IL-31 suppresses the expression of TARC/CCL17 in TNF-alpha/IFN-gamma-stimulated HaCaT keratinocytes

IL-31 suppresses the expression of TARC/CCL17 in TNF-alpha/IFN-gamma-stimulated HaCaT keratinocytes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article IL-31 suppresses the expression of TARC/CCL17 in TNF-alpha/IFN-gamma-stimulated HaCaT keratinocytes Momoka Iwamoto, Takuya Takafuji, Yoshihito Yamada, Tomoyuki Fujita This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6260398/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 15 You are reading this latest preprint version Abstract The most common symptom of atopic dermatitis (AD) is itching, which results in sleep disturbances and psychological stress, substantially reducing the quality of life of patients. Nemolizumab is a monoclonal antibody targeting interleukin 31 receptor A (IL-31RA) and mitigates the itching in AD by inhibiting the activity of IL-31, a cytokine mainly produced by T helper 2 cells. However, elevated levels of thymus and activation-regulated chemokine (TARC)/C–C motif chemokine ligand 17 (CCL17), a marker for disease severity in AD, were observed in some patients with AD following nemolizumab administration, although these patients almost did not exhibit an exacerbation of AD symptoms. This study aimed to elucidate the possible mechanism underlying the upregulation of CCL17 with nemolizumab treatment in some patients. We evaluated the effects of IL-31 and nemolizumab on the mRNA expression of CCL17 in TNF-alpha/IFN-gamma-stimulated HaCaT keratinocytes in vitro using qRT-PCR. In addition, we comprehensively analyzed the impact of IL-31 and nemolizumab on keratinocyte-related phenomena, using RNA sequencing. Our findings indicated that IL-31 suppressed the expression of CCL17 mRNA in keratinocytes, and nemolizumab reversed this suppressive effect. We further discovered that although oncostatin M suppressed the expression of CCL17 mRNA, its action was unaffected by nemolizumab. Furthermore, IL-31 affected keratinocyte differentiation, lipid metabolism, and inflammatory cytokine. Herein, we propose a possible mechanism by which nemolizumab counteracts the inhibitory effect of IL-31 on CCL17 expression, thus explaining the increased CCL17 levels following nemolizumab administration in some patients with AD. atopic dermatitis TARC/CCL17 keratinocyte HaCaT cell IL-31 Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Atopic dermatitis (AD) is a prevalent chronic inflammatory skin disease with a complex and heterogeneous etiology. Persistent itching is the most distressing symptom for patients with AD, which results in sleep disruption, anxiety, depression, and decreased productivity at work, thereby diminishing their quality of life [ 38 ]. Moreover, pruritus-induced scratching exacerbates the skin lesions and triggers the itch-scratch cycle, which underlies the pathogenesis of AD [ 22 , 49 ]. Thus, controlling pruritus is crucial in treating AD, and novel treatments are expected to improve efficacy and have fewer adverse effects. Interleukin-31 (IL-31) is a key cytokine that triggers pruritus via various cells including T helper 2 cells, macrophages, dendritic cells, eosinophils, mast cells, basophils, ILC2, as well as fibroblasts and epidermal keratinocytes [ 2 , 3 ]. The IL-31 receptor (IL-31R) is a heterodimer composed of IL-31RA and oncostatin M receptor β (OSMRb). It binds to IL-31, and transmits stimuli by activating downstream intracellular signaling pathways such as Janus kinase (JAK)/signal transducer and transcription activator, phosphatidylinositol 3‑kinase (PI3K)/protein kinase B (AKT) and mitogen-activated protein kinase (MAPK) [ 50 ]. Peripheral sensory neurons, epidermal keratinocytes, immune cells, and fibroblasts, express IL-31R that contribute to pruritus, inflammatory responses, skin barrier abnormalities, and fibrosis [ 7 , 10 ] IL-31 is substantially expressed in the skin lesions of patients with AD compared to healthy individuals, and a positive correlation has been established between serum IL-31 levels and disease severity [ 35 , 37 ]. These findings suggest that IL-31 signaling contributes to various pathological aspects of AD, including pruritus. Nemolizumab, a monoclonal antibody against IL-31RA, was developed to inhibit IL-31 signaling, thereby reducing itching in AD. Clinical trials have demonstrated the efficacy of nemolizumab in reducing key features of AD including pruritus in patients with AD who experienced moderate to severe itching, which was inadequately controlled by existing treatments [ 25 , 26 ]. Nemolizumab has also shown effectiveness in other diseases, such as prurigo nodularis [ 48 ]. However, adverse events, including worsening of AD and increased levels of thymus and activation-regulated chemokine (TARC)/C–C motif chemokine ligand 17 (CCL17), have been observed in some patients. Notably, increased CCL17 levels were not always accompanied by worsening AD symptoms [ 25 , 26 ]. The mechanism underlying the increased CCL17 serum levels post-nemolizumab administration as well as the direct and indirect effects of IL-31 on CCL17 expression remain to be investigated. Interestingly, the expression of CCL17 and other cytokines is increased in tape strips collected from patients with AD compared to healthy controls [ 13 , 20 ], indicating that these changes may reflect chemokine production from epidermal and immune cells. Moreover, AD-derived keratinocytes express high levels of IL-31R and produce various chemokines such as CCL17, in response to IL-31 expression [ 9 , 22 ]. Therefore, we focused on keratinocytes as one of the CCL17-producing cells and examined the effects of IL-31 on CCL17 expression with or without nemolizumab, followed by transcriptome analysis of HaCaT keratinocytes treated with IL-31 and nemolizumab to elucidate the overall impact of IL-31 and its regulation by nemolizumab, including increased CCL17 expression. 2. Materials and Methods 2.1 Cell culture and reagents The HaCaT human keratinocyte cell line was procured from Cell Lines Service (German Cancer Research Center, Heidelberg, Germany) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and penicillin/streptomycin (100 U/mL and 100 µg/mL, respectively) at 37°C in a humidified incubator containing 5% CO 2 . An equivalent of 31300 cells/cm 2 was seeded for 24 h to reach confluency before treatment with or without recombinant human tumor necrosis factor-alpha (TNF-α), IL-31, oncostatin M (R&D Systems, Minneapolis, MN, USA), interferon-gamma (IFN-γ) (PeproTech, Rocky Hill, NJ, USA) or nemolizumab (Chugai Pharmaceutical, Tokyo, Japan), and cultured for a further 72 h. 2.2 RNA isolation Total RNA was isolated from HaCaT cells using a QIA shredder and a RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. 2.3 Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) cDNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA). TaqMan Gene Expression Master Mix, TaqMan Gene Expression Assays, and TaqMan primer/probes were used for qRT-PCR on a QuantStudio 7 Flex Real-Time PCR System (Thermo Fisher Scientific), with the standard settings. The following sets of primers were purchased from Thermo Fisher Scientific: IL31RA (Hs00371172_m1), OSMR (Hs00384276_m1), IL-6 signal transducer (IL6ST: Hs00174360_m1), CCL17 (Hs00171074_m1), involucrin (IVL: Hs00846307_s1), keratin 10 (KRT10: Hs00166289_m1), CCL22 (Hs01574247_m1), IL-1β (Hs01555410_m1), thymic stromal lymphopoietin (TSLP: Hs00263639_m1), IL-33 (Hs04931857_m1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH: Hs02786624_g1). The expression of the target genes was normalized to that of GAPDH using the 2 − ΔΔCT method. 2.4 Library preparation and sequencing RNA sequencing (RNA-Seq) libraries were prepared for sequencing using SMART-Seq V4 ultra-low input RNA (TaKaRa Bio, Shiga, Japan) and Nextera XT DNA Library Prep Kit (Illumina, San Diego, CA, USA), according to the manufacturer’s instructions, and sequenced on the NovaSeq X Plus platform (Illumina). Differential expression was established based on at least a twofold difference in the expression levels (|log2 fold change ≥ 1|) and an adjusted p -value < 0.05 for statistical significance. Thereafter, gene set enrichment analysis (GSEA) was performed using with the R package clusterProfiler and ReactomePA. The canonical pathway, upstream regulator, disease, and biofunction analyses of the differentially expressed genes (DEGs) were conducted using Ingenuity Pathway Analysis (IPA, Qiagen) to compare the differences between the treatments. 2.5 Statistical analysis Data are expressed as the mean ± standard error of the mean (SEM). The differences between more than two groups were analyzed using Dunnett’s multiple comparison test, whereas those between two groups were analyzed using an unpaired t-test. All statistical analyses were performed using EXSUS software (CAC Croit Corp., Tokyo, Japan). A p -value < 0.05 indicated a statistically significant difference. 2.6 Data availability RNA-seq data have been deposited in the NCBI Gene Expression Omnibus (GEO) (public repository) and are accessible using the GEO series accession number GSE281127. 