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.

Baicalin induces Mrgprb2-dependent pseudo-allergy in mice 

Jue Wanga, Yongjing Zhanga, Delu Chea, Yingnan Zenga, Yuanyuan Wub, Qiaohong Qinc, Nan Wanga,*

a School of Pharmacy, Xi’an Jiaotong University, Xi’an, 710061, China
b Department of Respiratory and Critical Care Medicine, The Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an, 710061, China  c Institute of Basic and Translational Medicine, Xi’an Medical University, Xi’an, 710021, China

Baicalin, a component of traditional Chinese medicine, is one of the main compounds present in Scutellaria baicalensis Georgi. Pseudo-allergy induced by the injection of these medicines is a frequent adverse drug reaction. Therefore, elucidation of the anaphylactoid reaction of baicalin and its underlying mechanisms are important. Mast cells are primary effectors of allergic reactions, including pseudo-allergy. Studies have shown that Mrgprx2 in human mast cells is a specific receptor that is crucial for pseudo-allergic drug reactions, Mrgprb3 is the rat ortholog of human Mrgprx2, which in mice is designated as Mrgprb2. Here, we aimed to investigate baicalin- induced pseudo-allergy and the association of Mrgprb3 and Mrgprb2 with this effect. We examined the aller- genic effect of baicalin on RBL-2H3 cells and Mrgprb3-knockdown RBL-2H3 cells. Mrgprb2-expressing HEK293 cells and Mrgprb2-knockout mice were used to evaluate the role of Mrgprb2 in baicalin-induced allergy. Baicalin was found to dose-dependently induce pseudo-allergy both in vitro and in vivo. RBL-2H3 cells were activated by baicalin, whereas in Mrgprb3-knockout RBL-2H3 cells, baicalin showed a negligible effect on cell activation. Furthermore, baicalin activated the Mrgprb2-expressing HEK293 cells. Our data showed that baicalin did not induce allergy in Mpgprb2-knockout mice. We conclude that baicalin induces pseudo-allergy via Mrgprb2 in mice.

Keywords:Baicalin Pseudo-allergy Mrgprb2 Mrgprb3 Degranulation

Exploration of sensory and spinal neurons expressing gastrin-releasing peptide in itch and pain related behaviors

Devin M Barry ^1 2, Xue-Ting Liu ^1 2 3, Benlong Liu ^1 2, Xian-Yu Liu ^1 2, Fang Gao ^1 2, Xiansi Zeng ^1 2 4, Juan Liu ^1 2, Qianyi Yang ^1 2, Steven Wilhelm ^1 2, Jun Yin ^1 2, Ailin Tao ^1 3, Zhou-Feng Chen ^5 6 7 

Abstract

Gastrin-releasing peptide (GRP) functions as a neurotransmitter for non-histaminergic itch, but its site of action (sensory neurons vs spinal cord) remains controversial. To determine the role of GRP in sensory neurons, we generated a floxed Grp mouse line. We found that conditional knockout of Grp in sensory neurons results in attenuated non-histaminergic itch, without impairing histamine-induced itch. Using a Grp-Cre knock-in mouse line, we show that the upper epidermis of the skin is exclusively innervated by GRP fibers, whose activation via optogeneics and chemogenetics in the skin evokes itch- but not pain-related scratching or wiping behaviors. In contrast, intersectional genetic ablation of spinal Grp neurons does not affect itch nor pain transmission, demonstrating that spinal Grp neurons are dispensable for itch transmission. These data indicate that GRP is a neuropeptide in sensory neurons for non-histaminergic itch, and GRP sensory neurons are dedicated to itch transmission.

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

A vaccine targeting the RBD of the S protein of SARS-CoV-2 induces protective immunity

