KDM5B drives acute liver injury by impairing autophagy and activating cGAS-STING signaling via binding to ATG16L1

KDM5B drives acute liver injury by impairing autophagy and activating cGAS-STING signaling via binding to ATG16L1 | 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 Article KDM5B drives acute liver injury by impairing autophagy and activating cGAS-STING signaling via binding to ATG16L1 Lihua Qu, Haoxiang Ou, Yaoyao Ma, Jiangyue Wang, Jiaqi Xiao, Danxia Liu, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7430149/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 Acute liver injury (ALI) is a severe hepatic disorder characterized by high morbidity and mortality, presenting major challenges for clinical management. However, its underlying pathogenesis remains incompletely understood. Through analysis of four Gene Expression Omnibus (GEO) datasets using the Robust Rank Aggregation (RRA) method and RNA sequencing, the histone demethylase KDM5B was identified as a core upregulated gene in ALI. This finding was validated in carbon tetrachloride (CCl 4 )-induced murine ALI models, in which KDM5B expression was significantly elevated. Liver-specific knockdown of KDM5B via AAV-shKDM5B alleviated hepatic injury by reducing immune cell infiltration and inflammatory cytokine release, while enhancing autophagic flux. Mechanistically, KDM5B bound to the autophagy-related protein ATG16L1, disrupting the assembly of the ATG12-ATG5-ATG16L1 complex and impairing autophagosome maturation. This led to the accumulation of cytoplasmic DNA, which activated the cGAS-STING-TBK1-IRF3/NF-κB signaling pathway and amplified pro-inflammatory responses. This study revealed a protein-interaction-dependent mechanism by which KDM5B inhibited autophagy and triggered cGAS-STING signaling. It uncovered a novel mechanism underlying the vicious cycle of impaired autophagy and excessive inflammation in ALI, offering a molecular basis and potential therapeutic target involving the KDM5B-ATG16L1 axis. Biological sciences/Cell biology/Autophagy Biological sciences/Immunology/Innate immunity Acute liver injury Inflammation Autophagy KDM5B cGAS-STING Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Acute liver injury (ALI) is a common disorder that poses a significant threat to public health 1 . Its pathogenesis is complex, marked by a sudden onset and progressive deterioration. Critically, ALI often serves as the precursor to acute liver failure and can rapidly progress to liver failure 2 , which triggering systemic inflammatory responses, coagulopathy, multi-organ dysfunction, and potentially life-threatening conditions 3 , 4 . Notably, ALI can be induced by various etiological factors, such as viral infections (e.g., hepatitis B and C viruses), drug or toxin exposure (e.g., acetaminophen overdose) and autoimmune reactions 5 , 6 , 7 , 8 . Current therapeutic approaches primarily include pharmacological treatments, supportive care, and liver transplantation 9 . However, the scarcity of donor organs 10 , suboptimal efficacy of existing pharmacotherapies 11 , and an incomplete understanding of the underlying pathogenesis in many cases continue to hinder clinical management. Therefore, further investigation into the mechanisms driving ALI and the identification of novel therapeutic targets is urgently needed. Autophagy is an evolutionarily conserved lysosomal degradation pathway in eukaryotes that maintains cellular homeostasis and metabolic stability 12 . To date, at least 19 autophagy-related proteins have been identified as essential regulators of autophagosome formation. Initially discovered in yeast, these proteins are highly conserved across eukaryotic species 13 . During autophagy initiation, microtubule-associated protein 1 light chain 3 (LC3) and the ATG12-ATG5-ATG16L1 complex assemble on the phagophore. Interacting with the ATG12-ATG5 conjugate, ATG16L1 forms a dimeric complex that activates ATG3, thereby promoting the lipidation of LC3 with phosphatidylethanolamine. This process converts LC3B-I to LC3B-II, which is essential for autophagy activation 14 . Thus, the formation of the ATG12-ATG5-ATG16L1 complex is essential for autophagosome maturation. In the context of ALI, autophagy plays a dual role: homeostatic autophagy helps to eliminate damaged organelles and misfolded proteins, while dysregulated autophagy amplifies inflammation and induces cell death 15 . For example, macrophage-specific deletion of ATG16L1 in partial hepatectomy models reduces autophagic flux, activates inflammatory pathways, and impairs liver regeneration 16 . Similarly, hepatocyte-specific knockdown of ATG5 blocks autophagosome formation and aggravates lipopolysaccharide (LPS)-induced hepatic inflammation 17 . These findings highlight the essential anti-inflammatory role of autophagy 18 , 19 . However, the molecular mechanisms underlying autophagy dysregulation in ALI remain poorly understood and warrant further investigation. The cyclic GMP-AMP synthase–stimulator of interferon genes (cGAS-STING) pathway is a key innate immune signaling axis that has garnered considerable attention in recent years 20 . Its functions by sensing aberrant cytosolic DNA and initiating inflammatory responses 21 . Within this pathway, cGAS serves as the cytosolic DNA sensor. Upon detecting and binding to cytosolic DNA, cGAS catalyzes the production of the second messenger 2'3'-cyclic GMP-AMP (cGAMP). Subsequently, cGAMP binds to and activates the STING, which is located on the endoplasmic reticulum membrane 22 , 23 . Upon activation, STING undergoes a conformational change and recruits TANK-binding kinase 1 (TBK1), which phosphorylates interferon regulatory factor 3 (IRF3). This leads to the production of type I interferons (IFN-I) and pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) 24 . Dysregulated cGAS-STING signaling contributes to immunopathology in various hepatic inflammatory diseases 25 . In ALI, activation of this pathway promotes inflammatory cell infiltration and hepatocyte apoptosis 26 . Although the pro-inflammatory role of cGAS-STING signaling in ALI is well established 26 , the upstream molecular events leading to its activation remain incompletely understood. Lysine Demethylase 5B (KDM5B), a member of the Jumonji C (JmjC) domain-containing histone demethylase family, is best known for catalyzing the demethylation of histone H3 lysine 4 di- and tri-methylation (H3K4me2/3), thereby modulating transcriptional activity 27 . However, the function of KDM5B extends beyond histone demethylation. For example, a study in alcohol-exposed male mice showed that KDM5B can bind to the promoter region of hepatocyte nuclear factor 4 alpha (HNF4A) and exacerbate alcohol-induced hepatocyte injury through a mechanism independent of its histone demethylase function 28 . While KDM5B has been implicated in tumorigenesis, stem cell differentiation, and metabolic disorders 29 , 30 , its specific role and underlying mechanisms in the context of ALI remain unexplored. In this study, we found that KDM5B binds to the key autophagy-related protein ATG16L1, disrupting the formation of the ATG12-ATG5-ATG16L1 complex and thereby inhibiting autophagosome maturation in ALI. This autophagy impairment resulted in the accumulation of cytosolic DNA, which activated the cGAS-STING signaling pathway and triggered excessive inflammatory cytokine release, ultimately exacerbating hepatic injury. To validate this mechanism, we generated a hepatocyte-specific KDM5B knockdown mouse model. KDM5B knockdown significantly alleviated carbon tetrachloride (CCl 4 )-induced ALI, as evidenced by reduced immune cell infiltration, restoration of autophagic flux, and suppression of cGAS-STING pathway activation. Collectively, these findings uncovered a novel mechanism by which KDM5B regulated autophagy independently of its histone demethylase activity, providing a theoretical basis for targeting the KDM5B-ATG16L1 axis as a potential therapeutic strategy for ALI. Results GEO dataset-based differential gene expression analysis in ALI To identify potential genes related to ALI, we analyzed four Gene Expression Omnibus (GEO) datasets and identified 400 differentially expressed genes (DEGs). Robust Rank Aggregation (RRA) analysis further narrowed this to 40 significantly altered genes, including 20 upregulated and 20 downregulated genes. Notably, KDM5B was markedly upregulated in ALI patients compared to controls (Fig. 1 A-B). Additionally, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed enriched DEGs in innate immune and inflammatory pathways, including the cytosolic DNA-sensing pathway and the interleukin-17 (IL-17) signaling pathway (Fig. 1 C). KDM5B expression was significantly up-regulated in ALI To validate KDM5B as a key DEG in ALI, we established a murine model via intraperitoneal injection of CCl 4 and performed RNA sequencing on liver tissues collected 24 hours post-injection (Fig. 2 A). Volcano plot analysis identified 1,084 DEGs compared to controls, including 733 upregulated and 351 downregulated genes (Fig. 2 B). Heatmap analysis of the KDM family revealed a marked upregulation of KDM5B in CCl 4 -treated mice (Fig. 2 C), which was further confirmed by quantitative real-time PCR (qRT-PCR), showing significantly elevated KDM5B mRNA levels in CCl 4 -induced liver tissues (Fig. 2 D). To further assess KDM5B protein expression, immunohistochemistry and western blotting were performed on liver tissues collected at 24 and 48 hours after CCl 4 injection. Immunohistochemical staining showed a significant increase in KDM5B expression at 24 hours, followed by a decline at 48 hours (Fig. 2 E). Consistently, western blotting revealed a similar pattern, with peak KDM5B expression at 24 hours and a relative decrease at 48 hours compared to the controls (Fig. 2 F). To investigate KDM5B expression in different liver cell types, we isolated primary hepatocytes, lymphocytes, and macrophages from CCl 4 - treated mice. The qRT-PCR analysis showed that KDM5B expression was markedly elevated in all three cell types following CCl 4 administration (Fig. 2 G). Collectively, the findings indicate that both KDM5B mRNA and protein levels are significantly upregulated in CCl 4 -induced murine model of ALI. Liver-specific knockdown of KDM5B ameliorated CCl-induced ALI To explore the functional role of KDM5B in CCl 4 -induced ALI, we administered adeno-associated virus (AAV) vectors encoding either shRNA targeting KDM5B (AAV-shKDM5B) or KDM5B overexpression construct (AAV8-KDM5B) via tail vein injection. Two weeks post-injection, ALI was induced through intraperitoneal administration of CCl 4 (1 mL/kg), and liver tissues were collected 24 hours later for analysis (Fig. 3 A). Hematoxylin and eosin (H&E) staining revealed a significant reduction in necrotic liver areas in mice with liver-specific KDM5B knockdown compared to control groups (Fig. 3 B). Correspondingly, serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), both indicators of liver injury, were significantly decreased in the knockdown group (Fig. 3 C). Western blot analysis confirmed that