Viral proteins are differentially packaged into EVs from HTLV-1 infected cells
In this manuscript we sought to further investigate HTLV-1 EVs via the use of DUC, generating three distinct EV populations: 2 k, 10 k, and 100 k EVs. The advantage of utilizing DUC exclusively over iodixanol gradients is that DUC can result in a higher EV yield and does not require additional reagents that may interfere with downstream assays. We expected 2 k, 10 k, and 100 k EVs to resemble EVs sedimented in high-, mid-, and low-density Iodixanol fractions, respectively. Using Nanoparticle Tracking Analysis (NTA), we observed that the average diameter of 2 k EVs were about 189 nm, 10 k around 171 nm, and 100 k around 129 nm (Additional file 1: Fig. S1). These data support the hypothesis that 2 k EVs are denser than 10 k and 100 k EVs, also resembling EVs that would sediment in high-density fractions, as shown in our previous publication [35].
EVs play an important role in cell-to-cell communication, with potential roles in autocrine, paracrine, and endocrine signaling [35, 41, 62,63,64,65,66]. EV biogenesis may be connected to normal cellular processes, such as autophagy, by serving as intracellular “vehicles” that carry cargo targeted for degradation [38, 40, 67,68,69]. Additionally, we have shown that infection with viruses, such as HTLV-1 and HIV-1, cause the release of EVs with viral cargo (i.e., viral protein and non-coding RNA). In order to further understand the capacity of EVs to carry viral and human proteins, we performed proteomic analysis of the 2 k, 10 k, and 100 k EVs from HTLV-1 infected cells. EV samples were reduced by dithiotreitol, alkylated with iodoacetamide, trypsinised overnight, desalted using C-18 ZipTips, dried under nitrogen, and reconstituted with Formic Acid prior to injection into the Liquid Chromatography Tandem Mass Spectrometry (LCMS/MS) instrument. Acquired MS/MS spectra were searched against a fully tryptic HTLV-1 database (UniProt) with Proteome Discoverer 2.1. Data in Fig. 1a show the proteomic distribution of viral proteins. To qualitatively analyze the viral proteomic data, we evaluated the relative abundance of each protein present (“ + ”) within an EV type. The relative abundance of a protein was then indicated by “ + ” if the peptide hits were detected between 1 to 5 times, “ + + ” between 6 to 15 times, “ + + + ” between 16 to 29 times, and “ + + + + ” for 30 times and above. In 2 k EVs, the precursor polyprotein Gag-Pro-Pol (most frequent), Gag (p19), Envelope, and Tax proteins were detected (Gag-Pro-Pol ++++ > Gag++ > Envelope++ > Tax+). In 10 k EVs, Gag-Pro-Pol (most frequent), Gag (p19), Gag-Pol, Envelope, and Protease were detected (Gag-Pro-Pol +++ > Gag+++ > Gag-Pol++ > Envelope+ > Protease+). In 100 k EVs, Gag-Pro-Pol (most frequent), Gag, and Envelope were detected (Gag-Pro-Pol ++++ > Gag++ > Envelope+). All EVs contained Gag-Pro-Pol, Gag and Envelope related proteins. Unique viral proteins were detected in 2 k (i.e., Tax), 10 k (i.e., Protease), while 100 k EVs contained no unique viral proteins. Overall, this data suggests potential differential packaging of viral proteins into different EV types.
To further validate the presence of viral proteins in EVs we inspected EVs from two different HTLV-1 infected cell lines, HUT102 and ATL-16. Data in Fig. 1b show Western blot of EVs separated by DUC and observed that the Gag related matrix protein, p19 was present in 2 k, 10 k, and 100 k EVs from both HUT102 (lanes 1–3) and ATL-16 (Additional file 1: Fig. S2). Densitometry analysis confirmed higher abundance of p19 in the 10 k EVs from HUT102 (Fig. 1c). Next, we observed presence of Tax in 2 k, and more so, in 10 k EV from HUT102 cells. It is important to mention that Tax was also detected in the MS analysis, however it was present as a peptide fused to Gag (13 hits; see Additional file 2) and therefore considered as not significant. Detection of Tax in WB serves as a confirmation. In the case of EVs from ATL-16 cells, Tax also was predominantly present in the 10 k subpopulation. We also assayed for presence of the common EV marker, CD63, and observed different patterns of protein modification. In the case of HUT102, the 2 k and 10 k EVs contained glycosylated and unmodified CD63, however the 100 k EVs contained almost exclusively unmodified CD63. On the other hand, EVs from ATL-16 cells contained primarily glycosylated CD63, with exception of the 10 k EV subpopulation, which also contained unmodified CD63 (Additional file 1: Fig. S2). We have previously observed the existence of particular patterns of CD63 modifications. EVs isolated from HAM/TSP patient PBMCs were positive for both the glycosylated and unmodified CD63 populations, whereas those isolated from normal PBMC donors only contained unmodified CD63 [41]. Overall, our data suggests that both the 2 k and 10 k EVs from HTLV-1 infected cells have the potential to modulate cell responses conducive of disease progression, due to the detection of viral proteins (Gag/p19 and Tax) and of EV markers previously associated with advanced stages of HTLV-1 infection ((glycosylated (Gly-CD63) and unmodified CD63)). However, the 100 k EV population has unique and previously unseen characteristic in the context of HTLV-1, suggestive of a vesicle with the ability to interact with cells compared to 2 k/10 k EVs, but without the oncogenic effects of Tax.
