Interleukin-10 disrupts liver repair in acetaminophen-induced acute liver failure.

Introduction: Systemic levels of the anti-inflammatory cytokine interleukin 10 (IL-10) are highest in acetaminophen (APAP)-induced acute liver failure (ALF) patients with the poorest prognosis. The mechanistic basis for this counterintuitive finding is not known, as induction of IL-10 is hypothesized to temper the pathological effects of immune cell activation. Aberrant production of IL-10 after severe liver injury could conceivably interfere with the beneficial, pro-reparative actions of immune cells, such as monocytes. Methods: To test this possibility, we determined whether IL-10 levels are dysregulated in mice with APAP-induced ALF and further evaluated whether aberrant production of IL-10 prevents monocyte recruitment and/or the resolution of necrotic lesions by these cells. Results: Our studies demonstrate that in mice challenged with 300 mg/kg acetaminophen (APAP), a hepatotoxic dose of APAP that fails to produce ALF (i.e., APAP-induced acute liver injury; AALI), Ly6Chi monocytes were recruited to the liver and infiltrated the necrotic lesions by 48 hours coincident with the clearance of dead cell debris. At 72 hours, IL-10 was upregulated, culminating in the resolution of hepatic inflammation. By contrast, in mice treated with 600 mg/kg APAP, a dose that produces clinical features of ALF (i.e., APAP-induced ALF; AALF), IL-10 levels were markedly elevated by 24 hours. Early induction of IL-10 was associated with a reduction in the hepatic numbers of Ly6Chi monocytes resulting in the persistence of dead cell debris. Inhibition of IL-10 in AALF mice, beginning at 24 hours after APAP treatment, increased the hepatic numbers of monocytes which coincided with a reduction in the necrotic area. Moreover, pharmacologic elevation of systemic IL-10 levels in AALI mice reduced hepatic myeloid cell numbers and increased the area of necrosis. Discussion: Collectively, these results indicate that during ALF, aberrant production of IL-10 disrupts the hepatic recruitment of monocytes, which prevents the clearance of dead cell debris. These are the first studies to document a mechanistic basis for the link between high IL-10 levels and poor outcome in patients with ALF.

Assaying Spontaneous Network Activity and Cellular Viability Using Multi-Well Microelectrode Arrays.

Microelectrode array (MEA) technology is a neurophysiological method that allows for the measurement of spontaneous or evoked neural activity to determine chemical effects thereon. Following assessment of compound effects on multiple endpoints that evaluate network function, a cell viability endpoint in the same well is determined using a multiplexed approach. Recently, it has become possible to measure electrical impedance of cells attached to the electrodes, where greater impedance indicates greater number of cells attached. This would allow rapid and repeated assessments of cell health as the neural network develops in longer exposure assays without impacting cell health. Typically, the lactate dehydrogenase (LDH) assay for cytotoxity and CellTiter-Blue® (CTB) assay for cell viability are only performed at the end of the chemical exposure period because these assays involve lysing of the cells. Procedures describing the multiplexed methods in acute and network formation screening are included in this chapter.

Modulation of macrophage phenotype to treat liver fibrosis-Current approaches and future possibilities.

Liver fibrosis is a leading cause of death worldwide, accounting for approximately 2 million deaths annually. Despite its wide prevalence, there are currently no pharmacological therapies that directly reverse the fibrotic process in patients. Studies over the last decade have revealed that liver fibrosis is reversible in patients and in animal models. Further, studies aimed at elucidating the mechanism of fibrosis reversal have revealed that macrophages are central to this process. During resolution of fibrosis, proinflammatory macrophages shift phenotype to pro-resolution macrophages which produce matrix degrading enzymes and mediators that inactivate hepatic stellate cells, the cell type principally involved in matrix production during fibrosis development. Since fibrosis reversal begins when disease-causing macrophages transition to disease-reversing macrophages, studies have focused on identifying pharmacological agents that stimulate this process to occur. If successful, these "drugs" would constitute a first-in-class, macrophage-targeted therapeutic approach to reverse liver fibrosis. In the following review, we summarize the current approaches under investigation to modify macrophage phenotype for liver disease treatment. Further we discuss the potential of other approaches to identify novel macrophage-targeted drugs that modify the phenotype of these cells.

Differential regulation of hepatic physiology and injury by the TAM receptors Axl and Mer.

