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Keywords:

  • Neisseria meningitidis ;
  • heat-killed;
  • cytokines;
  • toll-like receptor 2;
  • toll-like receptor 4

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

Neisseria meningitidis may cause severe invasive disease. The carriage state of the pathogen is common, and the reasons underlying why the infection becomes invasive are not fully understood. The aim of this study was to compare the differences between invasive and carrier strains in the activation of innate immunity. The monocyte expression of TLR2, TLR4, CD14, and HLA-DR, cytokine production, and the granulocyte oxidative burst were analyzed after in vitro stimulation by heat-killed invasive (n = 14) and carrier (n = 9) strains of N. meningitidis. The expression of the cell surface markers in monocytes, the oxidative burst, and cytokine concentrations were measured using flow cytometry. Carrier strains stimulated a higher production of inflammatory cytokines and oxidative burst in granulocytes than invasive strains (all p < 0.001), whereas invasive strains significantly up-regulated TLR2, TLR4 (p < 0.001), and CD14 (p < 0.01) expression on monocytes. Conversely, the monocyte expression of HLA-DR was higher after the stimulation by carrier strains (p < 0.05) in comparison to invasive strains. The LPS inhibitor polymyxin B abolished the differences between the strains. Our findings indicate different immunostimulatory potencies of invasive strains of N. meningitidis compared with carrier strains.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

Neisseria meningitidis is a Gram-negative bacterium that is adapted for the human upper respiratory tract. Asymptomatic carriage occurs in 10–40% of healthy humans [1]. The colonization of the respiratory tract is transient and may lead to the acquisition of immunity to meningococci in adult populations. However, pathogenic strains of N. meningitidis can penetrate through the epithelial barrier to the bloodstream, leading to a severe invasive disease. Sepsis caused by N. meningitidis is associated with a high fatality rate. The mortality rate of invasive meningococcal disease (IMD) ranges from 3% for meningococcal meningitis to 55% in patients with fulminant meningococcemia [2]. Moreover, IMD survivors often suffer from serious consequences.

During IMD, the production of both pro- and anti-inflammatory cytokines increases, which may lead to the generalized dysregulation of inflammatory pathways. The severity of IMD is correlated with the release of pro-inflammatory cytokines due to the stimulatory effects of lipooligosaccharide (LOS) and meningococcal DNA levels in the plasma [3-6]. Cytokine production is also elicited by non-lipopolysaccharide (LPS) components of N. meningitidis [7].

The host defense response is determined through the recognition of N. meningitidis by immune cell receptors, leading to the production of cytokines, effective phagocytosis, and the activation of intracellular killing mechanisms. N. meningitidis is recognized by different Toll-like receptors (TLRs) on circulating monocytes. TLR4 recognizes LOS directly or in a complex with CD14, depending on concentration of LPS [7, 8]. TLR2 is mainly activated by bacterial non-LPS antigens, such as peptidoglycans, bacterial lipoproteins, and lipoteichoic acids [9]; however, some studies have demonstrated that TLR2 expression level may be affected after stimulation by LPS [10, 11].

Several studies have compared invasive and carrier strains according to genetic and phenotypic features [12-16]. However, there is only a limited number of studies, which compared invasive and carrier strains in the impact of cytokine production or oxidative burst [17-19]. In this study, we compared the stimulation of monocytes and granulocytes by heat-killed (HK) invasive and carrier N. meningitidis strains. The immune response was characterized by the production of inflammatory cytokines; changes in TLR2, TLR4, CD14, and HLA-DR surface expression in monocytes and the intensity of the oxidative burst in granulocytes.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

Bacterial strains

The 23 strains of N. meningitidis used in this study are described in Table 1. A total of 14 strains were obtained from patients with IMD: serogroup B: 285/05 B:1:NST, 171/05 B:15:P1.4, 126/05 B:4:P1.15, 113/04 B:22:P1.14, 109/05 B:NT:NST, 75/03 B:NT:P1.5, 24/05 B:4:P1.7, and 1/03 B:15:P1.4; and serogroup C: 185/02 C:2a:P1.2, 184/02 C:2a:P1.2,P1.5, 137/05 C:NT:P1.2,P1.5, 37/03 C:2a:P1.2, 30/05 C:21:NST, and 11/03 C:2a:P1.2,P1.5. Nine noninvasive strains were obtained from asymptomatic carriers: serogroup B: 255/01 B:NT:P1.5, 233/93 B:15:P1.4, 222/04 B:4:P1.15, and 14/03 B:4:P1.7; and serogroup C: 272/93 C:2a:P1.2, 145/99 C:2a:P1.2,P1.5, 133/96 C:21:P1.7, 23/01 C:2a:P1.2,P1.5, and 22/93 C:2a:P1.2 (from the strain collection of the National Reference Laboratory for Meningococcal Infections, National Institute of Public Health, Prague, Czech Republic).

