Toll/interleukin-1 receptor domain-containing adaptor protein (TIRAP) in Hepatic Stellate Cells: Beyond Membrane and Cytoplasmic Presence

Toll/interleukin-1 receptor domain-containing adaptor protein (TIRAP) in Hepatic Stellate Cells: Beyond Membrane and Cytoplasmic Presence | 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 Toll/interleukin-1 receptor domain-containing adaptor protein (TIRAP) in Hepatic Stellate Cells: Beyond Membrane and Cytoplasmic Presence Pramod Patidar, Rajat Atre, Mirza S. Baig This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8652622/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The Toll/interleukin-1 receptor domain-containing adaptor protein (TIRAP) is a key adaptor in Toll-like receptor signaling, classically functioning as a cytoplasmic bridging adaptor for Myeloid differentiation primary response 88 (MyD88) recruitment. While its role in inflammatory signaling is well established, its contribution to alcohol-induced liver fibrosis remains unclear. Using lipopolysaccharide (LPS) and ethanol-stimulated hepatic stellate cells (LX-2), we demonstrate, for the first time, nuclear translocation of TIRAP under alcoholic conditions. Confocal microscopy and nuclear–cytoplasmic fractionation confirmed TIRAP enrichment in the nucleus. Importantly, silencing of TIRAP in LX-2 significantly reduced the nuclear translocation and expression of fibrotic markers (such as α-SMA and collagen) in HSCs, suggesting a key role of nuclear TIRAP in driving fibrogenesis. Overall, these findings reveal a novel role for TIRAP beyond cytoplasmic signaling, suggesting potential nuclear functions that may regulate transcriptional programs driving inflammation and fibrosis. TIRAP MyD88 LPS Alcohol Hepatic Stellate Cell (HSC) and Fibrosis Figures Figure 1 Figure 2 Figure 3 Introduction The Toll-interleukin-1 receptor (TIR) domain-containing adaptor protein, also known as MyD88-adaptor-like (MAL), is a pivotal adaptor molecule in the innate immune system 1 . Functionally, TIRAP acts as a bridging adaptor for several Toll-like receptors (TLR) family members, most notably TLR2 and TLR4, and facilitates downstream signaling by recruiting MyD88 to activated receptors. Through this mechanism, it governs the activation of key transcription factors such as nuclear factor κB (NF-κB) and activator protein 1 (AP-1), thereby triggering the expression of pro-inflammatory cytokines, chemokines, and other immune mediators essential for host defense 2 . Since its discovery in 2001, TIRAP has been extensively studied and is now recognized as a major regulator of inflammatory signaling, largely owing to its ability to form complex protein–protein interaction networks through its conserved TIR domain 3 . Dysregulation of TIRAP whether through altered expression, genetic polymorphisms, or functional modification has been implicated in a broad spectrum of disorders, including autoimmune conditions, chronic inflammatory diseases, microbial infections, and even tumorigenesis 4 . In these pathological contexts, aberrant TIRAP-dependent signaling can exacerbate immune activation or impair the resolution of inflammation, underscoring its importance as both a homeostatic regulator and a potential therapeutic target. Traditionally, TIRAP has been described as a membrane-associated or cytoplasmic adaptor, functioning proximally to TLRs at the plasma membrane or endosomal compartments. Interestingly, a study has also suggested that TIRAP may interact with c-Jun, a subunit of the AP-1 transcription factor, particularly under conditions of LPS-dependent TLR4 activation 5 . This interaction highlights an additional layer of complexity in TIRAP-mediated signaling, suggesting that, beyond its role in bridging MyD88 recruitment, TIRAP may directly modulate transcription factor activity to fine-tune inflammatory responses. Building on this concept, we sought to investigate the role of TIRAP in the context of alcohol-induced liver injury and fibrosis. Chronic alcohol consumption is a well-established driver of hepatic inflammation, oxidative stress, and stellate cell activation, leading to progressive fibrosis 6 . While TLR signaling and particularly TLR4 have been strongly implicated in these processes, the contribution of TIRAP remains incompletely understood. Intriguingly, previous studies have implicated TIRAP as an important mediator in radiation-induced liver injury, further supporting its potential relevance in liver pathophysiology 7 . These prompted us to examine whether TIRAP functions as a critical signaling adaptor influencing transcriptional programs that drive inflammation and fibrosis in alcohol-related liver disease. Methods Cell culture -The LX-2 cell line was provided by (Dr. Sheikh Tasduq Abdullah, CSIR – Indian Institute of Integrative Medicine, Jammu). The cells were maintained in Dulbecco's Modified Eagle Medium (DMEM, Gibco ThermoFisher Scientific India) supplemented with 10% FBS (Gibco, ThermoFisher Scientific India) and 1% penicillin/streptomycin in an incubator at 37°C with 5% CO2. To mimic the alcoholic liver disease, HSCs were treated with LPS (1 µg/ml) and ethanol (25 mM). siRNA Transfection and TIRAP Silencing- LX-2 cells were