Dual function of MrgprB2 receptor-dependent neural immune axis in chronic pain

Yucui Jiang ^a1 , Fan Ye ^d1 , Jian Zhang ^b1 , Yun Huang ^b , Yingxin Zong ^b , Feiyan Chen ^a , Yan Yang ^b , Chan Zhu ^b , Tao Yang ^c, Guang Yu ^b , Zongxiang Tang ^b

^aSchool of Chinese Medicine, Nanjing University of Chinese Medicine, 138 Xianlin Road, Nanjing 210023, China
^bSchool of Medicine, Nanjing University of Chinese Medicine, 138 Xianlin Road, Nanjing 210023, China
^cState Key Laboratory of Organic Electronics and Information Displays & Institute of Advanced Materials (IAM), Nanjing University of Posts & Telecommunications, 9 Wenyuan Road, Nanjing 210023, China
^dCollege of Pharmacy, Jishou University, Jishou 416000, China

Received 26 March 2024, Revised 26 November 2024, Accepted 26 February 2025, Available online 28 February 2025.

Abstract

Introduction: Neuro-immune interactions have been recognized to be involved in the development of neuropathic pain induced by chemotherapeutic drugs (CINP). However, its role in pain resolution remains largely unknown, particularly concerning mast cells.

Objectives: To investigate the bidirectional modulation of mast cell Mas-related G protein-coupled receptor B2 (MrgprB2)-mediated neuro-immune interactions in CINP.

Methods: CINP model was established in wild-type mice, Mas-related G protein-coupled receptor D knockout (MrgprD^-/-) mice, mast cell-deficient mice, MrgprB2 knockout (MrgprB2^-/-) mice, and MrgprB2-Cre tdTomato mice. The role of MrgprB2 receptor in CINP was investigated by calcium imaging, cytokine antibody arrays, mining of single-cell sequencing databases, immunofluorescence, western blotting, co-immunoprecipitation (Co-IP), among other methodologies.

Results: We observed that cisplatin-induced allodynia was significantly inhibited in MrgprB2^-/- mice, which was attributed to the blockade of tryptase release and the suppression of upregulation of protease-activated receptor 2 (PAR2) expression in dorsal root ganglion (DRG). Thus, the activation of MrgprB2/Tryptase/PAR2 axis contributed to the development of cisplatin-induced pain. In addition, we also found that there was co-expression of PAR2 and MrgprD in DRG neurons. And activation of PAR2 can negatively regulate the expression of MrgprD, whether in a physiological state or in a chronic pain condition. Consequently, MrgprD expression was down-regulated by the activation of the MrgprB2/Tryptase/PAR2 axis during the later stages of CINP, which was associated with pain relief. Therefore, the activation of MrgprB2/Tryptase/PAR2 axis also contributed to the alleviation of cisplatin-induced pain. This finding was in line with the phenomenon that persistent stimulation by cisplatin did not cause a continuous increase in pain.

Conclusions: Our research elucidated the bidirectional modulation of MrgprB2-dependent neural immune axis in CINP. This study emphasized that MrgprB2 is a critical target for early intervention in CINP, and highlighted the necessity of considering the mechanism differences at different stages in pain management.

Keywords: CINP; MrgprB2 receptor; MrgprD receptor; PAR2 receptor.

S1P/S1PRs-TRPV4 axis is a novel therapeutic target for persistent pain and itch in chronic dermatitis

inyu Zhang1,2 | Yuan Zhou2 | ChangmingWang2 | JiahuiRen2 | YinWang2 |Pei Liu1 | WeimengFeng1 | XueLi2 | MingxinQi2 | YanYang2 |Chan Zhu2 | FangWang3 | YuxiangMa4 | ZongxiangTang2 | GuangYu1,2

1Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing
University of Chinese Medicine, Nanjing, China
2Key Laboratory for Chinese Medicine of Prevention and Treatment in Neurological Diseases, School of Medicine, Nanjing University of Chinese Medicine, Nanjing, China
3Department of Dermatology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
4School of Life Science, China Pharmaceutical University, Nanjing, China

Abstract
Background and Purpose: While pain and itch are both commonly associated with chronic dermatitis (CD), the molecular mechanisms underlying these debilitating symptoms is not well understood. This study aims to identify novel, endogenous compounds that mediate CD-associated pain and itch.

