Ballantine, Lucy Elizabeth
Transcriptomic analysis of peripheral blood monocytes and synovial macrophages in inflammatory arthritis.
PhD thesis, University of Glasgow.
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Background: Rheumatoid arthritis (RA) and psoriatic arthritis (PsA) are two distinct forms of chronic auto-immunity; understanding the transcriptomic profiles of key leukocyte subsets implicated in these arthritides could improve the diagnosis and treatment of patients. Current microarray analyses of samples derived from RA and PsA patients have examined the genetic profiles of whole blood or diseased tissue which, although informative, can mask the genetic contributions of individual cell types. Monocytes and macrophages are a cellular subset known to play a major role in PsA and RA through the production of pro-inflammatory chemokines, cytokines and destructive proteinases.
Aim: To define the transcriptome in CD14+ cells separated from the blood and synovial fluid of PsA and RA patients, and to then compare and contrast that signature in health and disease. Thereafter to define the relevant activities of selected novel moieties described in the foregoing analysis.
Methods & Results: The transcriptomic profiles of healthy, RA and PsA CD14+ blood cells were remarkably similar - few genes could distinguish diseased from healthy CD14+ cells. Comparison of the genetic signature of the RA and PsA synovial fluid CD14+ cells revealed that just over 50% of the differentially expressed genes were shared between the two disease groups. Furthermore, analysing the canonical pathways in the synovial fluid cells compared to the matched peripheral blood of both patient groups surprisingly revealed Liver X receptor (LXR) activation pathway as the most significantly upregulated pathway: this pathway has been previously shown by our group to play a pro-inflammatory role in arthritis.
Examination of specific upregulated mRNAs in the synovial fluid CD14+ cells from both disease types revealed two novel genes that had not previously been associated with arthritis, the lysosomal enzyme legumain and the cell surface molecule plexin A1. Legumain was demonstrated to be present in RA and PsA CD14+ cells by RNA and protein analysis and was physiologically active. Incubation of CD14+ cells with patient synovial fluid under hypoxic conditions also potentiated legumain expression. Plexin A1 was confirmed to be expressed at the mRNA level within RA synovium. siRNA knockdown of plexin A1 suggested that it may play a pro-inflammatory role within macrophages since subsequent treatment of these macrophages with LPS resulted in decreased TNFα production. However, investigations into the identity of the specific ligands for plexin A1 in arthritis, known as semaphorins, were inconclusive.
I finally generated microarray data to evaluate the transcriptome of macrophages activated via cel contact with activated T cells. Such cells shared only a small percentage of genes with those dysregulated in the RA and PsA synovial fluid derived CD14+ cells suggesting that this model at the time points chosen may not be an appropriate in vitro representation of articular macrophages. An imaging system of this in vitro model was also established to visualise the dynamic nature of the T cell – macrophage interactions and demonstrated that variables such as duration or method of T cell activation could alter the number and duration of interactions between the two cell types.
Conclusions: These studies demonstrate that the CD14+ cells isolated from the blood are similar transcriptomically between healthy controls and RA and PsA patients. The synovial fluid CD14+ cells from RA and PsA patients exhibit substantial overlap in terms of their genetic profile. Two novel molecules expressed by diseased patients namely plexin A1 and legumain have been identified and their preliminary characteristics in the context of synovitis have been defined.
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