Search

Alcohol use among older adults is on the rise. This increase is clinically relevant as older adults are at risk for increased morbidity and mortality from many alcohol-related chronic diseases compared to younger patients. However, little is known regarding the synergistic effects of alcohol and age. There are intriguing data suggesting that aging may lead to impaired intestinal barrier integrity and dysbiosis of the intestinal microbiome, which could increase susceptibility to alcohol's negative effects. To study the effects of alcohol in age we exposed aged and young mice to 3 days of moderate ethanol and evaluated changes in gut parameters. We found that these levels of drinking do not have obvious effects in young mice but cause significant alcohol-induced gut barrier dysfunction and expression of the pro-inflammatory cytokine TNFα in aged mice. Ethanol-induced downregulation of expression of the gut-protective antimicrobial peptides Defa-rs1, Reg3b, and Reg3g was observed in aged, but not young mice. Analysis of the fecal microbiome revealed age-associated shifts in microbial taxa, which correlated with intestinal and hepatic inflammatory gene expression. Taken together, these data demonstrate that age drives microbiome dysbiosis, while ethanol exposure in aged mice induces changes in the expression of antimicrobial genes important for separating these potentially damaging microbes from the intestinal lumen. These changes highlight potential mechanistic targets for prevention of the age-related exacerbation of effects of ethanol on the gut., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Cutaneous burn injury is one of the most devastating injuries one can obtain, with tissue damage extending beyond the skin wound to distal organs, including the gastrointestinal tract, liver, and lungs. Multiple organ failure is a leading cause of death after burn injury, resulting in excessive systemic and localized inflammation directly contributing to end organ damage. We postulated that the gut-liver-lung inflammatory axis underscores multiple organ failure in the context of burn injury and is hyper-activated when ethanol intoxication precedes burn. Mesenchymal stem cells (MSCs) are regenerative and anti-inflammatory, and MSC treatment has been shown to be beneficial in several immune disorders and injury models. Our objective was to determine whether intravenous infusion of exogenous bone marrow-derived MSCs could reduce post-burn and intoxication pulmonary, hepatic, and systemic inflammation. Vehicle- or ethanol- (1.6 g/kg) treated mice were subjected to sham or 15% total body surface area scald burn. One hour post-injury, mice were given 5 × 10 5 CFSE-labeled MSCs or phosphate-buffered saline intravenously (i.v.) and were euthanized 24 h later. We assessed circulating biomarkers of inflammation and liver damage, measured cytokine and chemokine production, and quantified apoptosis in lung and liver tissue. Compared to intoxicated and burned mice, those treated with MSCs had less cellularity, limited apoptosis, and a slight reduction in the pro-inflammatory cytokine interleukin-6 (IL-6) and the neutrophil chemokine, KC (CXCL1) in lung tissue. Mice with MSCs treatment had more dramatic anti-inflammatory effects on systemic and hepatic inflammation, as serum IL-6 levels were diminished by 43%, and il6 and kc expression in liver tissue were markedly reduced, as were biomarkers of liver damage, aspartate transaminase (AST) and alanine transaminase (AST), compared with intoxicated and burned mice. Taken together, our results suggest intravenous MSCs treatment can diminish systemic inflammation, lessen hepatic damage, and decrease liver and lung apoptosis and inflammation, indicating MSCs as a novel therapy for restoring homeostasis of multiple organ systems in intoxicated burn patients., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Dendritic cells (DCs) are a critical component of anti-tumor immunity due to their ability to induce a robust immune response to antigen (Ag). Alcohol was previously shown to reduce DC ability to present foreign Ag and promote pro-inflammatory responses in situations of infection and trauma. However the impact of alcohol exposure on generation of an anti-tumor response, especially in the context of generation of an immune vaccine has not been examined. In the clinic, DC vaccines are typically generated from autologous blood, therefore prior exposure to substances such as alcohol may be a critical factor to consider regarding the effectiveness in generating an immune response. In this study, we demonstrate for the first time that ethanol differentially affects DC and tumor Ag-specific T cell responses depending on sex. Signaling pathways were found to be differentially regulated in DC in females compared to males and these differences were exacerbated by ethanol treatment. DC from female mice treated with ethanol were unable to activate Ag-specific cytotoxic T cells (CTL) as shown by reduced expression of CD44, CD69, and decreased production of granzyme B and IFNγ. Furthermore, although FOXO3, an immune suppressive mediator of DC function, was found to be upregulated in DC from female mice, ethanol related suppression was independent of FOXO3. These findings demonstrate for the first time differential impacts of alcohol on the immune system of females compared to males and may be a critical consideration for determining the effectiveness of an immune based therapy for cancer in patients that consume alcohol., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

In this study, the role and fate of AMs were examined in pulmonary inflammation after intoxication and injury. Clinical evidence has revealed that half of all burn patients brought to the emergency department are intoxicated at the time of injury. This combined insult results in amplified neutrophil accumulation and pulmonary edema, with an increased risk of lung failure and mortality, relative to either insult alone. We believe that this excessive pulmonary inflammation, which also parallels decreased lung function, is mediated in part by AMs. Restoration of lung tissue homeostasis is dependent on the eradication of neutrophils and removal of apoptotic cells, both major functions of AMs. Thirty minutes after binge ethanol intoxication, mice were anesthetized and given a 15% total body surface area dorsal scald injury. At 24 h, we found a 50% decrease in the total number of AMs (P < 0.05) and observed a proinflammatory phenotype on the remaining lung AMs. Loss of AMs paralleled a 6-fold increase in the number of TUNEL + lung apoptotic cells (P < 0.05) and a 3.5-fold increase in the percentage of annexin V + apoptotic cells in BAL (P < 0.05), after intoxication and injury, relative to controls. In contrast to the reduction in the number of cells, AMs from intoxicated and injured mice had a 4-fold increase in efferocytosis (P < 0.05). In summary, these data suggest that loss of AMs may delay resolution of inflammation, resulting in the pulmonary complications and elevated mortality rates observed in intoxicated and burn-injured patients., (© Society for Leukocyte Biology.)

Annually, excessive alcohol use accounts for more than $220 billion in economic costs and 80,000 deaths, making excessive alcohol use the third leading lifestyle-related cause of death in the US. Patients with an alcohol-use disorder (AUD) also have an increased susceptibility to respiratory pathogens and lung injury, including a 2-4-fold increased risk of acute respiratory distress syndrome (ARDS). This review investigates some of the potential mechanisms by which alcohol causes lung injury and impairs lung immunity. In intoxicated individuals with burn injuries, activation of the gut-liver axis drives pulmonary inflammation, thereby negatively impacting morbidity and mortality. In the lung, the upper airway is the first checkpoint to fail in microbe clearance during alcohol-induced lung immune dysfunction. Brief and prolonged alcohol exposure drive different post-translational modifications of novel proteins that control cilia function. Proteomic approaches are needed to identify novel alcohol targets and post-translational modifications in airway cilia that are involved in alcohol-dependent signal transduction pathways. When the upper airway fails to clear inhaled pathogens, they enter the alveolar space where they are primarily cleared by alveolar macrophages (AM). With chronic alcohol ingestion, oxidative stress pathways in the AMs are stimulated, thereby impairing AM immune capacity and pathogen clearance. The epidemiology of pneumococcal pneumonia and AUDs is well established, as both increased predisposition and illness severity have been reported. AUD subjects have increased susceptibility to pneumococcal pneumonia infections, which may be due to the pro-inflammatory response of AMs, leading to increased oxidative stress., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Of the 450,000 burn patients each year, 50% have a positive blood alcohol content, and this predisposes them to worsened clinical outcomes. Despite high prevalence and established consequences, the mechanisms responsible for alcohol-mediated complications of postburn remote organ damage are currently unknown. To this end, mice received a single dose of alcohol (1.12 g/kg) or water by oral gavage and were subjected to a 15% total body surface area burn. Animals with a burn alone lost ∼5% of their body weight in 24 h, whereas intoxicated and burned mice lost only 1% body weight (P < 0.05) despite a 17% increase in hematocrit (P < 0.05) and a 57% increase in serum creatinine (P < 0.05) over burn injury alone. This retention of water weight despite increased dehydration suggests that intoxication at the time of a burn causes a shift in fluid compartments that may exacerbate end-organ ischemia and damage as evidenced by a 3-fold increase in intestinal bacterial translocation (P < 0.05), a 30% increase (P < 0.05) in liver weight-to-body weight ratio, and an increase in alveolar wall thickness over a burn alone. Furthermore, administration of the bradykinin antagonist HOE140 30 min after intoxication and burn restored fluid balance and alleviated end-organ damage. These findings suggest that alcohol potentiates postburn remote organ damage through shifts in fluid compartments mediated by bradykinin.

