Dissecting itch in atopic dermatitis through single-cell RNA sequencing of mouse dorsal root ganglia

Dissecting itch in atopic dermatitis through single-cell RNA sequencing of mouse dorsal root ganglia | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 30 July 2025 V1 Latest version Share on Dissecting itch in atopic dermatitis through single-cell RNA sequencing of mouse dorsal root ganglia Authors : Yun Kyung Jang 0000-0001-5731-7576 , Ji Hwan Moon , Jae-Sang Ryu , Seung Hwa Baek , Young Su Jang 0000-0003-0110-7574 , Dong Hyun Kim , Yoon Ji Bang , So-Jung Choi , Dong Keon Yon 0000-0003-1628-9948 , Seung Hae Kwon , Hyun Je Kim , and Jung Shin U [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.175386989.90717974/v1 611 views 334 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Background : Chronic itch is a debilitating symptom of atopic dermatitis (AD), yet the neuroimmune mechanisms underlying its persistence remain incompletely understood. Objective : We aimed to delineate the molecular and cellular alterations within the dorsal root ganglia (DRG) in AD Methods : We performed single-cell RNA sequencing of DRGs from AD mice and controls, followed by differential gene expression analysis, gene ontology enrichment, and ligand–receptor interaction mapping. Key transcriptomic findings were validated by RNA in situ hybridization, immunofluorescence staining, and flow cytometry. Results: AD DRGs exhibited expansion of sensory neuron clusters (PEP1, NP1–NP3), with NP3 notably enriched for protein translation pathways and itch-associated genes, including Il31ra , Osmr , Cysltr2 , Sst , Jak1 , and Stat3 , which were significantly reduced upon JAK inhibitor treatment. Among non-neuronal cells, neutrophils and eosinophils were markedly increased. Neutrophils were enriched in degranulation and immune response pathways, characterized by upregulation of Ifitm1 , Ifitm2 , Ifitm3 , Ifitm6 , Qsox1 , and Adam15 . Ligand–receptor interaction analysis revealed enhanced signaling via thrombospondin, oncostatin M, and chemokine pathways, indicating active neuroimmune crosstalk within the DRG microenvironment. Conclusion : These findings demonstrate that AD induces coordinated molecular reprogramming of sensory neurons and immune cells within the DRG, underscoring active neuroimmune interactions as a key mechanism driving chronic itch in AD. 0pt 2.5ex plus 1ex minus .2ex 1.5ex plus .2ex 0pt 2ex plus .5ex minus .2ex 1ex plus .2ex 0pt 1.5ex plus .5ex minus .2ex 0.8ex plus .2ex Article type: Original article Title: Dissecting itch in atopic dermatitis through single-cell RNA sequencing of mouse dorsal root ganglia Short title : Dissecting Itch Mechanisms in Atopic Dermatitis Yun Kyung Jang 1* , Ji Hwan Moon 2* , Jae-Sang Ryu 1 , Seung Hwa Baek 1 , Young Su Jang 1 , Dong Hyun Kim 1 , Yoon Ji Bang 3 , So-Jung Choi 3 , Dong Keon Yon 5 , Seung-Hae Kwon 6 , Hyun Je Kim 4 , Jung U Shin 1 1 Department of Dermatology, CHA Bundang Medical Center, CHA University School of Medicine, Seongnam, South Korea, 2 Translational Genomics Center, Samsung Medical Center, Seoul, South Korea, 3 Department of Biomedical Science, Seoul National University Graduate School, Seoul, South Korea, 4 Department of Microbiology and Immunology, Seoul National University College of Medicine, Seoul, South Korea, 5 Department of Pediatrics, Kyung Hee University Medical Center, Kyung Hee University College of Medicine, Seoul, South Korea, 6 Metropolitan Seoul Center, Korea Basic Science Institute, Seoul, South Korea Author contributions: Yun Kyung Jang contributed to interpretation of data and to drafting and reviewing the manuscript; Ji Hwan Moon was responsible for single-cell RNA-sequencing, functional analysis, visualizing and interpretation of data, and drafting the manuscript; Jae-Sang Ryu, Seung Hwa Baek, Seung Hae Kwon and Young Su Jang performed the experiments related to the study; Yi Joon Kim, Yoon Ji Bang, and So-Jung Choi contributed to the analysis and interpretation of data; Dong Hyun Kim, Dong Keon Yon and Hyun Je Kim involved in conceptualization and designing of the study; Jung U Shin involved in conceptualization of the study, designing study, interpreting data, editing of the draft, and review. All authors read and approved the final manuscript. 