid type A receptor activation and serotonin type 3 receptor inhibition were involved in ALLO-induced scratching. We next examined whether endogenously produced ALLO is involved in ethanol-induced scratching in atopic dermatitis mice, because ethanol administration increases ALLO in rodent brain. Acute ethanol administration increased brain ALLO levels, which coincided with increased scratching. Pre-treatment with finasteride, a synthetic ALLO inhibitor, suppressed ethanol-induced scratching and ALLO production in the brain. Collectively, our results demonstrated for the first time that ALLO administration caused marked scratching in atopic dermatitis mice, and ethanol-induced scratching may be mediated through endogenously produced brain ALLO.

miRNA-711 Binds and Activates TRPA1 Extracellularly to Evoke Acute and Chronic Pruritus

QingjianHan¹⁴DiLiu¹⁴MarinoConvertino²ZilongWang¹ChangyuJiang¹Yong HoKim¹XinLuo¹XinZhang¹AndreaNackley¹Nikolay V.Dokholyan²⁵Ru-RongJi¹³⁶

Summary

Increasing evidence suggests that extracellular miRNAs may serve as biomarkers of diseases, but the physiological relevance of extracellular 
miRNA  is unclear. We find that intradermal cheek injection of miR-711 induces TRPA1-depedent itch (scratching) without pain (wiping) in naive mice. Extracellular perfusion of miR-711 induces TRPA1 currents in both Trpa1-expressing heterologous cells and native sensory neurons through the core sequence GGGACCC. Computer simulations reveal that the core sequence binds several residues at the extracellular S5-S6 loop of TRPA1, which are critical for TRPA1 activation by miR-711 but not allyl isothiocyanate. Intradermal inoculation of human Myla cells induces lymphoma and chronic itch in immune-deficient mice, associated with increased serum levels of miR-711, secreted from cancer cells. Lymphoma-induced chronic itch is suppressed by miR-711 inhibitor and a blocking peptide that disrupts the miR-711/TRPA1 interaction. Our findings demonstrated an unconventional physiological role of extracellular naked miRNAs as itch mediators and ion channel modulators.

Neutrophils promote CXCR3-dependent itch in the development of atopic dermatitis.

Walsh CM¹, Hill RZ¹, Schwendinger-Schreck J¹, Deguine J¹, Brock EC¹, Kucirek N¹, Rifi Z¹, Wei J², Gronert K², Brem RB³, Barton GM¹, Bautista DM¹.

Abstract

Chronic itch remains a highly prevalent disorder with limited treatment options. Most chronic itch diseases are thought to be driven by both the nervous and immune systems, but the fundamental molecular and cellular interactions that trigger the development of itch and the acute-to-chronic itch transition remain unknown. Here, we show that skin-infiltrating neutrophils are key initiators of itch in atopic dermatitis, the most prevalent chronic itch disorder. Neutrophil depletion significantly attenuated itch-evoked scratching in a mouse model of atopic dermatitis. Neutrophils were also required for several key hallmarks of chronic itch, including skin hyperinnervation, enhanced expression of itch signaling molecules, and upregulation of inflammatory cytokines, activity-induced genes, and markers of neuropathic itch. Finally, we demonstrate that neutrophils are required for induction of CXCL10, a ligand of the CXCR3 receptor that promotes itch via activation of sensory neurons, and we find that that CXCR3 antagonism attenuates chronic itch.

KEYWORDS: 

immunology; inflammation; mouse; neuroscience