Antipruritic Effect of Ethyl Acetate Extract from Fructus cnidii in Mice with 2,4-Dinitrofluorobenzene-Induced Atopic Dermatitis.

Abstract

Atopic dermatitis (AD) is a common inflammatory skin disease characterized by intense pruritus and skin lesions. The exact cause of AD is not yet known and the available therapeutic strategies for AD are limited. Fructus cnidii is commonly used in traditional Chinese medicine as an herb for treating chronic itch. However, the mechanism underlying the antipruritic effects of Fructus cnidii is not well understood. In the present study, we investigated the antipruritic effect of locally administered ethyl acetate extract from Fructus cnidii (EAEFC) to 2,4-dinitrofluorobenzene- (DNFB-) induced AD in a mouse model. The scratching behavior, skin thickness, dermatitis score, weight, blood immunoglobulin E (IgE) level, and itch-related cytokine levels were subsequently monitored and evaluated. Results showed that EAEFC treatment attenuated the DNFB-induced AD-like symptoms by alleviating the skin lesions and decreasing the dermatitis score. Hematoxylin and eosin (H&E) and toluidine blue (TB) staining analyses demonstrated that EAEFC mitigated the DNFB-induced increase in skin thickness and prevented the infiltration of mast cells. Behavioral tests showed that EAEFC decreased the DNFB-induced acute and chronic scratching behaviors. Furthermore, EAEFC reduced the levels of itch-related cytokines, such as thymic stromal lymphopoietin (TSLP), interleukin- (IL-) 17, IL-33, and IL-31, and the DNFB-induced boost in serum IgE. Collectively, these results suggest that EAEFC is a potential therapeutic candidate for the treatment of chronic itch in AD.

The nonselective cation channel TRPV4 inhibits angiotensin II receptors

Nicholas W. Zaccor, Charlotte J. Sumner, Solomon H. Snyder

Abstract

G protein-coupled receptors (GPCRs) are a ubiquitously expressed family of receptor proteins that regulate many physiological functions and other proteins. They act through two dissociable signaling pathways, the exchange of GDP to GTP by linked G proteins and the recruitment of β-arrestins. GPCRs modulate several members of the transient receptor potential (TRP) channel family of non-selective cation channels. How TRP channels reciprocally regulate GPCR signaling is less well explored. Here, using an array of biochemical approaches, including immunoprecipitation and -fluorescence, calcium imaging, phosphate radiolabeling, and a β-Arrestin dependent luciferase assay, we characterize a GPCR-TRP channel pair, angiotensin II receptor type 1 (AT1R) and transient receptor potential vanilloid 4 (TRPV4), in primary murine choroid plexus epithelial cells and immortalized cell lines. We found that AT1R and TRPV4 are binding partners, and that activation of AT1R by angiotensin II (ANGII) elicits β-arrestin-dependent inhibition and internalization of TRPV4. Activating TRPV4 with endogenous and synthetic agonists inhibited ANGII-mediated G-protein associated second messenger accumulation, AT1R receptor phosphorylation and β-arrestin recruitment. We also noted that TRPV4 inhibits AT1R phosphorylation by activating the calcium-activated phosphatase calcineurin in a Ca2+/calmodulin dependent manner, preventing β-arrestin recruitment and receptor internalization. These findings suggest that when TRP channels and GPCRs are co-expressed in the same tissues, many of these channels can inhibit GPCR desensitization.

