Burn-induced hyperglycemia, hepatosteatosis, and in-sulin resistance are common complications observed in the burn patient population and are associated with poor outcomes, contributing significantly to morbidity and mortality (Bonab et al., 2010; Silver et al., 2008). Insulin resistance in liver, skeletal muscle, and adipose can persist even 3 weeks after burn injury (Carter et al., 2004; Cree and Wolfe, 2008; Thorell et al., 1999). Burn injury also drives systemic inflammation with elevations in pro-inflammatory cytokines and suppressed cell-mediated immunity, leading to multi-organ dysfunction (Marshall, 2000). One mechanism of systemic insulin resistance may be through adipose dysfunction and inflammation (Johnson et al., 2012). Macrophages are known to infiltrate adipose during states of metabolic stress, such as in obesity, and contribute to inflammation, uncontrolled lipolysis, as well as local and systemic insulin resistance (Johnson et al., 2012).
Clinical and laboratory studies have demonstrated that ethanol (EtOH) exposure prior to traumatic injury, such as a burn, markedly elevates systemic and tissue-specific inflammatory responses (Bird and Kovacs, 2008; Jung et al., 2011) and is associated with poorer outcomes (McGill et al., 1995). In the United States, half of the patients with burn-related injuries have alcohol in their system at the time of admission, and the vast majority of those subjects are binge drinkers rather than chronic alcoholics (Albright et al., 2009). It is well-established that alcohol increases the dysregulated inflammatory and immune response caused by burn in animal models and patients (reviewed in Bird and Kovacs, 2008). We and others have shown previously that neutrophils infiltrate the gut, lung, and site of injury after the combined insult (Bird et al., 2010a; Chen et al., 2013; Faunce et al., 1999; Li et al., 2011; Zahs et al., 2012, 2013). While the primary role of neutrophils is to clear pathogens, they often cause damage due to production of enzymes such as elastase, reactive oxygen species, and pro-inflammatory cytokines including interleukin-6 (IL-6), IL-1β, and tumor necrosis factor alpha (TNFα). Serum cytokines IL-6 and TNFα and tissue levels of KC and IL-6 are elevated in response to the dual insult of EtOH and burn injury compared with either injury alone (Chen et al., 2013; Li et al., 2011; Zahs et al., 2013).
Chronic EtOH has been shown to drive macrophage infiltration into adipose tissue, and it is associated with reduced fat mass due to up-regulated lipolysis (Kang et al., 2007; Zhong et al., 2012). To date, it is not known how EtOH exposure combined with burn injury affects the adipose microenvironment. Using an established murine model of binge EtOH exposure and burn injury, we demonstrated that the combined insults drove systemic and adipose inflammation 24 hours postinjury. Furthermore, we found that employing an episodic multiday binge EtOH exposure paradigm followed by burn injury potentiated adipose inflammation and induced macrophage infiltration, indicating that binge, and especially episodic binge EtOH exposure, followed by burn drives adipose inflammation that could contribute to systemic inflammation.
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- Materials and Methods
Half of burn patients admitted to the hospital consumed alcohol prior to sustaining their injuries (Albright et al., 2009; Nelson and Kolls, 2002; Thai et al., 1985). The vast majority of inebriated burn patients are not chronic alcohol abusers, but rather binge drinkers. Recent evidence suggests that binge drinking is on the rise with 1 in 6 Americans reporting an average of 4 binge-drinking episodes per month. Thus, gaining a better understanding about the effects of episodic binge alcohol exposure on postburn morbidity and mortality is of immediate public health relevance. Burn injury induces dramatic insulin resistance, hyperlipidemia, and hyperglycemia, contributing to elevated morbidity and mortality (Bonab et al., 2010; Carter et al., 2004). EtOH exacerbates burn-induced inflammation and impairs the immune response, thus increasing a patient's susceptibility to infection (Bird and Kovacs, 2008).
