Background and Aims In nonalcoholic fatty liver disease (NAFLD), fibrosis is the most important factor contributing to NAFLD‐associated morbidity and mortality. Prevention of progression and reduction in fibrosis are the main aims of treatment. Even in early stages of NAFLD, hepatic and systemic hyperammonemia is evident. This is due to reduced urea synthesis; and as ammonia is known to activate hepatic stellate cells, we hypothesized that ammonia may be involved in the progression of fibrosis in NAFLD. Approach and Results In a high‐fat, high‐cholesterol diet–induced rodent model of NAFLD, we observed a progressive stepwise reduction in the expression and activity of urea cycle enzymes resulting in hyperammonemia, evidence of hepatic stellate cell activation, and progressive fibrosis. In primary, cultured hepatocytes and precision‐cut liver slices we demonstrated increased gene expression of profibrogenic markers after lipid and/or ammonia exposure. Lowering of ammonia with the ammonia scavenger ornithine phenylacetate prevented hepatocyte cell death and significantly reduced the development of fibrosis both in vitro in the liver slices and in vivo in a rodent model. The prevention of fibrosis in the rodent model was associated with restoration of urea cycle enzyme activity and function, reduced hepatic ammonia, and markers of inflammation. Conclusions The results of this study suggest that hepatic steatosis results in hyperammonemia, which is associated with progression of hepatic fibrosis. Reduction of ammonia levels prevented progression of fibrosis, providing a potential treatment for NAFLD.

Precision cut liver slices (PCLSs) retain the structure and cellular composition of the native liver and represent an improved system to study liver fibrosis compared to two‐dimensional mono‐ or co‐cultures. The aim of this study was to develop a bioreactor system to increase the healthy life span of PCLSs and model fibrogenesis. PCLSs were generated from normal rat or human liver, or fibrotic rat liver, and cultured in our bioreactor. PCLS function was quantified by albumin enzyme‐linked immunosorbent assay (ELISA). Fibrosis was induced in PCLSs by transforming growth factor beta 1 (TGFβ1) and platelet‐derived growth factor (PDGFββ) stimulation ± therapy. Fibrosis was assessed by gene expression, picrosirius red, and α‐smooth muscle actin staining, hydroxyproline assay, and soluble ELISAs. Bioreactor‐cultured PCLSs are viable, maintaining tissue structure, metabolic activity, and stable albumin secretion for up to 6 days under normoxic culture conditions. Conversely, standard static transwell‐cultured PCLSs rapidly deteriorate, and albumin secretion is significantly impaired by 48 hours. TGFβ1/PDGFββ stimulation of rat or human PCLSs induced fibrogenic gene expression, release of extracellular matrix proteins, activation of hepatic myofibroblasts, and histological fibrosis. Fibrogenesis slowly progresses over 6 days in cultured fibrotic rat PCLSs without exogenous challenge. Activin receptor‐like kinase 5 (Alk5) inhibitor (Alk5i), nintedanib, and obeticholic acid therapy limited fibrogenesis in TGFβ1/PDGFββ‐stimulated PCLSs, and Alk5i blunted progression of fibrosis in fibrotic PCLS. Conclusion: We describe a bioreactor technology that maintains functional PCLS cultures for 6 days. Bioreactor‐cultured PCLSs can be successfully used to model fibrogenesis and demonstrate efficacy of antifibrotic therapies.

ObjectiveHepatic stellate cells (HSC) transdifferentiation into myofibroblasts is central to fibrogenesis. Epigenetic mechanisms, including histone and DNA methylation, play a key role in this process. Concerted action between histone and DNA-mehyltransferases like G9a and DNMT1 is a common theme in gene expression regulation. We aimed to study the efficacy of CM272, a first-in-class dual and reversible G9a/DNMT1 inhibitor, in halting fibrogenesis.DesignG9a and DNMT1 were analysed in cirrhotic human livers, mouse models of liver fibrosis and cultured mouse HSC. G9a and DNMT1 expression was knocked down or inhibited with CM272 in human HSC (hHSC), and transcriptomic responses to transforming growth factor-β1 (TGFβ1) were examined. Glycolytic metabolism and mitochondrial function were analysed with Seahorse-XF technology. Gene expression regulation was analysed by chromatin immunoprecipitation and methylation-specific PCR. Antifibrogenic activity and safety of CM272 were studied in mouse chronic CCl4 administration and bile duct ligation (BDL), and in human precision-cut liver slices (PCLSs) in a new bioreactor technology.ResultsG9a and DNMT1 were detected in stromal cells in areas of active fibrosis in human and mouse livers. G9a and DNMT1 expression was induced during mouse HSC activation, and TGFβ1 triggered their chromatin recruitment in hHSC. G9a/DNMT1 knockdown and CM272 inhibited TGFβ1 fibrogenic responses in hHSC. TGFβ1-mediated profibrogenic metabolic reprogramming was abrogated by CM272, which restored gluconeogenic gene expression and mitochondrial function through on-target epigenetic effects. CM272 inhibited fibrogenesis in mice and PCLSs without toxicity.ConclusionsDual G9a/DNMT1 inhibition by compounds like CM272 may be a novel therapeutic strategy for treating liver fibrosis.

