In vitro characterization of the microglial inflammatory response to Streptococcus suis, an important emerging zoonotic agent of meningitis

Abstract

Streptococcus suis is an important swine and human pathogen responsible for septicemia and meningitis. In vivo research in mice suggested that in the brain, microglia might be involved in activating the inflammatory response against S. suis. The aim of this study was to better understand the interactions between S. suis and microglia. Murine microglial cells were infected with a virulent wild-type strain of S. suis. Two isogenic mutants deficient at either capsular polysaccharide (CPS) or hemolysin production were also included. CPS contributed to S. suis resistance to phagocytosis and regulated the inflammatory response by hiding proinflammatory components from the bacterial cell wall, while the absence of hemolysin, a potential cytotoxic factor, did not have a major impact on S. suis interactions with microglia. Wild-type S. suis induced enhanced expression of Toll-like receptor 2 by microglial cells, as well as phosphotyrosine, protein kinase C, and different mitogen-activated protein kinase signaling events. However, cells infected with the CPS-deficient mutant showed overall stronger and more sustained phosphorylation profiles. CPS also modulated inducible nitric oxide synthase expression and further nitric oxide production from S. suis-infected microglia. Finally, S. suis-induced NF-κB translocation was faster for cells stimulated with the CPS-deficient mutant, suggesting that bacterial cell wall components are potent inducers of NF-κB. These results contribute to increase the knowledge of mechanisms underlying S. suis inflammation in the brain and will be useful in designing more efficient anti-inflammatory strategies for meningitis.

FIG. 1.

Phagocytosis of S. suis by murine microglial cells. (A) Kinetics of phagocytosis of S. suis strains (1 × 10⁶) by murine microglia after 15-, 30-, and 60-min infection times. *, P < 0.05 compared to phagocytosis levels obtained with the wild-type strain. (B) Effect of opsonization on phagocytosis at 30 min postinfection. Bacteria were nonopsonized (no serum) or preopsonized with 20% either normal or inactivated mouse serum. *, P < 0.05 compared to phagocytosis levels obtained with the wild-type strain; #, P < 0.05, indicating statistically significant differences between nonopsonized strains and their respective normal-serum- or inactivated-serum-opsonized counterparts. The numbers of internalized bacteria were determined by quantitative plating after 1 h of antibiotic treatment, and the results are expressed as CFU of recovered bacteria per ml (means plus SEM obtained from three independent experiments).

FIG. 2.

Interaction of murine microglial cells with S. suis. Microglia were infected with either the S. suis WT strain or the CPS⁻ mutant for 2 h. The cells were then washed, and bacteria were visualized with rabbit anti-S. suis serum and Alexa Fluor 488-conjugated goat anti-rabbit IgG (green), while phagolysosomes from microglial cells were evidenced with rat anti-LAMP1 antibody and Alexa Fluor 568-conjugated goat anti-rat IgG (red). The cell nuclei were stained with DAPI (blue). The images were examined with a confocal laser scanning microscope.

FIG. 3.

Comparative study of cytokine production: TNF-α (A), MCP-1 (B), IP-10 (C), IL-1β (D), and IL-6 (E). Murine microglial cells were incubated with the different S. suis strains (1 × 10⁶). The culture supernatants were harvested at 12 h poststimulation and analyzed for cytokine production by ELISA. The data are expressed as means plus SEM from at least three independent experiments. n.i., noninfected cells. *, P < 0.05, indicating significant differences from n.i. cells; #, P < 0.05, indicating significant differences from the WT S. suis strain.

FIG. 4.

Increase of TLR2 mRNA expression in murine microglial cells following S. suis infection. Microglia were stimulated for 1, 2, 4, and 8 h with 1 × 10⁶ S. suis bacteria. Total RNA was isolated from the microglia at the indicated time points and analyzed for TLR2 mRNA expression by real-time quantitative PCR as described in Materials and Methods. The levels of TLR2 gene expression following S. suis infection were calculated after the cycle thresholds against the β-actin and β₂ microglobulin housekeeping genes were normalized, using the 2^−ΔΔCt method. The results are presented as fold induction relative to noninfected microglia. *, P < 0.05, indicating significant differences between infected and noninfected cells; **, P < 0.001, indicating significant differences between microglia stimulated with WT S. suis and cells infected with the S. suis CPS⁻ mutant.The results are means plus SEM of three independent experiments.

