Adipose inflammation and macrophage infiltration after binge ethanol and burn injury - PubMed (original) (raw)

Adipose inflammation and macrophage infiltration after binge ethanol and burn injury

Yuanyuan Qin et al. Alcohol Clin Exp Res. 2014 Jan.

Abstract

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.

Keywords: Alcohol; Chemokine; Crown-Like Structure; Cytokine; Trauma.

Copyright © 2013 by the Research Society on Alcoholism.

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Figures

Figure 1

Figure 1. Interleukin-6 (IL-6) levels increase after single and episodic binge ethanol exposure and burn injury

Mice received single binge (A-B), episodic binge (C-D), or vehicle control. Thirty minutes after ethanol, mice were either subjected to burn or sham injury, sacrificed 24 hours later, and adipose tissue was isolated, as described in methods. A. IL-6 mRNA was measured by quantitative (q)PCR and normalized to 18S. N=16, 16, 18 and 19 for sham vehicle, sham ethanol, burn vehicle and burn ethanol, respectively. There was a significant interaction between burn and ethanol (p<0.05). *** P<0.001 versus (vs.) all groups. B. Whole adipose tissue was homogenized and IL-6 protein levels were determined and normalized to the total amount of protein in the homogenate. N=4, 3, 5 and 5 for sham vehicle, sham ethanol, burn vehicle and burn ethanol, respectively. No significant interaction was found between burn and ethanol. *P<0.05 vs. sham groups. C. After episodic binge and burn, IL-6 mRNA was measured by qPCR and normalized to 18S. N=7, 4, 7 and 10 for sham vehicle, sham ethanol, burn vehicle and burn ethanol, respectively. There was a significant interaction between burn and ethanol (p<0.05). ***P<0.001 vs. all groups. D. IL-6 protein levels were detected after episodic binge in adipose tissue and were normalized to total protein as above. N=7, 5, 12 and 10 for sham vehicle, sham ethanol, burn vehicle and burn ethanol, respectively. There was no significant interaction between burn and ethanol in episodic binge treatment.*P<0.05 vs. sham groups.

Figure 2

Figure 2. Adipose expression of neutrophil chemokine KC after single and episodic binge ethanol and burn injury

Mice received single (A-C) or episodic binge (D-F), or vehicle control and burn, as described above. A. Adipose tissue KC mRNA levels were measured by qPCR and normalized to 18S. N=20, 20, 27 and 21 for sham vehicle, sham ethanol, burn vehicle and burn ethanol, respectively. There was a significant interaction between burn and ethanol (p<0.05). ** P<0.01 vs. all other groups. B. Whole adipose tissue homogenate levels of KC protein were determined and normalized to the total protein. N=4, 3, 5 and 4 for sham vehicle, sham ethanol, burn vehicle and burn ethanol, respectively. No significant interaction was found between burn and ethanol. * P<0.05 vs. sham groups. C. Neutrophil elastase mRNA level was quantified by qPCR in adipose tissue and normalized to 18S. N=12, 11, 16 and 15 for sham vehicle, sham ethanol, burn vehicle and burn ethanol, respectively. There was no significant interaction between burn and ethanol. D. After episodic binge and burn, adipose tissue KC mRNA levels were measured by qPCR and normalized to 18S. N=6, 4, 7 and 8 for sham vehicle, sham ethanol, burn vehicle and burn ethanol, respectively. E. Whole adipose tissue KC protein levels were determined and normalized to total protein. N=7, 5, 12 and 10 for sham vehicle, sham ethanol, burn vehicle and burn ethanol, respectively. There was no interaction between burn and ethanol (p<0.05) in episodic binge treatment. *P<0.05 vs. sham vehicle. ^P<0.05 vs. sham ethanol group. F. Neutrophil marker elastase mRNA level was quantified after episodic binge or burn by qPCR in adipose tissue. N=7, 4, 7 and 13 for sham vehicle, sham ethanol, burn vehicle and burn ethanol, respectively. There was a significant interaction between burn and ethanol (p<0.05). *P<0.05 vs. sham ethanol. **P<0.001 vs. sham vehicle. ***P<0.001 vs. sham ethanol.

