Acid-sensing ion channel 3 is required for agmatine-induced histamine-independent itch in mice

Guo-Kun Zhou1†, Wen-Jing Xu1†, Yi Lu1†, Yan Zhou2†, Chen-Zhang Feng3†, Jiang-Tao Zhang1†, Shi-Yu Sun1, Ruo-Meng Wang2, Tong Liu1,4,5* and Bin Wu1*

Introduction: Itch is a common symptom of many skin and systemic diseases. Identifying novel endogenous itch mediators and the downstream signaling pathways involved will contribute to the development of new strategies for the treatment of chronic itch. In the present study, we adopted behavioral testing, patch clamp recording and metabonomics analysis to investigate the role of agmatine in itch and the underlying mechanism.

Methods: Behavioral analysis was used to evaluate the establishing of acute and chronic itch mice model, and to test the effects of different drugs or agents on mice itch behavior. Western blotting analysis was used to test the effect of agmatine on phosphorylation of ERK (p-ERK) expression in the spinal cord. Patch clamp recording was used to determine the effect agmatine on the excitability of DRG neurons and the role of ASIC3. Finally, the metabonomics analysis was performed to detect the concentration of agmatine in the affected skin under atopic dermatitis or psoriasis conditions.

Results: We fused a mouse model and found that an intradermal injection of agmatine (an endogenous polyamine) into the nape of the neck or cheek induced histamine-independent scratching behavior in a dose-dependent manner. In addition, the ablation of nociceptive C-fibers by resiniferatoxin (RTX) abolished agmatine-induced scratching behavior. However, agmatine-induced itch was not affected by the pharmacological inhibition of either transient receptor potential vanilloid 1 (TRPV1) or transient receptor potential ankyrin 1 (TRPA1); similar results were obtained from TRPV1^−/− or TRPA1^−/− mice. Furthermore, agmatine-induced itch was significantly suppressed by the administration of acid-sensing ion channel 3 (ASIC3) inhibitors, APETx2 or amiloride. Agmatine also induced the upregulation of p-ERK in the spinal cord; this effect was inhibited by amiloride. Current clamp recording showed that the acute perfusion of agmatine reduced the rheobase and increased the number of evoked action potentials in acute dissociated dorsal root ganglion (DRG) neurons while amiloride reversed agmatine-induced neuronal hyperexcitability. Finally, we identified significantly higher levels of agmatine in the affected skin of a mouse model of atopic dermatitis (AD) when compared to controls, and the scratching behavior of AD mice was significantly attenuated by blocking ASIC3.

Discussion: Collectively, these results provide evidence that agmatine is a novel mediator of itch and induces itch via the activation of ASIC3. Targeting neuronal ASIC3 signaling may represent a novel strategy for the treatment of itch.

KEYWORDS: itch, agmatine, ASIC3, atopic dermatitis, pain

Thymic stromal lymphopoietin controls hair growth

Jessica L. Shannon,1,2 David L. Corcoran,3 John C. Murray,1 Steven F. Ziegler,4,5 Amanda S. MacLeod,1,2,6

and Jennifer Y. Zhang1,7,*
1Department of Dermatology, Duke University, P.O. Box 103052, Durham, NC 27710, USA 2Department of Immunology, Duke University, Durham, NC 27710, USA
3Genomic and Computational Biology, Duke University, Durham, NC 27705, USA 4Benaroya Research Institute, Seattle, WA 98101, USA
5Department of Immunology, University of Washington, Seattle, WA 98195, USA 6Molecular Genetics and Microbiology, Duke University, Durham, NC 27710, USA 7Department of Pathology, Duke University Medical Center, Durham, NC 27710, USA

SUMMARY

Skin tissue regeneration after injury involves the production and integration of signals by stem cells residing in hair follicles (HFSCs). Much remains unknown about how specific wound-derived factors modulate stem cell contribution to hair growth. We demonstrate that thymic stromal lymphopoietin (TSLP) is produced in response to skin injury and during the anagen phase of the hair cycle. Intra- dermal injection of TSLP promoted wound-induced hair growth (WIHG), whereas neutralizing TSLP receptor (TSLPR) inhibited WIHG. Using flow cytometry and fluorescent immunostaining, we found that TSLP promoted proliferation of transit-amplifying cells. Lgr5CreER- mediated deletion of Tslpr in HFSCs inhibited both wound-induced and exogenous TSLP-induced hair growth. Our data highlight a novel function for TSLP in regulation of hair follicle activity during homeostasis and wound healing.

