Mast cell-5-HT-HTR2A axis involvement in chronic itch induced by SADBE

Abstract

Although 5-hydroxytryptamine (5-HT) contributes to pruritus associated with allergic contact dermatitis (ACD), the role of 5-HT derived from mast cells (MC) in chronic pruritus induced by squaric acid dibutyl ester (SADBE), and the expression and distribution of 5-HT2A receptor (HTR2A) in sensory neurons remain unclear. In this study, a SADBE-induced ACD mouse model was established to evaluate pruritus behavior, MC activation, and 5-HTproduction. The mechanism was verified through pharmacological intervention (MC stabilizer cromolyn, HTR2A antagonist Ketanserin) and FcεRIα-KO mice. It was found that SADBE triggered time-dependent MC recruitment (peaking at Day 14–21) and Mc-derived 5-HT release, which were associated with persistent pruritus. The intervention of MC stabilizer cromolyn and FcεRIα-KO mice confirmed MC/IgE-dependent 5-HT release, and inhibiting MC degranulation could reduce pruritus. Single-cell RNA sequencing and RNAscope in situ hybridization techniques revealed that HTR2A was mainly expressed in the NF3/PEP2/NP3 subsets of DRG neurons. The co-expression level of HTR2A and Nppb was relatively high, partially overlapping with TRPV1/TRPA1. HTR2A antagonists can relieve SADBE-induced pruritus. In conclusion, we have determined that the MC-5-HT-HTR2A axis is involved in chronic pruritus in SADBE-induced ACD, and targeting this axis provides a very promising therapeutic strategy.

Introduction

Allergic contact dermatitis (ACD), a prevalent inflammatory skin disease, is characterized by chronic pruritus that severely impairs patients’ quality of life. Several studies have shown that 5-HT levels are increased in areas of skin lesions in atopic dermatitis, with significant differences compared to normal skin,^1,2 suggesting that 5-HT is involved in the pathogenesis of atopic dermatitis. While serotonin (5-hydroxytryptamine, 5-HT) has been recognized as a key mediator of itch signaling, its cellular origins and receptor-specific pathways in chronic itch remain incompletely defined. ³

Platelet-derived 5-HT has been implicated in acute itch responses. ⁴ Although the importance of the platelet-derived 5-HT system has been emphasized in the acetone-ether-water (AEW)-induced chronic itch model of xeroderma and the imiquimod (IMQ)-induced to psoriasis model ⁵ ; however, emerging evidence suggests that mast cells, critical effector cells in allergic inflammation, may serve as an alternative source of 5-HT in chronic pruritic conditions. ⁶ Notably, squaric acid dibutyl ester (SADBE), a hapten widely used to model ACD, induces persistent itch in mice, similar to the symptoms of ACD in humans ⁷ ; yet the role of mast cell-derived 5-HT in this process is unknown.

5-HT receptors (5-hydroxytryptamine receptors) are a group of G protein-coupled receptors (GPCRs) and ligand-gated ion channels (LGICs) found in the central and peripheral nervous system, which can be classified into seven subtypes. ⁸ The 5-HT2A receptor (HTR2A), a G protein-coupled receptor, is a potential therapeutic target for pruritus due to its involvement in neuronal sensitization. ⁴ Despite this, the precise expression profile of HTR2A in sensory neurons – particularly within the dorsal root ganglion (DRG) – and its functional interplay with pruriceptive neuropeptides (e.g. Nppb) or ion channels (e.g. TRPV1/TRPA1) remain elusive. Resolving these questions is critical for understanding how 5-HTergic signaling drives chronic itch in ACD and for developing receptor-specific antipruritic therapies.

Here, we hypothesize that mast cell-derived 5-HT activates HTR2A-expressing sensory neurons via specific neuronal subpopulations, thereby perpetuating SADBE-induced chronic itch. This study aims to (1) delineate the contribution of mast cell-derived 5-HT to itch in SADBE-sensitized mice, (2) map the spatial and functional expression of HTR2A in DRG neurons, and evaluate the therapeutic potential of blocking the 5-HT/HTR2A axis.

