Isolated Kupffer Cells Research Articles

Hepatotoxicants induce cytokine imbalance in response to innate immune system

In recent years, attention has been paid to innate immune systems as mechanisms to initiate or promote drug-induced liver injury (DILI). Kupffer cells are hepatic resident macrophages and might be involved in the pathogenesis of DILI by release of pro- and anti-inflammatory mediators such as cytokines, chemokines, reactive oxygen species, and/or nitric oxides. The purpose of this study was to investigate alterations in mediator levels induced by hepatotoxic compounds in isolated Kupffer cells and discuss the relation between balance of each cytokine or chemokine and potential of innate immune-mediated DILI. Primary cultured rat Kupffer cells were treated with hepatotoxic (acetaminophen, troglitazone, trovafloxacin) or non-hepatotoxic (pioglitazone, levofloxacin) compounds with or without lipopolysaccharide (LPS). After 24 hr treatment, cell supernatants were collected and various levels of mediators released by Kupffer cells were examined. Although hepatotoxicants had no effect on the LPS-induced tumor necrosis factor-alpha (TNF-α) secretion, they enhanced the release of pro-inflammatory cytokine interleukin-1 beta (IL-1β) and suppressed the anti-inflammatory cytokines interleukin-6 (IL-6) and interleukin-10 (IL-10) induced by LPS. These cytokine shifts were not associated with switching the phenotypes of M1 and M2 macrophages in Kupffer cells. In conclusion, the present study suggested that the levels of some specific cytokines are affected by DILI-related drugs with LPS stimulation, and imbalance between pro- and anti-inflammatory cytokines, induced by the up-regulation of IL-1β and the down-regulation of IL-6 or IL-10, plays a key role in innate immune-mediated DILI.

Ron receptor-dependent gene regulation of Kupffer cells during endotoxemia

Ron receptor tyrosine kinase signaling in macrophages, including Kupffer cells and alveolar macrophages, suppresses endotoxin-induced proinflammatory cytokine/chemokine production. Further, we have also identified genes from Ron replete and Ron deplete livers that were differentially expressed during the progression of liver inflammation associated with acute liver failure in mice by microarray analyses. While important genes and signaling pathways have been identified downstream of Ron signaling during progression of inflammation by this approach, the precise role that Ron receptor plays in regulating the transcriptional landscape in macrophages, and particular in isolated Kupffer cells, has still not been investigated. Kupffer cells were isolated from wild-type (TK+/+) and Ron tyrosine kinase deficient (TK-/-) mice. Ex vivo, the cells were treated with lipopolysaccharide (LPS) in the presence or absence of the Ron ligand, hepatocyte growth factor-like protein (HGFL). Microarray and qRT-PCR analyses were utilized to identify alterations in gene expression between genotypes. Microarray analyses identified genes expressed differentially in TK+/+ and TK-/- Kupffer cells basally as well as after HGFL and LPS treatment. Interestingly, our studies identified Mefv, a gene that codes for the anti-inflammatory protein pyrin, as an HGFL-stimulated Ron-dependent gene. Moreover, lipocalin 2, a proinflammatory gene, which is induced by LPS, was significantly suppressed by HGFL treatment. Microarray results were validated by qRT-PCR studies on Kupffer cells treated with LPS and HGFL. The studies herein suggest a novel mechanism whereby HGFL-induced Ron receptor activation promotes the expression of anti-inflammatory genes while inhibiting genes involved in inflammation with a net effect of diminished inflammation in macrophages.

