Type I Interferon induces Keratinocytes Necroptosis via the ZBP1-MLKL Axis in Oral Lichen Planus

Type I Interferon induces Keratinocytes Necroptosis via the ZBP1-MLKL Axis in Oral Lichen Planus | 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 Type I Interferon induces Keratinocytes Necroptosis via the ZBP1-MLKL Axis in Oral Lichen Planus Xinke Jiang, Junjun Chen, Yilin Yao, Yirao Lai, Xiaojie Yang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7981446/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Apr, 2026 Read the published version in Inflammation → Version 1 posted 12 You are reading this latest preprint version Abstract Oral lichen planus (OLP) is a prevalent chronic inflammatory condition that affects the oral mucosa. Histologically, it is characterized by the liquefaction and degeneration of basal epithelial cells, indicating a disruption of the basal layer architecture, which may represent an early event in the disease’s pathogenesis. However, the molecular mechanisms underlying this epithelial damage remain poorly understood. In our study, histological staining and transmission electron microscopy revealed aberrant cell death in the basal epithelial layer of OLP tissues. Immunodetection demonstrated the presence of phosphorylated mixed lineage kinase domain-like protein (pMLKL), a key marker of necroptosis, specifically localized to basal keratinocytes. Notably, interferon type I (IFN-I), particularly IFNα, was significantly upregulated in OLP mucosa compared to healthy controls. The expression of Z-DNA binding protein 1 (ZBP1), an IFN-stimulated gene and an upstream regulator of necroptosis, was elevated in pMLKL-positive epithelial cells. In vitro stimulation with IFNα2a induced the expression of ZBP1 and pMLKL in HaCaT keratinocytes, while ZBP1 knockdown abrogated MLKL phosphorylation. Collectively, these results suggest that, in the context of increased IFN-I signaling, ZBP1 is aberrantly upregulated in the OLP epithelium, promoting necroptosis in basal keratinocytes. This necroptotic activity may contribute to the damage and disruption of the basal layer, providing novel insights into the pathogenesis of OLP and highlighting potential molecular targets for therapeutic intervention. oral lichen planus keratinocyte necroptosis mixed lineage kinase domain-like protein Z-DNA binding protein 1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Oral lichen planus (OLP) is a common chronic inflammatory disorder of the oral mucosa, affecting approximately 1.01% of the global population[ 1 , 2 ]. The characteristic histopathological features of OLP include band-like lymphocytic infiltration in the lamina propria and liquefactive degeneration of basal keratinocytes[ 1 , 3 , 4 ]. Recent evidence has demonstrated that keratinocytes actively regulate mucocutaneous inflammatory responses[ 5 , 6 ]. In the local microenvironment of OLP, keratinocytes can be activated by external stimuli, resulting in the abnormal secretion of inflammatory mediators[ 1 ]. These findings suggest that keratinocytes in OLP are not merely passive targets of inflammation but may actively participate in disease modulation or initiation. Notably, the abnormal death of basal keratinocytes may represent a critical pathogenic event. Current research indicates that activated CD8⁺ cytotoxic T lymphocytes within OLP lesions can induce apoptosis, a non-lytic process that does not release intracellular contents, in basal keratinocytes[ 1 , 7 ]. However, ultrastructural analyses using transmission electron microscopy have revealed ruptured plasma membranes and cytoplasmic vacuolization in basal cells of OLP lesions, suggesting leakage of intracellular contents[ 8 ]. These findings imply that non-apoptotic forms of programmed cell death may also be aberrantly activated in OLP. Necroptosis, the most common form of lytic cell death, is a highly regulated, pro-inflammatory process that occurs more rapidly than apoptosis and is associated with the release of pro-inflammatory cytokines and damage-associated molecular patterns (DAMPs)[ 9 , 10 ]. Upon activation, the necroptosis signaling cascade involves the phosphorylation of receptor-interacting serine/threonine-protein kinase 3 (RIPK3), which subsequently activates mixed lineage kinase domain-like protein (MLKL)[ 11 , 12 ]. Phosphorylated MLKL (pMLKL) oligomerizes and translocates to the plasma membrane, where it forms pores, ultimately leading to cell swelling and membrane rupture[ 13 , 14 ]. Therefore, investigating the mechanism of necroptosis in the abnormal death of OLP epithelium is of great significance. Recent studies have highlighted the aberrant activation of interferon type I (IFN-I) signaling in various autoimmune diseases, including systemic lupus erythematosus and psoriasis, where it contributes to disease progression and tissue injury[ 15 – 17 ]. Z-DNA binding protein 1 (ZBP1) is a cytoplasmic nucleic acid sensor and a well-characterized IFN-I–inducible gene[ 18 ]. It contains an N-terminal Zα domain that specifically recognizes and binds left-handed Z-form nucleic acids (Z-DNA and Z-RNA), thereby initiating necroptotic signaling[ 19 , 20 ]. Based on our previous observations of IFN-I pathway activation in OLP lesions, we hypothesize that elevated IFN-I expression in OLP leads to aberrant upregulation of ZBP1 in basal keratinocytes. This may, in turn, activate the ZBP1–MLKL signaling axis, ultimately triggering necroptosis. In this study, we present the first evidence of necroptotic cell death occurring in the basal keratinocytes of OLP lesions, providing a novel mechanistic perspective on the pathogenesis of OLP. Additionally, we elucidate the aberrant induction of the key regulatory molecule ZBP1 under elevated IFN-I signaling conditions, which subsequently leads to the phosphorylation of MLKL. Collectively, these findings offer new insights into the initiation of immune responses in OLP and propose potential molecular targets for future therapeutic interventions. Methods and materials Human mucosa sample collection A total of 30 patients with clinically and histopathologically confirmed OLP and 30 age- and sex-matched healthy controls were recruited from the Department of Oral Medicine at Shanghai Ninth People’s Hospital, China, between December 2022 and July 2024. All participants provided written informed consent prior to sample collection. OLP tissues were obtained through 8-mm punch biopsies of the lesional buccal mucosa. Control tissues were collected from excess mucosa removed during orthognathic surgery. Immediately after removal, one-third of each tissue specimen was fixed for histological analysis, while the remaining two-thirds were dissected to isolate the epithelial component for subsequent experiments. All biopsy procedures were conducted by experienced oral surgeons. The study protocol was approved by the Ethics Committee of Shanghai Ninth People’s Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine (No. SH9H-2021-T100-2) and adhered to the principles of the Declaration of Helsinki. Data acquisition, processing and analysis The single-cell RNA sequencing (scRNA-seq) dataset GSE211630 (5 OLP, 1 healthy mucosa) was obtained from the Gene Expression Omnibus (GEO). Count matrices were processed in Seurat (R): integrated, principal component analysis (PCA)-reduced, clustered (k-nearest neighbor (kNN) / shared nearest neighbor (SNN)), and visualized (Uniform Manifold Approximation and Projection (UMAP)/t-SNE). Cell clusters were annotated (SingleR, CellMarker, PanglaoDB, HCA) using marker genes (FindMarkers). Subpopulations were isolated, re-clustered, and analyzed. Differentially expressed genes (DEGs) underwent functional enrichment (ClusterProfiler/gseaplot2). Additionally, bulk RNA-seq was performed on fresh samples (3 OLP, 3 healthy epithelium). Raw reads underwent QC (FastQC), trimming, alignment (HISAT2/STAR), and quantification (Cufflinks/featureCounts/HTSeq). Data and clinical info were analyzed in RStudio: Principal component analysis (PCA) for outliers/variability, DEG identification (DESeq2), and visualization (heatmaps). Cell culture HaCaT cells, a human epidermal keratinocyte cell