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Hepcidin regulates systemic iron homeostasis. Suppression of hepcidin expression occurs physiologically in iron deficiency and increased erythropoiesis but is pathologic in thalassemia and hemochromatosis. Here we show that epigenetic events govern hepcidin expression. Erythropoiesis and iron deficiency suppress hepcidin via erythroferrone-dependent and -independent mechanisms, respectively, in vivo, but both involve reversible loss of H3K9ac and H3K4me3 at the hepcidin locus. In vitro, pan-histone deacetylase inhibition elevates hepcidin expression, and in vivo maintains H3K9ac at hepcidin-associated chromatin and abrogates hepcidin suppression by erythropoietin, iron deficiency, thalassemia, and hemochromatosis. Histone deacetylase 3 and its cofactor NCOR1 regulate hepcidin; histone deacetylase 3 binds chromatin at the hepcidin locus, and histone deacetylase 3 knockdown counteracts hepcidin suppression induced either by erythroferrone or by inhibiting bone morphogenetic protein signaling. In iron deficient mice, the histone deacetylase 3 inhibitor RGFP966 increases hepcidin, and RNA sequencing confirms hepcidin is one of the genes most differentially regulated by this drug in vivo. We conclude that suppression of hepcidin expression involves epigenetic regulation by histone deacetylase 3. Hepcidin controls systemic iron levels by inhibiting intestinal iron absorption and iron recycling. Here, Pasricha et al. demonstrate that the hepcidin-chromatin locus displays HDAC3-mediated reversible epigenetic modifications during both erythropoiesis and iron deficiency.

Two important signaling pathways in liver fibrosis are the PDGF- and TGFβ pathway and compounds inhibiting these pathways are currently developed as antifibrotic drugs. Testing antifibrotic drugs requires large numbers of animal experiments with high discomfort. Therefore, a method to study these drugs ex vivo was developed using precision-cut liver slices from fibrotic rat livers (fPCLS), representing an ex vivo model with a multicellular fibrotic environment. We characterized the fibrotic process in fPCLS from rat livers after 3 weeks of bile duct ligation (BDL) during incubation and tested compounds predominantly inhibiting the TGFβ pathway (perindopril, valproic acid, rosmarinic acid, tetrandrine and pirfenidone) and PDGF pathway (imatinib, sorafenib and sunitinib). Gene expression of heat shock protein 47 (Hsp47), α smooth muscle actin (αSma) and pro-collagen 1A1 (Pcol1A1) and protein expression of collagens were determined. During 48 hours of incubation, the fibrosis process continued in control fPCLS as judged by the increased gene expression of the three fibrosis markers, and the protein expression of collagen 1, mature fibrillar collagen and total collagen. Most PDGF-inhibitors and TGFβ-inhibitors significantly inhibited the increase in gene expression of Hsp47, αSma and Pcol1A1. Protein expression of collagen 1 was significantly reduced by all PDGF-inhibitors and TGFβ-inhibitors, while total collagen was decreased by rosmarinic acid and tetrandrine only. However, fibrillar collagen expression was not changed by any of the drugs. In conclusion, rat fPCLS can be used as a functional ex vivo model of established liver fibrosis to test antifibrotic compounds inhibiting the PDGF- and TGFβ signalling pathway.

The recent increase in new technologies to analyze host-pathogen interaction has fostered a race to develop new methodologies to assess these not only on the cellular level, but also on the tissue level. Due to mouse-other mammal differences, there is a desperate need to develop relevant tissue models that can more closely recapitulate the host tissue during disease and repair. Whereas organoids and organs-on-a-chip technologies have their benefits, they still cannot provide the cellular and structural complexity of the host tissue. Here, precision cut tissue slices (PCTS) may provide invaluable models for complex ex-vivo generated tissues to assess host-pathogen interaction as well as potential vaccine responses in a “whole organ” manner. In this mini review, we discuss the current literature regarding PCTS in veterinary species and advocate that PCTS represent remarkable tools to further close the gap between target identification, subsequent translation of results into clinical studies, and thus opening avenues for future precision medicine approaches.

Liver fibrosis is an exaggerated wound healing response defined by the excessive accumulation of extracellular matrix. This study investigated the antifibrotic potential of naringenin (NRG), asiatic acid (AA), and icariin (ICA) using murine and human precision‐cut liver slices (PCLS). These natural products have shown promise in animal models, but human data are lacking. In this study, PCLS prepared from male mouse liver tissue (mPCLS), healthy human liver tissue (hhPCLS), and cirrhotic human liver tissue (chPCLS) were cultured for 48 h with varying concentrations of the three compounds. Our findings indicate that NRG reduced collagen type 1 (COL1A1) expression in a concentration‐dependent manner in both mPCLS and chPCLS, decreased fibrosis‐related gene expression, and significantly lowered pro‐collagen type 1 (PCOL1A1) levels in the culture medium by 54 ± 21% (mPCLS) and 78 ± 35% (chPCLS). Furthermore, NRG effectively inhibited IL‐1β and TNF‐α in mPCLS and IL‐1β in chPCLS on both gene and protein levels. AA specifically reduced COL1A1 and PCOL1A1 in chPCLS, while ICA selectively downregulated Col1a1 and Acta2 gene expression in mPCLS. This study suggests NRG's potential as an effective antifibrotic agent, warranting further investigation into its mechanisms and therapeutic applications in liver fibrosis.

