The missing link between itch and inflammation in atopic dermatitis.

Atopic dermatitis is a common skin disease with high morbidity and is associated with severe itch and chronic skin inflammation. In this issue of Cell, Wilson et al. demonstrate that epithelial cells communicate directly with cutaneous sensory neurons via a cytokine to induce itch.

Physiology and Pathophysiology of Itch.

Itch is a topic to which everyone can relate. The physiological roles of itch are increasingly understood and appreciated. The pathophysiological consequences of itch impact quality of life as much as pain. These dynamics have led to increasingly deep dives into the mechanisms that underlie and contribute to the sensation of itch. When the prior review on the physiology of itching was published in this journal in 1941, itch was a black box of interest to a small number of neuroscientists and dermatologists. Itch is now appreciated as a complex and colorful Rubik's cube. Acute and chronic itch are being carefully scratched apart and reassembled by puzzle solvers across the biomedical spectrum. New mediators are being identified. Mechanisms blur boundaries of the circuitry that blend neuroscience and immunology. Measures involve psychophysics and behavioral psychology. The efforts associated with these approaches are positively impacting the care of itchy patients. There is now the potential to markedly alleviate chronic itch, a condition that does not end life, but often ruins it. We review the itch field and provide a current understanding of the pathophysiology of itch. Itch is a disease, not only a symptom of disease.

Mechanistic insights into the antipruritic effects of lebrikizumab, an anti-IL-13 mAb.

BACKGROUND: Atopic dermatitis is a chronic inflammatory skin disease with persistent and severe itch among its hallmark features. Significant increases in type 2 cytokines (ie, IL-4, IL-13, IL-31) have been documented in acute atopic dermatitis lesions and lead to multifaceted downstream effects, including inflammation, epidermal barrier dysfunction, and itch. OBJECTIVE: The primary objective of preclinical studies reported here was to test direct effects of IL-13 and an anti-IL-13 mAb, lebrikizumab, in a human dorsal root ganglion model in itch amplification, neuronal excitability, and transcriptional downstream targets. METHODS: Neuroactive effects were assessed via live cell calcium imaging, electric field stimulation, and RNA sequencing of human dorsal root ganglia stimulated with IL-13 alone or in combination with lebrikizumab. RESULTS: These preclinical findings suggest that IL-13 plays a direct enhancer role in multiple itch and neuroactive pathways as well as transcriptional downstream effects, and provide key insights into the mechanistic basis for lebrikizumab's anti-itch effects. CONCLUSION: IL-13 is a potent enhancer of neuronal responses to different itch stimuli, consistent with the neuroimmune axis contributing to chronic itch-associated inflammatory skin disease, and blockade of this cytokine pathway may provide a therapeutic approach.

Role of mast cells and basophils in pruritus.

To protect our body systems, there is a constant interactive conversation between the skin nervous and immune system. Important elements of this conversation in the skin include mast cells, basophils, and sensory nerve fibers. These cells employ a vast array of sensors that detect danger and react accordingly. This reaction, summarized as neurogenic inflammation, manifests at the conscious level as sensations including pain and itch. Here we provide a perspective on the blossoming knowledge that is illuminating connections between mast cells, basophils, and sensory nerve fibers in the mediation of itch. We discuss established mediators and receptors, in particular cytokine and neuropeptide pathways, upstream proteases, and proteinase-activated receptors, and the emerging role of mas-related G-protein-coupled receptors in itch.

Why do we itch?

Why do we have the sensation of itch and the associated scratching response? The conventional explanation is that arthropods and other environmental irritants elicit the sensation of itch that then leads to scratching and removal of the stimulus. This explanation is reasonable yet is too limiting for this fascinating sensory phenomenon and the associated somatic (voluntary) response of scratching. We delve into other explanations here. Some of these explanations fit within current areas of basic and clinical knowledge while others are speculative and may help to frame future discussions and study.

Activation of mas-related G-protein-coupled receptors by the house dust mite cysteine protease Der p1 provides a new mechanism linking allergy and inflammation.

Cysteine and serine proteases function via protease-activated and mas-related G-protein-coupled receptors (Mrgprs) to contribute to allergy and inflammation. Der p1 is a cysteine protease and major allergen from the house dust mite and is associated with allergic rhinitis and allergic asthma. Der p1 activates protease-activated receptor 2 and induces the release of the pro-inflammatory cytokine IL-6 from cells. However, the possibility that Der p1 acts on Mrgprs has not been considered. We report here that ratiometric calcium imaging reveals that Der p1 activates the human receptor MRGPRX1 and the mouse homolog MrgprC11, implicated previously in itch. Der p1 cleavage of N-terminal receptor peptides followed by site-directed mutagenesis of the cleavage sites links receptor activation to specific amino acid residues. Der p1 also induced the release of IL-6 from heterologous cells expressing MRGPRX1. In summary, activation of Mrgprs by the allergen Der p1 may contribute to inflammation.

