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Title: An optimised THP-1 cell line model for interrogating the effects of immune complexes on macrophage polarisation
Author: Re, Nikki Alexandra
ISNI:       0000 0004 7230 2083
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2017
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Full text unavailable from EThOS. Thesis embargoed until 01 Jun 2023
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Macrophages are a highly plastic group of cells which polarise into inflammatory or anti-inflammatory subtypes. Independent groups define macrophage subtypes differently, creating an inherent bias for subset-specific markers in the literature. Of additional interest, macrophages express Fc-Receptors and are hence activated by immune complexes (ICs); this has implications for these cells in IC-driven conditions such as rheumatoid arthritis. The acute myeloid leukemia derived THP-1 cell line provides a genetically identical baseline for studying the effects of ICs on macrophage phenotypes. However, a lack of consistency between published protocols on how to generate macrophages from these cells introduces technical issues. Hence the aims here were to identify a panel of macrophage polarisation markers that could be used to optimise a THP-1 cell line model, and to use this system to investigate the effects of immune complexes on monocyte and macrophage transcriptomes. M1 and M2a markers were isolated from publicly available primary macrophage datasets. THP-1 cell differentiation was optimised by titrating PMA concentration and recovery time. Concentrations of polarising cytokine and duration of cytokine exposure were varied to determine ideal polarisation conditions. RNA-seq was performed to validate the model and to test the effects of IgG1-HAGG on macrophage polarisation. Novel and established M1 and M2 markers were identified from public datasets. These transcripts were used to develop a THP-1 macrophage differentiation protocol; 5ng/ml PMA for 24h followed by 72h rest in media and subsequent polarisation with 20ng/ml IFNγ + 250ng/ml LPS (M1) or 30ng/ml IL-4 (M2a) for 48h. Comparisons of THP-1 macrophage and MDM datasets suggested that cell line and primary macrophages were similar. Changes in gene expression upon IC treatment appeared to be consistent for monocytes but not macrophages. IC treated Monocytes appeared to be enriched for some immune related genes, and an increase in target genes for specific transcription factors was reported; The IRF3 pathway was highlighted here. Overall, reliable macrophage polarisation markers have been identified, a THP-1 macrophage polarisation protocol mimicking primary cells has been developed and some interesting pathways have been highlighted in monocytes upon immune complex treatment.
Supervisor: Morgan, Ann ; Baxter, Euan ; Robinson, James ; Carr, Ian Sponsor: MRC
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available