Am J Physiol Gastrointest Liver Physiol 307: G711–G718, 2014. First published August 7, 2014; doi:10.1152/ajpgi.00185.2014.

An alteration of the gut-liver axis drives pulmonary inflammation after intoxication and burn injury in mice Michael M. Chen,2,3,4 Anita Zahs,2,3 Mary M. Brown,1,2 Luis Ramirez,1,2 Jerrold R. Turner,5 Mashkoor A. Choudhry,1,2,3,4 and Elizabeth J. Kovacs1,2,3,4 1

Department of Surgery, Loyola University Medical Center, Maywood, Illinois; 2Burn and Shock Trauma Research Institute, Loyola University Medical Center, Maywood, Illinois; 3Alcohol Research Program, Loyola University Medical Center, Maywood, Illinois; 4Loyola University Chicago Stritch School of Medicine, Maywood, Illinois; and 5Department of Pathology, University of Chicago, Chicago, Illinois Submitted 21 May 2014; accepted in final form 1 August 2014

Chen MM, Zahs A, Brown MM, Ramirez L, Turner JR, Choudhry MA, Kovacs EJ. An alteration of the gut-liver axis drives pulmonary inflammation after intoxication and burn injury in mice. Am J Physiol Gastrointest Liver Physiol 307: G711–G718, 2014. First published August 7, 2014; doi:10.1152/ajpgi.00185.2014.—Approximately half of all adult burn patients are intoxicated at the time of their injury and have worse clinical outcomes than those without prior alcohol exposure. This study tested the hypothesis that intoxication alters the gut-liver axis, leading to increased pulmonary inflammation mediated by burn-induced IL-6 in the liver. C57BL/6 mice were given 1.2 g/kg ethanol 30 min prior to a 15% total body surface area burn. To restore gut barrier function, the specific myosin light chain kinase inhibitor membrane-permeant inhibitor of kinase (PIK), which we have demonstrated to reduce bacterial translocation from the gut, was administered 30 min after injury. Limiting bacterial translocation with PIK attenuated hepatic damage as measured by a 47% reduction in serum alanine aminotransferase (P ⬍ 0.05), as well as a 33% reduction in hepatic IL-6 mRNA expression (P ⬍ 0.05), compared with intoxicated and burn-injured mice without PIK. This mitigation of hepatic damage was associated with a 49% decline in pulmonary neutrophil infiltration (P ⬍ 0.05) and decreased alveolar wall thickening compared with matched controls. These results were reproduced by prophylactic reduction of the bacterial load in the intestines with oral antibiotics before intoxication and burn injury. Overall, these data suggest that the gut-liver axis is deranged when intoxication precedes burn injury and that limiting bacterial translocation in this setting attenuates hepatic damage and pulmonary inflammation. ethanol; trauma; steatosis; IL-6; myosin light chain kinase AS THE MOST FREQUENTLY ABUSED substance in the United States, alcohol has a high association with unintentional injuries (49, 53) and is the third-leading cause of preventable death (37). The practice of consuming enough alcohol to bring the blood alcohol concentration to ⱖ0.08%, known as binge drinking, is an increasingly prevalent form of intoxication (39) and the characteristic consumption pattern among patients presenting with traumatic injury, including burns (46). Approximately half of adult burn patients are intoxicated at the time of admission, and these patients have worse clinical outcomes than individuals who sustain similar injuries without alcohol exposure (23, 29, 48). Specifically, they are twice as likely to acquire an infection, undergo 60% more surgical procedures, require more days on mechanical ventilation, and stay longer in the intensive care unit (6, 48). As a result of these complica-

