Lysophosphatidic acid activates nociceptors and causes pain or itch depending on the application mode in human skin

Miriam M Düll , Martina Stengel , Vivien Ries , Marion Strupf , Peter W Reeh , Andreas E Kremer , Barbara Namer 

Abstract

Lysophosphatidic acid (LPA) is involved in the pathophysiology of cholestatic pruritus and neuropathic pain. Slowly conducting peripheral afferent C-nerve fibers are crucial in the sensations of itch and pain. In animal studies, specialized neurons (“pruriceptors”) have been described, expressing specific receptors, eg, from the Mas-related G-protein-coupled receptor family. Human nerve fibers involved in pain signaling (“nociceptors”) can elicit itch if activated by focalized stimuli such as cowhage spicules. In this study, we scrutinized the effects of LPA in humans by 2 different application modes on the level of psychophysics and single nerve fiber recordings (microneurography). In healthy human subjects, intracutaneous LPA microinjections elicited burning pain, whereas LPA application through inactivated cowhage spicules evoked a moderate itch sensation. Lysophosphatidic acid microinjections induced heat hyperalgesia and hypersensitivity to higher electrical stimulus frequencies. Pharmacological blockade of transient receptor potential channel A1 or transient receptor potential channel vanilloid 1 reduced heat hyperalgesia, but not acute chemical pain. Microneurography revealed an application mode-dependent differential activation of mechanosensitive (CM) and mechanoinsensitive C (CMi) fibers. Lysophosphatidic acid microinjections activated a greater proportion of CMi fibers and more strongly than CM fibers; spicule application of LPA activated CM and CMi fibers to a similar extent but excited CM fibers more and CMi fibers less intensely than microinjections. In conclusion, we show for the first time in humans that LPA can cause pain as well as itch dependent on the mode of application and activates afferent human C fibers. Itch may arise from focal activation of few nerve fibers with distinct spatial contrast to unexcited surrounding afferents and a specific combination of activated fiber subclasses might contribute.

Keywords: Lysophosphatidic acid, C- fibers, Microneurography, Psychophysics, TRPV1, TRPA1, Itch, Neuropathic pain, Cholestatic pruritus

Mast cells instruct keratinocytes to produce thymic stromal lymphopoietin: Relevance of the tryptase/protease-activated receptor 2 axis

Davender Redhu, PhD,a Kristin Franke, PhD,a Marina Aparicio-Soto, PhD,a Vandana Kumari, PhD,a Kristijan Pazur, PhD,a Anja Illerhaus, PhD,b Karin Hartmann, MD,c,d Margitta Worm, MD,a and Magda Babina, PhDa Berlin and Cologne, Germany; and Basel, Switzerland

Background: Thymic stromal lymphopoietin (TSLP) promotes TH2 inflammation and is deeply intertwined with inflammatory dermatoses like atopic dermatitis. The mechanisms regulating TSLP are poorly defined.

Objective: We investigated whether and by what mechanisms mast cells (MCs) foster TSLP responses in the cutaneous environment.
Methods: Ex vivo and in vivo skin MC degranulation was induced by compound 48/80 in wild-type protease-activated receptor 2 (PAR-2)- and MC-deficient mice in the presence or absence of neutralizing antibodies, antagonists, or exogenous mouse MC protease 6 (mMCP6). Primary human keratinocytes and murine skin explants were stimulated with lysates/supernatants of human skin MCs, purified tryptase, or MC lysate diminished of tryptase. Chymase and histamine were also used. TSLP was quantified by ELISA, real-time quantitative PCR, and immunofluorescence staining.
Results: Mas-related G protein–coupled receptor X2 (Mrgprb2) activation elicited TSLP in intact skin, mainly in the epidermis. Responses were strictly MC dependent and relied on PAR-2. Complementarily, TSLP was elicited by tryptase in murine skin explants. Exogenous mMCP6 could fully restore responsiveness in MC-deficient murine skin explants. Conversely, PAR-2 knockout mice were unresponsive to mMCP6 while displaying increased responsiveness to other inflammatory pathways, such as IL-1a. Indeed, IL-1a acted in concert with tryptase. In primary human keratinocytes, MC-elicited TSLP generation was likewise abolished by tryptase inhibition or elimination. Chymase and histamine did not affect TSLP production, but histamine triggered IL-6, IL- 8, and stem cell factor.
Conclusion: MCs communicate with kerationocytes more broadly than hitherto suspected. The tryptase/PAR-2 axis is a crucial component of this cross talk, underlying MC-dependent stimulation of TSLP in neighboring kerationocytes. Interference specifically with MC tryptase may offer a treatment option for disorders initiated or perpetuated by aberrant TSLP, such as atopic dermatitis. (J Allergy Clin Immunol 2022;nnn:nnn-nnn.)

Key words: Atopic dermatitis, interleukin 1, keratinocytes, mast cells, PAR-2, TSLP, tryptase

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.

