A dual-fluorescence assay for gene delivery vehicle screening in macrophages with an inflammation-inducible reporter construct.

Background: 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.

Methods: 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 mRFP1 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. We compared gene delivery and inflammation of several clinically relevant viral vehicles and commercially available non-viral vehicles. Statistical analysis between groups was performed with a one-way ANOVA with post-hoc Tukey's test.

Results: The reporter macrophages demonstrated a dynamic range after LPS stimulation with an EC50 of 0.61 ng/mL that was highly predictive of TNF-α release. Flow cytometry revealed heterogeneity between groups but confirmed population level shifts in pro-inflammatory markers. Finally, we demonstrated utility of the reporter by showing divergent effects with various leading gene delivery vehicles.

Discussion: This screening technique developed here provides a dynamic, high-throughput screening technique for determining inflammatory response by mouse macrophages to specific stimuli. The method presented here provides insight into the inflammatory response in mouse macrophages to different viral and non-viral gene delivery methods and provides a tool for high-throughput screening of novel vehicles.

Supplementary information: The online version contains supplementary material available at 10.1186/s44330-025-00030-x.