Acute exposure to ethanol affects Toll-like receptor signaling and subsequent responses: an overview of recent studies.

Ethanol suppresses innate resistance to a variety of microbes, and findings of studies from both our laboratory and other laboratories indicate suppression of responses is mediated through two Toll-like receptors (TLRs): TLR3 and TLR4. In this article, we review recent findings from studies in our laboratory, indicating that ethanol also suppresses responses mediated through other TLRs. Considering the importance of TLR-mediated responses in innate immunity, this supports the possibility that suppression of these responses may constitute a major mechanism by which ethanol suppresses innate immunity. In addition, ethanol-induced changes in cellular signaling and in patterns of gene expression induced through TLR3 were examined in mouse peritoneal macrophages, and these results are reviewed in this article. Signaling through TLR3 was inhibited, and results of DNA microarray analysis supported the notion that inhibition of an interferon-related amplification loop might be responsible for suppression of gene expression for several effector molecules of innate immunity and inflammation not previously known to be altered by ethanol. Thus, ethanol alters responses through most or all mouse TLRs, and this suppresses expression of a wide range of innate immune mediators.     

Ethanol suppresses cytokine responses induced through Toll-like receptors as well as innate resistance to Escherichia coli in a mouse model for binge drinking.

Toll-like receptors (TLRs) recognize molecular patterns associated with pathogens and initiate various mechanisms that are critical in innate resistance to infection. It has been reported that acute administration of ethanol suppresses responses mediated through TLR3 and TLR4. However, it is not known whether this is also true for other TLRs. Ligands for TLR2/TLR6 (zymosan A), TLR5 (bacterial flagellin), TLR7 (R-848), and TLR9 (CpG DNA) were used to induce cytokine production in mice, and the effects of ethanol (6 g/kg by gavage) on this induction were determined. Because different cell types may be affected differently by ethanol, cytokines were measured in serum (as an indication of cytokines produced by a number of different cell types) and in peritoneal lavage fluid (as an indicator of cytokine production primarily by peritoneal macrophages). Ethanol significantly affected the concentration of at least one of the cytokines evaluated in serum or peritoneal lavage fluid [interleukin (IL)-6, IL-10, and IL-12 p40 subunit] induced by all TLR ligands tested. The results also supported the suggestion that serum and peritoneal cytokines were mostly derived from different cells types, which were affected differently by ethanol. To determine whether ethanol-induced changes in TLR responses were associated with suppression of innate resistance to infection, a model of experimental peritonitis with a nonpathogenic (indigenous) strain of Escherichia coli was developed. Ethanol significantly decreased host resistance to E. coli peritonitis. Thus, ethanol suppresses responses induced by TLR receptors in mice and in the same experimental system it suppresses resistance to infection.     

Alcohol and immunology: introduction to and summary of the 2003 Alcohol and Immunology Research Interest Group (AIRIG) meeting.

The 8th Meeting of the Alcohol and Immunology Research Interest Group (AIRIG) was held at Loyola University Medical Center, Maywood, Illinois, USA, on November 21, 2003. Reports from multiple laboratories reveal that the functional integrity of the immune system is of paramount importance to the survival of the individual after infection or injury. Evidence supports the idea that exposure to alcohol causes dysregulation of both the innate and the adaptive arms of the immune system. Gaining a better understanding of how alcohol interferes with normal inflammatory and immunoregulatory processes will aid researchers in the design of therapeutic interventions that can be used to improve these responses to better fight infection and maintain the health of the individual. At this meeting, nine speakers presented a summary of their recent work on the combined effects of ethanol and injury, infection, or inflammatory challenge. Topics were (1) T-cell activation after chronic ethanol ingestion in mice, (2) effect of ethanol consumption on the severity of acute viral-mediated pancreatitis, (3) ethanol and alveolar macrophage dysfunction, (4) impaired intestinal immunity and barrier function: a cause for enhanced bacterial translocation in alcohol intoxication and burn injury, (5) immune consequences of the combined insult of acute ethanol exposure and burn injury, (6) consequences of alcohol-induced dysregulation of immediate hemodynamic and inflammatory responses to trauma/hemorrhage, (7) regulation of tumor necrosis factor-alpha production by Kupffer cells after chronic exposure to ethanol, (8) acute exposure to ethanol and suppression of cytokine responses induced through Toll-like receptors, and (9) inhibition of antigen-presenting cell functions by alcohol: implications for hepatitis C virus infection. We anticipate that the work presented at the 8th Meeting of AIRIG, summarized in this article, and presented in the nine articles to follow in this Special Issue of Alcohol will stimulate ideas that will develop into research projects in these topical areas.     