MicroRNA-223 Acts as an Important Regulator to Kupffer Cells Activation at the Early Stage of Con A-Induced Acute Liver Failure via AIM2 Signaling Pathway

Background: Acute liver failure (ALF), known as a rapid and severe clinical syndrome, can induce multiple organ dysfunction and failure. It was noticed that Kupffer cells activation at the initial phase was involved in some intense inflammatory responses in the pathogenesis of ALF. However, detailed regulation mechanism of Kupffer cells activation during ALF is still obscured. Present study aimed to discover the potential regulator and explore deeper information of Kupffer cells activation at the early stage of ALF. Methods: The mouse model of ALF was established by Concanavalin A injection. Dynamic immunological statuses of Kupffer cells at the early stage of ALF were exhibited by detecting typical cytokines. The expression of inflammasome AIM2 was measured in both RNA and protein level. Its role of affecting Kupffer cells activation during ALF by inducing IL-1β production was identified by RNA interference in vitro. Moreover, the expression of miR-223 in vivo was measured by q-PCR and its role in regulating Kupffer cells activation during Con A induced ALF was determined by RNAs transfection. Results: Present study showed that mass production of IL-1β from isolated Kupffer cells in Con A treated mice might be the main driving force of Kupffer cells pro-inflammatory activation during ALF. The role of AIM2 in affecting pro-inflammatory activation of Kupffer cells by inducing IL-1β production was crucial to ALF. Further study found that miR-223 acted as a regulator in Kupffer cells activation at the early stage of ALF by influencing IL-1β production via AIM2 pathway. Conclusion: For the first time, this paper demonstrated that miR-223 acted to inhibit IL-1β production via AIM2 pathway, suppressing Kupffer cells pro-inflammatory activation at the early stage of ALF. Thus, it played an important role in the pathogenesis of ALF.

The Effects of Twist-2 on Liver Endotoxin Tolerance Induced by a Low Dose of Lipopolysaccharide

Endotoxin tolerance is an important mechanism for preventing uncontrolled inflammatory cytokine production in bacterial sepsis. However, its molecular mechanisms remain largely unknown. It was reported that Twist-2 protein was a negative regulator for cytokine signaling by repressing the nuclear factor (NF)-κB-dependent cytokine pathway. However, the relationship between Twist-2 and endotoxin tolerance is unclear. Endotoxin tolerance models of BABL/c mice and isolated Kupffer cells (KCs) were established to observe the changes of Twist-2 during endotoxin tolerance. Then, Twist-2 shRNA was used to specifically inhibit Twist-2 gene in KCs to further explore the role of Twist-2 in endotoxin tolerance. The expression of Twist-2 was analyzed by immunohistochemistry, reverse transcription polymerase chain reaction, and Western blotting, respectively. The responses to lipopolysaccharide were assessed by the activation of nuclear factor-κB and the production of tumor necrosis factor-α. The histopathologic changes in the liver of the non-endotoxin tolerance group were more serious than those of the endotoxin tolerance group. Endotoxin tolerance also led to less activation of nuclear factor-κB, lower expression levels of tumor necrosis factor-α mRNA, and more expression of Twist-2 than those of non-endotoxin tolerance group in liver and KCs. Moreover, the inhibitive effects partly weaken in KCs transfected with Twist-2 shRNA. Twist-2 was involved in endotoxin tolerance through inhibiting NF-κB trans-activation and cytokines transcriptional activities. It may be a new target for the clinical treatment of sepsis and other inflammatory diseases.

Rolipram Attenuates Bile Duct Ligation–Induced Liver Injury in Rats: A Potential Pathogenic Role of PDE4

Anti-inflammatory and antifibrotic effects of the broad spectrum phosphodiesterase (PDE) inhibitor pentoxifylline have suggested an important role for cyclic nucleotides in the pathogenesis of hepatic fibrosis; however, studies examining the role of specific PDEs are lacking. Endotoxemia and Toll-like receptor 4 (TLR4)-mediated inflammatory and profibrotic signaling play a major role in the development of hepatic fibrosis. Because cAMP-specific PDE4 critically regulates lipopolysaccharide (LPS)-TLR4-induced inflammatory cytokine expression, its pathogenic role in bile duct ligation-induced hepatic injury and fibrogenesis in Sprague-Dawley rats was examined. Initiation of cholestatic liver injury and fibrosis was accompanied by a significant induction of PDE4A, B, and D expression and activity. Treatment with the PDE4-specific inhibitor rolipram significantly decreased liver PDE4 activity, hepatic inflammatory and profibrotic cytokine expression, injury, and fibrosis. At the cellular level, in relevance to endotoxemia and inflammatory cytokine production, PDE4B was observed to play a major regulatory role in the LPS-inducible tumor necrosis factor (TNF) production by isolated Kupffer cells. Moreover, PDE4 expression was also involved in the in vitro activation and transdifferentiation of isolated hepatic stellate cells (HSCs). Particularly, PDE4A, B, and D upregulation preceded induction of the HSC activation marker α-smooth muscle actin (α-SMA). In vitro treatment of HSCs with rolipram effectively attenuated α-SMA, collagen expression, and accompanying morphologic changes. Overall, these data strongly suggest that upregulation of PDE4 expression during cholestatic liver injury plays a potential pathogenic role in the development of inflammation, injury, and fibrosis.

