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Effects of Multiday Ethanol Intoxication on Postburn Inflammation, Lung Function, and Alveolar Macrophage Phenotype

Curtis, Brenda J.∗,†; Boe, Devin M.∗,†,‡; Shults, Jill A.§; Ramirez, Luis§; Kovacs, Elizabeth J.∗,†,‡

Author Information
doi: 10.1097/SHK.0000000000001188



In the United States, an estimated 485,000 people have burn injuries warranting medical attention, annually, with approximately 10% requiring hospital admission (1). Severe burn is arguably the most detrimental injury one can sustain, as profound physiological derangements extend beyond the cutaneous injury itself and often times lead to multiple organ failure (MOF) and an increased risk for infection and sepsis (2). Nearly half of all burn patients living in the United States have a positive blood alcohol concentration (BAC) level at the time of hospital admission (3). Burn injury with recent alcohol consumption is associated with worsened adverse outcomes, including increased risk for nosocomial pneumonia infections, 3 times longer days of hospital admission, twice as many days requiring mechanical ventilation, and an increased rate of mortality, compared with patients who were not intoxicated before injury (4–6). Respiratory failure, acute respiratory distress syndrome (ARDS), and pulmonary infection are leading causes of postburn morbidity and mortality (7). Clinical and experimental evidence provides clear links connecting excessive and prolonged lung inflammation and oxidative stress after severe burn with ARDS and MOF, although the contributing pathophysiologic mechanisms remain largely undefined (8, 9).

Almost immediately after burn injury, local and systemic release of proinflammatory molecules, referred to the “cytokine storm,” causes damage to the delicate alveolar architecture, which is compounded by excessive neutrophil infiltration and retention in the interstitium, and extracellular fluid accumulation, which is worsened when ethanol intoxication precedes injury (10, 11). The pulmonary inflammatory response to distal burn is characterized by the release of proinflammatory and chemotactic molecules, leukocyte infiltration, oxidative stress, and extracellular fluid accumulation, yet the underlying pathophysiological mechanisms remain largely undescribed, and even less is known regarding the mechanism(s) by which alcohol intoxication augments this response. Preclinical animal model studies demonstrated that when alcohol intoxication precedes burn, pulmonary inflammation is exacerbated and prolonged and lung congestion, characterized by neutrophil infiltration, alveolar wall thickening, and edema, is worsened (9, 12).

Binge alcohol consumption is the most common drinking pattern in the United States, with an estimated 38 million Americans engaging in this hazardous behavior (13). It is defined by either a BAC at or above 0.08% or by the amount of alcoholic beverages one consumes in a 2-h time period (four drinks or more for women, five or more for men), and is the drinking pattern the vast majority of trauma patients, including burn victims, engaged in before injury (3). In addition, college student report a multiple day binge drinking pattern, with consecutive days of drinking over the weekend followed by relative abstinence during the week. (14). We previously demonstrated that mice subjected to our multiday episodic binge ethanol treatment paradigm, modeling college student drinking behavior, had increased pulmonary congestion and neutrophil infiltration, elevated lung levels of neutrophil chemoattractants, and impaired respiratory function, when compared with burn injury alone (11). In addition, survival studies showed that multiday ethanol treatment in the absence of injury was nonlethal, that mice exposed to multiday binge ethanol before burn had 48% survival at 7-d postburn, whereas mice exposed to burn alone had 84% survival. We also observed that animals that succumbed to the combined insult died between 24 and 72 h (11). Therefore, in this study we characterized this critical time frame by examining lung function and histology, measuring an array of immunomodulatory factors in both the blood and in lung tissue, quantifying pulmonary neutrophils, and characterizing alveolar macrophage phenotype. Our analyses suggest that multiday binge ethanol consumption before burn worsens lung dysfunction, exacerbates circulating and pulmonary levels of proinflammatory molecules, and alters alveolar macrophage phenotype, when compared with burn injury or ethanol intoxication alone.


Murine model of scald burn injury and multiday binge ethanol intoxication

C57BL/6 male mice were purchased from Jackson Laboratories (Bar Harbor, Maine) and housed in sterile microisolator cages under pathogen-free conditions in the Loyola University Medical Center Comparative Medicine facility for a minimum of 1 week before experimentation. We used our well-established murine model of scald burn injury and episodic binge ethanol intoxication, as described (11). Briefly, 8- to 10-week-old male mice weighing 20 to 27 g were injected intraperitoneally with ethanol (1.2 g/kg) or saline vehicle for 3 consecutive days, then unmanipulated for 4 days, and then injected with ethanol for an additional 3 consecutive days. This ethanol dosing strategy raises the BAC to 150 mg/dL, 30 min after injection. On the final day of treatment, 30 min after ethanol injection, mice were anesthetized (100 mg/kg ketamine and 10 mg/kg xylazine) (Webster Veterinary, Sterling, Mass), dorsa were shaved, and then mice were subjected to a 15% total body surface area (TBSA) full thickness, insensate scald burn injury (15). This was achieved by placing each mouse in a plastic template that exposed the shaved dorsum to a 92°C to 95°C (or room temperature for sham injury) water bath. All mice were treated with 1 mL of saline resuscitation fluid and cages were placed on warming pads during anesthesia recovery. Mice were euthanized using CO2 narcosis followed by exsanguination. Data presented herein are representative of two animal experiments, minimum. To avoid possible confounding factors caused by circadian rhythms, all animal experimentation was performed between 8 and 9 am. All protocols were approved by the Loyola University Chicago Institutional Animal Care and Use Committee.


