Gas6 Inhibits Toll-Like Receptor-Mediated Inflammatory Pathways in Mouse Microglia via Axl and Mer

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.

Single-Cell Transcriptome Profiling Reveals the Suppressive Role of Retinal Neurons in Microglia Activation Under Diabetes Mellitus

“…Our data suggested that OPN secreted by Muller and microglia could suppress TNF activation to impede excessive activation of microglia. Similarly, our data showed that GAS6 mediated cell-cell communication between amacrine and microglia was enhanced by hyperglycemia, and negatively regulated the pro-inflammatory via TAM receptors, Axl and Mer (Gilchrist et al, 2020; Figure 3E). CellChat predicted that hyperglycemia-induced GAS6 signaling was mediated by ligand GAS6 and both two receptors (Figure 3D), Ligand GAS6 was upregulated in microglia and amacrine, whereas the receptors were up-regulated in microglia (Figure 3F).…”

Glial Cells as Therapeutic Approaches in Brain Ischemia-Reperfusion Injury

“…Among these molecules, high mobility group box 1 (HMGB1) protein appears in the early stages of stroke and is recognized by several toll-like receptors (TLRs) like TLR2 or TLR4, which trigger an inflammatory response through the release of pro-inflammatory cytokines in an NF-κB-dependent process. In fact, TLR4 blockade has shown to be protective against brain ischemia with a reduction of the infarcted area, which could be due to a decrease in pro-inflammatory cytokines secreted by microglia [ 45 ]. Peroxiredoxins (Prdxs) constitute another important group of DAMPs with redox-activity.…”

Sex-specific transcriptome of spinal microglia in neuropathic pain due to peripheral nerve injury

“…Notably, previous studies have revealed that Lgals3 (Galectin 3) inhibition attenuates pain after nerve injury (Ma et al, 2016), Grn (Granulin) promotes peripheral nerve regeneration (Lim et al, 2012), Apoe polymorphisms are related to severity of peripheral neuropathy (Monastiriotis et al, 2013; Monastiriotis et al, 2012) and Ctsb (Cathepsin B) inhibition attenuates development of allodynia in inflammatory pain (Ma et al, 2016; Sun et al, 2012). Axl (AXL Receptor Tyrosine Kinase) signalling via its ligand growth arrest gene 6 ( Gas6 ) that is also upregulated after CCI, facilitates phagocytosis of apoptotic cells by microglia as well as increasing cellular migration and myelination and reducing apoptosis and toll-like receptor-mediated inflammation (Gilchrist et al, 2020; Goudarzi et al, 2020; Grommes et al, 2008). Bhlhe40 is a transcription factor that regulates myeloid cell activation and inflammation as well as self-renewal (Carey et al, 2020; Jarjour et al, 2019).…”