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. 2010 Jun;78(6):2857-67.
doi: 10.1128/IAI.01138-09. Epub 2010 Apr 5.

Toll-like Receptor 2- And MyD88-dependent Phosphatidylinositol 3-kinase and Rac1 Activation Facilitates the Phagocytosis of Listeria Monocytogenes by Murine Macrophages

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Toll-like Receptor 2- And MyD88-dependent Phosphatidylinositol 3-kinase and Rac1 Activation Facilitates the Phagocytosis of Listeria Monocytogenes by Murine Macrophages

Yanna Shen et al. Infect Immun. .
Free PMC article

Abstract

Toll-like receptors (TLRs) play a key role in the innate immune response by sensing bacterial ligands. The mechanisms involved in the TLR-mediated cytokine response are well established; however, the possible contribution of TLR-dependent recognition of bacteria to macrophage phagocytosis remains unclear. Listeria monocytogenes is an intracellular, parasitic, Gram-positive bacterium recognized mainly by TLR2. In this study, we investigated whether TLR2-dependent signaling is involved in the phagocytosis of L. monocytogenes by macrophages. We found no difference in the number of L. monocytogenes cells associating with wild-type (WT) and TLR2(-/-) macrophages 1 h after infection. However, the number of L. monocytogenes cells phagocytosed in TLR2(-/-) and MyD88(-/-) macrophages was significantly lower than that of WT macrophages. In addition, lipopolysaccharide (LPS) treatment restored impaired phagocytic activity of TLR2(-/-) macrophages but did not enhance the activity of MyD88(-/-) macrophages. The efficiency of phagocytosis was suppressed by inhibitors of phosphatidylinositol 3-kinase (PI3K) and the small Rho GTPases but not by cycloheximide. Moreover, functional activation of PI3K and Rac1 was impaired in TLR2(-/-) and MyD88(-/-) macrophages. In an in vivo infection model, we found significantly lower numbers of L. monocytogenes cells phagocytosed in peritoneal macrophages of TLR2(-/-) and MyD88(-/-) mice after intraperitoneal infection. Moreover, a lower number of bacteria were detected in the spleens of TLR2(-/-) mice 1 day after intravenous infection than in WT mice. These results clearly indicated that TLR2-MyD88-dependent signaling enhances the basal level of phagocytosis of L. monocytogenes by macrophages through activation of PI3K and Rac1, not by synthesis of proinflammatory cytokines or expression of phagocytic receptors.

