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Acute liver failure (ALF), characterized by the quick occurrence of disorder in liver, is a serious liver injury with extremely high mortality. Therefore, we investigated whether diallyl trisulfide (DATS), a natural product from garlic, protected against ALF in mice and studied underlying mechanisms. In the present study, lipopolysaccharide (LPS) (10 μg·kg−1)/D-galactosamine (D-gal) (500 mg·kg−1) was intraperitoneally injected to ICR mice to induce ALF. The mice were orally administered 20-, 40-, or 80-mg·kg−1 DATS) 1 h before LPS/D-gal exposure. Serum biochemical analyses and pathological study found that DATS pretreatment effectively prevented the ALF in LPS/D-gal-treated mice. Mechanistically, pretreatment of DATS inhibited the increase of the numbers of CD11b+ Kupffer cells and other macrophages in the liver, the release of tumor necrosis factor-α into the blood, and Caspase-1 activation induced by LPS/D-gal treatment in mice. Furthermore, DATS inhibited the activation of Caspase-3, downregulation of Bcl-2/Bax ratio, and increase of TUNEL positive staining. Altogether, our findings suggest that DATS exhibits hepatoprotective effects against ALF elicited by LPS/D-gal challenge, which probably associated with anti-inflammation and anti-apoptosis. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs) impact myeloid cell responsiveness to environmental cues such as pathogen components and metabolites. Although TLR protein expression in monocytes and tissue macrophages is thought to be optimized for microenvironments in each tissue, a comprehensive study has not been reported. We here examined protein expression of endogenous TLRs in tissue-resident myeloid cells. Neutrophils in peripheral blood, spleen, liver and lung expressed TLR2, TLR4 and TLR5 in all tissues. Ly6C+ MHC II‒ classical monocytes mature into Ly6C‒ MHC II+ monocyte-derived dendritic cells (moDCs) or Ly6C‒ MHC II‒ patrolling monocytes. These subsets were found in all the tissues studied. TLR2 and TLR4 were displayed on all of these subsets, regardless of location. In contrast, expression of endosomal TLRs did vary with tissues and subsets. moDCs expressed TLR9, but much less TLR7. In contrast, TLR7, not TLR3 or TLR9, was highly expressed in classical and patrolling monocytes. Tissue macrophages such as red pulp macrophages in the spleen, Kupffer cells in the liver, microglia in the brain, alveolar macrophages in the lung and adipose tissue macrophages all expressed TLR2, TLR4 and TLR3. TLR7 was also expressed in these tissue macrophages except Kupffer cells in the liver. TLR9 expression in tissue macrophages was much lower or hard to detect. These results suggest that expression of endosomal TLRs in myeloid cells is influenced by their differentiation status and tissue-specific microenvironments. [ABSTRACT FROM AUTHOR]

In the present study, intestinal tight junctions (TJs) and Kupffer cell polarization were investigated in an alcoholic steatohepatitis (ASH) mouse model to uncover the potential side effects of overexposure to fish oil or omega-3 fatty acids. The mice were fed ad libitum with a liquid diet containing ethanol and fish oil. In the meantime, ethanol was given every 5–7 days by gavage to simulate binge drinking. After the 7th binge, steatosis, necrosis, inflammatory infiltration, and bridging fibrosis were observed in the liver by histological staining. After the 13th binge, the inducers, markers and other downstream genes/proteins of the Kupffer cell M1/M2 phenotype in the liver, serum, and small intestine were analysed. The results suggested that a chronic high dosage of fish oil alone reduced the mRNA levels of most genes tested and showed a tendency to damage the intestinal zonula occludens-1 localization and reduce the number of M2 Kupffer cells. Meanwhile, the combination of fish oil and ethanol damaged the intestinal TJs, resulting in an increased endotoxin level in the liver. Gut-derived endotoxin polarized Kupffer cells to the M1 phenotype, whereas the number of cells with the M2 phenotype (markers: CD163 and CD206) was decreased. Interleukin-4 (IL-4), an M2 Kupffer cell inducer, was also decreased. Moreover, in vitro experiments showed that IL-4 reversed eicosapentaenoic acid-induced CD163 and CD206 mRNA suppression in RAW 264.7 cells. Overall, our results showed that a chronic high dosage of fish oil exacerbated gut–liver axis injury in alcoholic liver disease in mice, and endotoxin/IL-4-induced Kupffer cell polarization imbalance might play an important role in that process. [ABSTRACT FROM AUTHOR]