Effect of ethanol and aging on histamine release and membranes of mast cells

The effects of ethanol on histamine release from mast cells were compared to ethanol's effects on membrane order of mast cell membranes and synaptosomes in young (6 month) and old (24 month) Fischer 344 rats. Concanavalin A (con A) stimulated histamine release in a concentration dependent manner. Ethanol (10-500 mM) inhibited con A stimulated release while having no effect on nonstimulated release in both young and old rats. Ethanol's effect on membrane order of synaptosomes and mast cell plasma membranes was estimated by measuring the fluorescence polarization of diphenylhexatriene. Ethanol (10-500 mM) decreased the polarization of synaptosomes to the same degree in young and old rats. The polarization of mast cell membranes was also decreased by ethanol but to a greater degree than synaptosomes. The ethanol induced changes in polarization correlated (r2 = 0.99) with ethanol's inhibition of con A stimulated histamine release from mast cells. These findings suggest that mast cells may be more sensitive to membrane disordering by ethanol than synaptosomes. In addition, we have demonstrated that mast cells may be a useful model system for studying ethanol effects on stimulus-secretion coupling. No differences were found between rats 6 and 24 months for histamine release (with or without ethanol) or membrane order of mast cells or synaptosomes.     

Alcohol and inflammation and immune responses: summary of the 2005 Alcohol and Immunology Research Interest Group (AIRIG) meeting.

The 10th annual meeting of the Alcohol and Immunology Research Interest Group (AIRIG) was held at Loyola University Medical Center, Maywood, Illinois on November 18, 2005. The AIRIG meeting was held to exchange new findings and ideas regarding the profound suppressive effects of alcohol exposure on the immune system. The event consisted of five sessions, two of which featured plenary talks from invited speakers, two with oral presentations from selected abstracts, and a final poster session. Participants presented a range of novel information focused on ethanol-induced effects on innate and adaptive immunity after either acute or chronic exposure. In particular, participants offered insights into the negative effects of ethanol on the innate processes of adhesion, migration, inflammation, wound repair, and bone remodeling. Presentations also focused on the means by which ethanol disrupts activation of macrophages and dendritic cells (DC), especially stimulation mediated by Toll-like receptor ligands. Additional talks provided new data on the means by which ethanol suppresses adaptive immunity, with an emphasis on DC-mediated activation of T cells, effector T cell activity, and T cell-driven B cell responses.     

Inhibition of (Na+, K+)-ATPase by fluoride: evidence for a membrane adaptation to ethanol

Fluoride ions inhibit several membrane enzymes in a manner that is dependent on membrane fluidity. Inhibition of (Na+, K+)-ATPase by fluoride ions may be a model for membrane effects on (Na+, K+)-ATPase. Therefore, we have examined properties of fluoride inhibition relative to interactions with ethanol and to ligands that alter sensitivity of (Na+, K+)-ATPase to ethanol. Fluoride ion reduced the K0.5 and Hill coefficient for K+ activation of p-nitrophenylphosphatase. Ethanol decreased the Hill coefficient and apparent affinity for inhibition of phosphatase activity by fluoride ion while dimethylsulfoxide had the opposite effects. Chronic ethanol treatment in vivo, which produced behavioral tolerance, had effects on fluoride inhibition opposite to those of ethanol in vitro. Inhibition by fluoride therefore may provide a useful marker for physiologic or pharmacologic conditions that alter regulation of (Na+, K+)-ATPase by membrane properties.     

