Kupffer Cell Depletion Research Articles

Nuclear Factor High-mobility Group Box1 Mediating the Activation of Toll-like Receptor 4 Signaling in Hepatocytes in the Early Stage of Non-alcoholic Fatty Liver Disease in Mice

One of the challenges surrounding nonalcoholic fatty liver disease (NAFLD) is to discover the mechanisms that underlie the initiation of it. The aim of this study was to elucidate the effects of toll-like receptor 4 (TLR4) signaling in liver parenchymal cells during the early stage of NAFLD. Male TLR4-wildtype, TLR4-knockout, TLR2-knockout, MyD88-knockout, and TRIF-knockout mice were fed a normal diet or high-fat diet (HFD). Liver steatosis, alanine aminotrans-ferase levels, nuclear translocation of nuclear factor kappa B (NF-κB) (P65), macrophage accumulation, and neutrophil infiltration were assessed. Using Kupffer cell depletion or bone-marrow transplantation, we examined the potential role of Kupffer cells and myeloid infiltrating cells during the initiation of NAFLD. Immunohistochemistry and western blotting were implemented to determine the release of high-mobility group box1 (HMGB1). The neutral antibody against HMGB1 was used to block the activity of free HMGB1. Here, we report that the activation of TLR4 signaling in hepatocytes, accompanied with the relocation of P65 in nucleus, was proven to play an important role during the initiation of NAFLD. Importantly, HMGB1 releasing from hepatocytes in response to free fatty acid (FFA) infusion was first reported as the key molecule for the TLR4/MyD88 activation and cytokines expression in vitro and in vivo. Treatment with neutralizing antibody to HMGB1 protects against FFA-induced tumor necrosis factor alpha and interleukin-6 production. Conclusion: Our study supports the notion that TLR4/MyD88 signaling in liver parenchymal cells plays a pivotal role during the early progression of HFD-induced NAFLD, in which free HMGB1 served as a positive component mediating TLR4 activation.

Kupffer cell depletion prevents but has no therapeutic effect on metabolic and inflammatory changes induced by a high‐fat diet

We aimed to evaluate activation of macrophages in insulin-sensitive tissues (liver, adipose tissue, and muscles) under high-fat diet (HFD) and elucidate the role of Kupffer cells (KC) in HFD-induced insulin resistance. Tissue macrophage populations, insulin signaling, and sensitivity were evaluated in mice fed a HFD for 4 or 16 wk. Selective KC depletion was obtained by intravenous injections of liposome-encapsulated clodronate. Mice fed a HFD for 4 to 16 wk have hepatic and peripheral insulin resistance together with macrophage recruitment in the adipose tissue but not in the liver. Depletion of KC for the last 10 d of the 16 wk experiment fails to improve insulin sensitivity compared to PBS-treated animals. In contrast, preventive KC depletion prior to and during the 4 wk HFD attenuates the development of obesity, adiposity, adipose tissue inflammation (P<0.01 vs. PBS group), and insulin resistance (P<0.01). Interestingly, in mice fed a normal diet, prolonged KC depletion ameliorates insulin sensitivity and decreases adiposity without altering physiological body weight gain or food intake. Preventive and prolonged KC depletion ameliorates insulin sensitivity and prevents adipose tissue inflammation, suggesting a communication between the liver and the adipose tissue in the development of HFD-induced metabolic alterations.

Nuclear factor high-mobility group box1 mediating the activation of toll-like receptor 4 signaling in hepatocytes in the early stage of nonalcoholic fatty liver disease in mice