line, were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Corning) supplemented with 15% fetal bovine serum (FBS; Gibco) and Penicillin-Streptomycin (100 µg/mL penicillin and 100 U/mL streptomycin; Gibco). The cell line was authenticated through short tandem repeat sequence analysis and maintained in a cell culture incubator (Thermo) at 37°C with 5% CO 2 . IFNα2a (3SBIO) was stored at -4°C and diluted in culture medium to a concentration of 1500 IU/mL prior to application. Birinapant (MCE) was prepared as a 5 mM stock solution in DMSO, stored at -20°C, and applied at a final concentration of 5 µM after dilution in culture medium. Q-VD-OPH (MCE) was prepared as a 10 mM stock solution in DMSO, stored at -20°C, and applied at a final concentration of 10 µM following dilution in culture medium. HaCaT cells were genetically modified to overexpress or knockout target genes using lentiviral vectors. The cells were transduced with lentiviruses and selected with appropriate antibiotics to establish stable overexpression or knockout lines. All target sequences utilized for overexpression and knockout are provided in the supplementary materials. Quantitative real-time PCR (qPCR) Total RNA was extracted from samples using FreeZol Reagent (Vazyme) according to the manufacturer’s instructions. The concentration and purity of the RNA were assessed spectrophotometrically. Reverse transcription was conducted using HiScript III All-in-One RT SuperMix Perfect for qPCR (Vazyme) to synthesize complementary DNA (cDNA). Quantitative real-time PCR was performed with SYBR Green-based detection on a real-time PCR system (Thermo), and reactions were prepared according to the manufacturer’s recommendations. Relative gene expression was calculated using the 2^−ΔΔCt method with an appropriate endogenous control. The primer sequences are provided in the supplementary materials. Immunoblotting Cells and tissues were lysed using RIPA lysis buffer (Beyotime) supplemented with protease and phosphatase inhibitors (Beyotime) via a low-temperature grinder (Wonbio) at 4°C. The lysates were incubated on ice for 20 minutes and subsequently cleared by high-speed centrifugation at 12,000 rpm for 15 minutes at 4°C. After reserving 5% of the supernatants for the BCA assay, the remaining supernatants were denatured with SDS-PAGE sample loading buffer and then subjected to Western blotting (WB) analysis. The sources and dilutions of primary antibodies are detailed in the supplementary materials. Protein bands were visualized using an e-Blot touch imager (e-Blot). The intensity of each band was quantified and normalized against β-actin as an internal loading control using ImageJ software. Immunostaining, histology, and electron microscopy For electron microscopy, fresh tissue (∼2 mm³) was sequentially fixed in 2.5% glutaraldehyde and 1% osmium tetroxide (4°C), dehydrated, infiltrated, and embedded. Regions of interest were identified on toluidine blue-stained semi-thin sections (1 µm) by light microscopy. Ultrathin sections (∼50 nm) were examined by transmission electron microscopy (TEM) (FEI TF20). For histology and immunohistochemistry (IHC) analysis, hematoxylin and eosin (HE) staining and IHC were performed using commercial kits per manufacturer's instructions. For IHC/multiplex IHC (mIHC; TSA kit, Yuanxi), 4% paraformaldehyde-fixed, paraffin-embedded tissues were sectioned (3 µm), deparaffinized, underwent antigen retrieval, permeabilization, blocking, and incubation with primary antibodies followed by HRP-conjugated secondary antibodies and tyramide-fluorophores. For immunofluorescence (IF), cells on glass slides were fixed (4% paraformaldehyde), permeabilized, blocked, and incubated with primary and fluorophore-conjugated secondary antibodies. For imaging, all sections/slides were mounted in antifade reagent (Beyotime). Confocal microscopy (Zeiss LSM 700) and Zeiss ZEN software were used for fluorescence imaging and analysis. Primary antibody details are provided in Supplementary Materials. Statistical analysis Statistical analyses were performed using GraphPad Prism Software. Differences between groups were assessed using the Student’s t -test or one-way ANOVA. A p -value of less than 0.05 was considered statistically significant for all analyses. Results Enhanced epithelial cell death signaling in OLP We retrieved scRNA-seq datasets related to OLP from the GEO (Fig. 1 A). Dimensionality reduction was conducted using UMAP with dimensions set to 1:10 and point size at 0.5 (Fig. 1 B). Based on cluster-specific marker genes, the cells were classified into eight major populations (Fig. 1 C). UMAP plots and bar graphs indicated altered cellular composition across OLP subtypes and controls (Fig. 1 D, E), revealing a significant reduction of epithelial cells in erosive OLP (EOLP) compared to non-erosive OLP (NEOLP) and controls. This finding suggests excessive basal epithelial cell death may be involved in the pathogenesis of OLP. To further investigate pathological changes, HE staining of OLP and control tissues revealed disruption of the basement membrane and liquefactive degeneration of basal cells, which was more pronounced in EOLP (Fig. 1 F). TUNEL staining demonstrated a marked increase in cell death within OLP tissues, particularly in the basal layer (Fig. 1 G). mIHC illustrated disrupted epithelial structure, discontinuous expression of keratin 14 (K14), and dense lymphocytic infiltration (Fig. 1 H). TEM revealed plasma membrane rupture and cytoplasmic vacuolization in basal layer cells of OLP tissues (Fig. 1 I), suggesting the involvement of non-apoptotic programmed cell death. Necroptosis in OLP epithelial cells To investigate programmed cell death in the basal cells of OLP, we conducted Gene Set Enrichment Analysis (GSEA) on epithelial subpopulations derived from scRNA-seq data. This analysis revealed significant activation of necroptosis in the OLP epithelium (Fig. 2 A). To validate these findings, we analyzed lesional oral mucosa for cell death markers. Western blot analysis demonstrated the presence of cleaved CASPASE-3 (an apoptosis marker) and phosphorylated MLKL (a necroptosis marker) in the OLP epithelium, with semi-quantitative analysis confirming elevated levels of pMLKL compared to controls (Fig. 2 B), suggesting enhanced necroptosis. mIHC of paraffin-embedded sections detected cleaved CASPASE-3 and pMLKL in the basal layer, with no evidence of co-localization (Fig. 2 C), indicating distinct forms of cell death, with necroptosis being predominant. Additional IHC revealed that pMLKL signals were extensively distributed in degenerative basal keratinocytes (Fig. 2 D), thereby linking necroptosis to abnormal basal cell death in OLP. Activation of IFN-I pathway in OLP To investigate the activation of immune pathways in the epithelium of OLP, we analyzed scRNA-seq data from GEO. Epithelial cells from five OLP samples demonstrated significant activation of the IFN-I signaling pathway, characterized by the upregulation of multiple interferon-stimulated genes (ISGs). Notably, IRF7 and IFIH1 exhibited particularly strong transcriptional induction (Fig. 3 A). To validate these findings, qPCR of lesional mucosa from ten OLP patients compared to three controls confirmed increased expression of IFNA (Fig. 3 B), indicating a heightened IFN-I microenvironment that may contribute to immune dysregulation. Moreover, bulk RNA sequencing of isolated epithelial layers revealed an enrichment of IFN-I-related pathways through Gene Ontology (GO) analysis (Fig. 3 C), with five ISGs consistently upregulated (Fig. 3 D). To assess whether IFN-I induces necroptosis in keratinocytes, we treated HaCaT cells with IFNα2α. A positive control group received IFNα2α in combination with Q-VD-OPH and Birinapant. Immunofluorescence staining demonstrated distinct pMLKL-positive cells following treatment with IFNα2α (Fig. 3 E), although the number was lower than that observed in the positive control. This suggests that keratinocytes are sensitive to IFN-I-induced necroptotic signaling, and that keratinocyte necroptosis may play a role in the pathogenesis of OLP. ZBP1 expression and necroptotic signaling in OLP Given that IFN-I has been shown to promote necroptosis in keratinocytes, we subsequently