Myeloid Mixed Lineage Kinase 3 Contributes to Chronic Ethanol-Induced Inflammation and Hepatocyte Injury in Mice

Proinflammatory activity of hepatic macrophages plays a key role during progression of alcoholic liver disease (ALD). Since mixed lineage kinase 3 (MLK3)-dependent phosphorylation of JNK is involved in the activation of macrophages, we tested the hypothesis that myeloid MLK3 contributes to chronic ethanol-induced inflammatory responses in liver, leading to hepatocyte injury and cell death. Primary cultures of Kupffer cells, as well in vivo chronic ethanol feeding, were used to interrogate the role of MLK3 in the progression of liver injury. Phosphorylation of MLK3 was increased in primary cultures of Kupffer cells isolated from ethanol-fed rats compared to cells from pair-fed rats. Kupffer cells from ethanol-fed rats were more sensitive to LPS-stimulated cytokine production; this sensitization was normalized by pharmacological inhibition of MLK3. Chronic ethanol feeding to mice increased MLK3 phosphorylation robustly in F4/80⁺ Kupffer cells, as well as in isolated nonparenchymal cells. MLK3^−/− mice were protected from chronic ethanol-induced phosphorylation of MLK3 and JNK, as well as multiple indicators of liver injury, including increased ALT/AST, inflammatory cytokines, and induction of RIP3. However, ethanol-induced steatosis and hepatocyte apoptosis were not affected by MLK3. Finally, chimeric mice lacking MLK3 only in myeloid cells were also protected from chronic ethanol-induced phosphorylation of JNK, expression of inflammatory cytokines, and increased ALT/AST. MLK3 expression in myeloid cells contributes to phosphorylation of JNK, increased cytokine production, and hepatocyte injury in response to chronic ethanol. Our data suggest that myeloid MLK3 could be targeted for developing potential therapeutic strategies to suppress liver injury in ALD patients.Key words: Alcoholic liver disease (ALD), Kupffer cells, Necroptosis, Toll-like receptor 4 (TLR4), Cytokines     

Protective effect of quercetin, EGCG, catechin and betaine against oxidative stress induced by ethanol in vitro

There is a need for a nontoxic antioxidant agent to be identified which will prevent alcoholic liver disease (ALD) in alcoholic patients. We tested 4 candidate agents: quercetin, EGCG, catechin and betaine, all of which occur naturally in food. HepG2 cells overexpressing CYP2E1 were subjected to arachidonic acid, iron and 100 mM ethanol with or without the antioxidant agent. All the agents prevented oxidative stress and MDA/4HNE formation induced by ethanol, except for EGCG. Catechin prevented CYP2E1 induction by ethanol. All the agents tended to down-regulate the ethanol-induced increased expression of glutathionine peroxidase 4 (GPX4). All the agents, except catechin, tended to reduce the expression of SOD2 induced by ethanol. Heat shock protein 70 was up-regulated by ethanol alone and betaine tended to prevent this. All 4 agents down-regulated the expression of Gadd45b in the presence of ethanol, which could explain the mechanism of DNA demethylation associated with the up-regulation of the gene expression observed in experimental ALD. In conclusion, the in vitro model of oxidative stress induced by ethanol provided evidence that all 4 agents tested prevented some aspect of liver cell injury caused by ethanol.     

Ethanol-induced oxidative stress is mediated by p38 MAPK pathway in mouse hippocampal cells

It has been known that ethanol causes neuronal cell death through oxidative stress. Ethanol itself and reactive oxygen species (ROS) produced by ethanol modulate intracellular signaling pathways including mitogen-activated protein kinase (MAPK) cascades. This study was conducted to examine the impact of ethanol on MAPK signaling in HT22 cells. Ethanol (100 and 400 mM) caused activation of ERK, p38 MAPK, and JNK. ERK activation occurred in early time and p38 MAPK activation was evident when ERK activation was diminished. Specific inhibitor of p38 MAPK (SB203580) protected HT22 cells against ethanol, which was accompanied by an inhibition of ROS accumulation. However, inhibitors of ERK (U0126) and JNK (SP600125) had no effects on ethanol-induced neuronal cell death when they are treated with ethanol for 24 h. These results suggest that p38 MAPK may have important roles in ROS accumulation during ethanol-induced oxidative stress in HT22 cells.     

