ROLE OF INTEGRIN SIGNALING IN MACROPHAGE RECRUITMENT AND FUNCTIONAL PLASTICITY

Abstract

Macrophages are essential components of the innate immune system derived from the myeloid lineage, primarily known for their phagocytic abilities, aiding in the clearance of infections, removal of cellular debris and apoptotic cells, and strengthening host defense mechanisms. What's remarkable about macrophages is their adaptability to different environments, leading to the adoption of distinct phenotypes. These include the classically activated phenotype or M1 type, characterized by the secretion of pro-inflammatory cytokines like IL-12 and TNF-α. M1 macrophages exhibit heightened antigen-presenting capabilities and produce nitric oxide (NO) and other reactive oxygen (RO) intermediates. Conversely, alternatively activated macrophages or M2-type macrophages showcase an immunosuppressive function, secreting anti-inflammatory cytokines such as IL-10. They play pivotal roles in tissue remodeling, metastasis, and angiogenesis through the release of specific growth factors, vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-9. The activation of M2 macrophages is prompted by cytokines like IL-4, IL-13, or IL-10. Moreover, in tumor environments, macrophages infiltrate tumors and take on roles as tumor-associated macrophages (TAMs). TAMs predominantly exhibit characteristics akin to the M2 phenotype, although they may also display traits of M1 macrophages. Their diverse functional phenotypes within the tumor microenvironment significantly influence tumor progression and evasion of immune responses, highlighting their complex interplay with cancer cells and the immune system. Integrins, a family of cell adhesion receptors, have emerged as critical regulators of macrophage polarization. Notably, several reports have suggested that integrin signaling can be linked with the induction and maintenance of the immunosuppressive or M2-type functional phenotype of TAMs. Some of the latest reports show that integrins promote TAM adhesion to extracellular matrix components, triggering downstream signaling events that contribute to an immunosuppressive and tumorigenic microenvironment. Thus, understanding the complex role of monocytes/macrophages trafficking in the tumor microenvironment and the function of integrins in TAM polarization is critical for developing targeted therapeutic strategies that disrupt supportive interactions within the tumor milieu and boost anti-tumor immune responses. Although numerous studies have shown that different integrins play a role in immune cell function during cancer progression, growth, angiogenesis, and metastasis, to date, precise measurement of the integrin expression dynamics and their functional association with various functional subtypes of macrophages is not well defined. In this thesis, using a primary human monocyte-derived tumor supernatant-induced TAMs and a murine breast tumor model, explain the possible relationship between specific integrins and their roles in the adhesion, recruitment, and migration of monocytes, neutrophils, and macrophages from their progenitor site, bone marrow to different organs such as the peritoneum, tumor, and lungs in the breast tumor environment. In this study, a 4T1-induced BALB/c mouse model was used to investigate the role of various integrin molecules in monocyte, macrophage, neutrophils, and TAM polarization, migration over a 30-day period. The distribution of monocytes/macrophages and neutrophils was observed at various time points, including days 3, 7, 10, 15, 23, and 30. Cells migrate from the bone marrow to the blood and then to other organs, facilitated by a decrease in CXCR4 and an increase in CCR2. Integrin α5β1 was upregulated in CD11b+Ly6G-Ly6Chi monocytes and CD11b+Ly6G+ neutrophils in the bone marrow and blood after day 10 post-induction. Integrin αvβ3 levels were higher in F4/80+ macrophages in the peritoneum, tumor, and lungs. The neutrophils in the tumor and lungs have higher αvβ3 as compared to α5β1. Secondly, the work has evaluated the role of integrins and their ligands in macrophage polarization and programming into TAM during tumor progression. Monocytes infiltrate the tumor, differentiate into F4/80+ macrophages, and then polarize into M1 and M2 TAM. The data show that higher expression of integrin α5β1 was found on tumor-infiltrating monocytes (Ly6ChiMHCIIlow) and M1-TAMs (F4/80+Ly6ClowMHCIIhi), but higher levels of integrin αvβ3 were observed on M2-TAMs (F4/80+Ly6ClowMHCIIlow). Further analysis suggested that CD11b+Ly6G-F480+α5+ TAMs showed enhanced levels of inflammatory cytokines such as il-6 and tnf-α and CD11b+Ly6G-F480+αv+ TAMs demonstrated a pro-tumorigenic phenotype with higher levels of il-10, arg1, tgf-β transcripts. This explains the essential role of αvβ3 in the M2-type polarization in the breast tumor. On day 30 post-tumor induction, higher CD206, and MERTK expressing M2 TAMs in tumors and lungs exhibited upregulated expression of integrin αvβ3 and α5β1. To this end, various phenotypes of TAMs were generated from human blood-derived primary monocytes in vitro using breast (MDAMB-231, MCF-7) and pancreatic (Panc1, MiaPaca2) tumor supernatant. Blocking of integrins α5 and αv during macrophage differentiation in vitro reduced the expression of M2 markers like CD206 and CD163, as well as VEGF and arg1 transcripts. Next, bioinformatics tools were used to investigate the signaling molecules and pathways involved in monocyte and macrophage infiltration, migration, differentiation, and polarization of TAMs in association with integrins αvβ3 and α5β1 in breast cancer. This study examined RNA-seq data from the 4T1-induced murine breast tumor model from a GEO dataset (GSE195857). The dataset was divided into two categories: mammary gland macrophages (MGMs) from healthy mice vs. tumor-associated macrophages (TAMs) from tumors and bone marrow-derived monocytes from healthy (BMDM-Hs) vs. tumor-bearing mice (BMDM-Ts). The STRING database showed several genes, such as Focal adhesion Kinase (FAK, also known as ptk2) and Proto-oncogene protein tyrosine kinases (src, crk, lyn), which are involved in important signaling pathways controlling cell adhesion, motility, and survival are strongly associated with both integrins αv and α5. This suggests that FAK and Src-mediated signaling pathways may be involved in integrin-mediated immune cell infiltration and TAM reprogramming in the breast tumor microenvironment. The later part of the study aims to investigate the immunomodulatory effects of Homoeopathic medicines such as Arsenic album, Rhus Toxicodendron, Hepar sulphuris, and Bryonia alba of 30C potency on LPS-induced peripheral blood neutrophils and monocytes. LPS-stimulated neutrophils and monocytes showed significantly lower levels of pro-inflammatory cytokines (IL-6 and TNF-α) and pathogen recognition receptors (TLR-2, TLR-4) upon being treated with homeopathic medicines. Similarly, the presence of homoeopathic medicines reduced reactive oxygen species production and activation marker expression, such as CD44, CD69, and CD62L in stimulated cells. Stimulated cells showed different expression levels of cell adhesion receptors (e.g., integrin β1, β3, and αv), which regulate monocytes and neutrophil spreading. In conclusion, homoeopathic medicines have a significant effect on LPS-induced inflammatory responses in monocytes and neutrophils, providing empirical evidence for their beneficial effects. In summary, the presented thesis provides a comprehensive exploration of the intricate interplay between integrins and monocytes/macrophages within the context of breast cancer and inflammation. It also elucidates the roles of integrins in the migration of monocytes, neutrophils, and macrophages, emphasizing the dynamic changes in integrin expression during tumor progression, the influence of specific integrins on the differentiation and polarization of tumor-associated macrophage, and the molecular mechanisms associated with integrins in monocytes/macrophages.