explored potential regulators that mediate this process. ZBP1, a cytosolic nucleic acid sensor, is known to activate necroptosis via the RIPK3-MLKL pathway. Therefore, we examined its expression in the epithelium of OLP. Transcriptional and protein analyses revealed significantly elevated levels of ZBP1 in OLP compared to control samples (Fig. 4 A, B), indicating aberrant epithelial activation and a potential role in the pathogenesis of OLP. Additionally, mIHC staining demonstrated the colocalization of ZBP1, pRIPK3, and pMLKL in the basal epithelial cells of OLP (Fig. 4 C), suggesting that ZBP1 may mediate necroptosis through the activation of the RIPK3-MLKL pathway. In HaCaT cells, ZBP1 overexpression markedly increased pMLKL levels, while the RIPK3 inhibitor GSK-872 reduced pMLKL levels in a dose-dependent manner (Fig. 4 D), confirming that ZBP1 promotes necroptosis via the RIPK3-MLKL pathway. ZBP1-mediated necroptosis in keratinocytes under IFN-I stimulation To assess the expression of ZBP1 in keratinocytes following IFN-I stimulation, HaCaT cells were treated with IFNα2α for 24 hours and subsequently analyzed through transcriptome sequencing (Fig. 5 A). The transcription of ZBP1 was significantly elevated in the IFNα2α group (Fig. 5 B), and WB confirmed increased protein levels in both IFNα2α-treated and positive control cells across three biological replicates (Fig. 5 C). These results indicate that IFN-I promotes the upregulation of ZBP1 in keratinocytes. IF staining demonstrated colocalization of ZBP1 and pMLKL in IFNα2α-treated HaCaT cells. In contrast, ZBP1 knockout cells did not exhibit pMLKL expression upon IFNα2α treatment (Fig. 5 D), thereby confirming the necessity of ZBP1 for IFN-I–induced necroptosis. A schematic diagram was created to illustrate the proposed mechanism (Fig. 5 E). Discussion In OLP tissues, colloid bodies are frequently observed in both the basal and suprabasal layers. These structures have traditionally been attributed to keratinocyte apoptosis triggered by dysregulated immune responses, with CD8⁺ T cells identified as key effector cells. In addition to colloid bodies, liquefactive degeneration of basal keratinocytes is also evident. Both the previous report[ 8 ] and our ultrastructural analyses reveal plasma membrane disruption in basal cells, suggesting the involvement of a non-apoptotic form of programmed cell death. This process likely contributes to epithelial injury and basement membrane compromise, which in turn facilitates immune cell infiltration along chemokine gradients, thereby amplifying tissue damage in OLP. In healthy oral epithelium, K4 and K14 exhibit distinct spatial expression patterns, localized to the suprabasal and basal layers, respectively. In contrast, OLP lesions demonstrate an altered distribution, with K14 expression extending throughout the entire epithelial layer and a significant reduction in K4 expression. These changes indicate disrupted epithelial differentiation in OLP. The expanded K14 expression likely reflects basal cell hyperproliferation and impaired stratification, while the diminished K4 expression may suggest compromised functionality of terminally differentiated cells. A previous study has shown that epidermis-specific RIPK1 knockout in transgenic mice leads to keratinocyte necroptosis and skin inflammation, accompanied by epidermal hyperplasia and increased expression of K6 and K10 during early postnatal stages[ 21 ]. This hyperplastic phenotype parallels the suprabasal thickening observed in NEOLP lesions in our study. Collectively, these findings imply that aberrant epithelial proliferation and differentiation, potentially driven by necroptotic signaling, may contribute to disease progression in OLP. Recent studies on necroptosis have primarily concentrated on death receptor pathways, particularly the TNF receptor (TNFR) pathway. Under pro-apoptotic conditions, death receptors can initiate apoptosis; however, the inhibition of apoptosis may redirect the cellular response toward necroptosis[ 22 , 23 ]. In OLP, TNFα mRNA has been detected in lesional T cells, and serum TNFα levels are elevated[ 24 ]. Further research suggests that CD8⁺ T cells may secrete TNFα, which binds to TNFR1 on keratinocytes, thereby triggering the apoptosis of basal cells[ 25 ]. Clinically, TNFα inhibitors have been approved for the treatment of psoriasis[ 26 ]; however, case reports reveal the occurrence of psoriasis-like oral lesions following infliximab therapy, likely due to an imbalance between TNFα and IFNα[ 27 ]. Collectively, these findings indicate that apoptosis signaling may not be suppressed in OLP, and death receptor pathways may not be the primary drivers of basal cell necroptosis. Instead, IFNα may play a central role by altering the states of basal keratinocytes and promoting necroptosis. Given TNFα’s dual roles in cell death and immune regulation, future studies should aim to elucidate how to balance TNFα and IFNα activities to minimize epithelial injury and develop safer therapeutic options for OLP and related diseases. Emerging evidence suggests that aberrant IFN-I signaling is involved in the pathogenesis of various autoimmune diseases[ 16 , 17 ]. Under physiological conditions, basal IFN-I signaling, induced by endogenous stimuli such as commensal microbiota, supports the homeostatic expression of downstream genes and ensures preparedness for rapid necroptotic responses[ 28 ]. These findings are consistent with the immune environment observed in OLP lesions and raise the possibility that dysregulated IFN-I signaling similarly contributes to epithelial injury in OLP. Although this connection remains incompletely defined, accumulating data indicate a pathogenic role for IFN-I in promoting necroptosis and chronic inflammation in OLP[ 26 ]. Clinically, IFNα-based therapies for hepatitis B and C have been reported to exacerbate or even induce OLP, further supporting its potential pathogenic role[ 29 ]. However, a definitive causal relationship between IFNα and the onset of OLP remains unproven, highlighting the need for further investigation into IFN-I-mediated innate immune responses in this disease. In this study, we identified ZBP1 as a key mediator of IFN-I–induced necroptosis in OLP. ZBP1 is reported to regulate cell death, inflammation, and immunity[ 20 , 30 , 31 ]. Loss of ZBP1 alleviates epithelial inflammation driven by Adar1 Zα domain mutations or epidermal RIPK1 deletion, indicating the occurrence of necroptosis[ 32 – 35 ]. Consistent with these findings, our data suggest that ZBP1 acts downstream of IFN-I, promoting epithelial injury via the RIPK3–MLKL axis and sustaining local inflammation. The elevated levels of ADAR1p150 and pRIPK1 in OLP tissues and IFNα2α-treated keratinocytes further indicate that under certain IFN-I conditions, checkpoint control may be bypassed, allowing for necroptotic signaling. Although these findings provide preliminary mechanistic insights, they are primarily based on in vitro data, as there is currently no animal model of OLP. Future efforts will focus on establishing an appropriate murine model to validate the pathological role of ZBP1-mediated necroptosis in basal keratinocytes. To our knowledge, this study is the first to identify necroptosis in the basal epithelial cells of OLP and to reveal a novel mechanism involving aberrant activation of ZBP1 and subsequent RIPK3-MLKL signaling. Additionally, we elucidate the regulatory role of type I interferon (IFN-I) in promoting ZBP1 activation, providing new insights into the pathogenesis of OLP and highlighting ZBP1 as a promising target for therapeutic intervention. Declarations Competing Interests The authors declare no competing interests. Ethics Approval and Consent to Participate Ethical approval was obtained from the ethics committee of the Ninth People’s Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine (No. SH9H-2021-T100-2). Written informed consent was obtained from all participants before enrollment. Clinical trial number not applicable. Funding This work was supported by National Natural Science Foundation of China (Grant No. 82020108010, 82270976, and 82205200); Clinical Research Program of 9th People's Hospital, Shanghai Jiao Tong University School of Medicine (Grant No.202217). Author Contribution YFW, LS, and GYT contributed to the conception and design of the study. XKJ, JJC, YLY, and YRL were responsible for data acquisition. XKJ, XJY, and YWD conducted the analysis and interpretation of the data. XKJ and JJC drafted the original manuscript. JJC, YFW, and SL provided critical revisions. All authors reviewed the manuscript. 