Materials and methods

Animals

Six-week-old specific pathogen-free (SPF) male C57BL/6 mice were purchased from Zhuhai Biotest Company (Zhuhai, China). C57-FcεRIα knockout (KO) mice (6–8 weeks) were purchased from Gem Pharmatech Co, Ltd (Jiangsu, China) and provided by Dr. Shaoheng He from Jinzhou Medical University. All animals were fed with SPF-grade feed and distilled water and housed in an SPF-grade facility with a normal light-dark cycle at 24°C before the experiment. All animal experiments were conducted in accordance with the protocol approved by the Animal Research Committee of the Second Affiliated Hospital of Guangzhou Medical University (Approval No: A2020-026).

Contact allergic dermatitis (ACD) itch model

According to the method described in the literature, ⁹ a contact allergic dermatitis (ACD) model was established using SADBE (Cat. 339792, Sigma). One day before modeling, the hair on the neck and back of the mice was shaved to expose the skin. On the first day of the experiment, 25 μl of 1% SADBE in acetone was applied to the abdominal skin (2 × 3 cm) of the mice once a day for three consecutive days for sensitization. Five days later, 25 μl of 1% SADBE in acetone was applied to the neck and back of the ACD mice once a day for three consecutive days for local stimulation.

Mouse scratching behavior

The scratching behavior of the mice was observed to determine the itch phenotype. After modeling, the mice in the model group and the control group were placed in individual transparent observation cages, one mouse per cage, and video recorded for 1 h in a quiet environment. A scratching behavior was defined as the mouse lifting its hind leg to scratch the neck and back and then lowering it. The videos of the mice were observed and the number of scratching behaviors was counted and statistically analyzed. All behavioral tests were conducted by experimenters who were unaware of the experimental conditions.

Drug Administration: To block 5-HT-HTR2A signaling, Ketanserin (1 mg/kg; Cat. 74050-98-9, MedChenExpress) was intraperitoneally administered to the mice 30 min before each video recording. To reduce mast cell degranulation and 5-HT release, Cromolyn sodium (60 mg/kg; Cat. 1150502, Merck) was intraperitoneally administered to the mice 30 min before each video recording.

For the 5-HT injection experiment, 5-HT (Cat. H9523, Sigma) was dissolved in 0.9% physiological saline. 5-HT was injected into the neck and back (1.5 μg/μl, 50 μl). Mice in the control group received an equal volume of physiological saline injection.

Skin histopathology

At different time points after modeling, mice were intraperitoneally injected with 1% pentobarbital normal saline solution for anesthesia. The skin on the neck and back of the model mice and the control group mice was placed in a 4% paraformaldehyde solution and fixed at 4℃ overnight.

The skin samples were dehydrated with alcohol, removed with xylene, and then embedded in paraffin for sectioning. According to the manufacturer’s instructions, the sections were stained with H&E and toluidine blue, sealed, and then read using the fully automatic digital section scanning system PRECICE500B (Beijing Una Technology Co., LTD., China). H&E staining was used to observe the thickening of the mice epidermis and the infiltration of inflammatory cells. The skin thickness and the number of inflammatory cells were statistically analyzed using Image J software. Observe and count the number of intact (IMC) and degranulated (DMC) MCS in TB staining. The formula for the threshing rate of MC is MCD = DMCs/(IMCs + DMCs) × 100%.^10–12

Immunofluorescence staining

The paraffin sections were dewaxed and subjected to antigen retrieval. After washing with PBST, the sections were blocked with 3% goat serum in PBST for 30 min. The primary antibody was added and incubated overnight at 4°C in a wet box. The next day, after washing with PBST, the sections were incubated with fluorescently labeled secondary antibody at room temperature in the dark for 90 min. After PBST washing, the cell nuclei were stained with DAPI (1:2000, Invitrogen), followed by washing and air-drying before sealing the slides. The samples were then photographed and recorded using a confocal microscope (Zeiss LSM880). Mast cells were labeled with FITC-conjugated avidin (1:1000, Cat. 434411; Thermo Fisher Scientific). 5-HT (Serotonin) Rabbit Antibody (1:1000; Cat. 20080, ImmunoStar), and goat anti-rabbit Alexa 568 conjugated (1:200, Cat. A11011, Thermo Fisher) were used for labeling 5-HT.

RNAscope in situ hybridization (RNAscope ISH)

The bilateral C1-T1 DRG of mice were removed and fixed in 4% paraformaldehyde at 4°C overnight. After dehydration in sucrose solution, they were embedded in cryostat tissue matrix (OCT) and sectioned. RNAscope ISH assay was performed on DRG tissues as previously described. ¹³ The following probes consisting of 20 ZZ oligonucleotides and obtained from Advanced Cell Diagnostics were used:Nppb (Cat No. 425021), MrgprA3(Cat No.502041), Trpv1 (Cat No.313331), TrpA1 (Cat No.400211-C2).

