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

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

Inflammatory pathways amongst people living with HIV in Malawi differ according to socioeconomic status

Background: Non-communicable diseases (NCDs) are increased amongst people living with HIV (PLWH) and are driven by persistent immune activation. The role of socioeconomic status (SES) in immune activation amongst PLWH is unknown, especially in low-income sub-Saharan Africa (SSA), where such impacts may be particularly severe. Methods: We recruited Malawian adults with CD4<100 cells/ul two weeks after starting ART in the REALITY trial (NCT01825031), as well as volunteers without HIV infection. Clinical assessment, socioeconomic evaluation, blood draw for immune activation markers and carotid femoral pulse wave velocity (cfPWV) were carried out at 2- and 42-weeks post-ART initiation. Socioeconomic risk factors for immune activation and arterial stiffness were assessed using linear regression models. Results: Of 279 PLWH, the median (IQR) age was 36 (31–43) years and 122 (44%) were female. Activated CD8 T-cells increased from 70% amongst those with no education to 88% amongst those with a tertiary education (p = 0.002); and from 71% amongst those earning less than 10 USD/month to 87% amongst those earning between 100–150 USD/month (p = 0.0001). Arterial stiffness was also associated with higher SES (car ownership p = 0.003, television ownership p = 0.012 and electricity access p = 0.029). Conversely, intermediate monocytes were higher amongst those with no education compared to a tertiary education (12.6% versus 7.3%; p = 0.01) and trended towards being higher amongst those earning less than 10 USD/month compared to 100–150 USD/month (10.5% versus 8.0%; p = 0.08). Water kiosk use showed a protective association against T cell activation (p = 0.007), as well as endothelial damage (MIP1β, sICAM1 and sVCAM1 p = 0.047, 0.026 and 0.031 respectively). Conclusions: Socioeconomic risk factors for persistent inflammation amongst PLWH in SSA differ depending on the type of inflammatory pathway. Understanding these pathways and their socioeconomic drivers will help identify those at risk and target interventions for NCDs. Future studies assessing drivers of inflammation in HIV should include an SES assessment

Inflammatory phenotypes predict changes in arterial stiffness following antiretroviral therapy initiation

Abstract Background Inflammation drives vascular dysfunction in HIV, but in low-income settings causes of inflammation are multiple, and include infectious and environmental factors. We hypothesized that patients with advanced immunosuppression could be stratified into inflammatory phenotypes that predicted changes in vascular dysfunction on ART. Methods We recruited Malawian adults with CD4 &amp;lt;100 cells/μL 2 weeks after starting ART in the REALITY trial (NCT01825031). Carotid femoral pulse-wave velocity (cfPWV) measured arterial stiffness 2, 12, 24, and 42 weeks post–ART initiation. Plasma inflammation markers were measured by electrochemiluminescence at weeks 2 and 42. Hierarchical clustering on principal components identified inflammatory clusters. Results 211 participants with HIV grouped into 3 inflammatory clusters representing 51 (24%; cluster-1), 153 (73%; cluster-2), and 7 (3%; cluster-3) individuals. Cluster-1 showed markedly higher CD4 and CD8 T-cell expression of HLADR and PD-1 versus cluster-2 and cluster-3 (all P &amp;lt; .0001). Although small, cluster-3 had significantly higher levels of cytokines reflecting inflammation (IL-6, IFN-γ, IP-10, IL-1RA, IL-10), chemotaxis (IL-8), systemic and vascular inflammation (CRP, ICAM-1, VCAM-1), and SAA (all P &amp;lt; .001). In mixed-effects models, cfPWV changes over time were similar for cluster-2 versus cluster-1 (relative fold-change, 0.99; 95% CI, .86–1.14; P = .91), but greater in cluster-3 versus cluster-1 (relative fold-change, 1.45; 95% CI, 1.01–2.09; P = .045). Conclusions Two inflammatory clusters were identified: one defined by high T-cell PD-1 expression and another by a hyperinflamed profile and increases in cfPWV on ART. Further clinical characterization of inflammatory phenotypes could help target vascular dysfunction interventions to those at highest risk