Lung function parameters were measured at 24, 48, and 72 h using unrestrained whole body barometric plethysmography (Buxco Research Systems), as described (11). Briefly, mice were placed in the plethysmography chamber, allowed to acclimate, and then enhanced pause (Penh), minute volume (MVb), tidal volume (TVb), and breath frequency (f) measurements were recorded for 10 min, on a breath-by-breath basis. Two independent experiments were performed, n = 3 to 13 per group.

Lung histology

The upper right lobe was used for histopathologic analyses, as described (10). In summary, the lung lobe was gently inflated with 10% formalin, fixed overnight, paraffin-embedded, sectioned at 5 μm, and then stained using hemoxylin and eosin (H&E). Stained images were photographed at 400× (EVOS; Thermo Fisher Scientific) and examined in a blinded fashion. Representative images from two independent animal experiments are shown, n = 3 to 6 per group.

Cytokine/chemokine quantification

The middle right lobe was flash frozen in liquid nitrogen, stored at −80°C until use, and then homogenized in BioPlex cell lysis buffer (Bio-Rad, Hercules, Calif), according to manufacturer guidelines. All samples were assayed in duplicate. Results were normalized to total protein, as determined using the Bio-Rad protein assay (Bio-Rad). In some instances, concentrations for cytokines/chemokines with low levels were extrapolated from the standard curve, though still within the BioPlex assay limit of detection. Experiments were performed a minimum of 2 times, n = 12 to 22 per group, total. Data from one representative experiment are shown.

Enzymatic lung tissue dissociations and flow cytometry

The upper left lung lobe was cut into small pieces, transferred to a C-tube (Miltenyi Biotec, Auburn, Calif) containing digestion buffer (1 mg/mL of Collagenase D and 0.1 mg/mL DNase I [Roche, Indianapolis, Ind] in HBSS) and homogenized using a GentleMACS dissociator (Miltenyi Biotec), according to manufacturer guidelines. Single-cell suspensions were obtained by passing homogenates through 70 μm nylon cell strainers. Red blood cells were lysed in ACK lysis buffer (Life Technologies, Grand Island, NY) and remaining cells were counted using trypan blue exclusion of dead cells. Nonspecific binding to the Fcy II/III receptor was prevented by incubating 1 × 106 lung cells with anti-CD16/32 (clone 93; eBioscience, San Diego, Calif). Cells were immunostained with rat antimouse antibodies: CD45 e780 (clone30-F11; eBioscience), CD11b eFluor 450 (clone M1/70; eBioscience), Ly6G (Gr-1) PE Cy7-conjugated (clone RB6-8C5; eBioscience), CD206 PE, CD24 APC, CD64 PerCP, and MHC II v500. Antibody incubation was carried out for 30 min at 4oC. Cells were washed and fixed as described (16, 17). Flow experiments were performed using a BD Fortessa cytometer (BD Biosciences, San Jose, Calif) and data analyzed using Flow Jo FCS software (Tree Star Inc., Ashland, Ore). Experiments were performed at minimum of 2 times, n = 4 to 6 per group. Data from one representative experiment are shown.

Statistical analyses

Statistical analyses comparing four treatment groups (sham vehicle, sham ethanol, burn vehicle, and burn ethanol) were performed using one-way analysis of variance (ANOVA) with a Tukey's multiple comparison test. Significant differences are reported when P < 0.05. Data are presented as mean ± standard error of the mean (SEM), and graphs were generated using GraphPad Prism 5.02 software.