Renal fibrosis is a serious clinical problem forming the utmost cause of need for renal replacement therapy. No adequate preventive or curative therapy is available that can be clinically used to specifically target renal fibrosis. The search for new efficacious treatment strategies is therefore warranted. Although in vitro models using homogeneous cell populations have contributed to the understanding of the pathogenetic mechanisms involved in renal fibrosis, these models poorly mimic the complex in vivo milieu. Therefore, here we evaluated a precision-cut kidney slice (PCKS) model as a new, multicellular ex vivo model to study development of fibrosis and the prevention thereof using anti-fibrotic compounds. Precision-cut slices (200-300 µm thickness) were prepared from healthy C57BL/6 mouse kidneys using a Krumdieck tissue slicer. To induce changes mimicking the fibrotic process, slices were incubated with TGFβ1 (5 ng/ml) for 48 hours in the presence or absence of the anti-fibrotic cytokine IFNγ (1 µg/ml) or an IFNγ conjugate which is targeted to the PDGFRβ (PPB-PEG-IFNγ). Following culture, tissue viability (ATP-content) and expression of α-SMA, fibronectin, collagen I, and collagen III were determined using real-time PCR and immunohistochemistry. Slices remained viable up to 72 hours of incubation and no significant effects of TGFβ1 and IFNγ on viability were observed. TGFβ1 markedly increased α-SMA, fibronectin, and collagen I mRNA and protein expression levels. IFNγ and PPB-PEG-IFNγ significantly reduced TGFβ1-induced fibronectin, collagen I and collagen III mRNA expression which was confirmed by immunohistochemistry. The PKCS model is a novel tool to test the pathophysiology of fibrosis and to screen the efficacy of anti-fibrotic drugs ex vivo in a multicellular and pro-fibrotic milieu. Major advantage of the slice model is that it can be used not only for animal but also for (fibrotic) human kidney tissue.

Extracellular vesicles (EVs) are cell‐produced, membrane‐surrounded vesicles that harbour the biological features of donor cells. In the current study, we are the first to isolate and characterize EVs isolated from human precision‐cut liver slices (PCLS), obtained from both healthy and metabolic dysfunction‐associated steatohepatitis (MASH) cirrhotic livers. PCLS derived from patients can faithfully represent disease conditions in humans. EVs were isolated from human PCLS after incubating in normal medium or modified medium that mimics the pathophysiological environment of metabolic dysfunction associated liver disease (MASLD). MASH PCLS produced higher amounts of EVs compared to healthy PCLS (p < 0.001). Mass spectrometry revealed that around 300 proteins were significantly different in EVs derived from MASH PCLS versus healthy PCLS (FDR < 0.05), irrespective of the type of medium. Significantly changed EV proteins were largely involved in signalling receptor binding function and showed potential in promoting fibrosis. In the liver, these ligand‐associated receptors are highly expressed in hepatic stellate cells, and the MASH EVs functionally promoted the activation of hepatic stellate cells. Furthermore, the amounts of EpCAM and ITGA3 in EVs were positively associated with the progression of MASLD, which suggests the use of liver‐derived EVs as potential biomarkers for MASLD. Characterization of EVs derived from human PCLS may assist future studies in investigating the pathogenesis and identifying liver‐specific EVs as biomarkers of MASLD.

Fasting hypoglycemia is a severe and incompletely understood symptom of various inborn errors of metabolism (IEM). Precision-cut liver slices (PCLS) represent a promising model for studying glucose production ex vivo. This study quantified the net glucose production of human and murine PCLS in the presence of different gluconeogenic precursors. Dihydroxyacetone-supplemented slices from the fed mice yielded the highest rate, further stimulated by forskolin and dibutyryl-cAMP. Moreover, using 13 C isotope tracing, we assessed the contribution of glycogenolysis and gluconeogenesis to net glucose production over time. Pharmacological inhibition of the glucose 6-phosphate transporter SLC37A4 markedly reduced net glucose production and increased lactate secretion and glycogen storage, while glucose production was completely abolished in PCLS from glycogen storage disease type Ia and Ib patients. In conclusion, this study identifies PCLS as an effective ex vivo model to study hepatic glucose production and opens opportunities for its future application in IEM research and beyond. Ex vivo precision-cut liver slices allow glucose production quantification to model glycogen storage disease and inborn errors of metabolism.

