TLR7-let-7 Signaling Contributes to Ethanol-Induced Hepatic Inflammatory Response in Mice and in Alcoholic Hepatitis.

BACKGROUND: Toll-like receptor 7 (TLR7) is an endosomal TLR that is activated by single-stranded RNA, including endogenous microRNAs (e.g., let-7b). Increased hepatic expression of TLRs, microRNAs, and inflammatory mediators is linked to ethanol (EtOH) exposure and to alcoholic liver disease (ALD). ALD invovles chronic hepatic inflammation that can progress to alcoholic hepatitis (AH), a particularly severe form of ALD. This study aimed to investigate TLR7 expression in patients with different liver disease phenotypes and in mouse liver following alcohol exposure. METHODS: Hepatic mRNA expression was determined by RNA sequencing of liver tissue from patients with liver disease or normal liver tissue. Mice were exposed to subchronic EtOH followed by administration of the TLR7 agonist imiquimod. Primary human hepatocytes were exposed to EtOH or imiquimod in vitro. RESULTS: RNAseq analysis revealed that hepatic expression of TLR7 and let-7b microRNA, an endogenous TLR7 ligand, was significantly increased in AH patients. Hepatic expression of TLR7 and let-7b positively correlated with hepatic IL-8 mRNA expression. In mice, EtOH increased hepatic TLR7 mRNA expression and enhanced imiquimod-induced expression of the pro-inflammatory mediators TNFα, MCP-1, and iNOS. In vitro, EtOH significantly increased hepatocyte TLR7 mRNA and the TLR7 agonist, imiquimod, induced hepatocyte expression of TNFα and IL-8 mRNA. EtOH also increased the release of let-7b in microvesicles from hepatocytes, suggesting that EtOH can increase the expression of both the receptor and its endogenous ligand. CONCLUSIONS: These studies suggest that increased TLR7 signaling caused by increased expression of TLR7 and its endogenous ligand let-7b may contribute to the enhanced inflammatory response associated with AH.

Modeling the Kinetics of Integrin Receptor Binding to Hepatic Extracellular Matrix Proteins.

The composition of the extracellular matrix (ECM) proteins and the expression of their cognate receptors dictate cell behavior and dynamics. In particular, the interactions of ECM proteins with integrin receptors are key mediators of these cellular processes, playing a crucial role in the progression of several diseases of the liver, including inflammation, fibrosis/cirrhosis and cancer. This study establishes a modeling approach combining computation and experiments to evaluate the kinetics of integrin receptor binding to hepatic ECM proteins. ECM ligand concentration was derived from LC-MS/MS quantification of the hepatic ECM from mice exposed to chronic carbon tetrachloride (CCl4); receptor density was derived from published literature. Mathematical models for ECM-integrin binding kinetics that were developed incorporate receptor divalence and an aggregation scheme to represent clustering. The computer simulations reproduced positive cooperativity in the receptor aggregation model when the aggregation equilibrium constant (Ka) was positive and greater than Keq for divalent complex formation. Importantly, the modeling projected an increase in integrin binding for several receptors for which signaling is known to be increased after CCl4 exposure in the liver. The proposed modeling approach may be of use to elucidate the kinetics of integrin receptor binding to ECM proteins for homeostatic and diseased livers.

Plasminogen Activator Inhibitor-1 Is Critical in Alcohol-Enhanced Acute Lung Injury in Mice.

Chronic alcohol exposure is a clinically important risk factor for the development of acute respiratory distress syndrome, the most severe form of acute lung injury (ALI). However, the mechanisms by which alcohol sensitizes the lung to development of this disease are poorly understood. We determined the role of the antifibrinolytic protein plasminogen activator inhibitor-1 (PAI-1) in alcohol enhancement of experimental endotoxin-induced ALI. Wild-type, PAI-1-/-, and integrin ß3-/- mice were fed ethanol-containing Lieber-DeCarli liquid or a control diet for 6 weeks, followed by systemic LPS challenge. LPS administration triggered coagulation cascade activation as evidenced by increased plasma thrombin-antithrombin levels and pulmonary fibrin deposition. Ethanol-exposed animals showed enhanced PAI-1 expression and pulmonary fibrin deposition with coincident exaggeration of pulmonary inflammatory edematous injury. PAI-1 deficiency markedly reduced pulmonary fibrin deposition and greatly reduced inflammation and injury without impacting upstream coagulation. Interestingly, pulmonary platelet accumulation was effectively abolished by PAI-1 deficiency in ethanol/LPS-challenged mice. Moreover, mice lacking integrin αIIBß3, the primary platelet receptor for fibrinogen, displayed a dramatic reduction in early inflammatory changes after ethanol/LPS challenge. These results indicate that the mechanism whereby alcohol exaggerates LPS-induced lung injury requires PAI-1-mediated pulmonary fibrin accumulation, and suggest a novel mechanism whereby alcohol contributes to inflammatory ALI by enhancing fibrinogen-platelet engagement.