ND7/23 cell culture and calcium imaging

ND7/23 cells were obtained from the Stem Cell Research Center of the Chinese Academy of Sciences and were cultured in a 37°C, 5% CO2 incubator using DMEM medium (Gibco) supplemented with 10% FBS. For intracellular calcium ion concentration measurement, the cultured ND7/23 cells were placed in a 37°C incubator and treated with 5 μM Fura-2 (Cat. F1221, Invitrogen) for 30 min. The extracellular dye was washed away with calcium perfusion solution, and the calcium ion concentration was determined at room temperature using the F340/F380 ratio with a calcium imaging analysis system DMi8 (Leica, Switzerland). 5-HT (100 μM), calcium imaging buffer (1 l; g: 8.25 NaCl, 0.35 KCl, 0.1998 CaCl2, 0.114 MgCl2, 2.38 Hepes, and 1.8 glucose, pH 7.4).

DRG neuron cell culture and calcium imaging

The DRG of 2–3 weeks-old mice was collected in pre-cooled Neurobasal medium (Gibco). After removing the supernatant, 2 ml of enzymatic hydrolysate (papain (Worthington) dissolved in Neurobasal medium (45.6 mgP/ml)) was added and incubated at 37°C for 20 min. Wash three times with Neurobasal medium and centrifuge to remove the supernatant. Then add 2 ml of enzymatic hydrolysate (3 mg of type 2 collagenase (Roche) dissolved in Neurobasal medium), and incubate at 37°C for 20 min. Shake gently every 5 min during this period. After washing three times with Neurobasal medium and centrifuging to remove the supernatant, 0.2 ml of Neurobasal complete medium (10 ml formula: Mix 9.3 ml of Neurobasal medium, 0.2 ml of B27 (Gibco), 0.2 ml of horse serum (Gibco), 0.2 ml of FBS (Gibco), and 0.1 ml of GlutaMAX (Gibco) by pipetting). Inoculate the cell suspension onto a coverslip coated with polylysine (Sigma). About 40 min later, Neurobasal complete medium was added for culture. The DRG calcium imaging operation is the same as that of ND7/23 calcium imaging.

RNA extraction and qPCR analysis of ND7/23 cells

The cultured ND7/23 cells were placed in an incubator at 37℃ and stimulated with 5-HT (100 μM) for 1, 3, 6, and 24 h, respectively. RNA was extracted from the cells using TRIzol (Cat. 15596018, Invitrogen), and cDNA was prepared using PrimeScript RT Premix (Cat#RR036A, Takara, Kusatsu, Japan). All these operations were carried out according to the manufacturer’s instructions. qPCR was performed using SYBR Premix Ex Taq enzyme (Takara, Kusatsu, Japan), with three replicates for each sample.

FOS primer sequences:

qF: CCCGAGCTGGTGCATTACA;

qR: GAGGCCAGATGTGGATGCTT.

Statistical analysis

One-way ANOVA test followed by Dunnett’s multiple comparisons test was used to evaluate the behavior, number of inflammatory cells, number of mast cells, epidermal thickness and number of 5-HT⁺ cells of SADBE mice at different time points. The comparison between the two groups was conducted using the unpaired two-tailed student t-test. p value ≤ 0.05 is considered statistically significant. ns: p > 0.05; *p ≤ 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Result

SADBE modeling of ACD in mice

To assess chronic itch, we topically applied 1% SADBE to the nape of mice for three consecutive days and quantified scratching behavior. The number of hind-leg scratching bouts directed at the application site within 60 min was recorded. SADBE-treated mice began scratching on day 3, with the behavior peaking on day 7. This pruritic response remained significantly elevated until day 13 and did not return to baseline until day 28 (Figure 1(b)). Histopathological analysis of the skin revealed characteristic inflammatory changes, including hyperkeratosis, epidermal hyperplasia with downward elongation of rete ridges, and significant dermal inflammatory cell infiltration (Figure 1(c)). Measurements of epidermal thickness and inflammatory cell infiltration confirmed that these pathological features increased over time, peaked around day 14, and were sustained for up to 3 weeks (Figure 1(d) and (e)).