Is the Nervous System More Important Than the Immune System in Itch and Atopic Dermatitis?

Atopic dermatitis is a common cutaneous disorder characterized by severe itch, chronic inflammation, and increased nerve fiber density. It has been assumed that the neural changes are in response to ongoing inflammation. We used in vivo imaging over time of fluorescently labeled peripheral sensory nerves during epicutaneous sensitization to ovalbumin in an allergic mouse model of atopic dermatitis. Visualization of cutaneous nerve branches and blood vessels sequentially over months revealed that peripheral sensory nerves begin to pathfind within 48 hours of antigen exposure. Innervation density and arbor complexity of neuropeptidergic fibers in the skin increased within days. Neural sprouting preceded changes in vascularization, vascular permeability, and immune infiltration. Blocking neural activation during periods of sensitization prevented ovalbumin-induced changes in neural recruitment and pattern reorganization, as well as subsequent inflammatory infiltrates and scratching behavior. These data implicate different roles for recently identified itch molecules in modulating various steps in the inflammatory response. Thus, in contrast to the traditional view that neural changes are reactive to inflammation and scratching, these data suggest that allergic stimulation in a chronic eczema model requires neural recruitment and activation early in the process for the elaboration and maintenance of the inflammatory cascade.

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

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.

Peripheral NMDA Receptor/NO System Blockage Inhibits Itch Responses Induced by Chloroquine in Mice.

Intradermal administration of chloroquine (CQ) provokes scratching behavior in mice. Chloroquine-induced itch is histamine-independent and we have reported that the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway is involved in CQ-induced scratching behavior in mice. Previous studies have demonstrated that activation of N-methyl-d-aspartate receptors (NMDARs) induces NO production. Here we show that NMDAR antagonists significantly decrease CQ-induced scratching in mice while a non-effective dose of an NMDAR agonist potentiates the scratching behavior provoked by sub-effective doses of CQ. In contrast, combined pre-treatment with sub-effective doses of an NMDAR antagonist, MK-801, and the NO synthase inhibitor, L-N-nitro arginine methyl ester (L-NAME), decreases CQ-induced scrat-ching behavior. While intradermal administration of CQ significantly increases the concentration of intradermal nitrite, the end product of NO metabolism, effective doses of intraperitoneal and intradermal MK-801 significantly decrease intradermal nitrite levels. Likewise, administration of an effective dose of L-NAME significantly decreases CQ-induced nitrite production. We conclude that the NMDA/NO pathway in the skin modulates CQ-induced scratching behavior.

Maxadilan-simile expression in Nyssomyia neivai, a sandfly vector in an endemic region of Brazil, and its immunogenicity in patients with American tegumentary leishmaniasis.

BACKGROUND: Maxadilan (Max) is a salivary component in the sandfly Lutzomyia longipalpis (Lutz & Neiva 1912), a vector of visceral leishmaniasis. Max has a powerful vasodilatory effect and is a candidate vaccine that has been tested in experimental leishmaniasis. Nyssomyia neivai (Pinto 1926) is a vector of the pathogen responsible for American tegumentary leishmaniasis (ATL) in Brazil. OBJECTIVE: We searched for Max expression in Ny. neivai and for antibodies against Max in ATL patients. METHODS: cDNA and protein were extracted from the cephalic segment, including salivary glands, of Ny. neivai and analysed by polymerase chain reaction, DNA sequencing, and blotting assays. The results were compared with data obtained from Lu. longipalpis samples. We quantified antibodies against Max in serum samples from 41 patients with ATL (31 and 10 with the cutaneous and mucocutaneous forms, respectively) and 63 controls from the endemic northeastern region of São Paulo state, using enzyme-linked immunosorbent assay. FINDINGS: Recognition of a Max-simile peptide by specific antibodies confirmed expression of a Max sequence in Ny. neivai (GenBank EF601123.1). Compared to controls, patients with ATL presented higher levels of antibodies against Max (p = 0.004); 24.4% of the patients with ATL and 3.2% of the controls presented anti-Max levels above the cutoff index (p = 0.014). The anti-Max levels were not associated with the specific clinical form of ATL, leishmanin skin test response, absence or presence of amastigotes in histopathologic exam, results of indirect immunofluorescence testing for leishmaniasis, or duration of cutaneous form disease. MAIN CONCLUSION: High serum anti-Max levels did not protect patients against ATL, but confirmed previous natural exposure to Ny. neivai bites in this ATL endemic region.

GRPR/PI3Kγ: Partners in Central Transmission of Itch.