Address for reprint requests and other correspondence: E. J. Kovacs, Bldg. 110 Rm. 4232, 2160 South First Ave., Maywood, IL 60153 (e-mail: ekovacs@luc. edu). http://www.ajpgi.org

tions, burn patients with prior alcohol exposure have been identified as a patient population at high risk for death in the early stages postburn (43). With 1,000,000 burn injuries in the United States each year, resulting in 40,000 hospitalizations (1), this represents a substantial burden of morbidity, mortality, and socioeconomic cost that is worsened by alcohol abuse. Despite the prevalence and known repercussions of intoxication at the time of a burn injury, there is little difference in the treatment and management of burn patients with and without prior alcohol exposure. In fact, relatively little is known about the mechanisms by which alcohol affects the postburn response. The complicated natural history of burn injuries, as well as the complex and duration-dependent effects of alcohol, impacts nearly every major organ system (32). Therefore, the detrimental response to intoxication and burn injury likely encompasses aberrations, and potentially cross talk, between multiple organs. The studies reported here examine the role of the gut-liver axis after intoxication and burn injury by limiting bacterial translocation through pharmacological restoration of intestinal barrier function. This was accomplished by administration of an inhibitor of myosin light chain kinase (MLCK) after burn injury, which, as we previously demonstrated, decreases intestinal barrier damage and subsequent bacterial translocation (62). In a separate group of animals, the initial bacterial burden within the intestines was decreased before injury by prophylactic oral antibiotics. Finally, systemic effects of manipulating the gut-liver axis were investigated by examining pulmonary inflammation and IL-6 levels after intoxication and burn injury. MATERIALS AND METHODS

Animals. Male C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME) were housed in sterile micro-isolator cages under specific pathogen-free conditions in the Loyola University Chicago Comparative Medicine Facility. All experiments were conducted with approval of the Loyola Institutional Animal Care and Use Committee and strict accordance to the committee’s guidelines. All experiments were performed with 23- to 25-g mice at 8 –10 AM to avoid confounding factors related to circadian rhythms. Murine model of intoxication and burn injury. A murine model of a single binge ethanol exposure and burn injury was employed as described previously (19) with minor modifications. Briefly, mice were given a single intraperitoneal dose of ethanol (1.12 g/kg) that resulted in a blood ethanol concentration of 150 –180 mg/dl at 30 min or saline vehicle. The mice were then anesthetized (100 mg/kg ketamine and 10 mg/kg xylazine), the dorsum was shaved, and the animals were placed in a plastic template that exposed 15% of the total body surface area and subjected to a scald injury in a 90 –92°C

