Design and Validation of a Live Cell Reporter for Macrophage Activation

Abstract

Macrophages are phagocytic cells of the innate immune system, which defend the body against invading pathogens. These immune cells are capable of transiently adopting different polarization states suited to different phases of an infection. They exist in a naïve (M0) state in the absence of appropriate stimulus but adopt a classical (M1) activation state, a pro-inflammatory, highly microbicidal phenotype in response to microbial ligands and certain cytokines to combat current infections. Macrophages can repolarize to an alternative (M2) activation state, a largely anti-inflammatory phenotype, after an infection has been resolved. A plethora of microbial and plant-derived compounds have the potential to interfere with normal macrophage polarization and thereby compromise or enhance the innate immune response to infection. In order to study this process in live macrophages, a fluorescent reporter for M1 activation has been designed, engineered, and validated. The reporter has been successfully validated through a series of experiments where murine macrophages that have been stably transduced with the reporter construct were exposed to lipopolysaccharide (LPS) from gram-negative bacteria and the cytokine interferon-gamma (IFN-γ), which are known inducers of macrophage M1 polarization. It is demonstrated that the reporter expression is concurrent with that of the endogenous gene. Furthermore, data also suggest that the reporter is a suitable tool for investigating the immunomodulatory properties of plant-derived compounds, such as polysaccharides extracted from American ginseng.