Invariant natural killer T cells contribute to chronic-plus-binge ethanol-mediated liver injury by promoting hepatic neutrophil infiltration

Neutrophil infiltration is a hallmark of alcoholic steatohepatitis; however, the underlying mechanisms remain unclear. We previously reported that chronic-plus-binge ethanol feeding synergistically induces hepatic recruitment of neutrophils, which contributes to liver injury. In this paper, we investigated the roles of invariant natural killer T (iNKT) cells in chronic-plus-binge ethanol feeding-induced hepatic neutrophil infiltration and liver injury. Wild-type and two strains of iNKT cell-deficient mice (CD1d- and Jα18-deficient mice) were subjected to chronic-plus-binge ethanol feeding. Liver injury and inflammation were examined. Chronic-plus-binge ethanol feeding synergistically increased the number of hepatic iNKT cells and induced their activation, compared with chronic feeding or binge alone. iNKT cell-deficient mice were protected from chronic-plus-binge ethanol-induced hepatic neutrophil infiltration and liver injury. Moreover, chronic-plus-binge ethanol feeding markedly upregulated the hepatic expression of several genes associated with inflammation and neutrophil recruitment in wild-type mice, but induction of these genes was abrogated in iNKT cell-deficient mice. Importantly, several cytokines and chemokines (e.g., MIP-2, MIP-1, IL-4, IL-6 and osteopontin) involved in neutrophil infiltration were upregulated in hepatic NKT cells isolated from chronic-plus-binge ethanol-fed mice compared to pair-fed mice. Finally, treatment with CD1d blocking antibody, which blocks iNKT cell activation, partially prevented chronic-plus-binge ethanol-induced liver injury and inflammation. Chronic-plus-binge ethanol feeding activates hepatic iNKT cells, which play a critical role in the development of early alcoholic liver injury, in part by releasing mediators that recruit neutrophils to the liver, and thus, iNKT cells represent a potential therapeutic target for the treatment of alcoholic liver disease.     

CD44 disruption attenuates murine hepatic ischemia/reperfusion injury

Neutrophil adhesion and migration are critical in hepatic ischemia/reperfusion (I/R) injury. Despite very strong preclinical data, recent clinical trials failed to show a protective effect of anti-adhesion therapy in reperfusion injury. Therefore, the aim of this study was to assess the role of CD44 in neutrophil infiltration and liver injury from hepatic I/R. In this study, using a partial hepatic ischemic model in vivo, we determined the potential role of CD44 in neutrophil infiltration and liver injury from I/R. Reperfusion caused significant hepatocellular injury as it was determined by plasma ALT levels and liver histopathology. The injury was associated with a marked neutrophil recruitment and CD44 expression into the ischemic livers. Administration of anti-CD44 antibody to mice reduced the infiltration of neutrophil into the ischemic tissue, associated with liver function preservation. These results support crucial roles of CD44 in neutrophil recruitment and infiltration leading to liver damage in hepatic I/R injury. Moreover, they provide the rationale for targeting to CD44 as a potential therapeutic approach in liver I/R injury.     

Interleukin-4 (IL-4) and IL-13 suppress excessive neutrophil infiltration and hepatocyte damage during acute murine schistosomiasis japonica