Genome-wide association studies have implicated the TAM receptor tyrosine kinase (RTK) Mer in liver disease, yet our understanding of the role that Mer and its related RTKs Tyro3 and Axl play in liver homeostasis and the response to acute injury is limited. We find that Mer and Axl are most prominently expressed in hepatic Kupffer and endothelial cells and that as mice lacking these RTKs age, they develop profound liver disease characterized by apoptotic cell accumulation and immune activation. We further find that Mer is critical to the phagocytosis of apoptotic hepatocytes generated in settings of acute hepatic injury, and that Mer and Axl act in concert to inhibit cytokine production in these settings. In contrast, we find that Axl is uniquely important in mitigating liver damage during acetaminophen intoxication. Although Mer and Axl are protective in acute injury models, we find that Axl exacerbates fibrosis in a model of chronic injury. These divergent effects have important implications for the design and implementation of TAM-directed therapeutics that might target these RTKs in the liver.

Regulation of macrophage activation in the liver after acute injury: Role of the fibrinolytic system.

The liver functions, in part, to prevent exposure of the body to potentially harmful substances ingested in the diet. While it is highly efficient at accomplishing this, it is frequently prone to liver injury due to the biotransformation of xenobiotics into toxic metabolites. To counter this injury, the liver has evolved a unique capacity to rapidly and efficiently repair itself. Successful resolution of acute liver injury relies on hepatic macrophage populations that orchestrate the reparative response. After injury, Kupffer cells, the resident macrophages of the liver, become activated and secrete proinflammatory cytokines. These cytokines recruit other immune cells, including monocyte-derived macrophages, to the liver where they contribute to the repair process. Monocyte-derived macrophages traffic into the necrotic foci where they rapidly phagocytose dead cell debris. Simultaneous with this process, these cells change phenotype from a proinflammatory macrophage to a pro-restorative macrophage that produce pro-mitogenic growth factors and anti-inflammatory cytokines. Ultimately this process triggers resolution of inflammation, and along with proliferation of other hepatic cells, restores the liver architecture and function. While the mechanisms regulating specific macrophage functions during repair remain to be elucidated, recent studies indicate a key role for the fibrinolytic system in coordinating macrophage function during repair. In this review, we will highlight the function and role of hepatic macrophages in repair after acute liver injury, and will discuss the role of the fibrinolytic enzyme, plasmin, in regulation of these various processes.

Hypoxia upregulates Cxcl12 in hepatocytes by a complex mechanism involving hypoxia-inducible factors and transforming growth factor-ß.

INTRODUCTION: Cxcl12, or stromal-derived factor-1, is a chemokine produced by several hepatic cell types, including hepatocytes, after liver injury and surgical resection. Studies have revealed that Cxcl12 is important for regeneration of the liver after surgical resection and for development of liver fibrosis during chronic liver injury. While the function of Cxcl12 in the liver is well established, the mechanism by which Cxcl12 is upregulated is not fully understood. Because regions of hypoxia develop in the liver following injury, we tested the hypothesis that hypoxia upregulates Cxcl12 in hepatocytes by a hypoxia-inducible factor (HIF)-dependent mechanism. METHODS: To test this hypothesis, primary mouse hepatocytes were isolated from the livers of HIF-1α-deficient mice or HIF-1ß-deficient mice and exposed to 1% oxygen. Cxcl12 expression was increased following exposure of primary mouse hepatocytes to 1% oxygen. Previously we have shown, that in addition to HIFs, transforming growth factor-ß is required for upregulation of a subset of genes in hypoxic hepatocytes. To examine the role of TGF-ß in regulation of Cxcl12 during hypoxia, hepatocytes were pretreated with the TGF-ß receptor I inhibitor, SB431542. RESULTS: Upregulation of Cxcl12 by hypoxia was partially prevented in hepatocytes from HIF-1α-deficient mice and completely prevented in hepatocytes from HIF-1ß-deficient hepatocytes. This suggests that under hypoxic conditions, both HIF-1α and HIF-2α regulate Cxcl12 in hepatocytes. Pretreatment of hepatocytes with SB431542 completely prevented upregulation Cxcl12 by hypoxia. Further, treatment of hepatocytes with recombinant TGF-ß1 upregulated Cxcl12 in hepatocytes cultured in room air. CONCLUSION: Collectively, these studies demonstrate that hypoxia upregulates Cxcl12 in primary mouse hepatocytes by a mechanism that involves HIFs and TGF-ß.

Concentration-response evaluation of ToxCast compounds for multivariate activity patterns of neural network function.