Previous work by our laboratory has demonstrated that IL-6 is elevated in serum, lungs, and gut of animals exposed to EtOH and burn (Chen et al., 2013; Li et al., 2011; Zahs et al., 2013). We provide evidence herein that cytokines and chemokines are also elevated in adipose tissue suggesting that this tissue may be an additional source of circulating inflammatory mediators. Adipose tissue plays a critical role in maintaining systemic metabolic homeostasis, and adipose inflammation leads to metabolic dysregulation characterized by insulin resistance, hyperlipidemia, and hyperglycemia—all of which commonly occur in burn patients. We set out to examine the role of adipose inflammation in response to the combined insult of EtOH exposure and burn injury. We have demonstrated that mice singly or episodically exposed to EtOH alone did not mount a dramatic inflammatory response in adipose tissue in the absence of a secondary insult such as burn. However, in mice administered a single EtOH binge and burn injury, there was a dramatic elevation in the pro-inflammatory response in adipose tissue indicated by elevations of cytokine and chemokines IL-6, KC, and MCP-1, along with a blunting of adiponectin, an anti-inflammatory adipokine. IL-6 is primarily secreted by macrophages and T cells to stimulate the immune response, for example, during infection and after burn (Bird and Kovacs, 2008) and elevated serum IL-6 in injured patients is correlated with negative outcomes (Biffl et al., 1996). While we detected elevations in KC, we found that there was no change in neutrophil elastase in adipose tissue after single binge EtOH exposure, burn injury, or the combined insults. It is likely that 24 hours postinjury may not be the correct time point for the neutrophil composition of adipose to be dramatically regulated, unlike the neutrophil response in lung, skin, or gut. Furthermore, Nagy and colleagues reported down-regulation of the anti-inflammatory adipokine adiponectin in chronic EtOH-induced adipose inflammation, while Xu and colleagues showed that increasing adiponectin improves alcoholic fatty liver disease (Kang et al., 2007; Xu et al., 2003). We demonstrate dramatic blunting of adiponectin after single binge and burn, which may aid in driving the pro-inflammatory milieu. Finally, iNOS was down-regulated by single binge EtOH and burn injury. Interestingly, Syapin and colleagues have demonstrated that iNOS is inhibited by EtOH in glial cells (Sanchez et al., 2007). In other tissues, burn has been shown to elevate iNOS expression or have no effect (Babcock et al., 2012; Oppeltz et al., 2012). Hence, iNOS regulation by EtOH and burn in adipose needs further investigation. Taken together, our data support the findings that adipose tissue may be a source of some circulating cytokines after single binge EtOH and burn injury.
Binge drinking is not usually a single acute event; binge drinkers tend to consume alcohol in multiple binge episodes. Using an episodic binge model, we demonstrated that burn-induced inflammation was markedly elevated in mice given multiple exposures to binge levels of EtOH and that this occurred to a greater extent than following a single binge exposure. Similar to single binge, after episodic binge and subsequent burn, IL-6 and KC were elevated compared with either insult alone or sham vehicle controls. Despite elevations in KC protein in combined injury, neutrophil elastase mRNA level was dramatically reduced in burn EtOH groups relative to either insult alone. This was a surprising finding because other insults such as a 3 day exposure to a high-fat diet have been shown to induce neutrophil infiltration into adipose tissue (Elgazar-Carmon et al., 2008; Talukdar et al., 2012), which persisted for up to 90 days (Talukdar et al., 2012). Perhaps a more detailed study of episodic binge and burn injury over a time course might capture neutrophil infiltration in response to high levels of KC. Even with dramatic increases in IL-6, KC, and MCP-1 associated with episodic binge EtOH exposure, iNOS was blunted similar to a single binge. It is evident that while some production of pro-inflammatory cytokines or chemokines occurs in response to burn and EtOH, some immunosuppression is concurrent. Future studies will include examining the role of IL-6 in adipose inflammation as it has been shown to be both pro-inflammatory and anti-inflammatory in binge and burn-injured animals, and may mediate some of the divergent effects demonstrated.
Finally, we report for the first time that F4/80+ macrophages were detected in adipose crown-like structures and inflammatory loci resulting from episodic binge EtOH exposure and burn injury, likely due to elevations in MCP-1. MCP-1 and its receptor CCR2 have been shown to mediate macrophage infiltration into adipose tissue in response to obesity (Johnson et al., 2012), and these infiltrating macrophages often surround dying adipocytes forming crown-like structures (Johnson et al., 2012). Kang and colleagues (2007) have demonstrated infiltration of macrophages and production of pro-inflammatory cytokines, IL-6, MCP-1, and TNFα, in adipose tissue after chronic EtOH exposure for 4 weeks. Our data demonstrate that macrophage infiltration occurs within days after burn and EtOH exposure.
From our studies, it is evident that burn injury primarily drives inflammation in adipose tissue, and EtOH exposure prior to burn potentiates this response. The dramatic increase in magnitude of response between single and episodic binge exposure suggests that the driving factor in the inflammatory response is the frequency of the EtOH exposure, as the burn injury and time of sacrifice are identical in each group. Our findings support the relevance of adipose inflammatory response to EtOH and burn insult that warrants further investigation. Like EtOH, obesity is also associated with a prolonged increase in pro-inflammatory mediators, such as IL-6 and MCP-1, an impaired immune response, and an increased susceptibility to bacterial infection (Johnson et al., 2012; Milner and Beck, 2012). Work from our group and others over the past decade has linked adipose inflammation to obesity and insulin resistance (Johnson et al., 2012; Sampey et al., 2011, 2012).