Figure 1.

SADBE constructed mouse ACD model. (a) Flow chart of SADBE constructed mouse ACD model. (b) Quantification of scratching behavior during 60 min on different days after modeling. (c) Representative H&E-stained dorsal skin sections (scale bar = 50 μm). (d) Epidermal thickness analysis of dorsal skin sections. (e) Quantification of counts of inflammatory cell infiltration in dorsal skin sections. Values are shown as x ± s. One-way·ANOVA test in (b), (d), and·(e).

Blocking mast cell degranulation inhibits itching

Examination of mast cell populations in the skin revealed a time-dependent change following SADBE application. While the number of mast cells was unaltered during the first week, a significant increase was observed on days 14 and 21 (Figure 2(a) and (b)). The functional importance of mast cells was confirmed using the stabilizer Cromolyn. In SADBE-modeled mice, Cromolyn significantly reduced 5-HT⁺ cells in the skin of mice (Figure 2(d) and (e)) and inhibited mast cell degranulation (Figure 2(f) and (g)). Mouse scratch behavior counts showed that Cromolyn reduced scratch behavior by 39% (Figure 2(h)).

Figure 2.

Mast cell expression is increased in the late stage, and blocking MC degranulation can inhibit pruritus. (a) Staining results of skin mast cells in SADBE model mice, scales = 50 μm. (b) Mast cell count results of SADBE model mice. (c) Schematic diagram of SADBE model mice injected with Morley. (d) Immunofluorescence staining results of 5-HT⁺ cells in the skin of SADBE mice, scale = 50 μm. (e) Counting results of 5-HT⁺ cells in mouse skin. (f) The staining results of toluidine blue on the skin of SADBE mice, with scales = 50 μm. Thick arrows represent intact mast cells, while thin arrows represent degranulated mast cells. (g) Results of skin cell counting in the ACD model group of mice constructed by SADBE. (h) Count of scratch behavior in SADBE mice that inhibit mast cell degranulation. Values are shown as x ± s. One-way·ANOVA test in (b). Unpaired t test in (e), (g), and (h).

Blocking the degranulation mediated by FcεRI of mast cells can inhibit pruritus

To further explore the effect of 5-HT released by mast cell degranulation, we used FcεRIα-KO mice for SADBE modeling. FcεRI is the core receptor that mediates immediate allergic reactions in mast cells. ¹⁴ It triggers degranulation and the release of inflammatory mediators by recognizing the IgE-antigen complex. Knockout of this receptor can block IgE-dependent degranulation. It was found that after knocking out FcεRI, the scratching behavior of the mice was significantly reduced. ¹⁵ TB staining revealed that the degranulation rate of mast cells in knockout mice was also significantly reduced.

Building on the pharmacological evidence with Cromolyn, the genetic ablation of FcεRIα provides definitive genetic evidence that IgE-dependent mast cell degranulation is a key driver of chronic itch in our model. The concomitant reduction in both degranulation and scratching behavior in knockout mice strongly suggests that the pruritogens released through this pathway – which include, as we subsequently investigated, 5-HT – are essential for sustaining the itch-scratch cycle (Figure 3).

Figure 3.

Blocking the degranulation mediated by FcεRI of mast cells can inhibit pruritus caused by SADBE. (a) Schematic diagram of SADBE modeling for WT mice and FcεRIα-KO mice. (b) SADBE scratch behavior counts of WT mice and FcεRIα-KO mice. (c) Staining results of mouse skin mast cells, scale = 100 μm. (d) Degranulation rate of mast cells. Values are shown as x ± s. Unpaired t test in (b) and (d).

Increased amounts of mast cell-derived 5-HT in the skin

We next assessed the dynamics of 5-HT in the skin during SADBE-induced itch. Immunofluorescence analysis revealed that 5-HT levels were significantly elevated on days 7, 14, and 21 compared to baseline (Figure 4(a) and (b)). Given that peripheral 5-HT is primarily derived from mast cells, we therefore investigated their association. Co-staining analysis confirmed a significant co-localization of 5-HT with mast cells in the skin at the peak phase (day 14), suggesting that mast cells are a major source of 5-HT in this model and implicating mast cell-derived 5-HT in SADBE-induced pruritus (Figure 4(c) and (d)).

Figure 4.