Due to the importance of neutrophils and proinflammatory cytokines in schistosomal liver damage, we analyzed the mechanisms underlying neutrophil and proinflammatory responses in murine schistosomiasis japonica. We found that granulomatous inflammation around parasite eggs in the liver was greater in Schistosoma japonicum-infected IL-4-/- IL-13-/- (double-knockout [DKO]) mice than in infected wild-type (WT) mice at 6 weeks, but not at 8 weeks, postinfection, suggesting the importance of Th2 responses in these typical hepatic lesions. Infected DKO mice also showed increased neutrophil infiltration accompanying more severe pathology, as shown by the enhanced necrosis of hepatocytes. This was not likely due to a Th1/Th2 imbalance, because there was no detectable increase in gamma interferon (IFN-γ) production in these DKO mice. mRNA expression of interleukin-17A (IL-17A), proinflammatory cytokines, and the neutrophil chemoattractant CXCL2 in liver was higher in infected DKO mice than in WT mice. However, in IL-4-/- IL-13-/- IL-17A-/- (triple-knockout [TKO]) mice, the absence of IL-17A was associated with only marginal differences in schistosomal liver damage, suggesting that IL-17A is only partially responsible for neutrophil-driven hepatic damage. Furthermore, the expression of mRNAs encoding proinflammatory cytokines was not under the control of IL-17A in TKO mice. These findings indicate that IL-4 and IL-13 suppress excessive neutrophil recruitment, proinflammatory cytokine production, and hepatic damage during the acute stage of S. japonicum infection, suggesting that neutrophils and proinflammatory cytokines are mainly responsible for hepatocyte damage during acute murine schistosomiasis japonica. However, neutrophil induction and the production of proinflammatory cytokines were not due solely to IL-17A.     

Hydroxysafflor yellow A attenuates ischemia/reperfusion-induced liver injury by suppressing macrophage activation

Hydroxysafflor yellow A (HSYA), a major constituent in the hydrophilic fraction of the safflower plant, can retard the progress of hepatic fibrosis. However, the anti-inflammatory properties and the underlying mechanisms of HSYA on I/R-induced acute liver injury are unknown. Inhibiting macrophage activation is a potential strategy to treat liver ischemia/reperfusion (I/R) injury. In this study, we investigated the therapeutic effect of HSYA on liver I/R injury and the direct effect of HSYA on macrophage activation following inflammatory conditions. The therapeutic effects of HSYA on I/R injury were tested in vivo using a mouse model of segmental (70%) hepatic ischemia. The mechanisms of HSYA were examined in vitro by evaluating migration and the cytokine expression profile of the macrophage cell line RAW264.7 exposed to acute hypoxia and reoxygenation (H/R). Results showed that mice pretreated with HSYA had reduced serum transaminase levels, attenuated inflammation and necrosis, reduced expression of inflammatory cytokines, and less macrophage recruitment following segmental hepatic ischemia. In vitro HSYA pretreated RAW264.7 macrophages displayed reduced migratory response and produced less inflammatory cytokines. In addition, HSYA pretreatment down-regulated the expression of matrix matalloproteinase-9 and reactive oxygen species, and inhibited NF-κB activation and P38 phosphorylation in RAW264.7 cells. Thus, these data suggest that HSYA can reduce I/R-induced acute liver injury by directly attenuating macrophage activation under inflammatory conditions.     

Pulmonary and Hepatic Gene Expression following Cecal Ligation and Puncture: Monophosphoryl Lipid A Prophylaxis Attenuates Sepsis-Induced Cytokine and Chemokine Expression and Neutrophil Infiltration

Polymicrobial sepsis induced by cecal ligation and puncture (CLP) reproduces many of the pathophysiologic features of septic shock. In this study, we demonstrate that mRNA for a broad range of pro- and anti-inflammatory cytokine and chemokine genes are temporally regulated after CLP in the lung and liver. We also assessed whether prophylactic administration of monophosphoryl lipid A (MPL), a nontoxic derivative of lipopolysaccharide (LPS) that induces endotoxin tolerance and attenuates the sepsis syndrome in mice after CLP, would alter tissue-specific gene expression post-CLP. Levels of pulmonary interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), granulocyte colony-stimulating factor (G-CSF), IL-1 receptor antagonist (IL-1ra), and IL-10 mRNA, as well as hepatic IL-1β, IL-6, gamma interferon (IFN-γ), G-CSF, inducible nitric oxide synthase, and IL-10 mRNA, were reduced in MPL-pretreated mice after CLP compared to control mice. Chemokine mRNA expression was also profoundly mitigated in MPL-pretreated mice after CLP. Specifically, levels of pulmonary and hepatic macrophage inflammatory protein 1α (MIP-1α), MIP-1β, MIP-2, and monocyte chemoattractant protein-1 (MCP-1) mRNA, as well as hepatic IFN-γ-inducible protein 10 and KC mRNA, were attenuated in MPL-pretreated mice after CLP. Attenuated levels of IL-6, TNF-α, MCP-1, MIP-1α, and MIP-2 in serum also were observed in MPL-pretreated mice after CLP. Diminished pulmonary chemokine mRNA production was associated with reduced neutrophil margination and pulmonary myeloperoxidase activity. These data suggest that prophylactic administration of MPL mitigates the sepsis syndrome by reducing chemokine production and the recruitment of inflammatory cells into tissues, thereby attenuating the production of proinflammatory cytokines.     

