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.

Inhibition of mite-induced dermatitis, pruritus, and nerve sprouting in mice by the endothelin receptor antagonist bosentan

Makiko Kido-Nakahara1, Bing Wang, Fumitaka Ohno, Gaku Tsuji, Dugarmaa Ulzii, Masaki Takemura, Masutaka Furue, Takeshi Nakahara

Abstract

Background: Endothelin-1 (EDN1) can evoke histamine-independent pruritus in mammals and is
upregulated in the lesional epidermis of atopic dermatitis (AD). EDN1 increases the production of
interleukin 25 (IL-25) from keratinocytes to accelerate T helper type 2 immune deviation. Plasma EDN1
levels are positively correlated with the clinical severity and itch intensity of AD. Therefore, we
hypothesized that the inhibition of EDN1 might be useful for treating atopic inflammation and itch and
investigated the effects of the topical application of the EDN1 receptor antagonist bosentan on the skin
inflammation and itch in a murine AD model.
Methods: We analyzed the mite-induced AD-like NC/Nga murine model, which was topically applied with
bosentan or ethanol control every day for 3 weeks. We also subjected in vitro primary sensory neuron
culture systems to nerve elongation and branching assays after EDN1 stimulation.
Results: Topical application of bosentan significantly attenuated the development of mite-induced AD-like
skin inflammation, dermatitis scores, ear thickness, scratching bouts, and serum level of thymus and
activation‐regulated chemokine in NC/Nga mice. Bosentan application also significantly reduced the gene
expression of Il13, Il17, and Ifng in the treated lesions. Histologically, the number of infiltrated dermal
cells, the epidermal EDN1 expression, and the number of intraepidermal nerve fibers were significantly
inhibited upon bosentan application. While EDN1 significantly elongated the neurites of dorsal root
ganglion cells in a dose- and time-dependent manner, bosentan treatment attenuated this.
Conclusions: EDN1 plays a significant role in mite-induced inflammation and itch. Topical bosentan is a
potential protective candidate for AD.

Baicalin induces Mrgprb2-dependent pseudo-allergy in mice 

Jue Wanga, Yongjing Zhanga, Delu Chea, Yingnan Zenga, Yuanyuan Wub, Qiaohong Qinc, Nan Wanga,*

a School of Pharmacy, Xi’an Jiaotong University, Xi’an, 710061, China
b Department of Respiratory and Critical Care Medicine, The Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an, 710061, China  c Institute of Basic and Translational Medicine, Xi’an Medical University, Xi’an, 710021, China

Baicalin, a component of traditional Chinese medicine, is one of the main compounds present in Scutellaria baicalensis Georgi. Pseudo-allergy induced by the injection of these medicines is a frequent adverse drug reaction. Therefore, elucidation of the anaphylactoid reaction of baicalin and its underlying mechanisms are important. Mast cells are primary effectors of allergic reactions, including pseudo-allergy. Studies have shown that Mrgprx2 in human mast cells is a specific receptor that is crucial for pseudo-allergic drug reactions, Mrgprb3 is the rat ortholog of human Mrgprx2, which in mice is designated as Mrgprb2. Here, we aimed to investigate baicalin- induced pseudo-allergy and the association of Mrgprb3 and Mrgprb2 with this effect. We examined the aller- genic effect of baicalin on RBL-2H3 cells and Mrgprb3-knockdown RBL-2H3 cells. Mrgprb2-expressing HEK293 cells and Mrgprb2-knockout mice were used to evaluate the role of Mrgprb2 in baicalin-induced allergy. Baicalin was found to dose-dependently induce pseudo-allergy both in vitro and in vivo. RBL-2H3 cells were activated by baicalin, whereas in Mrgprb3-knockout RBL-2H3 cells, baicalin showed a negligible effect on cell activation. Furthermore, baicalin activated the Mrgprb2-expressing HEK293 cells. Our data showed that baicalin did not induce allergy in Mpgprb2-knockout mice. We conclude that baicalin induces pseudo-allergy via Mrgprb2 in mice.