One of the challenges surrounding nonalcoholic fatty liver disease (NAFLD) is to discover the mechanisms that underlie the initiation of it. The aim of the present study was to elucidate the effects of Toll-like receptor 4 (TLR4) signaling in liver parenchymal cells during the early stage of NAFLD. Male TLR4-wildtype, TLR4-knockout, TLR2-knockout, MyD88-knockout, and TRIF-knockout mice were fed a normal diet or high-fat diet (HFD). Liver steatosis, alanine aminotransferase levels, nuclear translocation of nuclear factor kappa B (NF-κB) (p65), macrophage accumulation, and neutrophil infiltration were assessed. Using Kupffer cell depletion or bone marrow transplantation, we examined the potential role of Kupffer cells and myeloid infiltrating cells during the initiation of NAFLD. Immunohistochemistry and western blotting were implemented to determine the release of high-mobility group box1 (HMGB1). The neutral-antibody against HMGB1 was used to block the activity of free HMGB1. Here we report that the activation of TLR4 signaling in hepatocytes, accompanied with the relocation of P65 in nucleus, was proven to play an important role during the initiation of NAFLD. Importantly, HMGB1 releasing from hepatocytes in response to free fatty acid (FFA) infusion was first reported as the key molecule for the TLR4/MyD88 activation and cytokines expression in vitro and in vivo. Treatment with neutralizing antibody to HMGB1 protects against FFA-induced tumor necrosis factor alpha and interleukin-6 production. Our study supports the notion that TLR4/MyD88 signaling in liver parenchymal cells plays a pivotal role during the early progression of HFD-induced NAFLD, in which free HMGB1 served as a positive component mediating TLR4 activation.

658. Enhancing Systemic Delivery of Recombinant Adeno-Associated Viruses through Kupffer Cell Depletion

658. Enhancing Systemic Delivery of Recombinant Adeno-Associated Viruses through Kupffer Cell Depletion

Systemic Effects of White Adipose Tissue Dysregulation and Obesity‐Related Inflammation

Systemic Effects of White Adipose Tissue Dysregulation and Obesity‐Related Inflammation

Kupffer cell depletion reduces hepatic inflammation and apoptosis but decreases survival in abdominal sepsis

During abdominal sepsis, the activation of hepatic Kupffer cells (KC) and its consequences are of central interest. This study evaluates the impact of selective KC depletion on hepatic microcirculation, cytokine release, and systemic alterations in the colon ascendens stent peritonitis (CASP), a model of polymicrobial abdominal sepsis. For KC depletion clodronate liposomes were injected 24 h before CASP surgery in female C57BL/6N mice. Three and 12 h after CASP, in-vivo fluorescence microscopy of the liver was performed. Analysis of hepatocellular apoptosis was conducted by immunohistochemistry. In addition, levels of tumor necrosis factor (TNF), IL-6, and IL-10 in the liver, lungs, spleen, and plasma were determined, and bacteriology and survival analysis were performed. CASP led to significant sinusoidal perfusion failure, increased leukocyte recruitment, hepatocellular apoptosis and increased levels of TNF, IL-6, and IL-10 in the liver and plasma. KC depletion before CASP significantly reduced leukocyte recruitment to the liver and hepatocellular apoptosis. IL-10 secretion decreased dramatically in the liver and plasma of KC-depleted septic mice. In contrast, TNF levels were clearly elevated after clodronate treatment. In the lung and spleen, a compensatory upregulation of IL-10 could be detected after KC depletion. Clodronate treatment resulted in a significant reduction in survival. The results indicate that KC depletion is locally protective in polymicrobial abdominal sepsis, as it reduces hepatic inflammation and apoptosis. These effects could be observed in the presence of clearly elevated TNF levels. However, the lack of IL-10 in KC-depleted mice resulted in a detrimental systemic proinflammation.

Evaluation of innate immunity and vector toxicity following inoculation of bovine, porcine or human adenoviral vectors in a mouse model

Nonhuman adenovirus (Ad) vectors derived from bovine Ad serotype 3 (BAd3) or porcine Ad serotype 3 (PAd3) can circumvent pre-existing immunity against human Ad (HAd). We have previously reported differential transduction of human and nonhuman cells by these Ad vectors, and their distinct receptor usage and biodistribution. To compare the induction of innate immunity, vector toxicity and vector uptake by Kupffer cells (KCs) following intravenous administration of PAd3, BAd3, or HAd5 vectors in mice, we determined mRNA expression levels of proinflammatory chemokines and cytokines, and Toll-like receptors (TLRs) in the liver and spleen. Tissue toxicity of these vectors was assessed by comparing serum levels of liver-specific enzymes, histopathology and Kupffer cell (KC) depletion. Compared to the HAd5 vector, PAd3 and BAd3 vectors were more potent stimulators of innate immune responses as indicated by enhanced mRNA expression of TLRs and proinflammatory chemokines and cytokine genes. Histopathological changes in the liver were most pronounced in HAd5-inoculated mice while BAd3- or PAd3-inoculated mice revealed mild histologic changes that were confined to early time points. Inoculation with HAd5 or PAd3 vectors resulted in a significant ( P < 0.05) decline of the number of KCs in the liver. Together, these results extend our previous observations regarding distinct in vivo biology of nonhuman and human Ad vectors.

