Cav3.2 T-type calcium channel mediates acute itch and contributes to chronic itch and inflammation in experimental atopic dermatitis

Ji-Woong Ahn ¹, Song-Ee Kim ², Do-Young Kim ³, Inhye Jeong ², Sohyun Kim ¹, Seungsoo Chung ⁴, Sang Eun Lee ⁵

• PMID: 37863387
• DOI: 10.1016/j.jid.2023.07.029

Abstract

Voltage-gated calcium channels regulate neuronal excitability. The Cav3.2 isoform of the T-type voltage-activated calcium channel is expressed in sensory neurons and is implicated in pain transmission. However, its role in itch remains unclear. Herein, we demonstrated that Cav3.2 is expressed by mechanosensory and peptidergic subsets of mouse dorsal root ganglion (DRG) neurons and colocalized with TRPV1 and receptors for type 2 cytokines. Cav3.2-positive neurons innervate human skin. A deficiency of Cav3.2 reduces histamine, IL-4/IL-13, and thymic stromal lymphopoietin-induced itch in mice. Cav3.2 channels were upregulated in the DRGs of an atopic dermatitis (AD)-like mouse model and mediated neuronal excitability. Genetic knockout of Cav3.2 or T-type calcium channel blocker mibefradil treatment reduced spontaneous and mechanically induced scratching behaviors and skin inflammation in an AD-like mouse model. Substance P and vasoactive intestinal polypeptide levels were increased in the trigeminal ganglia (TG) from AD-like mouse model, and genetic ablation or pharmacological inhibition of Cav3.2 reduced their gene expression. Cav3.2 knockout also attenuated the pathologic changes in ex vivo skin explants co-cultured with TG neurons from AD-induced mice. Our study identifies the role of Cav3.2 in both histaminergic and non-histaminergic acute itch. Cav3.2 channel also contributes to AD-related chronic itch and neuroinflammation.

Keywords: Cav3.2; T-type voltage-activated calcium channel; atopic dermatitis; itch; neuroinflammation; substance P; vasoactive intestinal polypeptide.

Pain and itch coding mechanisms of polymodal sensory neurons

Abstract

Pain and itch coding mechanisms in polymodal sensory neurons remain elusive. MrgprD+ neurons represent a major polymodal population and mediate both mechanical pain and nonhistaminergic itch. Here, we show that chemogenetic activation of MrgprD+ neurons elicited both pain- and itch-related behavior in a dose-dependent manner, revealing an unanticipated compatibility between pain and itch in polymodal neurons. While VGlut2-dependent glutamate release is required for both pain and itch transmission from MrgprD+ neurons, the neuropeptide neuromedin B (NMB) is selectively required for itch signaling. Electrophysiological recordings further demonstrated that glutamate synergizes with NMB to excite NMB-sensitive postsynaptic neurons. Ablation of these spinal neurons selectively abolished itch signals from MrgprD+ neurons, without affecting pain signals, suggesting a dedicated itch-processing central circuit. These findings reveal distinct neurotransmitters and neural circuit requirements for pain and itch signaling from MrgprD+ polymodal sensory neurons, providing new insights on coding and processing of pain and itch.

Presenter: Ju Hee Ryu

Satellite Glial Cells and Neurons in Trigeminal Ganglia Are Altered in an Itch Model in Mice

MeytalCohen1,2, RachelFeldman-Goriachnik1,2 and MenachemHanani1,2,*

1 LaboratoryofExperimentalSurgery,Hadassah-HebrewUniversityMedicalCenter,Jerusalem91240,Israel; meytal.cohen@mail.huji.ac.il(M.C.);rahel.gor@gmail.com(R.F.-G.)

2 FacultyofMedicine,TheHebrewUniversityofJerusalem,MountScopus,Jerusalem91240,Israel * Correspondence:hananim@cc.huji.ac.il;Tel.:+972-2-5844721

Abstract: Itch(pruritus) is a common chronic condition with a lifetime prevalence of over 20%. The mechanisms underlying itch are poorly understood,and its therapy is difficult. There is recent evidence that following nerve injury or inflammation, intercellular communications in sensory ganglia are augmented, which may lead to abnormal neuronal activity, and hence to pain, but there is no information on whether such changes take place in an itch model. We studied changes in neurons and satellite glial cells(SGCs)in trigeminal ganglia in an itch model in mice using repeated applications of 2,4,6-trinitro-1-chlorobenzene(TNCB) to the external ear over a period of 11days. Treated mice showed augmented scratching behavior as compared with controls during the application period and for several days afterward.Immunostaining for the activation marker glial fibrillary acidic protein SGCs was greater by about 35% after TNCB application, and gap junction-mediated coupling between neurons increased from about 2% to 13%. The injection of gap junction blockers reduced scratching behavior, suggesting that gap junctions contribute to itch. Calcium imaging studies showed increased responses of SGCs to the pain (and presumed itch )mediator ATP. We conclude that changes in both neurons and SGCs in sensory ganglia may play a role in itch.