Next, we investigated for the presence of autophagy proteins, as these may be suggestive of the biogenesis pathway generating each EV population. In Fig. 1b, we also probed for LC3-I/II, an important protein in the formation of the autophagosome, a membrane-bound structure responsible for encapsulating intracellular components. LC3-I elongates the pre-autophagosome’s membrane and promotes engulfment of components in a ubiquitin-like mechanism of targeting. Once the autophagosome is formed, LC3-II is normally detected, suggesting flux from LC3-I to LC3-II and advanced stages of autophagy, which could include degradation of cargo and secretion of vesicles [38, 70]. Therefore, LC3-I in the extracellular environment could be suggestive of high levels of cytoplasmic LC3-I resulting in secretion in EVs. However, LC3-II alone in the extracellular environment could indicate the secretion of vesicles derived from mature stages of autophagy. The autophagy protein p62, could be used to evaluate autophagy flux since it is essential to the activation of autophagy. Upon the p62-mediated recruitment of autophagy proteins and formation of mature autophagosomes, p62 levels decrease due to potential degradation [71].
Interestingly, our Western blot data revealed increased presence of LC3-II marker in 2 k EVs from HUT102 cells, but not in ATL-16 (Additional file 1: Fig. S2). In contrast, observations of 10 k EVs from HUT102 cells revealed increased LC3-I levels, whereas ATL-16 contained mostly LC3-II. In order to validate our observations, we probed for the p62 protein. Specific cytoplasmic targets, such as viral components (among others), may stimulate p62 via pattern recognition receptors (PRRs), initiating recruitment of autophagic machinery [38, 70, 72]. Activation of autophagy requires initial upregulation of p62; however, upon formation of the autophagosome, decreased p62 levels have been reported [70]. Here we found that HUT102/ATL-16 2 k EV did not contain p62, however both 10 k and 100 k (more abundant) contained p62.
Overall, 2 k EVs contained mature markers of autophagy as evidenced by presence of LC3-II and no p62, whereas 10 k and 100 k EVs contained markers of pre-autophagosomes (LC3-I and p62; early stages of autophagy). This data is suggestive that 2 k EVs may carry viral components that have undergone some degradation. On the other hand, 10 k EVs may potentially contain viral proteins targeted for autophagic degradation that may have been interrupted, resulting in secretion through the EV pathway.
Proteomic analysis of human proteins in EVs from infected cells
We next analyzed presence of human proteins in each EV population (2 k, 10 k, and 100 k EVs) via mass spectrometry. EV samples were processed as indicated previously for analysis of viral proteins, however acquired MS/MS spectra were searched against a fully tryptic indexed Homo sapiens database (UniProt) with Proteome Discoverer 2.1. The 2 k population contained 1,020 unique proteins of interest out of a total 1,614 proteins; 10 k population contained 56 unique proteins out of 606; and 100 k population contained 66 unique proteins out of 415 total proteins (Fig. 2a). Therefore, the 2 k population contains the highest variety of human proteins (2 k > 10 k > 100 k).