3. Results 3.1 TNF-α and IFN-γ stimulation increased the expression of CCL17 mRNA in HaCaT cells First, we evaluated the effects of TNF-α and IFN-γ stimulation on the expression of CCL17 in HaCaT cells. The relative expression mRNA of CCL17 was upregulated by stimulation with TNF-α or IFN-γ in a concentration-dependent manner (Fig. 1 a), as previously described [ 45 ]. Th1 cytokines, such as TNF-α and IFN-γ, are recognized for stimulating the production of CCL17 in epithelial cells, including the HaCaT keratinocyte cell line. Moreover, co-treatment with TNF-α and low concentrations of IFN-γ at 0.1 and 1 ng/mL increased the expression of CCL17 further compared to stimulation with each agent alone, but not at 10 and 100 ng/mL concentrations of IFN-γ. Conversely, co-stimulation upregulated the expression levels of IL-31 and OSM-associated receptor chains (IL-31RA, OSMR, IL-6ST) compared to stimulation with each agent individually (Fig. 1 b–d). 3.2 Effects of IL-31 and OSM on the expression of CCL17 mRNA Next, we investigated the effect of IL-31 and nemolizumab on the mRNA expression of CCL17 in response to the TNF-α- and IFN-γ-stimulated expression of CCL17 and IL-31 receptor. Consequently, CCL17 expression was downregulated by IL-31 treatment but was significantly reversed by the addition of nemolizumab (Fig. 2 a). A similar result was observed for the expression of CCL22, a chemokine related to AD [ 18 ] (Fig. S1). OSM is also a neuromodulator of itching, and the expression of OSM and OSMR is increased in AD skin [ 6 ]. Furthermore, similar to IL-31, OSM suppressed the expression of CCL17; however, this effect was not reversed by the addition of nemolizumab (Fig. 2 a). IL-31 treatment upregulated the expression of IL-31 and OSM-associated receptor chains, which was significantly reversed by nemolizumab addition, whereas OSM treatment also upregulated their expression, which was not reversed by nemolizumab addition (Fig. 2 b–d). These findings indicate that the IL-31/IL-31RA axis regulates the expression of CCL17 in keratinocytes. 3.3 Effects of IL-31 differentiation markers and inflammatory cytokine genes RNA-seq analysis was performed to examine the IL-31- and nemolizumab-induced gene expression changes. The principal component analysis (PCA) plots for the normalized abundance values revealed that 66.21% of the variance could be explained by the first two principal components (Fig. 3 a). The mRNA expression profile of the protein-coding genes between the four groups is shown by hierarchical clustering (Fig. 3 b). The influence of IL-31 or nemolizumab on gene expression profile is illustrated in the volcano plot (Fig. 3 c). DEGs with an adjusted p -value < 0.05 between the TNF-α + IFN-γ and TNF-α + IFN-γ + IL-31 treatments were related to the epidermal growth factor (EGF) receptor family, including amphiregulin (AREG), neuregulin 1 (NRG1), heparin-binding EGF-like growth factor and epiregulin (EREG) (Fig. 3 c). In addition, the differentiation markers keratin 13 (KRT13) and KRT15 were downregulated. The combined treatment with IL-31 and nemolizumab reversed the expression of several genes, such as NRG1 and EREG , compared to IL-31 treatment alone. As IL-31 is involved in the differentiation of keratinocytes [ 11 ], we examined the effects of this cytokine on the expression of differentiation markers (IVL and KRT10) by qRT-PCR. IL-31 decreased the mRNA expression of IVL and KRT10 , which was reversed by the expression of differentiation markers (Fig. 4 a). These results suggested that while IL-31 altered keratinocyte differentiation, nemolizumab could restore it. GSEA was used to determine whether the identified gene sets differed significantly between each group. The analysis was performed using Reactome terms for a comprehensive understanding of the biological importance of the perturbation by IL-31 and nemolizumab treatment in keratinocytes. The top ten upregulated and downregulated terms from GSEA presented (Fig. 3 d). Between the TNF-α + IFN-γ and TNF-α + IFN-γ + IL-31 treatments, the positively enriched terms included “Interleukin-4 and Interleukin-13 signaling” and “Signaling by Interleukin,” which was consistent with the expected results. The negatively enriched terms included “Fatty acid metabolism.” In contrast, between the TNF-α + IFN-γ + IL-31 and TNF-α + IFN-γ + IL-31 + nemolizumab treatments, the negatively enriched terms included “Signaling by Interleukin,” and the positively enriched terms included “Fatty acid metabolism.” Furthermore, network analysis was performed using IPA, which revealed IL-31 treatment activated the EGF and related pathways (Fig. 3 e). Conversely, nemolizumab treatment suppressed these pathways and inhibited the cytokine-related pathways, including TNF, IL-1A and IL-1B. In addition to IL-1B, the inflammatory cytokines TSLP and IL-33 are highly expressed in the skin lesions of patients with AD [ 9 ]. The IL-31-upregulated mRNA expression of each cytokine was suppressed by co-treatment with nemolizumab, which was confirmed by qRT-PCR (Fig. 4 b). These results suggest that nemolizumab suppresses multiple inflammatory cytokines by inhibiting the binding of IL-31 to IL-31RA and exerts an anti-inflammatory effect. 4. Discussion Nemolizumab administration improves pruritus in AD while also increasing the serum levels of CCL17 in some patients. However, there appears to be little correlation between increased CCL17 levels and AD symptom severity in these patients, a pattern consistent with previous reports on JAK inhibitors [ 5 , 15 , 26 ]. These underlying mechanisms have not yet been elucidated, thus necessitating further investigation to address the clinical aspects regarding the upregulated levels of CCL17 post-drug treatment. In this study, we assessed the effect of IL-31 on the expression of CCL17 in keratinocytes, which are one of the major sources of CCL17 production. In line with previous findings [ 45 ], we observed that the expression of CCL17 was elevated in HaCaT cells that were co-stimulated with TNF-α and IFN-γ but was reduced by the addition of IL-31. Interestingly, IL-31 stimulation increased the expression of its own receptor (IL31RA and OSMR) in the presence of TNF-α and IFN-γ. These results suggest that IL-31 stimulation may induce a positive feedback loop in the IL-31 signaling pathway, thus further driving the downregulation of CCL17 expression. The expression of TNF-α, IFN-γ, and IL-31 is elevated in the skin lesions of patients with AD [ 17 , 30 , 41 ]. Recent technological advances, such as single-cell RNA-seq, have elaborated on the sources of cytokine production in AD lesions and their involvement in the pathogenesis. Particularly, the interactions between different cell types and their roles in specific disease states can now be comprehensively understood [ 1 , 21 ]. TNF-α is produced by immune cells, such as macrophages, dendritic cells, and keratinocytes, which promotes skin inflammation and impairs the skin barrier function [ 16 ]. Similarly, IFN-γ is a cytokine secreted by Th1 cells, and plays a crucial role in regulating the inflammatory responses of AD [ 42 ]. The stimulation conditions in this in vitro study using TNF-α and IFN-γ reflect the disease state of patients with AD. Our findings suggest that the action of IL-31 suppresses the expression of CCL17, which is reversed by nemolizumab treatment. This may explain the upregulated levels of CCL17 in some of the patients with AD who are treated with nemolizumab. Nonetheless, the promotive effect of IL-31 on CCL17 expression is also reported in keratinocytes [ 14 ]. Therefore, IL-31 may exert varying effects on CCL17 expression depending on specific conditions. OSM belongs to the IL-6 family of cytokines and shares a common signaling receptor subunit (OSMR) with IL-31. It is known for its increased expression of OSM and OSMR in the skin lesion in patients with AD as well as increased production of OSM from skin-infiltrating T cells [ 6 ]. Moreover, OSM acts as a neuromodulator of itching in diseases such as psoriasis and cutaneous T cell lymphoma, which are characterized by chronic itching, including AD [ 43 ]. Furthermore, the intradermal administration of OSM-encoding adenovirus induces robust skin inflammation with epidermal hyperplasia and increases the expression of chemokines and cytokines in the ear, thus indicating OSM’s contribution to inflammatory responses [ 34 ]. In addition, OSM induces the production of CCL11 from fibroblasts and CCL21 from vascular endothelial cells [ 29 , 40 ]. These findings suggest that OSM exacerbates the inflammatory responses and itching in patients with AD. To the best of our knowledge, this study is the first to reveal the inhibitory effect of OSM on CCL17 expression, which is not reversed by nemolizumab addition. Another study hypothesized that nemolizumab-mediated inhibition of IL-31 signaling could facilitate the interaction between the free OSMR subunit i.e., OSMRb and gp130 (IL6ST) on the cell membrane, which result in an overabundance of functional receptors, thus enhancing the responsiveness to OSM [ 4 ]. However, this hypothesis was not supported in our experiments. Alternatively, we propose that OSM may act via a mechanism independent of the IL-31/IL-31RA axis, with variations depending on the cell type and stimulation conditions. Conversely, OSM released by monocytes in the skin suppresses the IL-31-stimulated itching by inhibiting IL-31RA expression in the dorsal root ganglia [ 39 ]. Therefore, IL-31 and OSM could regulate inflammatory mediators through their interaction and control itching in patients with AD. Bulk RNA-seq