Decreased Pulmonary Inflammation Following Ethanol and Burn Injury in Mice Deficient in TLR4 but not TLR2 Signaling

Clinical and animal model studies demonstrate that burn injury results in an enhanced systemic inflammatory response leading to multiple organ dysfunction syndrome and multiple organ failure (MOF) (Marshall, 2000; Moore and Moore, 1995). This dysregulated immune response in humans and mice can be characterized by higher levels of interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), prostaglandin-E2 (PGE2), and lower delayed-type hypersensitivity response and lymphocyte proliferation (Drost et al., 1993; Kowal-Vern et al., 1997; Zhou et al., 1992). The suppression of the immune response observed after burn is exaggerated with the addition of alcohol in patients (Howland and Hingson, 1987; Kowal-Vern et al., 1994; McGill et al., 1995) and also in animal models (Colantoni et al., 2000; Faunce et al., 1997, 1998; Fontanilla et al., 2000; Messingham et al., 2000). Currently, the major postinjury complications result from pulmonary failure (Solomkin, 1990). This phenomenon is likely a result of bacteria and endotoxin leaking from the gut to the lungs (Magnotti and Deitch, 2005), the increased risk of contact with pathogens from both the circulation and the airway, and the delicate architecture of the lung itself. In support of this hypothesis, the combined insult of ethanol and burn injury was shown to augment intestinal permeability and bacterial translocation (Choudhry et al., 2002; Kavanaugh et al., 2005). In animal models, increased numbers of neutrophils were observed in the lungs of both mice and rats following burn injury (Mester et al., 1994; Stengle et al., 1996) and also in animals receiving ethanol prior to burn (Bird et al., 2010; Li et al., 2007; Patel et al., 1999). Previous studies in this laboratory demonstrated increased pulmonary inflammation after ethanol and burn injury consisting of higher levels of neutrophil accumulation, edema, and chemokine production in the lungs (Bird et al., 2010; Patel et al., 1999), as well as in a model with acute ethanol, burn injury, and infection with Pseudomonas aeruginosa (Murdoch et al., 2008). Toll-like receptors (TLRs) recognize specific molecular patterns from both invading pathogens and host tissue proteins that are released at times of injury. Previous studies using other models of lung injury and/or infection demonstrated decreased inflammatory responses in mice lacking certain TLRs and the adaptor molecules used in their signaling pathways. Mice genetically deficient in TLR2 and TLR4 showed a decreased inflammatory response in the lung after bleomycin-induced injury compared to wild-type mice (Jiang et al., 2006). After burn injury, it was observed that stimulation of total splenocytes or purified splenic macrophages with TLR2 and TLR4 ligands, peptidoglycan, and lipopolysaccharide (LPS), respectively, resulted in enhanced production of pro-inflammatory cytokines (Cairns et al., 2008; Paterson et al., 2003). Administration of LPS to mice after burn resulted in increased pulmonary neutrophil accumulation, cytokine production, and mortality (Murphy et al., 2005). Microvascular leakage after burn injury was also attenuated in TLR4 knockout mice relative to their wild-type counterparts (Breslin et al., 2008). In addition to TLR4, TLR2 was shown to play a role in pulmonary inflammation. It was recently shown that blocking TLR2 signaling, using both TLR2 knockout mice and a neutralizing antibody against the receptor, decreased pulmonary inflammation and fibrosis compared to wild-type mice after intratracheal instillation of bleomycin (Yang et al., 2009) as well as in a model of pneumocystis pneumonia (Wang et al., 2008). Using a mouse model of blunt chest trauma injury, Hoth and colleagues demonstrated decreased neutrophil infiltrate and pro-inflammatory cytokine production in the lungs of TLR2 knockout mice (Hoth et al., 2007). Additionally, biologically relevant doses of alcohol (25 to 50 mM) were shown to up-regulate TLR2 in human airway epithelial cells in vitro, which in turn lead to significantly higher production of the neutrophil chemoattractant, IL-8 (Bailey et al., 2009). Therefore, it appears that TLR2 may also be important in the distal organ inflammation after injury and/or infection. The studies described later examine the role of TLR signaling in the aberrant pulmonary inflammation observed in mice treated with ethanol and burn injury. We observed increased neutrophil infiltration as well as increased production of neutrophil chemoattractant, KC, in the lungs of C57BL/6 (wild-type) mice at 24 hours after ethanol and burn injury compared to sham and burn-injured mice. Elevated pulmonary levels of pro-inflammatory mediators, IL-6 and KC, were also seen at this time point. Mice genetically deficient in TLR4 were protected against the excessive pulmonary inflammation seen in mice given ethanol and burn injury relative to either insult alone. In contrast, mice deficient in TLR2 had similar levels of leukocytes and chemokine production in their lungs as their wild-type counterparts after the combined insult. These data suggest a role for TLR4 signaling and not TLR2 in the inflammatory response in the lung after ethanol and burn injury.