Epithelia-sensory neuron crosstalk underlies cholestatic itch induced by lysophosphatidylcholine

Yong Chen, Zi-Long Wang, Michele Yeo, Qiao-Juan Zhang, Ana E. López- Romero, Hui-Ping Ding, Xin Zhang, Qian Zeng, Sara L. Morales-Lázaro, Carlene Moore, Ying-Ai Jin, Huang-He Yang, Johannes Morstein, Andrey Bortsov, Marcin Krawczyk, Frank Lammert, Manal Abdelmalek, Anna Mae Diehl, Piotr Milkiewicz, Andreas E. Kremer, Jennifer Y. Zhang, Andrea Nackley, Tony E. Reeves, Mei- Chuan Ko, Ru-Rong Ji, Tamara Rosenbaum, Wolfgang Liedtke

BACKGROUND & AIMS

Limited understanding of pruritus mechanisms in cholestatic liver diseases hinders development of anti-pruritic treatments. Previous studies implicated lysophosphatidic acid (LPA) as a potential mediator of cholestatic pruritus.

METHODS

Pruritogenicity of LPC, LPA’s precursor, was examined in naïve mice, cholestatic mice, and nonhuman primates. LPC’s pruritogenicity involving keratinocyte-TRPV4 was studied using genetic and pharmacological approaches, cultured keratinocytes, ion channel physiology and structural-computational modeling. Activation of pruriceptor-sensory neurons by microRNA-146a (miR-146a), secreted from keratinocytes, was identified by in-vitro and ex-vivo Ca²⁺-imaging assays. Sera from primary biliary cholangitis (PBC) patients were used for measuring the levels of LPC and miR-146a.

RESULTS

LPC was robustly pruritic in mice. TRPV4 in skin keratinocytes was essential for LPC-induced itch and itch in mice with cholestasis. 3D-structural modeling, site-directed mutagenesis and channel function analysis suggested a TRPV4 C-terminal motif for LPC binding and channel activation. In keratinocytes, TRPV4-activation by LPC induced extracellular release of miR-146a, which activated TRPV1⁺-sensory neurons to cause itch. Both LPC and miR-146a levels were elevated in sera of PBC patients with itch and correlated with itch intensity. Moreover, LPC and miR-146a were also increased in sera of cholestatic mice and elicited itch in nonhuman primates.

CONCLUSIONS

We identified LPC as a novel cholestatic pruritogen that induces itch through epithelia-sensory neuron crosstalk, whereby it directly activates skin keratinocyte-TRPV4, which rapidly release miR-146a to activate skin-innervating TRPV1⁺-pruriceptor sensory neurons. Our findings support the new concept of the skin, as a sensory organ, playing a critical role in cholestatic itch, beyond liver, peripheral sensory neurons and central neural pathways supporting pruriception.

The CysLT2R receptor mediates leukotriene C4-driven acute and chronic itch

ABSTRACT

Acute and chronic itch are burdensome manifestations of skinpathologies including allergic skin diseases and atopic dermatitis,but the underlying molecular mechanisms are not well understood.Cysteinyl leukotrienes (CysLTs), comprising LTC4,LTD4, and LTE4,areproduced by immune cells during type 2 inflammation. Here, weuncover a role for LTC4and its signaling through the CysLT receptor2 (CysLT2R) in itch.Cysltr2transcript is highly expressed in dorsalroot ganglia (DRG) neurons linked to itch in mice. We also detectedCYSLTR2in a broad population of human DRG neurons. Injection ofleukotriene C4(LTC4) or its nonhydrolyzable form NMLTC4,butnei-ther LTD4nor LTE4, induced dose-dependent itch but not pain be-haviors in mice. LTC4-mediated itch differed in bout duration andkinetics from pruritogens histamine, compound 48/80, and chloro-quine. NMLTC4-induced itch was abrogated in mice deficient forCysltr2or when deficiency was restricted to radioresistant cells. Itchwas unaffected in mice deficient forCysltr1,Trpv1, or mast cells(WShmice). CysLT2R played a role in itch in the MC903 mouse modelof chronic itch and dermatitis, but not in models of dry skin or com-pound 48/80- orAlternaria-induced itch. In MC903-treated mice,CysLT levels increased in skin over time, andCysltr2−/−mice showeddecreased itch in the chronic phase of inflammation. Collectively,our study reveals that LTC4acts through CysLT2R as its physiologicalreceptor to induce itch, and CysLT2R contributes to itch in a model ofdermatitis. Therefore, targeting CysLT signaling may be a promisingapproach to treat inflammatory itch.

