BioProject

Microglia are the resident mononuclear phagocytes of the CNS parenchyma and represent an initial line of defense against invading microorganisms. More...

Microglia are the resident mononuclear phagocytes of the CNS parenchyma and represent an initial line of defense against invading microorganisms. Microglia utilize Toll-like receptors (TLRs) for pathogen recognition and TLR2 specifically senses conserved motifs of gram-positive bacteria including lipoproteins, lipoteichoic acids, and peptidoglycan (PGN) leading to cytokine/chemokine production. Interestingly, primary microglia derived from TLR2 knockout (KO) mice over-expressed numerous IL-12 family members, including IL-12p40, IL-12p70, and IL-27 in response to intact S. aureus, but not the less structurally complex TLR2 ligands Pam3CSK4 or PGN. The ability of intact bacteria to augment IL-12 family member expression was specific for gram-positive organisms, since numerous gram-negative strains were unable to elicit exaggerated responses in TLR2 KO microglia. Inhibition of SYK or IRAK4 signaling did not impact heightened IL-12 family member production in S. aureus-treated TLR2 KO microglia, whereas PI3K, MAPK, and JNK inhibitors were all capable of restoring exaggerated cytokine expression to wild type levels. Additionally, elevated IL-12 production in TLR2 KO microglia was ablated by a TLR9 antagonist, suggesting that TLR9 drives IL-12 family member production following exposure to intact bacteria that remains unchecked in the absence of TLR2 signaling. Collectively, these findings indicate crosstalk between TLR2 and TLR9 pathways to regulate IL-12 family member production by microglia. The summation of TLR signals must be tightly controlled to ensure the timely cessation and/or fine tuning of cytokine signaling to avoid nonspecific bystander damage due to sustained IL-12 release. Overall design: TLR2 KO mice were backcrossed with C57BL/6 animals for a minimum of eight generations prior to use in these studies. Age- and sex-matched C57BL/6 mice were used as wild type (WT) controls. Primary mixed glial cultures were prepared from the cerebral corticies of neonatal mice (2-4 days of age) and microglia were harvested using a differential shaking technique with a purity of >98%. A USA300 community-acquired methicillin-resistant S. aureus (CA-MRSA) clinical isolate recovered from a patient with a fatal brain abscess was used to stimulate the microglia isolates. Bacterial strains were heat-inactivated and used to stimulate microglia at 107 colony forming units (cfu)/well for 6 and 12 hours time points. Three replicates of each mouse type (WT, TLR2 KO) at both time points 6 and 12 hours were used for the microarray experiments. Data was only usable for 2 replicates of the KO-12 hr group. Less...