Experimental Approach: Lesional skin of CD model mice was examined using unbiased metabolomic analysis to identify candidate pain or itch inducing compounds in CD. Sphingosine-1-phosphate (S1P) concentration in CD model skin was analysed using UPLC/MS/MS. Behaviour, calcium imaging and immunofluorescence staining were used to determine the pain and itch effects and mechanisms of the identified CD-related compounds.

Key Results: In the lesional skin of CD model mice, 136 compounds were significantly changed. These compounds are predominately associated with the sphingolipids metabolism pathway. S1P is significantly increased in the lesional skin . The TRPV4 channel was critical for S1P induced itch and pain. Sphingosine kinase 2 (SPHK2), the key enzyme controlling S1P synthesis, was significantly increased in lesional skin. ABC294640, a SPHK2 inhibitor, significantly decreased S1P concentration in lesional CD model skin, as well as in model associated epidermal hyperplasia and chronic pain and itch. In CD patients, SPHK2 expression and S1P concentration were significantly elevated compared to healthy control skin.

Conclusion and Implications: Our results indicate that, in CD, increased S1P induces chronic pain and itch partly through TRPV4. Inhibition of S1P synthesis or the S1P/S1P receptor-TRPV4 pathway are promising treatment strategies for CD associated pain and itch.

KEYWORDS
chronic dermatitis, itch, pain, S1P, TRPV4

The role of PAR2 in regulating MIF release in house dust mite-induced atopic dermatitis

Lingxuan Zhou¹Guohong Zhang¹Kai Zhang¹Ziyan Rao²Zhanli Tang³Yang Wang¹Jiahui Zhao^1,4*

• ¹Department of Dermatology, Peking University First Hospital, Beijing Key Laboratory of Molecular Diagnosis on Dermatoses, National Clinical Research Center for Skin and Immune Disease, National Medical Products Administration (NMPA) Key Laboratory for Quality Control and Evaluation of Cosmetics, Beijing, China
• ²Department of Biomedical Informatics, School of Basic Medical Sciences, State Key Laboratory of Vascular Homeostasis and Remodeling, Peking University, Beijing, China
• ³Department of Dermatology, Qilu Hospital of Shandong University, Jinan, China
• ⁴Chinese Institute for Brain Research (CIBR), Beijing, China

Atopic dermatitis (AD) is a chronic disease characterized by relapsed eczema and intractable itch, and is often triggered by house dust mites (HDM). PAR2 is a G-protein coupled receptor on keratinocytes and may be activated by HDM to affect AD processes. We first established a HDM-derived AD mouse model in wild-type (WT) and Par2^-/-mice. Single cell RNA sequencing of the diseased skins found a stronger cellular communication between the ligand macrophage migration inhibitory factor (MIF) from keratinocytes and its receptors on antigen-presenting cells, suggesting the critical role of MIF in AD. HDM-WT mice showed severer skin lesions and pathological changes with stronger immunofluorescence MIF signals in skin sections than HDM-Par2^-/- mice. Primary keratinocytes from WT mice stimulated with HDM or SLIGRL (PAR2 agonist) secreted more MIF in cultured medium and induced stronger immunofluorescence MIF signals than those from Par2^-/- mice. The skin section of HDM-WT mice showed higher immunofluorescence signals of P115 (relating to MIF secretion) and KIF13B (possibly relating to intracellular trafficking of MIF) than that of HDM-Par2^-/- mice. Acetylation of α-tubulin increased after stimulation by SLIGRL in WT keratinocytes but not in Par2^-/- keratinocytes. HDM-WT mice treated with the MIF antagonist ISO-1 displayed improvement of AD-like presentations and lower expressions of IL-4, IL-13, TSLP and Arg1 (a biomarker of M2 macrophage) mRNAs. We conclude that MIF is an important cytokine and is significantly increased in the AD model. PAR2 affects AD changes by regulating the expression, intracellular trafficking, and secretion of MIF in epidermis.

