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.

背景小胶质细胞是众所周知的神经发炎的关键调节因子，在多种神经退行性疾病中都有特征。这些细胞检查中枢神经系统，并在炎性条件下通过刺激toll样受体（TLR）而被“激活”，从而导致形态变化以及细胞因子的产生和释放。在本研究中，我们研究了相关TAM受体Mer和Axl及其配体Gas6在调节小胶质促炎性TNF-α的产生和小胶质形态方面的作用。

方法：通过TLR4激动剂，脂多糖（LPS）刺激或不刺激Gas6刺激野生型（WT），Mer ^-/-和Axl ^-/-背景的鼠小胶质细胞的原代培养。使用逆转录定量聚合酶链反应（RT-qPCR）检查TNF-α，Mer和Axl的基因表达，并使用酶联免疫吸附测定（ELISA）来测量小胶质细胞释放的TNF-α。进行了β-肌动蛋白和小胶质细胞标记物Iba1的免疫荧光染色，揭示了小胶质细胞的形态变化，并使用定量了细胞特征（面积，周长，费雷特直径，最小费雷特，圆度和长宽比）。图像 软件。

结果：在基础条件下，与野生型细胞相比，Axl ^-/-小胶质细胞中的TNF-α基因表达明显降低。但是，所有小胶质细胞培养物均以相似程度的TNF-α表达上调强烈响应LPS刺激。此外，当在Axl基因敲除细胞中时，Mer受体表达对LPS刺激的反应较小。Gas6的存在始终抑制WT，Mer ^-/-和Axl ^-/-小胶质细胞中LPS诱导的TNF-α上调。此外，Gas6还抑制LPS诱导的野生型细胞中小胶质细胞面积，周长和细胞圆度的变化。

结论：Gas6可以负面调节小胶质对LPS的促炎反应以及 通过通过TAM受体Axl和Mer的任何一种刺激其他TLR。这一发现表明TLR和TAM受体信号通路之间存在相互作用，并揭示了TAM配体Gas6的抗炎作用，它可能具有治疗潜力。