Phagocytosis of non-encapsulated and encapsulated Streptococcus pneumoniae by murine microglia is increased after stimulation with Toll-like receptors agonists

Objectives

Bacterial phagocytosis by microglia contributes to the resistance of the brain to infections. Microglial cells express Toll-like receptors (TLR) which can be stimulated by pathogen-associated molecular patterns (PAMPs). We hypothesized that PAMPs may stimulate microglia thereby increasing their ability to phagocytose Streptococcus pneumoniae.

Methods

Primary cultures of mouse microglia were exposed to TLR agonists: tripalmitoyl-S-glyceryl-cysteine (Pam3CSK4 at 0.1 μg/ml; TLR2), endotoxin (LPS at 0.01 μg/ml; TLR4) and oligonucleotides containing unmethylated cytosin-guanosin motifs (CpG at 1 μg/ml; TLR9) for 24 h. TLR agonists were used at the lowest concentrations inducing the maximum stimulation of microglia cells in terms of NO release. After stimulation, cultures were challenged with two S. pneumoniae strains: the encapsulated D39 or the unencapsulated R6 strains were added at a ratio of 100 bacteria per cell. Phagocytosis was left to proceed for 30 and 90 min at 37°C + 5% CO₂. For phagocytosis inhibition studies, 10 μM cytochalasin D (CD) was used. After washing, the microglial cultures were incubated in medium containing gentamicin (200 μg/ml) for 1 h to kill extracellular bacteria. Thereafter, cells were washed and lysed with distilled water. Viable intracellular bacteria were enumerated by quantitative plating of serial 10-fold dilutions. To monitor intracellular survival, microglia cells were allowed to ingest bacteria for 30 min. Then, incubation in medium with gentamicin was performed for 1 h. Thereafter, viable intracellular bacteria were determined at various time points by quantitative plating after cell lysis. Kruskall-Wallis test followed by Dunn's multiple comparisons test was performed to compare phagocytosis between groups (n ≥ 10); p < 0.05 was considered statistically significant.

Results

Unstimulated microglia ingested bacteria at a low rate. CpG at 1 μg/ml strongly increased the number of phagocytosed bacteria (p < 0.01 at 30 and 90 min). The bacterial uptake was enhanced after stimulation with Pam3CSK4 at 0.1 μg/ml (p < 0.05 after 90 min) and LPS at 0.01 μg/ml. The phagocytic rates were different for both strains: the uptake of the non-encapsulated R6 strain was approximately 10 times more rapid than the phagocytosis of the encapsulated D39 strain. CD inhibited phagocytosis > 90% in all groups. Intracellular survival assays showed that bacterial killing was similar among unstimulated microglia and cells stimulated with TLR agonists.

Conclusion

After stimulation with bacterial TLR agonists, phagocytosis of S. pneumoniae by microglial cells is increased. The uptake of the non-encapsulated R6 strain is 10 times quicker than the phagocytosis of the encapsulated D39 strain.