Abstract
The mechanism(s) by which cells can sense exogenous oxidants that may contribute to intracellular oxidative/nitrosative stress is not clear. The objective of this study was to determine how cells might respond to exogenous oxidants to potentially initiate, propagate and/or maintain inflammation associated with many human diseases through NF-κB activation. First, we used HEK-Blue cells that are stably transfected with mouse toll-like receptor 4 (mTLR4) or mouse TLR2. These cells also express optimized secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of a promoter inducible by NF-κB transcription factor. These cells were challenged with their respective receptor-specific ligands, different pro-oxidants and/or inhibitors that act at different levels of the receptor signaling pathways. A neutralizing antibody directed against TLR4 inhibited responses to both TLR4-specific agonist and a prooxidant, which confirmed that both agents act through TLR4. We used the level of SEAP released into the culture media due to NF-κB activation as a measure of TLR4 or TLR2 stimulation. Pro-oxidants evoked increased release of SEAP from HEK-Blue mTLR4 cells at a much lower concentration compared with release from the HEK-Blue mTLR2 cells. Specific TLR4 signaling pathway inhibitors and oxidant scavengers (anti-oxidants) significantly attenuated oxidant-induced SEAP release by TLR4 stimulation. Furthermore, a novel pro-oxidant that decays to produce the same reactants as activated phagocytes induced inflammatory pain responses in the mouse orofacial region with increased TLR4 expression, and IL-1β and TNFα tissue levels. EUK-134, a synthetic serum-stable scavenger of oxidative species decreased these effects. Our data provide in vitro and related in vivo evidence that exogenous oxidants can induce and maintain inflammation by acting mainly through a TLR4-dependent pathway, with implications in many chronic human ailments.
Highlights
Oxidative/nitrosative stress (ONS) induced by reactive oxygen species (ROS) and reactive nitrogen species (RNS) are said to be an important initiating factor in many human diseases with little or no effective treatment [1]
Our present in vitro results have shown that exogenous prooxidants can activate NF-kB through stimulation of TLR4 or TLR2 to induce secreted embryonic alkaline phosphatase (SEAP) release
We used these agents to determine to what extent the prooxidants they produce may be ‘sensed’ through TLR4 or TLR2 expressed on cell surface to activate NF-kB in HEK-Blue cells stably transfected with mouse toll-like receptor 4 (mTLR4) or mouse TLR2 (mTLR2)
Summary
Oxidative/nitrosative stress (ONS) induced by reactive oxygen species (ROS) and reactive nitrogen species (RNS) are said to be an important initiating factor in many human diseases with little or no effective treatment [1]. ONS may be caused by an imbalance in the generation and removal of ROS/RNS [2] These oxidative species are implicated in signal transduction and gene activation that may play a role in initiating, propagating and maintaining several disease states [3,4]. Substantial amounts of ROS (used in this manuscript to encompass RNS) are generated from endogenous (internal) sources as by-products of normal and essential metabolic reactions. It is not clear whether and how exogenous (external) oxidants may play a role in regulating the levels of endogenous oxidants, thereby increasing cellular ONS that contributes to the propagation and maintenance of different disease states. It is important to understand the mechanism(s) involved in cellular oxidant sensing because of the role of ONS in many lifethreatening diseases [9] including chronic pain [10]
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