seeded in 6-well plates at a density of 0.5 × 10 5 cells per well, and upon reaching 80% confluency, the cells were transfected with TIRAP-specific siRNA at concentrations of 50 nM. For transfection, TIRAP siRNA was diluted in 100 µL of serum-free media, and separately, 5 µL of Lipofectamine transfection reagent was diluted in 100 µL of serum-free media. Both solutions were combined and incubated at room temperature for 45 minutes to allow complex formation. The resulting siRNA-lipid complexes were then added to macrophages in wells containing 800 µL of serum-free media, and the cells were incubated with the complexes for 4–6 hours. Following this, 1 mL of complete medium supplemented with 2X serum concentration was added to each well, and incubation continued for an additional 24 hours. Immunofluorescence- LX-2 cells were seeded on coverslips in 12-well plates (0.5 × 10 5 cells/well). For fluorescence staining, after treatment, cells were washed with PBS and fixed in 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton-X 100 for 10 min. Then, the cells were incubated with 5% blocking reagent at room temperature to block nonspecific binding sites. After that, cells were incubated with primary antibodies against TIRAP (Santa Cruz, USA), p-TIRAP (Invitrogen, USA) overnight at 4°C, followed by incubation with FITC-conjugated Alexa Fluor 488 and Alexa Fluor 594. Nuclear counterstaining was performed using DAPI mounting medium (Thermo Fisher Scientific, USA) according to the manufacturer's instructions. Stained cells were analyzed using an Olympus confocal laser scanning microscope. Immunoblotting- Cells were collected and washed three times with phosphate-buffered saline (PBS) before being lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with protease and phosphatase inhibitors. Total protein concentration was quantified using the Bradford protein assay (Bio-Rad, USA). Equal amounts of protein were then resolved by SDS-PAGE and transferred onto nitrocellulose membranes (Bio-Rad, USA). The membranes were blocked with 5% BSA (HiMedia, India) for 1 hour at room temperature and subsequently incubated with the appropriate primary antibodies overnight at 4°C. Following primary antibody incubation, the membranes were washed three times with TBST and then incubated with horseradish peroxidase (HRP)–conjugated secondary antibodies for 1 hour at room temperature. The blots were then incubated with the chemiluminescence HRP Substrate (Bio-Rad, USA) and imaged using a gel doc (Image Quant LAS 4000, GE Healthcare, Uppsala, Sweden) following a second TBST wash. ImageJ (National Institutes of Health; doi.org/10.1038/nmeth.2089 ) was used to determine band intensities. The antibodies used were as follows: TIRAP (Santa Cruz, USA), p-TIRAP (Invitrogen, USA), β-Actin (Invitrogen, USA), & secondary HRP conjugated antibody. Nuclear cytoplasmic assay -The LX-2 cells were washed with PBS, and the cytoplasmic and nuclear proteins were extracted using the Nuclear/Cytosol Fractionation Kit (Bio Vision, USA) according to the manufacturer’s protocol. Protein concentration was estimated using the Bradford assay dye reagent (Bio-Rad, USA) according to the manufacturer’s instructions. Equal protein concentration was prepared in 4x protein loading buffer and resolved on a 12% SDS-PAGE gel, transferred onto nitrocellulose membrane (Bio-Rad, USA), blocked with 5% BSA (HiMedia, India) for 1 h at room temperature, and probed with primary antibodies at 4°C overnight. After incubation, the membrane was washed three times with TBST buffer and incubated with an HRP-conjugated secondary antibody (Invitrogen, USA) for 1 hour at room temperature. The blots were washed again with TBST and detected using the enhanced chemiluminescence HRP Substrate (BioRad, USA) using a Gel documentation system (Image Quant LAS 4000, GE Healthcare, Uppsala, Sweden). Band intensities were determined using ImageJ. Results and Discussion Ethanol and LPS Induce Nuclear Translocation of TIRAP in Hepatic Stellate Cells - To investigate whether TIRAP activation contributes to inflammation and fibrosis in alcohol-related liver disease, we employed LX-2 hepatic stellate cells as an experimental model. Initially, we examined whether ethanol exposure induces phosphorylation of TIRAP. LX-2 cells were treated with 25 mM ethanol for 24 hours and analyzed using a phospho-TIRAP–specific antibody (Fig. 1 A and B). As anticipated we found ethanol-dependent increase in TIRAP phosphorylation (activation) via both immunofluorescence as well as immunoblotting. Interestingly, at the cytosol or plasma membrane, immunocytochemical analysis revealed that the activation was found to be accompanied with pronounced nuclear localization of TIRAP. Distinct punctate nuclear staining was observed, clearly contrasting with its commonly described cytoplasmic distribution. This unexpected finding was further validated using nuclear–cytoplasmic fractionation assays, which demonstrated consistent enrichment of TIRAP in nuclear fractions accompanied by reduced levels in the cytoplasmic compartment (Fig. 1 C– 1 F). Additionally, combined treatment with LPS and ethanol further enhanced nuclear localization of TIRAP, mimicking