Non‐alcoholic steatohepatitis (NASH) and alcohol‐associated steatohepatitis (ASH) are the major cause of liver‐related mortality with limited therapeutic options available. In this study, we investigated the role of Src tyrosine kinase in the pathogenesis of (N)ASH. We examined Src kinase expression in livers from patients with NASH, ASH, cirrhosis and biliary atresia, and from preclinical mouse models: methionine choline deficient (MCD)‐diet‐induced NASH, Lieber–DeCarli ethanol (EtOH)‐diet‐induced ASH, carbon tetrachloride (CCl4)‐ and bile duct ligation (BDL)‐induced liver fibrosis. Functional studies, using Src kinase inhibitor KX2‐391, were performed in NASH and ASH mouse models, and in relevant in vitro models. Transcriptomic analysis revealed increased Src kinase expression in human and mouse diseased livers that positively correlated with disease progression. Src kinase inhibition ameliorated lipid biosynthesis (steatosis); monocytes/macrophages infiltration and inflammatory cytokines expression (inflammation) and hepatic stellate cells (HSCs) activation and collagen expression (fibrosis) in MCD‐diet and EtOH‐diet induced liver disease mouse models. In vitro Src inhibition attenuated FFA‐induced hepatocytic lipid accumulation, LPS/IFNγ‐induced nitric oxide release and expression of several inflammatory signatures in macrophages and TGFβ‐induced HSCs activation, contractility and collagen expression. Moreover, Src inhibition diminished gene expression of inflammatory markers in LPS/IFNγ‐stimulated BMDMs and LPS‐stimulated PCLS, and reduced FA accumulation, macrophage activation and collagen deposition in 3D human liver spheroids. Mechanistic studies further revealed that Src kinase mediated the effects through FAK/PI3K/AKT pathway. Our results demonstrate a multicellular and functional role of Src kinase highlighting Src kinase as a promising therapeutic target in (non)alcoholic steatohepatitis. Non‐alcoholic steatohepatitis (NASH) and alcohol‐associated steatohepatitis (ASH) are the major cause of liver‐related mortality with limited therapeutic options available. In this study, we investigated the role of Src tyrosine kinase in the pathogenesis of (N)ASH. Our results demonstrate a multicellular and functional role of Src kinase highlighting Src kinase as a promising therapeutic target in (non)alcoholic steatohepatitis.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a growing healthcare problem with limited therapeutic options. Progress in this field depends on the availability of reliable preclinical models. Human precision-cut liver slices (PCLSs) have been employed to replicate the initiation of MASLD, but a comprehensive investigation into MASLD progression is still missing. This study aimed to extend the current incubation time of human PCLSs to examine different stages in MASLD. Healthy human PCLSs were cultured for up to 96 h in a medium enriched with high sugar, high insulin, and high fatty acids to induce MASLD. PCLSs displayed hepatic steatosis, characterized by accumulated intracellular fat. The development of hepatic steatosis appeared to involve a time-dependent impact on lipid metabolism, with an initial increase in fatty acid uptake and storage, and a subsequent down-regulation of lipid oxidation and secretion. PCLSs also demonstrated liver inflammation, including increased pro-inflammatory gene expression and cytokine production. Additionally, liver fibrosis was also observed through the elevated production of pro-collagen 1a1 and tissue inhibitor of metalloproteinase-1 (TIMP1). RNA sequencing showed that the tumor necrosis factor alpha (TNFα) signaling pathway and transforming growth factor beta (TGFβ) signaling pathway were consistently activated, potentially contributing to the development of inflammation and fibrosis. In conclusion, the prolonged incubation of human PCLSs can establish a robust ex vivo model for MASLD, facilitating the identification and evaluation of potential therapeutic interventions.

Non-alcoholic fatty liver disease (NAFLD) is a common liver disorder closely related to metabolic syndrome. NAFLD can progress to an inflammatory state called non-alcoholic steatohepatitis (NASH), which may result in the development of fibrosis and hepatocellular carcinoma. To develop therapeutic strategies against NAFLD, a better understanding of the molecular mechanism is needed. Current in vitro NAFLD models fail to capture the essential interactions between liver cell types and often do not reflect the pathophysiological status of patients. To overcome limitations of commonly used in vitro and in vivo models, precision-cut liver slices (PCLSs) were used in this study. PCLSs, prepared from liver tissue obtained from male Wistar rats, were cultured in supraphysiological concentrations of glucose, fructose, insulin, and palmitic acid to mimic metabolic syndrome. Accumulation of lipid droplets was visible and measurable after 24 h in PCLSs incubated with glucose, fructose, and insulin, both in the presence and absence of palmitic acid. Upregulation of acetyl-CoA carboxylase 1 and 2, and of sterol responsive element binding protein 1c, suggests increased de novo lipogenesis in PCLSs cultured under these conditions. Additionally, carnitine palmitoyltransferase 1 expression was reduced, which indicates impaired fatty acid transport and disrupted mitochondrial β-oxidation. Thus, steatosis was successfully induced in PCLSs with modified culture medium. This novel ex vivo NAFLD model could be used to investigate the multicellular and molecular mechanisms that drive NAFLD development and progression, and to study potential anti-steatotic drugs.