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Microglia and macrophages are essential for homeostatic maintenance and innate immune response in the brain. They are the first line of defense against infections such as HIV/SIV in the brain. However, they are susceptible to infection and function as viral reservoirs even under effective viral suppression. While current antiretroviral regimens successfully suppress viremia and improve quality of life and lifespan, neurologic complications persist and are in part attributed to activated microglia. We sought to test the hypothesis that brain microglia return to a more homeostatic-like state when viremia is suppressed by combination antiretroviral therapy. Using the SIV-rhesus macaque model, we combined single-cell RNA sequencing, bioinformatics, and pathway analysis to compare gene expression profiles of brain myeloid cells under 4 conditions: uninfected, SIV infected, SIV infected with cART suppression, and SIV encephalitis (SIVE). Our study reveals greater myeloid diversity and an elevated proinflammatory state are associated with untreated SIV infection compared with uninfected animals. The development of encephalitis and suppression of viremia both reduced myeloid diversity. However, they had converse effects on the activation state of microglia and inflammation. Notably, suggestive of a restoration of a homeostatic state in microglia, gene expression and activation of pathways related to inflammation and immune response in cART-suppressed monkeys were most similar to that in uninfected monkeys. Untreated SIV infection shared characteristics, especially in brain macrophages to SIVE, with SIVE showing dramatic inflammation. In support of our hypothesis, our study demonstrates that cART indeed restores this key component of the brain's homeostatic state. Summary: ScRNA-seq of rhesus monkey microglia reveals clusters of cells in activated states in the setting of SIV infection, which is primarily reversed by suppressing viremia with combination antiretroviral therapy.

It is well recognized that not all individuals age equivalently, with functional dependence attributable, at least in part, to stress accumulated across the lifespan. Amongst these dependencies are age-related declines in cognitive function, which may be the result of impaired inhibitory processing (e.g., sensory gating). Herein, we examined the unique roles of life and biological stress on somatosensory gating dynamics in 74 adults (22-72 years old). Participants completed a sensory gating paired-pulse electrical stimulation paradigm of the right median nerve during magnetoencephalography (MEG) and data were subjected to advanced oscillatory and time-domain analysis methods. We observed separable mechanisms by which increasing levels of life and biological stress predicted higher oscillatory gating ratios, indicative of age-related impairments in inhibitory function. Specifically, elevations in life stress significantly modulated the neural response to the first stimulation in the pair, while elevations in biological stress significantly modulated the neural response to the second stimulation in the pair. In contrast, neither elevations in life nor biological stress significantly predicted the gating of time-domain neural activity in the somatosensory cortex. Finally, our study is the first to link stress-induced decline in sensory gating to cognitive dysfunction, suggesting that gating paradigms may hold promise for detecting discrepant functional trajectories in age-related pathologies in the future.

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.

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.

HIV-1 infection of macrophages is a multistep and multifactorial process that has been shown to be altered by exposure to methamphetamine (Meth). Identifying the resulting dynamic changes in the proteome of these cells, offers novel insights into the effect of Meth on HIV-1 infection. In this study, we measured the effect of Meth on a subset of the proteome of HIV-1-infected human monocyte-derived macrophages (hMDM) using Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra (SWATH-MS) and multiple reaction monitoring (MRM). Cells were exposed to Meth either prior to, or after infection, and then maintained throughout the duration of the experiment. In discovery phase we used SWATH-MS full unbiased profiling followed by bioinformatic analyses which were further validated using Multiple Reaction Monitoring (MRM). This is the first study to use MRM approach in such biological setting. We found that Meth affects expression of multiple molecular pathways including Rab proteins, which are master regulators of intracellular trafficking. Besides highly dynamic changes, our data show high donor-to-donor variability of responses. Our results shed new light on the molecular mechanisms underlying cellular pathology of hMDM resulting from viral infection and a drug of abuse — Meth.

Chronological age remains an imperfect measure of accumulated physiological stress. Biological measures of aging may provide key advantages, allowing scientists focusing on age-related functional changes to use metrics derived from epigenetic factors like DNA methylation (DNAm), which could provide greater precision. Here we investigated the relationship between methylation-based age and an essential cognitive function that is known to exhibit age-related decline: selective attention. We found that DNAm-age predicted selective attention abilities and fully mediated the relationship between selective attention and chronological age. Using neuroimaging with magnetoencephalography, we found that gamma activity in the anterior cingulate was robustly predicted by DNAm-derived biological age, revealing the neural dynamics underlying this DNAm age-related cognitive decline. Anterior cingulate gamma activity also significantly predicted behavior on the selective attention task, indicating its functional relevance. These findings suggest that DNAm age may be a better predictor of cognitive and brain aging than more traditional chronological metrics.

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.

Long-acting antiretrovirals can improve therapy and prevention for HIV-1 infection. Current long-acting cabotegravir (CAB LAP) can be administered every other month. Previously, we demonstrated that a myristoylated CAB prodrug encased in poloxamer 407 provided extended plasma drug concentrations. We now demonstrate that this first-generation nanoformulated prodrug can sustain plasma CAB concentrations above the protein-adjusted 90% inhibitory concentration for 4 months in rhesus macaques. A 2.5-fold extension in CAB half-life and a 1.6-fold increase in area under the concentration-time curve were observed compared with CAB LAP.

