Background: It is well appreciated that obtaining sufficient numbers of primary microglia for in vitro experiments has always been a challenge for scientists studying the biological properties of these cells. Supplementing culture medium with granulocyte-macrophage colony-stimulating factor ( GM-CSF) partially alleviates this problem by increasing microglial yield. However, GM-CSF has also been reported to transition microglia into a dendritic cell ( DC)-like phenotype and consequently, affect their immune properties. Methods: Although the concentration of GM-CSF used in our protocol for mouse microglial expansion ( 0.5 ng/ml) is at least 10-fold less compared to doses reported to affect microglial maturation and function (>= 5 ng/ml), in this study we compared the responses of microglia derived from mixed glial cultures propagated in the presence/absence of low dose GM-CSF to establish whether this growth factor significantly altered the immune properties of microglia to diverse bacterial stimuli. These stimuli included the gram-positive pathogen Staphylococcus aureus ( S. aureus) and its cell wall product peptidoglycan ( PGN), a Toll-like receptor 2 ( TLR2) agonist; the TLR3 ligand polyinosine-polycytidylic acid ( polyl: C), a synthetic mimic of viral double-stranded RNA; lipopolysaccharide ( LPS) a TLR4 agonist; and the TLR9 ligand CpG oligonucleotide ( CpG-ODN), a synthetic form of bacteria/viral DNA. Results: Interestingly, the relative numbers of microglia recovered from mixed glial cultures following the initial harvest were not influenced by GM-CSF. However, following the second and third collections of the same mixed cultures, the yield of microglia from GM-CSF-supplemented flasks was increased two-fold. Despite the ability of GM-CSF to expand microglial numbers, cells propagated in the presence/absence of GM-CSF demonstrated roughly equivalent responses following S. aureus and PGN stimulation. Specifically, the induction of tumor necrosis factor-alpha ( TNF-alpha), macrophage inflammatory protein-2 ( MIP-2/CXCL2), and major histocompatibility complex ( MHC) class II, CD80, CD86 expression by microglia in response to S. aureus were similar regardless of whether cells had been exposed to GM-CSF during the mixed culture period. In addition, microglial phagocytosis of intact bacteria was unaffected by GM-CSF. In contrast, upon S. aureus stimulation, CD40 expression was induced more prominently in microglia expanded in GM-CSF. Analysis of microglial responses to additional pathogen-associate molecular patterns ( PAMPs) revealed that low dose GM-CSF did not significantly alter TNF-alpha or MIP-2 production in response to the TLR3 and TLR4 agonists polyl: C or LPS, respectively; however, cells expanded in the presence of GM-CSF produced lower levels of both mediators following CpG-ODN stimulation. Conclusion: We demonstrate that low levels of GM-CSF are sufficient to expand microglial numbers without significantly affecting their immunological responses following activation of TLR2, TLR4 or TLR3 signaling. Therefore, low dose GM-CSF can be considered as a reliable method to achieve higher microglial yields without introducing dramatic activation artifacts.