Background & Aim: Diallyl trisulfide (DATS) has been verified to ameliorate hepatotoxicity induced by many drugs, but the protective actions of isoniazid (INH) and rifampicin (RFP) have not been reported. We attempted to elucidate the potential effects and mechanisms of DATS against INH&RFP-caused hepatotoxicity. Methods: Male Kunming mice weighing 18–22 g were divided into 6 groups. For the hepatic-protective study, DATS (10 mg per kg, 20 mg per kg, and 40 mg per kg bw, respectively) was orally administered two hours before the INH&RFP (100 mg per kg, 100 mg per kg bw, respectively) treatments. After 11 days of treatment, 10 mice in each group were taken for the carbon clearance test, while the other 10 mice were sacrificed for the collection of serum and livers for further measurements, including the levels of serum alanine aminotransferase (ALT), aspartate transaminase (AST) and total bilirubin (T.Bili), the liver index, and liver histopathological examination. Malondialdehyde (MDA), glutathione (GSH), and the level of interleukin 1-β (IL-1-β) were measured, the carbon clearance test was performed and the immunohistochemistry of F4/80 marker for activated Kupffer cells (KCs) was analyzed to investigate potential mechanisms. Results: DATS co-administration significantly inhibited the increase of liver index and elevation of serum ALT, AST and T.Bili levels induced by INH&RFP, as well as improved the hepatocellular structure. The further mechanistic studies demonstrated that DATS co-administration counteracted INH&RFP-induced oxidative stress in mice, which was illustrated by the restoration of GSH levels, and the reduction of MDA levels in the liver. Furthermore, DATS co-administration reactivated the KCs inhibited by INH&RFP, which was illustrated by the increase of carbon phagocytosis, and the restoration of the number of activated KCs and IL-1-β levels in the liver. Conclusion: DATS effectively protected the liver against INH&RFP-induced hepatotoxicity, which might be due to its antioxidant effect and enhancement of KCs' activities. [ABSTRACT FROM AUTHOR]

Alcohol downregulates IRAK‐M, SHIP1, SOCS1, and IL‐10, inhibitors of TLR4 signaling, via NF‐κB‐induced miR‐155 and HDAC11 in Kupffer cells. Inflammation promotes the progression of alcoholic liver disease. Alcohol sensitizes KCs to gut‐derived endotoxin (LPS); however, signaling pathways that perpetuate inflammation in alcoholic liver disease are only partially understood. We found that chronic alcohol feeding in mice induced miR‐155, an inflammatory miRNA in isolated KCs. We hypothesized that miR‐155 might increase the responsiveness of KCs to LPS via targeting the negative regulators of LPS signaling. Our results revealed that KCs that were isolated from alcohol‐fed mice showed a decrease in IRAK‐M, SHIP1, and PU.1, and an increase in TNF‐α levels. This was specific to KCs, as no significant differences were observed in these genes in hepatocytes. We found a causal effect of miR‐155 deficiency on LPS responsiveness, as KCs that were isolated from miR‐155 KO mice showed a greater induction of IRAK‐M, SHIP1, and suppressor of cytokine signaling 1 after LPS treatment. C/EBPβ, a validated miR‐155 target, stimulates IL‐10 transcription. We found a higher induction of C/EBPβ and IL‐10 in KCs that were isolated from miR‐155 KO mice after LPS treatment. Gain‐ and loss‐of‐function studies affirmed that alcohol‐induced miR‐155 directly regulates IRAK‐M, SHIP1, suppressor of cytokine signaling 1, and C/EBPβ, as miR‐155 inhibition increased and miR‐155 overexpression decreased these genes in LPS or alcohol‐pretreated wild‐type KCs. HDAC11, a regulator of IL‐10, was significantly increased and IL‐10 was decreased in KCs that were isolated from alcohol‐fed mice. Functionally, knockdown of HDAC11 with small interfering RNA resulted in an IL‐10 increase in LPS or alcohol‐pretreated Mϕ. We found that acetaldehyde and NF‐κB pathways regulate HDAC11 levels. Collectively, our results indicate that the alcohol‐induced responsiveness of KCs to LPS, in part, is governed by miR‐155 and HDAC11. [ABSTRACT FROM AUTHOR]