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Sensing of viral and endogenous RNA by ZBP1/DAI induces necroptosis. The EMBO journal 36:2529–2543. https://doi.org/10.15252/embj.201796476 Hao, Yu, Bo Yang, Jinke Yang, Xijuan Shi, Xing Yang, Dajun Zhang, and Dengshuai Zhao et al. 2022. ZBP1: A Powerful Innate Immune Sensor and Double-Edged Sword in Host Immunity. International Journal of Molecular Sciences 23:10224. https://doi.org/10.3390/ijms231810224 Lin, Juan, Snehlata Kumari, Chun Kim, Trieu-My Van, Laurens Wachsmuth, Apostolos Polykratis, and Manolis Pasparakis. 2016. RIPK1 counteracts ZBP1-mediated necroptosis to inhibit inflammation. Nature 540:124–128. https://doi.org/10.1038/nature20558 Newton, Kim, Katherine E., Allie Wickliffe, Debra L. Maltzman, Andreas Dugger, Victoria C. Strasser, Jennie R. Pham, and Lill et al. 2016. RIPK1 inhibits ZBP1-driven necroptosis during development. Nature 540:129–133. https://doi.org/10.1038/nature20559 Chen, Xin-Yu, Ying-Hong Dai, Xin-Xing Wan, Xi-Min Hu, Wen-Juan Zhao, Xiao-Xia Ban, Hao Wan, Kun Huang, Qi Zhang, and Kun Xiong. 2022. ZBP1-Mediated Necroptosis: Mechanisms and Therapeutic Implications. Molecules (Basel Switzerland) 28:52. https://doi.org/10.3390/molecules28010052 Hubbard, Nicholas W., M. Joshua, Megan Ames, Lan H. Maurano, Y. Chu, Kim, S. Somfleth, Nandan, Margo Gokhale, and Werner et al. 2022. ADAR1 mutation causes ZBP1-dependent immunopathology. Nature 607:769–775. https://doi.org/10.1038/s41586-022-04896-7 Additional Declarations No competing interests reported. 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1","display":"","copyAsset":false,"role":"figure","size":617279,"visible":true,"origin":"","legend":"\u003cp\u003eEnhanced epithelial cell death signaling in OLP. \u003cstrong\u003e(A)\u003c/strong\u003e Basic information on the single-cell sequencing dataset. \u003cstrong\u003e(B)\u003c/strong\u003e UMAP plot illustrating cell clustering from oral lichen planus (OLP) and healthy control (HC) samples. \u003cstrong\u003e(C)\u003c/strong\u003eViolin plot depicting the expression levels of marker genes across different cell clusters. \u003cstrong\u003e(D)\u003c/strong\u003e UMAP plot showing the distribution of cell clusters among the EOLP, NEOLP, and HC groups. \u003cstrong\u003e(E)\u003c/strong\u003e Bar chart representing the proportions of each cluster within the respective groups. \u003cstrong\u003e(F)\u003c/strong\u003e HE staining results for EOLP, NEOLP, and HC samples. Red boxes indicate magnified regions. Yellow dashed lines indicate the junction between the epithelium and the lamina propria, while yellow arrows indicate cells exhibiting liquefactive degeneration. \u003cstrong\u003e(G)\u003c/strong\u003e IF staining for TUNEL alongside corresponding HE staining results for OLP and HC samples. TUNEL labels fragmented DNA, while DAPI marks the cell nuclei. \u003cstrong\u003e(H)\u003c/strong\u003e mIHC staining results for K4/K14 in OLP and HC samples. K4/K14 mark the epithelial cells, and DAPI labels the cell nuclei. Yellow arrows denote cells with membrane rupture and loss of nuclear structure. \u003cstrong\u003e(I)\u003c/strong\u003e Electron microscopy image of the epithelial basal layer structure in OLP. Vacuolization in the cytoplasm and disruption of the basement membrane are observable.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7981446/v1/ade6526aa4fdb13094be5d0b.jpeg"},{"id":96246871,"identity":"d825dbc4-9853-4426-80ca-6477f5c64f41","added_by":"auto","created_at":"2025-11-19 07:26:48","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":317342,"visible":true,"origin":"","legend":"\u003cp\u003eNecroptosis in OLP epithelial cells.\u003cstrong\u003e (A) \u003c/strong\u003eGSEA results for the epithelial cluster in the single-cell sequencing dataset indicate significant differences in the enrichment of the necroptosis gene set. \u003cstrong\u003e(B)\u003c/strong\u003e WB results for programmed cell death markers in OLP and HC mucosal epithelial samples. \u003cstrong\u003e(C)\u003c/strong\u003e mIHC staining and corresponding HE staining results for apoptosis and necroptosis markers in OLP and HC samples. \u003cstrong\u003e(D)\u003c/strong\u003e pMLKL IHC staining results for OLP and HC samples. Red boxes indicate the magnified regions, and the yellow arrow points to a cell exhibiting liquefactive degeneration.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7981446/v1/e38d89baeff52eb7de6dbc36.jpeg"},{"id":96087180,"identity":"14abfff4-abef-4ddf-80c8-c2d3858059ef","added_by":"auto","created_at":"2025-11-17 12:40:23","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":277520,"visible":true,"origin":"","legend":"\u003cp\u003eActivation of IFN-I pathway in OLP. \u003cstrong\u003e(A)\u003c/strong\u003e Transcriptional level changes of several ISGs in the epithelial cluster of the single-cell sequencing dataset. \u003cstrong\u003e(B)\u003c/strong\u003eqPCR results for IFNA in OLP and HC mucosal samples. \u003cstrong\u003e(C)\u003c/strong\u003e GO enrichment analysis results for the epithelial tissue transcriptome sequencing data. \u003cstrong\u003e(D)\u003c/strong\u003eTranscriptional level changes of several ISGs in the epithelial tissue transcriptome sequencing data.\u003cstrong\u003e (E) \u003c/strong\u003eResults of IF staining (left) and the proportion of pMLKL-positive cells (right) in HaCaT cells following drug treatment.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7981446/v1/2e99a22bac6f49f13627aefc.jpeg"},{"id":96247804,"identity":"d64672b7-5a2f-4411-9f0d-65aa2d6446de","added_by":"auto","created_at":"2025-11-19 07:27:44","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":228538,"visible":true,"origin":"","legend":"\u003cp\u003eZBP1 expression and necroptotic signaling in OLP. \u003cstrong\u003e(A)\u003c/strong\u003e Changes in the transcriptional levels of ZBP1 in the epithelial tissue transcriptome sequencing data. \u003cstrong\u003e(B)\u003c/strong\u003eWB results of ZBP1 expression in mucosal epithelial samples from OLP and HC. \u003cstrong\u003e(C)\u003c/strong\u003emIHC staining results for necroptosis pathway-related markers in OLP and HC samples. \u003cstrong\u003e(D)\u003c/strong\u003e WB results of ZBP1 overexpression and the relative expression levels of pMLKL protein in HaCaT cells treated with drugs.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7981446/v1/2e5a42dabbd32435e0e65e9e.jpeg"},{"id":96087186,"identity":"0bf8643d-ba56-4d63-8202-81dfc06adb29","added_by":"auto","created_at":"2025-11-17 12:40:24","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":304634,"visible":true,"origin":"","legend":"\u003cp\u003eZBP1-mediated necroptosis under IFN-I stimulation in keratinocytes. \u003cstrong\u003e(A)\u003c/strong\u003e Basic information regarding the cell transcriptome sequencing dataset. \u003cstrong\u003e(B)\u003c/strong\u003eChanges in the transcriptional levels of ZBP1 within the cell transcriptome sequencing data. \u003cstrong\u003e(C)\u003c/strong\u003e WB results of ZBP1 expression in HaCaT cells following drug treatment. \u003cstrong\u003e(D)\u003c/strong\u003e Results of IF staining and the proportion of pMLKL/ZBP1-positive cells in HaCaT cells after treatment. \u003cstrong\u003e(E)\u003c/strong\u003eSchematic diagram illustrating the proposed mechanism.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7981446/v1/961eb564536e0d282b412123.jpeg"},{"id":106808905,"identity":"f97d116a-210f-4530-ba99-bdeaf79fed98","added_by":"auto","created_at":"2026-04-13 16:04:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2458763,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7981446/v1/b60c5eda-a949-4d51-a16c-1a4feb89fb55.pdf"},{"id":96248105,"identity":"356d3a00-19dc-4922-85c9-91b5b25cbab4","added_by":"auto","created_at":"2025-11-19 07:28:03","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":16218,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalmaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7981446/v1/7cc5ff2d5724b851eacd6bad.docx"},{"id":96249984,"identity":"d1386f66-aab6-4006-86f1-207336489722","added_by":"auto","created_at":"2025-11-19 07:36:58","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":335748,"visible":true,"origin":"","legend":"","description":"","filename":"RawimagesofWB.