Summary Microglia play a key role in brain development, normal homeostasis, and neurodegenerative disorders. Single-cell technologies have led to important findings about microglia, with many animal model studies using single-cell RNA sequencing (scRNA-seq), whereas most human specimen studies using archived frozen brains for single-nucleus RNA sequencing (snRNA-seq). However, microglia compose a small proportion of the total brain tissue; snRNAseq depletes expression of microglia activation genes that characterize many diseases. Here we examine the use of purified, cryopreserved microglia for scRNA-seq. Comparison of scRNA-seq on paired fresh and cryopreserved microglia from rhesus monkeys revealed a high level of correlation of gene expression between the two conditions. Disease-related genes were relatively unaffected, but an increase in immediate-early gene expression was present in cryopreserved cells. Regardless, changes in immediate-early gene expression are still detectable. Cryopreservation of microglia is a suitable procedure for prospectively archiving samples., Graphical abstract, Highlights • High-quality scRNA-seq can be performed on cryopreserved microglia • Cryopreservation of microglia does not affect disease-related gene expression • Expression of immediate-early genes (IEG) is increased in cryopreserved microglia • scRNA-seq of cyropreserved microglia reveals gene expression changes, including IEG, Molecular Neuroscience ; Cellular Neuroscience ; Transcriptomics

HIV persists in cellular reservoirs despite effective combined antiretroviral therapy (cART) and there is viremia flare up upon therapy interruption. Opioids modulate the immune system and suppress antiviral gene responses, which significantly impact people living with HIV (PLWH). However, the effect of opioids on viral reservoir dynamics remain elusive. Herein, we developed a morphine dependent SIVmac251 infected Rhesus macaque (RM) model to study the impact of opioids on HIV reservoirs. RMs on a morphine (or saline control) regimen were infected with SIVmac251. The cART was initiated in approximately half the animals five weeks post-infection, and morphine/saline administration continued until the end of the study. Among the untreated RM, we did not find any difference in plasma/CSF or in cell-associated DNA/RNA viral load in anatomical tissues. On the other hand, within the cART suppressed macaques, there was a reduction in cell-associated DNA load, intact proviral DNA levels, and in inducible SIV reservoir in lymph nodes (LNs) of morphine administered RMs. In distinction to LNs, in the CNS, the size of latent SIV reservoirs was higher in the CD11b+ microglia/macrophages in morphine dependent RMs. These results suggest that in the proposed model, morphine plays a differential role in SIV reservoirs by reducing the CD4+ T-cell reservoir in lymphoid tissues, while increasing the microglia/reservoir size in CNS tissue. The findings from this pre-clinical model will serve as a tool for screening therapeutic strategies to reduce/eliminate HIV reservoirs in opioid dependent PLWH.IMPORTANCE Identification and clearance of HIV reservoirs is a major challenge in achieving a cure for HIV. This is further complicated by co-morbidities that may alter the size of the reservoirs. There is an overlap between the risk factors for HIV and opioid abuse. Opiates have been recognized as prominent co-morbidities in HIV-infected populations. People infected with HIV also abusing opioids have immune modulatory effects and more severe neurological disease. However, the impact of opioid abuse on HIV reservoirs remains unclear. In this study, we used morphine dependent SIVmac251 infected rhesus macaque (RM) model to study the impact of opioids on HIV reservoirs. Our studies suggested that people with HIV who abuse opioids had higher reservoirs in CNS than the lymphoid system. Extrapolating the macaque findings in humans suggests that such differential modulation of HIV reservoirs among people living with HIV abusing opioids could be considered for future HIV cure research efforts.

HIV persists in cellular reservoirs despite effective anti-retroviral therapy with rebound of viremia upon therapy interruption. Opioids modulate the immune system and suppress antiviral gene responses, which significantly impact people living with HIV (PLWH). However, the effects of opioids on viral reservoir remains elusive. Herein, we describe a morphine dependent SIVmac251 infected Rhesus macaque (RM) model to study the impact of opioids on HIV reservoirs. RMs were ramped up with morphine (n=10) or saline (n=9) for two weeks to a final dosage of 5mg/kg administered twice daily, which was maintained for seven weeks, and then infected with SIVmac251. Combined anti-retroviral therapy (cART) was initiated in approximately half the animals in each group five weeks post-infection and morphine/saline administration continued for 10 months. Among drug naïve macaques, there were no differences in plasma/CSF viral load nor in cell-associated DNA/RNA loads. However, within the cART-suppressed macaques, there was a reduction in cell-associated DNA load, intact proviral DNA copy numbers, and inducible SIV reservoir in both peripheral blood and lymph nodes (LNs) of morphine-administered RMs compared to saline controls. Further, PBMCs of morphine administered RMs, a reduction in Th1 polarized CD4+ T cells and in LNs there was a reduction in the total Tfh and Th1 like Tfh cells were observed, indicating probably have impact on reduction of viral reservoirs. In distinction to PBMC and LNs, within the CNS size of latent SIV reservoirs was higher in the CD11b+ microglia/macrophages of morphine-dependent RMs. These data suggest that morphine plays a role in modulating SIV reservoirs, reduces the CD4+ T-cell reservoir in both peripheral blood and LNs, and increases microglia/macrophage reservoirs in CNS. These findings will aid in understanding of molecular mechanism(s) of opioid-mediated differential modulation of viral reservoirs and evaluation of therapeutic strategies to reduce/eliminate HIV reservoirs in opioid-dependent PLWH.Author summaryOpioids are commonly used as well as abused by HIV infected individuals, are known to suppress immune responses. However, their effects on modulating viral reservoir dynamics is not known. Here we developed a morphine dependent SIVmac251 infected rhesus macaque (RM) model to study the impact of opioids on HIV reservoirs and immune cells. We found that there was no difference in viral loads or cell-associated DNA/RNA loads among morphine dependent vs. saline treated control macaques. On the other hand, when macaques were treated with cART, there was a reduction in SIV reservoirs both in periphery and lymphoid tissues in morphine administered RMs, and the size of latent SIV reservoir was higher in the CNS CD11b+ microglia/macrophages as compared to control macaques. Therefore, these new data form macaque models suggest that PLWH who suffering from opioid use disorders have higher reservoirs in CNS as compared to lymphoid system. Thus, through understanding these reservoirs among PLWH who uses opioids are critical for better designing HIV cure strategies.

