Mitochondrial antiviral signaling protein enhances MASLD progression through the ERK/TNFα/NFκβ pathway

Mitochondrial antiviral signaling protein enhances MASLD progression through the ERK/TNFα/NFκβ pathway VISUAL ABSTRACT ORIGINAL ARTICLE Mitochondrial antiviral signaling protein enhances MASLD progression through the ERK/TNFα/NFκβ pathway Eva Nóvoa 1,2 |Natália da Silva Lima 1 |Maria J. Gonzalez-Rellan 1 | Maria D.P. Chantada-Vazquez 3 |Joanne Verheij 4 |Amaia Rodriguez 2,5 | Eva M. Esquinas-Roman 6 |Marcos F. Fondevila 1 |Mirja Koning 4 | Uxia Fernandez 1,2 |Alba Cabaleiro 1 |Tamara Parracho 1 | Jose Iglesias-Moure 1 |Samuel Seoane 1 |Begoña Porteiro 1 | Adriana Escudero 6 |Ana Senra 1 |Roman Perez-Fernandez 1 |Miguel López 1,2 | Miguel Fidalgo 6 |Diana Guallar 6 |Maria L. Martinez-Chantar 7 | Carlos Dieguez 1,5 |Marta Varela-Rey 6 |Vincent Prevot 8 | Markus Schwaninger 9 |Abraham Meijnikman 10 |Susana B. Bravo 2 | Gema Frühbeck 4,5 |Ruben Nogueiras 1,5,11 1 Department of Physiology, CIMUS, University of Santiago de Compostela, Santiago de Compostela, Spain 2 CIBER Fisiopatologia de la Obesidad y Nutrición (CIBERobn), A Coruña, Spain 3 Proteomic Unit, Health Research Institute of Santiago de Compostela (IDIS), Santiago de Compostela, A Coruña, Spain 4 Department of Pathology, Amsterdam University Medical Center, Amsterdam, The Netherlands 5 Department of Endocrinology & Nutrition, Metabolic Research Laboratory, Clínica Universidad de Navarra, University of Navarra, IdiSNA, Navarra, Spain 6 Gene Regulatory Control in Disease Laboratory, Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), Instituto de Investigación Sanitaria de Santiago de Compostela (IDIS), University of Santiago de Compostela, Santiago de Compostela, A Coruña, Spain 7 Liver Disease Lab, BRTA CIC bioGUNE, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Derio, Bizkaia, Spain 8 Univ. Lille, Inserm, CHU Lille, Laboratory of Development and Plasticity of the Neuroendocrine Brain, Lille Neuroscience & Cognition, UMR-S 1172, European Genomic Institute for Diabetes (EGID), Lille, France 9 Institute for Experimental and Clinical Pharmacology and Toxicology, University of Lübeck, Lübeck, Germany 10 Department of Internal and Experimental Vascular Medicine, Amsterdam University Medical Centers, Location AMC, Amsterdam, The Netherlands 11 Galician Agency of Innovation (GAIN), Xunta de Galicia, Santiago de Compostela, Spain Abstract Background and Aims: Mitochondrial antiviral signaling protein (MAVS) is a critical regulator that activates the host’s innate immunity against RNA Abbreviations: BA, bile acid; CDHFD, choline-deficient plus high-fat diet; MASH, metabolic dysfunction-associated steatohepatitis; MASLD, metabolic dysfunction– associated steatotic liver disease; MAVS, mitochondrial antiviral-signaling protein; MCD, methionineand choline-deficient; NAS, nonalcoholic fatty liver disease activity score; OA, oleic acid; OGT, O-linked N-acetylglucosamine (O-GlcNAc) transferase; WD, Western diet. Eva Nóvoa and Natália da Silva Lima equally contributed. Supplemental Digital Content is available for this article. Direct URL citations are provided in the HTML and PDF versions of this article on the journal's website, www.hepjournal.com. ------------------------------------------------------------------------------------------------------- This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. Received: 29 August 2023 | Accepted: 19 April 2024 DOI: 10.1097/HEP.0000000000000930 Hepatology. 2025;81:1535–1552. www.hepjournal.com | 1535 viruses, and its signaling pathway has been linked to the secretion of proinflammatory cytokines. However, the actions of MAVS on inflammatory pathways during the development of metabolic dysfunction–associated steatotic liver disease (MASLD) have been little studied. Approach and Results: Liver proteomic analysis of mice with genetically manipulated hepatic p63, a transcription factor that induces liver steatosis, revealed MAVS as a target downstream of p63. MAVS was thus further evaluated in liver samples from patients and in animal models with MASLD. Genetic inhibition of MAVS was performed in hepatocyte cell lines, primary hepatocytes, spheroids, and mice. MAVS expression is induced in the liver of both animal models and people with MASLD as compared with those without liver disease. Using genetic knockdown of MAVS in adult mice ameliorates diet-induced MASLD. In vitro, silencing MAVS blunts oleic and palmitic acid–induced lipid content, while its overexpression increases the lipid load in hepatocytes. Inhibiting hepatic MAVS reduces circulating levels of the proinflammatory cytokine TNFαand the hepatic expression of both TNFαand NFκβ. Moreover, the inhibition of ERK abolished the activation of TNFαinduced by MAVS. The posttranslational modification O-GlcNAcylation of MAVS is required to activate inflammation and to promote the high lipid content in hepatocytes. Conclusions: MAVS is involved in the development of steatosis, and its inhibition in previously damaged hepatocytes can ameliorate MASLD. INTRODUCTION Metabolic dysfunction–associated steatotic liver disease (MASLD) is a metabolic disorder marked by hepatic steatosis and at least 1 cardiometabolic risk factor, progressing from simple steatosis to steatohepatitis, fibrosis, cirrhosis, and HCC.