Exploring the Role of Topoisomerase II Beta in Macrophage Maturation and Pro-inflammatory Cytokine Production

Abstract

Although it is known that DNA topo II?is required for the regulation of transcription during neural development and differentiation, it is not clear whether the enzyme is required during differentiation of human monocytes into macrophages and/or the subsequent transcription of cytokine genes. To test this, a robust model of differentiation of monocyte-like cells into macrophage-like cells using U937 and HL-60 cells treated with phorbol 12-myristate 13-acetate (PMA) and Lipopolysaccharide (LPS) was validated. Differentiation was determined by morphological and growth characteristics and CD11b surface antigen expression as determined by flow cytometry. qRT-PCR was also used to measure mRNA transcript levels of key genes known to be up-regulated during monocyte differentiation and the secretion of pro-inflammatory cytokines produced by differentiated cells were measured using ELISA. siRNA topo II?knockdown did not hinder monocyte-like cells from undergoing differentiation, however experiments revealed a correlation between topo II?knockdown and secreted TNF?, with the latter decreasing when topo II?was reduced. This pattern was also noted when measuring IL-1?secretion. Similar results were seen using a Murine transgenic fibroblast cell line lacking topo II?, which when stimulated with LPS secreted significantly lower levels of IL-6 compared to the wild type cells. Thus topo II?expression is necessary for secretion of normal levels of the cytokines, TNF?, IL-1?and IL-6 in response to LPS at certain time points. In addition in the macrophage-like state of the two cell lines, the relative levels of the ?isoform (mRNA and protein) were shown to be significantly increased compared to ?, further outlining the importance of topo II?in the differentiated state. Chromatin immuno-precipitation followed by qPCR showed however that topo II?was not associated at three defined proximal promoter regions of either the TNF?and IL-1?genes, although further studies are required to rule out a direct association of topo II?with these and other regions of the genes. Down regulation of topo II?protein using the inhibitor ICRF-193 did not hinder monocyte-like cells from undergoing differentiation either. However, contrary to the knockdown results, a 6 h pre-treatment with 1 nM ICRF-193 increased TNF?levels in these cells, both at the mRNA and the protein level, along with a slight increase in secreted TNF?. NF-?B, EGR2, TLR4 and TLR2 transcript levels were also increased under these conditions. Thus further studies are required to determine if these increases are due to additional cellular effects of the drug or whether topo II?may play an inhibitory effect on transcription. Thus it is clear that topo II?plays an important role in expression of cytokines and understanding the exact nature of this requires further research that may yield potential new avenues for treatment of disease.

Date of Award	1 Jul 2014
Original language	English
Awarding Institution	Northumbria University
Supervisor	Kay Padget (Supervisor)