Toll-like receptors 1 and 6 are involved in TLR2-mediated macrophage activation by hepatitis C virus core and NS3 proteins

Hepatitis C virus (HCV) is a leading cause of end-stage liver disease through sustained inflammation of the liver produced by the host's immune system. The mechanism for HCV evasion or activation of the immune system is not clear. TLRs are cellular activators of the innate immune system. We recently reported that TLR2-mediated innate immune signaling pathways are activated by HCV core and NS3 proteins. TLR2 activation requires homo- or heterodimerization with TLR1 or TLR6. Here, we aimed to determine whether TLR2 coreceptors participated in cellular activation by HCV core or NS3 proteins. By designing small interfering RNAs targeted to TLR2, TLR1, and TLR6, we showed that knockdown of each of these receptors impairs pro- and anti-inflammatory cytokine activation by TLR-specific ligands as well as by HCV core and NS3 proteins in human embryonic kidney-TLR2 cells and in primary human macrophages. We found that HCV core and NS3 proteins induced TNF-alpha and IL-10 production in human monocyte-derived macrophages, which was impaired by TLR2, TLR1, and TLR6 knockdown. Contrary to human data, results from TLR2, TLR1, or TLR6 knockout mice indicated that the absence of TLR2 and its coreceptor TLR6, but not TLR1, prevented the HCV core and NS3 protein-induced peritoneal macrophage activation. In conclusion, TLR2 may use TLR1 and TLR6 coreceptors for HCV core- and NS3-mediated activation of macrophages and innate immunity in humans. These results imply that multiple pattern recognition receptors could participate in cellular activation by HCV proteins.     

Immunostimulatory role of mitochondrial DAMPs: alarming for pre‐eclampsia?

Mitochondria are critical signaling organelles that play an integral cellular role in the activation of diverse physiological responses to perturbation. Mitochondrial damage‐associated molecular patterns (DAMPs) act as redox signaling nodes synchronizing mitochondrial metabolism with triggering of inflammation. Oxidative stress and inflammation are implicated in the pathogenesis of pre‐eclampsia; however, the mechanisms involved in the novel crosstalk between these two pathogenic pathways are less well elucidated. In this review, we show that mitochondrial redox signals are paramount for regulating and maintaining the inflammatory response to danger signals. Mitochondrial DNA (mtDNA) represents a mitochondrial DAMP and is often liberated as signal of mitochondrial dysfunction. This review will explore the mechanistic role of mitochondrial DNA in directly coordinating adaptive changes in the maternal inflammatory status in pre‐eclampsia through recruitment of innate immune cells and subsequent cytokine production. Finally, we provide emerging evidence of elevated circulating mitochondrial DAMPs in pre‐eclampsia.     

Emerging role of redox dysregulation in alcoholic and nonalcoholic fatty liver disease

Fatty liver disease (FLD), associated with chronic alcohol consumption or obesity, is a serious medical problem. Strong evidence indicates that oxidative stress and dysregulation of redox-sensitive signaling pathways are central to the pathobiology of FLD. Herein, this Forum summarizes current knowledge regarding mechanisms of FLD from both clinical and experimental studies. Special emphasis is given to the role of redox biology disturbances in the initiation and progression of FLD from both chronic alcohol consumption and obesity. Focus areas in this Forum include discussions on the (i) multi-hit hypothesis; (ii) interaction of adipokines and redox signaling pathways; (iii) role of sub-cellular organelle systems (i.e., endoplasmic reticulum and mitochondria); and (iv) contribution of the innate immune system, in FLD. A state-of-the-art discussion is also included highlighting key lessons learned from experimental studies using rodent models of FLD.     