analysis revealed a direct effect of IL-31 on CCL17 expression, keratinocyte differentiation, lipid metabolism, and the regulation of inflammatory cytokines. In addition, IL-31 impairs the skin barrier function by inhibiting the expression of filaggrin [ 11 , 19 ]. The intradermal administration of IL-31 in mice induced epidermal thickening and increased transepidermal water loss [ 36 ]. EGF controls various biological actions, such as survival, migration, and proliferation, and plays a crucial role in regulating the skin barrier functions and inflammatory responses in AD [ 32 ]. Our RNA-seq analysis indicated that IL-31 treatment activated the EGF pathway. In a previous report, IL-31 increased the expression of the transcription factor EGR1 in keratinocytes [ 47 ] and EGF expression in bronchial epithelial cells [ 24 ], which suggests the involvement of IL-31 in EGF signaling. Conversely, EGF inhibitors upregulate the expression of CCL17 in keratinocytes [ 28 ], thus indicating that EGF signaling negatively regulates CCL17 expression, which is consistent with the effects of IL-31 treatment observed in this study. Although it is unclear whether the effect is direct or indirect, IL-31 treatment might modulate CCL17 expression through the EGF pathway. Moreover, we confirmed the downregulation of fatty acid metabolism pathways following IL-31 treatment. Previous studies have characterized AD by aberrant lipid metabolism, resulting in an impaired epidermal barrier, which contributes to inflammation and emphasizes the critical role of lipids in AD pathogenesis [ 33 ]. The effects of IL-31 on lipid metabolism have been investigated using three-dimensional tissue engineered human skin equivalents. The studies showed an increase in long-chain free fatty acids and a decrease in the content of ester-linked ω-hydroxy ceramides [ 12 ]. Although the role of IL-31 in lipid metabolism is yet, skin lipid metabolism is shown to affect the skin barrier function and immune responses; lipid alterations are becoming clearer with the development of omics technology [ 31 ]. Further basic and clinical studies on the regulation of lipid metabolism in the skin of patients with AD would provide in-depth insights into the involvement of IL-31 and pathogenesis via lipid alterations. The activation of IL-31 signaling produces several cytokines and chemokines in various cell types. In keratinocytes, IL-31 induces the production of IL-1α and regulates the skin barrier function [ 19 ]. In addition, inflammatory cytokines (IL-1β and IL-6) and chemokines (CXCL1/8/10 and CCL2/18) are released from eosinophils in response to IL-31 stimulation. This release is further enhanced when eosinophils are co-cultuered with either keratinocytes or fibroblasts [ 8 , 46 ]. Recently, IL-31 induces the production of inflammatory mediators (TNF-α, IL-6, CXCL8, and CCL2/5/22) in dendritic cells, and the production of IL-1β and IL-6 in macrophages [ 23 , 27 ]. These findings suggest that IL-31 exacerbates inflammation by promoting the production of cytokines. The transcriptome analysis of prurigo nodularis showed that nemolizumab treatment suppressed inflammatory cytokines, including IL-13 and IFN-γ [ 44 ], which suggests a similar possibility of the downregulation of cytokines in AD. In this study, the IL-31-induced production of inflammatory cytokines, such as IL-1β, TSLP and IL-33, was suppressed by nemolizumab treatment, thus indicating an anti-inflammatory effect of nemolizumab and improving AD pathology. Despite its novel findings, this study has some limitations. First, although increased IL-31 expression has been reported in the lesions of patients with AD [ 30 , 37 ], the concentration of IL-31 used in this study may not accurately reflect levels on lesional skin. Second, this study was performed using HaCaT keratinocyte monocultures only. The association between IL-31, EGF signaling, and lipid metabolism could not be fully elucidated by the transcriptome analysis. As AD has a complex and heterogeneous etiology, further research is needed to fully elucidate the mechanisms of IL-31 and nemolizumab treatment. In conclusion, this study demonstrated that IL-31 acts as a repressive regulator of the expression of CCL17 in keratinocytes in vitro, and nemolizumab treatment reverses this inhibitory effect, potentially upregulating the expression of CCL17 in the serum of patients with AD after nemolizumab administration. In addition to the direct effect on CCL17 expression, IL-31 also affected keratinocyte differentiation, lipid metabolism, and induced inflammatory cytokines, which suggests multiple roles of IL-31 in the pathogenesis of AD and the regulation of itching and inflammation. However, we performed the study using only HaCaT ketatinocyte to discover the mechanism of IL-31. To elucidate the clinical significance of IL-31 inhibition, future research should focus on the signaling pathway using samples isolated from patients with AD after nemolizumab administration. Declarations Competing interests The authors are employees of Maruho Co. Ltd. Supporting information Supplementary data associated with this article can be found in the online version. Funding This study was supported by Maruho Co., Ltd. Author Contribution M.I. and Y.Y. conceived and designed the study. M.I. conducted the experiments, collected the data, carried out the statistical analyses, and drafted the initial manuscript. M.I. and T.T. were involved in the data analysis and interpretation. T.F. and Y.Y. provided supervision throughout the study and contributed to the review and editing of the manuscript. All authors participated in drafting or critically revising the manuscript for important intellectual content and approved the final version to be published. Acknowledgements We greatly thank Takuya Yokoyama for support throughout the experiments. References Alkon N, Assen FP, Arnoldner T, Bauer WM, Medjimorec MA, Shaw LE et al (2023) Single-cell RNA sequencing defines disease-specific differences between chronic nodular prurigo and atopic dermatitis. J Allergy Clin Immunol 152(2):420-435. https://doi.org/10.1016/j.jaci.2023.04.019 Alkon N, Bauer WM, Krausgruber T, Goh I, Griss J, Nguyen V et al (2022) Single-cell analysis reveals innate lymphoid cell lineage infidelity in atopic dermatitis. 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N Engl J Med 383(2):141-150. https://doi.org/10.1056/NEJMoa1917006 Kasraie S, Niebuhr M, Werfel T (2010) Interleukin (IL)-31 induces pro-inflammatory cytokines in human monocytes and macrophages following stimulation with staphylococcal exotoxins. Allergy 65(6):712-721. https://doi.org/10.1111/j.1398-9995.2009.02255.x Komine M, Kakinuma T, Kagami S, Hanakawa Y, Hashimoto K, Tamaki K (2005) Mechanism of thymus- and activation-regulated chemokine (TARC)/CCL17 production and its modulation by roxithromycin. J Invest Dermatol 125(3):491-498. https://doi.org/10.1111/j.0022-202X.2005.23840.x Langdon C, Kerr C, Tong L, Richards CD (2003) Oncostatin M regulates eotaxin expression in fibroblasts and eosinophilic inflammation in C57BL/6 mice. J Immunol 170(1):548-555. https://doi.org/10.4049/jimmunol.170.1.548 Neis MM, Peters B, Dreuw A, Wenzel J, Bieber T, Mauch C et al (2006) Enhanced expression levels of IL-31 correlate with IL-4 and IL-13 in atopic and allergic contact dermatitis. J Allergy Clin Immunol 118(4):930-937. https://doi.org/10.1016/j.jaci.2006.07.015 Nowowiejska J, Baran A, Flisiak I (2023) Lipid Alterations and Metabolism Disturbances in Selected Inflammatory Skin Diseases. Int J Mol Sci 24(8). https://doi.org/10.3390/ijms24087053 Pastore S, Mascia F, Mariani V, Girolomoni G (2008) The epidermal growth factor receptor system in skin repair and inflammation. J Invest Dermatol 128(6):1365-1374. https://doi.org/10.1038/sj.jid.5701184 Pavel P, Blunder S, Moosbrugger-Martinz V, Elias PM, Dubrac S (2022) Atopic Dermatitis: The Fate of the Fat. Int J Mol Sci 23(4). https://doi.org/10.3390/ijms23042121 Pohin M, Guesdon W, Mekouo AA, Rabeony H, Paris I, Atanassov H et al (2016) Oncostatin M overexpression induces skin inflammation but is not required in the mouse model of imiquimod-induced psoriasis-like inflammation. Eur J Immunol 46(7):1737-1751. https://doi.org/10.1002/eji.201546216 Raap U, Wichmann K, Bruder M, Stander S, Wedi B, Kapp A, Werfel T (2008) Correlation of IL-31 serum levels with severity of atopic dermatitis. J Allergy Clin Immunol 122(2):421-423. https://doi.org/10.1016/j.jaci.2008.05.047 Singh B, Jegga AG, Shanmukhappa KS, Edukulla R, Khurana Hershey GH, Medvedovic M et al (2016) IL-31-Driven Skin Remodeling Involves Epidermal Cell Proliferation and Thickening That Lead to Impaired Skin-Barrier Function. PLoS One 11(8):e0161877. https://doi.org/10.1371/journal.pone.0161877 Sonkoly E, Muller A, Lauerma AI, Pivarcsi A, Soto H, Kemeny L et al (2006) IL-31: a new link between T cells and pruritus in atopic skin inflammation. J Allergy Clin Immunol 117(2):411-417. https://doi.org/10.1016/j.jaci.2005.10.033 Ständer S (2021) Atopic Dermatitis. N Engl J Med 384(12):1136-1143. https://doi.org/10.1056/NEJMra2023911 Suehiro M, Numata T, Saito R, Yanagida N, Ishikawa C, Uchida K et al (2023) Oncostatin M suppresses IL31RA expression in dorsal root ganglia and interleukin-31-induced itching. Front Immunol 14:1251031. https://doi.org/10.3389/fimmu.2023.1251031 Sugaya M, Fang L, Cardones AR, Kakinuma