The US Environmental Protection Agency's ToxCast program has generated toxicity data for thousands of chemicals but does not adequately assess potential neurotoxicity. Networks of neurons grown on microelectrode arrays (MEAs) offer an efficient approach to screen compounds for neuroactivity and distinguish between compound effects on firing, bursting, and connectivity patterns. Previously, single concentrations of the ToxCast Phase II library were screened for effects on mean firing rate (MFR) in rat primary cortical networks. Here, we expand this approach by retesting 384 of those compounds (including 222 active in the previous screen) in concentration-response across 43 network activity parameters to evaluate neural network function. Using hierarchical clustering and machine learning methods on the full suite of chemical-parameter response data, we identified 15 network activity parameters crucial in characterizing activity of 237 compounds that were response actives ("hits"). Recognized neurotoxic compounds in this network function assay were often more potent compared to other ToxCast assays. Of these chemical-parameter responses, we identified three k-means clusters of chemical-parameter activity (i.e., multivariate MEA response patterns). Next, we evaluated the MEA clusters for enrichment of chemical features using a subset of ToxPrint chemotypes, revealing chemical structural features that distinguished the MEA clusters. Finally, we assessed distribution of neurotoxicants with known pharmacology within the clusters and found that compounds segregated differentially. Collectively, these results demonstrate that multivariate MEA activity patterns can efficiently screen for diverse chemical activities relevant to neurotoxicity, and that response patterns may have predictive value related to chemical structural features.

Dichotomous Role of Plasmin in Regulation of Macrophage Function after Acetaminophen Overdose.

Kupffer cells and monocyte-derived macrophages are critical for liver repair after acetaminophen (APAP) overdose. These cells produce promitogenic cytokines and growth factors, and they phagocytose dead cell debris, a process that is critical for resolution of inflammation. The factors that regulate these dynamic functions of macrophages after APAP overdose, however, are not fully understood. We tested the hypothesis that the fibrinolytic enzyme, plasmin, is a key regulator of macrophage function after APAP-induced liver injury. In these studies, inhibition of plasmin in mice with tranexamic acid delayed up-regulation of proinflammatory cytokines after APAP overdose. In culture, plasmin directly, and in synergy with high-mobility group B1, stimulated Kupffer cells and bone marrow-derived macrophages to produce cytokines by a mechanism that required NF-κB. Inhibition of plasmin in vivo also prevented trafficking of monocyte-derived macrophages into necrotic lesions after APAP overdose. This prevented phagocytic removal of dead cells, prevented maturation of monocyte-derived macrophages into F4/80-expressing macrophages, and prevented termination of proinflammatory cytokine production. Our studies reveal further that phagocytosis is an important stimulus for cessation of proinflammatory cytokine production as treatment of proinflammatory, monocyte-derived macrophages, isolated from APAP-treated mice, with necrotic hepatocytes decreased expression of proinflammatory cytokines. Collectively, these studies demonstrate that plasmin is an important regulator of macrophage function after APAP overdose.

Screening the ToxCast phase II libraries for alterations in network function using cortical neurons grown on multi-well microelectrode array (mwMEA) plates.

Methods are needed for rapid screening of environmental compounds for neurotoxicity, particularly ones that assess function. To demonstrate the utility of microelectrode array (MEA)-based approaches as a rapid neurotoxicity screening tool, 1055 chemicals from EPA's phase II ToxCast library were evaluated for effects on neural function and cell health. Primary cortical networks were grown on multi-well microelectrode array (mwMEA) plates. On day in vitro 13, baseline activity (40 min) was recorded prior to exposure to each compound (40 µM). Changes in spontaneous network activity [mean firing rate (MFR)] and cell viability (lactate dehydrogenase and CellTiter Blue) were assessed within the same well following compound exposure. Following exposure, 326 compounds altered (increased or decreased) normalized MFR beyond hit thresholds based on 2× the median absolute deviation of DMSO-treated wells. Pharmaceuticals, pesticides, fungicides, chemical intermediates, and herbicides accounted for 86% of the hits. Further, changes in MFR occurred in the absence of cytotoxicity, as only eight compounds decreased cell viability. ToxPrint chemotype analysis identified several structural domains (e.g., biphenyls and alkyl phenols) significantly enriched with MEA actives relative to the total test set. The top 5 enriched ToxPrint chemotypes were represented in 26% of the MEA hits, whereas the top 11 ToxPrints were represented in 34% of MEA hits. These results demonstrate that large-scale functional screening using neural networks on MEAs can fill a critical gap in assessment of neurotoxicity potential in ToxCast assay results. Further, a data-mining approach identified ToxPrint chemotypes enriched in the MEA-hit subset, which define initial structure-activity relationship inferences, establish potential mechanistic associations to other ToxCast assay endpoints, and provide working hypotheses for future studies.

Assaying Spontaneous Network Activity and Cellular Viability Using Multi-well Microelectrode Arrays.

