MrgprA3⁺ Primary Sensory Neurons Mediate Acute Allergic Itch Responses in Atopic Dermatitis Model Mice

Abstract

Itch is a prominent symptom of atopic dermatitis (AD). However, the underlying mechanism remains complex and has not yet been fully elucidated. Mas-related G protein-coupled receptor A3 (MrgprA3) has emerged attention as a marker of primary sensory neurons that specifically transmit itch signals; however, its involvement in AD-related itch has not been extensively explored. In this study, we developed an AD itch mouse model by repeatedly applying house dust mite (HDM) extract to barrier-impaired skin via a special diet. To clarify the role of MrgprA3⁺ neurons in itch behavior in our AD model, we adopted a toxin receptor-mediated cell knockout strategy using transgenic mice in which the diphtheria toxin receptor (DTR) gene was placed under the control of the Mrgpra3 promoter. When the HDM extract was repeatedly applied to the face and back skin of special diet-fed mice, the mice exhibited AD-like dry and eczematous skin lesions accompanied by three types of itch-related behaviors:1) spontaneous scratching, 2) acute scratching after antigen challenge, and 3) light touch-evoked scratching. Upon diphtheria toxin administration, substantial depletion of DTR⁺/MrgprA3⁺ neurons was observed in the dorsal root ganglion. Ablation of MrgprA3⁺ neurons suppressed acute itch responses after HDM application, whereas spontaneous and touch-evoked itch behaviors remained unaffected. Our findings unequivocally demonstrate that in our AD model, MrgprA3⁺ primary sensory neurons mediate acute allergic itch responses, whereas these neurons are not involved in spontaneous itch or alloknesis.

INTRODUCTION

Itch is an unpleasant sensation that triggers an urge to scratch. While transient mild itch is not particularly problematic, chronic severe itch requires treatment. Atopic dermatitis (AD) is a common chronic skin disease characterized by severe itch.¹⁾ In AD, common and innocuous mechanical stimuli (e.g., contact with clothes) often elicit an itch sensation (called alloknesis), contributing to chronic itch.^2,3) The chronic nature of itch significantly reduces the QOL of patients with AD, and repetitive scratching exacerbates skin lesions.^4,5) Therefore, effective itch control is crucial for AD management. However, the intricate mechanisms underlying itch in AD are complex and not fully understood, leading to unmet needs in the treatment of AD.^6,7)

Several subtypes of Mas-related G protein-coupled receptors (Mrgpr) are exclusively expressed in primary sensory neurons, suggesting their critical role in sensory functions such as itch.⁸⁾ Among the Mrgpr subtypes, MrgprA3 has been shown to be the receptor responsible for itch caused by the antimalarial drug chloroquine.⁹⁾ Interestingly, ablation of MrgprA3⁺ neurons diminished chloroquine-induced itch responses, as well as those caused by other pruritogens. Furthermore, when mice exclusively expressing the transient receptor potential vanilloid 1 in MrgprA3⁺ neurons were treated with capsaicin, a pain-producing substance, they exhibited itch behavior instead of pain behavior.¹⁰⁾ These findings suggested that MrgprA3⁺ neurons play a specific role in the transmission of itch signals. Activation of MrgprA3 and MrgprA3⁺ neurons has been suggested to contribute to itch associated with dry skin and contact dermatitis.^10–16) However, it is not fully elucidated whether MrgprA3⁺ neurons are involved in itch in AD.

A unique diet-induced AD mouse model has been previously reported. HR-1 hairless mice fed a special diet (SD) lacking both polyunsaturated fatty acids and starch exhibited dry scaly skin followed by AD-like skin inflammation.¹⁷⁾ We recently showed that feeding SD to the C57BL/6 strain, which is commonly used in genetic studies, also caused dry skin with reduced skin barrier function and increased spontaneous scratching behavior, although only mild skin inflammation was observed (M. Fujii, unpublished observations). Type 2-immune responses as well as skin barrier dysfunction are involved in the pathogenesis of AD. Repeated application of extracts of house dust mites (HDM), environmental allergens involved in the pathogenesis of AD, to the skin of SD-fed C57BL/6 mice markedly worsened skin inflammation with gene expression profiles more similar to human AD, compared with other well-known AD models such as Flaky-tail mouse, ovalbumin-induced, oxazolone-induced, and NC/Nga mice (M. Fujii, unpublished observations).