STING pathway stimulation results in a differentially activated innate immune phenotype associated with low nitric oxide and enhanced antibody titers in young and aged mice

BackgroundOne of the most concerning public health issues, related to vaccination and disease prevention, is the inability to induce durable immune responses following a single-dose immunization. In this regard, the nature of the inflammatory environment induced by vaccine adjuvants can negatively impact the resulting immune response. To address these concerns, new strategies to vaccine design are needed in order to improve the outcomes of immune responses, particularly in immunologically disadvantaged populations.MethodsComparisons of the scope of innate immune activation induced by TLR agonists versus cyclic dinucleotides (CDNs) was performed. Their effects on the activation characteristics (e.g., metabolism, cytokine secretion) of bone marrow derived dendritic cells (BMDCs) were studied. In addition, the differential effects on in vivo induction of antibody responses were measured.ResultsAs compared to TLR ligands, the stimulation of BMDCs with CDNs induced distinctly different metabolic outcomes. Marked differences were observed in the production of nitric oxide (NO) and the cytokine BAFF. These distinct differences were correlated with improved (i.e., more rapid and persistent) vaccine antibody responses in both aged and young mice.ConclusionsOur results illustrate that the innate immune pathway targeted by adjuvants can critically impact the outcome of the immune response post-vaccination. Specifically, CDN stimulation of APCs induced an activation phenotype that was characterized by decreased innate effector molecule production (e.g., NO) and increased BAFF. This was attributed to the induction of an innate inflammatory environment that enabled the host to make the most of the existing B lymphocyte potential. The use of adjuvants that differentially engage mechanisms of innate immune activation would be particularly advantageous for the generation of robust, single dose vaccines. The results of this study demonstrated that CDNs induced differential innate activation and enhanced vaccine induced antibody responses in both young and aged mice.     

Immunostimulatory and antigen delivery properties of liposomes made up of total polar lipids from non-pathogenic bacteria leads to efficient induction of both innate and adaptive immune responses

Novel liposomes prepared from total polar lipids of non-pathogenic bacteria, viz. Leptospira biflexa serovar Potac (designated leptosomes) and Mycobacterium smegmatis (designated smegmosomes) were evaluated for their adjuvant effects with various antigen presenting cells (APCs), viz. murine macrophage cell line, J774A.1 and bone marrow derived dendritic cells (BMDCs). These liposomes induced strong membrane fusion as evident from resonance energy transfer (RET) assays and effectively transferred the fluorescent probe to the membrane of these APCs. Moreover, both vesicles caused significant activation of APCs as revealed by release of proinflammatory cytokines (IL-6, IL-12, TNF-α) and enhanced expression of co-stimulatory signals and maturation markers (CD80, CD86, MHCII), which was significantly higher for smegmosomes as compared to leptosomes. Additionally, activation of APCs by liposomes correlated with their ability to stimulate allospecific T cell proliferation and IFN-γ release. In contrast, conventional PC/chol liposomes failed to fuse and induced only a very low level of APC activation. Interestingly, the stimulatory activity of these lipid vesicles was restricted to APCs as they did not cause any significant activation or mitogenic effect on lymphocytes (B and T cells) in vitro. Overall, the activation of APCs by both leptosomes and smegmosomes correlated with activation of strong humoral and cell mediated immune responses in C57/BL6 mice to entrapped ovalbumin (OVA) and was significantly higher than those induced by conventional liposomes and alum, which failed to activate cytotoxic T lymphocytes (CTLs). Taken together these results demonstrate the adjuvant potential of these novel lipid vesicles that may simultaneously induce both innate and adaptive immune responses due to their immune stimulatory and antigen delivery properties.     

Mechanistic basis and preliminary practice of butyric acid and butyrate sodium to mitigate gut inflammatory diseases: a comprehensive review