Immunofluorescence analysis of mast cells with 5-HT in the skin of mice in the ACD model group constructed by SADBE. (a) Immunofluorescence staining results of 5-HT in the skin of mice in the ACD model group constructed by SADBE, scale bar = 50 μm. (b) Results of 5-HT⁺ cell counting in the skin of mice in the ACD model group constructed by SADBE. (c) Immunofluorescence coexpression staining of 5-HT and mast cells in dorsal skin sections. (d) Quantitative results of the expression of 5-HT with mast cells in dorsal skin sections. Values are shown as x ± s. One-way·ANOVA test in (b).

Involvement of the 5HT-HTR2A axis in SADBE-induced itch

We investigated whether 5-HT directly triggers itch and its underlying mechanism. Intradermal 5-HT injection indeed provoked significant scratching behavior (Figure 5(a) and (b)). In addition, after treating ND7/23 neuron cells with 5-HT, we could induce the expression of FOS in them (Figure 5(c)). Then, through calcium imaging experiments, we found that 5-HT could cause calcium influx in ND7/23 neuron cells and primary DRG neurons in mice, indicating direct activation of neurons (Figure 5(d) and (e)). To identify the receptor mediating this effect, we targeted HTR2A, a receptor previously implicated in itch. ⁴ Pharmacological blockade of HTR2A with Ketanserin effectively alleviated scratching in chronic itch mice by 33% (Figure 5(f) and (g)), supporting a role for the HTR2A pathway in 5-HT-mediated pruritus.

Figure 5.

5-HT can activate neurons and cause itching. (a) Schematic diagram of intradermal injection of 5-HT in mice. (b) Scratching behavior of mice after intradermal injection of 5-HT. (c) FOS expression level after 5-HT stimulation of ND7/23 cells. (d) Calcium ion level after 5-HT (100 μM) stimulation of ND7/23 cells and DRG neuron cells, scale = 50 μm. (e) Quantification of the peak calcium response (Δ340/380) in ND7/23 cells and primary DRG neurons upon 5-HT stimulation. (f) Diagram of SADBE model mice injected with Ketanserin. (g) Scratching behavior count in SADBE mice after inhibition of HTR2A. Values are shown as x ± s. One-way·ANOVA test in (c). Unpaired t test in (b), and (g).

Htr2a is expressed on pruritus related neurons in DRG

The stinging receptor is a receptor in the human body used to sense skin itching. The cytoplasm of these receptors is mainly distributed in the DRG and TG regions. The nerve endings of pruritus receptors are widely distributed in the skin. Through their connection with the nervous system, they enable us to sense the stimulation of itching. We used previously published single-cell sequencing data to study the expression profile of HTR2A in sensory neurons, thereby gaining a deeper understanding of its mechanism of action in pruritus symptoms.

Previous transcriptomic analyses have revealed different subgroups of DRG neurons. Based on different molecular markers, neurons can be classified into two peptideable neuron clusters (PEP1-2), three non-peptideable neuron clusters (NP1-3), five myelinated neuron clusters (NF1-5), and one tyrosine hydroxylase-positive neuron cluster (TH) ¹⁶ (Figure 6(a)).

Figure 6.

Htr2a is expressed on pruritus related neurons in DRG. (a) Classification and proportion of neurons of each subtype in DRG. (b) Expression of transcripts related to pruritus was aggregated into functional subsets based on single-cell RNA-seq data. Complete datasets and methods can be obtained in previous studies. ¹⁶

NP subsets may correspond to different pruritus receptor subtypes and functions, known as NP1, NP2, and NP3 neurons.^16,17 NP1 neurons express Mrgprd and Trpa1, which react to β -alanine and allyl isothiocyanate respectively to produce pruritus^17,18; NP2 neurons express Mrgpra3, ¹⁹ mediating non-histamine-dependent pruritus; NP3 neurons are a group of neurons that highly express the neuropeptides Nppb and Sst, both of which are related to the neurotransmission function of pruritus information.^19–22 Htr2a is mainly expressed in the NF3, NP3, and PEP2 subsets of sensory neurons (Figure 6(b)).

The expression characteristics of HTR2A in DRG neurons were analyzed by RNASCOPE

Mrgpra3 and Nppb are genes highly associated with pruritus, and Trpa1 and Trpv1 are two ion channels associated with pruritus. In order to confirm the existence of Htr2a transcripts in mouse DRG neurons, we further analyzed the co-expression of Htr2a and appellate genes by ISH.