AAV-shKDM5B effectively reduced hepatic KDM5B protein expression in CCl 4 -treated mice, indicating successful knockdown of KDM5B (Fig. 3 D). In contrast, liver-specific overexpression of KDM5B aggravated liver injury. H&E staining showed significantly enlarged necrotic areas (Fig. 3 E), accompanied by a marked increase in serum ALT and AST levels compared to controls (Fig. 3 F). Collectively, these findings demonstrate the critical role of KDM5B in the pathogenesis of CCl 4 -induced ALI. Liver-specific knockdown of KDM5B significantly mitigates liver injury, whereas its overexpression exacerbates hepatic damage. Liver-specific knockdown of KDM5B alleviated inflammation in CCl-induced ALI To examine the immunoregulatory role of KDM5B in CCl 4 -induced ALI, the impact of KDM5B modulation on hepatic inflammatory responses was assessed. Immunohistochemical staining revealed that CCl 4 treatment significantly increased infiltration of F4/80 + macrophages in the liver, which was markedly reduced by AAV-shKDM5B treatment. Conversely, AAV8-KDM5B overexpression further intensified macrophage infiltration (Fig. 4 A). Additionally, CCl 4 treatment markedly increased hepatic mRNA expression of pro-inflammatory cytokines (IL-6, IL-1β, IL-18, as well as TNF-α), the chemokine CXCL10, and interferon-stimulated genes (ISG15 and IFN-β). These increases were significantly suppressed in the AAV-shKDM5B group (Fig. 4 B-H). In contrast, AAV8-KDM5B treatment elevated the transcription of these inflammatory mediators (Fig. 4 I-L). Consistent with the mRNA data, enzyme-linked immunosorbent assay (ELISA) showed obviously decreased serum levels of pro-inflammatory cytokines in the KDM5B knockdown group (Fig. 4 M-P). Flow cytometry analysis further confirmed a decreased proportion of hepatic CD11b + F4/80 + macrophages in the KDM5B knockdown group (Fig. 4 Q). Collectively, these findings demonstrate that KDM5B promotes immune cell infiltration and upregulation of inflammatory mediators, thereby contributing to the pathogenesis of ALI. KDM5B promoted the pathological process of ALI by inhibiting autophagy Given the pivotal role of autophagy in ALI, we further explored whether KDM5B modulates autophagic activity. AML12 hepatocytes were transfected with shKDM5B plasmids to knock down KDM5B expression, followed by LPS treatment to induce stress. Autophagy-related markers were then assessed. qRT-PCR and western blotting revealed that LPS exposure significantly reduced the mRNA and protein levels of autophagy markers LC3-II/LC3-I and Beclin-1, while increasing the accumulation of the selective autophagy substrate p62/SQSTM1, indicating impaired autophagic flux. Notably, KDM5B knockdown effectively rescued LPS-induced autophagy inhibition, as evidenced by restored LC3-II/LC3-I and Beclin-1 expression and reduced p62/SQSTM1 levels (Fig. 5 A-D). Supporting these findings, transmission electron microscopy (TEM) showed a significant decrease in autophagosome numbers following LPS treatment, whereas KDM5B knockdown markedly increased autophagosome formation (Fig. 5 E). Confocal microscopy further corroborated these results, demonstrating diminished LC3 puncta formation in LPS-treated cells, which was significantly enhanced upon KDM5B knockdown (Fig. 5 F). Collectively, these findings indicate that KDM5B knockdown restores autophagic activity, suggesting that KDM5B contributes to ALI pathogenesis through inhibition of autophagy. KDM5B binding to ATG16L1 inhibited ATG12-ATG5-ATG16L1 complex formation and impaired autophagy in ALI To explore the potential molecular mechanism by which KDM5B suppressed the autophagy in ALI, we transfected AML12 hepatocytes with a Flag-tagged KDM5B expression plasmid. Flag-KDM5B was immunoprecipitated, and interacting proteins were identified via liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Fig. 6 A). Silver staining confirmed efficient expression and purification of Flag-KDM5B (Fig. 6 B). Bioinformatic analysis using the STRING database predicted a potential interaction between KDM5B and ATG16L1 (Fig. 6 C). Consistently, LC-MS/MS analysis identified ATG16L1 as one of the KDM5B-interacting proteins (Fig. 6 D). To validate this interaction, co-immunoprecipitation (Co-IP) assays were performed, confirming that KDM5B physically binds to ATG16L1 (Fig. 6 E-F). Additionally, confocal microscopy revealed strong spatial co-localization between KDM5B and ATG16L1 (Fig. 6 G). Further experiments demonstrated that overexpression of KDM5B significantly disrupted the interaction of ATG16L1 with the ATG12-ATG5 complex (Fig. 6 H), which is essential for autophagosome maturation. Moreover, ATG16L1 knockdown significantly reduced the LC3-II/LC3-I ratio, further confirming the essential role of ATG16L1 in autophagy regulation (Fig. 6 I). Collectively, these findings demonstrate that KDM5B binds to ATG16L1 and interferes with the assembly of the ATG12-ATG5-ATG16L1 complex, thereby impairing autophagosome maturation and contributing to the progression of ALI. Knockdown of KDM5B inhibited the cGAS-STING/NF-κB signaling pathways in ALI To further investigate the role of KDM5B in regulating autophagy and inflammation during ALI, we analyzed its connection to the cGAS-STING and NF-κB signaling pathways, both of which are central mediators of immune-inflammatory responses. Gene Set Enrichment Analysis (GSEA) revealed significant activation of the cytosolic DNA-sensing and NF-κB signaling pathways in CCl 4 -induced ALI, implicating their involvement in disease progression (Fig. 7 A-B). Western blotting demonstrated that LPS treatment markedly increased the expression of cGAS and enhanced the phosphorylation of STING, TBK1, IRF3, and NF-κB p65, indicating strong activation of inflammatory signaling pathways. Notably, knockdown of KDM5B significantly suppressed this activation (Fig. 7 C). Confocal laser scanning microscopy further confirmed these findings, showing that KDM5B knockdown significantly attenuated LPS-induced levels of phosphorylated STING, TBK1, and IRF3 (Fig. 7 D). Taken together, these results demonstrate that KDM5B knockdown attenuates activation of the cGAS-STING pathway and downstream NF-κB signaling, thereby reducing inflammatory cytokine production and mitigating the progression of ALI. Discussion This study identified a significant upregulation of KDM5B in CCl 4 -induced ALI and uncovered a novel pathogenic mechanism. Mechanistically, KDM5B binds to ATG16L1 and disrupts the formation of the ATG12-ATG5-ATG16L1 complex, impairing autophagosome maturation. This autophagy dysfunction leads to cytosolic DNA accumulation, activating the cGAS-STING signaling pathway and promoting excessive release of inflammatory mediators, thereby aggravating hepatic injury. These findings highlight KDM5B as a critical regulator of ALI and propose it as a potential molecular target for therapeutic intervention (Fig. 8 ). Emerging evidence has linked KDM5B to the progression of multiple hepatic diseases, including the promotion of tumorigenesis in hepatocellular carcinoma 31 and facilitation of fibrosis in alcoholic liver disease 32 . However, its role in ALI remained undefined. Through integrated bioinformatics and experimental validation, we found that KDM5B was markedly upregulated in CCl 4 -induced ALI and that its knockdown significantly alleviated liver injury, establishing KDM5B as a central driver of ALI pathogenesis. Immune cell infiltration and sustained inflammation are hallmark features of ALI 33 . Hepatocytes respond to injury by secreting type I interferon (IFN-β) 34 , which subsequently activates downstream interferon-stimulated genes (e.g., ISG15, contributing to immune modulation 35 . IFN-β also promotes the expression of chemokines like CXCL10, which recruits CD11b + myeloid cells to the liver, where they differentiate into F4/80 + macrophages 36 . These infiltrating macrophages amplify inflammation by releasing large quantities of pro-inflammatory cytokines, thus exacerbating liver damage 37 . Although the inflammatory pathways in ALI are well-documented, the upstream regulatory mechanisms remain poorly understood. Our findings demonstrated increased hepatic infiltration of CD11b + F4/80 + macrophages and elevated expression of CXCL10, ISG15, IFN-β, and key pro-inflammatory cytokines in ALI. Importantly, liver-specific knockdown of KDM5B significantly reduced macrophage infiltration and suppressed the expression of these inflammatory mediators. Collectively, this study revealed that KDM5B aggravated liver injury by impairing autophagy and enhancing immune cell recruitment and inflammatory cytokine production, thereby offering new insights into the molecular mechanisms of ALI and a promising therapeutic target. Autophagy is an essential cellular process that maintains homeostasis at both the cellular and organismal levels 38 . This multistep process is initiated by autophagosome formation, which is primarily regulated by members of the autophagy-related protein family 39 . Among them, ATG16L1 binds to the ATG12-ATG5 conjugate to form the ATG12-ATG5-ATG16L1 complex, which is critical for catalyzing LC3 lipidation and driving autophagosome maturation 40 . Recent studies have highlighted the role of epigenetic regulators, particularly histone-modifying enzymes, in modulating autophagy in liver diseases 41 . For instance, Wang et al. 42 reported that inhibition of the histone demethylase KDM8 activates autophagy and suppresses hepatocellular carcinoma growth. In line with this, our study identified KDM5B as another histone demethylase regulating autophagy in the context of ALI. Notably, KDM5B knockdown restored autophagic flux, as indicated by an increased LC3-II/LC3-I ratio, elevated Beclin-1 expression, and reduced accumulation of the autophagy substrate p62/SQSTM1. Importantly, we further demonstrated that KDM5B directly interacted with ATG16L1, disrupting the assembly of the ATG12-ATG5-ATG16L1 complex. This interference impaired LC3 lipidation, reduced autophagosome biogenesis, and ultimately inhibited autophagic flux. To explore the role of KDM5B in regulating inflammation during ALI, we performed GSEA, which revealed significant enrichment of the cGAS-STING and NF-κB signaling pathways. Notably, the cGAS-STING axis is pivotal for hepatic inflammatory responses by sensing cytosolic DNA and generating cGAMP, thereby activating TBK1 43, 44 . Activated TBK1 subsequently phosphorylates key components of the NF-κB signaling pathway 45 , leading to the transcription of pro-inflammatory genes expression. Our findings demonstrated that knockdown of KDM5B suppressed activation of the cGAS-STING pathway in macrophages, as evidenced by reduced phosphorylation of STING, TBK1, IRF3, and NF-κB p65. This attenuation of signaling results in dampened downstream inflammatory responses. Collectively, these results reveal a clear mechanistic link between KDM5B and the activation of cGAS-STING/NF-κB mediated inflammation in ALI. Collectively, this study established KDM5B as a critical modulator of ALI pathogenesis, mechanistically linking its regulation of autophagy and inflammatory signaling