T, Jaber SH, Blauvelt A, Hwang ST (2006) Oncostatin M enhances CCL21 expression by microvascular endothelial cells and increases the efficiency of dendritic cell trafficking to lymph nodes. J Immunol 177(11):7665-7672. https://doi.org/10.4049/jimmunol.177.11.7665 Szegedi K, Lutter R, Res PC, Bos JD, Luiten RM, Kezic S, Middelkamp-Hup MA (2015) Cytokine profiles in interstitial fluid from chronic atopic dermatitis skin. J Eur Acad Dermatol Venereol 29(11):2136-2144. https://doi.org/10.1111/jdv.13160 Szymanski U, Cios A, Ciepielak M, Stankiewicz W (2021) Cytokines and apoptosis in atopic dermatitis. Postepy Dermatol Alergol 38(2):1-13. https://doi.org/10.5114/ada.2019.88394 Tseng PY, Hoon MA (2021) Oncostatin M can sensitize sensory neurons in inflammatory pruritus. Sci Transl Med 13(619):eabe3037. https://doi.org/10.1126/scitranslmed.abe3037 Tsoi LC, Hacini-Rachinel F, Fogel P, Rousseau F, Xing X, Patrick MT et al (2022) Transcriptomic characterization of prurigo nodularis and the therapeutic response to nemolizumab. J Allergy Clin Immunol 149(4):1329-1339. https://doi.org/10.1016/j.jaci.2021.10.004 Tsuda T, Tohyama M, Yamasaki K, Shirakata Y, Yahata Y, Tokumaru S et al (2003) Lack of evidence for TARC/CCL17 production by normal human keratinocytes in vitro. J Dermatol Sci 31(1):37-42. https://doi.org/10.1016/s0923-1811(02)00138-x Wong CK, Leung KM, Qiu HN, Chow JY, Choi AO, Lam CW (2012) Activation of eosinophils interacting with dermal fibroblasts by pruritogenic cytokine IL-31 and alarmin IL-33: implications in atopic dermatitis. PLoS One 7(1):e29815. https://doi.org/10.1371/journal.pone.0029815 Yeo H, Ahn SS, Lee YH, Shin SY (2020) Regulation of pro-opiomelanocortin (POMC) gene transcription by interleukin-31 via early growth response 1 (EGR-1) in HaCaT keratinocytes. Mol Biol Rep 47(8):5953-5962. https://doi.org/10.1007/s11033-020-05668-0 Yokozeki H, Murota H, Matsumura T, Komazaki H, Nemolizumab JPSG (2024) Efficacy and safety of nemolizumab and topical corticosteroids for prurigo nodularis: Results from a randomised, double-blind, placebo-controlled, phase II/III clinical study in patients aged >/=13 years. Br J Dermatol. https://doi.org/10.1093/bjd/ljae131 Yosipovitch G, Berger T, Fassett MS (2020) Neuroimmune interactions in chronic itch of atopic dermatitis. J Eur Acad Dermatol Venereol 34(2):239-250. https://doi.org/10.1111/jdv.15973 Zhang Q, Putheti P, Zhou Q, Liu Q, Gao W (2008) Structures and biological functions of IL-31 and IL-31 receptors. Cytokine Growth Factor Rev 19(5-6):347-356. https://doi.org/10.1016/j.cytogfr.2008.08.003 Additional Declarations No competing interests reported. Supplementary Files Supplementarydata.docx Fig. S1 Effects of IL-31 and nemolizumab on the mRNA expression of CCL22 in HaCaT cells. HaCaT cells were stimulated with TNF-α (0.1 ng/mL) and IFN-γ (100 ng/mL) for 72 h. At the same time, cells were stimulated with IL-31 (100 and 500 ng/mL) or nemolizumab (100 µg/mL). The relative mRNA expression of CCL22 was measured by qRT-PCR, and GAPDH was used as the internal control. Data are presented as the mean ± SEM (n = 3). *** p < 0.001 vs. 0.1 ng/mL TNF-α + 100 ng/mL IFN-γ and $$ p < 0.01 vs. 0.1 ng/mL TNF-α + 100 ng/mL IFN-γ + 500 ng/mL IL-31. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 10 Feb, 2026 Reviews received at journal 26 Jan, 2026 Reviews received at journal 15 Jan, 2026 Reviews received at journal 11 Jan, 2026 Reviewers agreed at journal 10 Jan, 2026 Reviewers agreed at journal 09 Jan, 2026 Reviewers agreed at journal 08 Jan, 2026 Reviews received at journal 08 Jan, 2026 Reviewers agreed at journal 08 Jan, 2026 Reviews received at journal 08 Jan, 2026 Reviewers agreed at journal 08 Jan, 2026 Reviewers invited by journal 24 Mar, 2025 Editor assigned by journal 20 Mar, 2025 Submission checks completed at journal 20 Mar, 2025 First submitted to journal 19 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6260398","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":433376006,"identity":"f371bf54-6ca0-44a9-83d5-faeeb67ae057","order_by":0,"name":"Momoka Iwamoto","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEUlEQVRIiWNgGAWjYBACNhiDvYGB8QGDgQSYwwwVTMCtBSjFc4CB2YAoLXApoBY2CRifGbdSBgY+ieSnGz7+sGHgYT9jVnWjwIJBt/3sM+mCCgZ5fgaGZw+wOUwizezmjIQ0Bh6etLTbOUCHmZ1JN5OecYbBcGYDQ7oBNi3SCWa3eRIOM9hLMB+DaDmQxibN28aQYHCAIU0Cq5b0b2AtPBKMbcVgLeefEdKSYwbVwnyMGazlBiFb5N+U3ZyRBvZLsjRQC4/ZjWfM1jxnJAxnNmP3i3zP8W03PtiAQ8zwc86fOjmz82mMt3kqbOT52XvSsIUYDNQ3QBk8UBroJGaeNDw6sAP2YyRrGQWjYBSMguEIAOV7Um4/LEdWAAAAAElFTkSuQmCC","orcid":"","institution":"Maruho Co., Ltd","correspondingAuthor":true,"prefix":"","firstName":"Momoka","middleName":"","lastName":"Iwamoto","suffix":""},{"id":433376007,"identity":"33dc1bba-f082-416a-8ddf-c328f243ce62","order_by":1,"name":"Takuya Takafuji","email":"","orcid":"","institution":"Maruho Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Takuya","middleName":"","lastName":"Takafuji","suffix":""},{"id":433376008,"identity":"d8454a3d-5278-4519-8980-9b860b1869cb","order_by":2,"name":"Yoshihito Yamada","email":"","orcid":"","institution":"Maruho Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Yoshihito","middleName":"","lastName":"Yamada","suffix":""},{"id":433376009,"identity":"b80ddd88-8c75-41f3-9b0a-98b717fccb33","order_by":3,"name":"Tomoyuki Fujita","email":"","orcid":"","institution":"Maruho Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Tomoyuki","middleName":"","lastName":"Fujita","suffix":""}],"badges":[],"createdAt":"2025-03-19 10:08:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6260398/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6260398/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":80047833,"identity":"f26f8973-916d-40b0-802b-8fba87bd5902","added_by":"auto","created_at":"2025-04-07 09:58:25","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":70629,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cu\u003eEffects of TNF-α and IFN-γ on the mRNA expression of \u003c/u\u003e\u003cu\u003e\u003cem\u003eCCL17\u003c/em\u003e\u003c/u\u003e\u003cu\u003e, \u003c/u\u003e\u003cu\u003e\u003cem\u003eIL31RA\u003c/em\u003e\u003c/u\u003e\u003cu\u003e, \u003c/u\u003e\u003cu\u003e\u003cem\u003eOSMR\u003c/em\u003e\u003c/u\u003e\u003cu\u003e and \u003c/u\u003e\u003cu\u003e\u003cem\u003eIL6ST\u003c/em\u003e\u003c/u\u003e\u003cu\u003e in HaCaT cells.\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eHaCaT cells were stimulated with TNF-α (0.1, 1, 10 and 100 ng/mL) or IFN-γ (0.1, 1, 10 and 100 ng/mL) for 72 h. The relative expression of \u003cem\u003eCCL17\u003c/em\u003e (a), \u003cem\u003eIL31RA\u003c/em\u003e (b), \u003cem\u003eOSMR\u003c/em\u003e (c), and \u003cem\u003eIL6ST\u003c/em\u003e (d) mRNA was measured by qRT-PCR. \u003cem\u003eGAPDH\u003c/em\u003e was used as the internal control. Data are presented as the mean ± SEM (n = 3). *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, and ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001 vs. 0 ng/mL TNF-α + 0 ng/mL IFN-γ.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6260398/v1/027346f697dbb0b2d154a2e0.jpg"},{"id":80047831,"identity":"9bdf17fe-9a22-4d90-ad26-379dd7c46e35","added_by":"auto","created_at":"2025-04-07 09:58:17","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":89565,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cu\u003eEffects of IL-31, oncostatin M (OSM) and nemolizumab on the mRNA expression of \u003c/u\u003e\u003cu\u003e\u003cem\u003eCCL17, IL31RA, OSMR, and IL6ST\u003c/em\u003e\u003c/u\u003e\u003cu\u003e in HaCaT cells.\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eHaCaT cells were stimulated with TNF-α (0.1 ng/mL) and IFN-γ (100 ng/mL) for 72 h. At the same time, the cells were stimulated with IL-31 (4, 20, 100, and 500 ng/mL), OSM (0.1, 1, and 10 ng/mL), or nemolizumab (10 and 100 µg/mL). The relative mRNA expression of \u003cem\u003eCCL17\u003c/em\u003e (a), \u003cem\u003eIL31RA\u003c/em\u003e (b), \u003cem\u003eOSMR\u003c/em\u003e (c), and \u003cem\u003eIL6ST\u003c/em\u003e(d) was measured by qRT-PCR, and \u003cem\u003eGAPDH\u003c/em\u003e was used as the internal control. Data are represented as the mean ± SEM (n = 3). *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 vs. 0.1 ng/mL TNF-α + 100 ng/mL IFN-γ; %%%\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001 vs. 0.1 ng/mL TNF-α + 100 ng/mL IFN-γ + 4 ng/mL IL-31; \\\\\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, and \\\\\\\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 vs. 0.1 ng/mL TNF-α + 100 ng/mL IFN-γ + 20 ng/mL IL-31; #\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ##\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, and ###\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 vs. 0.1 ng/mL TNF-α + 100 ng/mL IFN-γ + 100 ng/mL IL-31; $$\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01 and $$$\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 vs. 0.1 ng/mL TNF-α + 100 ng/mL IFN-γ + 500 ng/mL IL-31.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6260398/v1/fcece011063e5d9a751ebab3.jpg"},{"id":80045213,"identity":"4c5837ad-d4af-4ce5-af1a-f6f5bf1034bb","added_by":"auto","created_at":"2025-04-07 09:42:13","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":493407,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cu\u003eTranscriptome analysis of keratinocytes treated with IL-31.