Involvement of ROS in NF-κB activation by LPS

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LPS is an endotoxin found in the outer membrane of Gram-negative bacteria. It activates host innate immunity by stimulating phagocytic cells (monocytes/macrophages and neutrophils) to produce proinflammatory cytokines like IL-1, IL-6 and TNF-α [126]. LPS is recognized by TLR4, a member of the TLR family that is involved in innate immunity and inflammation response. Upon the binding of TLR4 to LPS, the cytoplasmic region of TLR4 recruits MyD88, which links TLR4 to IRAK and TRAF6 that mediates NF-κB activation [127, 128]. CD14, which is expressed on the surface and in the cytoplasm (sCD14) of monocytes/macrophages and neutrophils, has also been reported to play a key role in the recognition of LPS and in downstream cytokine release, notably through its interaction with LBP (LPS-binding protein), which binds the lipid A region of LPS and aids LPS to dock at the TLR4 [129-131]. Involvement of ROS in NF-κB activation by TLR4 has been suggested using antioxidants. Pre-treatment of neutrophils with NAC or α-tocopherol prevented LPS-induced NF-κB activation and the production of pro-inflammatory cytokines [132], and NAC and DMSO were reported to block NF-κB activation and IL-8 secretion in monocyte-like THP-1 cells challenged with LPS [133]. Sanlioglu et al. [134] went one step further demonstrating for the first time that the activation of Rac1 and the subsequent production of ROS are key steps involved in NF-κB activation and TNF secretion in macrophages challenged with LPS. Using blocking antibodies, they also reported that the ROS-dependent Rac1 activation is independent of the CD14 receptor, suggesting that alternative pathways contribute to NF-κB activation by LPS [134]. The exact molecular source of ROS upon LPS challenge was recently discovered by Park et al. [135]. They showed that, in HEK293T cells, LPS-induced ROS generation and NF-κB activation are mediated by direct interaction of TLR4 with Nox4 (NADPH oxidase 4), a protein related to the phagocytic cells NAPPH oxidase 2 (Nox2), but only confirmed partially this role in the U937 monocytic cell line, suggesting that another Nox enzyme might be involved in LPS-induced NF-κB activation in U937 cells (Figure 6) [135]. Whether this local oxidative stress triggers activation of TRAF6, as with the IL-1β signalling pathway, is currently unknown. It should, however, be noted that ROS production after LPS challenge has been showed to mediate the formation of a complex between TRAF6 and the redox-sensitive ASK1, which, in turn, triggers p38 activation, another downstream target of LPS signalling [136].