HIV‐1 infection of KCs leads to dysregulated innate immune response to LPS, and may play a role in hepatic inflammation and fibrosis. End‐stage liver disease is a common cause of non‐AIDS‐related mortality in HIV+ patients, despite effective anti‐retroviral therapies (ARTs). HIV‐1 infection causes gut CD4 depletion and is thought to contribute to increased gut permeability, bacterial translocation, and immune activation. Microbial products drain from the gut into the liver via the portal vein where Kupffer cells (KCs), the resident liver macrophage, clear translocated microbial products. As bacterial translocation is implicated in fibrogenesis in HIV patients through unclear mechanisms, we tested the hypothesis that HIV infection of KCs alters their response to LPS in a TLR4‐dependent manner. We showed that HIV‐1 productively infected KCs, enhanced cell‐surface TLR4 and CD14 expression, and increased IL‐6 and TNF‐α expression, which was blocked by a small molecule TLR4 inhibitor. Our study demonstrated that HIV infection sensitizes KCs to the proinflammatory effects of LPS in a TLR4‐dependent manner. These findings suggest that HIV‐1‐infected KCs and their dysregulated innate immune response to LPS may play a role in hepatic inflammation and fibrosis and represent a novel target for therapy. [ABSTRACT FROM AUTHOR]

Objectives Emerging evidence indicates that Wnt/β-catenin pathway is linked to the fibrosis of different organs including liver fibrosis. β-Catenin promotes hepatic stellate cells ( HSCs) activation, a key event in the development of liver fibrosis, and has emerged as a novel mediator of fibrosis. Curcumin, a natural active ingredient derived from turmeric, possesses an inhibitory effect on liver fibrosis. This study is aimed to examine whether curcumin affects β-catenin expression/activity in HSCs and explores the underlying mechanisms. Methods The researchers used Western blot, real-time PCR, transfection assay and electrophoretic mobility shift assay and employed cultured HSCs and rat model of liver injury. Key findings Results showed that curcumin could reduce β-catenin protein level in HSCs in vitro and in vivo. Both β-catenin transactivation activity and DNA-binding activity were suppressed by curcumin. Moreover, nuclear β-catenin protein level was decreased by curcumin treatment. Further experiments suggested that delta-like homologue 1 contributed to curcumin inhibition of β-catenin transactivation activity in cultured HSCs. Conclusions Curcumin affects β-catenin pathway in HSCs and might suggest a possible new explanation for the effects of curcumin on HSC activation and liver fibrosis. [ABSTRACT FROM AUTHOR]

Objective: Only little is known about the mechanisms of action of corticosteroids in the treatment of inflammatory liver diseases. As there is increasing evidence that stimulation of the innate immune system plays an important pathogenetic role in these conditions, we hypothesized that steroids may interfere with the activation of the Toll-like receptor (TLR) system of the liver. Methods: To test this hypothesis, murine non-parenchymal liver cells (Kupffer cells, liver sinusoidal endothelial cells) and primary hepatocytes were stimulated with TLR 1–9 ligands in the presence or absence of dexamethasone. Expression of pro- and anti-inflammatory cytokines was determined by quantitative reverse transcription–PCR or ELISA, respectively. Nuclear factor ‘kappa-light-chain-enhancer' of activated B-cells (NF-κB) activation was assessed by western blot analysis. Results: TLR agonists induced the expression of pro- [tumor necrosis factor-α (TNF-α), IL-6, IL-1β, IFN-β] and anti-inflammatory cytokines [IL-10, transforming growth factor-β (TGF-β)], which was differentially modulated by steroid treatment. TNF-α and IL-6 expression was suppressed by dexamethasone, while IL-10 but not TGF-β was enhanced after TLR stimulation. IFN-β production induced by TLR 4 agonists but not TLR 3 agonists was inhibited by dexamethasone. TLR expression itself was down-regulated by steroid treatment in a cell type-specific manner. These effects were associated with suppression of the TLR-mediated activation of NF-κB. Conclusions: TLR signaling is modulated by corticosteroids in a cell type-specific fashion resulting in down-regulation of TLR expression, suppression of pro-inflammatory and up-regulation of anti-inflammatory cytokines. This represents an as yet unknown mechanism of action for corticosteroids that may at least in part explain their therapeutic effects in inflammatory liver diseases. [ABSTRACT FROM AUTHOR]

