Fibronectin stimulates Escherichia coli phagocytosis by microglial cells

Abstract number: P1230

Ribes S., Ebert S., Regen T., Adam N., Bunkowski S., Hanisch U.K., Hammerschmidt S., Nau R.

Objectives: Infections caused by Escherichia coli are common in the clinical setting and are still associated with high rates of mortality and long term sequelae despite antimicrobial therapy. Parenchymal microglia are one of the effective defence systems in the brain to remove invading bacteria contributing to the resistance of the brain. Microglia express Toll-like receptors (TLRs) that recognize invading pathogens as well as endogenous proteins at non-physiological concentrations such as fibronectin (Fn). Here, we hypothesized that the endogenous TLR4 ligand Fn might protect immunocompromised patients against infections by increasing the ability of microglial cells to phagocytose E. coli.

Methods: Primary cultures of mouse microglia were exposed to increasing concentrations of Fn (10, 50 or 100 mg/l) for 24 h. A control group of unstimulated cells was included in all experiments. After stimulation, supernatants were collected and stored at -80°C until measurement of cyto-/chemokine levels. Then, microglial cultures were challenged with either live E. coli DH5alpha or E. coli K1 at a ratio of 100 bacteria per cell. Phagocytosis was left to proceed for 30 and 90 min at 37°C. For phagocytosis inhibition studies, cytochalasin D (CD) was used at 10 microM. After bacterial exposure, microglial cultures were washed and incubated in medium containing gentamicin (200 mg/l) 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. ANOVA (followed by Bonferroni's multiple comparisons test) was performed to analyse differences between groups (n  12); p < 0.05 was considered statistically significant.

Results: The supernatants of unstimulated cells were devoid of measurable amounts of cyto-/chemokines. Unstimulated microglia ingested bacteria at a low rate. The endogenous TLR4-ligand Fn stimulated murine microglial cultures in a dose-dependent manner to secrete pro-inflammatory compounds and increase their ability to phagocytose E. coli DH5alpha (p < 0.05 after 30 and 90 min) and E. coli K1 (p < 0.05 after 90 min). CD blocked the entry of E. coli strains by 90%.

Conclusion: Fibronectin stimulates microglia to phagocytose bacteria in a dose-dependent manner. This approach could improve the brain resistance of immunocompromised patients against infections caused by E. coli.