to CCl 4 -induced liver injury. However, further investigation is required to elucidate the role of KDM5B across different pathological stages and its interactions with additional signaling pathways. This will be essential to fully define the functional landscape of KDM5B in ALI and to strengthen the molecular foundation for developing targeted therapeutic strategies. Conclusion In summary, our study identified KDM5B as a pivotal regulator of ALI. Mechanistically, KDM5B bound to ATG16L1 and disrupted the formation of the ATG12-ATG5-ATG16L1 complex, thereby impairing LC3 lipidation and autophagosome maturation. This autophagy defect led to cytoplasmic DNA accumulation, which activated the cGAS-STING-TBK1/NF-κB signaling axis. Therefore, CD11b + F4/80 + macrophages were recruited to the liver, and pro-inflammatory cytokines, including IL-6, IL-1β, IL-18, TNF-α, and CXCL10, were released, amplifying the inflammatory response and exacerbating ALI pathology. These findings not only elucidate a novel mechanism underlying ALI progression but also highlight the KDM5B-ATG16L1 axis as a promising therapeutic target for the treatment of ALI. Methods Animal model establishment and experimental procedures The 6-8-week-old male C57BL/6J mice were supplied by Wuhan Mouse Bailey Biotechnology Co., Ltd (Wuhan, China). Animals were housed under strictly controlled conditions at 20–22°C with 40–70% humidity. The experimental group was injected with adeno-associated virus AAV-shKDM5B or AAV8-KDM5B via the tail vein. Following a two-weeks period to ensure sufficient viral expression, ALI was induced via intraperitoneal administration of CCl 4 (1 mL/kg). Right before the injection, CCl 4 was meticulously diluted in olive oil (1:10, v/v) and aseptically filtered through 0.22 µm membranes. Hepatic tissues were harvested 24 hours post-induction. All study protocols were received approval from the Laboratory Animal Ethics Committee of Hubei Institute of Science and Technology. Hematoxylin-eosin (H&E) staining Murine liver tissues were fixed in 4% paraformaldehyde. Following fixation, the tissues were dehydrated through an ascending ethanol gradient series. Subsequently, the tissues were embedded in paraffin and sectioned at 5 µm. The sections were then stained with H&E. Histopathological alterations in the liver tissues were assessed using light microscopy. Immunohistochemistry Following fixation, liver tissues were paraffin-embedded, sectioned, dewaxed, and rehydrated. Antigen retrieval was performed using heated citric acid buffer (10 mM, pH 6.0). To quench endogenous peroxidase activity, tissue sections were treated with 3% hydrogen peroxide for 10 minutes. Nonspecific binding sites were blocked with 5% bovine serum albumin (BSA). Sections were then incubated with primary antibodies and maintained at 4°C overnight. The following day, matched secondary antibodies were applied, and incubation proceeded for 2 hours at ambient temperature. DAB chromogenic substrate was then applied, followed by hematoxylin counterstaining. Sections were permanently mounted with coverslips and examined under a light microscope. Isolation of primary hepatocytes Primary hepatocytes were isolated from C57BL/6J mice. After anesthesia was induced with 35% chloral hydrate, a thoracotomy was performed to expose the portal vein. Cannulation of the portal vein and inferior vena cava was established using indwelling needles. The liver was sequentially perfused with 25 mL calcium-free perfusion buffer followed by 0.05% collagenase IV solution (Worthington, LS004188). Enzymatic digestion was terminated when the liver exhibited softening, collapse, and persistent finger-pressure indentation. Following digestion, the liver was placed in a dish containing serum-free DMEM supplemented with 1% penicillin-streptomycin. After removing the Glisson's capsule, hepatocytes were released by gentle agitation and filtered through a 100-mesh sterile sieve (Biosharp, China). The cell suspension underwent multiple cycles of centrifugation (400× g, 3 minutes, 4°C) with washing and resuspension steps. Viable hepatocytes were finally resuspended in complete DMEM medium, counted, and plated. Transcriptome RNA sequencing Collected mouse liver tissues underwent homogenization in RNAiso Plus (Takara, Tokyo, Japan), followed by total RNA isolation. The KAPA mRNA-Seq Kit was utilized to generate sequencing libraries, processed samples underwent paired-end sequencing on an Illumina HiSeq X instrument. The resulting reads were mapped to the murine reference genome (GRCm39) using TopHat. DESeq2 was employed for the quantification and normalization of transcript-level read counts, enabling the identification of differentially expressed genes (DEGs). Cell culture and transfection procedure The murine hepatocyte line AML12 was commercially acquired from the ATCC (Manassas, VA, USA). RAW264.7 cells were obtained from Procell company (Wuhan, China). Cells were cultured in standard DMEM containing 10% FBS and 1% penicillin-streptomycin at 37°C within a 5% CO 2 humidified environment. Plasmids encoding shKDM5B were transfected into cells using the Neofect™ Transfection Reagent (Beijing, China) according to the manufacturer's protocol. After a 24-hours incubation, cells were exposed to 1 µg/mL LPS for 24 hours before being harvested. RNA extraction and real-time PCR Total RNA was extracted from liver tissues or cultured cells, respectively, using TRIzol Reagent (Thermo Fisher Scientific) following the manufacturer’s instructions. Subsequently, complementary DNA (cDNA) was synthesized from total RNA (1 µg) via the HiScript III RT SuperMix for qPCR kit (Vazyme, China). Amplification of quantitative PCR products was carried out employing the SYBR Green qPCR Master Mix (Vazyme, China) on a real-time thermocycler. Relative quantification of target genes was performed, with normalization against GAPDH and subsequent calculation by 2 −ΔΔCt analysis. Amplification primer sequences are listed in Table S1 . Western blotting Analysis From liver specimens or cell cultures, proteins were isolated utilizing RIPA lysis buffer. Subsequently, total protein quantification was performed using a BCA assay kit. Protein lysates were resolved on 12% SDS-PAGE gels and electrotransferred to PVDF membranes. Membranes were blocked with 5% skim milk for 1 hour at room temperature. Next, primary antibody incubation proceeded overnight at 4°C. Following three washes in TBST, membranes were incubated with secondary antibodies at room temperature for 1 h. Following comprehensive rinsing with TBST, the proteins were detected using an ECL substrate kit (Biosharp, China) and documented by ImageQuant LAS 500 chemiluminescence imaging system (Cytiva, USA). Lastly, signal intensities were processed via ImageJ software (NIH, USA). Details of the primary antibodies are listed in Table S2 . Isolation and Flow Cytometric Profiling of Hepatic Macrophages Small fragments were prepared from liver tissues and digested using RPMI 1640 medium containing 0.05% collagenase IV (Roche) and 0.01% trypsin inhibitor (Gibco, Thermo Fisher Scientific) at 37°C for 60 minutes. The tissue digest was filtered through a 40-µm cell strainer. Subsequently, the generated single-cell suspension underwent centrifugation, with the supernatant subsequently removed. Erythrocytes underwent hypotonic lysis, followed by centrifugation (800×g, 10 minutes, 4°C). The isolated cells were then resuspended in 40% Percoll and subjected to centrifugation at 2,000 rpm for 30 minutes. After aspiration of the supernatant fluid, the cell suspension was layered onto 80% Percoll gradient and centrifuged at ambient temperature. Subsequently, the pellet underwent two washing cycles, was resuspended in RPMI-1640 medium, and counted to achieve the desired cell density. Cells were then stained with anti-F4/80 (#123107, BioLegend) and anti-CD11b (#101211, BioLegend) antibodies in flow cytometry buffer for 45 minutes at 4°C in the dark. Analysis was performed using a flow cytometer with FlowJo software. Transmission electron microscopy analysis AML12 hepatocytes were fixed using 2.5% glutaraldehyde at 4°C for 2 hours, then subjected to 0.1 mol/L phosphate buffer. Post-fixation was performed utilizing 1% osmium tetroxide (OsO 4 ) for 2 hours. The cells were then dehydrated using a graded series of acetone and embedded in epoxy resin, polymerized, and sectioned. Ultrathin sections underwent double staining with 2% uranyl acetate and lead citrate, followed by examination of autophagosomes using transmission electron microscopy (HITA-CHI, Japan). Immunofluorescence analysis Cell specimens underwent fixation using 4% paraformaldehyde, permeabilization with Permeabilization Buffer (Servicebio, China), and blocking with 5% BSA (Biosharp, China). Following the blocking step, primary antibody incubation proceeded overnight at 4°C. Secondary antibodies and DAPI (Servicebio, China) were then added and incubated for 1 hour at room temperature in the dark. Imaging of the slides was performed using a laser scanning confocal microscope (Olympus FV3000), and ImageJ was applied for quantitative analysis of the images. Immunoprecipitation AML12 cells were lysed with IP lysis buffer (Beyotime, China) containing a protease inhibitor mix (ABclonal, China) over ice for 30 minutes. Following this, lysates were processed by centrifugation (12000× g, 15 minutes, 4°C), after which the supernatants were collected. Cell lysates were immunoprecipitated with specific antibodies overnight at 4°C. After that, Protein A/G magnetic beads were mixed with the lysates for 4 hours at 4°C to capture immunocomplexes. Finally, bound proteins were then eluted by boiling in loading buffer for 10 minutes at 100°C, followed by immunoblotting analysis. Mass Spectrometry Analysis of Hepatocytes Lysis of AML12 cells was performed on ice for 30 minutes with 500 µL pre-chilled IP lysis buffer. This buffer consisted of 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, and 0.5% sodium deoxycholate, and was supplemented with cocktails of protease and phosphatase inhibitors. Following lysis, the supernatant was incubated overnight at 4°C with 2 µg of anti-KDM5B antibody or cognate IgG control. Subsequently, pre-washed Protein A/G agarose beads were added to the antigen-antibody mixture and following a 4-hours rotation incubation at 4°C, beads were pelleted via brief centrifugation (1,000× g, 1 min). The bead-bound immunocomplexes were next washed thrice using ice-cold TBST buffer. Following the final wash, the beads were resuspended in 2× loading buffer and subjected to heat denaturation at 100°C for 10 minutes. The eluted proteins were subsequently resolved on SDS-PAGE gels. Post-electrophoresis, the gel band corresponding to the sample was excised. The extracted peptides underwent analysis via liquid chromatography-tandem mass spectrometry (LC-MS/MS). The MS/MS spectra obtained were searched against the mouse non-redundant protein database (NCBI), utilizing the Mascot algorithm from Matrix Science. Statistical analysis Statistical evaluations were conducted using GraphPad Prism