Typical antimicrobials induce mast cell degranulation and anaphylactoid reactions via MRGPRX2 and its murine homologue MRGPRB2

ABSTRACT

Mast cells are unique immune cells that function as sentinels in host defence reactions, including immediate hypersensitivity responses and allergic responses. The mast cell-specific receptor named MAS-related G protein-coupled receptor X2 (MRGPRX2) triggers mast-cell degranulation, a key process in anaphylactoid reactions. It is widely observed that antimicrobials can induce pseudo-allergic reactions (i.e. IgE-independent mechanism) with symptoms ranging from skin inflammation to life-threatening systemic anaphylaxis. However, their direct involvement and the mechanisms underlying anaphylactoid reactions caused by antimicrobials have not been demonstrated. Structurally different antimicrobials were screened by Ca2+ imaging using MRGPRX2 overexpressing HEK293 cells. MRGPRX2 related anaphylactoid reactions induced by these components were investigated by body temperature drop and mast cell degranulation assays. We showed that MRGPRX2 is involved in allergic-like reactions to three types of antimicrobials in a dose-dependent manner. However, mast cells lacking the receptor show reduced degranulation. Furthermore, mice without MAS-related G protein-coupled receptor B2 (the orthologous gene of MRGPRX2) exhibited reduced substance-induced inflammation. Interestingly, β-lactam and antiviral nucleoside analogues did not induce anaphylactic reactions, which were also observed in vitro. These results should alarm many clinicians that such drugs might induce anaphylactoid reactions and provide guidance on safe dosage of these drugs.

Keywords: Anaphylactoid reaction Antimicrobials Degranulation Mast Cells Mrgprx2

Spinal-cord-NLRP1-inflommsome-open-access-contributes-to-dry-skin-induced-chronic-itch-in-mice

Abstract
Background: Dry skin itch is one of the most common skin diseases and elderly people are believed to be particularly prone to it. The inflammasome has been suggested to play an important role in chronic inflammatory disorders including inflammatory skin diseases such as psoriasis. However, little is known about the role of NLRP1 inflammasome in dry skin-induced chronic itch.
Methods: Dry skin-induced chronic itch model was established by acetone-ether-water (AEW) treatment. Spontaneous scratching behavior was recorded by video monitoring. The expression of nucleotide oligomerization domain (NOD)-like receptor protein 1 (NLRP1) inflammasome complexes, transient receptor potential vanilloid type 1 (TRPV1), and the level of inflammatory cytokines were determined by western blot, quantitative real-time PCR, and enzyme-linked immunosorbent assay (ELISA) kits. Nlrp1a knockdown was performed by an adeno-associated virus (AAV) vector containing Nlrp1a-shRNA- eGFP infusion. H.E. staining was used to evaluate skin lesion.
Results: AEW treatment triggers spontaneous scratching and significantly increases the expression of NLRP1, ASC, and caspase-1 and the levels of IL-1β, IL-18, IL-6, and TNF-α in the spinal cord and the skin of mice. Spinal cord Nlrp1a knockdown prevents AEW-induced NLRP1 inflammasome assembly, TRPV1 channel activation, and spontaneous scratching behavior. Capsazepine, a specific antagonist of TRPV1, can also inhibit AEW-induced inflammatory response and scratching behavior. Furthermore, elderly mice and female mice exhibited more significant AEW-induced scratching behavior than young mice and male mice, respectively. Interestingly, AEW-induced increases in the expression of NLRP1 inflammasome complex and the levels of inflammatory cytokines were more remarkable in elderly mice and female mice than in young mice and male mice, respectively.
Conclusions: Spinal cord NLRP1 inflammasome-mediated inflammatory response contributes to dry skin-induced chronic itch by TRPV1 channel, and it is also involved in age and sex differences of chronic itch. Inhibition of NLRP1 inflammasome may offer a new therapy for dry skin itch.
Keywords: NLRP1 inflammasome, TRPV1, Chronic itch, Dry skin, Spinal cord

Local Ca2+ signals couple activation of TRPV1 and ANO1 sensory ion channels

Shihab Shah 1, Chase M Carver 2, Pierce Mullen 1, Stephen Milne 1, Viktor Lukacs 1, Mark S Shapiro 2, Nikita Gamper 3 4

