Elevated Abundance, Size, and MicroRNA Content of Plasma Extracellular Vesicles in Viremic HIV-1+ Patients: Correlations With Known Markers of Disease Progression

Abstract

Background: Because of factors only partly understood, the generalized elevated immune activation and inflammation characterizing HIV-1-infected patients are corrected incompletely with antiretroviral therapy (ART). Extracellular vesicles (EVs) including exosomes and microvesicles released by several cell types may contribute to immune activation and dysfunction. EV size, abundance, and content appear to differ according to infection phase, disease progression, and ART.

Methods: We examined whether the size of EVs and the abundance of exosomes in plasma are associated with cell and tissue activation as well as with viral production. Acetylcholinesterase-bearing (AChE+) exosomes in plasma were quantified using an AChE assay. EV size was analyzed using dynamic light scattering. Proteins and microRNAs present in EVs were detected by Western blot and real-time polymerase chain reaction, respectively.

Results: Exosomes were found more abundant in the plasma of ART-naive patients. EV size was larger in ART-naive than in ART-suppressed patients, elite controllers, or healthy control subjects. Both exosome abundance and EV sizes were inversely correlated with CD4/CD8 T-cell ratio and neutrophil, platelet, and CD4 T-cell counts and positively correlated with CD8 T-cell counts. A negative correlation was found between CD4 T-cell nadir and exosome abundance, but not EV size. Levels of miR-155 and miR-223 but not miR-92 were strongly correlated negatively with EV abundance and size in ART-naive patients.

Conclusions: Monitoring of circulating EVs and EV-borne microRNA is possible and may provide new insight into HIV-1 pathogenesis, disease progression, and the associated inflammatory state, as well as the efficacy of ART and the treatments intended to reduce immune activation.

FIGURE 1

Characterization of EVs. Plasma was obtained from healthy individuals (n = 16) and HIV-1–infected individuals (n = 17 ART-naive, n = 13 ART-suppressed, n = 13 elite controllers). ExoQuick was used to precipitate EV fraction. A, AChE activity measured for each patient. B, DLS (Malvern Zeta nanosizer) sizing of EVs after fixation with 2% paraformaldehyde. C, Distribution of particle mean size, based on DLS (black indicates healthy individuals). D, Western blot of proteins precipitated from pooled plasma of each group. E and F, Correlations of AChE+ MV abundance and EV size with CD4/CD8 ratio in all HIV-infected patients, reported as Spearman r and P values (2-tailed). Asterisks denote statistically significant differences (***P < 0.001).

FIGURE 2

Detection of selected microRNAs in plasma EVs and correlation with AChE activity and size of EVs. Plasma obtained from healthy individuals (n = 8) and HIV-1–infected individuals (n = 8 ART-naive, n = 8 ART-suppressed, n = 8 elite controllers) was treated with proteinase K, and ExoQuick was used to precipitate EV fraction. MicroRNA miR-155 (A), mir-223 (B), and miR-92 (C) present in exosomes and MVs were amplified as described in the “Materials and Methods” section. Panels (D–F) show the correlation between microRNA relative abundance and AChE activity for ART-naive patients. Panels (G–I) show the correlation between microRNA abundance and EV size for ART-naive patients. All correlations are reported as Spearman r and P values (2-tailed). Asterisks denote statistically significant differences (*P < 0.05, **P < 0.01). Panels (J and K) show representative electron microscopy images of exosomes. Panels (L and M) show plasma MV size distribution, based on DLS measurement. Samples in (K and M) were treated with proteinase K.