Keywords: itch, neuroimmune, atopic dermatitis, skin, inflammation

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.

The influence of adolescent nicotine exposure on ethanol intake and brain gene expression

PLoS One . 2018 Jun 18;13(6):e0198935. doi: 10.1371/journal.pone.0198935. eCollection 2018.

Constanza P Silva ¹, William J Horton ², Michael J Caruso ¹, Aswathy Sebastian ³, Laura C Klein ¹, Istvan Albert ³, Helen M Kamens ¹

• PMID: 29912970
• PMCID: PMC6005571
• DOI: 10.1371/journal.pone.0198935

Abstract

Nicotine and alcohol are often co-abused. Adolescence is a vulnerable period for the initiation of both nicotine and alcohol use, which can lead to subsequent neurodevelopmental and behavioral alterations. It is possible that during this vulnerable period, use of one drug leads to neurobiological alterations that affect subsequent consumption of the other drug. The aim of the present study was to determine the effect of nicotine exposure during adolescence on ethanol intake, and the effect of these substances on brain gene expression. Forty-three adolescent female C57BL/6J mice were assigned to four groups. In the first phase of the experiment, adolescent mice (PND 36-41 days) were exposed to three bottles filled with water or nicotine (200 μg/ml) for 22 h a day and a single bottle of water 2 h a day for six days. In the second phase (PND 42-45 days), the 4-day Drinking-in-the-Dark paradigm consisting of access to 20% v/v ethanol or water for 2h or 4h (the last day) was overlaid during the time when the mice did not have nicotine available. Ethanol consumption (g/kg) and blood ethanol concentrations (BEC, mg %) were measured on the final day and whole brains including the cerebellum, were dissected for RNA sequencing. Differentially expressed genes (DEG) were detected with CuffDiff and gene networks were built using WGCNA. Prior nicotine exposure increased ethanol consumption and resulting BEC. Significant DEG and biological pathways found in the group exposed to both nicotine and ethanol included genes important in stress-related neuropeptide signaling, hypothalamic-pituitary-adrenal (HPA) axis activity, glutamate release, GABA signaling, and dopamine release. These results replicate our earlier findings that nicotine exposure during adolescence increases ethanol consumption and extends this work by examining gene expression differences which could mediate these behavioral effects.

Transient receptor potential ankyrin 1 (TRPA1) positively regulates imiquimod‐induced, psoriasiform dermal inflammation in mice

YanZhou1,2 | DanHan1,2 BoWang4 | ZhenruiShi2 Samuel T. Hwang2 TaylorFollansbee3 | XuesongWu2 | SebastianYu2 | | DanT.Domocos3 | MirelaCarstens3 | EarlCarstens3 |

1Department of Dermatology, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi, China

2Department of Dermatology, University of California, Davis, California

3Department of Neurobiology, Physiology and Behavior, University of California, Davis, California

4Center for Translational Medicine, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi, China