Lung function after burn and intoxication

Whole body unrestrained plethysmography was used to examine breathing patters and respiratory function in mice subjected to multiday binge ethanol intoxication, burn injury, or the combined insult. In our previous study, we observed respiratory dysfunction, characterized by shallow breaths and a slower breathing rate, at 24 h postinjury in mice subjected to burn alone, which was exacerbated when intoxication preceded injury (11). Herein, we evaluated whether defective breathing parameters persisted over time, by collecting respiratory measurements at 24, 48, and 72 h after treatment. Mice subjected to multiday ethanol intoxication alone had no signs of impaired respiration at later time points. However, at 24 h, control (vehicle-treated, sham-injured) mice had a mean breath rate of 473.51 ± 21.5 breaths/min, which was reduced by 49% to 239.9 ± 42.5 breaths/min (P < 0.05) by burn alone, and by 60% to 190.8 ± 9.1 breaths/min (P < 0.05) when ethanol preceded injury (Fig. 1A). In addition, enhanced pause (Penh), a measure of airway resistance and bronchoconstriction, was increased nearly 7-fold to 5.5 ± 1.8 by burn injury, compared with a Penh of 0.7 ± 0.2 observed in controls. Mice treated with ethanol before burn had more than a 2-fold increase in Penh compared with those with burn injury alone (10.5 ± 0.5) which was an 11-fold increase compared with controls (P < 0.05, compared with sham vehicle and sham ethanol groups) (Fig. 1B). Tidal volume, indicative of inspiration/expiration volumes, was decreased 47% below control (0.08 ± 0.01 mL) (P < 0.05) in burn-injured mice and by 50% (0.08 ± 0.01 mL) in intoxicated, burned mice (P < 0.05) (Fig. 1C). Minute volume, the amount of air inhaled/exhaled per minute (Fig. 1D), showed similar patterns as both were reduced by burn injury, though statistical significance was only reached when comparing sham-injured, intoxicated mice to intoxicated, burned mice (P < 0.05). Sham-injured, vehicle-treated controls had a mean minute volume of 74.1 ± 15.8 mL/min which was reduced by 72% to 20.8 ± 6.6 mL/min by burn injury and by 80% to 15.1 ± 1.8 mL/min, when ethanol intoxication preceded burn (P < 0.05, compared with sham, ethanol-treated mice). At 48 h, control mice had a mean breath rate of 464.8 ± 14.0 breaths/min, which was reduced by 37% to 289.6 ± 43.1 breaths/min (P < 0.05) by burn alone, and by 58% to 196.8 ± 9.8 breaths/min (P < 0.05) when ethanol preceded injury (Fig. 1E). In addition, enhanced pause (Penh) was increased by 3-fold to 3.0 ± 0.9 by burn injury, compared with a Penh of 0.8 ± 0.1 observed in controls (P < 0.05). Mice treated with ethanol before burn had a 2-fold increase in Penh compared with those with burn injury alone (7.9 ± 0.5) (P < 0.05) which was a 9-fold increase compared with controls (P < 0.05) (Fig. 1F). Tidal volume (Fig. 1G) and minute volume (Fig. 1H) showed similar patterns, as both were reduced by burn alone (P < 0.05, compared with sham controls), and were further decreased in those with the combined insult (P < 0.05, compared with sham controls). Sham-injured, vehicle-treated controls had a mean tidal volume of 0.17 ± 0.02 mL which was reduced by 42% to 0.10 ± 0.02 mL by burn injury (P < 0.05) and by 53% to 0.08 ± 0.01, when ethanol intoxication preceded burn (P < 0.05). Similarly, sham-injured, nonintoxicated control mice had a mean minute volume of 79.0 ± 10.2 mL which was reduced by 59% to 32.1 ± 9.7 mL by burn injury (P < 0.05) and by 80% to 63.4 ± 1.6 in intoxicated, injured mice (P < 0.05). Mice surviving until 72 h had similar lung function across all groups (Fig. 1, I–L). Overall, these data indicate that burn injury alone causes respiratory dysfunction at 48 h postinjury, which is exacerbated when ethanol intoxication precedes burn. Although only 48% of the mice survived to 72 h (11), those that did had respiratory parameters similar to controls.

Fig. 1:
Lung function assessment:

Lung histology

Using our model of multiday binge ethanol intoxication followed by burn injury, our laboratory previously reported that 24 h after injury lungs from mice exposed to the combined insult had pulmonary edema and congestion, primarily due to an increase in neutrophil sequestration in the interstitium. There was a 10-fold increase in the number of neutrophils in intoxicated, burned mice compared with sham which was 1.5-fold above burn alone (11). Here, we report that this histopathology persisted over time. Groups of mice were euthanized at either 24 or 72 h after insult to examine lung histology. Similar to our previous findings, lung histology from animals treated with ethanol alone looked similar to vehicle-treated mice (11) (Fig. 2, A and B). Increased cellularity and alveolar wall thickening were observed in burned mice which was amplified in mice treated with multiday binge ethanol intoxication before burn, at 24 h after injury (Fig. 2, C and D). At 72 h postinsult, lungs from ethanol-treated animals appeared similar to controls (Fig. 2, E and F). Interestingly, the increased cellularity and pulmonary congestion observed in burned animals persisted to 72 h (Fig. 2G), but this was not observed in mice treated with ethanol before burn (Fig. 2H).

