Ribosomal S6 kinase 1 regulates inflammaging via the senescence secretome.

Inhibition of S6 kinase 1 (S6K1) extends lifespan and improves healthspan in mice, but the underlying mechanisms are unclear. Cellular senescence is a stable growth arrest accompanied by an inflammatory senescence-associated secretory phenotype (SASP). Cellular senescence and SASP-mediated chronic inflammation contribute to age-related pathology, but the specific role of S6K1 has not been determined. Here we show that S6K1 deletion does not reduce senescence but ameliorates inflammation in aged mouse livers. Using human and mouse models of senescence, we demonstrate that reduced inflammation is a liver-intrinsic effect associated with S6K deletion. Specifically, we show that S6K1 deletion results in reduced IRF3 activation; impaired production of cytokines, such as IL1ß; and reduced immune infiltration. Using either liver-specific or myeloid-specific S6K knockout mice, we also demonstrate that reduced immune infiltration and clearance of senescent cells is a hepatocyte-intrinsic phenomenon. Overall, deletion of S6K reduces inflammation in the liver, suggesting that suppression of the inflammatory SASP by loss of S6K could underlie the beneficial effects of inhibiting this pathway on healthspan and lifespan.

Detection of senescence using machine learning algorithms based on nuclear features.

Cellular senescence is a stress response with broad pathophysiological implications. Senotherapies can induce senescence to treat cancer or eliminate senescent cells to ameliorate ageing and age-related pathologies. However, the success of senotherapies is limited by the lack of reliable ways to identify senescence. Here, we use nuclear morphology features of senescent cells to devise machine-learning classifiers that accurately predict senescence induced by diverse stressors in different cell types and tissues. As a proof-of-principle, we use these senescence classifiers to characterise senolytics and to screen for drugs that selectively induce senescence in cancer cells but not normal cells. Moreover, a tissue senescence score served to assess the efficacy of senolytic drugs and identified senescence in mouse models of liver cancer initiation, ageing, and fibrosis, and in patients with fatty liver disease. Thus, senescence classifiers can help to detect pathophysiological senescence and to discover and validate potential senotherapies.

COPI vesicle formation and N-myristoylation are targetable vulnerabilities of senescent cells.

Drugs that selectively kill senescent cells (senolytics) improve the outcomes of cancer, fibrosis and age-related diseases. Despite their potential, our knowledge of the molecular pathways that affect the survival of senescent cells is limited. To discover senolytic targets, we performed RNAi screens and identified coatomer complex I (COPI) vesicle formation as a liability of senescent cells. Genetic or pharmacological inhibition of COPI results in Golgi dispersal, dysfunctional autophagy, and unfolded protein response-dependent apoptosis of senescent cells, and knockdown of COPI subunits improves the outcomes of cancer and fibrosis in mouse models. Drugs targeting COPI have poor pharmacological properties, but we find that N-myristoyltransferase inhibitors (NMTi) phenocopy COPI inhibition and are potent senolytics. NMTi selectively eliminated senescent cells and improved outcomes in models of cancer and non-alcoholic steatohepatitis. Our results suggest that senescent cells rely on a hyperactive secretory apparatus and that inhibiting trafficking kills senescent cells with the potential to treat various senescence-associated diseases.

Pre-RNA splicing in metabolic homeostasis and liver disease.

The liver plays a key role in sensing nutritional and hormonal inputs to maintain metabolic homeostasis. Recent studies into pre-mRNA splicing and alternative splicing (AS) and their effects on gene expression have revealed considerable transcriptional complexity in the liver, both in health and disease. While the contribution of these mechanisms to cell and tissue identity is widely accepted, their role in physiological and pathological contexts within tissues is just beginning to be appreciated. In this review, we showcase recent studies on the splicing and AS of key genes in metabolic pathways in the liver, the effect of metabolic signals on the spliceosome, and therapeutic intervention points based on RNA splicing.

Endothelial sensing of AHR ligands regulates intestinal homeostasis.

Endothelial cells line the blood and lymphatic vasculature, and act as an essential physical barrier, control nutrient transport, facilitate tissue immunosurveillance and coordinate angiogenesis and lymphangiogenesis1,2. In the intestine, dietary and microbial cues are particularly important in the regulation of organ homeostasis. However, whether enteric endothelial cells actively sense and integrate such signals is currently unknown. Here we show that the aryl hydrocarbon receptor (AHR) acts as a critical node for endothelial cell sensing of dietary metabolites in adult mice and human primary endothelial cells. We first established a comprehensive single-cell endothelial atlas of the mouse small intestine, uncovering the cellular complexity and functional heterogeneity of blood and lymphatic endothelial cells. Analyses of AHR-mediated responses at single-cell resolution identified tissue-protective transcriptional signatures and regulatory networks promoting cellular quiescence and vascular normalcy at steady state. Endothelial AHR deficiency in adult mice resulted in dysregulated inflammatory responses and the initiation of proliferative pathways. Furthermore, endothelial sensing of dietary AHR ligands was required for optimal protection against enteric infection. In human endothelial cells, AHR signalling promoted quiescence and restrained activation by inflammatory mediators. Together, our data provide a comprehensive dissection of the effect of environmental sensing across the spectrum of enteric endothelia, demonstrating that endothelial AHR signalling integrates dietary cues to maintain tissue homeostasis by promoting endothelial cell quiescence and vascular normalcy.

Nanodrug delivery systems for metabolic chronic liver diseases: advances and perspectives.