Hepatic ischemic preconditioning provides protection against distant renal ischemia and reperfusion injury in mice

We previously demonstrated that there are acute and delayed phases of renal protection against renal ischemia and reperfusion (IR) injury with renal ischemic preconditioning (IPC). This study assessed whether hepatic IPC could also reduce distant renal IR injury through the blood stream-mediated supply of reactive oxygen species (ROS). Male C57BL/6 mice were randomly divided into four groups: group I, sham operated including right nephrectomy; group II (IR), left renal ischemia for 30 min and reperfusion injury; group III (IPC-IR), hepatic ischemia for 10 min followed by 10 min of reperfusion before left renal IR injury; group IV (MPG - IPC + IR), pretreated with 100 mg/kg N-(2-mercaptopropionyl)-glycine (MPG) 15 min before hepatic IPC and left renal IR injury. Renal function, histopathologic findings, proinflammatory cytokines, and cytoprotective proteins were evaluated 15 min or 24 hr after reperfusion. Hepatic IPC attenuated the expression of proinflammatory cytokines, tumor necrosis factor α, intercellular adhesion molecule 1, and induced inducible nitric-oxide synthase, and the phosphorylation of Akt in the murine kidney. Renal function was better preserved in mice with hepatic IPC (group III) than groups II or IV. Hepatic IPC protects against distant renal IR injury through the blood stream-delivery of hepatic IPC-induced ROS, by inducing cytoprotective proteins, and by inhibiting inflammatory reactions.     

Blockade of IL-6 signaling exacerbates liver injury and suppresses antiapoptotic gene expression in methionine choline-deficient diet-fed db/db mice

Our previous study revealed that blockade of interleukin-6 (IL-6)-STAT3 signaling ameliorated liver injury, although hepatic STAT3(-/-) or GP130(-/-) mice have been reported to develop severe liver injury, in a murine methionine choline deficient (MCD) diet-induced model of non-alcoholic steatohepatitis (NASH). In this study, to determine whether profound blockade of IL-6-STAT3 signaling may still ameliorate liver injury, we studied db/db mice, which have impaired leptin-mediated STAT3 activation, using the MCD diet-induced NASH model. Male lean and db/db mice (6 weeks old) were fed either control chow or an MCD diet for 8 or 12 weeks. Half of the mice were treated with 15 mg/kg rat anti-mouse IL-6 receptor neutralizing antibody (MR16-1) intraperitoneally twice weekly, the remainder were injected with 15 mg/kg rat IgG as a control. Hepatic steatosis, injury, fibrosis, markers of lipid peroxidation/oxidant stress and antiapoptotic gene expression were evaluated. Plasma IL-6 levels were elevated in all groups of db/db mice. Although hepatic IL-6/ GP130 signaling was activated in chow-fed db/db mice, this was suppressed in MCD diet-fed db/db mice, accompanied by downregulation of hepatic IL-6 receptor and GP130 mRNA expression. MR16-1 treatment of MCD diet-fed db/db mice further repressed STAT3 activities and expression of STAT3-related antiapoptotic genes, such as Bcl-2 and Ref-1, but increased plasma-free fatty acid and hepatic markers of lipid peroxidation/oxidant stress, leading to increased liver injury, hepatocyte apoptosis and liver fibrosis. Although 'moderate' blockade of enhanced IL-6-STAT3 signaling may be beneficial in NASH, as we reported previously, these findings demonstrate that a profound defect in STAT3 activation is detrimental in terms of liver injury, hepatocyte apoptosis and liver fibrosis, indicating the hepato-protective role of IL-6 signaling in this severe NASH model.     

