Targeting of the peptide hormone relaxin to injured mouse liver, via a nanoparticle/gene therapy approach, switches pro-fibrotic hepatic macrophages to a restorative phenotype that orchestrates ...tissue repair.
Liver disease is a major global health-care problem, affecting an estimated 844 million people worldwide. Despite this substantial burden, therapeutic options for liver disease remain limited, in ...part owing to a paucity of detailed analyses defining the cellular and molecular mechanisms that drive these conditions in humans. Single-cell transcriptomic technologies are transforming our understanding of cellular diversity and function in health and disease. In this Review, we discuss how these technologies have been applied in hepatology, advancing our understanding of cellular heterogeneity and providing novel insights into fundamental liver biology such as the metabolic zonation of hepatocytes, endothelial cells and hepatic stellate cells, and the cellular mechanisms underpinning liver regeneration. Application of these methodologies is also uncovering critical pathophysiological changes driving disease states such as hepatic fibrosis, where distinct populations of macrophages, endothelial cells and mesenchymal cells reside within a spatially distinct fibrotic niche and interact to promote scar formation. In addition, single-cell approaches are starting to dissect key cellular and molecular functions in liver cancer. In the near future, new techniques such as spatial transcriptomics and multiomic approaches will further deepen our understanding of disease pathogenesis, enabling the identification of novel therapeutic targets for patients across the spectrum of liver diseases.
Little is known about the roles of myeloid cell subsets in kidney injury and in the limited ability of the organ to repair itself. Characterizing these cells based only on surface markers using flow ...cytometry might not provide a full phenotypic picture. Defining these cells at the single-cell, transcriptomic level could reveal myeloid heterogeneity in the progression and regression of kidney disease.
Integrated droplet- and plate-based single-cell RNA sequencing were used in the murine, reversible, unilateral ureteric obstruction model to dissect the transcriptomic landscape at the single-cell level during renal injury and the resolution of fibrosis. Paired blood exchange tracked the fate of monocytes recruited to the injured kidney.
A single-cell atlas of the kidney generated using transcriptomics revealed marked changes in the proportion and gene expression of renal cell types during injury and repair. Conventional flow cytometry markers would not have identified the 12 myeloid cell subsets. Monocytes recruited to the kidney early after injury rapidly adopt a proinflammatory, profibrotic phenotype that expresses
, before transitioning to become
macrophages that accumulate in late injury. Conversely, a novel
macrophage subset acts during repair.
Complementary technologies identified novel myeloid subtypes, based on transcriptomics in single cells, that represent therapeutic targets to inhibit progression or promote regression of kidney disease.
Complement receptor of immunoglobulin superfamily (CRIg) is expressed on liver macrophages and directly binds complement component C3b or Gram-positive bacteria to mediate phagocytosis. CRIg plays ...important roles in several immune-mediated diseases, but it is not clear how its pathogen recognition and phagocytic functions maintain homeostasis and prevent disease. We previously associated cytolysin-positive Enterococcus faecalis with severity of alcohol-related liver disease. Here, we demonstrate that CRIg is reduced in liver tissues from patients with alcohol-related liver disease. CRIg-deficient mice developed more severe ethanol-induced liver disease than wild-type mice; disease severity was reduced with loss of toll-like receptor 2. CRIg-deficient mice were less efficient than wild-type mice at clearing Gram-positive bacteria such as Enterococcus faecalis that had translocated from gut to liver. Administration of the soluble extracellular domain CRIg-Ig protein protected mice from ethanol-induced steatohepatitis. Our findings indicate that ethanol impairs hepatic clearance of translocated pathobionts, via decreased hepatic CRIg, which facilitates progression of liver disease.