version 9.0. Results are presented as mean ± SEM, derived from a minimum of three separate experiments. Differences between groups were assessed using one-way ANOVA, and subsequent multiple comparisons were performed using two-way ANOVA with Tukey's test. Statistical significance is indicated as * P < 0.05, ** P < 0.01, *** P < 0.001; n.s. represents non-significant results. Declarations Author contributions L.Q. and C.C. conceived and designed the study, including determining the core direction and experimental protocols. H.O. and Y.M. performed the in vivo and in vitro experiments, and drafted the manuscript with the assistance of other co-authors. J.X. and Q.Y. curated the data using statistical software and analytical methods. W.H. and X.Q. conducted the literature search and organized the references. J.W., D.L., X.Q., Y.B. and S.S. revised the language of the first draft. All the authors reviewed every aspect of the manuscript and approved the version as submitted. Funding This article was supported by the National Natural Science Foundation of China (No. 82302365), Natural Science Foundation of Hubei Province, China (No. 2024AFB517), Hubei University of Science and Technology Development Fund Project (No. BK202442) and Foundation of Hubei University of Science and Technology Science “Special Project on Diabetes and Angiopathy” (No.2024TNB05). Competing interests The authors declare no conflicts of interest. References Asrani SK, Devarbhavi H, Eaton J, Kamath PS. Burden of liver diseases in the world. J Hepatol 2019, 70 (1) : 151-171. Stravitz RT, Lee WM. Acute liver failure. Lancet 2019, 394 (10201) : 869-881. Bernal W, Auzinger G, Dhawan A, Wendon J. Acute liver failure. Lancet 2010, 376 (9736) : 190-201. Chung RT, Stravitz RT, Fontana RJ, Schiodt FV, Mehal WZ, Reddy KR , et al. Pathogenesis of liver injury in acute liver failure. Gastroenterology 2012, 143 (3) : e1-e7. Zuo J, Tian YX, An Q, Wu BY, Yang JR, Fan YC. 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Nat Chem Biol 2025, 21 (6) : 894-905. Chen R, Du J, Zhu H, Ling Q. The role of cGAS-STING signalling in liver diseases. JHEP Rep 2021, 3 (5) : 100324. Xu D, Tian Y, Xia Q, Ke B. The cGAS-STING Pathway: Novel Perspectives in Liver Diseases. Front Immunol 2021, 12: 682736. Zhang L, Wei X, Wang Z, Liu P, Hou Y, Xu Y , et al. NF-κB activation enhances STING signaling by altering microtubule-mediated STING trafficking. Cell Rep 2023, 42 (3) : 112185. Additional Declarations (Not answered) Supplementary Files Figure1supplemental.xls Figure1 supplemental Figure2supplemental.xlsx Figure2 supplemental OriginalWesternblot.pdf Original Western blot TableS1.docx Table S1 TableS2.docx Table S2 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-7430149","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":511466849,"identity":"2f0622fa-5f01-47e2-b4c2-7662f7e30829","order_by":0,"name":"Lihua 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Datasets and KEGG Analysis. A\u003c/strong\u003e Heatmap analysis of 40 significantly differentially expressed genes between ALI patients and normal controls. Red indicates upregulation and green indicates downregulation. \u003cstrong\u003eB\u003c/strong\u003e The log2 FC of 40 DEGs was calculated based on RRA of the 4 datasets. \u003cstrong\u003eC\u003c/strong\u003eKEGG enrichment analysis of key pathways in the integrated dataset.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7430149/v1/f20d70956a483e76dccae5bc.png"},{"id":91170755,"identity":"cf515fd2-ae65-4dc0-91f7-d2e7b60cd01b","added_by":"auto","created_at":"2025-09-12 11:34:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":393967,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eValidation of KDM5B expression in CCl\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e-induced ALI mouse model. A\u003c/strong\u003e Schematic diagram of the experiment in which mice were constructed as an ALI model by intraperitoneal injection of CCl\u003csub\u003e4\u003c/sub\u003e and liver tissues were obtained for RNA-seq after 24 hours. \u003cstrong\u003eB\u003c/strong\u003e Volcano plot of differentially expressed mRNAs between control and CCl\u003csub\u003e4\u003c/sub\u003e-induced mice. Red marks upregulated genes, and blue marks downregulated genes. \u003cstrong\u003eC\u003c/strong\u003e Heatmap analysis of KDM family gene expression patterns in control and CCl\u003csub\u003e4\u003c/sub\u003e-induced ALI mice. \u003cstrong\u003eD\u003c/strong\u003e qRT-PCR analysis of KDM family gene expression in control and CCl\u003csub\u003e4\u003c/sub\u003e-induced ALI mice. \u003cstrong\u003eE-F\u003c/strong\u003e Immunohistochemistry and western blotting analysis of KDM5B levels in hepatic tissues of CCl\u003csub\u003e4\u003c/sub\u003e-induced mice at distinct time points. \u003cstrong\u003eG\u003c/strong\u003e qRT-PCR was used to assess the levels of KDM5B mRNA in primary hepatocytes, lymphocytes, and macrophages of CCl\u003csub\u003e4\u003c/sub\u003e-induced mice, respectively. Results displayed as mean ± SEM (n = 3; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-7430149/v1/613b92ff714888f372ebfa1f.png"},{"id":91170759,"identity":"057eb30f-f32d-44cb-81c7-42d7b5293e17","added_by":"auto","created_at":"2025-09-12 11:34:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1318655,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKDM5B knockdown attenuates while its overexpression exacerbates CCl\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e-induced ALI. A \u003c/strong\u003eFlow chart of the experiment: tail vein injection of AAV-shKDM5B or AAV8-KDM5B adeno-associated virus was performed, followed by intraperitoneal CCl\u003csub\u003e4\u003c/sub\u003e administration (1 mL/kg) after 2 weeks, and livers were harvested at 24 hours post-induction. \u003cstrong\u003eB\u003c/strong\u003e H\u0026amp;E staining showing pathological changes in liver morphology across different groups (scale bar = 100 μm). The graph depicts quantitative analysis of the necrotic area. \u003cstrong\u003eC\u003c/strong\u003e Measurement of serum ALT and AST levels in various mouse groups. \u003cstrong\u003eD\u003c/strong\u003e Western blotting detection and quantitation of hepatic KDM5B in AAV-shKDM5B-treated mice. \u003cstrong\u003eE-F\u003c/strong\u003e H\u0026amp;E-stained liver sections (scale bar = 100 μm) with serum ALT and AST measurement in AAV8-KDM5B mice. Results displayed as mean ± SEM (n = 3; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-7430149/v1/239d49c9eb3eb174b65cec9a.png"},{"id":91172475,"identity":"a55ac4f0-3a7b-42c7-9b82-5fe03160d9d4","added_by":"auto","created_at":"2025-09-12 11:50:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1562896,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnockdown of KDM5B attenuates the inflammatory response to CCl\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e-induced ALI in mice. A\u003c/strong\u003e Immunohistochemistry staining for F4/80\u003csup\u003e+\u003c/sup\u003e macrophages in mouse liver tissues (scale bar = 100 μm). Quantification of macrophage infiltration is shown on the right. \u003cstrong\u003eB-H\u003c/strong\u003e qRT-PCR analysis of IL-6, IL-1β, IL-18, TNF-α, CXCL10, ISG15, and IFN-β mRNA expression in hepatic tissues of AAV-shKDM5B mice compared to control groups. \u003cstrong\u003eI-L\u003c/strong\u003e qRT-PCR analysis of IL-6, IL-1β, IL-18, TNF-α mRNA expression in hepatic tissues of AAV8-KDM5B mice compared to control groups. \u003cstrong\u003eM-P\u003c/strong\u003e ELISA analysis of serum levels of inflammatory cytokines IL-6, IL-1β, IL-18, and TNF-α in AAV-shKDM5B mice compared to control groups. \u003cstrong\u003eQ\u003c/strong\u003e Flow cytometry analysis of CD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003e macrophage population in liver tissues. Results displayed as mean ± SEM (n = 3; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-7430149/v1/4b9bb0e8c974250b0dc969ba.png"},{"id":91172012,"identity":"16bd21bc-a776-47a0-94cd-d8bb73f18c84","added_by":"auto","created_at":"2025-09-12 11:42:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1834314,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnockdown of KDM5B ameliorates LPS-induced impairment of autophagy.\u003c/strong\u003e \u003cstrong\u003eA-C\u003c/strong\u003e qRT-PCR was used to assess the mRNA levels of autophagy-related genes LC3, Beclin-1, and p62/SQSTM1 in AML12 cells. \u003cstrong\u003eD\u003c/strong\u003e Western blot analysis of LC3II/I, Beclin-1, and p62 protein levels in AML12 cells. \u003cstrong\u003eE\u003c/strong\u003e Transmission electron microscopy (TEM) observation of the morphology and number of autophagosomes in AML12 cells (scale bar = 5 µm), red arrows denote autophagosomes, and quantitative analysis of autophagosome number per cell is displayed on the right. \u003cstrong\u003eF\u003c/strong\u003e Laser confocal microscopy observation of LC3 expression in AML12 cells. Green fluorescence indicates autophagosomes (scale bar = 20 µm), with the number of autophagosomes per cell quantified on the right. Results displayed as mean ± SEM (n = 3; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-7430149/v1/febe87275b523502a980b3f8.png"},{"id":91172004,"identity":"f55edae4-8405-4dd8-9a36-8a3166b5394a","added_by":"auto","created_at":"2025-09-12 11:42:08","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1417042,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKDM5B binding to ATG16L1 impairs autophagy.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e Schematic diagram of the process of constructing a cell line stably expressing Flag-KDM5B in AML12 cells, trypsin digestion followed by mass spectrometry analysis. \u003cstrong\u003eB\u003c/strong\u003eSilver staining experiments to validate Flag-KDM5B expression. \u003cstrong\u003eC\u003c/strong\u003eConstruction of the KDM5B protein interaction network by STRING database. \u003cstrong\u003eD\u003c/strong\u003eMass spectrometry identification of the top 10 KDM5B-interacting proteins. \u003cstrong\u003eE-F\u003c/strong\u003eCo-IP experiments confirming the interaction between KDM5B and ATG16L1 from the perspective of KDM5B and ATG16L1, respectively. \u003cstrong\u003eG\u003c/strong\u003e Laser confocal microscopy was used to observe the co-localization of KDM5B and ATG16L1 in primary hepatocytes. \u003cstrong\u003eH\u003c/strong\u003e Overexpression of KDM5B in AML12 cell line was used to investigate how KDM5B overexpression affects the binding of ATG16L1 to the ATG12-ATG5 complex. \u003cstrong\u003eI\u003c/strong\u003e Knockdown of ATG16L1 was detected as a change in the level of the autophagy marker LC3II/I. Results displayed as mean ± SEM (n = 3; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-7430149/v1/0313e99663a640ac7199e1e4.png"},{"id":91170763,"identity":"68bb3bfc-af8a-4c9c-b34e-ae8e60923fb5","added_by":"auto","created_at":"2025-09-12 11:34:08","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2106575,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of KDM5B on cGAS-STING and NF-κB signaling pathways. A-B \u003c/strong\u003eGSEA showing the enrichment of cytoplasmic DNA-sensing pathway and NF-κB signaling pathway in CCl\u003csub\u003e4\u003c/sub\u003e-induced ALI. \u003cstrong\u003eC\u003c/strong\u003e Western blotting analysis of cGAS, p-STING, STING, p-TBK1, TBK1, p-IRF3, IRF3, p-p65, and p65 protein expression in RAW264.7 macrophages. \u003cstrong\u003eD\u003c/strong\u003e Laser confocal microscopy to detect the expression levels of p-STING, p-TBK1 and p-IRF3 in RAW264.7 macrophages. (Nuclei stained with DAPI; target proteins shown in red. Scale bar = 20 μm). Results displayed as mean ± SEM (n = 3; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001).