HIV-1 and methamphetamine alter galectins -1, -3, and -9 in human monocyte-derived macrophages.

Macrophages are key elements of the innate immune system. Their HIV-1 infection is a complex process that involves multiple interacting factors and various steps and is further altered by exposure of infected cells to methamphetamine (Meth), a common drug of abuse in people living with HIV. This is reflected by dynamic changes in the intracellular and secreted proteomes of these cells. Quantification of these changes poses a challenge for experimental design and associated analytics. In this study, we measured the effect of Meth on expression of intracellular and secreted galectins-1, -3, and -9 in HIV-1 infected human monocyte-derived macrophages (hMDM) using SWATH-MS, which was further followed by MRM targeted mass spectrometry validation. Cells were exposed to Meth either prior to or after infection. Our results are the first to perform comprehensive quantifications of galectins in primary hMDM cells during HIV-1 infection and Meth exposure a building foundation for future studies on the molecular mechanisms underlying cellular pathology of hMDM resulting from viral infection and a drug of abuse-Meth.

Sequence-specific extracellular microRNAs activate TLR7 and induce cytokine secretion and leukocyte migration.

Toll-like receptors (TLRs) can contribute to central nervous system disease pathologies via recognition of microRNAs (miRNAs); however, it remains to be determined which miRNAs are able to activate this signaling. Here we report that numerous miRNAs induced the production of tumor necrosis factor alpha in multiple myeloid cell types, including microglia, and that this effect was abolished in cells deficient in TLR7. Examination of closely related miRNAs that differed in their ability to activate TLR7 resulted in the identification of a motif (UGCUUAU) in miR-20a-5p and specific nucleotides (all the uridines and surprisingly the cytosine as well) in a key area of miR-20a-5p and miR-148b-3p that were vital for the secretion of cytokines via TLR7 stimulation. A 10-nucleotide sequence including this motif was identified to be the shortest single-stranded RNA to signal via TLR7. An miRNA containing this motif induced the secretion of multiple proinflammatory molecules, which was dependent on the phosphoinositide 3-kinase, mitogen-activated protein kinase, and nuclear factor kappa-light-chain-enhancer of activated B cell signaling pathways. Wild-type mice administered miR-20a-5p, which contained this motif, demonstrated increased leukocyte migration. This effect was significantly ameliorated in TLR7-knockout mice, and mice administered miR-20b-5p, in which the motif was mutated, did not exhibit leukocyte migration. We provide a detailed analysis of miRNAs that activate endosomal TLR7 and identify key nucleotide features of a sequence motif recognized by TLR7.

SWATH-MS and MRM: Quantification of Ras-related proteins in HIV-1 infected and methamphetamine-exposed human monocyte-derived macrophages (hMDM).

HIV-1 infection of macrophages is a multistep and multifactorial process that has been shown to be enhanced by exposure to methamphetamine (Meth). In this study, we sought to identify the underlying mechanisms of this effect by quantifying the effect of Meth on the proteome of HIV-1-infected macrophages using sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) approach. The analyses identified several members of the Rab family of proteins as being dysregulated by Meth treatment, which was confirmed by bioinformatic analyses that indicated substantial alteration of vesicular transport pathways. Validation of the SWATH-MS was performed using an MRM based approach, which confirmed that Meth exposure affects expression of the Rab proteins. However, the pattern of expression changes were highly dynamic, and displayed high donor-to-donor variability. Surprisingly a similar phenomenon was observed for Actin. Our results demonstrate that Meth affects vesicular transport pathways, suggesting a possible molecular mechanism underlying its effect on HIV infection hMDM and a potential broader effect of Meth on cellular homeostasis.

Methamphetamine Increases the Proportion of SIV-Infected Microglia/Macrophages, Alters Metabolic Pathways, and Elevates Cell Death Pathways: A Single-Cell Analysis.