0pt 2.5ex plus 1ex minus .2ex 1.5ex plus .2ex 0pt 2ex plus .5ex minus .2ex 1ex plus .2ex 0pt 1.5ex plus .5ex minus .2ex 0.8ex plus .2ex ORCID: Yun Kyung Jang 0000-0001-5731-7576 Ji Hwan Moon 0000-0002-0172-4428 Jae-Sang Ryu 0000-0002-8887-4698 Seung Hwa Baek 0000-0001-7391-4653 Young Su Jang 0000-0003-0110-7574 Dong Hyun Kim 0000-0003-3394-2400 Yoon Ji Bang 0000-0002-1539-6277 So-Jung Choi 0009-0005-9544-5233 Dong Keon Yon 0000-0003-1628-9948 Seung Hae Kwon 0000-0001-5554-9015 Hyun Je Kim 0000-0003-4467-0949 Jung U Shin 0000-0001-5259-6879 Corresponding author: Jung U Shin, M.D. Ph.D Department of Dermatology, CHA Bundang Medical Center, CHA University School of Medicine, Seongnam 13496, Korea Email: [email protected] Funding sources : This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. RS-2023-00208452). Conflicts of Interest : The authors have no conflict of interest to declare. Reprint requests: Jung U Shin, M.D. Ph.D Manuscript word count : 2347 words [excluding abstract, references, figures, tables] Abstract word count : 203 words References : 39 Figures: 5 / Supplement Figures: 4 Supplement Tables: 2 0pt 2.5ex plus 1ex minus .2ex 1.5ex plus .2ex 0pt 2ex plus .5ex minus .2ex 1ex plus .2ex 0pt 1.5ex plus .5ex minus .2ex 0.8ex plus .2ex Abstract Background : Chronic itch is a debilitating symptom of atopic dermatitis (AD), yet the neuroimmune mechanisms underlying its persistence remain incompletely understood. Objective : We aimed to delineate the molecular and cellular alterations within the dorsal root ganglia (DRG) in AD Methods : We performed single-cell RNA sequencing of DRGs from AD mice and controls, followed by differential gene expression analysis, gene ontology enrichment, and ligand–receptor interaction mapping. Key transcriptomic findings were validated by RNA in situ hybridization, immunofluorescence staining, and flow cytometry. Results: AD DRGs exhibited expansion of sensory neuron clusters (PEP1, NP1–NP3), with NP3 notably enriched for protein translation pathways and itch-associated genes, including Il31ra , Osmr , Cysltr2 , Sst , Jak1 , and Stat3 , which were significantly reduced upon JAK inhibitor treatment. Among non-neuronal cells, neutrophils and eosinophils were markedly increased. Neutrophils were enriched in degranulation and immune response pathways, characterized by upregulation of Ifitm1 , Ifitm2 , Ifitm3 , Ifitm6 , Qsox1 , and Adam15 . Ligand–receptor interaction analysis revealed enhanced signaling via thrombospondin, oncostatin M, and chemokine pathways, indicating active neuroimmune crosstalk within the DRG microenvironment. Conclusion : These findings demonstrate that AD induces coordinated molecular reprogramming of sensory neurons and immune cells within the DRG, underscoring active neuroimmune interactions as a key mechanism driving chronic itch in AD. Key words: atopic dermatitis; dorsal root ganglia; itch; neuroimmune interaction; single-cell RNA sequencing Introduction Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by relapsing eczematous lesions and persistent itch. Itch induces scratching behavior that disrupts skin barrier, promotes inflammation, and further intensifies pruritus, thereby creating a vicious cycle [1]. Itch also significantly impairs patients’quality of life [2]. Understanding and controlling itch is therefore essential for effective management of AD. Although multiple pruritogens and neuroimmune pathways have been identified, the molecular and cellular architecture underlying itch transmission within the dorsal root ganglia (DRG) remains poorly defined [3,4]. Sensory neurons, residing in the DRG, are central to itch perception. These neurons respond to pruritogens, including