Neuronal STING drives neuroimmune circuits underlying atopic itch

Background: Chronic itch is a hallmark symptom of atopic dermatitis (AD), but the molecular mechanisms within sensory neurons that underlie itch are still not fully understood. Methods: Transcriptomic profiling of dorsal root ganglia (DRG) from an AD-like mouse model with graded disease severity and scratching behavior was used to identify itch-associated molecules in sensory neurons. Pharmacological modulation of STING in control versus AD mice, and in wild-type versus STING- deficient mice, was used to determine the contribution of neuronal STING to atopic itch. Circulating cell-free DNA and IL-31 were examined as upstream modulators of neuronal STING signaling. Results: Tmem173 expression in DRG progressively increased with AD severity and scratching behavior. Pharmacological STING inhibition reduced pruritus and intraepidermal nerve fiber density and suppressed IL-6–STAT3–LCN2 and GRP–GRPR pathway activation in the spinal cord. In the periphery, STING activation upregulated TRPV1 and substance P expression and induced mast cell activation. Intrathecal STING agonist administration recapitulated scratching behavior and associated transcriptomic changes, whereas these effects were not observed in STING-deficient mice. Circulating cell-free DNA, a candidate endogenous activator of the cGAS–STING pathway, was elevated in both an AD-like mouse model and patients with AD, and IL-31 further amplified STING-induced responses.

These findings uncover a previously unrecognized role for neuronal STING in coordinating central and peripheral neuroimmune circuits that drive AD-associated itch. Article type: Original article Title: Neuronal STING drives neuroimmune circuits underlying atopic itch Short title: Neuronal STING in itch signaling Authors: Young Su Jang 1*, Jae-Sang Ryu 2*, Han Sai Lee 3*, Hyeongjin Na 4, A-ram Kim 2, Dong Keon Yon5, Dong Hyun Kim 2, Young Shin Song 6, Jung U Shin 2 1.Department of Biomedical Science, Graduate School, CHA University, Seongnam, Republic of Korea 2.Department of Dermatology, Bundang CHA Medical Center, CHA University School of Medicine, Seong- nam, Republic of Korea 3.Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Sci- ence and Technology, Seoul National University, Seoul, Republic of Korea 1 Posted on 28 Nov 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.176432425.50521984/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary. 4.Department of Medicine, CHA University School of Medicine, Seongnam, Republic of Korea 5.Center for Digital Health, Medical Science Research Institute, Kyung Hee University Medical Center, Kyung Hee University College of Medicine, Seoul, Republic of Korea 6.Department of Internal Medicine, Seoul Metropolitan Government Seoul National University Boramae Medical Center, Seoul National University College of Medicine, Seoul, Republic of Korea *These authors contributed equally to this work Corresponding author: Jung U Shin Department of Dermatology, CHA University College of Medicine, Bundang CHA Medical Center, Yatap-ro 59, Bundang-gu, Seongnam 13496, Republic of Korea E-mail: [email protected] Phone No: +82-31-780-5240 ORCIDs: Young Su Jang: https://orcid.org/0000-0003-0110-7574 Jae-Sang Ryu: https://orcid.org/0000-0002-8887-4698 Han Sai Lee: https://orcid.org/0000-0001-6337-0162 Hyeongjin Na: https://orcid.org/0009-0007-2221-0147 A-Ram Kim: https://orcid.org/0000-0002-5458-1985 Dong Keon Yon: https://orcid.org/0000-0003-1628-9948 Dong Hyun Kim: https://orcid.org/0000-0003-3394-2400 Young Shin Song: https://orcid.org/0000-0003-4603-1999 Jung U Shin: https://orcid.org/0000-0001-5259-6879 Acknowledgments: This work was supported by grants from the National Research Foundation of Korea (NRF), funded by the Ministry of Science and ICT (MSIT) of the Korean government (Nos. RS-2023-00208452 and RS- 2024-00440577), and by the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare (No. RS-2025-25459933). Competing interests The authors declare no competing interests. Total number of words: 3452/ 3500 (excluding the abstract, tables, figure legends and references)

Background: Chronic itch is a hallmark symptom of atopic dermatitis (AD), but the molecular mechanisms within sensory neurons that underlie itch are still not fully understood.

2 Posted on 28 Nov 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.176432425.50521984/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary. Transcriptomic profiling of dorsal root ganglia (DRG) from an AD-like mouse model with graded disease severity and scratching behavior was used to identify itch-associated molecules in sensory neurons. Pharma- cological modulation of STING in control versus AD mice, and in wild-type versus STING-deficient mice, was used to determine the contribution of neuronal STING to atopic itch. Circulating cell-free DNA and IL-31 were examined as upstream modulators of neuronal STING signaling.