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7981446/v1/ad49a3e02f4dfa26f396cbf8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Type I Interferon induces Keratinocytes Necroptosis via the ZBP1-MLKL Axis in Oral Lichen Planus","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOral lichen planus (OLP) is a common chronic inflammatory disorder of the oral mucosa, affecting approximately 1.01% of the global population[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The characteristic histopathological features of OLP include band-like lymphocytic infiltration in the lamina propria and liquefactive degeneration of basal keratinocytes[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Recent evidence has demonstrated that keratinocytes actively regulate mucocutaneous inflammatory responses[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In the local microenvironment of OLP, keratinocytes can be activated by external stimuli, resulting in the abnormal secretion of inflammatory mediators[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. These findings suggest that keratinocytes in OLP are not merely passive targets of inflammation but may actively participate in disease modulation or initiation. Notably, the abnormal death of basal keratinocytes may represent a critical pathogenic event.\u003c/p\u003e\u003cp\u003eCurrent research indicates that activated CD8⁺ cytotoxic T lymphocytes within OLP lesions can induce apoptosis, a non-lytic process that does not release intracellular contents, in basal keratinocytes[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, ultrastructural analyses using transmission electron microscopy have revealed ruptured plasma membranes and cytoplasmic vacuolization in basal cells of OLP lesions, suggesting leakage of intracellular contents[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. These findings imply that non-apoptotic forms of programmed cell death may also be aberrantly activated in OLP. Necroptosis, the most common form of lytic cell death, is a highly regulated, pro-inflammatory process that occurs more rapidly than apoptosis and is associated with the release of pro-inflammatory cytokines and damage-associated molecular patterns (DAMPs)[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Upon activation, the necroptosis signaling cascade involves the phosphorylation of receptor-interacting serine/threonine-protein kinase 3 (RIPK3), which subsequently activates mixed lineage kinase domain-like protein (MLKL)[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Phosphorylated MLKL (pMLKL) oligomerizes and translocates to the plasma membrane, where it forms pores, ultimately leading to cell swelling and membrane rupture[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Therefore, investigating the mechanism of necroptosis in the abnormal death of OLP epithelium is of great significance.\u003c/p\u003e\u003cp\u003eRecent studies have highlighted the aberrant activation of interferon type I (IFN-I) signaling in various autoimmune diseases, including systemic lupus erythematosus and psoriasis, where it contributes to disease progression and tissue injury[\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Z-DNA binding protein 1 (ZBP1) is a cytoplasmic nucleic acid sensor and a well-characterized IFN-I\u0026ndash;inducible gene[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. It contains an N-terminal Zα domain that specifically recognizes and binds left-handed Z-form nucleic acids (Z-DNA and Z-RNA), thereby initiating necroptotic signaling[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Based on our previous observations of IFN-I pathway activation in OLP lesions, we hypothesize that elevated IFN-I expression in OLP leads to aberrant upregulation of ZBP1 in basal keratinocytes. This may, in turn, activate the ZBP1\u0026ndash;MLKL signaling axis, ultimately triggering necroptosis.\u003c/p\u003e\u003cp\u003eIn this study, we present the first evidence of necroptotic cell death occurring in the basal keratinocytes of OLP lesions, providing a novel mechanistic perspective on the pathogenesis of OLP. Additionally, we elucidate the aberrant induction of the key regulatory molecule ZBP1 under elevated IFN-I signaling conditions, which subsequently leads to the phosphorylation of MLKL. Collectively, these findings offer new insights into the initiation of immune responses in OLP and propose potential molecular targets for future therapeutic interventions.\u003c/p\u003e"},{"header":"Methods and materials","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eHuman mucosa sample collection\u003c/h2\u003e\u003cp\u003e A total of 30 patients with clinically and histopathologically confirmed OLP and 30 age- and sex-matched healthy controls were recruited from the Department of Oral Medicine at Shanghai Ninth People\u0026rsquo;s Hospital, China, between December 2022 and July 2024. All participants provided written informed consent prior to sample collection. OLP tissues were obtained through 8-mm punch biopsies of the lesional buccal mucosa. Control tissues were collected from excess mucosa removed during orthognathic surgery. Immediately after removal, one-third of each tissue specimen was fixed for histological analysis, while the remaining two-thirds were dissected to isolate the epithelial component for subsequent experiments. All biopsy procedures were conducted by experienced oral surgeons. The study protocol was approved by the Ethics Committee of Shanghai Ninth People\u0026rsquo;s Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine (No. SH9H-2021-T100-2) and adhered to the principles of the Declaration of Helsinki.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eData acquisition, processing and analysis\u003c/h3\u003e\n\u003cp\u003eThe single-cell RNA sequencing (scRNA-seq) dataset GSE211630 (5 OLP, 1 healthy mucosa) was obtained from the Gene Expression Omnibus (GEO). Count matrices were processed in Seurat (R): integrated, principal component analysis (PCA)-reduced, clustered (k-nearest neighbor (kNN) / shared nearest neighbor (SNN)), and visualized (Uniform Manifold Approximation and Projection (UMAP)/t-SNE). Cell clusters were annotated (SingleR, CellMarker, PanglaoDB, HCA) using marker genes (FindMarkers). Subpopulations were isolated, re-clustered, and analyzed. Differentially expressed genes (DEGs) underwent functional enrichment (ClusterProfiler/gseaplot2).\u003c/p\u003e\u003cp\u003eAdditionally, bulk RNA-seq was performed on fresh samples (3 OLP, 3 healthy epithelium). Raw reads underwent QC (FastQC), trimming, alignment (HISAT2/STAR), and quantification (Cufflinks/featureCounts/HTSeq). Data and clinical info were analyzed in RStudio: Principal component analysis (PCA) for outliers/variability, DEG identification (DESeq2), and visualization (heatmaps).\u003c/p\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eHaCaT cells, a human epidermal keratinocyte cell line, were cultured in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM; Corning) supplemented with 15% fetal bovine serum (FBS; Gibco) and Penicillin-Streptomycin (100 \u0026micro;g/mL penicillin and 100 U/mL streptomycin; Gibco). The cell line was authenticated through short tandem repeat sequence analysis and maintained in a cell culture incubator (Thermo) at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. IFNα2a (3SBIO) was stored at -4\u0026deg;C and diluted in culture medium to a concentration of 1500 IU/mL prior to application. Birinapant (MCE) was prepared as a 5 mM stock solution in DMSO, stored at -20\u0026deg;C, and applied at a final concentration of 5 \u0026micro;M after dilution in culture medium. Q-VD-OPH (MCE) was prepared as a 10 mM stock solution in DMSO, stored at -20\u0026deg;C, and applied at a final concentration of 10 \u0026micro;M following dilution in culture medium. HaCaT cells were genetically modified to overexpress or knockout target genes using lentiviral vectors. The cells were transduced with lentiviruses and selected with appropriate antibiotics to establish stable overexpression or knockout lines. All target sequences utilized for overexpression and knockout are provided in the supplementary materials.