Next, we examined whether 2 k, 10 k, and 100 k EVs contained unique human proteins with roles in processes related to HTLV-1 pathogenesis. Therefore, we analyzed for functional protein interactions and generated a Protein–Protein Interaction (PPI) network using the Search Tool for the Retrieval of Interacting Genes (STRING) to predict relevant functional interactions [73]. The PPI network of 2 k EVs was enriched in proteins involved in Parkinson’s Disease, Phagosome formation, Cell Cycle, Ras GTPase, Helicase Superfamily Terminal Domains, and Ribosome Biogenesis (Fig. 2b). The 10 k EVs contained noninteractive proteins belonging to the family of Alpha-Actinin-4; Tubulin beta-2A chain; Dehydrogenase/reductase SDR family member 2; and Rab GDP dissociation inhibitor alpha family proteins (Fig. 2c). Finally, the 100 k EVs contained a PPI network interconnected by fibronection-1 (FN1) with proteins involved in PI3K-Akt signaling, ECM-receptor interaction; TGF-beta signaling, and Focal adhesion with a high degree of confidence (> 0.7) (Fig. 2d). Overall, the 2 k population contains several proteins with interconnected potential functions in disease (i.e., Parkinson’s) and in regulation of cell cycle, autophagy, euchromatin and gene activation, and protein synthesis/translation, and the 100 k EV proteins were all connected to FN1, involved in the support of cell migration, cell differentiation, and tissue repair [74]. The 10 k EVs contained noninteractive proteins with minimal complementary effects.
Finally, it has already been reported that EVs can modulate immune responses by promoting expression of cytokines [75], or of proteins important for immune cell interaction, such as upregulation of ICAM-1 [35, 76]. More importantly, EVs have the potential to carry cytokines (encapsulated or membrane-bound) and molecules, such as ICAM-1, denoting the importance EVs in pathogenesis [35, 75]. Additionally, the cytokine profile in EVs has been shown to be consistent with the type of cell or organ where it originates [77]. Using high throughput ELISA, we screened for cytokines encapsulated or present on the surface of EVs (2 k,10 k, and 100 k). Data in Fig. 2e show that the 2 k EVs encapsulated high-levels of the proinflammatory cytokine involved in macrophage activation, known as Macrophage Inflammatory Proteins (MIP-1a). On the other hand, 10 k EVs encapsulated the cell proliferation cytokine IL-18, and 100 k EVs encapsulated the chemokine RANTES (RANTES: regulated upon activation normally T-cell expressed and secreted). However, on the surface of EVs the predominant cytokine was IL-33, known for regulating gene expression and promoting secretion of IL-4, IL-5, IL-9 by T-helper, mast, eosinophils, and basophils cells [78, 79]. IL-33 was on the surface of all three EV populations (2 k, 10 k, and 100 k EVs). EVs from uninfected T-cells (CEM) and myeloids (U937) were also evaluated, and cytokine levels were low compared to HUT102 EVs, except for IL-33 (Fig. 2f). Overall, this suggests that EVs from HTLV-1 infected cells contain higher cytokine levels than EVs from uninfected cells, and that the levels of inflammatory molecules vary in or on the surface of EVs according to the EV subpopulation.
Functional effects of 2 k, 10 k and 100 k HTLV-1 EVs on uninfected recipient T-cells
We have recently shown that HUT102 EVs (HTLV-1 EVs) promote cell-to-cell contact between uninfected T-cells [35]. Here, we attempted to elucidate whether a particular HTLV-1 EV subtype (i.e., 2 k, 10 k, and 100 k) is responsible for the increased cell-to-cell contact previously observed. Various size EVs were labeled with BODIPY and used to treat uninfected CEM T-cells. Using fluorescent microscopy, we observed uptake of all size EVs 24-h post-treatment (Fig. 3a). All HTLV-1 EV subtypes promoted increased aggregation of CEM cells, with the largest agglutination with 10 k (21.33%), followed by 2 k (18.84%), and least with 100 k (12.52%) EVs (10 k > 2 k > 100 k EVs). Finally, CEM cell viability was unaffected by HTLV-1 EV treatment, with only a slight but significant increase when treated with 10 k HTLV-1 EVs (Fig. 3b; lane 4). Furthermore, this data suggests that HTLV-1 EVs from 2 k, 10 k, and 100 k populations have the potential to increase agglutination and cell-to-cell contact without causing cell death. Altogether, this data suggests that the increase in agglutination and cell-to-cell contact is a consequence of treatment with EVs, and not an artifact related to treatment and cell death.
HTLV-1 EVs from all three populations ubiquitously promoted increased cell-to-cell contact, with potential increased potency in 10 k EVs. Cell-to-cell contact may be mediated by cell surface proteins and adhesion molecules such as CD45, CD43, ICAM-1, and LFA-1 [21, 22]. We have recently shown that EVs from HTLV-1 infected cells (with and without transcriptional activation via irradiation) contain protein markers involved in mechanisms of viral transmission. More specifically, we have shown that HTLV-1 EVs contain proteins involved in the formation of VB (i.e., CD45) and VS (i.e., ICAM-1) and that neutralizing antibodies against these two proteins may decrease the agglutination and viral spread in cell lines and PBMCs [35].