In the present study, to clarify the specific role of MrgprA3⁺ neurons in itch behavior in our AD model, we adopted a toxin receptor-mediated cell knockout (TRECK) strategy to specifically and temporally ablate target cells.¹⁸⁾ Consequently, our results show that MrgprA3⁺ primary sensory neurons mediate antigen-induced acute itch responses in AD mice.

MATERIALS AND METHODS

Animals

Four-week-old female C57BL/6NCrSlc mice were purchased. Mrgpra3GFP-Cre (allele symbol: Tg(Mrgpra3-GFP/cre)#Xzd) mice were originally generated by Dr. Xinzhong Dong (Johns Hopkins School of Medicine) and their frozen sperm were obtained from Drs. Tsuda and Shiratori (Kyusyu University) for reconstitution of the mouse line through in vitro fertilization at the Bioscience Research Center of Kyoto Pharmaceutical University. Rosa26iDTR mice (allele symbol: Gt(ROSA)26Sortm1(HBEGF)Awai) were purchased from the Jackson Laboratory (Bar Harbor, ME, U.S.A.). Heterozygous Mrgpra3GFP-Cre mice and homozygous Rosa26iDTR mice were crossed, and the offspring were genotyped for Cre recombinase using DNA obtained from tail clips with the following primers: forward 5′-ATTGCTGTCACTTGGTCGTGGC-3′ and reverse 5′-GGAAAATGCTTCTGTCCGTTTGC-3′ (TaKaRa Bio, Otsu, Shiga, Japan). All mice were housed under standard conditions with free access to food and water and maintained at a temperature of 22 ± 1 °C under a 12-h light/dark cycle. All animal experiments were approved by the Ethics Committee of Animal Research at Kyoto Pharmaceutical University (Approval Number: PCOL-20-006).

HDM Ointment

Dermatophagoides farinae, one of the most common HDM, was cultured according to a previously described method,¹⁹⁾ with slight modifications. Briefly, mites were kept in ventilated flasks with a sterile animal diet (MF; Oriental Yeast, Tokyo, Japan) at 27 °C and 77% humidity. Mite bodies were separated from the culture media by flotation using a saturated sodium chloride solution or cyclohexane. The separated mite bodies were lyophilized and stored at −80 °C until use. Lyophilized mite bodies were stirred in 10 volumes of phosphate-buffered saline at 4 °C for 48 h, followed by centrifugation (4 °C, 16000 × g, 15 min). The supernatant was dialyzed against saline at 4 °C using a molecular porous membrane tube (Spectra/Por^®-3, MWCO 3500; Spectrum Laboratories, Rancho Dominguez, CA, U.S.A.), and the inner solution was collected as HDM extracts and stored at −80 °C until use. HDM extracts were then mixed with hydrophilic petrolatum (Maruishi Pharmaceutical, Osaka, Japan) at a concentration of HDM extract 300 µg protein/100 mg hydrophilic petrolatum, referred to as HDM ointment.

Induction of AD Symptoms

Four- to seven-week-old mice, weighing 18–20 g for males and 16–18 g for females, were fed an SD lacking both polyunsaturated fatty acids and starch (Product No. D03052309; Research Diets, New Brunswick, NJ, U.S.A.) to induce AD-like dry scaly skin.¹⁷⁾ The mice were fed a standard laboratory diet (AIN-76A, Product No. D10001; Research Diets) were used as normal controls. To exacerbate dermatitis symptoms, from 6 weeks after the start of SD feeding, HDM ointment (200 mg/animal) was applied to the face, ears, and upper back of the mouse once daily for 5 consecutive days, referred to as “round.” Three rounds of application were conducted at 9-d intervals.