Background: Exacerbation of cutaneous wound infections and delayed wound closure are frequent complications seen in alcohol exposed subjects who sustain injuries. We previously reported that acute alcohol exposure alters the early dermal inflammatory phase of wound healing and also several parameters of the proliferative wound healing phase in wounds from ethanol (EtOH)-treated mice for several days or weeks after EtOH exposure. Hence, it is likely that the cumulative defects arising in the early phases of the wound healing process directly contribute to the increased complications observed in intoxicated patients at the time of injury., Methods: C57BL/6 mice were given intraperitoneal EtOH (2.2 g/kg body weight) or vehicle (saline) EtOH using our episodic binge EtOH exposure protocol (3 days EtOH, 4 days off, 3 days EtOH) to yield a blood alcohol concentration (BAC) of 300 mg/dl at the time of wounding. Mice were subjected to six 3 mm full-thickness dorsal wounds and immediately treated topically with 10 μl of sterile saline (control) or diluted Staphylococcus aureus corresponding to 1 × 10(4) CFU/wound. Wounds were harvested at 24 hours post injury to evaluate wound area, neutrophil and macrophage accumulation, and the protein levels of cytokines, interleukin-6 (IL-6), IL-1β, and IL-10, and chemokines, macrophage inflammatory protein-2 (MIP-2) and MIP-1α, monocyte chemotactic protein-1 (MCP-1), and keratinocyte-derived chemokine (KC). The abundance and localization of cathelicidin-related antimicrobial peptide (CRAMP) and the kallikrein epidermal proteases (KLK5 and KLK7) were also determined., Results: Compared to control mice, EtOH-treated mice exhibited delayed wound closure, decreased macrophage accumulation, and impaired production of MIP-1α. Furthermore, skin from EtOH-treated mice demonstrated a reduction in the abundance of epidermal CRAMP and KLK7., Conclusions: These findings suggest that EtOH exposure hinders several distinct components of the innate immune response, including phagocyte recruitment and chemokine/cytokine and AMP production. Together, these effects likely contribute to delayed wound closure and enhanced infection severity observed in intoxicated patients., (Copyright © 2014 by the Research Society on Alcoholism.)

Objective: T-helper (Th)-17 lymphocytes play a crucial role in maintenance and regulation of gut immunity. Our laboratory has demonstrated that acute ethanol (EtOH) exposure before burn injury results in intestinal T cell suppression and enhanced bacterial translocation., Background: To extend these studies, we examined the effects of EtOH exposure and burn injury on Th17 responses within intestinal lymphoid Peyer's patches (PP). We further investigated whether restitution of interleukin (IL)-23 enhances PP cell IL-17 and IL-22 after EtOH and burn injury., Methods: Male mice, approximately 25 g, were gavaged with EtOH (2.9 mg/kg) before receiving an approximately 12.5% total body surface area full thickness burn. One day postinjury, PP mixed cells were cultured in the presence of plate-bound anti-CD3/soluble anti-CD28 in the presence or absence of IL-23 for 48 hours. Supernatants were harvested for IL-17 and IL-22 levels., Results: When combined with EtOH intoxication, burn injury significantly decreased IL-17 and IL-22, as compared with sham injury. IL-23 treatment successfully increased levels of IL-22 but not IL-17. This restoration was prevented when PP cells were treated with CH-223191, an aryl hydrocarbon receptor inhibitor. To further delineate the mechanism of differential IL-17 and IL-22 suppression, PP cells were treated with phorbol 12-myristate 13-acetate (PMA) and ionomycin, which signal via protein kinase C (PKC) and calcium flux. Treatment with PMA and ionomycin significantly prevented the decrease in IL-17 but not IL-22 after EtOH exposure and burn injury., Conclusions: These findings suggest that IL-23-mediated restoration of IL-22 is aryl hydrocarbon receptor dependent, whereas IL-17 requires activation of protein kinase C and intracellular calcium signaling.

Background: Ethanol (EtOH) exposure prior to traumatic injury, such as a burn, elevates systemic and local inflammatory responses and increases morbidity and mortality. Adipose is a large tissue mass that is often inflamed during obesity or other stresses, which disturbs metabolic homeostasis. To date, there has been little investigation into the inflammatory response of adipose tissue after combined EtOH exposure and burn injury., Methods: Two EtOH exposure regimens were utilized to examine the role of inflammation in adipose tissue after EtOH and burn injury. Mice were either given a single or episodic binge exposure to EtOH or saline followed by scald (burn) or sham injury 30 minutes later. Twenty-four hours post injury, serum and adipose tissue were collected for assessment of inflammatory mediators., Results: Single binge EtOH alone induced no inflammation in adipose when compared with sham vehicle-treated mice. However, single binge EtOH followed by burn injury induced significant elevations in mRNA and protein concentrations of pro-inflammatory mediators interleukin-6 (IL-6), KC, and monocyte chemoattractant protein 1 compared with either insult alone or sham vehicle group. Additionally, EtOH exposure and burn injury significantly blunted inducible nitric oxide synthase (iNOS), indicating a complex inflammatory response. Episodic binge EtOH exposure followed by burn injury exacerbated the postburn adipose inflammatory response. The magnitude of the episodic binge-induced inflammatory parameters postburn were 2- to 5-fold greater than the response detected after a single exposure of EtOH, indicating EtOH-induced potentiation of burn-induced inflammatory response. Finally, inflammatory loci and crown-like structures in adipose were significantly increased by episodic binge EtOH and burn injury., Conclusions: This is the first report of binge and burn-induced crown-like structure formation. Evidence presented herein suggests an important role for alcohol and burn as an additional mediator of adipose inflammation in postburn injury, a common complication in burn patients., (Copyright © 2013 by the Research Society on Alcoholism.)