Tmem173 expression in DRG progressively increased with AD severity and scratching behavior. Pharma- cological STING inhibition reduced pruritus and intraepidermal nerve fiber density and suppressed IL-6– STAT3–LCN2 and GRP–GRPR pathway activation in the spinal cord. In the periphery, STING activation upregulated TRPV1 and substance P expression and induced mast cell activation. Intrathecal STING ago- nist administration recapitulated scratching behavior and associated transcriptomic changes, whereas these effects were not observed in STING-deficient mice. Circulating cell-free DNA, a candidate endogenous acti- vator of the cGAS–STING pathway, was elevated in both an AD-like mouse model and patients with AD, and IL-31 further amplified STING-induced responses.

These findings uncover a previously unrecognized role for neuronal STING in coordinating central and peripheral neuroimmune circuits that drive AD-associated itch. Key words: atopic dermatitis; dorsal root ganglia; pruritus; neuroimmune crosstalk; STING

Itch is an unpleasant sensation that triggers the urge to scratch and serves as a protective mechanism against harmful environmental stimuli 1. In chronic inflammatory skin disorders such as atopic dermatitis (AD), however, persistent itch significantly compromises patients’ quality of life2. In AD, pruritus arises from the release of pruritogens that activate cutaneous sensory neurons and amplify immune responses 3,4. Key mediators implicated in AD-associated itch include interleukin (IL)-4, IL-13, IL-31, IL-33, thymic stromal lymphopoietin (TSLP), leukotriene C4, periostin, and proteases 5-10. Once activated by pruritogens, sensory neurons transmit itch signals to the spinal cord primarily through the gastrin-releasing peptide (GRP)–GRP receptor (GRPR) pathway 11-14, and the excitability of spinal GRPR+ neuron is enhanced by astrocyte-derived lipocalin 2 (LCN2), which is induced by IL-6 released from dorsal root ganglia (DRG) neurons 15-17. In the periphery, activated transient receptor potential vanilloid 1 (TRPV1)-expressing sensory neurons release substance P, which activates mast cells and further promotes type 2 inflammation 9,18. Furthermore, scratching behavior induced by itch disrupts the skin barrier and activates TRPV1+ sensory neurons, establishing a vicious cycle of neuroimmune activation 19. The stimulator of interferon genes (STING, encoded by TMEM173 ) is an endoplasmic reticulum-resident adaptor protein that is activated by cyclic dinucleotides, including cyclic GMP–AMP (cGAMP) synthesized by cyclic GMP-AMP synthase (cGAS) upon sensing mislocalized cytosolic double stranded DNA, leading to the induction of type I interferons (IFNs) and inflammatory cytokines 20-23. While STING has been extensively studied in antiviral immunity and cancer, recent pain models suggest that STING activation in DRG neurons modulates nociceptive signaling through NF- κB, TRPV1, and type I IFNs 24-26, but its contribution to pruritic signaling remains unknown. To elucidate novel molecular mechanisms underlying itch in AD, we analyzed the DRG transcriptome from an AD-like mouse model and identified Tmem173 as a candidate regulator of neuroimmune signal- ing. TMEM173 expression was progressively upregulated in accordance with disease severity and scratching behavior, and functional studies demonstrated that neuronal STING drives both central and peripheral itch pathways. Moreover, circulating cell-free DNA was increased in the plasma of both patients with AD and an AD-like mouse model, and IL-31 amplified STING-induced responses. These findings identify STING 3 Posted on 28 Nov 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.176432425.50521984/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary. as a pivotal regulator of itch pathways in AD and suggest its potential as a therapeutic target for chronic pruritus.

