MRP8/14 Is a Molecular Signature Triggered by Dopamine in HIV Latent Myeloid Targets That Increases HIV Transcription and Distinguishes HIV+ Methamphetamine Users with Detectable CSF Viral Load and Brain Pathology.

There is a significant overlap between HIV infection and substance-use disorders. Dopamine (DA) is the most abundantly upregulated neurotransmitter in methamphetamine abuse, with receptors (DRD1-5) that are expressed by neurons as well as by a large diversity of cell types, including innate immune cells that are the targets of HIV infection, making them responsive to the hyperdopaminergic environment that is characteristic of stimulant drugs. Therefore, the presence of high levels of dopamine may affect the pathogenesis of HIV, particularly in the brain. The stimulation of HIV latently infected U1 promonocytes with DA significantly increased viral p24 levels in the supernatant at 24 h, suggesting effects on activation and replication. Using selective agonists to different DRDs, we found that DRD1 played a major role in activating viral transcription, followed by DRD4, which increased p24 with a slower kinetic rate compared to DRD1. Transcriptome and systems biology analyses led to the identification of a cluster of genes responsive to DA, where S100A8 and S100A9 were most significantly correlated with the early increase in p24 levels following DA stimulation. Conversely, DA increased the expression of these genes' transcripts at the protein level, MRP8 and MRP14, respectively, which form a complex also known as calprotectin. Interestingly, MRP8/14 was able to stimulate HIV transcription in latent U1 cells, and this occurred via binding of the complex to the receptor for an advanced glycosylation end-product (RAGE). Using selective agonists, both DRD1 and DRD4 increased MRP8/14 on the surface, in the cytoplasm, as well as secreted in the supernatants. On the other hand, while DRD1/5 did not affect the expression of RAGE, DRD4 stimulation caused its downregulation, offering a mechanism for the delayed effect via DRD4 on the p24 increase. To cross-validate MRP8/14 as a DA signature with a biomarker value, we tested its expression in HIV+ Meth users' postmortem brain specimens and peripheral cells. MRP8/14+ cells were more frequently identified in mesolimbic areas such as the basal ganglia of HIV+ Meth+ cases compared to HIV+ non-Meth users or to controls. Likewise, MRP8/14+ CD11b+ monocytes were more frequent in HIV+ Meth users, particularly in specimens from participants with a detectable viral load in the CSF. Overall, our results suggest that the MRP8 and MRP14 complex may serve as a signature to distinguish subjects using addictive substances in the context of HIV, and that this may play a role in aggravating HIV pathology by promoting viral replication in people with HIV who use Meth.

Age-associated changes in microglia activation and Sirtuin-1- chromatin binding patterns.

The aging process is associated with changes in mechanisms maintaining physiology, influenced by genetics and lifestyle, and impacting late life quality and longevity. Brain health is critical in healthy aging. Sirtuin 1 (Sirt1), a histone deacetylase with silencing properties, is one of the molecular determinants experimentally linked to health and longevity. We compared brain pathogenesis and Sirt1-chromatin binding dynamics in brain pre-frontal cortex from 2 groups of elder rhesus macaques, divided by age of necropsy: shorter-lived animals (18-20 years old (yo)), equivalent to 60-70 human yo; and longer-lived animals (23-29 yo), corresponding to 80-100 human yo and modeling successful aging. These were compared with young adult brains (4-7 yo). Our findings indicated drastic differences in the microglia marker Iba1, along with factors influencing Sirt1 levels and activity, such as CD38 (an enzyme limiting NAD that controls Sirt1 activity) and mir142 (a microRNA targeting Sirt1 transcription) between the elder groups. Iba1 was lower in shorter-lived animals than in the other groups, while CD38 was higher in both aging groups compared to young. mir142 and Sirt1 levels were inversely correlated in longer-lived brains (>23yo), but not in shorter-lived brains (18-20 yo). We also found that Sirt1 binding showed signs of better efficiency in longer-lived animals compared to shorter-lived ones, in genes associated with nuclear activity and senescence. Overall, differences in neuroinflammation and Sirt1 interactions with chromatin distinguished shorter- and longer-lived animals, suggesting the importance of preserving microglia and Sirt1 functional efficiency for longevity.

