Reduced spontaneous itch in mouse models of cholestasis

Published in:- Scientific Reports (2021.03.17) IF~4.379

Jacqueline Langedijk, Ruth Bolier, Dagmar Tolenaars, Lysbeth ten Bloemendaal, Suzanne Duijst, Dirk de Waart, Ulrich Beuers, Piter Bosma & Ronald Oude Elferink

Abstract

Pruritus is one of the most distressing symptoms in cholestatic patients. Plasma autotaxin (ATX) activity correlates with the severity of pruritus in cholestatic patients, but the pathophysiology is unclear. To study pruritus in mice, we measured scratch activity in cholestatic Atp8b1 mutant mice, a model for Progressive Familial Intrahepatic Cholestasis type 1, and wild type mice (WT) with alphanaphthylisothiocyanate (ANIT)-induced cholestasis. To induce cholestasis, Atp8b1 mutant mice received a diet containing 0.1% cholic acid (CA) and WT mice were treated with ANIT. In these mice ATX was also overexpressed by transduction with AAV-ATX. Scratch activity was measured using an unbiased, electronic assay. Marked cholestasis was accomplished in both Atp8b1 mutant mice on a CA-supplemented diet and in ANIT-treatment in WT mice, but scratch activity was decreased rather than increased while plasma ATX activity was increased. Plasma ATX activity was further increased up to fivefold with AAV-ATX, but this did not induce scratch activity. In contrast to several reports two cholestatic mouse models did not display increased scratch activity as a measure of itch perception. Increasing plasma ATX activity by overexpression also did not lead to increased scratch activity in mice. This questions whether mice are suitable to study cholestatic itch.

TRPV1 and TRPA1 Channels Are Both Involved Downstream of Histamine-Induced Itch 

by Jenny Wilzopolski ^1,2,3,*, Manfred Kietzmann ¹, Santosh K. Mishra ², Holger Stark ⁴, Wolfgang Bäumer ^2,3 and Kristine Rossbach¹

¹Department of Pharmacology, Toxicology and Pharmacy, University of Veterinary Medicine Hannover, Foundation, 30559 Hannover, Germany

²Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27607, USA

³Department of Veterinary Medicine, Institute of Pharmacology and Toxicology, Freie Universität Berlin, 14195 Berlin, Germany

⁴Institute of Pharmaceutical and Medical Chemistry, Heinrich Heine University Düsseldorf, 40225 Duesseldorf, Germany

^*Author to whom correspondence should be addressed. Academic Editors: Emanuela Masini, Laura Lucarini and Alessandro AlaimoBiomolecules2021, 11(8), 1166; https://doi.org/10.3390/biom11081166Received: 13 July 2021 / Revised: 31 July 2021 / Accepted: 4 August 2021 / Published: 6 August 2021(This article belongs to the Special Issue New Developments in Histamine Research)

Abstract: Two histamine receptor subtypes (HR), namely H1R and H4R, are involved in the trans- mission of histamine-induced itch as key components. Although exact downstream signaling mechanisms are still elusive, transient receptor potential (TRP) ion channels play important roles in the sensation of histaminergic and non-histaminergic itch. The aim of this study was to investigate the involvement of TRPV1 and TRPA1 channels in the transmission of histaminergic itch. The potential of TRPV1 and TRPA1 inhibitors to modulate H1R- and H4R-induced signal transmission was tested in a scratching assay in mice in vivo as well as via Ca2+ imaging of murine sensory dorsal root ganglia (DRG) neurons in vitro. TRPV1 inhibition led to a reduction of H1R- and H4R- induced itch, whereas TRPA1 inhibition reduced H4R- but not H1R-induced itch. TRPV1 and TRPA1 inhibition resulted in a reduced Ca2+ influx into sensory neurons in vitro. In conclusion, these results indicate that both channels, TRPV1 and TRPA1, are involved in the transmission of histamine-induced pruritus.

Keywords: histamine; histamine H1 receptor; histamine H4 receptor; itch; signal transduction; TRPV1; TRPA1; dorsal root ganglion neurons (DRG); Ca2+-imaging

Dissecting the precise nature of itch-evoked scratching

Nivanthika K Wimalasena ¹, George Milner ², Ricardo Silva ³, Cliff Vuong ³, Zihe Zhang ¹, Diana M Bautista ⁴, Clifford J Woolf ⁵

Abstract

Itch is a discrete and irritating sensation tightly coupled to a drive to scratch. Acute scratching developed evolutionarily as an adaptive defense against skin irritants, pathogens, or parasites. In contrast, the itch-scratch cycle in chronic itch is harmful, inducing escalating itch and skin damage. Clinically and preclinically, scratching incidence is currently evaluated as a unidimensional motor parameter and believed to reflect itch severity. We propose that scratching, when appreciated as a complex, multidimensional motor behavior, will yield greater insight into the nature of itch and the organization of neural circuits driving repetitive motor patterns. We outline the limitations of standard measurements of scratching in rodent models and present new approaches to observe and quantify itch-evoked scratching. We argue that accurate quantitative measurements of scratching are critical for dissecting the molecular, cellular, and circuit mechanisms underlying itch and for preclinical development of therapeutic interventions for acute and chronic itch disorders.