Fig. 2:
Time course of lung histology after intoxication and burn injury:

Serum cytokine/chemokine levels at 24 h

Circulating levels of the proinflammatory cytokine, interleukin (IL)-6, anti-inflammatory cytokine, IL-10, as well as chemokines granulocyte-colony stimulating factor (G-CSF), KC (CXCL-1), and monocyte-chemoattractant protein (MCP)-1 (CCL2), were measured in serum collected from animals euthanized 24 h after intoxication and injury. Multiday binge ethanol intoxication alone had no effect on levels of any molecule measured. However, burn injury resulted in a nonstatistically significant 16-fold increase in serum levels of IL-6, raising the levels from 7.4 ± 1.1 pg/mL observed in sham-injured, vehicle-treated controls to 124.8 ± 26.5 pg/mL in burned mice. When mice were given ethanol before burn, IL-6 levels were increased to 351.9 pg/mL, which is 47-fold higher than controls and 14-fold higher than burn alone (P < 0.05, compared with all other groups) (Fig. 3A). Similar responses were observed with other immunomodulatory molecules, where burn-injured mice had heightened circulating levels of IL-10, G-CSF, KC, and MCP-1 which was further elevated in mice exposed to the combined insult. Compared with control levels of IL-10 (8.3 ± 3.9 pg/mL), burned animals had a nonstatistically significant 26-fold increase to 485.0 ± 150.6 pg/mL, and those given multiday binge ethanol before burn had a 37-fold increase to 670.7 ± 232.1 pg/mL (P < 0.05, compared with uninjured mice) (Fig. 3B). Although not statistically significant, burn injury dramatically increased the circulating level of G-CSF 86-fold to 28,752.0 ± 7,002.8 pg/mL, compared with 332.5 ± 60.7 pg/mL in controls. G-CSF levels were further increased by 301-fold to 100,318.7 pg/mL ± 28,284.2 pg/mL in mice exposed to the combined insult, when compared with controls (P < 0.05, compared with all other groups) (Fig. 3C). KC levels were 186.1 ± 90.0 pg/mL in control mice, which increased 4-fold to 878.0 ± 84.1 pg/mL in burned mice (P < 0.05), and was further elevated 5-fold higher than controls to 1,046.2 ± 177.6 pg/mL in mice where ethanol intoxication preceded burn (Fig. 3D). Lastly, levels of MCP-1 were elevated from 899.3 ± 212.1 pg/mL in sham-injured, vehicle-treated mice to 2,078.6 ± 599.0 pg/mL in mice exposed to the combined insult (P < 0.05, compared with sham-injured, ethanol-intoxicated mice) (Fig. 3E).

Fig. 3:
Serum cytokine and chemokine levels 24 hours:

Lung cytokine/chemokine levels at 24 h

We previously demonstrated that 24 h after intoxication and injury, mice with the combined insult had higher levels of the neutrophil chemoattractants, KC, and macrophage inflammatory protein (MIP-2, CXCL2) (11). To further investigate the lung inflammatory milieu at this time point, multiplex analyses were performed on lung tissue homogenates. Levels of the proinflammatory cytokines, IL-1β and TNF-α, and the anti-inflammatory cytokine, IL-10, were similar between groups (Fig. 4, A–C). However, a nonstatistically significant trend toward increased levels of IL-6 in lungs from burned mice was observed, raising levels from 2.39 ± 0.38 pg/mg protein in controls to 9.63 ± 5.5 pg/mg protein in injured mice. When mice were treated with ethanol before burn, IL-6 levels were 9-fold higher than controls, at 23.2 ± 4.7 pg/mg total protein (P < 0.05, compared with sham-injured, vehicle-treated mice) (Fig. 4D). Lung MCP-1 levels were more than doubled by burn injury, raising levels from 151.9 ± 18.7 pg/mg protein in controls to 400.7 ± 92.6 pg/mg total protein in burned mice, which was similar to the levels observed in mice given the combined insult, 433.9 ± 109.1 pg/mg total protein, though not statistically significant (Fig. 4E). Leukemia inhibitory factor (LIF) was elevated 2-fold from 2.33 ± 0.1 pg/mg total protein in control mice to 4.51 ± 0.4 pg/mg total protein by burn injury alone (P < 0.05), and further increased to 8.38 ± 1.3 pg/mg total protein when ethanol intoxication preceded burn (P < 0.05, compared with all other groups) (Fig. 4F).

