Substance P activates Mas-related G protein-coupled receptors to induce itch.

Azimi E1, Reddy VB1, Pereira PJ2, Talbot S3, Woolf CJ3, Lerner EA4.

Abstract

BACKGROUND: Substance P (SP) is linked to itch and inflammation through activation of receptors on mast cells and sensory neurons. There is increasing evidence that SP functions through Mas-related G protein-coupled receptors (Mrgprs) in addition to its conventional receptor, neurokinin-1.

OBJECTIVE: Because Mrgprs mediate some aspects of inflammation that had been considered mediated by neurokinin-1 receptor (NK-1R), we sought to determine whether itch induced by SP can also be mediated by Mrgprs.

METHODS: Genetic and pharmacologic approaches were used to evaluate the contribution of Mrgprs to SP-induced scratching behavior and activation of cultured dorsal root ganglion neurons from mice.

RESULTS: SP-induced scratching behavior and activation of cultured dorsal root ganglion neurons was dependent on Mrgprs rather than NK-1R.

CONCLUSION: We deduce that SP activates MrgprA1 on sensory neurons rather than NK-1R to induce itch.

KEYWORDS: Mas-related G protein–coupled receptors; Substance P; calcium imaging; dorsal root ganglion neurons; knockout mice; receptor antagonist

Substance P Release by Sensory Neurons Triggers Dendritic Cell Migration and Initiates the Type-2 Immune Response to Allergens

Caroline Perner, Cameron H. Flayer, Xueping Zhu, …, Ohn A. Chow, Isaac M. Chiu, Caroline L. Sokol

Abstract

Dendritic cells (DCs) of the cDC2 lineage initiate allergic immunity and in the dermis are marked by their expression of CD301b. CD301b+ dermal DCs respond to allergens encountered in vivo, but not in vitro. This suggests that another cell in the dermis may sense allergens and relay that information to activate and induce the migration of CD301b+ DCs to the draining lymph node (dLN). Using a model of cutaneous allergen exposure, we show that allergens directly activated TRPV1+ sensory neurons leading to itch and pain behaviors. Allergen-activated sensory neurons released the neuropeptide Substance P, which stimulated proximally located CD301b+ DCs through the Mas-related G-protein coupled receptor member A1 (MRGPRA1). Substance P induced CD301b+ DC migration to the dLN where they initiated T helper-2 cell differentiation. Thus, sensory neurons act as primary sensors of allergens, linking exposure to activation of allergic-skewing DCs and the initiation of an allergic immune response.

Protease-Activated Receptor-2 Regulates Neuro-Epidermal Communication in Atopic Dermatitis

Timo Buhl, Akihiko Ikoma, Cordula Kempkes, Ferda Cevikbas, Mathias Sulk, Joerg Buddenkotte, Tasuku Akiyama, Debbie Crumrine, Eric Camerer, Earl Carstens, Michael P Schön, Peter Elias, Shaun R Coughlin, Martin Steinhoff

Background: Activation of protease-activated receptor-2 (PAR2) has been implicated in inflammation, pruritus, and skin barrier regulation, all characteristics of atopic dermatitis(AD), as well as Netherton syndrome which has similar characteristics. However, understanding the precise role of PAR2 on neuro-immune communication in AD has been hampered by the lack of appropriate animal models.
Methods: We used a recently established mouse model with epidermal overexpression of PAR2 (PAR2OE) and littermate WT mice to study the impact of increased PAR2 expression in epidermal cells on spontaneous and house dust mite (HDM)-induced skin inflammation, itch, and barrier dysfunction in AD, in vivo and ex vivo.
Results: PAR2OE newborns displayed no overt abnormalities, but spontaneously developed dry skin, severe pruritus, and eczema. Dermatological, neurophysiological, and immunological analyses revealed the hallmarks of AD-like skin disease. Skin barrier defects were observed before onset of skin lesions. Application of HDM onto PAR2OE mice triggered pruritus and the skin phenotype. PAR2OE mice displayed an increased density of nerve fibers, increased nerve growth factor and endothelin-1 expression levels, alloknesis, enhanced scratching (hyperknesis), and responses of dorsal root ganglion
cells to non-histaminergic pruritogens.