74 Toll-Like Receptor (TLR) 2 in Kupffer Cells Plays a Crucial Role in the Development of Steatohepatitis in Mice

Background: Gut-derived bacterial products are candidates for a promoting factor to progress non-alcoholic steatohepatitis (NASH). Toll-like receptors are pattern recognition receptors that sense microbial products such as LPS. We have previously shown that TLR9, a receptor for bacterial DNA, plays an important role in the development of NASH. However, little information on TLR2, a receptor for Gram-positive bacterial products such as peptidoglycan, is available in that process. In addition, the role of TLR2 in Kupffer cells, hepatic macrophages, remain unknown. Materials and Methods: WT and TLR2 KO were fed choline-deficient (CDAA) or Choline-supplemented control (CSAA) diet for 22 weeks. Steatosis, inflammation and fibrosis were assessed by histological examination, quantitative real-time PCR and ELISA. To examine the role of Kupffer cells in the development of steatohepatitis, WT mice were injected clodronate to deplete Kupffer cells, followed by CDAA diet feeding for 2 weeks. To further explore whether Kupffer cells mediate TLR2-dependent inflammation and fibrosis, we generated TLR2-chimeric mice using a combination of Kupffer cell depletion, irradiation and bone-marrow transplantation, followed by CDAA diet feeding for 22 weeks. Results: After 22 weeks of CDAA diet feeding, WT mice showed typical steatohepatitis characterized by severe steatosis, inflammatory cell infiltration and hepatocyte ballooning as well as perisinusoidal fibrosis. In contrast, TLR2 KOmice exhibited less inflammatory cell infiltration and hepatocyte ballooning. NAFLD activity score in TLR2KO mice was significantly lower than that in WT mice. In addition, TLR2KO mice showed a significant reduction in fibrosis compared with WT mice. In Kupffer cells-depleted WT mice, diet-induced steatohepatitis and fibrosis were significantly suppressed compared with WT mice without Kupffer cell depletion. In chimeric mice, full reconstitution of Kupffer cells was achieved, but hepatic stellate cells were not replaced by bone marrow-derived cells. WT mice containing Tlr2deficient bone marrow showed suppressed steatohepatitis and fibrosis, whereas Tlr2-deficient mice containing wild-type bone marrow showed advanced steatohepatitis and fibrosis. Conclusion: TLR2 expression in Kupffer cells plays a crucial role in CDAA diet-induced steatohepatitis and fibrosis in mice.

Adenovirus infection results in alterations of insulin signaling and glucose homeostasis

Recombinant adenovirus (Ad) vectors can initiate an inflammatory response, limiting its use in gene therapy and basic research. Despite increased efforts to better understand Ad infection, little is known about how it affects cellular metabolic responses. In the current studies, we explored the effects of Ad vectors on insulin signaling molecules and glucose homeostasis. Nonreplicative Ad vectors were injected into rats through the tail vein, and at 4-13 days postinjection insulin signaling and glucose tolerance were examined. Ad vector infection significantly reduced total levels of the insulin receptor (IR), and insulin receptor substrates 1 and 2 (IRS-1, IRS-2) in the liver of rats, resulting in decreased insulin-induced tyrosine phosphorylation of IR, IRS-1, and IRS-2, and decreased interaction of IRS-1 and IRS-2 with phosphoinositide 3-kinase (PI3K). In addition, Ad infection resulted in impaired systemic glucose homeostasis, which recovered by 13 days, after the protein levels of IR, IRS-1, and IRS-2 had started to normalize. Expression of a TNF inhibitor or Kupffer cell depletion attenuated the Ad vector-induced decreases of insulin signaling molecules, indicating a potential role of Kupffer cell activation in this process. These studies provide evidence that systemic administration of Ad vectors can impair insulin signaling in liver, resulting in altered systemic glucose metabolism. Thus, effects of Ad vector infection on insulin action and glucose metabolism need to be considered when Ad vectors are used in research or gene therapy and may be more broadly applicable to other viral agents.