alcoholic liver disease conditions and confirming that this translocation is a reproducible and biologically regulated event. These observations expand the functional scope of TIRAP beyond its canonical cytoplasmic role. The presence of TIRAP within the nucleus suggests potential involvement in nuclear signaling mechanisms. Given that adaptor proteins rarely localize to the nucleus, this discovery raises the possibility that TIRAP may interact with transcriptional regulators, chromatin-associated proteins, or other nuclear partners, thereby influencing the expression of fibrosis-related and pro-inflammatory genes. TIRAP Knockdown Reduces Nuclear Localization and Suppresses Fibrogenic Marker Expression- To confirm the requirement of TIRAP for nuclear translocation under alcoholic conditions, TIRAP expression was silenced in LX-2 cells using siRNA. Knockdown of TIRAP markedly diminished its nuclear localization following ethanol and LPS exposure (Fig. 2 A), demonstrating that the observed nuclear accumulation is TIRAP-dependent. Next, to determine the functional relevance of nuclear TIRAP in fibrogenesis, we examined the expression of key extracellular matrix components and fibrotic markers. Confocal microscopy revealed that silencing TIRAP significantly reduced levels of α-SMA and collagen compared with control cells (Fig. 2 C and 2 D). Further decreased expression α-SMA and collagen was found at mRNA level by RT-PCR (Fig. 2 E). These findings indicate that nuclear TIRAP plays a critical role in activating fibrogenic signaling pathways in hepatic stellate cells. While the involvement of TLR4-driven inflammatory signaling in alcoholic liver disease (ALD) is well established, much less attention has been given to the role of adaptor proteins that fine-tune these pathways. In particular, the contribution of TIRAP in alcohol-induced liver inflammation remains largely unexplored. Most previous studies have emphasized receptor activation or downstream transcriptional responses, leaving adaptor-level regulation insufficiently understood. In this context, our study offers one of the earliest insights into how TIRAP may participate in the inflammatory processes associated with ALD. Our findings indicate that TIRAP functions as an important signaling node by interacting with multiple cytoplasmic proteins, thereby influencing key downstream pathways that drive hepatic inflammation. The observed alterations in TIRAP signaling under alcohol-induced conditions suggest that this adaptor plays a previously unrecognized role in shaping inflammatory outcomes in ALD. Taken together, these results extend the current understanding of TLR4 signaling complexity in alcoholic liver disease and point toward TIRAP as a promising, yet underappreciated, target for therapeutic intervention. Taken together, these results identify nuclear TIRAP as a previously unrecognized regulator of stellate-cell activation and fibrotic progression in alcohol-induced liver injury (Fig. 3 ). Traditionally viewed as a cytoplasmic adaptor mediating Toll-like receptor signaling, TIRAP now emerges as a potential modulator of nuclear processes, possibly through interactions with transcription factors such as c-Jun—known to regulate fibrogenic gene expression. By amplifying pro-inflammatory and pro-fibrotic signals from within the nucleus, nuclear TIRAP may represent a key checkpoint in alcoholic liver fibrosis. Future studies focusing on the nuclear interactome of TIRAP and its transcriptional targets will be essential to fully define its mechanistic role and therapeutic potential. Conclusion The study provides the first direct evidence of TIRAP translocating to the nucleus in a model of liver fibrosis, revealing a novel and previously unrecognized aspect of TIRAP biology. This nuclear localization suggests that TIRAP may have functions beyond its established role in cytoplasmic signaling, potentially influencing gene expression, chromatin dynamics, or other nuclear processes that contribute to fibrogenesis. The identification of nuclear TIRAP opens important questions regarding its regulatory mechanisms, interaction partners, and downstream effects in liver fibrosis. Future studies aimed at mapping TIRAP’s nuclear interactome and dissecting its functional consequences will be critical to determining whether TIRAP acts as a mechanistic driver of liver pathology. Ultimately, these insights could position TIRAP as a promising biomarker for alcoholic liver disease and a potential therapeutic target, paving the way for novel strategies to treat liver fibrosis and associated malignancies. Declarations Conflict of interest The authors declare no conflict of interest. Funding This work was supported by Department of Biotechnology (DBT), Government of India sponsored National Network Project to MSB (NNP-BT/PR40197/BTIS/137/68/2023). Author Contribution Conceptualization and supervision: MSB; Writing and editing: MSB, PP, and RA; Investigation: MSB, PP, and RA. Acknowledgments We thank the Indian Institute of Technology Indore (IITI) for providing the necessary facilities and support. We are genuinely thankful to Dr. Sheikh Tasduq Abdullah for providing the cell line. We are grateful to Khandu Wadhonkar and Kundan Solanki for their assistance with the research experiments. We also extend our sincere thanks to Alexander G. Obukhov for his invaluable comments and for reviewing the manuscript. References Fitzgerald KA, Palsson-McDermott EM, Bowie AG, et al. Mal (MyD88-adapter-like) is required for Toll-like receptor-4 signal transduction. Nature . 