Alcohol use disorders (AUDs), including alcohol abuse and dependence, and cigarette smoking are widely acknowledged and common risk factors for pneumococcal pneumonia. Reasons for these associations are likely complex but may involve an imbalance in pro- and anti-inflammatory cytokines within the lung. Delineating the specific effects of alcohol, smoking, and their combination on pulmonary cytokines may help unravel mechanisms that predispose these individuals to pneumococcal pneumonia. We hypothesized that the combination of AUD and cigarette smoking would be associated with increased bronchoalveolar lavage (BAL) proinflammatory cytokines and diminished anti-inflammatory cytokines, compared with either AUDs or cigarette smoking alone. Acellular BAL fluid was obtained from 20 subjects with AUDs, who were identified using a validated questionnaire, and 19 control subjects, matched on the basis of age, sex, and smoking history. Half were current cigarette smokers; baseline pulmonary function tests and chest radiographs were normal. A positive relationship between regulated and normal T cell expressed and secreted (RANTES) with increasing severity of alcohol dependence was observed, independent of cigarette smoking (P = 0.0001). Cigarette smoking duration was associated with higher IL-1β (P = 0.0009) but lower VEGF (P = 0.0007); cigarette smoking intensity was characterized by higher IL-1β and lower VEGF and diminished IL-12 (P = 0.0004). No synergistic effects of AUDs and cigarette smoking were observed. Collectively, our work suggests that AUDs and cigarette smoking each contribute to a proinflammatory pulmonary milieu in human subjects through independent effects on BAL RANTES and IL-1β. Furthermore, cigarette smoking additionally influences BAL IL-12 and VEGF that may be relevant to the pulmonary immune response.

Alcohol consumption leads to an exaggerated inflammatory response after burn injury. Elevated levels of interleukin-6 (IL-6) in patients are associated with increased morbidity and mortality after injury, and high systemic and pulmonary levels of IL-6 have been observed after the combined insult of ethanol exposure and burn injury. To further investigate the role of IL-6 in the pulmonary inflammatory response, we examined leukocyte infiltration and cytokine and chemokine production in the lungs of wild-type and IL-6 knockout mice given vehicle or ethanol (1.11 g/kg) and subjected to a sham or 15% total body surface area burn injury. Levels of neutrophil infiltration and neutrophil chemoattractants were increased to a similar extent in wild-type and IL-6 knockout mice 24 h after burn injury. When ethanol exposure preceded the burn injury, however, a further increase of these inflammatory markers was seen only in the wild-type mice. Additionally, signal transducer and activator of transcription-3 (STAT3) phosphorylation did not increase in response to ethanol exposure in the IL-6 knockout mice, in contrast to their wild-type counterparts. Visual and imaging analysis of alveolar wall thickness supported these findings and similar results were obtained by blocking IL-6 with antibody. Taken together, our data suggest a causal relationship between IL-6 and the excessive pulmonary inflammation observed after the combined insult of ethanol and burn injury., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Interleukin 6 (IL-6) is an inflammatory cytokine known to be elevated in chronic diseases and after insults such as trauma and infection. Although necessary for the development of B cells and Th17 cells, IL-6, at elevated levels, can also cause tissue damage and lead to a rise in inflammation. Previous work in our laboratory has shown that IL-6 is increased both systemically and in multiple organ systems including the ileum after ethanol exposure and burn injury. As this combined insult causes elevated intestinal morphological damage, tight junction protein localization alterations, and phosphorylated myosin light chain levels, we sought to determine the role of IL-6 in these intestinal responses using a model of binge ethanol exposure and burn injury. Interleukin 6 antibody treatment after the combined insult reduced morphological changes in the ileum, bacterial translocation, and phosphorylated myosin light chain levels relative to either injury alone. Zonula occludens protein 1 and occludin localization was also reestablished in wild-type mice given IL-6 antibody after ethanol and burn. Interleukin 6-knockout mice given ethanol and burn injury also had reduced intestinal damage; however, no changes in bacterial translocation or tight junction protein localization were observed as compared with similarly treated wild-type mice. These data suggest that IL-6 may have a role in intestinal tissue damage observed after the combined insult of binge ethanol exposure and burn injury, although complete loss of IL-6 does not seem to be beneficial in this model. Modulation of IL-6 may present a new option for preventing intestinal damage and associated inflammation after a combined insult of ethanol exposure and burn injury.

Alcohol consumption alters factors that modify gene expression without changing the DNA code (i.e., epigenetic modulators) in many organ systems, including the immune system. Alcohol enhances the risk for developing several serious medical conditions related to immune system dysfunction, including acute respiratory distress syndrome (ARDS), liver cancer, and alcoholic liver disease (ALD). Binge and chronic drinking also render patients more susceptible to many infectious pathogens and advance the progression of HIV infection by weakening both innate and adaptive immunity. Epigenetic mechanisms play a pivotal role in these processes. For example, alcohol-induced epigenetic variations alter the developmental pathways of several types of immune cells (e.g., granulocytes, macrophages, and T-lymphocytes) and through these and other mechanisms promote exaggerated inflammatory responses. In addition, epigenetic mechanisms may underlie alcohol's ability to interfere with the barrier functions of the gut and respiratory systems, which also contribute to the heightened risk of infections. Better understanding of alcohol's effects on these epigenetic processes may help researchers identify new targets for the development of novel medications to prevent or ameliorate alcohol's detrimental effects on the immune system.

Laboratory evidence suggests that intestinal permeability is elevated following either binge ethanol exposure or burn injury alone, and this barrier dysfunction is further perturbed when these insults are combined. We and others have previously reported a rise in both systemic and local proinflammatory cytokine production in mice after the combined insult. Knowing that long myosin light-chain kinase (MLCK) is important for epithelial barrier maintenance and can be activated by proinflammatory cytokines, we examined whether inhibition of MLCK alleviated detrimental intestinal responses seen after ethanol exposure and burn injury. To accomplish this, mice were given vehicle or a single binge ethanol exposure followed by a sham or dorsal scald burn injury. Following injury, one group of mice received membrane permeant inhibitor of MLCK (PIK). At 6 and 24 h postinjury, bacterial translocation and intestinal levels of proinflammatory cytokines were measured, and changes in tight junction protein localization and total intestinal morphology were analyzed. Elevated morphological damage, ileal IL-1β and IL-6 levels, and bacterial translocation were seen in mice exposed to ethanol and burn injury relative to either insult alone. This increase was not seen in mice receiving PIK after injury. Ethanol-exposed and burn-injured mice had reduced zonula occludens protein-1 and occludin localization to the tight junction relative to sham-injured mice. However, the observed changes in junctional complexes were not seen in our PIK-treated mice following the combined insult. These data suggest that MLCK activity may promote morphological and inflammatory responses in the ileum following ethanol exposure and burn injury.

Clinical and laboratory investigations have provided evidence that ethanol suppresses normal lung immunity. Our initial studies revealed that acute ethanol exposure results in transient suppression of phagocytosis of Pseudomonas aeruginosa by macrophages as early as 3 h after initial exposure. Focusing on mechanisms by which ethanol decreases macrophage Fcγ-receptor (FcγR) phagocytosis we targeted the study on the focal adhesion and cytoskeletal elements that are necessary for phagosome progression. Ethanol inhibited macrophage phagocytosis of IgG-coated bead recruitment of actin to the site of the phagosome, dampened the phosphorylation of vinculin, but had no effect on paxillin phosphorylation suggesting a loss in "phagosomal adhesion" maturation. Moreover, our observations revealed that FcγR-phagocytosis induced Rac activation, which was increased by only 50% in ethanol exposed cells, compared to 175% in the absence of ethanol. This work is the first to show evidence of the cellular mechanisms involved in the ethanol-induced suppression of FcγR-mediated phagocytosis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The ability of a pathogen to evade host immunity successfully, in contrast to the host's capacity to defend itself against a foreign invader, is a complex struggle, in which eradication of infection is dictated by a robust immunologic response. Often, there are external factors that can alter the outcome by tipping the scale to benefit pathogen establishment rather than resolution by the host's defense system. These external sources, such a cigarettes, alcohol, or environmental pollutants, can negatively influence the effectiveness of the immune system's response to a pathogen. The observed suppression of immune function can be attributed to dysregulated cytokine and chemokine production, the loss of migratory potential, or the inability to phagocytose pathogens by immune cells. This review will focus on the mechanisms involved during the toxin-induced suppression of phagocytosis. The accumulated data support the importance of studying the mechanisms of phagocytosis following exposure to these factors, in that this effect alone cannot only leave the host susceptible to infection but also promote alterations in many other macrophage functions necessary for pathogen clearance and restoration of homeostasis.