Abstract

ANO1 (TMEM16A) is a Ca2+-activated Cl− channel (CaCC) expressed in peripheral somatosensory neurons that are activated by painful (noxious) stimuli. These neurons also express the Ca2+-permeable channel and noxious heat sensor TRPV1, which can activate ANO1. Here, we revealed an intricate mechanism of TRPV1-ANO1 channel coupling in rat dorsal root ganglion (DRG) neurons. Simultaneous optical monitoring of CaCC activity and Ca2+ dynamics revealed that the TRPV1 ligand capsaicin activated CaCCs. However, depletion of endoplasmic reticulum (ER) Ca2+ stores reduced capsaicin-induced Ca2+ increases and CaCC activation, suggesting that ER Ca2+ release contributed to TRPV1-induced CaCC activation. ER store depletion by plasma membrane–localized TRPV1 channels was demonstrated with an ER-localized Ca2+ sensor in neurons exposed to a cell-impermeable TRPV1 ligand. Proximity ligation assays established that ANO1, TRPV1, and the IP3 receptor IP3R1 were often found in close proximity to each other. Stochastic optical reconstruction microscopy (STORM) confirmed the close association between all three channels in DRG neurons. Together, our data reveal the existence of ANO1-containing multichannel nanodomains in DRG neurons and suggest that coupling between TRPV1 and ANO1 requires ER Ca2+ release, which may be necessary to enhance ANO1 activation.

Bradykinin Is a Potent Pruritogen in Atopic Dermatitis: A Switch From Pain to Itch

Miwa Hosogi ¹, Martin Schmelz, Yoshiki Miyachi, Akihiko Ikoma

Abstract

Histamine, substance P, serotonin and bradykinin were applied by iontophoresis to lesional and visually non-lesional skin of 14 patients with atopic dermatitis, and normal skin of 15 healthy volunteers. Itch could be evoked by light stroking of skin with a cotton swab (alloknesis) in all lesional skin sites, but not in non-lesional or normal skin. Substances were applied in the same skin area before and 3 h after administration of placebo or antihistamine (olopatadine hydrochloride: H1-receptor-blocker). Intensities of itch and pain sensation and areas of flare and wheal were measured. All the substances induced significantly more intense itch in lesional skin than in non-lesional skin of patients. Even bradykinin, which evoked only weak itch and pain of similar intensities in non-lesional skin of patients and in healthy volunteers, induced intense itch in lesional skin, while the simultaneously increased pain did not suppress the itch sensation, indicating central sensitization. Histamine- and substance P-induced itch was almost completely suppressed by antihistamines, whereas bradykinin- and serotonin-induced itch was not. This suggests that substance P is a histamine-dependent pruritogen also in lesional skin under sensitized conditions but that bradykinin and serotonin are histamine-independent pruritogens in lesional skin. It is concluded that serotonin and bradykinin, classic endogenous algogens, can turn into potent histamine-independent pruritogens in lesional skin of atopic dermatitis.

Enhanced Thermal Sensitivity of TRPV3 in Keratinocytes Underlies Heat-Induced Pruritogen Release and Pruritus in AD

Seong Hoon Seo, Sohyun Kim, Song-Ee Kim, Seungsoo Chung and Sang Eun Lee

Abstract

Itch in atopic dermatitis (AD) is aggravated under warm conditions. Transient receptor potential vanilloid (TRPV) 3, a member of the thermosensitive transient receptor potential channels, is activated by innocuous heat and is abundantly expressed in keratinocytes. The potential role of TRPV3 in itch is illustrated in TRPV3 channelopathies of humans and mice. However, the role of TRPV3 in heat-induced itch in AD and the underlying mechanisms are unclear. Here we showed that keratinocytes isolated from patients with AD exhibit enhanced expression and heat sensitivity with hyperactive channel function of TRPV3. Heat stimulus induced enhanced secretion of thymic stromal lymphopoietin, nerve growth factor, and prostaglandin E2 by keratinocytes from patients with AD through TRPV3 activation. TRPV3 agonists increased thymic stromal lymphopoietin, nerve growth factor, prostaglandin E2, and IL-33 production in human keratinocytes and induced scratching behavior upon intradermal injection in mice. TRPV3 was upregulated in the skin of MC903-induced AD mouse model. Heat stimulation to MC903-treated mice increased scratching behavior and produced higher levels of thymic stromal lymphopoietin, nerve growth factor, prostaglandin E2, and IL-33 from the epidermis, which were attenuated by pharmacologic inhibition of TRPV3. Moreover, neutralization of thymic stromal lymphopoietin reduced heat-evoked scratching in MC903-challenged mice. These results suggest that TRPV3 is a potential therapeutic target for heat-induced itch in AD.