0193-1857/14 Copyright © 2014 the American Physiological Society

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water bath or a sham injury in room temperature water. The scald injury resulted in an insensate, full-thickness burn (20). The mice were then resuscitated with 1.0 ml of saline and allowed to recover on warming pads. The intoxicated and burn-injured mice were split into two groups and, at 30 min after injury, given the specific long MLCK inhibitor membrane-permeant inhibitor of kinase (PIK, 50 ␮M, 100 ␮l ip) or saline control, as described previously (62). A separate group of mice received prophylactic antibiotics [neomycin and polymyxin E (100 and 60 mg·kg⫺1·day⫺1, respectively)] via drinking water for 10 days before intoxication and burn injury. This resulted in a 3.67 log reduction in culturable aerobic fecal bacteria at the time of injury. Mice were euthanized by CO2 narcosis and exsanguinated 24 h after injury. The control arms of sham-vehicle, sham-ethanol, and burnvehicle are presented to demonstrate baseline results and provide injury context for the assessed parameters. The data from these control groups (see Figs. 1– 4) are consistent with previously reported results in intestines (62), liver (10), and lungs (7). As this study examines a therapeutic intervention for the established consequences of intoxication and burn injury in this animal model, we focus on animals subjected to ethanol intoxication and burn injury, with and without treatments. Tight junction localization and morphological damage in the ileum. Immunofluorescent staining was done as previously described (62) with minor modifications. Briefly, a small section of ileum (5 mm) was embedded in optimal cutting temperature (OCT) medium and frozen at ⫺80°C. The ileum was sectioned (8 ␮m) and stained with rabbit anti-zonula occludens protein-1 (ZO-1; Invitrogen, Carlsbad, CA) and then with goat anti-rabbit Alexa Fluor 488 (Invitrogen). Sections were further stained with fluorescent-conjugated phalloidin (actin) and Hoechst nuclear stain (Invitrogen). Images were taken at ⫻400 magnification. Bacterial translocation. Bacterial translocation was assessed as previously described (61, 62). Briefly, three to five mesenteric lymph nodes per mouse were removed and placed in cold RPMI medium and kept on ice. Mesenteric lymph nodes were separated from connective tissue and homogenized in RPMI medium using frosted glass slides. Homogenates were plated in triplicate on tryptic soy agar and placed in a 37°C incubator overnight. On the following day, colonies were counted, averaged, and divided by the total number of lymph nodes harvested. Histopathological examination of the liver. The whole liver was removed at the time of euthanization, weighed, and embedded in OCT medium or snap-frozen in liquid nitrogen. Frozen sections were cut at 7 ␮m, stained with Oil Red O, and then examined for the presence of fat droplets, as described previously (16). Briefly, sectioned tissue was rinsed in isopropyl alcohol and placed in a filtered working solution of Oil Red O for 20 min. The slides were rinsed in isopropyl alcohol and then in tap water, counterstained with hematoxylin for 1 min, and then serially rinsed in tap water, ammonia water (lithium carbonate), and finally tap water. Representative images were taken at ⫻400 magnification. Assessment of liver cytokines and triglyceride quantification. Liver tissue was homogenized in 1 ml of BioPlex cell lysis buffer according to the manufacturer’s instructions (Bio-Rad, Hercules, CA) and analyzed for cytokine production using an enzyme-linked immunosorbent assay (ELISA) for IL-6 (BD Biosciences, Franklin Lakes, NJ). The results were normalized to total protein using the Bio-Rad protein assay. A portion of the frozen liver was used in a triglyceride quantification kit according to the manufacturer’s instruction (Abcam, Cambridge, MA). Hepatic IL-6 mRNA expression. RNA was isolated from liver homogenate using the RNeasy Mini Kit (Qiagen, Valencia, CA), and cDNA was produced using the iScript cDNA synthesis kit (Bio-Rad). Quantitative real-time polymerase chain reaction was performed using the StepOne Plus Real-Time PCR Systems (Applied Biosystems, Carlsbad, CA), and data were analyzed by the 2⫺⌬⌬CT relative quantification method (33).

Serum measurements. At 24 h postinjury, mice were euthanized, and blood was collected via cardiac puncture and harvested for serum by centrifugation after clotting. Serum aliquots were used to measure IL-6 by ELISA (BD Biosciences) or liver transaminase levels using a DRI-CHEM 7000 (HESKA, Loveland, CO), as described previously (8). Histopathological examination of the lungs. The upper right lobe of the lung was inflated with 10% formalin and fixed overnight, as described previously (40). The lung was then embedded in paraffin, sectioned at 5 ␮m, and stained with hematoxylin and eosin. The sections were analyzed microscopically in a blinded fashion for the number of neutrophils in 10 high-power fields as a marker of inflammation. Representative images were taken at ⫻400 magnification. Ten high-power fields (⫻400 magnification) per animal were analyzed using the Java-based imaging program ImageJ (National Institutes of Health, Bethesda, MD). The images were converted to binary to differentiate lung tissue from air space and then analyzed for the percent area covered by lung tissue in each field of view, as described previously (7). KC analysis of lung homogenates. The right middle lung lobe was snap-frozen in liquid nitrogen. The tissues were then homogenized in 1 ml of BioPlex cell lysis buffer according to the manufacturer’s instructions (Bio-Rad). The homogenates were filtered and analyzed using an ELISA for KC (BD Biosciences), a neutrophil chemokine and murine analog for human IL-8. The results were normalized to total protein using the Bio-Rad protein assay. Statistical analysis. A Student’s t-test and Tukey’s post hoc test were used for statistical comparisons (GraphPad Instat) between intoxicated and burn-injured mice given saline and intoxicated and burn-injured mice treated with PIK or antibiotics; P ⬍ 0.05 was considered to be statistically significant. Values are means ⫾ SE. RESULTS