Differential contribution of complement receptor C5aR in myeloid and non-myeloid cells in chronic ethanol-induced liver injury in mice

BackgroundComplement is implicated in the development of alcoholic liver disease. C3 and C5 contribute to ethanol-induced liver injury; however, the role of C5a receptor (C5aR) on myeloid and non-myeloid cells to progression of injury is not known.MethodsC57BL/6 (WT), global C5aR-/-, myeloid-specific C5aR-/-, and non-myeloid-specific C5aR-/- mice were fed a Lieber-DeCarli diet (32% kcal EtOH) for 25 days. Cultured hepatocytes were challenged with ethanol, TNFα, and C5a.ResultsChronic ethanol feeding increased expression of pro-inflammatory mediators in livers of WT mice; this response was completely blunted in C5aR-/- mice. However, C5aR-/- mice were not protected from other measures of hepatocellular damage, including ethanol-induced increases in hepatic triglycerides, plasma alanine aminotransferase and hepatocyte apoptosis. CYP2E1 and 4-hydroxynonenal protein adducts were induced in WT and C5aR-/- mice. Myeloid-specific C5aR-/- mice were protected from ethanol-induced increases in hepatic TNFα, whereas non-myeloid-specific C5aR-/- displayed increased hepatocyte apoptosis and inflammation after chronic ethanol feeding. In cultured hepatocytes, cytotoxicity induced by challenge with ethanol and TNFα was completely eliminated by treatment with C5a in cells from WT, but not C5aR-/- mice. Further, treatment with C5a enhanced activation of pro-survival signal AKT in hepatocytes challenged with ethanol and TNFα.ConclusionTaken together, these data reveal a differential role for C5aR during ethanol-induced liver inflammation and injury, with C5aR on myeloid cells contributing to ethanol-induced inflammatory cytokine expression, while non-myeloid C5aR protects hepatocytes from death after chronic ethanol feeding.     

Hepatic and Cerebral Energy Production System in Rats with Acute and Chronic Ethanol Intoxication

We studied the effects of ethanol on the energy production system in the brain and liver in acute and chronic intoxications. Ethanol was found to inhibit mitochondrial respiratory chain in the liver. Acute ethanol intoxication results in uncoupling of oxidative phosphorylation. NAD-dependent respiration prevails in chronic intoxication. In the brain, ethanol exposure induces a compensated low-energy shift with activation of fast mitochondrial metabolic cluster and uncoupling of oxidative phosphorylation.     

Different effects of acute and chronic ethanol on LPS-induced cytokine production and TLR4 receptor behavior in mouse peritoneal macrophages

Both binge and chronic heavy drinking can adversely affect the immune system, but the effects seem to be at least partly dependent on the manner of ethanol (EtOH) consumption. Previous study results from several labs have clearly demonstrated that acute administration of EtOH interferes with innate immune responses. Specifically, EtOH has a general inhibitory effect on cytokine and chemokine production induced by various Toll-like receptor (TLR) ligands, and it suppresses signaling on several levels along the TLR signaling pathways. However, it is not clear whether chronic exposure to ethanol has the same effects or not. The purpose of this study was to investigate the difference between the effect of chronic versus acute EtOH exposure on LPS-induced cytokine production and clustering of components of the TLR4 complex, which is an important early signaling event. Some groups of mice received acute EtOH by oral gavage using our binge drinking model and/or chronic administration of EtOH at 20% (w/v) in the drinking water as the sole liquid source for 4 wk. The cellular distribution of CD14 and TLR4 were studied by confocal microscopy following exposure of peritoneal cells to LPS locally in vivo, and cytokine production in peritoneal fluid and serum was measured by ELISA after LPS injection via a tail vein. Chronic EtOH exposure did not consistently cause significant changes in LPS-induced cytokine production. However, mice previously exposed to chronic EtOH treatment became partially resistant to the suppressive effects of acute EtOH administration with regard to cytokine production. As we have reported previously, acute EtOH treatment suppressed the LPS-induced clustering of TLR4 and CD14 in peritoneal macrophages. However, peritoneal cells from mice treated with chronic EtOH exhibited a greater amount of intracellular expression of CD14 instead of CD14/TLR4 clustering on the membrane following LPS exposure. The results demonstrate different effects of chronic versus acute EtOH treatment on LPS-induced cytokine production in mice. Partial tolerance to the effect of acute EtOH administration caused by chronic EtOH treatment suggests a compensatory mechanism is induced by chronic EtOH administration. Acute EtOH exposure acts probably by disrupting the receptor clustering following LPS recognition, whereas adaptations induced by chronic EtOH treatment seem to involve alteration of LPS receptor expression.     