Keywords: high-speed recording; itch; pruritis; rodent models; scratching behavior.

Statin-boosted cellular uptake of penetratin due to reduced membrane dipole
potential

published in bioRxiv, 2020.08.04

Gyula Batta, Levente Kárpáti, Gabriela Fulaneto Henrique, Szabolcs Tarapcsák, Tamás Kovács, Florina Zákány, István M. Mándity, Peter Nagy

Abstract

Since cell penetrating peptides are promising tools for delivery of cargo into cells, factors limiting or facilitating their cellular uptake are intensely studied. Using labeling with pH-insensitive and pH-sensitive dyes we report that escape of penetratin from acidic endo-lysosomal compartments is retarded compared to its cellular uptake. The membrane dipole potential, known to alter transmembrane transport of charged molecules, is shown to be negatively correlated with the concentration of penetratin in the cytoplasmic compartment. Treatment of cells with therapeutically relevant concentrations of atorvastatin, an inhibitor of HMG-CoA reductase and cholesterol synthesis, significantly increased the release of penetratin from acidic endocytic compartments in two different cell types. This effect of atorvastatin correlated with its ability to decrease the membrane dipole potential. These results highlight the importance of the dipole potential in regulating cellular uptake of cell penetrating peptides and suggest a clinically relevant way of boosting this process.

Identification of the dog orthologue of human MAS-related G protein coupled receptor X2 (MRGPRX2) essential for drug-induced pseudo-allergic reactions

Published in scientifc reports, 30 september 2020

Eri Hamamura-Yasuno, Takuma Iguchi, Kazuyoshi Kumagai, Yoshimi tsuchiya&Kazuhiko Mori

Abstarct

MAS-related G protein coupled receptor-X2 (MRGPRX2), expressed in human mast cells, is associated with drug-induced pseudo-allergic reactions. Dogs are highly susceptible to drug-induced anaphylactoid reactions caused by various drugs; however, the distribution and physiological function of canine MRGPR family genes, including MRGPRX2, remain largely unknown. In the present study, we clarified the distribution of dog MRGPR family genes by real-time quantitative PCR and in situ hybridisation. We also investigated the stimulatory effects of various histamine-releasing agents, including fluoroquinolones, on HEK293 cells transiently transfected with dog MRGPR family genes to identify their physiological function. Dog MRGPRX2 and MRGPRG were distributed in a limited number of tissues, including the skin (from the eyelid, abdomen, and cheek), whereas MRGPRD and MRGPRF were extensively expressed in almost all tissues examined. Histochemical and in situ hybridisation analyses revealed that MRGPRX2 was expressed in dog connective tissue-type mast cells in the skin. Intracellular Ca²⁺ mobilisation assay revealed that HEK293 cells, expressing dog MRGPRX2 or human MRGPRX2, but not dog MRGPRD, MRGPRF, and MRGPRG, responded to histamine-releasing agents. Our results suggest that dog MRGPRX2 is the functional orthologue of human MRGPRX2 and plays an essential role in drug-induced anaphylactoid reactions in dogs.

Presented by Jong hyun Im

P17 induces chemotaxis and differentiation of monocytes via MRGPRX2-mediated mast cell–line activation

Journal of allergy and clinical immunology, ~14.11 (IF)

Karthi Duraisamy, Kailash Singh, Mukesh Kumar, Benjamin Lefranc, Elsa Bonnafe, Michel Treilhou, Jerome Leprince and Billy K. C. Chow, (Hong Kong, China; and Rouen and Albi, France).

Abstract

Background: P17, a peptide isolated from Tetramorium bicarinatum ant venom, is known to induce an alternative phenotype of human monocyte–derived macrophages via activation of an unknown G protein–coupled receptor (GPCR).

Objective: We sought to investigate the mechanism of action and the immunomodulatory effects of P17 mediated through MRGPRX2 (Mas related G protein–coupled receptor X2).