Conclusion: PAR2 in keratinocytes, activated by exogenous and endogenous proteases, is sufficient to drive barrier dysfunction, inflammation, and pruritus and sensitize skin to the effects of HDM in a mouse model that mimics human AD. PAR2 signaling in keratinocytes appears to be sufficient to drive several levels of neuro-epidermal communication, another feature of human AD.

Inhibition of mite-induced dermatitis, pruritus, and nerve sprouting in mice by the endothelin receptor antagonist bosentan

Makiko Kido-Nakahara1, Bing Wang, Fumitaka Ohno, Gaku Tsuji, Dugarmaa Ulzii, Masaki Takemura, Masutaka Furue, Takeshi Nakahara

Abstract

Background: Endothelin-1 (EDN1) can evoke histamine-independent pruritus in mammals and is
upregulated in the lesional epidermis of atopic dermatitis (AD). EDN1 increases the production of
interleukin 25 (IL-25) from keratinocytes to accelerate T helper type 2 immune deviation. Plasma EDN1
levels are positively correlated with the clinical severity and itch intensity of AD. Therefore, we
hypothesized that the inhibition of EDN1 might be useful for treating atopic inflammation and itch and
investigated the effects of the topical application of the EDN1 receptor antagonist bosentan on the skin
inflammation and itch in a murine AD model.
Methods: We analyzed the mite-induced AD-like NC/Nga murine model, which was topically applied with
bosentan or ethanol control every day for 3 weeks. We also subjected in vitro primary sensory neuron
culture systems to nerve elongation and branching assays after EDN1 stimulation.
Results: Topical application of bosentan significantly attenuated the development of mite-induced AD-like
skin inflammation, dermatitis scores, ear thickness, scratching bouts, and serum level of thymus and
activation‐regulated chemokine in NC/Nga mice. Bosentan application also significantly reduced the gene
expression of Il13, Il17, and Ifng in the treated lesions. Histologically, the number of infiltrated dermal
cells, the epidermal EDN1 expression, and the number of intraepidermal nerve fibers were significantly
inhibited upon bosentan application. While EDN1 significantly elongated the neurites of dorsal root
ganglion cells in a dose- and time-dependent manner, bosentan treatment attenuated this.
Conclusions: EDN1 plays a significant role in mite-induced inflammation and itch. Topical bosentan is a
potential protective candidate for AD.

Role of 5-HT1A and 5-HT3 receptors in serotonergic activation of sensory neurons in relation to itch and pain behavior in the rat

Dan Domocos, Tudor Selescu, Laura Cristina Ceafalan,  Mirela Iodi Carstens, Earl Carstens, Alexandru Babes

Abstract

Serotonin (5-hydroxytryptamine, 5-HT) released by platelets, mast cells, and immunocytes is a potent inflammatory mediator which modulates pain and itch sensing in the peripheral nervous system. The serotonergic receptors expressed by primary afferent neurons involved in these sensory functions are not fully identified and appear to be to a large extent species dependent. Moreover, the mechanisms through which 5-HT receptor activation is coupled to changes in neuronal excitability have not been completely revealed. Using a combination of in vitro (calcium and voltage imaging and patch-clamp) and in vivo behavioral methods, we used both male and female Wistar rats to provide evidence for the involvement of two 5-HT receptor subtypes, 5-HT1A and 5-HT3, in mediating the sustained and transient effects, respectively, of 5-HT on rat primary afferent neurons involved in pain and itch processing. In addition, our results are consistent with a model in which sustained serotonergic responses triggered via the 5-HT1A receptor are due to closure of background potassium channels, followed by membrane depolarization and action potentials, during which the activation of voltage-gated calcium channels leads to calcium entry. Our results may provide a better understanding of mammalian serotonergic itch signaling.

https://onlinelibrary.wiley.com/doi/full/10.1002/jnr.24633

Differential Coding of Itch and Pain by aSubpopulation of Primary Afferent Neurons

