HIV-infection and cocaine use regulate semen extracellular vesicles proteome and miRNAome in a manner that mediates strategic monocyte haptotaxis governed by miR-128 network.

BACKGROUND: Extracellular vesicles (EVs) are regulators of cell-cell interactions and mediators of horizontal transfer of bioactive molecules between cells. EV-mediated cell-cell interactions play roles in physiological and pathophysiological processes, which maybe modulated by exposure to pathogens and cocaine use. However, the effect of pathogens and cocaine use on EV composition and function are not fully understood. RESULTS: Here, we used systems biology and multi-omics analysis to show that HIV infection (HIV +) and cocaine (COC) use (COC +) promote the release of semen-derived EVs (SEV) with dysregulated extracellular proteome (exProtein), miRNAome (exmiR), and exmiR networks. Integrating SEV proteome and miRNAome revealed a significant decrease in the enrichment of disease-associated, brain-enriched, and HIV-associated miR-128-3p (miR-128) in HIV + COC + SEV with a concomitant increase in miR-128 targets-PEAK1 and RND3/RhoE. Using two-dimensional-substrate single cell haptotaxis, we observed that in the presence of HIV + COC + SEV, contact guidance provided by the extracellular matrix (ECM, collagen type 1) network facilitated far-ranging haptotactic cues that guided monocytes over longer distances. Functionalizing SEV with a miR-128 mimic revealed that the strategic changes in monocyte haptotaxis are in large part the result of SEV-associated miR-128. CONCLUSIONS: We propose that compositionally and functionally distinct HIV + COC + and HIV-COC- SEVs and their exmiR networks may provide cells relevant but divergent haptotactic guidance in the absence of chemotactic cues, under both physiological and pathophysiological conditions.

Alterations in Abundance and Compartmentalization of miRNAs in Blood Plasma Extracellular Vesicles and Extracellular Condensates during HIV/SIV Infection and Its Modulation by Antiretroviral Therapy (ART) and Delta-9-Tetrahydrocannabinol (Δ9-THC).

