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.

J Med Chem. 2022 Feb 24;65(4):3218-3228. doi: 10.1021/acs.jmedchem.1c01709.Epub 2022 Feb 4.

Thieno[2,3- d]pyrimidine-Based Positive Allosteric Modulators of Human Mas-Related G Protein-Coupled Receptor X1 (MRGPRX1)

Ilyas Berhane, Niyada Hin, Ajit G Thomas, Qian Huang, Chi Zhang, Vijayabhaskar Veeravalli, Ying Wu, Justin Ng, Jesse Alt, Camilo Rojas, Hiroe Hihara ¹, Mika Aoki ¹, Kyoko Yoshizawa ¹, Tomoki Nishioka ¹, Shuichi Suzuki ¹, Shao-Qiu He, Qi Peng, Yun Guan, Xinzhong Dong, Srinivasa N Raja, Barbara S Slusher, Rana Rais, Takashi Tsukamoto

• PMID: 35119273
• DOI: 10.1021/acs.jmedchem.1c01709

Abstract

Mas-related G protein-coupled receptor X1 (MRGPRX1) is a human sensory neuron-specific receptor and potential target for the treatment of pain. Positive allosteric modulators (PAMs) of MRGPRX1 have the potential to preferentially activate the receptors at the central terminals of primary sensory neurons and minimize itch side effects caused by peripheral activation. Using a high-throughput screening (HTS) hit, a series of thieno[2,3-d]pyrimidine-based molecules were synthesized and evaluated as human MRGPRX1 PAMs in HEK293 cells stably transfected with human MrgprX1 gene. An iterative process to improve potency and metabolic stability led to the discovery of orally available 6-(tert-butyl)-5-(3,4-dichlorophenyl)-4-(2-(trifluoromethoxy)phenoxy)thieno[2,3-d]pyrimidine (1t), which can be distributed to the spinal cord, the presumed site of action, following oral administration. In a neuropathic pain model induced by sciatic nerve chronic constriction injury (CCI), compound 1t(100 mg/kg, po) reduced behavioral heat hypersensitivity in humanized MRGPRX1 mice, demonstrating the therapeutic potential of MRGPRX1 PAMs in treating neuropathic pain.

Lancet. 2021 Oct 30;398(10311):1581-1592. doi: 10.1016/S0140-6736(21)01256-3.Epub 2021 Oct 28.

Efficacy and safety of maralixibat treatment in patients with Alagille syndrome and cholestatic pruritus (ICONIC): a randomised phase 2 study

Emmanuel Gonzales ¹, Winita Hardikar ², Michael Stormon ³, Alastair Baker ⁴, Loreto Hierro ⁵, Dorota Gliwicz ⁶, Florence Lacaille ⁷, Alain Lachaux ⁸, Ekkehard Sturm ⁹, Kenneth D R Setchell ¹⁰, Ciara Kennedy ¹¹, Alejandro Dorenbaum ¹², Jana Steinmetz ¹³, Nirav K Desai ¹⁴, Andrew J Wardle ¹⁵, Will Garner ¹⁵, Pamela Vig ¹⁵, Thomas Jaecklin ¹⁶, Etienne M Sokal ¹⁷, Emmanuel Jacquemin ¹⁸Affiliations expand

• PMID: 34755627
• DOI: 10.1016/S0140-6736(21)01256-3

Abstract

Background: Alagille syndrome is a rare genetic disease that often presents with severe cholestasis and pruritus. There are no approved drugs for management. Maralixibat, an apical, sodium-dependent, bile acid transport inhibitor, prevents enterohepatic bile acid recirculation. We evaluated the safety and efficacy of maralixibat for children with cholestasis in Alagille syndrome.

Methods: ICONIC was a placebo-controlled, randomised withdrawal period (RWD), phase 2b study with open-label extension in children (aged 1-18 years) with Alagille syndrome (NCT02160782). Eligible participants had more than three times the normal serum bile acid (sBA) levels and intractable pruritus. After 18 weeks of maralixibat 380 μg/kg once per day, participants were randomly assigned (1:1) to continue maralixibat or receive placebo for 4 weeks. Subsequently, all participants received open-label maralixibat until week 48. During the long-term extension (204 weeks reported), doses were increased up to 380 μg/kg twice per day. The primary endpoint was the mean sBA change during the RWD in participants with at least 50% sBA reduction by week 18. Cholestastic pruritus was assessed using observer-rated, patient-rated, and clinician-rated 0-4 scales. The safety population was defined as all participants who had received at least one dose of maralixibat. This trial was registered with ClinicalTrials.gov, NCT02160782, and is closed to recruitment.