One mechanism linking both obesity-induced inflammation and EtOH exposure that could drive insulin resistance is an increase in gut permeability, resulting in bacterial translocation into tissues to induce both organ-specific and systemic inflammation. We have previously reported on increased gut permeability and bacterial translocation in EtOH and burn models (Kavanaugh et al., 2005; Rendon et al., 2013; Zahs et al., 2012, 2013). In humans and murine models, elevated morbidity and mortality result after burn injury due to inflammation secondary to intestinal permeability and septicemia (Kavanaugh et al., 2005; Magnotti and Deitch, 2005; Messingham et al., 2002; Zahs et al., 2012, 2013), which is often followed by an exaggerated alcohol-induced suppression of immune function through a lower delayed-type hypersensitivity response and blunted lymphocyte proliferation (Choudhry et al., 2000; Faunce et al., 1998; Messingham et al., 2000, 2002). At 6 and 24 hours post EtOH plus burn injury, gut permeability is compromised, which could account for elevated leukocyte infiltration, and IL-1β and IL-6 in mice exposed to EtOH then burn as compared to either insult alone (Zahs et al., 2012, 2013). Gut bacteria also regulate obesity susceptibility and systemic inflammation in response to high-fat diet (Turnbaugh et al., 2006). Additionally, we have previously demonstrated the dependence of Toll-like receptor 4 (TLR4; but not TLR2) in EtOH and burn-induced lung pathology and pro-inflammatory response (Bird et al., 2010b). TLR4 has also been shown to be necessary for obesity-induced inflammation (Shi et al., 2006). Elevated gut permeability and bacterial translocation after the combined insult of EtOH and burn may be responsible for the rise in lipopolysaccharide (endotoxin) burden and a TLR4-dependent inflammatory response in adipose, similar to obesity.
A second mechanism regulating EtOH and burn-induced alterations in adipose inflammation is lipolysis and adipocyte apoptosis, which would release free fatty acids into the local microenvironment and circulation. In humans, chronic alcohol use correlated with reduced fat mass (Addolorato et al., 1997). Burn injury also results in loss of fat mass through apoptotic cell loss (Yasuhara et al., 2006). Duffy and colleagues (2009) demonstrated that insulin and glucose are elevated in patients postburn compared with healthy controls, with burn-induced increases in cytokine release from adipose tissue macrophages and circulating monocytes, which could interfere with insulin signaling (Duffy et al., 2009). In rodents, single binge and burn drive a transient microvesicular steatosis, while chronic alcohol exposure also leads to reduced fat mass and adipocyte size, along with an increase in hepatosteatosis and induction of systemic insulin resistance (Emanuele et al., 2009; Zhong et al., 2012). It has been shown that alcohol-driven lipolysis is not catecholamine-mediated (Kang and Nagy, 2006). EtOH has been shown to increase levels of phosphatase and tensin homolog (PTEN) and suppressor of cytokine signaling (SOCS3), which are important negative regulators of insulin signaling in both liver and adipose and can lead to elevated lipolysis and cytotoxicity (Shulga et al., 2005; Zhong et al., 2012). It is possible that saturated free fatty acids liberated by lipolysis, which are known to signal through TLRs during obesity-induced inflammation and insulin resistance (Suganami et al., 2007), may mediate a pro-inflammatory response after EtOH and burn exposure. Additionally, reduction in fat mass through apoptosis or lipolysis contributes to reduced insulin sensitivity because fat is redistributed from adipose to other metabolically sensitive tissues, such as the liver where hepatosteatosis ensues (Johnson et al., 2012). Liver IL-6 and other pro-inflammatory mediators increase with EtOH and/or burn exposure (Colantoni et al., 2000; Li et al., 2011). Emanuele and colleagues (2007) demonstrated single and/or combined injury increased hepatic ICAM-1, IL-1β, TNFα, and nuclear NF-κB, which can lead to insulin resistance. Indeed, we have previously reported that insulin administration to rodents after EtOH and burn, improves liver inflammation and microvesicular steatosis, demonstrating further evidence of links between metabolic homeostasis and inflammatory response (Emanuele et al., 2007).
Finally, a third mechanism linking EtOH intake to exacerbated inflammation is oxidative stress resulting from alcohol metabolism. Besides alcohol dehydrogenase, EtOH can also be metabolized through the microsomal EtOH-oxidizing system by cytochrome P4502E1 (CYP2E1), which has been shown to lead to increased oxidative stress (Nagy, 2004). CYP2E1 is mainly expressed in the liver, but also found in the white adipose tissues (Tang et al., 2012). Sebastian and colleagues (2011) demonstrated that CYP2E1 protein levels in adipose tissue were increased after chronic EtOH feeding. In our study, we failed to find a significant increase in mRNA or protein expression of CYP2E1 in adipose tissue of episodic binge, burned mice, or the combined exposure (data not shown). This may due to the type or length of exposure, as our treatment was shorter with either a 1-dose single binge or episodic binge constituting a total of 6 days of EtOH exposure.
Taken together, we report for the first time that there is an inflammatory response in adipose tissue after the combined insult of EtOH and burn injury, and that this response is augmented after episodic binge relative to a single EtOH exposure. While binge drinking leads to unintentional injuries such as falls, crashes, and burns, it may also lead to more insidious tissue inflammation as a comorbidity with obesity leading to insulin resistance. Future studies in lean versus obese rodents could yield further mechanistic insight into burn and EtOH-induced effects on local and systemic inflammation.