Improved innate immunity of endotoxin-tolerant mice increases resistance to Salmonella enterica serovar typhimurium infection despite attenuated cytokine response

Abstract

During infection with gram-negative bacteria, exposure of immune cells to lipopolysaccharide (LPS) from the bacterial cell membrane induces a rapid cytokine response which is essential for the activation of host defenses against the invading pathogens. Administration of LPS to mice induces a state of hyporesponsiveness, or tolerance, characterized by reduced cytokine production upon subsequent LPS challenge. In the model of experimental Salmonella enterica serovar Typhimurium infection of mice, we assessed the question of whether complete LPS tolerance induced by repetitive doses of LPS interfered with cytokine production and host defense against gram-negative bacteria. Although production of various cytokines in response to serovar Typhimurium was attenuated by LPS pretreatment, LPS-tolerant mice showed improved antibacterial activity, evidenced by a prolongation of survival and a continuously lower bacterial load. We attribute this protective effect to three independent mechanisms. (i) Peritoneal accumulation of leukocytes in the course of LPS pretreatment accounted for enhanced defense against serovar Typhimurium during the first 6 h of infection but not for decreased bacterial load in late-stage infection. (ii) LPS-tolerant mice had an increased capacity to recruit neutrophilic granulocytes during infection. (iii) LPS-tolerant mice showed threefold-increased Kupffer cell numbers, enhanced phagocytic activity of the liver, and strongly improved clearance of blood-borne serovar Typhimurium. These results demonstrate that despite attenuated cytokine response, acquired LPS tolerance is associated with enhanced resistance to infections by gram-negative bacteria and that this effect is mainly mediated by improved effector functions of the innate immune system.

FIG. 1

LPS-tolerant mice show reduced peak levels of TNF-α, IFN-γ, and IL-6 in plasma, liver, and spleen after serovar Typhimurium infection. BALB/c mice were rendered tolerant by daily i.p. or i.v. injections of 1 mg of serovar Abortus equi LPS/kg for 3 days. Twenty-four hours after the last LPS injection, control (n = 9) and LPS-tolerant (n = 6) mice were infected i.p. with serovar Typhimurium (10⁷ bacteria/kg) and killed 3 h after infection for determination of cytokines. Data are expressed as means ± SEM. Dunnett's test was performed after one-way ANOVA with P of <0.05 (∗) and <0.01 (∗∗) versus control. Plasma IFN-γ was below the detection limit (nd).

FIG. 2

Time course of bacterial load in peritoneal lavage fluid and blood of control and LPS-tolerant mice during serovar Typhimurium infection (10⁷ bacteria/kg i.p.). Data are shown as means ± SEM (n = 3). For statistical analysis, the unpaired two-sided Student's t test was performed for each time point with log-transformed data. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001 (all versus control).

FIG. 3

Time course of peritoneal cell numbers in control and LPS-tolerant mice infected with serovar Typhimurium (10⁷ bacteria/kg i.p.). Total peritoneal cell numbers are expressed as means ± SEM (n = 3). For statistical analysis, log-transformed data were tested by the unpaired two-sided Student's t test. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001 (all versus control).

FIG. 4

Effect of different LPS administration routes for tolerance induction on time course of bacterial load. Control and LPS-tolerant mice were infected i.p. with serovar Typhimurium (10⁷ bacteria/kg). The data are means ± SEM (n = 9 for controls and n = 6 for the LPS groups). For statistical analysis, an unpaired two-tailed Student's t test was done after one-way ANOVA of log-transformed data to compare the three groups at each time point individually. ∗, P < 0.05 for LPS i.p. versus control; ∗∗, P < 0.01 for LPS i.p. versus control; ∗∗∗, P < 0.001 for LPS i.p. versus control; †, P < 0.05 for LPS i.v. versus control; ††, P < 0.01 for LPS i.v. versus control; †††, P < 0.001 for LPS i.v. versus control; ‡, P < 0.05 for LPS i.v. versus LPS i.p.; ††, P < 0.01 for LPS i.v. versus LPS i.p.; ‡‡‡, P < 0.001 for LPS i.v. versus LPS i.p.

FIG. 5

Effect of LPS tolerance on blood clearance of bacteria and phagocytic activity in liver and spleen. Control and LPS-tolerant mice were infected i.v. with serovar Typhimurium (10⁸ bacteria/kg). The data are calculated as percent recovery of the inoculum and expressed as means ± SEM (n = 6). For statistical analysis, the unpaired Student's t test with log-transformed data was performed. ∗, P < 0.05 versus control; ∗∗, P < 0.01 versus control; ∗∗∗, P < 0.001 versus control).

FIG. 6

Clearance of serovar Typhimurium in control (co) and LPS-tolerant (LPS) mice after macrophage depletion with Cl₂MBP liposomes. LPS tolerance was induced by daily i.p. administration of 1 mg of serovar Abortus equi LPS/kg for 3 days. Twenty-four, 48, and 71 h after the last LPS injection, liposomes (+) or pyrogen-free saline (−) was injected i.v. One hour after the last injection of liposomes, mice were infected i.v. with serovar Typhimurium (10⁸ bacteria/kg). Ten minutes after injection of serovar Typhimurium, viable bacteria were determined in blood and liver homogenates and calculated as percent recovery of the inoculum. Pooled data from three experiments are expressed as means + SEM, with 7 to 14 mice per group. For statistical analysis, the Bonferroni test for selected groups was done after one-way ANOVA. ∗, P < 0.05; ∗∗∗, P < 0.001 versus saline control (co −); †††, P < 0.001 versus LPS control (LPS −).