The artificial surface-induced whole blood inflammatory reaction revealed by increases in a series of chemokines and growth factors is largely complement dependent

Exposing blood to an artificial surface results in a systemic inflammatory response, including cytokine release and complement activation. We studied the artificial surface-induced inflammation in human whole blood using an extensive panel of inflammatory mediators including proinflammatory cytokines, chemokines and growth-factors and investigated the role of the complement system in the induction of this response. Using multiplex technology, 27 different inflammatory mediators were measured after circulating blood for 4 hours in polyvinyl chloride tubing. The C3 inhibitor compstatin was used to block complement activation. A significant (p < 0.05) increase in 14 of the 27 mediators was induced by the surface, of which 7 were chemokines (IL-8, MCP-1, MIP-1alpha, MIP-1beta, RANTES, eotaxin and IP-10) and 5 were growth-factors (G-CSF, GM-CSF, VEGF, PDGF and FGF). The traditional proinflammatory cytokines like IL-1beta, TNFalpha and IL-6 were not induced, although IL-6, as well as IL-15 and IL-17 increased if the surface was coated with highly bioincompatible laminaran. Inhibition of complement activation with compstatin significantly (p < 0.05) reduced the formation of 12 of the 14 mediators. For 10 of the 12 mediators, the inhibition was by 2/3 or more, for the remaining two the inhibition was more moderate. A highly biocompatible heparin-coated PVC surface was used as negative control and completely abolished the whole inflammatory response. The artificial surface PVC markedly induced a broad spectrum of chemokines and growth-factors, which was largely dependent on activation of complement.     

Dexamethasone Pretreatment Attenuates Lung and Kidney Injury in Cholestatic Rats Induced by Hepatic Ischemia/Reperfusion

Hepatic ischemia followed by reperfusion (IR) results in mild to severe organ injury, in which tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) seem to be involved. Thus, we aim to assess the influence of hepatic ischemia/reperfusion injury on remote organs in addition to cholestasis and consider the possible efficacy of steroid pretreatment in reducing the injury. A common bile duct ligation model was done on 24 male Sprague–Dawley rats. After 7 days, the rats were divided randomly into control group, IR group, and dexamethasone (DEX) group. The IR group showed significant increases in serum alanine aminotransferase, aspartate aminotransferase, and creatinine levels compared with the control and DEX groups. By ELISA techniques, higher levels of TNF-α and IL-1β in lung and kidney tissues were measured in the IR group than in the control and DEX groups, these were verified by immunohistochemistry. The lung histology of the IR group rats showed neutrophil infiltration, interstitial edema, and alveolar wall thickening. Kidney histology of the IR group rats showed vacuolization of the proximal tubular epithelial cells and tubular dilatation with granular eosinophilic casts. Better morphological aspects were observed in the DEX-pretreated animals. Minimal lesions were observed in the control. The results suggest that hepatic ischemia/reperfusion injury in cholestatic rats induced lung and kidney injuries. Pretreatment with dexamethasone reduced the IR-induced injury in addition to cholestasis.     

ST2, an IL-1R family member, attenuates inflammation and lethality after intestinal ischemia and reperfusion

Ischemia reperfusion injury is characterized by local and systemic inflammation leading to considerable mortality. Previously, we have reported that soluble T1/ST2 (sST2), a member of the IL-1 receptor gene family, inhibits LPS-induced macrophage proinflammatory cytokine production. Here, we report the therapeutic effect of sST2-Fc in a murine model of intestinal ischemia reperfusion-induced injury. Administration of sST2-Fc fusion protein i.v., 10 min before reperfusion, reduced the production of TNF-alpha dose-dependently in the intestine and in the lungs. The sST2-Fc treatment with the highest dose (100 mug) resulted in inhibited vascular permeability, neutrophilia, and hemorrhage in the intestine and the lungs compared with controls treated with normal IgG. This was associated with down-regulated tissue levels of proinflammatory cytokines, markedly reduced serum TNF-alpha levels, and increased survival of mice from the sST2-Fc-treated group after ischemia and reperfusion injury. The beneficial effect of sST2-Fc treatment was associated with elevated IL-10 production in intestine and lung. sST2-Fc was not able to prevent the inflammatory response associated with intestinal ischemia and reperfusion in IL-10-deficient mice, suggesting that sST2 exerts its anti-inflammatory effect in a IL-10-dependent manner. These results also demonstrate that sST2-Fc may provide a novel, complementary approach in treating ischemic reperfusion injury.     

