A re‐innervated in vitro skin model of non‐histaminergic itch and skin neurogenic inflammation: PAR2‐, TRPV1‐ and TRPA1‐agonist induced functionality

N. Lebonvallet, J. W. Fluhr, C. Le Gall‐Ianotto, R. Leschiera, M. Talagas, J.‐L. Carré, A. Reux, L. Misery, A. Bataille, C. Brun3, T. Oddos3, J.‐P. Pennec4

• Published in:- Skin Health and Disease

Abstract

Background: Skin, and epidermis, is innervated by sensory nerve fibres. Interactions between them and signal transduction are only partially eluci- dated in physiological/pathological conditions, especially in pruritus.

Objectives: To study the mechanisms involved in pruritus in vitro, we developed a skin explant model re‐innervated by sensory neurons.

Methods: This model is based on the co‐culture of human skin explants and sensory neurons from dorsal root ganglia of rats. Innervation and the expression of protease activated receptor 2 (PAR2), transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential ankyrin one (TRPA1) was analysed by immunostaining. The response of the model to TRPV1, PAR2 and TRPA1 agonists was analysed by patch‐clamp, qPCR and enzyme‐linked immunosorbent assay.

Results: After 5 days of re‐innervating nerve fibres was evidenced in the epidermis. Re‐innervation was correlated with decrease of epidermal thickness and the number of apoptotic cells in the tissue. The major actors of non‐histaminergic itch (PAR‐2, thymic stromal lymphopoietin [TSLP], TSLP‐R, TRPA1 and TRPV1) were expressed in neurons and/or epidermal cells of skin explants. After topical exposure of TRPV1‐(Capsaicin), TRPA1‐ (Polygodial) and PAR2‐agonist (SLIGKV‐NH2) activation of reinnervating neurons could be shown in patch‐clamp analysis. The release of TSLP was increased with capsaicin or SLIGKV but decreased with polygodial. Release of CGRP was increased by capsaicin and polygodial but decreased with SLIGKV. Activation by SLIGKV showed a decrease of VEGF; polygodial induced an increase of TSLP, Tumour necrosis factor (TNF) and nerve growth factor and capsaicin lead to a decrease of sema3 and TNF expression.

Conclusion: The present model is suitable for studying itch and neurogenic inflammation pathways in vitro. We observed that activation of TRPV1, TRPA1 and PAR‐2 leads to different response profiles in re‐innervated skin explants.

The role of PTEN in primary sensory neurons in processing itch and thermal information in mice

Published in :- Cell reports ; I.F.:- 9.423

Abstract

PTEN is known as a tumor suppressor and plays essential roles in brain development. Here, we report that PTEN in primary sensory neurons is involved in processing itch and thermal information in adult mice. Dele- tion of PTEN in the dorsal root ganglia (DRG) is achieved in adult Drg11-CreER: PTENflox/flox (PTEN CKO) mice with oral administration of tamoxifen, and CKO mice develop pathological itch and elevated itch responses on exposure to various pruritogens. PTEN deletion leads to ectopic expression of TRPV1 and MrgprA3 in IB4+ non-peptidergic DRG neurons, and the TRPV1 is responsive to capsaicin. Importantly, the elevated itch re- sponses are no longer present in Drg11-CreER: PTENflox/flox: TRPV1flox/flox (PTEN: TRPV1 dCKO) mice. In addi- tion, thermal stimulation is enhanced in PTEN CKO mice but blunted in dCKO mice. PTEN-involved regulation of itch-related gene expression in DRG neurons provides insights for understanding molecular mechanism of itch and thermal sensation at the spinal level.

Slick Potassium Channels Control Pain and Itch in Distinct Populations of Sensory and Spinal Neurons in Mice

Published in :- Journal of Anesthesiology, I.F.- 7.89

Abstract

Background: Slick, a sodium-activated potassium channel, has been recently identified in somatosensory pathways, but its functional role is poorly understood. The authors of this study hypothesized that Slick is involved in processing sensations of pain and itch.

Methods: Immunostaining, in situ hybridization, Western blot, and real-time quantitative reverse transcription polymerase chain reaction were used to investigate the expression of Slick in dorsal root ganglia and the spinal cord. Mice lacking Slick globally (Slick–/–) or conditionally in neurons of the spinal dorsal horn (Lbx1-Slick–/–) were assessed in behavioral models.

