Hepatology
Abstract
Metabolic dysfunction-associated steatotic liver disease (MASLD) is a global health concern with limited interventions. While the role of gut bacteria in MASLD has been extensively studied, the contribution of gut fungi remains largely unexplored. This study investigates the impact of fungal dysbiosis and the role of CARD9, a key adaptor protein in fungal sensing on gut-liver axis dysfunction in MASLD. Patients with advanced liver fibrosis exhibited distinct mycobiota profiles. Using a CARD9-deficient mouse model subjected to high-fat high-glucose/fructose feeding, we observed exacerbated liver injury and fibrosis accompanied by fungal dysbiosis, paralleling our findings in human patients. Beyond its established expression in myeloid cells, CARD9 was also detected in intestinal enterocytes where its expression was diminished under metabolic stress. Intestinal organoids with CARD9 inhibition had reduced expression of antimicrobial Reg3g, the tight junction protein ZO-1, and the antifungal enteroendocrine hormone PYY. These findings suggest that CARD9 maintains gut barrier integrity, preventing microbial translocation and subsequent liver injury and fibrosis. Our results provide new insights into the interplay between fungal dysbiosis, gut barrier dysfunction, and MASLD, and identify CARD9 as a key protein within this axis.
Authors
Vijay Pandyarajan, So Yeon Kim, Takashi Tsuchiya, Selena Liu, Sadam H. Bhat, Jieun Kim, David M. Underhill, Mazen Noureddin, Shelly C. Lu, Ekihiro Seki
Abstract
Fontan-associated liver disease (FALD) is a frequent complication in single ventricle patients palliated with the Fontan operation. FALD severity can impact clinical decisions; however, the pathophysiology of FALD progression is unknown. Single-cell spatial transcriptomics (ST) was performed on liver explant tissue sections from FALD patients with early (n=1) and advanced fibrosis (n=1) using CosMxTM Spatial Molecular Imaging with in-situ hybridization of 6000 genes. Immunofluorescence for liver zonation and cellular stress markers was performed to confirm protein expression based on ST analysis in additional FALD tissues (n=18). Unbiased clustering yielded 12 liver cell types, comprising six subtypes of hepatocytes. FALD with advanced fibrosis demonstrated expansion of mid-zonal hepatocytes, accompanied by loss of zonal markers characteristic of canonical pericentral and periportal hepatocytes. A subset of hepatocytes in advanced FALD demonstrated increased cellular stress and a redundant zonal phenotype, which we have termed zonally ambiguous and stressed hepatocytes. CellChat analysis revealed that ectopic WNT2 signaling is likely driving disrupted hepatocyte zonation. To corroborate these bioinformatic findings, we performed immunofluorescence staining of FALD specimens, which confirmed a disruption of liver zonation, and a significant increase in heat shock protein 70 (HSP70). Lastly, HSP70 expression strongly correlated with the Congestive Hepatic Fibrosis (CHF) score. Thus, single-cell ST has identified a unique population of hepatocytes with features of cellular stress and redundant zonal gene expression specific to advanced FALD. Further studies on hepatocyte metabolic function in Fontan patients will lead to a greater understanding of FALD development and progression during chronic maladaptation.
Authors
Brandon M. Lehrich, Jordann N. Lewis, Vik Meadows, Lori Schmitt, Mylarappa B. Ningappa, Jia-Jun Liu, Silvia Liu, Catherine K. Gestrich, Victor O. Morell, Rakesh Sindhi, Satdarshan P. Monga, Anita Saraf
Abstract
Mitochondrial retrograde signaling plays crucial roles in maintaining metabolic homeostasis via regulating genome modification and oxidative responsive gene expression. In this study, we identified GCN5L1, a protein localized in both mitochondria and cytoplasm, and demonstrated its specific translocation from mitochondria to cytoplasm during lipid overload and high-fat diet feeding. Using transcriptome and proteome analyses, we identified that cytoplasmic GCN5L1 binds to and promotes the acetylation of PPARγ at lysine 289 (K289). This acetylation protected PPARγ from ubiquitination-mediated degradation by proteasome. GCN5L1 translocation enhanced protein stability of PPARγ and subsequently promoted lipid accumulation in both cultured cells and murine models. Our study further reveals that PPARγ-K289 mutation reduces the ubiquitination of PPARγ and exacerbates liver steatosis in mice. These findings unveil a mitochondrial retrograde signaling during lipid overload, which regulates the crucial lipogenic transcriptional factor. This discovery elucidates an unrecognized mitochondrial function and mechanism underlying hepatic lipid synthesis.