Both substance use disorder and HIV infection continue to affect many individuals. Both have untoward effects on the brain, and the two conditions often co-exist. In the brain, macrophages and microglia are infectable by HIV, and these cells are also targets for the effects of drugs of abuse, such as the psychostimulant methamphetamine. To determine the interaction of HIV and methamphetamine, we isolated microglia and brain macrophages from SIV-infected rhesus monkeys that were treated with or without methamphetamine. Cells were subjected to single-cell RNA sequencing and results were analyzed by statistical and bioinformatic analysis. In the animals treated with methamphetamine, a significantly increased proportion of the microglia and/or macrophages were infected by SIV. In addition, gene encoding functions in cell death pathways were increased, and the brain-derived neurotropic factor pathway was inhibited. The gene expression patterns in infected cells did not cluster separately from uninfected cells, but clusters comprised of microglia and/or macrophages from methamphetamine-treated animals differed in neuroinflammatory and metabolic pathways from those comprised of cells from untreated animals. Methamphetamine increases CNS infection by SIV and has adverse effects on both infected and uninfected microglia and brain macrophages, highlighting the dual and interacting harms of HIV infection and drug abuse on the brain.

Interactions of Monocytes, HIV, and ART Identified by an Innovative scRNAseq Pipeline: Pathways to Reservoirs and HIV-Associated Comorbidities.

HIV reservoirs persist despite successful antiretroviral therapy (ART) and are a major obstacle to the eradication and cure of HIV. The mature monocyte subset, CD14+CD16+, contributes to viral reservoirs and HIV-associated comorbidities. Only a subset of monocytes harbors HIV (HIV+), while the rest remain uninfected, exposed cells (HIVexp). We developed an innovative single cell RNA sequencing (scRNAseq) pipeline that detects HIV and host transcripts simultaneously, enabling us to examine differences between HIV+ and HIVexp mature monocytes. Using this, we characterized uninfected, HIV+, and HIVexp primary human mature monocytes with and without ART. We showed that HIV+ mature monocytes do not form their own cluster separately from HIVexp but can be distinguished by significant differential gene expression. We found that ART decreased levels of unspliced HIV transcripts potentially by modulating host transcriptional regulators shown to decrease viral infection and replication. We also identified and characterized mature monocyte subpopulations differentially impacted by HIV and ART. We identified genes dysregulated by ART in HIVexp monocytes compared to their uninfected counterpart and, of interest, the junctional protein ALCAM, suggesting that ART impacts monocyte functions. Our data provide a novel method for simultaneous detection of HIV and host transcripts. We identify potential targets, such as those genes whose expression is increased in HIV+ mature monocytes compared to HIVexp, to block their entry into tissues, preventing establishment/replenishment of HIV reservoirs even with ART, thereby reducing and/or eliminating viral burden and HIV-associated comorbidities. Our data also highlight the heterogeneity of mature monocyte subsets and their potential contributions to HIV pathogenesis in the ART era.IMPORTANCE HIV enters tissues early after infection, leading to establishment and persistence of HIV reservoirs despite antiretroviral therapy (ART). Viral reservoirs are a major obstacle to the eradication and cure of HIV. CD14+CD16+ (mature) monocytes may contribute to establishment and reseeding of reservoirs. A subset of monocytes, consisting mainly of CD14+CD16+ cells, harbors HIV (HIV+), while the rest remain uninfected, exposed cells (HIVexp). It is important to identify cells harboring virus to eliminate reservoirs. Using an innovative single-cell RNA sequencing (scRNAseq) pipeline to detect HIV and host transcripts simultaneously, we characterized HIV+ and HIVexp primary human mature monocytes with and without ART. HIV+ mature monocytes are not a unique subpopulation but rather can be distinguished from HIVexp by differential gene expression. We characterized mature monocyte subpopulations differently impacted by HIV and ART, highlighting their potential contributions to HIV-associated comorbidities. Our data propose therapeutic targets to block HIV+ monocyte entry into tissues, preventing establishment and replenishment of reservoirs even with ART.

Secreted Metabolome of Human Macrophages Exposed to Methamphetamine.

