FACS to Identify Immune Subsets in Mouse Brain and Spleen.

Flow cytometry enables the multi-parametric quantification of cell types, especially in immunophenotyping of unique immune cell subsets that can either contribute to or ameliorate pathology. For tissues to be used in such analyses, single-cell suspensions must be created. Here we describe protocols for preparing single-cell suspensions of mouse spleen and brain tissue, as well as the steps for fluorescently activated cell staining/sorting (FACS). Specifically, this protocol enables the isolation of lymphocytes for the study of immune responses during various diseases, such as long-term neuroinflammation following ischemic stroke.

Plasma protein alterations during human large vessel stroke: A controlled comparison study.

BACKGROUND: Stroke is a major cause of death and disability in the United States. Mechanical thrombectomy (MT) and tissue plasminogen activator are the current treatments for ischemic stroke, which have improved clinical outcomes. Despite these treatments, functional and cognitive deficits still occur demonstrating a need for predictive biomarkers for beneficial clinical outcomes which can be used as therapeutic targets for pharmacotherapy. The aim of this study compares the proteomic expression of systemic arterial blood collected at the time of MT to those from a matched cerebrovascular disease (CVD) control cohort. METHODS: The Blood And Clot Thrombectomy Registry And Collaboration (BACTRAC) (clinicaltrials.gov NCT03153683) collects and banks arterial blood, both distal and proximal to the thrombus, from ischemic stroke subjects undergoing MT. Arterial blood from patients undergoing a diagnostic angiogram was also collected and banked as CVD controls. Changes in cardiometabolic and inflammatory proteins between stroke and CVD controls were analyzed via Olink Proteomics. RESULTS: Proteins including ARTN, TWEAK, HGF, CCL28, FGF-5, CXCL9, TRANCE and GDNF were found to be decreased in stroke subjects when compared to CVD controls. CXCL1, CCL5, OSM, GP1BA, IL6, MMP-1, and CXCL5 were increased in stroke subjects when compared to CVD controls. These proteins were also significantly correlated to stroke outcome metrics such as NIHSS, infarct volume and MoCA scoring. CONCLUSION: Overall, acute stroke patients had an increase in inflammatory proteins with a decrease in trophic proteins systemically compared to matched CVD controls. Using our CVD controls, proteins of interest were directly compared to stroke patients with the same cerebrovascular risk factors instead of statistically controlling for comorbidities. The novel methodology of matching an arterial blood CVD control group to a stroke group, as well as controlling for age and comorbid status add to the literature on prognostic stroke biomarkers, which are specific targets for future therapeutics.

Influence of BMI on adenosine deaminase and stroke outcomes in mechanical thrombectomy subjects.

BACKGROUND: Emergent Large Vessel Occlusion (ELVO) strokes are ischemic vascular events for which novel biomarkers and therapies are needed. The purpose of this study is to investigate the role of Body Mass Index (BMI) on protein expression and signaling at the time of ELVO intervention. Additionally, we highlight the protein adenosine deaminase (ADA), which is a deaminating enzyme that degrades adenosine, which has been shown to be neuroprotective in ischemia. We investigate the relationship between ADA and BMI, stroke outcomes, and associated proteomic networks which might aid in personalizing prognosis and future treatment of ELVO stroke. METHODS: The Blood And Clot Thrombectomy And Collaboration (BACTRAC) study is a continually enrolling tissue bank (clinicaltrials.gov NCT03153683) and registry from stroke patients undergoing mechanical thrombectomy (MT). N â€‹= â€‹61 human carotid plasma samples were analyzed for inflammatory and cardiometabolic protein expression by Olink Proteomics. Statistical analyses used t-tests, linear, logistic, and robust regressions, to assess the relationship between BMI, proteomic expression, and stroke-related outcomes. RESULTS: The 61 subjects studied were broken into three categories: normal weight (BMI 18.5-24.9) which contained 19 subjects, overweight (BMI 25-30) which contained 25 subjects, and obese (BMI ≥30) which contained 17 subjects. Normal BMI group was a significantly older population (mean 76 years) when compared to overweight (mean 66 years) and obese (mean 61 years) with significance of p â€‹= â€‹0.041 and p â€‹= â€‹0.005, respectively. When compared to normal weight and overweight categories, the obese category had significantly higher levels of adenosine deaminase (ADA) expression (p â€‹= â€‹0.01 and p â€‹= â€‹0.039, respectively). Elevated levels of ADA were found to have a significant positive correlation with both infarct volume and edema volume (p â€‹= â€‹0.013 and p â€‹= â€‹0.041, respectively), and were associated with a more severe stroke (NIHSS on discharge) and greater stroke related disability (mRS on discharge) with significance of p â€‹= â€‹0.053 and p â€‹= â€‹0.032, respectively. CONCLUSIONS: When examined according to BMI, subjects undergoing MT for ELVO demonstrate significant differences in the expression of certain plasma proteins, including ADA. Levels of ADA were found to be significantly higher in the obese population when compared to normal or overweight groups. Increased levels of ADA in the obese group were predictive of increased infarct volume, edema volume, and worse NIHSS scores and mRS at discharge. These data provide novel biomarker candidates as well as treatment targets while increasing the personalization of stroke prognosis and treatment.

