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Title: Functional expression of endogenous ligands of TLR4 and associated inflammatory markers in monocytes and endothelial cells during normotensive pregnancy, and pregnancy complicated by pre-eclampsia
Author: Al-ofi, Ebtisam
ISNI:       0000 0004 5348 6921
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2014
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A leading cause of pregnancy-related mortality and morbidity is PE (PE). The innate immune system recognises pathogens through transmembrane proteins called toll-like receptors (TLRs). Lipopolysaccharide (LPS) is recognised by TLR4 and peptidoglycan (PDG) is recognised by TLR2. However, other endogenous ligands such as heparan sulfate, hyaluronan, fibrinogen, fibronectin, and HMGB1 can stimulate TLR4 and trigger innate immunology. Thus, in Chapter 1 the pathophysiology of PE women is described with special emphasis on immune cells (monocytes/macrophages), inflammatory responses, and endothelial cell inflammation. Also, the expression of TLR in the foeto-maternal interface and PE was explained. To further characterise the systemic effects of PE on circulating cells, monocyte subpopulations in normal pregnant (NP) and PE patients were analysed using flow cytometry (Chapter 3). We demonstrated for the first time that non-classical CD14lowCD16+ monocytes were significantly increased in women with PE, and these women also displayed irregular expression of several chemokine receptors and antigen presentation molecules. The most striking phenotypic difference in cell surface molecules was the marked upregulation of TLR4 expression, where both CD14highCD16+ and CD14lowCD16+ monocytes demonstrated higher levels in PE patients than their NP counterparts. In Chapter 4, we reported that stimulation of PE monocytes with bacterial TLR4 ligands resulted in profound secretion of various cytokines, in comparison to NP controls. In addition to LPS and PDG, fibrinogen was the only endogenous ligand that stimulated PE monocytes, producing inflammatory cytokines. The interactions between PE/NP monocytes and endothelial cells were examined. The inflammatory responses following stimulation with TLR2 and 4 ligands were investigated in monocyte-HUVEC co-cultures by CBA (Chapter 5). Co-culture of PE monocytes with endothelial cells demonstrated profound secretion of inflammatory cytokines (IL-6) and chemokines (IL-8 and MCP-1), in comparison to NP controls. However, production of the anti-inflammatory cytokine (IL-10) was lower in PE cocultures. Additionally, we showed that stimulation of PE co-cultures with TLR ligands led to an abnormal disturbance in cytokines and chemokines as compared to NP cocultures. In Chapter 6, the effects of TLR ligands on the expression levels of angiogenic (vascular endothelial growth factor: VEGF) and anti-angiogenic factors (soluble fmslike tyrosine kinase 1: sFlt-1), and adhesion molecules (vascular endothelial adhesion molecule 1: VCAM-1) were examined with CBA and ELISA in PE/NP co-cultures. VEGF production was significantly lower for PE monocyte-HUVEC co-cultures compared to NP controls. The expression level of sFlt-1 from HUVECs was downregulated when co-cultured with monocytes from NP women; down-regulation of sFlt-1 by NP monocytes was significantly higher than for PE monocytes. Stimulation of monocyte-HUVEC co-cultures by bacterial ligands, induced sFlt-1 production in NP and PE cultures. Interestingly, exposure of PE monocytes co-cultured to fibrinogen resulted in down-regulation of sFlt-1 production, whilst this was unchanged for NP controls. Compared to NP, PE monocytes induced higher expression levels of VCAM-1 released by endothelial cells. Furthermore, exposure of PE monocytes and HUVEC cocultures to TLR ligands, led to an exaggerated VCAM-1 response compared to NP controls. In Chapter 7, plasma levels of endogenous ligands were measured with ELISA; and fibrinogen was investigated in placentae of PE and NP women, using immunohistochemistry. Fibrinogen, fibronectin, and HMGB1 levels were higher in the plasma of PE women compared to NP women, whilst heparan sulfate levels did not differ. Also, placental expression of fibrinogen was lower in PE women than in NP. In summary, this thesis examined the physiology and innate immunology of human monocytes, and their interaction with vascular endothelial cells in the pathogenesis of hypertensive pregnancy disorders. It investigated the possible role of the endogenous ligands of TLR2 and TLR4 in this common pregnancy disorder that still contributes considerable maternal and foetal deaths and disease worldwide.
Supervisor: Anumba, Dilly Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available