In this follow-up study, we investigated the abundance and compartmentalization of blood plasma extracellular miRNA (exmiRNA) into lipid-based carriers-blood plasma extracellular vesicles (EVs) and non-lipid-based carriers-extracellular condensates (ECs) during SIV infection. We also assessed how combination antiretroviral therapy (cART), administered in conjunction with phytocannabinoid delta-9-tetrahydrocannabinol (THC), altered the abundance and compartmentalization of exmiRNAs in the EVs and ECs of SIV-infected rhesus macaques (RMs). Unlike cellular miRNAs, exmiRNAs in blood plasma may serve as minimally invasive disease indicators because they are readily detected in stable forms. The stability of exmiRNAs in cell culture fluids and body fluids (urine, saliva, tears, cerebrospinal fluid (CSF), semen, blood) is based on their association with different carriers (lipoproteins, EVs, and ECs) that protect them from the activities of endogenous RNases. Here, we showed that in the blood plasma of uninfected control RMs, significantly less exmiRNAs were associated with EVs compared to the level (30% higher) associated with ECs, and that SIV infection altered the profile of EVs and ECs miRNAome (Manuscript 1). In people living with HIV (PLWH), host-encoded miRNAs regulate both host and viral gene expression, which may serve as indicators of disease or treatment biomarkers. The profile of miRNAs in blood plasma of PLWH (elite controllers versus viremic patients) are different, indicating that HIV may alter host miRNAome. However, there are no studies assessing the effect of cART or other substances used by PLWH, such as THC, on the abundance of exmiRNA and their association with EVs and ECs. Moreover, longitudinal exmiRNA profiles following SIV infection, treatment with THC, cART, or THC+cART remains unclear. Here, we serially analyzed miRNAs associated with blood plasma derived EVs and ECs. Methods: Paired EVs and ECs were separated from EDTA blood plasma of male Indian rhesus macaques (RMs) in five treatment groups, including VEH/SIV, VEH/SIV/cART, THC/SIV, THC/SIV/cART, or THC alone. Separation of EVs and ECs was achieved with the unparalleled nano-particle purification tool ─PPLC, a state-of-the-art, innovative technology equipped with gradient agarose bead sizes and a fast fraction collector that allows high resolution separation and retrieval of preparative quantities of sub-populations of extracellular structures. Global miRNA profiles of the paired EVs and ECs were determined with RealSeq Biosciences (Santa Cruz, CA) custom sequencing platform by conducting small RNA (sRNA)-seq. The sRNA-seq data were analyzed using various bioinformatic tools. Validation of key exmiRNA was performed using specific TaqMan microRNA stem-loop RT-qPCR assays. Results: We investigated the effect of cART, THC, or both cART and THC together on the abundance and compartmentalization of blood plasma exmiRNA in EVs and ECs in SIV-infected RMs. As shown in Manuscript 1 of this series, were in uninfected RMs, ~30% of exmiRNAs were associated with ECs, we confirmed in this follow up manuscript that exmiRNAs were present in both lipid-based carriers-EVs and non-lipid-based carriers-ECs, with 29.5 to 35.6% and 64.2 to 70.5 % being associated with EVs and ECs, respectively. Remarkably, the different treatments (cART, THC) have distinct effects on the enrichment and compartmentalization pattern of exmiRNAs. In the VEH/SIV/cART group, 12 EV-associated and 15 EC-associated miRNAs were significantly downregulated. EV-associated miR-206, a muscle-specific miRNA that is present in blood, was higher in the VEH/SIV/ART compared to the VEH/SIV group. ExmiR-139-5p that was implicated in endocrine resistance, focal adhesion, lipid and atherosclerosis, apoptosis, and breast cancer by miRNA-target enrichment analysis was significantly lower in VEH/SIV/cART compared to VEH/SIV, irrespective of the compartment. With respect to THC treatment, 5 EV-associated and 21 EC-associated miRNAs were significantly lower in the VEH/THC/SIV. EV-associated miR-99a-5p was higher in VEH/THC/SIV compared to VEH/SIV, while miR-335-5p counts were significantly lower in both EVs and ECs of THC/SIV compared to VEH/SIV. EVs from SIV/cART/THC combined treatment group have significant increases in the count of eight (miR-186-5p, miR-382-5p, miR-139-5p and miR-652, miR-10a-5p, miR-657, miR-140-5p, miR-29c-3p) miRNAs, all of which were lower in VEH/SIV/cART group. Analysis of miRNA-target enrichment showed that this set of eight miRNAs were implicated in endocrine resistance, focal adhesions, lipid and atherosclerosis, apoptosis, and breast cancer as well as cocaine and amphetamine addiction. In ECs and EVs, combined THC and cART treatment significantly increased miR-139-5p counts compared to VEH/SIV group. Significant alterations in these host miRNAs in both EVs and ECs in the untreated and treated (cART, THC, or both) RMs indicate the persistence of host responses to infection or treatments, and this is despite cART suppression of viral load and THC suppression of inflammation. To gain further insight into the pattern of miRNA alterations in EVs and ECs and to assess potential cause-and-effect relationships, we performed longitudinal miRNA profile analysis, measured in terms of months (1 and 5) post-infection (MPI). We uncovered miRNA signatures associated with THC or cART treatment of SIV-infected macaques in both EVs and ECs. While the number of miRNAs was significantly higher in ECs relative to EVs for all groups (VEH/SIV, SIV/cART, THC/SIV, THC/SIV/cART, and THC) longitudinally from 1 MPI to 5 MPI, treatment with cART and THC have longitudinal effects on the abundance and compartmentalization pattern of exmiRNAs in the two carriers. As shown in Manuscript 1 where SIV infection led to longitudinal suppression of EV-associated miRNA-128-3p, administration of cART to SIV-infected RMs did not increase miR-128-3p but resulted in longitudinal increases in six EV-associated miRNAs (miR-484, miR-107, miR-206, miR-184, miR-1260b, miR-6132). Furthermore, administration of cART to THC treated SIV-infected RMs resulted in a longitudinal decrease in three EV-associated miRNAs (miR-342-3p, miR-100-5p, miR181b-5p) and a longitudinal increase in three EC-associated miRNAs (miR-676-3p, miR-574-3p, miR-505-5p). The longitudinally altered miRNAs in SIV-infected RMs may indicate disease progression, while in the cART Group and the THC Group, the longitudinally altered miRNAs may serve as biomarkers of response to treatment. Conclusions: This paired EVs and ECs miRNAome analyses provided a comprehensive cross-sectional and longitudinal summary of the host exmiRNA responses to SIV infection and the impact of THC, cART, or THC and cART together on the miRNAome during SIV infection. Overall, our data point to previously unrecognized alterations in the exmiRNA profile in blood plasma following SIV infection. Our data also indicate that cART and THC treatment independently and in combination may alter both the abundance and the compartmentalization of several exmiRNA related to various disease and biological processes.