Pulmonary infections are a major cause of mortality in the critically ill burn patient. Alcohol consumption before burn increases the risk of pulmonary infection. Previously, we have shown an elevated mortality and lung pathology in mice given ethanol before burn and intratracheal infection relative to controls. Here we examine the cellular composition at 24 and 48 h in the circulation and the alveoli of infected mice given alcohol and burn. At 24 h after injury, blood neutrophils obtained from mice exposed to ethanol before burn and infection were 2-fold above those of the experimental controls (P < 0.05). By 48 h, the number of circulating neutrophils decreased and was comparable to levels found in untreated animals. Moreover, at 24 h, bronchoalveolar lavage cells obtained from all treatment groups had similar frequencies and contained 80% neutrophils regardless of treatment. In contrast, the following day, neutrophils were elevated 2-fold only in the alveoli of infected burn animals and 5-fold when ethanol preceded the injury (P < 0.05). These data were confirmed by immunofluorescence microscopy using a neutrophil-specific marker (P < 0.05). Levels of neutrophil chemoattractants, KC and macrophage inflammatory protein 2, and the cytokine, IL-1β, were 2-fold greater in the lungs of infected mice given burn, regardless of ethanol exposure, relative to infected sham injured animals (P < 0.05). Like the number of neutrophils, by the second day after injury, KC and macrophage inflammatory protein 2 remained 5-fold higher in the animals given ethanol, burn, and infection, when compared with other groups (P < 0.05). A similar pattern was seen for pulmonary levels of IL-1β (P < 0.05). Additionally, a reduction in neutrophil apoptosis was observed at the 24-h time point in infected mice exposed to ethanol and burn (P < 0.05). Targeting proinflammatory mediators in mice exposed to ethanol before burn and infection may help alleviate prolonged neutrophil accumulation in the lungs.

Tissue injury owing to acute and chronic alcohol consumption has extensive medical consequences, with the level and duration of alcohol exposure affecting both the magnitude of injury and the time frame to recovery. While the understanding of many of the molecular processes disrupted by alcohol has advanced, mechanisms of alcohol-induced tissue injury remain a subject of intensive research. Alcohol has multiple targets, as it affects diverse cellular and molecular processes. Some mechanisms of tissue damage as a result of alcohol may be common to many tissue types, while others are likely to be tissue specific. Here, we present a discussion of the alcohol-induced molecular and cellular disruptions associated with injury or recovery from injury in bone, muscle, skin, and gastric mucosa. In every case, the goal of characterizing the sites of alcohol action is to devise potential measures for protection, prevention, or therapeutic intervention., (Copyright © 2010 by the Research Society on Alcoholism.)

Background: Clinical and laboratory evidence suggests that alcohol consumption prior to burn injury leads to dysregulated immune function and subsequent higher rates of morbidity and mortality. Our laboratory previously observed higher levels of pro-inflammatory cytokines and leukocyte infiltration in the lungs of mice following ethanol and burn injury. To understand the mechanism of the increased inflammatory response, we looked at different signaling initiators of inflammation including toll-like receptors 2 and 4 (TLR2 and 4) pathways., Methods: Wild-type, TLR2, and TLR4 knockout mice were treated with vehicle or a single binge dose of ethanol (1.11 g/kg) and subsequently given a sham or burn injury. Twenty-four hours postinjury, systemic and pulmonary levels of pro-inflammatory cytokines were quantified, and differences in neutrophil infiltration were determined by histological examination., Results: Higher numbers of neutrophils were observed in the lungs of wild-type mice following the combined insult of ethanol and burn injury relative to either injury alone. This increase in leukocyte accumulation was absent in the TLR4 knockout mice. Circulating levels of IL-6 and tumor necrosis factor-α were also elevated in wild-type mice but not in TLR4 knockout mice. Consistent with these findings, pulmonary levels of KC and IL-6 were increased in wild-type mice following burn and ethanol compared to burn injury alone as well as to their TLR4 knockout counterparts. In contrast, TLR2 knockout mice displayed similar levels, to wild-type mice, of neutrophil infiltration as well as IL-6 and KC in the lung., Conclusions: These data suggest that TLR4 signaling is a crucial contributory component in the exuberant inflammation after ethanol and burn injury. However, TLR2 does not appear to play a vital role in the aberrant pulmonary inflammation., (Copyright © 2010 by the Research Society on Alcoholism.)

The morbidity and mortality resulting from alcohol-related diseases globally impose a substantive cost to society. To minimize the financial burden on society and improve the quality of life for individuals suffering from the ill effects of alcohol abuse, substantial research in the alcohol field is focused on understanding the mechanisms by which alcohol-related diseases develop and progress. Since ethical concerns and inherent difficulties limit the amount of alcohol abuse research that can be performed in humans, most studies are performed in laboratory animals. This article summarizes the various laboratory models of alcohol abuse that are currently available and are used to study the mechanisms by which alcohol abuse induces organ damage and immune defects. The strengths and weaknesses of each of the models are discussed. Integrated into the review are the presentations that were made in the symposium "Methods of Ethanol Application in Alcohol Model-How Long is Long Enough" at the joint 2008 Research Society on Alcoholism (RSA) and International Society for Biomedical Research on Alcoholism (ISBRA) meeting, Washington, DC, emphasizing the importance not only of selecting the most appropriate laboratory alcohol model to address the specific goals of a project but also of ensuring that the findings can be extrapolated to alcohol-induced diseases in humans.

Clinical and laboratory evidence suggests that alcohol consumption dysregulates immune function. Burn patients who consume alcohol before their injuries demonstrate higher rates of morbidity and mortality, including acute respiratory distress syndrome, than patients without alcohol at the time of injury. Our laboratory observed higher levels of proinflammatory cytokines and leukocyte infiltration in the lungs of mice after ethanol exposure and burn injury than with either insult alone. To understand the mechanism of the increased pulmonary inflammatory response in mice treated with ethanol and burn injury, we investigated the role of intercellular adhesion molecule (ICAM)-1. Wild-type and ICAM-1 knockout (KO) mice were treated with vehicle or ethanol and subsequently given a sham or burn injury. Twenty-four hours postinjury, lungs were harvested and analyzed for indices of inflammation. Higher numbers of neutrophils were observed in the lungs of wild-type mice after burn and burn with ethanol treatment. This increase in pulmonary inflammatory cell accumulation was significantly lower in the KO mice. In addition, levels of KC, interleukin-1beta, and interleukin-6 in the lung were decreased in the ICAM-1 KO mice after ethanol exposure and burn injury. Interestingly, no differences were observed in serum or lung tissue content of soluble ICAM-1 24 hours postinjury. These data suggest that upregulation of adhesion molecules such as ICAM-1 on the vascular endothelium may play a critical role in the excessive inflammation seen after ethanol exposure and burn injury.