2001;413:78–83. Bernard NJ, O’Neill LA. Mal, more than a bridge to MyD88. IUBMB Life . 2013;65:777–786. Rajpoot S, Wary KK, Ibbott R, et al. TIRAP in the Mechanism of Inflammation. Front Immunol . 2021;12:697588. Belhaouane I, Hoffmann E, Chamaillard M, Brodin P, Machelart A. Paradoxical Roles of the MAL/Tirap Adaptor in Pathologies. Front Immunol . 2020;11:569127. Srivastava M, Saqib U, Banerjee S, et al. Inhibition of the TIRAP-c-Jun interaction as a therapeutic strategy for AP1-mediated inflammatory responses. International Immunopharmacology . 2019;71:188–197. Fujii H. Fibrogenesis in alcoholic liver disease. WJG . 2014;20:8048. Chen Y, Zhou P, Deng Y, et al. ALKBH5‐mediated m 6 A demethylation of TIRAP mRNA promotes radiation‐induced liver fibrosis and decreases radiosensitivity of hepatocellular carcinoma. Clinical & Translational Med . 2023;13:e1198. Schulien I, Hockenjos B, Schmitt-Graeff A, et al. The transcription factor c-Jun/AP-1 promotes liver fibrosis during non-alcoholic steatohepatitis by regulating Osteopontin expression. Cell Death Differ . 2019;26:1688–1699. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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-8652622","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":588475456,"identity":"544b6eb0-254c-444a-8eb7-a44713301895","order_by":0,"name":"Pramod Patidar","email":"","orcid":"","institution":"Indian Institute of Technology Indore (IITI)","correspondingAuthor":false,"prefix":"","firstName":"Pramod","middleName":"","lastName":"Patidar","suffix":""},{"id":588475457,"identity":"820f36d3-c7e7-4b92-94dc-8c37f7f47ec4","order_by":1,"name":"Rajat Atre","email":"","orcid":"","institution":"Indian Institute of Technology Indore (IITI)","correspondingAuthor":false,"prefix":"","firstName":"Rajat","middleName":"","lastName":"Atre","suffix":""},{"id":588475461,"identity":"30eabe0b-e7b5-4078-86de-db1482bd0323","order_by":2,"name":"Mirza S. Baig","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuElEQVRIiWNgGAWjYFACHgaGBAMGOTZmBoYDID4bkVoMjNmYmUnRwsBgkNjAwEyks3T7zx5geFDwJ72Pnf/gAYYaOwY+6Qb8Wsxu5CWAHJbbBnbYsWQGNpkDhLTwGCBpYQMiiQQCWs6fAWtJh3j/HzFaDuSAtSSAtTC2EaMF6JcDCQbGhkCHGRxI7EvmIcJhZw8+/PFHTl6+/+DjDx++2cnJzyCgBQQOwFkJ0GgaBaNgFIyCUUAhAAC6zjgtynRK9QAAAABJRU5ErkJggg==","orcid":"","institution":"Indian Institute of Technology Indore (IITI)","correspondingAuthor":true,"prefix":"","firstName":"Mirza","middleName":"S.","lastName":"Baig","suffix":""}],"badges":[],"createdAt":"2026-01-20 19:54:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8652622/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8652622/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102745934,"identity":"57e65470-107f-4c93-afa9-3357c1fbcf06","added_by":"auto","created_at":"2026-02-16 08:54:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":801828,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eTIRAP phosphorylation and nuclear translocation in hepatic stellate cells (HSCs), HSCs were exposed to ethanol (25mM) and LPS (1 μg/ml), for 24 hrs and TIRAP nuclear translocation was analyzed by (A) Confocal microscopy images showing the cellular phosphorylation of TIRAP by immunostaining with their specific primary antibodies for phosphor-TIRAP. The secondary antibodies were Alexa Fluor 594-tagged (red) for phosphor-TIRAP (B)Cellular phosphorylation of TIRAP by western blotting with their specific primary antibodies for TIRAP, phosphor-TIRAP and secondary antibody (C and D) Confocal microscopy images showing the nuclear translocation of TIRAP by immunostaining with their specific primary antibodies for TIRAP. The secondary antibodies were Alexa Fluor 488-tagged (green) for TIRAP. (E and F) Nuclear cytoplasmic assay. All data are representative of three independent experiments and are presented as the mean ± SD, (One-way Anova test). ****P≤ 0.00001; ***P≤ 0.0001; **P ≤ 0.001; *P ≤ 0.01.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8652622/v1/2143d33023b56e24d710423b.png"},{"id":102428765,"identity":"1c5a4dd4-e087-4f80-a481-a607fea86d70","added_by":"auto","created_at":"2026-02-11 14:58:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":843694,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eTIRAP nuclear translocation in hepatic stellate cells (HSCs), HSCs were exposed to ethanol (25mM) and LPS (1 μg/ml), for 24 hrs and TIRAP nuclear translocation was analyzed in TIRAP silencing condition by (A and B) Nuclear cytoplasmic assay. (C and D) Confocal microscopy images showing the cellular expression of collagen and α-SMA by immunostaining with their specific primary antibodies for collagen and α-SMA. The secondary antibodies were Alexa Fluor 594-tagged (red) for collagen and Alexa Fluor 488-tagged (green) for α-SMA (E) mRNA expression of α-SMA and collagen via RT-PCR. All data are representative of three independent experiments and are presented as the mean ± SD, (One-way Anova test). ****P≤ 0.00001; ***P≤ 0.0001; **P ≤ 0.001; *P ≤ 0.01.