The HMGB1/RAGE axis triggers neutrophil-mediated injury amplification following necrosis

In contrast to microbially triggered inflammation, mechanisms promoting sterile inflammation remain poorly understood. Damage-associated molecular patterns (DAMPs) are considered key inducers of sterile inflammation following cell death, but the relative contribution of specific DAMPs, including high–mobility group box 1 (HMGB1), is ill defined. Due to the postnatal lethality of Hmgb1-knockout mice, the role of HMGB1 in sterile inflammation and disease processes in vivo remains controversial. Here, using conditional ablation strategies, we have demonstrated that epithelial, but not bone marrow–derived, HMGB1 is required for sterile inflammation following injury. Epithelial HMGB1, through its receptor RAGE, triggered recruitment of neutrophils, but not macrophages, toward necrosis. In clinically relevant models of necrosis, HMGB1/RAGE-induced neutrophil recruitment mediated subsequent amplification of injury, depending on the presence of neutrophil elastase. Notably, hepatocyte-specific HMGB1 ablation resulted in 100% survival following lethal acetaminophen intoxication. In contrast to necrosis, HMGB1 ablation did not alter inflammation or mortality in response to TNF- or FAS-mediated apoptosis. In LPS-induced shock, in which HMGB1 was considered a key mediator, HMGB1 ablation did not ameliorate inflammation or lethality, despite efficient reduction of HMGB1 serum levels. Our study establishes HMGB1 as a bona fide and targetable DAMP that selectively triggers a neutrophil-mediated injury amplification loop in the setting of necrosis

Rapid production of human liver scaffolds for functional tissue engineering by high shear stress oscillation-decellularization

The development of human liver scaffolds retaining their 3-dimensional structure and extra-cellular matrix (ECM) composition is essential for the advancement of liver tissue engineering. We report the design and validation of a new methodology for the rapid and accurate production of human acellular liver tissue cubes (ALTCs) using normal liver tissue unsuitable for transplantation. The application of high shear stress is a key methodological determinant accelerating the process of tissue decellularization while maintaining ECM protein composition, 3D-architecture and physico-chemical properties of the native tissue. ALTCs were engineered with human parenchymal and non-parenchymal liver cell lines (HepG2 and LX2 cells, respectively), human umbilical vein endothelial cells (HUVEC), as well as primary human hepatocytes and hepatic stellate cells. Both parenchymal and non-parenchymal liver cells grown in ALTCs exhibited markedly different gene expression when compared to standard 2D cell cultures. Remarkably, HUVEC cells naturally migrated in the ECM scaffold and spontaneously repopulated the lining of decellularized vessels. The metabolic function and protein synthesis of engineered liver scaffolds with human primary hepatocytes reseeded under dynamic conditions were maintained. These results provide a solid basis for the establishment of effective protocols aimed at recreating human liver tissue in vitro

All-In-One: Advanced preparation of Human Parenchymal and Non-Parenchymal Liver Cells

BACKGROUND & AIMS: Liver cells are key players in innate immunity. Thus, studying primary isolated liver cells is necessary for determining their role in liver physiology and pathophysiology. In particular, the quantity and quality of isolated cells are crucial to their function. Our aim was to isolate a large quantity of high-quality human parenchymal and non-parenchymal cells from a single liver specimen. METHODS: Hepatocytes, Kupffer cells, liver sinusoidal endothelial cells, and stellate cells were isolated from liver tissues by collagenase perfusion in combination with low-speed centrifugation, density gradient centrifugation, and magnetic-activated cell sorting. The purity and functionality of cultured cell populations were controlled by determining their morphology, discriminative cell marker expression, and functional activity. RESULTS: Cell preparation yielded the following cell counts per gram of liver tissue: 2.0+/-0.4x107 hepatocytes, 1.8+/-0.5x106 Kupffer cells, 4.3+/-1.9x105 liver sinusoidal endothelial cells, and 3.2+/-0.5x105 stellate cells. Hepatocytes were identified by albumin (95.5+/-1.7%) and exhibited time-dependent activity of cytochrome P450 enzymes. Kupffer cells expressed CD68 (94.5+/-1.2%) and exhibited phagocytic activity, as determined with 1mum latex beads. Endothelial cells were CD146+ (97.8+/-1.1%) and exhibited efficient uptake of acetylated low-density lipoprotein. Hepatic stellate cells were identified by the expression of alpha-smooth muscle actin (97.1+/-1.5%). These cells further exhibited retinol (vitamin A)-mediated autofluorescence. CONCLUSIONS: Our isolation procedure for primary parenchymal and non-parenchymal liver cells resulted in cell populations of high purity and quality, with retained physiological functionality in vitro. Thus, this system may provide a valuable tool for determining liver function and disease

Contribution of Underlying Connective Tissue Cells to Taste Buds in Mouse Tongue and Soft Palate