Methamphetamine signals transcription of IL1ß and TNFα in a reactive oxygen species-dependent manner and interacts with HIV-1 Tat to decrease antioxidant defense mechanisms.

Methamphetamine (Meth) abuse is a common HIV co-morbidity that is linked to aggravated Central Nervous System (CNS) inflammation, which accentuates HIV- associated neurological disorders, triggered both directly or indirectly by the drug. We used the well-established human innate immune macrophage cell line system (THP1) to demonstrate that Reactive Oxygen Species (ROS) immediately induced by Meth play a role in the increased transcription of inflammatory genes, in interaction with HIV-1 Tat peptide. Meth and Tat, alone and together, affect early events of transcriptional activity, as indicated by changes in RNA polymerase (RNAPol) recruitment patterns throughout the genome, via ROS-dependent and -independent mechanisms. IL1ß (IL1ß) and TNF α (TNFα), two genes with defining roles in the inflammatory response, were both activated in a ROS-dependent manner. We found that this effect occurred via the activation of the activator protein 1 (AP-1) comprising cFOS and cJUN transcription factors and regulated by the SRC kinase. HIV-1 Tat, which was also able to induce the production of ROS, did not further impact the effects of ROS in the context of Meth, but promoted gene activity independently from ROS, via additional transcription factors. For instance, HIV-1 Tat increased NFkB activation and activated gene clusters regulated by Tata box binding peptide, ING4 and IRF2. Importantly, HIV-1 Tat decreased the expression of anti-oxidant genes, where its suppression of the detoxifying machinery may contribute to the aggravation of oxidative stress induced by ROS in the context of Meth. Our results provide evidence of effects of Meth via ROS and interactions with HIV Tat that promote the transcription of inflammatory genes such as IL1ß and TNFα.

Polygenic networks in peripheral leukocytes indicate patterns associated with HIV infection and context-dependent effects of cannabis use.

In spite of suppressive antiretroviral therapies (ART), Human Immunodeficiency Virus (HIV)-infected subjects still experience the consequences of viral persistence and chronic inflammation. In the brain, where most HIV-1 targets are of innate immune origin, neurological and cognitive impairments are detectable and enhanced by highly prevalent substance use disorders. Cannabis is one of the most prevalent substances among HIV+ â€‹subjects, compared to non-infected populations, either prescribed for improving various symptoms or used recreationally, as well as a component of polysubstance use. The mechanisms by which addictive substances and HIV interact are multifactorial and poorly understood. Importantly, the HIV brain target cells, macrophages and microglia, express receptors to neurotransmitters elevated by such drugs, and express receptors to cannabinoids, particularly CB2R. We have tested a panel of 784 transcripts associated with neurological disorders, digitally multiplexed and detectable in peripheral blood cells from a small cohort (n â€‹= â€‹102) of HIV-positive (HIV+) and HIV-negative (HIV-) specimens, stratified based on criteria of lifetime (LT) dependence of cannabis (CAN+) or not (CAN-). Demographic homogeneity and low incidence of co-morbidities helped increase power and allowed the identification of key differences consistent with HIV infection, cannabis exposure, or their interactions. A small percentage of these subjects used cannabis as well as other drugs. The data was analyzed using robust systems and visualization strategies to detect orchestrated patterns in gene networks connected based on molecular interfaces with higher power than in single genes. We found that the effects of cannabis differed drastically between HIV- and HIV+ â€‹groups, particularly in gene networks playing a role in inflammation, neurodegeneration, apoptosis and leukocyte adhesion and transmigration. At the level of individual genes, we identified detrimental effects that were associated with polysubstance use as a covariate, particularly methamphetamine. Transcription factor usage predictions suggest that the effects of cannabis are associated with transcriptional co-regulation at the gene promoters by multiple factors that vary by context. Overall, we have found that the effects of cannabis may be context-dependent, with potential benefits in the context of HIV reflected by improvements in cognition, but in the absence of the polysubstance use component.

The COVID-19 Pandemic: Reflections of Science, Person, and Challenge in Academic Research Settings.