Type 2 cytokine-JAK1 signaling is involved in the development of dry-skin induced mechanical alloknesis

Yui Toyosawa ^ab, Eriko Komiya ^ac, Eriko Komiya ^ac, Takahide Kaneko ^b, Yasushi Suga ^b, Mitsutoshi Tominaga^a, Kenji Takamori ^aba

^aJuntendo Itch Research Center (JIRC), Institute for Environmental and Gender- Specific Medicine, Juntendo University Graduate School of Medicine, 2-1-1 Tomioka, Urayasu, Chiba 279-0021, Japan
^bDepartment of Dermatology, Juntendo University Urayasu Hospital, 2-1-1 Tomioka, Urayasu, Chiba 279-0021, Japan
^cDepartment of Functional Morphology, Faculty of Pharmacy, Juntendo University, 6-8-1 Hinode, Urayasu, Chiba 279-0013, Japan

Received 8 March 2024, Revised 2 October 2024, Accepted 18 October 2024, Available online 22 October 2024.

Abstract

Background

Mechanical alloknesis (m-alloknesis) is itch hypersensitivity induced by normally innocuous stimuli. It is sometimes observed in dry skin based itch-related diseases such as atopic dermatitis (AD), and often triggers the vicious itch-scratch cycle. The acetone-ether and water (AEW) mouse model mimics dry skin induced m-alloknesis, yet its underlying mechanism remains unclear. Janus kinase (JAK) inhibitors are used to treat AD, but their effects on m-alloknesis are not fully known.

Objective

To reveal the effects of various oral JAK inhibitors on m-alloknesis and their action points, using AEW model.

Methods

AEW model was prepared by treatment with a mixture of acetone-ether, and they were orally administrated a JAK1/2 inhibitor baricitinib, a selective JAK1 inhibitor abrocitinib, or a JAK2 selective inhibitor AZ960, and evaluated m-alloknesis score as the total number of scratching responses in 30 mechanical stimulations. To further elucidate the mechanism of action, IL-4, IL-13 or thymic stromal lymphopoietin (TSLP) or their neutralizing antibodies were also applied to mice. In addition, the levels of these cytokines in mouse skin were measured using multiple immunoassays.

Results

All of JAK inhibitors effectively reduced m-alloknesis, with abrocitinib demonstrating the most significant inhibition. The neutralizing antibodies against IL-4, IL-13, and TSLP inhibited m-alloknesis in AEW mice. Intradermal administration of IL-4, IL-13, or TSLP induced m-alloknesis, and abrocitinib effectively mitigated each cytokine-induced response. Highly sensitive assays detected IL-4, IL-13, IL-31 and TSLP in AEW-treated skin, with TSLP levels significantly increased.

Conclusion

Type 2 cytokine-JAK1 signaling is involved in the development of m-alloknesis in dry skin.

Abbreviations

AD, atopic dermatitis; AEW, acetone-ether and water; IL, interleukin; ILC2, group 2 innate lymphoid cells; JAK, Janus kinase; m-alloknesis, mechanical alloknesis; NT, non-treated; SC, stratum corneum; STAT, signal transducer and activator of transcription; TEWL, transepidermal water loss; Th2, type 2 T helper cells; TSLP, thymic stromal lymphopoietin; W, water

Keywords

atopic dermatitis, dry skin, JAK inhibitors, mechanical alloknesis,mechanical itch, Th2 cytokines

Nociceptive transient receptor potential ankyrin 1 (TRPA1) in sensory neurons are targets of the antifungal drug econazole

Kaoru Kasuya1, Kenji Takahashi1,2, Miho Hashimoto2 and Toshio Ohta1,2*

Abstract

Background Econazole is a widely used imidazole derivative antifungal for treating skin infections. The molecular

targets for its frequent adverse effects of skin irritation symptoms, such as pruritus, burning sensation, and pain, have

not been clarified. Transient receptor potential (TRP) channels, non-selective cation channels, are mainly expressed in

peripheral sensory neurons and serve as sensors for various irritants.

