Br J Pharmacol. 2023 Mar 16. doi: 10.1111/bph.16072. Online ahead of print.

Molecular mechanisms of MrgprA3-independent activation of the transient receptor potential ion channels TRPA1 and TRPV1 by chloroquine

Tabea C Fricke ¹, Sebastian Pantke ¹, Bjarne Lüttmann ¹, Frank G Echtermeyer ¹, Christine Herzog ¹, Mirjam J Eberhardt ¹, Andreas Leffler ¹

• PMID: 36928865
• DOI: 10.1111/bph.16072

Abstract

Background and purpose: Itch associates several pathologies and is a common drug-induced side effect. Chloroquine (CQ) was reported to induce itch by activating the Mas-related G protein-coupled receptor MrgprA3 and subsequently TRPA1. In this study we demonstrate that CQ employs at least two MrgprA3-independent mechanisms to activate or sensitize TRPA1 and TRPV1.

Experimental approach: Patch Clamp and calcium-imaging were utilized to examine effects of CQ on TRPA1 and TRPV1 expressed in HEK-293T cells.

Key results: In calcium-imaging, CQ induces a concentration-dependent but MrgprA3-independent activation of TRPA1 and TRPV1. While CQ itself inhibits TRPA1 and TRPV1 in patch clamp recordings, co-application of CQ and UVA-light evokes membrane currents through both channels. This effect is inhibited by the reducing agent dithiothreitol (DTT) and reduced on mutants lacking cysteine residues accounting for reactive oxygen species (ROS)-sensitivity. The combination of CQ and UVA-light triggers an accumulation of intracellular ROS, removes fast inactivation of voltage-gated sodium currents and activates TRPV2. On the other hand, CQ is a weak base and induces intracellular alkalosis. Intracellular alkalosis can activate TRPA1 and TRPV1, and CQ applied at alkaline pH-values indeed activates both channels.

Conclusion and implications: Our data reveal novel pharmacological properties of CQ allowing activation of TRPA1 and TRPV1 via photosensitization as well as intracellular alkalosis. These findings add complexity to the commonly accepted dogma that CQ-induced itch is specifically mediated by MrgprA3 coupling to TRPA1.

Keywords: chloroquine; histamine; itch; oxidative stress; sensory neuron.

Microglia–neuron interactions promote chronic itch via the NLRP3-IL-1β-GRPR axis

Allergy 2023 Mar 6. doi: 10.1111/all.15699. Online ahead of print.

Xueting Liu ¹, Yanmei Wang ¹, Yueling Zeng ¹, De Wang ¹, Yuhuan Wen ¹, Limin Fan ¹, Ying He ¹, Junyan Zhang ¹, Weimin Sun ¹, Yongping Liu ¹, Ailin Tao ¹

• PMID: 36876522
• DOI: 10.1111/all.15699

Abstract

Background: Spinal astrocytes contribute to chronic itch via sensitization of itch-specific neurons expressing gastrin-releasing peptide receptor (GRPR). However, whether microglia-neuron interactions contribute to itch remains unclear. In this study, we aimed to explore how microglia interact with GRPR⁺neurons and promote chronic itch.

Methods: RNA sequencing, quantitative real-time PCR, western blot, immunohistochemistry, RNAscope ISH, pharmacologic and genetic approaches were performed to examine the roles of spinal NLRP3 (The NOD-like receptor family, pyrin-containing domain 3) inflammasome activation and IL-1β-IL1R1 signaling in chronic itch. Grpr-eGFP and Grpr KO mice were used to investigate microglia-GRPR⁺neuron interactions.

Results: We observed NLRP3 inflammasome activation and IL-1β production in spinal microglia under chronic itch conditions. Blockade of microglial activation and the NLRP3/caspase-1/IL-1β axis attenuated chronic itch and neuronal activation. Type 1 IL-1 receptor (IL-1R1) was expressed in GRPR⁺neurons, which are essential for the development of chronic itch. Our studies also find that IL-1β⁺microglia are localized in close proximity to GRPR⁺ neurons. Consistently, intrathecal injection of IL1R1 antagonist or exogenous IL-1β indicate that the IL-1β-IL-1R1 signaling pathway enhanced the activation of GRPR⁺ neurons. Furthermore, our results demonstrate that the microglial NLRP3/caspase-1/IL-1β axis contributes to several different chronic itches triggered by small molecules and protein allergens from the environment and drugs.

Conclusion: Our findings reveal a previously unknown mechanism in which microglia enhances the activation of GRPR⁺ neurons through the NLRP3/caspase-1/IL-1β/IL1R1 axis. These results will provide new insights into the pathophysiology of pruritus and novel therapeutic strategies for patients with chronic itch.

Keywords: IL-1β; NLRP3 inflammasome; chronic itch; gastrin-releasing peptide receptor; microglia.

© 2023 European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.

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.