SIV Infection Regulates Compartmentalization of Circulating Blood Plasma miRNAs within Extracellular Vesicles (EVs) and Extracellular Condensates (ECs) and Decreases EV-Associated miRNA-128.

Background: This is Manuscript 1 of a two-part Manuscript of the same series. Here, we present findings from our first set of studies on the abundance and compartmentalization of blood plasma extracellular microRNAs (exmiRNAs) into extracellular particles, including blood plasma extracellular vesicles (EVs) and extracellular condensates (ECs) in the setting of untreated HIV/SIV infection. The goals of the study presented in this Manuscript 1 are to (i) assess the abundance and compartmentalization of exmiRNAs in EVs versus ECs in the healthy uninfected state, and (ii) evaluate how SIV infection may affect exmiRNA abundance and compartmentalization in these particles. Considerable effort has been devoted to studying the epigenetic control of viral infection, particularly in understanding the role of exmiRNAs as key regulators of viral pathogenesis. MicroRNA (miRNAs) are small (~20-22 nts) non-coding RNAs that regulate cellular processes through targeted mRNA degradation and/or repression of protein translation. Originally associated with the cellular microenvironment, circulating miRNAs are now known to be present in various extracellular environments, including blood serum and plasma. While in circulation, miRNAs are protected from degradation by ribonucleases through their association with lipid and protein carriers, such as lipoproteins and other extracellular particles-EVs and ECs. Functionally, miRNAs play important roles in diverse biological processes and diseases (cell proliferation, differentiation, apoptosis, stress responses, inflammation, cardiovascular diseases, cancer, aging, neurological diseases, and HIV/SIV pathogenesis). While lipoproteins and EV-associated exmiRNAs have been characterized and linked to various disease processes, the association of exmiRNAs with ECs is yet to be made. Likewise, the effect of SIV infection on the abundance and compartmentalization of exmiRNAs within extracellular particles is unclear. Literature in the EV field has suggested that most circulating miRNAs may not be associated with EVs. However, a systematic analysis of the carriers of exmiRNAs has not been conducted due to the inefficient separation of EVs from other extracellular particles, including ECs. Methods: Paired EVs and ECs were separated from EDTA blood plasma of SIV-uninfected male Indian rhesus macaques (RMs, n = 15). Additionally, paired EVs and ECs were isolated from EDTA blood plasma of combination anti-retroviral therapy (cART) naïve SIV-infected (SIV+, n = 3) RMs at two time points (1- and 5-months post infection, 1 MPI and 5 MPI). Separation of EVs and ECs was achieved with PPLC, a state-of-the-art, innovative technology equipped with gradient agarose bead sizes and a fast fraction collector that allows high-resolution separation and retrieval of preparative quantities of sub-populations of extracellular particles. Global miRNA profiles of the paired EVs and ECs were determined with RealSeq Biosciences (Santa Cruz, CA) custom sequencing platform by conducting small RNA (sRNA)-seq. The sRNA-seq data were analyzed using various bioinformatic tools. Validation of key exmiRNAs was performed using specific TaqMan microRNA stem-loop RT-qPCR assays. Results: We showed that exmiRNAs in blood plasma are not restricted to any type of extracellular particles but are associated with lipid-based carriers-EVs and non-lipid-based carriers-ECs, with a significant (~30%) proportion of the exmiRNAs being associated with ECs. In the blood plasma of uninfected RMs, a total of 315 miRNAs were associated with EVs, while 410 miRNAs were associated with ECs. A comparison of detectable miRNAs within paired EVs and ECs revealed 19 and 114 common miRNAs, respectively, detected in all 15 RMs. Let-7a-5p, Let-7c-5p, miR-26a-5p, miR-191-5p, and let-7f-5p were among the top 5 detectable miRNAs associated with EVs in that order. In ECs, miR-16-5p, miR-451, miR-191-5p, miR-27a-3p, and miR-27b-3p, in that order, were the top detectable miRNAs in ECs. miRNA-target enrichment analysis of the top 10 detected common EV and EC miRNAs identified MYC and TNPO1 as top target genes, respectively. Functional enrichment analysis of top EV- and EC-associated miRNAs identified common and distinct gene-network signatures associated with various biological and disease processes. Top EV-associated miRNAs were implicated in cytokine-cytokine receptor interactions, Th17 cell differentiation, IL-17 signaling, inflammatory bowel disease, and glioma. On the other hand, top EC-associated miRNAs were implicated in lipid and atherosclerosis, Th1 and Th2 cell differentiation, Th17 cell differentiation, and glioma. Interestingly, infection of RMs with SIV revealed that the brain-enriched miR-128-3p was longitudinally and significantly downregulated in EVs, but not ECs. This SIV-mediated decrease in miR-128-3p counts was validated by specific TaqMan microRNA stem-loop RT-qPCR assay. Remarkably, the observed SIV-mediated decrease in miR-128-3p levels in EVs from RMs agrees with publicly available EV miRNAome data by Kaddour et al., 2021, which showed that miR-128-3p levels were significantly lower in semen-derived EVs from HIV-infected men who used or did not use cocaine compared to HIV-uninfected individuals. These findings confirmed our previously reported finding and suggested that miR-128 may be a target of HIV/SIV. Conclusions: In the present study, we used sRNA sequencing to provide a holistic understanding of the repertoire of circulating exmiRNAs and their association with extracellular particles, such as EVs and ECs. Our data also showed that SIV infection altered the profile of the miRNAome of EVs and revealed that miR-128-3p may be a potential target of HIV/SIV. The significant decrease in miR-128-3p in HIV-infected humans and in SIV-infected RMs may indicate disease progression. Our study has important implications for the development of biomarker approaches for various types of cancer, cardiovascular diseases, organ injury, and HIV based on the capture and analysis of circulating exmiRNAs.