Neutrophil Depletion Blocks Early Collagen Degradation in Repairing Cholestatic Rat Livers

Biliary obstruction results in a well-characterized cholestatic inflammatory and fibrogenic process; however, the mechanisms and potential for liver repair remain unclear. We previously demonstrated that Kupffer cell depletion reduces polymorphonuclear cell (neutrophil) (PMN) and matrix metalloproteinase (MMP)8 levels in repairing liver. We therefore hypothesized that PMN-dependent MMP activity is essential for successful repair. Male Sprague-Dawley rats received reversible biliary obstruction for 7 days, and the rat PMN-specific antibody RP3 was administered 2 days before biliary decompression (repair) and continued daily until necropsy, when liver underwent morphometric analysis, immunohistochemistry, quantitative RT-PCR, and in situ zymography. We found that RP3 treatment did not reduce Kupffer cell or monocyte number but significantly reduced PMN number at the time of decompression and 2 days after repair. RP3 treatment also blocked resorption of type I collagen. In addition, biliary obstruction resulted in increased expression of MMP3, MMP8, and tissue inhibitor of metalloproteinase 1. Two days after biliary decompression, both MMP3 and tissue inhibitor of metalloproteinase 1 expression declined toward sham levels, whereas MMP8 expression remained elevated and was identified in bile duct epithelial cells by immunohistochemistry. PMN depletion did not alter the hepatic expression of these genes. Conversely, collagen-based in situ zymography demonstrated markedly diminished collagenase activity following PMN depletion. We conclude that PMNs are essential for collagenase activity and collagen resorption during liver repair, and speculate that PMN-derived MMP8 or PMN-mediated activation of intrinsic hepatic MMPs are responsible for successful liver repair.

Kupffer cells participate in 2-butoxyethanol-induced liver hemangiosarcomas

2-Butoxyethanol increases hemangiosarcomas selectively in male mouse liver after chronic inhalation through mechanisms that have not fully been elucidated. Hemolysis, a primary toxic effect associated with 2-butoxyethanol exposure in rodents, increased hemosiderin (iron) deposition in Kupffer cells in the liver. These findings, along with the induction of hepatic neoplastic lesions, led to our hypothesis that the induction hemangiosarcomas by 2-butoxyethanol is due to the activation of Kupffer cells, subsequent to hemolysis, that results in the induction of DNA synthesis in target cells (endothelial cells); allowing for the selective proliferation of preneoplastic target cells and/or the promotion of new initiated cells. The present studies were conducted to determine whether Kupffer cells contributed to 2-butoxyethanol-induced endothelial DNA synthesis in the liver, thereby determining whether a linkage exists between these events. Male B6C3F1 mice were treated with 450 and 900 mg/kg 2-butoxyethanol (via daily gavage; 5×/week) for 7 days in the presence or absence of Kupffer cell depletion (via clodronate-encapsulated liposomes). 2-Butoxyethanol (450 and 900 mg/kg/day) increased the number of F4/80 stained cells (Kupffer cells) compared to controls (∼1.3- and ∼1.6-fold over control, respectively). Clodronate liposome treatment reduced the number of Kupffer cells by >90%, as assessed by F4/80 immunohistochemistry. Increased hemolysis, measured by increases in relative spleen weights and decreased hematocrit was confirmed in 2-butoxyethanol treated mice. The percentage of iron-stained endothelial cells increased by ∼11-fold over control, and endothelial cell DNA synthesis increased ∼1.7-fold over control in 2-butoxyethanol exposed mice. Importantly, Kupffer cell depletion reduced 2-butoxyethanol-induced iron staining and hepatic endothelial cell DNA synthesis. These studies provide evidence supporting the hypothesis that the Kupffer cell modulates 2-butoxyethanol-induced endothelial cell DNA synthesis, and therefore may contribute to hemangiosarcoma induction by 2-butoxyethanol.

