Objective:

We have documented an impaired neutrophil migration toward the infectious focus in severe sepsis. This phenomenon appears to be mediated by nitric oxide, the release of which is stimulated by circulating inflammatory cytokines released by immune cells after stimulation by bacteria and/or their products. Toll-like receptor 4 (TLR4) is the major recognition receptor for lipopolysaccharide, a component of Gram-negative bacterial cell walls. In the present study, we investigated whether TLR4 is involved in the failure of neutrophil migration in mice subjected to polymicrobial or Gram-negative sepsis.

Design:

Controlled animal study.

Setting:

University research laboratory.

Subjects:

Male C3H/HeJ (TLR4-deficient) and C3H/HePas (TLR4-normal) mice.

Interventions:

Mice were subjected to sublethal or lethal polymicrobial sepsis, both induced by cecal ligation and puncture or intraperitoneal polymicrobial inoculation, and subjected to sublethal Gram-negative sepsis induced by intraperitoneal Salmonella typhimurium inoculation (GNI). Survival was monitored for 5 days. In separate experiments, mice were killed 6 hrs after sepsis induction, and intraperitoneal neutrophil migration, bacteremia, lung neutrophil sequestration, and levels of cytokines, chemokines, and nitrate were evaluated.

Measurements and Results:

TLR4-deficient (C3H/HeJ) mice presented incapacity to promote neutrophil recruitment to the infectious site after sublethal GNI, resulting in high mortality. However, TLR4 signaling is not essential to display neutrophil migration in sublethal polymicrobial sepsis induced by both cecal ligation and puncture and polymicrobial inoculation models, but surprisingly, it is crucial to establish the impairment of neutrophil migration in lethal polymicrobial sepsis, since TLR4-deficient mice that underwent lethal cecal ligation and puncture or polymicrobial inoculation did not present failure of neutrophil migration to infectious focus. As a consequence, these animals presented low bacteremia and a high survival rate and did not display systemic inflammation, determined by high levels of circulating cytokines and lung neutrophil sequestration and chemokine production.

Conclusion:

These results highlight the harmful role of TLR4 signaling in polymicrobial severe sepsis.

During an infection, a complex cascade of events is initiated after the invasion of the host by pathogenic microorganisms (1). In this context, neutrophils play a crucial role in orchestrating the host defense. They are the first cells that migrate to the infectious site and are able to kill microorganisms by producing bactericidal agents, including reactive oxygen and nitrogen species (2). In addition, these cells also release cytokines and chemokines, which enhance the recruitment and activation of both themselves and other immune cells (3, 4). Hence, the neutrophils are extremely important for local control of bacterial growth and, consequently, for the prevention of bacterial dissemination. In fact, our previous studies showed that lethal sepsis induced by cecal ligation and puncture (CLP) or Staphylococcus aureus inoculation is associated with impaired neutrophil migration to sites of infection (5–8). Moreover, intravital microscopy revealed a decreased rolling and adhesion of neutrophils to endothelium from the venules surrounding the infectious site during lethal sepsis (6). The impaired neutrophil migration was associated with high mortality and increased numbers of bacteria in peritoneal exudate and blood. Conversely, in sublethal sepsis, the neutrophil migration was not suppressed and the bacterial infection was restricted to the peritoneal cavity; consequently, no significant mortality was observed.