Findings: Between Oct 28, 2014, and Aug 14, 2015, 31 participants (mean age 5·4 years [SD 4·25]) were enrolled and 28 analysed at week 48. Of the 29 participants who entered the randomised drug withdrawal period, ten (34%) were female and 19 (66%) were male. In the RWD, participants switched to placebo had significant increases in sBA (94 μmol/L, 95% CI 23 to 164) and pruritus (1·7 points, 95% CI 1·2 to 2·2), whereas participants who continued maralixibat maintained treatment effect. This study met the primary endpoint (least square mean difference -117 μmol/L, 95% CI -232 to -2). From baseline to week 48, sBA (-96 μmol/L, -162 to -31) and pruritus (-1·6 pts, -2·1 to -1·1) improved. In participants who continued to week 204 (n=15) all improvements were maintained. Maralixibat was generally safe and well tolerated throughout. The most frequent adverse events were gastrointestinal related. Most adverse events were self-limiting in nature and mild-to-moderate in severity.

Interpretation: In children with Alagille syndrome, maralixibat is, to our knowledge, the first agent to show durable and clinically meaningful improvements in cholestasis. Maralixibat might represent a new treatment paradigm for chronic cholestasis in Alagille syndrome.

Funding: Mirum Pharmaceuticals.

Copyright © 2021 Elsevier Ltd. All rights reserved.

Nat Commun 2021 Apr 12;12(1):2185. doi: 10.1038/s41467-021-22479-4.

Therapeutic B-cell depletion reverses progression of Alzheimer’s disease

Ki Kim ^# 1, Xin Wang ^# 1, Emeline Ragonnaud ^# 1, Monica Bodogai ^# 1, Tomer Illouz ^2 3 4, Marisa DeLuca ¹, Ross A McDevitt ⁵, Fedor Gusev ⁶, Eitan Okun ^2 3 4, Evgeny Rogaev ^6 7 8 9, Arya Biragyn ¹⁰

• PMID: 33846335
• PMCID: PMC8042032
• DOI: 10.1038/s41467-021-22479-4

Abstract

The function of B cells in Alzheimer’s disease (AD) is not fully understood. While immunoglobulins that target amyloid beta (Aβ) may interfere with plaque formation and hence progression of the disease, B cells may contribute beyond merely producing immunoglobulins. Here we show that AD is associated with accumulation of activated B cells in circulation, and with infiltration of B cells into the brain parenchyma, resulting in immunoglobulin deposits around Aβ plaques. Using three different murine transgenic models, we provide counterintuitive evidence that the AD progression requires B cells. Despite expression of the AD-fostering transgenes, the loss of B cells alone is sufficient to reduce Aβ plaque burden and disease-associated microglia. It reverses behavioral and memory deficits and restores TGFβ⁺ microglia, respectively. Moreover, therapeutic depletion of B cells at the onset of the disease retards AD progression in mice, suggesting that targeting B cells may also benefit AD patients.

Nat Commun. 2020 Dec 11;11(1):6363. doi: 10.1038/s41467-020-19931-2.

Effect of gut microbiota on depressive-like behaviors in mice is mediated by the endocannabinoid system

Grégoire Chevalier ¹, Eleni Siopi ², Laure Guenin-Macé ³, Maud Pascal ^1 2, Thomas Laval ³, Aline Rifflet ⁴, Ivo Gomperts Boneca ⁴, Caroline Demangel ³, Benoit Colsch ⁵, Alain Pruvost ⁵, Emeline Chu-Van ⁵, Aurélie Messager ⁵, François Leulier ⁶, Gabriel Lepousez ², Gérard Eberl ⁷, Pierre-Marie Lledo ⁸

• PMID: 33311466
• PMCID: PMC7732982
• DOI: 10.1038/s41467-020-19931-2

Abstract

Depression is the leading cause of disability worldwide. Recent observations have revealed an association between mood disorders and alterations of the intestinal microbiota. Here, using unpredictable chronic mild stress (UCMS) as a mouse model of depression, we show that UCMS mice display phenotypic alterations, which could be transferred from UCMS donors to naïve recipient mice by fecal microbiota transplantation. The cellular and behavioral alterations observed in recipient mice were accompanied by a decrease in the endocannabinoid (eCB) signaling due to lower peripheral levels of fatty acid precursors of eCB ligands. The adverse effects of UCMS-transferred microbiota were alleviated by selectively enhancing the central eCB or by complementation with a strain of the Lactobacilli genus. Our findings provide a mechanistic scenario for how chronic stress, diet and gut microbiota generate a pathological feed-forward loop that contributes to despair behavior via the central eCB system.