Tmem173 expression increased progressively with AD severity and scratching behavior To investigate transcriptomic alterations associated with itch signaling in AD, we performed RNA sequencing of DRG isolated from the NC/Nga mouse model of AD, in which AD-like dermatitis was induced by topical application of Dermatophagoides farinae body extract. Based on SCORAD scores at week 6, mice were categorized into mild and severe AD groups ( Fig. 1A, B ), and mice with severe AD showed significantly higher TEWL and scratching behavior than those with mild AD ( Fig. 1C, D ). Hierarchical clustering of differentially expressed genes (DEGs) revealed three distinct clusters ( Fig. 1E ). Cluster 1 consisted of genes that were downregulated in AD, without a clear severity-dependent gradient. Cluster 2 included genes whose expression was lowest in severe AD compared with normal (NOR) or mild AD; among these, 17 genes showed a progressive decrease from NOR controls to severe AD (Supplementary Table 1 ). Cluster 3 consisted of genes that were more highly expressed in severe AD than in NOR or mild AD, and 24 of these genes were progressively upregulated with increasing disease severity (Supplementary Table 1) . Gene ontology (GO) analysis of genes upregulated in the more severe condition across pairwise comparisons (normal vs. mild AD, normal vs. severe AD, and mild AD vs. severe AD) demonstrated enrichment of biological processes related to responses to external stimuli and innate immune activation (Fig. 1F ). Tmem173, which encodes STING , was one of the most significantly upregulated genes in the DRG of AD mice ( Supplementary Table 1 ). Both Tmem173 expression and the STING pathway activity score increased in parallel with disease severity ( Fig. 1G ). Volcano plots further demonstrated a significant up- regulation of Tmem173 in both mild and severe AD groups compared with NOR controls (Fig. 1H ). These transcriptomic findings were validated by qRT-PCR, which showed elevated expression of Tmem173 and its downstream targets, Ifna1 and Ifnb1 , in the DRG of AD mice ( Fig. 1I ). The upregulation of Tmem173 was also observed in the DRG of other itch mouse models, including diphenylcyclopropenone (DCP)-treated and MC903-treated mice (Supplementary Fig. 1). Additionally, Cyp26a1 , Fam150b , Bdnf , andSteap1, implicated in neuronal sensitization and oxidative stress 27-31, were among the disease severity-associated genes and showed a strong correlation with Tmem173 (Supplementary Fig. 2, Supplementary Table 1 ). To assess whether sensory neurons directly respond to STING activation, primary mouse DRG neurons were treated with the STING agonist ADU-S100, which induced a dose-dependent increase in Ifna1 and Ifnb1 expression (Supplementary Fig. 3 ). STING inhibition alleviates pruritus and suppresses spinal itch signaling pathways To determine whether STING contributes to AD-associated itch, we intraperitoneally administered the STING inhibitor H-151 to AD mice. STING inhibition reduced erythema and scaling ( Fig. 2A ), and decreased SCORAD scores and TEWL ( Fig. 2B, C ). Among clinical parameters, scratching behavior ex- hibited the most pronounced reduction following H-151 treatment (Fig. 2D ). Histological analysis revealed reduced epidermal thickness and intraepidermal nerve fiber density in H-151-treated mice ( Fig. 2E–G ). In addition, mRNA expression of Bdnf , Cyp26a1 , and Steap1 was downregulated following H-151 treatment (Supplementary Fig. 4 ). While STING inhibition significantly reduced itch responses, Th2 cytokine levels in the skin remained unchanged and serum total IgE was paradoxically increased ( Supplementary Fig. 5 ), suggesting that itch suppression was unlikely to be mediated by systemic immune suppression. Next, we investigated whether STING inhibition modulates GRP–GRPR signaling in the ascending itch pathway. In the spinal dorsal horn (SDH), GRP expression was significantly elevated in AD mice and was reduced by H-151 treatment ( Fig. 2H ). In our RNA-seq data, Il6 , a cytokine released into the spinal cord that enhances GRP–GRPR signaling 15-17, was positively correlated with Tmem173 (Supplementary Fig. 6 ), and its elevated levels in AD DRG were reduced by STING inhibition ( Fig. 2I). Furthermore, 4 Posted on 28 Nov 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.176432425.50521984/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary. IL-6 and phosphorylated STAT3 (p-STAT3) were markedly increased in the spinal cord of AD mice, with p-STAT3 localized primarily to GFAP + astrocytes, and both were attenuated by STING inhibition ( Fig. 2J ). Expression of LCN2, a downstream mediator induced by astrocytic STAT3 activation, was upregulated in the spinal cord of AD mice and was reduced by H-151 ( Fig. 2K ). Together, these findings indicate that neuronal STING contributes to AD-associated itch by activating the IL-6–STAT3–LCN2 axis and enhancing GRP–GRPR signaling. STING activation drives TRPV1–substance P-dependent mast cell activation TRPV1+ sensory neurons release substance P, which activates mast cells via Mrgprb2 9,18. In our transcrip- tomic analysis, Tmem173 expression was positively correlated with both Trpv1 and Tac1 (Supplementary Fig. 6 ). The AD-induced increase in TRPV1 and substance P expression in the DRG was attenuated by H-151 (Fig. 3A, B ). Immunofluorescence staining revealed co-localization of phosphorylated STING (p- STING) with TRPV1 and substance P, both of which were increased in AD DRG and reduced by H-151 treatment (Fig. 3C, D ). Consistently, dermal mast cell infiltration was significantly increased in AD mice and was markedly