Kupffer cell activation is a causal factor for hepatic insulin resistance

Recruited adipose tissue macrophages contribute to chronic and low-grade inflammation causing insulin resistance in obesity. Similarly, we hypothesized here that Kupffer cells, the hepatic resident macrophages, play a pathogenic role in hepatic insulin resistance induced by a high-fat diet. Mice were fed a normal diet or high-fat diet for 3 days. Kupffer cell activation was evaluated by immunohistochemistry and quantitative RT-PCR. Insulin sensitivity was assessed in vivo by hyperinsulinemic-euglycemic clamp and insulin-activated signaling was investigated by Western blot. Liposome-encapsulated clodronate was injected intravenously to deplete macrophages prior to a short-term exposure to high-fat diet. Here, we characterized a short-term high-fat diet model in mice and demonstrated early hepatic insulin resistance and steatosis concurrent with Kupffer cell activation. We demonstrated that selective Kupffer cell depletion obtained by intravenous clodronate, without affecting adipose tissue macrophages, was sufficient to enhance insulin-dependent insulin signaling and significantly improve hepatic insulin sensitivity in vivo in this short-term high-fat diet model. Our study clearly shows that hepatic macrophage response participates to the onset of high-fat diet-induced hepatic insulin resistance and may therefore represent an attractive target for prevention and treatment of diet- and obesity-induced insulin resistance.

Kupffer Cells Promote Hepatic Steatosis Via Interleukin-1β–Dependent Suppression of Peroxisome Proliferator-Activated Receptor α Activity

Kupffer cells have been implicated in the pathogenesis of various liver diseases. However, their involvement in metabolic disorders of the liver, including fatty liver disease, remains unclear. The present study sought to determine the impact of Kupffer cells on hepatic triglyceride storage and to explore the possible mechanisms involved. To that end, C57Bl/6 mice rendered obese and steatotic by chronic high-fat feeding were treated for 1 week with clodronate liposomes, which cause depletion of Kupffer cells. Loss of expression of marker genes Cd68, F4/80, and Clec4f, and loss of Cd68 immunostaining verified almost complete removal of Kupffer cells from the liver. Also, expression of complement components C1, the chemokine (C-C motif) ligand 6 (Ccl6), and cytokines interleukin-15 (IL-15) and IL-1beta were markedly reduced. Importantly, Kupffer cell depletion significantly decreased liver triglyceride and glucosylceramide levels concurrent with increased expression of genes involved in fatty acid oxidation including peroxisome proliferator-activated receptor alpha (PPARalpha), carnitine palmitoyltransferase 1A (Cpt1alpha), and fatty acid transport protein 2 (Fatp2). Treatment of mice with IL-1beta decreased expression of PPARalpha and its target genes, which was confirmed in primary hepatocytes. Consistent with these data, IL-1beta suppressed human and mouse PPARalpha promoter activity. Suppression of PPARalpha promoter activity was recapitulated by overexpression of nuclear factor kappaB (NF-kappaB) subunit p50 and p65, and was abolished upon deletion of putative NF-kappaB binding sites. Finally, IL-1beta and NF-kappaB interfered with the ability of PPARalpha to activate gene transcription. Our data point toward important cross-talk between Kupffer cells and hepatocytes in the regulation of hepatic triglyceride storage. The effect of Kupffer cells on liver triglycerides are at least partially mediated by IL-1beta, which suppresses PPARalpha expression and activity.