[1]Its pathogenesis involves genetic, dietary, microbiota, and inflammatory factors. MASLD affects 25% of the global population, driven by obesity and sedentary lifestyles, making it a major cause of chronic liver disease.[2]Understanding the multiple and complex molecular pathways implicated in MASLD onset and progression is a major priority. One of the numerous molecules reported to be involved in this disease is the transcription factor p63, which is in the family comprising p53, p63, and p73.[3]The TAp63 isoform, which includes the transactivation domain, is elevated in the liver of animal models and patients with obesity and MASLD.[4]TAp63-induced hepatic fat content is mediated by the activation of IKKβand endoplasmic reticulum stress.[4]Of note, inflammation is one of the hallmarks of metabolic dysfunction-associated steatohepatitis (MASH).[5] The prevailing notion is that abnormal fatty acid accumulation in hepatocytes is considered the “first hit”in MASLD, followed by multiple hits that contribute in parallel to disease progression.[6]One of these hits is inflammation, as proinflammatory cytokines damage hepatocytes that are sensitized by their organelle’s stress in MASLD, although the mechanisms by which the different hits lead to MASLD remain largely unknown. Mitochondrial dysfunction also plays a pivotal role in the pathogenesis of MASLD, contributing to oxidative stress, impaired lipid metabolism, and inflammation within hepatocytes. Dysfunctional mitochondria exacerbate the proinflammatory milieu in MASLD and activation of inflammatory signaling pathways, such as NF-κβ and inflammasome activation. Mitochondrial antiviralsignaling protein (MAVS) is localized mainly in the mitochondrial outer membrane, although it has also been detected in peroxisome and mitochondrial-associated endoplasmic reticulum membranes.[7,8]MAVS (also known as virus-induced signaling adaptor or IFNβ promoter stimulator protein-1) is essential for antiviral innate immunity.[9]Upon viral recognition, cytosolic proteins such as retinoic acid-inducible gene I and melanoma differentiation–associated protein 5 activate MAVS, initiating an immune response through interferon regulatory factors 3 and 7 (IRF3/7) and NFκβ transcription factors. This leads to the expression and secretion of proinflammatory cytokines and antiviral genes.[9,10]As activation of MAVS signaling upon RNA virus infection also leads to an increase of glucose metabolic pathways, MAVS is considered a key factor in linking glucose metabolism to antiviral innate Correspondence Natália da Silva Lima, Department of Physiology, Research Centre of Molecular Medicine and Chronic Diseases (CIMUS), Instituto de Investigación Sanitaria de Santiago de Compostela, Universidad de Santiago de Compostela (USC), Santiago de Compostela 1706, Spain. Email: natalia. [email protected] Ruben Nogueiras, Department of Physiology, Research Centre of Molecular Medicine and Chronic Diseases (CIMUS), Instituto de Investigación Sanitaria de Santiago de Compostela, Universidad de Santiago de Compostela (USC), Santiago de Compostela 1706, Spain. Email: [email protected] 1536 | HEPATOLOGY 7.5 AAV8-TAp63 vs AAV8-GFP AAV8-TAp63α vs AAV8-GFP shRNA p63α vs sh scrambled TCA cycle and respiratory electron chain TCA cycle and respiratory electron chain Immune system p value p value (-log10) others (3%) % proteins peroxisome (7%) Golgi (7%) ER (12%) nucleus (19%) mitochondrion (52%) protein numbe r 1 1×10-1 1×10-2 1×10-3 1×10-4 1×10-5 21 2-3 1 Pathway Gene expression/transcription 0123456 Cell-cell communication Metabolism of proteins Signal transduction Metabolism Metabolism of RNA Autophagy Vesicle-mediated transport Fatty acid metabolism Fatty acid metabolism Amino acid metabolism Amino acid metabolism MAVS MAVS shRNA p63 vs sh scrambled 0.0 0 400000 STD AAV8 GFP AAV8-TAp63 AAV8-TAp63 * 300000 Liver protein levels (A.U.) 200000 100000 MAVS 0 600000 CDHFD sh scrambled shRNA p63 Lentiv-shp63 * 400000 Liver protein levels (A.U.) 200000 MAVS MAVS MAVS -1 0 1 -log10 (p value) log (Fold Change) 2.5 5.0 12 0 -1 0 1 -log10 (p value) log (Fold Change) Cellular component 4 8 (A) 10 STD AAV8-GFP STD AAV8-TAp63 * 4 2 0 p63 mRNA expression (Fold change) 6 8 2.5 ** 1.0 0.5 0.0 MAVS protein levels (% of control) 1.5 2.0 2.5 * 1.0 0.5 0.0 p63 mRNA expression (Fold change) 1.5 2.0 1.5 *** 1.0 0.5 0.0 MAVS protein levels (% of control) 3* 0 MAVS mRNA expression (Fold change) 1 2 8* 4 2 0 MAVS mRNA expression (Fold change) 6 (E) 3** *** ** 1 2 0 MAVS mRNA expression (Fold change) 2.0 STD CDHFD 6 weeks STD HFD 6 weeks 0.5 1.0 1.5 0.05 0.0 MAVS mRNA expression (Fold change) 3 STD MCD 4 weeks 1 2 0 MAVS mRNA expression (Fold change) 15 Chow diet WD 9 weeks 5 10 0 MAVS mRNA expression (Fold change) (G) CDHFD sh scrambled AAV8-GFP AAV8-TAp63 MAVS 75 kDa GAPDH 37 kDa sh scrambled shp63 MAVS 75 kDa GAPDH 37 kDa CDHFD shp63 (F) (C) (D) (B) MAVS ENHANCES MASLD PROGRESSION | 1537 immunity.