Antimicrobial protein REG3A and signaling networks are predictive of stroke outcomes.

Regenerating Family Member 3 Alpha (REG3A) is a multifunctional protein with antimicrobial activity, and primarily secreted by the intestine and pancreas. Studies have shown an increased expression of REG3A in systemic inflammatory responses to acute injury and infection, but studies investigating REG3A during the pathogenesis of ischemic stroke are limited. The aims of this study were to examine the associations between arterial expression of REG3A and other arterial inflammatory proteins implicated in stroke pathogenesis, as well as associations between REG3A and markers of poor outcome for ischemic stroke. The University of Kentucky Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC) protocol (clinicaltrials.gov NCT03153683) utilizes thrombectomy to isolate intracranial arterial blood (i.e. distal to thrombus) and systemic arterial blood (i.e. carotid). Samples were analyzed by Olink Proteomics for N = 42 subjects. Statistical analyses of plasma proteins included 2-sample t-tests, spearman and biserial correlations, and robust regression models to elucidate network signaling and association to clinical outcomes. Results indicated that levels of systemic REG3A were positively correlated with inflammatory proteins interleukin IL6 (R = 0.344, p = 0.030) and IL17C (R = 0.468, p = 0.002). 2-sided t- tests examining differences of systemic REG3A within quartiles of NIHSS admission score depicted significant differences between quartiles. Those with NIHSS scores corresponding to moderate and moderate-severe neurofunctional deficits had significantly higher levels of systemic REG3A compared to those with NIHSS scores corresponding to mild and mild-moderate neurofunctional deficits (p = 0.016). STRING analyses of proteins in each robust regression model demonstrated substantial networking between REG3A and other systemic proteins highly relevant to ischemic stroke. The present study provides novel data on systemic REG3A in the context of ischemic stroke. These results demonstrate the influential role of REG3A regarding surrogate functional and radiographic outcomes of stroke severity. Additionally, they provide novel insight into the role of REG3A and related proteins during the complex neuroinflammatory process of ischemic stroke. These data provide a foundation for future studies to investigate REG3A and related networking proteins as potential biomarkers with prognostic potential, as well as potential therapeutic targets.

Isolation and identification of leukocyte populations in intracranial blood collected during mechanical thrombectomy.

Using standard techniques during mechanical thrombectomy, the Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC) protocol (NCT03153683) isolates intracranial arterial blood distal to the thrombus and proximal systemic blood in the carotid artery. We augmented the current protocol to study leukocyte subpopulations both distal and proximal to the thrombus during human stroke (n = 16 patients), and from patients with cerebrovascular disease (CVD) undergoing angiography for unrelated conditions (e.g. carotid artery stenosis; n = 12 patients). We isolated leukocytes for flow cytometry from small volume (<1 mL) intracranial blood and systemic blood (5-10 mL) to identify adaptive and innate leukocyte populations, in addition to platelets and endothelial cells (ECs). Intracranial blood exhibited significant increases in T cell representation and decreases in myeloid/macrophage representation compared to within-patient carotid artery samples. CD4+ T cells and classical dendritic cells were significantly lower than CVD controls and correlated to within-patient edema volume and last known normal. This novel protocol successfully isolates leukocytes from small volume intracranial blood samples of stroke patients at time of mechanical thrombectomy and can be used to confirm preclinical results, as well as identify novel targets for immunotherapies.

Delayed diapedesis of CD8 T cells contributes to long-term pathology after ischemic stroke in male mice.