Macrophages comprise a major component of the human innate immune system that is involved in maintaining homeostasis and responding to infections or other insults. Besides cytokines and chemokines, macrophages presumably influence the surrounding environment by secreting various types of metabolites. Characterization of secreted metabolites under normal and pathological conditions is critical for understanding the complex innate immune system. To investigate the secreted metabolome, we developed a novel workflow consisting of one Reverse Phase (RP) C18 column linked in tandem with a Cogent cholesterol-modified RP C18. This system was used to compare the secreted metabolomes of human monocyte-derived macrophages (hMDM) under normal conditions to those exposed to methamphetamine (Meth). This new experimental approach allowed us to measure 92 metabolites, identify 11 of them as differentially expressed, separate and identify three hydroxymethamphetamine (OHMA) isomers, and identify a new, yet unknown metabolite with a m/z of 192. This study is the first of its kind to address the secreted metabolomic response of hMDM to an insult by Meth. Besides the discovery of novel metabolites secreted by macrophages, we provide a novel methodology to investigate metabolomic profiling.

Multimodal Theranostic Nanoformulations Permit Magnetic Resonance Bioimaging of Antiretroviral Drug Particle Tissue-Cell Biodistribution.

RATIONALE: Long-acting slow effective release antiretroviral therapy (LASER ART) was developed to improve patient regimen adherence, prevent new infections, and facilitate drug delivery to human immunodeficiency virus cell and tissue reservoirs. In an effort to facilitate LASER ART development, "multimodal imaging theranostic nanoprobes" were created. These allow combined bioimaging, drug pharmacokinetics and tissue biodistribution tests in animal models. METHODS: Europium (Eu3+)- doped cobalt ferrite (CF) dolutegravir (DTG)- loaded (EuCF-DTG) nanoparticles were synthesized then fully characterized based on their size, shape and stability. These were then used as platforms for nanoformulated drug biodistribution. RESULTS: Folic acid (FA) decoration of EuCF-DTG (FA-EuCF-DTG) nanoparticles facilitated macrophage targeting and sped drug entry across cell barriers. Macrophage uptake was higher for FA-EuCF-DTG than EuCF-DTG nanoparticles with relaxivities of r2 = 546 mM-1s-1 and r2 = 564 mM-1s-1 in saline, and r2 = 850 mM-1s-1 and r2 = 876 mM-1s-1 in cells, respectively. The values were ten or more times higher than what was observed for ultrasmall superparamagnetic iron oxide particles (r2 = 31.15 mM-1s-1 in saline) using identical iron concentrations. Drug particles were detected in macrophage Rab compartments by dual fluorescence labeling. Replicate particles elicited sustained antiretroviral responses. After parenteral injection of FA-EuCF-DTG and EuCF-DTG into rats and rhesus macaques, drug, iron and cobalt levels, measured by LC-MS/MS, magnetic resonance imaging, and ICP-MS were coordinate. CONCLUSION: We posit that these theranostic nanoprobes can assess LASER ART drug delivery and be used as part of a precision nanomedicine therapeutic strategy.

Sirtuin 1-Chromatin-Binding Dynamics Points to a Common Mechanism Regulating Inflammatory Targets in SIV Infection and in the Aging Brain.

Microglia and macrophages are the main non-neuronal subsets of myeloid origin in the brain, and are critical regulators in neurodegenerative disorders, where inflammation is a key factor. Since HIV infection results in neurological perturbations that are similar to those in aging, we examined microglial and infiltrating myeloid subsets in the search for changes that might resemble the ones in aging. For that, we used the SIV infection in rhesus macaques to model neuroAIDS. We found that Sirt-1, a molecule that impacts survival and health in many models, was decreased in cell preparations containing a majority of microglia and myeloid cells from the brain of infected macaques. The role of Sirt-1 in neuroAIDS is unknown. We hypothesized that Sirt-1 silencing functions are affected by SIV. Mapping of Sirt-1 binding patterns to chromatin revealed that the number of Sirt-1-bound genes was 29.6% increased in myeloid cells from infected animals with mild or no detectable neuropathology, but 51% was decreased in severe neuropathology, compared to controls. Importantly, Sirt-1-bound genes in controls largely participate in neuroinflammation. Promoters of type I IFN pathway genes IRF7, IRF1, IFIT1, and AIF1, showed Sirt-1 binding in controls, which was consistently lost after infection, together with higher transcription. Loss of Sirt-1 binding was also found in brains from old uninfected animals, suggesting a common regulation. The role of Sirt-1 in regulating these inflammatory markers was confirmed in two different in vitro models, where Sirt-1 blockage modulated IRF7, IRF1 and AIF1 levels both in human macrophage cell lines and in human blood-derived monocytes from various normal donors, stimulated with a TLR9 agonist. Our data suggests that Sirt-1-inflammatory gene silencing is disturbed by SIV infection, resembling aging in brains. These findings may impact our knowledge on the contribution of myeloid subsets to the neurological consequences of HIV infection, aggravated and overlapping with the aging process.