Hyperthermia differentially regulates TLR4 and TLR2-mediated innate immune response

Fever influences multiple parameters of the immune response. However, the mechanisms by which fever manipulates immune response remain undefined. Here we present the evidences that fever range hyperthermia differentially regulates immune response to lipopolysaccharide (LPS) and lipoteichoic acids (LTA) through modulating Toll-like receptor (TLR) signaling. Pretreatment with 39.5 degrees C temperature enhanced LPS, but not LTA, induced NF-kappaB activation and TNF-alpha, IL-6 production in human macrophages. Consistently, expression of TLR4, but not TLR2, was up-regulated by 39.5 degrees C treatment. The increase in LPS-induced cytokine production was inhibited by TLR4-blocking antibody, indicating the enhancement of LPS-induced cytokine production by 39.5 degrees C pretreatment was TLR4-dependent. Pretreatment of mice with 39.5 degrees C temperature also enhanced LPS, but not LTA, induced TNF-alpha and IL-6 production in vivo. These results support the concept that fever range hyperthermia might activate innate immune response by promoting TLR4 expression and signaling, providing a possible mechanistic explanation for the function of fever in regulating innate immune responses.     

PI3K and negative regulation of TLR signaling

Excessive immune responses are detrimental to the host and negative feedback regulation is crucial for the maintenance of immune-system integrity. Recent studies have shown that phosphoinositide 3-kinase (PI3K) is an endogenous suppressor of interleukin-12 (IL-12) production triggered by Toll-like receptor (TLR) signaling and limits excessive Th1 polarization. Unlike IRAK-M (IL-1 receptor-associated kinase-M) and SOCS-1 (suppressor of cytokine signaling-1) that are induced by TLR signaling and function during the second or continuous exposure to stimulation, PI3K functions at the early phase of TLR signaling and modulates the magnitude of the primary activation. Thus, PI3K, IRAK-M and SOCS-1 have unique roles in the gate-keeping system, preventing excessive innate immune responses.     

Gamma interferon production by hepatic NK T cells during Escherichia coli infection is resistant to the inhibitory effects of oxidative stress

The reductive-oxidative status of tissues regulates the expression of many inflammatory genes that are induced during gram-negative bacterial infections. The cytokine gamma interferon (IFN-gamma) is a potent stimulus for host inflammatory gene expression, and oxidative stress has been shown to inhibit its production in mice challenged with Escherichia coli bacteria. The objective of the present study was to characterize the cells that produced IFN-gamma in a mouse bacterial peritonitis model and determine the effects of oxidative stress on their activation. The liver contained large numbers of IFN-gamma-expressing lymphocytes following challenge with viable E. coli bacteria. The surface phenotypes of IFN-gamma-expressing hepatic lymphocytes were those of natural killer (NK) cells (NK1.1(+) CD3(-)), conventional T cells (NK1.1(-) CD3(+)), and NK T cells (NK1.1(+) CD3(+)). Treating mice with diethyl maleate to deplete tissue thiols significantly impaired IFN-gamma production by NK cells, conventional T cells, and CD1d-restricted NK T cells in response to E. coli challenge. However, IFN-gamma expression by a subset of NK T cells, which did not bind alpha-galactosylceramide-CD1d tetramers, was resistant to the inhibitory effects of tissue oxidative stress. Stress-resistant IFN-gamma-expressing cells were also predominantly CD8(+) and bore gamma delta T-cell antigen receptors. The residual IFN-gamma response by NK T cells may explain previous reports of hepatic gene expression following gram-negative bacterial challenge in thiol-depleted mice. The finding also demonstrates that innate immune cells differ significantly in their responses to altered tissue redox status.     

