Decreased pulmonary inflammation following ethanol and burn injury in mice deficient in TLR4 but not TLR2 signaling

Abstract

Background: Clinical and laboratory evidence suggests that alcohol consumption prior to burn injury leads to dysregulated immune function and subsequent higher rates of morbidity and mortality. Our laboratory previously observed higher levels of pro-inflammatory cytokines and leukocyte infiltration in the lungs of mice following ethanol and burn injury. To understand the mechanism of the increased inflammatory response, we looked at different signaling initiators of inflammation including toll-like receptors 2 and 4 (TLR2 and 4) pathways.

Methods: Wild-type, TLR2, and TLR4 knockout mice were treated with vehicle or a single binge dose of ethanol (1.11 g/kg) and subsequently given a sham or burn injury. Twenty-four hours postinjury, systemic and pulmonary levels of pro-inflammatory cytokines were quantified, and differences in neutrophil infiltration were determined by histological examination.

Results: Higher numbers of neutrophils were observed in the lungs of wild-type mice following the combined insult of ethanol and burn injury relative to either injury alone. This increase in leukocyte accumulation was absent in the TLR4 knockout mice. Circulating levels of IL-6 and tumor necrosis factor-α were also elevated in wild-type mice but not in TLR4 knockout mice. Consistent with these findings, pulmonary levels of KC and IL-6 were increased in wild-type mice following burn and ethanol compared to burn injury alone as well as to their TLR4 knockout counterparts. In contrast, TLR2 knockout mice displayed similar levels, to wild-type mice, of neutrophil infiltration as well as IL-6 and KC in the lung.

Conclusions: These data suggest that TLR4 signaling is a crucial contributory component in the exuberant inflammation after ethanol and burn injury. However, TLR2 does not appear to play a vital role in the aberrant pulmonary inflammation.

Copyright © 2010 by the Research Society on Alcoholism.

Figures

Fig. 1

Decreased levels of circulating pro-inflammatory cytokines in TLR4 knockout mice. Levels of tumor necrosis factor-α and IL-6 in serum were quantified by Invitrogen multiplex assay. *p < 0.05 compared to all other groups using 1-way ANOVA. N = 3 per group. Representative data from 1 of 2 studies performed are shown as mean concentration ± SEM.

Fig. 2

Decreased pulmonary inflammation following ethanol and burn injury in TLR4 KO mice. Lung sections from WT, TLR4 KO, and TLR2 KO mice were examined for degree of inflammation 24 hours after ethanol and burn injury. Representative micrographs of hematoxylin and eosin (H & E) lung sections are shown. All images are at 200× magnification.

Fig. 3

Increased leukocyte infiltration in the lungs following ethanol and burn injury of WT and TLR2 KO mice. Enlarged representative images of lungs WT, TLR4 KO, and TLR2 KO mice 24 hours after exposure to ethanol and burn injury (400×). Neutrophils are indicated by arrows.

Fig. 4

Increased Gr-1-positive cells and myeloperoxidase (MPO) activity after ethanol and burn injury in the lungs of wild-type mice. (A) Lung sections from wild-type mice were stained by flurochrome-conjugated antibodies against Gr-1 (green) to detect the presence of neutrophils. Representative micrographs from all 4 treatment groups are shown. All images are at 200× magnification. (B) Representative lung sections from wild-type (WT), TLR4 KO, and TLR2 KO mice treated with ethanol and burn injury. Green staining indicates neutrophils. (C) MPO activity was measured in lung homogenates from wild-type animals. The data are represented as MPO activity per mg of protein. N = 7 to 12 per group. *p < 0.05 compared to sham animals, #p < 0.05 compared to burn vehicle animals.

Fig. 5

No differences in circulating white blood cells in TLR4 knockout mice after ethanol and burn injury. Total and differential cell counts in whole blood were obtained using a Hemavet 750 machine. Mean values are listed as thousands of cells per microliter of blood ± SEM. *p < 0.05 compared to all sham animals, #p < 0.05 compared to sham ethanol wild-type only by 1-way ANOVA. N = 3 to 6 per group.

Fig. 6

Decreased levels of pro-inflammatory cytokines and chemokines in TLR4 knockout mice. Levels of KC and IL-6 in lung homogenates were quantified by multiplex bead array. Cytokine concentrations were normalized to total protein in the sample and shown as mean concentration ± SEM. *p < 0.05 compared to all groups using 1-way ANOVA. N = 3 to 6 per group.

Fig. 7

No difference in pulmonary inflammation between wild-type and TLR2 knockout mice after ethanol and burn injury. Levels of KC (A) and IL-6 (B) were quantified in lung homogenates by multiplex bead array. Cytokine concentrations were normalized to total protein in the sample and shown as mean concentration ± SEM. *p < 0.05 compared to sham groups by 1-way ANOVA comparing all 8 groups. N = 4 to 10 per group.