Marker-Independent Assessment of Molecular Activation Patterns in Macrophages

Abstract

Tissue repair, regeneration, and fibrosis are processes regulated by inflammatory monocytes and monocyte-derived macrophages (MDMs) that circulate in peripheral blood or reside in tissue. After a traumatic injury, monocytes and macrophages undergo significant phenotypic and functional changes that allow them to play essential roles in the initiation, maintenance, and resolution stages of tissue repair. Macrophages are also highly sensitive to physical stimuli in their environment and sense, for example, the degree of stiffness of the surrounding extracellular matrix. This makes them promising targets in biomaterial research, as the macrophage response is a crucial factor for long-term implant survival and performance. In addition, designing implant materials in a way that reduces inflammation and facilitates tissue integration has the potential to significantly reduce surgical costs and increase the quality of life for many patients. The research on how the physical properties of implant materials influence macrophage biology is still in its infancy. This work investigates how alternative analysis methods to classical immunological techniques can help overcome the current challenges in macrophage biomaterial research. It was therefore investigated how standard methods like flow cytometry perform in identifying macrophage phenotype after detachment from extracellular matrix-mimicking biomaterials. Our findings show that the detachment of adherent macrophages from a substrate induces significant bias depending on the analyzed surface antigens. Raman microspectroscopy (RM) is a non-invasive spectroscopic method that does not require fixation or antibody staining of biological samples. RM was therefore implemented as an alternative for single cell analysis of adherent macrophages. It was shown that macrophage activation can be robustly identified based on distinct Raman fingerprint spectra and that this method can be employed on macrophages adherent to biomaterial substrates to identify activation and phenotype in a marker-independent manner. Lipid Raman spectra were found to be significantly altered between macrophage phenotypes, making lipids an ideal target for the identification of macrophage polarization using RM. Lastly, it was investigated, if the myeloid leukemia-derived monocytic cell line THP-1 shows similar molecular activation when compared to primary MDMs as identified by RM. THP-1 protein and phospholipid levels were significantly altered by proinflammatory activation in THP-1 macrophages while MDMs also showed altered nucleic acid and non-membrane intracellular lipid composition. Altogether the findings of this thesis will contribute to a faster and more efficient development of regenerative biomaterials.