A growing literature suggests a relationship between HIV-infection and a molecular profile of age acceleration. However, despite the widely known high prevalence of HIV-related brain atrophy and HIV-associated neurocognitive disorder (HAND), epigenetic age acceleration has not been linked to HIV-related changes in structural MRI. We applied morphological MRI methods to study the brain structure of 110 virally suppressed participants with HIV infection and 122 uninfected controls age 22–72. All participants were assessed for cognitive impairment, and blood samples were collected from a subset of 86 participants with HIV and 83 controls to estimate epigenetic age. We examined the group-level interactive effects of HIV and chronological age and then used individual estimations of epigenetic age to understand the relationship between age acceleration and brain structure. Finally, we studied the effects of HAND. HIV-infection was related to gray matter reductions, independent of age. However, using epigenetic age as a biomarker for age acceleration, individual HIV-related age acceleration was associated with reductions in total gray matter. HAND was associated with decreases in thalamic and hippocampal gray matter. In conclusion, despite viral suppression, accentuated gray matter loss is evident with HIV-infection, and greater biological age acceleration specifically relates to such gray matter loss.

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., 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.

Long-acting cabotegravir (CAB) extends antiretroviral drug administration from daily to monthly. However, dosing volumes, injection site reactions and health-care oversight are obstacles towards a broad usage. The creation of poloxamer-coated hydrophobic and lipophilic CAB prodrugs with controlled hydrolysis and tissue penetrance can overcome these obstacles. To such ends, fatty acid ester CAB nanocrystal prodrugs with 14, 18 and 22 added carbon chains were encased in biocompatible surfactants named NMCAB, NM2CAB and NM3CAB and tested for drug release, activation, cytotoxicity, antiretroviral activities, pharmacokinetics and biodistribution. Pharmacokinetics studies, performed in mice and rhesus macaques, with the lead 18-carbon ester chain NM2CAB, showed plasma CAB levels above the protein-adjusted 90% inhibitory concentration for up to a year. NM2CAB, compared with NMCAB and NM3CAB, demonstrated a prolonged drug release, plasma circulation time and tissue drug concentrations after a single 45 mg per kg body weight intramuscular injection. These prodrug modifications could substantially improve CAB’s effectiveness. Nanoformulated long-acting cabotegravir prodrugs are shown to be capable of extending the native drug’s antiretroviral activity, biodistribution and pharmacokinetics for up to 12 months in mice and rhesus macaques.

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.

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.

Traumatic brain injury (TBI) is an important health concern and effective treatment strategies remain elusive. Understanding the complex multicellular response to TBI may provide new avenues for intervention. In the context of TBI, cell-cell communication is critical. One relatively unexplored form of cell-cell communication in TBI is extracellular vesicles (EVs). These membrane-bound vesicles can carry many different types of cargo between cells. Recently, miRNA in EVs have been shown to mediate neuroinflammation and neuronal injury. To explore the role of EV-associated miRNA in TBI, we isolated EVs from the brain of injured mice and controls, purified RNA from brain EVs, and performed miRNA sequencing. We found that the expression of miR-212 decreased, while miR-21, miR-146, miR-7a, and miR-7b were significantly increased with injury, with miR-21 showing the largest change between conditions. The expression of miR-21 in the brain was primarily localized to neurons near the lesion site. Interestingly, adjacent to these miR-21-expressing neurons were activated microglia. The concurrent increase in miR-21 in EVs with the elevation of miR-21 in neurons, suggests that miR-21 is secreted from neurons as potential EV cargo. Thus, this study reveals a new potential mechanism of cell-cell communication not previously described in TBI.

HIV-infected individuals are living longer on antiretroviral therapy, but many patients display signs that in some ways resemble premature aging. To investigate and quantify the impact of chronic HIV infection on aging, we report a global analysis of the whole-blood DNA methylomes of 137 HIV+ individuals under sustained therapy along with 44 matched HIV− individuals. First, we develop and validate epigenetic models of aging that are independent of blood cell composition. Using these models, we find that both chronic and recent HIV infection lead to an average aging advancement of 4.9 years, increasing expected mortality risk by 19%. In addition, sustained infection results in global deregulation of the methylome across >80,000 CpGs and specific hypomethylation of the region encoding the human leukocyte antigen locus (HLA). We find that decreased HLA methylation is predictive of lower CD4 / CD8 T cell ratio, linking molecular aging, epigenetic regulation, and disease progression.