Results: The authors found Slick to be enriched in nociceptive Aδ-fibers and in populations of interneurons in the spinal dorsal horn. Slick–/– mice, but not Lbx1-Slick–/– mice, showed enhanced responses to noxious heat in the hot plate and tail-immersion tests. Both Slick–/– and Lbx1-Slick–/– mice demonstrated prolonged paw licking after capsaicin injection (mean ± SD, 45.6±30.1s [95% CI, 19.8 to 71.4]; and 13.1±16.1s [95% CI, 1.8 to 28.0]; P = 0.006 [Slick–/– {n = 8} and wild-type {n = 7}, respectively]), which was paralleled by increased phosphorylation of the neuronal activity marker extracellular signal–regulated kinase in the spinal cord. In the spi- nal dorsal horn, Slick is colocalized with somatostatin receptor 2 (SSTR2), and intrathecal preadministration of the SSTR2 antagonist CYN-154806 pre- vented increased capsaicin-induced licking in Slick–/– and Lbx1-Slick–/– mice. Moreover, scratching after intrathecal delivery of the somatostatin analog octreotide was considerably reduced in Slick–/– and Lbx1-Slick–/– mice (Slick–/– [n = 8]: 6.1 ± 6.7 bouts [95% CI, 0.6 to 11.7]; wild-type [n =8]: 47.4 ± 51.1 bouts [95% CI, 4.8 to 90.2]; P = 0.039).

Conclusions: Slick expressed in a subset of sensory neurons modulates heat-induced pain, while Slick expressed in spinal cord interneurons inhibits capsaicin-induced pain but facilitates somatostatin-induced itch.

Cell. 2021 Jul 8;184(14):3762-3773.e10. doi: 10.1016/j.cell.2021.05.017. Epub 2021 Jun 15.

Sneezing reflex is mediated by a peptidergic pathway from nose to brainstem

Fengxian Li ¹, Haowu Jiang ¹, Xiaolei Shen ¹, Weishan Yang ¹, Changxiong Guo ¹, Zhiyao Wang ¹, Maolei Xiao ¹, Lian Cui ², Wenqin Luo ², Brian S Kim ³, Zhoufeng Chen ⁴, Andrew J W Huang ⁵, Qin Liu ⁶

• PMID: 34133943
• DOI: 10.1016/j.cell.2021.05.017

Abstract

Sneezing is a vital respiratory reflex frequently associated with allergic rhinitis and viral respiratory infections. However, its neural circuit remains largely unknown. A sneeze-evoking region was discovered in both cat and human brainstems, corresponding anatomically to the central recipient zone of nasal sensory neurons. Therefore, we hypothesized that a neuronal population postsynaptic to nasal sensory neurons mediates sneezing in this region. By screening major presynaptic neurotransmitters/neuropeptides released by nasal sensory neurons, we found that neuromedin B (NMB) peptide is essential for signaling sneezing. Ablation of NMB-sensitive postsynaptic neurons in the sneeze-evoking region or deficiency in NMB receptor abolished the sneezing reflex. Remarkably, NMB-sensitive neurons further project to the caudal ventral respiratory group (cVRG). Chemical activation of NMB-sensitive neurons elicits action potentials in cVRG neurons and leads to sneezing behavior. Our study delineates a peptidergic pathway mediating sneezing, providing molecular insights into the sneezing reflex arc.

Keywords: caudal ventral respiratory group; nasal sensory neurons; neuropeptide; sneeze; sneeze-evoking region.

ZBTB20 in nociceptive neurons of the trigeminal ganglia regulates pruritus

Xin Jia, Meng-Han Dai, An-Jing Ren, Ting-Ting Wang, Weiping J. Zhang and Ling Zhang

Published in :-Frontiers of Medicine (2021.Mar.04) Impact factor:-5.02

Abstract

Recent studies have shown that ZBTB20, a zinc-finger protein containing transcription factor, is highly expressed in small-diameter primary sensory neurons in mice, and modulates pain through regulating TRP channels. However, whether ZBTB20 regulates itch sensation has not been demonstrated. In this study, small-diameter primary sensory
neuron-specific ZBTB20 knockout (PN-ZB20KO) mice were used to investigate the role of ZBTB20 in the regulation of itch sensation. First, both histamine-dependent and non-histamine-dependent itch behaviors induced by injection of histamine and chloroquine (CQ) into the cheek were significantly diminished in PN-ZB20KO mice. Second, double immunohistochemistry showed that ZBTB20 was mainly expressed in CGRP-labeled small peptidergic neurons and was expressed at low levels in IB4-labeled small non-peptidergic and NF200-labeled large neurons in the trigeminal ganglia (TG). ZBTB20 was also expressed in most TRPV1+ and TRPA1+ neurons and to a lesser extent in TRPM8+ neurons in the TG. Furthermore, cheek injection of histamine and CQ enhanced the mRNA expression of TRPV1 and TRPA1 but not TRPM8 in the TG. Moreover, TRPV1 and TRPA1 knockout (KO) mice exhibited attenuation of itch behavior induced by histamine and CQ, respectively. Finally, silencing endogenous ZBTB20 with recombinant lentivirus expressing a short hairpin RNA against ZBTB20 (LV-shZBTB20) in TG neurons attenuated histamine- and non-histamine-induced itch and downregulated TRP channels in the TG. Our study suggests that ZBTB20 plays an important role in mediating itch in small primary sensory neurons.