Vascular endothelial growth factor antagonist modulates leukocyte trafficking and protects mouse livers against ischemia/reperfusion injury

Although hypoxia stimulates the expression of vascular endothelial growth factor (VEGF), little is known of the role or mechanism by which VEGF functions after ischemia and reperfusion (I/R) injury. In this report, we first evaluated the expression of VEGF in a mouse model of liver warm ischemia. We found that the expression of VEGF increased after ischemia but peaked between 2 and 6 hours after reperfusion. Mice were treated with a neutralizing anti-mouse VEGF antiserum (anti-VEGF) or control serum daily from day -1 (1 day before the initiation of ischemia). Treatment with anti-VEGF significantly reduced serum glutaminic pyruvic transaminase levels and reduced histological evidence of hepatocellular damage compared with controls. Anti-VEGF also markedly decreased T-cell, macrophage, and neutrophil accumulation within livers and reduced the frequency of intrahepatic apoptotic terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling-positive cells. Moreover, there was a reduction in the expression of pro-inflammatory cytokines (tumor necrosis factor-alpha and interferon-gamma), chemokines (interferon-inducible protein-10 and monocyte chemoattractant protein-1) and adhesion molecules (E-selectin) in parallel with enhanced expression of anti-apoptotic genes (Bcl-2/Bcl-xl and heme oxygenase-1) in anti-VEGF-treated animals. In conclusion, hypoxia-inducible VEGF expression by hepatocytes modulates leukocyte trafficking and leukocyte-induced injury in a mouse liver model of warm I/R injury, demonstrating the importance of endogenous VEGF production in the pathophysiology of hepatic I/R injury.     

Neutralization of Groα and Macrophage Inflammatory Protein-2 Attenuates Renal Ischemia/Reperfusion Injury

Previous studies have provided strong evidence for a role for neutrophils in mediating pathology during reperfusion of ischemic tissues. CXC chemokines including interleukin-8, KC/Gro alpha, and macrophage inflammatory protein (MIP)-2, direct neutrophils to tissue sites of inflammation. In the current study we tested the efficacy of antibodies to KC/Gro alpha and MIP-2 in inhibiting neutrophil infiltration into kidneys during reperfusion after 1 hour of warm ischemia using a mouse model. KC mRNA and protein were produced within 3 hours after reperfusion of the ischemic kidneys. MIP-2 mRNA and protein were twofold to fourfold lower than KC and were at low levels until 9 hours after reperfusion. Only 60% of mice subjected to ischemia/reperfusion injury survived to day 3 after reperfusion. Treatment with rabbit neutralizing antibodies to both KC and MIP-2 inhibited neutrophil infiltration into ischemic kidneys during reperfusion, restored renal function as assessed by decreased serum creatinine and urea nitrogen levels to near normal levels, and resulted in complete survival of treated animals. Finally, treatment with both antibodies significantly reduced histologically graded pathology of kidneys subjected to ischemia/reperfusion injury. Collectively, the results indicate the efficacy of neutralizing the chemokines directing neutrophils into ischemic kidneys during reperfusion to inhibit this infiltration and attenuate the resulting pathology.     