Cannabidiol protects against hepatic ischemia/reperfusion injury by attenuating inflammatory signaling and response, oxidative/nitrative stress, and cell death

Ischemia/reperfusion (I/R) is a pivotal mechanism of liver damage after liver transplantation or hepatic surgery. We have investigated the effects of cannabidiol (CBD), the nonpsychotropic constituent of marijuana, in a mouse model of hepatic I/R injury. I/R triggered time‐dependent increases/changes in markers of liver injury, hepatic oxidative/nitrative stress, mitochondrial dysfunction, inflammation, stress signaling, and cell death. CBD significantly reduced the extent of liver inflammation, oxidative/nitrative stress, and cell death and also attenuated the bacterial endotoxin‐triggered NF‐κB activation and TNF‐α production in isolated Kupffer cells, likewise the adhesion molecule expression in primary human liver sinusoidal endothelial cells stimulated with TNF‐α and attachment of human neutrophils to the activated endothelium. These protective effects were preserved in CB2 knockout mice and were not prevented by CB1/2 antagonists in vitro. Thus, CBD may represent a novel, protective strategy against I/R injury by attenuating key inflammatory pathways and oxidative/nitrative tissue injury, independent of classical CB1/2 receptors.

The mechanism of protective effects of branched‐chain amino acids on hepatic ischemia/reperfusion‐induced liver injury in rats

We determined whether there is a protective effect of branched‐chain amino acid (BCAA) on hepatic ischemia/reperfusion (IR)‐induced acute liver injury. Wister rats were divided into four groups: simple laparotomy with vehicle; simple laparotomy with BCAA; IR (30 min clamp) with vehicle; and IR with BCAA. Serum liver function tests and the gene expression of adhesion molecules and endothelin‐1 in the liver were examined. In the in vivo study, portal venous pressure, leukocyte adhesion, and hepatic microcirculation were evaluated. Furthermore, Kupffer cells were isolated and cultured with BCAA in lipopolysaccharide (LPS). Increased levels of liver function tests, the expression of adhesion molecules and endothelin‐1, portal venous pressure, enhanced leukocyte adhesion and deteriorated hepatic microcirculation following IR were significantly attenuated by BCAA treatment. In the experiment using isolated Kupffer cells, the expression of inflammatory cytokine, and endothelin‐1 in response to LPS stimulation was attenuated by BCAA. Oral administration of BCAA has excellent therapeutic potential for IR‐induced liver injury. These effects may result from the direct attenuation of Kupffer cell activation.

Cytochrome P4502E1 inhibitor, chlormethiazole, decreases lipopolysaccharide‐induced inflammation in rat Kupffer cells with ethanol treatment

