Toll-like receptor 4 signaling: A common pathway for interactions between prooxidants and extracellular disulfide high mobility group box 1 (HMGB1) protein-coupled activation.

摘要: Necrotic cells passively release HMGB1, which can stimulate TLR4 in an autocrine fashion to potentially initiate "sterile" inflammation that maintains different disease states. We have shown prooxidants induce NF-κB activation through stimulation. examined whether enhance HMGB1-induced signaling activation. used LPS-EK as a specific agonist for TLR4, and PPC SIN-1 situ sources ROS. As model systems, we HEK-Blue (stably transfected with mouse TLR4), RAW-Blue™ (derived from murine RAW 264.7 macrophages) primary macrophages TLR4-KO mice. Both RAW-Blue express optimized secreted embryonic alkaline phosphatase (SEAP) reporter under the control of promoter inducible by NF-κB. treated HMGB1 alone and/or conjunction inhibitors using SEAP measure increased TNFα IL-6 released TLR4-WT, but not macrophages. Pro-oxidants release, quantified ELISA. both fluorescence microscopy imaging flow cytometry quantify expression intracellular TLR4-neutralizing antibody decreased prooxidant-induced release. Prooxidants promoted determined TNF-α, accumulation iROS. (Box A), anti-HMGB1 anti-TLR4-neutralizing pAbs inhibited activation, A) pAb had no effect on The present results confirm proinflammatory effects activating signaling.