Burn injury is a significant and severe representation of critical illness. Nearly, 50% of patients admitted to hospitals for burn injuries have detectable levels of ethanol in their circulations and these patients have poorer clinical outcomes than burned individuals without measurable circulating ethanol. We report here data on a clinically relevant form of hepatic injury, the development of microvesicular steatosis, in a murine model wherein animals were either given ethanol or saline, and were subjected to burn or sham injury. Because better glycemic control with insulin has been shown in clinical studies to impart major clinical benefit, an additional group of burn ethanol animals were treated with insulin. Insulin significantly reduced blood glucose in injured animals to levels no different from those seen in animals that were neither ethanol exposed nor burned. A single intraperitoneal injection of ethanol was insufficient to raise blood alanine aminotransferase (ALT), measured as an index of liver injury. However, burn injury led to significant increases in ALT at 24 and 48 hours, which had returned to preinjury levels by 7 days. This ALT rise was completely prevented with insulin treatment. A single injection of ethanol did not evoke increased microvesicular steatosis but did potentiate the ability of burn to do so at 24 hours after injury. The burn induced increase in microvesicular steatosis was also seen at 48 hours, but had subsided by 7 days. The increased microvesicular steatosis was prevented by insulin therapy. Thus, ethanol potentiates the ability of burn to cause acute liver injury, which is completely preventable by insulin therapy. These findings may have substantial clinical significance and suggest this model may be useful for the study of the mechanisms of hepatic injury as well as the mechanisms, probably multiple, of insulin action in this setting.

Elevated blood alcohol content (BAC) on admission is associated with poorer outcomes, larger burns and more inhalation injury. This study's purpose was to examine the effects of alcohol through a matched case-controlled study, measuring early and extended markers of clinical outcomes. The hypothesis was that patients with an elevated admission BAC would require more resuscitation and have a longer hospital stay. Admissions 16 to 75 years of age with 15 to 75% TBSA and admission BACs were identified. Patients with BAC >30 mg/dl (BAC+, cases) were matched with patients with undetectable BAC (BAC-, controls), according to age, sex, TBSA, inhalation injury and mechanism. Screening identified 258 patients, 146 with admission BACs. Twenty-seven had a BAC > or = 30 mg/dl. There were 24 matched pairs. At 24 hours, BAC+ group had larger acute physiology and chronic health evaluation II scores (23.33 vs 18.75, P < .05), fluid requirements (5.25 vs 3.82 L (cc/kg/TBSA), P < .05), and base deficit (11.15 vs 7.15, P < .05). The duration of mechanical ventilation (14.85 vs 4.23 days, P < .05), intensive care unit length of stay (22.85 vs 9.38, P < .05), hospital length of stay (28.95 vs 15.68, P < .05), and mean hospital charges ($239,507 vs $144,598, P < .05) were increased in the BAC+ patients. Despite matched baseline injury characteristics, elevated BAC was associated with poorer short term and extended clinical outcomes, illustrating the impact of alcohol intoxication on physiologic derangement after burn injury.

Clinical and experimental evidence demonstrates that ethanol exposure prior to injury alters local and systemic inflammatory responses, increasing morbidity and mortality. Moreover, the aberrant inflammatory responses can directly and indirectly lead to the poor prognosis after injury by altering leukocyte infiltration into the wound site and remote organs and by suppressing immunity leading to increased susceptibility to opportunistic infections. Recent studies from our laboratory have focused on inflammatory responses at the wound site and in other distal organs after exposure to acute ethanol and burn injury. This combined insult leads to increased mortality after dermal or intratracheal pseudomonas infection, relative to infected mice given ethanol or burn injury alone. The increased mortality in mice given ethanol and burn injury parallels elevated serum levels of proinflammatory cytokines, IL-6 and TNF-alpha, marked infiltration of leukocytes into the lung and gut, as well as immunosuppression at the sites of infection. Bacterial translocation from the gut is likely to be responsible, in part, for the aberrant accumulation of leukocytes in the lungs of ethanol-exposed, burn-injured mice. Additionally, other factors, such as expression of adhesion molecules, increased chemokine production, and leakiness of the vascular endothelium, may also be involved.

Physiological angiogenesis is regulated by various factors, including signaling through vascular endothelial growth factor (VEGF) receptors. We previously reported that a single dose of ethanol (1.4 g/kg), yielding a blood alcohol concentration of 100 mg/dl, significantly impairs angiogenesis in murine wounds, despite adequate levels of VEGF, suggesting direct effects of ethanol on endothelial cell signaling (40). To examine the mechanism by which ethanol influences angiogenesis in wounds, we employed two different in vitro angiogenesis assays to determine whether acute ethanol exposure (100 mg/dl) would have long-lasting effects on VEGF-induced capillary network formation. Ethanol exposure resulted in reduced VEGF-induced cord formation on collagen and reduced capillary network structure on Matrigel in vitro. In addition, ethanol exposure decreased expression of endothelial VEGF receptor-2, as well as VEGF receptor-2 phosphorylation in vitro. Inhibition of ethanol metabolism by 4-methylpyrazole partially abrogated the effect of ethanol on endothelial cell cord formation. However, mice treated with t-butanol, an alcohol not metabolized by alcohol dehydrogenase, exhibited no change in wound vascularity. These results suggest that products of ethanol metabolism are important factors in the development of ethanol-induced changes in endothelial cell responsiveness to VEGF. In vivo, ethanol exposure caused both decreased angiogenesis and increased hypoxia in wounds. Moreover, in vitro experiments demonstrated a direct effect of ethanol on the response to hypoxia in endothelial cells, as ethanol diminished nuclear hypoxia-inducible factor-1alpha protein levels. Together, the data establish that acute ethanol exposure significantly impairs angiogenesis and suggest that this effect is mediated by changes in endothelial cell responsiveness to both VEGF and hypoxia.

Both clinical and experimental data have linked acute ethanol exposure to increased susceptibility to infection as well as increased morbidity and mortality after injury. Macrophages play an integral role in the innate immune system and are important in priming the adaptive immune system. In this study, we investigated the effect of a single in vivo exposure of macrophages to physiologically relevant levels of ethanol (1.2 and 2.9 g/kg) followed by ex vivo stimulation with lipopolysaccharide (LPS) or bacteria. Our study confirms the work of others showing that a single administration of ethanol suppresses the production of tumor necrosis factor-alpha(TNF-alpha), interleukin-6 (IL-6), and IL-12 in response to LPS. There was no effect of ethanol on LPS induction of cytokine production at 30 min after treatment. In contrast, at 3 h, both doses of ethanol exposure decreased ex vivo TNF-alpha production by splenic and alveolar macrophages. Interestingly, the higher dose of ethanol resulted in sustained suppression of LPS-induced TNF-alpha production at 3 and 6 h after ethanol administration, as well as decreased IL-6 and IL-12 production after 6 h, as compared to control (saline-treated groups). Alveolar macrophages behaved similarly at 3 h after ethanol treatment. LPS-stimulated production of TNF-alpha and IL-6 was reduced at 3 h after ethanol administration, when compared with the saline-treated animals. Alveolar macrophages stimulated for 3 h with bacteria also showed decreased TNF-alpha and IL-6 production after harvested from mice given 2.9 g/kg ethanol for 3 h. This time point and high dose of ethanol also resulted in decreased Pseudomonas aeruginosa phagocytosis by alveolar macrophages. Taken together, we conclude that the effects of physiological levels of ethanol are dose dependent, have effects that last after ethanol is cleared from the circulation, and can affect multiple macrophage functions.

