Graduation Date

Fall 12-15-2017

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Biochemistry & Molecular Biology

First Advisor

Dr. Kaustubh Datta

Abstract

Macrophages are extremely heterogeneous and highly plastic hematopoietic cells that reside in all tissues and act as a bridge between the innate and adaptive arms of the immune responses. Besides, they undertake a wide array of housekeeping functions like, clearance of cellular debris that arise due to regular turnover in tissues, iron homeostasis, immune surveillance as well as tissue repair processes post inflammation. They are also causally associated with several clinical conditions, including cancer where the infiltration of macrophages contribute to disease progression, metastasis and therapy resistance, and thereby poor clinical outcome.

Neuropilins (NRPs) are non-tyrosine kinase cell surface glycoproteins expressed in all vertebrates and widely conserved across species. The two isoforms, NRP1 and NRP2 are mainly known for their role as co-receptors for class III Semaphorins and for members of the vascular endothelial growth factor (VEGF) family of molecules. Both NRP1 and NRP2 are overexpressed in many cancers, exert pleiotropic effects in various aspects of cancer pathobiology and correlate with the stage and grade of the disease and poor survival. Intriguingly, additional immunoregulatory roles for NRPs have been reported in myeloid and lymphoid cells, in normal physiological as well as different pathological conditions, including cancer, various immunological disorders, and bone diseases. In comparison to NRP1, the overall comprehensive function of NRP2 in macrophages and factors that govern those functions is not well known. Therefore, the enveloping goal of this dissertation is to comprehensively understand the role and function of NRP2 in macrophages and their implication in tumor associated macrophages in solid tumor.

In the first part of the dissertation, we primarily investigated the expression pattern of NRP2 during the differentiation of macrophages under inflammatory (M1) and immunosuppressive (M2) polarizing conditions. We observed although NRP2 is not expressed at a detectable level in monocytes, its expression is upregulated during the differentiation of monocytes precursors to either M1 or M2 type mature macrophages. We identified a novel function of NRP2 in regulating phagocytosis in macrophages by modulating the levels of Rab5+ early phagosomes and Rab7+ late phagosomes. In NRP2 depleted macrophages clearance of bacteria or yeast components was delayed and these cells exhibited elevated number or early phagosomes and decreased number of late phagosomes.

Since pancreatic cancer (PC) typifies the complex architecture of a tumor stroma and tumor associated macrophages are abundantly present here, the second part of the dissertation is aimed at elucidating the implication of NRP2 regulated phagocytosis in TAMs in this lethal disease. The expression NRP2 in TAMs in human and mouse PC tissues was assessed. Due to nutrient limiting conditions and rapid proliferation of tumor cells in the TME, many of the cancer cells undergo apoptosis. TAMs efferocytose these dying tumor cells, an event that polarizes them further to pro-tumoral type and contribute to relapse and therapy resistance. We observed that in the absence of NRP2, clearance of apoptotic cells was delayed. The implication of this observation in PC progression was tested using a subcutaneous tumor model. Upon depletion of NRP2 in TAMs, tumors were smaller. Of note, NRP2 depletion did not affect TAM recruitment to, or angiogenesis but recruited and activated anti-tumor CD8 cells into the TME. Further, RNA-Seq analysis of CD11b+ TAMs isolated from tumors indicated that many genes associated with either endosomal/phagosomal maturation, recycling and exocytosis, immune modulation and leukocyte recruitment and activation are deregulated following NRP2 depletion. Further, NRP2 depletion downregulated many of the immunosuppressive genes and cytokines in macrophages that may be important for either suppression of T cell response or induce Treg formation. The data further suggested that NRP2 is important for efferocytosis and maintaining TAM-like phenotype in intratumoral macrophages, thus helping to create a tumorigenic immunosuppressive environment in the TME.

Taken together, studies in this dissertation document a requirement for NRP2 in macrophage phagocytosis. Further, regulation of efferocytosis in TAMs by NRP2 have implications in TAM functions and tumor progression. Hence, targeting NRP2 axis in TAMs may help re-engineer the immunosuppressive stroma in the TME and emerge as potential therapy in combination with chemotherapy or other adjuvant therapies.

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