cytokines, such as IL-4, IL-13, and IL-31, as well as histamine and proteases, and release neuropeptides, including substance P, calcitonin gene-related peptide, somatostatin, and natriuretic peptide B (NPPB), to transmit signals to the spinal cord and modulate inflammation in the skin [5-10]. Recent advances in single-cell RNA sequencing (scRNA-seq) have revealed the cellular heterogeneity of the DRG, identifying distinct sensory neuron subtypes and diverse non-neuronal populations, including satellite glial cells, endothelial cells, and infiltrating immune cells [10-12]. Sensory neurons can be subclassified into peptidergic (PEP1, 2), non-peptidergic (NP 1-3), C- low-threshold mechanoreceptors (C-LTMRs), and neurofilament (NF 1-4), and TRMP8 high nerve fibers. Satellite glial cells provide structural and metabolical support to neurons, while the DRG vasculature facilitates molecular exchange [13,14]. Although macrophages, neutrophils, and B cells have been identified within the DRG has been recognized [15,16], their functional roles in AD-associated itch remain largely unexplored. Here, we perform single-cell transcriptomic and functional profiling of DRGs in a murine model of AD. We identify an enrichment of NP3 neurons with transcriptional signatures associated with pruritus, accompanied by immune cell infiltration and activation, particularly of neutrophils and eosinophils. Ligand–receptor interaction analysis reveals enhanced neuroimmune communication mediated by oncostatin M, thrombospondin, and chemokine signaling pathways. These findings uncover a previously unrecognized neuroimmune axis within the DRG and highlight potential targets for chronic itch in AD. 0pt 2.5ex plus 1ex minus .2ex 1.5ex plus .2ex 0pt 2ex plus .5ex minus .2ex 1ex plus .2ex 0pt 1.5ex plus .5ex minus .2ex 0.8ex plus .2ex AD-induced mouse model 0pt 2.5ex plus 1ex minus .2ex 1.5ex plus .2ex 0pt 2ex plus .5ex minus .2ex 1ex plus .2ex 0pt 1.5ex plus .5ex minus .2ex 0.8ex plus .2ex Male NC/Nga mice (5 weeks old; Charles River Laboratories, Yokohama, Japan) were used to establish an AD model. Dorsal hair was removed and 200 μL of 4% sodium dodecyl sulfate (SDS; Biosesang, Seongnam, Korea) was applied to disrupt the skin barrier. After 2 hours, 100 mg of Dermatophagoides farinae body extract ointment (BiostirAD; Biostir Inc., Kobe, Japan) was applied to the dorsal skin and ears. This protocol was repeated twice weekly for six weeks. For pharmacological intervention, upadacitinib (5 mg/kg in PBS, MedChemExpress, NJ, USA;) was administered intraperitoneally twice daily for four consecutive days. DRG dissociation and single-cell preparation Thoracic DRGs were enzymatically dissociated in 3 mg/mL collagenase type I (Sigma, St. Louis, MO, USA) in Basal Medium Eagle (BME; GIBCO, Australia) at 37°C for 90 min, followed by 1 mg/mL trypsin (Sigma) for 15 min. The reaction was stopped with 1 mg/mL soybean trypsin inhibitor and 0.2 mg/mL DNase I (Roche Diagnostics GmbH, Mannheim, Germany), and cells were resuspended in BME for subsequent analysis. Single-cell RNA sequencing and data processing Single-cell RNA sequencing (scRNA-seq) was performed by ROKIT Genomics (Seoul, Korea), using the BD Rhapsody WTA Analysis Pipeline v1.9.1, with alignment to the mouse reference genome (GRCm38). Unique molecular identifier (UMI)–based expression matrices were generated. Further analysis was conducted using the Seurat R package (v4.9.9) in R software (v4.2.3). Cells expressing 200–3,000 genes with <25% mitochondrial gene content were included. Mitochondrial genes were identified using the MitoCarta3.0 database. Data were log-normalized and integrated using the Harmony algorithm (v0.1.1) to minimize batch effects. Clustering and cell type annotation Principal component analysis (PCA) was performed using the top 2,000 variable genes, and the first 41 principal components were used to generate a UMAP plot. Clustering was conducted using the Seurat’s Louvain algorithm (resolution = 1.5). Cell types