Innate immunity and inflammation in sepsis: mechanisms by which acute ethanol exposure alters the course of sepsis and the effect to TLR4 signaling

Introduction

Alcohol consumption is a significant risk factor for mortality in patients with sepsis. Alcohol is the most widely abused substance worldwide and numerous studies have revealed that it has widespread effects on the immune system and leaves abusers at increased risk of a variety of infections. An increased predisposition to infection among patients with alcohol use problems may also mediate an association with sepsis.

Objective

The present study was carried out to investigate the mechanisms by which acute ethanol exposure alters the course of sepsis and the effect of TLR4 signaling.

Methods

Two different strains of mice, C3H/HeJ (TLR4-mutants) and C3H/HeOuJ (wildtype), were treated with a dosage of 6 g/kg ethanol, which yields a blood-ethanol concentration of ~0.4%, similar to the blood-ethanol levels that occur in ethanol-dependent humans. Viable, indigenous Escherichia coli, log-phase, grown in LB broth was administered intraperitoneally. The dosage of E. coli was 2 × 10⁸ per mouse, which serves as a model for loss of intestinal integrity and release of bacteria in large numbers. Blood samples were obtained retro-orbitally while the animal was under halothane anesthesia. After euthanasia, peritoneal lavage was performed and samples of this fluid were used to quantify bacteria by making serial dilutions in LB agar, and for cell-counting, for cytospin and cytokine and chemokine study. Spleen was also harvested from all the mice for carrying out bacterial quantification, RNA analysis, and flow-cytometry analysis.

Results

Ethanol administration decreases resistance to E. coliand causes a decrease in the ability to clear bacteria both from the peritoneal cavity as well as the spleen. At early time points, ethanol also suppresses the production of proinflammatory cytokines (for example, IL-1, IL-17, IFNγ, TNFα, and so forth) and chemokines (for example, Eotaxin, RANTES, MIP-1, MIG, LIX, and so forth). Most (80 to 90%) of the cells in the peritoneal cavity were found to be macrophages (full of bacteria) and hardly any neutrophils could be found. See Figure 1.

Figure 1

Survival of TLR4 mutant (HeJ) versus wildtype mice.

Conclusions

Ethanol decreases clearance of bacteria in the peritoneal cavity and increases mortality. Ethanol also decreases production of most proinflammatory cytokines and chemokines. A large number of macrophages in the peritoneal fluid indicates decreased attraction of neutrophils to the peritoneal cavity, decreased clearance of bacteria by macrophages and neutrophils in the peritoneal cavity, and, hence, increased mortality. TLR4 is dispensable for survival in E. coli sepsis but it also contributes to lethality in wildtype mice. Although TLRs have been implicated as an important element of host defense against infections, evidence indicates that these receptors may also play a crucial role in the pathophysiology of sepsis.