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.

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

Antipruritic Effect of Ethyl Acetate Extract from Fructus cnidii in Mice with 2,4-Dinitrofluorobenzene-Induced Atopic Dermatitis.

Abstract

Atopic dermatitis (AD) is a common inflammatory skin disease characterized by intense pruritus and skin lesions. The exact cause of AD is not yet known and the available therapeutic strategies for AD are limited. Fructus cnidii is commonly used in traditional Chinese medicine as an herb for treating chronic itch. However, the mechanism underlying the antipruritic effects of Fructus cnidii is not well understood. In the present study, we investigated the antipruritic effect of locally administered ethyl acetate extract from Fructus cnidii (EAEFC) to 2,4-dinitrofluorobenzene- (DNFB-) induced AD in a mouse model. The scratching behavior, skin thickness, dermatitis score, weight, blood immunoglobulin E (IgE) level, and itch-related cytokine levels were subsequently monitored and evaluated. Results showed that EAEFC treatment attenuated the DNFB-induced AD-like symptoms by alleviating the skin lesions and decreasing the dermatitis score. Hematoxylin and eosin (H&E) and toluidine blue (TB) staining analyses demonstrated that EAEFC mitigated the DNFB-induced increase in skin thickness and prevented the infiltration of mast cells. Behavioral tests showed that EAEFC decreased the DNFB-induced acute and chronic scratching behaviors. Furthermore, EAEFC reduced the levels of itch-related cytokines, such as thymic stromal lymphopoietin (TSLP), interleukin- (IL-) 17, IL-33, and IL-31, and the DNFB-induced boost in serum IgE. Collectively, these results suggest that EAEFC is a potential therapeutic candidate for the treatment of chronic itch in AD.

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.

Typical antimicrobials induce mast cell degranulation and anaphylactoid reactions via MRGPRX2 and its murine homologue MRGPRB2

ABSTRACT

Mast cells are unique immune cells that function as sentinels in host defence reactions, including immediate hypersensitivity responses and allergic responses. The mast cell-specific receptor named MAS-related G protein-coupled receptor X2 (MRGPRX2) triggers mast-cell degranulation, a key process in anaphylactoid reactions. It is widely observed that antimicrobials can induce pseudo-allergic reactions (i.e. IgE-independent mechanism) with symptoms ranging from skin inflammation to life-threatening systemic anaphylaxis. However, their direct involvement and the mechanisms underlying anaphylactoid reactions caused by antimicrobials have not been demonstrated. Structurally different antimicrobials were screened by Ca2+ imaging using MRGPRX2 overexpressing HEK293 cells. MRGPRX2 related anaphylactoid reactions induced by these components were investigated by body temperature drop and mast cell degranulation assays. We showed that MRGPRX2 is involved in allergic-like reactions to three types of antimicrobials in a dose-dependent manner. However, mast cells lacking the receptor show reduced degranulation. Furthermore, mice without MAS-related G protein-coupled receptor B2 (the orthologous gene of MRGPRX2) exhibited reduced substance-induced inflammation. Interestingly, β-lactam and antiviral nucleoside analogues did not induce anaphylactic reactions, which were also observed in vitro. These results should alarm many clinicians that such drugs might induce anaphylactoid reactions and provide guidance on safe dosage of these drugs.

Keywords: Anaphylactoid reaction Antimicrobials Degranulation Mast Cells Mrgprx2

Spinal-cord-NLRP1-inflommsome-open-access-contributes-to-dry-skin-induced-chronic-itch-in-mice

Abstract
Background: Dry skin itch is one of the most common skin diseases and elderly people are believed to be particularly prone to it. The inflammasome has been suggested to play an important role in chronic inflammatory disorders including inflammatory skin diseases such as psoriasis. However, little is known about the role of NLRP1 inflammasome in dry skin-induced chronic itch.
Methods: Dry skin-induced chronic itch model was established by acetone-ether-water (AEW) treatment. Spontaneous scratching behavior was recorded by video monitoring. The expression of nucleotide oligomerization domain (NOD)-like receptor protein 1 (NLRP1) inflammasome complexes, transient receptor potential vanilloid type 1 (TRPV1), and the level of inflammatory cytokines were determined by western blot, quantitative real-time PCR, and enzyme-linked immunosorbent assay (ELISA) kits. Nlrp1a knockdown was performed by an adeno-associated virus (AAV) vector containing Nlrp1a-shRNA- eGFP infusion. H.E. staining was used to evaluate skin lesion.
Results: AEW treatment triggers spontaneous scratching and significantly increases the expression of NLRP1, ASC, and caspase-1 and the levels of IL-1β, IL-18, IL-6, and TNF-α in the spinal cord and the skin of mice. Spinal cord Nlrp1a knockdown prevents AEW-induced NLRP1 inflammasome assembly, TRPV1 channel activation, and spontaneous scratching behavior. Capsazepine, a specific antagonist of TRPV1, can also inhibit AEW-induced inflammatory response and scratching behavior. Furthermore, elderly mice and female mice exhibited more significant AEW-induced scratching behavior than young mice and male mice, respectively. Interestingly, AEW-induced increases in the expression of NLRP1 inflammasome complex and the levels of inflammatory cytokines were more remarkable in elderly mice and female mice than in young mice and male mice, respectively.
Conclusions: Spinal cord NLRP1 inflammasome-mediated inflammatory response contributes to dry skin-induced chronic itch by TRPV1 channel, and it is also involved in age and sex differences of chronic itch. Inhibition of NLRP1 inflammasome may offer a new therapy for dry skin itch.
Keywords: NLRP1 inflammasome, TRPV1, Chronic itch, Dry skin, Spinal cord

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.