Behrang Sharif, Ariel R. Ase, Alfredo Ribeiro-da-Silva, Philippe Séguéla

Itch and pain are distinct unpleasant sensations that can be triggered from the same receptive fields in the skin, raising the question of how pruriception and nociception are coded and discriminated. Here, we tested the multimodal capacity of peripheral first-order neurons, focusing on the genetically defined subpopulation of mouse C-fibers that express the chloroquine receptor MrgprA3. Using optogenetics, chemogenetics, and pharmacology, we assessed the behavioral effects of their selective stimulation in a wide variety of conditions. We show that metabotropic Gq-linked stimulation of these C-afferents, through activation of native MrgprA3 receptors or DREADDs, evokes stereotypical pruriceptive rather than nocifensive behaviors. In contrast, fast ionotropic stimulation of these same neurons through light-gated cation channels or native ATP-gated P2X3 channels predominantly evokes nocifensive rather than pruriceptive responses. We conclude that C-afferents display intrinsic multimodality, and we provide evidence that optogenetic and chemogenetic interventions on the same neuronal populations can drive distinct behavioral outputs.

The nonselective cation channel TRPV4 inhibits angiotensin II receptors

Nicholas W. Zaccor, Charlotte J. Sumner, Solomon H. Snyder

Abstract

G protein-coupled receptors (GPCRs) are a ubiquitously expressed family of receptor proteins that regulate many physiological functions and other proteins. They act through two dissociable signaling pathways, the exchange of GDP to GTP by linked G proteins and the recruitment of β-arrestins. GPCRs modulate several members of the transient receptor potential (TRP) channel family of non-selective cation channels. How TRP channels reciprocally regulate GPCR signaling is less well explored. Here, using an array of biochemical approaches, including immunoprecipitation and -fluorescence, calcium imaging, phosphate radiolabeling, and a β-Arrestin dependent luciferase assay, we characterize a GPCR-TRP channel pair, angiotensin II receptor type 1 (AT1R) and transient receptor potential vanilloid 4 (TRPV4), in primary murine choroid plexus epithelial cells and immortalized cell lines. We found that AT1R and TRPV4 are binding partners, and that activation of AT1R by angiotensin II (ANGII) elicits β-arrestin-dependent inhibition and internalization of TRPV4. Activating TRPV4 with endogenous and synthetic agonists inhibited ANGII-mediated G-protein associated second messenger accumulation, AT1R receptor phosphorylation and β-arrestin recruitment. We also noted that TRPV4 inhibits AT1R phosphorylation by activating the calcium-activated phosphatase calcineurin in a Ca2+/calmodulin dependent manner, preventing β-arrestin recruitment and receptor internalization. These findings suggest that when TRP channels and GPCRs are co-expressed in the same tissues, many of these channels can inhibit GPCR desensitization.

Local Ca2+ signals couple activation of TRPV1 and ANO1 sensory ion channels

Shihab Shah 1, Chase M Carver 2, Pierce Mullen 1, Stephen Milne 1, Viktor Lukacs 1, Mark S Shapiro 2, Nikita Gamper 3 4

Abstract

ANO1 (TMEM16A) is a Ca2+-activated Cl− channel (CaCC) expressed in peripheral somatosensory neurons that are activated by painful (noxious) stimuli. These neurons also express the Ca2+-permeable channel and noxious heat sensor TRPV1, which can activate ANO1. Here, we revealed an intricate mechanism of TRPV1-ANO1 channel coupling in rat dorsal root ganglion (DRG) neurons. Simultaneous optical monitoring of CaCC activity and Ca2+ dynamics revealed that the TRPV1 ligand capsaicin activated CaCCs. However, depletion of endoplasmic reticulum (ER) Ca2+ stores reduced capsaicin-induced Ca2+ increases and CaCC activation, suggesting that ER Ca2+ release contributed to TRPV1-induced CaCC activation. ER store depletion by plasma membrane–localized TRPV1 channels was demonstrated with an ER-localized Ca2+ sensor in neurons exposed to a cell-impermeable TRPV1 ligand. Proximity ligation assays established that ANO1, TRPV1, and the IP3 receptor IP3R1 were often found in close proximity to each other. Stochastic optical reconstruction microscopy (STORM) confirmed the close association between all three channels in DRG neurons. Together, our data reveal the existence of ANO1-containing multichannel nanodomains in DRG neurons and suggest that coupling between TRPV1 and ANO1 requires ER Ca2+ release, which may be necessary to enhance ANO1 activation.