To investigate the role of Cytochrome P4502E1 in sensitizing Kupffer cells to lipopolysaccharide (LPS)-mediated inflammation after ethanol induction. Sprague-Dawley rats were fed a liquid ethanol diet, control diet or ethanol diet supplemented with CYP2E1 inhibitor, chlormethiazole (CMZ), for 4 weeks. Hepatic CYP2E1 protein, nuclear factor-kappa B (NF-κB) p65 protein and tumor necrosis factor (TNF)-α mRNA were measured. In vitro, isolated Kupffer cells from control rats were exposed to ethanol with different CMZ concentration; CYP2E1 expression and reactive oxygen species (ROS) generation were compared. The identified CMZ concentration was further utilized to evaluate the role of CYP2E1 on the sensitization of ethanol-induced Kupffer cell to LPS. The effect of LPS alone was tested in controlled Kupffer cells without ethanol. TNF-α, nuclear NF-κB p65 and cytoplasm IκB-α were monitored for all groups. Ethanol feeding increased hepatic CYP2E1 level, nuclear accumulation of NF-κB p65 and TNF-α expression in rats. These changes were inhibited by CMZ supplementation. In cultured Kupffer cells, increased CYP2E1 content and ROS production by in vitro ethanol induction were dose-dependently inhibited by CMZ. Compared with LPS alone, the ethanol induction group produced significantly more TNF-α, nuclear NF-κB p65 and less cytoplasm IκB-α under LPS stimuli. CMZ abolished the effects of ethanol on LPS-stimulated NF-κB translocation and TNF-α generation in Kupffer cells. In cultured Kupffer cell, using CMZ as inhibitor, ethanol-induced CYP2E1 overexpression was proved to contribute to the sensitization of Kupffer cells to LPS stimuli, with amplification of ROS production and activation of NF-κB, resulting in increased TNF-α production.

Protective effects of branched-chain amino acids on hepatic ischemia-reperfusion-induced liver injury in rats: a direct attenuation of Kupffer cell activation

We determined whether there is a protective effect of branched-chain amino acid (BCAA) on hepatic ischemia-reperfusion (I/R)-induced acute liver injury. Wister rats were divided into the following four groups: simple laparotomy with vehicle; simple laparotomy with BCAA (1 g/kg body wt orally); I/R (30 min clamp) with vehicle; and I/R with BCAA. Serum liver function tests and the gene expression of adhesion molecules (intercellular adhesion molecule and vascular cell adhesion molecule) and vasoconstrictor-related genes (endothelin-1) in the liver were examined. In the in vivo study, portal venous pressure, leukocyte adhesion, and hepatic microcirculation were evaluated. Furthermore, Kupffer cells were isolated and cultured with various concentrations of BCAA in the presence or absence of lipopolysaccharide (LPS). Increased levels of liver function tests following I/R were significantly attenuated by BCAA treatment. The increased expression of adhesion molecules and endothelin-1 was also significantly attenuated by BCAA treatment. Moreover, increased portal venous pressure, enhanced leukocyte adhesion, and deteriorated hepatic microcirculation following I/R were all improved by BCAA treatment. In the experiment using isolated Kupffer cells, the expression of interleukin-6, interleukin-1β, and endothelin-1 in response to LPS stimulation was attenuated by BCAA in a dose-dependent fashion. These results indicate that perioperative oral administration of BCAA has excellent therapeutic potential to reduce I/R-induced liver injury. These beneficial effects may result from the direct attenuation of Kupffer cell activation under stressful conditions.

Kupffer cells express a unique combination of phenotypic and functional characteristics compared with splenic and peritoneal macrophages

The immunostimulatory role of Kupffer cells in various inflammatory liver diseases is still not fully understood. In this study, phenotypic and functional aspects of Kupffer cells from healthy C57BL/6 mice were analyzed and compared with those of splenic and peritoneal macrophages to generate a blueprint of the cells under steady-state conditions. In the mouse liver, only one population of Kupffer cells was identified as F4/80(high)CD11b(low) cells. We observed that freshy isolated Kupffer cells are endocytic and show a relatively high basal ROS content. Interestingly, despite expression of TLR mRNA on Kupffer cells, ligation of TLR4, TLR7/8, and TLR9 resulted in a weak induction of IL-10, low or undetectable levels of IL-12p40 and TNF, and up-regulation of CD40 on the surface. Kupffer cells and splenic macrophages show functional similarities, in comparison with peritoneal macrophages, as reflected by comparable levels of TLR4, TLR7/8, and TLR9 mRNA and low or undetectable levels of TNF and IL-12p40 produced upon TLR ligation. The unique, functional characteristics of Kupffer cells, demonstrated in this study, suggest that Kupffer cells under steady-state conditions are specialized as phagocytes to clear and degrade particulates and only play a limited immunoregulatory role via the release of soluble mediators.