[11]Interestingly, O-GlcNAcylation is a highly regulated, reversible posttranslational modification involved in a wide variety of metabolic signals and cellular processes,[12,13]and this modification has been reported to be essential for the host antiviral innate immunity of MAVS.[11,14] In this work, we used unbiased proteomics to analyze the liver of mice with manipulated TAp63 levels. We identified that MAVS is regulated by TAp63: it is upregulated after TAp63 activation and downregulated after p63αinhibition. MAVS activation was consistent across various MASLD animal models and in human MASLD liver samples. In both in vitro and in vivo settings, MAVS overexpression increased hepatocyte lipid accumulation, while MAVS inhibition relieved TAp63and diet-induced steatosis. These effects on lipid metabolism were mediated through the ERK/TNFα/NFκβ pathway, as ERK, TNFα,orNFκβ inhibition attenuated MAVSinduced lipid accumulation. Furthermore, mutating the O-GlcNAcylated residue Thr373 on MAVS hindered its effects, underscoring the importance of O-GlcNAcylation in MAVS-mediated inflammation and lipid accumulation. METHODS Animals and diets Animal protocols were approved by the Committee at the University of Santiago de Compostela and received humane care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals.” Cohort of patients with NASH for western blot analysis All reported investigations were carried out in accordance with the principles of the Declaration of Helsinki, as revised in 2013 and approved by the Hospital’s Ethical Committee responsible for research (protocol 2021.005). Written informed consent was obtained from all the participants. Anthropometric, biochemical, and clinical characteristics of patients are shown in Supplemental Table S2, http://links.lww.com/HEP/I457. For further details, see Supporting Participants and Methods. Lists of primers and antibodies used are shown in Supplemental Tables S3 and S4. Uncropped western blots are shown in Supplemental Figure S10, http://links.lww.com/HEP/I457. RESULTS MAVS is positively regulated by p63 and increases in diet-induced animal models of MASLD Hepatic TAp63 overexpression induces steatosis, while p63 inhibition ameliorates diet-induced steatosis.[4]Proteomic analyses in these models revealed novel pathways and regulators of hepatic lipid accumulation (Figures 1A–D). Volcano plots show multiple changes in hepatic protein levels upon TAp63 manipulation (Figures 1A, B). A protein interactome network displayed the TCA cycle, respiratory electron chain, fatty acid, and amino acid metabolism alterations after overexpression and knockdown of p63 (Figure 1C). We specifically searched for proteins oppositely expressed in mice following the overexpression or knockdown of p63; we found that p63 positively regulated 43 proteins. Analyzing this protein set revealed a significant overrepresentation (44%) of metabolism-related proteins, with mitochondria identified as the most affected cell component (Figure 1D). Notably, MAVS was induced by TAp63 and downregulated by p63 inhibition, prompting further investigation into its role. Corroborating our proteomic results, both MAVS mRNA expression and protein levels increased upon TAp63 induction in the liver (Figure 1E), while they were reduced upon hepatic p63αinhibition in dietinduced obese mice (Figure 1F). MAVS expression was assessed in livers from mouse models of dietFIGURE 1 Hepatic MAVS is upregulated in different in vivo and in vitro models of MASLD and MASH. (A, B) Volcano plots of hepatic protein expression determined by LC-MS/MS proteomics of (A) mice fed a standard diet (STD), with TAp63 overexpressed specifically in liver compared to the control group (n =3 per group), or (B) mice fed a choline-deficient high-fat diet (CDHFD) for 12 weeks, with hepatic p63 downregulated as compared to the control group (n =3 per group). Values obtained for MAVS are represented in the graph. (C) Protein-protein interaction network of deregulated proteins involved in metabolism according to the STRING database. Node color indicates the 3 main affected functions: TCA and respiratory electron chain (red), fatty acid metabolism (green), and amino acid metabolism (blue). MAVS is indicated in pink. (D) Reactome pathway classification of deregulated proteins in the liver of mice from (A) and (B), showing the number of proteins included in each category and the associated FDR. The same proteins were also classified according to the