Methods We investigated the effect of econazole on TRP channel activation by measuring intracellular calcium

concentration ([Ca2+]i) through fluorescent ratio imaging in mouse dorsal root ganglion (DRG) neurons isolated from

wild-type, TRPA1(−/−) and TRPV1(−/−) mice, as well as in heterologously TRP channel-expressed cells. A cheek injection

model was employed to assess econazole-induced itch and pain in vivo.

Results Econazole evoked an increase in [Ca2+]i, which was abolished by the removal of extracellular Ca2+ in mouse

DRG neurons. The [Ca2+]i responses to econazole were suppressed by a TRPA1 blocker but not by a TRPV1 blocker.

Attenuation of the econazole-induced [Ca2+]i responses was observed in the TRPA1(−/−) mouse DRG neurons but was

not significant in the TRPV1(−/−) neurons. Econazole increased the [Ca2+]i in HEK293 cells expressing TRPA1 (TRPA1-

HEK) but not in those expressing TRPV1, although at higher concentrations, it induced Ca2+ mobilization from

intracellular stores in untransfected naïve HEK293 cells. Miconazole, which is a structural analog of econazole, also

increased the [Ca2+]i in mouse DRG neurons and TRPA1-HEK, and its nonspecific action was larger than econazole.

Fluconazole, a triazole drug failed to activate TRPA1 and TRPV1 in mouse DRG neurons and TRPA1-HEK. Econazole

induced itch and pain in wild-type mice, with reduced responses in TRPA1(−/−) mice.

Conclusions These findings suggested that the imidazole derivatives econazole and miconazole may induce skin

irritation by activating nociceptive TRPA1 in the sensory neurons. Suppression of TRPA1 activation may mitigate the

adverse effects of econazole.

Keywords Antifungal, Heterologous expression, Intracellular Ca2+ concentration, Nociceptor, Sensory neuron,

Transient receptor potential channel

LPS exacerbates TRPV4-mediated itch through the intracellular TLR4-PI3K signalling

Yanping Hao1,2,3 | Liyan Wu1,2 | Yuhui Wang4 | Dongmei Shan1,2 | Yifei Liu1 | Jing Feng1,2 | Yi Chang3 | Ting Wang1,2,5,6

J Cell Mol Med  2024 Jul;28(13):e18509. doi: 10.1111/jcmm.18509.

• ¹Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China.
• ²University of Chinese Academy of Sciences, Beijing, China.
• ³Yangpu Hospital, School of Medicine, Tongji University, Shanghai, China.
• ⁴Department of Anesthesiology, Plastic Surgery Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
• ⁵Yunnan Key Laboratory of Gastrodia and Fungi Symbiotic Biology, Zhaotong University, Zhaotong, Yunnan, China.
• ⁶Yunnan Engineering Research Center of Green Planting and Processing of Gastrodia, Zhaotong University, Zhaotong, Yunnan, China.

PMID: 38957035 PMCID: PMC11220342 DOI: 10.1111/jcmm.18509

Abstract

Pruritus is often accompanied with bacterial infections, but the underlying mechanism is not fully understood. Although previous studies revealed that lipopolysaccharides (LPS) could directly activate TRPV4 channel and TRPV4 is involved in the generation of both acute itch and chronic itch, whether and how LPS affects TRPV4-mediated itch sensation remains unclear. Here, we showed that LPS-mediated TRPV4 sensitization exacerbated GSK101-induced scratching behaviour in mice. Moreover, this effect was compromised in TLR4-knockout mice, suggesting LPS acted through a TLR4-dependent mechanism. Mechanistically, LPS enhanced GSK101-evoked calcium influx in mouse ear skin cells and HEK293T cells transfected with TRPV4. Further, LPS sensitized TRPV4 channel through the intracellular TLR4-PI3K-AKT signalling. In summary, our study found a modulatory role of LPS in TRPV4 function and highlighted the TLR4-TRPV4 interaction in itch signal amplification.