Fig. 4:
Lung inflammatory immunomodulator levels at 24 hours:

Lung cytokine levels at 72 h and 7 days

As we observed increased cellularity and pulmonary congestion that persisted out to 72 h in burned mice, we characterized the inflammatory milieu at this time point and in lungs from mice surviving to 7 days postinsult using lung tissue homogenates in multiplex bead arrays. We observed no statistically significant differences in IL-1β, TNF-α, IL-10, IL-6, MCP-1, and KC at 72 h (Fig. 5, A–F) or at 7 days (Supplemental Table 1,

Fig. 5:
Lung cytokine levels 72 h after injury:

Alveolar macrophage phenotype

Alveolar macrophages play a critical role in innate immune responses by first initiating inflammation and then switching phenotype to promote resolution through efferocytosis of dead cells and the production of anti-inflammatory, proresolving molecules. Flow cytometry was used to assess alveolar macrophage phenotype by characterizing cell surface receptor levels. Using enzymatically dissociated lung tissue, which contains a heterogeneous population of lung cell types, including leukocytes, the mean fluorescent intensity (MFI) of proinflammatory, classical activation markers, macrophage receptor with collagenous structure (MARCO), CD11b, and MHC Class II, and the anti-inflammatory marker, CD206, was determined in CD45+CD11c+CD24-CD64+F4/80+ alveolar macrophages. Consistent with our previous findings using a single binge ethanol intoxication before burn injury model (12) when compared with vehicle-treated, sham-injured mice, MARCO MFI was 2 times higher in alveolar macrophages from burned mice 24 h after injury, regardless of prior multiday binge ethanol intoxication (P < 0.05) (Fig. 6A). However, the MFI of CD11b increased by 110% in burn-injured mice (P < 0.05) (Fig. 6B) and minor increases of CD206 MFI were observed in all treatment groups, though no statistically significant differences were observed at this time point (Fig. 6C). At 72 h after injury, MHC Class II MFI was nearly identical across groups (Fig. 6D). In contrast, the MFI of CD11b and CD206 were increased by 70% and 130%, respectively, on alveolar macrophages from intoxicated burn mice compared with uninjured controls (P < 0.05), indicating an alternatively activated phenotype (Fig. 6, E and F).

Fig. 6:
Flow cytometry analyses of alveolar macrophage cell surface receptors.


Uncontrolled inflammation underlies sepsis, multiple organ failure, and death after burn injury. Inflammatory mediators are also key contributors to ARDS development, regardless of etiology. Cytokine levels can serve as biomarkers of injury severity and predictors of mortality. For example, serum IL-6 and MCP-1 levels are elevated in patients with severe burn injury and high levels are correlated with mortality (18). Consistent with these findings, at 24 h after injury, we observed elevated circulating levels of IL-6 and MCP-1 in burned mice, and even higher levels when multiple days of ethanol intoxication preceded injury (Fig. 3). In addition, the concentration of G-CSF in burn patient serum is elevated and is positively correlated with burn size (19). In this study, increased G-CSF levels observed in burned mice were exacerbated when intoxication preceded injury. Increased circulating levels of the chemokine IL-8 in septic/systemic inflammatory response syndrome (SIRS) patients is also predictive of morbidity and mortality (20). As described herein, KC, the murine IL-8 homologue, was increased in serum from burned mice. The additional insult of multiday intoxication before burn raised levels slightly higher than isolated burn injury alone. Recently, the serum TNF-α/IL-10 ratio was inversely correlated with injury severity, burn size, and predictive of hypersusceptibility to repeated infection in burn patients (21), further demonstrating the potential of inflammatory mediators as predictive biomarkers. Lastly, here we report elevated serum IL-10 concentrations in burn-injured mice, which was further escalated by multiday binge ethanol intoxication, highlighting the deleterious consequences of ethanol consumption on postburn inflammation and resolution.

In addition to measuring cytokine/chemokine levels in mouse serum, we also examined levels in lung tissue homogenates, 24 and 72 h after intoxication and injury (Figs. 4 and 5). Levels of the proinflammatory mediators, IL-1β and TNFα, as well as the anti-inflammatory molecule, IL-10, were similar across groups, over time. Conversely, IL-6 and MCP-1 levels were both elevated lungs of intoxicated, burned mice, 24 h after injury, demonstrating that in addition to elevated circulating proinflammatory molecules, the local pulmonary inflammatory milieu in mice exposed to the combined insult was more severe. Moreover, burned mice had increased leukemia inhibitory factor (LIF) levels and mice exposed to the combined insult had levels nearly 2 times higher than isolated injury. LIF, an acute phase protein and member of the IL-6 family of cytokines, acts to promote tissue homeostasis and limit pneumonic lung injury in mouse models (22), yet increased levels have been correlated with poor outcomes, as LIF levels are elevated in BAL fluid from ARDS patients (23) and in the serum of patients with significant burns (TBSA > 20%), with the highest levels observed in those who did not survive (24). These observations further highlight the complexity of maintaining pulmonary homeostasis after burn injury, requiring initial dampening of inflammation to prevent tissue damage while not suppressing the ability of the immune system to respond to pathogen attack. Our observation that lung levels of IL-1β, TNFα, IL-10, KC, MCP-1, and IL-6 are not different from controls 72 h postinjury could be interpreted such that early inflammation has been resolved by this time point, regardless of treatment. However, given that nearly 50% of the mice treated with multiple days of ethanol intoxication before burn died within the first 72 h, it is highly likely that uncontrolled/excessive inflammation contributed to mortality. Our results from a small pilot study are in support of this notion, as mice euthanized 48 h after injury and intoxication have dramatic alveolar wall thickening, congestion, and leukocyte infiltrates (Curtis and Kovacs, unpublished observation). In addition, the plethysmography results presented herein show lung dysfunction 48 h postintoxication and injury, with increased Penh values indicative of bronchoconstriction and airway resistance, and fewer, shallow breaths per minute (Fig. 1). Furthermore, we previously demonstrated that impaired lung function was correlated with an increased number of neutrophils (11). Ineffective neutrophil clearance prolongs inflammation, contributing to increased capillary endothelial cell permeability and pulmonary edema, likely contributing to the 50% mortality previously observed in mice treated with multiple days of ethanol before burn injury (11).