Here we sought to further investigate expression levels of proteins involved in HTLV-1 transmission into recipient uninfected cells (CEM; intracellular proteins), EVs from uninfected cells (CEM EVs; EV-associated proteins), infected donor cells (HUT102 cells; intracellular proteins), and EVs from HUT102 cells (HTLV-1 EVs; EV-associated proteins). When using Western blot analysis, intracellular protein expression levels of CD45, CD43 (binding partner of CD45), ICAM-1, and LFA-1 (binding partner of ICAM-1) were significantly elevated in HTLV-1 infected HUT102 cells (Fig. 3c; lane 2) compared to uninfected CEM cells (Fig. 3c; lane 1). Intracellular viral proteins were also probed as controls for infection, with bands for Tax, gp61, gp46, and p19 present only in the intracellular material from HUT102 cells (Fig. 3c; lanes 2). Moreover, the tetraspanin and EV marker, CD63 [80], showed equal protein expression levels between HUT102 and CEM cells. We next investigated the EV-associated protein levels in HTLV-1 EVs and CEM EVs. Cell protein markers and adhesion molecules present in VBs (CD45 only) or involved with the formation of VS (ICAM-1 and LFA-1) were almost exclusively present in HTLV-1 EVs (Fig. 3c; lane 4), with traces of CD45 and LFA-1 present in CEM EVs (Fig. 3c; lane 3). Not surprisingly, viral proteins Tax, gp61, and p19 were present only in HTLV-1 EVs, as previously observed [35]. However, an interesting observation was made regarding an EV marker protein, where the unmodified band for CD63 was expressed in both HTLV-1 and CEM EVs, but the modified, glycosylated form of CD63 (Gly-CD63) was only observed in HTLV-1 EVs (Fig. 3c; lane 4 vs. lane 3).
Separation of HTLV-1 EVs (2 k,10 k, and 100 k) would allow further characterization of which EV population is responsible for promoting cell-to-cell contact and viral spread. Data in Fig. 3d shows that CD45 was abundant in 100 k EVs from CEM and HUT102 (lanes 5 and 8). However, CD43 was not seen in EVs obtained from both cell lines (lanes 3–8). EVs from infected cells had high expression of ICAM-1 in 2 k and 100 k (lanes 6 and 8) and less in 10 k EVs (lane 7). Control EVs (CEM EVs) did not contain detectable ICAM-1 (lanes 3–5). Similarly, LFA-1 was detected in 2 k, 10 k, and 100 k EVs, but with reduced signal in 10 k EVs and no detectable signal in Control EVs. We also assessed for presence of viral proteins and observed their presence only in HTLV-1 EVs. We observed increased expression of gp46 and p19 in 2 k and 100 k (lanes 6 & 8) compared to 10 K (lane 7). Finally, Gly-CD63, typically found in EVs from infected cells ((see [41, 54]), was observed primarily in 2 k and 10 k, but not in 100 k EVs or in Control EVs.
Altogether, these data suggest that HTLV-1 infected cells have an upregulated expression of proteins important for viral transmission via mechanisms of VB (CD45 and CD43) and VS (ICAM-1 and LFA-1) which are also overexpressed extracellularly on HTLV-1 EVs in the case of CD45, ICAM-1 and LFA-1. Furthermore, the presence of HTLV-1 EVs and their cargo may promote cell-to-cell contact and subsequent viral transmission. HTLV-1 EVs may potentially adhere and "decorate" uninfected cell surfaces, facilitating agglutination without compromising cell viability.
Inhibition of ICAM-1 and CD45 via small interfering RNA prevents packaging into HTLV-1 EVs and cell-to-cell contact
We have shown that the mechanisms of cell-to-cell contact in HTLV-1 infection may be mediated by EVs, especially during enhanced viral spread [35]. More specifically, we have shown that EVs from infected cells carry proteins known to be involved in cell-to-cell contact (ICAM-1 and CD45) and that neutralization with antibodies protected cells from enhanced viral spread [35]. Here, we attempted to go one step further and validated these observations by inhibiting translation of ICAM-1 and CD45 from the infected donor cells using small interfering RNA (siRNA). EVs from siRNA (scrambled, CD45, and ICAM-1) treated donor cells were isolated, as described previously (Fig. 3a), and protein levels evaluated using Western blot analysis (Additional file 1: Fig. S3). Addition of the scrambled siRNA (lane 1) showed baseline levels of CD45, ICAM-1, and Actin proteins in these EVs. In addition, CD45 and ICAM-1 levels were suppressed when treated siRNA against CD45 (lane 2) and siRNA against ICAM-1 (lane 3), respectively. We next used these EVs to treat recipient uninfected cells (Fig. 3e). We observed that EVs from infected cells treated with siRNA against ICAM-1 reduced cell aggregation by 6.97% and siRNA against CD45 by 7.40% (Fig. 3e). As expected, positive and negative controls showed a total aggregation at 43.80% and background at 1.94%, respectively [35]. We also examined the uninfected CEM target cells (Recipient cells) by performing a Western blot probing for p19 in the WCE of recipient cells (Fig. 3f). The p19 levels were less abundant in CEM cells treated with EVs from infected cells treated with siRNA against ICAM-1 and CD45, suggesting that inhibiting ICAM-1 or CD45 resulted in a potential decrease of viral spread into target cells. Altogether, this data suggests that the virus may use intracellular pathways to promote packaging of ICAM-1 and CD45 into EVs, that would subsequently enhance cellular aggregation and viral spread.
