Effects of low dose GM-CSF on microglial inflammatory profiles to diverse pathogen-associated molecular patterns (PAMPs).
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 (polyI: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 polyI:C or LPS, respectively; however, cells expanded in the presence of GM-CSF produced lower levels of both mediators following CpG-ODN stimulation.


CONCLUSIONS
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.
Esen
Nilufer
N
Department of Neurobiology and Developmental Sciences, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA. EsenNilufer@uams.edu <EsenNilufer@uams.edu>
Kielian
Tammy
T
D000818:Animals; D002478:Cells, Cultured; D018920:Coculture Techniques / Q000379:methods; D004306:Dose-Response Relationship, Immunologic; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000008:administration & dosage*; D018836:Inflammation Mediators / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D017628:Microglia / Q000378:metabolism* / Q000382:microbiology / Q000473:pathology*; D051192:Receptors, Pattern Recognition / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology; D013211:Staphylococcus aureus / Q000276:immunology; D051195:Toll-Like Receptor 2 / Q000502:physiology; D051196:Toll-Like Receptor 3 / Q000502:physiology; D051197:Toll-Like Receptor 4 / Q000502:physiology
D018836:Inflammation Mediators; D051192:Receptors, Pattern Recognition; C519703:TLR3 protein, mouse; C493486:Tlr2 protein, mouse; C493487:Tlr4 protein, mouse; D051195:Toll-Like Receptor 2; D051196:Toll-Like Receptor 3; D051197:Toll-Like Receptor 4; D016178:Granulocyte-Macrophage Colony-Stimulating Factor