Figures

FIG. 1.
FIG. 1.
Difference in the phagocytosis of Listeria monocytogenes (LM) between WT and TLR2−/− macrophages. (A) WT macrophages were incubated with 40 μg ml−1 anti-TLR2 MAb (IgG1) or mouse control IgG for 1 h and infected with L. monocytogenes at an MOI of 20 for 1 h. Cells were then treated with 20 μg ml−1 gentamicin for 30 min, and the number of phagocytosed bacteria was determined. (B) WT, TLR2−/−, and TLR4−/− macrophages were infected with L. monocytogenes at an MOI of 20 for 1 h. The number of phagocytosed bacteria was determined after treatment with gentamicin for 30 min. (C and D) WT and TLR2−/− macrophages were infected with L. monocytogenes as mentioned above, and the number of associating L. monocytogenes cells (C) and the mean phagocytic index (D) were determined. The mean phagocyte index was calculated as follows: (number of phagocytosed L. monocytogenes cells/number of associating L. monocytogenes cells) × 100. (E) Bone marrow-derived macrophages from WT and TLR2−/− macrophages were infected with L. monocytogenes at an MOI of 20 for 1 h, and the number of phagocytosed bacteria was determined. (F) WT and TLR2−/− macrophages were cultured with polystyrene beads for 2 h at a bead-to-cell ratio of 50 to 1. After being washed, cells were fixed with 1% paraformaldehyde and the number of internalized beads was counted under fluorescence microscope. Data are expressed as the means ± standard deviations (SD) of results for triplicate cultures. Similar results were obtained in three independent experiments. *, P < 0.05.
FIG. 2.
FIG. 2.
Fluorescence microscopic analysis for the phagocytosis of L. monocytogenes (LM). (A) WT and TLR2−/− macrophages were cultured with CFSE-labeled L. monocytogenes at an MOI of 50 for 1 h. After being washed, cells were fixed and incubated with rabbit anti-Listeria Ab and anti-rabbit IgG Ab conjugated with Alexa Fluor 594 to stain adherent L. monocytogenes to macrophages. According to the procedure, phagocytosed L. monocytogenes (green spots) was distinguished from adherent L. monocytogenes (yellow spots) in merged fluorescence images. (B and C) After infected L. monocytogenes cells were stained with rabbit anti-Listeria Ab and Alexa Fluor 594-conjugated anti-rabbit IgG Ab, the numbers of associating L. monocytogenes and adherent L. monocytogenes cells were counted in 500 WT and TLR2−/− macrophages, and the number of phagocytosed L. monocytogenes cells was determined. The phagocytic index (a percentage) was calculated as (number of phagocytosed L. monocytogenes cells/number of associating L. monocytogenes cells) × 100. The average number of associating L. monocytogenes cells (B) and the mean phagocyte index (C) are depicted. *, P < 0.05. (D) Columns depict the exact distribution of the phagocytic index in WT and TLR2−/− macrophages. The experiment was repeated three times, and data are expressed as the means ± standard errors for results of three independent experiments. (E) WT and TLR2−/− macrophages were infected with CFSE-labeled L. monocytogenes at an MOI of 50 for 1 h and incubated with CTC for 15 min. Cells were fixed and treated with rabbit anti-Listeria Ab and Alexa Fluor 350-conjugated anti-rabbit IgG Ab. The numbers of viable and dead bacteria inside macrophages were counted. The viability of intracellular bacteria (%) was calculated as follows: (number of viable L. monocytogenes cells/number of viable and dead L. monocytogenes cells) × 100.
FIG. 3.
FIG. 3.
Difference in the phagocytosis of L. monocytogenes (LM) between WT and MyD88−/− macrophages. (A) WT and MyD88−/− macrophages were infected with L. monocytogenes at an MOI of 20 for 1 h. After being washed, cells were treated with gentamicin for 30 min and the number of phagocytosed bacteria was determined. Data are expressed as the means ± standard deviations (SD) for results of triplicate cultures. Similar results were obtained in three independent experiments. *, P < 0.05. (B) WT and MyD88−/− macrophages were infected with CFSE-labeled L. monocytogenes at an MOI of 50 for 1 h. The numbers of phagocytosed L. monocytogenes and adherent L. monocytogenes cells were evaluated after immunostaining. Data are expressed as the means ± SD for results of triplicate cultures. Similar results were obtained in two independent experiments. *, P < 0.05. (C) WT and MyD88−/− macrophages were incubated with polystyrene beads for 2 h. After being washed, cells were fixed and the number of internalized beads was counted. (D) WT and MyD88−/− macrophages were infected with CFSE-labeled L. monocytogenes at an MOI of 50 for 1 h and treated similarly to those shown in Fig. 2E. The viability of intracellular bacteria was calculated as a percentage. (E) WT, TLR2−/−, and MyD88−/− macrophages were stained with FITC-conjugated anti-TLR2 MAb (IgG2b) or control IgG2b, and the level of TLR2 expression was analyzed by FACS. The histogram with the bold line shows the level of TLR2 expression on the three types of macrophages. The hatched area represents the basal fluorescent intensity in cells treated with control Ab. (F) Whole PEC and adherent PEC obtained from WT (red), TLR2−/− (blue), and MyD88−/− (green) mice were stained with PE-conjugated anti-F4/80 MAb (IgG2a). The expression of F4/80 was analyzed by FACS. The hatched area represents the basal fluorescent intensity in cells treated with control IgG2a.
