| dc.description.abstract | Macrophages are involved in the pathogenesis of many inflammatory diseases including fibrosis, asthma, and atherosclerosis. These macrophages possess remarkable plasticity which allows them to respond to diverse microenvironments, polarizing towards either pro-inflammatory (M1) or anti-inflammatory (M2) phenotype. Cytosolic phospholipase A2 (cPLA2α) enzymes cleave phospholipids at the sn-2 position to yield free fatty acid and lysophospholipids. They play an important role in inflammation and inflammatory diseases. Many cytokines which act upon macrophages are known to affect the activity of cPLA2α enzymes, however, it is unclear what role this plays in the polarization process. In this thesis, we investigated the role of cPLA2α and associated downstream signaling pathways during macrophage polarization using THP-1 cells and provided experimental data to assist with the development of a computational model for studying macrophage polarization.
We first showed that the macrophages derived from the THP-1 cells were able to polarize to M1 and M2a phenotype in response to interferon (IFN)-γ plus lipopolysaccharide (LPS), and IL-4 respectively. However, the cells did not polarize towards an M2c phenotype in response to IL-10. We further showed that resting the cells for 48h after differentiation was sufficient for optimal polarization towards M1 and M2a phenotype. We showed that M1 macrophages were able to responds to IL-4 stimulation, showing some capacity for switching between M1 and M2 phenotype. In response to M1 stimulation, PTGS2 and PLA2G4C were upregulated, and we measured increased prostaglandin (PG)E2 in cell supernatants. ALOX15 was upregulated in M2a polarization, but we were unable to measure any 15-Hydroxyeicosatetraenoic acid (15-HETE). Inhibition of cPLA2α using AVX002, AVX420, or Pyrrophenone, and inhibition of COX enzymes using either Naproxen or Celecoxib did not affect the polarization towards either M1 or M2a phenotypes and did not affect response to IL-4 during switching from M1 and M2a phenotypes.
The computational model developed to study macrophage polarization predicted that PGE2 stimulation would inhibit M1, and promote M2 polarization. We concluded from experimental data that inhibiting endogenous PGE2 release did not alter macrophage polarization, however in agreement with the computational findings, exogenous PGE2 applied during M1 polarization suppress the expression of M1 marker genes. PGE2 stimulation during M2a polarization did augments the induction of some M2 marker genes (TGM2 and SOCS1), but also inhibited the expression of others (ALOX15 and MRC1).
In summary, we showed that THP-1 derived macrophages polarized to M1 and M2a but not M2c phenotype; inhibiting cPLA2α or endogenous PGE2 production using COX inhibitors did not affect polarization. Exogenous PGE2 on the other hand appeared to supress M1, and partially augment M2a polarization. | |