Abstract

Transient receptor potential ankyrin 1 (TRPA1), a membrane protein ion channel, is known to mediate itch and pain in skin. The function of TRPA1, however, in psori‐ asiform dermatitis (PsD) is uncertain. Herein, we found that expression of TRPA1 is highly up‐regulated in human psoriatic lesional skin. To study the role of TRPA1 in PsD, we assessed Psoriasis Severity Index (PSI) scores, transepidermal water loss (TEWL), skin thickness and pathology, and examined dermal inflammatory infiltrates, Th17‐related genes and itch‐related genes in c57BL/6 as wild‐type (WT) and TRPA1 gene knockout (KO) mice following daily application of topical IMQ cream for 5 days. Compared with WT mice, clinical scores, skin thickness change and TEWL scores were similar on day 3, but were significantly decreased on day 5 in IMQ‐treated TRPA1 KO mice (vs WT mice), suggesting reduced inflammation and skin barrier de‐ fects. Additionally, the relative area of epidermal Munro’s microabscesses and mRNA levels of neutrophil inducible chemokines (S100A8, S100A9 and CXCL1) were de‐ creased in the treated skin of TRPA1 KO mice, suggesting that neutrophil recruitment was impaired in the KO mice. Furthermore, mast cells, CD31+ blood vascular cells, CD45+ leukocytes and CD3+ T cells were all reduced in the treated skin of TRPA1 KO mice. Lastly, mRNA expression levels of IL‐1β, IL‐6, IL‐23, IL‐17A, IL‐17F and IL‐22 were decreased in TRPA1 KO mice. In summary, these results suggest a key role for TRPA1 in psoriasiform inflammation and raising its potential as a target for therapeu‐ tic intervention.

KEYWORDS

imiquimod, inflammation, itch, mice, psoriasis, TRPA1

Spinal Inhibitory Ptf1a-Derived Neurons Prevent Self-Generated Itch

Augusto Escalante , Rüdiger Klein

Abstract

Chronic itch represents an incapacitating burden on patients suffering from a spectrum of diseases. Despite recent advances in our understanding of the cells and circuits implicated in the processing of itch information, chronic itch often presents itself without an apparent cause. Here, we identify a spinal subpopulation of inhibitory neurons defined by the expression of Ptf1a, involved in gating mechanosensory information self-generated during movement. These neurons receive tactile and motor input and establish presynaptic inhibitory contacts on mechanosensory afferents. Loss of Ptf1a neurons leads to increased hairy skin sensitivity and chronic itch, partially mediated by the classic itch pathway involving gastrin-releasing peptide receptor (GRPR) spinal neurons. Conversely, chemogenetic activation of GRPR neurons elicits itch, which is suppressed by concomitant activation of Ptf1a neurons. These findings shed light on the circuit mechanisms implicated in chronic itch and open novel targets for therapy developments.

Keywords: DREADD receptors; GRPR; Ptf1a; dorsal horn interneurons; intersectional genetics; itch; spinal cord.

A Group of Cationic Amphiphilic Drugs Activates MRGPRX2 and Induces Scratching Behavior in Mice

Katharina Wolf, PhD#*a , Helen Kühn, PhD#a , Felicitas Boehm, MSca , Lisa Gebhardt, MSca , Markus Glaudo, MSc , Konstantin Agelopoulos, PhDb , Sonja Ständer, MDb , Philipp Ectors, PhDc , Dirk Zahn, PhDc , Yvonne K. Riedel, MScd , Dominik Thimm, PhDd , Christa E. Müller, PhDd , Sascha Kretschmann, PhDe , Anita N. Kremer, MD, PhDe , Daphne Chienf, BSc, Nathachit Limjunyawongf , PhD, Qi Pengf , Xinzhong Dong, PhDf , Pavel Kolkhir, MDg,h , Jörg Scheffel, PhDh , Mia Lykke Søgaard, MSca , Benno Weigmann, PhDa , Markus F. Neurath, MDa,i, Tomasz Hawro, MD, PhDh , Martin Metz, MDh , Michael J.M. Fischer, MDj , Andreas E. Kremer, MD, PhD*a

ABSTRACT

Background: Mas gene-related G protein-coupled receptors (MRGPRs) are a GPCR family responsive to various exogenous and endogenous agonists, playing a fundamental role in pain and itch sensation. The primate-specific family member MRGPRX2 and its murine orthologue MRGPRB2 are expressed by mast cells, mediating IgE-independent signaling and pseudo-allergic drug reactions. Objectives: Therefore, knowledge about the function and regulation of MRGPRX2/MRGPRB2 is of major importance in prevention of drug hypersensitivity reactions and drug-induced pruritus.

