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Title: Characterization of the orphan chemokines CCL18 and CXCL4 : potential players in atherosclerosis
Author: Hussain, Khansa
ISNI:       0000 0004 9350 2738
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2018
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Atherosclerosis is an inflammatory reaction of the arteries, dependent upon monocyte recruitment into the sub-endothelial space. There, monocytes differentiate or polarize into macrophages which play pivotal roles in disease progression. Differentiation is a hallmark of monocytes and is driven by cytokines signalling. Chemokines are a family of cytokines released during inflammation, chiefly to recruit leukocytes from the circulation. CCL18 and CXCL4 are two poorly characterised chemokines present in atherosclerotic lesions. CXCL4 enhances the survival of monocytes and drives their differentiation into so-called “M4” macrophages with a unique transcriptome. CCL18 is reported to be amongst the genes actively transcribed in M4 macrophages. We hypothesized that CCL18 may play a role in the development of atherosclerosis and set out to further characterise the effects of CCL18 on monocyte function. A main experimental aim was to identify the CCL18 receptor, which despite conflicting reports in the literature, remains undiscovered. As studies commenced, a brief report was published identifying the chemokine receptor CCR8 as a functional CCL18 receptor. We therefore initially focussed on CCR8 to determine its role in CCL18 signalling. Several CCR8-expressing cell lines were used to probe CCL18 responses by a variety of different assays, including ligand binding, chemotaxis and receptor endocytosis. Although robust responses to CCL1, the principal CCR8 ligand were seen, no significant responses to CCL18 were observed. We therefore conclude that the specific receptor for CCL18 remains unidentified. We subsequently examined the responses of monocytes to CCL18. Specifically, we examined the ability of freshly isolated monocytes to bind CCL18 on their surface and 4 to migrate in response to CCL18. Although we found that CCL18 could bind specifically to monocytes, the cells were unable to navigate CCL18 gradients, suggesting that the chemokine may not recruit monocytes in vivo. However, we did find that CCL18 was an efficacious monocyte survival factor, enhancing monocyte survival in serum-free media. To better understand the relationship between M4 differentiation and CCL18 production, we examined the properties of M4 macrophages, directly comparing them to M0 macrophages cultured in the presence of macrophage colony-stimulating factor. M4 macrophages rapidly increased in size and were significantly bigger than M0 macrophages following 7 days of culture. Profiling of cellular supernatants found that enhanced secretion of CCL22 but not CCL18 was a characteristic early event in M4 polarization. We also observed that M4 macrophages scavenged significantly lower levels of oxidised low density lipoprotein (oxLDL) than M0 macrophages throughout polarization, which correlated with lower expression of CD36 mRNA, a well-known oxLDL scavenger. To further illuminate the M4 polarization process, we also examined the expression of 84 transcriptional factors by quantitative polymerase chain reaction (qPCR) and identified two transcription factors as being significantly upregulated during M4 polarization. In summary, the work described here has enhanced our understanding of the effects of CCL18 and CXCL4 on monocyte function, with potential relevance to the atherosclerotic process. Translating these findings into novel therapeutic approaches should be a key goal of future research.
Supervisor: Pease, James Sponsor: Jāmiʻat al-Malik Saʻūd
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available