Neisseria meningitidis capsular polysaccharides induce inflammatory responses via TLR2 and TLR4-MD-2 - PubMed (original) (raw)

Neisseria meningitidis capsular polysaccharides induce inflammatory responses via TLR2 and TLR4-MD-2

Susu M Zughaier. J Leukoc Biol. 2011 Mar.

Abstract

CPS are major virulence factors in infections caused by Neisseria meningitidis and form the basis for meningococcal serogroup designation and protective meningococcal vaccines. CPS polymers are anchored in the meningococcal outer membrane through a 1,2-diacylglycerol moiety, but the innate immunostimulatory activity of CPS is largely unexplored. Well-established human and murine macrophage cell lines and HEK/TLR stably transfected cells were stimulated with CPS, purified from an endotoxin-deficient meningococcal serogroup B NMB-lpxA mutant. CPS induced inflammatory responses via TLR2- and TLR4-MD-2. Meningococcal CPS induced a dose-dependent release of cytokines (TNF-α, IL-6, IL-8, and CXCL10) and NO from human and murine macrophages, respectively. CPS induced IL-8 release from HEK cells stably transfected with TLR2/6, TLR2, TLR2/CD14, and TLR4/MD-2/CD14 but not HEK cells alone. mAb to TLR2 but not an isotype control antibody blocked CPS-induced IL-8 release from HEK-TLR2/6-transfected cells. A significant reduction in TNF-α and IL-8 release was seen when THP-1- and HEK-TLR4/MD-2-CD14- but not HEK-TLR2- or HEK-TLR2/6-transfected cells were stimulated with CPS in the presence of Eritoran (E5564), a lipid A antagonist that binds to MD-2, and a similar reduction in NO and TNF-α release was also seen in RAW 264.7 cells in the presence of Eritoran. CD14 and LBP enhanced CPS bioactivity, and NF-κB was, as anticipated, the major signaling pathway. Thus, these data suggest that innate immune recognition of meningococcal CPS by macrophages can occur via TLR2- and TLR4-MD-2 pathways.

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Figures

Figure 1.

Figure 1.. Meningococcal serogroup B CPS polymers purified from a LOS-deficient (lpxA) mutant (CPS-lpxA) induced inflammatory cytokine (TNF-α and IL-6) and chemokine (IP-10 and IL-8) release from human macrophages.

THP-1 cells (250×103/well) in 96-well plates were induced with serogroup B CPS-lpxA polymers (50–1.56 μg/ml) overnight. Cytokine and chemokine release was quantified by ELISA. (A) TNF-α; (B) IL-6; (C) IP-10 (CXCL10); and (D) IL-8 release. Unstimulated macrophages incubated simultaneously were used as the control. Error bars represent

sd

from the mean of quadruplicate readouts. The results are representative of four independent experiments.

Figure 2.

Figure 2.. Meningococcal CPS from strains causing invasive disease induce IL-8 release via TLR2 and TLR4-MD-2.

Stably transfected HEK293 cells seeded in a 96-well plate at 250 × 103/well with (A) TLR2/6, (B) TLR2-CD14, or (C) TLR4-MD-2-CD14 were induced with a 5-μg dose of meningococcal CPS polymers from serogroups A (NMA), B (NMB), C (NMC), Y (NMY), and W135. MAPS is the vaccine-grade meningococcal serotype A CPS polymer used. Meningococcal LOS (NMB LOS) was used at 1 ng/ml (∼0.56 pmole) as a control. Unstimulated macrophages incubated simultaneously were also used as a control. IL-8 release was measured by ELISA. Error bars represent

sd

from the mean of quadruplicate readouts. The results are representative of three independent experiments.

Figure 3.

Figure 3.. Meningococcal CPS polymers activate TLR2- and TLR4-MD-2-mediated IL-8 responses.

