Seminal Plasma-Derived Extracellular-Vesicle Fractions from HIV-Infected Men Exhibit Unique MicroRNA Signatures and Induce a Proinflammatory Response in Cells Isolated from the Female Reproductive Tract

Abstract

The continuing spread of HIV/AIDS is predominantly fueled by sexual exposure to HIV-contaminated semen. Seminal plasma (SP), the liquid portion of semen, harbors a variety of factors that may favor HIV transmission by facilitating viral entry into host cells, eliciting the production of proinflammatory cytokines, and enhancing the translocation of HIV across the genital epithelium. One important and abundant class of factors in SP is extracellular vesicles (EVs), which, in general, are important intercellular signal transducers. Although numerous studies have characterized blood plasma-derived EVs from both uninfected and HIV-infected individuals, little is known about the properties of EVs from the semen of HIV-infected individuals. We report here that fractionated SP enriched for EVs from HIV-infected men induces potent transcriptional responses in epithelial and stromal cells that interface with the luminal contents of the female reproductive tract. Semen EV fractions from acutely infected individuals induced a more proinflammatory signature than those from uninfected individuals. This was not associated with any observable differences in the surface phenotypes of the vesicles. However, microRNA (miRNA) expression profiling analysis revealed that EV fractions from infected individuals exhibit a broader and more diverse profile than those from uninfected individuals. Taken together, our data suggest that SP EVs from HIV-infected individuals exhibit unique miRNA signatures and exert potent proinflammatory transcriptional changes in cells of the female reproductive tract, which may facilitate HIV transmission.IMPORTANCE Seminal plasma (SP), the major vehicle for HIV, can modulate HIV transmission risk through a variety of mechanisms. Extracellular vesicles (EVs) are extremely abundant in semen, and because they play a key role in intercellular communication pathways and immune regulation, they may impact the likelihood of HIV transmission. However, little is known about the properties and signaling effects of SP-derived EVs in the context of HIV transmission. Here, we conduct a phenotypic, transcriptomic, and functional characterization of SP and SP-derived EVs from uninfected and HIV-infected men. We find that both SP and its associated EVs elicit potent proinflammatory transcriptional responses in cells that line the genital tract. EVs from HIV-infected men exhibit a more diverse repertoire of miRNAs than EVs from uninfected men. Our findings suggest that EVs from the semen of HIV-infected men may significantly impact the likelihood of HIV transmission through multiple mechanisms.

Keywords: epithelial cells; extracellular vesicles; female reproductive tract; human immunodeficiency virus; miRNAs; seminal plasma; stromal fibroblasts; transcription.

FIG 1

Characterization of EVs in the MV and exosome fractions of SP from uninfected versus HIV-infected individuals. (A) EV particle counts in the MV fractions, exosome fractions, and original preprocessed seminal plasma were determined by nanoparticle tracking analysis. (B) The mean size distribution of particles in the MV fractions, exosome fractions, and original preprocessed seminal plasma was determined by nanoparticle tracking analysis. (C) The levels of the non-EV-associated protein albumin in the MV fractions, exosome fractions, and original preprocessed seminal plasma were determined by ELISA. P values were determined by the Friedman repeated-measures ANOVA and Dunn’s multiple-comparison post hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (D) MV counts in the MV fractions used in this study, as determined using an LSR II flow cytometer. n.s., not significant, as determined by an unpaired Student's t test.

FIG 2

Principal-component analysis and hierarchical clustering of eSFs or eECs treated with rSP or the MV fraction from uninfected or HIV-infected individuals. (A, B) PCA of RNA-seq data sets from eSFs or eECs exposed to vehicle, 1% rSP, or 1% the MV fraction from uninfected or HIV-infected individuals. Samples originating from the same eSF donor are connected by lines. (C, D) Heatmap showing differentially expressed genes (DEGs) between the treatment conditions in eSFs or eECs. The numbers refer to genital tissue donor numbers.

FIG 3

Validation of select genes induced by SP from HIV-infected individuals. Culture supernatants from eSFs (A, B, C) or eECs (C) were assessed for the protein levels of CCL2 (A), VEGF (B), CXCL1 (C), or HGF (D). Treatment conditions are indicated on the x axis, and each eSF or eEC donor is depicted with a different color. The dotted line shows the levels of the protein under vehicle-treated conditions. The numbers above the bars indicate the fold induction relative to the level of induction for the corresponding vehicle-treated condition. All donors were treated under the exact same conditions, as indicated. When these paired data sets from all 3 donors were combined and analyzed using paired Student’s t tests, there was a significant induction of CCL2 by HIV-infected SP (P < 0.05), VEGF by uninfected SP (P < 0.05), VEGF by HIV-infected SP (P < 0.05), and HGF by uninfected SP (P < 0.05).