were annotated based on marker genes identified by Seurat’s FindMarkers function (log2 fold change ≥ 0.25; expressed in ≥25% of cells) and verified based on published references. Differential expression and enrichment analysis Differentially expressed genes (DEGs) between AD and control groups were identified for each cluster using Seurat (log2 fold change ≥ 0.25; p < 0.05). Gene set enrichment analysis (GSEA) was performed using the enrichment platform. Gene ontology (GO) terms with adjusted p-values < 0.05 were considered significant and visualized using dot and bar plots. Immunofluorescence staining 0pt 2.5ex plus 1ex minus .2ex 1.5ex plus .2ex 0pt 2ex plus .5ex minus .2ex 1ex plus .2ex 0pt 1.5ex plus .5ex minus .2ex 0.8ex plus .2ex DRGs were fixed in 4% paraformaldehyde (Sigma), embedded in OCT compound (Sakura Finetek, Torrance, CA), and cryosectioned at 14 μm using a Leica CM3050S cryostat. Sections were incubated overnight at 4°C with anti-Ly6G (ab238132, 1:500; Abcam, Cambridge, UK) and anti-NeuN (ab104224, 1:200; Abcam), followed by Alexa Fluor–conjugated secondary antibodies (A11008, Alexa 488; A21428, Alexa 555; Invitrogen, Carlsbad, CA, USA). Nuclei were counterstained with DAPI (D1306, Invitrogen). Images were acquired using a Zeiss Axio Scan.Z1 microscope and multiplex multiphoton molecular imaging system (Stellaris 8, Leica, metropolitan Seoul Center, KBSI). RNAscope in situ hybridization Multiplex fluorescent in situ hybridization was performed using the RNAscope Kit (Advanced Cell Diagnostics, Newark, CA, USA). Frozen DRG sections were processed using a HybEZ System (Advanced Cell Diagnostics) at 40°C. Sections were fixed and dehydrated. Probe sets included Tubb3-C1, Jak1-C2, Il31ra-C3, Osmr-C1, and Cysltr2-C2. Fluorophores (Opal520, Opal570, Opal650; Akoya Biosciences, USA) were used for visualization. Imaging was performed using a Leica Stellaris 8 confocal microscope. 0pt 2.5ex plus 1ex minus .2ex 1.5ex plus .2ex 0pt 2ex plus .5ex minus .2ex 1ex plus .2ex 0pt 1.5ex plus .5ex minus .2ex 0.8ex plus .2ex Flow cytometry DRG tissues were dissociated in cold DPBS (Welgene, Daegu, Korea) supplemented with 1.5% fetal bovine serum (FBS; Welgene) and filtered through a 100 μm nylon mesh (SPL Life Sciences, Pocheon, Korea). Cells were stained with anti-CD45.2-FITC, anti-CD11b-PE/Cy7, and anti-Ly6G-PerCP/Cy5.5 (BioLegend, San Diego, CA, USA). Dead cells were excluded using Zombie Aqua™ (BioLegend). Flow cytometry was conducted using a CytoFLEX system (Beckman Coulter, Brea, CA, USA), and data were analyzed using FlowJo software (Treestar Inc., Ashland, OR, USA). 0pt 2.5ex plus 1ex minus .2ex 1.5ex plus .2ex 0pt 2ex plus .5ex minus .2ex 1ex plus .2ex 0pt 1.5ex plus .5ex minus .2ex 0.8ex plus .2ex Results Upregulation of itch-related genes in the NP3 neurons of AD mouse DRG ScRNA-seq was performed on DRGs from Dermatophagoides farinae –induced AD mice and control mice (n = 3 per group). UMAP-based dimensionality reduction identified distinct cellular clusters, with neurons marked by Tubb3 expression (Fig. 1A). Clustering analysis identified nine neuronal and ten non-neuronal subpopulations, including peptidergic nociceptors (PEP1, PEP2), non-peptidergic (NP1–3), neurofilament (NF1–5), type C low-threshold mechanoreceptors (cLTMR1), and a putative cLTMR2 (p_cLTMR2) among neurons (Fig. 1B, Fig. S1). Non-neuronal clusters consisted of satellite glial cells, Schwann cells, endothelial cells, pericytes, fibroblasts, and multiple immune cell, including neutrophils, macrophages, and B cells (Fig. 1B, Fig. S1). Compared to controls, AD mice exhibited a relative enrichment of PEP1, NP1, NP2, and NP3 neuronal populations (Fig. 1C). Gene expression profiling revealed significant upregulation of itch-associated receptors ( Htr1a , Htr1f , Cysltr2 , S1pr1 , Trpv1 , Osmr ), neuropeptides ( Tac1 , Nmb , Sst ), and JAK/STAT pathway components ( Jak1 , Stat3) in NP3 neurons from AD mice (Fig. 1D, Fig. S2A-C). Volcano plot analysis further