cellular component using the FunRich tool. The size of the circumference is proportional to the number of proteins deregulated in the metabolic pathway. On the other hand, the color indicates the significance of the pvalue, that is, red is more significant than blue. (E, F) mRNA levels of p63 and MAVS, as well as MAVS protein levels, in mice in the conditions as in (A) and (B) (n =4–8 per group). (G) MAVS mRNA in the liver of mice fed an STD, a high-fat diet (HFD) (n =4–6), a choline-deficient plus high-fat diet (CDHFD) (n =8–10), a methionine-and-choline-deficient (MCD) diet (n =5–6 per group), and a Western diet (WD) (n =8). HPRT and GAPDH were used to normalize mRNA and protein levels, respectively. Data are presented as mean ±SEM; ∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001, Student ttest. Abbreviations: MASH, metabolic dysfunction-associated steatohepatitis; MASLD, metabolic dysfunction–associated steatotic liver disease; MASH, metabolic dysfunction-associated steatohepatitis; MAVS, mitochondrial antiviral-signaling protein. 1538 | HEPATOLOGY induced MASLD and MASH, including mice fed a highfat diet for 6 weeks, a choline-deficient plus high-fat diet (CDHFD) for 6 weeks, a methionineand cholinedeficient (MCD) diet for 4 weeks, or a Western diet (WD) for 9 weeks. In all 3 animal models, MAVS mRNA was elevated (Figure 1G), suggesting a consistent increase in MAVS expression in preclinical models of MASLD. r = 0.53 p = 0.0144 4** (A) (B) 3 MAVS mRNA expression (Fold change) 2 1 Non MASLD MASLD Non MASLD MASLD 0 4 (C) 3 MAVS mRNA expression 2 1 0 02 NAS score 468 2500 * * * *** ** (D) 1500 2000 MAVS mRNA Normalized Counts 1000 500 0 n=10 n=51 n=34 n=53 n=54 n=14 Control MASLD MASH F0-F1 MASH F2 MASH F3 MASH F4 r = 0.67 p = 0.0022 4 3 MAVS mRNA expression 2 1 0 0 100 Serum triglycerides 200 300 400 r = 0.01 p = 0.9437 4 3 MAVS mRNA expression 2 1 0 30 40 BMI 50 60 4** 3 MAVS protein levels (% of control) 2 1 0 1.674082402 MAFLD 1.711372745 2.164456228 2.504299404 3.171719437 3.388166625 0.84023987 1.89852008 1.265705729 1.275834447 0.983566104 Ab Cell Signalling Non MASLD MASLD MAVS 75 kDa GAPDH 37 kDa Non MAFLD 0.967690865 0.857686226 1.092995909 1.098060895 0.957285837 0.527910376 1.508679207 1.00612458 FIGURE 2 MAVS is increased in the liver of patients with MASLD. (A) MAVS mRNA expression in the liver of patients without MASLD (nonMASLD) (n =11) or MASLD (n =8). (B) MAVS protein levels in the liver of patients without MASLD (non-MASLD) (n =9) or MASLD (n =10). (C) Correlation between NAS score, serum triglycerides, and BMI with MAVS mRNA levels. (D) Normalized DESeq2 counts for MAVS mRNA expression across the GSE135251 data set composed of liver samples from control patients and patients with 5 grades of MASLD progression. HPRT and GAPDH were used to normalize mRNA and protein levels, respectively. Lines indicate splicing in the same gel. Data are presented as mean ±SEM; ∗∗p<0.01, Student’sttest. Abbreviations: MASLD, metabolic dysfunction–associated steatotic liver disease; MAVS, mitochondrial antiviral-signaling protein; NAS, nonalcoholic fatty liver disease activity score. MAVS ENHANCES MASLD PROGRESSION | 1539 10 Control THLE2 cells THLE2 cells 50 μm 50 μm50 μm 50 μm siMAVS Control siMAVS OA OA + siMAVS Control siMAVS TAp63 TAp63 + siMAVS OA + siMAVSOA OA + siMAVSOA OA+PA OA+PA shMAVS *** *** *** ** ** *** *** *** 8 6 4 2 0 Oil Red O Staining Area (% control) 10 Control siMAVS OA OA + siMAVS *** *** *** *** * 8 6 4 2 0 Oil Red O Staining Area (% control) 5shControl Vehicle shControl Oleic + Palmitic Acid shMAVS Oleic + Palmitic Acid *** ** 3 4 1 2 0 Biotracker Staining Area (% of control) 4 3 1 2 0 Biotracker Staining Area (% of control) Control pMAVS ** 2.5 2.0 1.5 1.0 0.5 0.0 Oil Red O Staining Area (% control) Control pMAVS *** 2.5 2.0 1.5 1.0 0.5 0.0 Oil Red O Staining Area (% control) Control pMAVS *** 8 6 4 2 0 Oil Red O Staining Area (% control) (A) (C) (E) Control THLE2 cellsPrimary Hepatocytes Primary Hepatocytes SpheroidsSpheroids 50 μm 50 μm50 μm 50 μm50 μm 50 μm 100 μm 100 μm 100 μm 100 μm 100 μm 50 μm 50 μm 50 μm50 μm 50 μm 50 μm siMAVS Control Control Control pMAVS Control pMAVS Control pMAVS siMAVS pTAp63 + siMAVSpTAp63 (B) (D) (F) (G) 1540 | HEPATOLOGY MAVS is increased in the liver of people with MASLD MAVS expression was evaluated in liver biopsies from people with MASLD (nonalcoholic fatty liver disease activity score [NAS] ≥3) or mild-MASLD livers (NAS ≤2) (Supplemental Table S2, http://links.lww.com/HEP/ I457). MAVS mRNA expression was significantly higher in the livers of people with MASLD than in those without the disease (Figure 2A). This result agrees with a report that also found increased MAVS gene expression in people with NASH.[15]However, another study described that protein levels of MAVS were significantly downregulated in people with MASLD.