Methods: To identify the GPCR for P17, we screened 314 GPCRs. Upon identification of MRGPRX2, a battery of in silico, in vitro, ex vivo, and in vivo assays along with the receptor mutation studies were performed. In
particular, to investigate the immunomodulatory actions, we used b-hexosaminidase release assay, cytokine releases, quantification of mRNA expression, cell migration and differentiation assays,
immunohistochemical labeling, hematoxylin and eosin, and immunofluorescence staining.

Results: P17 activated MRGPRX2 in a dose-dependent manner in b-arrestin recruitment assay. In LAD2 cells, P17 induced calcium and b-hexosaminidase release. Quercetin- and short hairpin RNA–mediated knockdown of MRGPRX2 reduced P17- evoked b-hexosaminidase release. In silico and in vitro mutagenesis studies showed that residue Lys8 of P17 formed a cation-p interaction with the Phe172 of MRGPRX2 and [Ala8
] P17 lost its activity partially. P17 activated LAD2 cells to recruit THP-1 and human monocytes in Transwell migration assay, whereas MRGPRX2-impaired LAD2 cells cannot. In addition, P17-treated LAD2 cells stimulated differentiation of THP-1 and human monocytes, as indicated by the enhanced expression of macrophage markers cluster of differentiation 11b and TNF-α by quantitative RT-PCR. Immunohistochemical and immunofluorescent staining suggested monocyte recruitment in mice ears injected with P17.

Conclusion: Our data provide novel structural information regarding the interaction of P17 with MRGPRX2 and intracellular pathways for its immunomodulatory action.

Supplementary datas:-

Analysis of TRPV channel activation by stimulation of FCεRI and MRGPR receptors in mouse peritoneal mast cells

A. Sol ́ıs-Lo ́ pez1, U. Kriebs1, A. Marx1, S. Mannebach2, W. B. Liedtke3, M. J. Caterina4, M. Freichel1, V. V. Tsvilovskyy1*

1 PharmakologischesInstitut,Ruprecht-Karls-Universita ̈tHeidelberg,Heidelberg,Baden-Wu ̈rttemberg, Germany, 2 Experimentelle und Klinische Pharmakologie und Toxikologie, Universita ̈t des Saarlandes, Homburg, Saarland, Germany, 3 Department of Neurology, School of Medicine Duke University, Durham, North Carolina, United States of America, 4 Departments of Neurosurgery, Biological Chemistry, and Neuroscience, Neurosurgery Pain Research Institute, The Johns Hopkins School of Medicine, Baltimore, Maryland, United States of America

Abstract

The activation of mast cells (MC) is part of the innate and adaptive immune responses and depends on Ca2+ entry across the plasma membrane, leading to the release of preformed inflammatory mediators by degranulation or by de novo synthesis. The calcium conducting channels of the TRPV family, known by their thermo and osmotic sensitivity, have been pro- posed to be involved in the MC activation in murine, rat, and human mast cell models. So far, immortalized mast cell lines and nonspecific TRPV blockers have been employed to charac- terize the role of TRPV channels in MC. The aim of this work was to elucidate the physiological role of TRPV channels by using primary peritoneal mast cells (PMCs), a model of connective tissue type mast cells. Our RT-PCR and NanoString analysis identified the expression of TRPV1, TRPV2, and TRPV4 channels in PMCs. For determination of the functional role of the expressed TRPV channels we performed measurements of intracellular free Ca2+ concentra- tions and beta-hexosaminidase release in PMCs obtained from wild type and mice deficient for corresponding TRPV1, TRPV2 and TRPV4 in response to various receptor-mediated and physical stimuli. Furthermore, substances known as activators of corresponding TRPV-chan- nels were also tested using these assays. Our results demonstrate that TRPV1, TRPV2, and TRPV4 do not participate in activation pathways triggered by activation of the high-affinity receptors for IgE (FcεRI), Mrgprb2 receptor, or Endothelin-1 receptor nor by heat or osmotic stimulation in mouse PMCs.

The TRPM3 ion channel mediates nociception but not itch evoked by endogenous pruritogenic mediators

Biochemical Pharmacology, ~ 5.0 (IF)

BalázsKelemen, SilviaPinto, NawooKim, ErikaLisztes, MartinHanyicska, AnitaVladár, AttilaOláh, ZsófiaPénzes, BrianShu, JorisVriens, TamásBíró, TiborRohács, ThomasVoets, Balázs IstvánTóth

