Serum amyloid A1 directly engages TLR4/MD-2 to trigger neuroinflammatory signaling in microglia cultures

Introduction: Serum amyloid A1 (SAA1) is an inducible acute-phase protein produced in the liver and released in response to environmental and inflammatory stimuli, making it a reliable clinical biomarker of acute inflammation. In addition to its hepatic origin, SAA1 has been detected in axonal myelin sheaths in multiple sclerosis and Alzheimer's disease, suggesting that it may influence cells of the central nervous system (CNS) through several receptors, including Toll like receptors (TLRs). Aim: We aimed to clarify the role of TLR4 in mediating SAA1-induced inflammation in CNS cells and to elucidate the molecular mechanisms of SAA1 interaction with the TLR4/MD-2 complex. Methods: We used an engineered TLR4 reporter cell system to assess TLR4 activation by SAA1. In primary microglia cultures, we evaluated the effects of SAA1 on pro-inflammatory mediators, with or without pharmacological inhibitors, including the TLR4 inhibitor CLI-095, curcumin, and L48H37. Biochemical and computational approaches were used to investigate direct interactions between SAA1 and the TLR4/MD-2 complex. Results: SAA1 activates the TLR4/MD-2 complex in a concentration-dependent manner. This effect was abolished by the TLR4 inhibitor CLI-095. Curcumin and L48H37, which bind to the hydrophobic pocket of MD-2, reduced SAA1-induced release of interleukin-1β, tumor necrosis factor-α, and nitric oxide, as well as NF-κB activation in microglia. Biochemical and computational studies confirmed a direct interaction between SAA1 and TLR4/MD-2. Conclusions: SAA1 directly engages TLR4/MD-2 to trigger neuroinflammatory responses. Targeting this axis may attenuate SAA1-driven neuroinflammation and offers potential therapeutic strategies for neurodegenerative disorders.