Alveolar macrophages are a complex cell type able to differentiate between inhaled particulates, innocuous antigens, and commensal microbes, preventing inappropriate inflammatory responses. However, they can also be activated by a variety of stimuli, triggering a proinflammatory immune response to pathogenic microorganisms. As such, alveolar macrophages are key regulators of lung homeostasis after injury. Involved in neutrophil recruitment and efferocytosis as well as the production of reparative and proresolving molecules, understanding alveolar macrophage fate is a principal component to deciphering the complex mechanisms contributing to the excessive and prolonged pulmonary response seen after intoxication and burn. Both ethanol intoxication and burn injury are known to independently alter alveolar macrophage phenotype. Ethanol intoxication alone causes immunosuppression through a variety of mechanisms, including impaired immune cell phagocytic activity, reduced ability to respond to chemotactic stimuli, and diminished capacity to present antigen (25, 26). Alveolar macrophages isolated from mice exposed to a chronic ethanol consumption treatment paradigm undergo oxidative stress (25) and exhibit impaired phagocytosis of Pseudomonas aeruginosa after a single in vivo binge ethanol exposure or when treated with ethanol in vitro(26, 27). Conflicting results regarding alveolar macrophage responsiveness to infectious stimuli after burn have been reported. Alveolar macrophages from burn patients with inhalation injury have increased chemotaxis toward casein and zymosan-activated serum in vitro(28) and are primed for an overexuberant proinflammatory response after TLR2 or TLR4 activation (29, 30). In contrast, alveolar macrophage hyporesponsiveness to endotoxin was correlated with mortality in burn victims with inhalation injury (31). In a mouse model of burn injury and pulmonary infection, alveolar macrophage phagocytic activity was diminished after burn rendering cells incapable of phagocytosing and clearing P. aeruginosa, leading to sepsis and mortality (32). As nearly 50% of burn patients are intoxicated at the time of injury (14), it is imperative not only to understand how intoxication or injury alone alters alveolar macrophage phenotype but also to study the combined effect.

Alveolar macrophage homeostatic maintenance of the lung inflammatory milieu is facilitated in part through cell surface receptor expression. The scavenger receptor, MARCO, binds unopsonized bacteria and particulate matter and its activation promotes the clearance of apoptotic cells via efferocytosis (33). Hence, MARCO activation mediates clearance of lung pathogens, yet at the same time it can block inflammatory responses through efferocytosis. In addition, CD11b contributes to complement receptor-3-mediated efferocytosis of iC3b-opsonized apoptotic cells (34). Conversely, the mannose receptor, CD206, selectively binds to glycosylated lipids and proteins found on pathogen surfaces as well as to unopsonized bacteria, suppressing a proinflammatory response to commensals (35), and promotes tissue repair and resolution. In this study, we observed increased MARCO activation at 24 h postburn, regardless of ethanol intoxication, indicative of macrophage activation. Furthermore, at 24 h postinjury, CD11b levels were increased in burn-injured mice, but not in those treated with ethanol before burn. However, at 72 h, CD11b levels remained elevated after burn, but were highest in intoxicated, burned mice. CD206 levels were also significantly elevated in mice treated with ethanol before burn. Speculatively, these findings could indicate that alveolar macrophages from intoxicated, burned mice have a proinflammatory phenotype at 24 h, yet their ability to efferocytose apoptotic cells is diminished due to reduced or delayed CD11b upregulation. Moreover, this response has been mounted by the 72 h time point. Our observation that CD206 is only upregulated at 72 h in intoxicated, burned mice suggests that the lung damage in mice exposed to the combined insult triggers a more profound reparative, anti-inflammatory response; and the anti-inflammatory nature of alternatively activated macrophages may render them less capable of mounting an effective immune response to infectious pathogens.