Limiting bacterial translocation attenuates hepatosteatosis after intoxication and burn injury. At 24 h after intoxication and burn injury, intestinal villi demonstrated a loss of tight junction integrity (Fig. 1A), a 62-fold increase in bacterial translocation (Fig. 1B), and a 14-fold elevation in hepatic triglycerides (see Fig. 3D) compared with sham-injured animals. PIK treatment after intoxication and burn injury restored tight junction complexes as demonstrated by colocalization of ZO-1 and actin (Fig. 1A). This corresponded to a 71% decrease (P ⬍ 0.05) in bacterial translocation (Fig. 1B) and a 75% reduction (P ⬍ 0.05) in liver triglycerides (Fig. 1D) compared with saline-treated animals subjected to the same injury. Antibiotic prophylaxis failed to restore tight junction complexes in the terminal ileum (Fig. 1A) but did reduce bacterial translocation by 93% (P ⬍ 0.05; Fig. 1B), which corresponded to an 84% decrease (P ⬍ 0.05) in liver triglycerides (Fig. 1D) relative to intoxicated and burn-injured mice given saline. The attenuation of steatosis after intoxication and burn injury in PIK- or antibiotic-treated animals was evident upon histological examination of Oil Red O-stained images (Fig. 1C). PIK or antibiotic treatment reduces hepatic damage after intoxication and burn injury. Mice given ethanol and subjected to burn injury were found to have a ⬎25-fold increase in serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT), as well as 1.3-fold increase in liver weight-to-body weight ratio, compared with sham-injured animals (Fig. 2). PIK treatment after the combined insult alleviated hepatic damage as measured by a 49% reduction (P ⬍ 0.05) in serum AST, a 47% decrease (P ⬍ 0.05) in serum ALT, and a 24% lower (P ⬍ 0.05) liver weight-to-body weight ratio than

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intoxicated and burn-injured animals given saline (Fig. 2). Analogous to PIK treatment, antibiotic prophylaxis reduced serum AST by 44% (P ⬍ 0.05), ALT by 43% (P ⬍ 0.05), and liver weight-to-body weight ratio by 23% (P ⬍ 0.05) relative to animals given saline after ethanol and burn injury (Fig. 2). Hepatic and systemic levels of IL-6 are reduced by limiting bacterial translocation after intoxication and burn injury. At 24 h after intoxication and burn injury, an ⬃60-fold increase in hepatic mRNA expression (Fig. 3B) corresponded to a 10-fold increase in IL-6 protein levels in the liver (Fig. 3A) and serum (Fig. 3C) compared with sham-injured mice. After the combined insult of intoxication and burn injury, PIK treatment led to a 33% reduction (P ⬍ 0.05) in hepatic mRNA expression (Fig. 3B), a 71% decrease (P ⬍ 0.05) in IL-6 protein levels in the liver (Fig. 3A), and 70% lower (P ⬍ 0.05) serum IL-6 (Fig. 3C). Similarly, in mice that received antibiotic prophylaxis, hepatic IL-6 was reduced by 51% (P ⬍ 0.05), IL-6 mRNA expression by 45% (P ⬍ 0.05), and serum IL-6 by 68% (P ⬍ 0.05) compared with intoxicated and burn-injured mice given saline.

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Fig. 1. Decreasing bacterial translocation attenuated hepatosteatosis 24 h after intoxication and burn injury. A: tight junction protein localization was assessed with antibody staining against zonula occludens protein-1 (ZO-1; green), phalloidin (red), and nuclei (blue) after treatment with saline vehicle, myosin light chain kinase (MLCK) inhibition with membrane-permeant inhibitor of kinase (PIK), or prophylactic antibiotics (Abx). Representative villi are shown at ⫻400 magnification. B: bacterial translocation to mesenteric lymph nodes after intoxication and burn injury reported as number of colony-forming units (CFU). C: liver sections stained with Oil Red O to visualize triglyceride accumulation at ⫻400 magnification. D: triglycerides in liver homogenates after intoxication and burn injury. *P ⬍ 0.05 vs. PIK and Abx (by Student’s t-test). Values are means ⫾ SE; n ⫽ 4 – 6 animals per group. Dashed line, sham-vehicle; dotted line, sham-ethanol; dashed-dotted line, burn-vehicle.