Table 1. List of N. meningitidis strains used in this study
No. of strainStatusSpecimenSerogroupsPhenotypeHypervirul.STClonal complex (cc)
  1. Status: IMD = invasive meningococcal disease; CAR = carrier; Specimen: TH = throat swab; HE = hemoculture; CSF = cerebrospinal fluid; Phenotype: serogroup: serotype: subtype; NT, NST = serotype unassigned; subtype unassigned; Hypervirulent lineages: Y = yes; N = no; ST = sequence type; Clonal complex: UA = clonal complex unassigned.

0285/05IMDCSFBB:1:NSTY112cc41/44
0185/02IMDHECC:2a:P1.2Y11cc11
0184/02IMDHECC:2a:P1.2,P1.5Y11cc11
0171/05IMDHEBB:15:P1.4N5126UA
0137/05IMDCSFCC:NT:P1.2,P1.5Y11cc11
0126/05IMDCFBB:4:P1.15Y33cc32
0113/04IMDHEBB:22:P1.14Y1001cc18
0109/05IMDHEBB:NT:NSTN919UA
0075/03IMDHEBB:NT:P1.5Y269cc269
0037/03IMDCSFCC:2a:P1.2Y11cc11
0030/05IMDCSFCC:21:NSTY3346cc41/44
0024/05IMDHEBB:4:P1.7Y112cc41/44
0011/03IMDHECC:2a:P1.2,P1.5Y11cc11
0001/03IMDCSFBB:15:P1.4Y1403cc41/44
0272/93CARTHCC:2a:P1.2Y11cc11
0255/01CARTHBB:NT:P1.5N6165UA
0233/93CARTHBB:15:P1.4N107UA
0222/04CARTHBB:4:P1.15Y110cc41/44
0145/99CARTHCC:2a:P1.2,P1.5Y2994cc11
0133/96CARTHCC:21:P1.7N261cc53
0023/01CARTHCC:2a:P1.2,P1.5Y11cc11
0022/93CARTHCC:2a:P1.2Y11cc11
0014/03CARTHBB:4:P1.7N669UA

N. meningitidis strains were cultivated overnight on Mueller–Hinton chocolate agar plates at 37 °C in a 5% CO2 atmosphere and heat-inactivated for 40 min at 70 °C. The N. meningitidis strains were characterized using molecular methods (http://neisseria.org/nm/typing). The concentration of N. meningitidis in the media was determined by measuring the absorbance at 530 nm using a UV–Visible spectrophotometer (Ultrospec 2000; Pharmacia Biotech, Cambridge, UK) [20].

Purified LPS

Lipopolysaccharides from Escherichia coli O26:B6 L2762 were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Monoclonal antibodies

Fluorescein isothiocyanate (FITC)-conjugated anti-human CD14 clone MφP9 and allophycocyanin (APC)-conjugated anti-human HLA-DR clone L243 (G46-6) were purchased from BD Biosciences (San Jose, CA, USA). Phycoerythrin (PE)-conjugated anti-human TLR2 clone TL2.1 and PE-conjugated anti-human TLR4 clone HTA125 were obtained from eBiosciences (San Diego, CA, USA).

Whole-blood experiments

The protocol of in vitro experiments with peripheral blood was approved by the Ethics Committee of the Na Bulovce Hospital, Prague. Heparinized peripheral blood was collected from healthy volunteers into S-Monovette Lithium Heparin tubes (Sarstedt, France). In total, 500 μL of the sample was incubated with 107 bacteria/mL or LPS (100 ng/mL) at 37 °C in a 5% CO2 atmosphere for 24 h. In some experiments, 5 μg/mL polymyxin B (PMB) was added. The surface expression of TLR2, TLR4, CD14, and HLA-DR was measured after the incubation period. The monoclonal antibodies were added to 100 μL of blood and incubated for 30 min at room temperature and protected from light. Red blood cells were lysed after the incubation of samples with FACS lysing solution (Becton Dickinson, Mountain View, CA, USA) for 10 min, and the cells were pelleted through centrifugation (1 000 × g for 5 min at 20 °C). The cells were washed twice in ice-cold wash buffer (Cell Wash; Becton Dickinson), resuspended in 1% paraformaldehyde (Cell Fix; Becton Dickinson), and immediately analyzed. The surface expression of monocytes was measured as the mean fluorescence intensity (MFI). Flow cytometry was performed using FACSCaliburTM and CellQuest software (all Becton Dickinson).

Cytokine production

The stimulated blood was centrifuged (at 1 400 × g and 20 °C for 15 min), and the plasma was stored in aliquots at −80 °C. The concentrations of interleukin (IL)-1β, IL-6, IL-8, IL-10, IL-12, and tumor necrosis factor α (TNFα) in the plasma were quantified using the CBATM assay Human Inflammatory Kit (BD Biosciences, Heidelberg, Germany) and BD LSRFortessaTM (Becton Dickinson); the detection limit of the method was 20 pg/mL.

