Contribution of phagocytosis and intracellular sensing for cytokine production by Staphylococcus aureus-activated macrophages

Abstract

Toll-like receptors (TLRs) are involved in the sensing of microbially derived compounds. We analyzed the contribution of these receptors to cytokine production by macrophages following stimulation with whole bacteria. Using knockout mice, we confirmed that the TLR4 and TLR2 contribution was predominant in the induction of tumor necrosis factor alpha and interleukin-10 by gram-negative bacteria. In contrast, the absence of TLR2 and/or TLR4 or TLR9 did not affect the response to gram-positive bacteria. In the absence of TLR2, phagocytosis was essential for cytokine production in response to heat-killed Staphylococcus aureus (HKSA). Because intracellular sensing was important in the absence of TLR2, we evaluated the contribution of Nod1 and Nod2, intracytoplasmic sensors of peptidoglycan-derived muropeptides, to the response to HKSA. By transfecting RAW 264.7 macrophages with dominant negative (DN) forms of Nod1 and Nod2, we showed that both molecules inhibited NF-kappaB activation in response to HKSA. The unexpected interference of DN Nod1 in the response of macrophages to gram-positive bacteria was confirmed with a Nod2 agonist (muramyl dipeptide) in transfection experiments with HEK293T cell. Taken together, these results show the contribution of phagocytosis and Nod molecules to the response to HKSA in macrophages and also identify possible cross talk between Nod1 and Nod2.

FIG. 1.

TNF-α and IL-10 production by peritoneal macrophages from C57BL/6 and TLR2/TLR4^−/− mice. Macrophages were stimulated with LPS from E. coli (100 ng/ml), Pam3CysSK4 (1 μg/ml), or an oligonucleotide containing the CpG motif (6 μg/ml) or with various heat-killed bacteria at 1 × 10⁷ bacteria/ml. Ec, Escherichia coli; Nm, Neisseria meningitidis; Sp, Streptococcus pyogenes. The presence of TNF-α (A and B) or IL-10 (C and D) in culture supernatants was assessed by ELISA. Results represent the means ± SEM of five independent experiments. *, P < 0.05 for TLR2 and TLR4 double knockout mice versus C57BL/6 mice (Mann-Whitney U test).

FIG. 2.

TNF-α and IL-10 production by peritoneal macrophages from C57BL/6 and MyD88^−/− mice. Macrophages were stimulated with Pam3CysSK4 (1 μg/ml), HKSA at 1 × 10⁷ bacteria/ml, or poly(I:C) (50 μg/ml). The presence of TNF-α or IL-10 in culture supernatants was assessed by ELISA. Data are the means ± SEM of eight independent experiments. *, P < 0.05 for MyD88^−/− mice versus C57BL/6 mice (Mann-Whitney U test); #, P < 0.05 for unstimulated cells versus stimulated HKSA cells in MyD88^−/− macrophages (Wilcoxon signed-rank test). ND, not detectable.

FIG. 3.

Role of phagocytosis in cytokine production by peritoneal macrophages from C57BL/6 and TLR2/TLR4^−/− mice. The inhibition of phagocytosis by cytochalasin D was checked by flow cytometry (A). Cytochalasin D (Cyto.D) (3 μM) was added to macrophages from C57BL/6 mice 20 min before incubation with Alexa 488-labeled S. aureus for 1 h. In other experiments, macrophages were stimulated for 24 h with heat-killed Staphylococcus aureus with protein A (HKSA) or without protein A (− prot A) (10 μg/ml, equivalent to 1 × 10⁷ bacteria/ml). The presence of TNF-α (B), IL-10 (C), or IL-6 (D) in culture supernatants was assessed by ELISA. Results represent the means ± SEM of four independent experiments. *, P < 0.05 versus cells without the addition of cytochalasin D (Wilcoxon signed-rank test). MFI, mean fluorescence intensity.

FIG. 4.

TNF-α and IL-10 production by peritoneal macrophages from C57BL/6 and TLR9^−/− mice. Macrophages were stimulated with Pam3CysSK4 (1 μg/ml), an oligonucleotide containing the CpG motif (6 μg/ml), or HKSA at 1 × 10⁷ bacteria/ml. The presence of TNF-α or IL-10 in culture supernatants was assessed by ELISA. Results represent the means ± SEM of five independent experiments. *, P < 0.05 for TLR9^−/− mice versus C57BL/6 mice (Mann-Whitney U test).

FIG. 5.

Role of phagocytosis and intracellular Nod proteins in the recognition of Staphylococcus aureus (HKSA). (A) Effect of phagocytosis inhibition by cytochalasin D (Cyto.D) on TNF-α production by peritoneal macrophages from C57BL/6 mice after 4 h or 24 h of stimulation by HKSA. Each line represents an individual experiment. *, P < 0.05 compared to cells without cytochalasin D (Wilcoxon signed-rank test). The macrophage cell line RAW 264.7 was transfected with an NF-κB-dependent luciferase reporter and (B) 250 ng of DN forms of MyD88, Nod1, or Nod2 or (C) 250 ng of a plasmid coding for β-galactosidase. The luciferase activity was measured after stimulation for 4 h with 10⁷ bacteria/ml of HKSA. The activation (n-fold) was determined and compared to that of unstimulated cells transfected with an equivalent amount of empty vector (pcDNA3). The results are the means ± SEM of five independent experiments. *, P < 0.05 compared to cells transfected with pcDNA3 and stimulated with HKSA (Wilcoxon signed-rank test).

FIG. 6.

Inhibition of NF-κB activation via Nod2 by overexpression of DN Nod1. (A) HEK293T cells were cotransfected with an NF-κB-dependent luciferase reporter, Nod2, and DN Nod1 or TLR2 expression plasmids. Simultaneously, the cells were stimulated with 100 nM MDP or 1 μg/ml Pam3CysSK4. The luciferase activity was measured, and NF-κB activation compared to that of unstimulated cells transfected with empty vector was determined. (B) HEK293T cells were cotransfected with an NF-κB-dependent luciferase reporter, Nod1, and DN Nod1 or TLR2 expression plasmids to check the specificity of DN Nod1. Simultaneously, the cells were stimulated with 100 nM M-Tri_DAP or 1 μg/ml Pam3CysSK4. The luciferase activity was measured, and NF-κB activation compared to that unstimulated cells transfected with empty vector was determined. The results are the means ± SEM of five independent experiments. *, P < 0.05 (Wilcoxon signed-rank test).