A novel sphingosylphosphorylcholine and sphingosine-1-phosphate receptor 1 antagonist, KRO-105714, for alleviating atopic dermatitis

Sae-Bom Yoon ^# 1, Chang Hoon Lee ^# 1, Hyun Young Kim ¹, Daeyoung Jeong ¹, Moon Kook Jeon ¹, Sun-A Cho ², Kwangmi Kim ³, Taeho Lee ⁴, Jung Yoon Yang ¹, Young-Dae Gong ⁵, Heeyeong Cho ^1 6

PMID: 32514255, DOI: 10.1186/s12950-020-00244-6

Abstract

Background: Atopic dermatitis (eczema) is a type of inflammation of the skin, which presents with itchy, red, swollen, and cracked skin. The high global incidence of atopic dermatitis makes it one of the major skin diseases threatening public health. Sphingosylphosphorylcholine (SPC) and sphingosine-1-phosphate (S1P) act as pro-inflammatory mediators, as an angiogenesis factor and a mitogen in skin fibroblasts, respectively, both of which are important biological responses to atopic dermatitis. The SPC level is known to be elevated in atopic dermatitis, resulting from abnormal expression of sphingomyelin (SM) deacylase, accompanied by a deficiency in ceramide. Also, S1P and its receptor, sphingosine-1-phosphate receptor 1 (S1P1) are important targets in treating atopic dermatitis.

Results: In this study, we found a novel antagonist of SPC and S1P1, KRO-105714, by screening 10,000 compounds. To screen the compounds, we used an SPC-induced cell proliferation assay based on a high-throughput screening (HTS) system and a human S1P1 protein-based [³⁵S]-GTPγS binding assay. In addition, we confirmed the inhibitory effects of KRO-105714 on atopic dermatitis through related cell-based assays, including a tube formation assay, a cell migration assay, and an ELISA assay on inflammatory cytokines. Finally, we confirmed that KRO-105714 alleviates atopic dermatitis symptoms in a series of mouse models.

Conclusions: Taken together, our data suggest that SPC and S1P1 antagonist KRO-105714 has the potential to alleviate atopic dermatitis.

Keywords: Antagonist; Anti-inflammatory; Atopic dermatitis; High-throughput screening; Sphingosine-1-phosphate receptor 1; Sphingosylphosphorylcholine.

Presenter: Song Da-Eun

J Invest Dermatol. 2022 Mar;142(3 Pt A):594-602. doi: 10.1016/j.jid.2021.07.178.Epub 2021 Sep 1.

Specific β-Defensins Stimulate Pruritus through Activation of Sensory Neurons

Pang-Yen Tseng ¹, Mark A Hoon ²

• PMID: 34480893
• DOI: 10.1016/j.jid.2021.07.178

Abstract

Pruritus is a common symptom of dermatological disorders and has a major negative impact on QOL. Previously, it was suggested that human β-defensin peptides elicit itch through the activation of mast cells. In this study, we investigated in more detail the mechanisms by which β-defensins induce itch by defining the receptors activated by these peptides in humans and mice, by establishing their action in vivo, and by examining their expression in inflammatory dermal diseases. We found that elevated expression of DEFB103 is highly correlated with skin lesions in psoriasis and atopic dermatitis. We showed that the peptide encoded by this gene and related genes activate Mas-related G protein-coupled receptors with different potencies that are related to their charge density. Furthermore, we establish that although these peptides can activate mast cells, they also activate sensory neurons, with the former cells being dispensable for itch reactions in mice. Together, our studies highlight that specific β-defensins are likely endogenous pruritogens that can directly stimulate sensory neurons.

Presenter: Lee Gi-Baek

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.

Exploring neuronal mechanisms involved in the scratching behavior of a mouse model of allergic contact dermatitis by transcriptomics

Boyu Liu, Ruixiang Chen, Jie Wang, Yuanyuan Li, Chengyu Yin, Yan Tai, Huimin Nie, Danyi Zeng, Junfan Fang, Junying Du, Yi Liang, Xiaomei Shao, Jianqiao Fang , Boyi Liu 

Abstract

Background: Allergic contact dermatitis (ACD) is a common skin condition characterized by contact hypersensitivity to allergens, accompanied with skin inflammation and a mixed itch and pain sensation. The itch and pain dramatically affects patients’ quality of life. However, still little is known about the mechanisms triggering pain and itch sensations in ACD.

Methods: We established a mouse model of ACD by sensitization and repetitive challenge with the hapten oxazolone. Skin pathological analysis, transcriptome RNA sequencing (RNA-seq), qPCR, Ca²⁺ imaging, immunostaining, and behavioral assay were used for identifying gene expression changes in dorsal root ganglion innervating the inflamed skin of ACD model mice and for further functional validations.

Results: The model mice developed typical ACD symptoms, including skin dryness, erythema, excoriation, edema, epidermal hyperplasia, inflammatory cell infiltration, and scratching behavior, accompanied with development of eczematous lesions. Transcriptome RNA-seq revealed a number of differentially expressed genes (DEGs), including 1436-DEG mRNAs and 374-DEG-long noncoding RNAs (lncRNAs). We identified a number of DEGs specifically related to sensory neuron signal transduction, pain, itch, and neuroinflammation. Comparison of our dataset with another published dataset of atopic dermatitis mouse model identified a core set of genes in peripheral sensory neurons that are exclusively affected by local skin inflammation. We further found that the expression of the pain and itch receptor MrgprD was functionally upregulated in dorsal root ganglia (DRG) neurons innervating the inflamed skin of ACD model mice. MrgprD activation induced by its agonist β-alanine resulted in exaggerated scratching responses in ACD model mice compared with naïve mice.