Skin Dryness and Barrier Function

At least one week prior to the evaluation of skin symptoms, the hair on the back of the mice was removed. Skin dryness and barrier function were evaluated after the final application of HDM ointment. Skin dryness was scored as follows: (0) none, (1) slightly dry, (2) obviously dry with scaling, or (3) severely dry with extensive scaling. Transepidermal water loss (TEWL) on the dorsal skin was measured as an index of barrier function using Tewameter^® TM210 (Courage + Khazaka Electronic, Cologne, Germany).

Itch-Related Scratching Behavior

At least two weeks before measuring scratching behavior, a small magnet (1 mm diameter, 3 mm length) was implanted subcutaneously into both hind paws under isoflurane anesthesia. Before and after the final HDM application, scratching behavior was measured using an automatic measuring apparatus (MicroAct™; Neuroscience, Tokyo, Japan). Mice were placed in an observation chamber (11 cm diameter, 18 cm high) surrounded by a round coil for at least 10 min of acclimatation, and data were acquired for 1 h with the following settings: frequency: 10–30 Hz, minimum duration: 0.5 s, minimum beat: 5 times, beat/duration: 5 Hz, fill short gap: 0.1 s, peak: 0.1–2.5 V. After data acquisition, the cumulative duration of scratching behavior was calculated using MicroAct software (version 2.12).

In some experiments, scratching behavior was assessed by video analysis instead of using MicroAct, following previously reported methods.^20,21) Mice were placed in vinyl chloride cages for at least 10 min and their behavior was video-recorded for 1 h. The cumulative duration of scratching behavior was determined using an in-house counter.

On the day after the final HDM application, touch-evoked itch was evaluated according to a previously described method,²²⁾ with slight modifications. Mice were individually placed in a housing cage, acclimatized for 15 min, and then subjected to innocuous mechanical stimuli using a 0.7 mN von Frey filament, applied four times to different sites on the upper back skin. Each stimulus was defined as a positive response when the mouse immediately exhibited a site-directed scratching. The set of von Frey stimuli was repeated three times at intervals of several minutes, and the mean value per set was calculated as the touch-evoked itch score.

All measurements of itch-related scratching behavior were taken during the daytime hours (9:00–18:00).

Immunohistochemistry

At the time of sacrifice, the mice were transcardially perfused with saline and 4% paraformaldehyde under isoflurane anesthesia. Dorsal root ganglia (DRG) of the cervical (C1–C3) regions were carefully harvested and post-fixed with 4% paraformaldehyde for 2 h. Subsequently, the DRG were embedded in OCT Compound (Sakura Finetek Japan, Tokyo, Japan) and cut into 8 µm-thick sections. The sections were blocked with medium containing 2% normal goat serum. Anti-green fluorescent protein (GFP) polyclonal antibody (1 : 500 dilution; Thermo Fisher Scientific, Waltham, MA, U.S.A.) was applied overnight at 4 °C. The sections were then incubated with goat anti-rabbit immunoglobulin G (IgG) (H + L), a highly cross-adsorbed secondary antibody, Alexa Fluor 488 (1 : 500 dilution; Thermo Fisher Scientific) for 1 h at room temperature in the dark. Subsequently, anti-HB-EGF polyclonal antibody, Alexa Fluor 647 conjugated (1 : 100 dilution; Bioss Antibodies, Woburn, MA, U.S.A.), was applied for 1 h at room temperature in the dark. After washing, the cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (NucBlue™ Fixed Cell Stain ReadyProbes™ reagent; Thermo Fisher Scientific) for 5 min at room temperature in the dark and sealed with 90% glycerol/10% phosphate-buffered saline. Cells exhibiting green and red fluorescence were considered GFP⁺ and DTR⁺ cells, respectively, and cell counts were determined using fluorescence microscopy.