reduced by STING inhibition (Fig. 3E ). To determine whether STING activation in DRG neurons promotes mast cell activation, primary mouse DRG neurons were stimulated with ADU-S100 and co-cultured with mast cells. ADU-S100 induced substance P release from DRG neurons ( Fig. 3F ), and conditioned media from these neurons enhanced mast cell chemotaxis ( Fig. 3G) , upregulated Tpsab1 and Tpsb2 (encoding tryptase β1 and β2) (Fig. 3H ), and increased tryptase release ( Fig. 3I) . Neuronal STING-induced mast cell activation was abolished by QWF, an antagonist of Mrgprb2 ( Fig. 3G–I ). To- gether, these findings indicate that STING activation in sensory neurons promotes mast cell activation via TRPV1–substance P-dependent neuroimmune crosstalk. Neuronal STING activation is sufficient to induce pruritus responses To investigate whether STING activation in sensory neurons is sufficient to elicit pruritus, we intrathecally administered STING agonist ADU-S100 to wild-type (WT) and STING-deficient (STING gt/gt ; GT) mice once daily for three consecutive days. In WT mice, ADU-S100 induced STING phosphorylation in DRG neurons (Supplementary Fig. 7), increased scratching behavior (Fig. 4A ), and promoted dermal mast cell infiltration (Fig. 4B ). These effects were not observed in STING gt/gt mice (Fig. 4A, B; Supplementary Fig. 7 ). In addition, DRGs from ADU-S100-treated WT mice exhibited elevated expression of Ifna1, Ifnb1, Il6, andTac1 , along with increased substance P release, none of which were observed in STING gt/gt mice (Fig. 4C, D ). Immunofluorescence analysis further confirmed that TRPV1 and substance P expression was increased following STING activation and was abolished in STING gt/gt mice (Fig. 4E, F ), indicating that STING activation in sensory neurons is sufficient to drive pruritus responses. Circulating cell-free DNA as a potential STING activator and IL-31 as a signaling amplifier We measured circulating cell-free DNA, an endogenous agonist of the cGAS–STING pathway 32-34. Circu- lating cell-free DNA levels were significantly increased in the plasma of an AD-like mice ( Fig. 5A ) and in patients with AD ( Fig. 5B ). We next investigated whether AD-associated cytokines modulate STING signaling in DRG neurons. Primary mouse DRG neurons were treated with ADU-S100, either alone or in combination with IL-4 or IL-31. ADU-S100 treatment, alone or with cytokines, increased p-STING expres- sion (Fig. 5C ). Although co-treatment with IL-4 did not significantly change p-STING intensity compared with ADU-S100 alone, co-treatment with IL-31 markedly enhanced the signal intensity of p-STING ( Fig. 5C ). Phosphorylated TBK1 (p-TBK1) exhibited a similar pattern; ADU-S100 increased p-TBK1 expression, and IL-31 co-treatment further augmented p-TBK1 intensity ( Fig. 5D ). Additionally, IL-31 co-treatment further upregulated Ifna1, Ifnb1 , and Il6 expression (Fig. 5E ), and significantly increased IL-6 release compared with ADU-S100 alone (Fig. 5F ). These findings indicate that IL-31 potentiates STING signaling in DRG neurons, and may thereby amplify neuroimmune responses in AD.

The cGAS–STING pathway is a cytosolic DNA-sensing mechanism that activates type I IFN responses 5 Posted on 28 Nov 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.176432425.50521984/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary. upon detection of mislocalized cytosolic double-stranded DNA 20-23. In addition to its established role in cellular senescence, inflammatory diseases, and antiviral and tumor immunity 35, recent studies have begun to elucidate its function in sensory neurons. Activation of the STING pathway in sensory neurons can be triggered by nerve injury or cellular stress 36. Previous studies using neuropathic and inflammatory pain models have implicated a context-dependent role of STING in nociceptive signaling, mediated through NF- κB, TRPV1, and type I IFNs24-26. While some reports have demonstrated that STING activation exacerbates neuroinflammation and chronic pain via proinflammatory cytokines 24,26, others have shown that STING- induced type I IFNs can suppress nociceptive signaling 25,37. Notably, Manon et al. 37 recently reported that STING activation reduces nociceptor excitability by downregulating TRPV1 and upregulating KchIP1 expression. However, the role of the STING pathway in itch signaling remains largely unknown. To elucidate molecular changes in sensory neurons associated with pruritus, we performed RNA sequencing of DRG tissue from the NC/Nga AD-like mouse model. At week 6, mice were stratified into mild and severe groups based on their SCORAD scores, and the severe group exhibited markedly increased scratching behav- ior. Transcriptomic analysis revealed that Tmem173 expression was progressively upregulated across NOR control, mild, and severe AD groups, and was positively correlated with genes involved in pruritus, neuronal sensitization, and oxidative stress. Pharmacological STING inhibition attenuated scratching behavior, re- duced intraepidermal nerve fiber density, and downregulated itch-associated genes in the DRG. Interestingly, although STING inhibition reduced pruritus, it led to an increase in serum IgE levels without altering Th2 cytokine expression in the skin. This may reflect a shift in the Th1/Th2 balance, wherein suppression of Th1 responses by STING inhibition reduces counter-regulatory control of Th2-driven IgE production 38. These findings suggest that STING-mediated regulation of itch is not primarily dependent on systemic immune modulation but rather operates through neuroimmune signaling mechanisms. In AD, pruritogens