Key Points Question Are DNA methylation–based metrics of biological age associated with cortical thickness, and does a biological age acceleration index capture unique changes in cortical thinning compared with chronological age measures? Findings In this cross-sectional study of 82 healthy adults, both biological and chronological age were associated with widespread cortical thinning, but biological age acceleration was also associated with additional changes in cortical morphological features. Older biological age relative to chronological age was associated with accentuated thinning within prefrontal and temporal regions. Meaning These findings suggest that DNA methylation–based biological age may yield additional insight into healthy and pathological cortical aging compared with chronological age alone., This cross-sectional study investigates the association of DNA methylation–based biological age with cortical thickness and assesses whether a biological age acceleration index compared with chronological age captures unique variance in cortical thinning., Importance Magnetic resonance imaging (MRI) studies of aging adults have shown substantial intersubject variability across various brain metrics, and some of this variability is likely attributable to chronological age being an imprecise measure of age-related change. Accurately quantifying one’s biological age could allow better quantification of healthy and pathological changes in the aging brain. Objective To investigate the association of DNA methylation (DNAm)–based biological age with cortical thickness and to assess whether biological age acceleration compared with chronological age captures unique variance in cortical thinning. Design, Setting, and Participants This cross-sectional study used high-resolution structural brain MRI data collected from a sample of healthy aging adults who were participating in a larger ongoing neuroimaging study that began in May 2014. This population-based study accrued participants from the greater Omaha, Nebraska, metropolitan area. One hundred sixty healthy adults were contacted for the MRI component, 82 of whom participated in both DNAm and MRI study components. Data analysis was performed from March to June 2019. Main Outcomes and Measures Vertexwise cortical thickness, DNAm-based biological age, and biological age acceleration compared with chronological age were measured. A pair of multivariable regression models were computed in which cortical thickness was regressed on DNAm-based biological age, controlling for sex in the first model and also controlling for chronological age in the second model. Results Seventy-nine adult participants (38 women; mean [SD] age, 43.82 [14.50] years; age range, 22-72 years) were included in all final analyses. Advancing biological age was correlated with cortical thinning across frontal, superior temporal, inferior parietal, and medial occipital regions. In addition, biological age acceleration relative to chronological age was associated with cortical thinning in orbitofrontal, superior and inferior temporal, somatosensory, parahippocampal, and fusiform regions. Specifically, for every 1 year of biological age acceleration, cortical thickness would be expected to decrease by 0.024 mm (95% CI, −0.04 to −0.01 mm) in the left orbitofrontal cortex (partial r, −0.34; P = .002), 0.014 mm (95% CI, −0.02 to −0.01 mm) in the left superior temporal gyrus (partial r, −0.36; P = .001), 0.015 mm (95% CI, −0.02 to −0.01 mm) in the left fusiform gyrus (partial r, −0.38; P = .001), 0.015 mm (95% CI, −0.02 to −0.01 mm) in the right fusiform gyrus (partial r, −0.43; P

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., Author Summary HIV associated neurocognitive disorder (HAND) are neurological disorders caused due to the entry of HIV infection in the brain. HIV-1 does not directly infect central or peripheral neurons, however, virus-infected cells of the monocyte/macrophage lineage maintain a low-level HIV infection in the CNS. "Indirect effects" of macrophage activation–such as dysregulation of cytokines and chemokines, free-radical (oxidative stress) injury, and secretion of soluble factors that are potently neurotoxic–have been implicated as effectors of nervous system injury in HIV. Here, we report that extracellular vesicles released from macrophages can enhance neurotoxicity. Using a nonhuman primate model of HAND, simian immunodeficiency virus encephalitis (SIVE), we find that exosomes isolated from SIVE brains contain,microRNAs, including miR-21, that can serve as ligands to the key immune regulatory receptors, toll-like receptors, and can elicit neurotoxicity. We provide in vitro evidence for such an effect, and that the toxicity can be mediated by necroptosis. Thus, our study provides insights into other potential neurotoxic mechanisms by which HIV infection in the brain could harm neuronal health.

Traumatic brain injury (TBI) produces profound and lasting neuroinflammation that has both beneficial and detrimental effects. Recent evidence has implicated microRNAs (miRNAs) in the regulation of inflammation both in the periphery and the CNS. We examined the expression of inflammation associated miRNAs in the context of TBI using a mouse controlled cortical impact (CCI) model and found increased levels of miR-21, miR-223 and miR-155 in the hippocampus after CCI. The expression of miR-155 was elevated 9-fold after CCI, an increase confirmed by in situ hybridization (ISH). Interestingly, expression of miR-155 was largely found in neuronal nuclei as evidenced by co-localization with DAPI in MAP2 positive neurons. In miR-155 knock out (KO) mice expression of type I interferons IFNα and IFNβ, as well as IFN regulatory factor 1 and IFN-induced chemokine CXCL10 was decreased after TBI relative to wild type (WT) mice. Unexpectedly, miR-155 KO mice had increased levels of microglial marker Iba1 and increased neuronal degeneration as measured by fluoro-jade C (FJC) staining, suggesting a neuroprotective role for miR-155 in the context of TBI. This work demonstrates a role for miR-155 in regulation of the IFN response and neurodegeneration in the aftermath of TBI. While the presence of neuronal nuclear miRNAs has been described previously, their importance in disease states is relatively unknown. Here, we show evidence of dynamic regulation and pathological function of a nuclear miRNA in TBI.

