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Updated: Aug 25, 2026

NF-κB-dependent Luciferase Activation and Quantification of Gene Expression in Salmonella Infected Tissue Culture Cells
Published on: January 12, 2020
Reactive oxygen and nitrogen species differentially regulate Toll-like receptor 4-mediated activation of NF-kappa B
Kieran A Ryan1, Michael F Smith, Michael K Sanders
1Digestive Health Center of Excellence, University of Virginia, Charlottesville, Virginia 22908, USA.
Abstract:
Toll-like receptor 4 (TLR4) has been identified as a transmembrane protein involved in the host innate immune response to gram-negative bacterial lipopolysaccharide (LPS). Upon activation by LPS recognition, the TIR domain of TLR4 signals through MyD88 to activate the nuclear factor kappa B (NF-kappa B) pathway, a critical regulator of many proinflammatory genes, including interleukin-8 (IL-8). Emerging evidence suggests that reactive oxygen species (ROS) can contribute to diverse signaling pathways, including the LPS-induced cascade. In the present study we investigated the role of ROS in TLR-mediated signaling. Purified Escherichia coli LPS, a highly specific TLR4 agonist, elicited an oxidative burst in the monocyte-like cell line THP-1 in a time- and dose-dependent manner. This oxidative burst was shown to be dependent on the presence of TLR4 through transfection studies in HEK cells, which do not normally express this protein, and with bone marrow-derived macrophages from C3H/HeJ mice, which express a mutated TLR4 protein. LPS-stimulated IL-8 expression could be blocked by the antioxidants N-acetyl-L-cysteine and dimethyl sulfoxide at both the protein and mRNA levels. These antioxidants also blocked LPS-induced IL-8 promoter transactivation as well as the nuclear translocation of NF-kappa B. These data provide evidence that ROS regulate immune signaling through TLR4 via their effects on NF-kappa B activation.
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