Keywords: itch, TRPA1, TRPV1, ZBTB20, trigeminal ganglia, pain, pruritus

A plant-derived TRPV3 inhibitor suppresses pain and itch

PUBLISHED IN:- JOURNAL OF BRITISH PHARMACOLOGY. DOI: 10.1111/BPH.15390

Abstract

Background and Purpose: Itching is the most frequent pathology in dermatology that has significant impacts on people’s mental health and social life. Transient receptor potential vanilloid 3 (TRPV3) channel is a promising target for treating pruritus. However, few selecetive and potent antagonists have been reported. This study was designed to identify selective TRPV3 antagonist and elucidate its anti-pruritus pharmacology.

Experimental Approach: FlexStation and calcium fluorescence imaging were conducted to track the functional compounds. Whole-cell patch clamp was used to record itch-related ion channel currents. Homologous recombination and site-directed mutagenesis were employed to construct TRPV3 channel chimeras and point mutations for exploring pharmacological mechanism. Mouse models were used for in vivo anti-pruritus assay.

Key Results: An acridone alkaloid (citrusinine-II) was purified and characterized from Atalantia monophylla. It directly interacts with Y564 within S4 helix of TRPV3 to selectively inhibit the channel with a half maximal inhibitory concentration (IC50) of 12.43 μM. Citrusinine-II showed potential efficacy to attenuate both chronic and acute itch. Intradermal administration of citrusinine-II (143 ng/skin site) nearly completely inhibited itch behaviours. It also shows significant analgesic effects. Little side effects of the compound are observed.

Conclusion and Implications: By acting as a selective and potent inhibitor of TRPV3 channel, citrusinine-II shows valuable therapeutic effects in pruritus animal models and is a promising candidate drug and/or lead molecule for the development of anti- pruritus drugs.

KEYWORDS: citrusinine-II, Inhibition, Itch, Pain, TRPV3

Reduced spontaneous itch in mouse models of cholestasis

Published in:- Scientific Reports (2021.03.17) IF~4.379

Jacqueline Langedijk, Ruth Bolier, Dagmar Tolenaars, Lysbeth ten Bloemendaal, Suzanne Duijst, Dirk de Waart, Ulrich Beuers, Piter Bosma & Ronald Oude Elferink

Abstract

Pruritus is one of the most distressing symptoms in cholestatic patients. Plasma autotaxin (ATX) activity correlates with the severity of pruritus in cholestatic patients, but the pathophysiology is unclear. To study pruritus in mice, we measured scratch activity in cholestatic Atp8b1 mutant mice, a model for Progressive Familial Intrahepatic Cholestasis type 1, and wild type mice (WT) with alphanaphthylisothiocyanate (ANIT)-induced cholestasis. To induce cholestasis, Atp8b1 mutant mice received a diet containing 0.1% cholic acid (CA) and WT mice were treated with ANIT. In these mice ATX was also overexpressed by transduction with AAV-ATX. Scratch activity was measured using an unbiased, electronic assay. Marked cholestasis was accomplished in both Atp8b1 mutant mice on a CA-supplemented diet and in ANIT-treatment in WT mice, but scratch activity was decreased rather than increased while plasma ATX activity was increased. Plasma ATX activity was further increased up to fivefold with AAV-ATX, but this did not induce scratch activity. In contrast to several reports two cholestatic mouse models did not display increased scratch activity as a measure of itch perception. Increasing plasma ATX activity by overexpression also did not lead to increased scratch activity in mice. This questions whether mice are suitable to study cholestatic itch.

P17 induces chemotaxis and differentiation of monocytes via MRGPRX2-mediated mast cell–line activation

Journal of allergy and clinical immunology, ~14.11 (IF)

Karthi Duraisamy, Kailash Singh, Mukesh Kumar, Benjamin Lefranc, Elsa Bonnafe, Michel Treilhou, Jerome Leprince and Billy K. C. Chow, (Hong Kong, China; and Rouen and Albi, France).

Abstract

Background: P17, a peptide isolated from Tetramorium bicarinatum ant venom, is known to induce an alternative phenotype of human monocyte–derived macrophages via activation of an unknown G protein–coupled receptor (GPCR).

Objective: We sought to investigate the mechanism of action and the immunomodulatory effects of P17 mediated through MRGPRX2 (Mas related G protein–coupled receptor X2).

Methods: To identify the GPCR for P17, we screened 314 GPCRs. Upon identification of MRGPRX2, a battery of in silico, in vitro, ex vivo, and in vivo assays along with the receptor mutation studies were performed. In
particular, to investigate the immunomodulatory actions, we used b-hexosaminidase release assay, cytokine releases, quantification of mRNA expression, cell migration and differentiation assays,
immunohistochemical labeling, hematoxylin and eosin, and immunofluorescence staining.