In spring of 2021, the Society on NeuroImmune Pharmacology (SNIP) organized a virtual workshop on the coronavirus disease 2019 (COVID-19). The daylong event's fourth and final symposium, "Well-being and reflections," offered a glimpse at the pandemic's impact on the lives of our scientists and educators. This manuscript includes a brief summary of the symposium, a transcription of our incoming president Dr. Santosh Kumar's lecture, titled "Intervention and improved well-being of basic science researchers during the COVID-19 era: a case study," and the panel discussion that followed, "Reflection and sharing," featuring Drs. Jean M. Bidlack, Sylvia Fitting, Santhi Gorantla, Maria Cecilia G. Marcondes, Loyda M. Melendez, and Ilker K. Sariyer. The conclusion of this manuscript includes comments from SNIP's president Dr. Sulie L. Chang and our Chief Editor, Dr. Howard E. Gendelman. Drs. Sowmya Yelamanchili and Jeymohan Joseph co-chaired the symposium.

Detection of H3K4me3 Identifies NeuroHIV Signatures, Genomic Effects of Methamphetamine and Addiction Pathways in Postmortem HIV+ Brain Specimens that Are Not Amenable to Transcriptome Analysis.

Human postmortem specimens are extremely valuable resources for investigating translational hypotheses. Tissue repositories collect clinically assessed specimens from people with and without HIV, including age, viral load, treatments, substance use patterns and cognitive functions. One challenge is the limited number of specimens suitable for transcriptional studies, mainly due to poor RNA quality resulting from long postmortem intervals. We hypothesized that epigenomic signatures would be more stable than RNA for assessing global changes associated with outcomes of interest. We found that H3K27Ac or RNA Polymerase (Pol) were not consistently detected by Chromatin Immunoprecipitation (ChIP), while the enhancer H3K4me3 histone modification was abundant and stable up to the 72 h postmortem. We tested our ability to use HeK4me3 in human prefrontal cortex from HIV+ individuals meeting criteria for methamphetamine use disorder or not (Meth +/-) which exhibited poor RNA quality and were not suitable for transcriptional profiling. Systems strategies that are typically used in transcriptional metadata were applied to H3K4me3 peaks revealing consistent genomic activity differences in regions where addiction and neuronal synapses pathway genes are represented, including genes of the dopaminergic system, as well as inflammatory pathways. The resulting comparisons mirrored previously observed effects of Meth on suppressing gene expression and provided insights on neurological processes affected by Meth. The results suggested that H3K4me3 detection in chromatin may reflect transcriptional patterns, thus providing opportunities for analysis of larger numbers of specimens from cases with substance use and neurological deficits. In conclusion, the detection of H3K4me3 in isolated chromatin can be an alternative to transcriptome strategies to increase the power of association using specimens with long postmortem intervals and low RNA quality.

Macrophages and brown adipocytes cross-communicate to modulate a thermogenic program following methamphetamine exposure.

Hyperthermia is a potentially lethal side-effect of Methamphetamine (Meth), a stimulant drug. Activation of non-shivering thermogenesis in brown adipose tissue (BAT) is partly responsible for Meth-induced rise in temperature, with contributing sympathetic neurotransmitters, such as norepinephrine (NE), and reactive oxygen species (ROS). However, the mechanisms controlling the development of a molecular thermogenic program in brown adipocytes (BA) following Meth are unknown. We hypothesize that Meth and NE affect BAT cells, BA and macrophages, to modify their physiology and interactions, with consequences to thermogenic genes. We also hypothesize that ROS play a critical role in signaling transcription of thermogenic genes and their regulatory components. Using primary BA and macrophage cultures, we measured Meth and NE interference with physiological and phenotypic measures that are relevant to thermogenesis in BAT. Meth caused both BA and macrophages to decrease mitochondrial maximal capacity and increase ROS. In BA, the thermogenic genes UCP1, PPARγ, PGC1α and GADD45γ were transcriptionally increased by Meth in a ROS-dependent manner. In macrophages, Meth increased oxidative stress response and caused a predominance of M2 subset markers. BA transcriptional changes in response to Meth and NE were significantly controlled by macrophages. The results suggest that BA and macrophages respond to Meth and NE, with effects on mitochondrial functions and transcription of genes involved in thermogenesis. ROS-dependent signals in BA and cellular interactions between BA and macrophages synergize to regulate the BAT environment and control critical pathways leading to Meth-hyperthermia.