highlighted the increased expression of Osmr , Sst , Nppb , Jak1 , Il31ra , Stat3 in NP3 (Fig. 1E). The top 20 upregulated and downregulated DEGs are listed in Table S1. Gene ontology analysis demonstrated enrichment of pathways related to protein targeting to the membrane, endoplasmic reticulum localization, and peptide biosynthesis, indicating enhanced translational activity in NP3 neurons (Fig. 1F). In contrast, NP1, NP2, and PEP1 clusters showed relatively modest transcriptional changes without alterations in itch-related mediators or receptors (Fig. 1D, Fig. S2A–C, Fig. S3A, B). JAK inhibitor reduces expression of pruritogenic genes RNAscope in situ hybridization confirmed elevated expression of Il31ra in DRG neurons from AD mice (Fig. 2A), with Jak1 and Osmr showing a similar increasing trend (Fig. 2B). Il31ra signals were particularly localized to small-diameter neurons, consistent with NP3 characteristics. Treatment with the selective JAK1 inhibitor upadacitinib significantly reduced scratching behavior in AD mice (Fig. 2C) and led to downregulation of Il31ra , Jak1 , Osmr , and Cysltr2 expression in the DRG (Fig. 2A, B). These findings support the involvement of JAK-STAT signaling downstream of IL-31 and OSM receptors in mediating chronic itch and suggest that NP3 neurons are direct targets of this pathway. Neutrophil infiltration and transcriptional activation in the DRG of AD mice In non-neuronal populations, neutrophils and eosinophils were more abundant in AD DRGs (Fig. 3A). Flow cytometry confirmed enhanced infiltration of CD45⁺ immune cells (Fig. 3B), with a significant increase in Ly6G⁺CD11b⁺ neutrophils (Fig. 3C). Immunofluorescence staining showed accumulation of Ly6G⁺ cells around neuronal structures, suggesting active immune cell recruitment into the DRG in response to peripheral inflammation (Fig. S4). Transcriptomic profiling of neutrophils showed upregulation of interferon-inducible genes ( Ifitm1–3, Ifitm6 ), oxidative stress regulators ( Qsox1 ), and migration-associated genes ( Adam15 ) in AD mice (Fig. 3D). The top 20 upregulated and downregulated DEGs are listed in Table S2. GO enrichment analysis of DEGs revealed activation of pathways related to neutrophil degranulation , chemokine -mediated signaling, and innate immune response (Fig. 3E). These transcriptional changes suggest that neutrophils in the DRG adopt a functionally activated phenotype, potentially contributing to local neuroinflammation and sensitization of sensory neurons in AD. Enhanced immune cell interactions in the DRG of AD mice Cell–cell interaction analysis using the CellChat algorithm revealed globally increased intercellular communication in AD DRGs, with heightened signaling among neurons, immune cells, fibroblasts, and endothelial cells. In particular, enhanced signaling was observed involving neutrophils and eosinophils (Fig. 4). Three major ligand–receptor signaling axes were enriched in AD: thrombospondin (THBS), oncostatin M (OSM), and C-X-C motif chemokines (CXCL) (Fig. 5A–F). THBS signaling from neutrophils, eosinophils, pericytes, and fibroblasts to NP1 and NP3 neuronal subtypes was notably elevated in the AD mice (Fig. 5A). Similarly, OSM signaling from neutrophils and eosinophils to NP1/NP3 neurons was similarly increased (Fig. 5B). CXCL signaling was increased from pericytes, fibroblasts, and endothelial cells to infiltrating neutrophils and eosinophils (Fig. 5C). Ligand–receptor pair analysis revealed Thbs1 – Cd47 as the principal axis in THBS signaling (Fig. 5D), Osm – Osmr and Osm – Lifr in OSM signaling (Fig. 5E), and Cxcl1 / Cxcl2 – Cxcr2 and Cxcl12 – Cxcr4 in CXCL signaling (Fig. 5F). These findings indicate that immune cell–mediated signaling is amplified in the DRG of AD mice, with potential contributions to the modulation of sensory neurons and the development of chronic itch. Discussion Sensory neurons in the DRG transmit diverse sensations, including pain, itch, temperature, and proprioception, and are broadly classified