Characterizing the Dynamics of Macrophage Polarization and Signaling
Forsmo, James Edward
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Apart from their primary functions in innate immunity and phagocytosis, macrophages are critical regulators of inflammation through their ability to adopt polarization states, a spectrum of phenotypes in which macrophages change their morphology and produce large amounts of pro/anti-inflammatory cytokines and biomolecules. Dysregulated macrophage polarization has been implicated in numerous chronic inflammatory diseases. However, as a result of the tightly-regulated nature of macrophage polarization, it is difficult to design immunomodulatory strategies for modulating macrophage polarization without a robust understanding of the temporal dynamics involved. As a result, there is a need for a deeper understanding of polarization dynamics that can be used in conjunction with computational modeling to develop better immunomodulatory strategies. In this thesis, RAW 264.7 murine macrophages are subject to a number of different stimulation strategies in order to temporally characterize how these cells translate inputs (such as pro-inflammatory cytokines and biomolecules) into a key marker of pro-inflammatory polarization output (iNOS expression over time). Specifically, I measured iNOS expression primarily through immunocytochemistry performed in either 96-well microwell plates or in PDMS microfluidic devices for three-dimensional (3D) culture experiments. Performing polarization experiments in microfluidic devices revealed that 3D culture environments exhibit differences in polarization dynamics. In particular, I found that the M1 response to lipopolysaccharide (LPS) stimulation is always transient regardless of whether macrophages are re-stimulated with fresh LPS-containing medium. Subsequently, I investigated re-stimulating with an M1 cytokine that activates different pathways than LPS to upregulate pro-inflammatory genes, referred to in this thesis as an orthogonal stimulus. Interestingly, further re-stimulation with an orthogonal stimulus, which upregulates iNOS through a separate pathway, such as interferon-γ, can extend M1 polarization and reach greater levels of iNOS expression than any single stimulus alone, in an order and dose-dependent fashion. I also showed that culturing macrophages in a three-dimensional environment within microfluidic devices delays their response to LPS but exhibits the same critical maximum of iNOS expression, suggesting a potential role for the extracellular matrix in regulating polarization dynamics. This thesis contributes novel findings to our understanding of macrophage polarization and proposes new avenues of investigation into the dynamics of other important cytokines in both 2D and 3D culture environments. These results may inform the development of novel models for understanding the temporal dynamics of macrophage polarization and direct further work into modulating polarization states for the treatment of inflammatory diseases.