Methamphetamine and Cannabis: A Tale of Two Drugs and their Effects on HIV, Brain, and Behavior.

HIV infection and drug use intersect epidemiologically, and their combination can result in complex effects on brain and behavior. The extent to which drugs affect the health of persons with HIV (PWH) depends on many factors including drug characteristics, use patterns, stage of HIV disease and its treatment, comorbid factors, and age. To consider the range of drug effects, we have selected two that are in common use by PWH: methamphetamine and cannabis. We compare the effects of methamphetamine with those of cannabis, to illustrate how substances may potentiate, worsen, or even buffer the effects of HIV on the CNS. Data from human, animal, and ex vivo studies provide insights into how these drugs have differing effects on the persistent inflammatory state that characterizes HIV infection, including effects on viral replication, immune activation, mitochondrial function, gut permeability, blood brain barrier integrity, glia and neuronal signaling. Moving forward, we consider how these mechanistic insights may inform interventions to improve brain outcomes in PWH. This review summarizes literature from clinical and preclinical studies demonstrating the adverse effects of METH, as well as the potentially beneficial effects of cannabis, on the interacting systemic (e.g., gut barrier leakage/microbial translocation, immune activation, inflammation) and CNS-specific (e.g., glial activation/neuroinflammation, neural injury, mitochondrial toxicity/oxidative stress) mechanisms underlying HIV-associated neurocognitive disorders.

Sex differences and Tat expression affect dopaminergic receptor expression and response to antioxidant treatment in methamphetamine-sensitized HIV Tat transgenic mice.

Methamphetamine (Meth) abuse is a common HIV comorbidity. Males and females differ in their patterns of Meth use, associated behaviors, and responses, but the underlying mechanisms and impact of HIV infection are unclear. Transgenic mice with inducible HIV-1 Tat protein in the brain (iTat) replicate many neurological aspects of HIV infection in humans. We previously showed that Tat induction enhances the Meth sensitization response associated with perturbation of the dopaminergic system, in male iTat mice. Here, we used the iTat mouse model to investigate sex differences in individual and interactive effects of Tat and Meth challenge on locomotor sensitization, brain expression of dopamine receptors (DRDs) and regulatory adenosine receptors (ADORAs). Because Meth administration increases the production of reactive oxygen species (ROS), we also determined whether the effects of Meth could be rescued by concomitant treatment with the ROS scavenger N-acetyl cysteine (NAC). After Meth sensitization and a 7-day abstinence period, groups of Tat+ and Tat-male and female mice were challenged with Meth in combination with NAC. We confirmed that Tat expression and Meth challenge suppressed DRD mRNA and protein in males and females' brains, and showed that females were particularly susceptible to the effects of Meth on D1-like and D2-like DRD subtypes and ADORAs. The expression of these markers differed strikingly between males and females, and between females in different phases of the estrous cycle, in a Tat -dependent manner. NAC attenuated Meth-induced locomotor sensitization and preserved DRD expression in all groups except for Tat + females. These data identify complex interactions between sex, Meth use, and HIV infection on addiction responses, with potential implications for the treatment of male and female Meth users in the context of HIV, especially those with cognitive disorders.

Systems Biology Analysis of the Antagonizing Effects of HIV-1 Tat Expression in the Brain over Transcriptional Changes Caused by Methamphetamine Sensitization.

