Dissecting Acute Drug-Induced Hepatotoxicity and Therapeutic Responses of Steatotic Liver Disease Using Primary Mouse Liver and Blood Cells in a Liver-On-A-Chip Model.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is hallmarked by hepatic steatosis, cell injury, inflammation, and fibrosis. This study elaborates on a multicellular biochip-based liver sinusoid model to mimic MASLD pathomechanisms and investigate the therapeutic effects of drug candidates lanifibranor and resmetirom. Mouse liver primary hepatocytes, hepatic stellate cells, Kupffer cells, and endothelial cells are seeded in a dual-chamber biocompatible liver-on-a-chip (LoC). The LoC is then perfused with circulating immune cells (CICs). Acetaminophen (APAP) and free fatty acids (FFAs) treatment recapitulate acute drug-induced liver injury and MASLD, respectively. As a benchmark for the LoC, multiplex immunofluorescence on livers from APAP-injected and dietary MASLD-induced mice reveals characteristic changes on parenchymal and immune cell populations. APAP exposure induces cell death in the LoC, and increased inflammatory cytokine levels in the circulating perfusate. Under FFA stimulation, lipid accumulation, cellular damage, inflammatory secretome, and fibrogenesis are increased in the LoC, reflecting MASLD. Both injury conditions potentiate CIC migration from the perfusate to the LoC cellular layers. Lanifibranor prevents the onset of inflammation, while resmetirom decreases lipid accumulation in hepatocytes and increases the generation of FFA metabolites in the LoC. This study demonstrates the LoC potential for functional and molecular evaluation of liver disease drug candidates.

Multiplex Immunostaining to Spatially Resolve the Cellular Landscape in Human and Mouse Livers.

Histological techniques based on tissue colorations (e.g., hematoxylin-eosin, Sirius red) and immunostaining remain gold standard methodologies for diagnostic or phenotyping purposes in liver disease research and clinical hepatology. With the development of -omics technologies, greater information can be extracted from tissue sections. We describe a sequential immunostaining protocol consisting of repetitive cycles of immunostaining and chemically induced antibody stripping that can be readily applied to various formalin-fixed tissues (liver or other organs, mouse or human) and does not require specific equipment or commercial kits. Importantly, the combination of antibodies can be adapted according to specific clinical or scientific needs.

The contradictory roles of macrophages in non-alcoholic fatty liver disease and primary liver cancer-Challenges and opportunities.

Chronic liver diseases from varying etiologies generally lead to liver fibrosis and cirrhosis. Among them, non-alcoholic fatty liver disease (NAFLD) affects roughly one-quarter of the world population, thus representing a major and increasing public health burden. Chronic hepatocyte injury, inflammation (non-alcoholic steatohepatitis, NASH) and liver fibrosis are recognized soils for primary liver cancer, particularly hepatocellular carcinoma (HCC), being the third most common cause for cancer-related deaths worldwide. Despite recent advances in liver disease understanding, therapeutic options on pre-malignant and malignant stages remain limited. Thus, there is an urgent need to identify targetable liver disease-driving mechanisms for the development of novel therapeutics. Monocytes and macrophages comprise a central, yet versatile component of the inflammatory response, fueling chronic liver disease initiation and progression. Recent proteomic and transcriptomic studies performed at singular cell levels revealed a previously overlooked diversity of macrophage subpopulations and functions. Indeed, liver macrophages that encompass liver resident macrophages (also named Kupffer cells) and monocyte-derived macrophages, can acquire a variety of phenotypes depending on microenvironmental cues, and thus exert manifold and sometimes contradictory functions. Those functions range from modulating and exacerbating tissue inflammation to promoting and exaggerating tissue repair mechanisms (i.e., parenchymal regeneration, cancer cell proliferation, angiogenesis, fibrosis). Due to these central functions, liver macrophages represent an attractive target for the treatment of liver diseases. In this review, we discuss the multifaceted and contrary roles of macrophages in chronic liver diseases, with a particular focus on NAFLD/NASH and HCC. Moreover, we discuss potential therapeutic approaches targeting liver macrophages.

Interleukin-18 signaling promotes activation of hepatic stellate cells in mouse liver fibrosis.