Methods: To identify novel MRGPR (ant)agonists, we screened a library of pharmacologically active compounds utilizing a high-throughput calcium mobilization assay. Identified hit compounds were analyzed for their pseudo-allergic and pruritogenic effects in mice and human.

Results: We found a class of commonly used drugs activating MRGPRX2 which consists to a large extent of antidepressants, antiallergic drugs, and antipsychotics. Three-dimensional pharmacophore modeling revealed structural similarities of the identified agonists, classifying them as cationic amphiphilic drugs. Mast cell activation was investigated using the three representatively selected antidepressants clomipramine, paroxetine, and desipramine. Indeed, we could show a concentration-dependent activation and MRGPRX2-dependent degranulation of the human mast cell line LAD2. Furthermore, clomipramine, paroxetine, and desipramine were able to induce degranulation of human skin and murine peritoneal mast cells. These substances elicited dose-dependent scratching behavior upon intradermal injection in C57BL/6 mice but less in MRGPRB2-mutant mice as well as wheal-and-flare reactions upon intradermal injections in humans.

Conclusion: Our results contribute to the characterization of structure-activity relationships and functionality of MRGPRX2 ligands and facilitate prediction of adverse reactions like drug induced pruritus to prevent severe drug hypersensitivity reactions.

Genetic priming of sensory neurons in mice that overexpress PAR2 enhances allergen responsiveness

Joao M. Braza,1, Todd Demboa,1, Alexandra Charruyerb, Ruby Ghadiallyb,c, Marlys S. Fassettc,d, and Allan I. Basbauma,2

aDepartment of Anatomy, University of California, San Francisco, CA 94158; bDivision of Dermatology, San Francisco Veteran’s Administration Medical Center, San Francisco, CA 94121; cDepartment of Dermatology, University of California, San Francisco, CA 94158; and dDepartment of Microbiology and Immunology, University of California, San Francisco, CA 94158

Contributed by Allan I. Basbaum, January 13, 2021 (sent for review October 14, 2020; reviewed by Diana M. Bautista and Earl Carstens)

Pruritus is a common symptom of inflammatory skin conditions, including atopic dermatitis (AD). Although primary sensory neu- rons that transmit pruritic signals are well-cataloged, little is known about the neuronal alterations that occur as a result of skin disruption in AD. To address this question, we examined the mo- lecular and behavioral consequences of challenging Grhl3PAR2/+ mice, which overexpress PAR2 in suprabasal keratinocytes, with serial topical application of the environmental allergen house dust mite (HDM). We monitored behavior and used RNA sequencing, qPCR, and in situ hybridization to evaluate gene expression in tri- geminal ganglia (TG), before and after HDM. We found that nei- ther Grhl3PAR2/+ nor wild-type (WT) mice exhibited spontaneous scratching, and pruritogen-induced acute scratching did not differ. In contrast, HDM exacerbated scratching in Grhl3PAR2/+ mice. Despite the absence of scratching in untreated Grhl3PAR2/+ mice, several TG genes in these mice were up-regulated compared to WT. HDM treat- ment of the Grhl3PAR2/+ mice enhanced up-regulation of this set of genes and induced additional genes, many within the subset of TG neurons that express TRPV1. The same set of genes was up- regulated in HDM-treated Grhl3PAR2/+ mice that did not scratch, but at lesser magnitude. Finally, we recorded comparable transcrip- tional changes in IL31Tg mice, demonstrating that a common ge- netic program is induced in two AD models. Taken together, we conclude that transcriptional changes that occur in primary sensory neurons in dermatitis-susceptible animals underlie a genetic priming that not only sensitizes the animal to chronic allergens but also contributes to pruritus in atopic skin disease.