Microelectrode array (MEA) technology is a neurophysiological method that allows for the spontaneous measure of activity in neural cultures and determination of drug and chemical effects thereon. Recent introduction of multi-well MEA (mwMEA) formats have dramatically increased the throughput of this technology, allowing more efficient compound screening. Rapid characterization of compounds for neuroactivity or neurotoxicity hazard evaluation following acute, chronic, or developmental exposures ideally would also consider compound effects on cell health, and to do so in the same well requires a multiplexed approach. Procedures describing the multiplexed method to acute and developmental screening are described in this chapter.

Effects of an environmentally-relevant mixture of pyrethroid insecticides on spontaneous activity in primary cortical networks on microelectrode arrays.

Pyrethroid insecticides exert their insecticidal and toxicological effects primarily by disrupting voltage-gated sodium channel (VGSC) function, resulting in altered neuronal excitability. Numerous studies of individual pyrethroids have characterized effects on mammalian VGSC function and neuronal excitability, yet studies examining effects of complex pyrethroid mixtures in mammalian neurons, especially in environmentally relevant mixture ratios, are limited. In the present study, concentration-response functions were characterized for five pyrethroids (permethrin, deltamethrin, cypermethrin, ß-cyfluthrin and esfenvalerate) in an in vitro preparation containing cortical neurons and glia. As a metric of neuronal network activity, spontaneous mean network firing rates (MFR) were measured using microelectorde arrays (MEAs). In addition, the effect of a complex and exposure relevant mixture of the five pyrethroids (containing 52% permethrin, 28.8% cypermethrin, 12.9% ß-cyfluthrin, 3.4% deltamethrin and 2.7% esfenvalerate) was also measured. Data were modeled to determine whether effects of the pyrethroid mixture were predicted by dose-addition. At concentrations up to 10µM, all compounds except permethrin reduced MFR. Deltamethrin and ß-cyfluthrin were the most potent and reduced MFR by as much as 60 and 50%, respectively, while cypermethrin and esfenvalerate were of approximately equal potency and reduced MFR by only ∼20% at the highest concentration. Permethrin caused small (∼24% maximum), concentration-dependent increases in MFR. Effects of the environmentally relevant mixture did not depart from the prediction of dose-addition. These data demonstrate that an environmentally relevant mixture caused dose-additive effects on spontaneous neuronal network activity in vitro, and is consistent with other in vitro and in vivo assessments of pyrethroid mixtures.

In vitro screening of silver nanoparticles and ionic silver using neural networks yields differential effects on spontaneous activity and pharmacological responses.

Silver nanoparticles (AgNPs) are used in a wide range of consumer and medical products because of their antimicrobial and antifungal properties, and can translocate to the brain following exposure. Therefore, to screen AgNPs for potential impacts on human health, it is essential to examine neural function. The present study examined AgNPs (3 citrate coated, 3 PVP coated; 10-75nm) and AgNO3 effects on spontaneous and pharmacologically-induced neural network function in rat primary cortical cells on multi-well microelectrode array (mwMEA) plates. Baseline activity (1h) was recorded prior to exposure to non-cytotoxic concentrations of AgNPs and AgNO3 (0.08-0.63 and 0.08-1.7µg/ml, respectively). Changes in number of total extracellularly-recorded action potential spikes (total spikes; TS) and active electrodes (AE), relative to controls, were assessed 1, 24, and 48h after exposure to AgNP suspensions or AgNO3. After the 48h recording, the response to a pharmacological challenge with the GABAA antagonist, bicuculline (BIC), was assessed. Only two particles altered neural network function. Citrate coated 10nm AgNP caused concentration-related increases in AEs at 24h. After BIC treatment, PVP coated 75nm AgNP caused concentration-dependent increases in AE. AgNO3 effects differed from AgNPs, causing a concentration-related decrease in AEs at 24 and 48h, and a concentration-related decrease in TS following BIC challenge. Importantly, the direction of AgNO3 effects on neural activity was opposite those of 10nm Ag citrate at concentrations up to 0.63µg/ml, and different from 75nm Ag PVP, indicating ionic silver does not mediate these effects. These results demonstrate that non-cytotoxic concentrations of 10nm citrate- and 75nm PVP-coated Ag NPs alter neural network function in vitro, and should be considered for additional neurotoxicity hazard characterization.

In vitro screening of metal oxide nanoparticles for effects on neural function using cortical networks on microelectrode arrays.