Taste buds, the sensory organs for taste, have been described as arising solely from the surrounding epithelium, which is in distinction from other sensory receptors that are known to originate from neural precursors, i.e., neural ectoderm that includes neural crest (NC). Our previous study suggested a potential contribution of NC derived cells to early immature fungiform taste buds in late embryonic (E18.5) and young postnatal (P1-10) mice. In the present study we demonstrated the contribution of the underlying connective tissue (CT) to mature taste buds in mouse tongue and soft palate. Three independent mouse models were used for fate mapping of NC and NC derived connective tissue cells: (1) P0-Cre/R26-tdTomato (RFP) to label NC, NC derived Schwann cells and derivatives; (2) Dermo1-Cre/RFP to label mesenchymal cells and derivatives; and (3) Vimentin-CreER/mGFP to label Vimentin-expressing CT cells and derivatives upon tamoxifen treatment. Both P0-Cre/RFP and Dermo1-Cre/RFP labeled cells were abundant in mature taste buds in lingual taste papillae and soft palate, but not in the surrounding epithelial cells. Concurrently, labeled cells were extensively distributed in the underlying CT. RFP signals were seen in the majority of taste buds and all three types (I, II, III) of differentiated taste bud cells, with the neuronal-like type III cells labeled at a greater proportion. Further, Vimentin-CreER labeled cells were found in the taste buds of 3-month-old mice whereas Vimentin immunoreactivity was only seen in the CT. Taken together, our data demonstrate a previously unrecognized origin of taste bud cells from the underlying CT, a conceptually new finding in our knowledge of taste bud cell derivation, i.e., from both the surrounding epithelium and the underlying CT that is primarily derived from NC

Cholemic nephropathy causes acute kidney injury and is accompanied by loss of aquaporin 2 in collecting ducts

Impairment of renal function often occurs in patients with liver disease. Hepatorenal syndrome is a significant cause of acute kidney injury (AKI) in cirrhotic patients (HRS-AKI, type 1). Causes of non-HRS AKI include cholemic nephropathy (CN), a disease that is characterized by intratubular bile casts and tubular injury. As data on patients with CN is mostly obtained from case reports or autopsy studies, we aimed to investigate the frequency and clinical course of CN. We identified 149 patients who underwent kidney biopsy between 2000 to 2016 at the Department of Gastroenterology, Hepatology and Endocrinology. Of these, 79 had a history of liver disease and deterioration of renal function. When applying recent EASL criteria 45 of the 79 patients (57%) presented with AKI, whereas 34 patients (43%) had chronic kidney disease (CKD) (43%). Renal biopsy revealed the diagnosis of CN in 8 of the 45 patients with AKI (17.8%), whereas none of the patients with CKD was diagnosed with CN. Univariate analysis identified serum bilirubin, alkaline phosphatase and urinary bilirubin and urobilinogen as predictive factors for the diagnosis of CN. Histological analysis of AKI patients with normal bilirubin, elevated bilirubin and the diagnosis of CN revealed loss aquaporin 2 (AQP2) expression in collecting ducts in patients with elevated bilirubin and CN. Biopsy related complications requiring medical intervention occurred in four of 79 patients (5.1%). In conclusion, CN is a common finding in patients with liver disease, AKI and highly elevated bilirubin. Loss of AQP2 in AKI patients with elevated bilirubin and CN might be the result of toxic effects of cholestasis and be in part responsible for the impairment of renal function

The balancing act of the liver: tissue regeneration versus fibrosis

Epithelial cell loss alters a tissue's optimal function and awakens evolutionarily adapted healing mechanisms to reestablish homeostasis. Although adult mammalian organs have a limited regeneration potential, the liver stands out as one remarkable exception. Following injury, the liver mounts a dynamic multicellular response wherein stromal cells are activated in situ and/or recruited from the bloodstream, the extracellular matrix (ECM) is remodeled, and epithelial cells expand to replenish their lost numbers. Chronic damage makes this response persistent instead of transient, tipping the system into an abnormal steady state known as fibrosis, in which ECM accumulates excessively and tissue function degenerates. Here we explore the cellular and molecular switches that balance hepatic regeneration and fibrosis, with a focus on uncovering avenues of disease modeling and therapeutic intervention.LCE is jointly funded by a Wellcome Trust Four-Year PhD Studentship with the Stem Cell Biology and Medicine Programme and a Wellcome Cambridge Trust Scholarship. MH is a Wellcome Trust Sir Henry Dale Fellow and is jointly funded by the Wellcome Trust and the Royal Society (104151/Z/14/Z). This work is partially funded by an H2020 grant awarded to MH (LSMF4LIFE)

11Beta‐hydroxysteroid dehydrogenase‐1 deficiency or inhibition enhances hepatic myofibroblast activation in murine liver fibrosis