\u0026nbsp;\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-7430149/v1/4be0171c77957b9c812e73ba.png"},{"id":91172478,"identity":"bfb07bc1-6417-4b13-8905-f75e0eadb502","added_by":"auto","created_at":"2025-09-12 11:50:08","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1217427,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMolecular mechanism of KDM5B mediating autophagy and inflammation in ALI.\u003c/strong\u003e In CCl\u003csub\u003e4\u003c/sub\u003e-induced ALI, KDM5B expression is upregulated, and KDM5B binds to ATG16L1, disrupting the formation of ATG12-ATG5-ATG16L1 complex and inhibiting autophagosome maturation. This leads to cytoplasmic DNA accumulation and subsequent activation of the cGAS-STING cascade, which in turn evoked the generation of pro-inflammatory mediators and intensified hepatic injury.\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-7430149/v1/4ac50a415fa08d767d7f5575.png"},{"id":93417956,"identity":"ce42ff6c-9a35-4862-a393-eb7e32d63d11","added_by":"auto","created_at":"2025-10-13 15:38:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11045940,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7430149/v1/47756d4b-57bc-448a-9c50-0fd0e3c1e161.pdf"},{"id":91170754,"identity":"2b0598cc-83c1-4b29-84f2-c79d575c50ed","added_by":"auto","created_at":"2025-09-12 11:34:07","extension":"xls","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12233,"visible":true,"origin":"","legend":"Figure1 supplemental","description":"","filename":"Figure1supplemental.xls","url":"https://assets-eu.researchsquare.com/files/rs-7430149/v1/baa38eb4bc809a310fbed4c5.xls"},{"id":91172007,"identity":"076dff65-40bb-48f0-bc22-20eda15d34bb","added_by":"auto","created_at":"2025-09-12 11:42:08","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":7566946,"visible":true,"origin":"","legend":"Figure2 supplemental","description":"","filename":"Figure2supplemental.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7430149/v1/954eaa62afa83a69b9a8eb6a.xlsx"},{"id":91173922,"identity":"19c3cbc6-8f2a-4805-9403-1dfad439b20d","added_by":"auto","created_at":"2025-09-12 11:58:08","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":195864,"visible":true,"origin":"","legend":"Original Western blot","description":"","filename":"OriginalWesternblot.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7430149/v1/1eb7565585dd06071c747337.pdf"},{"id":91172476,"identity":"286d3c47-c328-4fcd-ab6e-743fff715462","added_by":"auto","created_at":"2025-09-12 11:50:08","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":16967,"visible":true,"origin":"","legend":"\u003cp\u003eTable S1\u003c/p\u003e","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7430149/v1/e488b053d0b345dfab9324d5.docx"},{"id":91172001,"identity":"4e042de5-6912-4c24-a60a-8c6ff1a74f39","added_by":"auto","created_at":"2025-09-12 11:42:07","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":31608,"visible":true,"origin":"","legend":"Table S2","description":"","filename":"TableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7430149/v1/fd71f70d8732a3bfc5b1c9a6.docx"}],"financialInterests":"(Not answered)","formattedTitle":"KDM5B drives acute liver injury by impairing autophagy and activating cGAS-STING signaling via binding to ATG16L1","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAcute liver injury (ALI) is a common disorder that poses a significant threat to public health \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Its pathogenesis is complex, marked by a sudden onset and progressive deterioration. Critically, ALI often serves as the precursor to acute liver failure and can rapidly progress to liver failure \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, which triggering systemic inflammatory responses, coagulopathy, multi-organ dysfunction, and potentially life-threatening conditions \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Notably, ALI can be induced by various etiological factors, such as viral infections (e.g., hepatitis B and C viruses), drug or toxin exposure (e.g., acetaminophen overdose) and autoimmune reactions \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Current therapeutic approaches primarily include pharmacological treatments, supportive care, and liver transplantation \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. However, the scarcity of donor organs \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, suboptimal efficacy of existing pharmacotherapies \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, and an incomplete understanding of the underlying pathogenesis in many cases continue to hinder clinical management. Therefore, further investigation into the mechanisms driving ALI and the identification of novel therapeutic targets is urgently needed.\u003c/p\u003e\u003cp\u003eAutophagy is an evolutionarily conserved lysosomal degradation pathway in eukaryotes that maintains cellular homeostasis and metabolic stability \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. To date, at least 19 autophagy-related proteins have been identified as essential regulators of autophagosome formation. Initially discovered in yeast, these proteins are highly conserved across eukaryotic species \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. During autophagy initiation, microtubule-associated protein 1 light chain 3 (LC3) and the ATG12-ATG5-ATG16L1 complex assemble on the phagophore. Interacting with the ATG12-ATG5 conjugate, ATG16L1 forms a dimeric complex that activates ATG3, thereby promoting the lipidation of LC3 with phosphatidylethanolamine. This process converts LC3B-I to LC3B-II, which is essential for autophagy activation \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Thus, the formation of the ATG12-ATG5-ATG16L1 complex is essential for autophagosome maturation. In the context of ALI, autophagy plays a dual role: homeostatic autophagy helps to eliminate damaged organelles and misfolded proteins, while dysregulated autophagy amplifies inflammation and induces cell death \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. For example, macrophage-specific deletion of ATG16L1 in partial hepatectomy models reduces autophagic flux, activates inflammatory pathways, and impairs liver regeneration \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Similarly, hepatocyte-specific knockdown of ATG5 blocks autophagosome formation and aggravates lipopolysaccharide (LPS)-induced hepatic inflammation \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. These findings highlight the essential anti-inflammatory role of autophagy \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. However, the molecular mechanisms underlying autophagy dysregulation in ALI remain poorly understood and warrant further investigation.\u003c/p\u003e\u003cp\u003eThe cyclic GMP-AMP synthase\u0026ndash;stimulator of interferon genes (cGAS-STING) pathway is a key innate immune signaling axis that has garnered considerable attention in recent years \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Its functions by sensing aberrant cytosolic DNA and initiating inflammatory responses \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Within this pathway, cGAS serves as the cytosolic DNA sensor. Upon detecting and binding to cytosolic DNA, cGAS catalyzes the production of the second messenger 2'3'-cyclic GMP-AMP (cGAMP). Subsequently, cGAMP binds to and activates the STING, which is located on the endoplasmic reticulum membrane \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Upon activation, STING undergoes a conformational change and recruits TANK-binding kinase 1 (TBK1), which phosphorylates interferon regulatory factor 3 (IRF3). This leads to the production of type I interferons (IFN-I) and pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Dysregulated cGAS-STING signaling contributes to immunopathology in various hepatic inflammatory diseases \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. In ALI, activation of this pathway promotes inflammatory cell infiltration and hepatocyte apoptosis \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Although the pro-inflammatory role of cGAS-STING signaling in ALI is well established \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, the upstream molecular events leading to its activation remain incompletely understood.\u003c/p\u003e\u003cp\u003eLysine Demethylase 5B (KDM5B), a member of the Jumonji C (JmjC) domain-containing histone demethylase family, is best known for catalyzing the demethylation of histone H3 lysine 4 di- and tri-methylation (H3K4me2/3), thereby modulating transcriptional activity \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. However, the function of KDM5B extends beyond histone demethylation. For example, a study in alcohol-exposed male mice showed that KDM5B can bind to the promoter region of hepatocyte nuclear factor 4 alpha (HNF4A) and exacerbate alcohol-induced hepatocyte injury through a mechanism independent of its histone demethylase function \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. While KDM5B has been implicated in tumorigenesis, stem cell differentiation, and metabolic disorders \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, its specific role and underlying mechanisms in the context of ALI remain unexplored.\u003c/p\u003e\u003cp\u003eIn this study, we found that KDM5B binds to the key autophagy-related protein ATG16L1, disrupting the formation of the ATG12-ATG5-ATG16L1 complex and thereby inhibiting autophagosome maturation in ALI. This autophagy impairment resulted in the accumulation of cytosolic DNA, which activated the cGAS-STING signaling pathway and triggered excessive inflammatory cytokine release, ultimately exacerbating hepatic injury. To validate this mechanism, we generated a hepatocyte-specific KDM5B knockdown mouse model. KDM5B knockdown significantly alleviated carbon tetrachloride (CCl\u003csub\u003e4\u003c/sub\u003e)-induced ALI, as evidenced by reduced immune cell infiltration, restoration of autophagic flux, and suppression of cGAS-STING pathway activation. Collectively, these findings uncovered a novel mechanism by which KDM5B regulated autophagy independently of its histone demethylase activity, providing a theoretical basis for targeting the KDM5B-ATG16L1 axis as a potential therapeutic strategy for ALI.