Phosphoryl moieties of lipid A from Neisseria meningitidis and N. gonorrhoeae lipooligosaccharides play an important role in activation of both MyD88- and TRIF-dependent TLR4-MD-2 signaling pathways

Abstract

We have previously shown that the lipooligosaccharide (LOS) from Neisseria meningitidis and N. gonorrhoeae engages the TLR4-MD-2 complex. In this study, we report that LOS from different meningococcal and gonococcal strains have different potencies to activate NF-κB through TLR4-MD-2 and that the relative activation can be correlated with ion abundances in MALDI-TOF mass spectrometry that are indicative of the number of phosphoryl substituents on the lipid A (LA) component of the LOS. The LOSs from three of the strains, meningococcal strain 89I and gonococcal strains 1291 and GC56, representing high, intermediate, and low potency on NF-κB activation, respectively, differently activated cytokine expression through the TLR4-MD-2 pathway in monocytes. In addition to induction of typical inflammatory cytokines such as TNF-α, IL-1β, and IL-6, MIP-1α and MIP-1β also were significantly higher in cells treated with 89I LOS, which had the most phosphoryl substitutions on the LA compared with 1291 LOS and GC56 LOS. We found that LOS activated both the MyD88- and TRIF-dependent pathways through NF-κB and IFN regulatory factor 3 transcription factors, respectively. Moreover, LOS induced the expression of costimulatory molecule CD80 on the surfaces of monocytes via upregulation of IFN regulatory factor 1. These results suggest that phosphoryl moieties of LA from N. meningitidis and N. gonorrhoeae LOSs play an important role in activation of both the MyD88- and TRIF-dependent pathways. Our findings are consistent with the concept that bacteria modulate pathogen-associated molecular patterns by expression of phosphoryl moieties on the LA to optimize interactions with the host.

Figure 1

LOS variously activates NF-κB. HEK-293 NF-κB reporter cells (20,000 cells per well) were distributed in 96-well plates and treated with LOS (10 ng/ml) for 18 h as described in the Materials and Methods. SEAP activity was measured at OD 620 nm. The results presented are the mean ± SD from at least 3 independent experiments.

Figure 2

The relative SEAP activity in HEK293 NF-κB reporter cells is positively correlated with increasing numbers of phosphoryl substituents on the LA. Negative-ion MALDI-TOF MS spectra were acquired of intact LOS from eight Neisserial strains. Ion abundance ratios were calculated from the relative intensity of fragment ion peaks at m/z 1793 for LA molecules with 3 phosphoryl substituents plus the relative intensity at m/z 1836 for LA molecules with 2 phosphoryls and a single PEA group, divided by the relative intensity at m/z 1713 for LA molecules with 2 phosphoryl groups lacking additional phosphoryl substitution. The ion abundance ratios were plotted versus the induction of NF-κB in the HEK293 cells, and the positive correlation (r = 0.87) between the two sets of values was statistically significant (p < 0.001). The inset presents the positive correlation between the ion abundance ratios for 3 strains selected as representative of low (GC56), intermediate (1291), and high (89I) induction potential for SEAP activity (r = 0.98; p < 0.001).

Figure 3

Cytokine induction in THP-1 cells by whole bacteria and LOS. Whole bacteria and LOS of strains 89I, 1291, and GC56 were used to infect THP-1 cells for 18 h. LOS was added at a final concentration of 100 ng/ml. Supernatants were harvested for the determination of TNF- α concentration. The results presented are the mean ± SD from at least 3 independent experiments. *Cytokine expression was significantly higher when cells were stimulated with 89I bacteria and LOS compared with that of 1291 and GC56 (p < 0.001).

Figure 4

Dose-response of THP-1 monocytes to LOS. PMA-differentiated THP-1 cells were treated with 89I, 1291, or GC56 LOS at concentrations ranging from 10 pg/ml to 1 μg/ml. Cells were incubated for 18 h and supernatants were analyzed for TNF-α expression. The data points represent the mean ± SD of three independent experiments.

Figure 5

Immunoblot for NF-κB and IRF-3 phosphorylation following LOS treatment. THP-1 cells (2 × 10⁶) were distributed into each well of the 6-well plates and were stimulated with 100 ng/ml of 89I, 1291, or GC56 LOS for 0, 15, 30, 60, 120, and 240 min. After washing with PBS, cells were lysed by the direct addition of SDS sample buffer and applied to a 10% SDS polyacrylamide gel. The phosphorylated and non-phosphorylated proteins were detected by immunoblotting with antibody directed against either phosphor-p65, phosphor-IRF-3, p65 or IRF-3. Panel A, western immunoblots of NF-κB and IRF-3 phosphorylation following treatment by 89I, 1291, and GC56. Panels B and C, the relative intensity of each band in the immunoblots was determined using densitometry and the ratios of the phosphorylated bands to the non-phosphorylated bands were calculated. Data are presented as fold increases of treated compared with untreated samples, NF-κB (B) and IRF-3(C) phosphorylation. Data are representative of at least three independent experiments. * p < 0.05 indicates significance for comparisons between 89I and 1291 or GC56 LOS.

Figure 6

Cytokine profiles of human monocytes after LOS treatment. Human monocytes freshly isolated from buffy coat were aliquoted at 1 × 10⁶ cells per well in 6-well plates and treated with 1 μg/ml of LOS as described in the Materials and Methods. After 18 h stimulation, 50 μl of the supernatants were analyzed using a 30-plex kit for cytokine expression. A, 19 cytokines expressed at concentrations less than 4000 pg/ml. B, 4 cytokines expressed at concentrations greater than 4000 pg/ml. The results presented are the mean ± SEM of 3 independent experiments deriving from three individual donors. C, induction of IFN-β in THP-1 cells assayed by ELISA. The results presented are the mean ± SD of 3 independent experiments. * p < 0.05 for comparisons between 89I and 1291 or GC56 LOS.

Figure 7

Differential induction of the costimulatory molecule CD80 on monocytes by LOS. Monocytes were aliquoted to 1 × 10⁶ cells per well in 6-well plates and treated with LOS (1 μg/ml) with or without anti-TLR4 antibody (10 μg/ml). After 18 h stimulation, the cells were harvested and analyzed for CD80 expression by flow cytometry as described in Material and Methods. A, CD80 expression after 89I, 1291, and GC56 LOS treatment in the absence or presence of pretreatment with anti-TLR4 antibody for 30 min. Filled histogram indicates untreated cells and open histogram indicates cells treated with LOS or anti-TLR4 plus LOS. B, CD80 expression induced by 89I LOS, phosphoryl-PEA (PPEA) hexaacyl LA, and phosphoryl (P) hexaacyl LA. Filled histogram indicates untreated cells and open histogram indicates cells treated with LOS or LA.

Figure 8

IRF-1 and IRF-7 expression in THP-1 cells following LOS treatment. THP-1 cells were plated (1 × 10⁶ cells per well) in a 6-well plate and stimulated with LOS at 100 ng/ml for 8 or 18 h. After washing with PBS, cells were lysed with SDS sample buffer and the cell lysates were applied to a 10% SDS polyacrylamide gel. Immunoblots were performed with antibody against IRF-1, IRF-7, or actin. A, western immunoblot detection of IRF-7 and IRF-1 in response to 89I, 1291, or GC56. B, the relative intensity of each band in the immunoblots was determined using densitometry and the ratios of the IRF-1 and IRF-7 bands to the corresponding actin bands were calculated. Data are presented as fold increases of treated compared with untreated samples. Data are representative of at least three independent experiments. * p < 0.05 indicates significance for comparisons between 89I and 1291 or GC56 LOS.