Ligand recognition and G protein coupling of the human itch receptor MRGPRX1

Abstract
MRGPRX1, a Mas-related GPCR (MRGPR), is a key receptor for itch perception and targeting MRGPRX1 may have potential to treat both chronic itch and pain. Here we report cryo-EM structures of the MRGPRX1-Gi1 and MRGPRX1-Gq trimers in complex with two peptide ligands, BAM8-22 and CNF-Tx2. These structures reveal a shallow orthosteric pocket and its conformational plasticity for sensing multiple different peptidic itch allergens. Distinct from MRGPRX2, MRGPRX1 contains a unique pocket feature at the extracellular ends of TM3 and TM4 to accommodate the peptide C-terminal “RF/RY” motif, which could serve as key mechanisms for peptidic allergen recognition. Below the ligand binding pocket, the G6.48XP6.50F6.51G6.52X(2)F/W6.55 motif is essential for the inward tilting of the upper end of TM6 to induce receptor activation. Moreover, structural features inside the ligand pocket and on the cytoplasmic side of MRGPRX1 are identified as key elements for both Gi and Gq signaling.
Collectively, our studies provide structural insights into understanding itch sensation, MRGPRX1 activation, and downstream G protein signaling.

Presenter: Ju Hee Ryu

Keratinocyte-derived defensins activate neutrophil-specific receptors Mrgpra2a/b to prevent skin dysbiosis and bacterial infection

Abstract
Healthy skin maintains a diverse microbiome and a potent immune system to fight off infections. Here, we discovered that the epithelial-cell-derived antimicrobial peptides defensins activated orphan G-protein-coupled receptors (GPCRs) Mrgpra2a/b on neutrophils. This signaling axis was required for effective neutrophil-mediated skin immunity and microbiome homeostasis. We generated mutant mouse lines lacking the entire Defensin (Def) gene cluster in keratinocytes or Mrgpra2a/b. Def and Mrgpra2 mutant animals both exhibited skin dysbiosis, with reduced microbial diversity and expansion of Staphylococcus species. Defensins and Mrgpra2 were critical for combating S. aureus infections and the formation of neutrophil abscesses, a hallmark of antibacterial immunity. Activation of Mrgpra2 by defensin triggered neutrophil release of IL-1b and CXCL2 which are vital for proper amplification and propagation of the antibacterial immune response. This study demonstrated the importance of epithelial-neutrophil signaling via the defensin-Mrgpra2 axis in maintaining healthy skin ecology and promoting antibacterial host defense.

Presenter: Ji Eun Cha

Kallikrein 7 Promotes Atopic Dermatitis-Associated Itch Independently of Skin Inflammation

Abtract

Atopic dermatitis (AD) is a highly prevalent, itchy inflammatory skin disorder that is thought to arise from a combination of skin barrier defect and immune dysregulation. Kallikreins (KLK), a family of serine proteases with a diverse array of homeostatic functions, including skin desquamation and innate immunity, are hypothesized to contribute to AD pathogenesis. However, their precise role in AD has not been clearly defined. In this study, RNA sequencing analyses identified KLK7 as the most abundant and differentially expressed KLK in both human AD and murine AD-like skin. Further, in mice, Klk7 expression was localized to the epidermis in both steady state and inflammation. Unexpectedly, KLK7 was dispensable for the development of AD-associated skin inflammation. Instead, KLK7 was selectively required for AD-associated chronic itch. Even without the alleviation of skin inflammation, KLK7-deficient mice exhibited significantly attenuated scratching, compared with littermate controls, after AD-like disease induction. Collectively, our findings indicate that KLK7 promotes AD-associated itch independently from skin inflammation and reveal a previously unrecognized epidermal-neural mechanism of AD associated itch.