Knockout or inhibition of USP30 protects dopaminergic neurons in a Parkinson’s disease mouse model

Mutations in SNCA, the gene encoding α-synuclein (αSyn), cause familial Parkinson’s disease (PD) and aberrant αSyn is a key pathological hallmark of idiopathic PD. This α-synucleinopathy leads to mitochondrial dysfunction, which may drive dopaminergic neurodegeneration. PARKIN and PINK1, mutated in autosomal recessive PD, regulate the preferential autophagic clearance of dysfunctional mitochondria (“mitophagy”) by inducing ubiquitylation of mitochondrial proteins, a process counteracted by deubiquitylation via USP30. Here we show that loss of USP30 in Usp30 knockout mice protects against behavioral deficits and leads to increased mitophagy, decreased phospho-S129 αSyn, and attenuation of SN dopaminergic neuronal loss induced by αSyn. These observations were recapitulated with a potent, selective, brain-penetrant USP30 inhibitor, MTX115325, with good drug-like properties. These data strongly support further study of USP30 inhibition as a potential disease-modifying therapy for PD.</p

Knockout or inhibition of USP30 protects dopaminergic neurons in a Parkinson’s disease mouse model

Acknowledgements: We thank Dr Laura Parton for project management/co-leadership of MTX115325, providing the foundation for Mission compound studies and MTX115325 development in PD. We also thank Drs. Krutika Joshi and Veronique VanderHorst of BIDMC for their support with slides scanning for TH+ neuronal counting and TH+ fiber densitometry. We thank Matthew Jacobsen for the pathology assessment of mouse livers and Nirav Prakas Patel for early work on mouse ESCs. We would like to thank Prof. Maria Grazia Spillantini for her valuable comments on the manuscript. Research in the D.K.S. lab is funded by an NINDS grant (R21NS109408), the Weston Brain Institute and the Owens Foundation. Research in the G.B. lab is supported by the UK Dementia Research Institute, that receives contributions from UK DRI Ltd, the UK MRC, the Alzheimer’s Society, and Alzheimer’s Research UK as well as a grant from the Romanian Ministry of Research, Innovation and Digitization (no. PNRR-III-C9-2022-I8-66; contract 760114). IGG was funded by a grant from the Medical Research Council, UK (MC_UU_00018/2). Research in the S.P.J. lab is funded by Cancer Research UK Discovery grant (DRCPGM\100005), and ERC Synergy grant DDREAMM (855741). This project has received funding to SJ from CRUK programme grant C6/A11224, C6/A18796 and Wellcome Investigator Award (206388/Z/17/Z) together with core infrastructure funding by Cancer Research UK (C6946/A24843) and Wellcome (WT203144). The Sanger Mouse Genetics Project was supported by the Wellcome Trust (098051). CRUK supports D.J.A.Funder: G.B. lab is supported by the UK Dementia Research Institute, that receives contributions from UK DRI Ltd, the UK MRC, the Alzheimer’s Society, and Alzheimer’s Research UK as well as a grant from the Romanian Ministry of Research, Innovation and Digitization (no. PNRR-III-C9-2022-I8-66; contract 760114).AbstractMutations in SNCA, the gene encoding α-synuclein (αSyn), cause familial Parkinson’s disease (PD) and aberrant αSyn is a key pathological hallmark of idiopathic PD. This α-synucleinopathy leads to mitochondrial dysfunction, which may drive dopaminergic neurodegeneration. PARKIN and PINK1, mutated in autosomal recessive PD, regulate the preferential autophagic clearance of dysfunctional mitochondria (“mitophagy”) by inducing ubiquitylation of mitochondrial proteins, a process counteracted by deubiquitylation via USP30. Here we show that loss of USP30 in Usp30 knockout mice protects against behavioral deficits and leads to increased mitophagy, decreased phospho-S129 αSyn, and attenuation of SN dopaminergic neuronal loss induced by αSyn. These observations were recapitulated with a potent, selective, brain-penetrant USP30 inhibitor, MTX115325, with good drug-like properties. These data strongly support further study of USP30 inhibition as a potential disease-modifying therapy for PD.</jats:p