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.

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.

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.

Background Methamphetamine (Meth) abuse is a major health problem linked to the aggravation of HIV- associated complications, especially within the Central Nervous System (CNS). Within the CNS, Meth has the ability to modify the activity/function of innate immune cells and increase brain viral loads. Here, we examined changes in the gene expression profile of neuron-free microglial cell preparations isolated from the brain of macaques infected with the Simian Immunodeficiency Virus (SIV), a model of neuroAIDS, and exposed to Meth. We aimed to identify molecular patterns triggered by Meth that could explain the detection of higher brain viral loads and the development of a pro-inflammatory CNS environment in the brain of infected drug abusers. Results We found that Meth alone has a strong effect on the transcription of genes associated with immune pathways, particularly inflammation and chemotaxis. Systems analysis led to a strong correlation between Meth exposure and enhancement of molecules associated with chemokines and chemokine receptors, especially CXCR4 and CCR5, which function as co-receptors for viral entry. The increase in CCR5 expression was confirmed in the brain in correlation with increased brain viral load. Conclusions Meth enhances the availability of CCR5-expressing cells for SIV in the brain, in correlation with increased viral load. This suggests that Meth is an important factor in the susceptibility to the infection and to the aggravated CNS inflammatory pathology associated with SIV in macaques and HIV in humans. Electronic supplementary material The online version of this article (doi:10.1186/s12865-016-0145-0) contains supplementary material, which is available to authorized users.

Secretion of pro-inflammatory molecules by astrocytes after alcohol treatment was shown to be associated with neuroinflammation. We hypothesized that activation of cytosolic phospholipase A2 (cPLA2) and cyclooxygenase (COX-2) by ethanol in astrocytes enhanced the secretion of inflammatory agents via the interactive tyrosine phosphorylation of toll-like receptor 4 (TLR4) and Src kinase. To test this hypothesis, we treated primary human astrocytes with 20 mM ethanol for 48 h at 37°C. Ethanol exposure elevated cytochrome P450-2E1 activity, reactive oxygen species levels, and secretion of prostaglandin E2 (PGE2) in these cells. Secretion of PGE2 was associated with induction of cPLA2 activity and protein content as well as COX-2 protein level in a Src phosphorylation-dependent manner that occurred by enhanced transcription. Immunoprecipitation and Western blot analyses indicated that the interactive tyrosine phosphorylation of TLR4–Src complex at the cell membrane triggered the activation of cPLA2 and COX-2 in the cytoplasm through a Src signaling intermediate. Inhibition of ethanol metabolism, blockage of Src activity, or inactivation of TLR4 prevented the activation of cPLA2 and COX-2 as well as diminished PGE2 production, suggesting that interactive phosphorylation of TLR4–Src regulated the pro-inflammatory response in astrocytes. Experiments with small interfering RNA knockdown of TLR4 in human astrocytes confirmed that silencing expression also abolished the interactive phosphorylation of both TLR4 and Src in the presence of ethanol.

Objective Poor penetration of anti-retroviral therapy across the blood brain barrier (BBB) poses an impediment on control of HIV-1 infection in brain macrophages. PPARγ, a member of the nuclear receptors family, regulates important physiological functions (including anti-inflammatory effects) in response to ligand-mediated activation. Since PPARγ agonists are rapidly absorbed by oral administration and efficiently permeate the BBB we hypothesized that PPARγ stimulation may suppress HIV-1 replication.

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.

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-α-induced proteins, interferon (IFN)-inducible genes, intercellular adhesion molecule-1, transcription factors of the nuclear factor-κB 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.

Polychlorinated biphenyls (PCBs) have been demonstrated to modulate marine mammal immune functions; however, the underlying mechanisms involved are poorly understood. Cytosolic calcium (Ca2+) is an important second messenger involved in numerous leukocyte functions. The direct effects of in vitro exposure to PCBs on Ca2+ mobilization were evaluated in leukocytes isolated from bottlenose dolphins, beluga whales, and B6C3F1 mice. Concentration- and time-response experiments with three non-coplanar PCBs (138, 153, 180), one coplanar PCB (169), and TCDD were tested. Exposure to the three non-coplanar PCBs significantly increased cytosolic Ca2+ in dolphin neutrophils, while PCB 180 significantly increased cytosolic Ca2+ in beluga neutrophils. Two non-coplanar PCBs (138 and 153) significantly increased Ca2+ in beluga monocytes, yet the response was delayed compared to that in neutrophils. Neither PCBs nor TCDD increased cytosolic Ca2+ in mouse neutrophils or monocytes. In experiments with Ca2+-free medium, only PCB 153 increased cytosolic Ca2+ in dolphin neutrophils, though the increase was less than that observed with Ca2+-supplemented medium, suggesting that extracellular Ca2+ was the predominant source for the rise in cytosolic Ca2+. Furthermore, in cells incubated with Ca2+-free medium, a significant increase in cytosolic Ca2+ was induced by thapsigargin following PCB exposure, indicating that intracellular Ca2+ was available, yet not mobilized by the PCBs, and further suggesting that PCBs mobilize extracellular Ca2+. These results demonstrate for the first time the direct effects of non-coplanar PCBs on Ca2+ mobilization in marine mammals, which may be involved in the modulation of phagocytosis previously observed in these species.