Kupffer cells are resident macrophages of the liver and play an important role in its normal physiology and homeostasis as well as participating in the acute and chronic responses of the liver to toxic compounds. Activation of Kupffer cells directly or indirectly by toxic agents results in the release of an array of inflammatory mediators, growth factors, and reactive oxygen species. This activation appears to modulate acute hepatocyte injury as well as chronic liver responses including hepatic cancer. Understanding the role Kupffer cells play in these diverse responses is key to understanding mechanisms of liver injury. Idiosyncratic drug-induced liver disease results in morbidity and mortality, impacting severely on the development of new pharmacological agents. Modulation of the response of Kupffer cells by drugs has been suggested as a cause for the idiosyncratic response. Similarly, liver damage seen in chronic ethanol consumption appears to be modulated by Kupffer cell activation. More recent evidence has noted a contributory role of Kupffer cell activation in the process of hepatic carcinogenesis. Several nongenotoxic carcinogens, for example, activate Kupffer cells resulting in the release of cytokines and/or reactive oxygen species that induce hepatocyte cell proliferation and may enhance clonal expansion of preneoplastic cells leading to neoplasia. Kupffer cells therefore appear to play a central role in the hepatic response to toxic and carcinogenic agents. Taken together, the data presented in this symposium illustrate to the toxicologist the central role played by Kupffer cells in mediating hepatotoxicity. [ABSTRACT FROM AUTHOR]

Hepatic stellate cell (HSC) activation is a key event in the natural process of wound healing as well as in fibrosis development in liver. Current in vitro models for HSC activation contribute significantly to the understanding of HSC biology and fibrogenesis but still fall far short of recapitulating in vivo intercellular functional and anatomic relationships. In addition, when cultured on uncoated plastic, HSC spontaneously activate, which makes HSC activation difficult to regulate or analyze. We have examined whether the use of precision-cut liver slices might overcome these limitations. Liver slices (8 mm diameter, 250 μm thickness) were generated from normal rat liver and incubated for 3 or 16 h with increasing doses of carbon tetrachloride (CCl4). Rat liver slices remained viable during incubation, as shown by minimal enzyme leakage. Expression of markers for HSC activation and the onset of fibrogenesis in the liver slices was studied using real-time PCR and Western blotting. In unstimulated liver slices, mRNA and protein levels of desmin, heat shock protein 47, and αB-crystallin remained constant, indicating quiescence of HSC, whereas Krüppel-like factor 6 expression was increased. In contrast, incubation with CCl4 led to a time- and dose-dependent increase in mRNA expression of all markers and an increased αB-crystallin protein expression. In conclusion, we have developed a technique to induce activation of quiescent HSC in rat liver slices. This model permits the study of toxicity-induced HSC activation within a physiological milieu, not only in animal but ultimately also in human tissue, and could contribute to the reduction of animal experiments. [ABSTRACT FROM AUTHOR]