 Br J Dermatol. 2018 Sep;179(3):669-678. doi: 10.1111/bjd.16498.Epub 2018 Jun 21.

Itch in dermatomyositis: the role of increased skin interleukin-31

H J Kim ^1 2 3, M Zeidi ^1 2, D Bonciani ^1 2 4, S M Pena ², J Tiao ^1 2, S Sahu ^1 2, V P Werth ^1 2

• PMID: 29494763
• PMCID: PMC6119520
• DOI: 10.1111/bjd.16498

Abstract

Background: Interleukin (IL)-31 is implicated in pruritus associated with pruritic skin diseases like atopic dermatitis. Although pruritus is a prominent feature in dermatomyositis (DM), few studies have evaluated the pathogenesis of DM-associated itch.

Objectives: To establish the prevalence of itch in DM, and to investigate the role of IL-31 in DM-related itch.

Methods: Pruritus and disease activity of DM were evaluated by a visual analogue scale (VAS) and the Cutaneous Disease and Activity Severity Index (CDASI), respectively. Expression of IL-31 and IL-31 receptor alpha (IL-31RA) in lesional DM, nonlesional DM and healthy control skin was evaluated by quantitative reverse-transcriptase polymerase chain reaction and immunofluorescence. Flow cytometry was performed on skin cells isolated from lesional DM skin to identify cellular sources of IL-31 in DM.

Results: Among 191 patients with DM, 50·8% had moderate-to-severe itch, and itch was correlated with increased cutaneous severity (r = 0·34). In patients with itchy DM, gene expression of IL31 and IL31RA in lesional skin was upregulated compared with nonlesional skin and healthy control skin. IL31 mRNA expression positively correlated with VAS itch score (r = 0·67). On immunofluorescence, immunoreactivity for IL-31 and IL-31RA was stronger in lesional skin. Flow cytometry showed that lesional DM skin contained significantly more IL-31-producing cells, and CD4⁺ cells were the most common cell type. Lenabasum, an emerging treatment for DM, significantly downregulated IL-31 from CpG-stimulated peripheral blood mononuclear cells.

Conclusions: Increased skin IL-31 may play a role in DM-associated itch, and ongoing trials will evaluate the effects of systemic treatment on IL-31 and itch in DM.

Transl Psychiatry. 2019 Aug 5;9(1):189. doi: 10.1038/s41398-019-0525-3.

Fecal microbiota transplantation alleviated Alzheimer’s disease-like pathogenesis in APP/PS1 transgenic mice

Jing Sun ¹, Jingxuan Xu ², Yi Ling ¹, Fangyan Wang ³, Tianyu Gong ⁴, Changwei Yang ⁴, Shiqing Ye ⁴, Keyue Ye ⁴, Dianhui Wei ⁴, Ziqing Song ⁴, Danna Chen ⁴, Jiaming Liu ^5 6

• PMID: 31383855
• PMCID: PMC6683152
• DOI: 10.1038/s41398-019-0525-3

Abstract

Alzheimer’s disease (AD) is the most common dementia in the elderly. Treatment for AD is still a difficult task in clinic. AD is associated with abnormal gut microbiota. However, little is known about the role of fecal microbiota transplantation (FMT) in AD. Here, we evaluated the efficacy of FMT for the treatment of AD. We used an APPswe/PS1dE9 transgenic (Tg) mouse model. Cognitive deficits, brain deposits of amyloid-β (Aβ) and phosphorylation of tau, synaptic plasticity as well as neuroinflammation were assessed. Gut microbiota and its metabolites short-chain fatty acids (SCFAs) were analyzed by 16S rRNA sequencing and ¹H nuclear magnetic resonance (NMR). Our results showed that FMT treatment could improve cognitive deficits and reduce the brain deposition of amyloid-β (Aβ) in APPswe/PS1dE9 transgenic (Tg) mice. These improvements were accompanied by decreased phosphorylation of tau protein and the levels of Aβ40 and Aβ42. We observed an increases in synaptic plasticity in the Tg mice, showing that postsynaptic density protein 95 (PSD-95) and synapsin I expression were increased after FMT. We also observed the decrease of COX-2 and CD11b levels in Tg mice after FMT. We also found that FMT treatment reversed the changes of gut microbiota and SCFAs. Thus, FMT may be a potential therapeutic strategy for AD.