Functional effects of HTLV-1 EVs on angiogenesis and inflammation
We have recently used an in vitro angiogenesis assay to examine the effects of stem cell EVs on the formation of vascular structures [43]. The assay consists of a mixture of mesenchymal stem cells (MSCs) and GFP expressing human aortic endothelial cells (AECs) that were cultured together to investigate the effects of distinct HTLV-1 EV populations. The AECs in this co-culture develop vascular-like tubules, which may resemble those produced by endothelial cells in the blood–brain barrier (BBB). We utilized this system to evaluate the functional effects of 2 k, 10 k, and 100 k HTLV-1 EVs on tubular formation or deterioration. Using an angiogenesis assay, data in Fig. 4a showed differential effects of 2 k, 10 k, and 100 k HTLV-1 EVs on tubular formation. At 3 days post-treatment, 2 k and 10 k EVs showed deterioration, as evidenced by shorter and thinner tubules when compared to control (Fig. 4a; Day 3, GFP panels). In contrast, 100 k HTLV-1 EVs displayed longer tubules of consistent thickness, similar to control images. Similar results were observed at day 6 with further deterioration of tubules when using 2 k and 10 k (Fig. 4a; Day 6, GFP panels). The changes in tubule integrity were quantitated by measurement of fluorescent signal on tubules, displayed as a percentage of the total covered area (Fig. 4b). Altogether, these data suggest that 2 k and 10 k EVs have detrimental effects on cells (i.e., Mesenchymal stem cells and epithelial cells), potentially making them contribute to HTLV-1 pathogenesis, such as in the case of HAM/TSP. These negative effects could be due to the high abundance of viral proteins/RNA and human proteins in these two fractions. On the other hand, 100 k EVs may have certain protective and growth effects. Further research is needed to elucidate the mechanism of this observed protection.
HTLV-1 EVs have previously been shown to be internalized by MSCs, resulting in increased proliferation and activation of NF-κB pathway [81]. We therefore examined whether HTLV-1 EVs alone are sufficient to enter (from the angiogenesis assay) and allow viral replication in MSCs. The RT-qPCR analysis was performed on MSCs (Fig. 4c; lane 1) treated with various EVs (lanes 2–4) for 9 days. The starting 2 k, 10 k, and 100 k EV material contained env RNA at approximately 5 × 104 copies/mL, 5 × 104 copies/mL, and 5 × 103 copies/mL, respectively (lane 3). However, env RNA levels in MSCs treated with 2 k, 10 k, and 100 k EVs showed no increase of newly synthesized RNA (lane 4). We also performed additional RT-qPCR analysis in biological triplicate to evaluate the levels of genomic RNA in EVs and observed the highest levels in 2 k and 10 k EVs (Additional file 1: Fig. S4). These data suggest that while HTLV-1 EVs may have detrimental effects on recipient cells and adjacent bystander cells (i.e., endothelial cells), it does not establish a new productive infection in these cells.
