Differential activation of human and mouse Toll-like receptor 4 by the adjuvant candidate LpxL1 of Neisseria meningitidis

Abstract

Neisseria meningitidis LpxL1 lipopolysaccharide (LPS) bearing penta-acylated lipid A is considered a promising adjuvant candidate for inclusion in future N. meningitidis vaccines, as it elicits a markedly reduced endotoxic response in human macrophages relative to that in wild-type (hexa-acylated) LPS, while it is an equally effective adjuvant in mice. As dendritic cells (DC) and Toll-like receptors (TLR) are regarded as central mediators in the initiation of an immune response, here we evaluated the ability of LpxL1 LPS to mature and to activate human DC and examined its TLR4-/MD-2-activating properties. Unexpectedly, purified LpxL1 LPS displayed minimal human DC-stimulating properties compared to wild-type LPS. Although whole bacteria induced DC maturation and activation irrespective of their type of LPS, the LpxL1 mutant failed to activate the human recombinant TLR4/MD-2 complex expressed in HeLa cells. Similarly, purified LpxL1 LPS was unable to activate human TLR4/MD-2 and it even acted as an antagonist of wild-type LPS. Both wild-type and LpxL1 LPSs activated the murine TLR4/MD-2 complex, consistent with their abilities to induce maturation and activation of murine DC. Assays with cells transfected with different combinations of human and murine TLR4 and MD-2 indicated that TLR4 was a more-major determinant of the LPS response than MD-2. The species-specific activation of the TLR4/MD-2 complex by LpxL1 LPS may have an impact on the use of LpxL1 LPS as an adjuvant and the use of murine immunization models in human meningococcal vaccine development.

FIG. 1.

Maturation and activation of DC stimulated with purified LPS derived from N. meningitidis H44/76 and the LpxL1 mutant. imDC were incubated with the indicated LPS preparations for 20 h and analyzed for expression of maturation markers CD83, CD86, and CD40 by FACS (A) and production of IL-10, IL-12p70, and TNF-α by ELISA (B). Representative results (A) and the mean ± SEM values (B) of three separate experiments with three different donors are shown. unstim, unstimulated; nd, not determined; bd, below detection level.

FIG. 2.

Interaction of the N. meningitidis wild type and the LpxL1 mutant with DC. (A) ImDC were incubated with the PFA-fixed wild type, H44/76, or the LpxL1 mutant at 1:50 and 1:200 ratios for 20 h and analyzed for the production of IL-10 and IL-12p70 by ELISA. unstim; unstimulated; bd, below detection level; *, P < 0.05. (B and C) imDC were incubated with the PFA-fixed, FITC-labeled wild type, H44/76, or the LpxL1 mutant at 1:50 and 1:200 ratios. At the indicated time points, DC were PFA fixed, washed, and analyzed by FACS (B) and by confocal microscopy (C) (2 h; 1:200 ratio is shown). The mean ± SEM values (A and B) and representative results (C) of three separate experiments with three different donors are shown.

FIG. 3.

Differential activation of hTLR4/hMD-2 by H44/76 and LpxL1. HeLa 57A cells transfected with hTLR4, hMD-2, and hCD14 were stimulated with purified H44/76 LPS or LpxL1 LPS (A) or PFA-fixed N. meningitidis H44/76 or the LpxL1 mutant (B) at the indicated concentrations. After 5 h, NF-κB luciferase activities, indicated in RLU, were measured. The values are the means ± SEMs of at least three experiments.

FIG. 4.

Inhibition of LPS-induced activation of TLR4/MD-2 by LpxL1 LPS. HeLa 57A cells transfected with hTLR4, hMD-2, and hCD14 were stimulated with purified H44/76 LPS, LpxL1 LPS, or mixtures of these LPS preparations at ratios of 1:1 and 1:10 (1 = 10 ng/ml). After 5 h of incubation, NF-κB luciferase activities were measured. Values are expressed in RLU and represent the means ± SEMs for the results of at least three experiments.

FIG. 5.

Activation of mTLR4/mMD-2 by H44/76 and the LpxL1 mutant. HeLa 57A cells transfected with mTLR4, mMD-2, and mCD14 were stimulated with purified H44/76 LPS or LpxL1 LPS (A) or PFA-fixed N. meningitidis H44/76 or the LpxL1 mutant (B) at the indicated concentrations. After 5 h, NF-κB luciferase activities were measured. Values are expressed in RLU and represent the means ± SEMs for the results of at least three experiments.

FIG. 6.

Species-specific recognition of N. meningitidis LpxL1 LPS. HeLa 57A cells transfected with hTLR4 (A) or mTLR4 (B) in combination with human or murine MD-2 and CD14 were stimulated with 10 ng/ml of H44/76 LPS or LpxL1 LPS. After 5 h of incubation, NF-κB luciferase activities were measured. Values are expressed in RLU and are the means ± SEMs for the results of at least three experiments.