Author: Adewunmi Olumuyiwa Adelaja

Publisher:

Published: 2020

Total Pages: 196

ISBN-13:

Macrophages are ubiquitous tissue-resident cells that are essential for tissue homeostasis and function. Macrophages initiate, coordinate, and resolve the inflammatory response to pathogens, as well as coordinate tissue repair programs. The inflammatory program of macrophages is largely controlled by the inducible transcription factor NF B. The temporal pattern of NF B activity (signaling dynamics) regulates the immune response of macrophages to a diverse set of ligands. The extent to which NF B signaling dynamics are stimulus-specific is not known. Furthermore, the functions of macrophages are regulated by the cytokine milieu. The effect that cytokine milieu may have on the stimulus specificity of NF B signaling dynamics in primary macrophages has not been reported. In Chapter 2, I examined the specificity of the temporal pattern of NF B nuclear translocation in response to diverse ligands associated with host, bacteria and viruses. Using macrophages isolated from knockin mice that express a fluorescent NF B fusion protein at endogenous levels, I measured and tracked nuclear NF B in individual cells over many hours using an automated image acquisition and analysis workflow. Using supervised machine learning, I quantified the stimulus specificity of NF B signaling dynamics by measuring the performance of ligand classification using NF B activity alone. Then, I tested the hypothesis that NF B signaling dynamics are less stimulus-specific in macrophages from a mouse model of a systemic autoimmune disease (Sj gren's syndrome (S.S.)). My results indicated that oscillatory characteristics that define host-associated and virus-associated ligands are greatly diminished. Close examination of results showed that the sensitivity of classifying host-associated ligands is nearly abolished in SS macrophages. Furthermore, dose response studies of NF B signaling dynamics revealed that the dose specificity of bacterium-associated ligands, but not host- and virus-associated ligands, are diminished in S.S. macrophages. In Chapter 3, I explored the effect of the cytokine milieu on the stimulus specificity of NF B signaling dynamics. Using time-lapse, live cell microscopy, I examined the effect of IFN , IL-4, and TNF conditioning on stimulus specificity of host-, virus-, and bacterium associated ligands. Supervised machine learning revealed that NF B response to virus-associated ligands and bacterium-associated ligands are less distinguishable in the context of IFN conditioning. In contrast, NF B responses to host-associated and pathogen-associated ligands is more distinguishable in the context of IFN conditioning. Examination of NF B dynamics in IFN conditioned macrophages revealed a loss of oscillatory character in response to virus-associated ligands but not to host-associated ligands. Since host-associated and virus-associated ligands induce predominantly oscillatory dynamics in na ve macrophages, abrogation of oscillatory character in response to virus-associated but not to host-associated ligands in IFN conditioning makes the NF B oscillations a distinguishing hallmark of host-associated ligands, at the expense of distinguishing virus-associated ligands from bacterium-associated ligands. The results showed that NF B dynamics are more stimulus-specific in the context of IL-4 conditioning. Close examination of the NF B dynamics showed IL-4 conditioning diminishes responsiveness of NF B translocation to virus-associated ligands, while it preserves the responsiveness to bacterium-associated and host-associated ligands and differentially enhances peak prominence of NF B to bacterium-associated ligands but not to host-associated ligands. Finally, interrogating the effects of TNF conditioning on stimulus specificity of NF B dynamics revealed that bacterium-associated ligands are nearly indistinguishable in the absence of constitutive tonic TNF. Further, NF B response to host-associated and bacterium-associated ligands are less distinguishable. In the absence of constitutive, tonic TNF, oscillatory characteristics of NF B signaling are abolished, which means that host- and bacterium-associated ligands both induce non-oscillatory NF B signaling, whereas NF B responsiveness to virus-associated ligands is nearly abolished. Furthermore, the absence of constitutive, tonic TNF and feedforward, paracrine TNF abrogate the dose specificity of NF B signaling in response to bacteria-associated and host-associated ligands, respectively. In conclusion, the work presented in this dissertation shows that the stimulus-specificity of NF B signaling in macrophages is greatly diminished in a murine model of Sj gren's Syndrome, an autoimmune disorder and that cytokine milieu control the specificity of NF B signaling in macrophages. These findings suggest that modulation of NF B signaling in macrophages by IFN , IL-4, and TNF signaling pathways may yield fruitful pharmaceutical targets for treating autoimmune and infectious diseases.