Keywords: LPS; PI3K; TLR4; TRPV4; itch sensitization.

Neuronal BST2: A Pruritic Mediator alongside Protease-Activated Receptor 2 in the IL-27eDriven Itch Pathway

Yanqing Li1, Weiwei Chen1, Xingyun Zhu1, Huiyuan Mei1, Martin Steinhoff2,3,4,5,6,7,
Joerg Buddenkotte2,3,4, Jinhai Wang1, Wenhao Zhang1, Zhenghui Li8, Xiaolong Dai1, Chunxu Shan9, Jiafu Wang9,10 and Jianghui Meng9,10

1School of Life Sciences, Henan University, Henan, China; 2Department of Dermatology and Venereology, Hamad Medical Corporation, Doha, Qatar; 3Translational Research Institute, Academic Health System, Hamad Medical Corporation, Doha, Qatar; 4Dermatology Institute, Academic Health System, Hamad Medical Corporation, Doha, Qatar; 5Department of Dermatology, Weill Cornell Medicine-Qatar, Doha, Qatar; 6College of Medicine, Qatar University, Doha, Qatar; 7Israel Englander Department of Dermatology, Weill Cornell Medicine, New York, New York, USA; 8Department of Neurosurgery, The Third Affiliated Hospital of Zhengzhou University, Zhengzhou, China; and 9School of Biotechnology, Faculty of Science and Health, Dublin City University, Dublin, Ireland 10These authors contributed equally as senior authors.\

Correspondence: Jianghui Meng, School of Biotechnology, Faculty of Science and Health, Dublin City University, Glasnevin Avenue, Dublin 9, Ireland. E-mail: Jianghui.meng@dcu.ie

Abbreviations: AD, atopic dermatitis; HC, healthy control; LAD, lesional atopic dermatitis; mTGN, murine trigeminal ganglionic neuron; PAR2, pro- tease-activated receptor 2; phKC, primary human keratinocyte; STAT, signal transducer and activator of transcription; Th, T helper

Received 29 October 2023; revised 11 January 2024; accepted 27 January 2024; accepted manuscript published online XXX; corrected proof published online XXX

Chronic itch is a common and complex symptom often associated with skin diseases such as atopic dermatitis (AD). Although IL-27 is linked to AD, its role and clinical significance in itch remain undefined. We sought to investigate IL-27 function in itch using tissue-specific transgenic mice, various itch models, behavior scoring, RNA sequencing, and cytokine/kinase array. Our findings show that IL-27 receptors were overexpressed in human AD skin. Intradermal IL-27 injection failed to directly induce itch in mice but upregulated skin protease- activated receptor 2 (PAR2) transcripts, a key factor in itch and AD. IL-27 activated human keratinocytes, increasing PAR2 transcription and activity. Coinjection of SLIGRL (PAR2 agonist) and IL-27 in mice heightened PAR2-mediated itch. In addition, IL-27 boosted BST2 transcription in sensory neurons and keratinocytes. BST2 was upregulated in AD skin, and its injection in mice induced itch-like response. BST2 colocalized with sensory nerve branches in AD skin from both human and murine models. Sensory neurons released BST2, and mice with sensory neuronespecific BST2 knockout displayed reduced itch responses. Overall, this study provides evidence that skin IL-27/PAR2 and neuronal IL-27/BST2 axes are implicated in cutaneous inflammation and pruritus. The discovery of neuronal BST2 in pruritus shed light on BST2 in the itch cascade.

Keywords: Atopic dermatitis, BST2, IL-27, itch, PAR2
Journal of Investigative Dermatology (2024) -, -e-; doi:10.1016/j.jid.2024.01.025

20-HETE mediated TRPV1 activation drives allokinesis via MrgprA3+ neurons in chronic dermatitis

Guang Yu1,2*, Pei Liu1*, Xiaobao Huang3*, Mingxin Qi2, Xue Li2, Weimeng Feng1, Erxin Shang1, Yuan Zhou2, Changming Wang2, Yan Yang2, Chan Zhu2, Fang Wang3, Zongxiang Tang2, Jinao Duan1

1. Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine, Nanjing, China.
2. Key Laboratory for Chinese Medicine of Prevention and Treatment in Neurological Diseases, School of Medicine, Nanjing University of Chinese Medicine, Nanjing, China.
3. Department of Dermatology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China.