\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003e(a) Principal component analysis (PCA) plot of the transcriptome by the treatment type (control, TNF-α/IFN-γ stimulation with or without IL-31 (500 ng/mL) and nemolizumab (100 µg/mL)). Each dot in the PCA plot represents a different sample and each color represents different sample annotations. The PC1 and PC2 axis labels indicate the percentage of variance explained by each of these components. (b) A heatmap of the RNA-seq expression z-scores computed for the protein-coding genes. The genes (rows) are clustered using the Pearson’s correlation distance and complete linkage hierarchical clustering. The color code shows the row z-score, with the red color indicating the higher expression of a gene and the blue indicating the lower expression of a gene. (c) Differently expressed genes (DEGs) in a volcano plot with an adjusted \u003cem\u003ep\u003c/em\u003e-value cutoff of 0.05 (NS: not significant, FC: fold-change, Log2FC: significant by |log2FC \u0026gt; 1 or \u0026lt; −1|, \u003cem\u003ep\u003c/em\u003e-value: significant by adjusted \u003cem\u003ep\u003c/em\u003e-value \u0026lt; 0.05; \u003cem\u003ep\u003c/em\u003e-value and Log2FC: significant by both adjusted \u003cem\u003ep\u003c/em\u003e-value \u0026lt; 0.05 and |log2FC \u0026gt; 1 or \u0026lt; −1|). (d) Gene set enrichment analysis (GSEA) of the most enriched gene sets of all the detected genes in each group. The GSEA analysis RidgePlot of top ten significantly enriched Reactome pathway of the gene sets. (e) Ingenuity Pathway Analysis (IPA) graphical summaries of core analysis, including the canonical pathways, upstream regulators, diseases, and biological functions.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6260398/v1/19a07f51b023ac81d6814cea.jpg"},{"id":80047826,"identity":"52c6b3bc-5101-4d96-b984-5d58c4ba8608","added_by":"auto","created_at":"2025-04-07 09:58:13","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":51296,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cu\u003eEffects of IL-31 on differentiation and inflammatory genes\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003e(a), (b) HaCaT cells were stimulated with TNF-α (0.1 ng/mL) and IFN-γ (100 ng/mL) for 72 h. At the same time, cells were stimulated with IL-31 (100 and 500 ng/mL) or nemolizumab (100 µg/mL). The relative mRNA expression of \u003cem\u003eIVL\u003c/em\u003e, \u003cem\u003eKRT10\u003c/em\u003e, \u003cem\u003eIL-1β\u003c/em\u003e, \u003cem\u003eTSLP\u003c/em\u003e and \u003cem\u003eIL-33\u003c/em\u003e was measured by qRT-PCR, and \u003cem\u003eGAPDH\u003c/em\u003e was used as the internal control. Data are represented as the mean ± SEM (n = 3). **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 vs. 0.1 ng/mL TNF-α + 100 ng/mL IFN-γ; #\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ##\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, and ###\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 vs. 0.1 ng/mL TNF-α + 100 ng/mL IFN-γ + 100 ng/mL IL-31; $\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, $$\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, and $$$\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 vs. 0.1 ng/mL TNF-α + 100 ng/mL IFN-γ + 500 ng/mL IL-31.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6260398/v1/77b88b79ec960240d5db610f.jpg"},{"id":80048624,"identity":"60af6917-fe49-4534-9a51-e52bfcefba7c","added_by":"auto","created_at":"2025-04-07 10:06:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1360856,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6260398/v1/12293623-ffca-441c-9940-2493337d4fec.pdf"},{"id":80045208,"identity":"e3ca25f6-52be-441e-b005-5d7cf6160006","added_by":"auto","created_at":"2025-04-07 09:42:13","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":48021,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cu\u003e\u003cstrong\u003eFig. S1\u003c/strong\u003e\u003c/u\u003e\u003cu\u003e Effects of IL-31 and nemolizumab on the mRNA expression of \u003c/u\u003e\u003cu\u003e\u003cem\u003eCCL22\u003c/em\u003e\u003c/u\u003e\u003cu\u003e in HaCaT cells.\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eHaCaT cells were stimulated with TNF-α (0.1 ng/mL) and IFN-γ (100 ng/mL) for 72 h. At the same time, cells were stimulated with IL-31 (100 and 500 ng/mL) or nemolizumab (100 µg/mL). The relative mRNA expression of \u003cem\u003eCCL22\u003c/em\u003e was measured by qRT-PCR, and \u003cem\u003eGAPDH\u003c/em\u003e was used as the internal control. Data are presented as the mean ± SEM (n = 3). ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 vs. 0.1 ng/mL TNF-α + 100 ng/mL IFN-γ and $$\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 vs. 0.1 ng/mL TNF-α + 100 ng/mL IFN-γ + 500 ng/mL IL-31.\u003c/p\u003e","description":"","filename":"Supplementarydata.docx","url":"https://assets-eu.researchsquare.com/files/rs-6260398/v1/efb12d10092ab193d231d375.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"IL-31 suppresses the expression of TARC/CCL17 in TNF-alpha/IFN-gamma-stimulated HaCaT keratinocytes","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAtopic dermatitis (AD) is a prevalent chronic inflammatory skin disease with a complex and heterogeneous etiology. Persistent itching is the most distressing symptom for patients with AD, which results in sleep disruption, anxiety, depression, and decreased productivity at work, thereby diminishing their quality of life [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Moreover, pruritus-induced scratching exacerbates the skin lesions and triggers the itch-scratch cycle, which underlies the pathogenesis of AD [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Thus, controlling pruritus is crucial in treating AD, and novel treatments are expected to improve efficacy and have fewer adverse effects.\u003c/p\u003e \u003cp\u003eInterleukin-31 (IL-31) is a key cytokine that triggers pruritus via various cells including T helper 2 cells, macrophages, dendritic cells, eosinophils, mast cells, basophils, ILC2, as well as fibroblasts and epidermal keratinocytes [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The IL-31 receptor (IL-31R) is a heterodimer composed of IL-31RA and oncostatin M receptor β (OSMRb). It binds to IL-31, and transmits stimuli by activating downstream intracellular signaling pathways such as Janus kinase (JAK)/signal transducer and transcription activator, phosphatidylinositol 3‑kinase (PI3K)/protein kinase B (AKT) and mitogen-activated protein kinase (MAPK) [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Peripheral sensory neurons, epidermal keratinocytes, immune cells, and fibroblasts, express IL-31R that contribute to pruritus, inflammatory responses, skin barrier abnormalities, and fibrosis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] IL-31 is substantially expressed in the skin lesions of patients with AD compared to healthy individuals, and a positive correlation has been established between serum IL-31 levels and disease severity [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. These findings suggest that IL-31 signaling contributes to various pathological aspects of AD, including pruritus.\u003c/p\u003e \u003cp\u003eNemolizumab, a monoclonal antibody against IL-31RA, was developed to inhibit IL-31 signaling, thereby reducing itching in AD. Clinical trials have demonstrated the efficacy of nemolizumab in reducing key features of AD including pruritus in patients with AD who experienced moderate to severe itching, which was inadequately controlled by existing treatments [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Nemolizumab has also shown effectiveness in other diseases, such as prurigo nodularis [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. However, adverse events, including worsening of AD and increased levels of thymus and activation-regulated chemokine (TARC)/C\u0026ndash;C motif chemokine ligand 17 (CCL17), have been observed in some patients. Notably, increased CCL17 levels were not always accompanied by worsening AD symptoms [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The mechanism underlying the increased CCL17 serum levels post-nemolizumab administration as well as the direct and indirect effects of IL-31 on CCL17 expression remain to be investigated.\u003c/p\u003e \u003cp\u003eInterestingly, the expression of CCL17 and other cytokines is increased in tape strips collected from patients with AD compared to healthy controls [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], indicating that these changes may reflect chemokine production from epidermal and immune cells. Moreover, AD-derived keratinocytes express high levels of IL-31R and produce various chemokines such as CCL17, in response to IL-31 expression [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Therefore, we focused on keratinocytes as one of the CCL17-producing cells and examined the effects of IL-31 on CCL17 expression with or without nemolizumab, followed by transcriptome analysis of HaCaT keratinocytes treated with IL-31 and nemolizumab to elucidate the overall impact of IL-31 and its regulation by nemolizumab, including increased CCL17 expression.