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8652622/v1/12bbaaec666a7d6c53ad935f.png"},{"id":102745890,"identity":"b7f0cc95-ca5d-46b9-85cb-280bb6e23d79","added_by":"auto","created_at":"2026-02-16 08:54:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":147937,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eSchematic showing TIRAP translocation from the membrane (m-TIRAP) and cytoplasm (c-TIRAP) to the nucleus in hepatic stellate cells in response to LPS and ethanol (ETOH) treatment.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8652622/v1/b78436e614de07a01e7c6dc2.png"},{"id":106455120,"identity":"be537d9e-575a-4b60-9ec6-e69c1c1c4cdb","added_by":"auto","created_at":"2026-04-08 17:40:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2126451,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8652622/v1/f64e3d13-61d2-4578-9aff-12b71c1152ed.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Toll/interleukin-1 receptor domain-containing adaptor protein (TIRAP) in Hepatic Stellate Cells: Beyond Membrane and Cytoplasmic Presence","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe Toll-interleukin-1 receptor (TIR) domain-containing adaptor protein, also known as MyD88-adaptor-like (MAL), is a pivotal adaptor molecule in the innate immune system\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Functionally, TIRAP acts as a bridging adaptor for several Toll-like receptors (TLR) family members, most notably TLR2 and TLR4, and facilitates downstream signaling by recruiting MyD88 to activated receptors. Through this mechanism, it governs the activation of key transcription factors such as nuclear factor κB (NF-κB) and activator protein 1 (AP-1), thereby triggering the expression of pro-inflammatory cytokines, chemokines, and other immune mediators essential for host defense\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Since its discovery in 2001, TIRAP has been extensively studied and is now recognized as a major regulator of inflammatory signaling, largely owing to its ability to form complex protein\u0026ndash;protein interaction networks through its conserved TIR domain\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Dysregulation of TIRAP whether through altered expression, genetic polymorphisms, or functional modification has been implicated in a broad spectrum of disorders, including autoimmune conditions, chronic inflammatory diseases, microbial infections, and even tumorigenesis\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. In these pathological contexts, aberrant TIRAP-dependent signaling can exacerbate immune activation or impair the resolution of inflammation, underscoring its importance as both a homeostatic regulator and a potential therapeutic target. Traditionally, TIRAP has been described as a membrane-associated or cytoplasmic adaptor, functioning proximally to TLRs at the plasma membrane or endosomal compartments. Interestingly, a study has also suggested that TIRAP may interact with c-Jun, a subunit of the AP-1 transcription factor, particularly under conditions of LPS-dependent TLR4 activation\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. This interaction highlights an additional layer of complexity in TIRAP-mediated signaling, suggesting that, beyond its role in bridging MyD88 recruitment, TIRAP may directly modulate transcription factor activity to fine-tune inflammatory responses.\u003c/p\u003e \u003cp\u003eBuilding on this concept, we sought to investigate the role of TIRAP in the context of alcohol-induced liver injury and fibrosis. Chronic alcohol consumption is a well-established driver of hepatic inflammation, oxidative stress, and stellate cell activation, leading to progressive fibrosis\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. While TLR signaling and particularly TLR4 have been strongly implicated in these processes, the contribution of TIRAP remains incompletely understood. Intriguingly, previous studies have implicated TIRAP as an important mediator in radiation-induced liver injury, further supporting its potential relevance in liver pathophysiology\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. These prompted us to examine whether TIRAP functions as a critical signaling adaptor influencing transcriptional programs that drive inflammation and fibrosis in alcohol-related liver disease.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003eCell culture\u003c/b\u003e -The LX-2 cell line was provided by (Dr. Sheikh Tasduq Abdullah, CSIR \u0026ndash; Indian Institute of Integrative Medicine, Jammu). The cells were maintained in Dulbecco's Modified Eagle Medium (DMEM, Gibco ThermoFisher Scientific India) supplemented with 10% FBS (Gibco, ThermoFisher Scientific India) and 1% penicillin/streptomycin in an incubator at 37\u0026deg;C with 5% CO2. To mimic the alcoholic liver disease, HSCs were treated with LPS (1 \u0026micro;g/ml) and ethanol (25 mM).