OBJECTIVE: Stroke is a debilitating disorder with significant annual mortality and morbidity rates worldwide. Immune cells are recruited to the injured brain within hours after stroke onset and can exhibit either protective or detrimental effects on recovery. However, immune cells, including CD8 T cells, persist in the injured brain for weeks, suggesting a longer-term role for the adaptive immune system during functional recovery. The aim of this study was to determine if the delayed secondary diapedesis of CD8 T cells into the ischemic brain negatively impacts functional recovery after transient ischemic stroke in male mice. RESULTS: Mice exhibited an increased number of leukocytes in the ipsilesional hemispheres at 14 days (3-fold; p < 0.001) and 30 days (2.2-fold; p = 0.02) after transient middle cerebral artery occlusion (tMCAo) compared to 8 days post-tMCAo, at which time acute neuroinflammation predominantly resolves. Moreover, mice with higher ipsilesional CD8 T cells at 30 days (R2 = 0.52, p < 0.01) exhibited worse functional recovery. To confirm a detrimental role of chronic CD8 T cell diapedesis on recovery, peripheral CD8 T cells were depleted beginning 10 days post-tMCAo. Delayed CD8 T cell depletion improved motor recovery on the rotarod (F(1,28) = 4.264; p = 0.048) compared to isotype control-treated mice. CD8 T cell-depleted mice also exhibited 2-fold (p < 0.001) reduced leukocyte infiltration at 30 days post-tMCAo. Specifically, macrophage, neutrophil, and CD4 T cell numbers were reduced in the ipsilesional hemisphere of the CD8 T cell-depleted mice independent of inflammatory status of the post-stroke CNS (e.g. microglial phenotype and cytokine production). RNAseq identified a unique profile for brain infiltrating CD8 T cells at 30 days post-tMCAo, with 46 genes differentially expressed relative to CD8 T cells at 3 days post-tMCAo. CONCLUSION: Our data reveal a role for CD8 T cells in the chronic phase post-stroke that can be therapeutically targeted. We demonstrate long-term CD8 T cell recruitment into the ipsilesional hemisphere that affects both immune cell numbers present in the injured brain and functional recovery through one month after stroke onset.

Intracranial VCAM1 at time of mechanical thrombectomy predicts ischemic stroke severity.

BACKGROUND: Emergent large vessel occlusion (ELVO) strokes are devastating ischemic vascular events for which novel treatment options are needed. Using vascular cell adhesion molecule 1 (VCAM1) as a prototype, the objective of this study was to identify proteomic biomarkers and network signaling functions that are potential therapeutic targets for adjuvant treatment for mechanical thrombectomy. METHODS: The blood and clot thrombectomy and collaboration (BACTRAC) study is a continually enrolling tissue bank and registry from stroke patients undergoing mechanical thrombectomy. Plasma proteins from intracranial (distal to clot) and systemic arterial blood (carotid) were analyzed by Olink Proteomics for N=42 subjects. Statistical analysis of plasma proteomics used independent sample t tests, correlations, linear regression, and robust regression models to determine network signaling and predictors of clinical outcomes. Data and network analyses were performed using IBM SPSS Statistics, SAS v 9.4, and STRING V11. RESULTS: Increased systemic (p<0.001) and intracranial (p=0.013) levels of VCAM1 were associated with the presence of hypertension. Intracranial VCAM1 was positively correlated to both infarct volume (p=0.032; r=0.34) and edema volume (p=0.026; r=0.35). The %∆ in NIHSS from admittance to discharge was found to be significantly correlated to both systemic (p=0.013; r = -0.409) and intracranial (p=0.011; r = -0.421) VCAM1 levels indicating elevated levels of systemic and intracranial VCAM1 are associated with reduced improvement of stroke severity based on NIHSS from admittance to discharge. STRING-generated analyses identified biologic functional descriptions as well as function-associated proteins from the predictive models of infarct and edema volume. CONCLUSIONS: The current study provides novel data on systemic and intracranial VCAM1 in relation to stroke comorbidities, stroke severity, functional outcomes, and the role VCAM1 plays in complex protein-protein signaling pathways. These data will allow future studies to develop predictive biomarkers and proteomic targets for drug development to improve our ability to treat a devastating pathology.

Proteomic changes in intracranial blood during human ischemic stroke.

BACKGROUND: Since 2015, mechanical thrombectomy has been the standard treatment for emergent large vessel occlusion ischemic stroke. OBJECTIVE: To investigate, using the previously published Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC) protocol (clinicaltrials.gov NCT03153683), how the protein expression of a patient's intracranial blood during ischemic stroke compares with the protein expression of their systemic arterial blood in order to better understand and treat stroke. METHODS: Plasma samples from 25 subjects underwent proteomic analysis, where intracranial protein expression was compared with systemic protein levels. Data including sex, comorbidities, infarct volume, and infarct time were included for each subject. RESULTS: A majority of important proteins had a lower expression in intracranial blood than in systemic arterial blood. Proteins with the most significant changes in expression were: endopeptidase at -0.26 (p<0.0001), phospholipid transfer protein (PLTP) at -0.26 (p=0.0005), uromodulin (UMOD) at -0.14 (p=0.002), ficolin-2 (FCN2) at -0.46 (p=0.005), C-C motif chemokine 19 (CCL19) at -0.51 (p<0.0001), C-C motif chemokine 20 (CCL20) at -0.40 (p<0.0001), fibroblast growth factor 21 at -0.37 (p=0.0002), and C-C motif chemokine (CCL23) at -0.43 (p=0.0003). CONCLUSIONS: Evaluation of proteomic changes in the intravascular space of a cerebral infarct in progress in human subjects suggested that changes in proteins such PLTP, fetuin-B (FETUB), and FCN2 may be involved in atherosclerotic changes, and chemokines such as CCL23 are known to play a role in the Th2 autoimmune response. These data provide a scientific springboard for identifying clinically relevant biomarkers for diagnosis/prognosis, and targets for much needed neuroprotective/neuroreparative pharmacotherapies.