Abstract

During the molecular transduction of itch, the stimulation of pruriceptors on sensory fibers leads to the activation or sensitization of ion channels, which results in a consequent depolarization of the neurons. These ion channels mostly belong to the transient receptor potential (TRP) channels, which are involved in nociception and thermosensation. In particular, TRPV1 and TRPA1 were described in the transduction of both thermal nociception as well as histaminergic and non-histaminergic itch. The thermosensitive TRPM3 plays an indispensable role in heat nociception together with TRPV1 and TRPA1. However, the role of TRPM3 in the development of pruritus has not been studied yet. Therefore, in this study we aimed at investigating the potential role of TRPM3 in the transduction of pruritus and pain by investigating itch- and nociception-related behavior of Trpm3^+/+ and Trpm3^−/− mice, and by studying the activation of somatosensory neurons isolated from trigeminal ganglia upon application of algogenic and pruritogenic substances. Activators of TRPM3 evoked only nocifensive responses, but not itch in Trpm3^+/+ animals, and these nocifensive responses were abolished in the Trpm3^−/− strain. Histamine and endogenous non-histaminergic pruritogens induced itch in both Trpm3^+/+and Trpm3^−/− mice to a similar extent. Genetic deletion or pharmacological blockade diminished TRPM3 mediated Ca²⁺ responses of sensory neurons, but did not affect responses evoked by pruritogenic substances. Our results demonstrate that, in contrast to other thermosensitive TRP channels, TRPM3 selectively mediates nociception, but not itch sensation, and suggest that TRPM3 is a promising candidate to selectively target pain sensation.

Quorum sensing between bacterial species on the skin protects against epidermal injury in atopic dermatitis

Michael R. Williams, Stephen K. Costa, Livia S. Zaramela, Shadi Khali, Daniel A. Todd, Heather L. Winter

published in Science Translational Medicine, 01 May 2019

Abstract

Colonization of the skin by Staphylococcus aureus is associated with exacerbation of atopic dermatitis (AD), but any direct mechanism through which dysbiosis of the skin microbiome may influence the development of AD is unknown. Here, we show that proteases and phenol-soluble modulin α (PSMα) secreted by S. aureus lead to endogenous epidermal proteolysis and skin barrier damage that promoted inflammation in mice. We further show that clinical isolates of different coagulase-negative staphylococci (CoNS) species residing on normal skin produced autoinducing peptides that inhibited the S. aureus agr system, in turn decreasing PSMα expression. These autoinducing peptides from skin microbiome CoNS species potently suppressed PSMα expression in S. aureus isolates from subjects with AD without inhibiting S. aureus growth. Metagenomic analysis of the AD skin microbiome revealed that the increase in the relative abundance of S. aureus in patients with active AD correlated with a lower CoNS autoinducing peptides to S. aureus ratio, thus overcoming the peptides’ capacity to inhibit the S. aureus agr system. Characterization of a S. hominis clinical isolate identified an autoinducing peptide (SYNVCGGYF) as a highly potent inhibitor of S. aureus agr activity, capable of preventing S. aureus–mediated epithelial damage and inflammation on murine skin. Together, these findings show how members of the normal human skin microbiome can contribute to epithelial barrier homeostasis by using quorum sensing to inhibit S. aureus toxin production.

MRGPRX2 Is the Codeine Receptor of Human Skin Mast Cells: Desensitization through β-Arrestin and Lack of Correlation with the FcεRI Pathway

Magda Babina, Zhao Wang, Saptarshi Roy, Sven Guhl, Kristin Franke, Metin Artuc, Hydar Ali and Torsten Zuberbier

Published in: Journal of Investigative Dermatology

DOI: 10.1016/j.jid.2020.09.017

Abstract

Codeine stimulates skin mast cells and is therefore used in skin tests and as an inducer of experimental itch. MRGPRX2 responds to various drugs, including opioids, to elicit pseudoallergic reactions, but whether it represents the main opiate receptor of skin mast cells remains unknown. By combining a number of approaches, including the silencing of MRGPRX2, we now report that MRGPRX2 is indeed the dominant codeine receptor of dermal mast cells. Activation by codeine displayed profound subject variability and correlated with secretion elicited by compound 48/80 or substance P but not by FcεRI aggregation. Degranulation by codeine was attenuated by stem cell factor, whereas the opposite was found for FcεRI. Compound 48/80 or codeine alone was able to achieve maximum MRGPRX2 activation. MRGPRX2 was rapidly internalized on codeine binding in a b arrestin-1‒dependent manner. Codeine-triggered b-arrestin activation was also established by the Tango assay. Prestimulation with MRGPRX2 agonists (but not C3a or FcεRI aggregation) resulted in refractoriness to further stimulation by the same or another MRGPRX2 ligand (cross desensitization). This was duplicated in a cell line (RBL-MRGPRX2). Collectively, codeine degranulates skin mast cells through MRGPRX2, at which it acts as a balanced ligand. It has yet to be determined whether codeine-induced refractoriness could be exploited to desensitize MRGPRX2 to prevent severe pseudoallergic reactions.