Authors
Jiaqi Zhang, Danni Wang, Qiqi Tang, Yaoshu Yue, Xin Lu, Xiuya Hu, Yitong Han, Jiarun Chen, Zihan Wang, Xue Bai, Kai Zhang, Yongsheng Chang, Longhao Sun, Lu Zhu, Lingdi Wang
Abstract
Serine-rich splicing factor 3 (SRSF3) is crucial for the metabolic functions of the liver. The genetic deletion of SRSF3 in mouse hepatocytes impairs hepatic lipid and glucose metabolism and leads to fibrosis and formation of hepatocellular adenoma that progresses to hepatocellular carcinoma. SRSF3 protein is proteosomally degraded in metabolic-dysfunction associated fatty liver disease (MAFLD) and metabolic-dysfunction-associated steatohepatitis (MASH). We show here that depleting SRSF3 protein in hepatocytes promoted R-loop accumulation and increased DNA damage in the liver. Prevention of SRSF3 degradation in vivo protected hepatocytes from DNA double-strand breaks in mice with MASH. This protection extended to other DNA-damaging agents such as camptothecin, palmitic acid, or hydrogen peroxide when tested on HepG2 cells in vitro. SRSF3 interacted with TRIM28 and MDC1, which are components of the ATM DNA-damage repair complex, and knockdown of any of these 3 proteins reduced the expression of the other 2 proteins, suggesting they form a functional complex. Lastly, by preventing degradation of SRSF3, we were able to reduce tumors in a diethyl-nitrosamine–induced (DEN-induced) model of cirrhotic HCC. These findings suggest that maintenance of SRSF3 protein stability is crucial for preventing DNA damage and protecting liver from early metabolic liver disease and progression to HCC.
Authors
Panyisha Wu, Manasi Das, Yanting Wang, Yichun Ji, Yuli Wu, Deepak Kumar, Lily J. Jih, Nicholas J.G. Webster
Abstract
Neutrophils play a pivotal role in the progression of metabolic dysfunction–associated steatohepatitis (MASH) by mediating inflammatory responses. However, the heterogeneity of neutrophil subsets in MASH and their specific contributions to disease progression remain unclear. In this study, analysis of liver biopsies from 265 patients revealed a strong association between elevated neutrophil counts and MASH severity, particularly fibrosis. Five distinct neutrophil subsets were identified in human liver tissue, with PAD4+ neutrophils serving as key drivers in MASH progression. Mechanistically, PAD4+ neutrophils generate neutrophil extracellular traps (NETs) and activate hepatic stellate cells via the TAOK1-dependent MAPK signaling pathway. Inhibition of PAD4+ neutrophils in vivo attenuated the progression of liver fibrosis without exacerbating liver injury. Collectively, these findings elucidate the pivotal involvement of PAD4+ neutrophils in MASH progression and identify them as promising therapeutic targets for mitigating fibrosis and inflammation.
Authors
Jiajia Shen, Shanshan Huang, Yaohui Wang, Qingyuan Wang, Shibo Lin, Wei Guan, Yingyun Gong, Yiming Si, Ming Zhao, Hongwen Zhou, Hui Liang
Abstract
Next generation sequencing can identify previously uncharacterized gene expression patterns in disease. Beyond differentially expressed genes analysis, we investigated the ability of within-population diversity (α-diversity) of the transcriptome to reveal additional biological information in alcohol-associated liver disease (ALD), comparing Differential Shannon diversity (DSD) to transcriptome heterogeneity changes. RNA sequencing data from normal livers and patients with early ALD and severe AH were analyzed. α-diversity indices and Percent Shannon Diversity of a gene, which refers to this gene's contribution to total Shannon entropy, were calculated. Ingenuity pathway analysis identified canonical pathways determined by differentially expressed genes (DEG) and DSD approaches. ALD significantly decreased hepatic transcriptome α-diversity correlating with increased relative contribution of select genes. These changes were driven by lower abundance gene expression loss. DEG and DSD analyses showed overlapping genes and canonical pathways, but DSD also identified additional genes and pathways not highlighted by DEG, including fatty acid oxidation, extracellular matrix degradation, and cholesterol metabolism pathways that may represent additional therapeutic targets. Importantly, DSD more effectively identified differences between ASH and AH. Overall, α-diversity analysis revealed that ALD progressively reduces transcriptome heterogeneity, and that DSD provides complementary insights into disease mechanisms missed by standard approaches.