Overdose of acetaminophen (APAP) causes severe centrilobular hepatic necrosis in humans and experimental animals. Here, to explore its mechanism, we administered APAP at subtoxic (150 mg/kg ip) and toxic (500 mg/kg ip) doses to overnight fasted mice. Animals were sacrificed at different time points from 15 min to 4 h postinjection. We assessed liver toxicity by plasma ALT activity and by electron microscopy. Using nylon filter arrays and RTQPCR, we performed genomics analysis in liver. We ran proteomics on liver mitochondrial subfractions using the newly developed quantitative fluorescent 2D-DIGE© method (Amersham Pharmacia Biotech UK Limited). As soon as 15 min postinjection, centrilobular hepatocyte mitochondria were already slightly enlarged and GSH total content dropped by a third at top dose. GM-CSF mRNA, which is a granulocyte specific gene likely coming from resident Kupffer cells, was also induced to its maximum of 3-fold at both doses. Chaperone proteins Hsp10 and Hsp60 were readily decreased by half in mitochondria at both doses, most likely by leaking into cytoplasm. Although APAP is known as an apoptotic trigger, no apoptosis was observed at any time point. Most of the protein changes in mitochondria were present at 15 min postinjection, thus preceding most of the gene regulations. The decrease of ATP synthase subunits and β-oxidation pathway proteins indicated a loss of energy production. As the morphology of mitochondria was also affected very early at top dose, we concluded that APAP toxicity was a direct action of its known reactive metabolite NAPQI, rather than a consequence of gene regulation. However, the latter will either worsen the toxicity or lead toward cell recovery depending on the cellular damage level. [ABSTRACT FROM PUBLISHER]

Background: Sepsis and endotoxaemia occur frequently in biliary obstruction. Impaired Kupffer cell endocytosis is implicated in these events. Tumour necrosis factor and interleukin 6, secreted by Kupffer cells, are important mediators of sepsis. Kupffer cell clearance of endotoxin and secretion of cytokines in experimental obstructive jaundice were investigated. Methods: Wistar rats were randomized to bile duct ligation, sham operation or control. Groups (n = 8) were studied 1 and 3 weeks after operation. Kupffer cell function was assessed using in situ hepatic perfusion. Results: Clearance of endotoxin was significantly depressed 1 week (median (interquartile range) 20·3 (10·5–27·1) per cent) and 3 weeks (22·1 (20·2–23·2) per cent) after bile duct ligation compared with that in respective sham animals (35·5 (29·9–41·6) and 40·9 (37·7–47·0) per cent) and controls (39·5 (37·3–46·8) per cent). Secretion of tumour necrosis factor was significantly greater 1 week (1113·7 (706·5–1436·8) pg/ml) and 3 weeks (1118·2 (775·7–1484·1) pg/ml) following bile duct ligation compared with that in respective sham animals (114·3 (0–178·5) and 107·6 (63·7–166·4) pg/ml) and controls (0 (0–20·7) pg/ml). Interleukin 6 was not secreted by sham or control animals but was present in the perfusate from jaundiced animals at 1 and 3 weeks (52·5 (9·9–89·5) and 66·2 (60·2–193·1) pg/ml). Conclusion: These data demonstrate simultaneous impairment of Kupffer cell clearance of endotoxin and increased secretion of proinflammatory cytokines in experimental obstructive jaundice. These diverse responses may contribute to the development of sepsis-related complications in biliary obstruction. [ABSTRACT FROM AUTHOR]

Gut-derived endotoxins (lipopolysaccharide, LPS) complexed to LPS-binding protein (LBP) activate liver Kupffer cells via their CD14 receptor. Pro-inflammatory cytokines are released and this is postulated to promote liver injury. We previously demonstrated enhanced expression of CD14 endotoxin receptor after 2 weeks of alcohol administration. A similar result, based on 6 weeks of ethanol treatment, was recently reported and suggested to correlate with alcohol-induced liver injury. To establish whether this occurs prior to or after the initiation of damage, we investigated the temporal effect of continuous ethanol exposure on the expression of CD14 and the associated LBP. In addition, we studied the effect of treatment with gadolinium chloride (GdCl3) that inactivates Kupffer cells and alleviates alcohol-induced liver damage. The amount of CD14 and LBP mRNA, as determined by reverse transcriptase–polymerase chain reaction (RT–PCR), was unchanged 4–8 h after intragastric ethanol administration. However, after 24–48 h of repeated ethanol administration, CD14 and LBP mRNA both increased significantly and reached a level similar to that observed after 6 weeks of ethanol exposure by liquid diet. Immunostaining experiments with ED2 antibody demonstrated that GdCl3 efficiently inactivated Kupffer cells. However, there was no concomitant reduction in the expression of CD14 mRNA, suggesting that compensatory infiltration by ED2-negative, but CD14-positive, macrophages had occurred. Our results demonstrate that soon after the initiation of ethanol exposure, i.e. within 24–48 h, the hepatic expression of both the CD14 receptor and LBP is increased. This suggests that these increases could contribute to the initiation of alcoholic damage rather than being a consequence of the injury. [ABSTRACT FROM PUBLISHER]