Diphtheria Toxin (DTX)

DTX was purified from Corynebacterium diphtheriae according to a previous report.¹⁸⁾ DTX was provided by Dr. Michiko Saito (Kyoto Pharmaceutical University) and stored at −80 °C until use. After the second round of HDM application, DTX (50 µg/kg) was administered intraperitoneally four times at 2-d intervals. Mice in the control group were administered saline. The dose of DTX used was determined based on the results of preliminary experiments.

Statistical Analysis

Data were analyzed using GraphPad Prism (version 8.0; GraphPad Software, San Diego, CA, U.S.A.) and are presented as individual values and mean ± standard error (S.E.) for each group. Comparisons between two groups were performed using either the unpaired Student’s t-test or Mann–Whitney U-test for parametric and non-parametric data, respectively. For multiple group comparisons, one-way ANOVA with Dunnett’s multiple comparison test or Kruskal–Wallis test with Dunn’s multiple comparison test was used for parametric and non-parametric data, respectively. Differences were considered statistically significant at p < 0.05.

RESULTS

Development of AD-Like Symptoms and Itch-Related Scratching Behavior in SD-Fed and HDM-Treated C57BL/6 Mice

To induce AD-like symptoms, female C57BL/6 mice were fed an SD, and after six weeks, HDM ointment was repeatedly applied, as illustrated in Fig. 1A. The SD-fed and HDM-treated mice exhibited dry, scaly skin, and eczematous lesions (Fig. 1B), resembling that of human AD. Our recent study demonstrated that gene expression profiles in the skin of SD-fed and HDM-treated C57BL/6 mice were similar to those of patients with AD (M. Fujii, unpublished observations). Therefore, this mouse is hereafter referred to as the “AD” mouse. Consistent with the gross findings, the skin dryness scores and TEWL were significantly higher in AD mice than in untreated normal mice (Fig. 1C).

Fig. 1. Atopic Dermatitis (AD) Mouse Model Using Special Diet (SD) and House Dust Mite (HDM) Allergens

(A) Protocol for induction of AD symptoms. Four-week-old female C57BL/6 mice were fed SD deficient in both polyunsaturated fatty acids (PUFA) and starch to induce skin barrier dysfunction. After six weeks of SD feeding, HDM ointment (600 µg protein/200 mg ointment/animal) was applied to the face, ears, and upper back of the mice. The five-day consecutive application (round) was repeated three times at 9-d intervals. A normal diet (ND) was provided as a negative control, and no HDM were applied. (B) Gross appearance of untreated ND-fed mouse (left) and SD-fed and HDM-treated (right) mouse on the day after final HDM application. SD-fed and HDM-treated mice exhibited AD-like scaly eczematous lesions; thus “untreated ND-fed” mouse and “SD-fed and HDM-treated” one are hereafter referred simply to as “normal” and “AD” mouse, respectively. (C) Skin dryness score and transepidermal water loss (TEWL) on the day after final HDM application. (D) Itch-related scratching behavior before (spontaneous) and immediately after the final HDM challenge was analyzed using MicroAct. On the day after the final HDM application, touch-evoked scratching was assessed using a von Frey filament (bending force, 0.7 mN). Data represent individual values and mean ± S.E. n = 5. * p < 0.05, ** p < 0.01, and *** p < 0.001, unpaired Student’s t-test for the data of TEWL and scratching before and after HDM challenge, or Mann–Whitney U-test for the data of skin dryness score and touch-evoked scratching.

To evaluate itch responses, scratching behavior was determined before and after the final application of HDM ointment. In AD mice, scratching behavior was significantly increased even before the HDM challenge (Fig. 1D), indicating an increased spontaneous itch. Immediately after the HDM challenge, robust scratching behavior was observed in AD mice (Fig. 1D), suggesting the presence of acute allergic itch. Furthermore, touch-evoked scratching was assessed the day after the final HDM challenge, and the itch scores were significantly higher in AD mice (Fig. 1D), indicating the development of alloknesis, similar to that observed in human AD. These results suggested that our AD model has multiple complex itch mechanisms associated with AD.