Phagocytosis assay

The phagocytic activity of the granulocytes was measured as the respiratory (oxidative) burst with the FagoFlowTM Kit (Exbio, Prague, Czech Republic). The heparinized peripheral blood was stimulated by the invasive and carrier strains of N. meningitidis. After the ingestion of bacteria, reactive intermediates within phagocytes oxidize dihydrorhodamine 123 (DHR123) into fluorescent rhodamine 123, which is detected with a flow cytometer in the FITC channel (525 nm).

Statistical evaluation

The differences between the groups were evaluated with a one-way ANOVA with a level of significance p-value < 0.05. The statistical analyses were performed with SigmaStat® 3.0 software (Jandel Scientific, San Rafael, CA, USA).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

Cytokine secretion in whole blood induced by invasive and colonizing strains of N. meningitidis

The intensity of cytokine secretion after stimulation by invasive and carrier strains is shown in Fig. 1A–C. The cytokine production induced by both invasive and carrier strains was significantly up-regulated compared with unstimulated controls. The production of TNFα, IL-1β, IL-6, IL-8, and IL-10 after stimulation by carrier strains was significantly higher (all p < 0.001) compared with invasive strains.

image

Figure 1. Production of TNFα, IL-6, IL-10, and oxidative burst in granulocytes after stimulation by 14 heat-inactivated invasive and 9 carrier meningococcal strains, which were incubated in human whole blood for 24 h. (1A) TNFα production; (1B) IL-1 0 production; (1C) IL-6 production; (1D) oxidative burst. Box plots represent medians and interquartile ranges, and the whiskers show 5th/95th percentile ranges. The data are representative of three independent experiments. image (gray) Blank, n = 3; image (light gray) LPS, n = 3; ■ (black) Invasive (invasive strains), n = 42; □ (white) Carrier (carrier strains), n = 27. The differences among control, LPS, and meningococcal strains were evaluated with a one-way ANOVA. Significance between invasive and carrier strains: ***p < 0.001.

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PMB treatment did not affect the production of the analyzed cytokines (TNFα, IL-6, and IL-10) in the control samples. In LPS-stimulated samples, PMB inhibited the production of all of the cytokines (all p < 0.001). Interestingly, in both of the groups of samples stimulated with N. meningitidis, only IL-6 production was inhibited after PMB was added (p < 0.001). Conversely, the levels of TNFα and IL-10 were not affected by PMB after stimulation by carrier strains and even increased after stimulation by invasive strains compared with samples without PMB (p < 0.001). Nevertheless, no difference in cytokine production was observed between the carrier and invasive groups after PMB treatment.

Expression of TLR2, TLR4, CD14, and HLA-DR on monocytes

The changes in the surface expression of TLR2, TLR4, CD14, and HLA-DR in monocytes after stimulation by invasive and carrier strains of N. meningitidis are presented in Fig. 2A–D. Invasive strains significantly up-regulated TLR2 expression compared with carrier strains (p < 0.001) and unstimulated controls (p < 0.001). Stimulation by carrier strains and LPS did not increase TLR2 expression compared with the controls.

image

Figure 2. Surface expression of TLR2, TLR4, HLA-DR, and CD14 on monocytes after stimulation by 14 heat-inactivated invasive and 9 carrier meningococcal strains, which were incubated in whole human blood for 24 h. (2A) TLR2 expression; (2B) TLR4 expression; (2C) HLA-DR expression; (2D) CD14 expression. The data are representative of three independent experiments. Box plots represent medians and interquartile ranges, and the whiskers show 5th/95th percentile ranges. image (gray) Blank, n = 3; image (light gray) LPS, n = 3; ■ (black) Invasive (invasive strains), n = 42; □ (white) Carrier (carrier strains), n = 27. The differences between invasive and carrier meningococcal strains were evaluated with a one-way ANOVA. Significance between invasive and carrier strains: ***p < 0.001; **p < 0.01; *p<0.05.

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Unlike invasive strains, carrier strains and LPS caused a significant decrease in TLR4 expression compared with the controls (p < 0.001 and 0.01, respectively). Furthermore, all of the stimulants caused a significant decrease in CD14 expression compared with the controls, and a difference was also observed between the invasive and carrier strains (p < 0.01). HLA-DR expression was up-regulated after stimulation, and carrier strains induced a slightly higher expression of HLA-DR compared with invasive strains (p < 0.05). A significant negative correlation was observed between TLR2 and HLA-DR expression after stimulation by all of the meningococcal strains (ρ = −0.740; p < 0.001).