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Pulmonary inflammation after intoxication and burn injury is attenuated with PIK or antibiotic treatment. Intoxication and burn injury substantially increased alveolar wall thickness upon visual examination (Fig. 4A) compared with sham-injured animals. Imaging software was employed to quantify the amount of lung tissue vs. air space as a measure of alveolar wall thickness, as described previously (8). A threefold increase in lung tissue area in intoxicated and burn-injured mice compared with sham-injured mice (Fig. 4B) was accompanied by a ⬎10-fold increase in neutrophil infiltration (Fig. 4C) and pulmonary KC levels (Fig. 4D). PIK treatment, as well as antibiotics, lessened pulmonary congestion and cellularity upon visual examination (Fig. 4A). Consistent with visual findings, PIK treatment decreased pulmonary congestion by 23% (P ⬍ 0.05) compared with matched controls, and, similarly, antibiotic prophylaxis lessened congestion by 29% (P ⬍ 0.05; Fig. 4B). Neutrophil accumulation was reduced by 49% (P ⬍ 0.05) with PIK treatment and by 43% (P ⬍ 0.05) with antibiotics compared with intoxicated and burn-injured mice given

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Fig. 2. Liver damage 24 h after intoxication and burn injury is diminished when bacterial translocation is inhibited. A–C: serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and liver weightto-total body weight ratio after treatment with saline vehicle, MLCK inhibition (PIK), or prophylactic antibiotics (Abx). *P ⬍ 0.05 vs. PIK and Abx (by Student’s t-test). Values are means ⫾ SE; n ⫽ 4 – 6 animals per group. Dashed line, sham-vehicle; dotted line, sham-ethanol; dashed-dotted line, burn-vehicle.

saline (Fig. 4C). Finally, levels of the neutrophil chemokine KC in lung homogenates were reduced by 48% (P ⬍ 0.05) and 41% in PIK- and antibiotic-treated animals, respectively, compared with intoxicated and burn-injured mice given saline (Fig. 4D).

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Fig. 3. IL-6 production is decreased with PIK or antibiotic treatment 24 h after intoxication and burn injury. A: IL-6 protein levels in liver homogenates. B: IL-6 mRNA expression levels in liver reported as fold expression over sham-injured animals. C: serum IL-6 levels. *P ⬍ 0.05 vs. PIK and antibiotic (Abx) (by Student’s t-test). Values are means ⫾ SE; n ⫽ 4 – 6 animals per group. Dashed line, sham-vehicle; dotted line, sham-ethanol; dashed-dotted line, burn-vehicle.

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Fig. 4. Pulmonary inflammation 24 h after intoxication and burn injury is attenuated by restoration of the gut-liver axis. A: lung histology of intoxicated and burn-injured mice treated with PIK or prophylactic antibiotics (Abx). Sections were stained with hematoxylin and eosin and viewed at ⫻400 magnification. B: pulmonary congestion quantified using imaging software to calculate lung tissue area in 10 fields of view. C: neutrophil [polymorphonuclear leukocyte (PMN)] quantification in 10 high-power fields of view of intoxicated and burn-injured mice. D: pulmonary KC levels. *P ⬍ 0.05 vs. PIK and Abx (by Student’s t-test). Values are means ⫾ SE; n ⫽ 4 – 6 animals per group. Dashed line, sham-vehicle; dotted line, sham-ethanol; dashed-dotted line, burn-vehicle.