FIG 4

Representative dot plots showing an uninfected control and a sample treated with HIV in the presence of vehicle alone (bottom row) or samples infected with HIV in the presence of rSP, the MV fraction, or the exosome fraction (top row). (B) Graphs showing the levels of GFP, CD25, and HLA-DR on activated T cells from uninfected and HIV-infected individuals between the vehicle-alone condition and the rSP treatment conditions. Results are gated on live, singlet CD3⁺ CD8⁻ cells. (C) Representative histogram plots showing the cell surface expression of CD25⁺ and HLA-DR on T cells infected or not with HIV and in the absence or presence of rSP.

FIG 5

Detection and characterization of EVs for their absolute count, relative size, and cell of origin. (A) SSC height (SSC-H) dot plot showing instrument sensitivity to beads 100 to 1,000 nm in diameter, a range used to set EV gates on the LSR II flow cytometer. (B) Representative plots of the MV fraction gated for size as described in the legend to panel A and sorted according to various phenotypic markers. (C) Scatterplots of MV concentration (log₁₀ transformed) in uninfected versus acutely infected individuals according to the phenotypic markers examined. Although there were significant differences in the levels of semen MVs expressing CD16⁺ and CD46⁺ between uninfected and HIV-infected individuals (*, P < 0.05 by the Mann-Whitney test), these results were no longer significant after multiple-testing correction using the Benjamini-Hochberg procedure. (D) Scatterplots of the concentrations of MVs (log₁₀ transformed) from 10 chronically HIV-infected individuals with paired samples before and after initiation of ART. P values were determined by the Wilcoxon matched-pairs signed-rank test. *, P < 0.05; **, P < 0.01 These results remained significant after correcting for multiple testing with an FDR of 0.1.

FIG 6

t-SNE analysis of flow cytometric data for the MV fraction from uninfected and HIV-infected individuals. The expression levels of the indicated antigen are shown as heat plots, while individual EVs are represented as dots on the t-SNE. The colors correspond to arcsinh-transformed expression values for each given marker analyzed, with lower expression levels being shown in blue and higher expression levels being shown in red. Shown are the results for 3 representative individuals of each group of participant samples. The patterns of the t-SNE were similar between EVs from uninfected individuals and EVs from infected individuals, suggesting no global differences in the expression patterns of the antigens examined in this study. Shown are data sets corresponding to EVs stained for markers of lymphocytes, monocytes, and B cells (A), prostasome markers (B), and neutrophils and platelets (C).

FIG 7

Hierarchical clustering analysis and PCA of miRNAs from an EV-enriched fraction from uninfected and HIV-infected individuals. (A) Cluster dendrogram of EV-associated miRNAs, determined using standardized Euclidean distances with complete link hierarchical clustering. (B) (Left) PCA plot showing that sample variance is higher among uninfected individuals (red) than among HIV-infected individuals (blue); (right) box plot analysis of principal component 1 (PC1) demonstrating the higher variance among the uninfected EV specimens.

FIG 8

Differential expression analysis of miRNAs from an EV-enriched fraction from uninfected and HIV-infected individuals. (A) Frequency distribution of EV-associated miRNAs for uninfected and HIV-infected individuals. Each miRNA is represented by the number of TPM. (B) Venn diagrams showing common gene expression between the miRNAs from uninfected and HIV-infected individuals. (C) Heatmap showing the Z-scores for the top miRNAs differentially expressed between uninfected and HIV-infected individuals (FDR < 0.05). miRNAs were ranked by the fold change in expression (in parentheses), and samples were ranked by median expression. Each column represents an individual participant, and each row represents a single miRNA. The color scale shown on the top illustrates the relative expression levels of the indicated miRNAs across all samples.

FIG 9

Schematic of generation of the microvesicle (MV) fraction, exosome fraction, and reconstituted seminal plasma (rSP) from semen. Semen samples from uninfected and HIV-infected men were centrifuged at 400 to 600 × g, after which 250 μl of the supernatant was removed as a source of SP. The SP was then centrifuged through a 0.65-μm-pore-size filter, followed by a second round of centrifugation through a 0.22-μm-pore-size filter. The flowthrough from the final centrifugation was saved as the exosome fraction. The retentate from the final centrifugation was resuspended in 250 μl PBS and used as the MV fraction. To generate rSP, the MV fraction was resuspended in the exosome fraction.