Ablation of MrgprA3⁺ Sensory Neurons by TRECK

To generate mice with DTR expression in MrgprA3⁺ neurons using the Cre/loxP system, heterozygous transgenic mice expressing Cre recombinase and eGFP under the Mrgpra3 promoter were crossed with homozygous transgenic mice whose DTR expression was blocked by a loxP-flanked stop sequence. This allowed for the generation of littermates expressing (+) or not expressing (−) DTR in MrgprA3⁺ neurons (Fig. 2A). The expression of MrgprA3 and DTR in the DRG of Cre⁺ littermates was assessed using double immunohistochemical staining with anti-GFP and anti-DTR antibodies. Almost all GFP⁺ cells (98.5%) coexpressed DTR (Fig. 2B). Therefore, Cre⁺ and Cre⁻ littermates are hereafter referred to as DTR⁺ and DTR⁻ mice, respectively.

Fig. 2. Ablation of MrgprA3⁺ Neurons by Toxin Receptor-Mediated Cell Knockout

(A) Protocol for the generation of transgenic mice carrying the diphtheria toxin receptor (DTR) in MrgprA3⁺ neurons. Heterozygous Mrgpra3GFP-Cre mice were crossed with homozygous Rosa26iDTR mice to generate littermates expressing (+) or not expressing (−) DTR in MrgprA3⁺ neurons. (B) Dorsal root ganglia (DRG) sections from Cre⁺ mice stained with antibodies against GFP (green) and DTR (red). Yellow represents merge and white arrows represent positive cells. Scale bar represents 50 µm. (C) Effect of diphtheria toxin (DTX) on the number of GFP⁺/DTR⁺ DRG cells. After the second round of HDM application, saline or DTX (50 µg/kg) was intraperitoneally administered to AD mice four times at 2-d intervals. Images show representative DRG sections from saline-(left) and DTX-treated (right) mice. Data represent individual values and mean ± S.E. n = 3. * p < 0.05, unpaired Student’s t-test.

To ablate MrgprA3⁺ neurons by TRECK, DTX was administered to DTR⁺ mice. GFP⁺/DTR⁺ DRG cells in DTX-treated mice were markedly decreased compared with those in saline-treated control mice (Fig. 2C), indicating the successful DTX-induced conditional ablation of MrgprA3⁺ sensory neurons in DTR⁺ mice.

Effects of Ablation of MrgprA3⁺ Neurons on Skin Symptoms and Itch Responses in AD Mice

When male DTR⁺ mice were fed SD and treated with HDM, similar AD symptoms were observed (Fig. 3A). To ablate MrgprA3⁺ neurons, DTR⁺ AD mice were administered either saline or DTX, and the degree of skin symptoms and scratching behavior were compared. There were no apparent differences in gross findings, skin dryness scores, or TEWL between the saline- and DTX-treated groups (Figs. 3A, B). However, the analysis of scratching behavior showed that DTX administration significantly attenuated acute scratching immediately after the HDM challenge, but it had no effect on spontaneous or touch-evoked scratching (Fig. 3C).

Fig. 3. Effects of DTX Administration on Skin Symptoms and Itch Responses in Male DTR⁺ Mice

Male DTR⁺ AD mice were administered saline or DTX (50 µg/kg). Normal control mice were administered saline. (A) Gross appearance of saline-(left) and DTX-treated (right) mice on the day after the final HDM application. (B) Skin dryness score and TEWL on the day after final HDM application. (C) Itch-related scratching behavior before (spontaneous) and immediately after the final HDM challenge was analyzed using video recording. On the day after the final HDM application, touch-evoked scratching was assessed using a von Frey filament (bending force, 0.7 mN). Data represent individual values and mean ± S.E. n = 4 (normal + saline), 7 (AD + saline), and 8 (AD + DTX) mice. * p < 0.05, ** p < 0.01, and *** p < 0.001, one-way ANOVA with Dunnett’s multiple comparison test for the data of TEWL and scratching before and after HDM challenge, or Kruskal–Wallis test with Dunn’s multiple comparison test for the data of skin dryness score and touch-evoked scratching.

