Activation of toll-like receptor 2 (TLR2) and TLR4/MD2 by Neisseria is independent of capsule and lipooligosaccharide (LOS) sialylation but varies widely among LOS from different strains

Abstract

Lipooligosaccharide (LOS) structure and capsular polysaccharide of Neisseria meningitidis each greatly influence the virulence of the organism and the quality of host innate immune responses. In this study, we found that production of the proinflammatory cytokine tumor necrosis factor (TNF) by a human monocyte-derived cell line (THP-1) exposed to strains of N. meningitidis lacking capsule and/or with truncated LOS was similar to that elicited by the isogenic wild-type strain. These mutants also exhibited no difference in induction of the interleukin-8 (IL-8) promoter in a transfected HeLa cell system of Toll-like receptor 2 (TLR2) and TLR4/MD2 signaling. However, purified LOS from diverse strains of Neisseria (both N. meningitidis and N. gonorrhoeae) caused widely variant levels of IL-8 promoter induction in cells expressing MD2 that correlated with the production of TNF from THP-1 cells. These data suggest that although modification of the oligosaccharide chain of LOS and/or absence of capsule do not affect cell signaling mediated by TLR4/MD2, fine-structural differences in the LOS do influence signaling through TLR4/MD2 and, through this pathway, influence some of the proinflammatory responses elicited by Neisseria.

FIG. 1.

Proinflammatory response of THP-1 cells to N. meningitidis. The magnitude of TNF release in response to live wild-type (wt) N. meningitidis and mutants with truncated LOS (galE), without capsule (siaD), or without capsule together with truncated LOS (cps) was measured by ELISA. Each bar represents the median and interquartile range of three separate experiments. P > 0.05 (Kruskal-Wallis test).

FIG. 2.

Response of MD2-transfected HeLa cells to paraformaldehyde-fixed N. meningitidis as measured by induction of the IL-8 promoter. Cells were challenged with N. meningitidis mutants with truncated LOS (galE) (A), without capsule (siaD) (B), or without capsule together with truncated LOS (cps) (C), and the results were compared with the response of the wild-type (wt) strain. The median and interquartile range of IL-8 promoter induction of transfected HeLa cells are shown (n = 6). P > 0.05 (Kruskal-Wallis test).

FIG. 3.

Response of TLR2-transfected HeLa cells to fixed N. meningitidis as measured by induction of the IL-8 promoter. Cells were challenged with N. meningitidis mutants with truncated LOS (galE) (A), without capsule (siaD) (B), or without capsule together with truncated LOS (cps) (C), and the results were compared with the response of the wild-type (wt) strain. The median and interquartile range of IL-8 promoter induction of transfected HeLa cells are shown (n = 6). P > 0.05 (Kruskal-Wallis test).

FIG. 4.

Proinflammatory cytokine (TNF) production by THP-1 cells in response to LOS from Neisseria. Cells were challenged with 200 ng of LOS isolated from N. meningitidis, N. gonorrhoeae, E. coli, or S. enterica serovar Minnesota Re595 for 6 h, after which TNF concentrations in the supernatants were determined by ELISA. The neisserial LOS are ranked in order of decreasing oligosaccharide chain length (where known). Each bar represents the mean of quadruplicate data points ± the standard deviation.

FIG. 5.

Response of MD2-transfected HeLa cells to LOS from Neisseria as measured by induction of the IL-8 promoter. Cells were challenged at 10 ng/ml with LOS isolated from N. meningitidis, N. gonorrhoeae, E. coli, or S. enterica serovar Minnesota Re595, and the response of MD2-transfected cells was compared with the response of empty-vector-transfected cells. The neisserial LOS are ranked in order of decreasing oligosaccharide chain length (where known). The data represent the median and interquartile range of transfected HeLa cells (n = 3). Significant variation between strains was found (P < 0.01 [Kruskal-Wallis test]).

FIG. 6.

Correlation of signaling through TLR4/MD2 and production of TNF from THP-1 cells in response to LOS from different strains of Neisseria. The data used for this analysis derive from the TNF concentrations in Fig. 4 and the relative luciferase activities in Fig. 5. Spearman's rank correlation coefficient ρ = 0.867 (P < 0.01).

FIG. 7.

Dose-related response of MD2-transfected HeLa cells to LOS from two strains of N. gonorrhoeae. IL-8 promoter induction in transfected HeLa cells stimulated by a range of concentrations of the two LOS is shown. The data points represent the median and interquartile range of three pools of transfected HeLa cells.