Conclusions: We identified the molecular changes and cellular pathways in peripheral sensory ganglia during ACD that might participate in neurogenic inflammation, pain, and itch. We further revealed that the pain and itch receptor MrgprD is functionally upregulated in DRG neurons, which might contribute to peripheral pain and itch sensitization during ACD. Thus, targeting MrgprD may be an effective method for alleviating itch and pain in ACD.

Keywords: Allergic contact dermatitis; Itch; Pain; RNA-seq; Sensory neurons.

The online version contains supplementary material available at https://cmbl.biomedcentral.com/articles/10.1186/s11658-022-00316-w

Anoctamin 1/TMEM16A in pruritoceptors is essential for Mas-related G protein receptor-dependent itch

Kim, Hyesu^1,3,7; Kim, Hyungsup^1,7; Cho, Hawon²; Lee, Byeongjun¹; Lu, Huan-Jun¹; Kim, Kyungmin¹; Chung, Sooyoung¹; Shim, Won-Sik⁴; Shin, Young Kee³; Dong, Xinzhong⁵; Wood, John N⁶; Oh, Uhtaek^1,3,*Author InformationPAIN: February 08, 2022 – Volume – Issue –doi: 10.1097/j.pain.0000000000002611

Abstract

Itch is an unpleasant sensation that evokes a desire to scratch. Pathologic conditions such as allergy or atopic dermatitis produce severe itching sensation. Mas-related G protein receptors (Mrgprs) are receptors for many endogenous pruritogens. However, signaling pathways downstream to these receptors in dorsal root ganglion (DRG) neurons are not yet understood. We found that Anoctamin 1 (ANO1), a Ca²⁺-activated chloride channel, is a transduction channel mediating Mrgprs-dependent itch signals. Genetic ablation of Ano1 in DRG neurons displayed a significant reduction in scratching behaviors in response to acute and chronic Mrgprs-dependent itch models and the epidermal hyperplasia induced by dry skin. In-vivo Ca²⁺ imaging and electrophysiological recording revealed that chloroquine and other agonists of Mrgpr receptors excited DRG neurons via ANO1. More importantly, the overexpression of Ano1 in DRG neurons of Ano1-deficient mice rescued the impaired itching observed in Ano1-deficient mice. These results demonstrate that ANO1 mediates the Mrgprs-dependent itch signaling in pruriceptors and provides clues to treating pathologic itch syndromes.

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.

mMrgprA3/mMrgprC11/hMrgprX1: potential therapeutic targets for allergic contact dermatitis induced pruritus in mice and human

Fengxian Li , Changming Wang , Danyou Hu , Xinyu Zhang , Ran Shen , Yuan Zhou , Yan Yang , Chan Zhu , Zongxiang Tang , Guang Yu 

•Department of Anesthesiology, Zhujiang Hospital of Southern Medical University, Guangzhou, Guangdong, China •School of Medicine & Holistic Integrative Medicine, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China •Key Laboratory for Chinese Medicine of Prevention and Treatment in Neurological Diseases, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China

Abstract

Background: Although the Mas-related G-protein-coupled receptors (Mrgprs) play essential roles in itch detection, their contribution to allergic contact dermatitis (ACD) associated itch remains unclear.

Objectives: To investigate whether Mrgprs are involved in ACD and whether Mrgprs can be identified as potential therapeutic targets.

Methods: Mrgpr-clusterΔ^-/- mice and human MrgprX1 (hMrgprX1) transgenic mice were applied to evalute the function of Mrgprs in oxazolone-induced ACD.

Results: Utilizing ACD model, we find that Mrgpr-clusterΔ^-/- mice display significantly reduced pruritus. Among 12 Mrgprs deleted in Mrgpr-clusterΔ^-/- mice, the expression of MrgprC11 and MrgprA3 are significantly increased in ACD model, which also innervate the skin and spinal cord at higher-than-normal densities, the proportions of dorsal root ganglia neurons responding to bovine adrenal medulla peptide 8-22 and chloroquine are also remarkably increased in ACD model and result in enhancing itch behavior. To study the function of human Mrgprs in ACD-induced itch, we utilize hMrgprX1 transgenic mice, which rescues the severe itch defect of Mrgpr-clusterΔ^-/- mice in ACD model. Remarkably, pharmacological blockade of hMrgprX1 significantly attenuates ACD itch in hMrgprX1 transgenic mouse.

Conclusions: Our study provides the first evidence that Mrgprs are involved in ACD induced chronic itch, which provides new avenues for itch management in ACD. This article is protected by copyright. All rights reserved.

Keywords: Allergic contact dermatitis; Mrgprs; pruritus; sensory neurons.