The phenotype and function of monocyte microparticles, and their characterisation in paediatric HIV infection

Luckhurst, Natasha (2019) The phenotype and function of monocyte microparticles, and their characterisation in paediatric HIV infection. (PhD thesis), Kingston University, .


37 million people are currently living with HIV worldwide. With the development of Antiretroviral Therapy (ART) AIDS mortality has reduced, with cardiovascular disease (CVD) now the leading cause of HIV deaths. HIV associated CVD has been linked to chronic immune activation through the association of inflammatory biomarkers, clinical events and asymptomatic atherosclerosis; even from a young age. Microparticles (MPs), markers of cellular activation and injury, are also elevated in adults with HIV, and demonstrate the capacity to contribute to inflammation, endothelial dysfunction and coagulation. Monocytes play a key role in atherosclerosis initiation and display an activated, pro-inflammatory phenotype in paediatric HIV. Monocytic MPs (MMPS) are elevated in adults with HIV, thus the aim of this thesis was to quantify MMPS in HIV infected children and investigate their effects on monocytic function. A novel negative isolation method was first optimised to enable the study of human monocyte function 'ex vivo'. This isolation procedure allowed the negative enrichment of all three circulating monocyte subsets (Classical, Non-classical and Intermediate), with high purity and minimal activation. Extracted monocytes displayed minimal alterations in marker expression while retaining their phagocytic, migratory and cytokine secretion function 'ex vivo'. Using a monocytic cell line (THP-1), MP release under different stimulatory and apoptotic conditions was investigated. Under stimulatory conditions with cytokines and a calcium ionophore (A23187), MPs were released in a concentration-dependent manner, with endotoxin stimulation resulting in the highest quantity. Furthermore, MPs displayed a similar trend in surface marker expression in comparison to their parent cell. Next, we investigated the effects of THP-1 derived MMPS on human monocyte function employing the isolation method established. MMPS evoked the release of pro-inflammatory cytokines in addition to enhancing CD11b expression, adherence and transendothelial migration. This work demonstrates that MMPS activates monocytes and enhances endothelial migration, presenting a mechanism that contributes to atherosclerosis pathogenesis. Finally, MMPS and MPs from an endothelial, platelet and T cell origin were enumerated in children with HIV infection pre/post ART initiation and healthy sex-age-matched controls. Plasma samples were collected during the CHAPAS-3 CV sub-study, from which we analysed 16 children receiving ART (≥2 years), 11 treatment-naïve children and 15 controls. Circulating MPs from an endothelial, platelet and T cell origin found in children with HIV infection normalised to levels found in healthy controls, following ART initiation and experience. MMPS were elevated within the treatment-naïve cohort and remained elevated despite treatment intervention; this elevation was also observed in the treatment-experienced cohort. The persistence of elevated MMPS indicates consistent monocyte activation despite successful viral suppression, supporting the activated phenotype reported in literature. This data combined with the functional implications of MMPS on monocytic function described in this thesis elucidates a mechanism that contributes to elevated vascular inflammation reported within this population.

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