Fever-like thermal conditions regulate the activation of maturing dendritic cells

Fever is one of the most frequent clinical signs encountered in pathology, especially with respect to infectious diseases. It is currently thought that the role of fever on immunity is limited to activation of innate immunity; however, its relevance to activation of adaptive immunity remains unclear. Dendritic cells (DCs) that behave as sentinels of the immune system provide an important bridge between innate and adaptive immunity. To highlight the role of fever on adaptive immunity, we exposed murine bone marrow-derived lipopolysaccharide (LPS)- or live bacteria-maturing DCs over a 3-h period to 37 degrees C or to fever-like thermal conditions (39 degrees C or 40 degrees C). At these three temperatures, we measured the kinetics of cytokine production and the ability of DCs to induce an allogeneic mixed lymphocyte reaction. Our results show that short exposure of DCs to temperatures of 39 degrees C or 40 degrees C differentially increased the secretion of interleukin (IL)-12p70 and decreased the secretion of IL-10 and tumor necrosis factor alpha by maturing DCs. These fever-like conditions induced a regulation of cytokine production at the single-cell level. In addition, short-term exposed LPS-maturing DCs to 39 degrees C induced a stronger reaction with allogeneic CD4(+) T cells than maturing DCs incubated at 37 degrees C. These results provide evidence that temperature regulates cytokine secretion and DC functions, both of which are of particular importance in bacterial diseases.     

Effect of Ozone Exposure on Intracellular Glutathione Redox State in Cultured Human Airway Epithelial Cells

Intracellular oxidation and reduction (redox state) correspond closely to the surrounding environment. Most environmental factors affecting this balances such as oxidants, ultraviolet light, radioactive emissions, infections, and allergic reactions represent oxidative stress upon cells. We examined intracellular redox state after oxidative stress upon cultured human airway epithelial cells (Calu-3) by measuring reduced (GSH) or oxidized (GSSG) glutathione. We studied cytokine production, which is related to glutathione redox regulation, in response to ozone and also evaluated the effect of pretreatment with an ethyl ester of reduced glutathione (GSH-OEt) on cytokines. Ozone exposure (3.0 ppm, 3 min) time-dependently changed the redox state, while increasing production of interleukin(IL)-8 and IL-6, mRNA and protein. Treatment with GSH-OEt before ozone suppressed IL-8, but stimulated IL-6 production. Thus, oxidative stress affects intracellular glutathione redox state, in airway epithelial cells, activating signals to increase production of cytokine, modulation that may exacerbate respiratory symptoms.     

Pro-oxidative diesel exhaust particle chemicals inhibit LPS-induced dendritic cell responses involved in T-helper differentiation

BACKGROUND: Epidemiologic studies show that exposure to ambient particulate matter leads to asthma exacerbation. Diesel exhaust particles (DEPs), a model pollutant, act as an adjuvant for allergic sensitization. Increasing evidence shows that this effect could be mediated by an effect on dendritic cells (DCs). OBJECTIVE: Our aim was to elucidate the mechanism by which pro-oxidative DEP chemicals change DC function so that these antigen-presenting cells strengthen the immune response to an experimental allergen. METHODS: We exposed murine bone marrow-derived DCs and a homogeneous myeloid DC line, BC1, to DEPs and organic extracts made from these particles to determine how the induction of oxidative stress affects cellular maturation, cytokine production, and activation of antigen-specific T cells. RESULTS: DEP extracts induced oxidative stress in DCs. This change in redox equilibrium interfered in the ability of Toll-like receptor agonists to induce the expression of maturation receptors (eg, CD86, CD54, and I-A(d)) and IL-12 production. This perturbation of DC function was accompanied by decreased IFN-gamma and increased IL-10 induction in antigen-specific T cells. The molecular basis for the perturbation of DC function is the activation of a nuclear factor-erythroid 2 (NF-E2)-related factor 2-mediated signaling pathway that suppresses IL-12 production. NF-E2-related factor 2 deficiency abrogates the perturbation of DC function by DEPs. CONCLUSION: These data provide the first report that pro-oxidative DEP chemicals can interfere in T(H)1-promoting response pathways in a homogeneous DC population and provide a novel explanation for the adjuvant effect of DEPs on allergic inflammation. CLINICAL IMPLICATIONS: These data clarify the adjuvant effect of particulate air pollutants in allergic inflammatory disease.