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eGEO dataset-based differential gene expression analysis in ALI\u003c/h2\u003e\u003cp\u003eTo identify potential genes related to ALI, we analyzed four Gene Expression Omnibus (GEO) datasets and identified 400 differentially expressed genes (DEGs). Robust Rank Aggregation (RRA) analysis further narrowed this to 40 significantly altered genes, including 20 upregulated and 20 downregulated genes. Notably, KDM5B was markedly upregulated in ALI patients compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B). Additionally, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed enriched DEGs in innate immune and inflammatory pathways, including the cytosolic DNA-sensing pathway and the interleukin-17 (IL-17) signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eKDM5B expression was significantly up-regulated in ALI\u003c/h3\u003e\n\u003cp\u003eTo validate KDM5B as a key DEG in ALI, we established a murine model via intraperitoneal injection of CCl\u003csub\u003e4\u003c/sub\u003e and performed RNA sequencing on liver tissues collected 24 hours post-injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Volcano plot analysis identified 1,084 DEGs compared to controls, including 733 upregulated and 351 downregulated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Heatmap analysis of the KDM family revealed a marked upregulation of KDM5B in CCl\u003csub\u003e4\u003c/sub\u003e-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), which was further confirmed by quantitative real-time PCR (qRT-PCR), showing significantly elevated KDM5B mRNA levels in CCl\u003csub\u003e4\u003c/sub\u003e-induced liver tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). To further assess KDM5B protein expression, immunohistochemistry and western blotting were performed on liver tissues collected at 24 and 48 hours after CCl\u003csub\u003e4\u003c/sub\u003e injection. Immunohistochemical staining showed a significant increase in KDM5B expression at 24 hours, followed by a decline at 48 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Consistently, western blotting revealed a similar pattern, with peak KDM5B expression at 24 hours and a relative decrease at 48 hours compared to the controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). To investigate KDM5B expression in different liver cell types, we isolated primary hepatocytes, lymphocytes, and macrophages from CCl\u003csub\u003e4\u003c/sub\u003e- treated mice. The qRT-PCR analysis showed that KDM5B expression was markedly elevated in all three cell types following CCl\u003csub\u003e4\u003c/sub\u003e administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Collectively, the findings indicate that both KDM5B mRNA and protein levels are significantly upregulated in CCl\u003csub\u003e4\u003c/sub\u003e-induced murine model of ALI.\u003c/p\u003e\n\u003ch3\u003eLiver-specific knockdown of KDM5B ameliorated CCl-induced ALI\u003c/h3\u003e\n\u003cp\u003eTo explore the functional role of KDM5B in CCl\u003csub\u003e4\u003c/sub\u003e-induced ALI, we administered adeno-associated virus (AAV) vectors encoding either shRNA targeting KDM5B (AAV-shKDM5B) or KDM5B overexpression construct (AAV8-KDM5B) via tail vein injection. Two weeks post-injection, ALI was induced through intraperitoneal administration of CCl\u003csub\u003e4\u003c/sub\u003e (1 mL/kg), and liver tissues were collected 24 hours later for analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Hematoxylin and eosin (H\u0026amp;E) staining revealed a significant reduction in necrotic liver areas in mice with liver-specific KDM5B knockdown compared to control groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Correspondingly, serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), both indicators of liver injury, were significantly decreased in the knockdown group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Western blot analysis confirmed that AAV-shKDM5B effectively reduced hepatic KDM5B protein expression in CCl\u003csub\u003e4\u003c/sub\u003e-treated mice, indicating successful knockdown of KDM5B (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). In contrast, liver-specific overexpression of KDM5B aggravated liver injury. H\u0026amp;E staining showed significantly enlarged necrotic areas (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), accompanied by a marked increase in serum ALT and AST levels compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Collectively, these findings demonstrate the critical role of KDM5B in the pathogenesis of CCl\u003csub\u003e4\u003c/sub\u003e-induced ALI. Liver-specific knockdown of KDM5B significantly mitigates liver injury, whereas its overexpression exacerbates hepatic damage.\u003c/p\u003e\n\u003ch3\u003eLiver-specific knockdown of KDM5B alleviated inflammation in CCl-induced ALI\u003c/h3\u003e\n\u003cp\u003eTo examine the immunoregulatory role of KDM5B in CCl\u003csub\u003e4\u003c/sub\u003e-induced ALI, the impact of KDM5B modulation on hepatic inflammatory responses was assessed. Immunohistochemical staining revealed that CCl\u003csub\u003e4\u003c/sub\u003e treatment significantly increased infiltration of F4/80\u003csup\u003e+\u003c/sup\u003e macrophages in the liver, which was markedly reduced by AAV-shKDM5B treatment. Conversely, AAV8-KDM5B overexpression further intensified macrophage infiltration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Additionally, CCl\u003csub\u003e4\u003c/sub\u003e treatment markedly increased hepatic mRNA expression of pro-inflammatory cytokines (IL-6, IL-1β, IL-18, as well as TNF-α), the chemokine CXCL10, and interferon-stimulated genes (ISG15 and IFN-β). These increases were significantly suppressed in the AAV-shKDM5B group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-H). In contrast, AAV8-KDM5B treatment elevated the transcription of these inflammatory mediators (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI-L). Consistent with the mRNA data, enzyme-linked immunosorbent assay (ELISA) showed obviously decreased serum levels of pro-inflammatory cytokines in the KDM5B knockdown group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eM-P). Flow cytometry analysis further confirmed a decreased proportion of hepatic CD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003e macrophages in the KDM5B knockdown group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eQ). Collectively, these findings demonstrate that KDM5B promotes immune cell infiltration and upregulation of inflammatory mediators, thereby contributing to the pathogenesis of ALI.\u003c/p\u003e\n\u003ch3\u003eKDM5B promoted the pathological process of ALI by inhibiting autophagy\u003c/h3\u003e\n\u003cp\u003eGiven the pivotal role of autophagy in ALI, we further explored whether KDM5B modulates autophagic activity. AML12 hepatocytes were transfected with shKDM5B plasmids to knock down KDM5B expression, followed by LPS treatment to induce stress. Autophagy-related markers were then assessed. qRT-PCR and western blotting revealed that LPS exposure significantly reduced the mRNA and protein levels of autophagy markers LC3-II/LC3-I and Beclin-1, while increasing the accumulation of the selective autophagy substrate p62/SQSTM1, indicating impaired autophagic flux. Notably, KDM5B knockdown effectively rescued LPS-induced autophagy inhibition, as evidenced by restored LC3-II/LC3-I and Beclin-1 expression and reduced p62/SQSTM1 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-D). Supporting these findings, transmission electron microscopy (TEM) showed a significant decrease in autophagosome numbers following LPS treatment, whereas KDM5B knockdown markedly increased autophagosome formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Confocal microscopy further corroborated these results, demonstrating diminished LC3 puncta formation in LPS-treated cells, which was significantly enhanced upon KDM5B knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Collectively, these findings indicate that KDM5B knockdown restores autophagic activity, suggesting that KDM5B contributes to ALI pathogenesis through inhibition of autophagy.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eKDM5B binding to ATG16L1 inhibited ATG12-ATG5-ATG16L1 complex formation and impaired autophagy in ALI\u003c/h2\u003e\u003cp\u003eTo explore the potential molecular mechanism by which KDM5B suppressed the autophagy in ALI, we transfected AML12 hepatocytes with a Flag-tagged KDM5B expression plasmid. Flag-KDM5B was immunoprecipitated, and interacting proteins were identified via liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Silver staining confirmed efficient expression and purification of Flag-KDM5B (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Bioinformatic analysis using the STRING database predicted a potential interaction between KDM5B and ATG16L1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Consistently, LC-MS/MS analysis identified ATG16L1 as one of the KDM5B-interacting proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). To validate this interaction, co-immunoprecipitation (Co-IP) assays were performed, confirming that KDM5B physically binds to ATG16L1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE-F). Additionally, confocal microscopy revealed strong spatial co-localization between KDM5B and ATG16L1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). Further experiments demonstrated that overexpression of KDM5B significantly disrupted the interaction of ATG16L1 with the ATG12-ATG5 complex (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH), which is essential for autophagosome maturation. Moreover, ATG16L1 knockdown significantly reduced the LC3-II/LC3-I ratio, further confirming the essential role of ATG16L1 in autophagy regulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI). Collectively, these findings demonstrate that KDM5B binds to ATG16L1 and interferes with the assembly of the ATG12-ATG5-ATG16L1 complex, thereby impairing autophagosome maturation and contributing to the progression of ALI.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eKnockdown of KDM5B inhibited the cGAS-STING/NF-κB signaling pathways in ALI\u003c/h3\u003e\n\u003cp\u003eTo further investigate the role of KDM5B in regulating autophagy and inflammation during ALI, we analyzed its connection to the cGAS-STING and NF-κB signaling pathways, both of which are central mediators of immune-inflammatory responses. Gene Set Enrichment Analysis (GSEA) revealed significant activation of the cytosolic DNA-sensing and NF-κB signaling pathways in CCl\u003csub\u003e4\u003c/sub\u003e-induced ALI, implicating their involvement in disease progression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-B). Western blotting demonstrated that LPS treatment markedly increased the expression of cGAS and enhanced the phosphorylation of STING, TBK1, IRF3, and NF-κB p65, indicating strong activation of inflammatory signaling pathways. Notably, knockdown of KDM5B significantly suppressed this activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Confocal laser scanning microscopy further confirmed these findings, showing that KDM5B knockdown significantly attenuated LPS-induced levels of phosphorylated STING, TBK1, and IRF3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Taken together, these results demonstrate that KDM5B knockdown attenuates activation of the cGAS-STING pathway and downstream NF-κB signaling, thereby reducing inflammatory cytokine production and mitigating the progression of ALI.