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.