Liver and Skin Histopathology in Adults With Acid Sphingomyelinase Deficiency (Niemann-Pick Disease Type B)

Acid sphingomyelinase deficiency (ASMD) is a lysosomal storage disorder characterized by the pathologic accumulation of sphingomyelin (SM) in multiple cell types, and occurs most prominently within the liver, spleen, and lungs, leading to significant clinical disease. Seventeen ASMD patients underwent a liver biopsy during baseline screening for a phase 1 trial of recombinant human acid sphingomyelinase (rhASM) in adults with Niemann-Pick disease type B. Eleven of the 17 were enrolled in the trial and each received a single dose of rhASM and underwent a repeat liver biopsy on day 14. Biopsies were evaluated for fibrosis, SM accumulation, and macrophage infiltration by light and electron microscopy. When present, fibrosis was periportal and pericellular, predominantly surrounding affected Kupffer cells. Two baseline biopsies exhibited frank cirrhosis. SM was localized to isolated Kupffer cells in mildly affected biopsies and was present in both Kupffer cells and hepatocytes in more severely affected cases. Morphometric quantification of SM storage in liver biopsies ranged from 4% to 44% of the microscopic field. Skin biopsies were also performed at baseline and day 14 to compare the SM distribution in a peripheral tissue with that of liver. SM storage was present at lower levels in multiple cell types of the skin, including dermal fibroblasts, macrophages, vascular endothelial cells, vascular smooth muscle cells, and Schwann cells. This phase 1 trial of rhASM in adults with ASMD provided a unique opportunity for a prospective assessment of hepatic and skin pathology in this rare disease and their potential usage as pharmacodynamic biomarkers.

A new cannabinoid CB2 receptor agonist HU‐910 attenuates oxidative stress, inflammation and cell death associated with hepatic ischaemia/reperfusion injury

Cannabinoid CB(2) receptor activation has been reported to attenuate myocardial, cerebral and hepatic ischaemia-reperfusion (I/R) injury. We have investigated the effects of a novel CB(2) receptor agonist ((1S,4R)-2-(2,6-dimethoxy-4-(2-methyloctan-2-yl)phenyl)-7,7-dimethylbicyclo[2.2.1]hept-2-en-1-yl)methanol (HU-910) on liver injury induced by 1 h of ischaemia followed by 2, 6 or 24 h of reperfusion, using a well-established mouse model of segmental hepatic I/R. Displacement of [(3) H]CP55940 by HU-910 from specific binding sites in CHO cell membranes transfected with human CB(2) or CB(1) receptors (hCB(1/2) ) yielded K(i) values of 6 nM and 1.4 µM respectively. HU-910 inhibited forskolin-stimulated cyclic AMP production by hCB(2) CHO cells (EC(50) = 162 nM) and yielded EC(50) of 26.4 nM in [(35) S]GTPγS binding assays using hCB(2) expressing CHO membranes. HU-910 given before ischaemia significantly attenuated levels of I/R-induced hepatic pro-inflammatory chemokines (CCL3 and CXCL2), TNF-α, inter-cellular adhesion molecule-1, neutrophil infiltration, oxidative stress and cell death. Some of the beneficial effect of HU-910 also persisted when given at the beginning of the reperfusion or 1 h after the ischaemic episode. Furthermore, HU-910 attenuated the bacterial endotoxin-triggered TNF-α production in isolated Kupffer cells and expression of adhesion molecules in primary human liver sinusoidal endothelial cells stimulated with TNF-α. Pretreatment with a CB(2) receptor antagonist attenuated the protective effects of HU-910, while pretreatment with a CB(1) antagonist tended to enhance them. HU-910 is a potent CB(2) receptor agonist which may exert protective effects in various diseases associated with inflammation and tissue injury. This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.