Background Orthotopic liver transplantation (OLT) is accompanied by local and systemic manifestations of the ischaemia–reperfusion syndrome. Local effects are mediated in part through changes in intracellular calcium levels in Kupffer cells. Arachidonic acid metabolites mediate increases in intracellular calcium concentration and thus potentiate the effect of free radicals. This study was carried out to characterize white blood cell (WBC) calcium changes as a mediator for white cell activation in human OLT. Methods Twenty consecutive patients had OLT using standard surgery and anaesthesia techniques. Blood samples were drawn for estimation of WBC cytosolic calcium content at induction of anaesthesia, 5 min before graft reperfusion and 15 min after reperfusion. The rate of rise in intracellular calcium concentration after the addition of a calcium chloride 1 mmol l-1 solution to the extracellular milieu was used as an estimate of membrane calcium permeability. Results Both extracellular (P=0·0002) and intracellular (P=0·0008) calcium concentrations rose with time. However, at no time was there a correlation between extracellular and intracellular calcium levels or rate of calcium influx (r2=0·002, P=0·78). There was a significant increase in intracellular calcium concentration (P=0·0008) and in the rate of rise of intracellular calcium levels (P=0·0009) after reperfusion. Conclusion There was a significant increase in circulating monocyte membrane permeability for calcium and cytosolic calcium concentration following reperfusion in human OLT. This was independent of extracellular calcium concentration. These results are consistent with WBC activation by reperfusion and could be implicated in the systemic reperfusion syndrome seen in OLT in humans. [ABSTRACT FROM AUTHOR]

SummaryBackground: Gram-negative sepsis and its sequelae frequently complicate invasive procedures in patients with obstructive jaundice. In response to endotoxin, Kupffer cells secrete tumour necrosis factor (TNF), a pivotal early mediator of sepsis. An investigation was carried out into the specific role of Kupffer cell TNF secretion following endotoxin challenge in obstructive jaundice. Methods: Survival following intraperitoneal administration of endotoxin (2·0, 0·02 and 0·0002 mg per 100 g) was determined in rats following bile duct ligation (BDL) or sham operation. Plasma TNF concentration was quantified following endotoxin administration (0·0002 mg per 100 g) at 1, 2 and 6 h. Subsequently, the effect of Kupffer cell blockade by gadolinium chloride on survival and plasma TNF concentration was assessed. Results: Jaundiced animals showed a significantly increased mortality rate following intraperitoneal injection of endotoxin 2·0 mg per 100 g (BDL 100 per cent versus sham 0 per cent) and 0·02 mg per 100 g (BDL 70 per cent versus sham 0 per cent; P = 0·002, Fisher’s exact test). Median plasma TNF concentration was significantly greater in jaundiced animals 1 h after endotoxin administration (BDL 943 (interquartile range (i.q.r.) 211–3900) pg/ml versus sham 64 (i.q.r. 47–127) pg/ml; P = 0·002, Mann–Whitney U test). Kupffer cell blockade with gadolinium chloride increased the survival rate following endotoxin administration in BDL animals (BDL-GdCl3 100 per cent versus BDL-saline 40 per cent; P = 0·0003, Fisher’s exact test) and decreased median plasma levels of TNF (BDL-GdCl3 88 (i.q.r. 0–1065) pg/ml versus BDL-saline 16 550 (1255–29 360) pg/ml; P = 0·002, Mann–Whitney U test). Conclusion: Kupffer cell blockade improved survival and suppressed systemic TNF activity after endotoxin challenge. In obstructive jaundice, hypersecretion of... [ABSTRACT FROM AUTHOR]