In order to further examine the potential mechanisms of cellular damage, we sought to examine for the presence of damage associated molecular patterns (DAMPs) in HTLV-1 EVs. Some of the most common DAMPs are cytoplasmic organelles and nuclear components located in the extracellular environment, normally as a consequence of disease or cellular damage, leading to proinflammatory responses [82]. More recently, DAMPs in the form of heat shock proteins, DNA, histones, and nuclear proteins, such as High Mobility Group 1 [83,84,85], have been found associated with EVs from cells responding to potential infection and from cancer cells [86, 87]. Interestingly, proteomic analysis revealed presence of the HMGB1 and heat shock proteins almost exclusively in the 2 k EV population, and others in the field have previously made similar observations [88]. In Fig. 4d we show a representative Western blot where the 2 k EV population (lane 1) shows strong bands for histones H3, H2A/B, and H4, with histone modifications present in all samples. The 10 k EVs (lane 2) contained high levels of histones H3, H2A, and H1, and trace levels of H2B and H4. It is important to note that 2 k and 10 k EVs also showed varying histone modifications present in H3, H2A, H2B, and H1 proteins (compare lanes 1 and 2). The 100 k EVs (lane 3) contained no detectable histones. We hypothesized that if the histones are bound to DNA, and carried by the EVs bound to their surface, then use of a proteinase K and DNase/RNase cocktail would allow digestion of DNA, RNA and proteins (histones). However, if cargo is encapsulated in EVs, then the digestion cocktail would not have effects on the EV cargo. Additionally, we used freeze/thaw to rupture EV membranes and release encapsulated cargo. In Fig. 4e using GAPDH DNA as a generic marker of nucleosome (DNA + histones), we observed most of the nucleosomes outside of the 2 k EVs (Fig. 4e; left panel), and some nucleosomes inside 10 k EVs (Fig. 4e, center panel). Interestingly, we observed presence of DNA inside the 100 k EV, however, since we could not detect histones in 100 k EVs (Fig. 4e; lane 3, right panel), we concluded that the DNA is mostly free in the 100 k EV. These results indicate differences in 2 k, 10 k, and 100 k EV composition, which may regulate angiogenesis through various DAMPs.
HTLV-1 EVs promote differential expression of IL-8, IL-6, and RANTES in CNS related cells
In order to examine the potential role of HTLV-1 EVs in progression of neurodegeneration as observed in HAM/TSP patients, it is important to evaluate the BBB, composed of brain microvascular endothelial cells, pericytes, astrocytes, and together with the microglia/macrophage and neurons form the neurovascular unit. The hypothesis was whether HTLV-1 EVs were able to cross the BBB, then recipient cells in the CNS would respond to viral cargo (i.e., Tax and Env gp61/46) by secretion of cytokines involved in inflammation. We have recently shown that HTLV-1 EVs are capable of increasing proviral DNA in the brain of NOG mice and elicit effects on recipient cells (i.e. agglutination and viral transmission) [35]. In Fig. 5, individual cultures of cells typically involved in neuroinflammation such as astrocytes (CCF-STTG1), monocytic cell-derived macrophages (MDM; THP-1 and U937), and neurons (SHSY-5Y) were incubated with HTLV-1 EVs (2 k, 10 k, and 100 k). Western blot analysis was performed on the supernatant of each culture and probed for the presence of IL-8, IL-6, and RANTES. We observed an abundance of IL-8 from astrocytes treated with 2 k (lane 3), 100 k EVs (lane 5), and a less intense band but still present for 10 k EVs (lane 4); however, IL-6 was mostly absent (Fig. 5a). Data in Fig. 5b show that MDM cells ubiquitously expressed IL-8 (lanes 1–5), however the highest abundance was upon 2 k treatment (lane 3). Trace levels of IL-6 were observed in all conditions (lanes 1–4), except upon 100 k EV treatment (lane 5) and absence of RANTES across all treatments were observed (data not shown). In contrast, neurons did not express IL-8 or RANTES (data not shown), but they expressed the highest levels of background IL-6 in the controls (lanes 1 and 2). Neurons have been shown to express IL-6 under normal physiological conditions [89] and, therefore this higher background levels of IL-6 in controls is expected. Interestingly, the IL-6 levels decreased the most upon treatment with 2 k EVs compared to lane 2 (Fig. 5c). Finally, macrophages did not express either cytokine upon treatment with EVs (Fig. 5d). Overall, both astrocytes and MDM (THP-1) cells showed increased expression of IL-8, a cytokine shown to be involved in cell migration, angiogenesis, and metastasis in cancer cells [90,91,92]. Next, we performed Western blots to detect RANTES, IL-8, GAPDH, and ACTIN. In Fig. 5e, we observed that EVs elicited expression of RANTES in Astrocytes and monocytic cell-derived dendritic cells (mDCs). Addition of RNase A resulted in a slight decrease in the expression of RANTES. Addition of DNase I abolished expression of RANTES in Astrocytes and almost completely in mDCs. IL-8 expression was slightly increased upon addition of EVs treated with RNase A and DNase I in Astrocytes, but it was completely absent in all lanes for mDCs. GAPDH levels were affected for 2 k EVs in Astrocytes, and for 10 k > 2 k EVs for mDCs. These findings suggest that the EV cargo is sensitive to DNase I, potentially due to presence of nucleosome fragments that can serve as DAMPs. The depletion of EV-associated DNA or RNA has the potential to reduce inflammation.