TRPC3 channel gating by lipids requires localization at the ER/PM junctions defined by STIM1

Abstract

TRPC3, a member of the transient receptor potential (TRP) superfamily of cation channels, is a lipid-regulated, Ca2+-
permeable channel that mediates essential components of the receptor evoked Ca2+ signal. The modes and mechanisms by which lipids regulate TRPC3 and other members of the TRPC channel family are not well understood. Here, we report that PI(4,5)P2 regulates TRPC3 in three independent modes. PLC-dependent hydrolysis generates diacylglycerol (DAG) that interacts with lipid-binding site 2 in the channel pore. PI(4,5)P2 interacts with lipid site 1 to inhibit TRPC3 opening and regulate access of DAG to the pore lipid site 2. PI(4,5)P2 is required for regulating pore ionic selectivity by receptor stimulation. Notably, the activation and regulation of TRPC3 by PI(4,5)P2 require recruitment of TRPC3 to the ER/PM junctions at a PI(4,5)P2-rich domain. Accordingly, we identified an FFAT site at the TRPC3 N terminal loop within the linker helices that envelope the C-terminus pole helix. The FFAT site interacts with the ER-resident VAPB to recruit TRPC3 to the ER/PM junctions and control its receptor-mediated activation. The TRPC3’s lipid interacting sites are fully conserved in TRPC6 and TRPC7 and in part in other TRPC channels. These findings inform on multiple modes of regulation of ion channels by lipids that may be relevant to diseases affected by aberrant TRPC channel functions.

Periostin activates distinct modules of inflammation and itching downstream of the type 2 inflammation pathway

Satoshi Nunomura ¹, Daisuke Uta ², Isao Kitajima ³, Yasuhiro Nanri ⁴, Kosuke Matsuda ², Naoko Ejiri ³, Midori Kitajima ³, Hitoshi Ikemitsu ⁴, Misaki Koga ⁴, Sayaka Yamamoto ⁴, Yuko Honda ⁴, Hironobu Takedomi ⁴, Tsugunobu Andoh ⁵, Simon J Conway ⁶, Kenji Izuhara ⁷

Abstract

Atopic dermatitis (AD) is a chronic relapsing skin disease accompanied by recurrent itching. Although type 2 inflammation is dominant in allergic skin inflammation, it is not fully understood how non-type 2 inflammation co-exists with type 2 inflammation or how type 2 inflammation causes itching. We have recently established the FADS mouse, a mouse model of AD. In FADS mice, either genetic disruption or pharmacological inhibition of periostin, a downstream molecule of type 2 inflammation, inhibits NF-κB activation in keratinocytes, leading to downregulating eczema, epidermal hyperplasia, and infiltration of neutrophils, without regulating the enhanced type 2 inflammation. Moreover, inhibition of periostin blocks spontaneous firing of superficial dorsal horn neurons followed by a decrease in scratching behaviors due to itching. Taken together, periostin links NF-κB-mediated inflammation with type 2 inflammation and promotes itching in allergic skin inflammation, suggesting that periostin is a promising therapeutic target for AD.

Keywords: CP: Immunology; atopic dermatitis; integrin; itching; neutrophil; periostin.

Presenter: Kim Hyein

Yan Chena,c,1, Yuran Songb,c,1 , Huadong Wangd,1 , Yiyun Zhangc,e, Xinyu Huc,f, Kaikai Wangc,e, Yingjin Lua, Zoutao Zhangg, Shuai Lia,c , Anan Lig, Lan Baoa,e,f, Fuqiang Xud, Changlin Lia,c,h,2, and Xu Zhanga,c,e,2