[16] To assess whether gene expression correlated with protein levels in our cohort samples, we used 2 different antibodies (Figure 2B, Supplemental Figures S1 and S2, http://links.lww.com/HEP/I457 and Supplemental Materials and Methods, http://links.lww.com/HEP/ I457). In line with mRNA expression, anti-MAVS AbCellSig showed that protein levels of MAVS were also augmented in the liver of people with MASLD (Figure 2B) and positively correlated with NAS score and serum triglycerides, showing no association with BMI (Figure 2C). We also analyzed MAVS expression in a public data set (GEO accessions GSE135251), which provides bulk RNA sequencing results from samples of 206 patients with MASLD at various fibrosis stages and 10 healthy liver controls.[17]The analysis indicated that the MAVS mRNA is significantly upregulated in the liver of people with both MASLD and MASH at different stages (MASH F1–F4) (Figures 2D, E). The analysis of this cohort supports our data, indicating that MAVS levels are increased in MASLD. MAVS increases lipid content in human hepatocytes Since both gene expression and protein levels of MAVS were upregulated in the liver of people with MASLD, we studied its impact on hepatocytes. MAVS mRNA expression rose in human hepatic THLE2 cells treated with oleic acid (OA) (Supplemental Figure S3A, http:// links.lww.com/HEP/I457). As expected, OA treatment augmented intracellular lipid content; however, siRNAmediated MAVS silencing reduced OA-induced lipid storage (Figure 3A and Supplemental Figure S3B, http://links.lww.com/HEP/I457). As reported,[4]TAp63 overexpression increased the lipid droplets in hepatocytes, but this effect was blunted when MAVS was silenced (Figure 3B). Next, we ectopically overexpressed MAVS (Supplemental Figure S3C, http://links.lww.com/HEP/I457) and found an increased lipid content in THLE2 cells (Figure 3C). Parallel treatment with OA and loss-of-function and gain-offunction experiments in primary hepatocytes showed identical results (Figures 3D, E and Supplemental Figures S3D–F, http://links.lww.com/HEP/I457). We also obtained similar results using HepG2 cells: silencing MAVS reduced the OA-induced lipid content while overexpressing MAVS increased the lipid load (Supplemental Figures S3G, H, http://links.lww.com/ HEP/I457). Next, a more complex fatty acid mixture was used, combining palmitate acid and OA in THLE2 cells, which caused a significant increase in fatty acid concentration (Supplemental Figure S4A, http://links. lww.com/HEP/I457). We employed OA and palmitic acid in a 3D AML12 cell spheroid model. MAVS overexpression and silencing were confirmed (Supplemental Figure S4B, http:// links.lww.com/HEP/I457). Oleic acid treatment raised fatty acid levels, mitigated by MAVS silencing (Supplemental Figure S4C, http://links.lww.com/HEP/I457). Oleic acid and palmitate treatment increased lipid content, attenuated by MAVS silencing (Figure 3F). MAVS overexpression in spheroids increased lipid content (Figure 3G). Thus, MAVS manipulation in 3D models mirrors findings in 2D models. Inhibition of hepatic MAVS ameliorates CDHFD-induced, MCD-induced, and WDinduced MASLD We next investigated the in vivo relevance of these findings in mice subjected to either (i) a CDHFD (45% kcal from fat) for 16 weeks; (ii) an MCD diet for 4 weeks; or a WD (45% kJ fat, 15% kJ protein, 43% kJ carbohydrates, and 1.25% cholesterol) for 9 weeks (Supplemental Table S1, http://links.lww.com/HEP/I457). Mice received tail vein injections of a lentivirus encoding for a scrambled shRNA or shRNA against MAVS to decrease MAVS expression as described.[18] Injections were administered either 8 weeks into the FIGURE 3 MAVS regulates lipid accumulation in human hepatic cell lines and in mouse primary hepatocytes. (A–C) Representative Oil Red O staining and Oil Red Area of (A) THLE2 cells downregulating MAVS (siMAVS) treated with OA or vehicle for 24 hours (n =18–30 per group); (B) TAp63 upregulated in THLE2 cells after MAVS silencing (n =12–23 per group); and (C) THLE2 cells with overexpression of MAVS (n =16–18 per group). (D, E) Representative Oil Red O staining and Oil Red Area of murine primary hepatocytes with MAVS downregulated treated with OA or vehicle for 24 hours (D) or with MAVS overexpressed (E) (n =5–6 per group). (F) Spheroids shControl and shMAVS treated with 0.5 mM OA and 0.25 mM palmitic acid. (G) Spheroids with MAVS overexpressed. Data are presented as mean ±SEM; ∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001, using Student ttest (C, E, and G) or one-way ANOVA followed by a Bonferroni multiple comparison test (A, B, D, and F). Abbreviations: MAVS, mitochondrial antiviral-signaling protein; OA, oleic acid. MAVS ENHANCES MASLD PROGRESSION | 1541 < 5% CDHFD sh scrambled (n=10) CDHFD shMAVS (n=8) 5% - 33% > 33% - 66% > 66% No foci None None Few balloon cells Perisinusoidal or periportal Fibrosis stage Liver injury Lobular inflammation Steatose grade Many cells/prominent