One factor in the development of neuroAIDS is the increase in the migration of pro-inflammatory CD8 T cells across the blood–brain barrier. Typically these cells are involved with keeping the viral load down. However, the persistence of above average numbers of CD8 T cells in the brain, not necessarily specific to viral peptides, is facilitated by the upregulation of IL15 from astrocytes, in the absence of IL2, in the brain environment. Both IL15 and IL2 are common gamma chain (γc) cytokines. Here, using the non-human primate model of neuroAIDS, we have demonstrated that exposure to methamphetamine, a powerful illicit drug that has been associated with HIV exposure and neuroAIDS severity, can cause an increase in molecules of the γc system. Among these molecules, IL15, which is upregulated in astrocytes by methamphetamine, and that induces the proliferation of T cells, may also be involved in driving an inflammatory phenotype in innate immune cells of the brain. Therefore, methamphetamine and IL15 may be critical in the development and aggravation of central nervous system immune-mediated inflammatory pathology in HIV-infected drug abusers.

The effects of organochlorines (OC) on the immune systems of marine mammals and humans are poorly understood. One important innate immune function of peripheral blood neutrophils and monocytes is the respiratory burst, which generates reactive oxygen species (ROS) used to kill engulfed microorganisms. The present study characterized the immunomodulatory potential for mixtures of OCs, compared to that of individual OCs, on the respiratory burst in several marine mammals, humans, and B6C3F1 mice. The effects of three non-coplanar polychlorinated biphenyls (PCBs) (138, 153, 180), one coplanar PCB (169), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) and all possible mixtures were tested upon in vitro exposure for 1 h, and their effects on the generation of a respiratory burst were measured by flow cytometry. The final concentration for each congener, alone or in a mixture, was 5 ppm for PCBs and 0.05 ppb for TCDD. Both significant enhancement and suppression of the respiratory burst occurred in all species tested, but the pattern was different between species and cell types (neutrophils vs. monocytes). Both coplanar and non-coplanar OCs were involved in the modulation of the respiratory burst. Regression analysis was not able to elucidate which OCs were involved in modulating the responses, highlighting the difficulty of developing models to predict the immunotoxic effects attributed to OC mixtures. The traditional mouse model and toxic equivalency (TEQ) approach both failed to consistently predict the toxicity of OCs in all species tested, questioning their applicability in the risk assessment process for all species. Elucidating the relative sensitivities to the immunomodulatory effects of OC mixtures between different species may have important implications for risk assessment as well as conservation and management strategies.

A significant mortality of the American lobster (Homarus americanus) in western Long Island Sound in 1999 seriously impacted the harvest of this species. Whereas the exact cause or causes of the mortality are still not precisely known, dead and dying lobsters in the initial phase of the die-off were diagnosed with infections with paramoeba, a newly recognized disease condition of lobsters. Because the immune system represents the first line of defense against disease-causing agents and is one of the most sensitive systems to environmental stressors, this study is aimed at the development of new assays to quantify the immune system of lobsters. Flow cytometry allowed the discrimination of different populations of hemocytes based on their relative size and complexity. Also using flow cytometry, natural killer (NK) cell-like activity and its stimulation by human recombinant interleukin 2 (IL-2) are described for the first time in lobsters, as is the expression of TLR2, a pattern recognition receptor...

The eastern oyster is an economically and ecologically important species whose vitality is threatened by the protozoal parasite Perkinsus marinus. To better understand which cellular defense mechanisms impart resistance to P. marinus, resistant (Crassostrea gigas) and susceptible (Crassostrea virginica) oyster species were challenged by an experimental infection with P. marinus and their cellular responses were quantified and compared. Both in vivo and in vitro infection trials measured hemocyte phagocytosis, respiratory burst, apoptosis at 1, 3 and 7 days postinfection (in vivo) or 1-h postco-incubation (in vitro). Total parasite body burden concentrations were also measured at the end of in vivo infections. Infections were significantly more severe in C. virginica than C. gigas at 3 and 7 days postinfection confirming the resistance of C. gigas and validating the experimental model. There was more phagocytosis in infected C. virginica than infected C. gigas three days postinfection. In vitro, C...

Oyster culture, which is a large part of the North American aquaculture industry, is greatly hindered by the parasite Perkinsus marinus. The different oyster defense mechanisms and how the parasite seems to evade them are not fully understood, and the role of apoptosis as an oyster defense mechanism remains unclear. Apoptosis of eastern oyster (Crassostrea virginica) hemocyte subpopulations (granulocytes and hyalinocytes) was quantified at the single cell level using flow cytometric Annexin-V and TUNEL assays. The influences of habitat salinity (540 mOsm [18.9‰] and 910 mOsm [31.9‰] for oysters and 488 mOsm [17.1‰] and 810 mOsm [28.4‰] for P. marinus) and temperature (14.5°C and 25°C for oysters, 14.4°C and 22.5°C for P. marinus) on apoptosis frequencies were evaluated experimentally on the host hemocytes and the parasite. Apoptosis frequency was higher in granulocytes than hyalinocytes, regardless of temperature or salinity. Salinity affected apoptosis of granulocytes and hyalinocytes, with more...