* Guang Yu, Pei Liu and Xiaobao Huang are co-first authors.

Corresponding author: Guang Yu, E-mail: yuguang@njucm.edu.cn; Zongxiang Tang, E-mail: tangzxlab@njucm.edu.cn; Jinao Duan, E-mail: dja@njucm.edu.cn (J.D.).

© The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions.

Received: 2023.04.12; Accepted: 2024.01.26; Published: 2024.02.04

Abstract
Rationale: Noxious stimuli are often perceived as itchy in patients with chronic dermatitis (CD); however, itch and pain mechanisms of CD are not known.

Methods: TRPV1 involvement in CD was analyzed using a SADBE induced CD-like mouse model, and several loss- and gain-of-function mouse models. T rigeminal TRPV1 channel and Mrgpr A3+ neuron functions were analyzed by calcium imaging and whole-cell patch-clamp recordings. Lesional CD-like skin from mice were analyzed by unbiased metabolomic analysis. 20-HETE availability in human and mouse skin were determined by LC/MS and ELISA. And finally, HET0016, a selective 20-HETE synthase inhibitor, was used to evaluate if blocking skin TRPV1 activation alleviates CD-associated chronic itch or pain.

Results: While normally a pain inducing chemical, capsaicin induced both itch and pain in mice with CD condition. DREADD silencing of MrgprA3+ primary sensory neurons in these mice selectively decreased capsaicin induced scratching, but not pain-related wiping behavior. In the mice with CD condition, MrgprA3+ neurons showed elevated ERK phosphorylation. Further experiments showed that MrgprA3+ neurons from MrgprA3;Braf mice, which have constitutively active BRAF in MrgprA3+ neurons, were significantly more excitable and responded more strongly to capsaicin. Importantly, capsaicin induced both itch and pain in MrgprA3;Braf mice in an MrgprA3+ neuron dependent manner. Finally, the arachidonic acid metabolite 20-HETE, which can activate TRPV1, was significantly elevated in the lesional skin of mice and patients with CD. Treatment with the selective 20-HETE synthase inhibitor HET0016 alleviated itch in mice with CD condition.

Conclusion: Our results demonstrate that 20-HETE activates TRPV1 channels on sensitized MrgprA3+ neurons, and induces allokinesis in lesional CD skin. Blockade of 20-HETE synthesis or silencing of TRPV1-MrgprA3+ neuron signaling offers promising therapeutic strategies for alleviating CD-associated chronic itch.

Keywords: 20-HETE, TRPV1, allokinesis, MrgprA3+ neurons, chronic dermatitis

IL-31–generating network in atopic dermatitis comprising macrophages, basophils, thymic stromal lymphopoietin, and periostin

Takashi Hashimoto, MD, PhD,a Hiroo Yokozeki, MD, PhD,b Hajime Karasuyama, MD, PhD,c and Takahiro Satoh, MD, PhDa Tokorozawa and Tokyo, Japan

From a the Department of Dermatology, National Defense Medical College, Tokorozawa, and b the Department of Dermatology, Graduate School of Medical and Dental Sciences, and c the Inflammation, Infection and Immunity Laboratory, Advanced Research Institute, Tokyo Medical and Dental University (TMDU), Tokyo. This study was partially supported by Japan Society for the Promotion of Science (JSPS) KAKENHI Grant-in-Aid for Scientific Research (C) (grant numbers 17K16328, 19K08743, and 22K08395 to T.H. and 19K08805 and 22K08444 to T.S.). Disclosure of potential conflict of interest: The authors declare that they have no relevant conflicts of interest. Received for publication July 18, 2022; revised October 22, 2022; accepted for publication November 11, 2022. Available online November 19, 2022. Corresponding author: Takashi Hashimoto, MD, PhD, Department of Dermatology, National Defense Medical College, 3-2, Namiki, Tokorozawa, Saitama 359-8513, Japan. E-mail: hashderm@ndmc.ac.jp. The CrossMark symbol notifies online readers when updates have been made to the article such as errata or minor corrections 0091-6749/$36.00 2022 American Academy of Allergy, Asthma & Immunology https://doi.org/10.1016/j.jaci.2022.11.009

