ROLE OF ADHESION PROTEIN FIBULIN7 IN MODULATION OF MACROPHAGE FUNCTIONS

Abstract

Macrophages are phagocytic innate immune cells that belong to the myeloid lineage. These cells play a vital role in clearing off infections, thus contributing to host defense. In addition to phagocytosis, other functions performed by macrophages include, removal of cell debris and apoptotic cells. Because of their remarkable cellular plasticity, macrophages acquire different phenotypes depending on their surrounding microenvironment. The two extremes of functionally polarized phenotypes achieved by macrophages are M1/classically activated, and M2/alternatively activated macrophages classified in vitro. Classical or M1 macrophages express pro-inflammatory cytokines such as IL-12, TNF-α, and are generated in the presence of cytokine IFN-γ. Such macrophages possess enhanced antigen-presenting capacity and produce nitric oxide (NO) and other reactive oxygen intermediates (ROI). On the other hand, M2 macrophages are immunosuppressive in function and produce anti-inflammatory cytokines such as IL-10. They have an essential role in enhanced tissue remodeling, secretion of angiogenesis promoting factors. In contrast to M1 macrophages, M2 macrophages are generated in the presence of cytokines IL-4, IL-13, or IL-10. In addition to M1 and M2 phenotypes, the macrophages that infiltrate into various tumors such as breast, lungs, glioblastoma, where they secrete tumorpromoting factors such as VEGF and other growth factors, are known as tumor-associated macrophages (TAMs). Such macrophages primarily resemble the M2 phenotype but could have characteristics of M1 as well. Fibulin7 (Fbln7) is the most recently identified member of the fibulin family of secreted glycoproteins expressed in developing tooth, bone, cartilage as well as immune-privileged locations such as eye and placenta. Previous studies have demonstrated that the C-terminal fragment of Fbln7 protein (Fbln7-C) has anti-angiogenic activity both in vitro and in vivo. Still, a lot remains to be studied about its physiological and pathological functions. The research presented in the thesis has focused on the roles of Fbln7 and its C-terminal fragment, Fibulin-C, in the regulation of macrophage functions under inflammatory and cancer conditions. To this end, various phenotypes of macrophages were generated, including differentiation of THP-1 and human blood-derived primary monocytes into macrophages; generation of M1 and M2 polarized macrophage; differentiation and polarization of monocytes into breast tumor supernatant derived TAMs in vitro. Besides, we have also studied the surface charge potential on various phenotypes of macrophage using zeta potential measurement. Specific surface molecules could identify distinct functional phenotypes of macrophages, and studies have shown that modulation of surface charges could alter the phagocytic function of macrophages. Our results show that activation of macrophages with Lipopolysaccharide (LPS) or IL-1β or polarization into the M1 phenotype resulted in a significant decrease in the zeta potential compared to freshly isolated monocytes, unstimulated macrophages, and M2 polarized macrophages. Interestingly, as the phagocytosis starts at 15 mins and 45 mins post-initiation, the surface potential significantly increases, possibly due to the rearrangement of cell surface molecules. Furthermore, in LPS induced systemic inflammation model, bone marrow cells isolated after 2 h of LPS injection showed a significant reduction in zeta potential compared to naïve cells. These data suggest that the expression of different protein molecules on macrophages under different environments may contribute to the zeta potential and that the technique could be useful in monitoring macrophage functional phenotypes. The work presented in the second part of the thesis shows that both full length (Fbln7- FL) and the C-terminal fragment, Fbln7-C could inhibit the differentiation of M-CSF–induced monocyte-derived macrophages. This was demonstrated by morphological studies of change in the surface expression of mannose receptor (CD206), major histocompatibility complex-II, and CD11b. Both the proteins with a stronger effect with Fbln7-C reduced the inflammatory cytokine and ROS production from primary monocytes in vitro. Additionally, in an LPS induced systemic inflammation model, Fbln7-C and Fbln7-FL reduced the infiltration of immune cells, such as neutrophils and macrophages, into the inflamed peritoneum. However, only Fbln7-C could improve the survival of septic animals. These results indicated that Fbln7 and Fbln7-C could have therapeutic potential for inflammatory diseases via modulation of immune cell functions. Finally, in the last part of the thesis, studies were performed to investigate the potential of Fbln7-C as a modulator of tumor-associated macrophages (TAMs), and it's potential as an anticancer therapeutic. Our in vitro data shows that Fbln7-C could inhibit the tumor cell line (MDAMB- 231) supernatant induced reprogramming of human monocytes-derived TAMs. This was concluded based on the finding that there was higher expression of pERK1/2 and pSTAT1 molecules, and reduced expression of CD206 protein and arg1, ido, and vegf transcripts in monocytes cultured in the presence of Fbln7-C compared to media only controls. Interestingly, Fbln7-C treated TAMs retained their M1 like phenotype even after the removal of Fbln7-C, and their secretome demonstrated anti-cancer activities. Furthermore, in a 4T1 induced murine breast tumor model, intravenous administration of Fbln7-C, following the appearance of measurable tumors, significantly reduced the growth and weight of the tumors. Detailed phenotypic analysis of the infiltrated monocyte/macrophage populations (F480+Ly6G-CD11b+) at day 23 post-induction showed a higher percentage of inflammatory monocytes (Ly6ChiCD11b+) and a delayed differentiation into anti-inflammatory TAMs as evident by their reduced levels of CD206 expression. These data suggested that Fbln7-C could regulate the tumor environment-induced macrophage reprogramming and has the potential for cancer therapeutics. To summarize, the above study indicated that Fbln7-C or its bioactive fragments could be explored as a potent immune-modulator in inflammation and cancers.