A key event featured in the early stage of chronic gut inflammatory diseases is the disordered recruitment and excess accumulation of immune cells in the gut lamina propria. This process is followed by the over-secretion of pro-inflammatory factors and the prolonged overactive inflammatory responses. Growing evidence has suggested that gut inflammatory diseases may be mitigated by butyric acid (BA) or butyrate sodium (NaB). Laboratory studies show that BA and NaB can enhance gut innate immune function through G-protein-mediated signaling pathways while mitigating the overactive inflammatory responses by inhibiting histone deacetylase. The regulatory effects may occur in both epithelial enterocytes and the immune cells in the lamina propria. Prior to further clinical trials, comprehensive literature reviews and rigid examination concerning the underlying mechanism are necessary. To this end, we collected and reviewed 197 published reports regarding the mechanisms, bioactivities, and clinical effects of BA and NaB to modulate gut inflammatory diseases. Our review found insufficient evidence to guarantee the safety of clinical practice of BA and NaB, either by anal enema or oral administration of capsule or tablet. The safety of clinical use of BA and NaB should be further evaluated. Alternatively, dietary patterns rich in “fruits, vegetables and beans” may be an effective and safe approach to prevent gut inflammatory disease, which elevates gut microbiota-dependent production of BA. Our review provides a comprehensive reference to future clinical trials of BA and NaB to treat gut inflammatory diseases.     

Ethanol and cytokine secretion.

Cytokines are regulatory polypeptides secreted during the generation of an immune or inflammatory response by lymphocytes, cells of the monocyte/macrophage series, and a variety of other cell types. Alterations in the production, site of action, or metabolism of cytokines by exogenous factors, such as ethanol (EtOH), may have deleterious effects on the immune system as a whole. EtOH has been implicated in the onset of a variety of immune defects in vivo including effects on the production of cytokines critically involved in inflammatory responses (tumor necrosis factor, interleukin 1 and interleukin 6). In this review, we examine current knowledge regarding the effects of EtOH on the release of cytokines in humans and in animal models, in vitro and in vivo, which may help to elucidate the adverse actions of EtOH on mammalian immune systems.     

临床实用的新思路和新方法：体外血液生命系统药物治疗个体化试验

目的建立体外静脉注射药品测试系统，以确定药物注射后是否引发血液中快速先天免疫炎症反应．保证避免可以致死的天然免疫炎症反应的发生。方法采用体外血液生命系统药物治疗个体化试验，设计为三组：新药实验组：0．2毫升新药加到o．6枸橼酸抗凝的新鲜正常人全血中；阳性对照组：0．2毫升（热灭活）大肠杆菌悬液（浓度为6×10^9），加到o．6毫升枸橼酸抗凝的新鲜新鲜正常人全血中；阴性对照组：0．2毫升生理盐水加入新鲜的0．6毫升枸橼酸抗凝全血中。充分混匀．37℃孵育1小时，2000转／分离心5分钟；测定与统计各组IL-8／IL-10和IL-8／DARC指标。按上述公式．求出新药实验组和阳性对照组的激活率（药物的影响率）。结果根据新药实验组和阳性对照组的激活率的比较，可判定新药是否具有免疫原活性。结论此测试系统操作简单，只需清洁操作，不需无菌操作，也不需加人工培养液，但注意点是要新鲜血，采血后应尽快做实验，而且必须是枸橼酸抗凝，不能灭活补体系统，也就是说不能用EDTA抗凝，否则血液失去生命信息完整的网络关系。只要认真按以上要求，细心认真操作，一定能成功。此创新方法对疾病治疗研究有帮助。     

Wine modifies the effects of alcohol on immune cells of mice

Ethanol may be detrimental to immune cells due to the generation of free radicals during detoxification. If this is true, then alcoholic beverages that contain antioxidants, like red wine, should be protective against immune cell damage. We investigated this by giving mice either a red muscadine wine (Vitis rotundifolia), a cabernet sauvignon (Vitis vinifera), ethanol (all at 6% alcohol) or water in the water bottles as the sole fluid for 8 wk. Plasma antioxidant capacity was measured with alphaalpha-diphenyl-beta-picrylhydrazyl and was more than doubled in the mice that consumed wine compared to control mice that consumed water or ethanol. Cytochrome P450-2E1 levels and glutathione-S-transferase activity were modified in such a way as to be interpreted as protective. An immune response was elicited by an intraperitoneal injection of lipopolysaccharide. Later (24 h), natural killer cells and T-lymphocytes derived from the circulation were quantitated in the leukocyte fraction by flow cytometry. Ethanol consumption, as ethanol, significantly suppressed baseline cell numbers relative to the other groups. However, the mice that consumed the same amount of alcohol as wine had baseline cell numbers not different from the water-consuming controls. The lymphocyte response to lipopolysaccharide challenge was inhibited in the mice that consumed ethanol, but was normal in those that consumed the same amount of alcohol in the form of wine. We conclude that there are phytochemicals acting as antioxidants and impacting on the detoxification pathway in the wine that offset the detrimental effects of ethanol on immunity.     

