GRPR/Extracellular SignaleRegulated Kinase and NPRA/Extracellular SignaleRegulated Kinase Signaling Pathways Play a Critical Role in Spinal Transmission of Chronic Itch

Abstract

Intractable or recurrent chronic itch greatly reduces the patients’ QOL and impairs their daily activities. In this study, we investigated whether there are certain key signaling molecules downstream of the recently identified peptides mediating itch in the spinal cord. RNA sequencing analysis of mouse spinal cord in chronic itch models induced by squaric acid dibutylester and imiquimod showed that extracellular signaleregulated kinase (ERK) 1/2 cascade is the most significantly upregulated gene cluster in both models. In four different mouse models of chronic itch, sustained ERK phosphorylation was detected mainly in spinal neurons, and MAPK/ERK kinase inhibitors significantly inhibited chronic itch in these models. Phosphorylated ERK was observed in the interneurons expressing the receptors of different neuropeptides for itch, including gastrin-releasing peptide receptor, natriuretic peptide receptor A, neuromedin B receptor, and sst2A. Blocking gastrin-releasing peptide receptor and natriuretic peptide receptor A by genetic approaches or toxins in mice significantly attenuated or ablated spinal phosphorylated ERK. When human embryonic kidney 293T cells transfected with these receptors were exposed to their respective agonists, ERK was the most significantly activated intracellular signaling molecule. Together, our work showed that phosphorylated ERK is a unique marker for itch signal transmission in the spinal cord and an attractive target for the treatment of chronic itch.

Behavioral characterization of a CRISPR-generated TRPA1 knockout rat in models of pain, itch, and asthma

Rebecca M. Reese1,4, Michelle Dourado1,4, Keith Anderson 3, Søren Warming 3, Kimberly L. Stark1, Alessia Balestrini2, eric Suto2, Wyne Lee2, Lorena Riol-Blanco2, Shannon D. Shields1 & David H. Hackos1*

The transient receptor potential (TRP) superfamily of ion channels has garnered significant attention by the pharmaceutical industry. In particular, TRP channels showing high levels of expression in sensory neurons such as TRPV1, TRPA1, and TRPM8, have been considered as targets for indications where sensory neurons play a fundamental role, such as pain, itch, and asthma. Modeling these indications in rodents is challenging, especially in mice. The rat is the preferred species for pharmacological studies
in pain, itch, and asthma, but until recently, genetic manipulation of the rat has been technically challenging. Here, using CRISPR technology, we have generated a TRPA1 KO rat to enable more sophisticated modeling of pain, itch, and asthma. We present a detailed phenotyping of the TRPA1
KO rat in models of pain, itch, and asthma that have previously only been investigated in the mouse. With the exception of nociception induced by direct TRPA1 activation, we have found that the TRPA1 KO rat shows apparently normal behavioral responses in multiple models of pain and itch. Immune cell infiltration into the lung in the rat OVA model of asthma, on the other hand, appears to be dependent on TRPA1, similar to was has been observed in TRPA1 KO mice. Our hope is that the TRPA1 KO rat will become a useful tool in further studies of TRPA1 as a drug target.

Allantoin induces pruritus by activating MrgprD in chronic kidney disease 

Yan Yang, Yulin Sun, Donglang Guan, Dan Chen, Dijun Wang, Tongtong Liu, Meixiao Sheng, Tao Jing, Shi Jun, Chan Zhu, Guang Yu, Xinzhong Dong, Zongxiang Tang

Abstract

Chronic kidney disease is a disease with decreased, irreversible renal function. Pruritus is the most common skin symptom in patients with chronic kidney disease, especially in end-stage renal disease (AKA chronic kidney disease-associated pruritus [CKD-aP]); however, the underlying molecular and neural mechanism of the CKD-aP in patients remains obscure. Our data show that the level of allantoin increases in the serum of CKD-aP and CKD model mice. Allantoin could induce scratching behavior in mice and active DRG neurons; the calcium influx and the action potential were significantly reduced in DRG neurons of MrgprD KO or TRPV1 KO mice. U73122, an antagonist of PLC, could also block calcium influx in DRG neurons induced by allantoin. Thus, our results concluded that allantoin plays an important role in CKD-aP, mediated by MrgprD and TrpV1, in CKD patients.