Dose-dependent IL-8 release from stably transfected HEK293 cells induced with meningococcal serogroup B CPS-lpxA polymers overnight. (A) HEK/TLR4-MD-2-CD14 stably transfected cells were induced with serogroup B CPS-lpxA or a membrane phospholipid extract designated PL-lpxA from the same strain. NMB LOS and Pam3CSK4 were used as controls. (B) HEK/TLR2/6 stably transfected cells were stimulated with CPS-lpxA polymers. Rhizobium LPS and Pam3CSK4 were used as controls. IL-8 release was measured by the ELISA method. Error bars represent

sd

from the mean of quadruplicate readouts. The results are representative of three independent determinations.

Figure 4.

Figure 4.. Blocking of TLR2 and TLR4 with antibodies reduced meningococcal CPS polymer-induced cellular activation.

(A) TNF-α release from THP-1 cells blocked with TLR2 and TLR4 antibodies for 30 min prior to the addition of serogroup B CPS-lpxA (50 μg/ml), the phospholipid (sham control) extraction PL-lpxA (50 μg/ml), NMB LOS (1 ng/ml), or Pam3CSK4 (5 μg/ml) ± E5564 (1 μg/ml), the synthetic TLR4-MD-2 inhibitor, and incubated overnight. (B) IL-8 release from HEK-TLR2/6 stably transfected cells stimulated with CPS in the presence or absence of anti-TLR2 blocking antibody. TNF-α and IL-8 release was measured by ELISA. The results are representative of two independent experiments.

Figure 5.

Figure 5.. MD-2 is required for meningococcal CPS recognition via TLR4.

(A) HEK/TLR4-MD-2-CD14 stably transfected cells were induced with serogroup B CPS-lpxA, with or without 1 μg/ml E5564, a synthetic lipid A antagonist that binds MD-2, in 10% FBS. NMB LOS was used as the control. (B) HEK/TLR4-MD-2-CD14 stably transfected cells were induced with serogroup B CPS-lpxA, with or without 0.5 μg/ml E5564 in serum-free conditions. (C) HEK/TLR4 stably transfected cells alone induced with serogroup B CPS-lpxA, with or without exogenous sMD and sCD14 or 1 μg/ml E5564. (D) HEK/TLR2/6 stably transfected cells induced with CPS-lpxA in serum-free and 10% FBS conditions in the presence or absence of 1 μg/ml E5564. The results are representative of two independent experiments.

Figure 6.

Figure 6.. CD14 and LBP enhanced meningococcal CPS bioactivity.

Dose-dependent IL-8 release from HEK/TLR2–6 stably transfected cells induced with serogroup B CPS-lpxA in serum-free (SF) or 10% FBS conditions, with or without exogenous rCD14 or rLBP added at 20 ng/ml. The results are representative of two independent experiments.

Figure 7.

Figure 7.. Inflammatory signaling transcription factors are induced by meningococcal CPS-lpxA polymers.

(A) Inducible transcription factors dual luciferase reporters (see Materials and Methods) were transfected into HEK/TLR4-MD-2-CD14 stably transfected cells and then stimulated with serogroup B meningococcal CPS-lpxA polymers (100 and 10 μg doses) for 5 h. Inducible transcription factor firefly luciferase activity was normalized to the constitutively expressed Renilla luciferase reporter. (B) Inducible transcription factor dual luciferase reporters transfected into HEK/TLR2/6 stably transfected cells and then stimulated with serogroup B meningococcal CPS-lpxA (100 μg doses) for 5 h. Inducible transcription factor firefly luciferase activity was normalized to constitutively expressing the Renilla luciferase reporter. Negative control is transfected with the noninducible firefly luciferase reporter. Positive control is a mixture of constitutively expressing GFP and the firefly luciferase construct. This experiment is representative of two independent determinations. RLU, Relative light unit.

Figure 8.

Figure 8.. TIRAP and TRAM adaptor proteins are required for meningococcal CPS-induced IL-8 release.

HEK/TLR2/6 stably transfected cells were transiently transfected with 0.5 μg DN-TIRAP or DN-TRAM constructs. An empty vector was used as a control in the presence of CPS-lpxA, and tranfection buffer designated CPS-superfect only. Unstimulated cells HEK/TLR2/6-ve (negative) were used as a control for DN-construct but without CPS_-lpxA_.

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