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study identified a significant upregulation of KDM5B in CCl\u003csub\u003e4\u003c/sub\u003e-induced ALI and uncovered a novel pathogenic mechanism. Mechanistically, KDM5B binds to ATG16L1 and disrupts the formation of the ATG12-ATG5-ATG16L1 complex, impairing autophagosome maturation. This autophagy dysfunction leads to cytosolic DNA accumulation, activating the cGAS-STING signaling pathway and promoting excessive release of inflammatory mediators, thereby aggravating hepatic injury. These findings highlight KDM5B as a critical regulator of ALI and propose it as a potential molecular target for therapeutic intervention (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eEmerging evidence has linked KDM5B to the progression of multiple hepatic diseases, including the promotion of tumorigenesis in hepatocellular carcinoma \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e and facilitation of fibrosis in alcoholic liver disease \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. However, its role in ALI remained undefined. Through integrated bioinformatics and experimental validation, we found that KDM5B was markedly upregulated in CCl\u003csub\u003e4\u003c/sub\u003e-induced ALI and that its knockdown significantly alleviated liver injury, establishing KDM5B as a central driver of ALI pathogenesis.\u003c/p\u003e\u003cp\u003eImmune cell infiltration and sustained inflammation are hallmark features of ALI \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Hepatocytes respond to injury by secreting type I interferon (IFN-β) \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, which subsequently activates downstream interferon-stimulated genes (e.g., ISG15, contributing to immune modulation \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. IFN-β also promotes the expression of chemokines like CXCL10, which recruits CD11b\u003csup\u003e+\u003c/sup\u003e myeloid cells to the liver, where they differentiate into F4/80\u003csup\u003e+\u003c/sup\u003e macrophages \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. These infiltrating macrophages amplify inflammation by releasing large quantities of pro-inflammatory cytokines, thus exacerbating liver damage \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Although the inflammatory pathways in ALI are well-documented, the upstream regulatory mechanisms remain poorly understood. Our findings demonstrated increased hepatic infiltration of CD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003e macrophages and elevated expression of CXCL10, ISG15, IFN-β, and key pro-inflammatory cytokines in ALI. Importantly, liver-specific knockdown of KDM5B significantly reduced macrophage infiltration and suppressed the expression of these inflammatory mediators. Collectively, this study revealed that KDM5B aggravated liver injury by impairing autophagy and enhancing immune cell recruitment and inflammatory cytokine production, thereby offering new insights into the molecular mechanisms of ALI and a promising therapeutic target.\u003c/p\u003e\u003cp\u003eAutophagy is an essential cellular process that maintains homeostasis at both the cellular and organismal levels \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. This multistep process is initiated by autophagosome formation, which is primarily regulated by members of the autophagy-related protein family \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Among them, ATG16L1 binds to the ATG12-ATG5 conjugate to form the ATG12-ATG5-ATG16L1 complex, which is critical for catalyzing LC3 lipidation and driving autophagosome maturation \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Recent studies have highlighted the role of epigenetic regulators, particularly histone-modifying enzymes, in modulating autophagy in liver diseases \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. For instance, Wang \u003cem\u003eet al.\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e reported that inhibition of the histone demethylase KDM8 activates autophagy and suppresses hepatocellular carcinoma growth. In line with this, our study identified KDM5B as another histone demethylase regulating autophagy in the context of ALI. Notably, KDM5B knockdown restored autophagic flux, as indicated by an increased LC3-II/LC3-I ratio, elevated Beclin-1 expression, and reduced accumulation of the autophagy substrate p62/SQSTM1. Importantly, we further demonstrated that KDM5B directly interacted with ATG16L1, disrupting the assembly of the ATG12-ATG5-ATG16L1 complex. This interference impaired LC3 lipidation, reduced autophagosome biogenesis, and ultimately inhibited autophagic flux.\u003c/p\u003e\u003cp\u003eTo explore the role of KDM5B in regulating inflammation during ALI, we performed GSEA, which revealed significant enrichment of the cGAS-STING and NF-κB signaling pathways. Notably, the cGAS-STING axis is pivotal for hepatic inflammatory responses by sensing cytosolic DNA and generating cGAMP, thereby activating TBK1\u003csup\u003e43, 44\u003c/sup\u003e. Activated TBK1 subsequently phosphorylates key components of the NF-κB signaling pathway \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e, leading to the transcription of pro-inflammatory genes expression. Our findings demonstrated that knockdown of KDM5B suppressed activation of the cGAS-STING pathway in macrophages, as evidenced by reduced phosphorylation of STING, TBK1, IRF3, and NF-κB p65. This attenuation of signaling results in dampened downstream inflammatory responses. Collectively, these results reveal a clear mechanistic link between KDM5B and the activation of cGAS-STING/NF-κB mediated inflammation in ALI.\u003c/p\u003e\u003cp\u003eCollectively, this study established KDM5B as a critical modulator of ALI pathogenesis, mechanistically linking its regulation of autophagy and inflammatory signaling to CCl\u003csub\u003e4\u003c/sub\u003e-induced liver injury. However, further investigation is required to elucidate the role of KDM5B across different pathological stages and its interactions with additional signaling pathways. This will be essential to fully define the functional landscape of KDM5B in ALI and to strengthen the molecular foundation for developing targeted therapeutic strategies.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, our study identified KDM5B as a pivotal regulator of ALI. Mechanistically, KDM5B bound to ATG16L1 and disrupted the formation of the ATG12-ATG5-ATG16L1 complex, thereby impairing LC3 lipidation and autophagosome maturation. This autophagy defect led to cytoplasmic DNA accumulation, which activated the cGAS-STING-TBK1/NF-κB signaling axis. Therefore, CD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003e macrophages were recruited to the liver, and pro-inflammatory cytokines, including IL-6, IL-1β, IL-18, TNF-α, and CXCL10, were released, amplifying the inflammatory response and exacerbating ALI pathology. These findings not only elucidate a novel mechanism underlying ALI progression but also highlight the KDM5B-ATG16L1 axis as a promising therapeutic target for the treatment of ALI.\u003c/p\u003e"},{"header":"Methods","content":"\u003ch2\u003eAnimal model establishment and experimental procedures\u003c/h2\u003e\u003cp\u003eThe 6-8-week-old male C57BL/6J mice were supplied by Wuhan Mouse Bailey Biotechnology Co., Ltd (Wuhan, China). Animals were housed under strictly controlled conditions at 20–22°C with 40–70% humidity. The experimental group was injected with adeno-associated virus AAV-shKDM5B or AAV8-KDM5B via the tail vein. Following a two-weeks period to ensure sufficient viral expression, ALI was induced via intraperitoneal administration of CCl\u003csub\u003e4\u003c/sub\u003e (1 mL/kg). Right before the injection, CCl\u003csub\u003e4\u003c/sub\u003e was meticulously diluted in olive oil (1:10, v/v) and aseptically filtered through 0.22 µm membranes. Hepatic tissues were harvested 24 hours post-induction. All study protocols were received approval from the Laboratory Animal Ethics Committee of Hubei Institute of Science and Technology.\u003c/p\u003e\u003ch2\u003eHematoxylin-eosin (H\u0026amp;E) staining\u003c/h2\u003e\u003cp\u003eMurine liver tissues were fixed in 4% paraformaldehyde. Following fixation, the tissues were dehydrated through an ascending ethanol gradient series. Subsequently, the tissues were embedded in paraffin and sectioned at 5 µm. The sections were then stained with H\u0026amp;E. Histopathological alterations in the liver tissues were assessed using light microscopy.\u003c/p\u003e\u003ch2\u003eImmunohistochemistry\u003c/h2\u003e\u003cp\u003eFollowing fixation, liver tissues were paraffin-embedded, sectioned, dewaxed, and rehydrated. Antigen retrieval was performed using heated citric acid buffer (10 mM, pH 6.0). To quench endogenous peroxidase activity, tissue sections were treated with 3% hydrogen peroxide for 10 minutes. Nonspecific binding sites were blocked with 5% bovine serum albumin (BSA). Sections were then incubated with primary antibodies and maintained at 4°C overnight. The following day, matched secondary antibodies were applied, and incubation proceeded for 2 hours at ambient temperature. DAB chromogenic substrate was then applied, followed by hematoxylin counterstaining. Sections were permanently mounted with coverslips and examined under a light microscope.\u003c/p\u003e\u003ch2\u003eIsolation of primary hepatocytes\u003c/h2\u003e\u003cp\u003ePrimary hepatocytes were isolated from C57BL/6J mice. After anesthesia was induced with 35% chloral hydrate, a thoracotomy was performed to expose the portal vein. Cannulation of the portal vein and inferior vena cava was established using indwelling needles. The liver was sequentially perfused with 25 mL calcium-free perfusion buffer followed by 0.05% collagenase IV solution (Worthington, LS004188). Enzymatic digestion was terminated when the liver exhibited softening, collapse, and persistent finger-pressure indentation. Following digestion, the liver was placed in a dish containing serum-free DMEM supplemented with 1% penicillin-streptomycin. After removing the Glisson's capsule, hepatocytes were released by gentle agitation and filtered through a 100-mesh sterile sieve (Biosharp, China). The cell suspension underwent multiple cycles of centrifugation (400× g, 3 minutes, 4°C) with washing and resuspension steps. Viable hepatocytes were finally resuspended in complete DMEM medium, counted, and plated.