Ivy Allie, Bess Shelby N, Agrawal Shilpi, Kochar Varun, Stokes Abbey L, Muldoon Timothy J, Nelson Christopher E
Department of Biomedical Engineering, University of Arkansas, 120 John A. White Jr. Engineering Hall, Fayetteville, AR 72701 USA.
Cell and Molecular Biology Program, University of Arkansas, Fayetteville, AR USA.
BMC Methods. 2025;2(1):8. doi: 10.1186/s44330-025-00030-x. Epub 2025 May 8.
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.
背景：巨噬细胞是再生医学和癌症免疫治疗等各种应用中有前景的治疗靶点。由于其可塑性，巨噬细胞能在最小的环境变化下从非激活状态转变为激活状态。为使巨噬细胞在各自应用中发挥有效作用，有必要筛选表型变化以阐明细胞对不同递送载体、疫苗、小分子及其他刺激的反应。 方法：我们基于核因子κB（NF-κB）的激活创建了一种针对巨噬细胞的灵敏且动态的高通量筛选方法。对于该报告基因，我们将mRFP1荧光基因置于炎症启动子的控制之下，该启动子招募NF-κB反应元件以在巨噬细胞的炎症反应期间促进表达。我们基于巨噬细胞炎症反应的关键标志物对炎症报告基因进行了表征，这些标志物包括肿瘤坏死因子-α（TNF-α）细胞因子释放以及对炎症和非炎症细胞表面标志物的免疫染色。我们比较了几种临床相关病毒载体和市售非病毒载体的基因递送和炎症情况。组间统计分析采用单因素方差分析及事后Tukey检验。 结果：报告巨噬细胞在脂多糖（LPS）刺激后显示出动态范围，半数有效浓度（EC50）为0.61 ng/mL，这与TNF-α释放高度相关。流式细胞术揭示了各组之间的异质性，但证实了促炎标志物在群体水平上的变化。最后，我们通过展示各种领先基因递送载体的不同作用证明了该报告基因的实用性。 讨论：此处开发的这种筛选技术提供了一种动态的高通量筛选技术，用于确定小鼠巨噬细胞对特定刺激的炎症反应。本文介绍的方法深入了解了小鼠巨噬细胞对不同病毒和非病毒基因递送方法的炎症反应，并为新型载体的高通量筛选提供了一种工具。 补充信息：在线版本包含可在10.1186/s44330-025-00030-x获取的补充材料。
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