Pamoic Acid-Induced Peripheral GPR35 Activation Improves Pruritus and Dermatitis

Chaeeun Kim ¹, Yerin Kim ¹, Ji Yeon Lim ¹, Minseok Kim ¹, Haiyan Zheng ¹, Miri Kim ¹, Sun Wook Hwang ¹

Affiliations expand

• PMID: 37501600
• DOI: 10.1111/bph.16201

Abstract

Background and purpose: Pruritic dermatitis is a disease with a considerable unmet need for treatment and appears to present with not only epidermal but also peripheral neuronal complications. Here we propose a novel pharmacologic modulation targeting both peripheral dorsal root ganglion (DRG) sensory neurons and skin keratinocytes. GPR35 is an orphan G-protein-coupled receptor expressed in DRG neurons and has been predicted to downregulate neuronal excitability when activated. Modulator information is currently increasing for GPR35 and pamoic acid (PA), a salt-forming agent for drugs, has been shown to be an activator solely specific for GPR35. Here we investigated its effect on dermatitic pathology.

Experimental approach: We confirmed GPR35 expression in peripheral neurons and tissues. The effect of PA treatment was pharmacologically evaluated in cultured cells in vitro and in in vivo animal models for acute and chronic pruritus.

Key results: Local PA application mitigated acute non-histaminergic itch and consistently, obstructed DRG neuronal responses. Keratinocyte fragmentation under dermatitic simulation was also dampened following PA incubation. Chronic pruritus in 1-chloro-2,4-dinitrobenzene (DNCB) and psoriasis models was also moderately but significantly reversed by the repeated applications of PA. Dermatitic scores in the DNCB and psoriatic models were also improved by its application, indicating that it is beneficial for mitigating disease pathology.

Conclusions and implications: Our findings suggest that pamoic acid activation of peripheral GPR35 can contribute to the improvement of pruritus and its associated diseases.

Keywords: GPR35; Pamoic acid; dermatitis; itch; neuron.

Microglia are involved in regulating histamine-dependent and non-dependent itch transmissions with distinguished signal pathways

Yuxiu Yang, Bin Mou, Qi-Ruo Zhang, Hong-Xue Zhao, Jian-Yun Zhang, Xiao Yun, Ming-Tao Xiong, Ying Liu, Yong U Liu, Haili Pan, Chao-Lin Ma, Bao-Ming Li, Jiyun Peng

https://doi.org/10.1002/glia.24438

Abstract

Although itch and pain have many similarities, they are completely different in perceptual experience and behavioral response. In recent years, we have a deep understanding of the neural pathways of itch sensation transmission. However, there are few reports on the role of non-neuronal cells in itch. Microglia are known to play a key role in chronic neuropathic pain and acute inflammatory pain. It is still unknown whether microglia are also involved in regulating the transmission of itch sensation. In the present study, we used several kinds of transgenic mice to specifically deplete CX3CR1+ microglia and peripheral macrophages together (whole depletion), or selectively deplete microglia alone (central depletion). We observed that the acute itch responses to histamine, compound 48/80 and chloroquine were all significantly reduced in mice with either whole or central depletion. Spinal c-fos mRNA assay and further studies revealed that histamine and compound 48/80, but not chloroquine elicited primary itch signal transmission from DRG to spinal Npr1- and somatostatin-positive neurons relied on microglial CX3CL1-CX3CR1 pathway. Our results suggested that microglia were involved in multiple types of acute chemical itch transmission, while the underlying mechanisms for histamine-dependent and non-dependent itch transmission were different that the former required the CX3CL1-CX3CR1 signal pathway.

Keywords: CX3CR1; Nppb; TRPV1; c-fos; histamine; itch; microglia.

TRPV3-ANO1 interaction positively regulates wound healing in keratinocytes

Yu Yamanoi1,2,3, Jing Lei1,2, Yasunori Takayama4, Shigekuni Hosogi5, Yoshinori Marunaka6,7 &Makoto Tominaga1,2

Communications Biology volume 6, Article number: 88 (2023) Cite this article

Abstract

Transient receptor potential vanilloid 3 (TRPV3) belongs to the TRP ion channel super family and functions as a nonselective cation channel that is highly permeable to calcium. This channel is strongly expressed in skin keratinocytes and is involved in warmth sensation, itch, wound healing and secretion of several cytokines. Previous studies showed that anoctamin1 (ANO1), a calcium-activated chloride channel, was activated by calcium influx through TRPV1, TRPV4 or TRPA1 and that these channel interactions were important for TRP channel-mediated physiological functions. We found that ANO1 was expressed by normal human epidermal keratinocytes (NHEKs). We observed that ANO1 mediated currents upon TRPV3 activation of NHEKs and mouse skin keratinocytes. Using an in vitro wound-healing assay, we observed that either a TRPV3 blocker, an ANO1 blocker or low chloride medium inhibited cell migration and proliferation through p38 phosphorylation, leading to cell cycle arrest. These results indicated that chloride influx through ANO1 activity enhanced wound healing by keratinocytes.