Figure 3

Figure 3. Episodic binge ethanol and burn injury drives crown like structure formation

A. Representative immunohistochemical images of macrophage marker F4/80-positive staining in adipose tissue from vehicle, episodic binge, sham or burn injured mice (10X, N= 4,5, 5 and 7 for sham vehicle, sham ethanol, burn vehicle and burn ethanol, respectively). Inflammatory loci (arrow) and crown like structure (*, and inset) are indicated. Ten randomly selected 10× fields were assessed for F4/80+ crown like structures (B, P<0.01 vs. both vehicle groups, P<0.05 vs. sham ethanol) and inflammatory loci (C, *P<0.05, ** P<0.01, ***P<0.001 vs. sham vehicle mice and ^ P<0.05 vs. sham ethanol). No significant interaction was found between burn and ethanol.

Figure 4

Figure 4. Adipose chemokine levels of monocyte chemotactic protein-1 (MCP-1) and adiponectin were inversely regulated after single and episodic binge ethanol and burn injury

Mice received single (A-C) or episodic binge (D-F), or vehicle control and burn. A. Adipose tissue MCP-1 and 18S mRNA levels were measured. N=16, 16, 20 and 19 for sham vehicle, sham ethanol, burn vehicle and burn ethanol, respectively. * P<0.05 vs. vehicle. B. MCP-1 protein levels were examined and normalized to total protein. N=4, 3, 3 and 4 for sham vehicle, sham ethanol, burn vehicle and burn ethanol, respectively. *P<0.05 vs. sham ethanol, **P<0.01 vs. sham vehicle. C. Adipose tissue adiponectin and 18S mRNA levels were measured. N=8, 8, 9 and 8 for sham vehicle, sham ethanol, burn vehicle and burn ethanol, respectively. *P<0.05 vs. burn vehicle, ***P<0.001 vs. sham vehicle. D. In adipose tissue from mice exposed to episodic binge or burn, MCP-1 and 18S mRNA levels were measured. N=6, 4, 6 and 13 for sham vehicle, sham ethanol, burn vehicle and burn ethanol, respectively. E. MCP-1 protein was normalized to the total amount of protein lysate. N=7, 4, 12 and 11 for sham vehicle, sham ethanol, burn vehicle and burn ethanol, respectively. F. After episodic binge, adipose tissue adiponectin and 18S mRNA levels were measured. N=7, 4, 7 and 14 for sham vehicle, sham ethanol, burn vehicle and burn ethanol, respectively. No significant interactions between burn and ethanol were found with single or episodic binge treatment for MCP-1 and adiponectin levels.

Figure 5

Figure 5. Single and episodic binge followed by burn injury failed to upregulate pro-inflammatory mediators

Mice received single (A-D) or episodic binge (E-H), or vehicle control and burn. A-C. Adipose tissue TNFα, IL-1β, or IL-10 was measured and normalized to total protein. N=4, 3, 5 and 5 for sham vehicle, sham ethanol, burn vehicle and burn ethanol, respectively. D. Adipose tissue iNOS mRNA level was measured by qPCR . N=12, 12, 15 and 15 for sham vehicle, sham ethanol, burn vehicle and burn ethanol, respectively. ***P<0.001 vs. sham groups. ^^^P<0.001 vs. sham groups. E-G. After episodic binge and burn, adipose tissue levels of TNFα, IL-1β, or IL-10 were measured and normalized to total protein. N=6, 5, 12 and 11 for sham vehicle, sham ethanol, burn vehicle and burn ethanol, respectively. H. Adipose tissue iNOS mRNA level was measured after episodic binge by qPCR as above. N=7, 4, 7 and 13 for sham vehicle, sham ethanol, burn vehicle and burn ethanol, respectively. *P<0.05 vs. sham ethanol. There were no significant interactions between burn and ethanol detected.

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