Long-Term Low-Dose Delta-9-Tetrahydrocannbinol (THC) Administration to Simian Immunodeficiency Virus (SIV) Infected Rhesus Macaques Stimulates the Release of Bioactive Blood Extracellular Vesicles (EVs) that Induce Divergent Structural Adaptations and Signaling Cues.

Blood extracellular vesicles (BEVs) carry bioactive cargo (proteins, genetic materials, lipids, licit, and illicit drugs) that regulate diverse functions in target cells. The cannabinoid drug delta-9-tetrahydrocannabinol (THC) is FDA approved for the treatment of anorexia and weight loss in people living with HIV. However, the effect of THC on BEV characteristics in the setting of HIV/SIV infection needs to be determined. Here, we used the SIV-infected rhesus macaque model of AIDS to evaluate the longitudinal effects of THC (THC/SIV) or vehicle (VEH/SIV) treatment in HIV/SIV infection on the properties of BEVs. While BEV concentrations increased longitudinally (pre-SIV (0), 30, and 150 days post-SIV infection (DPI)) in VEH/SIV macaques, the opposite trend was observed with THC/SIV macaques. SIV infection altered BEV membrane properties and cargo composition late in infection, since i) the electrostatic surface properties (zeta potential, ζ potential) showed that RM BEVs carried negative surface charge, but at 150 DPI, SIV infection significantly changed BEV ζ potential; ii) BEVs from the VEH/SIV group altered tetraspanin CD9 and CD81 levels compared to the THC/SIV group. Furthermore, VEH/SIV and THC/SIV BEVs mediated divergent changes in monocyte gene expression, morphometrics, signaling, and function. These include altered tetraspanin and integrin ß1 expression; altered levels and distribution of polymerized actin, FAK/pY397 FAK, pERK1/2, cleaved caspase 3, proapoptotic Bid and truncated tBid; and altered adhesion of monocytes to collagen I. These data indicate that HIV/SIV infection and THC treatment result in the release of bioactive BEVs with potential to induce distinct structural adaptations and signaling cues to instruct divergent cellular responses to infection.