FGF13 is required for histamine-induced itch sensation by interaction with NaV1.7

Abstract

Itch can be induced by activation of small-diameter dorsal root ganglion (DRG) neurons which express abundant intracellular fibroblast growth factor 13 (FGF13). Although FGF13 is revealed to be essential for heat nociception, its role in mediating itch remains to be investigated. Here, we reported that loss of FGF13 in mouse DRG neurons impaired the histamine-induced scratching behavior. Calcium imaging showed that the percentage of histamine-responsive DRG neurons was largely decreased in FGF13-deficient mice, and consistently, electrophysiological recording exhibited that histamine failed to evoke action potential firing in most DRG neurons from these mice. Given that the reduced histamine-evoked neuronal response was caused by knockdown of FGF13 but not by FGF13A deficiency, FGF13B was supposed to mediate this process. Furthermore, overexpression of histamine type 1 receptor H1R, but not H2R, H3R nor H4R, increased the percentage of histamine-responsive DRG neurons, and the scratching behavior in FGF13-deficient mice was highly reduced by selective activation of H1R, suggesting that H1R is mainly required for FGF13-mediated neuronal response and scratching behavior induced by histamine. However, overexpression of H1R failed to rescue the histamine-evoked neuronal response in FGF13-deficient mice. Histamine enhanced the FGF13 interaction with NaV1.7. Disruption of this interaction by a membrane-permeable competitive peptide, GST-Flag-NaV1.7CT-TAT, reduced the percentage of histamine-responsive DRG neurons, and impaired the histamine-induced scratching, indicating that the FGF13/NaV1.7 interaction is a key molecular determinant in the histamine-induced itch sensation. Therefore, our study reveals a novel role of FGF13 in mediating itch sensation via the interaction of NaV1.7 in peripheral nervous system.

Substance P activates Mas-related G protein-coupled receptors to induce itch.

Azimi E1, Reddy VB1, Pereira PJ2, Talbot S3, Woolf CJ3, Lerner EA4.

Abstract

BACKGROUND: Substance P (SP) is linked to itch and inflammation through activation of receptors on mast cells and sensory neurons. There is increasing evidence that SP functions through Mas-related G protein-coupled receptors (Mrgprs) in addition to its conventional receptor, neurokinin-1.

OBJECTIVE: Because Mrgprs mediate some aspects of inflammation that had been considered mediated by neurokinin-1 receptor (NK-1R), we sought to determine whether itch induced by SP can also be mediated by Mrgprs.

METHODS: Genetic and pharmacologic approaches were used to evaluate the contribution of Mrgprs to SP-induced scratching behavior and activation of cultured dorsal root ganglion neurons from mice.

RESULTS: SP-induced scratching behavior and activation of cultured dorsal root ganglion neurons was dependent on Mrgprs rather than NK-1R.

CONCLUSION: We deduce that SP activates MrgprA1 on sensory neurons rather than NK-1R to induce itch.

KEYWORDS: Mas-related G protein–coupled receptors; Substance P; calcium imaging; dorsal root ganglion neurons; knockout mice; receptor antagonist

Substance P Release by Sensory Neurons Triggers Dendritic Cell Migration and Initiates the Type-2 Immune Response to Allergens

Caroline Perner, Cameron H. Flayer, Xueping Zhu, …, Ohn A. Chow, Isaac M. Chiu, Caroline L. Sokol

Abstract

Dendritic cells (DCs) of the cDC2 lineage initiate allergic immunity and in the dermis are marked by their expression of CD301b. CD301b+ dermal DCs respond to allergens encountered in vivo, but not in vitro. This suggests that another cell in the dermis may sense allergens and relay that information to activate and induce the migration of CD301b+ DCs to the draining lymph node (dLN). Using a model of cutaneous allergen exposure, we show that allergens directly activated TRPV1+ sensory neurons leading to itch and pain behaviors. Allergen-activated sensory neurons released the neuropeptide Substance P, which stimulated proximally located CD301b+ DCs through the Mas-related G-protein coupled receptor member A1 (MRGPRA1). Substance P induced CD301b+ DC migration to the dLN where they initiated T helper-2 cell differentiation. Thus, sensory neurons act as primary sensors of allergens, linking exposure to activation of allergic-skewing DCs and the initiation of an allergic immune response.

