Stages of meningococcal sepsis simulated in vitro, with emphasis on complement and Toll-like receptor activation

Abstract

The clinical presentation of meningococcal disease is closely related to the number of meningococci in the circulation. This study aimed to examine the activation of the innate immune system after being exposed to increasing and clinically relevant concentrations of meningococci. We incubated representative Neisseria meningitidis serogroup B (ST-32) and serogroup C (ST-11) strains and a lipopolysaccharide (LPS)-deficient mutant (the 44/76 lpxA mutant) in human serum and whole blood and measured complement activation and cytokine secretion and the effect of blocking these systems. HEK293 cells transfected with Toll-like receptors (TLRs) were examined for activation of NF-kappaB. The threshold for cytokine secretion and activation of NF-kappaB was 10(3) to 10(4) meningococci/ml. LPS was the sole inflammation-inducing molecule at concentrations up to 10(5) to 10(6) meningococci/ml. The activation was dependent on TLR4-MD2-CD14. Complement contributed to the inflammatory response at >or=10(5) to 10(6) meningococci/ml, and complement activation increased exponentially at >or=10(7) bacteria/ml. Non-LPS components initiated TLR2-mediated activation at >or=10(7) bacteria/ml. As the bacterial concentration exceeded 10(7)/ml, TLR4 and TLR2 were increasingly activated, independent of CD14. In this model mimicking human disease, the inflammatory response to N. meningitidis was closely associated with the bacterial concentration. Therapeutically, CD14 inhibition alone was most efficient at a low bacterial concentration, whereas addition of a complement inhibitor may be beneficial when the bacterial load increases.

FIG. 1.

Complement activation (mean and scatter plot) by twofold increasing concentrations of N. meningitidis in individual serum samples from 10 healthy adults, measured by the soluble TCC level. TCC was significantly increased compared with the basal level at 3 × 10⁷ bacteria/ml and higher, as calculated by one-way ANOVA with repeated measurements and Bonferroni posttest analysis (P < 0.05). AU, arbitrary units; NmB, strain 44/76.

FIG. 2.

Cytokine secretion (means and standard deviations [SD]) in whole blood from five healthy adults at 10-fold increasing concentrations of wild-type (NmB) and LPS-deficient (NmLPS−) N. meningitidis. P values represent overall differences between the two bacterial strains and were calculated by two-way ANOVA with repeated measurements.

FIG. 3.

Cytokine secretion (means and SD) in whole blood incubated with 10-fold increasing concentrations of wild-type N. meningitidis (NmB). Blood from eight donors was incubated with 10⁵ to 10⁷ bacteria/ml, while blood from five donors was incubated with 10⁴ and 10⁸ bacteria/ml. The effects of inhibiting CD14 (blocking antibody) and complement (C3 level) separately and together were investigated. Statistical significance was calculated at each concentration of meningococci by one-way ANOVA with repeated measurements and Bonferroni posttest analysis. *, P < 0.05; **, P < 0.01; ***, P < 0.001. The significance of the results with combined inhibition is specified only if it is different from that of the results with separate inhibition.

FIG. 4.

Cytokine secretion (means and SD) in whole blood from five healthy adults incubated with 10-fold increasing concentration of LPS-deficient N. meningitidis (NmLPS−). The effects of inhibiting CD14 (blocking antibody) and complement (C3 level) separately and together were investigated. Statistical significance was calculated at each concentration of meningococci by one-way ANOVA with repeated measurements and Bonferroni posttest analysis. *, P < 0.05; **, P < 0.01.

FIG. 5.

Cytokine secretion (means and SD) in whole blood incubated with wild-type (NmB) and LPS-deficient (NmLPS−) N. meningitidis after inhibition of complement by compstatin (C3 level) versus a C5aR antagonist. No significant differences were found between the two inhibitors by a paired t test.

FIG. 6.

Relative upregulation (means and ranges) of NF-κB luciferase activity in HEK293 cells. HEK293 cells expressing TLR4/MD2 alone or in combination with CD14 were incubated with wild-type N. meningitidis (NmB) (A) or purified N. meningitidis LPS (B) at 10-fold increasing concentrations. (C) HEK293 cells expressing TLR2 alone or in combination with CD14 were incubated with the LPS-deficient N. meningitidis strain (NmLPS−). The TLR-negative cells were incubated with 10⁸ bacteria/ml or 1 μg (33,000 EU) LPS/ml. (D) HEK293 cells expressing TLR9 were incubated with 10⁸ bacteria/ml or 1 μg (33,000 EU) LPS/ml.