ballooning < 2 foci per 200 x field 6 2 0 0 5 3 3 4 1 5 3 2 2 3 3 2 0 1 5 5 9 8 Lentiv-shMAVS (A) (B) (D) (E) (G) (C) shMAVS End 2.0 CDHFD sh scrambled MCD sh scrambled MCD shMAVS CDHFD shMAVS ** 1.5 1.0 MAVS mRNA expression (Fold change) 0.5 0.0 2.5 2.0 ** 1.5 1.0 Oil Red O Staining Area (% Control) 0.5 0.0 2.0 * 1.5 1.0 Sirius Red Area (% Control) 0.5 0.0 2.0 *** 1.5 1.0 MAVS mRNA expression (Fold change) 0.5 0.0 1.5 1.0 Sirius Red Area (% of control) 0.5 0.0 150 ** ** * 2.0 1.0 1.5 Collagen1 IHC Area (% control) 0.5 0.0 *** ** * 100 ALT (U/L) 50 0 (I) 5 3 4 Liver mRNA expression (Fold change) 2 1 0 150 100 AST (U/L) 50 0 150 * 100 50 ALT (U/L) 0 2.5 * * 2.0 1.5 1.0 0.5 Liver mRNA expression (Fold change) 0.0 Col1α1 Col1α2 ColIII Col1α1 Col1α2 ColIII 80 60 20 40 AST (U/L) 0 WT CDHFD CD-HFD CDHFD sh scrambled 50 μm HEOil Red O StainingSirius red 50 μm 50 μm 50 μm 50 μm 50 μm CDHFD shMAVS (H) HESirius RedCollagen 1 IHC MCD sh scrambled 50 μm 50 μm 50 μm 50 μm 50 μm 50 μm MCD shMAVS 8 weeks 8 weeks shMAVS End MCD MCD STD diet 4 weeks 4 weeks Lentiv-shMAVS (F) WT 1542 | HEPATOLOGY Disruption of BA homeostasis is closely linked to the progression of MASLD, contributing to dysregulated energy balance, increased liver inflammation, and fibrosis. Elevated levels of BA, observed in individuals with NASH, suggest a correlation between toxic BA levels and MASH development.[32,33]Therefore, our proteomic data may suggest that the MAVS-induced liver steatosis is somehow mediated by BAs. Indeed, future studies are needed to elucidate the precise interaction between MAVS and BAs, along with the functional relevance of BA as mediators of the deleterious effects of MAVS in the liver. O-GlcNAcylation of MAVS induces hepatocyte lipid content and inflammation O-GlcNAcylation of MAVS is crucial in mediating its different actions,[11,14]and we have recently linked O-GlcNAcylation to MASLD development.[34]We investigated whether this posttranslational modification modulates MAVS effects on inflammation and steatosis. Coimmunoprecipitation analysis showed increased O-GlcNAcylated MAVS in the livers of MCD diet–fed mice (Figure 7A) and CDHFD-fed mice (Figure 7B). Thus, we treated primary hepatocytes with O-(2-acetamido-2deoxy-D-glucopyransylidene)-amino-N-phenylcarbamate (PUGNAc), an inhibitor of O-GlcNAcase, the enzyme that prevents the removal of O-GlcNAc form target proteins and therefore increases protein O-GlcNAc levels,[35]and found that PUGNAc increased MAVS protein levels (Figure 7C). We then treated primary hepatocytes with OSMI-1, an inhibitor of O-GlcNAc transferase that catalyzes the addition of O-GlcNAc to target proteins and thus decreases protein O-GlcNAc levels,[36]which reduced MAVS protein levels (Figure 7D). We ectopically overexpressed MAVS and, after 24 hours, treated them with OSMI-1. As in experiments described above, MAVS favored fatty acid accumulation; however, the treatment with OSMI-1 blunted MAVS-induced hepatocyte lipid content (Figure 7E), and this was associated with lower levels of TNFα(Figure 7F) and NFκβ (Figure 7G). Finally, to gain insight into the functional consequences of O-GlcNAcylation of MAVS, we identified MAVS O-GlcNAcylation sites using the software https:// www.oglcnac.mcw.edu/. Thr373 was the site identified in mice and therefore, we mutated Thr373 to Ala. Wildtype MAVS increased lipid content and secreted TNFα and NFκβ protein levels in primary hepatocytes. In contrast, the T373A-MAVS mutant completely lost its capacity to exert those effects (Figures 7H–J). Overall, these data demonstrate that O-GlcNAcylation in the Thr373 is required for the action of MAVS on hepatic inflammation and fatty acid content. DISCUSSION In this work, we have identified the role of MAVS on fatty acid metabolism and its implications in the development of MASLD. MAVS is elevated in the liver of animal models as well as of people with MASLD. Moreover, our in vitro and in vivo genetic functional studies indicated that overexpression of MAVS induces lipid deposition while its silencing alleviates steatosis. To our knowledge, the regulation of MAVS in human MASLD has been previously assessed in only 2 studies. The first study showed that MAVS gene expression is increased in the liver of people with NASH as compared with a non-NASH control group[15]; however, the second study found that the MAVS protein levels are reduced in the liver of people living with MASLD.