The transient receptor potential (TRP) channel Trpv1 has been shown to be involved in histamine-dependent pruritus. ²³ RNAscope ISH analysis confirmed that 43.5% of Htr2a⁺ neurons were Trpv1⁺, while 55.8% of Trpv1⁺ neurons were Htr2a⁺ (Figure 7(a)). Trpa1 is considered to be an ion channel required for histamine-independent pruritus, ²⁴ and RNAscope ISH analysis confirmed that most Htr2a⁺ neurons (73.2%) were Trpa1⁺, while 70.5% of Trpa1⁺ neurons were Htr2a⁺ (Figure 7(b)). We found a remarkably high degree of co-localization between Htr2a and Nppb, the vast majority of Nppb+ neurons (89.8%) expressed Htr2a, while a substantial proportion of Htr2a+ neurons (41.2%) were also positive for Nppb (Figure 7(d)). The results of single cell sequencing showed that Mrgpra3 mainly expressed two subgroups of NP2, while Htr2a was not expressed in NP2. RNAscope ISH analysis confirmed that Mrgpra3 had little overlap with Htr2a, where only 4.45% of Htr2a neurons were Mrgpra3⁺ neurons (Figure 7(c)).

Figure 7.

RNAscope ISH analysis of Htr2a expression characteristics in DRG. (a–d) Triple/double labeling ISH of DRG frozen sections was performed using RNAscope ISH, Scale = 50 μm. (a) Htr2a, red; Trpv1, green; DAPI, blue. (b) Htr2a, red; TRPA1, green; DAPI, blue. (c) Htr2a, red; Mrgpra3, green; DAPI, blue. (d) Htr2a, red; Nppb, green; DAPI, blue. Similar results were obtained from at least three animals.

Discussion

Chronic itch is a common symptom of skin diseases characterized by a long lasting itchy sensation on the skin, which seriously affects the quality of life of patients. Despite its widespread clinical occurrence, the pathogenesis of chronic itch remains incompletely understood. The SADBE mouse model is an animal model used to study cutaneous immune diseases. ²⁵ By administering SADBE to the dorsal skin of mice, a T-cell-mediated immune response can be induced that mimics the immune features of human skin allergic diseases. This model has the advantages of high reproducibility and simplicity, and is widely used to study allergic diseases such as contact dermatitis. ⁷

Mast cells play an important role in the pathogenesis of chronic itch. Mast cells are a class of immune cells distributed in human skin, mucous membranes and other tissues, and are important participants in antigen-specific and non-specific immunity of the organism. In chronic itch, the number and activation state of mast cells are significantly increased. ²⁶ It has been found that patients with chronic itch have an increased number of mast cells in their skin tissues, accompanied by an infiltration of inflammatory cells in the tissues. ²⁷ Mast cell activation releases a variety of inflammatory mediators, such as histamine,^28,29 5-hydroxytryptamine, ⁶ and IL-31, ³⁰ which play an important role in the pathogenesis of chronic itch. In addition, the degree of activation of 5-hydroxytryptamine and mast cells is positively correlated with the severity of itch. ³¹ Our data also showed that SADBE modeled mice developed itchy symptoms that were maintained for a longer period of time, and that the level of 5-HT expression was not high in the early days, and it took until the 14th day to reach the peak, and then it remained at this high level until the 21st day. This suggests that 5-HT may play a minor role in the spontaneous itching that occurs in the initial stages of ACD. Our data found that in the SADBE model, the number of mast cells was not high in the early stages and needed to increase until the second week, and then the number of mast cells reached a peak by day 21. At day 14, there was a significant co-expression of mast cells with 5-HT, with 149 of the 164 mast cells counted expressing 5-HT and 84.6% of the 176 5-HT positive cells counted were mast cells. This indicates that 5-HT expression increased significantly in the later stages (day 14–21) of the ACD model constructed by SADBE, and most of the 5-HT was of mast cell origin, suggesting that mast cell-derived 5-HT may play an important role in maintaining prolonged pruritus. Although the mast cell-derived 5-HT had not reached its peak at the time of peak itchiness in mice, the increase and maintenance of the amount of mast cell-derived 5-HT may be the reason why the itchy symptom has been sustained in SADBE mice. Inflammatory mediators released by mast cells may also induce abnormal sensitization of peripheral nerve endings, leading to over-transmission of itch. ²⁶

