Extracellular vesicles from HTLV-1 infected cells modulate target cells and viral spread

Abstract

Background: The Human T-cell Lymphotropic Virus Type-1 (HTLV-1) is a blood-borne pathogen and etiological agent of Adult T-cell Leukemia/Lymphoma (ATLL) and HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). HTLV-1 has currently infected up to 10 million globally with highly endemic areas in Japan, Africa, the Caribbean and South America. We have previously shown that Extracellular Vesicles (EVs) enhance HTLV-1 transmission by promoting cell-cell contact.

Results: Here, we separated EVs into subpopulations using differential ultracentrifugation (DUC) at speeds of 2 k (2000×g), 10 k (10,000×g), and 100 k (100,000×g) from infected cell supernatants. Proteomic analysis revealed that EVs contain the highest viral/host protein abundance in the 2 k subpopulation (2 k > 10 k > 100 k). The 2 k and 10 k populations contained viral proteins (i.e., p19 and Tax), and autophagy proteins (i.e., LC3 and p62) suggesting presence of autophagosomes as well as core histones. Interestingly, the use of 2 k EVs in an angiogenesis assay (mesenchymal stem cells + endothelial cells) caused deterioration of vascular-like-tubules. Cells commonly associated with the neurovascular unit (i.e., astrocytes, neurons, and macrophages) in the blood-brain barrier (BBB) showed that HTLV-1 EVs may induce expression of cytokines involved in migration (i.e., IL-8; 100 k > 2 k > 10 k) from astrocytes and monocyte-derived macrophages (i.e., IL-8; 2 k > 10 k). Finally, we found that EVs were able to promote cell-cell contact and viral transmission in monocytic cell-derived dendritic cell. The EVs from both 2 k and 10 k increased HTLV-1 spread in a humanized mouse model, as evidenced by an increase in proviral DNA and RNA in the Blood, Lymph Node, and Spleen.

Conclusions: Altogether, these data suggest that various EV subpopulations induce cytokine expression, tissue damage, and viral spread.

Fig. 1

Tax and p19 viral proteins are present in EV populations derived from HTLV-1 infected cells. a Viral protein distribution was evaluated in different EV populations (i.e., 2 k, 10 k, and 100 k) derived from HUT102 cells. Mass spectrometry data was searched against a fully tryptic HTLV-1 database (UniProt) via Proteome Discoverer 2.1. b Different EV populations (2 k, 10 k, and 100 k) of HUT102 were obtained via differential ultracentrifugation and 30 μg was loaded into each lane. Viral proteins (p19 and Tax), EV marker (CD63), autophagy proteins (LC3-I, LC3-II, and p62) and Actin were detected using Western blot. c Quantification of Western blot bands by densitometry analysis

Fig. 2

Proteomic analysis of Human proteins in HTLV-1 EVs. a Venn diagram of 2 k, 10 k, and 100 k EVs showing total number of proteins identified (in parenthesis) and unique proteins (underneath total proteins). Mass spectrometry data was searched against fully tryptic indexed Homo sapiens database (UniProt) via Proteome Discoverer 2.1. Next, Protein–Protein Interaction (PPI) network was evaluated for unique proteins in b 2 k, c 10 k, and d 100 k EVs. Predicted PPIs were analyzed by STRING to determine the pathway interaction of proteins in each EV. PPIs were analyzed from STRING to determine the pathway enriched in each EV population. In the PPI network, nodes represent Proteins (circles), Edges represent Protein–Protein Associations (lines), and color represent related pathway or family of protein (colored circles). High confidence levels of 0.7–0.9 were used for this analysis. e HTLV-1 EVs (2 k, 10 k, and 100 k) and f control EVs (CEM and U937) were evaluated using a Luminex platform for presence of encapsulated or surface-bound cytokines. Only 9 representative EVs were selected out of a panel of 33 cytokines. Red boxes show the most abundant cytokines across the different EV populations

Fig. 3

EV subpopulations enhance cell-to-cell contact, which is abolished by ICAM-1 or CD45 siRNA. a Fluorescent Microscopy was used to track BODIPY™ 493/503 labeled EVs (2 k at 1: 6,200 cell to EV; 10 k at 1: 20,000; 100 k at 1: 8,200, and CEM at 1: 8,866) added to CEM recipient cells. Analysis was performed 24 h post-treatment and quantified total and aggregated cells. Scale bar (100 μM). b Cell viability of recipient cells (CEM; 5 × 10⁵ cells/mL, in biological triplicates) incubated with HTLV-1 2 k, 10 k, 100 k, and total EV populations (CEM and HUT102). c Cells and corresponding total EVs (CEM and HUT102), d as well as subpopulations (2 k, 10 k and 100 k EVs), were analyzed via Western blot for cell surface proteins (CD45, CD43, ICAM-1, LFA-1), HTLV-1 proteins (gp46, and p19), EVs (CD63), and Actin. e The 10 k and 100 k EVs were isolated (single 100,000 × g spin) from HUT102 cells transfected cells with siRNA against ICAM-1, CD45, or scramble (for 3 days). All samples were labeled with BODIPY and then incubated with CEM cells at a ratio of 1:10,000 cell to EV for 3 days. Total HUT102 EVs was used as positive control for enhanced cell-to-cell contact and CEM cells alone or with scrambled siRNA as a negative control. The images were taken by Fluorescent Microscopy. f HTLV-1 donor cells (HUT102) were irradiated (IR; 10 Gy) to induce cell cycle arrest and treat recipient cells (CEM) in fresh exo-free media for 4 days. Western blot analysis was performed for p19 and Actin. Statistical analyses were performed using two-tailed Student’s t test, “*” for p ≤ 0.05 and “**” for p ≤ 0.01