[16]In our study, we found that both mRNA and protein levels of MAVS are significantly upregulated in people with MASLD, with MAVS transcripts being positively associated with NAS and serum triglyceride levels. Two critical points could explain these apparently discrepant results. First, MASLD characterization and sample size of the cohorts differed: we used 18 samples from people with obesity and MASLD (in fibrosis stage 1 or 2) diagnosed according to BruntKleiner’s criteria[37]; in contrast, in the previous report, 8 samples with an NAS ≥3 are classified as MASLD, while those with NAS ≤2 without steatosis are classified as non-MASLD,[18]and the potential impact of dyslipidemia on MAVS expression was not analyzed. Second, 2 different antibodies were used: while both antibodies showed a clear reduction in protein levels after silencing MAVS in a human hepatic cell line, the antibody used in the previous study did not show differences when MAVS was overexpressed, and when tested in human samples questions about its reliability emerge (Supplemental Figure S1, http://links.lww.com/ HEP/I457). Finally, different protocols for protein FIGURE 7 O-GlcNAcylation of MAVS regulates lipid accumulation. (A) O-GlcNAcylated levels of MAVS in the liver of mice fed STD and MCD diet (n =4 per group). (B) O-GlcNAcylated levels of MAVS in the liver of mice control and CDHFD (n =6 per group). (C, D) MAVS protein levels of primary hepatocyte cells in the absence or presence of PUGNAc 75 µM and OSMI-1 100 µM for 24 hours (n =6 per group). (E, F, G) Representative Oil Red O staining, TNFαrelease to cell supernatant, and NFκβ protein levels of primary hepatocytes cells with MAVS overexpression treated with OSMI-1 100 µM or vehicle for 24 hours (n =4–6 per group). (H, I, J) Representative Oil Red O staining, TNFαrelease to cell supernatant, and NFκβ protein levels of primary hepatocyte cells with MAVS overexpression and MAVS Mutant Thr373 overexpression (n = 4–8 per group). GAPDH was used to normalize protein levels. Data are presented as mean ±SEM; *p<0.05, **p<0.01, ***p<0.001, Student t test (A–D) and one-way ANOVA followed by a Bonferroni multiple comparison test (E–J). Abbreviations: CDHFD, choline-deficient plus high-fat diet; MAVS, mitochondrial antiviral-signaling protein; MCD, methionineand choline-deficient. MAVS ENHANCES MASLD PROGRESSION | 1549 extraction were likely used, which may affect the quality of the western blots. Further studies will be necessary to clarify these issues. Indeed, the observed discrepancies may stem from the utilization of small cohorts in both studies. To substantiate our findings across a broader spectrum, we leveraged a publicly accessible data set from what we believe to be one of the largest cohorts available, which offers comprehensive transcriptomic data.[17]The analysis indicated that the mRNA expression of MAVS is significantly upregulated in the liver of people with both MASLD and MASH at different stages (MASH F1–F4), supporting that MAVS levels are increased in MASLD. In addition to its regulation, the other key question is whether the inactivation of MAVS is beneficial or detrimental to the liver. To address this question, a study using mouse models with either MAVS global knockout or hepatocyte-specific MAVS knockout reported that these mice were more prone to developing diet-induced NAFLD.[16]In contrast, we found in this study that adult-onset MAVS inhibition in the whole liver or specifically in hepatocytes protected mice against diet-induced MASLD. However, there is a fundamental difference between the 2 studies: while the earlier work disrupted MAVS from embryonic stages, we knocked down MAVS in adult stages after liver damage had already been induced. Therefore, the deletion of MAVS seems to exert very different actions in mice depending on the time of the genetic intervention and liver status: its deletion is detrimental at early stages in healthy conditions, but it is beneficial when the mice have already developed MASLD. MAVS is widely recognized as playing an essential role in antiviral innate immunity, whereby it mediates the activation of NFκB and interferons and the induction of interferons in response to viral infection in multiple cell types, prompting the secretion of proinflammatory cytokines.[9,38]For instance, the deletion of MAVS abolishes the induction of IFN-I and other proinflammatory cytokines by respiratory syncytial virus.[39]Inflammation is also one of the hallmarks of MASH and is considered paramount for disease progression.[40,41]Under conditions of stress, the expression of inflammatory cytokines, such as TNFα, is increased in hepatocytes.[42]The current dogma is that both hepatocytes and Kupffer cells are the primary sources of TNFαproduction in the initiating stage of MASH, whereas infiltrated monocytes and macrophages later contribute to the vicious cycle of TNFαsignaling cascade in MASH progression.