\u003c/p\u003e \u003cp\u003e \u003cb\u003esiRNA Transfection and TIRAP Silencing-\u003c/b\u003e LX-2 cells were seeded in 6-well plates at a density of 0.5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well, and upon reaching 80% confluency, the cells were transfected with TIRAP-specific siRNA at concentrations of 50 nM. For transfection, TIRAP siRNA was diluted in 100 \u0026micro;L of serum-free media, and separately, 5 \u0026micro;L of Lipofectamine transfection reagent was diluted in 100 \u0026micro;L of serum-free media. Both solutions were combined and incubated at room temperature for 45 minutes to allow complex formation. The resulting siRNA-lipid complexes were then added to macrophages in wells containing 800 \u0026micro;L of serum-free media, and the cells were incubated with the complexes for 4\u0026ndash;6 hours. Following this, 1 mL of complete medium supplemented with 2X serum concentration was added to each well, and incubation continued for an additional 24 hours.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunofluorescence-\u003c/b\u003e LX-2 cells were seeded on coverslips in 12-well plates (0.5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well). For fluorescence staining, after treatment, cells were washed with PBS and fixed in 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton-X 100 for 10 min. Then, the cells were incubated with 5% blocking reagent at room temperature to block nonspecific binding sites. After that, cells were incubated with primary antibodies against TIRAP (Santa Cruz, USA), p-TIRAP (Invitrogen, USA) overnight at 4\u0026deg;C, followed by incubation with FITC-conjugated Alexa Fluor 488 and Alexa Fluor 594. Nuclear counterstaining was performed using DAPI mounting medium (Thermo Fisher Scientific, USA) according to the manufacturer's instructions. Stained cells were analyzed using an Olympus confocal laser scanning microscope.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunoblotting-\u003c/b\u003e Cells were collected and washed three times with phosphate-buffered saline (PBS) before being lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with protease and phosphatase inhibitors. Total protein concentration was quantified using the Bradford protein assay (Bio-Rad, USA). Equal amounts of protein were then resolved by SDS-PAGE and transferred onto nitrocellulose membranes (Bio-Rad, USA). The membranes were blocked with 5% BSA (HiMedia, India) for 1 hour at room temperature and subsequently incubated with the appropriate primary antibodies overnight at 4\u0026deg;C. Following primary antibody incubation, the membranes were washed three times with TBST and then incubated with horseradish peroxidase (HRP)\u0026ndash;conjugated secondary antibodies for 1 hour at room temperature. The blots were then incubated with the chemiluminescence HRP Substrate (Bio-Rad, USA) and imaged using a gel doc (Image Quant LAS 4000, GE Healthcare, Uppsala, Sweden) following a second TBST wash. ImageJ (National Institutes of Health; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003edoi.org/10.1038/nmeth.2089\u003c/span\u003e\u003cspan address=\"10.1038/nmeth.2089\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to determine band intensities. The antibodies used were as follows: TIRAP (Santa Cruz, USA), p-TIRAP (Invitrogen, USA), β-Actin (Invitrogen, USA), \u0026amp; secondary HRP conjugated antibody.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNuclear cytoplasmic assay\u003c/b\u003e -The LX-2 cells were washed with PBS, and the cytoplasmic and nuclear proteins were extracted using the Nuclear/Cytosol Fractionation Kit (Bio Vision, USA) according to the manufacturer\u0026rsquo;s protocol. Protein concentration was estimated using the Bradford assay dye reagent (Bio-Rad, USA) according to the manufacturer\u0026rsquo;s instructions. Equal protein concentration was prepared in 4x protein loading buffer and resolved on a 12% SDS-PAGE gel, transferred onto nitrocellulose membrane (Bio-Rad, USA), blocked with 5% BSA (HiMedia, India) for 1 h at room temperature, and probed with primary antibodies at 4\u0026deg;C overnight. After incubation, the membrane was washed three times with TBST buffer and incubated with an HRP-conjugated secondary antibody (Invitrogen, USA) for 1 hour at room temperature. The blots were washed again with TBST and detected using the enhanced chemiluminescence HRP Substrate (BioRad, USA) using a Gel documentation system (Image Quant LAS 4000, GE Healthcare, Uppsala, Sweden). Band intensities were determined using ImageJ.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e \u003cb\u003eEthanol and LPS Induce Nuclear Translocation of TIRAP in Hepatic Stellate Cells\u003c/b\u003e- To investigate whether TIRAP activation contributes to inflammation and fibrosis in alcohol-related liver disease, we employed LX-2 hepatic stellate cells as an experimental model. Initially, we examined whether ethanol exposure induces phosphorylation of TIRAP. LX-2 cells were treated with 25 mM ethanol for 24 hours and analyzed using a phospho-TIRAP\u0026ndash;specific antibody (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and B). As anticipated we found ethanol-dependent increase in TIRAP phosphorylation (activation) via both immunofluorescence as well as immunoblotting. Interestingly, at the cytosol or plasma membrane, immunocytochemical