The Construction of a Plasmid for Detecting the Pathway of Arginine Metabolism in Human Macrophages: a Real-Time Assessment of Macrophage Polarity
Nancy Bigley (Committee Chair), Thomas Brown (Committee Member), Barbara Hull (Committee Member)
Master of Science (MS)
Macrophages are "first responders", innate immune system cells which quickly arrive to a site of infection and injury, consuming cell debris and foreign matter and recruiting other immune system cells to the area. While historically they have been thought to react uniformly to all challenges, the discovery of toll-like receptors has shown that macrophages actually work closely with the adaptive immune system in fine-tuning the immune response. Furthermore, it has recently been discovered that macrophages can become polarized to one of two subtypes-M1 or M2.
M1 macrophages are efficient producers of reactive oxygen species, nitrogen intermediates, and inflammatory cytokines. They are especially effective at mediating resistance against intracellular parasites and tumors. Arginine metabolism in M1 macrophages is characterized by high levels of inducible nitric oxide synthetase (iNos), and this is used as a marker for polarization of macrophages to the M1 phenotype. M2 macrophages, by contrast, produce anti-inflammatory molecules, have high levels of scavenger, mannose, and galactose-type receptors, and arginine metabolism is shifted to production of ornithine and polyamines via arginase. Arginase, encoded by the ARG1 gene, is considered to be one of the hallmarks of the M2 phenotype, and is one of the most specific markers used to determine polarization to that phenotype.
Polarization to one phenotype or another is not permanent, and macrophages can be polarized directly from one state to the other directly by addition of appropriate cytokines (IFNy, LPS, TNFa for M1, IL-4, IL-13, IL-10, TGFb for M2). The state of macrophage polarization can be determined by examining a population of macrophages for tell-tale products of one state or another (ROS, RNS, TNFa, IL-1, IL-6, IL-12, or IL-23 for M1, IL-10, TGFb, PDGF, VEGF, EGF, and arginase for M2). Determining macrophage polarization has implications in health outcomes- M1 macrophages excel at fighting parasites and fighting tumorous growth, while M2 macrophages assist in wound healing and nerve re-growth. The problem is that the methods of detecting macrophage polarization-flow cytometry, western blots, or ELISA tests, are not real time and kill the cells involved. This paper describes the theory and methods behind creating a plasmid which combines the promoter for genes whose transcription indicates the M1 or M2 phenotype with a GFP or RFP, when transfected into a colony of RAW macrophages, will enable realtime, quantitative visualization of the production of inducible nitric oxide synthetase or arginase, markers for the M1 and M2 phenotype. A clearer understanding of macrophage polarity in the course of illness, wounding, and cancer might lead to diagnostic or therapeutic discoveries in those areas.
Department or Program
Microbiology and Immunology
Year Degree Awarded
Copyright 2012, all rights reserved. This open access ETD is published by Wright State University and OhioLINK.