Abstract
BackgroundThe 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.ResultsHere, 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.ConclusionsAltogether, these data suggest that various EV subpopulations induce cytokine expression, tissue damage, and viral spread.
Highlights
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)
The advantage of utilizing differential ultracentrifugation (DUC) exclusively over iodixanol gradients is that DUC can result in a higher Extracellular Vesicles (EVs) yield and does not require additional reagents that may interfere with downstream assays
These data support the hypothesis that 2 k EVs are denser than 10 k and 100 k EVs, resembling EVs that would sediment in high-density fractions, as shown in our previous publication [35]
Summary
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 Carib‐ bean and South America. HTLV-1 infects 10 million globally with pockets of high endemicity in the Caribbean, South America, Western and Southern Africa, Iran, Japan, and Australia [2]. HTLV-1 was first found in 1979 and published in 1980 [3, 4] and is the causative agent of an aggressive cancer known as Adult T-cell Leukemia/ Lymphoma (ATLL) [5,6,7]. HTLV-1 causes a set of progressive neuroinflammatory conditions known as HTLV-1-associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP) [8, 9]. Reports of 33.6% prevalence in a cohort of 1889 indigenous Australian patients [18] and the lack of global awareness
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