Immunodetection of cyclooxygenase-2 (COX-2) is restricted to tissue macrophages in normal rat liver and to recruited mononuclear phagocytes in liver injury and cholangiocarcinoma

It has been suggested that cyclooxygenase-2 (COX-2)-mediated prostaglandin synthesis is associated with liver inflammation and carcinogenesis. The aim of this study is to identify the cellular source of COX-2 expression in different stages, from acute liver injury through liver fibrosis to cholangiocarcinoma (CC). We induced in rats acute and “chronic” liver injury (thioacetamide (TAA) or carbon tetrachloride (CCl4)) and CC development (TAA) and assessed COX-2 gene expression in normal and damaged liver tissue by RT-PCR of total RNA. The cellular localization of COX-2 protein in liver tissue was analyzed by immunohistochemistry as well as in isolated rat liver cells by Western blotting. The findings were compared with those obtained in human cirrhotic liver tissue. The specificity of the antibodies was tested by 2-DE Western blot and mass spectrometric identification of the positive protein spots. RT-PCR analysis of total RNA revealed an increase of hepatic COX-2 gene expression in acutely as well as “chronically” damaged liver. COX-2-protein was detected in those ED1+/ED2+ cells located in the non-damaged tissue (resident tissue macrophages). In addition COX-2 positivity in inflammatory mononuclear phagocytes (ED1+/ED2−), which were also present within the tumoral tissue was detected. COX-2 protein was clearly detectable in isolated Kupffer cells as well as (at lower level) in isolated “inflammatory” macrophages. Similar results were obtained in human cirrhotic liver. COX-2 protein is constitutively detectable in liver tissue macrophages. Inflammatory mononuclear phagocytes contribute to the increase of COX-2 gene expression in acute and chronic liver damage induced by different toxins and in the CC microenvironment.Electronic supplementary materialThe online version of this article (doi:10.1007/s00418-011-0889-9) contains supplementary material, which is available to authorized users.

Role of IL-17A in Neutrophil Recruitment and Hepatic Injury after Warm Ischemia–Reperfusion Mice

Recent evidence suggests that IL-17A regulates neutrophil-dependent organ injury. Accordingly, the purpose of this study was to determine the role of IL-17A in neutrophil recruitment after ischemia-reperfusion (I/R) and in subsequent liver injury. Two mouse models including wild-type and IL-17A knockout mice were evaluated for I/R injury. The medial largest lobe of the liver was clamped for 90 min. In another set of experiments, recombinant mouse (rm)IL-17A homodimer or rmIL-17A/F heterodimer were administered to knockout mice before I/R, and liver injury was investigated. Isolated Kupffer cells were incubated with rmIL-17A or rmIL-17F, and production of TNF-α was measured. Studies evaluating the extent of liver injury as measured by serum transaminase levels demonstrated similar levels in the acute phase (6 h) in these two models. In contrast, in the subacute phase (20 h) after I/R, both serum transaminase levels and percent of hepatic necrosis were significantly reduced in the knockout mice compared with the wild-type mice. This reduction in liver injury seen in the knockout mice was associated with suppression of chemokine and adhesion molecule expression and reduction in infiltration of neutrophils into the liver. Administration of rmIL-17A homodimer, but not IL-17A/F heterodimer, increased liver injury in the subacute phase of I/R in KO mice. TNF-α production by isolated Kupffer cells increased significantly in the cells incubated with rmIL-17A compared with rmIL-17F. These results indicate that IL-17A is a key regulator in initiating neutrophil-induced inflammatory responses and hepatic injury in the subacute phase after reperfusion.

Endothelial NO/cGMP/VASP Signaling Attenuates Kupffer Cell Activation and Hepatic Insulin Resistance Induced by High-Fat Feeding

