Dendritic cell activation and cytokine production induced by group B Neisseria meningitidis: interleukin-12 production depends on lipopolysaccharide expression in intact bacteria

Abstract

Interactions between dendritic cells (DCs) and microbial pathogens are fundamental to the generation of innate and adaptive immune responses. Upon stimulation with bacteria or bacterial components such as lipopolysaccharide (LPS), immature DCs undergo a maturation process that involves expression of costimulatory molecules, HLA molecules, and cytokines and chemokines, thus providing critical signals for lymphocyte development and differentiation. In this study, we investigated the response of in vitro-generated human DCs to a serogroup B strain of Neisseria meningitidis compared to an isogenic mutant lpxA strain totally deficient in LPS and purified LPS from the same strain. We show that the parent strain, lpxA mutant, and meningococcal LPS all induce DC maturation as measured by increased surface expression of costimulatory molecules and HLA class I and II molecules. Both the parent and lpxA strains induced production of tumor necrosis factor alpha (TNF-alpha), interleukin-1alpha (IL-1alpha), and IL-6 in DCs, although the parent was the more potent stimulus. In contrast, high-level IL-12 production was only seen with the parent strain. Compared to intact bacteria, purified LPS was a very poor inducer of IL-1alpha, IL-6, and TNF-alpha production and induced no detectable IL-12. Addition of exogenous LPS to the lpxA strain only partially restored cytokine production and did not restore IL-12 production. These data show that non-LPS components of N. meningitidis induce DC maturation, but that LPS in the context of the intact bacterium is required for high-level cytokine production, especially that of IL-12. These findings may be useful in assessing components of N. meningitidis as potential vaccine candidates.

FIG. 1

Representative flow cytometric profiles of surface phenotypic markers on day 8 DCs stimulated with medium, LPS, or the N. meningitidis H44/76 parent or lpxA strain. Day 7 DC cultures were incubated for 24 h with medium, 100 ng of meningococcal LPS, or 10⁷CFU of parent or lpxA organisms per ml. Open histograms show staining of appropriate isotype-matched controls. Solid histograms show staining of the antibody raised against the indicated surface marker. The data are representative of five separate experiments.

FIG. 2

Dose-dependent cytokine production in DCs in response to the parent H44/76 strain, the lpxA strain, and meningococcal LPS. DCs were stimulated with either medium, 100 ng of LPS, or 10⁵ to 10⁷ CFU of H44/76 parent or lpxA strain per ml in the presence of 10 μg of brefeldin A per ml. Intracellular cytokine production was assessed after 24 h. Open histograms represent staining in response to the parent strain. Solid histograms show responses to the lpxA mutant or LPS as indicated. Dashed lines show staining in unstimulated DCs. The data are representative of six experiments yielding comparable results.

FIG. 3

Addition of exogenous LPS only partially restores IL-1α, TNF-α, and IL-6 induction in DCs in response to the lpxA mutant. DCs were stimulated with 100 ng of LPS per ml, 10⁷CFU of parent or lpxA bacteria per ml, or 10⁷ CFU of lpxA strain plus 100 per ml ng of LPS per ml in the presence of 10 μg of brefeldin A per ml. Intracellular IL-6, TNF-α, and IL-1α production was assessed after 24 h. Results are presented as mean MFI ± standard error from three separate experiments.

FIG. 4

Addition of exogenous LPS does not reconstitute the ability of the lpxA strain to induce IL-12 production. DCs were stimulated with 100 ng of LPS per ml, 10⁷ CFU of parent or lpxA strain per ml, or 10⁷ CFU of the lpxA strain plus 100 ng of LPS per ml in the presence of 10 μg of brefeldin A per ml. Intracellular IL-12 production was assessed after culture for 24 h. The data are representative of three independent experiments.