School of Molecular & Cell Biology (ETDs)

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Differential expression analysis of PMA and 1,25(OH)2D3-induced monocyte-to-macrophage differentiation in THP-1 cells
(University of the Witwatersrand, Johannesburg, 2023-09) Perumal, Kelda Chloe; Meyer, Vanessa; Gentle, Nikki
The process of monocyte-to-macrophage differentiation is studied in vitro through the use of promonocytic model cell lines, such as the THP-1 cell line, where commonly used differentiation inducing agents include phorbol-12-myristate-13-acetate (PMA) and the active metabolite of vitamin D3, (1,25(OH)2D3; VD3). While both induce differentiation, differences in their mechanisms of action, as well as how the end states of the differentiation process differ, are not well understood. Therefore, this study used computational approaches to compare the effects of PMA and VD3 on the differentiation of monocytes into macrophages, using the promonocytic THP-1 cell line. Through the use of RNA-sequencing, gene expression was quantified in differentiated and undifferentiated THP-1 cells, treated with both PMA and VD3. Differential gene expression analysis was performed to determine genes that were differentially expressed as a result of either treatment relative to the untreated cells. This was followed by over-representation analysis to determine the pathways and processes in which the differentially expressed genes (DEGs) were involved. PMA treatment (3 989 DEGs) resulted in more changes in expression relative to VD3 treatment, where only 384 genes were found to be differentially expressed in response to treatment with VD3. Only TFE3, KIT and TRIB1 were observed to be crucial to the process of differentiation, irrespective of treatment. Apart from this, the treatments were observed to largely involve different biological pathways, resulting in cells that were phenotypically distinct from each other at the transcriptional level. This included changes observed in the expression of genes encoding transcription factors known to be involved in the differentiation process, such as CEBPA, GATA2, IRF8 and PU.1, as well as those encoding surface markers representative of monocytes and macrophages, such as CD14, CD64 and CD11b. The expression patterns observed here indicate that, at least at the concentrations and time points included in this study, PMA and VD3 induce macrophage-like cells that are at different stages of differentiation and are not comparable to either each other or primary macrophages. Furthermore, key differences observed in the expression of genes encoding pathogen recognition receptors and cytokines suggest that which differentiation inducing agent is used may have important implications for these cells’ capacity to recognise pathogens and produce cytokines. The findings of this study therefore emphasise that it is crucial to carefully consider the choice of differentiation-inducing agent when using THP-1 cells as an experimental system for studying monocyte-to-macrophage differentiation.
Differential Gene Expression Analysis of PMA Treated Pro-monocytic Cell Lines
(University of the Witwatersrand, Johannesburg, 2023) Kama, Asavela Olona; Meyer, Vanessa; Gentle, Nikki
HL-60, THP-1, and U937 are model cell lines that can undergo myeloid differentiation in vitro, allowing the study of myeloid cell function in drug metabolism, cytotoxicity, and the aetiology of infections. However, the differentiated end-state of these cells is not well characterised. Moreover, cell line-specific differences in the level of gene expression may confound results obtained from such studies. The aim of this study was thus to compare changes in gene expression between HL-60, THP-1, and U937 cells in response to the differentiation agent, phorbol 12-myristate 13-acetate (PMA), 48 hours after treatment. Gene expression profiles were compared across all three cell lines prior to and post-PMA treatment. Differential gene expression analysis between treated and untreated cells was performed using DESeq2 (v 4.2). Gene over-representation analysis was performed using cluster Profiler (v 4.0). HL-60, THP-1, and U937 cells had similar expression profiles prior to PMA treatment, but different sets of genes were significantly differentially expressed in these cell lines 48 h after treatment with PMA. A total of 475 genes were consistently differentially expressed across all cell lines. These genes were found to be involved in phagosome formation and cell cycle transition. HL-60, THP-1, and U937 cells had 944, 1231, and 624 uniquely differentially expressed genes, respectively. These genes were predominantly involved in energy metabolism and pathogen recognition. Overall, THP-1 cells showed greater potential to detect viruses, while U937 cells showed greater potential to detect bacteria. From this, it can be concluded that while all three cell lines did indeed undergo myeloid differentiation, the macrophage-like cell state produced in each case differed between cell lines.