Enhanced Thermal Sensitivity of TRPV3 in Keratinocytes Underlies Heat-Induced Pruritogen Release and Pruritus in AD

Seong Hoon Seo, Sohyun Kim, Song-Ee Kim, Seungsoo Chung and Sang Eun Lee

Abstract

Itch in atopic dermatitis (AD) is aggravated under warm conditions. Transient receptor potential vanilloid (TRPV) 3, a member of the thermosensitive transient receptor potential channels, is activated by innocuous heat and is abundantly expressed in keratinocytes. The potential role of TRPV3 in itch is illustrated in TRPV3 channelopathies of humans and mice. However, the role of TRPV3 in heat-induced itch in AD and the underlying mechanisms are unclear. Here we showed that keratinocytes isolated from patients with AD exhibit enhanced expression and heat sensitivity with hyperactive channel function of TRPV3. Heat stimulus induced enhanced secretion of thymic stromal lymphopoietin, nerve growth factor, and prostaglandin E2 by keratinocytes from patients with AD through TRPV3 activation. TRPV3 agonists increased thymic stromal lymphopoietin, nerve growth factor, prostaglandin E2, and IL-33 production in human keratinocytes and induced scratching behavior upon intradermal injection in mice. TRPV3 was upregulated in the skin of MC903-induced AD mouse model. Heat stimulation to MC903-treated mice increased scratching behavior and produced higher levels of thymic stromal lymphopoietin, nerve growth factor, prostaglandin E2, and IL-33 from the epidermis, which were attenuated by pharmacologic inhibition of TRPV3. Moreover, neutralization of thymic stromal lymphopoietin reduced heat-evoked scratching in MC903-challenged mice. These results suggest that TRPV3 is a potential therapeutic target for heat-induced itch in AD.

Abstract

Mast cells are a heterogeneous group of immune cells. The simplest and commonly accepted classification divides them in two groups according to their protease content. We have compared the action of diverse secretagogues on bone marrow derived (BMMC) and peritoneal (PMC) mast cells which represent classical models of mucosal and connective tissue type mast cells in mice. Whereas, antigen stimulation of the FcεRI receptors was similarly effective in triggering elevations of free intracellular Ca2+ concentration ([Ca2+]i) in both BMMC and PMC, robust [Ca2+]i rise following Endothelin-1 stimulation was observed only in a fraction of BMMC. Leukotriene C4 activating cysteinyl leukotriene type I receptors failed to evoke [Ca2+]i rise in either mast cell model. Stimulation of the recently identified target of many small-molecule drugs associated with systemic pseudo-allergic reactions, Mrgprb2, with compound 48/80, a mast cell activator with unknown receptor studied for many years, triggered Ca2+ oscillations in BMMC and robust [Ca2+]i rise in PMCs similarly to that evoked by FcεRI stimulation. [Ca2+]i rise in PMC could also be evoked by other Mrgprb2 agonists such as Tubocurarine, LL-37, and Substance P. The extent of [Ca2+]i rise correlated with mast cell degranulation. Expression analysis of TRPC channels as potential candidates mediating agonist evoked Ca2+ entry revealed the presence of transcripts of all members of the TRPC subfamily of TRP channels in PMCs. The amplitude and AUC of compound 48/80-evoked [Ca2+]i rise was reduced by ~20% in PMC from Trpc1/4/6−/− mice compared to Trpc1/4−/− littermatched control mice, whereas FcεRI-evoked [Ca2+]i rise was unaltered. Whole-cell patch clamp recordings showed that the reduction in compound 48/80-evoked [Ca2+]i rise in Trpc1/4/6−/− PMC was accompanied by a reduced amplitude of Compound 48/80-induced cation currents which exhibited typical features of TRPC currents. Together, this study demonstrates that PMC are an appropriate mast cell model to study mechanisms of Mrgprb2 receptor-mediated mast cell activation, and it reveals that TRPC channels contribute at least partially to Mrgprb2-mediated mast cellactivation but not following FcεRI stimulation. However, the channels conducting most of the Ca2+ entry in mast cells triggered by Mrgprb2 receptor stimulation remains to be identified.