Phenotyping primary human microglia: tight regulation of LPS responsiveness
Abstract
Much is still unknown about mechanisms underlying the phenotypical and functional versatility of human microglia. Therefore, we developed a rapid procedure to isolate pure microglia from postmortem human brain tissue and studied their immediate ex vivo phenotype and responses to key inflammatory mediators. Microglia were isolated, along with macrophages from the choroid plexus by tissue dissociation, density gradient separation, and selection with magnetic microbeads. By flow cytometry, microglia were identified by a CD11b+CD45 dim phenotype and a smaller size compared with CD11b+CD45 high macrophages. Interestingly, white matter microglia from donors with peripheral inflammation displayed elevated CD45 levels and increased size and granularity, but were still distinct from macrophages. The phenotype of isolated microglia was further specified by absent surface expression of CD14, CD200 receptor, and mannose receptor (MR, CD206), all of which were markedly expressed by macrophages. Microglia stimulated immediately after isolation with LPS and IFNγ failed to upregulate TNFα or CCR7. Notably, responsiveness to LPS and IFNγ was clearly instigated in microglia after overnight preculture, which coincided with a strong upregulation of CD14. Culture of microglia with IL‐4 resulted in the induction of HLA‐DR and CCL18 but not MR, whereas culture with dexamethasone did induce MR, in addition to CD163 and CCL18. In conclusion, this study demonstrates phenotypic changes of microglia associated with peripheral inflammation, and reveals tight regulation of responses to LPS and IFNγ as well as distinct microglial responses to IL‐4 and glucocorticoids. These findings are of high relevance to studies on human microglia functioning in health and disease. © 2012 Wiley Periodicals, Inc.
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Genes previously identified to be associated specifically with microglia but not with blood monocytes showed similar expression in co-pMG and fetalMG.
“As a set, genes previously identified to be associated specifically with microglia but not with blood monocytes (Bennett et al., 2016, Butovsky et al., 2014, Melief et al., 2012) (Figure 2C) showed similar expression in co-pMG and fetalMG.”
From A highly efficient human pluripotent stem cell microglia model displays a neuronal-co-culture-specific expression profile and inflammatory response (2017) · cites this paper as Melief et al., 2012
pMacpre, pMac, and pMGL also expressed high levels of most of these genes, along with many of those identified by Melief et al. (2012).
“Notably, pMacpre, pMac, and pMGL also expressed high levels of most of these genes, along with many of those identified by Melief et al. (2012).”
From A highly efficient human pluripotent stem cell microglia model displays a neuronal-co-culture-specific expression profile and inflammatory response (2017) · cites this paper as Melief et al., 2012
These cells do not express a convincing microglia signature.
“Moreover, these cells do not express a convincing microglia signature (Butovsky et al., 2014, Melief et al., 2012).”
From A highly efficient human pluripotent stem cell microglia model displays a neuronal-co-culture-specific expression profile and inflammatory response (2017) · cites this paper as Melief et al., 2012
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- Authors
- Melief, J., Koning, N., Schuurman, K. G., Van De Garde, M. D. B., Smolders, J., Hoek, R. M., Van Eijk, M., Hamann, J., & Huitinga, I.
- Venue
- Glia
- Publisher
- Wiley
- Published
- 2012-06-27
- Volume
- 60
- Issue
- 10
- Pages
- 1506-1517
- ISSN
- 0894-1491
- 1098-1136
- Cited by
- 132
- References
- 5
- Language
- en
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