HTLV-1 viral transmission is enhanced by 100 k HTLV-1 EVs in monocytic cell-derived dendritic cells
DCs have been shown to play an important role in viral transmission of HTLV-1 in mucosal tissues during breastfeeding and sexual intercourse [93]. We have shown that HTLV-1 infected cells secrete EVs containing viral proteins and RNA, with functional roles in modulation of cell-to-cell contact and viral transmission on uninfected cell lines, PBMCs, and in humanized infected NOG mice [35]. In Fig. 6, we evaluated the effects of EVs in monocytic cell (THP-1)-derived dendritic cells (mDCs) via use of flow cytometry, microscopy, RT-qPCR, and cell viability assays. The experimental design and timeline were described in detail in Fig. 6a. We performed FACS analysis on a 5-day old co-culture of mDCs with HUT102 EVs (2 k, 10 k, and 100 k EVs), CEM EVs, or no EVs (as control) to evaluate EV uptake by recipient cells (Fig. 6b). The live mDC populations were gated and denoted by “P1” (Control; Fig. 6b), which represented 33.7% of the total measured events (10,000 events total). The dead cell mDC population (P2), representing 49.9% of the total measured events, was identified and gated based on the low fluorescein isothiocyanate (FITC) and forward scatter (FSC) light intensity (data not shown); however, this is also observed in the side-scatter (SSC) versus FSC plots in Fig. 6b. The resulting Dot plots showed a 13.7% (CEM EVs), 11.7% (2 k EVs), 11.4% (10 k EVs) and 5.8% (100 k EVs) increase in the P1 mDC population, suggesting that EV treatment caused increases in granularity and size. The size increase is as also potentially due to increased cell aggregation, as observed previously. Additionally, we observed in histograms display an increase in fluorescence in the P1 population, as evidenced by the shift to right on the y-axis (FITC-A). This suggests that mDCs are uptaking or binding to all EVs types, with a slightly higher affinity for HTLV-1 EVs. In Fig. 6c (Day 5), we show that HTLV-1 EVs also caused increased cell agglutination in mDCs at day 5 post-treatment. Interestingly, we observed that treatment with 2 k HTLV-1 EVs yielded the highest level of cell clusters 78.3% (p-value ≤ 0.001) (Fig. 6c; Day 5, black circles), followed by 100 k EVs 45.7% (p-value ≤ 0.001), and the least with 10 k EVs 30% (p-value ≤ 0.05). Treatment with all populations of HTLV-1 EVs (All HTLV-1 EV; positive control) yielded an increase in agglutination (68.8%; p-value ≤ 0.001) as expected and treatment with EVs from uninfected CEM cells (All CEM EVs; negative control; 21.9%) had no significant increase. Here, virus was introduced by adding HTLV-1 infected cells (HUT102; donor cells) treated with ionizing radiation (IR; 10 Gy) to inhibit cellular replication in donor cells.
Nine days after initial treatment with EVs, RT-qPCR was performed to examine if particular EV populations also yielded changes in HTLV-1 env RNA levels on recipient mDCs (Fig. 6c; Day 9; upper panel). As expected, uninfected recipient mDCs had no HTLV-1 env RNA (lane 1) and donor cells alone (lane 2; Control) or with mDCs (lane 2; Recipient) resulted in about 1,360 env RNA copies/mL. The treatment with 2 k HTLV-1 EVs showed a twofold increase (3,085 copies/mL) in env RNA (lane 3; Recipient) compared to recipient cells with donor cells; however, 2 k HTLV-1 EVs alone (lane 3; Control) was not significantly different from RNA levels in recipient mDCs. The treatment with 10 k HTLV-1 also resulted in increased RNA levels (2,050 copies/mL; lane 4) compared to recipient cells with donor cells, with no significant difference between control and recipient mDCs. Interestingly, HTLV-1 env RNA levels in 100 k EVs where the lowest among all EV populations, with only 49 copies/mL (lane 5; Control). However, pretreatment with 100 k EVs resulted in a statistically significant increase of HTLV-1 env RNA by 2-logs to 2,411 copies/mL in the co-culture of mDCs with donor cells (lane 5; Recipient). The treatment with All HTLV-1 EVs also yielded higher levels of RNA in mDCs (3,347 copies/mL) than in control (2,496 copies/mL; lane 6). Finally, use of All CEM EVs caused a statistically significant increase in env RNA (lane 7; Recipient) similar to 100 k EV treatment. Overall, the data suggests that 100 k HTLV-1 EVs facilitate HTLV-1 Viral transmission in mDCs, despite observations of 2 k HTLV-1 EVs causing highest agglutination of mDCs. The 100 k HTLV-1 EVs are composed of less densely packed particles, of potentially smaller size, characteristics which may have facilitate uptake by mDCs and prime them for infection.
