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

BACKGROUND Due to 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 characterise a novel megakaryocyte NF-κB reporter cell line in order 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 GPIb and integrin β3 . Meg-01R cells responded to various inflammatory molecules as measured by NF-κB-dependent bioluminescence. For example, inflammatory molecules such as TNFα 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 LPS (non-ultrapure) although it was not able to react to ultrapure LPS due to the lack of sufficient TLR4 molecules on their surface. For the first time, we report the development and characterisation 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.