activate their respective receptors on peripheral sensory nerve fibers, and the resulting signals are transmitted to the spinal cord3,4. GRP, released from primary afferent neurons, activates GRPR- expressing excitatory interneurons in the SDH 13,14,39. Additionally, astrocyte-derived LCN2, upregulated by the IL-6–STAT3 signaling pathway, further potentiates excitatory synaptic transmission in the spinal cord15-17. Augmented GRP-GRPR signaling can lead to hyperexcitability of spinal itch circuits, thereby lowering the threshold for itch perception 11,12. In our AD-like mouse model, we observed STING-dependent increases in GRP, IL-6, p-STAT3, and LCN2 expression in the spinal cord, as well as elevated IL-6 and type I IFNs in the DRG, supporting a model in which neuronal STING activation drives an ascending itch signaling pathway. Chronic AD is characterized by a vicious cycle of neuroimmune crosstalk, in which interactions between sensory neurons and immune cells amplify inflammation and itch signaling. TRPV1-expressing sensory neurons not only transmit itch signals to the spinal cord, but also secrete neuropeptides, such as substance P, which is associated with disease severity 9,40,41. Substance P influences the activity of Mrgprb2 + mast cells (or MRGPRX2 in humans) 42, and in turn, mast cell-derived mediators such as tryptase and chymase further stimulate sensory neurons, thereby potentiating neurogenic inflammation 43,44. Building upon these findings, we found that STING activation in DRG neurons acts as an upstream regulator of substance P release from TRPV1 + sensory neurons. The AD-induced increases in TRPV1 and substance P expression in the DRG were reduced by STING inhibition. In addition, intrathecal administration of a STING agonist enhanced TRPV1 and substance P expression and promoted mast cell infiltration in the skin, and these effects were abolished in STINGgt/gt mice. In vitro , conditioned media from STING-activated DRG neurons significantly increased mast cell chemotaxis and tryptase release via Mrgprb2. Together, these findings indicate that STING activation in DRG neurons plays a pivotal role in regulating TRPV1–substance P– Mrgprb2-dependent neuroimmune crosstalk in AD. Pathogen-derived nucleic acids as well as self-DNA released from dying or injured cells, are recognized by cGAS, leading to the production of cGAMP and subsequent activation of STING 20-23. Extracellular self- DNA, including circulating cell-free DNA, can also access the cytosolic cGAS–STING pathway after uptake through endocytic or phagocytic mechanisms and subsequent release into the cytosol 32-34. In this study, 6 Posted on 28 Nov 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.176432425.50521984/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary. we observed significantly increased levels of circulating cell-free DNA in the plasma of patients with AD and an AD-like mouse model. Given the relative permeability of the blood–DRG barrier 45, circulating DNA or DNA-containing complexes may access the DRG microenvironment, and contribute to STING activation in DRG neurons. These findings are consistent with recent work implicating circulating cell- free DNA, particularly mitochondrial DNA, in the systemic immunopathology of AD, in which elevated plasma cell-free mtDNA has been reported and shown to correlate with disease severity 46. Moreover, we identified IL-31 as a potent amplifier of STING signaling in sensory neurons. Co-stimulation with IL-31 and a STING agonist synergistically enhanced the phosphorylation of STING and TBK1 and upregulated the expression of type I IFNs and IL-6. IL-31 binds to a heterodimeric receptor composed of IL-31RA and OSMRβ, which is expressed on TRPV1 + and TRPA1+ sensory neurons and transduces signals via STAT3. Since STAT3 regulates transcription ofTmem173 and Il6 47,48 , IL-31-induced STAT3 activation may further amplify STING-driven responses by upregulating both STING expression and its downstream cytokine, IL-6 8. Despite these findings, several limitations remain to be addressed. STING is expressed in multiple cell types, including immune cells and central nervous system neurons. For instance, STING activation in SDH has been shown to reduce opioid-induced and chronic itch via the TBK1–IRF3–type I IFN signaling cascade in mice49. It remains possible that intrathecal administration of a STING agonist in our study may have also influenced spinal STING activation, in addition to its effects on DRG neurons. Furthermore, we observed increased systemic Th2 responses, evidenced by elevated serum IgE levels, following STING inhibition. This finding suggests that systemic modulation of STING may have immunological side effects, potentially limiting its therapeutic utility via systemic administration. In addition, although circulating DNA and IL-31 may act as upstream activators of STING in sensory neurons, this proposed mechanistic link requires functional validation. In summary, our findings identify neuronal STING as a central modulator of AD-associated itch and neu- roimmune crosstalk. Specifically, neuronal STING activation integrates spinal IL-6–STAT3–LCN2 signaling with peripheral TRPV1–substance P-mediated mast cell activation, collectively contributing to pruritus and skin inflammation. Increased circulating cell-free DNA and IL-31 in AD may activate and potentiate the neuronal STING pathway, providing a mechanistic link by which peripheral Th2 inflammation can amplify danger-sensing pathways in sensory neurons. Together, these findings underscore a previously unrecognized role for STING in regulating itch signaling in AD.