Extensive evidence indicates that ethanol (alcohol) has immunomodulatory properties. Many of its effects on innate immune response are dose dependent, with acute or moderate use associated with attenuated inflammatory responses, and heavy ethanol consumption linked with augmentation of inflammation. Ethanol may modify innate immunity via functional alterations of the cells of the innate immune system. Mounting evidence indicates that ethanol can diversely affect antigen recognition and intracellular signaling events, which include activation of mitogen activated protein kinases, and NFkappaB, mediated by Toll-like receptors, leading to altered inflammatory responses. The mechanism(s) underlying these changes may involve dose-dependent effects of ethanol on the fluidity of cell membrane, resulting in interference with the timely assembly or disassembly of lipid rafts. Ethanol could also modify cell activation by specific interactions with cell membrane molecules.

Thermal injury is often associated with previous ethanol exposure, and close to 50% of patients admitted to a burn unit have a potentially high blood ethanol level. Cellular mechanisms by which ethanol and/or burn affect the hypothalamic-pituitary-gonadal (HPG) axis are not entirely understood. However, it is known that the proinflammatory cytokines, tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6 influence negatively on the endocrine functions of the HPG. We report a time course study (6, 12, 24, and 48 hours) of the effects of ethanol, burn, or the combination of burn/ethanol on proinflammatory cytokines of the hypothalamus, pituitary and testes of male C57Bl/6 mice. We found that there were highly significant increases in each of these cytokines caused by ethanol, burn, and burn/ethanol compared with sham/vehicle (P < .001). This was true in hypothalamus, pituitary, and testes. Because these cytokines generally reduce reproductive function, it may be that proinflammatory cytokines of HPG axis mediate the deleterious effects of burn and/or ethanol on mammalian reproduction.

Infectious complications are a major cause of mortality in trauma patients. Burn patients with prior ethanol exposure have a worse prognosis than those who sustain injury but had not been drinking. We examined pulmonary infection and lung pathology in mice given ethanol (1.2 g/kg) 30 minutes before being subjected to 13 to 15% total body surface area scald burn followed by intratracheal inoculation with Pseudomonas aeruginosa (1-2 x 10(3) colony-forming units [CFUs]). Survival was monitored for up to 48 hours. Sham control groups had 100% survival after intratracheal infection regardless of ethanol exposure. Infected burned animals had 55% survival; however, survival of infected mice exposed to ethanol and burn injury was significantly lower (27%, P < .0001). When pulmonary infection was evaluated, the lungs of sham groups were negative for bacterial colonies. In addition, at 24 hours there were no significant differences in lung CFUs from infected burned animals regardless of ethanol exposure (3.0 x 10(4)). However, pulmonary bacterial content significantly decreased (1.2 x 10, P < .02) at 48 hours in mice given burn injury alone, where CFUs from the lungs of mice exposed to ethanol prior to burn did not decline (5.4 x 10(5)). At the same time point, lungs from animals given ethanol and burn injury had about a 2-fold (P < .02) increase in leukocyte infiltration and vascular congestion, as well as decreased pulmonary oxygen saturation (82.8%, P < .02), when compared with other treatment groups. In summary, ethanol exposure in postburn intratracheal infection results in the inability to clear pulmonary infection marked by a prolonged pulmonary leukocyte accumulation and a decrease in pulmonary function.

Acute alcohol administration has minimal effects on basal immune function. However, when the immune system is challenged, acute alcohol administration alters the immune system's response. In the first 3 h after infection or traumatic injury, the presence of alcohol is associated with a decreased inflammatory response. This defect lasts long after the alcohol is cleared. Conversely, by 48 h after traumatic injury, the presence of alcohol is associated with a heightened inflammatory response. Aside from its in vivo actions, systemic administration of alcohol also alters the ex vivo response of immune cells, resulting in a decreased production of multiple cytokines after stimulation by lipopolysaccharide, concanavilin A, zymosan, and CpG DNA. Here, we describe a standardized model of acute administration of ethanol to mice used to study both the in vivo and ex vivo responses of immune cells to ethanol.

Background: Recent studies suggest that ethanol use imposes a greater risk of trauma-associated intestinal injury than trauma alone. The initiating and regulatory factors for multiple organ dysfunction syndromes are not well defined, yet evidence points to the gut as a possible trigger of the systemic inflammatory cascade as well as a potential source of cytokines. In the current study, we hypothesized that ethanol administration would alter cytokine levels and intestinal infiltration by neutrophils within the ileum of mice exposed to burn injury (15% total body surface of dorsal skin)., Methods: Ileal samples were collected for histological assessment, myeloperoxidase quantitation and the protein presence of tumor necrosis factor alpha (TNFalpha), interleukin (IL-) 6, macrophage inflammatory protein-2 (MIP-2; CXCL2) and the anti-inflammatory cytokine, IL-10. Additional ileal tissue samples were examined for localization of the IL-6 immunoreactivity., Results: We did not detect statistically significant cytokine/chemokine differences (MIP-2 and IL-10) between sham control and treatment conditions at either 2 or 24 hours. However, there was a significant decrease in TNFalpha at 24 hours in both burn injury alone and in combination with ethanol treatment conditions (p < 0.05). In addition, there was an increase in IL-6 levels at 24 hours in intestinal tissue obtained from mice subjected to a combination of acute ethanol and burn injury, compared to the mice receiving burn or sham injury (p < 0.001). Ileal homogenate increases in IL-6 at 24 hours were concurrent with decreased villus height in the ileum, but no discernable changes in neutrophil infiltration (myeloperoxidase activity levels) at either 2 or 24 hours. Additional immunocytochemical localization studies of ileal tissue revealed that there was a substantial increase of IL-6 in intestinal enterocytes subjected to both burn injury alone, or in combination with acute ethanol exposure., Conclusions: The present study suggests that acute ethanol exposure combined with burn injury enhances levels of IL-6 protein in the ileum. The enhanced levels of ileal IL-6 are likely due to enterocyte production of the cytokine.

In recent, landmark clinical trials, insulin to maintain euglycemia in critically ill patients improved clinical outcomes, including decreased all-cause mortality. Novel antiinflammatory effects of insulin have recently been described. Thermal injury is an excellent model of critical illness. The addition of ethanol to the model is of great clinical relevance because nearly 50% of the patients admitted to hospitals for burn injuries have ethanol in their circulation. Utilizing a murine model of critical illness (ethanol and skin burn), we tested the hypothesis that insulin treatment in ethanol-exposed, burn-injured mice reduced hepatic inflammation, a potential mechanism for the benefit of insulin. Adult male C57BL/6 mice were given a single intraperitoneal injection of ethanol or saline, were given a 15% total body full-thickness skin burn, or were sham-burned and killed 24 hours later. In each group, half the animals were given subcutaneous injections of the long-lasting basal insulin glargine; the other half, the appropriate vehicle. Hepatic inflammatory markers, including polymorphonuclear infiltration, a chemokine, an important adhesion molecule, proinflammatory cytokines, and nuclear factor kappaB, were measured, and all were increased by ethanol and/or burn. These increases were prevented by insulin. An antiinflammatory cytokine was reduced by ethanol and/or burn. Insulin prevented this decrease. Thus, insulin has a substantial antiinflammatory effect, and this may underlie its dramatic clinical benefit in critical illness.