into NP, PEP, and NF subtypes based on molecular profiles [4,8-10]. NP neurons express markers such as IB4 and P2rx3 , function as nociceptors together with PEP neurons, which are characterized by Tac1 and Calca expression [10,17,18]. NF neurons, marked by Nefh and Pvalb, contribute to proprioception via muscles and joint innervation [11]. Among these subtypes, NP3 neurons, enriched for Il31ra and Cysltr2, have been implicated in chronic inflammatory and serotonin-mediated itch [10]. IL-31, a key pruritogen in AD, signals through an IL-31RA and OSMRβ receptor complex that activates the JAK-STAT pathway, with STAT3 serving as both a downstream effector and transcriptional regulator of Il31ra and Osmr [19,20]. In addition, cysteinyl leukotrienes, acting through CysLTR2, contribute to chronic itch pathogenesis [21]. Our scRNA-seq analysis revealed an expansion of NP3 neurons in the DRG of AD mice, characterized by enriched translational activity and increased expression of Il31ra , Osmr , Cysltr2 , and key JAK-STAT components, including Jak1 and Stat3 . These findings were validated by RNA in situ hybridization. Furthermore, pharmacologic inhibition of JAK1 suppressed Il31ra , Jak1 , and Osmr expression and attenuated scratching behavior, supporting a central role for JAK-STAT signaling in NP3 neuron-mediated itch. NP3 neurons also showed elevated expression of the neuropeptide genes, Nppb and Sst , which are involved in ascending itch transmission [5,22,23]. NPPB transmits peripheral itch signals to the spinal cord by activating GRP-expressing interneurons [24], while somatostatin contributes to pruritus by disinhibiting the itch circuit via suppression of dynorphin-expressing inhibitory interneurons [3,25]. Together, these findings suggest that NP3 neurons act both as peripheral sensors of immune-derived pruritogens and as key relays for central itch transmission. Neuroimmune interactions have been increasingly recognized as key contributors to inflammatory skin diseases such as AD and allergic contact dermatitis [26,27]. Neuropeptides released from sensory neurons modulate local immune responses, while cytokines from keratinocytes and immune cells enhance neuronal excitability and pruritus. Although these bidirectional interactions are well documented in the skin, their extent within the DRG remain poorly understood. The DRG harbors various non-neuronal cells, including satellite glial cells, fibroblasts, endothelial cells, pericytes, and infiltrating immune cells. Although DRG-associated neutrophils have been implicated in neuropathic pain [28,29], their role in inflammation-induced itch has not been investigated. Our single-cell analysis revealed increased frequencies of neutrophils and eosinophils in the DRGs of AD mice. Neutrophils were enriched for degranulation and immune response pathways and upregulated of interferon-stimulated genes ( Ifitm1–3, Ifitm6 ), suggesting activation of type I and II interferon signaling. Additionally, genes such as Qsox1 and Adam15 , associated with immune activation and leukocyte migration, were elevated. Consistent with previous reports implicating skin-infiltrating neutrophils in itch initiation through neuronal activation and pruritic pathway upregulation [30], our study further reveals that DRG neutrophils also exhibit significant transcriptional changes, suggesting potential local contribution to itch pathogenesis. Beyond transcriptional changes, cell–cell communication analysis revealed enhanced signaling between neutrophils, eosinophils, and other DRG cell types in AD. CXCL signaling, originating predominantly from pericytes, fibroblasts, and endothelial cells, was increased and involved ligand–receptor pairs such as CXCL1/2–CXCR2 and CXCL12–CXCR4, likely facilitating granulocyte infiltration into the DRG [31-34]. Recruited neutrophils and eosinophils exhibited enhanced THBS and OSM signaling directed towards NP1 and NP3 neuronal subtypes. THBS1–CD47 signaling, implicated in inflammation, vascular