In summary, our results demonstrate that multiday ethanol intoxication before burn contributes to increased mortality through additive effects of two insults. The observed pulmonary edema, leukocyte congestion, and impaired respiration are likely caused by exacerbated inflammatory responses early after injury which may be due, in part, to the ineffectiveness of alveolar macrophages to resolve inflammation. Our finding that alveolar macrophages from intoxicated, burned mice have elevated levels of CD206 at 72 h, evidence of a heightened anti-inflammatory phenotype, may represent an underlying mechanism driving the increased susceptibility to infection observed in intoxicated burn patients.


1. ABA, A. B. A. (2016) Burn Incidence Fact Sheet, Burn Incidence and Treatment in the United States: 2016.
2. Aikawa N, Shinozawa Y, Ishibiki K, Abe O, Yamamoto S, Motegi M, Yoshii H, Sudoh M. Clinical analysis of multiple organ failure in burned patients. Burns Incl Therm Inj 13:103–109, 1987.
3. Howland J, Hingson R. Alcohol as a risk factor for injuries or death due to fires and burns: review of the literature. Public Health Rep 102:475–483, 1987.
4. Silver GM, Albright JM, Schermer CR, Halerz M, Conrad P, Ackerman PD, Lau L, Emanuele MA, Kovacs EJ, Gamelli RL. Adverse clinical outcomes associated with elevated blood alcohol levels at the time of burn injury. J Burn Care Res 29:784–789, 2008.
5. Griffin R, Poe AM, Cross JM, Rue LW 3rd, McGwin G Jr. The association between blood alcohol level and infectious complications among burn patients. J Burn Care Res 30:395–399, 2009.
6. Grobmyer SR, Maniscalco SP, Purdue GF, Hunt JL. Alcohol, drug intoxication, or both at the time of burn injury as a predictor of complications and mortality in hospitalized patients with burns. J Burn Care Rehabil 17:532–539, 1996.
7. Steinvall I, Bak Z, Sjoberg F. Acute respiratory distress syndrome is as important as inhalation injury for the development of respiratory dysfunction in major burns. Burns 34:441–451, 2008.
8. Turnage RH, Nwariaku F, Murphy J, Schulman C, Wright K, Yin H. Mechanisms of pulmonary microvascular dysfunction during severe burn injury. World J Surg 26:848–853, 2002.
9. Bird MD, Kovacs EJ. Organ-specific inflammation following acute ethanol and burn injury. J Leukoc Biol 84:607–613, 2008.
10. Patel PJ, Faunce DE, Gregory MS, Duffner LA, Kovacs EJ. Elevation in pulmonary neutrophils and prolonged production of pulmonary macrophage inflammatory protein-2 after burn injury with prior alcohol exposure. Am J Respir Cell Mol Biol 20:1229–1237, 1999.
11. Shults JA, Curtis BJ, Chen MM, O’Halloran EB, Ramirez L, Kovacs EJ. Impaired respiratory function and heightened pulmonary inflammation in episodic binge ethanol intoxication and burn injury. Alcohol 49:713–720, 2015.
12. Shults JA, Curtis BJ, Boe DM, Ramirez L, Kovacs EJ. Ethanol intoxication prolongs post-burn pulmonary inflammation: role of alveolar macrophages. J Leukoc Biol 100:1037–1045, 2016.
13. Centers for Disease and Prevention. Vital signs: binge drinking prevalence, frequency, and intensity among adults—United States, 2010. MMWR Morb Mortal Wkly Rep 61:14–19, 2012.
14. Hoeppner BB, Barnett NP, Jackson KM, Colby SM, Kahler CW, Monti PM, Read J, Tevyaw T, Wood M, Corriveau D, et al. Daily college student drinking patterns across the first year of college. J Stud Alcohol Drugs 73:613–624, 2012.
15. Faunce DE, Llanas JN, Patel PJ, Gregory MS, Duffner LA, Kovacs EJ. Neutrophil chemokine production in the skin following scald injury. Burns 25:403–410, 1999.
16. Boehmer ED, Goral J, Faunce DE, Kovacs EJ. Age-dependent decrease in Toll-like receptor 4-mediated proinflammatory cytokine production and mitogen-activated protein kinase expression. J Leukoc Biol 75:342–349, 2004.
17. Murdoch EL, Karavitis J, Deburghgraeve C, Ramirez L, Kovacs EJ. Prolonged chemokine expression and excessive neutrophil infiltration in the lungs of burn-injured mice exposed to ethanol and pulmonary infection. Shock 35:403–410, 2011.
18. Hur J, Yang HT, Chun W, Kim JH, Shin SH, Kang HJ, Kim HS. Inflammatory cytokines and their prognostic ability in cases of major burn injury. Ann Lab Med 35:105–110, 2015.