Ethanol Inhibition of Phagocytosis and Superoxide Anion Production by Microglia

Ethanol consumption has been associated with aberrant immune responses resulting in increased susceptibility to infection including opportunistic infections of the central nervous system. We have investigated the effects of chronic ethanol treatment on phagocytosis and production of superoxide anion by microglia. Phagocytosis of radiolabeled opsonized E. coli was markedly suppressed by treating microglia with ethanol. The unstimulated synthesis of superoxide anion was not altered by ethanol treatment of microglia, but ethanol treatment effectively suppressed phorbol-12 myristate-13 acetate-stimulated microglia superoxide anion production. The results indicate that ethanol inhibition of microglia function may play a role in increased susceptibility for central nervous system infections, particularly in immunocompromised subjects.     

The effects of chronic ethanol administration on rat liver and erythrocyte lipid composition: modulatory role of 'evening primrose oil'.

The effects of chronic administration of ethanol on the lipid composition of erythrocytes and liver of rats were investigated. Ethanol was chronically administered alone or in combination with Evening Primrose Oil (containing 10% v/v gamma-linolenic acid) in a nutritionally balanced milk diet. Chronic administration of ethanol alone significantly decreased the content of arachidonic acid in erythrocyte membranes, whereas the concomitant administration of Evening Primrose Oil reversed this effect. The triacylglycerol content was significantly increased in the microsomal fraction of the liver after chronic ethanol administration. Ethanol also significantly increased the ratio of cholesterol:cholesteryl esters in the microsomal fraction. The arachidonic acid content of the whole liver fraction was significantly reduced after chronic administration of ethanol, whereas concomitant administration of Evening Primrose Oil did not reverse this effect. The administration of Evening Primrose Oil during chronic ethanol intake may have beneficial effects, as it reverses some of the effects of ethanol on erythrocyte and hepatocyte membrane lipids which may be detrimental to health.     

The role of Toll-like receptor 2 in microbial disease and immunity

Cells expressing Toll-like receptor (TLR), TLR2 in association with TLR1, TLR6 or some other unknown co-receptor can respond upon interaction with a large variety of microbial ligands. The variety of TLR2 ligands is the greatest among all the TLRs and this is due to the heterodimerization needed for TLR2 mediated responses. Like other TLRs, TLR2 signaling induces antigen presenting cell activation, pro-inflammatory cytokine production and increased expression of co-stimulatory ligand expression. These events are important for induction of innate immune responses and improved acquired immunity. There is strong suggestive evidence that alteration or lack of TLR2 function in vivo may correlate to decreased immune protection from pathogens that contain TLR2 ligands, but more work needs to be performed to strengthen this correlation.     

Ethanol, oxidative stress, and cytokine-induced liver cell injury.

Both clinical findings and results of experiments with animal models of alcoholic hepatitis have shown the importance of cytokine-mediated cell-cell interactions in the onset of ethanol-induced liver damage. Proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 beta (IL-1 beta), and interleukin-6, are released from Kupffer cells or infiltrating neutrophils and macrophages and elicit defensive responses in parenchymal cells, including activation of apoptosis. Reactive oxygen species (ROS) and reactive nitrogen species (RNS), generated in response to cytokine-induced stress signals in parenchymal cells and also by activation of Kupffer cells and inflammatory cells, further mobilize cellular defense mechanisms. When these defensive responses are overwhelmed cells may die by necrosis, further stimulating inflammatory responses and infiltration of neutrophils. Chronic ethanol intake (i.e., many years of heavy alcohol use in human patients, several weeks or months in experimental animals) enhances the damaging consequences of these events through a variety of mechanisms. The formation of cytokines in the liver is stimulated by increasing circulating levels of endotoxin and by enhancing the responsiveness of Kupffer cells to such stimuli. In addition, ethanol promotes oxidative stress, both by increased formation of ROS and by depletion of oxidative defenses in the cell. Furthermore, liver cells from ethanol-treated animals are more susceptible to the cytotoxic effects of TNF-alpha and other cytokines than cells from control animals. Mitochondria play a critical role in the apoptotic response, and alterations in mitochondrial function after chronic ethanol treatment may contribute to enhanced cell death by apoptosis or necrosis. How the shift in the balance of cytokine-induced defensive and damage responses in hepatocytes contributes to the liver injury that occurs in alcoholic hepatitis remains poorly characterized and should be a rewarding area for future studies.     