Kupffer Cell and Interleukin-12–Dependent Loss of Natural Killer T Cells in Hepatosteatosis

Hepatosteatosis is associated with increased expression of tumor necrosis factor alpha (TNF-alpha) and interleukin (IL)-12, major T helper (Th) 1 cytokines, and reduced hepatic natural killer T (NKT) cell numbers. The relationship between lipid accumulation, cytokine expression, and hepatic NKT cells is not known. This study was conducted to assess the role of IL-12 in the development of hepatic steatosis and its potential impact on liver NKT cells. Male C57Bl/6 wildtype (WT) and IL-12-deficient (IL-12(-/-)) mice were fed a choline-deficient diet (CDD) for 0, 10, or 20 weeks. CDD led to marked hepatosteatosis, reduced hepatic but not splenic NKT cell numbers and function, and increased hepatic expression of the T(h)1-type cytokines IL-12, interferon gamma (IFN-gamma), and TNF-alpha in WT mice. The absence of IL-12 resulted in similar CDD-induced hepatosteatosis, but preserved hepatic NKT cells and significantly reduced hepatic IFN-gamma and TNF-alpha expression. Treatment of CDD-fed mice with lipopolysaccharide led to a significant increase in hepatic IL-12 expression, and Kupffer cell (KC) depletion reduced liver IL-12 expression and restored NKT cells in CDD-induced fatty liver. Interestingly, KCs from CDD-fed mice failed to produce increased quantities of IL-12 upon activation in vitro when compared to similarly treated KCs from control fed mice, suggesting that secondary factors in vivo promote heightened IL-12 production. Finally, human livers with severe steatosis showed a substantial decrease in NKT cells. Hepatosteatosis reduces the numbers of hepatic NKT cells in a KC-and IL-12-dependent manner. Our results suggest a pivotal and multifunctional role of KC-derived IL-12 in the altered immune response in steatotic liver, a process that is likely active within human nonalcoholic fatty liver disease.

Role of Acid Sphingomyelinase of Kupffer Cells in Cholestatic Liver Injury in Mice

Kupffer cells, resident tissue macrophages of the liver, play a key role in the regulation of hepatic inflammation, hepatocyte death, and fibrosis that characterize liver diseases. However, it is controversial whether Kupffer cells promote or protect from liver injury. To explore this issue we examined the role of Kupffer cells in liver injury, cell death, regeneration, and fibrosis on cholestatic liver injury in C57BL/6 mice using a model of partial bile duct ligation (BDL), in which animals do not die and the effects of BDL can be compared between injured ligated lobes and nonligated lobes. In cholestatic liver injury, the remaining viable cells represented tolerance for tumor necrosis factor alpha (TNF-alpha)-induced hepatocyte apoptosis and regenerative features along with AKT activation. Inhibition of AKT by adenovirus expressing dominant-negative AKT abolished the survival and regenerative properties in hepatocytes. Moreover, Kupffer cell depletion by alendronate liposomes increased hepatocyte damage and the sensitivity of TNF-alpha-induced hepatocyte apoptosis in ligated lobes. Kupffer cell depletion decreased hepatocyte regeneration and liver fibrosis with reduced AKT activation. To investigate the impact of acid sphingomyelinase (ASMase) in Kupffer cells, we generated chimeric mice that contained ASMase-deficient Kupffer cells and -sufficient hepatocytes using a combination of Kupffer cell depletion, irradiation, and the transplantation of ASMase-deficient bone marrow cells. In these mice, AKT activation, the tolerance for TNF-alpha-induced apoptosis, and the regenerative responses were attenuated in hepatocytes after BDL. Kupffer cells have a protective role for hepatocyte damage and promote cell survival, liver regeneration, and fibrosis in cholestatic liver disease. Kupffer cell-derived ASMase is crucial for AKT activation of hepatocytes that is required for the survival and regenerative responses.

