Abstract
Background: Microglia are well known key regulators of neuroinflammation which feature in multiple neurodegenerative disorders. These cells survey the CNS and, under inflammatory conditions, become “activated” through stimulation of toll-like receptors (TLRs), resulting in changes in morphology and production and release of cytokines. In the present study, we examined the roles of the related TAM receptors, Mer and Axl, and of their ligand, Gas6, in the regulation of microglial pro-inflammatory TNF-α production and microglial morphology.Methods: Primary cultures of murine microglia of wild-type (WT), Mer−/− and Axl−/− backgrounds were stimulated by the TLR4 agonist, lipopolysaccharide (LPS) with or without pre-treatment with Gas6. Gene expression of TNF-α, Mer, and Axl was examined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA) was used to measure TNF-α release from microglia. Immunofluorescence staining of β-actin and the microglial marker Iba1 was performed to reveal microglial morphological changes, with cellular characteristics (area, perimeter, Feret’s diameter, minimum Feret, roundness, and aspect ratio) being quantified using ImageJ software.Results: Under basal conditions, TNF-α gene expression was significantly lower in Axl−/− microglia compared to WT cells. However, all microglial cultures robustly responded to LPS stimulation with the upregulation of TNF-α expression to similar degrees. Furthermore, Mer receptor expression was less responsive to LPS stimulation when in Axl knockout cells. The presence of Gas6 consistently inhibited the LPS-induced upregulation of TNF-α in WT, Mer−/− and Axl−/− microglia. Moreover, Gas6 also inhibited LPS-induced changes in the microglial area, perimeter length, and cell roundness in wild-type cells.Conclusion: Gas6 can negatively regulate the microglial pro-inflammatory response to LPS as well as via stimulation of other TLRs, acting through either of the TAM receptors, Axl and Mer. This finding indicates an interaction between TLR and TAM receptor signaling pathways and reveals an anti-inflammatory role for the TAM ligand, Gas6, which could have therapeutic potential.
Highlights
Microglia are well known key regulators of neuroinflammation which feature in multiple neurodegenerative disorders
We have used single receptor knockout microglia to study both cytokine release in response to pro-inflammatory stimulation and morphological effects, with and without the influence of ligand stimulation by Gas6. We discovered that both Mer and Axl play important roles in inflammatory resolution and that Gas6 can inhibit a broad range of toll-like receptors (TLRs)-mediated inflammatory induction of TNF-α as well as associated morphological changes
Axl−/− microglia displayed significantly lower TNF-α gene expression when compared with WT microglia, whereas Mer−/− cells displayed no such difference (Figure 1A)
Summary
Microglia are well known key regulators of neuroinflammation which feature in multiple neurodegenerative disorders These cells survey the CNS and, under inflammatory conditions, become “activated” through stimulation of toll-like receptors (TLRs), resulting in changes in morphology and production and release of cytokines. Microglia undergo morphological changes upon inflammatory stimulation The transition from their surveillant state to a more ‘‘classically’’ activated phenotype has been well characterized with cells switching from a ramified, extended morphology to a more rounded, amoeboid structure (Djukic et al, 2006; Tam and Ma, 2014; Michell-Robinson et al, 2015; Arcuri et al, 2017). It is this molecular and phenotypic regulation of microglia that may become inefficient during neurodegeneration as chronic inflammation takes hold (Song and Colonna, 2018)
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