OBJECTIVEProinflammatory activation of Kupffer cells is implicated in the effect of high-fat feeding to cause liver insulin resistance. We sought to determine whether reduced endothelial nitric oxide (NO) signaling contributes to the effect of high-fat feeding to increase hepatic inflammatory signaling and if so, whether this effect 1) involves activation of Kupffer cells and 2) is ameliorated by increased NO signaling.RESEARCH DESIGN AND METHODSEffect of NO/cGMP signaling on hepatic inflammation and on isolated Kupffer cells was examined in C57BL/6 mice, eNos−/− mice, and Vasp−/− mice fed a low-fat or high-fat diet.RESULTSWe show that high-fat feeding induces proinflammatory activation of Kupffer cells in wild-type mice coincident with reduced liver endothelial nitric oxide synthase activity and NO content while, conversely, enhancement of signaling downstream of endogenous NO by phosphodiesterase-5 inhibition protects against high fat–induced inflammation in Kupffer cells. Furthermore, proinflammatory activation of Kupffer cells is evident in eNos−/− mice even on a low-fat diet. Targeted deletion of vasodilator-stimulated phosphoprotein (VASP), a key downstream target of endothelially derived NO, similarly predisposes to hepatic and Kupffer cell inflammation and abrogates the protective effect of NO signaling in both macrophages and hepatocytes studied in a cell culture model.CONCLUSIONSThese results collectively imply a physiological role for endothelial NO to limit obesity-associated inflammation and insulin resistance in hepatocytes and support a model in which Kupffer cell activation during high-fat feeding is dependent on reduced NO signaling. Our findings also identify the NO/VASP pathway as a novel potential target for the treatment of obesity-associated liver insulin resistance.

Cannabinoid CB2 receptors protect against alcoholic liver disease by regulating Kupffer cell polarization in mice

Activation of Kupffer cells plays a central role in the pathogenesis of alcoholic liver disease. Because cannabinoid CB2 receptors (CB2) display potent anti-inflammatory properties, we investigated their role in the pathogenesis of alcoholic liver disease, focusing on the impact of CB2 on Kupffer cell polarization and the consequences on liver steatosis. Wild-type (WT) mice fed an alcohol diet showed an induction of hepatic classical (M1) and alternative (M2) markers. Cotreatment of alcohol-fed mice with the CB2 agonist, JWH-133, decreased hepatic M1 gene expression without affecting the M2 profile. In keeping with this, genetic ablation of CB2 enhanced hepatic induction of M1 gene signature and blunted the induction of M2 markers. CB2 also modulated alcohol-induced fatty liver, as shown by the reduction of hepatocyte steatosis in JWH-133-treated mice and its enhancement in CB2-/- animals. Studies in isolated Kupffer cells and cultured macrophages further demonstrated that CB2 inhibits M1 polarization and favors the transition to an M2 phenotype. In addition, conditioned-medium experiments showed that preventing M1 polarization in CB2-activated macrophages protects from lipid accumulation in hepatocytes. Heme oxygenase-1 (HO-1) mediated the anti-inflammatory effects of CB2 receptors. Indeed, alcohol-fed mice treated with JWH-133 showed increased hepatic expression of macrophage HO-1, as compared to vehicle-treated counterparts. In keeping with this, JWH-133 induced HO-1 expression in cultured macrophages, and the HO-1 inhibitor, zinc protoporphyrin, blunted the inhibitory effect of JWH-133 on lipopolysaccharide-induced nuclear factor-kappa B activation and M1 polarization. Altogether, these findings demonstrate that CB2 receptors display beneficial effects on alcohol-induced inflammation by regulating M1/M2 balance in Kupffer cells, thereby reducing hepatocyte steatosis via paracrine interactions between Kupffer cells and hepatocytes. These data identify CB2 agonists as potential therapeutic agents for the management of alcoholic liver disease.

Cannabidiol protects against hepatic ischemia/reperfusion injury by attenuating inflammatory signaling and response, oxidative/nitrative stress, and cell death