FIG. 4.
FIG. 4.
The effect of LPS and cycloheximide on the phagocytosis of L. monocytogenes (LM) by TLR2−/− and MyD88−/− macrophages. (A) WT and TLR2−/− macrophages were infected with L. monocytogenes at an MOI of 20 in the presence or absence of 100 ng ml−1 Pam3CSK4 or 100 ng ml−1 LPS for 1 h, and the number of phagocytosed L. monocytogenes cells was determined. Data are expressed as the means ± standard deviations (SD) for results of triplicate cultures. *, P < 0.05. (B) WT, TLR2−/−, and MyD88−/− macrophages were infected with L. monocytogenes at an MOI of 20 in the presence or absence of 100 ng ml−1 LPS for 1 h, and the number of phagocytosed L. monocytogenes cells was counted. Data are expressed as the means ± SD for results of triplicate cultures. *, P < 0.05. (C) WT and TLR2−/− macrophages were infected with L. monocytogenes at an MOI of 20 for 1 h in the presence or absence of CHX, and the number of phagocytosed bacteria was determined. Data are expressed as the means ± SD for results of triplicate cultures. Similar results were obtained in three independent experiments.
FIG. 5.
FIG. 5.
The effect of LY294002 on the phagocytosis of L. monocytogenes (LM) and difference in the phosphorylation of Akt between WT, TLR2−/−, and MyD88−/− macrophages after L. monocytogenes infection. (A) WT macrophages were infected with L. monocytogenes at an MOI of 20 in the presence of graded concentrations of LY294002, a PI3K inhibitor, and the number of phagocytosed bacteria was determined. Data are expressed as the means ± standard deviations (SD) for results of triplicate cultures. (B) WT, TLR2−/−, and MyD88−/− macrophages were infected with L. monocytogenes at an MOI of 20 for 1 h in the presence or absence of 20 μM LY294002, and the number of phagocytosed bacteria was determined. Data are expressed as the means ± SD for results of triplicate cultures. Similar results were obtained in two independent experiments. *, P < 0.05. (C) WT, TLR2−/−, and MyD88−/− macrophages were infected with L. monocytogenes at an MOI of 20 for 15 and 30 min. Cells were lysed, and phosphorylated Akt was evaluated by Western blotting. Total Akt was used as a loading control.
FIG. 6.
FIG. 6.
The effect of toxin B on the phagocytosis of L. monocytogenes (LM) and difference in the phosphorylation of Rho family GTPase proteins between WT, TLR2−/−, and MyD88−/− macrophages after L. monocytogenes infection. (A) WT, TLR2−/−, and MyD88−/− macrophages were infected with L. monocytogenes at an MOI of 20 for 1 h in the presence or absence of 40 ng ml−1 toxin B, and the number of phagocytosed bacteria was determined. Data are expressed as the means ± standard deviations (SD) for results of triplicate cultures. Similar results were obtained in two independent experiments. *, P < 0.05. (B) WT, TLR2−/−, and MyD88−/− macrophages were infected with L. monocytogenes at an MOI of 20 for 15 and 30 min. The GTP-bound forms of Rac1 and Cdc42 were precipitated using glutathione S-transferase (GST)-bound p21-activated kinase 1 (PAK1) and quantified by Western blotting using anti-Cdc42 (IgG1) and anti-Rac1 (IgG2b) MAbs. Total Rac1 and Cdc42 in the cell lysate were used as a loading control.
FIG. 7.
FIG. 7.
The effect of LY294002 and toxin B on the expression of TLR2 on macrophages. WT, TLR2−/−, and MyD88−/− macrophages were incubated with 20 μM LY294002 or 40 ng ml−1 toxin B for 1 h and treated with anti-CD16/32 MAb (IgG2b), followed by staining with FITC-conjugated anti-TLR2 MAb (IgG2b) or control IgG2b. The level of TLR2 expression was analyzed by FACS. The histogram with the red line shows the level of TLR2 expression on macrophages without treatment, the blue line represents the level of TLR2 expression on macrophages treated with LY294002, and the green line shows the level of TLR2 expression on macrophages treated with toxin B. The hatched area represents the basal fluorescent intensity in cells treated with control IgG2b.
FIG. 8.
FIG. 8.
Difference in the phagocytosis of L. monocytogenes (LM) and bacterial burden between WT and TLR2−/− mice after L. monocytogenes infection in vivo. (A) WT, TLR2−/−, and MyD88−/− mice were injected i.p. with 3 ml of 3% thioglycolate medium and infected i.p. with 1 × 108 CFU of L. monocytogenes 4 days later. Five minutes after infection, PECs were collected, and the number of phagocytosed L. monocytogenes was determined. *, P < 0.05. (B) WT and TLR2−/− mice were infected intravenously with 104 CFU of L. monocytogenes. The spleens were removed 1 day after infection, and the number of viable bacteria in spleen was determined. *, P < 0.05.

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