Functional Genetic Assays of Immunometabolic Reporters

Characterizing and identifying neuroinflammatory states is crucial in developing treatments for neurodegenerative diseases. Microglia, the resident immune cells of the brain, regulate inflammation and play a vital role in maintaining brain health by producing cytokines, performing phagocytosis, and inducing or reducing inflammation. These functional states can be described by specific patterns of gene expression called transcriptional programs, which are determined by the activity of a set of key transcription factors that have mostly been identified. Thus, an assay for transcription factor activity could reveal the state of the microglial cells and neuroinflammation across the brain. This research developed an assay that uses a transcription factor dependent reporter to indicate which transcriptional programs are activated in the cell when exposed to different stimuli. The prototype assay quantifies nuclear factor kappa B (NF-kB) response in cultured human cells. NF-kB is a well-characterized transcription factor associated with inflammatory pathways in most cells, including microglia. The reporter construct contains an NF-kB specific responsive element that can induce fluorescence/luminescence upon activation of the transcription factor. In an iterative refinement, a dual response fluorescent reporter was developed, which uses a secondary constitutively fluorescent reporter for built-in normalization of the responsive element for microscopy studies. With further refinement, this modular system will serve as a template for less understood transcriptional enhancers allowing for rapid, low-cost assays of neuroimmune regulators and potential in vivo applications in the study of neuroinflammation.