Whole-blood incubation with the Neisseria meningitidis lpxL1 mutant induces less pro-inflammatory cytokines than the wild type, and IL-10 reduces the MyD88-dependent cytokines

Abstract

Levels of bacterial LPS, pro-inflammatory cytokines and IL-10 are related to the severity of meningococcal septicaemia. Patients infected with a Neisseria meninigitidis lpxL1 mutant ( Nm-mutant) with penta-acylated lipid A present with a milder meningococcal disease than those infected with hexa-acylated Nm wild type ( Nm-wt). The aim was to compare the pro-inflammatory responses after ex vivo incubation with the heat-inactivated Nm-wt or the Nm-mutant in citrated whole blood, and the modulating effects of IL-10. Concomitantly, we measured intracellular IL-6, IL-8 and TNF-α to elucidate which cell types were responsible for the pro-inflammatory responses. Incubation with Nm-wt (10⁶/ml;10⁷/ml;10⁸/ml) resulted in a dose-dependent increase of the MyD88-dependent pro-inflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α), which were mainly derived from monocytes. In comparison, only 10⁸/ml of the Nm-mutant significantly increased the concentration of these cytokines. The MyD88-independent cytokines (IP-10, RANTES) were evidently increased after incubation with the Nm-wt but were unaffected by the Nm-mutant. Co-incubation with IL-10 significantly reduced the concentrations of the MyD88-dependent cytokines induced by both the Nm-wt and the Nm-mutant, whereas the MyD88-independent cytokines were almost unaffected. In summary, the Nm-mutant is a weaker inducer of the MyD88-dependent/independent cytokines than the Nm-wt in whole blood, and IL-10 attenuates the Nm-stimulated increase in MyD88-dependent pro-inflammatory cytokines.

Keywords: IL-10; MyD88; Neisseria meningitidis; lpxL1; pro-inflammatory cytokines; whole blood.

Figure 1.

Time course of cytokine plasma levels. Whole blood from four donors was incubated (0, 2, 4, 6 and 12 h) with N. meningitidis wt (10⁷/ml) (red line), N. meningitidis lpxL1mutant (10⁷/ml) (blue line) or vehicle (no bacteria) (black line), plasma collected and measurements of cytokines performed. The concentrations [median (range)] (pg/ml) of the (a–d) MyD88-dependent cytokines IL-1β, IL-6, IL-8 and TNF-α (e, f), the MyD88-independent cytokines IP-10 and RANTES, and (g) the anti-inflammatory cytokine IL-10 are presented. The y-axis is presented in a log scale. *Significant differences in cytokine levels between the N. meningitidis wt and vehicle or between the N. meningitidis lpxL1 mutant and vehicle (P < 0.05). ns: non-significant differences.

Figure 2.

Plasma cytokine levels released upon increasing concentrations of bacteria. Whole blood from six donors was incubated for 4 h with increasing concentrations of N. meningitidis wt (wild type), N. meningitidis lpxL1 mutant (lpxL1) or vehicle (no bacteria), plasma collected and measurements of cytokines performed. The concentrations [median (range)] (pg/ml) of (a–d) the MyD88-dependent cytokines IL-1β, IL-6, IL-8 and TNF-α (e, f), the MyD88-independent cytokines IP-10 and RANTES, and (g) the anti-inflammatory IL-10 are presented. *(Top of charts) indicates significant differences in cytokine levels between the wt and vehicle or between the mutant lpxL1 and vehicle (P < 0.05). *(Below charts) indicates significant differences in cytokine levels between the wt and the mutant lpxL1, P < 0.05. ns: non-significant differences.

Figure 3.

Intracellular cytokine levels in different cell types. Whole blood from eight donors was incubated for 4 h with N. meningitidis wt (10⁸/ml), N. meningitidis lpxL1mutant (lpxL1) (10⁸/ml) or vehicle (no bacteria), and flow cytometry measurements of intracellular cytokines performed. In (a) the gating strategy used to identify the different types of WBCs is shown. WBCs are shown in gate I, CD3⁺ lymphocytes in gate III, granulocytes in gate IV and CD14⁺ monocytes in gate V. The cytokine fluorescence intensity (FI) levels of IL-6, IL-8 and TNF-α for monocytes, lymphocytes and granulocytes are displayed in overlay histograms of one representative donor, with lines in black (vehicle), red (10⁸/ml N. meningitidis wt) and blue (10⁸/ml N. meningitidis lpxL1 mutant). In (b) intracellular levels of IL-6, IL-8 and TNF-α in monocytes, lymphocytes and granulocytes are presented as median (range) of MFI. *(Top of charts) indicates significant differences in intracellular cytokine MFI between the wt and vehicle or between the lpxL1 mutant and vehicle (P < 0.05). *(Below charts) indicates significant differences between the wt and the lpxL1 mutant (P < 0.05). ns: non-significant differences.

Figure 3.

Intracellular cytokine levels in different cell types. Whole blood from eight donors was incubated for 4 h with N. meningitidis wt (10⁸/ml), N. meningitidis lpxL1mutant (lpxL1) (10⁸/ml) or vehicle (no bacteria), and flow cytometry measurements of intracellular cytokines performed. In (a) the gating strategy used to identify the different types of WBCs is shown. WBCs are shown in gate I, CD3⁺ lymphocytes in gate III, granulocytes in gate IV and CD14⁺ monocytes in gate V. The cytokine fluorescence intensity (FI) levels of IL-6, IL-8 and TNF-α for monocytes, lymphocytes and granulocytes are displayed in overlay histograms of one representative donor, with lines in black (vehicle), red (10⁸/ml N. meningitidis wt) and blue (10⁸/ml N. meningitidis lpxL1 mutant). In (b) intracellular levels of IL-6, IL-8 and TNF-α in monocytes, lymphocytes and granulocytes are presented as median (range) of MFI. *(Top of charts) indicates significant differences in intracellular cytokine MFI between the wt and vehicle or between the lpxL1 mutant and vehicle (P < 0.05). *(Below charts) indicates significant differences between the wt and the lpxL1 mutant (P < 0.05). ns: non-significant differences.