Development and characterization of a novel, megakaryocyte NF-κB reporter cell line for investigating inflammatory...

Essentials An easily detectable readout in megakaryocyte cell lines will enhance inflammatory research in these cells. Here, we report the development and characterization of a novel megakaryocyte NF-κB-reporter cell line (Meg-01R). Multiple inflammatory molecules modulate NF-κB activity in Meg-01R cells. Meg-01R cells respond to small molecule inhibitors such as IMD0354 and C87 that are known to inhibit NF-κB activity upon stimulation with TNFα. ABSTRACT: Background Because of the difficulties in acquiring large numbers of megakaryocytes, the impact of inflammatory responses on these cells and their ability to produce fully functional platelets under various pathological conditions has not been investigated in detail. Objectives The primary objective of this study is to develop and functionally characterize a novel megakaryocyte nuclear factor κB (NF-κB) reporter cell line to determine the effects of various inflammatory molecules on megakaryocytes and their signalling pathways. Methods A Meg-01-NF-κB-GFP-Luc (Meg-01R) cell line was developed by inserting a reporter NF-κB-GFP-Luc cassette into normal Meg-01 cells to produce luciferase following activation of NF-κB to enable easy detection of pro-inflammatory and reparative signalling. Results and conclusions Meg-01 and Meg-01R cells have comparable characteristics, including the expression of both GPIbα and integrin β₃ . Meg-01R cells responded to various inflammatory molecules as measured by NF-κB-dependent bioluminescence. For example, inflammatory molecules such as tumor necrosis factor-α and Pam3CSK4 increased NF-κB activity, whereas an antimicrobial peptide, LL37, reduced its activity. Meg-01R cells were also found to be sensitive to inhibitors (IMD0354 and C87) of inflammatory pathways. Notably, Meg-01R cells were able to respond to lipopolysaccharide (LPS; non-ultrapure), although it was not able to react to ultrapure LPS because of the lack of sufficient TLR4 molecules on their surface. For the first time, we report the development and characterization of a novel megakaryocyte NF-κB reporter cell line (Meg-01R) as a robust tool to study the inflammatory responses/signalling of megakaryocytes upon stimulation with a broad range of inflammatory molecules that can affect NF-κB activity.