Central nervous system-penetrating antiretrovirals impair energetic reserve in striatal nerve terminals.

The use of antiretroviral (ARV) drugs with central nervous system (CNS) penetration effectiveness (CPE) may be useful in the treatment of HIV-associated neurocognitive disorder (HAND) as well as targeting a CNS reservoir in strategies to achieve a functional cure for HIV. However, increased cognitive deficits are linked to at least one of these drugs (efavirenz). As mitochondrial dysfunction has been found with a number of ARVs, and as such can affect neuronal function, the objective of this study was to assess the effects of ARV with high CPE for toxicological profiles on presynaptic nerve terminal energy metabolism. This subcellular region is especially vulnerable in that a constant supply of ATP is required for the proper maintenance of neurotransmitter release and uptake supporting proper neuronal function. We evaluated the effects of acute treatment with ten different high CPE ARVs from five different drug classes on rat cortical and striatal nerve terminal bioenergetic function. While cortical nerve terminal bioenergetics were not altered, striatal nerve terminals exposed to efavirenz, nevirapine, abacavir, emtricitabine, zidovudine, darunavir, lopinavir, raltegravir, or maraviroc (but not indinavir) exhibit reduced mitochondrial spare respiratory capacity (SRC). Further examination of efavirenz and maraviroc revealed a concentration-dependent impairment of striatal nerve terminal maximal mitochondrial respiration and SRC as well as a reduction of intraterminal ATP levels. Depletion of ATP at the synapse may underlie its dysfunction and contribute to neuronal dysfunction in treated HIV infection.

MiR-21 in Extracellular Vesicles Leads to Neurotoxicity via TLR7 Signaling in SIV Neurological Disease.

Recent studies have found that extracellular vesicles (EVs) play an important role in normal and disease processes. In the present study, we isolated and characterized EVs from the brains of rhesus macaques, both with and without simian immunodeficiency virus (SIV) induced central nervous system (CNS) disease. Small RNA sequencing revealed increased miR-21 levels in EVs from SIV encephalitic (SIVE) brains. In situ hybridization revealed increased miR-21 expression in neurons and macrophage/microglial cells/nodules during SIV induced CNS disease. In vitro culture of macrophages revealed that miR-21 is released into EVs and is neurotoxic when compared to EVs derived from miR-21-/- knockout animals. A mutation of the sequence within miR-21, predicted to bind TLR7, eliminates this neurotoxicity. Indeed miR-21 in EV activates TLR7 in a reporter cell line, and the neurotoxicity is dependent upon TLR7, as neurons isolated from TLR7-/- knockout mice are protected from neurotoxicity. Further, we show that EVs isolated from the brains of monkeys with SIV induced CNS disease activates TLR7 and were neurotoxic when compared to EVs from control animals. Finally, we show that EV-miR-21 induced neurotoxicity was unaffected by apoptosis inhibition but could be prevented by a necroptosis inhibitor, necrostatin-1, highlighting the actions of this pathway in a growing number of CNS disorders.

Quantitative proteomics reveals oxygen-dependent changes in neuronal mitochondria affecting function and sensitivity to rotenone.