Methamphetamine (Meth) abuse is common among humans with immunodeficiency virus (HIV). The HIV-1 regulatory protein, trans-activator of transcription (Tat), has been described to induce changes in brain gene transcription that can result in impaired reward circuitry, as well as in inflammatory processes. In transgenic mice with doxycycline-induced Tat protein expression in the brain, i.e., a mouse model of neuroHIV, we tested global gene expression patterns induced by Meth sensitization. Meth-induced locomotor sensitization included repeated daily Meth or saline injections for seven days and Meth challenge after a seven-day abstinence period. Brain samples were collected 30 min after the Meth challenge. We investigated global gene expression changes in the caudate putamen, an area with relevance in behavior and HIV pathogenesis, and performed pathway and transcriptional factor usage predictions using systems biology strategies. We found that Tat expression alone had a very limited impact in gene transcription after the Meth challenge. In contrast, Meth-induced sensitization in the absence of Tat induced a global suppression of gene transcription. Interestingly, the interaction between Tat and Meth broadly prevented the Meth-induced global transcriptional suppression, by maintaining regulation pathways, and resulting in gene expression profiles that were more similar to the controls. Pathways associated with mitochondrial health, initiation of transcription and translation, as well as with epigenetic control, were heavily affected by Meth, and by its interaction with Tat in anti-directional ways. A series of systems strategies have predicted several components impacted by these interactions, including mitochondrial pathways, mTOR/RICTOR, AP-1 transcription factor, and eukaryotic initiation factors involved in transcription and translation. In spite of the antagonizing effects of Tat, a few genes identified in relevant gene networks remained downregulated, such as sirtuin 1, and the amyloid precursor protein (APP). In conclusion, Tat expression in the brain had a low acute transcriptional impact but strongly interacted with Meth sensitization, to modify effects in the global transcriptome.

Dopamine and its receptors play a role in the modulation of CCR5 expression in innate immune cells following exposure to Methamphetamine: Implications to HIV infection.

The Human Immunodeficiency Virus (HIV) infects cells in the Central Nervous System (CNS), where the access of antiretrovirals and antibodies that can kill the virus may be challenging. As a result of the early HIV entry in the brain, infected individuals develop inflammation and neurological deficits at various levels, which are aggravated by drugs of abuse. In the non-human primate model of HIV, we have previously shown that drugs of abuse such as Methamphetamine (Meth) increase brain viral load in correlation with a higher number of CCR5-expressing myeloid cells. CCR5 is a chemokine receptor that may be involved in increasing inflammation, but also, it is a co-receptor for viral entry into target cells. CCR5-expressing myeloid cells are the main targets of HIV in the CNS. Thus, the identification of factors and mechanisms that impact the expression of CCR5 in the brain is critical, as changes in CCR5 levels may affect the infection in the brain. Using a well-characterized in vitro system, with the THP1 human macrophage cell line, we have investigated the hypothesis that the expression of CCR5 is acutely affected by Meth, and examined pathways by which this effect could happen. We found that Meth plays a direct role by regulating the abundance and nuclear translocation of transcription factors with binding sites in the CCR5 promoter. However, we found that the main factor that modifies the CCR5 gene promoter at the epigenetic level towards transcription is Dopamine (DA), a neurotransmitter that is produced primarily in brain regions that are rich in dopaminergic neurons. In THP1 cells, the effect of DA on innate immune CCR5 transcription was mediated by DA receptors (DRDs), mainly DRD4. We also identified a role for DRD1 in suppressing CCR5 expression in this myeloid cell system, with potential implications for therapy. The effect of DA on innate immune CCR5 expression was also detectable on the cell surface during acute time-points, using low doses. In addition, HIV Tat acted by enhancing the surface expression of CCR5, in spite of its poor effect on transcription. Overall, our data suggests that the exposure of myeloid cells to Meth in the context of presence of HIV peptides such as Tat, may affect the number of HIV targets by modulating CCR5 expression, through a combination of DA-dependent and-independent mechanisms. Other drugs that increase DA may affect similar mechanisms. The implications of these epigenetic and translational mechanisms in enhancing HIV infection in the brain and elsewhere are demonstrated.

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.

Modeling the Function of TATA Box Binding Protein in Transcriptional Changes Induced by HIV-1 Tat in Innate Immune Cells and the Effect of Methamphetamine Exposure.