Ischemia/reperfusion (I/R) is a pivotal mechanism of liver damage after liver transplantation or hepatic surgery. We have investigated the effects of cannabidiol (CBD), the nonpsychotropic constituent of marijuana, in a mouse model of hepatic I/R injury. I/R triggered time-dependent increases/changes in markers of liver injury (serum transaminases), hepatic oxidative/nitrative stress (4-hydroxy-2-nonenal, nitrotyrosine content/staining, and gp91phox and inducible nitric oxide synthase mRNA), mitochondrial dysfunction (decreased complex I activity), inflammation (tumor necrosis factor α (TNF-α), cyclooxygenase 2, macrophage inflammatory protein-1α/2, intercellular adhesion molecule 1 mRNA levels; tissue neutrophil infiltration; nuclear factor κB (NF-κB) activation), stress signaling (p38MAPK and JNK), and cell death (DNA fragmentation, PARP activity, and TUNEL). CBD significantly reduced the extent of liver inflammation, oxidative/nitrative stress, and cell death and also attenuated the bacterial endotoxin-triggered NF-κB activation and TNF-α production in isolated Kupffer cells, likewise the adhesion molecule expression in primary human liver sinusoidal endothelial cells stimulated with TNF-α and attachment of human neutrophils to the activated endothelium. These protective effects were preserved in CB2 knockout mice and were not prevented by CB1/2 antagonists in vitro. Thus, CBD may represent a novel, protective strategy against I/R injury by attenuating key inflammatory pathways and oxidative/nitrative tissue injury, independent of classical CB1/2 receptors.

Myeloperoxidase and elastase are only expressed by neutrophils in normal and in inflammed liver

Myeloperoxidase (MPO) is involved in acute and chronic inflammatory diseases. The source of MPO in acute liver diseases is still a matter of debate. Therefore, we analysed MPO-gene expression on sections from normal and acutely damaged [carbon tetrachloride-(CCl4) or whole liver γ-Irradiation] rat liver by immunohistochemistry, real time PCR and Western blot analysis of total RNA and protein. Also total RNA and protein from isolated Kupffer cells, hepatic stellate cells, Hepatocytes, endothelial cells and neutrophil granulocytes (NG) was analysed by real time PCR and Western blot, respectively. Sections of acutely injured human liver were prepared for MPO and CD68 immunofluorescence double staining. In normal rat liver MPO was detected immunohistochemically and by immunofluorescence double staining only in single NG. No MPO was detected in isolated parenchymal and non-parenchymal cell populations of the normal rat liver. In acutely damaged rat liver mRNA of MPO increased 2.8-fold at 24 h after administration of CCl4 and 3.3-fold at 3 h after γ-Irradiation and MPO was detected by immunofluorescence double staining only in elastase (NE) positive NGs but not in macrophages (ED1 or CD68 positive cells). Our results demonstrate that, increased expression of MPO in damaged rat and human liver is due to recruited elastase positive NGs.

PP-047 Method to isolate Kupffer cells from rats

PP-047 Method to isolate Kupffer cells from rats

Apobec-1 Complementation Factor Modulates Liver Regeneration by Post-transcriptional Regulation of Interleukin-6 mRNA Stability

Apobec-1 complementation factor (ACF) is the RNA binding subunit of a core complex that mediates C to U RNA editing of apolipoprotein B (apoB) mRNA. Targeted deletion of the murine Acf gene is early embryonic lethal and Acf(-/-) blastocysts fail to implant and proliferate, suggesting that ACF plays a key role in cell growth and differentiation. Here we demonstrate that heterozygous Acf(+/-) mice exhibit decreased proliferation and impaired liver mass restitution following partial hepatectomy (PH). To pursue the mechanism of impaired liver regeneration we examined activation of interleukin-6 (IL-6) a key cytokine required for induction of hepatocyte proliferation following PH. Peak induction of hepatic IL-6 mRNA abundance post PH was attenuated >80% in heterozygous Acf(+/-) mice, along with decreased serum IL-6 levels. IL-6 secretion from isolated Kupffer cells (KC) was 2-fold greater in wild-type compared with heterozygous Acf(+/-) mice. Recombinant ACF bound an AU-rich region in the IL-6 3'-untranslated region with high affinity and IL-6 mRNA half-life was significantly shorter in KC isolated from Acf(+/-) mice compared with wild-type controls. These findings suggest that ACF regulates liver regeneration following PH at least in part by controlling the stability of IL-6 mRNA. The results further suggest a new RNA target and an unanticipated physiological function for ACF beyond apoB RNA editing.