Mitochondria are implicated in a variety of degenerative disorders and aging. Mitochondria are responsive to the oxygen in their environment, yet tissue culture is performed at atmospheric (21%) oxygen and not at physiological (1-11%) oxygen levels found in tissues. We employed imaging of mitochondrial probes, mass spectrometry, Western blots, and ATP assays of the human neuroblastoma cell-line SH-SY5Y and imaging of mitochondrial probes in human primary neurons under standard nonphysiological oxygen conditions (atmospheric) and under physiological oxygen levels in the nervous system to assess the impact of oxygen on mitochondrial function. SH-SY5Y cells cultured in physiological 5% oxygen exhibited the lowest reactive oxygen species (ROS) production, indicating that culture at 5% oxygen is favored; these results were mimicked in primary human cells. Mass spectrometric analysis revealed extensive mitochondrial proteomic alterations in SH-SY5Y cells based on oxygen culture condition. Among these, the rotenone-sensitive subunit of complex I NDUFV3 was increased in cells cultured at 5% oxygen. Rotenone is a Parkinson's disease-linked toxin, and correspondingly SH-SY5Y cells cultured at 5% oxygen also exhibited over 10 times greater sensitivity to rotenone than those cultured in atmospheric, 21%, oxygen. Our results indicate that neuronal mitochondria are responsive to oxygen levels and produce differential responses under different oxygen levels.

Preclinical pharmacokinetics and tissue distribution of long-acting nanoformulated antiretroviral therapy.

Long-acting injectable nanoformulated antiretroviral therapy (nanoART) was developed with the explicit goal of improving medicine compliance and for drug targeting of viral tissue reservoirs. Prior nanoART studies completed in humanized virus-infected mice demonstrated sustained antiretroviral responses. However, the pharmacokinetics (PK) and tissue distribution of nanoART were not characterized. To this end, the PK and tissue distribution of nanoformulated atazanavir (ATV) and ritonavir (RTV) injected subcutaneously or intramuscularly in mice and monkeys were evaluated. Fourteen days after injection, ATV and RTV levels were up to 13-, 41-, and 4,500-fold higher than those resulting from native-drug administration in plasma, tissues, and at the site of injection, respectively. At nanoART doses of 10, 50, 100, and 250 mg/kg of body weight, relationships of more- and less-than-proportional increases in plasma and tissue levels with dose increases were demonstrated with ATV and RTV. Multiple-dose regimens showed serum and tissue concentrations up to 270-fold higher than native-drug concentrations throughout 8 weeks of study. Importantly, nanoART was localized in nonlysosomal compartments in tissue macrophages, creating intracellular depot sites. Reflective data were obtained in representative rhesus macaque studies. We conclude that nanoART demonstrates blood and tissue antiretroviral drug levels that are enhanced compared to those of native drugs. The sustained and enhanced PK profile of nanoART is, at least in part, the result of the sustained release of ATV and RTV from tissue macrophases and at the site of injection.

Methamphetamine administration targets multiple immune subsets and induces phenotypic alterations suggestive of immunosuppression.

Methamphetamine (Meth) is a widely abused stimulant and its users are at increased risk for multiple infectious diseases. To determine the impact of meth on the immune system, we utilized a murine model that simulates the process of meth consumption in a typical addict. Our phenotypic analysis of leukocytes from this dose escalation model revealed that meth affected key immune subsets. Meth administration led to a decrease in abundance of natural killer (NK) cells and the remaining NK cells possessed a phenotype suggesting reduced responsiveness. Dendritic cells (DCs) and Gr-1(high) monocytes/macrophages were also decreased in abundance while Gr-1(low) monocytes/macrophages appear to show signs of perturbation. CD4 and CD8 T cell subsets were affected by methamphetamine, both showing a reduction in antigen-experienced subsets. CD4 T cells also exhibited signs of activation, with increased expression of CD150 on CD226-expressing cells and an expansion of KLRG1(+), FoxP3(-) cells. These results exhibit that meth has the ability to disrupt immune homeostasis and impact key subsets of leukocytes which may leave users more vulnerable to pathogens.

Peroxisome proliferator-activated receptor-gamma activation suppresses HIV-1 replication in an animal model of encephalitis.

