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