Interestingly, when cell viability was evaluated on three biological replicates, it was consistently observed that addition of EVs affected viability of mDCs (Fig. 6c, Day 9; lower panel). The 2 k HTLV-1 EV population caused the smallest decrease in cell viability (lane 3). Both 10 k and 100 k HTLV-1 EVs significantly decreased cell viability (lanes 4 and 5, respectively). We also observed that addition of All HTLV-1 EVs (Total EVs) and All CEM EVs (Control EVs) also caused a decrease in viability (lanes 6 and 7, respectively). A higher cell viability would result in an increased number of cells, which could explain the observed increased agglutination of mDCs upon 2 k treatment. The decrease in viability may suggests higher cellular stress, potentially resulting in cellular conditions that would promote increased viral transcription, such as in the case of 100 k HTLV-1 EVs (Fig. 6c; Day 9, lane 5). Collectively, these data indicate that all three populations HTLV-1 EVs enhance viral spread in mDCs, however, with increased efficiency upon pretreatment with the 100 k EV population.
In vivo priming with select HTLV-1 EV populations increases susceptibility of specific tissues to HTLV-1 infection
HTLV-1 EVs had differential effects on uninfected recipient cells (i.e., mDCs). The 2 k EV populations caused more elevated numbers of cell agglutination compared to 10 k and 100 k. However, we observed increased susceptibility to infection on uninfected mDCs upon priming with 100 k HTLV-1 EVs. Other EV populations (i.e., 2 k and 10 k) also resulted in elevated HTLV-1 env RNA levels on newly infected cells, when compared to HUT102 donor cells. Since, all HTLV-1 EVs showed potential to cause increased cell-to-cell contact and increases of HTLV-1 env RNA, we examined the effects of each EV population on a humanized NOG mouse model, as described previously [35].
Twelve humanized NOG (hu-NOG) mice were initially treated with 2 k, 10 k, or 100 k HTLV-1 EVs (10 μg in 250 L of PBS) and 5 days later treated with HUT102 donor cells (10 Gy; IRed). Peripheral blood and tissues ((i.e., Liver, Spleen, Lymph Node (L.N.), and Brain)) were collected three weeks later and processed for qPCR analysis of proviral DNA and RT-qPCR analysis of env RNA in each tissue. In Fig. 7a, we show that HTLV-1 proviral DNA in the Blood of NOG mice was increased by 2 k, 10 k, and 100 k HTLV-1 EVs. However, 2 k EVs caused the most consistent increase, with statistical significance (t-test: p-value ≤ 0.05). The Spleen showed a potential increase in proviral DNA when animals were pre-treated with 2 k EVs, but not with 10 k or 100 k EVs. The Liver seemed less susceptible to pretreatment with EVs and HTLV-1 infection, as no increase over control was observed, for 2 k (NOG 4–6), 10 k (NOG 7–9), and 100 k (NOG 10–12) EVs. HTLV-1 proviral DNA in the L.N. was increased with 2 k EVs only and, finally, proviral DNA levels in the Brain were increased above control when treated with 100 k EVs. The overall trend in viral DNA spread in vivo was 2 k > 10 k > 100 k. However, tissues such as the Brain showed potential for 100 k EVs to elicit increased viral transmission (100 k > 2 k > 10 k).
We next further examined the tissues of the twelve humanized NOG mice by evaluating the levels of viral RNA transcription in vivo to determine if a particular EV population resulted in productive or non-productive infection. In Fig. 7b, we observed that the Blood of NOG mice treated with EVs had a significant average increase of 1.7 and 4.6-fold higher env RNA levels with 2 k and 10 k, respectively. No changes to env RNA were observed in mice treated with 100 k EVs. An eightfold and fivefold average increase of env RNA in the Spleen was observed for 2 k and 10 k EVs, respectively. No significant change was observed with 100 k EV pretreatment in the Spleen. The Liver and L.N. was not noticeably affected by EVs, except for a decrease in env RNA in the liver and increase in the L.N. with 2 k EV pretreatment. Finally, the Brain was not affected by EV pretreatment, except for a 2.9-fold increase in 10 k EV treated mice. Overall, the 10 k HTLV-1 EV population showed increased potential to mediate productive viral transmission in the Blood, and 2 k EVs in the Spleen and potentially L.N.