C-Jun Terminal Kinase-2 Gene Deleted Mice Overexpress Hemeoxygenase-1 and Are Protected From Hepatic Ischemia Reperfusion Injury

Targeted deletion of c-jun amino terminal kinase-2 (jnk-2) upregulates the activator protein-1 transcription factor system. We hypothesized that this would lead to induction of hemeoxygenase-1 (HO-1) and confer protection from hepatic ischemia reperfusion injury. Wild-type and jnk-2 -/- animals were subjected to hepatic ischemia reperfusion insults in two models: a total hepatic ischemia model involving timed Pringle maneuver, and a partial hepatic ischemia model involving selective occlusion of the portal pedicle supplying the left hepatic lobe. Optimal durations of injury were calibrated for each model. After 24 hr, animals were killed, and blood and tissues were collected for alanine aminotransferase, histologic injury scoring, and other analyses. Before total or partial hepatic ischemia reperfusion insults, some animals were subject to HO-1 inhibition with chromium mesoporphyrin IX or Kupffer cell depletion with liposomal clodronate. Bone marrow-derived monocytes were grown from hemopoietic progenitors taken from wild-type and jnk-2 -/- mice before stimulation with lipopolysaccharide and measurement of tumour necrosis factor-alpha production. Jnk-2 -/- animals were protected from hepatic ischemia reperfusion injury. HO-1 expression and activity was elevated in jnk-2 -/- animals (2.2-fold; P=0.006). Most HO-1 was expressed in Kupffer cells. Inhibition of HO-1 in jnk-2 -/- animals led to the loss of protection from ischemia. Depletion of Kupffer cells using liposomal clodronate led to loss of hepatic HO-1 expression and much more severe injury in wild-type and jnk-2 -/- animals. In vitro studies of cultured macrophages demonstrated reduced tumour necrosis factor-alpha secretion after lipopolysaccharide stimulus, an effect lost after HO-1 inhibition.

Systemic adenoviral gene delivery to orthotopic murine breast tumors with ablation of coagulation factors, thrombocytes and Kupffer cells

Rapid clearance of adenoviruses from blood by macrophage lineage cells of the liver and spleen, and binding to platelets, hinder their successful systemic use for cancer gene therapy. Vitamin K dependent coagulation factors are important mediators for the adenovirus liver tropism. Here we aim to determine the effects of coagulation factor, thrombocyte and liver macrophage (Kupffer cell) ablation on biodistribution of serotype 5 adenoviruses in mice with orthotopic breast tumors. Prior to intravenous injection of adenoviruses, mice bearing orthotopic breast tumors were pretreated with warfarin to inhibit vitamin K dependent coagulation factor synthesis, an anti-platelet antibody causing thrombocytopenia or an inhibitor of the Kupffer cell scavenger receptor or their combination. Virus availability in blood after injection was determined from blood samples and transgene expression in tissues analyzed 72 hours afterwards with In Vivo Imaging and luciferase assays. Warfarin pretreatment reduced gene delivery to liver, spleen and lung. Kupffer cell ablation increased persistence of adenoviruses in blood but didn't affect biodistribution significantly. Depletion of Kupffer cells combined with thrombocytopenia doubled the systemic gene delivery of 5/3 chimeric adenovirus to tumors (p < 0.05). Triple ablation of platelets, Kupffer cells and coagulation factors increased the tumor to liver ratio of systemic adenovirus gene delivery by 81% (p < 0.05). Depletion of coagulation factors can reduce transduction of liver, spleen and lung. Kupffer cell depletion is the most feasible method of increasing amount adenovirus in systemic blood flow and in combination with ablation of thrombocytes can increase the transduction of adenovirus to tumors.

Tissue-resident Macrophages Protect the Liver From Ischemia Reperfusion Injury via a Heme Oxygenase-1-Dependent Mechanism

Kupffer cells are the resident macrophage population of the liver and have previously been implicated in the pathogenesis of hepatic ischemia-reperfusion injury (IRI). Kupffer cells are the major site of expression of hepatic heme oxygenase-1 (HO-1), which has been shown to have anti-inflammatory actions and to protect animals and cells from oxidative injury. Kupffer cells and circulating monocytes were selectively ablated using liposomal clodronate (LC) in the CD11b DTR mouse before induction of hepatic ischemia. Kupffer cell depletion resulted in loss of HO-1 expression and increased susceptibility to hepatic IRI, whereas ablation of circulating monocytes did not affect IRI phenotype. Targeted deletion of HO-1 rendered mice highly susceptible to hepatic IRI. In vivo, HO-1 deletion resulted in pro-inflammatory Kupffer cell differentiation characterized by enhanced Ly6c and MARCO (macrophage receptor with collagenous structure) expression as well as decreased F4/80 expression, mirrored by an expansion in immature circulating monocytes. In vitro, HO-1 inhibition throughout macrophage differentiation led to increased cell numbers, and pro-inflammatory Ly6c+ CD11c- F4/80- phenotype. These data support a critical role for tissue-resident macrophages in homeostasis following ischemic injury, and a co-dependence of HO-1 expression and tissue-resident macrophage differentiation.