A hallmark of chronic liver injury is fibrosis, with accumulation of extracellular matrix orchestrated by activated hepatic stellate cells (HSCs). Glucocorticoids limit HSC activation in vitro, and tissue glucocorticoid levels are amplified by 11beta‐hydroxysteroid dehydrogenase‐1 (11βHSD1). Although 11βHSD1 inhibitors have been developed for type 2 diabetes mellitus and improve diet‐induced fatty liver in various mouse models, effects on the progression and/or resolution of liver injury and consequent fibrosis have not been characterized. We have used the reversible carbon tetrachloride‐induced model of hepatocyte injury and liver fibrosis to show that in two models of genetic 11βHSD1 deficiency (global, Hsd11b1⁻/⁻, and hepatic myofibroblast‐specific, Hsd11b1fl/fl/Pdgfrb‐cre) 11βHSD1 pharmacological inhibition in vivo exacerbates hepatic myofibroblast activation and liver fibrosis. In contrast, liver injury and fibrosis in hepatocyte‐specific Hsd11b1fl/fl/albumin‐cre mice did not differ from that of controls, ruling out 11βHSD1 deficiency in hepatocytes as the cause of the increased fibrosis. In primary HSC culture, glucocorticoids inhibited expression of the key profibrotic genes Acta2 and Col1α1, an effect attenuated by the 11βHSD1 inhibitor [4‐(2‐chlorophenyl‐4‐fluoro‐1‐piperidinyl][5‐(1H‐pyrazol‐4‐yl)‐3‐thienyl]‐methanone. HSCs from Hsd11b1–/– and Hsd11b1fl/fl/Pdgfrb‐cre mice expressed higher levels of Acta2 and Col1α1 and were correspondingly more potently activated. In vivo [4‐(2‐chlorophenyl‐4‐fluoro‐1‐piperidinyl][5‐(1H‐pyrazol‐4‐yl)‐3‐thienyl]‐methanone administration prior to chemical injury recapitulated findings in Hsd11b1–/– mice, including greater fibrosis. Conclusion: 11βHSD1 deficiency enhances myofibroblast activation and promotes initial fibrosis following chemical liver injury; hence, the effects of 11βHSD1 inhibitors on liver injury and repair are likely to be context‐dependent and deserve careful scrutiny as these compounds are developed for chronic diseases including metabolic syndrome and dementia. (Hepatology 2018;67:2167‐2181)

Single-Cell Transcriptomics Uncovers Zonation of Function in the Mesenchyme during Liver Fibrosis.

Iterative liver injury results in progressive fibrosis disrupting hepatic architecture, regeneration potential, and liver function. Hepatic stellate cells (HSCs) are a major source of pathological matrix during fibrosis and are thought to be a functionally homogeneous population. Here, we use single-cell RNA sequencing to deconvolve the hepatic mesenchyme in healthy and fibrotic mouse liver, revealing spatial zonation of HSCs across the hepatic lobule. Furthermore, we show that HSCs partition into topographically diametric lobule regions, designated portal vein-associated HSCs (PaHSCs) and central vein-associated HSCs (CaHSCs). Importantly we uncover functional zonation, identifying CaHSCs as the dominant pathogenic collagen-producing cells in a mouse model of centrilobular fibrosis. Finally, we identify LPAR1 as a therapeutic target on collagen-producing CaHSCs, demonstrating that blockade of LPAR1 inhibits liver fibrosis in a rodent NASH model. Taken together, our work illustrates the power of single-cell transcriptomics to resolve the key collagen-producing cells driving liver fibrosis with high precision

PAK proteins and YAP-1 signalling downstream of integrin beta-1 in myofibroblasts promote liver fibrosis

Fibrosis due to extracellular matrix (ECM) secretion from myofibroblasts complicates many chronic liver diseases causing scarring and organ failure. Integrin-dependent interaction with scar ECM promotes pro-fibrotic features. However, the pathological intracellular mechanism in liver myofibroblasts is not completely understood, and further insight could enable therapeutic efforts to reverse fibrosis. Here, we show that integrin beta-1, capable of binding integrin alpha-11, regulates the pro-fibrotic phenotype of myofibroblasts. Integrin beta-1 expression is upregulated in pro-fibrotic myofibroblasts in vivo and is required in vitro for production of fibrotic ECM components, myofibroblast proliferation, migration and contraction. Serine/threonine-protein kinase proteins, also known as P21-activated kinase (PAK), and the mechanosensitive factor, Yes-associated protein 1 (YAP-1) are core mediators of pro-fibrotic integrin beta-1 signalling, with YAP-1 capable of perpetuating integrin beta-1 expression. Pharmacological inhibition of either pathway in vivo attenuates liver fibrosis. PAK protein inhibition, in particular, markedly inactivates the pro-fibrotic myofibroblast phenotype, limits scarring from different hepatic insults and represents a new tractable therapeutic target for treating liver fibrosis