Abstract

The novel Coronavirus SARS-CoV-2 causes a respiratory illness called COVID-19 leading to a pandemic. An effective preventive vaccine against this virus is urgently needed. As the most critical step during infection, SARS CoV-2 uses its Spike protein receptor-binding domain (S- RBD) to engage with the host cell receptor angiotensin converting enzyme 2 (ACE2)1,2. Here we showed that a recombinant vaccine comprising residues 319-545 of the S-RBD could induce a potent functional antibody response in the immunized mice, rabbits and non-human primates (Macaca mulatta) as early as 7 or 14 days after a single dose injection. The sera from the immunized animals blocked RBD binding to ACE2 expressed on the cell surface and neutralized the infection by SARS-CoV-2 pseudovirus and live SARS-CoV-2 in vitro. Importantly, the vaccination also provided protection in non-human primates from SARS-CoV-2 challenge in vivo. The elevated RBD-specific antibodies were also found in the sera from patients with COVID-19. Several immune pathways and CD4 T lymphocytes were implicated in the induction of the vaccine antibody response. Our finding highlights the importance of the RBD domain in the SARS-CoV-2 vaccine design and provides the rationale for the development of a protective vaccine through the induction of antibody against the RBD domain.

Periostin Activation of Integrin Receptors on Sensory Neurons Induces Allergic Itch

Santosh K. Mishra,1,2,3,4,9,* Joshua J. Wheeler,1,2 Saumitra Pitake,1 Huiping Ding,5 Changyu Jiang,7 Tomoki Fukuyama,1 Judy S. Paps,8 Patrick Ralph,1 Jacob Coyne,1 Michelle Parkington,1 Jennifer DeBrecht,1 Lauren C. Ehrhardt-Humbert,1 Glenn P. Cruse,1,2 Wolfgang Ba ̈ umer,1,6 Ru-Rong Ji,7 Mei-Chuan Ko,5 and Thierry Olivry2,8
1Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA 2Comparative Medicine Institute, North Carolina State University, Raleigh, NC, USA

3The WM Keck Behavioral Center, North Carolina State University, Raleigh, NC, USA
4Program in Genetics, North Carolina State University, Raleigh, NC, USA
5Department of Physiology and Pharmacology, Wake Forest School of Medicine, Winston-Salem, NC, USA
6Institute of Pharmacology and Toxicology, Department of Veterinary Medicine, Freie Universita ̈ t Berlin, Berlin, Germany 7Duke University, Durham, NC, USA
8Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA 9Lead Contact
*Correspondence: skmishra@ncsu.edu

SUMMARY

Chronic allergic itch is a common symptom affecting millions of people and animals, but its pathogenesis is not fully explained. Herein, we show that periostin, abundantly expressed in the skin of patients with atopic dermatitis (AD), induces itch in mice, dogs, and monkeys. We identify the integrin aVb3 expressed on a subset of sensory neurons as the periostin receptor. Using pharmacological and ge- netic approaches, we inhibited the function of neuronal integrin aVb3, which significantly reduces periostin-induced itch in mice. Furthermore, we show that the cytokine TSLP, the application of AD- causing MC903 (calcipotriol), and house dust mites all induce periostin secretion. Finally, we establish that the JAK/STAT pathway is a key regulator of periostin secretion in keratinocytes. Altogether, our results identify a TSLP-periostin reciprocal activation loop that links the skin to the spinal cord via periph- eral sensory neurons, and we characterize the non- canonical functional role of an integrin in itch.

https://doi.org/10.1016/j.celrep.2020.03.036

Irritant-evoked activation and calcium modulation of the TRPA1 receptor

Jianhua Zhao, John V. Lin King, Candice E. Paulsen, Yifan Cheng & David Julius

Abstract

The transient receptor potential ion channel TRPA1 is expressed by primary afferent nerve fibres, in which it functions as a low-threshold sensor for structurally diverse electrophilic irritants, including small volatile environmental toxicants and endogenous algogenic lipids¹. TRPA1 is also a ‘receptor-operated’ channel whose activation downstream of metabotropic receptors elicits inflammatory pain or itch, making it an attractive target for novel analgesic therapies². However, the mechanisms by which TRPA1 recognizes and responds to electrophiles or cytoplasmic second messengers remain unknown. Here we use strutural studies and electrophysiology to show that electrophiles act through a two-step process in which modification of a highly reactive cysteine residue (C621) promotes reorientation of a cytoplasmic loop to enhance nucleophilicity and modification of a nearby cysteine (C665), thereby stabilizing the loop in an activating configuration. These actions modulate two restrictions controlling ion permeation, including widening of the selectivity filter to enhance calcium permeability and opening of a canonical gate at the cytoplasmic end of the pore. We propose a model to explain functional coupling between electrophile action and these control points. We also characterize a calcium-binding pocket that is highly conserved across TRP channel subtypes and accounts for all aspects of calcium-dependent TRPA1 regulation, including potentiation, desensitization and activation by metabotropic receptors. These findings provide a structural framework for understanding how a broad-spectrum irritant receptor is controlled by endogenous and exogenous agents that elicit or exacerbate pain and itch.

https://www.nature.com/articles/s41586-020-2480-9.pdf

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.

