Novel Therapeutic Targets in Liver Fibrosis

Liver fibrosis is end-stage liver disease that can be rescued. If irritation continues due to viral infection, schistosomiasis and alcoholism, liver fibrosis can progress to liver cirrhosis and even cancer. The US Food and Drug Administration has not approved any drugs that act directly against liver fibrosis. The only treatments currently available are drugs that eliminate pathogenic factors, which show poor efficacy; and liver transplantation, which is expensive. This highlights the importance of clarifying the mechanism of liver fibrosis and searching for new treatments against it. This review summarizes how parenchymal, nonparenchymal cells, inflammatory cells and various processes (liver fibrosis, hepatic stellate cell activation, cell death and proliferation, deposition of extracellular matrix, cell metabolism, inflammation and epigenetics) contribute to liver fibrosis. We highlight discoveries of novel therapeutic targets, which may provide new insights into potential treatments for liver fibrosis.

A cell based assay for evaluating binding and uptake of an antibody using hepatic nonparenchymal cells

Evaluation of the binding and uptake of an antibody in liver non-parenchymal cells (NPC), including liver sinusoidal endothelial cells, is important for revealing its pharmacokinetic (PK) behavior, since NPC has important roles in eliminating an antibody from the blood via the Fc fragment of IgG receptor IIB (FcγRIIB). However, there is currently no in vitro quantitative assay using NPC. This study reports on the development of a cell-based assay for evaluating the binding and uptake of such an antibody using liver NPC of mice and monkeys. In mice, the FcγRIIB-expressing cells were identified in the CD146-positive and CD45-negative fraction by flow cytometry. A titration assay was performed to determine the PK parameters, and the obtained parameter was comparable to that determined by the fitting of the in vivo PK. This approach was also extended to NPC from monkeys. The concentration-dependent binding and uptake was measured to determine the PK parameters using monkey NPC, the FcγRIIB-expressing fraction of which was identified by CD31 and CD45. The findings presented herein demonstrate that the in vitro liver NPC assay using flow cytometry is a useful tool to determine the binding and uptake of biologics and to predict the PK.

Metabolic regulation of the lysosomal cofactor bis(monoacylglycero)phosphate in mice

Bis(monoacylglycero)phosphate (BMP), also known as lysobisphosphatidic acid, is a phospholipid that promotes lipid sorting in late endosomes/lysosomes by activating lipid hydrolases and lipid transfer proteins. Changes in the cellular BMP content therefore reflect an altered metabolic activity of the endolysosomal system. Surprisingly, little is known about the physiological regulation of BMP. In this study, we investigated the effects of nutritional and metabolic factors on BMP profiles of whole tissues and parenchymal and nonparenchymal cells. Tissue samples were obtained from fed, fasted, 2 h refed, and insulin-treated mice, as well as from mice housed at 5°C, 22°C, or 30°C. These tissues exhibited distinct BMP profiles that were regulated by the nutritional state in a tissue-specific manner. Insulin treatment was not sufficient to mimic refeeding-induced changes in tissue BMP levels, indicating that BMP metabolism is regulated by other hormonal or nutritional factors. Tissue fractionation experiments revealed that fasting drastically elevates BMP levels in hepatocytes and pancreatic cells. Furthermore, we observed that the BMP content in brown adipose tissue strongly depends on housing temperatures. In conclusion, our observations suggest that BMP concentrations adapt to the metabolic state in a tissue- and cell-type-specific manner in mice. Drastic changes observed in hepatocytes, pancreatic cells, and brown adipocytes suggest that BMP plays a role in the functional adaption to nutrient starvation and ambient temperature.

Application of single nuclei RNA sequencing to assess the hepatic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin

Cell-specific transcriptional responses are lost in the averages of bulk RNA sequencing. We performed single nuclei RNA sequencing (snSeq) on frozen liver samples from male C57BL/6 mice in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Approximately 19,907 hepatic genes were detected across 16,015 sequenced nuclei from control and treated samples. Eleven cell-(sub)types were identified including distinct hepatocyte sub-populations, consistent with the cell diversity of the liver. TCDD increased macrophages from 0.5% to 24.7%, while neutrophils were only present in treated samples. The number of differentially expressed genes correlated with the basal expression level of Ahr. In addition to expected functional enrichments within each cell-(sub)type, RAS signaling was enriched in nonparenchymal cells. snSeq also identified a Kupffer cell subtype highly expressing Gpnmb, consistent with a dietary NASH model. Overall, snSeq distinguished cell-specific transcriptional changes and population shifts consistent with the hepatotoxicity of TCDD.