The gastrin-releasing peptide (GRP) and its receptor (GRPR) are important components of itch transmission. Upstream, but not downstream, aspects of GRPR signaling have been investigated extensively. We hypothesize that GRPR signals in part through the PI3Kγ/Akt pathway. We used pharmacological, electrophysiological, and behavioral approaches to further evaluate GRPR downstream signaling pathways. Our data show that GRP directly activates small-size capsaicin-sensitive DRG neurons, an effect that translates into transient calcium flux and membrane depolarization (∼ 20 mV). GRPR activation also induces Akt phosphorylation, a proxy for PI3Kγ activity, in ex vivo naive mouse spinal cords and in GRPR transiently expressing HEK293 cells. The intrathecal injection of GRP led to intense scratching, an effect largely reduced by either GRPR antagonists or PI3Kγ inhibitor. Scratching behavior was also induced by the intrathecal injection of an Akt activator. In a dry skin model of itch, we show that GRPR blockade or PI3Kγ inhibition reversed the scratching behavior. Altogether, these findings are highly suggestive that GRPR is expressed by the central terminals of DRG nociceptive afferents, which transmit itch via the PI3Kγ/Akt pathway. SIGNIFICANCE STATEMENT: Itch is the most common symptom of the skin and is related to noncutaneous diseases. It severely impairs patients' quality of life when it becomes chronic and there is no specific or effective available therapy, mainly because itch pathophysiology is not completely elucidated. Our findings indicate that the enzyme PI3Kγ is a key central mediator of itch transmission. Therefore, we suggest PI3Kγ as an attractive target for the development of new anti-pruritic drugs. With this study, we take a step forward in our understanding of the mechanisms underlying the central transmission of itch sensation.

Are itch and scratching the nausea and vomiting of skin?

The physiologic similarities between itch and nausea may not be evident initially, but they share the role of repelling irritants and toxins from the body by inducting scratching and vomiting, respectively. In addition, itch and nausea frequently occur together in certain conditions such as uraemia. Here we show that the mechanisms underlying itch and nausea overlap and that advances in either field may influence the identification of novel drug targets, particularly for itch.

Protease-activated receptors and itch.

Protease-activated receptors (PARs) have been implicated in a variety of physiological functions, as well as somatosensation and particularly itch and pain. Considerable attention has focused on PARs following the finding they are upregulated in the skin of atopic dermatitis patients. The present review focuses on recent studies showing that PARs are critically involved in itch and sensitization of itch. PARs are expressed by diverse cell types including primary sensory neurons, keratinocytes, and immune cells and are activated by proteases that expose a tethered ligand. Endogenous proteases are also released from diverse cell types including keratinocytes and immune cells. Exogenous proteases released from certain plants and insects contacting the skin can also induce itch. Increased levels of proteases in the skin contribute to inflammation that is often accompanied by chronic itch which is not predominantly mediated by histamine. The neural pathway signaling itch induced by activation of PARs is distinct from that mediating histamine-induced itch. In addition, there is evidence that PARs play an important role in sensitization of itch signaling under conditions of chronic itch. These recent findings suggest that PARs and other molecules involved in the itch-signaling pathway are good targets to develop novel treatments for most types of chronic itch that are poorly treated with antihistamines.

Cowhage can induce itch in the atopic dog.

Itch is a cardinal symptom of atopic dermatitis in humans and dogs. Until now, experimental induction of itch in dogs has proven difficult. The objectives of this study were to determine whether protease-rich spicules, protein extracts and the protease mucunain of the tropical legume cowhage provoked itch and inflammation when rubbed onto canine skin. Native spicules variably induced itch manifestations in about half of the dogs, while challenges with protease-deactivated spicules remained negative. The epicutaneous application of cowhage extract and mucunain after microneedle roller usage also induced pruritus and inflammation. Importantly, there was an interindividual inconsistency in pruritus and inflammation induction and also marked differences in pruritus intensity after challenge. In conclusion, cowhage spicules, protein-rich extracts and mucunain can all induce pruritus and inflammation in dogs as in other species, but the inconsistency of provocation is currently a limitation of this challenge type for future studies of pruritus in dogs.

Understanding the pathophysiology of itch.

Itch is the most common symptom described by our patients. Treating this symptom can be challenging. A revolution is ongoing in understanding the pathophysiology of itch and will allow this challenge to be met. The present authors review and update the current understanding of the pathophysiology of itch.

Cryoneurolysis with Injectable Ice Slurry Modulates Mechanical Skin Pain.

