Differential effects of Gram-positive versus Gram-negative bacteria on NOSII and TNFalpha in macrophages: role of TLRs in synergy between the two

Abstract

1. Gram-negative and Gram-positive bacteria are sensed by Toll-like receptor (TLR)4 and TLR2, respectively. TLR4 recruits MyD88 and TRIF, whereas TLR2 recruits MyD88 without TRIF. NOSII and TNFalpha are central genes in innate immunity and are thought to be differentially regulated by the MyD88 versus TRIF signalling pathways. Here, we have used Gram-positive Staphylococcus aureus, Gram-negative Escherichia coli and highly selective TLR ligands to establish the precise relationship between TLR2, TLR1, TLR6 and TLR4 for NOSII versus TNFalpha induction. 2. In murine macrophages at 24 h, E. coli or LPS (TLR4) induced NO and TNFalpha release. In contrast, S. aureus (TLR2/TLR1/TLR6) or Pam(3)CSK4 (TLR2/TLR1), or FSL-1 and LTA (TLR2/TLR6) induced TNFalpha without an effect on NO. 3. At later time points (48-72 h), S. aureus induced NO release. The ability of S. aureus, but not E. coli or LPS, to induce NO release was inhibited by anti-TNFalpha-binding antibodies. 4. At 24 h, LPS synergised with TLR2 ligands to induce NO release and NOSII protein expression. LPS also induced the expression of TLR2 gene expression without affecting levels of TLR4. 5. Using cells from TLR2(-/-) or TLR4(-/-) mice, the ability of LPS to synergise with S. aureus or Pam(3)CSK4 was found to be dependent on both TLR2 and TLR4. 6. These observations are the first to clearly delineate the role of separately activating TLR2 and TLR4 in the induction of NOSII and TNFalpha genes compared with their coinduction when both receptor pathways are activated.

Figure 1

Effect of heat-killed Gram-positive and Gram-negative bacteria stimulation for 24 h on nitric oxide and TNF-α production from J774.2 macrophages. J774.2 macrophages were treated with either a Gram-positive bacteria S. aureus or Gram-negative bacteria E. coli (10⁷–10⁹ CFU ml⁻¹) for 24 h after which levels of (a) nitrite and (b) TNF-α were determined. The data represent the mean±s.e.m.; n=9. Statistical differences (compared with control) are denoted by ^* and are determined where P<0.05 as calculated by ANOVA followed by Dunnet's post-test.

Figure 2

Effect of bacterial PAMPs on NO and TNF-α production from J774.2 macrophages over 24 h. J774.2 macrophages were treated with LPS (1 μg ml⁻¹), LTA (10 μg ml⁻¹), peptidoglycan (10 μg ml⁻¹), Pam₃CSK4 (0.3 μg ml⁻¹) or FSL-1 (1 μg ml⁻¹) for 24 h, after which levels of (a) nitrite and (b) TNF-α were determined. The data represent the mean±s.e.m.; n=9. Statistical differences (compared with control) are denoted by ^* and are determined where P<0.05 as calculated by ANOVA followed by Dunnet's post-test.

Figure 3

Effect of continued incubation with S. aureus on NO production by J774 macrophages. Macrophages were stimulated with LPS (0.01 μg ml⁻¹), S. aureus (3 × 10⁸ CFU ml⁻¹) or TNF-α (10 ng ml⁻¹) for (a) 24, (b) 48 or (c) 72 h. The data represent the mean±s.e.m.; n=6. Statistical differences (compared with control) are denoted by ^* and are determined where P<0.05 as calculated by ANOVA followed by Dunnet's post-test.

Figure 4

Effect of LPS and S. aureus (SA) alone and in combination with NOSII induction at 24 and 48 h in J774 macrophages. Macrophages were treated with LPS at 1 or 0.01 μg ml⁻¹ or with S. aureus (10⁸ CFU ml⁻¹) for (a) 24 or (b) 48 h. NOSII-like immunoreactivity migrated at the predicted molecular weight of 130 kDa.

Figure 5

Effect of anti-TNF-α-binding antibodies on NO release by J774 macrophages stimulated with LPS (0.1 μg ml⁻¹) or S. aureus (3 × 10⁸ CFU ml⁻¹) for 72 h. Data are the mean±s.e.m. from n=3 experiments. Data were normalised to control and differences in responses calculated using one-sample t-test. Statistical differences (compared with control) are denoted by ^* and are determined where P<0.05.

Figure 6

Synergistic effect of LPS in combination with Gram-positive bacteria (S. aureus) or other TLR2 ligands on NO production from J774.2 macrophages at 24 h. J774 macrophages were costimulated for 24 h with different concentrations of LPS and (a) S. aureus (10⁸ CFU ml⁻¹), (b) LTA (10 μg ml⁻¹), (c) FSL-1 (1 μg ml⁻¹) or (d) Pam₃CSK4 (0.3 μg ml⁻¹). The data represent the mean±s.e.m. for at least n=9 experiments. Statistical differences (compared with control) are denoted by ^* and are determined where P<0.05 as calculated by two-way ANOVA.

Figure 7

Synergistic effect of LPS in combination with Gram-positive bacteria (S. aureus) or other TLR2 ligands on TNF-α production from J774.2 macrophages at 24 h. J774 macrophages were costimulated for 24 h with different concentrations of LPS and (a) S. aureus (10⁸ CFU ml⁻¹), (b) LTA (10 μg ml⁻¹), (c) FSL-1 (1 μg ml⁻¹) or (d) Pam₃CSK4 (0.3 μg ml⁻¹). The data represent the mean±s.e.m. for at least n=9 experiments. Statistical differences (compared with control) are denoted by ^* and are determined where P<0.05 as calculated by two-way ANOVA.

Figure 8

Effect of TLR2 or TLR4 ligands on the expression of TLR2 and TLR4 gene. J774.2 macrophages were treated for 3 h with LPS (1 μg ml⁻¹), Pam₃CSK4 (0.3 μg ml⁻¹), S. aureus (10⁸ CFU ml⁻¹) or E. coli (10⁸ CFU ml⁻¹) and levels of (a) TLR2 mRNA and (b) TLR4 mRNA were determined by RT-PCR. Levels of TLR4 and TLR2 mRNA levels were corrected by expressing data as a ratio of GAPDH in each sample. The data are the mean±s.e.m. for n=6. Statistical differences (compared with control) are denoted by ^* and are determined where P<0.05 as calculated by one-way ANOVA.

Figure 9

Effect of threshold concentrations of LPS (0.01 μg ml⁻¹), Pam₃CSK4 (0.03 μg ml⁻¹) or S. aureus (10⁸ CFU ml⁻¹) stimulated for 24 h on NO production by bone marrow-derived macrophages from (a) wild-type mice, (b) TLR2^−/− mice and (c) TLR4^−/− mice. TNF-α levels were measured in the same samples of bone marrow-derived macrophages stimulated for 24 h with threshold concentrations of LPS (0.01 μg ml⁻¹), Pam₃CSK4 (0.03 μg ml⁻¹) or S. aureus (10⁸ CFU ml⁻¹) from (d) wild-type mice, (e) TLR2^−/− mice and (f) TLR4^−/− mice. The data are the mean±s.e.m. for n=9. Statistical differences (compared with control; that is, no treatment) are denoted by ^* and are determined where P<0.05 as calculated by one-way ANOVA.