Experimental animals Four-week-old male NC/Nga and C57BL/6 mice were obtained from Orient Bio Inc. (Seongnam, Korea). STINGgt/gt mice were kindly provided by Professor Chan Kim (CHA University, Seongnam, Korea) and maintained on a C57BL/6 genetic background. All animal procedures were approved by the Institutional Animal Care and Use Committee of CHA University (IACUC-230037) and conducted in accordance with the Animal Welfare Act and institutional guidelines for animal care and use. Patients Plasma samples were collected from healthy controls and patients with AD under protocols approved by the Institutional Review Board of Bundang CHA Medical Center (IRB no. 2024-02-008), with written informed consent from all participants. Demographic and clinical characteristics, including sex, age, and EASI scores, for each group are summarized in Supplementary Table 2 . AD mouse model After shaving the dorsal skin of mice, AD-like skin lesions were induced by topical application of 100 mg Bio- stirAD (freeze-driedDermatophagoides farinae extract ointment; Biostir Inc., Kobe, Japan) to the depilated dorsal skin twice weekly for 6 weeks. To impair the skin barrier, 200 μL of 4% sodium dodecyl sulfate (SDS; 7 Posted on 28 Nov 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.176432425.50521984/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary. Biosesang, Seongnam, Korea, #BS003a) was applied to the dorsal skin and ears 2 h prior to each ointment application. AD severity was evaluated weekly using a modified SCORAD index, which scores erythema/hemorrhage, scarring/dryness, edema, and excoriation/erosion on a scale of 0 (none) to 3 (severe). Total scores (0–12) were calculated by summing four symptom scores. Mice with scores of 4–7 were defined as mild AD and those with 8–12 as severe AD. RNA extraction, library preparation, and sequencing DRGs from 11 mice (4 normal, 4 mild AD, and 3 severe AD) were collected, and total RNA was extracted using the NucleoSpin RNA/Protein Kit (Macherey-Nagel, Bethlehem, PA, USA, #740933). RNA concen- tration was measured with the Quant-iT RiboGreen RNA Assay Kit (Invitrogen, San Diego, CA, USA, #R11490), and RNA integrity was evaluated using the TapeStation RNA ScreenTape system (Agilent Tech- nologies, Santa Clara, CA, USA, #5067-5576). All samples had an RNA integrity number (RIN) > 7.0 and were used for library preparation. Libraries were constructed using 1 μg of total RNA per sample with the TruSeq Stranded mRNA Sample Prep Kit (Illumina, San Diego, CA, USA, #20020595). Final libraries were amplified by PCR, quantified with the KAPA Library Quantification Kit (KAPA BIOSYS- TEMS, Wilmington, MA, USA, #KK4854), and quality-checked using the TapeStation D1000 ScreenTape (Agilent Technologies, #5067-5582). Paired-end (2 × 100 bp) sequencing was conducted on the Illumina NovaSeq platform by Macrogen Inc. (Seoul, Korea). RNA-seq preprocessing, alignment, and differential expression analysis Raw reads were assessed using FastQC (v0.12.1), trimmed with Trimmomatic (v0.39), aligned to the mouse

genome using STAR (v2.7.4a), and quantified with RSEM (v1.3.1) 50-52. Read counts were nor- malized and used for principal component analysis and differential expression analysis with the DESeq2 package53. Genes were considered differentially expressed if they met the following criteria: |log2 fold change| [?] 1 and adjusted p-value < 0.05 (Wald test, Benjamini-Hochberg correction). DEGs were visualized using ggpubr (v0.6.0) as bar plots, box plots, volcano plots, and correlation plots. Heatmaps were generated using the ComplexHeatmap package54. Gene ontology enrichment analysis Gene Ontology Biological Process (GOBP) enrichment analysis was performed using the gProfiler R package55,56. Genes upregulated in the more severe conditions across comparisons (NOR vs. mild AD, NOR vs. severe AD, and mild AD vs. severe AD) were used as input gene sets. Significance was determined using a false discovery rate (FDR)-adjusted P-value (Benjamini–Hochberg method). Statistical analysis Data are presented as mean +- s.e.m. Statistical analyses were conducted using GraphPad Prism version 6.0 (GraphPad Software, San Diego, CA, USA). Depending on the experimental design, unpaired two-tailed Student’s t-test, one-way ANOVA, two-way ANOVA, or two-way repeated measures ANOVA were used, followed by appropriate post hoc multiple comparisons tests as indicated in the figure legends. A P-value < 0.05 was considered statistically significant. Data availability All data supporting the findings of this study are included within the manuscript and its Supplementary In- formation files. The RNA-seq data generated in this study have been deposited in the NCBI Gene Expression Omnibus under accession GSE303287. Author contributions Y.S.J. designed and performed the experiments and wrote the original draft of the manuscript. J.-S.R. performed animal experiments and histological analyses. H.S.L. and Y.S.S. conducted bioinformatics and statistical analyses, curated the data, and prepared the visualizations. H.N. and A.-R.K. assisted with 8 Posted on 28 Nov 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.176432425.50521984/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary. experimental design and data interpretation. D.K.Y. contributed to data interpretation and manuscript editing. D.H.K. supervised the project. J.U.S. interpreted the data, edited the manuscript, supervised the study, and acquired funding.