Development of a Cationic Amphiphilic Helical Peptidomimetic (B18L) As A Novel Anti-Cancer Drug Lead.

BST-2 is a novel driver of cancer progression whose expression confers oncogenic properties to breast cancer cells. As such, targeting BST-2 in tumors may be an effective therapeutic approach against breast cancer. Here, we sought to develop potent cytotoxic anti-cancer agent using the second-generation BST-2-based anti-adhesion peptide, B18, as backbone. To this end, we designed a series of five B18-derived peptidomimetics. Among these, B18L, a cationic amphiphilic α-helical peptidomimetic, was selected as the drug lead because it displayed superior anti-cancer activity against both drug-resistant and drug-sensitive cancer cells, with minimal toxicity on normal cells. Probing mechanism of action using molecular dynamics simulations, biochemical and membrane biophysics studies, we observed that B18L binds BST-2 and possesses membranolytic characteristics. Furthermore, molecular biology studies show that B18L dysregulates cancer signaling pathways resulting in decreased Src and Erk1/2 phosphorylation, increased expression of pro-apoptotic Bcl2 proteins, caspase 3 cleavage products, as well as processing of the caspase substrate, poly (ADP-ribose) polymerase-1 (PARP-1), to the characteristic apoptotic fragment. These data indicate that through the coordinated regulation of membrane, mitochondrial and signaling events, B18L executes cancer cell death and thus has the potential to be developed into a potent and selective anti-cancer compound.

Human Immunodeficiency Virus (HIV) Infection and Use of Illicit Substances Promote Secretion of Semen Exosomes that Enhance Monocyte Adhesion and Induce Actin Reorganization and Chemotactic Migration.

Semen exosomes (SE) from HIV-uninfected (HIV-) individuals potently inhibit HIV infection in vitro. However, morphological changes in target cells in response to SE have not been characterized or have the effect of HIV infection or the use of illicit substances, specifically psychostimulants, on the function of SE been elucidated. The objective of this study was to evaluate the effect of HIV infection, psychostimulant use, and both together on SE-mediated regulation of monocyte function. SE were isolated from semen of HIV- and HIV-infected (HIV+) antiretroviral therapy (ART)-naive participants who reported either using or not using psychostimulants. The SE samples were thus designated as HIV-Drug-, HIV-Drug+, HIV+Drug-, and HIV+Drug+. U937 monocytes were treated with different SEs and analyzed for changes in transcriptome, morphometrics, actin reorganization, adhesion, and chemotaxis. HIV infection and/or use of psychostimulants had minimal effects on the physical characteristics of SE. However, different SEs had diverse effects on the messenger RNA signature of monocytes and rapidly induced monocyte adhesion and spreading. SE from HIV infected or psychostimulants users but not HIV-Drug- SE, stimulated actin reorganization, leading to the formation of filopodia-like structures and membrane ruffles containing F-actin and vinculin that in some cases were colocalized. All SE stimulated monocyte chemotaxis to HIV secretome and activated the secretion of matrix metalloproteinases, a phenotype exacerbated by HIV infection and psychostimulant use. SE-directed regulation of cellular morphometrics and chemotaxis depended on the donor clinical status because HIV infection and psychostimulant use altered SE function. Although our inclusion criteria specified the use of cocaine, humans are poly-drug and alcohol users and our study participants used psychostimulants, marijuana, opiates, and alcohol. Thus, it is possible that the effects observed in this study may be due to one of these other substances or due to an interaction between different substances.