Nanomedicines are revolutionizing healthcare as recently demonstrated by the Pfizer/BioNTech and Moderna COVID-2019 vaccines, with billions of doses administered worldwide in a safe manner. Nonalcoholic fatty liver disease is the most common noncommunicable chronic liver disease, posing a major growing challenge to global public health. However, due to unmet diagnostic and therapeutic needs, there is great interest in the development of novel translational approaches. Nanoparticle-based approaches offer novel opportunities for efficient and specific drug delivery to liver cells, as a step toward precision medicines. In this review, the authors highlight recent advances in nanomedicines for the generation of novel diagnostic and therapeutic tools for nonalcoholic fatty liver disease and related liver diseases.

Chronic liver diseases are a growing concern for global public health since they can affect up to 25% of the global adult population. Currently, there is no effective treatment or cure for these diseases. Nanometer-sized capsules can be loaded with drugs and more accurately deliver these drugs to their sites of action. They help improve the availability of medicines to the liver and have the potential to reduce their side effects. Here, the authors discuss recent advances to explain how nanotechnology can help improve the benefits of existing medicines for liver disease therapy.

RGD density along with substrate stiffness regulate hPSC hepatocyte functionality through YAP signalling.

Human pluripotent stem cell-derived hepatocytes (hPSC-Heps) may be suitable for treating liver diseases, but differentiation protocols often fail to yield adult-like cells. We hypothesised that replicating healthy liver niche biochemical and biophysical cues would produce hepatocytes with desired metabolic functionality. Using 2D synthetic hydrogels which independently control mechanical properties and biochemical cues, we found that culturing hPSC-Heps on surfaces matching the stiffness of fibrotic liver tissue upregulated expression of genes for RGD-binding integrins, and increased expression of YAP/TAZ and their transcriptional targets. Alternatively, culture on soft, healthy liver-like substrates drove increases in cytochrome p450 activity and ureagenesis. Knockdown of ITGB1 or reducing RGD-motif-containing peptide concentration in stiff hydrogels reduced YAP activity and improved metabolic functionality; however, on soft substrates, reducing RGD concentration had the opposite effect. Furthermore, targeting YAP activity with verteporfin or forskolin increased cytochrome p450 activity, with forskolin dramatically enhancing urea synthesis. hPSC-Heps could also be successfully encapsulated within RGD peptide-containing hydrogels without negatively impacting hepatic functionality, and compared to 2D cultures, 3D cultured hPSC-Heps secreted significantly less fetal liver-associated alpha-fetoprotein, suggesting furthered differentiation. Our platform overcomes technical hurdles in replicating the liver niche, and allowed us to identify a role for YAP/TAZ-mediated mechanosensing in hPSC-Hep differentiation.

Liver RBFOX2 regulates cholesterol homeostasis via Scarb1 alternative splicing in mice.

RNA alternative splicing (AS) expands the regulatory potential of eukaryotic genomes. The mechanisms regulating liver-specific AS profiles and their contribution to liver function are poorly understood. Here, we identify a key role for the splicing factor RNA-binding Fox protein 2 (RBFOX2) in maintaining cholesterol homeostasis in a lipogenic environment in the liver. Using enhanced individual-nucleotide-resolution ultra-violet cross-linking and immunoprecipitation, we identify physiologically relevant targets of RBFOX2 in mouse liver, including the scavenger receptor class B type I (Scarb1). RBFOX2 function is decreased in the liver in diet-induced obesity, causing a Scarb1 isoform switch and alteration of hepatocyte lipid homeostasis. Our findings demonstrate that specific AS programmes actively maintain liver physiology, and underlie the lipotoxic effects of obesogenic diets when dysregulated. Splice-switching oligonucleotides targeting this network alleviate obesity-induced inflammation in the liver and promote an anti-atherogenic lipoprotein profile in the blood, underscoring the potential of isoform-specific RNA therapeutics for treating metabolism-associated diseases.

Phosphorylation of RXRα mediates the effect of JNK to suppress hepatic FGF21 expression and promote metabolic syndrome.

The cJun NH2-terminal kinase (JNK) signaling pathway in the liver promotes systemic changes in metabolism by regulating peroxisome proliferator-activated receptor α (PPARα)-dependent expression of the hepatokine fibroblast growth factor 21 (FGF21). Hepatocyte-specific gene ablation studies demonstrated that the Mapk9 gene (encoding JNK2) plays a key mechanistic role. Mutually exclusive inclusion of exons 7a and 7b yields expression of the isoforms JNK2α and JNK2ß. Here we demonstrate that Fgf21 gene expression and metabolic regulation are primarily regulated by the JNK2α isoform. To identify relevant substrates of JNK2α, we performed a quantitative phosphoproteomic study of livers isolated from control mice, mice with JNK deficiency in hepatocytes, and mice that express only JNK2α or JNK2ß in hepatocytes. We identified the JNK substrate retinoid X receptor α (RXRα) as a protein that exhibited JNK2α-promoted phosphorylation in vivo. RXRα functions as a heterodimeric partner of PPARα and may therefore mediate the effects of JNK2α signaling on Fgf21 expression. To test this hypothesis, we established mice with hepatocyte-specific expression of wild-type or mutated RXRα proteins. We found that the RXRα phosphorylation site Ser260 was required for suppression of Fgf21 gene expression. Collectively, these data establish a JNK-mediated signaling pathway that regulates hepatic Fgf21 expression.