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No reuse without permission. — https://doi.org/10.22541/au.176432425.50521984/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary. Figure legends Fig. 1. RNA sequencing analysis reveals Tmem173 as an atopic itch-related gene in the DRG of AD mice (A) Representative images of dorsal skin lesions at week 6. (B) SCORAD, (C) TEWL, and (D) scratching behavior (n = 4 per group). Data are presented as mean +- s.e.m. and analyzed using two-way repeated- measures ANOVA in (B, C) and one-way ANOVA in (D), followed by Tukey’s test. *P < 0.05, **P < 0.01, ****P < 0.0001 vs. mild AD (B, C); *P < 0.05,****P < 0.0001 vs. NOR, ####P < 0.0001 vs. mild AD (D). (E) Hierarchical clustering heatmap of DEGs in NOR ( n = 4, gray), mild AD ( n = 4, pink), and severe AD ( n = 3, red). Colors indicate relative expression (yellow, high; blue, low). (F) Lollipop plot showing GO: Biological Process terms for cluster 3 analyzed using gProfiler2. Colors indicate false discovery rate (FDR), and x-axis shows –log10 FC. (G) Box plots showing Tmem173 expression and STING pathway activity (Reactome: STING-mediated induction of host immune responses), based on variance-stabilized DESeq2 data. Each dot represents one sample from NOR, mild AD, or severe AD. Data are presented as medians with IQR and were analyzed by unpaired two-tailed t-test. (H) Volcano plots of DEGs comparing NOR with mild AD and severe AD (adjusted P < 0.05; |log2FC| [?] 1). (I) Tmem173, Ifna1, and Ifnb1 mRNA expression in the DRG (n = 6 per group). Data were analyzed using unpaired two-tailed t-test (** P < 0.01, ****P < 0.0001 vs. NOR). Fig. 2. STING inhibition alleviates pruritus and suppresses spinal itch signaling AD mice were treated intraperitoneally with vehicle or 10 mg/kg H-151 three times per week for six weeks. (A) Representative images of dorsal skin lesions after treatment. (B) SCORAD and (C) TEWL ( n = 8 per group). Data are presented as mean ± s.e.m. and were analyzed by two-way repeated-measures ANOVA with Tukey’s test. **P < 0.01, ****P < 0.0001 vs. AD. (D) Quantification of scratching behavior (n = 8 per group). (E) Representative images of H&E staining and PGP9.5 expression in the skin. Nuclei were counterstained with DAPI. Scale bars, 50 μm. (F) Epidermal thickness ( n = 7 per group) and (G) intraepidermal nerve fiber density ( n = 6 per group) were quantified from x20 images. (H, left) Western blot analysis of GRP in the spinal cord. β-actin served as a loading control. (H, right) Representative immunofluorescence images of GRP expression in the spinal dorsal horn (SDH). Scale bars, 50 μm. (I, left) Il6 mRNA expression ( n = 6 per group) and (I, right) Western blot analysis of IL-6 in the DRG. (J, left) Western blot analysis of IL-6, p-STAT3 (Tyr705), and STAT3 in the spinal cord, and (J, right) representative immunofluorescence images for GFAP and p-STAT3 in the SDH. Scale bars, 50 μm. (K, left) Lcn2 mRNA expression (n = 4 per group) and (K, right) Western blot analysis of LCN2 in the spinal cord. Data were analyzed by one-way ANOVA with Tukey’s test. *P < 0.05, ***P < 0.001, ****P < 0.0001 vs. NOR; ###P < 0.001,####P < 0.0001 vs. AD (D-G, I, K). Fig. 3. Neuronal STING activation drives mast cell activation via the TRPV1–substance P axis (A) Trpv1 and Tac1 mRNA expression in the DRG ( n = 6 per group). (B, left) Western blot analysis of TRPV1 in the DRG. β-actin was used as a loading control. (B, right) ELISA for substance P in the DRG (n = 4 per group). (C, D) Representative immunofluorescence images showing colocalization of p-STING with TRPV1 (C) or substance P (D) in the DRG. NeuN labels neuronal nuclei. Scale bars, 50 μm. (E, left) Representative images of toluidine blue staining in the