Abstract

Mast cells are a heterogeneous group of immune cells. The simplest and commonly accepted classification divides them in two groups according to their protease content. We have compared the action of diverse secretagogues on bone marrow derived (BMMC) and peritoneal (PMC) mast cells which represent classical models of mucosal and connective tissue type mast cells in mice. Whereas, antigen stimulation of the FcεRI receptors was similarly effective in triggering elevations of free intracellular Ca2+ concentration ([Ca2+]i) in both BMMC and PMC, robust [Ca2+]i rise following Endothelin-1 stimulation was observed only in a fraction of BMMC. Leukotriene C4 activating cysteinyl leukotriene type I receptors failed to evoke [Ca2+]i rise in either mast cell model. Stimulation of the recently identified target of many small-molecule drugs associated with systemic pseudo-allergic reactions, Mrgprb2, with compound 48/80, a mast cell activator with unknown receptor studied for many years, triggered Ca2+ oscillations in BMMC and robust [Ca2+]i rise in PMCs similarly to that evoked by FcεRI stimulation. [Ca2+]i rise in PMC could also be evoked by other Mrgprb2 agonists such as Tubocurarine, LL-37, and Substance P. The extent of [Ca2+]i rise correlated with mast cell degranulation. Expression analysis of TRPC channels as potential candidates mediating agonist evoked Ca2+ entry revealed the presence of transcripts of all members of the TRPC subfamily of TRP channels in PMCs. The amplitude and AUC of compound 48/80-evoked [Ca2+]i rise was reduced by ~20% in PMC from Trpc1/4/6−/− mice compared to Trpc1/4−/− littermatched control mice, whereas FcεRI-evoked [Ca2+]i rise was unaltered. Whole-cell patch clamp recordings showed that the reduction in compound 48/80-evoked [Ca2+]i rise in Trpc1/4/6−/− PMC was accompanied by a reduced amplitude of Compound 48/80-induced cation currents which exhibited typical features of TRPC currents. Together, this study demonstrates that PMC are an appropriate mast cell model to study mechanisms of Mrgprb2 receptor-mediated mast cell activation, and it reveals that TRPC channels contribute at least partially to Mrgprb2-mediated mast cellactivation but not following FcεRI stimulation. However, the channels conducting most of the Ca2+ entry in mast cells triggered by Mrgprb2 receptor stimulation remains to be identified.

IL-37 is protective in allergic contact dermatitis through mast cell inhibition 

Weihua Lia, Fengmin Dingb, Yi Zhaia, Wenting Taob, Jing Bic, Hong Fanc, Nina Yind,

Zhigang Wangc,⁎

a Department of Cardiology, Affiliated Liyuan Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430077, China b School of Basic Medical Sciences, Hubei University of Chinese Medicine, Wuhan 430065, China
c Department of Pathogen Biology, School of Basic Medical Sciences, Hubei University of Chinese Medicine, Wuhan 430065, China
d Department of Anatomy, School of Basic Medical Sciences, Hubei University of Chinese Medicine, Wuhan 430065, China