[43]Our results indicate that MAVS induces the expression and release of TNFαin hepatocytes and its downregulated transcription factor NFκβ, and that this inflammatory signaling pathway is an important mediator of the adverse effects caused by MAVS, as its inhibition blunted MAVS-induced steatosis. Since TNFαis known to be modulated by kinases such as p38 MAPK, ERK, and JNK,[25–29]we tested whether these factors could regulate the actions of MAVS on TNFαactivation. The data indicate that ERK is a key player since its inhibition blunted MAVS-induced TNFα elevation. Although our study focused on hepatocytes, our results do not discard that in vivo, at least part of the effects could also be mediated by TNFαproduced in other cells (eg, Kupffer cell, macrophages, or monocytes). Many studies have shown that MAVS can be regulated by posttranscriptional and posttranslational modifications, which affect its function in promoting innate immune responses (reviewed in Ren et al[44]). One posttranslational modification is glycosylation by O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT), which activates MAVS and is crucial for its antiviral response.[11]Our lab has recently demonstrated that O-GlcNAcylation is also an important feature in the development of MASLD, as OGT inhibits mitochondrial function, favoring the storage of fatty acids in hepatocytes, while the genetic knockdown of OGT in animal models of MASLD alleviates the disease.[34]In the present study, we found that MAVS is hyper-O-GlcNAcylated in the liver of mice with MASLD and that the pharmacological inhibition of OGT blunted MAVSinduced lipid storage. This is because the suppression of O-GlcNAcylation ameliorated MAVS-induced NFκβ expression and TNFαsecretion in hepatocytes. Importantly, when MAVS was mutated in Thr373, the actions of MAVS on inflammation and lipid accumulation did not occur. Therefore, our results indicate that this posttranslational modification regulates the harmful action of MAVS in hepatocytes. In summary, our findings show that: (a) MAVS expression is increased in the liver of mouse models of MASLD and of people living with MASLD; (b) MAVS overexpression induced lipid accumulation; (c) inhibition of MAVS in the whole liver or specifically in hepatocytes of adult mice ameliorated TAp63α-induced and dietinduced liver steatosis; (d) the steatotic action of MAVS is mediated by ERK/TNFα/NFκβ; and (e) the posttranslational modification O-GlNAcylation is critical for MAVS-induced inflammation and lipid storage. Notably, we present preliminary functional data demonstrating that silencing MAVS genetically or inhibiting its O-GlcNAcylation through pharmacological means effectively prevents steatosis and MASLD development. Overall, our results point toward MAVS as a molecule implicated in the development of steatosis. DATA AVAILABILITY STATEMENT The data associated with this paper are available upon request to the corresponding author. AUTHOR CONTRIBUTIONS Eva Nóvoa and Natália da Silva Lima designed the study, performed the experiments, interpreted the 1550 | HEPATOLOGY results, and wrote the manuscript. Maria J. GonzalezRellan, Amaia Rodriguez, Marcos F. Fondevila, Maria D.P. Chantada-Vazquez, Uxia Fernandez, Tamara Parracho, Begoña Porteiro, Adriana Escudero, and Ana Senra performed the experiments. Miguel López, Miguel Fidalgo, Diana Guallar, Maria L. MartinezChantar, Carlos Dieguez, Vincent Prevot, Markus Schwaninger, Susana B. Bravo, and Gema Frühbeck supervised experiments and edited the manuscript. Ruben Nogueiras wrote the manuscript and acquired funding. All authors critically reviewed and approved the manuscript. FUNDING INFORMATION This work was supported by grants from FEDER/ Ministerio de Ciencia, Innovación y UniversidadesAgencia Estatal de Investigación (Maria L. MartinezChantar: PID2020-117116RB-I00; Carlos Dieguez: BFU2017-87721; and Ruben Nogueiras: PID2021126096NB-I00 and RED2018-102379-T); Xunta de Galicia (Ruben Nogueiras: 2021-CP085 and 2020PG0157); Fundación BBVA (to Ruben Nogueiras); Subprograma Retos Colaboración RTC2019-007125-1 (to Maria L. Martinez-Chantar); Proyectos Investigación en Salud DTS20/00138 (to Maria L. Martinez-Chantar); Proyectos Investigación en Salud (Maria L. MartinezChantar: DTS20/00138); Fundación La Caixa (Ruben Nogueiras). This research also received funding from the European Community’s H2020 Framework Programme (ERC Synergy Grant-2019-WATCH810331, to Ruben Nogueiras, Vincent Prevot, and Markus Schwaninger). The Centro de Investigación Biomédica en Red (CIBER) de Fisiopatología de la Obesidad y Nutrición (CIBERobn) and the Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) are initiatives of the Instituto de Salud Carlos III (ISCIII) of Spain, which is supported by FEDER funds. The authors thank MINECO for the Severo Ochoa Excellence Accreditation bioGUNE (SEV-2016-0644) to CIC. CONFLICTS OF INTEREST Maria D.P. Chantada-Vazquez received grants from Fundación Instituto de Investigación Sanitaria de Santiago de Compostela (IDIS). The remaining authors have no conflicts to report. REFERENCES 1. Rinella ME, Lazarus JV, Ratziu V, Francque SM, Sanyal AJ, Kanwal F, et al. A multisociety Delphi consensus statement on new fatty liver disease nomenclature. Hepatology. 2023;78: 1966–86. 2. Younossi ZM, Golabi P, Paik JM, Henry A, Van Dongen C, Henry L. 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