\u003c/p\u003e\n\u003ch3\u003eQuantitative real-time PCR (qPCR)\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from samples using FreeZol Reagent (Vazyme) according to the manufacturer\u0026rsquo;s instructions. The concentration and purity of the RNA were assessed spectrophotometrically. Reverse transcription was conducted using HiScript III All-in-One RT SuperMix Perfect for qPCR (Vazyme) to synthesize complementary DNA (cDNA). Quantitative real-time PCR was performed with SYBR Green-based detection on a real-time PCR system (Thermo), and reactions were prepared according to the manufacturer\u0026rsquo;s recommendations. Relative gene expression was calculated using the 2^\u0026minus;ΔΔCt method with an appropriate endogenous control. The primer sequences are provided in the supplementary materials.\u003c/p\u003e\n\u003ch3\u003eImmunoblotting\u003c/h3\u003e\n\u003cp\u003eCells and tissues were lysed using RIPA lysis buffer (Beyotime) supplemented with protease and phosphatase inhibitors (Beyotime) via a low-temperature grinder (Wonbio) at 4\u0026deg;C. The lysates were incubated on ice for 20 minutes and subsequently cleared by high-speed centrifugation at 12,000 rpm for 15 minutes at 4\u0026deg;C. After reserving 5% of the supernatants for the BCA assay, the remaining supernatants were denatured with SDS-PAGE sample loading buffer and then subjected to Western blotting (WB) analysis. The sources and dilutions of primary antibodies are detailed in the supplementary materials. Protein bands were visualized using an e-Blot touch imager (e-Blot). The intensity of each band was quantified and normalized against β-actin as an internal loading control using ImageJ software.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eImmunostaining, histology, and electron microscopy\u003c/h2\u003e\u003cp\u003eFor electron microscopy, fresh tissue (\u0026sim;2 mm\u0026sup3;) was sequentially fixed in 2.5% glutaraldehyde and 1% osmium tetroxide (4\u0026deg;C), dehydrated, infiltrated, and embedded. Regions of interest were identified on toluidine blue-stained semi-thin sections (1 \u0026micro;m) by light microscopy. Ultrathin sections (\u0026sim;50 nm) were examined by transmission electron microscopy (TEM) (FEI TF20). For histology and immunohistochemistry (IHC) analysis, hematoxylin and eosin (HE) staining and IHC were performed using commercial kits per manufacturer's instructions. For IHC/multiplex IHC (mIHC; TSA kit, Yuanxi), 4% paraformaldehyde-fixed, paraffin-embedded tissues were sectioned (3 \u0026micro;m), deparaffinized, underwent antigen retrieval, permeabilization, blocking, and incubation with primary antibodies followed by HRP-conjugated secondary antibodies and tyramide-fluorophores. For immunofluorescence (IF), cells on glass slides were fixed (4% paraformaldehyde), permeabilized, blocked, and incubated with primary and fluorophore-conjugated secondary antibodies. For imaging, all sections/slides were mounted in antifade reagent (Beyotime). Confocal microscopy (Zeiss LSM 700) and Zeiss ZEN software were used for fluorescence imaging and analysis. Primary antibody details are provided in Supplementary Materials.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed using GraphPad Prism Software. Differences between groups were assessed using the Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test or one-way ANOVA. A \u003cem\u003ep\u003c/em\u003e-value of less than 0.05 was considered statistically significant for all analyses.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eEnhanced epithelial cell death signaling in OLP\u003c/h2\u003e\u003cp\u003eWe retrieved scRNA-seq datasets related to OLP from the GEO (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Dimensionality reduction was conducted using UMAP with dimensions set to 1:10 and point size at 0.5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Based on cluster-specific marker genes, the cells were classified into eight major populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). UMAP plots and bar graphs indicated altered cellular composition across OLP subtypes and controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, E), revealing a significant reduction of epithelial cells in erosive OLP (EOLP) compared to non-erosive OLP (NEOLP) and controls. This finding suggests excessive basal epithelial cell death may be involved in the pathogenesis of OLP.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further investigate pathological changes, HE staining of OLP and control tissues revealed disruption of the basement membrane and liquefactive degeneration of basal cells, which was more pronounced in EOLP (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). TUNEL staining demonstrated a marked increase in cell death within OLP tissues, particularly in the basal layer (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). mIHC illustrated disrupted epithelial structure, discontinuous expression of keratin 14 (K14), and dense lymphocytic infiltration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). TEM revealed plasma membrane rupture and cytoplasmic vacuolization in basal layer cells of OLP tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI), suggesting the involvement of non-apoptotic programmed cell death.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eNecroptosis in OLP epithelial cells\u003c/h2\u003e\u003cp\u003eTo investigate programmed cell death in the basal cells of OLP, we conducted Gene Set Enrichment Analysis (GSEA) on epithelial subpopulations derived from scRNA-seq data. This analysis revealed significant activation of necroptosis in the OLP epithelium (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). To validate these findings, we analyzed lesional oral mucosa for cell death markers. Western blot analysis demonstrated the presence of cleaved CASPASE-3 (an apoptosis marker) and phosphorylated MLKL (a necroptosis marker) in the OLP epithelium, with semi-quantitative analysis confirming elevated levels of pMLKL compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), suggesting enhanced necroptosis. mIHC of paraffin-embedded sections detected cleaved CASPASE-3 and pMLKL in the basal layer, with no evidence of co-localization (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), indicating distinct forms of cell death, with necroptosis being predominant. Additional IHC revealed that pMLKL signals were extensively distributed in degenerative basal keratinocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), thereby linking necroptosis to abnormal basal cell death in OLP.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eActivation of IFN-I pathway in OLP\u003c/h2\u003e\u003cp\u003eTo investigate the activation of immune pathways in the epithelium of OLP, we analyzed scRNA-seq data from GEO. Epithelial cells from five OLP samples demonstrated significant activation of the IFN-I signaling pathway, characterized by the upregulation of multiple interferon-stimulated genes (ISGs). Notably, IRF7 and IFIH1 exhibited particularly strong transcriptional induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). To validate these findings, qPCR of lesional mucosa from ten OLP patients compared to three controls confirmed increased expression of IFNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), indicating a heightened IFN-I microenvironment that may contribute to immune dysregulation. Moreover, bulk RNA sequencing of isolated epithelial layers revealed an enrichment of IFN-I-related pathways through Gene Ontology (GO) analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), with five ISGs consistently upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo assess whether IFN-I induces necroptosis in keratinocytes, we treated HaCaT cells with IFNα2α. A positive control group received IFNα2α in combination with Q-VD-OPH and Birinapant. Immunofluorescence staining demonstrated distinct pMLKL-positive cells following treatment with IFNα2α (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), although the number was lower than that observed in the positive control. This suggests that keratinocytes are sensitive to IFN-I-induced necroptotic signaling, and that keratinocyte necroptosis may play a role in the pathogenesis of OLP.