With evidence from a large number of animal models and clinical trials, it is now beyond debate that liver fibrosis and even cirrhosis are potentially reversible if the underlying cause can be ...successfully eliminated. However, in a significant proportion of patients cure of the underlying disease may not result in fibrosis regression or a significant reduction of the risk for hepatocellular carcinoma development. Understanding of the mechanistic pathways and regulatory factors that characterize matrix remodeling and architectural repair during fibrosis regression may provide therapeutic approaches to induce or accelerate regression as well as novel diagnostic tools. Recent seminal observations have determined that in resolving liver fibrosis a significant proportion of hepatic stellate cell-myofibroblasts (HSC-MFs) can revert to a near quiescent phenotype. Hepatic macrophages derived from inflammatory monocytes may contribute to fibrosis resolution through an in situ phenotypic switch mediated by phagocytosis. Emerging therapeutic approaches include deletion or inactivation of HSC-MFs, modulation of macrophage activity and autologous cell infusion therapies. Novel noninvasive diagnostic tests such as serum and imaging markers responsive to extracellular matrix degradation are being developed to evaluate the clinical efficacy of antifibrotic interventions.
During chronic injury a population of bipotent hepatic progenitor cells (HPCs) become activated to regenerate both cholangiocytes and hepatocytes. Here we show in human diseased liver and mouse ...models of the ductular reaction that Notch and Wnt signaling direct specification of HPCs via their interactions with activated myofibroblasts or macrophages. In particular, we found that during biliary regeneration, expression of Jagged 1 (a Notch ligand) by myofibroblasts promoted Notch signaling in HPCs and thus their biliary specification to cholangiocytes. Alternatively, during hepatocyte regeneration, macrophage engulfment of hepatocyte debris induced Wnt3a expression. This resulted in canonical Wnt signaling in nearby HPCs, thus maintaining expression of Numb (a cell fate determinant) within these cells and the promotion of their specification to hepatocytes. By these two pathways adult parenchymal regeneration during chronic liver injury is promoted.
Although macrophages are widely recognized to have a profibrotic role in inflammation, we have used a highly tractable CCl 4 -induced model of reversible hepatic fibrosis to identify and characterize ...the macrophage phenotype responsible for tissue remodeling: the hitherto elusive restorative macrophage. This CD11B hi F4/80 int Ly-6C lo macrophage subset was most abundant in livers during maximal fibrosis resolution and represented the principle matrix metalloproteinase (MMP) -expressing subset. Depletion of this population in CD11B promoter–diphtheria toxin receptor (CD11B-DTR) transgenic mice caused a failure of scar remodeling. Adoptive transfer and in situ labeling experiments showed that these restorative macrophages derive from recruited Ly-6C hi monocytes, a common origin with profibrotic Ly-6C hi macrophages, indicative of a phenotypic switch in vivo conferring proresolution properties. Microarray profiling of the Ly-6C lo subset, compared with Ly-6C hi macrophages, showed a phenotype outside the M1/M2 classification, with increased expression of MMPs, growth factors, and phagocytosis-related genes, including Mmp9, Mmp12, insulin-like growth factor 1 (Igf1), and Glycoprotein (transmembrane) nmb (Gpnmb). Confocal microscopy confirmed the postphagocytic nature of restorative macrophages. Furthermore, the restorative macrophage phenotype was recapitulated in vitro by the phagocytosis of cellular debris with associated activation of the ERK signaling cascade. Critically, induced phagocytic behavior in vivo, through administration of liposomes, increased restorative macrophage number and accelerated fibrosis resolution, offering a therapeutic strategy to this orphan pathological process.
