Investigating platelet function and immune activation in HIV-infection

[摘要] ENGLISH ABSTRACT: IntroductionIn the era of antiretroviral therapy (ART), people living with Human Immunodeficiency Virus(HIV) now have prolonged life spans. An emerging trend of non- acquired immune deficiencysyndrome (AIDS) related complications now prevails in the aging HIV infected population.Increased levels of inflammation and chronic immune activation are associated with HIVinfection. In the era of ART people living with HIV are at an increased risk of cardiovasculardisease (CVD). Platelets play a pivotal role in both inflammation and immune activation andupon activation platelets degranulate and secrete various inflammatory, coagulatory andadhesion molecules. Activated platelets express surface P-selectin (CD62P) and are a keycomponent of the coagulation pathway and serve as a link between inflammation andthrombosis. Activated platelets have been implicated in inflammatory and cardiovascular diseaseand have been identified as immune cells that play a crucial role in pathogen recognition andmodulation of immune cells during infections. Several antiviral and antibacterial properties ofplatelet alpha granule contents have been established. Platelet aggregometry remains the mostwidely used technique to evaluate platelet function even though this technique is limited by manypre-analytical variables. Platelet flow cytometry on the other hand offers a rapid measurement ofplatelet function in their physiological environment with minimal artefactual activation. Fewstudies have however reported on standardized methods to evaluate platelet function in thecontext of HIV. Platelet function remains unclear and data on HIV infected treatment naïveindividuals remains scarce. The aim of this project was to examine the relationship betweenplatelet function and immune activation in patients with HIV Materials and methodsThis study consisted of five sub-studies, firstly platelet indices and levels of platelet activationwere determined in a cohort of 330 participants (185 HIV infected ARV naïve and 145 uninfectedhealthy controls) using; flow cytometry and haemotology analyzers. The relationship betweenthese indices and markers of platelet activation, disease progression and immune activationwere assessed. Furthermore, levels of platelet activation and aggregation were evaluated in acohort of 82 participants (41 HIV infected (ARV naïve) individuals and 41 uninfected healthycontrols), using a novel whole blood flow cytometry based functional assay. These baselinelevels were then correlated with markers of immune activation and disease progression in HIV. In a subsequent study, platelet function in a cohort consisting of 58 HIV infected (ARV naïve)and 38 uninfected controls was evaluated using flow cytometry. Platelet response wasmeasured post stimulation with adenosine diphosphate (ADP) at concentrations known to inducereversible (0.04mM) and irreversible (0.2mM) platelet aggregation. In order to assess plateletfunction in HIV, platelet response was evaluated in a cohort consisting of 58 HIV infected (ARVnaïve) and 38 uninfected controls. Platelets were activated using varying concentrations of ADP,arachidonic acid (AA) and collagen and platelet function was measured using flow cytometry.Levels of circulating platelet leukocyte aggregates (PLAs) were also measured using flowcytometry in a cohort consisting of 35 HIV-infected (ARV naïve) individuals and 32 uninfectedhealthy controls. Associations between PLAs, immune activation and disease progression in HIVinfected individuals were determined. The final study evaluated platelet aggregates, plateletderived microparticles (PMPs) and microparticles (MPs) in a cohort consisting of 46 HIV infected(ARV-naïve) and 40 uninfected healthy controls. Associations between MPs, PMPs, plateletaggregates and markers of immune activation and disease progression were evaluated. ResultsHIV infected individuals showed decreased mean platelet volume levels (HIV mean 7.91 ± 0.85vs. 8.52 ± 1.12, p<0.0001) that directly correlated with CD4 counts (r=-0.2898, p=0.0075) andviral load (r=0.2680, p=0.0177). Platelet distribution width (PDW) levels directly correlated(r=0.3455, p=0.0362) with active coagulation and inversely correlated (r=-0.3666, p=0.0463) withplatelet aggregation. HIV infected individuals showed increased levels of platelet activation(%CD62P median 11.33[5.96-29.36] vs. control group 2.48[1.56-6.04], p=0.0001). In HIV,platelet function is retained and platelets showed increased response to submaximalconcentrations of endogenous agonists. HIV infected individuals showed increased levels ofcirculating platelet monocyte aggregates (25.26[16.16-32.28] vs. control group 14.12[8.36-18.83], p=0.0001) that directly correlated with markers of immune activation; %CD38/8(r=0.54624, p=0.0155), viral load (r=0.633, p<0.009). Furthermore we report on increased levelsof circulating MPs (median %MPs 1.7[0.95-2.83] vs. Control group 1.12[0.63-1.57], p=0.0160);PMPs (median %PMPs 26.64[11.33-36.62] vs. Control group 20.02[18.08-26.08], p=0.0133);activated PMPs (median CD62P MFI 3.81[3.46-4.54] vs. Control group 3.41[3.16-3.6],p=0.0037) and platelet aggregates (Median %CD62P 14.10[5.49-39.94] vs. Control group0.17[0.10-10.99], p= 0.0097) in HIV infected asymptomatic individuals. ConclusionThis study supports the potential use of the MPV and PDW as readily available markers ofplatelet activation and immune activation in HIV. We also showed elevated levels of activatedplatelets in HIV infected individuals that were hyper responsive to endogenous agonists in aconcentration dependent manner. Platelet flow cytometry is a rapid and valuable technique inthe evaluation of platelet function in HIV. The measurement of platelet function using flowcytometry allows the evaluation of platelet signalling pathways that may be modified in HIVinfected individuals. Lastly we describe an optimized whole blood flow cytometry based assayfor the evaluation of circulating microparticles (MPs), platelet derived microparticles (PMPs) andlevels of activated platelets and aggregates which mimics the in vivo physiological environmentof MPs. To the best of our knowledge, this study is the first to report on a novel approach inevaluating platelet function in HIV using a series of optimised whole blood flow cytometry basedplatelet assays. In addition, minimal work has been performed previously on platelet function inthe context of HIV-infection; and particularly in a cohort of asymptomatic, untreated patients asdefined for this study.

[发布机构] Stellenbosch University