Increased activation, Nuclear factor kappa B (NF-kB)

The NF-kB pathway consists of a series of events where the transcription factors of the NF-kB family play a key role. The proinflammatory cytokine (IL-1beta) can be activated by NF-kB , including Reactive Oxygen Species produced by  NADPH oxidase (NOX). Upon pathway activation, the IKK complex will be phosphorylated, which in turn phosphorylates IkBa. There, this transcription factor can express pro-inflammatory and pro-fibrotic genes. This can be achieved by ROS, IKK enhancer or nuclear translocation enhancer. 

NF-kB transcriptional activity: Beta lactamase reporter gene assay (Miller et al. 2010). NF-kB transcription: Lentiviral NF-kB GFP reporter with flow cytometry (Moujalled et al. 2012)

NF-κB translocation: RelA-GFP reporter assay (Wink et al 2017)

IκBa phosphorylation: Western blotting (Miller et al. 2010)

NF-κB p65 (Total/Phospho) ELISA

ELISA for IL-6, IL-8, and Cox

The ROS directly influences NF-κB signalling, resulting in differential production of cytokines and chemokines (McKay and Cidlowski, 1999). NIn accordance with the OECD AOP Handbook, the pathway begins with increased levels of reactive oxygen species (ROS), serving as the Molecular Initiating Event (MIE), which subsequently triggers the Activation of the NF-κB Signaling Pathway . This activation, in turn, directly influences the expression of genes involved in the Differential Production of Cytokines and Chemokines , culminating in the regulation of Pro-Inflammatory Responses Transcriptionally Mediated by NF-κB (. The resultant exaggerated and dysregulated pro-inflammatory response contributes to chronic inflammation and tissue damage, representing the Adverse Outcome (AO). This sequence of events is underpinned by the works of McKay and Cidlowski (1999) and aligns with the guidelines set forth in the OECD AOP Handbook.F-κB regulates pro-inflammatory responses that are transcriptionally mediated by NF‑κB.

McKay LI, Cidlowski JA. Molecular control of immune/inflammatory responses: interactions between nuclear factor-kappa B and steroid receptor-signaling pathways. Endocr Rev. 1999 Aug;20(4):435-59.

Miller SC, Huang R, Sakamuru S, Shukla SJ, Attene-Ramos MS, Shinn P, Van Leer D, Leister W, Austin CP, Xia M. Identification of known drugs that act as inhibitors of NF-kappaB signaling and their mechanism of action. Biochem Pharmacol. 2010 May 1;79(9):1272-80.

Moujalled DM, Cook WD, Lluis JM, Khan NR, Ahmed AU, Callus BA, Vaux DL. In mouse embryonic fibroblasts, neither caspase-8 nor cellular FLICE-inhibitory protein (FLIP) is necessary for TNF to activate NF-κB, but caspase-8 is required for TNF to cause cell death, and induction of FLIP by NF-κB is required to prevent it. Cell Death Differ. 2012 May;19(5):808-15.

Wink S, Hiemstra S, Herpers B, van de Water B. High-content imaging-based BAC-GFP toxicity pathway reporters to assess chemical adversity liabilities. Arch Toxicol. 2017 Mar;91(3):1367-1383.