A Dual-Fluorescence Assay for Gene Delivery Vehicle Screening in Macrophages with an Inflammation-Inducible Reporter Construct

Macrophages are a promising target for therapeutics in various applications such as regenerative medicine and immunotherapy for cancer. Due to their plastic nature, macrophages can switch from a non-activated state to activated with the smallest environmental change. For macrophages to be effective in their respective applications, screening for phenotypic changes is necessary to elucidate the cell response to different delivery vehicles, vaccines, small molecules, and other stimuli. We created a sensitive and dynamic high-throughput screening method for macrophages based on the activation of NF-κB. For this reporter, we placed an mCherry fluorescence gene under the control of an inflammatory promoter, which recruits NF-κB response elements to promote expression during the inflammatory response in macrophages. We characterized the inflammatory reporter based on key markers of an inflammatory response in macrophages including TNF-α cytokine release and immunostaining for inflammatory and non-inflammatory cell surface markers. With the inflammatory reporter, we also were able to create an LPS dose curve to determine the dynamic range of the reporter and determine the sensitivity of the reporter to stimulus through timepoint analysis of stimulus vs. non-stimulus treated reporter cells. We then used the reporter cell line to determine both delivery efficiency and inflammatory response to different viral and non-viral gene delivery vehicles. Thie screening technique developed here provides a dynamic, high-throughput screening technique for determining inflammatory response by mouse macrophages to specific stimuli and provides insight into the inflammatory response of mouse macrophages to different viral and non-viral gene delivery methods.