Fig. 4

Functional Effects of HTLV-1 EVs on Angiogenesis and Inflammation. a An angiogenesis assay in technical and biological triplicate was used to determine the effect of distinct HTLV-1 EVs (2 k, 10 k, and 100 k) on tubular formation in a co-culture of mesenchymal stem cells (MSCs) and aortic endothelial cells (AEC). EV-treated cells (1:2000 recipient cell to EV ratio). Positive control cells received complete, undiluted medium. Additional dose of EV treatment was given to the cells at day 4. Representative images were taken at days 3 and 6 showing tubular formation in response to the indicated treatment. b The image processing software WIMASIS was used to calculate the percentage of area covered by tubules (n = 3) on day 3 and day 6. c RT-qPCR results showing env RNA copy numbers of mesenchymal stem cells treated with CEM EVs (control) and different populations of HTLV-1 EVs: 2 k (left panel), 10 k (middle panel), and 100 k (right panel). A set of dashed black vertical lines (---) were used to indicate baseline env RNA copy numbers. A set of dashed red vertical lines ( ) were used to indicate the levels of starting material, suggestive of the minimum env RNA copy numbers necessary for EVs to increase vial spread in MSc. d Western blot analysis for core histones (H3, H2A, H2B, and H4), linker histone (H1) and actin in HUT102 EVs (2 k, 10 k, and 100 k). e GAPDH DNA levels (representative of nucleosomes) in 2 k, 10 k, and 100 k HUT102 EVs treated with proteinase K and DNase/RNase were evaluated by was quantitated by q-PCR. A two-tailed student t-test was used to evaluate statistical significance with “**” for p-values ≤ 0.01, indicating the level of significance relative to untreated (Control) samples

Fig. 5

HTLV-1 EVs promote differential expression of IL-8, IL-6, and RANTES in CNS related cells. HTLV-1 EVs (2 k, 10 k, and 100 k; HUT102) were used to treat a CCF-STTG1, b MDM (THP-1 + PMA), c SHSY-5Y, and d MDM (U937 + PMA). Supernatants were collected 5 days after, enriched for EVs using nanotrap nanoparticles (NT80/82), and evaluated for presence of proinflammatory cytokines (IL-8 and IL-6) via Western blot analysis. HTLV-1 EVs (2 k, 10 k, and 100 k; HUT102) were exposed to none (untreated control), RNase A, or DNase I and then used to treat e CCF-STTG1 (Astrocytes; upper left panel) and mDCs (THP-1 cells + GM-CSF (50 ng/mL) + IL-4 (1000 U/mL) + TNF-α (20 ng/mL) + ionomycin (200 ng/mL); upper right panel). After 5 days of incubation, supernatants were collected and incubated with NT80/82 for EV enrichment. Western blots analysis was performed to determine protein expression of IL-8, RANTES, GAPDH, and Actin. Densitometry analysis of the RANTES bands for astrocytes (lower left panel) and mDCs (lower right panel) was carried out using ImageJ analysis software and subtracting the background of each membrane

Fig. 6

HTLV-1 100 k EVs enhance viral transmission in dendritic cells. a A diagram representing a timeline of the experimental design and treatment conditions from day 0 to day 9. b Flow cytometry analysis of the EV uptake by mDCs was performed in biological duplicate. Dot plots (upper panels) contain a P1 (red) and P2 (green) gated mDC populations, where the P1 was used to evaluate percent change (% ), increases in size and morphology, of P1 control compared to EV treatment. The control mDC populations in P1 and P2 are 33.7% and 49.9%, respectively. Histograms (lower panel) show increase in fluorescence intensity (right shift) as a function of EV uptake by mDCs. c Recipient mDCs were incubated for 5 days with various populations of HTLV-1 EVs (2 k, 10 k, and 100 k EVs) and CEM EVs (control EVs) at a ratio of 1 cell to 10,000 EVs. This was followed by microscopic analysis. Images are representative of three independent experiments. Following microscopy, recipient cells were then treated with irradiated HUT102 cells (HTLV-1 Donor Cells; 10 Gy) and fresh exo-free media for 4 additional days. RNA was isolated from recipient cell pellets and quantitated by RT-qPCR for viral env RNA. The RNA levels for the EV input are denoted by blue bars (control), while recipient cells treated with the input EVs by the orange bars (Recipient). The recipient mDC viability was analyzed at day 9 (cultured in biological triplicates). Vertical dashed bar (---) separates additional control EVs on the right-hand side for Total EVs (from HUT102) and Control EVs (from CEM). A two-tailed student t-test was used to compare control cells with recipient cells and to evaluate statistical significance with “*” for p-values ≤ 0.05, “**” for p-values ≤ 0.01, and “***” for p-values ≤ 0.001, indicating the level of significance

Fig. 7

HTLV-1 2 k EVs promote enhanced viral spread. a Presence of proviral DNA (using qPCR for env region) from blood and tissues from multiple organs (Spleen, Liver, L.N., and Brain) of NOG mice (n = 12) treated with control (NOG 1–3), 2 k (NOG 4–6), 10 k (NOG 7–9), and 100 k (NOG 10–12) EVs followed by HUT102 donor cells (IRed) treatment 5 days later. b Presence of RNA (using RT-qPCR env RNA) from blood and tissues of NOG mice (n = 12) treated with control (NOG 1–3), 2 k (NOG 4–6), 10 k (NOG 7–9), and 100 k (NOG 10–12) EVs followed with HUT102 donor cells (IRed) treatment 5 days later. A two-tailed student t-test was used to evaluate statistical significance with “*” for p-values ≤ 0.05, “**” for p-values ≤ 0.01, and “***” for p-values ≤ 0.001, indicating the level of significance compared to control