\u003c/p\u003e\u003ch2\u003eTranscriptome RNA sequencing\u003c/h2\u003e\u003cp\u003eCollected mouse liver tissues underwent homogenization in RNAiso Plus (Takara, Tokyo, Japan), followed by total RNA isolation. The KAPA mRNA-Seq Kit was utilized to generate sequencing libraries, processed samples underwent paired-end sequencing on an Illumina HiSeq X instrument. The resulting reads were mapped to the murine reference genome (GRCm39) using TopHat. DESeq2 was employed for the quantification and normalization of transcript-level read counts, enabling the identification of differentially expressed genes (DEGs).\u003c/p\u003e\u003ch2\u003eCell culture and transfection procedure\u003c/h2\u003e\u003cp\u003eThe murine hepatocyte line AML12 was commercially acquired from the ATCC (Manassas, VA, USA). RAW264.7 cells were obtained from Procell company (Wuhan, China). Cells were cultured in standard DMEM containing 10% FBS and 1% penicillin-streptomycin at 37°C within a 5% CO\u003csub\u003e2\u003c/sub\u003e humidified environment. Plasmids encoding shKDM5B were transfected into cells using the Neofect™ Transfection Reagent (Beijing, China) according to the manufacturer's protocol. After a 24-hours incubation, cells were exposed to 1 µg/mL LPS for 24 hours before being harvested.\u003c/p\u003e\u003ch2\u003eRNA extraction and real-time PCR\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted from liver tissues or cultured cells, respectively, using TRIzol Reagent (Thermo Fisher Scientific) following the manufacturer’s instructions. Subsequently, complementary DNA (cDNA) was synthesized from total RNA (1 µg) via the HiScript III RT SuperMix for qPCR kit (Vazyme, China). Amplification of quantitative PCR products was carried out employing the SYBR Green qPCR Master Mix (Vazyme, China) on a real-time thermocycler. Relative quantification of target genes was performed, with normalization against GAPDH and subsequent calculation by 2\u003csup\u003e−ΔΔCt\u003c/sup\u003e analysis. Amplification primer sequences are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003ch2\u003eWestern blotting Analysis\u003c/h2\u003e\u003cp\u003eFrom liver specimens or cell cultures, proteins were isolated utilizing RIPA lysis buffer. Subsequently, total protein quantification was performed using a BCA assay kit. Protein lysates were resolved on 12% SDS-PAGE gels and electrotransferred to PVDF membranes. Membranes were blocked with 5% skim milk for 1 hour at room temperature. Next, primary antibody incubation proceeded overnight at 4°C. Following three washes in TBST, membranes were incubated with secondary antibodies at room temperature for 1 h. Following comprehensive rinsing with TBST, the proteins were detected using an ECL substrate kit (Biosharp, China) and documented by ImageQuant LAS 500 chemiluminescence imaging system (Cytiva, USA). Lastly, signal intensities were processed via ImageJ software (NIH, USA). Details of the primary antibodies are listed in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e.\u003c/p\u003e\u003ch2\u003eIsolation and Flow Cytometric Profiling of Hepatic Macrophages\u003c/h2\u003e\u003cp\u003eSmall fragments were prepared from liver tissues and digested using RPMI 1640 medium containing 0.05% collagenase IV (Roche) and 0.01% trypsin inhibitor (Gibco, Thermo Fisher Scientific) at 37°C for 60 minutes. The tissue digest was filtered through a 40-µm cell strainer. Subsequently, the generated single-cell suspension underwent centrifugation, with the supernatant subsequently removed. Erythrocytes underwent hypotonic lysis, followed by centrifugation (800×g, 10 minutes, 4°C). The isolated cells were then resuspended in 40% Percoll and subjected to centrifugation at 2,000 rpm for 30 minutes. After aspiration of the supernatant fluid, the cell suspension was layered onto 80% Percoll gradient and centrifuged at ambient temperature. Subsequently, the pellet underwent two washing cycles, was resuspended in RPMI-1640 medium, and counted to achieve the desired cell density. Cells were then stained with anti-F4/80 (#123107, BioLegend) and anti-CD11b (#101211, BioLegend) antibodies in flow cytometry buffer for 45 minutes at 4°C in the dark. Analysis was performed using a flow cytometer with FlowJo software.\u003c/p\u003e\u003ch2\u003eTransmission electron microscopy analysis\u003c/h2\u003e\u003cp\u003eAML12 hepatocytes were fixed using 2.5% glutaraldehyde at 4°C for 2 hours, then subjected to 0.1 mol/L phosphate buffer. Post-fixation was performed utilizing 1% osmium tetroxide (OsO\u003csub\u003e4\u003c/sub\u003e) for 2 hours. The cells were then dehydrated using a graded series of acetone and embedded in epoxy resin, polymerized, and sectioned. Ultrathin sections underwent double staining with 2% uranyl acetate and lead citrate, followed by examination of autophagosomes using transmission electron microscopy (HITA-CHI, Japan).\u003c/p\u003e\u003ch2\u003eImmunofluorescence analysis\u003c/h2\u003e\u003cp\u003eCell specimens underwent fixation using 4% paraformaldehyde, permeabilization with Permeabilization Buffer (Servicebio, China), and blocking with 5% BSA (Biosharp, China). Following the blocking step, primary antibody incubation proceeded overnight at 4°C. Secondary antibodies and DAPI (Servicebio, China) were then added and incubated for 1 hour at room temperature in the dark. Imaging of the slides was performed using a laser scanning confocal microscope (Olympus FV3000), and ImageJ was applied for quantitative analysis of the images.\u003c/p\u003e\u003ch2\u003eImmunoprecipitation\u003c/h2\u003e\u003cp\u003eAML12 cells were lysed with IP lysis buffer (Beyotime, China) containing a protease inhibitor mix (ABclonal, China) over ice for 30 minutes. Following this, lysates were processed by centrifugation (12000× g, 15 minutes, 4°C), after which the supernatants were collected. Cell lysates were immunoprecipitated with specific antibodies overnight at 4°C. After that, Protein A/G magnetic beads were mixed with the lysates for 4 hours at 4°C to capture immunocomplexes. Finally, bound proteins were then eluted by boiling in loading buffer for 10 minutes at 100°C, followed by immunoblotting analysis.\u003c/p\u003e\u003ch2\u003eMass Spectrometry Analysis of Hepatocytes\u003c/h2\u003e\u003cp\u003eLysis of AML12 cells was performed on ice for 30 minutes with 500 µL pre-chilled IP lysis buffer. This buffer consisted of 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, and 0.5% sodium deoxycholate, and was supplemented with cocktails of protease and phosphatase inhibitors. Following lysis, the supernatant was incubated overnight at 4°C with 2 µg of anti-KDM5B antibody or cognate IgG control. Subsequently, pre-washed Protein A/G agarose beads were added to the antigen-antibody mixture and following a 4-hours rotation incubation at 4°C, beads were pelleted via brief centrifugation (1,000× g, 1 min). The bead-bound immunocomplexes were next washed thrice using ice-cold TBST buffer. Following the final wash, the beads were resuspended in 2× loading buffer and subjected to heat denaturation at 100°C for 10 minutes. The eluted proteins were subsequently resolved on SDS-PAGE gels. Post-electrophoresis, the gel band corresponding to the sample was excised. The extracted peptides underwent analysis via liquid chromatography-tandem mass spectrometry (LC-MS/MS). The MS/MS spectra obtained were searched against the mouse non-redundant protein database (NCBI), utilizing the Mascot algorithm from Matrix Science.\u003c/p\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eStatistical evaluations were conducted using GraphPad Prism version 9.0. Results are presented as mean ± SEM, derived from a minimum of three separate experiments. Differences between groups were assessed using one-way ANOVA, and subsequent multiple comparisons were performed using two-way ANOVA with Tukey's test. Statistical significance is indicated as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; n.s. represents non-significant results.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003eL.Q. and C.C. conceived and designed the study, including determining the core direction and experimental protocols. H.O. and Y.M. performed the in vivo and in vitro experiments, and drafted the manuscript with the assistance of other co-authors. J.X. and Q.Y. curated the data using statistical software and analytical methods. W.H. and X.Q. conducted the literature search and organized the references. J.W., D.L., X.Q., Y.B. and S.S. revised the language of the first draft. All the authors reviewed every aspect of the manuscript and approved the version as submitted.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis article was supported by the National Natural Science Foundation of China (No. 82302365), Natural Science Foundation of Hubei Province, China (No. 2024AFB517), Hubei University of Science and Technology Development Fund Project (No. BK202442) and Foundation of Hubei University of Science and Technology Science \u0026ldquo;Special Project on Diabetes and Angiopathy\u0026rdquo; (No.2024TNB05).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAsrani SK, Devarbhavi H, Eaton J, Kamath PS. 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However, its underlying pathogenesis remains incompletely understood. Through analysis of four Gene Expression Omnibus (GEO) datasets using the Robust Rank Aggregation (RRA) method and RNA sequencing, the histone demethylase KDM5B was identified as a core upregulated gene in ALI. This finding was validated in carbon tetrachloride (CCl\u003csub\u003e4\u003c/sub\u003e)-induced murine ALI models, in which KDM5B expression was significantly elevated. Liver-specific knockdown of KDM5B via AAV-shKDM5B alleviated hepatic injury by reducing immune cell infiltration and inflammatory cytokine release, while enhancing autophagic flux. Mechanistically, KDM5B bound to the autophagy-related protein ATG16L1, disrupting the assembly of the ATG12-ATG5-ATG16L1 complex and impairing autophagosome maturation. This led to the accumulation of cytoplasmic DNA, which activated the cGAS-STING-TBK1-IRF3/NF-κB signaling pathway and amplified pro-inflammatory responses. This study revealed a protein-interaction-dependent mechanism by which KDM5B inhibited autophagy and triggered cGAS-STING signaling. It uncovered a novel mechanism underlying the vicious cycle of impaired autophagy and excessive inflammation in ALI, offering a molecular basis and potential therapeutic target involving the KDM5B-ATG16L1 axis.\u003c/p\u003e","manuscriptTitle":"KDM5B drives acute liver injury by impairing autophagy and activating cGAS-STING signaling via binding to ATG16L1","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-12 11:34:02","doi":"10.21203/rs.3.rs-7430149/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":"a34cf2ba-d528-4882-8fc5-2551d75a8d2d","owner":[],"postedDate":"September 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":54322058,"name":"Biological sciences/Cell biology/Autophagy"},{"id":54322059,"name":"Biological sciences/Immunology/Innate immunity"}],"tags":[],"updatedAt":"2025-10-13T15:30:40+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-12 11:34:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7430149","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7430149","identity":"rs-7430149","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}