Cinnamaldehyde elicits itch behavior via TRPV1and TRPV4 but not TRPA1

Domocos, Dan PhD^a,b; Follansbee, Taylor PhD^a; Nguyen, Amanda BS^a; Nguyen, Tony BS^a; Carstens, Mirela I. BS^a; Carstens, Earl PhD^a,Author Information Itch: July-September 2020 – Volume 5 – Issue 3 – p e36doi: 10.1097/itx.0000000000000036

Abstract

Introduction: 

Cinnamaldehyde (CA) elicits itch sensation in humans. We investigated if CA elicits scratching behavior in mice and determined the roles for TRPV1, TRPA1, and TRPV4.

Materials and Methods: 

Scratching behavior elicited by intradermal injection of CA was assessed in wildtype (WT) mice and knockout (KO) mice lacking TRPV1, TRPA1, TRPV4, or deficient in mast cells. We also assessed scratching and wet dog shakes elicited by low-threshold mechanical stimulation of skin treated topically with CA or vehicle. Using calcium imaging we tested if CA activates dorsal root ganglion (DRG) neurons of each genotype.

Results: 

Intradermal cheek injection of CA elicited dose-dependent hindlimb scratch bouts, with fewer forelimb wipes and facial groom bouts that were not dose-dependent. CA elicited significantly fewer scratch bouts in TRPV1and TRPV4 KO mice, but not TRPA1KOs, compared with WTs. There were no sex differences across genotypes. The histamine H1 antagonist cetirizine did not affect CA-evoked scratching, which was normal in mast cell deficient mice, indicating lack of histamine involvement. Scores for alloknesis were significantly greater following topical application of CA compared with vehicle. Post-CA alloknesis scores were significantly higher in TRPV4KOs of both sexes and in female TRPV1 and TRPA1KOs, compared with WTs. Low threshold mechanical stimuli also elicited significantly more wet dog shakes in mice treated topically with 20% CA, with significantly fewer in TRPV1, TRPA1, and TRPV4KOs compared with WTs. In calcium imaging studies, CA excited 24% of WT DRG cells, significantly fewer (11.5%) in cells from TRPV4KOs, and none in TRPA1KOs. Responses of cells of all genotypes exhibited significant sensitization to repeated CA stimulation. Sensitization was significantly enhanced by IL-4, which itself excited 16% of WT DRG cells and none from TRPA1KOs.

Discussion: 

The results indicate that TRPA1 is dispensable for CA-evoked scratching, which depends partly on TRPV1 and TRPV4.

A spinal neural circuitry for converting touch to itch sensation

Sihan Chen, Xiao-Fei Gao, Yuxi Zhou, Ben-Long Liu, Xian-Yu Liu, Yufen Zhang, Devin M Barry, Kun Liu, Yingfu Jiao, Rita Bardoni, Weifeng Yu , Zhou-Feng Chen

Abstract

Touch and itch sensations are crucial for evoking defensive and emotional responses, and light tactile touch may induce unpleasant itch sensations (mechanical itch or alloknesis). The neural substrate for touch-to-itch conversion in the spinal cord remains elusive. We report that spinal interneurons expressing Tachykinin 2-Cre (Tac2^Cre) receive direct Aβ low threshold mechanoreceptor (LTMR) input and form monosynaptic connections with GRPR neurons. Ablation or inhibition markedly reduces mechanical but not acute chemical itch nor noxious touch information. Chemogenetic inhibition of Tac2^Cre neurons also displays pronounced deficit in chronic dry skin itch, a type of chemical itch in mice. Consistently, ablation of gastrin-releasing peptide receptor (GRPR) neurons, which are essential for transmitting chemical itch, also abolishes mechanical itch. Together, these results suggest that innocuous touch and chemical itch information converge on GRPR neurons and thus map an exquisite spinal circuitry hard-wired for converting innocuous touch to irritating itch.