Cutaneous pain is a common symptom of skin disease, and available therapies are inadequate. We developed a neural selective and injectable method of cryoneurolysis with ice slurry, which leads to a long-lasting decrease in mechanical pain. The aim of this study is to determine whether slurry injection reduces cutaneous pain without inducing the side effects associated with conventional cryoneurolysis. Using the rat sciatic nerve, we examined the effects of slurry on nerve structure and function in comparison with the effects of a Food and Drug Administration‒approved cryoneurolysis device (Iovera). Coherent anti-Stokes Raman scattering microscopy and immunofluorescence staining were used to investigate histological effects on the sciatic nerve and on downstream cutaneous nerve fibers. Complete Freund's Adjuvant model of cutaneous pain was used to study the effect of the slurry on reducing pain. Structural changes in myelin induced by slurry were comparable with those induced by Iovera, which uses much colder temperatures. Compared with that of Iovera, the decrease in mechanical pain due to slurry was less profound but lasted longer without signs of dysesthesia. Slurry did not cause a reduction of epidermal nerve fibers or a change in thermal pain sensitivity. Slurry-treated rats showed reduced cutaneous mechanical pain in response to Complete Freund's Adjuvant. Slurry injection can be used to successfully reduce cutaneous pain without causing dysesthesia.

Maxadilan, the Lutzomyia longipalpis vasodilator, drives plasma leakage via PAC1-CXCR1/2-pathway.

Experiments were designed to determine if the vasodilatory peptides maxadilan and pituitary adenylate cyclase-activating peptide (PACAP-38) may cause plasma leakage through activation of leukocytes and to what extent these effects could be due to PAC1 and CXCR1/2 receptor stimulation. Intravital microscopy of hamster cheek pouches utilizing FITC-dextran and rhodamine, respectively, as plasma and leukocyte markers was used to measure arteriolar diameter, plasma leakage and leukocyte accumulation in a selected area (5mm(2)) representative of the hamster cheek pouch microcirculation. Our studies showed that the sand fly vasodilator maxadilan and PACAP-38 induced arteriolar dilation, leukocyte accumulation and plasma leakage in postcapillary venules. The recombinant mutant of maxadilan M65 and an antagonist of CXCR1/2 receptors, reparixin, and an inhibitor of CD11b/CD18 up-regulation, ropivacaine, inhibited all these effects as induced by maxadilan. Dextran sulfate, a complement inhibitor with heparin-like anti-inflammatory effects, inhibited plasma leakage and leukocyte accumulation but not arteriolar dilation as induced by maxadilan and PACAP-38. In vitro studies with isolated human neutrophils showed that maxadilan is a potent stimulator of neutrophil migration comparable with fMLP and leukotriene B(4) and that M65 and reparixin inhibited such migration. The data suggest that leukocyte accumulation and plasma leakage induced by maxadilan involves a mechanism related to PAC1- and CXCR1/2-receptors on leukocytes and endothelial cells.

Amelioration of Compound 48/80-Mediated Itch and LL-37-Induced Inflammation by a Single-Stranded Oligonucleotide.

Numerous inflammatory skin disorders display a high prevalence of itch. The Mas-related G protein coupled receptor X2 (MRGPRX2) has been shown to modulate itch by inducing non-IgE-mediated mast cell degranulation and the release of endogenous inducers of pruritus. Various substances collectively known as basic secretagogues, which include inflammatory peptides and certain drugs, can trigger MRGPRX2 and thereby induce pseudo-allergic reactions characterized by histamine and protease release as well as inflammation. Here, we investigated the capacity of an immunomodulatory single-stranded oligonucleotide (ssON) to modulate IgE-independent mast cell degranulation and, more specifically, its ability to inhibit the basic secretagogues compound 48/80 (C48/80)-and LL-37 in vitro and in vivo. We examined the effect of ssON on MRGPRX2 activation in vitro by measuring degranulation in a human mast cell line (LAD2) and calcium influx in MRGPRX2-transfected HEK293 cells. To determine the effect of ssON on itch, we performed behavioral studies in established mouse models and collected skin biopsies for histological analysis. Additionally, with the use of a rosacea mouse model and RT-qPCR, we investigated the effect on ssON on LL-37-induced inflammation. We reveal that both mast cell degranulation and calcium influx in MRGPRX2 transfected HEK293 cells, induced by the antimicrobial peptide LL-37 and the basic secretagogue C48/80, are effectively inhibited by ssON in a dose-dependent manner. Further, ssON demonstrates a capability to inhibit LL-37 and C48/80 activation in vivo in two mouse models. We show that intradermal injection of ssON in mice is able to block itch induced via C48/80 in a dose-dependent manner. Histological staining revealed that ssON inhibits acute mast cell degranulation in murine skin treated with C48/80. Lastly, we show that ssON treatment ameliorates LL-37-induced inflammation in a rosacea mouse model. Since there is a need for new therapeutics targeting non-IgE-mediated activation of mast cells, ssON could be used as a prospective drug candidate to resolve itch and inflammation in certain dermatoses.