19. Kim HS, Kim JH, Yim H, Kim D. Changes in the levels of interleukins 6, 8, and 10, tumor necrosis factor alpha, and granulocyte-colony stimulating factor in Korean burn patients: relation to burn size and postburn time. Ann Lab Med 32:339–344, 2012.
20. Hack CE, Hart M, van Schijndel RJ, Eerenberg AJ, Nuijens JH, Thijs LG, Aarden LA. Interleukin-8 in sepsis: relation to shock and inflammatory mediators. Infect Immun 60:2835–2842, 1992.
21. Tsurumi A, Que YA, Ryan CM, Tompkins RG, Rahme LG. TNF-alpha/IL-10 ratio correlates with burn severity and may serve as a risk predictor of increased susceptibility to infections. Front Public Health 4:216, 2016.
22. Traber KE, Symer EM, Allen E, Kim Y, Hilliard KL, Wasserman GA, Stewart CL, Jones MR, Mizgerd JP, Quinton LJ. Myeloid-epithelial cross talk coordinates synthesis of the tissue-protective cytokine leukemia inhibitory factor during pneumonia. Am J Physiol Lung Cell Mol Physiol 313:L548–L558, 2017.
23. Jorens PG, De Jongh R, Bossaert LL, De Backer W, Herman AG, Pollet H, Bosmans E, Taupin JL, Moreau JF. High levels of leukaemia inhibitory factor in ARDS. Cytokine 8:873–876, 1996.
24. Akita S, Akino K, Ren SG, Melmed S, Imaizumi T, Hirano A. Elevated circulating leukemia inhibitory factor in patients with extensive burns. J Burn Care Res 27:221–225, 2006.
25. Yeligar SM, Harris FL, Hart CM, Brown LA. Ethanol induces oxidative stress in alveolar macrophages via upregulation of NADPH oxidases. J Immunol 188:3648–3657, 2012.
26. Karavitis J, Murdoch EL, Gomez CR, Ramirez L, Kovacs EJ. Acute ethanol exposure attenuates pattern recognition receptor activated macrophage functions. J Interferon Cytokine Res 28:413–422, 2008.
27. Karavitis J, Murdoch EL, Deburghgraeve C, Ramirez L, Kovacs EJ. Ethanol suppresses phagosomal adhesion maturation, Rac activation, and subsequent actin polymerization during FcgammaR-mediated phagocytosis. Cell Immunol 274:61–71, 2012.
28. Riyami BM, Kinsella J, Pollok AJ, Clark C, Stevenson RD, Reid WH, Campbell D, Gemmell CG. Alveolar macrophage chemotaxis in fire victims with smoke inhalation and burns injury. Eur J Clin Invest 21:485–489, 1991.
29. Oppeltz RF, Rani M, Zhang Q, Schwacha MG. Burn-induced alterations in toll-like receptor-mediated responses by bronchoalveolar lavage cells. Cytokine 55:396–401, 2011.
30. Wright MJ, Murphy JT. Smoke inhalation enhances early alveolar leukocyte responsiveness to endotoxin. J Trauma 59:64–70, 2005.
31. Davis CS, Albright JM, Carter SR, Ramirez L, Kim H, Gamelli RL, Kovacs EJ. Early pulmonary immune hyporesponsiveness is associated with mortality after burn and smoke inhalation injury. J Burn Care Res 33:26–35, 2012.
32. Davis KA, Santaniello JM, He LK, Muthu K, Sen S, Jones SB, Gamelli RL, Shankar R. Burn injury and pulmonary sepsis: development of a clinically relevant model. J Trauma 56:272–278, 2004.
33. Palecanda A, Paulauskis J, Al-Mutairi E, Imrich A, Qin G, Suzuki H, Kodama T, Tryggvason K, Koziel H, Kobzik L. Role of the scavenger receptor MARCO in alveolar macrophage binding of unopsonized environmental particles. J Exp Med 189:1497–1506, 1999.
34. Mevorach D, Mascarenhas JO, Gershov D, Elkon KB. Complement-dependent clearance of apoptotic cells by human macrophages. J Exp Med 188:2313–2320, 1998.
35. Zhang J, Tachado SD, Patel N, Zhu J, Imrich A, Manfruelli P, Cushion M, Kinane TB, Koziel H. Negative regulatory role of mannose receptors on human alveolar macrophage proinflammatory cytokine release in vitro. J Leukoc Biol 78:665–674, 2005.

Alcohol; alternative activation; cytokines; injury; neutrophils; resolution; ARDS; acute respiratory distress syndrome; BAC; blood alcohol concentration; BAL; bronchoalveolar lavage; CD; cluster of differentiation; Penh; enhanced pause; H&E; hemoxylin and eosin; IF; immunofluorescence; IL; interleukin; CXCL-1; KC; LIF; leukemia inhibitory protein; LPS; lipopolysaccharide; MCP-1; macrophage chemoattractant protein-1; MIP-2; macrophage inflammatory protein-2; MFI; mean fluorescence intensity; MOF; multiple organ failure; SIRs; systemic inflammatory response syndrome; TBSA; total body surface area; TNF-α; tumor necrosis factor-alpha

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