Document Type

Thesis

Degree Name

Master of Science (MSc)

Department

Biology

Program Name/Specialization

Integrative Biology

Faculty/School

Faculty of Science

First Advisor

Dr. Sarah Poynter

Advisor Role

Principal Investigator

Abstract

Immunostimulatory nucleic acids, such as long double-stranded RNA (ds)RNA, can stimulate innate immune responses in a non-sequence specific manner. These molecules are recognized by pattern-recognition receptors in the cytoplasm, endosome, and on the cell surface. Activation leads to the production of mediators of innate immune pathways, including type I interferon and proinflammatory cytokines. Although these receptors are present in all cells, their role in non-immune cells is often overlooked due to their lower level of responsiveness. Nanocarriers can enhance these nucleic acid-mediated responses, allowing for exploration of innate immune pathways in non-immune cells. Nanoparticles as carriers of nucleic acids can improve cellular delivery, protect from nuclease degradation, and concentrate nucleic acids, enabling multivalent cellular presentation for a more potent response. In my thesis, poly inosinic:cytidylic acid (poly I:C), a commonly used dsRNA in pathway research and therapeutics, was delivered to cells using a previously explored carrier, nanophytoglycogen (NDX). Pathways surrounding uptake, signalling molecules, and overall immune responses were explored. Two non-immune human cell lines were used, immortalized human fibroblasts (THF) and embryonic kidney cells (HEK-293). In THF cells, immunofluorescent analysis demonstrated that NDX-delivery significantly enhanced poly I:C uptake and demonstrated the involvement of class A scavenger receptors (SRAs). Lysosomal staining indicated that poly I:C-NDX is present in low-pH vesicles after uptake. THF cells mount an enhanced immune response to poly I:C-NDX compared to poly I:C, measured using qRT-PCR for a classic interferon-stimulated gene, IFIT-1. Using THF cells lacking TLR-3, an endosomal receptor, and the type I interferon receptor, IFNAR, we observed a significant decrease of type I IFN response after treatment with poly I:C-NDX, demonstrating the reliance on these receptors. Knock-down of RIG-I-MDA5 or IPS-1 did not significantly change responses to poly I:C-NDX. HEK-293 cells modified with reporter assays for proinflammatory and type I interferon pathways were used to further explore cytokine outcomes in which cells with or without TLR3-overexpresison were tested. All cells produced both interferon and proinflammatory responses to poly I:C and poly I:C-NDX. Cells overexpressing TLR-3 had a significantly improved response to poly I:C-NDX compared to poly I:C, further indicating its importance in the ability for cells to robustly respond to the nanocarrier. The data from this thesis will contribute to the knowledge base surrounding nanoparticle-mediated dsRNA recognition and innate immune pathway activation in non-immune cells, leading to a better understanding of the fundamental mechanisms of innate immunity in vertebrates and contributing to potential future antiviral and anticancer therapeutics.

Convocation Year

2024

Convocation Season

Fall

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