Abstract

Pain and itch are distinct sensations arousing evasion and compulsive desire for scratching, respectively. It’s unclear whether they could invoke different neural networks in the brain. Here, we use the type 1 herpes simplex virus H129 strain to trace the neural networks derived from two types of dorsal root ganglia (DRG) neurons: one kind of polymodal nociceptors containing galanin (Gal ) and one type of pruriceptors expressing neurotensin (Nts). The DRG microinjection and immunosuppression were performed in transgenic mice to achieve a successful tracing from specific types of DRG neurons to the primary sensory cortex. About one-third of nuclei in the brain were labeled. More
than half of them were differentially labeled in two networks. For the ascending pathways, the spinothalamic tract was absent in the network derived from Nts-expressing pruriceptors, and the two networks shared the spinobulbar projections but occupied different subnuclei. As to the motor systems, more neurons in the primary motor cortex and red nucleus of the somatic motor system participated in the Gal-containing nociceptor-derived network, while more neurons in the nucleus of the solitary tract (NST) and the dorsal motor nucleus of vagus nerve (DMX) of the emotional motor system was found in the Nts-expressing pruriceptor-derived network. Functional validation of differentially labeled nuclei by c-Fos test and chemogenetic inhibition suggested the red nucleus in facilitating the response to noxious heat and the NST/DMX in regulating the histamine-induced scratching. Thus, we reveal the organization of neural networks in a DRG neuron type-dependent manner for processing pain and itch.

Presenter: Gi Baek Lee

Yongfeng Liu ^# 1 2, Can Cao ^# 1, Xi-Ping Huang ^1 2, Ryan H Gumpper ¹, Moira M Rachman ³, Sheng-Luen Shih ^1 2, Brian E Krumm ¹, Shicheng Zhang ¹, Brian K Shoichet ³, Jonathan F Fay ^4 5, Bryan L Roth ^6 7 8

• PMID: 36302898
• DOI: 10.1038/s41589-022-01173-6

Abstract

The human MAS-related G protein-coupled receptor X1 (MRGPRX1) is preferentially expressed in the small-diameter primary sensory neurons and involved in the mediation of nociception and pruritus. Central activation of MRGPRX1 by the endogenous opioid peptide fragment BAM8-22 and its positive allosteric modulator ML382 has been shown to effectively inhibit persistent pain, making MRGPRX1 a promising target for non-opioid pain treatment. However, the activation mechanism of MRGPRX1 is still largely unknown. Here we report three high-resolution cryogenic electron microscopy structures of MRGPRX1-Gαq in complex with BAM8-22 alone, with BAM8-22 and ML382 simultaneously as well as with a synthetic agonist compound-16. These structures reveal the agonist binding mode for MRGPRX1 and illuminate the structural requirements for positive allosteric modulation. Collectively, our findings provide a molecular understanding of the activation and allosteric modulation of the MRGPRX1 receptor, which could facilitate the structure-based design of non-opioid pain-relieving drugs.

Ursolic acid downregulates thymic stromal lymphopoietin through the blockade of intracellular calcium/caspase‐1/NF‐κB signaling cascade in HMC‐1 cells

PHIL-DONG MOON1,2*, NA-RA HAN1*, JIN SOO LEE1, HYUNG-MIN KIM1 and HYUN-JA JEONG3

1department of Pharmacology, college of Korean Medicine, 2center for converging Humanities, Kyung Hee University, Seoul 02447; 3department of Food Science and Technology and Research Institute for Basic Science, Hoseo University, Asan, chungnam 31499, Republic of Korea

Received december 14, 2018; Accepted March 20, 2019 dOI: 10.3892/ijmm.2019.4144

Thymic stromal lymphopoietin (TSLP) plays an important role in allergic disorders, including atopic dermatitis and asthma. Ursolic acid (UA) has various pharmacological properties, such as antioxidant, anti‐inflammatory and anti- cancer. However, the effect of UA on TSLP regulation has not been fully elucidated. The aim of the present study was to analyze how UA regulates the production of TSLP in the human mast cell line HMc-1. Enzyme-linked immunosorbent assay, quantitative polymerase chain reaction analysis, western blotting, caspase-1 assay and fluorescent measurements of intracellular calcium levels were conducted to analyze the regulatory effects of UA. The results revealed that UA inhib- ited TSLP production and mRNA expression. In addition, UA reduced the activation of nuclear factor-κB and degradation of IκBα. caspase-1 activity was increased by exposure to phorbol myristate acetate plus calcium ionophore, whereas it was reduced by UA. Finally, UA treatment prevented an increase in intracellular calcium levels. These results indicated that UA may be a useful agent for the treatment and/or prevention of atopic and inflammatory diseases, and its effects are likely mediated by TSLP downregulation.