BACKGROUND AND AIMS: Nucleotide-binding oligomerization domain-like receptor-family pyrin domain-containing 3 (NLRP3) inflammasome activation has been shown to result in liver fibrosis. Mechanisms and downstream signaling remain incompletely understood. Here, we studied the role of IL-18 in hepatic stellate cells (HSCs), and its impact on liver fibrosis. APPROACH AND RESULTS: We observed significantly increased serum levels of IL-18 (128.4 pg/ml vs. 74.9 pg/ml) and IL-18 binding protein (BP; 46.50 ng/ml vs. 15.35 ng/ml) in patients with liver cirrhosis compared with healthy controls. Single cell RNA sequencing data showed that an immunoregulatory subset of murine HSCs highly expresses Il18 and Il18r1 . Treatment of cultured primary murine HSC with recombinant mouse IL-18 accelerated their transdifferentiation into myofibroblasts. In vivo , IL-18 receptor-deficient mice had reduced liver fibrosis in a model of fibrosis induced by HSC-specific NLRP3 overactivation. Whole liver RNA sequencing analysis from a murine model of severe NASH-induced fibrosis by feeding a choline-deficient, L-amino acid-defined, high fat diet showed that genes related to IL-18 and its downstream signaling were significantly upregulated, and Il18-/- mice receiving this diet for 10 weeks showed protection from fibrotic changes with decreased number of alpha smooth muscle actin-positive cells and collagen deposition. HSC activation triggered by NLRP3 inflammasome activation was abrogated when IL-18 signaling was blocked by its naturally occurring antagonist IL-18BP. Accordingly, we observed that the severe inflammatory phenotype associated with myeloid cell-specific NLRP3 gain-of-function was rescued by IL-18BP. CONCLUSIONS: Our study highlights the role of IL-18 in the development of liver fibrosis by its direct effect on HSC activation identifying IL-18 as a target to treat liver fibrosis.

The arginine methyltransferase PRMT7 promotes extravasation of monocytes resulting in tissue injury in COPD.

Extravasation of monocytes into tissue and to the site of injury is a fundamental immunological process, which requires rapid responses via post translational modifications (PTM) of proteins. Protein arginine methyltransferase 7 (PRMT7) is an epigenetic factor that has the capacity to mono-methylate histones on arginine residues. Here we show that in chronic obstructive pulmonary disease (COPD) patients, PRMT7 expression is elevated in the lung tissue and localized to the macrophages. In mouse models of COPD, lung fibrosis and skin injury, reduced expression of PRMT7 associates with decreased recruitment of monocytes to the site of injury and hence less severe symptoms. Mechanistically, activation of NF-κB/RelA in monocytes induces PRMT7 transcription and consequential mono-methylation of histones at the regulatory elements of RAP1A, which leads to increased transcription of this gene that is responsible for adhesion and migration of monocytes. Persistent monocyte-derived macrophage accumulation leads to ALOX5 over-expression and accumulation of its metabolite LTB4, which triggers expression of ACSL4 a ferroptosis promoting gene in lung epithelial cells. Conclusively, inhibition of arginine mono-methylation might offer targeted intervention in monocyte-driven inflammatory conditions that lead to extensive tissue damage if left untreated.

Inhibition of LTßR signalling activates WNT-induced regeneration in lung.

Lymphotoxin ß-receptor (LTßR) signalling promotes lymphoid neogenesis and the development of tertiary lymphoid structures1,2, which are associated with severe chronic inflammatory diseases that span several organ systems3-6. How LTßR signalling drives chronic tissue damage particularly in the lung, the mechanism(s) that regulate this process, and whether LTßR blockade might be of therapeutic value have remained unclear. Here we demonstrate increased expression of LTßR ligands in adaptive and innate immune cells, enhanced non-canonical NF-κB signalling, and enriched LTßR target gene expression in lung epithelial cells from patients with smoking-associated chronic obstructive pulmonary disease (COPD) and from mice chronically exposed to cigarette smoke. Therapeutic inhibition of LTßR signalling in young and aged mice disrupted smoking-related inducible bronchus-associated lymphoid tissue, induced regeneration of lung tissue, and reverted airway fibrosis and systemic muscle wasting. Mechanistically, blockade of LTßR signalling dampened epithelial non-canonical activation of NF-κB, reduced TGFß signalling in airways, and induced regeneration by preventing epithelial cell death and activating WNT/ß-catenin signalling in alveolar epithelial progenitor cells. These findings suggest that inhibition of LTßR signalling represents a viable therapeutic option that combines prevention of tertiary lymphoid structures1 and inhibition of apoptosis with tissue-regenerative strategies.