Identifying Markers of Differentiation in Monocyte-Derived-Macrophages
(University of the Witwatersrand, Johannesburg, 2024-08) Gibson, Matthew Leo; Cronjé, Marianne; Gentle, Nikki
The importance of monocytes and monocyte-derived macrophages (MDMs) in both adaptive and innate immunity makes their study a topic of interest. Monocytes differentiate into macrophages through transcriptomic alterations, resulting in extensive changes in gene expression. Macrophage colony stimulating factor (M-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF) are the two primary cytokines that stimulate this differentiation, and are known to cause partial polarisation towards the M2 and M1 macrophage subtypes, respectively. However, the degree to which this polarisation takes place is not well-characterised. Therefore, this study aimed to use a computational approach to identify the differences and similarities in gene expression changes in macrophages induced with M-CSF and GM-CSF. RNA sequencing data for three human donors was obtained through EBI and used to quantify gene expression changes associated with M-CSF or GM-CSF treatment. Differential gene expression analysis was performed to identify the genes that were differentially expressed as a result of either treatment relative to the untreated monocytes. Over-representation analysis was used to determine the biological processes in which the differentially expressed genes (DEGs) were involved. Finally, transcription factors were identified within the lists of DEGs, as well as the genes encoding their known protein-protein interacting partners. Treatment with M-CSF and GM-CSF induced 4 072 and 4 399 DEGs, respectively, 2 734 of which were common. An examination of these DEGs revealed that the resultant macrophages lacked changes in expression of genes commonly associated with the M1 and M2 polarisation states. An investigation of the DEGs involved in myeloid cell differentiation and the regulation of inflammatory response revealed CCR2, IGF1 and INHBA to be inversely regulated by the two treatments. Furthermore, nine uniquely differentially expressed transcription factors involved in these biological processes were identified, each of which may be contributing to the lack of complete polarisation following differentiation. These results revealed that M-CSF and GM-CSF-induced macrophages, in the absence of activation, experience highly similar gene expression changes and lack changes in the expression of key polarisation marker genes.
Immunomodulation of the innate immune system: The role of vitamin D in the context of monocytes and macrophages
(University of the Witwatersrand, Johannesburg, 2024-07) Mol, Bronwyn Ashleigh; Gentle, Nikki; Meyer, Vanessa
Macrophages are widely distributed cells of the innate immune system with essential roles in homeostasis and disease. Despite concerted efforts, several aspects of macrophage origin, biology, and functionality remain poorly understood. To gain a deeper understanding of these cells, a physiologically relevant, but practical model is required. In vitro, macrophages are principally generated from primary monocytes and monocyte-like cell lines through a natural process referred to as monocyte-to-macrophage differentiation. Monocyte-like cell lines have several practical advantages over the use of primary monocytes with the most commonly employed monocyte-like cell lines being THP-1 and U937 cells. Despite their frequent use, no standardised protocol is employed in the differentiation of monocyte-like cell lines to macrophages. Naturally, this results in large discrepancies and a lack of comparability between studies. Furthermore, many of these protocols are not physiologically relevant and produce macrophages that are not responsive to downstream stimuli. 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the biologically active form of vitamin D3, is a recognised immunomodulator that shows pronounced genomic and non-genomic effects in immune cells. It is also reported as an inducer of monocyte-to-macrophage differentiation, though heavily debated, and a potential macrophage polarisation agent. Despite this, there is relatively little information concerning the role of 1,25(OH)2D3 in monocyte-to-macrophage differentiation and macrophage biology. This study aimed to develop a more physiologically relevant differentiation protocol for the monocyte-like THP-1 and U937 cell lines. This model was then used to investigate the role of 1,25(OH)2D3 in monocyte-to-macrophage differentiation and macrophage biology. Assessment of morphological features and the macrophage markers, CD11b and CD14, indicated that in both THP-1 and U937 cells, differentiation induced using a combination of 5 nM of phorbol 12-myristate 13-acetate (PMA) and 10 nM 1,25(OH)2D3 over 96 hours produced the most mature macrophages. It was observed that 1,25(OH)2D3 alone