Kunhi Ryu , Yunkyung Heo , Yechan Lee , Hyejin Jeon , Wan Namkung 

Abstract

Chloroquine (CQ) is an antimalaria drug that has been widely used for decades. However, CQ-induced pruritus remains one of the major obstacles in CQ treatment for uncomplicated malaria. Recent studies have revealed that MrgprX1 plays an essential role in CQ-induced itch. To date, a few MrgprX1 antagonists have been discovered, but they are clinically unavailable or lack selectivity. Here, a cell-based high-throughput screening was performed to identify novel antagonists of MrgprX1, and the screening of 2543 compounds revealed two novel MrgprX1 inhibitors, berbamine and closantel. Notably, berbamine potently inhibited CQ-mediated MrgprX1 activation (IC₅₀ = 1.6 μM) but did not alter the activity of other pruritogenic GPCRs. In addition, berbamine suppressed the CQ-mediated phosphorylation of ERK1/2. Interestingly, CQ-induced pruritus was significantly reduced by berbamine in a dose-dependent manner, but berbamine had no effect on histamine-induced, protease-activated receptors 2-activating peptide-induced, and deoxycholic acid-induced itch in mice. These results suggest that berbamine is a novel, potent, and selective antagonist of MrgprX1 and may be a potential drug candidate for the development of therapeutic agents to treat CQ-induced pruritus.

Keywords: Chloroquine; MrgprA3; MrgprX1; antagonist; berbamine; itch.

Presenter: Hye In Kim

Department of Neuroscience, Perelman School of Medicine, University of Pennsylvania, Philadelphia, United States; 2
Graduate Group in Biostatistics, University of California Davis, Davis, United States; 3 Howard Hughes Medical Institute,
Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Harvard Medical School, Boston, United States; 4 Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, United States; 5 Department of Molecular and Cellular Biology, University of California Davis, Davis,
United States; 6 Department of Mathematics, University of California Davis, Davis, United States

Abstract

Mice are the most commonly used model animals for itch research and for development of anti-itch drugs. Most laboratories manually quantify mouse scratching behavior to assess itch intensity. This process is labor-intensive and limits large-scale genetic or drug screenings. In this study, we developed a new system, Scratch-AID (Automatic Itch Detection), which could automatically identify and quantify mouse scratching behavior with high accuracy. Our system included a custom-designed videotaping box to ensure high-quality and replicable mouse behavior recording and a convolutional recurrent neural network trained with frame-labeled mouse scratching behavior videos, induced by nape injection of chloroquine. The best trained network achieved 97.6% recall and 96.9% precision on previously unseen test videos. Remarkably, Scratch-AID could reliably identify scratching behavior in other major mouse itch models, including the acute cheek model, the histaminergic model, and a chronic itch model. Moreover, our system detected significant differences in scratching behavior between control and mice treated with an anti-itch drug. Taken together, we have established a novel deep learning-based system that could replace manual quantification for mouse scratching behavior in different itch models and for drug screening.

Presenter: Gi Baek Lee

Ningyong Xu, Donna L. Cioffi, Mikhail Alexeyev, Thomas C. Rich, and Troy Stevens

Abstract

Orai1 interacts with transient receptor potential protein of the canonical subfamily (TRPC4) and contributes to calcium selectivity of the endothelial cell store-operated calcium entry current (ISOC). Orai1 silencing increases sodium permeability and decreases membraneassociated calcium, although it is not known whether Orai1 is an important determinant of cytosolic sodium transitions. We test the hypothesis that, upon activation of store-operated calcium entry channels, Orai1 is a critical determinant of cytosolic sodium transitions. Activation of store-operated calcium entry channels transiently increased cytosolic calcium and sodium, characteristic of release from an intracellular store. The sodium response occurred more abruptly and returned to baseline more rapidly than did the transient calcium
rise. Extracellular choline substitution for sodium did not inhibit the response, although 2-aminoethoxydiphenyl borate and YM-58483 reduced it by 50%. After this transient response, cytosolic sodium continued to increase due to influx through activated store-operated calcium entry channels. The magnitude of this sustained increase in cytosolic sodium was greater when experiments were conducted in low extracellular calcium and when Orai1 expression was silenced; these two interventions were not additive, suggesting a common mechanism. 2-Aminoethoxydiphenyl borate and YM-58483 inhibited the sustained increase in cytosolic sodium, only in the presence of Orai1. These studies demonstrate that sodium permeates activated store-operated calcium entry channels, resulting in an increase in cytosolic sodium; the magnitude of this response is determined by Orai1.