In contrast, male DTR⁻ AD mice treated with the same dose of DTX showed no suppression of acute scratching immediately after HDM challenge (Fig. 4C), suggesting that the antipruritic effect of DTX in DTR⁺ AD mice cannot be attributed to its toxic effects. Furthermore, these experiments were conducted with female mice, yielding similar results (Supplementary sFigs. 1, 2).

Fig. 4. Effects of DTX Administration on Skin Symptoms and Itch Responses in Male DTR⁻ Mice

Male DTR⁻ AD mice were administered saline or DTX (50 µg/kg). Normal control mice were administered saline. (A) Gross appearance of saline-(left) and DTX-treated (right) mice on the day after final HDM application. (B) Skin dryness score and TEWL on the day after final HDM application. (C) Itch-related scratching behavior before (spontaneous) and immediately after the final HDM challenge was analyzed using video recording. On the day after the final HDM application, touch-evoked scratching was assessed using a von Frey filament (bending force, 0.7 mN). Data represent individual values and mean ± S.E. n = 3 (normal + saline), 12 (AD + saline), and 12 (AD + DTX) mice. * p < 0.05, ** p < 0.01, and *** p < 0.001, one-way ANOVA with Dunnett’s multiple comparison test for the data of TEWL and scratching before and after HDM challenge, or Kruskal–Wallis test with Dunn’s multiple comparison test for the data of skin dryness score and touch-evoked scratching.

DISCUSSION

This study investigated the role of MrgprA3⁺ neurons in itch in our AD model. Consistent with the results of a previous study,¹⁰⁾ DTX administration ablated MrgprA3⁺ sensory neurons (Fig. 2). In this situation, the acute scratching response immediately after the HDM challenge was significantly attenuated (Fig. 3, Supplementary sFig. 1). This suggests that MrgprA3⁺ sensory neurons play a crucial role in mediating acute allergic itch in this AD model. Although the involvement of MrgprA3 itself and MrgprA3⁺ neurons in itch in various acute itch and dermatitis-associated itch has been reported elsewhere, considering the similarities and differences with our results will help to further clarify the complex mechanism of intractable itch in chronic diseases, such as AD.

Reddy and Lerner recently showed that HDM antigens directly activate human receptor MRGPRX1 and its mouse homolog MrgprC11.²³⁾ They used Der p1 from Dermatophagoides pteronyssinus as the HDM antigen, although we used a crude extract of D. farinae. This study also showed that, unlike Der p1, Der f1 (the major antigen of D. farinae) could not activate either MrgprA3 or MrgprC11.²³⁾ Furthermore, our unpublished results using hairless mice showed that 1) intradermal injection of the HDM extract did not induce scratching behavior in normal mice and 2) intradermal injection of the supernatant from which skin cells were isolated from mice suffering from AD and treated in vitro with the HDM extract caused scratching behavior (M. Fujii, unpublished observations). The present study revealed that ablation of MrgprA3⁺ neurons significantly suppressed antigen-induced acute scratching behavior in sensitized mice following repeated application of HDM extract. Therefore, based on these findings, we speculate that the antigens in the HDM extract do not directly stimulate sensory neurons, but itch is caused mainly by pruritogen(s) released from the skin cells of sensitized mice. However, in sensitized mice with AD, the direct action of HDM extract to induce itch cannot be completely ruled out. Therefore, it is necessary to investigate the neural responsiveness of sensitized mice using DRG cells in the future.