analysis revealed that the activation was found to be accompanied with pronounced nuclear localization of TIRAP. Distinct punctate nuclear staining was observed, clearly contrasting with its commonly described cytoplasmic distribution. This unexpected finding was further validated using nuclear\u0026ndash;cytoplasmic fractionation assays, which demonstrated consistent enrichment of TIRAP in nuclear fractions accompanied by reduced levels in the cytoplasmic compartment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u0026ndash;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Additionally, combined treatment with LPS and ethanol further enhanced nuclear localization of TIRAP, mimicking alcoholic liver disease conditions and confirming that this translocation is a reproducible and biologically regulated event.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese observations expand the functional scope of TIRAP beyond its canonical cytoplasmic role. The presence of TIRAP within the nucleus suggests potential involvement in nuclear signaling mechanisms. Given that adaptor proteins rarely localize to the nucleus, this discovery raises the possibility that TIRAP may interact with transcriptional regulators, chromatin-associated proteins, or other nuclear partners, thereby influencing the expression of fibrosis-related and pro-inflammatory genes.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTIRAP Knockdown Reduces Nuclear Localization and Suppresses Fibrogenic Marker Expression-\u003c/b\u003e To confirm the requirement of TIRAP for nuclear translocation under alcoholic conditions, TIRAP expression was silenced in LX-2 cells using siRNA. Knockdown of TIRAP markedly diminished its nuclear localization following ethanol and LPS exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), demonstrating that the observed nuclear accumulation is TIRAP-dependent. Next, to determine the functional relevance of nuclear TIRAP in fibrogenesis, we examined the expression of key extracellular matrix components and fibrotic markers. Confocal microscopy revealed that silencing TIRAP significantly reduced levels of α-SMA and collagen compared with control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Further decreased expression α-SMA and collagen was found at mRNA level by RT-PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). These findings indicate that nuclear TIRAP plays a critical role in activating fibrogenic signaling pathways in hepatic stellate cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhile the involvement of TLR4-driven inflammatory signaling in alcoholic liver disease (ALD) is well established, much less attention has been given to the role of adaptor proteins that fine-tune these pathways. In particular, the contribution of TIRAP in alcohol-induced liver inflammation remains largely unexplored. Most previous studies have emphasized receptor activation or downstream transcriptional responses, leaving adaptor-level regulation insufficiently understood. In this context, our study offers one of the earliest insights into how TIRAP may participate in the inflammatory processes associated with ALD. Our findings indicate that TIRAP functions as an important signaling node by interacting with multiple cytoplasmic proteins, thereby influencing key downstream pathways that drive hepatic inflammation. The observed alterations in TIRAP signaling under alcohol-induced conditions suggest that this adaptor plays a previously unrecognized role in shaping inflammatory outcomes in ALD. Taken together, these results extend the current understanding of TLR4 signaling complexity in alcoholic liver disease and point toward TIRAP as a promising, yet underappreciated, target for therapeutic intervention.\u003c/p\u003e \u003cp\u003eTaken together, these results identify nuclear TIRAP as a previously unrecognized regulator of stellate-cell activation and fibrotic progression in alcohol-induced liver injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Traditionally viewed as a cytoplasmic adaptor mediating Toll-like receptor signaling, TIRAP now emerges as a potential modulator of nuclear processes, possibly through interactions with transcription factors such as c-Jun\u0026mdash;known to regulate fibrogenic gene expression. By amplifying pro-inflammatory and pro-fibrotic signals from within the nucleus, nuclear TIRAP may represent a key checkpoint in alcoholic liver fibrosis. Future studies focusing on the nuclear interactome of TIRAP and its transcriptional targets will be essential to fully define its mechanistic role and therapeutic potential.