remodeling, and synapse formation [35,36], and OSM signaling via gp130/OSMRβ or gp130/LIFRβ complexes, which sensitizes sensory neurons to pruritogens [37-39], were both elevated. Collectively, these findings indicate complex multicellular neuroimmune interactions within the DRG, wherein CXCL-mediated chemokine signaling promotes granulocyte infiltration and neutrophil- and eosinophil-derived THBS and OSM signals modulate sensory neuron activity. This pro-pruritic microenvironment may contribute to the development and maintenance of chronic itch in AD, highlighting the DRG as a potential therapeutic target. In conclusion, although our study is limited by a small sample size and lack of direct functional validation, it provides novel insights into the neuroimmune landscape of the DRG in AD. Single-cell transcriptomic profiling identified NP3 neurons as key mediators of chronic itch, alongside increased immune cell infiltration and intercellular signaling via THBS, OSM, and CXCL pathways. A deeper understanding of these neuroimmune interactions may advance our knowledge of itch transmission mechanisms and inform therapeutic strategies for AD-associated pruritus. Reference 1. Mack MR, Kim BS. The Itch-Scratch Cycle: A Neuroimmune Perspective. Trends Immunol. 2018;39(12):980-991. 2. Nomura T, Honda T, Kabashima K. 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Sci Transl Med. 2021;13(619):eabe3037. Figure legends Figure 1. Single-cell transcriptomic profiling reveals enrichment and transcriptional activation of NP3 neurons in dorsal root ganglia (DRG) of mice with atopic dermatitis (AD). (A) UMAP plot of single-cell RNA-sequencing data from dorsal root ganglia of control and AD mice (n = 3 per group), with neurons identified by Tubb3 expression. (B) Cell type annotation of neuronal and non-neuronal clusters based on marker genes. (C) Relative frequencies of neuronal and non-neuronal clusters in control and AD mice. (D) Expression patterns of itch-related receptors, neuropeptides, and JAK-STAT signaling molecules in neuronal subtypes. (E) Volcano plot of differentially expressed genes in NP3 neurons. (F) Gene ontology (GO) enrichment analysis of NP3-specific differentially expressed genes, showing upregulation of pathways associated with protein translation and peptide biosynthesis. Statistical comparisons were performed using two-sided Wilcoxon rank-sum test with Bonferroni correction. Figure 2. JAK1 inhibition reduces expression of itch-related signaling genes and alleviates scratching behavior in AD mice. (A) Representative RNAscope fluorescence in situ hybridization images of Il31ra and Jak1 expression in the DRG of control, AD mice, and upadacitinib-treated mice. (B) Expression patterns of Osmr and Cysltr2 in the same groups. (C) Quantification of scratching behavior across groups. Data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test (*p < 0.05, **p < 0.01, ***p < 0.001 ). Figure 3. Neutrophil infiltration and gene expression profiling in the DRG of AD mice. (A) Bar graph showing the proportion of neutrophils and eosinophils among non-neuronal clusters in the DRG of AD mice (n = 3 per group). (B) Representative flow cytometry plots (left) and quantification (right) of CD45⁺ immune cell populations in control (n = 4) and AD (n = 5) mice. (C) Representative plots and quantification of CD45⁺CD11b⁺Ly6G⁺ neutrophils in the DRG of control and AD mice. (D) Volcano plot of differentially expressed genes in DRG-infiltrating neutrophils from AD versus control mice. (E) Gene ontology (GO) enrichment analysis of neutrophil-specific differentially expressed genes. Data are presented as mean ± SEM. Statistical comparisons were performed using unpaired two-tailed t-tests for flow cytometry data and two-sided Wilcoxon rank-sum tests with Bonferroni correction for GO analysis ( *p < 0.05, **p < 0.01 ). Figure 4. Cell-cell communication networks in the DRG of control and AD mice. Circle plots depicting intercellular communication