Effect of opioid receptor blockade on acetaldehyde self-administration and ERK phosphorylation in the rat nucleus accumbens

We have previously shown that acetaldehyde (ACD), the first metabolite of ethanol, regulates its motivational properties and possesses reinforcing effects by itself. A large and still growing body of evidence indicates that the endogenous opioidergic system plays a critical role in the motivational effects of ethanol and suggests a role for extracellular signal-regulated kinase (ERK) in these effects of both ethanol and ACD. The present study was undertaken to examine if opioid-mediated mechanisms are involved in the reinforcing properties of ACD and in ACD-elicited ERK activation. To this end, Wistar rats were trained to orally self-administer ACD (0.2%) by nose poking. Responses on active nose poke caused delivery of ACD solution, whereas responses on inactive nose poke had no consequences. The effect of pretreatment with a nonselective opioid receptor antagonist, naltrexone (NTX), was evaluated during (1) maintenance of ACD self-administration, (2) deprivation effect after ACD extinction, and (3) ACD self-administration under a progressive-ratio schedule of reinforcement. Additionally, we tested the effect of NTX on saccharin (0.05%) reinforcement, as assessed by oral self-administration, and on ACD-elicited ERK phosphorylation in the nucleus accumbens (Acb), as assessed by immunohistochemistry. Finally, we examined the effect of a μ₁-selective opioid receptor antagonist, naloxonazine (NLZ), on the maintenance phase of ACD and saccharin self-administration. The results indicate that NTX (0.4–0.8 mg/kg) reduced the maintenance, the deprivation effect, and the break points of ACD self-administration without suppressing saccharin self-administration. Moreover, NTX decreased ACD-elicited ERK activation in the Acb shell and core. NLZ (10–15 mg/kg) reduced the maintenance phase of ACD self-administration without interfering with saccharin self-administration, whereas both NTX and NLZ failed to modify responses on inactive nose poke indicating the lack of a nonspecific behavioral activation. Overall, these results indicate that the opioid system is implicated in the reinforcing properties of ACD and suggest an involvement of ERK. The finding that NTX and NLZ reduce ACD but not saccharin self-administration indicates that these effects are specific to ACD.     

Alcohol and liver cancer.

Hepatocellular carcinoma is the eighth most frequent cancer in the world, accounting for approximately 500,000 deaths per year. Unlike many malignancies, hepatocellular carcinoma occurs predominantly within the context of known risk factors, with hepatic cirrhosis being the most common precursor to the development of hepatocellular carcinoma. After ethanol ingestion, the liver represents the major site of metabolism. Ethanol metabolism by alcohol dehydrogenase leads to the generation of acetaldehyde and free radicals that bind rapidly to numerous cellular targets, including components of cell signaling pathways and DNA. In addition to direct DNA damage, acetaldehyde depletes glutathione, an antioxidant involved in detoxification. Chronic ethanol abuse leads to induction of hepatocyte microsomal cytochrome P450 2E1, an enzyme that metabolizes ethanol to acetaldehyde and, in doing so, causes further free radical production and aberrant cell function. Cytochrome P450 2E1-dependent ethanol metabolism is also associated with activation of procarcinogens, changes in cell cycle, nutritional deficiencies, and altered immune system responses. The identification of oxidative stress in mediating many deleterious effects of ethanol in the liver has led to renewed interest in the use of dietary antioxidants as therapeutic agents. Included in this group are S-adenosyl-L-methionine and plant-derived flavanoids.