Increasing evidence has supported the general hypothesis that organochlorines (OC) can produce immunotoxic effects in marine mammals. One important innate defense mechanism is phagocytosis, the ability of cells to ingest extracellular macromolecules. The present study is aimed at characterizing the immunomodulatory potential of mixtures of OCs on phagocytosis compared to that of individual compounds in different species of marine mammals and mice, the traditional model to study mammalian immunotoxicity. The ability of peripheral blood neutrophils and monocytes to engulf fluorescent microspheres was evaluated using flow cytometry. The immunomodulatory effects of three non-coplanar polychlorinated biphenyl (PCB) congeners, 138, 153, 180, one coplanar PCB, 169, as well as 2,3,7,8-TCDD, and all possible mixtures (26) were tested upon in vitro exposure. All species were not equally sensitive to the adverse effects of OCs on either neutrophils or monocytes phagocytosis. With the exception of harbor seals, all mixtures that significantly modulated neutrophil or monocyte phagocytosis contained at least one non-coplanar PCB. Regression analysis revealed that the non-coplanar congeners, more than the coplanar congeners, explained the variability in phagocytosis. Dendrograms revealed that phylogeny could not predict immunotoxicity. The currently used toxic equivalency (TEQ) approach and the traditional mouse model both failed to predict experimentally induced immunomodulatory effects in marine mammals tested, leading us to question the reliability of both TEQs and mouse model in risk assessment of OC mixtures. Testing the relative sensitivity to immunomodulatory effects of contaminants and contaminant mixtures between different species of marine mammals may have important implications for risk assessment as well as conservation and management strategies.

Human immunodeficiency virus (HIV) accesses the brain early in infection and can lead to neurocognitive disorders. The brain can also serve as a viral reservoir, but how virus is controlled in the brain is unknown. To examine this, CD8-depleting monoclonal antibody was injected into the cerebrospinal fluid of rhesus monkeys with chronic simian immunodeficiency virus (SIV) infection. This treatment led to the rapid increase of SIV in the brain. Virus in the brain is maintained by active suppression from the host immune system. This dynamic interaction can be manipulated in efforts to control and eradicate virus from the brain and other reservoirs.

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.

Mitochondrial dysfunction is implicated in a majority of neurodegenerative disorders and much study of neurodegenerative disease is done on cultured neurons. In traditional tissue culture, the oxygen level that cells experience is dramatically higher (21%) than in vivo conditions (1–11%). These differences can alter experimental results, especially, pertaining to mitochondria and oxidative metabolism. Our results show that primary neurons cultured at physiological oxygen levels found in the brain showed higher polarization, lower rates of ROS production, larger mitochondrial networks, greater cytoplasmic fractions of mitochondria and larger mitochondrial perimeters than those cultured at higher oxygen levels. Although neurons cultured in either physiological oxygen or atmospheric oxygen exhibit significant increases in mitochondrial reactive oxygen species (ROS) production when treated with the human immunodeficiency virus (HIV) virotoxin trans-activator of transcription, mitochondria of neurons cultured at physiological oxygen underwent depolarization with dramatically increased cell death, whereas those cultured at atmospheric oxygen became hyperpolarized with no increase in cell death. Studies with a second HIV virotoxin, negative regulation factor (Nef), revealed that Nef treatment also increased mitochondrial ROS production for both the oxygen conditions, but resulted in mitochondrial depolarization and increased death only in neurons cultured in physiological oxygen. These results indicate a role for oxidative metabolism in a mechanism of neurotoxicity during HIV infection and demonstrate the importance of choosing the correct, physiological, culture oxygen in mitochondrial studies performed in neurons.

Methamphetamine (METH) abuse is known to be associated with an inordinate rate of infections. Although many studies have described the association of METH exposure and immunosuppression, so far the underlying mechanism still remains elusive. In this study, we present evidence that METH exposure resulted in mitochondrial oxidative damage and caused dysfunction of primary human T cells. METH treatment of T lymphocytes led to a rise in intracellular calcium levels that enhanced the generation of reactive oxygen species. TCR-CD28 linked calcium mobilization and subsequent uptake by mitochondria in METH-treated T cells correlated with an increase in mitochondrion-derived superoxide. Exposure to METH-induced mitochondrial dysfunction in the form of marked decrease in mitochondrial membrane potential, increased mitochondrial mass, enhanced protein nitrosylation and diminished protein levels of complexes I, III, and IV of the electron transport chain. These changes paralleled reduced IL-2 secretion and T cell proliferative responses after TCR-CD28 stimulation indicating impaired T cell function. Furthermore, antioxidants attenuated METH-induced mitochondrial damage by preserving the protein levels of mitochondrial complexes I, III, and IV. Altogether, our data indicate that METH can cause T cell dysfunction via induction of oxidative stress and mitochondrial injury as underlying mechanism of immune impairment secondary to METH abuse.