5-HT in the skin is currently thought to originate mainly from mast cells and platelets.^32,33 A study by Luo et al. ⁵ reported that platelet-derived 5-HT plays an important role in itch in the AEW or SADBE models, whereas mast cell-derived 5-HT did not contribute to these two models of chronic itch. In the present study, our data revealed that mast cell-derived 5-HT was not expressed in the early stage, but the expression increased significantly after day 7, and mast cell-derived 5-HT was present for a long period of time, which may play an important role in maintaining prolonged itching. As for sodium cromoglycate, it is an organic compound mainly used as an anti-allergic drug its mechanism of action is that it can stabilize the cell membrane of mast cells and prevent mast cell degranulation, thus inhibiting the release of allergic reaction mediators such as histamine, 5-hydroxytryptophan, and slow-reacting substances and thus blocking the adverse effects of allergic reaction mediators on tissues. We were able to effectively alleviate itching in the SADBE mouse model after using ammonium tryptophan to inhibit the release of 5-HT from mast cells. Therefore, we believe that mast cell-derived 5-HT is involved in itching in the SADBE model, and that inhibitory therapeutic strategies targeting mast cells may provide new ideas for the treatment of chronic itch. The reason for the different conclusions is that Our findings on the critical role of mast cell (MC)-derived 5-HT in the late phase of SADBE-induced chronic itch both complement and extend previous work in the field. Notably, Luo et al. ⁵ reported that platelet-derived 5-HT, rather than MC-derived 5-HT, played a dominant role in chronic itch models including SADBE and AEW. Our data do not necessarily contradict these findings, but rather offer a critical temporal refinement to the model. We demonstrate that while MC infiltration and MC-derived 5-HT release are indeed minimal in the early stages (Days 0–7), they become significant and sustained in the mid-to-late phases (peaking at D14-21) of the SADBE response. This suggests a model where platelet-derived 5-HT may drive the initial acute itch phase, consistent with Luo et al., whereas MC-derived 5-HT is pivotal for perpetuating the chronic phase. This temporal distinction, previously overlooked, could explain the apparent discrepancy and highlight the complexity of pruritogenic mechanisms over time. Our use of FcεRIα-KO mice and cromolyn specifically targeting MC degranulation provides direct functional evidence for this MC-dependent late-phase mechanism. Furthermore, our study significantly advances the understanding of the neuronal side of this axis by precisely mapping HTR2A expression to specific pruriceptive DRG neuron subsets (NP3, PEP2, NF3) and revealing its striking co-localization with Nppb – a key mediator of mast cell-induced itch. ²² This positions HTR2A not as a general itch receptor, but as a specific nexus for neuro-immune crosstalk in chronic ACD, offering a more precise therapeutic target for interrupting persistent itch signaling.

5-HT acts as a neurotransmitter involved in a variety of physiological and behavioral modulations. In the skin and central nervous system, there are multiple 5-HT receptors involved in the perception and modulation of itch. 5-HT can modulate the excitability of sensory nerve endings and influence the pathway of itch signaling through binding to receptors such as HTR1, HTR2, and HTR3. Experimental studies have shown that injection of 5-HT or 5-HT receptor agonists may induce pruritic behavior in mice.^34,35 This phenomenon was confirmed by our results, in which mice spontaneously developed itchy behavior after intradermal injection of 5-HT, suggesting that itch in the mouse model with SADBE may be related to the release of 5-HT. And a similar phenomenon can be observed in humans, where injection of 5-HT can cause itching in normal skin. ³⁶ Clinically, 5-hydroxytryptamine may be increased in a variety of itch-related disorders, thereby affecting the perception and development of itch. A variety of 5-hydroxytryptamine reuptake inhibitors and receptor antagonists have been used to alleviate itch caused by certain disorders, including cholestatic itch,^37,38 various dermatologic conditions such as itchy rash simplex, steatotic eczema, and psoriasis, ³⁹ pruritus associated with true erythrocytosis,^40,41 and itch caused by certain cancers. ⁴²

HTR2A is a subtype of the 5-HT receptor family, which is widely distributed in the central nervous system and peripheral tissues and plays an important role in a variety of physiological and behavioral processes.HTR2A also plays a role in the regulation of itch, and when the skin or mucous membranes are stimulated, 5-HT can increase the excitability of the sensory nerve endings by binding to HTR2A, which in turn enhances itch signaling that triggers the sensation of itching. ⁴ The use of HTR2A receptor antagonists has been shown to be effective in inhibiting the 5-HT-induced itch response, ⁴³ and we showed a significant reduction in itchy behavior in SADBE mice using an HTR2A inhibitor in a SADBE mouse model, Akiyama et al. ³⁵ also observed this phenomenon in a mouse model of chronic dry pruritus.