Results: P17 activated MRGPRX2 in a dose-dependent manner in b-arrestin recruitment assay. In LAD2 cells, P17 induced calcium and b-hexosaminidase release. Quercetin- and short hairpin RNA–mediated knockdown of MRGPRX2 reduced P17- evoked b-hexosaminidase release. In silico and in vitro mutagenesis studies showed that residue Lys8 of P17 formed a cation-p interaction with the Phe172 of MRGPRX2 and [Ala8
] P17 lost its activity partially. P17 activated LAD2 cells to recruit THP-1 and human monocytes in Transwell migration assay, whereas MRGPRX2-impaired LAD2 cells cannot. In addition, P17-treated LAD2 cells stimulated differentiation of THP-1 and human monocytes, as indicated by the enhanced expression of macrophage markers cluster of differentiation 11b and TNF-α by quantitative RT-PCR. Immunohistochemical and immunofluorescent staining suggested monocyte recruitment in mice ears injected with P17.

Conclusion: Our data provide novel structural information regarding the interaction of P17 with MRGPRX2 and intracellular pathways for its immunomodulatory action.

Supplementary datas:-

Quorum sensing between bacterial species on the skin protects against epidermal injury in atopic dermatitis

Michael R. Williams, Stephen K. Costa, Livia S. Zaramela, Shadi Khali, Daniel A. Todd, Heather L. Winter

published in Science Translational Medicine, 01 May 2019

Abstract

Colonization of the skin by Staphylococcus aureus is associated with exacerbation of atopic dermatitis (AD), but any direct mechanism through which dysbiosis of the skin microbiome may influence the development of AD is unknown. Here, we show that proteases and phenol-soluble modulin α (PSMα) secreted by S. aureus lead to endogenous epidermal proteolysis and skin barrier damage that promoted inflammation in mice. We further show that clinical isolates of different coagulase-negative staphylococci (CoNS) species residing on normal skin produced autoinducing peptides that inhibited the S. aureus agr system, in turn decreasing PSMα expression. These autoinducing peptides from skin microbiome CoNS species potently suppressed PSMα expression in S. aureus isolates from subjects with AD without inhibiting S. aureus growth. Metagenomic analysis of the AD skin microbiome revealed that the increase in the relative abundance of S. aureus in patients with active AD correlated with a lower CoNS autoinducing peptides to S. aureus ratio, thus overcoming the peptides’ capacity to inhibit the S. aureus agr system. Characterization of a S. hominis clinical isolate identified an autoinducing peptide (SYNVCGGYF) as a highly potent inhibitor of S. aureus agr activity, capable of preventing S. aureus–mediated epithelial damage and inflammation on murine skin. Together, these findings show how members of the normal human skin microbiome can contribute to epithelial barrier homeostasis by using quorum sensing to inhibit S. aureus toxin production.

MRGPRX2 Is the Codeine Receptor of Human Skin Mast Cells: Desensitization through β-Arrestin and Lack of Correlation with the FcεRI Pathway

Magda Babina, Zhao Wang, Saptarshi Roy, Sven Guhl, Kristin Franke, Metin Artuc, Hydar Ali and Torsten Zuberbier

Published in: Journal of Investigative Dermatology

DOI: 10.1016/j.jid.2020.09.017

Abstract

Codeine stimulates skin mast cells and is therefore used in skin tests and as an inducer of experimental itch. MRGPRX2 responds to various drugs, including opioids, to elicit pseudoallergic reactions, but whether it represents the main opiate receptor of skin mast cells remains unknown. By combining a number of approaches, including the silencing of MRGPRX2, we now report that MRGPRX2 is indeed the dominant codeine receptor of dermal mast cells. Activation by codeine displayed profound subject variability and correlated with secretion elicited by compound 48/80 or substance P but not by FcεRI aggregation. Degranulation by codeine was attenuated by stem cell factor, whereas the opposite was found for FcεRI. Compound 48/80 or codeine alone was able to achieve maximum MRGPRX2 activation. MRGPRX2 was rapidly internalized on codeine binding in a b arrestin-1‒dependent manner. Codeine-triggered b-arrestin activation was also established by the Tango assay. Prestimulation with MRGPRX2 agonists (but not C3a or FcεRI aggregation) resulted in refractoriness to further stimulation by the same or another MRGPRX2 ligand (cross desensitization). This was duplicated in a cell line (RBL-MRGPRX2). Collectively, codeine degranulates skin mast cells through MRGPRX2, at which it acts as a balanced ligand. It has yet to be determined whether codeine-induced refractoriness could be exploited to desensitize MRGPRX2 to prevent severe pseudoallergic reactions.