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eZBP1 expression and necroptotic signaling in OLP\u003c/h2\u003e\u003cp\u003eGiven that IFN-I has been shown to promote necroptosis in keratinocytes, we subsequently explored potential regulators that mediate this process. ZBP1, a cytosolic nucleic acid sensor, is known to activate necroptosis via the RIPK3-MLKL pathway. Therefore, we examined its expression in the epithelium of OLP. Transcriptional and protein analyses revealed significantly elevated levels of ZBP1 in OLP compared to control samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B), indicating aberrant epithelial activation and a potential role in the pathogenesis of OLP. Additionally, mIHC staining demonstrated the colocalization of ZBP1, pRIPK3, and pMLKL in the basal epithelial cells of OLP (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), suggesting that ZBP1 may mediate necroptosis through the activation of the RIPK3-MLKL pathway. In HaCaT cells, ZBP1 overexpression markedly increased pMLKL levels, while the RIPK3 inhibitor GSK-872 reduced pMLKL levels in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), confirming that ZBP1 promotes necroptosis via the RIPK3-MLKL pathway.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eZBP1-mediated necroptosis in keratinocytes under IFN-I stimulation\u003c/h2\u003e\u003cp\u003eTo assess the expression of ZBP1 in keratinocytes following IFN-I stimulation, HaCaT cells were treated with IFNα2α for 24 hours and subsequently analyzed through transcriptome sequencing (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The transcription of ZBP1 was significantly elevated in the IFNα2α group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), and WB confirmed increased protein levels in both IFNα2α-treated and positive control cells across three biological replicates (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). These results indicate that IFN-I promotes the upregulation of ZBP1 in keratinocytes.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIF staining demonstrated colocalization of ZBP1 and pMLKL in IFNα2α-treated HaCaT cells. In contrast, ZBP1 knockout cells did not exhibit pMLKL expression upon IFNα2α treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), thereby confirming the necessity of ZBP1 for IFN-I\u0026ndash;induced necroptosis. A schematic diagram was created to illustrate the proposed mechanism (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE).\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn OLP tissues, colloid bodies are frequently observed in both the basal and suprabasal layers. These structures have traditionally been attributed to keratinocyte apoptosis triggered by dysregulated immune responses, with CD8⁺ T cells identified as key effector cells. In addition to colloid bodies, liquefactive degeneration of basal keratinocytes is also evident. Both the previous report[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and our ultrastructural analyses reveal plasma membrane disruption in basal cells, suggesting the involvement of a non-apoptotic form of programmed cell death. This process likely contributes to epithelial injury and basement membrane compromise, which in turn facilitates immune cell infiltration along chemokine gradients, thereby amplifying tissue damage in OLP.\u003c/p\u003e\u003cp\u003e In healthy oral epithelium, K4 and K14 exhibit distinct spatial expression patterns, localized to the suprabasal and basal layers, respectively. In contrast, OLP lesions demonstrate an altered distribution, with K14 expression extending throughout the entire epithelial layer and a significant reduction in K4 expression. These changes indicate disrupted epithelial differentiation in OLP. The expanded K14 expression likely reflects basal cell hyperproliferation and impaired stratification, while the diminished K4 expression may suggest compromised functionality of terminally differentiated cells. A previous study has shown that epidermis-specific RIPK1 knockout in transgenic mice leads to keratinocyte necroptosis and skin inflammation, accompanied by epidermal hyperplasia and increased expression of K6 and K10 during early postnatal stages[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. This hyperplastic phenotype parallels the suprabasal thickening observed in NEOLP lesions in our study. Collectively, these findings imply that aberrant epithelial proliferation and differentiation, potentially driven by necroptotic signaling, may contribute to disease progression in OLP.\u003c/p\u003e\u003cp\u003eRecent studies on necroptosis have primarily concentrated on death receptor pathways, particularly the TNF receptor (TNFR) pathway. Under pro-apoptotic conditions, death receptors can initiate apoptosis; however, the inhibition of apoptosis may redirect the cellular response toward necroptosis[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In OLP, TNFα mRNA has been detected in lesional T cells, and serum TNFα levels are elevated[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Further research suggests that CD8⁺ T cells may secrete TNFα, which binds to TNFR1 on keratinocytes, thereby triggering the apoptosis of basal cells[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Clinically, TNFα inhibitors have been approved for the treatment of psoriasis[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]; however, case reports reveal the occurrence of psoriasis-like oral lesions following infliximab therapy, likely due to an imbalance between TNFα and IFNα[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Collectively, these findings indicate that apoptosis signaling may not be suppressed in OLP, and death receptor pathways may not be the primary drivers of basal cell necroptosis. Instead, IFNα may play a central role by altering the states of basal keratinocytes and promoting necroptosis. Given TNFα\u0026rsquo;s dual roles in cell death and immune regulation, future studies should aim to elucidate how to balance TNFα and IFNα activities to minimize epithelial injury and develop safer therapeutic options for OLP and related diseases.\u003c/p\u003e\u003cp\u003eEmerging evidence suggests that aberrant IFN-I signaling is involved in the pathogenesis of various autoimmune diseases[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Under physiological conditions, basal IFN-I signaling, induced by endogenous stimuli such as commensal microbiota, supports the homeostatic expression of downstream genes and ensures preparedness for rapid necroptotic responses[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. These findings are consistent with the immune environment observed in OLP lesions and raise the possibility that dysregulated IFN-I signaling similarly contributes to epithelial injury in OLP. Although this connection remains incompletely defined, accumulating data indicate a pathogenic role for IFN-I in promoting necroptosis and chronic inflammation in OLP[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Clinically, IFNα-based therapies for hepatitis B and C have been reported to exacerbate or even induce OLP, further supporting its potential pathogenic role[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. However, a definitive causal relationship between IFNα and the onset of OLP remains unproven, highlighting the need for further investigation into IFN-I-mediated innate immune responses in this disease.