Background: IL-31 is a type 2 cytokine involved in the itch sensation in atopic dermatitis (AD). The cellular origins of IL-31 are generally considered to be TH2 cells. Macrophages have also been implicated as cellular sources of IL-31. Objective: We sought to determine the expression of IL-31 by macrophages and to elucidate the productive mechanisms and contributions to itch in AD skin lesions. Methods: Expression of IL-31 by macrophages, expressions of thymic stromal lymphopoietin (TSLP) and periostin, and presence of infiltrating basophils in human AD lesions were examined through immunofluorescent staining, and correlations were assessed. Furthermore, mechanisms of inducing IL-31– expressing macrophages were analyzed in an MC903-induced murine model for AD in vivo and in mouse peritoneal macrophages ex vivo.

Results: A significant population of IL-311 cells in human AD lesions was that of CD681 cells expressing CD163, an M2 macrophage marker. The number of IL-311/CD681 cells correlated with epidermal TSLP, dermal periostin, and the number of dermal-infiltrating basophils. In the MC903-induced murine AD model, significant scratching behaviors with enhanced expressions of TSLP and periostin were observed, accompanied by massive infiltration of basophils and IL-311/ MOMA-21/Arg-11 cells. Blockade of IL-31 signaling with anti– IL-31RA antibody or direct depletion of macrophages by clodronate resulted in attenuation of scratching behaviors. Toeffectively reduce lesional IL-311 macrophages and itch, basophil depletion was essential in combination with TSLP- and periostin-signal blocking. Murine peritoneal macrophages produced IL-31 when stimulated with TSLP, periostin, and basophils.

Conclusions: A network comprising IL-31–expressing macrophages, TSLP, periostin, and basophils plays a significant role in AD itch. (J Allergy Clin Immunol 2023;151:737-46.)

Key words: Atopic dermatitis, basophil, IL-31, itch, macrophage, periostin, thymic stromal lymphopoietin

Discovery of a Small Molecule Activator of Slack (Kcnt1) Potassium Channels That Significantly Reduces Scratching in Mouse Models of Histamine-Independent and Chronic Itch

Annika Balzulat, W. Felix Zhu, Cathrin Flauaus, Victor Hernandez-Olmos, Jan Heering, Sunesh Sethumadhavan, Mariam Dubiel, Annika Frank, Amelie Menge,
Maureen Hebchen, Katharina Metzner, Ruirui Lu, Robert Lukowski, Peter Ruth,
Stefan Knapp, Susanne Müller, Dieter Steinhilber, Inga Hänelt, Holger Stark, Ewgenij Proschak,* and Achim Schmidtko*

ABSTRACT
Various disorders are accompanied by histamine-independent itching, which is often resistant to the currently available therapies. Here, it is reported that the pharmacological activation of Slack (Kcnt1, KNa1.1), a potassium channel highly expressed in itch-sensitive sensory neurons, has therapeutic potential for the treatment of itching. Based on the Slack-activating antipsychotic drug, loxapine, a series of new derivatives with improved pharmacodynamic and pharmacokinetic profiles is designed that enables to validate Slack as a pharmacological target in vivo. One of these new Slack activators, compound 6, exhibits negligible dopamine D2 and D3 receptor binding, unlike loxapine. Notably, compound 6 displays potent on-target antipruritic activity in multiple mouse models of acute histamine-independent and chronic itch without motor side effects. These properties make compound 6 a lead molecule for the development of new antipruritic therapies targeting Slack.