OBJECTIVE: Poor penetration of antiretroviral therapy across the blood-brain barrier poses an impediment on control of HIV-1 infection in brain macrophages. Peroxisome proliferator-activated receptor (PPAR)-gamma, a member of the nuclear receptors family, regulates important physiological functions (including anti-inflammatory effects) in response to ligand-mediated activation. As PPARgamma agonists are rapidly absorbed by oral administration and efficiently permeate the blood-brain barrier, we hypothesized that PPARgamma stimulation may suppress HIV-1 replication. DESIGN AND METHODS: We investigated the effect of PPARgamma ligand (rosiglitazone) on HIV-1 replication in human monocyte-derived macrophages and in vivo using a murine model (immunodeficient mice reconstituted with human lymphocytes and intracerebrally inoculated with HIV-1 infected macrophages) of HIV-1 encephalitis. RESULTS: Treatment with rosiglitazone caused a significant decrease of virus infection in macrophages. PPARgamma stimulation inhibited virus replication by modulating NF-kappaB activation in a receptor-dependent manner, leading to downregulation of HIV-1 long terminal repeat (LTR) promoter activity and suppression of HIV-1 replication. These effects were PPARgamma specific as PPARgamma silencing or addition of PPARgamma antagonist abolished effects of PPARgamma stimulation on HIV-1 LTR and virus replication. Using a murine model for HIV-1 encephalitis, we demonstrated that PPARgamma ligand suppressed HIV-1 replication in macrophages in brain tissue and reduced viremia by 50%. CONCLUSION: In vitro data delineated the novel mechanism by which PPARgamma activation suppresses HIV-1 replication, and in vivo findings underscored the ability of PPARgamma agonists to reduce HIV-1 replication in lymphocytes and brain macrophages, thus offering a new therapeutic intervention in brain and systemic infection.

Effects of organochlorines, individually and in mixtures, on B-cell proliferation in marine mammals and mice.

Organochlorines (OC) are lipophilic and stable, and therefore accumulate in tissues of top predators, such as marine mammals. While the immunomodulatory effects of individual OC have been studied in lab animals, their effects in other species (such as marine mammals) and the possible interactions between chemicals in mixtures are not well understood. This study investigated the immunomodulatory effects of four polychlorinated biphenyls (PCB, IUPAC numbers 138, 153, 169, and 180), as well as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), individually and in mixtures, in marine mammals and mice. Mitogen-induced B lymphocyte proliferation was mostly modulated by non-coplanar PCBs, for which general mechanisms underlying toxicity are poorly understood. Simple additive effects of OC in mixtures were found only in mice, while both synergistic and antagonistic interactions between OC were found in marine mammals. The toxic equivalency (TEQ) approach, which is currently used to assess the dioxin-like toxicity of OC mixtures, failed to predict immunotoxicity in mice and marine mammals, likely due to the complexity of interactions between OC and effects via dioxin-independent pathways. The commonly used mouse model failed to predict the immunotoxicity due to OC in the marine mammals tested. In addition, clustering data suggested that phylogeny might not help predict the toxicity of OC. Lymphoproliferative response was modulated in most species tested suggesting the possibility of increased susceptibility to infectious diseases in these animals. These findings may be helpful in more accurately characterizing the immunotoxic potential of OC in different target species and help in more relevant risk assessment.

HIV-1 activates proinflammatory and interferon-inducible genes in human brain microvascular endothelial cells: putative mechanisms of blood-brain barrier dysfunction.

The mechanisms underlying blood-brain barrier (BBB) dysfunction seen in human immunodeficiency virus 1 (HIV-1) infection are poorly understood; however, they are believed to be caused by interactions of human brain microvascular endothelial cells (HBMEC) with virus-infected macrophages. Using a transwell system and Affymetrix arrays, we investigated HIV-1-induced genomic changes in HBMEC after coculture with HIV-1-infected or -uninfected monocyte-derived macrophages (MDM). Differentially expressed genes were determined by linear modeling and then were grouped by hierarchical clustering. Compared to HBMEC cocultured with noninfected MDM, 184 probe sets corresponding to 84 genes were differentially expressed in HBMEC cocultured with HIV-infected MDM. Genes activated in HIV-1 MDM-exposed HBMEC included proinflammatory cytokines and chemokines, tumor necrosis factor-alpha-induced proteins, interferon (IFN)-inducible genes, intercellular adhesion molecule-1, transcription factors of the nuclear factor-kappaB family, and signal transducer and activator of transcription 1. Analysis of molecular networks and canonical pathways associated with differentially expressed genes suggest that HIV-1 causes BBB impairment by mechanisms involving inflammation, cytokine, and IFN signaling in HBMEC.