ACAT1/SOAT1 blockade suppresses LPS‐mediated neuroinflammation by modulating the fate of toll‐like receptor 4 in microglia

Abstract

Background: Cholesterol is essential for growth and maintenance of mammalian cells. It is stored as cholesteryl esters by the enzymes acyl-CoA:cholesterol acyltransferases 1 & 2 (ACAT 1 & 2) (Sterol O-acyltransferase 1 & 2; SOATs in GenBank). ACAT1 blockade (A1B) in macrophages ameliorates various pro-inflammatory responses elicited by lipopolysaccharides (LPS) or by cholesterol loading. In mouse and human brains, Acat1 expression dominates over Acat2 and Acat1 is elevated in many neurodegenerative diseases and in acute neuroinflammation. However, the possible effects of ACAT1 blockade in neuroinflammation, regulated by mediators such as Toll-Like Receptor 4 (TLR4), has not been studied. Methods: We conducted LPS-induced acute neuroinflammation experiments in control vs myeloid specific or neuron specific Acat1 knockout ( KO) mice. Furthermore, we evaluated LPS-induced neuroinflammation in the microglial cell line N9 with or without pre-treatment of the small molecule ACAT1-specific inhibitor K-604. Biochemical and microscopy assays were used to monitor inflammatory responses and the fate of TLR4. Results: In vivo studies revealed that Acat1 inactivation in myeloid cell lineage, but not in neurons, markedly attenuated LPS-induced activation of various pro-inflammatory response genes in hippocampus and cortex. Studies in cell culture showed that pre-incubating cells with K-604 significantly ameliorated the pro-inflammatory responses induced by LPS. In cells acutely treated with LPS (for 30 min), pre-incubation with K-604 significantly increased the endocytosis of TLR4, the major transmembrane signaling receptor that mediates LPS-dependent acute neuroinflammation. In cells chronically treated with LPS (for 24-48 hrs), pre-incubation with K-604 significantly decreased the total TLR4 protein content, presumably due to enhanced trafficking of TLR4 to the lysosomes for degradation. For ex vivo evidence, we isolated microglia from adult mice, and found that in mice without LPS stimulation, myeloid Acat1 inactivation altered cellular distribution of TLR4; in mice with LPS stimulation, myeloid Acat1 inactivation decreased the cellular content of TLR4. Conclusion: Blocking ACAT1 in mouse microglia alters the fate of TLR4 and suppresses its ability to participate in pro-inflammatory signaling cascade in response to LPS.