Abstract

Background: Chronic itch is a highly debilitating symptom among patients with inflammatory skin diseases. Recent studies have revealed that gastrin-releasing peptide (GRP) and its receptor (gastrin-releasing peptide receptor [GRPR]) in the spinal dorsal horn (SDH) play a central role in itch transmission.

Objective: We aimed to investigate whether GRP-GRPR signaling is altered in SDH neurons in a mouse model of chronic itch and to determine the potential mechanisms underlying these alterations.

Methods: Patch-clamp recordings from enhanced green fluorescent protein (EGFP)–expressing (GRPR1) SDH neurons were used to examine GRP-GRPR signaling in spinal cord slices obtained from Grpr-EGFP mice. Immunohistochemical, genetic (gene expression and editing through adeno-associated virus vectors), and behavioral approaches were also used for in vivo experiments.

Results: We observed potentiation of GRP-evoked excitation in the GRPR1 SDH neurons of mice with contact dermatitis, without concomitant changes in GRPR expression. Interestingly, increases in excitation were attenuated by suppressing the reactive state of SDH astrocytes, which are known to be reactive in patients with chronic itch conditions. Furthermore, CRISPR-Cas9–mediated astrocyte-selective in vivo editing of a gene encoding lipocalin-2 (LCN2), an astrocytic factor implicated in chronic itch, suppressed increases in GRP-induced excitation of GRPR1 neurons, repetitive scratching, and skin damage in mice with contact dermatitis. Moreover, LCN2 potentiated GRP-induced excitation of GRPR1 neurons in normal mice.

Conclusion: Our findings indicate that, under chronic itch conditions, the GRP-induced excitability of GRPR1 SDH neurons is enhanced through a non–cell-autonomous mechanism involving LCN2 derived from reactive astrocytes.

Key words: Contact dermatitis, chronic itch, gastrin-releasing peptide, gastrin-releasing peptide receptor, spinal dorsal horn neurons, astrocytes, lipocalin 2, CRISPR-Cas9, patch-clamp recordings

Protease-Activated Receptor-2 Regulates Neuro-Epidermal Communication in Atopic Dermatitis

Timo Buhl, Akihiko Ikoma, Cordula Kempkes, Ferda Cevikbas, Mathias Sulk, Joerg Buddenkotte, Tasuku Akiyama, Debbie Crumrine, Eric Camerer, Earl Carstens, Michael P Schön, Peter Elias, Shaun R Coughlin, Martin Steinhoff

Background: Activation of protease-activated receptor-2 (PAR2) has been implicated in inflammation, pruritus, and skin barrier regulation, all characteristics of atopic dermatitis(AD), as well as Netherton syndrome which has similar characteristics. However, understanding the precise role of PAR2 on neuro-immune communication in AD has been hampered by the lack of appropriate animal models.
Methods: We used a recently established mouse model with epidermal overexpression of PAR2 (PAR2OE) and littermate WT mice to study the impact of increased PAR2 expression in epidermal cells on spontaneous and house dust mite (HDM)-induced skin inflammation, itch, and barrier dysfunction in AD, in vivo and ex vivo.
Results: PAR2OE newborns displayed no overt abnormalities, but spontaneously developed dry skin, severe pruritus, and eczema. Dermatological, neurophysiological, and immunological analyses revealed the hallmarks of AD-like skin disease. Skin barrier defects were observed before onset of skin lesions. Application of HDM onto PAR2OE mice triggered pruritus and the skin phenotype. PAR2OE mice displayed an increased density of nerve fibers, increased nerve growth factor and endothelin-1 expression levels, alloknesis, enhanced scratching (hyperknesis), and responses of dorsal root ganglion
cells to non-histaminergic pruritogens.

Conclusion: PAR2 in keratinocytes, activated by exogenous and endogenous proteases, is sufficient to drive barrier dysfunction, inflammation, and pruritus and sensitize skin to the effects of HDM in a mouse model that mimics human AD. PAR2 signaling in keratinocytes appears to be sufficient to drive several levels of neuro-epidermal communication, another feature of human AD.