Background: Alcohol consumption is involved in over half of all trauma-related injuries. These patients are known to exhibit a higher incidence of mortality and morbidity following injury compared with patients not exposed to ethanol. As studies from our laboratory demonstrated that ethanol exposure impairs re-epithelialization and angiogenesis after dermal wounding and because the earlier inflammatory phase of wound healing is likely to influence later responses, we chose to examine neutrophil infiltration and chemokine and proinflammatory cytokine levels in the skin following administration of a dermal excisional wound., Methods: BALB/c mice were given ethanol at a dose designed to increase blood alcohol concentration to 100 to 120 mg/dL at 30 minutes after treatment. Mice were then subjected to a full-thickness excisional wound. Wounds from ethanol and saline-treated mice were collected within the first 24 hours postinjury to assess neutrophil infiltration and myeloperoxidase (MPO) activity, neutrophil chemoattractant macrophage inflammatory protein-2 (MIP-2) and KC levels, and proinflammatory cytokine interleukin-1 beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) levels., Results: At 12 and 24 hours after injury, MPO in wounds from ethanol-exposed mice was significantly reduced compared with wounds from vehicle-treated animals. Despite this, histological examination of wounds did not reveal a difference in neutrophil infiltration between the 2 groups. Peak levels of MIP-2 and KC observed at 12 hours postinjury were decreased in wounds from ethanol-treated mice by 32 and 45%, respectively, relative to wounds from control mice. Levels of TNFalpha and IL-1beta (potent inducers of MIP-2 and KC, as well as neutrophil activation) were also assessed. Levels of TNFalpha were not elevated in either group after injury. However, IL-1beta demonstrated significantly lower peak levels at 6 hours postinjury in wounds from ethanol-treated mice, 58% less than wounds from controls., Conclusion: These studies reveal that early dermal inflammatory responses including MPO activity, production of MIP-2, KC, and IL-1beta are impaired in mice given ethanol before injury, which may also have detrimental affects on later stages of wound healing.

Background: Many trauma patients have been consuming alcohol at the time of injury. Although high concentrations of alcohol are correlated with poor outcome, few studies have examined the effects of low levels of alcohol. We examined the effects of low alcohol exposure after burn injury using a murine model., Methods: Three- and 18-month-old mice were given ethanol or saline 30 minutes before a 15% total body surface area burn injury. Twenty-four hours after injury, cellular immune responses, including delayed-type hypersensitivity response and splenocyte proliferation were examined, along with production of interferon-gamma, interleukin (IL)-2, and IL-4., Results: Alcohol administration resulted in a significant increase in interferon-gamma in the aged, but not young, burn-injured mice. Likewise, slight increases in IL-2, IL-4, and the delayed-type hypersensitivity response were observed., Conclusion: Low levels of ethanol at the time of injury are associated with partial restoration of immune responses in aged mice.

Acute ethanol exposure represents an increased risk factor for morbidity and mortality associated with surgical or traumatic injury. Despite clinical observations suggesting that ethanol exposure before injury alters tissue repair processes, little direct evidence about the mechanism by which ethanol affects the wound healing process is available. In this study, excisional wounds from female BALB/c mice with or without circulating ethanol levels of 100 mg/dl were used to assess wound closure, angiogenesis, and collagen content. Ethanol exposure resulted in a significant but transient delay in wound closure at day 2 postwounding (28 +/- 4% vs. 17 +/- 1%). In addition, total collagen content was significantly reduced by up to 37% in wounds from ethanol-treated mice compared with controls. The most significant effect of ethanol exposure on wounds was on vascularity because angiogenesis was reduced by up to 61% in wounds from ethanol-treated mice. The reduction in vessel density occurred despite near-normal levels of proangiogenic factors VEGF and FGF-2, suggesting a direct effect of ethanol exposure on endothelial cell function. Further evidence for a direct effect was observed in an in vitro angiogenesis assay because the exposure of endothelial cells to ethanol reduced angiogenic responsiveness to just 8.33% of control in a cord-forming assay. These studies provide novel information regarding the effect of a single dose of ethanol on multiple parameters of the wound healing process in vivo and suggest a potential mechanism by which ethanol impairs healing after traumatic injury.

Nearly 50% of the patients admitted to hospitals for burn injuries have detectable levels of alcohol (EtOH) in their circulation. In fact, EtOH is often a causal factor in their injury. It is well known that EtOH as well as burn injury disrupt function of the hypothalamic-pituitary-gonadal (HPG) axis. The cellular mechanisms by which EtOH and/or burn impacts on the HPG are not entirely understood. In the studies reported here, we tested the hypothesis that these injuries mediated their effects by local hypothalamic inflammation. Young adult male mice were subjected to either a 15% total body surface area, full thickness scald, to EtOH, or to both and compared to appropriate controls. They were sacrificed 48 h later. EtOH and burn, as well as the combined injury, consistently and impressively reduced serum testosterone, while increasing hypothalamic concentrations of all three of the pro-inflammatory cytokines, TNFalpha, IL-1beta, and IL-6. In general, the increases induced by burn were greater than those caused by EtOH and the effect of the combined insult was not additive. Hypothalamic concentrations of LHRH were also increased. The data are consistent with the idea that EtOH and/or burn, as models of critical illness, medicate their hypothalamic suppressive effects via increase in pro-inflammatory cytokines.

Previous studies from our laboratory have shown that acute alcohol (EtOH) ingestion prior to burn injury enhances intestinal bacterial translocation. This study tested if increased intestinal bacterial translocation in alcohol and burn injured rats is due to an overgrowth in intestinal bacteria. We determined if the translocation was accompanied with alterations in intestinal permeability and immune cell population. Rats (225-250 g) were gavaged with alcohol to achieve a blood EtOH level in the range of 100 mg/dl prior to burn or sham injury (25% total body surface area). Two days after injury, we found that acute alcohol ingestions prior to burn injury results in a significant increase in bacterial counts in small intestine. The increase in intestinal bacterial counts accompanied a significant increase in intestinal permeability. Finally, immunohistochemical analysis revealed a substantial (p<0.05) loss of both T cell and dendritic cells in intestine of alcohol and burn injured rats compared with intestine of rats receiving either burn or sham injury. Altogether, results presented in this manuscript suggest that increase in intestinal bacterial growth along with alterations in intestinal permeability and immune status contribute to the increase in bacterial translocation observed in alcohol and burn injured rats.

