Induction of endotoxin tolerance enhances bacterial clearance and survival in murine polymicrobial sepsis

Abstract

The fundamental mechanisms that underlie endotoxin tolerance remain to be elucidated, and the clinical significance of endotoxin tolerance in the context of active systemic infection remains in question. We hypothesized that the endotoxin tolerance phenotype would result in decreased inflammation at the expense of altered bacterial clearance and, thus, higher mortality in a murine model of polymicrobial sepsis induced by cecal ligation and puncture (CLP). Endotoxin tolerance was induced in C57Bl/6 mice with 5 mg/kg LPS or vehicle 18 h before subsequent CLP. Lung tissue, peritoneal fluid, and blood were collected at 1, 3, 6, and 18 h after surgery for subsequent analysis. Peritoneal macrophages were isolated for ex vivo phagocytosis assay. In separate experiments, mice were allowed to recover, and survival was monitored for 7 days. Endotoxin tolerance attenuated plasma TNF-alpha and IL-6 at 6 h after CLP. Peritoneal fluid cytokines were significantly attenuated as well. Endotoxin tolerance significantly improved bacterial clearance in both blood and peritoneal fluid after CLP. Similarly, ex vivo phagocytosis by primary peritoneal macrophages and RAW264.7 murine peritoneal macrophages was significantly improved after induction of the endotoxin tolerance phenotype. Contrary to our original hypothesis, we conclude that endotoxin tolerance significantly attenuates the host inflammatory response, augments bacterial clearance, and improves survival in this murine model of polymicrobial sepsis.

Fig. 1. LPS preconditioning attenuates the host systemic inflammatory response after CLP

C57Bl/6 mice (n = 3 at each time point per group) were preconditioned with either 5 mg/kg LPS or vehicle (0.9% saline) × 18 h before subsequent CLP or sham surgery. Plasma cytokines (A, TNF-α; B, IL-6; C, IL-10) were measured at 1, 3, 6, and 18 h after CLP. *P < 0.05 saline/CLP vs. saline/sham and LPS/CLP; ^†P < 0.05 saline/CLP vs. saline/sham only; ^‡P < 0.05 LPS/CLP vs. LPS/sham.

Fig. 2. LPS preconditioning attenuates the host systemic inflammatory response after CLP

C57Bl/6 mice (n = 3 at each time point per group) were preconditioned with either 5 mg/kg LPS or vehicle (0.9% saline) × 18 h before subsequent CLP or sham surgery. Peritoneal fluid cytokines (A, TNF-α; B, IL-6; C, IL-10) were measured at 1, 3, 6, and 18 h after CLP. *P < 0.05 saline/CLP vs. saline/sham and LPS/CLP; ^†P < 0.05 saline/CLP vs. saline/sham only; ^‡P < 0.05 LPS/CLP vs. LPS/sham.

Fig. 3. LPS preconditioning inhibits IKK kinase activity in the lung after CLP

C57Bl/6 mice (n = 3 per group) were preconditioned with either 5 mg/kg LPS or saline × 18 h before subsequent CLP or sham surgery. Lung tissue was harvested at 18 h after CLP, and IKK kinase activity was determined via kinase assay. A representative assay is shown (A) with image quantitative analysis of all mice (B). *P < 0.05 saline/CLP vs. saline/sham; ^†P < 0.05 saline/CLP vs. LPS/CLP.

Fig. 4. LPS preconditioning is associated with lower bacterial counts in the peripheral blood (A) and peritoneal fluid (B)

C57Bl/6 mice (n = 5 per group) were preconditioned with either 5 mg/kg LPS or vehicle (0.9% saline) × 18 h before subsequent CLP or sham surgery. Peripheral blood and peritoneal fluid samples were obtained 24 h after CLP and cultured in nutrient agar at 37°C for 24 h. *P < 0.05 saline/CLP vs. saline/sham; ^†P < 0.05 saline/CLP vs. LPS/CLP.

Fig. 5. LPS preconditioning does not significantly decrease peripheral blood or peritoneal WBC counts after CLP

C57Bl/6 mice (n = 8 per group) were preconditioned with either 5 mg/kg LPS or vehicle (0.9% saline) × 18 h before subsequent CLP or sham surgery. Peripheral blood and peritoneal fluid samples were obtained 24 h after CLP, and WBC counts (A) and PMN (B) were measured using Coulter counter.

Fig. 6. LPS preconditioning significantly increases bacterial clearance by augmenting phagocytosis

Ex vivo phagocytosis was performed using primary peritoneal macrophages cultured from mice at 18 h after preconditioning with either LPS (5 mg/kg) or vehicle (0.9% saline). Peritoneal macrophages were then treated with fluorescein-tagged S. aureus and E. coli. *P < 0.05 compared with vehicle.

Fig. 7. LPS preconditioning significantly increases bacterial clearance by augmenting phagocytosis

RAW264.7 mouse peritoneal macrophages were preconditioned with either LPS (10 ng/mL) or vehicle (0.9% saline) for 18 h before subsequent treatment with either fluorescein-tagged S. aureus or E. coli. *P < 0.05 compared with vehicle.

Fig. 8. Kaplan-Meier survival curves after CLP

C57Bl/6 mice (n = 30 per group) were preconditioned with either LPS (5 mg/kg, i.p.) or vehicle 18 h before CLP or sham surgery. Survival was monitored for 7 days. LPS preconditioning significantly improved survival after CLP (P < 0.01).