CCR7-Mediated Dendritic Cell Migration: A Multilevel Analysis of Metabolic and Regulatory Mechanisms

Abstract

Regulated cellular motility is a defining characteristic of immune cell functionality, which necessitates the intricate coordination of the actin cytoskeleton, metabolic dynamics, and the expression of cell surface receptors. While the cytoskeletal role has been extensively characterised, the underlying mechanisms governing receptor expression and metabolic turnover remain inadequately elucidated. The <em>CC-chemokine receptor 7</em> (CCR7) is a pivotal protein that facilitates immune cell trafficking, orchestrating <em>dendritic cell</em> (DC) migration to lymphatic organs and mediating homeostatic T cell homing. Despite the established significance of CCR7 in directed migration, the precise mechanisms through which CCR7 modulates this process within the complex tissue microenvironment remain contentious. <br> This thesis aims to investigate the multifaceted regulation of CCR7-mediated DC migration by examining its associated metabolic adaptations, variations in cell surface expression, and the downstream signalling pathways involved. <br> Chapter 3 focuses on lipid metabolism during DC maturation and migration. Specifically, the metabolic fates of exogenous alkyne-tagged <em>fatty acids</em> (FA) in both immature and mature DCs were scrutinised. Lipidomic analysis indicated a marked increase in ceramides and <em>triacylglycerides</em> (TAG) in mature DCs. Notably, the integration of alkyne-tagged FAs primarily occurred within TAGs, and a subsequent decrease in TAG content was observed following DC migration. This finding implies that mature DCs utilise TAGs as an energy reservoir to support energy-intensive processes such as migration. <br> In Chapter 4, the mechanisms governing CCR7 surface presentation were investigated. A genome-wide CRISPR knockout screen was employed to identify novel regulators of CCR7 surface expression. This screening effort unveiled several potential positive and negative regulators, with particular emphasis on the positive regulators. Among these, the prostaglandin <em>E2 receptor 4</em> (EP4) emerged as a key factor controlling CCR7 surface expression through an as-yet undefined mechanism. Other promising candidates, such as <em>Resistance to inhibitors of cholinesterase 8</em> (Ric8) and <em>C1Galt1 Specific Chaperon 1</em> (Cosmc), were also identified, suggesting their potential roles in regulating CCR7 through direct G-protein interaction or post-translational modifications, respectively. <br> Chapter 5 elucidates the downstream signalling and transcriptional responses initiated upon CCR7 activation. A comprehensive RNA sequencing analysis was performed to identify new downstream signalling pathways activated by CCR7 engagement. The results indicated that chemokine stimulation induces a restricted and maturation-specific transcriptional response without a clear activation of a specific pathway. However, β1,3-<em>N</em>-acetylglucosaminyltransferase 2 (B3gnt2) was recognised as a consistently differentially expressed gene across all here tested maturation conditions. This gene was implicated as a potential negative regulator of CCR7 surface expression, highlighting its potential central role in modulating DC trafficking. <br> In summary, this thesis offers a detailed examination of the metabolic and regulatory mechanisms that underpin CCR7-mediated DC migration. The findings enrich our understanding of lipid metabolism's role in DC functionality and the dynamic regulation of CCR7 surface expression. These insights bear significant implications for understanding immune cell trafficking and its relevance in both health and disease contexts.