Nanoparticles (NPs) may translocate to the brain following inhalation or oral exposures, yet higher throughput methods to screen NPs for potential neurotoxicity are lacking. The present study examined effects of 5 CeO2 (5- 1288 nm), and 4 TiO2 (6-142 nm) NPs and microparticles (MP) on network function in primary cultures of rat cortex on 12 well microelectrode array (MEA) plates. Particles were without cytotoxicity at concentrations ≤50 µg/ml. After recording 1 h of baseline activity prior to particle (3-50 µg/ml) exposure, changes in the total number of spikes (TS) and # of active electrodes (#AEs) were assessed 1, 24, and 48 h later. Following the 48 h recording, the response to a challenge with the GABAA antagonist bicuculline (BIC; 25 µM) was assessed. In all, particles effects were subtle, but 69 nm CeO2 and 25 nm TiO2 NPs caused concentration-related decreases in TS following 1 h exposure. At 48 h, 5 and 69 nm CeO2 and 25 and 31 nm TiO2 decreased #AE, while the two MPs increased #AEs. Following BIC, only 31 nm TiO2 produced concentration-related decreases in #AEs, while 1288 nm CeO2 caused concentration-related increases in both TS and #AE. The results indicate that some metal oxide particles cause subtle concentration-related changes in spontaneous and/or GABAA receptor-mediated neuronal activity in vitro at times when cytotoxicity is absent, and that MEAs can be used to screen and prioritize nanoparticles for neurotoxicity hazard.

A multiplexed assay for determination of neurotoxicant effects on spontaneous network activity and viability from microelectrode arrays.

Microelectrode array (MEA) recordings are increasingly being used as an in vitro method to detect and characterize the ability of drugs, chemicals and particles to cause neurotoxicity. While compound effects on spontaneous network activity are easily determined by MEA recordings, compound cytotoxicity is not routinely assessed, particularly within the same network from which recordings are collected. With the advent of higher-throughput 48 and 96 well MEA systems, rapid and simple methods to measure compound effects on cell health are required to facilitate efficient compound screening using MEAs. The present experiments sought to develop a multiplexed approach that allows measurement of network activity and cell health in the same MEA well. Primary cultures from rat cortex were exposed to six different compounds (glyphosate, ß-cyfluthrin, domoic acid, tributyltin, lindane and fipronil). Effects of these compounds (0.03-100 µM) on spontaneous network activity (mean firing rate; MFR), cellular metabolic activity (Cell Titer Blue™ (CTB) assay) and lactate dehydrogenase (LDH) release were determined in the same well following a 60-min exposure. Glyphosate elicited no effect on MFR, LDH release or CTB reduction. Tributyltin caused concomitant decreases in MFR and CTB reduction and increases LDH release, while domoic acid and ß-cyfluthrin decreased MFR in a concentration-dependent manner without altering either LDH release or CTB reduction. By contrast, lindane and fipronil did not alter LDH release or CTB reduction, but caused biphasic alterations in MFR, with increases in MFR at lower concentrations followed by decreases at higher concentrations. These results demonstrate a simple and rapid method for the simultaneous determination of test compound effects on spontaneous electrical activity and cell health from the same network, and will facilitate rapid screening of compounds for potential neurotoxicity.

Effect of treatment media on the agglomeration of titanium dioxide nanoparticles: impact on genotoxicity, cellular interaction, and cell cycle.

The widespread use of titanium dioxide (TiO2) nanoparticles in consumer products increases the probability of exposure to humans and the environment. Although TiO2 nanoparticles have been shown to induce DNA damage (comet assay) and chromosome damage (micronucleus assay, MN) in vitro, no study has systematically assessed the influence of medium composition on the physicochemical characteristics and genotoxicity of TiO2 nanoparticles. We assessed TiO2 nanoparticle agglomeration, cellular interaction, induction of genotoxicity, and influence on cell cycle in human lung epithelial cells using three different nanoparticle-treatment media: keratinocyte growth medium (KGM) plus 0.1% bovine serum albumin (KB); a synthetic broncheoalveolar lavage fluid containing PBS, 0.6% bovine serum albumin and 0.001% surfactant (DM); or KGM with 10% fetal bovine serum (KF). The comet assay showed that TiO2 nanoparticles induced similar amounts of DNA damage in all three media, independent of the amount of agglomeration, cellular interaction, or cell-cycle changes measured by flow cytometry. In contrast, TiO2 nanoparticles induced MN only in KF, which is the medium that facilitated the lowest amount of agglomeration, the greatest amount of nanoparticle cellular interaction, and the highest population of cells accumulating in S phase. These results with TiO2 nanoparticles in KF demonstrate an association between medium composition, particle uptake, and nanoparticle interaction with cells, leading to chromosomal damage as measured by the MN assay.