Xanthohumol suppresses inflammatory response to warm ischemia–reperfusion induced liver injury

Liver ischemia/reperfusion (I/R) leads to formation of reactive oxygen species (ROS), which cause hepatic injury and initiate an inflammatory response, which is a critical problem after liver surgery and transplantation. Xanthohumol, the major prenylated chalcone found in hops, has been discussed for its anti-inflammatory and ROS-scavenging properties, and thus, we aimed to investigate the effect of xanthohumol in a model of warm I/R liver injury. Xanthohumol was applied to BALB/c mice orally at a dose of 1 mg/g body weight for 5 days before I/R-injury was induced by clamping the vascular blood supply to the median and left lateral liver lobe for 1 h followed by a 6 h period of reperfusion. At this time, HPLC analysis revealed hepatic xanthohumol levels of approximately 2 μM, a concentration which has been shown to inhibit inflammatory effects in vitro. Assessment of hepatic HMOX1 expression, hepatic glutathione content and immunohistochemical analysis for proteins conjugated with the reactive aldehyde 4-hydroxynonenal indicated that I/R-induced oxidative stress was significantly inhibited in xanthohumol-fed compared to control mice. Histological analysis, TUNEL staining and determination of transaminase serum levels revealed no significant effects of xanthohumol on acute hepatocellular injury. However, at the same time point, pretreatment with xanthohumol almost completely blunted the I/R-induced AKT and NFκB activation and the expression of the proinflammatory genes IL-1alpha, IL-6, MCP-1 and ICAM-1, which are known to play a crucial role in the subacute phase of I/R-induced liver damage. In conclusion, these data indicate the potential of xanthohumol application to prevent adverse inflammatory responses to I/R-induced liver damage such as after surgical liver resection or transplantation.     

Fibronectin-alpha 4 beta 1 integrin-mediated blockade protects genetically fat Zucker rat livers from ischemia/reperfusion injury

We tested a hypothesis that interactions between fibronectin (FN), the major extracellular matrix component, and its integrin alpha 4 beta 1 receptor is important in the development of ischemia/reperfusion injury of steatotic liver transplants. We examined the effect of connecting segment-1 (CS1) peptide-facilitated blockade of FN-alpha 4 beta 1 interaction in a well-established steatotic rat liver model of ex vivo cold ischemia followed by iso-transplantation. In this model, CS1 peptides were administered through the portal vein of steatotic Zucker rat livers before and after cold ischemic storage. Lean Zucker recipients of fatty liver transplants received an additional 3-day course of CS1 peptides after transplant. CS1 peptide therapy significantly inhibited the recruitment of T lymphocytes, neutrophil activation/infiltration, and repressed the expression of proinflammatory tumor necrosis factor-alpha and interferon-gamma. Moreover, it resulted in selective inhibition of inducible nitric oxide synthase expression, peroxynitrite formation, and hepatic necrosis. Importantly, CS1 peptide therapy improved function/histological preservation of steatotic liver grafts, and extended their 14-day survival in lean recipients from 40% in untreated to 100% in CS1-treated OLTs. Thus, CS1 peptide-mediated blockade of FN-alpha 4 beta 1 interaction protects against severe ischemia/reperfusion injury experienced otherwise by steatotic OLTs. These novel findings document the potential of targeting FN-alpha 4 beta 1 in vivo interaction to increase the transplant donor pool through modulation of marginal steatotic livers.     

Chemotaxis and activation of particle-challenged human monocytes in response to monocyte migration inhibitory factor and C-C chemokines.

Cytokines that regulate monocyte migration were found in membrane tissue surrounding loosened prosthetic implants. Monocyte migration inhibition factor (MIF) is able to inhibit macrophage migration. Monocyte chemoattractant protein (MCP) and macrophage inflammatory protein (MIP) are potent macrophage chemoattractants. These cytokines may be expressed as part of the foreign body response to prosthetic particulate debris. Chemotaxis analysis and macrophage activation experiments were performed to determine the effects of MIF, MCP-1, and MIP-1alpha on macrophage migration and activation in vitro. We demonstrated that MIF had its maximal migration inhibitory effect for unchallenged and particle challenged macrophages at 1 ng/mL. MCP-1 and MIP-1alpha stimulated macrophage chemotaxis maximally at 1 to 10 ng/mL. Dose-response studies with MIF, MCP-1, and MIP-1alpha demonstrated that these cytokines did not modulate activation of unchallenged or particle challenged macrophages as evaluated by IL-6 and TNF-alpha release. However, these cytokines do not appear to affect macrophage release of proinflammatory mediators in vitro.