Fibroblasts persist within fibrotic scar tissue and exhibit considerable phenotypic and functional plasticity. Herein, we hypothesized that scar-associated fibroblasts may be a source of stress-induced inflammatory exacerbations and pain. To test this idea, we used a human model of surgery-induced fibrosis, total knee arthroplasty (TKA). Using a combination of tissue protein expression profiling and bioinformatics, we discovered that many months after TKA, the fibrotic joint exists in a state of unresolved chronic inflammation. Moreover, the infrapatellar fat pad, a soft tissue that becomes highly fibrotic in the post-TKA joint, expresses multiple inflammatory mediators, including the monocyte chemoattractant, chemokine (C-C motif) ligand (CCL) 2, and the innate immune trigger, IL-1α. Fibroblasts isolated from the post-TKA fibrotic infrapatellar fat pad express the IL-1 receptor and on exposure to IL-1α polarize to a highly inflammatory state that enables them to stimulate the recruitment of monocytes. Blockade of fibroblast CCL2 or its transcriptional regulator NF-κB prevented IL-1α–induced monocyte recruitment. Clinical investigations discovered that levels of patient-reported pain in the post-TKA joint correlated with concentrations of CCL2 in the joint tissue, such that the chemokine is effectively a pain biomarker in the TKA patient. We propose that an IL-1α–NF-κB–CCL2 signaling pathway, operating within scar-associated fibroblasts, may be therapeutically manipulated for alleviating inflammation and pain in fibrotic joints and other tissues.

This is a repository copy of c-Rel orchestrates energy-dependent epithelial and macrophage reprogramming in fibrosis.

Age-related chronic inflammation promotes cellular senescence, chronic disease, cancer, and reduced lifespan. In this study, we wanted to explore the effects of a moderate exercise regimen on inflammatory liver disease and tumorigenesis. We used an established model of spontaneous inflammaging, steatosis, and cancer (nfkb1 2/2 mouse) to demonstrate whether 3 mo of moderate aerobic exercise was sufficient to suppress liver disease and cancer development. Interventional exercise when applied at a relatively late disease stage was effective at reducing tissue inflammation (liver, lung, and stomach), oxidative damage, and cellular senescence, and it reversed hepatic steatosis and prevented tumor development. Underlying these benefits were transcriptional changes in enzymes driving the conversion of tryptophan to NAD + , this leading to increased hepatic NAD + and elevated activity of the NAD +-dependent deacetylase sirtuin. Increased SIRT activity was correlated with enhanced deacetylation of key transcriptional regulators of inflammation and metabolism, NF-kB (p65), and PGC-1a. We propose that moderate exercise can effectively reprogram pre-established inflammatory and metabolic pathologies in aging with the benefit of prevention of disease.

Reporting of In Vivo Experiments AST Aspartate aminotransferase αSMA α-smooth muscle actin ATG16L Autophagy Related 16L ATG7 Autophagy Related 7 AAV Adeno-associated virus BODIPY 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene BNIP BCL2 interacting protein 3 BNIP3L/NIX BCL2 interacting protein 3 like BBVA Banco Bilbao Vizcaya Argentaria°C Celsius Degrees CCPG1 Cell-Cycle Progression Gene 1 CMA Chaperon Mediated Autophagy Co-IP Co-Immuno Precipitation Col1a1 Type IA1 collagen CPT1 Carnitine PalmitoylTransferase I Ctrl Control Financial support: This work was supported by C0120R3166, C0245R4032 and BH182173 from Newcastle University. MG-M is a Sara Borrell Postdoctoral fellow (CD18/00203

Objectives NF-κB regulates genes that control inflammation, cell proliferation, differentiation and survival. Dysregulated NF-κB signalling alters normal skin physiology and deletion of cRel limits bleomycin-induced skin fibrosis. This study investigates the role of cRel in modulating fibroblast phenotype in the context of SSc. Methods Fibrosis was assessed histologically in mice challenged with bleomycin to induce lung or skin fibrosis. RNA sequencing and pathway analysis was performed on wild type and Rel−/− murine lung and dermal fibroblasts. Functional assays examined fibroblast proliferation, migration and matrix production. cRel overexpression was investigated in human dermal fibroblasts. cRel immunostaining was performed on lung and skin tissue sections from SSc patients and non-fibrotic controls. Results cRel expression was elevated in murine lung and skin fibrosis models. Rel−/− mice were protected from developing pulmonary fibrosis. Soluble collagen production was significantly decreased in fibroblasts lacking cRel while proliferation and migration of these cells was significantly increased. cRel regulates genes involved in extracellular structure and matrix organization. Positive cRel staining was observed in fibroblasts in human SSc skin and lung tissue. Overexpression of constitutively active cRel in human dermal fibroblasts increased expression of matrix genes. An NF-κB gene signature was identified in diffuse SSc skin and nuclear cRel expression was elevated in SSc skin fibroblasts. Conclusion cRel regulates a pro-fibrogenic transcriptional programme in fibroblasts that may contribute to disease pathology. Targeting cRel signalling in fibroblasts of SSc patients could provide a novel therapeutic avenue to limit scar formation in this disease.