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The viral TLR3 agonist poly(I:C) stimulates phagocytosis and intracellular killing of Escherichia coli by microglial cells

Abstract number: P1950

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

Objectives:Escherichia coli K1 meningitis is associated with a high rate of mortality and long term sequelae despite antimicrobial therapy, especially in pediatric patients. Microglial cells, the resident phagocytes in the central nervous system, express Toll-like receptors (TLR) that mediate the innate immune response upon recognition of invading pathogens. Polyinosine–polycytidylic acid [poly(I:C)] is a TLR3 agonist structurally similar to viral double-stranded RNA. Here, we show that a viral TLR3 agonist can stimulate microglial cells and increase their phagocytic activity and intracellular killing of E. coli K1, a pathogenic encapsulated bacterial strain.

Methods: Primary cultures of murine microglia were exposed to poly(I:C) at 0.1 or 10 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. Microglial cultures were co-incubated with live E. coli K1 for phagocytosis and intracellular killing assays 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. For intracellular killing assays, cells were incubated with bacteria for 90 min. After bacterial exposure, microglial cultures were washed and cultured in medium containing gentamicin (200 mg/l). At various time points, 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 geqslant R: gt-or-equal, slanted11); 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. Poly(I:C) stimulated murine microglial cultures in a dose-dependent manner to secrete TNF-a and CXCL1 and increase their ability to phagocytose (p < 0.05 after 30 min, p < 0.01 after 90 min) and kill intracellular E. coli K1. CD blocked the bacterial uptake by geqslant R: gt-or-equal, slanted90%.

Conclusions: Stimulation of microglia with a viral agonist of the TLR system such as poly(I:C) could increase the resistance of the brain to bacterial infections. This may be a promising approach to protect the brain of septicemic patients from meningitis, cerebritis and brain abscess.

Session Details

Date: 10/04/2010
Time: 00:00-00:00
Session name: Abstracts 20th European Congress of Clinical Microbiology and Infectious Diseases
Subject:
Location: Vienna, Austria, 10 - 13 April 2010
Presentation type:
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