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe study provides the first direct evidence of TIRAP translocating to the nucleus in a model of liver fibrosis, revealing a novel and previously unrecognized aspect of TIRAP biology. This nuclear localization suggests that TIRAP may have functions beyond its established role in cytoplasmic signaling, potentially influencing gene expression, chromatin dynamics, or other nuclear processes that contribute to fibrogenesis. The identification of nuclear TIRAP opens important questions regarding its regulatory mechanisms, interaction partners, and downstream effects in liver fibrosis. Future studies aimed at mapping TIRAP\u0026rsquo;s nuclear interactome and dissecting its functional consequences will be critical to determining whether TIRAP acts as a mechanistic driver of liver pathology. Ultimately, these insights could position TIRAP as a promising biomarker for alcoholic liver disease and a potential therapeutic target, paving the way for novel strategies to treat liver fibrosis and associated malignancies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by Department of Biotechnology (DBT), Government of India sponsored National Network Project to MSB (NNP-BT/PR40197/BTIS/137/68/2023).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization and supervision: MSB; Writing and editing: MSB, PP, and RA; Investigation: MSB, PP, and RA.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eWe thank the Indian Institute of Technology Indore (IITI) for providing the necessary facilities and support. We are genuinely thankful to Dr. Sheikh Tasduq Abdullah for providing the cell line. We are grateful to Khandu Wadhonkar and Kundan Solanki for their assistance with the research experiments. We also extend our sincere thanks to Alexander G. Obukhov for his invaluable comments and for reviewing the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFitzgerald KA, Palsson-McDermott EM, Bowie AG, et al. Mal (MyD88-adapter-like) is required for Toll-like receptor-4 signal transduction. \u003cem\u003eNature\u003c/em\u003e. 2001;413:78\u0026ndash;83. \u003c/li\u003e\n\u003cli\u003eBernard NJ, O\u0026rsquo;Neill LA. Mal, more than a bridge to MyD88. \u003cem\u003eIUBMB Life\u003c/em\u003e. 2013;65:777\u0026ndash;786. \u003c/li\u003e\n\u003cli\u003eRajpoot S, Wary KK, Ibbott R, et al. TIRAP in the Mechanism of Inflammation. \u003cem\u003eFront Immunol\u003c/em\u003e. 2021;12:697588. \u003c/li\u003e\n\u003cli\u003eBelhaouane I, Hoffmann E, Chamaillard M, Brodin P, Machelart A. Paradoxical Roles of the MAL/Tirap Adaptor in Pathologies. \u003cem\u003eFront Immunol\u003c/em\u003e. 2020;11:569127. \u003c/li\u003e\n\u003cli\u003eSrivastava M, Saqib U, Banerjee S, et al. Inhibition of the TIRAP-c-Jun interaction as a therapeutic strategy for AP1-mediated inflammatory responses. \u003cem\u003eInternational Immunopharmacology\u003c/em\u003e. 2019;71:188\u0026ndash;197. \u003c/li\u003e\n\u003cli\u003eFujii H. Fibrogenesis in alcoholic liver disease. \u003cem\u003eWJG\u003c/em\u003e. 2014;20:8048. \u003c/li\u003e\n\u003cli\u003eChen Y, Zhou P, Deng Y, et al. ALKBH5‐mediated m\u003csup\u003e6\u003c/sup\u003e A demethylation of TIRAP mRNA promotes radiation‐induced liver fibrosis and decreases radiosensitivity of hepatocellular carcinoma. \u003cem\u003eClinical \u0026amp; Translational Med\u003c/em\u003e. 2023;13:e1198. \u003c/li\u003e\n\u003cli\u003eSchulien I, Hockenjos B, Schmitt-Graeff A, et al. The transcription factor c-Jun/AP-1 promotes liver fibrosis during non-alcoholic steatohepatitis by regulating Osteopontin expression. \u003cem\u003eCell Death Differ\u003c/em\u003e. 2019;26:1688\u0026ndash;1699. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"TIRAP, MyD88, LPS, Alcohol, Hepatic Stellate Cell (HSC), and Fibrosis","lastPublishedDoi":"10.21203/rs.3.rs-8652622/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8652622/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe Toll/interleukin-1 receptor domain-containing adaptor protein (TIRAP) is a key adaptor in Toll-like receptor signaling, classically functioning as a cytoplasmic bridging adaptor for Myeloid differentiation primary response 88 (MyD88) recruitment. While its role in inflammatory signaling is well established, its contribution to alcohol-induced liver fibrosis remains unclear. Using lipopolysaccharide (LPS) and ethanol-stimulated hepatic stellate cells (LX-2), we demonstrate, for the first time, nuclear translocation of TIRAP under alcoholic conditions. Confocal microscopy and nuclear\u0026ndash;cytoplasmic fractionation confirmed TIRAP enrichment in the nucleus. Importantly, silencing of TIRAP in LX-2 significantly reduced the nuclear translocation and expression of fibrotic markers (such as α-SMA and collagen) in HSCs, suggesting a key role of nuclear TIRAP in driving fibrogenesis. Overall, these findings reveal a novel role for TIRAP beyond cytoplasmic signaling, suggesting potential nuclear functions that may regulate transcriptional programs driving inflammation and fibrosis.\u003c/p\u003e","manuscriptTitle":"Toll/interleukin-1 receptor domain-containing adaptor protein (TIRAP) in Hepatic Stellate Cells: Beyond Membrane and Cytoplasmic Presence","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-11 14:58:15","doi":"10.21203/rs.3.rs-8652622/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8687db41-49b7-41d9-8f32-c8e7d6623661","owner":[],"postedDate":"February 11th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-08T17:39:42+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-11 14:58:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8652622","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8652622","identity":"rs-8652622","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}