The hepatic "matrisome" responds dynamically to injury: Characterization of transitional changes to the extracellular matrix in mice.

The extracellular matrix (ECM) consists of diverse components that work bidirectionally with surrounding cells to create a responsive microenvironment. In some contexts (e.g., hepatic fibrosis), changes to the ECM are well recognized and understood. However, it is becoming increasingly accepted that the hepatic ECM proteome (i.e., matrisome) responds dynamically to stress well before fibrosis. The term "transitional tissue remodeling" describes qualitative and quantitative ECM changes in response to injury that do not alter the overall architecture of the organ; these changes in ECM may contribute to early disease initiation and/or progression. The nature and magnitude of these changes to the ECM in liver injury are poorly understood. The goals of this work were to validate analysis of the ECM proteome and compare the impact of 6 weeks of ethanol diet and/or acute lipopolysaccharide (LPS). Liver sections were processed in a series of increasingly rigorous extraction buffers to separate proteins by solubility. Extracted proteins were identified using liquid chromatography/tandem mass spectrometry (LC-MS/MS). Both ethanol and LPS dramatically increased the number of matrisome proteins ∼25%. The enhancement of LPS-induced liver damage by ethanol preexposure was associated with unique protein changes. CONCLUSION: An extraction method to enrich the hepatic ECM was characterized. The results demonstrate that the hepatic matrisome responds dynamically to both acute (LPS) and chronic (ethanol) stresses, long before more-dramatic fibrotic changes to the liver occur. The changes to the mastrisome may contribute, at least in part, to the pathological responses to these stresses. It is also interesting that several ECM proteins responded similarly to both stresses, suggesting a common mechanism in both models. Nevertheless, there were responses that were unique to the individual and combined exposures. (Hepatology 2017;65:969-982).

Potential Role of the Gut/Liver/Lung Axis in Alcohol-Induced Tissue Pathology.

Both Alcoholic Liver Disease (ALD) and alcohol-related susceptibility to acute lung injury are estimated to account for the highest morbidity and mortality related to chronic alcohol abuse and, thus, represent a focus of intense investigation. In general, alcohol-induced derangements to both organs are considered to be independent and are often evaluated separately. However, the liver and lung share many general responses to damage, and specific responses to alcohol exposure. For example, both organs possess resident macrophages that play key roles in mediating the immune/inflammatory response. Additionally, alcohol-induced damage to both organs appears to involve oxidative stress that favors tissue injury. Another mechanism that appears to be shared between the organs is that inflammatory injury to both organs is enhanced by alcohol exposure. Lastly, altered extracellular matrix (ECM) deposition appears to be a key step in disease progression in both organs. Indeed, recent studies suggest that early subtle changes in the ECM may predispose the target organ to an inflammatory insult. The purpose of this chapter is to review the parallel mechanisms of liver and lung injury in response to alcohol consumption. This chapter will also explore the potential that these mechanisms are interdependent, as part of a gut-liver-lung axis.

Chronic Alcohol Exposure Enhances Lipopolysaccharide-Induced Lung Injury in Mice: Potential Role of Systemic Tumor Necrosis Factor-Alpha.

BACKGROUND: It is well known that liver and lung injury can occur simultaneously during severe inflammation (e.g., multiple organ failure). However, whether these are parallel or interdependent (i.e., liver-lung axis) mechanisms is unclear. Previous studies have shown that chronic ethanol (EtOH) consumption greatly increases mortality in the setting of sepsis-induced acute lung injury (ALI). The potential contribution of subclinical liver disease in driving this effect of EtOH on the lung remains unknown. Therefore, the purpose of this study was to characterize the impact of chronic EtOH exposure on concomitant liver and lung injury. METHODS: Male mice were exposed to EtOH-containing Lieber-DeCarli diet or pair-fed control diet for 6 weeks. Some animals were administered lipopolysaccharide (LPS) 4 or 24 hours prior to sacrifice to mimic sepsis-induced ALI. Some animals received the tumor necrosis factor-alpha (TNF-α)-blocking drug, etanercept, for the duration of alcohol exposure. The expression of cytokine mRNA in lung and liver tissue was determined by quantitative PCR. Cytokine levels in the bronchoalveolar lavage fluid and plasma were determined by Luminex assay. RESULTS: As expected, the combination of EtOH and LPS caused liver injury, as indicated by significantly increased levels of the transaminases alanine aminotransferase/aspartate aminotransferase in the plasma and by changes in liver histology. In the lung, EtOH preexposure enhanced pulmonary inflammation and alveolar hemorrhage caused by LPS. These changes corresponded with unique alterations in the expression of pro-inflammatory cytokines in the liver (i.e., TNF-α) and lung (i.e., macrophage inflammatory protein-2 [MIP-2], keratinocyte chemoattractant [KC]). Systemic depletion of TNF-α (etanercept) blunted injury and the increase in MIP-2 and KC caused by the combination of EtOH and LPS in the lung. CONCLUSIONS: Chronic EtOH preexposure enhanced both liver and lung injury caused by LPS. Enhanced organ injury corresponded with unique changes in the pro-inflammatory cytokine expression profiles in the liver and the lung.