was not capable of inducing differentiation, yet when combined with PMA, greatly enhanced macrophage features. THP-1 cells are the most widely employed monocyte-like cell line, and are proposed to be the most reflective of primary monocytes. In this study these cells were shown to be more responsive to the effects of 1,25(OH)2D3 than their U937 counterparts. As such, RNA-sequencing was used to explore the efficacy of the proposed differentiation protocols and the influence of 1,25(OH)2D3 on macrophage biology in THP-1 cells. Differential gene expression analysis confirmed that the most effective differentiation protocol was the combination of 5 nM PMA with 10 nM 1,25(OH)2D3 when considering macrophage associated features including transcription factor usage, adhesion, phagocytosis, and cytokine and cytokine receptor expression. This protocol also produced THP-1-derived macrophages that showed increased expression of genes considered to be primary macrophage markers. These results also suggested that THP-1 cells differentiated with neither PMA nor PMA with 1,25(OH)2D3 were likely to represent fully polarised macrophages. 1,25(OH)2D3 treatment of THP-1 monocytes and THP-1-derived macrophages produced distinct gene expression profiles with considerably less overlap than expected. Though 1,25(OH)2D3 treatment often affected similar biological processes in both cell types, the genes within these processes found to be differentially expressed in each cell line were often distinct. For example, in THP-1- derived macrophages, but not THP-1 monocytes, 1,25(OH)2D3 treatment resulted in the increased expression of genes encoding numerous antibacterial peptides, several small GTPases and their regulators. Additionally, several type I interferon response related proteins showed decreased expression, while expression of cytokines and cytokine receptors was variable. This, taken together with the morphological work, indicates two potential roles for 1,25(OH)2D3 in macrophages. Firstly, a protective role as it suggests the potential to prime an antibacterial response, while still balancing inflammatory responses and protecting against autoinflammation induced by aberrant type I interferon response. Secondly, a potential role in determining the morphological features, clearly demonstrated through microscopy, and further suggested by the differential expression of a variety of small GTPases and their regulators.
Using ChIP-seq and Gene Expression Microarray data to explore transcriptional dysregulation of PXDN and PXDNL in cardiovascular diseases
(University of the Witwatersrand, Johannesburg, 2024) Naidoo, Shiven; Gentle, Nikki; Mavri-Damelin, Demetra
Background: Cardiovascular diseases (CVDs) remain one of the leading causes of death globally. The genes PXDN and PXDNL are both expressed in the cardiovascular system, and their dysregulation has been linked to various disorders, including CVDs, but little is known of their transcriptional regulation in the cardiovascular system or their roles in CVD pathogenesis. Methods: This study developed two custom bioinformatics pipelines in R to mine and analyse ChIP-seq data from ChIP-Atlas and gene expression microarray data from the Gene Expression Omnibus (GEO). The first pipeline used ChIPseeker to identify regulatory transcription factors (TFs) of PXDN and PXDNL in cardiovascular cells and tissues. ChIP-seq data from 400 experiments across 63 TFs was filtered to isolate TFs with high confidence binding peaks in the promoter and first intron of PXDN and PXDNL. The second pipeline used R Bioconductor packages to explore the expression profiles of PXDN, PXDNL, and their TFs in seven microarray datasets across three CVD-related contexts: cardiomyopathies, heart failure and TNF-α stimulation. Results and discussion: This study identified 27 TFs binding to PXDN and 18 TFs binding to PXDNL in cardiovascular cells. Sixteen of these TFs were shared by both PXDN and PXDNL, suggesting potential coregulatory mechanisms in cardiovascular cells where they are both expressed. Unique TFs were also identified for PXDN (11) and PXDNL (2). Differential gene expression analysis revealed no significant change in expression (log2FC > 0.5; p.adj < 0.05) for PXDN, PXDNL and many of their identified TFs in the CVD-related conditions investigated, suggesting that changes at the transcript level may not contribute to the progression of these conditions. Conclusions: This study advances our understanding of the transcriptional regulation of PXDN and PXDNL in healthy cardiovascular cells as well as their expression levels in the investigated CVD-related contexts. This study also contributes a bioinformatics pipeline which can be further developed and applied to analysing data from ChIP-Atlas and GEO. Future research can elucidate the roles of each TF in regulating PXDN and PXDNL in healthy and diseased cell lines