Jellyfish stings pose a significant threat to humans in coastal areas worldwide, with venomous jellyfish species stinging millions of individuals annually. Nemopilema nomurai is one of the largest ...jellyfish species, with numerous tentacles rich in nematocysts. N. nomurai venom (NnV) is a complex mixture of proteins, peptides, and small molecules that serve as both prey-capture and defense mechanisms. Yet, the molecular identity of its cardiorespiratory and neuronal toxic components of NnV has not been clearly identified yet. Here, we isolated a cardiotoxic fraction, NnTP (Nemopilema nomurai toxic peak), from NnV using chromatographic methods. In the zebrafish model, NnTP exhibited strong cardiorespiratory and moderate neurotoxic effects. LC-MS/MS analysis identified 23 toxin homologs, including toxic proteinases, ion channel toxins, and neurotoxins. The toxins demonstrated a synergistic effect on the zebrafish, leading to altered swimming behavior, hemorrhage in the cardiorespiratory region, and histopathological changes in organs such as the heart, gill, and brain. These findings provide valuable insights into the mechanisms underlying the cardiorespiratory and neurotoxic effects of NnV, which could be useful in developing therapeutic strategies for venomous jellyfish stings.
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•This work presents the isolation of key cardiotoxic components (NnTP) from N. nomurai venom by chromatographic methods.•NnTP treatment induces a notable hemorrhage in cardiopulmonary region and poor swimming behavior in Danio rerio model.•Significant histopathological changes were also observed from the heart, gill, and brain tissues upon NnTP injection.•Using LC-MS/MS, 23 toxin homologs were identified from NnTP, including toxic proteinases and ion channel toxins.
Monocytes are among the major myeloid cells that respond to Toxoplasma, a ubiquitous foodborne that infects ≥ 1 billion people worldwide, in human peripheral blood. As such, a molecular understanding ...of human monocyte-Toxoplasma interactions can expedite the development of novel human toxoplasmosis control strategies. Current molecular studies on monocyte-Toxoplasma interactions are based on average cell or parasite responses across bulk cell populations. Although informative, population-level averages of monocyte responses to Toxoplasma have sometimes produced contradictory results, such as whether CCL2 or IL12 define effective monocyte responses to the parasite. Here, we used single-cell dual RNA sequencing (scDual-Seq) to comprehensively define, for the first time, the monocyte and parasite transcriptional responses that underpin human monocyte-Toxoplasma encounters at the single cell level. We report extreme transcriptional variability between individual monocytes. Furthermore, we report that Toxoplasma-exposed and unexposed monocytes are transcriptionally distinguished by a reactive subset of CD14
CD16
monocytes. Functional cytokine assays on sorted monocyte populations show that the infection-distinguishing monocytes secrete high levels of chemokines, such as CCL2 and CXCL5. These findings uncover the Toxoplasma-induced monocyte transcriptional heterogeneity and shed new light on the cell populations that largely define cytokine and chemokine secretion in human monocytes exposed to Toxoplasma.
•We have developed Danio rerio animal model for the toxinological investigation of Nemopilema nomurai (NnV) jellyfish venom.•Cardiopulmonary tissues showed severe histopathological changes which are ...accompanied with the associated biochemical alterations.•NnV elicits neurotoxicity in zebrafish model with the modulations of swimming behavior and brain acetylcholinesterase activity.•Hence, we propose, Danio rerio as an alternative vertebrate model for the jellyfish venom toxinological research.
Nemopilema nomurai venom (NnV) is severely toxic to many organisms. However, the mechanism of its poisoning has not been properly understood yet. The present work demonstrates that zebrafish (Danio rerio) is an alternative vertebrate model for studying NnV jellyfish venom for the first time. In this model, NnV appears to cause severe hemorrhage and inflammation in cardiopulmonary regions of zebrafish. NnV also altered the swimming behavior of zebrafish accompanied by a significant downregulation of acetylcholinesterase (AChE) activity in brain tissues. Histopathological changes observed for various organs of D. rerio caused by NnV corresponded to an increase in lactate dehydrogenase (LDH) activity in tissues. NnV also significantly altered glutathione S-transferase (GST) activity in cardiopulmonary and brain tissues of D. rerio. SDS-PAGE revealed many protein bands of NnV of various sizes after silver staining. Taken together, these results indicate that Danio rerio can be a useful alternative animal model for jellyfish venom toxicology studies. Findings of the present study also suggest that Danio rerio could be used to develop an effective treatment strategy and discover the mechanism of action of jellyfish venom envenomation.
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