skin. Scale bars, 50 μm. (E, right) Quantification of mast cell numbers from x20 images ( n = 3 per group). Data are presented as mean ± s.e.m. and were analyzed by one-way ANOVA with Tukey’s test. **P < 0.01, ****P < 0.0001 vs. NOR;##P < 0.01,####P < 0.0001 vs. AD (A, B, E). (F) Primary mouse DRG neurons were stimulated with ADU-S100 (ADU) 60 μM for 1 h, and supernatant substance P levels were measured ( n = 4 per group). Data were analyzed using an unpaired two-tailed t-test. (G–I) Mast cells were incubated for 24 h with vehicle or ADU-treated DRG conditioned media with or without QWF. (G) Transwell migration assay of mast cells. ( n = 5 per group). (H) Tpsab1 and Tpsb2 mRNA expression ( n = 3 per group). (I) Concentration of tryptase in the supernatant (n = 4 per group). Data were analyzed by one-way ANOVA with Tukey’s test. * P < 0.05, **P 12 Posted on 28 Nov 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.176432425.50521984/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary. < 0.01, ***P < 0.001, **** P < 0.0001 (F–I). Fig. 4. Neuronal STING activation induces pruritus responses (A) Scratching behavior in wild-type (WT) and STING gt/gt (GT) mice following intrathecal administration of PBS or 30 μM ADU for three consecutive days (n = 8 per group). (B, left) Representative images of H&E and toluidine blue staining in the skin. Scale bars, 50 μm. (B, right) Quantification of mast cell numbers from x20 images (n = 5 per group). (C) mRNA expression of Ifna1 , Ifnb1, Il6 , and Tac1 (n = 7 per group) in the DRG, (D) ELISA for substance P ( n = 7 per group), and (E, F) representative immunofluorescence images of TRPV1 (E) and substance P (F) expression in the DRG. NeuN labels neuronal nuclei. Scale bars, 50 μm. Data are presented as mean ± s.e.m. and were analyzed by two-way ANOVA with Tukey’s test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. PBS-treated WT; ##P < 0.01,###P < 0.001,####P < 0.0001 vs. ADU-treated WT (A–D). Fig. 5. Circulating cell-free DNA is elevated in AD and IL-31 enhances STING signaling (A) Plasma concentrations of cell-free (cf) DNA were quantified in NOR and AD mice ( n = 9 per group) and in (B) healthy controls (HC; n = 10) and patients with AD ( n = 13). Data are presented as mean ± s.e.m. and were analyzed using an unpaired two-tailed t-test. * P < 0.05, ****P < 0.0001. (C–E) Primary mouse DRG neurons were treated for 1 h with PBS, 60 μM ADU-S100 (ADU), 60 μM ADU + 50 ng/mL IL-4, or 60 μM ADU + 50 ng/mL IL-31. (C, left) MFI of p-STING was quantified from x100 images ( n = 19 neurons per condition). (C, right) Representative immunofluorescence images of p-STING expression. (D, left) MFI of p-TBK1 ( n = 18 neurons per condition). (D, right) Representative immunofluorescence images of p-TBK1 expression. NeuN labels neuronal nuclei. Scale bars, 5 μm. (E) mRNA expression of Ifna1, Ifnb1, and Il6 (n = 8 per group). (F) Supernatant IL-6 levels collected at 24 h after treatment under the conditions described above ( n = 8 per group). Data were analyzed by one-way ANOVA with Tukey’s test. ** P < 0.01, ***P < 0.001, ****P < 0.0001 vs. PBS; ##P < 0.01, ####P < 0.0001 vs. ADU (C-F). 13 Posted on 28 Nov 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.176432425.50521984/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary. 14 Posted on 28 Nov 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.176432425.50521984/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary. 15 Posted on 28 Nov 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.176432425.50521984/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary. 16 Posted on 28 Nov 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.176432425.50521984/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary. 17