- Authors: Ansaldi, D., Hod, E. A., Stellari, F., Kim, J. B., Lim, E., Roskey, M., Francis, K. P., Singh, R., Zhang, N.
- Year: 2011
- Journal: PLoS ONE 6 e25093
- Applications: in vivo / DNA / in vivo-jetPEI
50 µg of an NF-kB-luc reporter and 7 µl of in vivo-JetPEI were each diluted into 200 µl with 5% glucose (N/P ratio of 7). The two solutions were then mixed and incubated for 15 minutes at room temperature. The entire mixture (400 µl) was injected intravenously into albino C57BL/6 mice.
NF-kappaB activation is a critical signaling event in the inflammatory response and has been implicated in a number of pathological lung diseases. To enable the assessment of NF-kappaB activity in the lungs, we transfected a luciferase based NF-kappaB reporter into the lungs of mice or into Raw264.7 cells in culture. The transfected mice showed specific luciferase expression in the pulmonary tissues. Using these mouse models, we studied the kinetics of NF-kappaB activation following exposure to lipopolysaccharide (LPS). The Raw264.7 cells expressed a dose-dependent increase in luciferase following exposure to LPS and the NF-kappaB reporter mice expressed luciferase in the lungs following LPS challenge, establishing that bioluminescence imaging provides adequate sensitivity for tracking the NF-kappaB activation pathway. Interventions affecting the NF-kappaB pathway are promising clinical therapeutics, thus we further examined the effect of IKK-2 inhibition by MLN120B and glycogen synthase kinase 3 beta inhibition by TDZD-8 on NF-kappaB activation. Pre-treatment with either MLN120B or TDZD-8 attenuated NF-kappaB activation in the pulmonary tissues, which was accompanied with suppression of pro-inflammatory chemokine MIP-1ss and induction of anti-inflammatory cytokine IL-10. In summary, we have established an imaging based approach for non-invasive and longitudinal assessment of NF-kappaB activation and regulation during acute lung injury. This approach will potentiate further studies on NF-kappaB regulation under various inflammatory conditions.