Keywords:Baicalin Pseudo-allergy Mrgprb2 Mrgprb3 Degranulation

Exploration of sensory and spinal neurons expressing gastrin-releasing peptide in itch and pain related behaviors

Devin M Barry ^1 2, Xue-Ting Liu ^1 2 3, Benlong Liu ^1 2, Xian-Yu Liu ^1 2, Fang Gao ^1 2, Xiansi Zeng ^1 2 4, Juan Liu ^1 2, Qianyi Yang ^1 2, Steven Wilhelm ^1 2, Jun Yin ^1 2, Ailin Tao ^1 3, Zhou-Feng Chen ^5 6 7 

Abstract

Gastrin-releasing peptide (GRP) functions as a neurotransmitter for non-histaminergic itch, but its site of action (sensory neurons vs spinal cord) remains controversial. To determine the role of GRP in sensory neurons, we generated a floxed Grp mouse line. We found that conditional knockout of Grp in sensory neurons results in attenuated non-histaminergic itch, without impairing histamine-induced itch. Using a Grp-Cre knock-in mouse line, we show that the upper epidermis of the skin is exclusively innervated by GRP fibers, whose activation via optogeneics and chemogenetics in the skin evokes itch- but not pain-related scratching or wiping behaviors. In contrast, intersectional genetic ablation of spinal Grp neurons does not affect itch nor pain transmission, demonstrating that spinal Grp neurons are dispensable for itch transmission. These data indicate that GRP is a neuropeptide in sensory neurons for non-histaminergic itch, and GRP sensory neurons are dedicated to itch transmission.

Role of 5-HT1A and 5-HT3 receptors in serotonergic activation of sensory neurons in relation to itch and pain behavior in the rat

Dan Domocos, Tudor Selescu, Laura Cristina Ceafalan,  Mirela Iodi Carstens, Earl Carstens, Alexandru Babes

Abstract

Serotonin (5-hydroxytryptamine, 5-HT) released by platelets, mast cells, and immunocytes is a potent inflammatory mediator which modulates pain and itch sensing in the peripheral nervous system. The serotonergic receptors expressed by primary afferent neurons involved in these sensory functions are not fully identified and appear to be to a large extent species dependent. Moreover, the mechanisms through which 5-HT receptor activation is coupled to changes in neuronal excitability have not been completely revealed. Using a combination of in vitro (calcium and voltage imaging and patch-clamp) and in vivo behavioral methods, we used both male and female Wistar rats to provide evidence for the involvement of two 5-HT receptor subtypes, 5-HT1A and 5-HT3, in mediating the sustained and transient effects, respectively, of 5-HT on rat primary afferent neurons involved in pain and itch processing. In addition, our results are consistent with a model in which sustained serotonergic responses triggered via the 5-HT1A receptor are due to closure of background potassium channels, followed by membrane depolarization and action potentials, during which the activation of voltage-gated calcium channels leads to calcium entry. Our results may provide a better understanding of mammalian serotonergic itch signaling.

https://onlinelibrary.wiley.com/doi/full/10.1002/jnr.24633

A vaccine targeting the RBD of the S protein of SARS-CoV-2 induces protective immunity

Abstract

The novel Coronavirus SARS-CoV-2 causes a respiratory illness called COVID-19 leading to a pandemic. An effective preventive vaccine against this virus is urgently needed. As the most critical step during infection, SARS CoV-2 uses its Spike protein receptor-binding domain (S- RBD) to engage with the host cell receptor angiotensin converting enzyme 2 (ACE2)1,2. Here we showed that a recombinant vaccine comprising residues 319-545 of the S-RBD could induce a potent functional antibody response in the immunized mice, rabbits and non-human primates (Macaca mulatta) as early as 7 or 14 days after a single dose injection. The sera from the immunized animals blocked RBD binding to ACE2 expressed on the cell surface and neutralized the infection by SARS-CoV-2 pseudovirus and live SARS-CoV-2 in vitro. Importantly, the vaccination also provided protection in non-human primates from SARS-CoV-2 challenge in vivo. The elevated RBD-specific antibodies were also found in the sera from patients with COVID-19. Several immune pathways and CD4 T lymphocytes were implicated in the induction of the vaccine antibody response. Our finding highlights the importance of the RBD domain in the SARS-CoV-2 vaccine design and provides the rationale for the development of a protective vaccine through the induction of antibody against the RBD domain.