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Cell culture and reagents\u003c/h2\u003e \u003cp\u003eThe HaCaT human keratinocyte cell line was procured from Cell Lines Service (German Cancer Research Center, Heidelberg, Germany) and cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) supplemented with 10% fetal bovine serum and penicillin/streptomycin (100 U/mL and 100 \u0026micro;g/mL, respectively) at 37\u0026deg;C in a humidified incubator containing 5% CO\u003csub\u003e2\u003c/sub\u003e. An equivalent of 31300 cells/cm\u003csup\u003e2\u003c/sup\u003e was seeded for 24 h to reach confluency before treatment with or without recombinant human tumor necrosis factor-alpha (TNF-α), IL-31, oncostatin M (R\u0026amp;D Systems, Minneapolis, MN, USA), interferon-gamma (IFN-γ) (PeproTech, Rocky Hill, NJ, USA) or nemolizumab (Chugai Pharmaceutical, Tokyo, Japan), and cultured for a further 72 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 RNA isolation\u003c/h2\u003e \u003cp\u003eTotal RNA was isolated from HaCaT cells using a QIA shredder and a RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR)\u003c/h2\u003e \u003cp\u003ecDNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA). TaqMan Gene Expression Master Mix, TaqMan Gene Expression Assays, and TaqMan primer/probes were used for qRT-PCR on a QuantStudio 7 Flex Real-Time PCR System (Thermo Fisher Scientific), with the standard settings. The following sets of primers were purchased from Thermo Fisher Scientific: IL31RA (Hs00371172_m1), OSMR (Hs00384276_m1), IL-6 signal transducer (IL6ST: Hs00174360_m1), CCL17 (Hs00171074_m1), involucrin (IVL: Hs00846307_s1), keratin 10 (KRT10: Hs00166289_m1), CCL22 (Hs01574247_m1), IL-1β (Hs01555410_m1), thymic stromal lymphopoietin (TSLP: Hs00263639_m1), IL-33 (Hs04931857_m1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH: Hs02786624_g1). The expression of the target genes was normalized to that of \u003cem\u003eGAPDH\u003c/em\u003e using the 2\u003csup\u003e\u0026minus;\u0026thinsp;ΔΔCT\u003c/sup\u003e method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Library preparation and sequencing\u003c/h2\u003e \u003cp\u003eRNA sequencing (RNA-Seq) libraries were prepared for sequencing using SMART-Seq V4 ultra-low input RNA (TaKaRa Bio, Shiga, Japan) and Nextera XT DNA Library Prep Kit (Illumina, San Diego, CA, USA), according to the manufacturer\u0026rsquo;s instructions, and sequenced on the NovaSeq X Plus platform (Illumina). Differential expression was established based on at least a twofold difference in the expression levels (|log2 fold change\u0026thinsp;\u0026ge;\u0026thinsp;1|) and an adjusted \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for statistical significance. Thereafter, gene set enrichment analysis (GSEA) was performed using with the R package clusterProfiler and ReactomePA. The canonical pathway, upstream regulator, disease, and biofunction analyses of the differentially expressed genes (DEGs) were conducted using Ingenuity Pathway Analysis (IPA, Qiagen) to compare the differences between the treatments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Statistical analysis\u003c/h2\u003e \u003cp\u003eData are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). The differences between more than two groups were analyzed using Dunnett\u0026rsquo;s multiple comparison test, whereas those between two groups were analyzed using an unpaired t-test. All statistical analyses were performed using EXSUS software (CAC Croit Corp., Tokyo, Japan). A \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicated a statistically significant difference.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Data availability\u003c/h2\u003e \u003cp\u003eRNA-seq data have been deposited in the NCBI Gene Expression Omnibus (GEO) (public repository) and are accessible using the GEO series accession number GSE281127.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 TNF-α and IFN-γ stimulation increased the expression of \u003cem\u003eCCL17\u003c/em\u003e mRNA in HaCaT cells\u003c/h2\u003e \u003cp\u003eFirst, we evaluated the effects of TNF-α and IFN-γ stimulation on the expression of CCL17 in HaCaT cells. The relative expression mRNA of \u003cem\u003eCCL17\u003c/em\u003e was upregulated by stimulation with TNF-α or IFN-γ in a concentration-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), as previously described [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Th1 cytokines, such as TNF-α and IFN-γ, are recognized for stimulating the production of CCL17 in epithelial cells, including the HaCaT keratinocyte cell line. Moreover, co-treatment with TNF-α and low concentrations of IFN-γ at 0.1 and 1 ng/mL increased the expression of CCL17 further compared to stimulation with each agent alone, but not at 10 and 100 ng/mL concentrations of IFN-γ. Conversely, co-stimulation upregulated the expression levels of IL-31 and OSM-associated receptor chains (IL-31RA, OSMR, IL-6ST) compared to stimulation with each agent individually (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eb\u0026ndash;d).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Effects of IL-31 and OSM on the expression of \u003cem\u003eCCL17\u003c/em\u003e mRNA\u003c/h2\u003e \u003cp\u003eNext, we investigated the effect of IL-31 and nemolizumab on the mRNA expression of \u003cem\u003eCCL17\u003c/em\u003e in response to the TNF-α- and IFN-γ-stimulated expression of CCL17 and IL-31 receptor. Consequently, CCL17 expression was downregulated by IL-31 treatment but was significantly reversed by the addition of nemolizumab (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). A similar result was observed for the expression of CCL22, a chemokine related to AD [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] (Fig. S1). OSM is also a neuromodulator of itching, and the expression of OSM and OSMR is increased in AD skin [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Furthermore, similar to IL-31, OSM suppressed the expression of CCL17; however, this effect was not reversed by the addition of nemolizumab (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). IL-31 treatment upregulated the expression of IL-31 and OSM-associated receptor chains, which was significantly reversed by nemolizumab addition, whereas OSM treatment also upregulated their expression, which was not reversed by nemolizumab addition (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eb\u0026ndash;d). These findings indicate that the IL-31/IL-31RA axis regulates the expression of CCL17 in keratinocytes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Effects of IL-31 differentiation markers and inflammatory cytokine genes\u003c/h2\u003e \u003cp\u003eRNA-seq analysis was performed to examine the IL-31- and nemolizumab-induced gene expression changes. The principal component analysis (PCA) plots for the normalized abundance values revealed that 66.21% of the variance could be explained by the first two principal components (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The mRNA expression profile of the protein-coding genes between the four groups is shown by hierarchical clustering (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). The influence of IL-31 or nemolizumab on gene expression profile is illustrated in the volcano plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). DEGs with an adjusted \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 between the TNF-α\u0026thinsp;+\u0026thinsp;IFN-γ and TNF-α\u0026thinsp;+\u0026thinsp;IFN-γ\u0026thinsp;+\u0026thinsp;IL-31 treatments were related to the epidermal growth factor (EGF) receptor family, including amphiregulin (AREG), neuregulin 1 (NRG1), heparin-binding EGF-like growth factor and epiregulin (EREG) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). In addition, the differentiation markers keratin 13 (KRT13) and KRT15 were downregulated. The combined treatment with IL-31 and nemolizumab reversed the expression of several genes, such as \u003cem\u003eNRG1\u003c/em\u003e and \u003cem\u003eEREG\u003c/em\u003e, compared to IL-31 treatment alone. As IL-31 is involved in the differentiation of keratinocytes [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], we examined the effects of this cytokine on the expression of differentiation markers (IVL and KRT10) by qRT-PCR. IL-31 decreased the mRNA expression of \u003cem\u003eIVL\u003c/em\u003e and \u003cem\u003eKRT10\u003c/em\u003e, which was reversed by the expression of differentiation markers (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). These results suggested that while IL-31 altered keratinocyte differentiation, nemolizumab could restore it.\u003c/p\u003e \u003cp\u003eGSEA was used to determine whether the identified gene sets differed significantly between each group. The analysis was performed using Reactome terms for a comprehensive understanding of the biological importance of the perturbation by IL-31 and nemolizumab treatment in keratinocytes. The top ten upregulated and downregulated terms from GSEA presented (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Between the TNF-α\u0026thinsp;+\u0026thinsp;IFN-γ and TNF-α\u0026thinsp;+\u0026thinsp;IFN-γ\u0026thinsp;+\u0026thinsp;IL-31 treatments, the positively enriched terms included \u0026ldquo;Interleukin-4 and Interleukin-13 signaling\u0026rdquo; and \u0026ldquo;Signaling by Interleukin,\u0026rdquo; which was consistent with the expected results. The negatively enriched terms included \u0026ldquo;Fatty acid metabolism.