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

Abstract

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

DNA damage mediates changes in neuronal sensitivity induced by the inflammatory mediators, MCP-1 and LPS, and can be reversed by enhancing the DNA repair function of APE1

“…Because we found that the effects of LPS could be attenuated by a CCR2 antagonist, we suspect that the maintenance of hypersensitivity induced by LPS is mediated through activation of TLR4 and subsequent upregulation of MCP-1/CCL2. This finding was intriguing because activation of TLR4 elicits the generation of ROS/RNS in macrophages (Qin et al, 2005a, Zhang et al, 2015), yet in neurons TLR4 activation cannot maintain sensitivity without activation of the CCR2. Therefore, we propose that the quantitative, spatial and temporal aspects of ROS/RNS generation are critical for inducing DNA damage and will be studied further.…”

HBx inhibits HMGB1 expression and active oxygen production in LO2 cells through the NF‐κB signaling pathway

“…These results suggest that these two stimulants can stimulate cells from both ends of the signaling pathway ( Figure 3) to induce changes in the nodes of the pathway. Based on these results 15,16 and previous reports, 17,18 the investigators speculated that the overexpression of HBx could inhibit the activity of NF-κB in normal hepatocytes, and NF-κB, HMGB1 and ROS were mutually regulated, TLR4 is also involved in regulation. Therefore, the investigators conclude that HBx suppresses the expression of HMGB1 and the production of ROS through NF-κB signaling pathway.…”

Modulation of neuronal excitability by immune mediators in epilepsy

“…In fact, only the disulfide (oxidized) isoform of this molecule activates TLR4 and promotes seizures but not the reduced form, which has instead chemoattractive properties [35,36]. …”