146 Chapter 5 in the shape, and transmigration of these cells were shown, but no mechanistic conclusions could be drawn on why these changes occurred and that is why I proceeded to further investigate this system by applying differential proteomics and metabolic analysis. The proteome data highlighted differences in the expression of proteins involved in the glycolytic pathway across all culture conditions. Following this finding, it was decided to investigate whether the glycolytic function was altered by measuring the enzymatic activity of the altered glycolytic enzymes and by performing extracellular flux analysis. Interestingly, no differences were observed in glycolytic function, but it was observed that there were opposite trends between protein expression and protein activity (when protein expression was higher, the activity was lower, and vice versa) which explains why no functional differences were observed. Altogether, our results highlight not only the necessity to investigate immunometabolism in a tissue-specific framework but also that multiple experimental approaches are necessary to understand how macrophages respond under specific conditions. CHARACTERIZATION OF TISSUE-RESIDENT MACROPHAGES Macrophages are highly versatile immune cells that can exhibit diverse characteristics and functions depending on their microenvironment. It is important to characterize macrophages to understand how they respond under different circumstances, especially during inflammatory responses. As was exemplified in Chapter 2 macrophage characterization involves various techniques, each providing different insights into their behavior. However, no single technique can provide a complete picture of macrophage responses, which is why I think that it is necessary to use a combination of techniques to gain a more complete understanding of their behavior.
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