Transient receptor potential vanilloid 4 (TRPV4) channel as a target of crotamiton and its bimodal effects

Hiroki Kittaka1 & Yu Yamanoi1,2,3 & Makoto Tominaga1,2,4

Received: 21 January 2017 / Revised: 2 May 2017 / Accepted: 12 May 2017 / Published online: 13 June 2017 # Springer-Verlag Berlin Heidelberg 2017

Abstract The sensation of itching can be defined as Ban un- pleasant cutaneous sensation that provokes a desire to scratch.^ The perception of itching is not critical for the main- tenance of life, but persistent itching can be extremely irritat- ing and decreases the quality of life. Crotamiton (N-ethyl-o- crotonotoluidide) has been used as an anti-itch agent for humans for around 70 years. In spite of the long use of crotamiton, its mechanism of action remains unknown. We hypothesized that crotamiton might have effects on transient receptor potential (TRP) channels expressed in the peripheral nervous system and the skin. We first examined the effects of crotamiton on TRP channels by whole-cell patch-clamp re- cordings. We found that crotamiton strongly inhibited TRPV (vanilloid) 4 channels followed by large currents after crotamiton washout. In mice, crotamiton inhibited itch- related behaviors induced by a TRPV4-selective agonist (GSK1016790A). We biophysically investigated the large TRPV4 currents after crotamiton washout. Comparing single-channel open probabilities and current amplitudes of TRPV4, increases in both parameters were found to contribute to the large washout currents of TRPV4. Because the change in current amplitudes suggested pore dilation of TRPV4, we examined this possibility with cation replacement experiments and by measuring changes in reversal potentials. Greater cat- ion influxes and changes in reversal potentials upon crotamiton washout were observed, suggesting that the TRPV4 pore dilated in its uninhibited state. From these re- sults, we identified the molecular target of crotamiton as TRPV4 and demonstrated pore dilation of TRPV4 upon crotamiton washout.

Keywords Crotamiton . TRPV4 . Pore dilation . Itch

Osthole, a Natural Plant Derivative Inhibits MRGPRX2 Induced Mast Cell Responses

Abstract

Mast cells are tissue-resident innate immune cells known for their prominent role in mediating allergic reactions. MAS-related G-protein coupled receptor-X2 (MRGPRX2) is a promiscuous G-protein coupled receptor (GPCR) expressed on mast cells that are activated by several ligands that share cationic and amphipathic properties. Interestingly, MRGPRX2 ligands include certain FDA-approved drugs, antimicrobial peptides, and neuropeptides. Consequently, this receptor has been implicated in causing mast cell-dependent pseudo-allergic reactions to these drugs and chronic inflammation associated with asthma, urticaria and rosacea in humans. In the current study, we examined the role of osthole, a natural plant coumarin, in regulating mast cell responses when activated by the MRGPRX2 ligands, including compound 48/80, the neuropeptide substance P, and the cathelicidin LL-37. We demonstrate that osthole attenuates both the early (Ca2C mobilization and degranulation) and delayed events (chemokine/cytokine production) of mast cell activation via MRGPRX2 in vitro. Osthole also inhibits MrgprB2- (mouse ortholog of human MRGPRX2) dependent inflammation in in vivo mouse models of pseudo-allergy. Molecular docking analysis suggests that osthole does not compete with the MRGPRX2 ligands for interaction with the receptor,
but rather regulates MRGPRX2 activation via allosteric modifications. Furthermore, flow cytometry and confocal microscopy experiments reveal that osthole reduces both surface and intracellular expression levels of MRGPRX2 in mast cells. Collectively, our data demonstrate that osthole inhibits MRGPRX2/MrgprB2-induced mast cell responses and provides a rationale for the use of this natural compound as a safer alternative treatment for pseudo-allergic reactions in humans.

Keywords: mast cells, pseudo-allergic reactions, osthole, MAS-related G-protein coupled receptor-X2, MrgprB2