\u0026rdquo; In contrast, between the TNF-α\u0026thinsp;+\u0026thinsp;IFN-γ\u0026thinsp;+\u0026thinsp;IL-31 and TNF-α\u0026thinsp;+\u0026thinsp;IFN-γ\u0026thinsp;+\u0026thinsp;IL-31\u0026thinsp;+\u0026thinsp;nemolizumab treatments, the negatively enriched terms included \u0026ldquo;Signaling by Interleukin,\u0026rdquo; and the positively enriched terms included \u0026ldquo;Fatty acid metabolism.\u0026rdquo; Furthermore, network analysis was performed using IPA, which revealed IL-31 treatment activated the EGF and related pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). Conversely, nemolizumab treatment suppressed these pathways and inhibited the cytokine-related pathways, including TNF, IL-1A and IL-1B. In addition to IL-1B, the inflammatory cytokines TSLP and IL-33 are highly expressed in the skin lesions of patients with AD [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The IL-31-upregulated mRNA expression of each cytokine was suppressed by co-treatment with nemolizumab, which was confirmed by qRT-PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). These results suggest that nemolizumab suppresses multiple inflammatory cytokines by inhibiting the binding of IL-31 to IL-31RA and exerts an anti-inflammatory effect.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eNemolizumab administration improves pruritus in AD while also increasing the serum levels of CCL17 in some patients. However, there appears to be little correlation between increased CCL17 levels and AD symptom severity in these patients, a pattern consistent with previous reports on JAK inhibitors [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. These underlying mechanisms have not yet been elucidated, thus necessitating further investigation to address the clinical aspects regarding the upregulated levels of CCL17 post-drug treatment. In this study, we assessed the effect of IL-31 on the expression of CCL17 in keratinocytes, which are one of the major sources of CCL17 production. In line with previous findings [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], we observed that the expression of CCL17 was elevated in HaCaT cells that were co-stimulated with TNF-α and IFN-γ but was reduced by the addition of IL-31. Interestingly, IL-31 stimulation increased the expression of its own receptor (IL31RA and OSMR) in the presence of TNF-α and IFN-γ. These results suggest that IL-31 stimulation may induce a positive feedback loop in the IL-31 signaling pathway, thus further driving the downregulation of CCL17 expression.\u003c/p\u003e \u003cp\u003eThe expression of TNF-α, IFN-γ, and IL-31 is elevated in the skin lesions of patients with AD [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Recent technological advances, such as single-cell RNA-seq, have elaborated on the sources of cytokine production in AD lesions and their involvement in the pathogenesis. Particularly, the interactions between different cell types and their roles in specific disease states can now be comprehensively understood [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. TNF-α is produced by immune cells, such as macrophages, dendritic cells, and keratinocytes, which promotes skin inflammation and impairs the skin barrier function [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Similarly, IFN-γ is a cytokine secreted by Th1 cells, and plays a crucial role in regulating the inflammatory responses of AD [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The stimulation conditions in this in vitro study using TNF-α and IFN-γ reflect the disease state of patients with AD. Our findings suggest that the action of IL-31 suppresses the expression of CCL17, which is reversed by nemolizumab treatment. This may explain the upregulated levels of CCL17 in some of the patients with AD who are treated with nemolizumab. Nonetheless, the promotive effect of IL-31 on CCL17 expression is also reported in keratinocytes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Therefore, IL-31 may exert varying effects on CCL17 expression depending on specific conditions.\u003c/p\u003e \u003cp\u003eOSM belongs to the IL-6 family of cytokines and shares a common signaling receptor subunit (OSMR) with IL-31. It is known for its increased expression of OSM and OSMR in the skin lesion in patients with AD as well as increased production of OSM from skin-infiltrating T cells [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Moreover, OSM acts as a neuromodulator of itching in diseases such as psoriasis and cutaneous T cell lymphoma, which are characterized by chronic itching, including AD [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Furthermore, the intradermal administration of OSM-encoding adenovirus induces robust skin inflammation with epidermal hyperplasia and increases the expression of chemokines and cytokines in the ear, thus indicating OSM\u0026rsquo;s contribution to inflammatory responses [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In addition, OSM induces the production of CCL11 from fibroblasts and CCL21 from vascular endothelial cells [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. These findings suggest that OSM exacerbates the inflammatory responses and itching in patients with AD. To the best of our knowledge, this study is the first to reveal the inhibitory effect of OSM on CCL17 expression, which is not reversed by nemolizumab addition. Another study hypothesized that nemolizumab-mediated inhibition of IL-31 signaling could facilitate the interaction between the free OSMR subunit i.e., OSMRb and gp130 (IL6ST) on the cell membrane, which result in an overabundance of functional receptors, thus enhancing the responsiveness to OSM [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, this hypothesis was not supported in our experiments. Alternatively, we propose that OSM may act via a mechanism independent of the IL-31/IL-31RA axis, with variations depending on the cell type and stimulation conditions. Conversely, OSM released by monocytes in the skin suppresses the IL-31-stimulated itching by inhibiting IL-31RA expression in the dorsal root ganglia [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Therefore, IL-31 and OSM could regulate inflammatory mediators through their interaction and control itching in patients with AD.\u003c/p\u003e \u003cp\u003eBulk RNA-seq analysis revealed a direct effect of IL-31 on CCL17 expression, keratinocyte differentiation, lipid metabolism, and the regulation of inflammatory cytokines. In addition, IL-31 impairs the skin barrier function by inhibiting the expression of filaggrin [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The intradermal administration of IL-31 in mice induced epidermal thickening and increased transepidermal water loss [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. EGF controls various biological actions, such as survival, migration, and proliferation, and plays a crucial role in regulating the skin barrier functions and inflammatory responses in AD [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Our RNA-seq analysis indicated that IL-31 treatment activated the EGF pathway. In a previous report, IL-31 increased the expression of the transcription factor EGR1 in keratinocytes [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] and EGF expression in bronchial epithelial cells [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], which suggests the involvement of IL-31 in EGF signaling. Conversely, EGF inhibitors upregulate the expression of CCL17 in keratinocytes [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], thus indicating that EGF signaling negatively regulates CCL17 expression, which is consistent with the effects of IL-31 treatment observed in this study. Although it is unclear whether the effect is direct or indirect, IL-31 treatment might modulate CCL17 expression through the EGF pathway.\u003c/p\u003e \u003cp\u003eMoreover, we confirmed the downregulation of fatty acid metabolism pathways following IL-31 treatment. Previous studies have characterized AD by aberrant lipid metabolism, resulting in an impaired epidermal barrier, which contributes to inflammation and emphasizes the critical role of lipids in AD pathogenesis [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The effects of IL-31 on lipid metabolism have been investigated using three-dimensional tissue engineered human skin equivalents. The studies showed an increase in long-chain free fatty acids and a decrease in the content of ester-linked ω-hydroxy ceramides [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Although the role of IL-31 in lipid metabolism is yet, skin lipid metabolism is shown to affect the skin barrier function and immune responses; lipid alterations are becoming clearer with the development of omics technology [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Further basic and clinical studies on the regulation of lipid metabolism in the skin of patients with AD would provide in-depth insights into the involvement of IL-31 and pathogenesis via lipid alterations.