Neuro-cognitive deficits, neuronal injury, and neurodegeneration are well documented in alcoholics, yet the underlying mechanisms remain elusive. Oxidative damage of mitochondria and cellular proteins intertwines with the progression of neuroinflammation and neurological disorders initiated by alcohol abuse. Here, we present the evidence that metabolism of ethanol in primary human neurons by alcohol dehydrogenase (ADH) or cytochrome P450-2E1 (CYP2E1) generates reactive oxygen species (ROS) and nitric oxide (NO) via induction of NADPH/xanthine oxidase (NOX/XOX) and nitric oxide synthase (NOS) in human neurons. The acetaldehyde-mediated increase in NOX, XOX, or NOS activity is regulated as a transcriptional rather than a translational process. Marked increase in the lipid peroxidation product (4-hydroxynonenal) and enhanced ROS generation coincides with decreased neuronal viability and diminished expression of neuronal marker (neurofilaments). Novel quantitative methods of ROS and NO detection help dissect the mechanisms of alcohol-induced neurodegeneration. Uncovering the basic mechanisms of oxidative neuronal injury will serve as the basis for development of new therapies.

Under inflammatory conditions (including HIV-1 encephalitis and multiple sclerosis), activated brain endothelium enhances the adhesion and transmigration of monocytes across the blood-brain barrier (BBB). Synthetic ligands that activate the peroxisome proliferator-activated receptors (PPARs) have anti-inflammatory properties, and PPAR stimulation prevents the interaction of leukocytes with cytokine stimulated-endothelium. However, the mechanism underlying these effects of PPAR ligands and their ability to intervene with leukocyte adhesion and migration across brain endothelial cells has yet to be explored. For the first time, using primary human brain endothelial cells (BMVEC), we demonstrated that monocyte adhesion and transendothelial migration across inflamed endothelium were markedly reduced by PPARγ activation. In contrast to non-brain-derived endothelial cells, PPARα activation in the BMVEC had no significant effect on monocyte-endothelial interaction. Previously, our work indicated a critical role of Rho GTPases (like RhoA) in BMVEC to control migration of HIV-1 infected monocytes across BBB. In this study, we show that in the BMVEC PPARγ stimulation prevented activation of two GTPases, Rac1 and RhoA, which correlated with decreased monocyte adhesion to and migration across brain endothelium. Relevant to HIV-1 neuropathogenesis, enhanced adhesion and migration of HIV-1 infected monocytes across the BBB were significantly reduced when BMVEC were treated with PPARγ agonist. These findings indicate that Rac1 and RhoA inhibition by PPARγ agonists could be a new approach for treatment of neuroinflammation by preventing monocyte migration across the BBB.

Marine mammals bioaccumulate various environmental contaminants such as organochlorines (OCs), which biomagnify via the food web. While the immunomodulatory effects of individual OCs have been studied, the effects of mixtures are not well understood. The immunomodulatory effects of polychlorinated biphenyl (PCB) 138, 153, 169, and 180 as well as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and all possible mixtures were examined in marine mammals and mice. Lymphocyte proliferation was significantly modulated by OCs in all species tested, mostly by non-coplanar PCBs, as shown using regression analyses. Correlation analyses showed significant correlations (interpreted as additive effects) between OCs in mice, killer whales, and Steller sea lions. Nonadditive synergistic and antagonistic interactions between OCs were detected in most of the species tested. Toxic equivalency (TEQ) values used for OC toxicity assessment failed to predict the immunomodulatory effects measured in mice and marine mammals. The commonly used mouse model failed to predict immunomodulatory effects in other species. Clustering data suggested that phylogeny does not predict toxicity of OCs. Overall, our data suggest the presence of species-specific sensitivities to different mixtures, in which OCs interactions may be complex and that may exert their effects through dioxinlike or dioxin-independent pathways. Lastly, lymphocyte proliferation, an important part of adaptive immunity, was significantly modulated in mice and marine mammals, suggesting the possibility of increased susceptibility to diseases. These findings will be useful to better characterize the risk associated with OC exposure and possibly lead to new conservation and management strategies.

Organochlorine (OC) contaminants, notably polychlorinated biphenyls (PCBs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), are ubiquitous in all ecosystems and found in the tissues of humans and wildlife. Although the immunotoxicity of coplanar, dioxinlike PCBs is well documented, the adverse effects exerted by non-coplanar, non-dioxinlike PCBs have received little attention. Direct causal relationship between PCB and dioxin exposure and the observed detrimental effects on the immune system has yet to be fully established in humans. The immunomodulatory potential of toxic coplanar PCB 169 and TCDD and abundant non-coplanar PCBs 138, 153, and 180 on human leukocyte phagocytosis, an important innate immune function that initiates the clearance of pathogens, was tested upon in vitro exposure. Mixture and concentration-response experiments demonstrated a suppression of phagocytosis by non-coplanar PCBs suggesting a previously unrecognized aryl hydrocarbon receptor (AhR)-independent pathway. Regression analysis revealed that reduction of phagocytosis was mostly explained by the non-coplanar congeners. The effects on phagocytosis could not be accurately predicted by either the currently used toxic equivalence (TEQ) approach or the mouse model, thus undermining the use of the traditional models in the risk assessment for OC mixtures containing non-coplanar congeners. Our results are cause for concern as they suggest an AhR-independent pathway through which non-coplanar PCBs modulate phagocytosis, the immune system's first line of defense, possibly increasing the risk to developing infectious disease.