Characterization of DEFA1A3 gene expression in human blood cells
Publication Date
December 11, 2019
Creator
Abstract
Alpha-defensins are small cationic peptides important in the immune response as they have antibacterial and virus properties and regulate the production of proinflammatory cytokines. Alpha-defensin 1-3 peptides are encoded in the copy number variable DEFA1A3 gene at chromosome 8p23.1 and are believed to be possible contributors to various inflammatory and autoimmune diseases. Here, we have answered the previously unresolved biological questions of establishing in which blood cell populations DEFA1A3 is expressed, assessing the DEFA1A3 mRNA expression levels and correlating the presence of DEFA1A3 mRNA with the presence of alpha-defensin 1-3 peptides. This project used a platelet-enriched plasma and density-gradient centrifugation protocols to separate platelets, granulocytes and peripheral blood mononuclear cells (PBMCs). PBMC subpopulations – T cells (CD3+), B cells (CD19+), monocytes (CD14+), natural killer cells (NK cells, CD56+) and other minor PBMC subsets – were further purified by a fluorescence activated cell sorting (FACS) procedure. Our results from DEFA1A3-specific polymerase chain reaction (PCR), quantitative-real time polymerase chain reaction (qRT-PCR) and immunocytochemistry, reconfirmed the presence of DEFA1A3 mRNA in granulocytes, which contributed to 71.4% of the whole blood DEFA1A3 gene expression levels, and their corresponding alpha-defensin 1-3 peptides respectively. We have also demonstrated the presence of DEFA1A3 mRNA, its contribution to the whole blood DEFA1A3 gene expression levels and the presence of alpha-defensin 1-3 peptides in platelets (1.4%) and in PBMCs (27.2%) – distributed between T cells (0.8%), B cells (1.0%), monocytes (3.4%), NK cells (83.8%) and the unstained fraction (11.0%). In conclusion, DEFA1A3 mRNA is present in all unstimulated white blood cells and platelets and can be associated with the presence of alpha-defensin 1-3 peptides. In whole blood, granulocytes are the major contributors to the DEFA1A3 gene expression levels; however, surprisingly the minor CD56+ NK cell subpopulation accounts for the majority of the DEFA1A3 gene expression levels attributed to PBMCs.
Item Type
ethesis
Thesis Type
MRes
Supervisors
Subjects (LC)
Associated Schools / Departments
School of Life Sciences
eprints ID
59496
UoN Repository URI
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