Date of Award

2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Cellular, Molecular and Biomedical Sciences

First Advisor

Eyal Amiel

Abstract

Known for their irregular shape and dendritic-like protrusions, dendritic cells (DCs) are the cellular link between innate and adaptive immunity. Upon encountering microbial products or pathogen-associated molecules through Toll-like receptors (TLRs), DCs undergo extensive metabolic and functional changes that support migration through the lymphatic architecture to secondary lymphoid organs where they initiate T lymphocyte-mediated adaptive immune responses. Along the way, DCs produce metabolites and inflammatory mediators helping to orchestrate and fine-tune effective antimicrobial defense mechanisms and subsequent immune responses to pathogens.DC activation is marked by: (I) metabolic reprogramming favoring glycolysis for generation of biosynthetic metabolic intermediates and (II) widespread gene expression changes including induction of key metabolic enzymes, such as inducible nitric oxide synthase (iNOS), a potent producer of toxic gaseous molecule nitric oxide (NO). NO acts first as a potent antimicrobial agent; however, is also linked to inhibition of several vital cellular activities including mitochondrial respiration. Inhibition of mitochondrial respiration has been shown to occur at discrete concentrations of activating stimulus and are directly linked to levels of iNOS expression and NO production, a phenomenon we have termed the mitochondrial respiration threshold. In this first part of this work, we used Real-Time Extracellular Flux Analysis to experimentally establish the NO-dependent mitochondrial respiration threshold; whereas the last part focuses on how this threshold is regulated. We explored the efficacy of two different iNOS inhibitors in blocking the iNOS reaction kinetically in real time and determined the temporal relationship between acute metabolic reprogramming and NO-mediated sustained metabolic reprogramming kinetically in single real-time assay. To explore the cellular regulation of the mitochondrial respiration threshold, we characterized the contribution of UDP-glucose as a modulator of NO-mediated mitochondrial respiration inhibition via the P2Y14 receptor in LPS-stimulated DCs. We demonstrated DCs in the presence of UDP-glucose enhance their proinflammatory profile through iNOS and IL-1, providing evidence for a new NO-dependent regulatory axis in DCs. Our studies provide novel applications for detection of NO-mediated metabolic effects in DCs and offer temporal insight into key metabolic events within DC activation. Overall, this work highlights the nuances of biological thresholds and metabolite signaling in activating situations, thus adding new ways to better model physiologically relevant myeloid cell encounters with pathogens.

Language

en

Number of Pages

260 p.

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Available for download on Tuesday, June 03, 2025

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