The specific mediators that trigger acute allergic itch through MrgprA3⁺ neurons remain unclear. In another study involving hairless mice induced with SD and HDM, several observations were made: 1) an acute scratching response did not occur, except for HDM, but only during the final application; 2) scratching peaked 10 min after the HDM challenge; and 3) the antihistamine olopatadine did not suppress this response (M. Fujii, unpublished observations). These findings suggest that there may be mediator(s) other than histamine that are acutely produced and released upon antigen challenge, which could be responsible for this type of itch. Serotonin is a pruritogen produced by mast cells upon antigen challenge. However, serotonin receptors (Htr1f and Htr2a) have been reported to be expressed mainly in a subpopulation of non-peptidergic nociceptors that do not express MrgprA3.²⁴⁾

Chloroquine has been found to directly activate MrgprA3.⁹⁾ However, the endogenous agonists of MrgprA3 remain unknown. Previous studies have shown that MrgprA3⁺ neurons express various pruriceptors.^9,10,14,24) Therefore, it remains unclear whether the mediator(s) responsible for acute allergic itch directly activate MrgprA3 or indirectly activate MrgprA3⁺ neurons via other pruriceptors. Interestingly, a recent study showed that GPR15L released from inflamed keratinocytes in pruritic diseases such as psoriasis and AD can directly activate MrgprA3 in sensory neurons and induce itch in mice.²⁵⁾ Further studies using Mrgpr knockout mice are required to clarify the mechanism underlying acute allergic itch in our AD model.

More recently, Yang et al. reported that dictamnine, a component of traditional Chinese medicine, suppressed itch behavior and skin inflammation in a mouse model of dinitrofluorobenzene (DNFB)-induced AD by directly blocking MrgprA3.¹⁶⁾ This study revealed the involvement of MrgprA3 activation in DNFB-induced AD. However, in this study, it was not determined when the drug and DNFB treatments were performed to evaluate scratching behavior; therefore, it is unclear whether the scratching behavior was spontaneous or acute immediately after antigen challenge. Since MrgprA3⁺ neurons express other pruriceptors, their loss would have a broader impact on itch than blockade of MrgprA3 activation. Nevertheless, our present results showed that in the HDM-induced AD model, ablation of MrgprA3⁺ neurons suppressed only acute allergic itch, but not spontaneous itch or alloknesis. In addition, neither skin barrier dysfunction nor dermatitis symptoms were improved (Fig. 3, Supplementary sFig. 1). This discrepancy may be due to the differences in the pathogenic mechanisms of each AD model. In other words, the mechanism of itch in our AD model is multifactorial and distinct mechanisms that are dependent and independent of MrgprA3⁺ neurons may coexist.

The ablation of MrgprA3⁺ neurons did not affect spontaneous or touch-evoked scratching (Fig. 3, Supplementary sFig. 1). These findings suggest that the neurons responsible for spontaneous and touch-evoked itches in our AD model are distinct from those of MrgprA3⁺ neurons. Our results are partly consistent with reports showing that touch-evoked itch is mediated by low-threshold mechanoreceptive afferents that either innervate Merkel cells or express Toll-like receptor 5.^26,27) In contrast, a recent study showed that MrgprA3⁺ sensory neurons do play a role in mediating pruritogen-induced alloknesis.²⁸⁾ As ongoing (spontaneous) itch and alloknesis (mechanical or touch-evoked itch) are central to chronic itch,²⁹⁾ further studies are needed to fully elucidate the mechanisms underlying the different itch responses in our model.

In conclusion, the present study has shed light on the role of MrgprA3⁺ sensory neurons in mediating acute allergic itch in our AD model. However, it is important to note that these neurons did not appear to be involved in spontaneous itch or alloknesis in our model. Similar to our mouse model, the mechanisms underlying itch in human AD may be complex and multifactorial. Further elucidation of the mechanisms underlying itch in chronic disease conditions may lead to the development of new therapeutic drugs for severe intractable itch.

Acknowledgments

We thank Yuma Yasui, Masahiro Adachi, Takako Kamiya, Seita Nobuta, and Kazunori Yoshikawa, Takato Otsuka, and Taisei Enomoto (Kyoto Pharmaceutical University) for experimental support. This work was supported by JSPS KAKENHI (JP16K09000 and 19K07333 to M.F.), Kyoto Pharmaceutical University Fund for the Promotion of Scientific Research Grant (18J02 to M.F.), and Shimizu Foundation for Immunology and Neuroscience Grant for 2017 (to M.F.).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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