Oligofructose protects against arsenic-induced liver injury in a model of environment/obesity interaction.

Arsenic (As) tops the ATSDR list of hazardous environmental chemicals and is known to cause liver injury. Although the concentrations of As found in the US water supply are generally too low to directly damage the liver, subhepatotoxic doses of As sensitize the liver to experimental NAFLD. It is now suspected that GI microbiome dysbiosis plays an important role in development of NALFD. Importantly, arsenic has also been shown to alter the microbiome. The purpose of the current study was to test the hypothesis that the prebiotic oligofructose (OFC) protects against enhanced liver injury caused by As in experimental NAFLD. Male C57Bl6/J mice were fed low fat diet (LFD), high fat diet (HFD), or HFD containing oligofructose (OFC) during concomitant exposure to either tap water or As-containing water (4.9ppm as sodium arsenite) for 10weeks. HFD significantly increased body mass and caused fatty liver injury, as characterized by an increased liver weight-to-body weight ratio, histologic changes and transaminases. As observed previously, As enhanced HFD-induced liver damage, which was characterized by enhanced inflammation. OFC supplementation protected against the enhanced liver damage caused by As in the presence of HFD. Interestingly, arsenic, HFD and OFC all caused unique changes to the gut flora. These data support previous findings that low concentrations of As enhance liver damage caused by high fat diet. Furthermore, these results indicate that these effects of arsenic may be mediated, at least in part, by GI tract dysbiosis and that prebiotic supplementation may confer significant protective effects.

Olanzapine activates hepatic mammalian target of rapamycin: new mechanistic insight into metabolic dysregulation with atypical antipsychotic drugs.

Olanzapine (OLZ), an effective treatment of schizophrenia and other disorders, causes weight gain and metabolic syndrome. Most studies to date have focused on the potential effects of OLZ on the central nervous system's mediation of weight; however, peripheral changes in liver or other key metabolic organs may also play a role in the systemic effects of OLZ. Thus, the purpose of this study was to investigate the effects of OLZ on hepatic metabolism in a mouse model of OLZ exposure. Female C57Bl/6J mice were administered OLZ (8 mg/kg per day) or vehicle subcutaneously by osmotic minipumps for 28 days. Liver and plasma were taken at sacrifice for biochemical analyses and for comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry metabolomics analysis. OLZ increased body weight, fat pad mass, and liver-to-body weight ratio without commensurate increase in food consumption, indicating that OLZ altered energy expenditure. Expression and biochemical analyses indicated that OLZ induced anaerobic glycolysis and caused a pseudo-fasted state, which depleted hepatic glycogen reserves. OLZ caused similar effects in cultured HepG2 cells, as determined by Seahorse analysis. Metabolomic analysis indicated that OLZ increased hepatic concentrations of amino acids that can alter metabolism via the mTOR pathway; indeed, hepatic mTOR signaling was robustly increased by OLZ. Interestingly, OLZ concomitantly activated AMP-activated protein kinase (AMPK) signaling. Taken together, these data suggest that disturbances in glucose and lipid metabolism caused by OLZ in liver may be mediated, at least in part, via simultaneous activation of both catabolic (AMPK) and anabolic (mammalian target of rapamycin) pathways, which yields new insight into the metabolic side effects of this drug.

Acute alcohol-induced liver injury.

Alcohol consumption is customary in most cultures and alcohol abuse is common worldwide. For example, more than 50% of Americans consume alcohol, with an estimated 23.1% of Americans participating in heavy and/or binge drinking at least once a month. A safe and effective therapy for alcoholic liver disease (ALD) in humans is still elusive, despite significant advances in our understanding of how the disease is initiated and progresses. It is now clear that acute alcohol binges not only can be acutely toxic to the liver, but also can contribute to the chronicity of ALD. Potential mechanisms by which acute alcohol causes damage include steatosis, dysregulated immunity and inflammation, and altered gut permeability. Recent interest in modeling acute alcohol exposure has yielded new insights into potential mechanisms of acute injury, which also may well be relevant for chronic ALD. Recent work by this group on the role of PAI-1 and fibrin metabolism in mediating acute alcohol-induced liver damage serve as an example of possible new targets that may be useful for alcohol abuse, be it acute or chronic.