FIG. 5.

Time course of increase in nitric oxide production (A) and iNOS expression (B) by murine microglial cells treated with S. suis. The heat-killed S. suis WT or CPS⁻ strain (1 × 10⁹) was incubated with microglia for 6, 12, 24, and 48 h. (A) Microglial supernatants were collected to measure nitric oxide production by the Griess reaction method. The data are expressed as the means plus SEM (in μM/ml) of three independent experiments. *, P < 0.05, indicating significant differences versus WT S. suis. n. i., noninfected cells. (B) Representative Western blot analysis of murine microglial extracts using an iNOS-specific antibody. Blotting with anti-α-actin antibody was used as a loading control.

FIG. 6.

S. suis-induced levels of tyrosine phosphorylation (p-Tyr) (A) and serine phosphorylation (p-PKC) (B). Murine microglial cells were infected for 15 or 30 min or 1, 2, or 4 h with either the WT strain or its CPS⁻ mutant (1 × 10⁶ bacteria). Cell lysates (total proteins) from noninfected cells and infected cells were subjected to Western blotting. p-Tyr and p-PKC protein levels were revealed by using anti-p-Tyr (clone 4G10) monoclonal antibody or anti-phospho-(Ser) PKC substrate antibody, respectively. The results are representative of three individual experiments.

FIG. 7.

Time course of phosphorylation of MAPKs in murine microglial cells. The cells were infected with either the S. suis WT strain or its CPS⁻ mutant (1 × 10⁶ bacteria). Cell extracts were recovered at 15 and 30 min and 1, 2, and 4 h postincubation and were subjected to Western blot analysis using antibodies specific for phospho-MAPKs (p-ERK, p-JNK, and p-p38). Following analysis, the blots were stripped and reprobed with an antibody specific for ERK, JNK, or p38 to verify uniformity in gel loading. The results are representative of three independent experiments. n. i., noninfected cells.

FIG. 8.

Pharmacologic inhibition of MAPKs. Murine microglial cells were treated with various inhibitors 1 h prior to infection with the S. suis WT strain or its CPS⁻ mutant (1 × 10⁶ bacteria). Apigenin (50 μM), SP600125 (50 μM), and SB203580 (75 μM) inhibit ERK 1/2, JNK, and p38, respectively. The inhibitors were all used at maximal subcytotoxic doses for a total of 13 h. (A) To confirm inhibition of MAPK phosphorylation, cell extracts were recovered after 2 h (p-ERK and p-JNK) or 4 h (p-p38) of bacterium-cell contact and then analyzed by Western blotting using specific antibodies for each of the proteins tested. The results are representative of three independent experiments. (B and C) To evidence inhibition in cytokine production, cells were infected for 12 h, and the supernatant was recovered for detection of TNF-α (B) and MCP-1 (C) production by ELISA. The data are expressed as means plus SEM from three independent experiments. *, P < 0.05, indicating significant differences from cells treated with MAPK inhibitors.

FIG. 9.

S. suis activates nuclear factor NF-κB in murine microglial cells. The cells were infected with either the S. suis WT strain or its CPS⁻ mutant (1 × 10⁶ cells) for 0.5 to 12 h. Noninfected cells were used as negative controls. LPS (1 μg/ml) served as a positive control. The cells were lysed, and nuclear extracts were subjected to EMSA. The presence of NF-κB-activated proteins in the cell nuclei was demonstrated by binding to oligonucleotide probes containing a single copy of the NF-κB motif 5′-AGT TGA GGG GAC TTT CCC AGG C-3′ end labeled with [γ-³²P]ATP. The binding reaction mixtures were electrophoresed on native 4% polyacrylamide gels to separate bound and unbound DNA probe. sp, specific probe; nsp, nonspecific probe.