Massive Release of CD9+ Microvesicles in Human Immunodeficiency Virus Infection, Regardless of Virologic Control

Abstract

Background: The role of extracellular vesicles (EVs) in human immunodeficiency virus (HIV) pathogenesis is unknown. We examine the cellular origin of plasma microvesicles (MVs), a type of ectocytosis-derived EV, the presence of mitochondria in MVs, and their relationship to circulating cell-free mitochondrial deoxyribonucleic acid (ccf-mtDNA) in HIV-infected patients and controls.

Methods: Five participant groups were defined: 30 antiretroviral therapy (ART)-naive; 30 ART-treated with nondetectable viremia; 30 elite controllers; 30 viremic controllers; and 30 HIV-uninfected controls. Microvesicles were quantified and characterized from plasma samples by flow cytometry. MitoTrackerDeepRed identified MVs containing mitochondria and ccf-mtDNA was quantified by real-time polymerase chain reaction.

Results: Microvesicle numbers were expanded at least 10-fold in all HIV-infected groups compared with controls. More than 79% were platelet-derived MVs. Proportions of MVs containing mitochondria (22.3% vs 41.6%) and MV mitochondrial density (706 vs 1346) were significantly lower among HIV-infected subjects than controls, lowest levels for those on ART. Microvesicle numbers correlated with ccf-mtDNA levels that were higher among HIV-infected patients.

Conclusions: A massive release of platelet-derived MVs occurs during HIV infection. Some MVs contain mitochondria, but their proportion and mitochondrial densities were lower in HIV infection than in controls. Platelet-derived MVs may be biomarkers of platelet activation, possibly reflecting pathogenesis even in absence of HIV replication.

Keywords: HIV; elite controllers; extracellular vesicles; microvesicles; mitochondria.

Figure 1.

Gating strategy to characterize microvesicles (MVs) by flow cytometry. (a) Microvesicles less than 1 μM were gated using size reference beads and CD9 expression. Representative contour plots showing side scatter (SSC) and forward scatter (FSC) or CD9 labeling of MVs from antiretroviral therapy-exposed human immunodeficiency virus-infected donors (n = 30); (b) CD3 staining on plasma-derived MVs or peripheral blood-derived T cells; CD14 staining on plasma-derived MVs or monocytes, and CD41a and CD61 staining on plasma-derived MVs or platelets; (c) coexpression of CD11b and CD16 on MVs and CD66b staining on plasma-derived MVs subsets or peripheral blood-derived neutrophils.

Figure 2.

Microvesicles (MVs) contain mitochondria. (a) Representative contour plots showing side scatter (SSC) and MitoTrackerDeepRed labeling of platelets and MVs from human immunodeficiency virus (HIV)-uninfected controls (n = 30) or MVs from antiretroviral therapy (ART)-exposed HIV-infected donors (n = 30); (b) transmission electron microscopy visualization of plasma-derived MVs with (black arrow) and without (white arrow) mitochondria from an ART-exposed HIV-infected donor (representative of 2 donors).

Figure 3.

Plasma circulating cell-free mitochondrial deoxyribonucleic acid (ccf-mtDNA) levels among the study groups. Whiskers represent median values ± interquartile range. Statistical significance was determined by Mann-Whitney U test to compare 2 groups, and Kruskal-Wallis test was used to compare more than 2 groups. ***, P > .001. EC, elite controllers; VC, viremic controllers.

Figure 4.

Correlation between plasma circulating cell-free mitochondrial deoxyribonucleic acid (ccf-mtDNA) levels: (a) microvesicles (MVs) count; (b) mito⁺ MVs count; (c) platelet-derived mito⁺ MVs count; (d) neutrophil-derived mito⁺ MVs count. Statistical correlation was determined using Spearman’s correlation analysis and was expressed as Rho-Spearman’s coefficient; P value.