\u003c/p\u003e \u003cp\u003eThe activation of IL-31 signaling produces several cytokines and chemokines in various cell types. In keratinocytes, IL-31 induces the production of IL-1α and regulates the skin barrier function [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In addition, inflammatory cytokines (IL-1β and IL-6) and chemokines (CXCL1/8/10 and CCL2/18) are released from eosinophils in response to IL-31 stimulation. This release is further enhanced when eosinophils are co-cultuered with either keratinocytes or fibroblasts [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Recently, IL-31 induces the production of inflammatory mediators (TNF-α, IL-6, CXCL8, and CCL2/5/22) in dendritic cells, and the production of IL-1β and IL-6 in macrophages [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. These findings suggest that IL-31 exacerbates inflammation by promoting the production of cytokines. The transcriptome analysis of prurigo nodularis showed that nemolizumab treatment suppressed inflammatory cytokines, including IL-13 and IFN-γ [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], which suggests a similar possibility of the downregulation of cytokines in AD. In this study, the IL-31-induced production of inflammatory cytokines, such as IL-1β, TSLP and IL-33, was suppressed by nemolizumab treatment, thus indicating an anti-inflammatory effect of nemolizumab and improving AD pathology.\u003c/p\u003e \u003cp\u003eDespite its novel findings, this study has some limitations. First, although increased IL-31 expression has been reported in the lesions of patients with AD [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], the concentration of IL-31 used in this study may not accurately reflect levels on lesional skin. Second, this study was performed using HaCaT keratinocyte monocultures only. The association between IL-31, EGF signaling, and lipid metabolism could not be fully elucidated by the transcriptome analysis. As AD has a complex and heterogeneous etiology, further research is needed to fully elucidate the mechanisms of IL-31 and nemolizumab treatment.\u003c/p\u003e \u003cp\u003eIn conclusion, this study demonstrated that IL-31 acts as a repressive regulator of the expression of CCL17 in keratinocytes in vitro, and nemolizumab treatment reverses this inhibitory effect, potentially upregulating the expression of CCL17 in the serum of patients with AD after nemolizumab administration. In addition to the direct effect on CCL17 expression, IL-31 also affected keratinocyte differentiation, lipid metabolism, and induced inflammatory cytokines, which suggests multiple roles of IL-31 in the pathogenesis of AD and the regulation of itching and inflammation. However, we performed the study using only HaCaT ketatinocyte to discover the mechanism of IL-31. To elucidate the clinical significance of IL-31 inhibition, future research should focus on the signaling pathway using samples isolated from patients with AD after nemolizumab administration.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors are employees of Maruho Co. Ltd.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eSupporting information\u003c/h2\u003e \u003cp\u003eSupplementary data associated with this article can be found in the online version.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study was supported by Maruho Co., Ltd.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM.I. and Y.Y. conceived and designed the study. M.I. conducted the experiments, collected the data, carried out the statistical analyses, and drafted the initial manuscript. M.I. and T.T. were involved in the data analysis and interpretation. T.F. and Y.Y. provided supervision throughout the study and contributed to the review and editing of the manuscript. All authors participated in drafting or critically revising the manuscript for important intellectual content and approved the final version to be published.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe greatly thank Takuya Yokoyama for support throughout the experiments.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlkon N, Assen FP, Arnoldner T, Bauer WM, Medjimorec MA, Shaw LE et al (2023) Single-cell RNA sequencing defines disease-specific differences between chronic nodular prurigo and atopic dermatitis. J Allergy Clin Immunol 152(2):420-435. https://doi.org/10.1016/j.jaci.2023.04.019\u003c/li\u003e\n\u003cli\u003eAlkon N, Bauer WM, Krausgruber T, Goh I, Griss J, Nguyen V et al (2022) Single-cell analysis reveals innate lymphoid cell lineage infidelity in atopic dermatitis. 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Br J Dermatol. https://doi.org/10.1093/bjd/ljae131\u003c/li\u003e\n\u003cli\u003eYosipovitch G, Berger T, Fassett MS (2020) Neuroimmune interactions in chronic itch of atopic dermatitis. J Eur Acad Dermatol Venereol 34(2):239-250. https://doi.org/10.1111/jdv.15973\u003c/li\u003e\n\u003cli\u003eZhang Q, Putheti P, Zhou Q, Liu Q, Gao W (2008) Structures and biological functions of IL-31 and IL-31 receptors. Cytokine Growth Factor Rev 19(5-6):347-356. https://doi.org/10.1016/j.cytogfr.2008.08.003\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-dermatological-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Archives of Dermatological Research](https://www.springer.com/journal/403)","snPcode":"403","submissionUrl":"https://submission.nature.com/new-submission/403/3","title":"Archives of Dermatological Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"atopic dermatitis, TARC/CCL17, keratinocyte, HaCaT cell, IL-31","lastPublishedDoi":"10.21203/rs.3.rs-6260398/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6260398/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe most common symptom of atopic dermatitis (AD) is itching, which results in sleep disturbances and psychological stress, substantially reducing the quality of life of patients. Nemolizumab is a monoclonal antibody targeting interleukin 31 receptor A (IL-31RA) and mitigates the itching in AD by inhibiting the activity of IL-31, a cytokine mainly produced by T helper 2 cells. However, elevated levels of thymus and activation-regulated chemokine (TARC)/C\u0026ndash;C motif chemokine ligand 17 (CCL17), a marker for disease severity in AD, were observed in some patients with AD following nemolizumab administration, although these patients almost did not exhibit an exacerbation of AD symptoms. This study aimed to elucidate the possible mechanism underlying the upregulation of CCL17 with nemolizumab treatment in some patients. We evaluated the effects of IL-31 and nemolizumab on the mRNA expression of \u003cem\u003eCCL17\u003c/em\u003e in TNF-alpha/IFN-gamma-stimulated HaCaT keratinocytes in vitro using qRT-PCR. In addition, we comprehensively analyzed the impact of IL-31 and nemolizumab on keratinocyte-related phenomena, using RNA sequencing. Our findings indicated that IL-31 suppressed the expression of \u003cem\u003eCCL17\u003c/em\u003e mRNA in keratinocytes, and nemolizumab reversed this suppressive effect. We further discovered that although oncostatin M suppressed the expression of \u003cem\u003eCCL17\u003c/em\u003e mRNA, its action was unaffected by nemolizumab. Furthermore, IL-31 affected keratinocyte differentiation, lipid metabolism, and inflammatory cytokine. Herein, we propose a possible mechanism by which nemolizumab counteracts the inhibitory effect of IL-31 on CCL17 expression, thus explaining the increased CCL17 levels following nemolizumab administration in some patients with AD.\u003c/p\u003e","manuscriptTitle":"IL-31 suppresses the expression of TARC/CCL17 in TNF-alpha/IFN-gamma-stimulated HaCaT keratinocytes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-07 09:42:08","doi":"10.21203/rs.3.rs-6260398/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-10T19:57:04+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-26T09:07:20+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-15T11:19:50+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-12T04:39:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"183501365967144221604867614455326459575","date":"2026-01-11T02:07:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"56996762950137929925899717844726110800","date":"2026-01-09T06:43:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"94978915506511875755658836440387435326","date":"2026-01-09T00:39:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-08T22:43:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"296871524058049815287004724539790060805","date":"2026-01-08T21:49:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-08T20:51:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"273445763871616350678531129392353893747","date":"2026-01-08T19:46:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-24T10:30:17+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-20T09:06:50+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-20T09:03:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Dermatological Research","date":"2025-03-19T09:56:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-dermatological-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Archives of Dermatological Research](https://www.springer.com/journal/403)","snPcode":"403","submissionUrl":"https://submission.nature.com/new-submission/403/3","title":"Archives of Dermatological Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a766d19f-4f2d-428f-9731-f4fdc89d06a5","owner":[],"postedDate":"April 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-02-20T03:53:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-07 09:42:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6260398","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6260398","identity":"rs-6260398","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}