We found that HTR2A was expressed in a variety of neurons in the DRG, and from the results of single-cell sequencing, we found that HTR2A was mainly expressed in the neurons of NF3, PEP2, and NP3. NP3 is a group of neurons that specifically expresses Nppb, sst, and Nts, and it has been found that Nppb-positive neurons are the sensors for mast cell-induced itch. ³⁰ Most NPPB-expressing neurons co-express HTR2A in human neurons; however, HTR2A expression is not restricted to NPPB neurons. When HTR2A is activated in neurons of the DRG, it may affect the processing and perception of itch signals. The processing of itch signals by the nervous system may be modulated, resulting in the perceived itch sensation becoming more intense. The results of a study by Akiyama et al. ³⁵ found that DRG cells from dry skinned mice could be activated by 5-HT. This may regulate the release of neuropeptides through the activation of HTR2A, leading to itch signaling, but relevant experiments are still needed to demonstrate whether it is possible to activate neurons by binding to HTR2A, prompting the release of neuropeptides leading to itch.

Although this study focuses on the mast cell-5-hydroxytryptame-HTR2A axis that plays a dominant role in SADBE-induced chronic pruritus, other neuropeptide signaling pathways may play a key initiating role in the early stages of model establishment. In the early stage, sensory neurons directly irritated by allergens may have played a pioneering role. Among them, B-type natriuretic peptide (BNP, encoded by the Nppb gene) and gastrin-releasing peptide (GRP), two neuropeptides closely related to “prury-specific” conduction, may be the key mediators for initiating the initial prury-specific signal. Authoritative studies have confirmed that the GRP-GRPR signaling pathway at the spinal cord level is the core downstream pathway for conducting histamine-independent pruritus^44,45; The primary sensory neurons expressing BNP (Nppb) are widely regarded as the “initiation neurons” that transmit signals from the periphery to the spinal cord in various acute and chronic pruritus models.^20,46 Our published data confirm that the transcriptional levels of GRP and NPPB in the DRG of the SADBE model are upregulated in the early stage of the disease (Day 3). After blocking the GRP-GRPR or BNP NPRA pathway, the scratching behavior of SADBE mice is reduced.^7,9 This strongly suggests that the activation of GRP and NPPB signaling pathways is a component of the early pruritus response. SADBE, as a strong sensitizer, directly activates specific sensory nerve endings in the skin in the early stage, promoting the priority activation of GRP⁺ neurons and Nppb⁺ neuron subsets and the release of GRP and BNP respectively, transmitting the itching signal to the spinal cord. This further activates the itch-specific conduction circuits within the spinal cord, including GRPR⁺ neurons and NPRA⁺ neurons.

Despite the compelling evidence presented herein, our study has several limitations that warrant consideration. First, we have delineated a crucial peripheral mechanism, the potential central (spinal and supraspinal) contributions of 5-HT and HTR2A signaling to the chronic itch cycle in this model were not examined. It is plausible that central sensitization mechanisms also play a role in sustaining the chronic itch state. Second, the use of the ND7/23 cell line, while valuable for in vitro proof-of-concept, may not fully recapitulate the complexity of mature sensory neurons in vivo. Finally, although we demonstrate a strong correlation and functional blockade, further research employing neuron-specific Htr2a knockout models is needed to unequivocally confirm that the anti-pruritic effect of ketanserin is mediated solely through its action on sensory neurons. Addressing these limitations in future work will be essential for a more comprehensive understanding of chronic itch pathogenesis and for validating the full therapeutic potential of targeting the MC-5-HT-HTR2A axis.

Conclusion

Our findings redefine mast cell-5-HT as a sustained pruritogen in ACD and position HTR2A as a nexus for neuroimmune crosstalk. By elucidating receptor localization and downstream pathways, this study paves the way for precision therapies targeting the 5-HT/HTR2A axis in chronic itch.