PMB added to control samples did not affect surface marker expression. However, PMB significantly changed the expression of the surface antigens after stimulation by LPS: TLR2, TLR4, and CD14 were up-regulated and HLA-DR was down-regulated compared with the experiments without PMB (p < 0.001). In the samples stimulated with invasive and carrier strains, the addition of PMB led to TLR2, TLR4, and CD14 up-regulation. Conversely, HLA-DR expression was down-regulated compared with samples without PMB (p < 0.001). The expression of the surface molecules did not differ between the groups after PMB treatment.

Effectiveness of phagocytosis

The effectiveness of phagocytosis, measured as the oxidative burst in granulocytes, is presented in Fig. 1D. The intensity of the oxidative burst was significantly increased after stimulation by meningococcal strains compared with the control and LPS-stimulated cells. Moreover, carrier strains induced a higher oxidative burst compared with invasive strains (p < 0.001). PMB treatment eliminated the difference between the groups.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

In this study, the differences in the immunostimulatory activity between invasive and carrier strains of N. meningitidis were investigated.

We demonstrated that carrier strains caused a stronger activation of innate immunity when compared to the invasive strains. We found more intense production of inflammatory cytokines: TNFα, IL-1β, IL-6, IL-8, and IL-10 after stimulation by carrier strains in comparison to invasive strains. The differences in the cytokine production induced by the invasive and carrier strains may have important clinical implications, because the cytokine levels in the bloodstream play an important role in the elimination of meningococci and the IMD outcome [21]. It is worth noting that these differences in the activation of innate immunity were confirmed even in isolates showing identical classical and molecular characteristics, such as identical serogroups, phenotypes, and sequence type. Similar differences in TNFα and IL-6 production were shown after stimulation by meningococcal strains causing septic shock, chronic meningococcemia, and carrier strains [17]. On the other hand, in the human nasopharynx, carriage isolates of meningococci reduce inflammatory response and induction of apoptosis more intensively than invasive strains [19]. Although this finding is in contrast to our observation, we suppose that the intensity of immune response to invasive and carrier strains depend on a different niche.

Carrier strains caused in our experimental model a more intense oxidative burst in granulocytes in comparison to invasive strains. Our results are in agreement with a study that demonstrated a high efficiency of phagocytosis of live carrier strains of N. meningitidis [18]. Although HK meningococci may be phagocytized differently than live bacteria, the oxidative burst does not differ between live and HK bacteria [22]. The oxidative burst may be influenced by TNFα [23], which intense production was observed after the stimulation by carrier strains in our experimental model. According to previous studies, it is disputed if oxidative burst is independent or affected by meningococcal LOS [24, 25].

We observed that TLR2 and TLR4 expressions on monocytes differ significantly after stimulation by invasive and carrier strains after 24 h. Carrier strains caused TLR4 down-regulation in comparison to invasive strains and control, as well. In contrast, invasive strains up-regulated TLR2 in comparison to carrier strains and control, as well. It was shown that OMV up-regulate TLR2 and TLR4 expression 6 h after stimulation [26]. However, TLR4 expression is progressively down-regulated due to the LPS tolerance [27]. We assume that our results indicate that the invasive strains affect TLR2 more intensively than the carrier strains and vice versa carrier strains affect TLR4 expression more intensively than invasive strains. It is possible to conclude that the expression of these TLR receptors may be influenced by surface composition of meningococci; TLR2 is affected especially by meningococcal proteins, [28-31] whereas TLR4 is affected by LOS and its conformation [14, 32, 33].

We used PMB for evaluating a role of LOS in our experimental model. PMB was previously used in several studies about meningococci with conflicting results [26, 34-38]. We observed that PMB eliminated most of the differences between the invasive and carrier strains in cytokine production and surface expression, and oxidative burst, as well. However, our results cannot explain the clear influence of PMB.

There are certain limitations to our study. Unlike the HK bacteria used in this study, live meningococci modify their surface characteristics in the bloodstream, and also their density depends on several factors [5]. Moreover, the phenotypic features of isolated strains may be changed by the procedures of cultivation and inactivation [39]. In addition, we used blood from healthy volunteers, whose phenotype could affect the immune reactions. It is well known that several polymorphisms may affect the immune reaction to meningococci [40].

In conclusion, a stronger activation of innate immunity by the carrier strains compared with the invasive strains of N. meningitidis may explain why invasive strains escape local inflammatory response in respiratory tract, while carrier strains are contained in the mucous membranes. Whether or not observed changes between carrier and invasive strains reflect the difference in the protein composition and LOS structure of N. meningitidis should be further evaluated.

The study was supported by grant PRVOUK/P24/LF1/3 and grant SVV-2012-264506. The authors thank Dr Jitka Kalmusová for help with bacterial suspension preparation and Dr Robert Munford, NIH, Bethesda, USA for the critical review.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References
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