ABSTRACT

Allergic contact dermatitis (ACD), characterized predominantly by erythema, vesiculation, and pruritus, is a T cell-mediated skin inflammatory condition. Among immune cells involved in ACD, mast cells (MCs) play an essential role in its pathogenesis. As an inhibitor of proinflammatory IL-1 family members, interleukin 37 (IL-37) has been shown to ameliorate inflammatory responses in various allergic diseases. In this study, we assessed the immunomodulatory effect of IL-37 on allergic inflammation using a 2,4-dinitrofluorobenzene (DNFB)-induced ACD rat model and isolated rat peritoneal mast cells (RPMCs). Systematic application of IL-37 significantly relieved ear swelling, reduced inflammatory cell infiltration, decreased inflammatory cytokine production (TNF- α, IL-1β, IFN-γ, and IL-13), inhibited MC recruitment, lowered IgE levels, and reduced IL-33 production in the local ear tissues with DNFB challenge. Additionally, RPMCs isolated from ACD rats with IL-37 intervention showed downregulation of IL-6, TNF-α, IL-13, and MCP-1 production following IL-33 stimulation, and reduction of β-hexosaminidase and histamine release under DNP-IgE/HSA treatment. Moreover, IL-37 treatment also significantly restrained NF-κB activation and P38 phosphorylation in ACD RPMCs. SIS3, a specific Smad3 in- hibitor, abolished the suppressive effects of IL-37 on MC-mediated allergic inflammation, suggesting the parti- cipation of Smad3 in the anti-ACD effect of IL-37. These findings indicated that IL-37 protects against IL-33- regulated MC inflammatory responses via inhibition of NF-κB and P38 MAPK activation accompanying the regulation of Smad3 in rats with ACD.

Cross-talk between Human Spinal Cord μ-opioid Receptor 1Y Isoform and Gastrin-releasing Peptide Receptor Mediates Opioid-induced Scratching Behavior.

Liu XY, Ginosar Y, Yazdi J, Hincker A, Chen ZF.

Abstract

BACKGROUND: 

Although spinal opioids are safe and effective, pruritus is common and distressing. The authors previously demonstrated in mouse spinal cord that interactions between μ-opioid receptor isoform 1D and gastrin releasing peptide receptor mediate morphine-induced scratch. The C-terminal of 1D inhibits morphine-induced scratch without affecting analgesia. The authors hypothesize that human spinal cord also contains itch-specific μ-opioid receptor isoforms which interact with gastrin releasing peptide receptor.

METHODS: 

Reverse transcription polymerase chain reaction was performed on human spinal cord complimentary DNA from two humancadavers. Calcium responses to morphine (1 μM) were examined using calcium imaging microscopy on human cells (HEK293) coexpressing gastrin releasing peptide receptor and different human μ-opioid receptor isoforms. The authors assessed morphine-induced scratching behavior and thermal analgesia in mice following intrathecal injection of morphine (0.3 nmol) and a transactivator of transcription peptide designed from C-terminal sequences of 1Y isoform (0, 0.1, and 0.4 nmol).

RESULTS: 

The authors demonstrated 1Y expression in the spinal cord dorsal horn. Morphine administration evoked a calcium response (mean ± SD) (57 ± 13 nM) in cells coexpressing both gastrin releasing peptide receptor and the 1Y isomer. This was blocked by 10 μM naltrexone (0.7 ± 0.4 nM; P < 0.0001), 1 μM gastrin-releasing peptide receptor antagonist (3 ± 2 nM; P < 0.0001), or 200 μM 1Y-peptide(2 + 2 nM; P < 0.0001). In mice, 0.4 nmol 1Y-peptide significantly attenuated morphine-induced scratching behaviors (scratching bouts, vehicle vs. 1Y-peptide) (92 ± 31 vs. 38 ± 29; P = 0.011; n = 6 to 7 mice per group), without affecting morphine antinociception in warm water tail immersion test (% of maximum possible effect) (70 ± 21 vs. 67 ± 22; P = 0.80; n = 6 mice per group).

CONCLUSIONS: 

Human μ-opioid receptor 1Y isomer is a C-terminal splicing variant of Oprm1 gene identified in human spinal cord. Cross-talk between 1Y and gastrin releasing peptide receptor is required for mediating opioid-induced pruritus. Disrupting the cross talkmay have implications for therapeutic uncoupling of desired analgesic effects from side effects of opioids.