Regulation of liver hepcidin expression by alcohol in vivo does not involve Kupffer cell activation or TNF-α signaling

Alcohol downregulates hepcidin expression in the liver leading to an increase in intestinal iron transport and liver iron storage. We have previously demonstrated that alcohol-mediated oxidative stress is involved in the inhibition of hepcidin transcription by alcohol in vivo. Kupffer cells and TNF-alpha play a key role in alcohol-induced liver injury. The aim of this study was to define their involvement in the regulation of hepcidin expression by alcohol. Kupffer cells were inactivated or depleted by employing gadolinium chloride and liposomes containing clodronate, respectively. Rats pair fed with the alcohol-Lieber-DeCarli diet for 6 wk and mice fed with 20% ethanol in the drinking water for 1 wk were used as experimental models. Interestingly, alcohol downregulated hepcidin expression in the livers of rats and mice independent of gadolinium chloride or clodronate treatment. One week of alcohol treatment was sufficient to induce a significant increase in TNF-alpha levels and phosphorylation of NF-kappaB subunit p65. The neutralization of TNF-alpha by specific antibodies inhibited p65 phosphorylation. However, neither the neutralization of TNF-alpha nor the lack of TNF-alpha receptor expression reversed alcohol-induced suppression of liver hepcidin expression. The level of alcohol-induced ROS in the liver was also undiminished following Kupffer cell inactivation or depletion. Our results demonstrate that alcohol-induced Kupffer cell activation and TNF-alpha signaling are not involved in the suppression of liver hepcidin expression by alcohol-mediated oxidative stress in vivo. Therefore, these findings suggest that alcohol acts within hepatocytes to suppress hepcidin expression and thereby influences iron homeostasis.

Reciprocal Activation Between CD4+ T Cells and Kupffer Cells During Hepatic Ischemia-Reperfusion

Mechanisms mediating CD4+ T-cell recruitment during alloantigen-independent hepatic ischemia-reperfusion (I/R) remain not fully understood. We hypothesized that Kupffer cells activate CD4+ T-cells in the postischemic liver, by the release of free oxygen radicals and cytokines. Recruitment of freshly isolated and fluorescence-labeled CD4+ T-cell was analyzed after hepatic I/R (90/30-120 min) using intravital microscopy in sham-operated mice, in mice after hepatic I/R and in postischemic groups after Kupffer cell depletion, after treatment with antioxidant glutathione, in interleukin (IL)-6-/- mice; and in wild-type mice after infusion of tumor necrosis factor (TNF) receptor-1-/-CD4+ T-cells. Using flow cytometry and immunohistochemistry, we assessed whether Kupffer cell-derived mediators activate CD4+ T-cells and sinusoidal endothelial cells. The clearance kinetics of fluorescence-labeled latex beads was determined as a marker of Kupffer cell activity in vivo. I/R-induced accumulation of CD4+ T-cells in hepatic sinusoids was significantly attenuated on Kupffer cell depletion, after scavenging of free radicals and after interruption of the IL-6- and TNF-alpha-dependent pathways. These mediators directly activate CD4+ T-cells and up-regulated the expression of T cell-relevant adhesion molecules on sinusoidal endothelial cells. Postischemic activity of Kupffer cells was significantly impaired in wild-type mice, and was even more depressed in CD4-/- animals. Kupffer cells trigger recruitment of CD4+ T-cells in the postischemic liver by the release of reactive oxygen species, IL-6, and TNF-alpha. These mediators are capable of activating CD4+ T-cells and sinusoidal endothelial cells. CD4+ T-cells, in turn, influence the activation of Kupffer cells.