Innate immune cells are targets of HIV-1 infection in the Central Nervous System (CNS), generating neurological deficits. Infected individuals with substance use disorders as co-morbidities, are more likely to have aggravated neurological disorders, higher CNS viral load and inflammation. Methamphetamine (Meth) is an addictive stimulant drug, commonly among HIV+ individuals. The molecular basis of HIV direct effects and its interactions with Meth in host response, at the gene promoter level, are not well understood. The main HIV-1 peptide acting on transcription is the transactivator of transcription (Tat), which promotes replication by recruiting a Tata-box binding protein (TBP) to the virus long-terminal repeat (LTR). We tested the hypothesis that Tat can stimulate host gene expression through its ability to increase TBP, and thus promoting its binding to promoters that bear Tata-box binding motifs. Genes with Tata-box domains are mainly inducible, early response, and involved in inflammation, regulation and metabolism, relevant in HIV pathogenesis. We also tested whether Tat and Meth interact to trigger the expression of Tata-box bearing genes. The THP1 macrophage cell line is a well characterized innate immune cell system for studying signal transduction in inflammation. These cells are responsive to Tat, as well as to Meth, by recruiting RNA Polymerase (RNA Pol) to inflammatory gene promoters, within 15 min of stimulation (1). THP-1 cells, including their genetically engineered derivatives, represent valuable tools for investigating monocyte structure and function in both health and disease, as a consistent system (2). When differentiated, they mimic several aspects of the response of macrophages, and innate immune cells that are the main HIV-1 targets within the Central Nervous System (CNS). THP1 cells have been used to characterize the impact of Meth and resulting neurotransmitters on HIV entry (1), mimicking the CNS micro-environment. Integrative consensus sequence analysis in genes with enriched RNA Pol, revealed that TBP was a major transcription factor in Tat stimulation, while the co-incubation with Meth shifted usage to a distinct and diversified pattern. For validating these findings, we engineered a THP1 clone to be deficient in the expression of all major TBP splice variants, and tested its response to Tat stimulation, in the presence or absence of Meth. Transcriptional patterns in TBP-sufficient and deficient clones confirmed TBP as a dominant transcription factor in Tat stimulation, capable of inducing genes with no constitutive expression. However, in the presence of Meth, TBP was no longer necessary to activate the same genes, suggesting promoter plasticity. These findings demonstrate TBP as mechanism of host-response activation by HIV-1 Tat, and suggest that promoter plasticity is a challenge imposed by co-morbid factors such as stimulant drug addiction. This may be one mechanism responsible for limited efficacy of therapeutic approaches in HIV+ Meth abusers.

Methamphetamine abuse affects gene expression in brain-derived microglia of SIV-infected macaques to enhance inflammation and promote virus targets.

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.

The cross-talk of HIV-1 Tat and methamphetamine in HIV-associated neurocognitive disorders.

Antiretroviral therapy has dramatically improved the lives of human immunodeficiency virus 1 (HIV-1) infected individuals. Nonetheless, HIV-associated neurocognitive disorders (HAND), which range from undetectable neurocognitive impairments to severe dementia, still affect approximately 50% of the infected population, hampering their quality of life. The persistence of HAND is promoted by several factors, including longer life expectancies, the residual levels of virus in the central nervous system (CNS) and the continued presence of HIV-1 regulatory proteins such as the transactivator of transcription (Tat) in the brain. Tat is a secreted viral protein that crosses the blood-brain barrier into the CNS, where it has the ability to directly act on neurons and non-neuronal cells alike. These actions result in the release of soluble factors involved in inflammation, oxidative stress and excitotoxicity, ultimately resulting in neuronal damage. The percentage of methamphetamine (MA) abusers is high among the HIV-1-positive population compared to the general population. On the other hand, MA abuse is correlated with increased viral replication, enhanced Tat-mediated neurotoxicity and neurocognitive impairments. Although several strategies have been investigated to reduce HAND and MA use, no clinically approved treatment is currently available. Here, we review the latest findings of the effects of Tat and MA in HAND and discuss a few promising potential therapeutic developments.

Phenotypic changes in the brain of SIV-infected macaques exposed to methamphetamine parallel macrophage activation patterns induced by the common gamma-chain cytokine system.

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.

Osteopontin expression in the brain triggers localized inflammation and cell death when immune cells are activated by pertussis toxin.