networks inferred by CellChat analysis in control (left) and AD (right) DRGs. Figure 5. Immune–neuronal signaling pathways in the DRG of AD mice. (A) THBS signaling network showing Thbs1–Cd47 interactions from neutrophils, eosinophils, pericytes, and fibroblasts to NP1 and NP3 neurons. (B) OSM signaling network highlighting OSM–OSMR and OSM–LIFR interactions from neutrophils and eosinophils to NP1 and NP3 neurons. (C) Chemokine signaling network showing CXCL1/2–CXCR2 and CXCL12–CXCR4 interactions from pericytes, fibroblasts, and endothelial cells to neutrophils and eosinophils. Communication strength was inferred using CellChat based on ligand–receptor expression levels and interaction probabilities. Figure S1. Clustering and annotation of neuronal and non-neuronal subpopulations in the DRG. Uniform manifold approximation and projection (UMAP) plot showing single-cell clustering from DRGs of control and AD mice. A total of nine neuronal clusters (PEP1–2, NP1–3, NF1–5, cLTMR1–2) and ten non-neuronal clusters (satellite glial cells, Schwann cells, endothelial cells, fibroblasts, and various immune cell types) were identified based on marker gene expression. Figure S2. Expression of itch-related receptors, neuropeptides, and JAK-STAT signaling molecules in neuronal subtypes. (A) Dot plots showing expression of itch-related receptors ( Htr1a , Htr1f , Cysltr2 , S1pr1 , Trpv1 , and Osmr ), with predominant enrichment in NP3 neurons. (B) Expression of neuropeptides ( Tac1 , Nmb , and Sst ), showing highest levels in NP3 neurons. (C) Expression of JAK/STAT signaling molecules ( Jak1 and Stat3 ), preferentially expressed in NP3 neurons. Figure S3. Differential gene expression and pathway enrichment in NP1, NP2, and PEP1 neuronal clusters. (A) Volcano plots showing differentially expressed genes in NP1 (left), NP2 (middle), and PEP1 (right) clusters comparing AD and control mice. (B) Gene ontology (GO) enrichment analysis of differentially expressed genes in NP1 (top), NP2 (middle), and PEP1 (bottom) clusters. Figure S4. Increased neutrophil infiltration in the DRG of AD mice detected by Immunofluorescence staining. Representative immunofluorescence images of DRG sections from control (upper) and AD (lower) mice stained with anti-Ly6G (red; neutrophil marker), anti-NeuN (green; neuronal marker), and DAPI (blue; nuclear stain). High-magnification views of the boxed regions are shown in the right panels. Scale bar: 50 μm. Information & Authors Information Version history V1 Version 1 30 July 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords animal models atopic dermatitis Authors Affiliations Yun Kyung Jang 0000-0001-5731-7576 CHA University Bundang Medical Center View all articles by this author Ji Hwan Moon Samsung Medical Center View all articles by this author Jae-Sang Ryu CHA University Bundang Medical Center View all articles by this author Seung Hwa Baek CHA University Bundang Medical Center View all articles by this author Young Su Jang 0000-0003-0110-7574 CHA University Bundang Medical Center View all articles by this author Dong Hyun Kim CHA University Bundang Medical Center View all articles by this author Yoon Ji Bang Seoul National University Graduate School Department of Biomedical Science View all articles by this author So-Jung Choi Seoul National University Graduate School Department of Biomedical Science View all articles by this author Dong Keon Yon 0000-0003-1628-9948 Kyung Hee University Medical Center View all articles by this author Seung Hae Kwon Korea Basic Science Institute Seoul Center View all articles by this author Hyun Je Kim Seoul National University College of Medicine View all articles by this author Jung Shin U [email protected] CHA University Bundang Medical Center View all articles by this author Metrics & Citations Metrics Article Usage 611 views 334 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Yun Kyung Jang, Ji Hwan Moon, Jae-Sang Ryu, et al. 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