The cell biology of the FcRn-albumin recycling system

Abstract

Human serum albumin (HSA) is the most abundant protein in plasma and has an exceptionally long circulatory half-life of around three weeks in humans. The enhanced half-life properties of HSA result from the selective interaction with the neonatal Fc receptor (FcRn) in acidic endosomes, which protects endocytosed albumin from lysosomal degradation and mediates recycling back to the plasma membrane. Endothelial and innate immune cells are considered the most relevant cells for FcRn-mediated albumin homeostasis in vivo. However, little is known about FcRn-albumin cell biology in physiologically relevant primary cells and the spatiotemporal aspects of the FcRn-albumin interaction within intracellular endosomes. My studies have used cell biological and biophysical approaches to examine FcRn-albumin interactions and trafficking in primary macrophages and endothelial cells. <br /> Here, I used two independent biophysical approaches to visualise the intracellular receptor-ligand interactions within globular endosomes and tubular transport carriers of primary macrophages. Firstly, fluorescence lifetime imaging microscopy (FLIM) of Förster resonance energy transfer (FRET) and secondly, raster image correlation spectroscopy (RICS) to monitor the diffusion kinetics of single fluorescent-labelled HSA molecules. Based on these analyses, I identified an interaction between FcRn and albumin within intracellular endosomes, and emerging tubules, in human FcRn-expressing macrophages. Furthermore, I detected a higher population of immobile, FcRn-bound wildtype HSA molecules within the lumen of endosomal structures compared to the non-FcRn binding rHSAH464Q mutant. My findings revealed the kinetics of FcRn-albumin binding within endosomal structures for recruitment into transport carriers for recycling. <br /> To investigate FcRn-albumin cell biology in physiologically relevant primary endothelial cells, I established cell lines of primary human vascular endothelial cells from the outgrowth in culture of blood endothelial precursors known as blood outgrowth endothelial cells (BOECs). My observations show that these endothelial cell lines internalised fluorescent-labelled HSA efficiently via fluid phase macropinocytosis. Intracellular HSA molecules co-localised with FcRn in endosomal structures potentially allowing the interaction of the receptor with its ligand. Wildtype HSA, but not the non-FcRn binding rHSAH464Q mutant, was sorted into FcRn-positive tubular transport carriers, that are likely to mediate recycling of endocytosed HSA back to the plasma membrane. These findings support the proposed contribution of vascular endothelial cells to albumin homeostasis in vivo. <br /> Understanding the underlying mechanisms of FcRn-albumin cell biology and the contribution of different cell types to albumin homeostasis is important for the design and generation of half-life extended albumin fusion proteins for the treatment of serum protein-related diseases such as hemophilia A (HemA). Despite exhibiting enhanced pharmacological properties, to date, very few albumin fusion protein therapeutics have been approved for the treatment of human patients. In particular for HemA, the treatment using recombinant coagulation factor VIII (FVIII) products is aggravated by the frequent development of inhibitory antibodies against FVIII in HemA patients which subsequently have to undergo highly expensive and burdensome immune tolerance induction protocols. <br /> In this study, I have established an imaging flow cytometry-based antigen uptake assay to investigate the internalisation of FVIII-Albumin fusion proteins by FVIII-specific B cells expanding the knowledge about how albumin fusion proteins might contribute to immune tolerance induction towards FVIII in vivo. Additionally, I established two in vitro protocols which, in combination, allow the generation of high numbers of FVIII-specific regulatory T cells. These antigen-specific Tregs have the potential to suppress immune responses against recombinant FVIII in vivo and represent an alternative approach to facilitate immune tolerance towards FVIII in HemA patients. <br /> In summary, this thesis has revealed the fundamental aspects of FcRn-albumin cell biology and trafficking in primary macrophages and endothelial cells, and potential strategies for immune tolerance induction using FVIII-Albumin fusion proteins in the context of HemA.