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dynamic, involving potential complications in every major organ system, including cardiac impairment (26), renal dysfunction (45), intestinal damage (9), hepatic derangements (27), and pulmonary failure (42). It is unlikely, given the permeating effects of burn injury and alcohol, that a single cell type or molecule is responsible for the multisystem sequelae observed in burn patients who were intoxicated at the time of their injury. Rather, the involvement of several organs, each influencing the response of others, seems plausible and is supported by several findings reported here. Emerging evidence points to a strong relationship between intestinal microbiota and the liver, which is proposed to regulate a myriad of human diseases (11, 47, 51, 52, 55). Independently, burn injury (23) and alcohol (22, 51) have also been shown to alter aspects of the gut-liver axis, but the combination of the two insults is relatively unexplored, despite its prevalence in the trauma population (39, 46). We previously reported that antecedent ethanol exposure in mice potentiates postburn intestinal damage (8, 61, 62), hepatosteatosis (10, 15),

and IL-6 production (8, 18, 32, 61) and exacerbates pulmonary inflammation (4, 5, 7, 8) compared with intoxication or burn injury alone. Here, through the use of two separate approaches, we demonstrate that gut-derived bacteria, or bacterial products, may play a causative, yet reversible, role in the well-established ability of ethanol to worsen postburn systemic inflammation. The intestinal barrier is normally maintained by multiple types of junction complexes, including the tight junction proteins ZO-1 and occludin. In the present study we chose to investigate only ZO-1 localization as a representation of tight junction integrity, but we previously verified similar results with occludin in this animal model (21, 58). In agreement with these studies, PIK treatment restored intestinal barrier function after injury (Fig. 1A), and we now report the benefits of this treatment in terms of the hepatic and pulmonary responses to intoxication and burn injury. The finding that PIK treatment dampened steatosis development after intoxication and burn injury (Fig. 1) suggests that the aberrant hepatic response is

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caused, at least in part, by the translocation of intestinal bacteria. Furthermore, decreasing intestinal bacterial load before injury alleviated these parameters to an equal extent. These findings do not rule out the possible influence of other etiologies of steatosis, such as oxidative stress (44) or changes in fat metabolism (27), but are supported by data from autopsies of burn patients, which reveal an association between fatty liver infiltration and bacterial translocation from the intestines (2). Similarly, in a hemorrhagic shock model, gut sterilization of rats was found to confer hepatic protection (24). The degree of steatosis in our intoxicated and burn-injured mice paralleled other markers of hepatic damage as measured by serum AST and ALT and relative size of the liver after injury (Fig. 2). Severe derangement of AST and ALT in burn patients is a harbinger for complications and death (41), and the liver can increase its size by 225% after burn injury alone (28). The liver is also the major site of ethanol metabolism and toxicity, which are known to sensitize the liver to gut-derived lipopolysaccharide (LPS) (57–59). Ethanol priming of the liver to postburn intestinal LPS (17) may help explain our findings that intoxication potentiated postburn hepatic damage that was attenuated with either PIK or antibiotic treatment (Fig. 2). The etiology of acute liver injury in the setting of intoxication and burn injury is of significant clinical concern, as the liver is a central player in regulating the inflammatory response that is thought to be responsible for the worsened prognosis (35). An important player in any inflammatory response, IL-6 is particularly elevated in thermally injured patients (54, 60) and correlates to increased mortality risk after injury (14). Moreover, we and others have reported that antecedent intoxication significantly raises IL-6 levels of burn-injured mice (8, 10, 32) and that reduction of IL-6 attenuates pulmonary inflammation (7) and gut permeability (61), possibly serving as a link between the gut, liver, and lung in this common clinical scenario. The present studies demonstrate that hepatic IL-6 production at the mRNA and protein levels can be manipulated through limitation of hepatic exposure to intestinal bacteria (Fig. 3, A and B). Furthermore, this correlates to IL-6 levels in the serum (Fig. 3C), indicating that the liver could be the source of systemic IL-6 when burn injury is preceded by ethanol exposure. This finding is consistent with studies of other trauma models where the liver is considered the source of systemic IL-6 after injury (25). Additionally, Li et al. (32) found elevated IL-6 in the plasma, ileum, liver, and lungs of mice exposed to ethanol and subjected to burn injury, with the highest concentration of IL-6 in the liver. IL-6 production after intoxication and burn injury in the liver may be initiated through the interaction between gut-derived LPS and Toll-like receptor 4 (TLR4) on Kupffer cells. Residing in the sinusoids of the liver, Kupffer cells are tissue-fixed macrophages that survey the blood for circulating antigen and mobilize an immune response. After burn injury, there is an increase in circulating LPS (22), which, through binding to TLR4, stimulates Kupffer cells to express inflammatory genes and produce cytokines such as IL-6 (12). Ethanol exposure alters TLR4 signaling in a variety of ways, including changes in CD14 expression (17), lipid rafts (13), and mitogen-activated protein kinases (30, 31), which can increase IL-6 production after exposure to LPS. Furthermore, the absence of TLR4 in the setting of intoxication and burn injury significantly decreases circulating IL-6 (5). More experiments would