Upregulation of osteopontin (OPN) is a characteristic of central nervous system pathologies. However, the role of OPN in inflammation is still controversial, since it can both prevent cell death and induce the migration of potentially damaging inflammatory cells. To understand the role of OPN in inflammation and cell survival, we expressed OPN, utilizing an adenoviral vector, in the caudoputamen of mice deficient in OPN, using beta-galactosidase- (ß-gal-) expressing vector as control. The tissue pathology and the expression of proinflammatory genes were compared in both treatments. Interestingly, inflammatory infiltrate was only found when the OPN-vector was combined with a peripheral treatment with pertussis toxin (Ptx), which activated peripheral cells to express the OPN receptor CD44v6. Relative to ß-gal, OPN increased the levels of inflammatory markers, including IL13Rα1, CXCR3, and CD40L. In Ptx-treated OPN KOs, apoptotic TUNEL+ cells surrounding the OPN expression site increased, compared to ß-gal. Together, these results show that local OPN expression combined with a peripheral inflammatory stimulus, such as Ptx, may be implicated in the development of brain inflammation and induction of cell death, by driving a molecular pattern characteristic of cytotoxicity. These are characteristics of inflammatory pathologies of the CNS in which OPN upregulation is a hallmark.

TLR signaling controls lethal encephalitis in WNV-infected brain.

Toll-like receptors (TLRs) are known to be activated in Central Nervous System (CNS) viral infections and are recognized to be a critical component in innate immunity. Several reports state a role for particular TLRs in various CNS viral infections. However, excessive TLR activation was previously reported by us in correlation with a pathogenic, rather than a protective, outcome, in a model of SIV encephalitis. Here we aimed at understanding the impact of TLR-mediated pathways by evaluating the early course of pathogenesis in the total absence of TLR signaling during CNS viral infections. We utilized a mouse model of sublethal West Nile virus (WNV) infection. WNV is an emerging neurotropic flavivirus, and a significant global cause of viral encephalitis. The virus was peripherally injected into animals that simultaneously lacked two key adapter molecules of TLR signaling, MyD88 and TRIF. On day 2 pi (post infection), MyD88/Trif-/- mice showed an increased susceptibility to WNV infection, and revealed an impairment in innate immune cytokines, when compared to wild type mice (WT). By day 6 pi, there was an increase in viral burden and robust expression of inflammatory cytokines as well as higher cell infiltration into the CNS in MyD88/Trif-/-, when compared to infected WT. A drastic increase in microglia activation, astrogliosis, and inflammatory trafficking were also observed on day 6 pi in MyD88/Trif-/-. Our observations show a protective role for TLR signaling pathways in preventing lethal encephalitis at early stages of WNV infection.

ROS and Sympathetically Mediated Mitochondria Activation in Brown Adipose Tissue Contribute to Methamphetamine-Induced Hyperthermia.

Methamphetamine (Meth) abuse has been shown to induce alterations in mitochondrial function in the brain as well as to induce hyperthermia, which contributes to neurotoxicity and Meth-associated mortality. Brown adipose tissue (BAT), a thermogenic site known to be important in neonates, has recently regained importance since being identified in significant amounts and in correlation with metabolic balance in human adults. Given the high mitochondrial content of BAT and its role in thermogenesis, we aimed to investigate whether BAT plays any role in the development of Meth-induced hyperthermia. By ablating or denervating BAT, we identified a partial contribution of this organ to Meth-induced hyperthermia. BAT ablation decreased temperature by 0.5°C and reduced the length of hyperthermia by 1 h, compared to sham-operated controls. BAT denervation also affected the development of hyperthermia in correlation with decreased the expression of electron transport chain molecules, and increase on PCG1a levels, but without affecting Meth-induced uncoupling protein 1 upregulation. Furthermore, in isolated BAT cells in culture, Meth, but not Norepinephrine, induced H2O2 upregulation. In addition, we found that in vivo Reactive Oxygen Species (ROS) play a role in Meth hyperthermia. Thus, sympathetically mediated mitochondrial activation in the BAT and Meth-induced ROS are key components to the development of hyperthermia in Meth abuse.