itch | dermatitis | trigeminal neurons | PAR2 | RNA sequencing

Low-Threshold Mechanosensitive VGLUT3-Lineage Sensory Neurons Mediate Spinal Inhibition of Itch by Touch

Kent Sakai, Kristen M. Sanders, Shing-Hong Lin, Darya Pavlenko, Hideki Funahashi, Taisa Lozada, Shuanglin Hao, Chih-Cheng Chen and Tasuku Akiyama

Abstract

Innocuous mechanical stimuli, such as rubbing or stroking the skin, relieve itch through the activation of low-threshold mechanoreceptors. However, the mechanisms behind this inhibition remain unknown. We presently investigated whether stroking the skin reduces the responses of superficial dorsal horn neurons to pruritogens in male C57BL/6J mice. Single-unit recordings revealed that neuronal responses to chloroquine were enhanced during skin stroking, and this was followed by suppression of firing below baseline levels after the termination of stroking. Most of these neurons additionally responded to capsaicin. Stroking did not suppress neuronal responses to capsaicin, indicating state-dependent inhibition. Vesicular glutamate transporter 3 (VGLUT3)-lineage sensory nerves compose a subset of low-threshold mechanoreceptors. Stroking-related inhibition of neuronal responses to chloroquine was diminished by optogenetic inhibition of VGLUT3-lineage sensory nerves in male and female Vglut3-cre/NpHR-EYFPmice. Conversely, in male and female Vglut3-cre/ChR2-EYFP mice, optogenetic stimulation of VGLUT3-lineage sensory nerves inhibited firing responses of spinal neurons to pruritogens after the termination of stimulation. This inhibition was nearly abolished by spinal delivery of the κ-opioid receptor antagonist nor-binaltorphimine dihydrochloride, but not the neuropeptide Y receptor Y1 antagonist BMS193885. Optogenetic stimulation of VGLUT3-lineage sensory nerves inhibited pruritogen-evoked scratching without affecting mechanical and thermal pain behaviors. Therefore, VGLUT3-lineage sensory nerves appear to mediate inhibition of itch by tactile stimuli.

Significance Statement 

Rubbing or stroking the skin is known to relieve itch. We investigated the mechanisms behind touch-evoked inhibition of itch in mice. Stroking the skin reduced the activity of itch-responsive spinal neurons. Optogenetic inhibition of VGLUT3-lineage sensory nerves diminished stroking-evoked inhibition, and optogenetic stimulation of VGLUT3-lineage nerves inhibited pruritogen-evoked firing. Together, our results provide a mechanistic understanding of touch-evoked inhibition of itch.

Abstract

Itch is an evolutionarily conserved sensation that facilitates expulsion of pathogens and noxious stimuli from the skin. However, in organ failure, cancer, and chronic inflammatory disorders such as atopic dermatitis (AD), itch becomes chronic, intractable, and debilitating. In addition to chronic itch, patients often experience intense acute itch exacerbations. Recent discoveries have unearthed the neuroimmune circuitry of itch, leading to the development of anti-itch treatments. However, mechanisms underlying acute itch exacerbations remain overlooked. Herein, we identify that a large proportion of patients with AD harbor allergen-specific immunoglobulin E (IgE) and exhibit a propensity for acute itch flares. In mice, while allergen-provoked acute itch is mediated by the mast cell-histamine axis in steady state, AD-associated inflammation renders this pathway dispensable. Instead, a previously unrecognized basophil-leukotriene (LT) axis emerges as critical for acute itch flares. By probing fundamental itch mechanisms, our study highlights a basophil-neuronal circuit that may underlie a variety of neuroimmune processes.