Intermittent Starvation Extends the Functional Lifetime of Primary Human Hepatocyte Cultures

Primary human hepatocyte (PHH) cultures have become indispensable to mitigate the risk of adverse drug reactions in human patients. In contrast to dedifferentiating monocultures, coculture with nonparenchymal cells maintains PHH functions for 2–4 weeks. However, because the functional lifespan of PHHs in vivo is 200–400 days, it is desirable to further prolong PHH functions in vitro toward modeling chronic drug exposure and disease progression. Fasting has benefits on the longevity of organisms and the health of tissues such as the liver. We hypothesized that a culturing protocol that mimics dynamic fasting/starvation could activate starvation pathways and prolong PHH functional lifetime. To mimic starvation, serum and hormones were intermittently removed from the culture medium of micropatterned cocultures (MPCCs) containing PHHs organized onto collagen domains and surrounded by 3T3-J2 murine fibroblasts. A weekly 2-day starvation optimally prolonged PHH functional lifetime for 6+ weeks in MPCCs versus a decline after 3 weeks in nonstarved controls. The 2-day starvation also enhanced the functions of PHH monocultures for 2 weeks, suggesting direct effects on PHHs. In MPCCs, starvation activated 5' adenosine monophosphate-activated protein kinase (AMPK) and restricted fibroblast overgrowth onto PHH islands, thereby maintaining hepatic polarity. The effects of starvation on MPCCs were partially recapitulated by activating AMPK using metformin or growth arresting fibroblasts via mitomycin-C. Lastly, starved MPCCs demonstrated lower false positives for drug toxicity tests and higher drug-induced cytochrome-P450 activities versus nonstarved controls even after 5 weeks. In conclusion, intermittent serum/hormone starvation extends PHH functional lifetime toward enabling clinically relevant drug screening.

Liver X Receptors Regulate Cholesterol Metabolism and Immunity in Hepatic Nonparenchymal Cells

Excess dietary cholesterol intake and the dysregulation of cholesterol metabolism are associated with the pathogenesis and progression of nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, and fibrosis. Hepatic accumulation of free cholesterol induces activation of nonparenchymal cells, including Kupffer cells, macrophages, and hepatic stellate cells, which leads to persistent inflammation and fibrosis. The nuclear receptors liver X receptor α (LXRα) and LXRβ act as negative regulators of cholesterol metabolism through the induction of hepatocyte cholesterol catabolism, excretion, and the reverse cholesterol transport pathway. Additionally, LXRs exert an anti-inflammatory effect in immune cell types, such as macrophages. LXR activation suppresses acute hepatic inflammation that is mediated by Kupffer cells/macrophages. Acute liver injury, diet-induced steatohepatitis, and fibrosis are exacerbated by significant hepatic cholesterol accumulation and inflammation in LXR-deficient mice. Therefore, LXRs regulate hepatic lipid metabolism and immunity and they are potential therapeutic targets in the treatment of hepatic inflammation that is associated with cholesterol accumulation.

The Crosstalk between Fat Homeostasis and Liver Regional Immunity in NAFLD

The liver is well known as the center of glucose and lipid metabolism in the human body. It also functions as an immune organ. Previous studies have suggested that liver nonparenchymal cells are crucial in the progression of NAFLD. In recent years, NAFLD’s threat to human health has been becoming a global issue. And by far, there is no effective treatment for NAFLD. Liver nonparenchymal cells are stimulated by lipid antigens, adipokines, or other factors, and secreted immune factors can alter the expression of key proteins such as SREBP-1c, ChREBP, and PPARγ to regulate lipid metabolism, thus affecting the pathological process of NAFLD. Interestingly, some ncRNAs (including miRNAs and lncRNAs) participate in the pathological process of NAFLD by changing body fat homeostasis. And even some ncRNAs could regulate the activity of HSCs, thereby affecting the progression of inflammation and fibrosis in the course of NAFLD. In conclusion, immunotherapy could be an effective way to treat NAFLD.

The Matrine Derivate MASM Inhibits Recruitment of Gr1hi Monocyte and Alleviates Liver Injury

Backgrounds: (6aS, 10S, 11aR, 11bR, 11cS)-10-methylaminododecahydro-3a, 7a-diaza-benzo (de) anthracene-8-thione (MASM), a novel derivative of matrine, exhibits better anti-inflammatory activity. This study was designed to evaluate the protective effect of MASM on acute and chronic liver injuries and explore the possible mechanisms. Methods: Acute and chronic liver injury models were established by the CCl4 intraperitoneal injection and the protective effect of MASM was assessed by biochemical and histological examination. The infiltration of different monocyte subsets into the liver was characterized and analyzed by flow cytometry. The in vitro effect of MASM on liver nonparenchymal cells was evaluated by real-time PCR and transwell chemotaxis assays. Results: Administration of MASM markedly attenuated acute liver injury and liver fibrosis induced by CCl4 injection. Meanwhile, the infiltrations of Gr1hi monocytes in injured livers and induced hepatic expression of monocyte chemoattractant protein-1 (MCP-1) were greatly inhibited. Cellular experiments demonstrated that MASM not only decreased the expression of MCP-1 but also inhibited its chemotactic activity. Conclusions: This study demonstrates that the protective effect of MASM on liver injury could be contributed to the suppression of Gr1hi monocyte infiltration to the liver and the inhibition of MCP-1 production and activity. These findings provide new insights into the protective role of MASM in liver injury.