Effects of Cadmium on Neutrophil Biology and the Potential Role in Autoantigen Formation in Rheumatoid Arthritis
Date: 2 March 2020
University of Exeter
Doctor of Philosophy in Medical Studies
Rheumatoid arthritis (RA) is an autoimmune disorder for which there is no known cause. The disease is characterized by progressive joint destruction, mediated by breakdown of immune tolerance by immune system dysfunction, influenced by several factors. This results in the development of self-reactive proteins, called autoantigens. In ...
Rheumatoid arthritis (RA) is an autoimmune disorder for which there is no known cause. The disease is characterized by progressive joint destruction, mediated by breakdown of immune tolerance by immune system dysfunction, influenced by several factors. This results in the development of self-reactive proteins, called autoantigens. In RA, the dominant autoantibodies are directed to modified host proteins that are citrullinated and carbamylated. These modifications are driven by peptidyl-arginine deiminase (PAD) and myeloperoxidase (MPO), which are found in the greatest quantity in neutrophils, the major white blood cell of the immune system. The primary anti-microbial activity of these cells is characterized by several distinct functions: phagocytosis, degranulation and neutrophil extracellular trap (NET) production. NETosis is a unique cell death mechanism in which neutrophils unwind chromatin fibres from histone proteins, and through packaging with antimicrobial peptides, are released from the cell to ensnare and kill bacteria. These cells have been previously suggested as a driver in RA pathogenesis. The toxic heavy metal, cadmium, has been suggested in recent years as a potential contributing factor in RA, although the effect of cadmium exposure on neutrophil biology and function has not been fully explored. This thesis aimed to investigate serum autoantigens in RA, and explore the potential interplay between cadmium and neutrophil function in order to discern any dysfunctional processes that could promote autoantigen development. Primary neutrophils were incubated with cadmium chloride and the effect on essential cellular functions was examined. Cadmium did not modify the phagocytic capacity of neutrophils, although it increased intracellular calcium levels. Cadmium also enhanced the processes of the reactive oxygen species production and NETosis upon co-stimulation with phorbol 12-myristate 13-acetate (PMA) - an activator of protein kinase C. Serum samples from 10 healthy control donors, 18 patients with RA, 4 patients with bronchiectasis (BR) and 4 patients with bronchiectasis and RA (BRRA) were analysed by western blotting to identify citrullinated proteins. Bands of interest were excised and identified using mass spectrometry. The heavy chain of immunoglobulin G, in addition to alpha-1-antitrypsin were found to be citrullinated in all samples, though in a much greater extent in RA patients. In addition, the same proteins also had a greater level of carbamylation than citrullination. Taken together, these modifications provide novel evidence into how post-translational modifications may play a role in rheumatoid factor development in RA patients. As both PAD and MPO require elevations in the cytosolic calcium level to function, the effect of cadmium on neutrophil function may play a priming role in the cell, such that a second stimulus may give rise to a more exaggerated response during inflammation and a more permissive environment for autoantigen development. Future work should examine the effect on MPO and PAD release after a period of cell priming with cadmium, and direct effects on immunoglobulin carbamylation/citrullination. This may allow greater insight into potential pathways through which RA may develop.
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