Effects of low dose GM-CSF on microglial inflammatory profiles to diverse pathogen-associated molecular patterns (PAMPs).

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