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Oxidant-induced TLR4 translocation in murine macrophages is Src kinase dependent
Critical Care volume 11, Article number: P44 (2007)
Multiorgan failure is a major cause of late mortality following trauma. Oxidative stress generated during shock/resuscitation (S/R) contributes to tissue injury by priming the immune system for an exaggerated response to subsequent inflammatory stimuli such as lipopolysaccharide (LPS): the so-called 'two-hit hypothesis'. The mechanisms of oxidant-induced cell priming, however, remain poorly elucidated. Our group has previously reported a role in this priming process for translocation of the LPS receptor TLR4 to the plasma membrane . We have also previously shown that oxidant priming reprograms LPS signaling in macrophages to a Src-dependent pathway leading to PI3k activation . Taken together, we hypothesized that Src activation may play a role in oxidant-induced TLR4 translocation.
Materials and methods
Wild-type (WT) and triple Src (hck/fgr/lyn-/-) knockout (KO) mice were subjected to hemorrhagic shock and resuscitation to generate oxidative stress in vivo. Alveolar macrophages (AMs) were then retrieved by bronchoalveolar lavage and analyzed for TLR4 translocation by immunofluorescence staining and flow cytometry. In a separate in vitro experiment, AMs from WT and KO mice were exposed to 200 μM hydrogen peroxide for 60 minutes and similarly analyzed by flow cytometry for surface expression of TLR4.
In vivo, S/R induced visible translocation of TLR4 to the plasma membrane of AMs from WT mice as seen by immunofluorescence. This effect was completely inhibited in the Src KO animals, and the observation was corroborated quantitatively by flow cytometry (see Figure 1). In order to confirm the role of oxidative stress in S/R-induced TLR4 translocation, AMs from WT and KO mice were exposed to hydrogen peroxide in vitro. Hydrogen peroxide caused an increase in surface expression of TLR4 in WT AMs by a ratio of 1.23 compared with WT control. This effect was once again inhibited in the Src KO AMs, where the ratio was 0.95 compared with WT control.
Oxidative stress induces TLR4 translocation to the cell surface of macrophages in a Src-dependent manner. Since cellular responsiveness to LPS is known to correlate with surface levels of TLR4, this novel finding may direct future therapies in modulating oxidant-induced cellular priming and subsequent organ failure.
Powers KA, Szaszi K, Khadaroo RG, et al.: Oxidative stress generated by hemorrhagic shock recruits Toll-like receptor 4 to the plasma membrane in macrophages. J Exp Med 2006, 203: 1951-1961. 10.1084/jem.20060943
Khadaroo RG, Kapus A, Powers KA, et al.: Oxidative stress reprograms lipopolysaccharide signaling via Src kinase-dependent pathway in RAW 264.7 macrophage cell line. J Biol Chem 2003, 278: 47834-47841. 10.1074/jbc.M302660200
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Tawadros, P., Wang, Z., Birch, S. et al. Oxidant-induced TLR4 translocation in murine macrophages is Src kinase dependent. Crit Care 11, P44 (2007). https://doi.org/10.1186/cc6023
- Alveolar Macrophage
- Hemorrhagic Shock
- PI3k Activation
- Receptor TLR4
- Exaggerated Response