\u003c/p\u003e\u003cp\u003eIn this study, we identified ZBP1 as a key mediator of IFN-I\u0026ndash;induced necroptosis in OLP. ZBP1 is reported to regulate cell death, inflammation, and immunity[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Loss of ZBP1 alleviates epithelial inflammation driven by Adar1 Zα domain mutations or epidermal RIPK1 deletion, indicating the occurrence of necroptosis[\u003cspan additionalcitationids=\"CR33 CR34\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Consistent with these findings, our data suggest that ZBP1 acts downstream of IFN-I, promoting epithelial injury via the RIPK3\u0026ndash;MLKL axis and sustaining local inflammation. The elevated levels of ADAR1p150 and pRIPK1 in OLP tissues and IFNα2α-treated keratinocytes further indicate that under certain IFN-I conditions, checkpoint control may be bypassed, allowing for necroptotic signaling. Although these findings provide preliminary mechanistic insights, they are primarily based on in vitro data, as there is currently no animal model of OLP. Future efforts will focus on establishing an appropriate murine model to validate the pathological role of ZBP1-mediated necroptosis in basal keratinocytes.\u003c/p\u003e\u003cp\u003eTo our knowledge, this study is the first to identify necroptosis in the basal epithelial cells of OLP and to reveal a novel mechanism involving aberrant activation of ZBP1 and subsequent RIPK3-MLKL signaling. Additionally, we elucidate the regulatory role of type I interferon (IFN-I) in promoting ZBP1 activation, providing new insights into the pathogenesis of OLP and highlighting ZBP1 as a promising target for therapeutic intervention.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting Interests\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eEthics Approval and Consent to Participate\u003c/h2\u003e\u003cp\u003e Ethical approval was obtained from the ethics committee of the Ninth People\u0026rsquo;s Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine (No. SH9H-2021-T100-2). Written informed consent was obtained from all participants before enrollment.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eClinical trial number\u003c/h2\u003e\u003cp\u003enot applicable.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by National Natural Science Foundation of China (Grant No. 82020108010, 82270976, and 82205200); Clinical Research Program of 9th People's Hospital, Shanghai Jiao Tong University School of Medicine (Grant No.202217).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eYFW, LS, and GYT contributed to the conception and design of the study. XKJ, JJC, YLY, and YRL were responsible for data acquisition. XKJ, XJY, and YWD conducted the analysis and interpretation of the data. XKJ and JJC drafted the original manuscript. JJC, YFW, and SL provided critical revisions. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data supporting the findings of this study are available within the paper and its supplementary information.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eEl-Howati, Asma, Martin H., Helen E. Thornhill, and Colley, Craig Murdoch. 2022. Immune mechanisms in oral lichen planus. \u003cem\u003eOral Diseases\u003c/em\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/odi.14142\u003c/span\u003e\u003cspan address=\"10.1111/odi.14142\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGonz\u0026aacute;lez-Moles, Miguel, Saman \u0026Aacute;ngel, and Warnakulasuriya. 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ADAR1 mutation causes ZBP1-dependent immunopathology. \u003cem\u003eNature\u003c/em\u003e 607:769\u0026ndash;775. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41586-022-04896-7\u003c/span\u003e\u003cspan address=\"10.1038/s41586-022-04896-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"inflammation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ifla","sideBox":"Learn more about [Inflammation](https://www.springer.com/journal/10753)","snPcode":"10753","submissionUrl":"https://submission.nature.com/new-submission/10753/3","title":"Inflammation","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"oral lichen planus, keratinocyte, necroptosis, mixed lineage kinase domain-like protein, Z-DNA binding protein 1","lastPublishedDoi":"10.21203/rs.3.rs-7981446/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7981446/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOral lichen planus (OLP) is a prevalent chronic inflammatory condition that affects the oral mucosa. Histologically, it is characterized by the liquefaction and degeneration of basal epithelial cells, indicating a disruption of the basal layer architecture, which may represent an early event in the disease\u0026rsquo;s pathogenesis. However, the molecular mechanisms underlying this epithelial damage remain poorly understood. In our study, histological staining and transmission electron microscopy revealed aberrant cell death in the basal epithelial layer of OLP tissues. Immunodetection demonstrated the presence of phosphorylated mixed lineage kinase domain-like protein (pMLKL), a key marker of necroptosis, specifically localized to basal keratinocytes. Notably, interferon type I (IFN-I), particularly IFNα, was significantly upregulated in OLP mucosa compared to healthy controls. The expression of Z-DNA binding protein 1 (ZBP1), an IFN-stimulated gene and an upstream regulator of necroptosis, was elevated in pMLKL-positive epithelial cells. In vitro stimulation with IFNα2a induced the expression of ZBP1 and pMLKL in HaCaT keratinocytes, while ZBP1 knockdown abrogated MLKL phosphorylation. Collectively, these results suggest that, in the context of increased IFN-I signaling, ZBP1 is aberrantly upregulated in the OLP epithelium, promoting necroptosis in basal keratinocytes. This necroptotic activity may contribute to the damage and disruption of the basal layer, providing novel insights into the pathogenesis of OLP and highlighting potential molecular targets for therapeutic intervention.\u003c/p\u003e","manuscriptTitle":"Type I Interferon induces Keratinocytes Necroptosis via the ZBP1-MLKL Axis in Oral Lichen Planus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-17 12:40:19","doi":"10.21203/rs.3.rs-7981446/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-18T08:54:40+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-26T17:09:20+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-16T01:43:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-12T00:37:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"338692441233166150910447552321900213542","date":"2025-11-07T14:51:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"335877583658762836676817004967735109767","date":"2025-11-06T15:59:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"13446457039524487000801515690579198387","date":"2025-11-06T14:59:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"225942292618408865583035442988014498683","date":"2025-11-05T15:33:12+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-05T14:47:31+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-03T10:27:16+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-03T10:24:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"Inflammation","date":"2025-10-29T15:16:16+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"inflammation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ifla","sideBox":"Learn more about [Inflammation](https://www.springer.com/journal/10753)","snPcode":"10753","submissionUrl":"https://submission.nature.com/new-submission/10753/3","title":"Inflammation","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"dd3a1c67-233d-4394-bb8e-37327409ae94","owner":[],"postedDate":"November 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-13T16:01:26+00:00","versionOfRecord":{"articleIdentity":"rs-7981446","link":"https://doi.org/10.1007/s10753-026-02497-4","journal":{"identity":"inflammation","isVorOnly":false,"title":"Inflammation"},"publishedOn":"2026-04-06 15:57:53","publishedOnDateReadable":"April 6th, 2026"},"versionCreatedAt":"2025-11-17 12:40:19","video":"","vorDoi":"10.1007/s10753-026-02497-4","vorDoiUrl":"https://doi.org/10.1007/s10753-026-02497-4","workflowStages":[]},"version":"v1","identity":"rs-7981446","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7981446","identity":"rs-7981446","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}