be needed to conclusively demonstrate the role of Kupffer cells in this setting, but evidence from other models of trauma supports the idea that Kupffer cells may be the systemic source of IL-6 after injury (25). Increased serum IL-6 is linked to poor survival in patients with acute respiratory distress syndrome (ARDS) (34), although whether IL-6 plays a causative role or is simply a marker of mortality risk remains to be conclusively demonstrated. Similarly, alcohol abuse is also associated with a worsened ARDS prognosis (56) and is a common complication after a thermal injury (50). ARDS is characterized by inflammation and edema in the lung parenchyma, leading to impaired gas exchange. Similarly, an increase in alveolar wall thickness in the combined injury shown in Fig. 4A is consistent with previously reported results (4, 5, 7, 8, 38). The degree of reduction in pulmonary inflammation observed with PIK treatment (Fig. 4) matches the level of reduction found in previous studies of IL-6-deficient mice subjected to the same combined injury (7), suggesting that the pulmonary benefit in PIK-treated animals may be related to decreased IL-6 levels. The parallel results in antibiotic-treated mice imply that the pulmonary benefits of PIK treatment stem from the limitation of bacterial translocation, and not from a direct action of PIK on the lungs. The studies described above demonstrate a clear and reversible interaction between bacterial translocation from the intestines and the hepatic response to burn injury and ethanol exposure. Interestingly, both PIK and antibiotic treatments reduced the amount of bacterial translocation to the extent observed after burn injury alone (Fig. 1B) (8, 61, 62), and the subsequent responses in the liver and lungs matched those observed in postburn injuries without ethanol exposure (Figs. 2 and 4) (3, 7, 8). These results may suggest that intoxication worsens postburn outcome through alterations in the gut-liver axis, since the additional damage observed with antecedent ethanol exposure can be reversed by restoration of gut-liver cross talk to levels seen after burn injury alone. Clinically, there are no accurate predictors for increased burn risk among alcohol abusers, and indiscriminate antibiotic prophylaxis in this population may raise numerous issues, including antimicrobial stewardship, drug compliance, alteration of endogenous flora, drug interactions, and cost. Therefore, an ideal therapeutic in this setting could be administered after the injury. PIK is still an experimental research drug, and, unfortunately, to our knowledge, there are no licensed drugs that act on MLCK inhibition. However, we believe that our data demonstrate a proof of concept that can be taken into human trials as drugs become available. GRANTS This research was supported by National Institute of Alcohol Abuse and Alcoholism Grants R01 AA-012034 (E. J. Kovacs), F30 AA-022856 (M. M. Chen), T32 AA-013527 (E. J. Kovacs), and F31 AA-019913 (A. Zahs) and by the Illinois Excellence in Academic Medicine Grant, the Margaret A. Baima Endowment Fund for Alcohol Research, the Dr. Ralph and Marian C. Falk Medical Research Trust, and the Loyola University Chicago Stritch School of Medicine MD/PhD program. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors.

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