Authors
Sudrishti Chaudhary, Jia-Jun Liu, Silvia Liu, Marissa Di, Juliane I. Beier, Ramon Bataller, Josepmaria Argemi, Panayiotis V. Benos, Gavin E. Arteel
Abstract
Iron regulatory protein 1 (IRP1) is a post-transcriptional regulator of cellular iron metabolism. In mice, loss of IRP1 causes polycythemia through translational de-repression of hypoxia-inducible factor 2α (HIF2α) mRNA, which increases renal erythropoietin production. Here we show that Irp1-/- mice develop fasting hypoglycemia and are protected against high-fat diet–induced hyperglycemia and hepatic steatosis. Discovery-based proteomics of Irp1-/- livers revealed a mitochondrial dysfunction signature. Seahorse flux analysis in primary hepatocytes and differentiated skeletal muscle myotubes confirmed impaired respiratory capacity, with a shift from oxidative phosphorylation to glycolytic ATP production. This metabolic rewiring was associated with enhanced insulin sensitivity and increased glucose uptake in skeletal muscle. Under metabolic stress, IRP1 deficiency altered the redox balance of mitochondrial iron, resulting in inefficient energy production and accumulation of amino acids and metabolites in skeletal muscle, rendering them unavailable for hepatic gluconeogenesis. These findings identify IRP1 as a critical regulator of systemic energy homeostasis.
Authors
Wen Gu, Nicole Wilkinson, Carine Fillebeen, Darren Blackburn, Korin Sahinyan, Eric Bonneil, Tao Zhao, Zhi Luo, Vahab Soleimani, Vincent Richard, Christoph H. Borchers, Albert Koulman, Benjamin Jenkins, Bernhard Michalke, Hans Zischka, Judith Sailer, Vivek Venkataramani, Othon Iliopoulos, Gary Sweeney, Kostas Pantopoulos
Abstract
Recombinant hepatitis B surface antigen (rHBsAg) vaccine with various adjuvants fails to break T and B cell tolerance in hosts with chronic hepatitis B (CHB). This study aims to explore the mechanisms to break immune tolerance that allows the host to respond to rHBsAg, achieving a cure for CHB. We engineered an anti–PD-L1–IFN-α (aPD-L1–IFN-α) heterodimeric fusion protein to allow rHBsAg to rejuvenate T and B cell responses in hepatitis B virus–tolerant (HBV-tolerant) mice. S.c. coimmunization with aPD-L1–IFN-α and rHBsAg significantly enhanced antigen uptake and maturation of both macrophage and dendritic cell (DC) subsets in draining lymph nodes. Macrophages drove early B cell activation, while cDC1s primed CD8+ T cells, breaking tolerance and leading to both B cell and cytotoxic T lymphocyte (CTL) differentiation. This strategy elicited not only anti-HBsAg neutralizing antibodies but also HBsAg-specific CD8+ T cell responses, achieving a functional cure without systemic toxicity. The efficacy of the aPD-L1–IFN-α adjuvant depended on both PD-L1 cis-targeting and IFN-α receptor signaling in antigen-presenting cells. These findings establish aPD-L1–IFN-α as a translatable adjuvant to break the strong tolerance induced by CHB, providing a dual-pathway strategy to induce HBV-specific T and B cell responses.
Authors
Chao-Yang Meng, Yong Liang, Longxin Xu, Hongjia Li, Jingya Guo, Hairong Xu, Fan Wang, Yang-Xin Fu, Hua Peng
Abstract
Toll-like receptor 7 (TLR7) agonists are promising immunostimulatory agents for the treatment of chronic infections and cancer. However, their systemic toxicity remains a challenge. In this study, SA-5, a novel liver-targeted, orally available TLR7 agonist, was evaluated for pharmacokinetics, safety, and efficacy in young and aged macaques across 1–10 mg/kg repeated doses. Safety was evaluated through hematologic, biochemical, and flow cytometric profiling, while efficacy was assessed via IFN-α production, gene expression of interferon-stimulated genes, and plasmacytoid dendritic cell activation. A principal component analysis (PCA)-based composite scoring system was used to integrate multimodal parameters. SA-5 induced dose-dependent type I IFN with limited systemic inflammation, with 3 mg/kg showing optimal balance. SA-5 had comparable immunostimulatory activity to GS-9620 but with reduced adverse biomarker shifts. In aged macaques, efficacy was maintained with modestly increased safety responses. These findings support SA-5 as a safer next-generation TLR7 agonist effective across age groups, highlighting integrated biomarker profiling in preclinical immunomodulatory drug development.
Authors
Shokichi Takahama, Takahiro Tomiyama, Sachiyo Yoshio, Yuta Nagatsuka, Hirotomo Murakami, Takuto Nogimori, Mami Kochi, Shoko Ochiai, Hidenori Kimura, Akihisa Fukushima, Tatsuya Kanto, Takuya Yamamoto
Abstract
Authors
Carolina M. Larrain, Jack H. Victory, Priyanka P. Desai, Lindsay R. Friedman, Hannah Stepp, Rachel Ashe, Kirsten Remmert, Surajit Sinha, Emily C. Smith, Nicole Russell, Tracey Pu, Alyssa V. Eade, Justine F. Burke, Jason Ho, Michael B. Yaffe, David E. Kleiner, Keith Schmidt, William D. Figg, Jonathan M. Hernandez
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