Ethanol is known to increase susceptibility to infections, in part, by suppressing macrophage function. Through TLRs, macrophages recognize pathogens and initiate inflammatory responses. In this study, we investigated the effect of acute ethanol exposure on murine macrophage activation mediated via TLR2, TLR4, and TLR9. Specifically, the study focused on the proinflammatory cytokines IL-6 and TNF-alpha and activation of p38 and ERK1/2 MAPKs after a single in vivo exposure to physiologically relevant level of ethanol followed by ex vivo stimulation with specific TLR ligands. Acute ethanol treatment inhibited IL-6 and TNF-alpha synthesis and impaired p38 and ERK1/2 activation induced by TLR2, TLR4, and TLR9 ligands. We also addressed the question of whether ethanol treatment modified activities of serine/threonine-specific, tyrosine-specific phosphatases, and MAPK phosphatase type 1. Inhibitors of three families of protein phosphatases did not restore ethanol-impaired proinflammatory cytokine production nor p38 and ERK1/2 activation. However, inhibitors of serine/threonine protein phosphatase type 1 and type 2A significantly increased IL-6 and TNF-alpha levels, and prolonged activation of p38 and ERK1/2 when triggered by TLR4 and TLR9 ligands. In contrast, with TLR2 ligand stimulation, TNF-alpha production was reduced, whereas IL-6 levels, and p38 and ERK1/2 activation were not affected. In conclusion, acute ethanol exposure impaired macrophage responsiveness to multiple TLR agonists by inhibiting IL-6 and TNF-alpha production. Mechanism responsible for ethanol-induced suppression involved inhibition of p38 and ERK1/2 activation. Furthermore, different TLR ligands stimulated IL-6 and TNF-alpha production via signaling pathways, which showed unique characteristics.

We and others have investigated the effects of acute and chronic ethanol (EtOH) administration on function of the hypothalamic-pituitary-gonadal (HPG) axis in female rats, consistently finding EtOH to be detrimental. There are now substantial data that pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNFalpha) and interleukin 6 (IL-6), have anti-reproductive effects. If EtOH increased levels of these cytokines, such data would be consistent with, though not necessarily prove, a cytokine mediated mechanism for EtOH's deleterious effects on reproduction. Young adult female Sprague Dawley rats were used. In the experiment reported here, the Lieber DeCarli diet was used, with animals fed a 36% EtOH containing diet or pair fed an identical diet which contained dextrimaltose instead of EtOH. This was done for 4 to 6 weeks. TNFalpha and IL-6 were measured in the hypothalamus, pituitary, and ovary by ELISA. EtOH exposure resulted in significant increases in TNFalpha and IL-6 in hypothalami, pituitaries, and ovaries. The data reported here are the first to show consistent stimulatory effects of EtOH exposure on cytokines in the reproductive axis of female rats. Because the effects of these cytokines are generally anti-reproductive, these data provide a rational for more rigorous testing of the notion that part of EtOH's deleterious HPG effects may be due to such immuno-endocrine interactions.

Recent studies from our laboratory have suggested that acute alcohol ingestion prior to burn injury enhances gut bacterial translocation by suppressing T cell-mediated intestinal immune defense. To determine the mechanism responsible for suppressed T cell function, we examined the activation of mitogen-activated protein kinases (MAPK) members p-38 and ERK-1/2. Both p-38 and ERK-1/2 are known to play a significant role in the T cell proliferation and their cytokine production. Rats were gavaged with ethanol to achieve a blood alcohol level of approximately 100 mg/dl, before they were subjected to a 25% total body surface area burn injury. Two days after injury, rats were sacrificed and mesenteric lymph node (MLN) T cells were isolated and their ability to proliferate in response to anti-CD3 was determined. For p-38 and ERK-1/2 determination, T cells were divided into two groups. Cells in one group were stimulated with anti-CD3 for 3 min and lysed. The cells in the second group were cultured for approximately 18 h in the presence of anti-CD3 and lysed. MAPK status in 18-h cultured cells allowed us to determine whether or not the changes in p-38 and ERK-1/2 are transient or persist in the proliferating cells. Two days after injury, anti-CD3-mediated MLN T cell proliferation was more suppressed in rats gavaged with alcohol prior to burn injury compared to rats receiving either burn injury alone or sham-injured rats regardless of their exposure. Western blot analyses showed significant inhibition of ERK-1/2 phosphorylation in both freshly isolated and 18-h cultured T cells from alcohol and burn-injured rats compared to the sham rat T cells. The inhibition of p-38 phosphorylation in T cells derived from alcohol and burn-injured rats was found to be transient as no significant difference in p-38 phosphorylation was noted between the 18 h incubated MLN T cells of sham and alcohol and burn-injured rats. Taken together, our findings suggest that low levels of ERK-1/2 activation is likely to play a significant role in MLN T cell proliferative suppression in alcohol and burn-injured rats.

A good deal of clinical evidence supports the idea that ethanol exposure is a causative factor in the occurrence of burn or other traumatic injury. In addition, more recent evidence reveals that individuals who sustain injury while under the influence of ethanol suffer from increased morbidity and mortality compared with those with comparable injuries who did not consume ethanol. Many of the complications seen in ethanol-exposed, burn-injured subjects result from depressed immune responses, which render the host unable to fight off infectious organisms. Both injury and ethanol exposure independently affect cellular immune responses, including delayed-type hypersensitivity and splenocyte proliferative responses, and the combined insult of ethanol exposure and injury acts in conjunction to increase further the magnitude and duration of immunosuppression. It is interesting that these immune responses can be restored experimentally in male, but not in female, mice by administration of low, proestrous levels of estrogen. The complexity of the responses after injury in ethanol-exposed subjects is multiplied when the sex of the subjects is added to the equation. This is due, in part, to the effect of the combined insult of injury and ethanol on the production of gonadal steroid hormones in males and females and the direct effects of those hormones on cytokine gene expression in sensitive cell types such as the macrophage. Evidence seems to indicate that cellular immune responses after ethanol exposure and burn injury differ in kinetics and magnitude for male and female subjects, and, hence, the therapeutic interventions to treat burn-injured patients should take into account both sex and ethanol exposure.

Acute ethanol consumption has been linked to an increase in infectious complications in trauma and burn patients. Ethanol modifies production of a variety of macrophage-derived immunoregulatory mediators. Lipopolysaccharide (LPS), a potent stimulator of inflammatory responses in macrophages, activates several intracellular signaling pathways, including mitogen-activated protein kinases (MAPK). In the current study, we investigated the effect of acute ethanol exposure on in vivo activation of p38 and extracellularly regulated kinases 1 and 2 (ERK1/2) MAPK in murine macrophages and the corresponding, LPS-stimulated interleukin (IL)-6 production. We demonstrated that a single dose of ethanol transiently down-regulated p38 and ERK1/2 activation levels (3-24 h after treatment) and impaired IL-6 synthesis. Ethanol-related reduction in IL-6 production was not further affected by the presence of inhibitors of p38 and ERK1/2 (SB 202190 and PD 98059, respectively). These results demonstrate that acute ethanol exposure can impair macrophage IL-6 production and indicate that this effect may result from ethanol-induced alterations in intracellular signaling through p38 and ERK1/2.

Background: The female liver is more sensitive to the toxic effect of chronic alcohol intake than the male liver. The aim of the study was to compare the influence of gender and sex hormonal status on apoptosis and cell proliferation following chronic ethanol intake., Methods: Male and female rats were pair fed for 8 weeks a liquid diet containing 36% of their total daily calories as ethanol (ETOH group) or sucrose (control group). Liver samples were analyzed for apoptosis and hepatocyte proliferation by immunohistochemistry. The hepatic production of factors able to influence cell death and proliferation, such as tumor necrosis factor alpha (TNFalpha) and interleukin 6 (IL-6) were determined., Results: In both male and female rats, ethanol intake promoted apoptosis in the liver. This effect of ethanol was more evident in female than male rat livers. Hepatic TNFalpha levels, which promote apoptosis, are significantly more elevated in female than in male livers. Hepatic IL-6 production, which promotes hepatocyte proliferation, was induced by ethanol only in males, but not female animals., Conclusion: This observed difference in cytokine responses may contribute to the enhanced sensitivity of female liver to EtOH-induced injury.