Distinctly perturbed metabolic networks underlie differential tumor tissue damages induced by immune modulator ß-glucan in a two-case ex vivo non-small-cell lung cancer study.

Cancer and stromal cell metabolism is important for understanding tumor development, which highly depends on the tumor microenvironment (TME). Cell or animal models cannot recapitulate the human TME. We have developed an ex vivo paired cancerous (CA) and noncancerous (NC) human lung tissue approach to explore cancer and stromal cell metabolism in the native human TME. This approach enabled full control of experimental parameters and acquisition of individual patient's target tissue response to therapeutic agents while eliminating interferences from genetic and physiological variations. In this two-case study of non-small-cell lung cancer, we performed stable isotope-resolved metabolomic (SIRM) experiments on paired CA and NC lung tissues treated with a macrophage activator ß-glucan and (13)C6-glucose, followed by ion chromatography-Fourier transform mass spectrometry (IC-FTMS) and nuclear magnetic resonance (NMR) analyses of (13)C-labeling patterns of metabolites. We demonstrated that CA lung tissue slices were metabolically more active than their NC counterparts, which recapitulated the metabolic reprogramming in CA lung tissues observed in vivo. We showed ß-glucan-enhanced glycolysis, Krebs cycle, pentose phosphate pathway, antioxidant production, and itaconate buildup in patient UK021 with chronic obstructive pulmonary disease (COPD) and an abundance of tumor-associated macrophages (TAMs) but not in UK049 with no COPD and much less macrophage infiltration. This metabolic response of UK021 tissues was accompanied by reduced mitotic index, increased necrosis, and enhaced inducible nitric oxide synthase (iNOS) expression. We surmise that the reprogrammed networks could reflect ß-glucan M1 polarization of human macrophages. This case study presents a unique opportunity for investigating metabolic responses of human macrophages to immune modulators in their native microenvironment on an individual patient basis.

Methodology and effects of repeated intranasal delivery of DNSP-11 in a rat model of Parkinson's disease.

BACKGROUND: To circumvent the challenges associated with delivering large compounds directly to the brain for the treatment of Parkinson's disease (PD), non-invasive procedures utilizing smaller molecules with protective and/or restorative actions on dopaminergic neurons are needed. NEW METHOD: We developed a methodology for evaluating the effects of a synthetic neuroactive peptide, DNSP-11, on the nigrostriatal system using repeated intranasal delivery in both normal and a unilateral 6-hydroxydopamine (6-OHDA) lesion rat model of PD. RESULTS: Normal rats repeatedly administered varying doses of DNSP-11 intranasally for 3 weeks exhibited a significant increase in dopamine (DA) turnover in both the striatum and substantia nigra (SN) at 300µg, suggestive of a stimulative effect of the dopaminergic system. Additionally, a protective effect was observed following repeated intranasal administration in 6-OHDA lesioned rats, as suggested by: a significant decrease in d-amphetamine-induced rotation at 2 weeks; a decrease in DA turnover in the lesioned striatum; and an increased sparing of tyrosine hydroxylase (TH) positive (+) neurons in a specific sub-region of the lesioned substantia nigra pars compacta (SNpc). Finally, tracer studies showed (125)I-DNSP-11 distributed diffusely throughout the brain, including the striatum and SN, as quickly as 30min after a single intranasal dose. COMPARISON WITH EXISTING METHODS: The results of bilateral intranasal administration of DNSP-11 are compared to our unilateral single infusion studies to the brain in rats. CONCLUSIONS: These studies support that DNSP-11 can be delivered intranasally and maintain its neuroactive properties in both normal rats and in a unilateral 6-OHDA rat model of PD.

Dynamic changes in dopamine neuron function after DNSP-11 treatment: effects in vivo and increased ERK 1/2 phosphorylation in vitro.

Glial cell-line derived neurotrophic factor (GDNF) has demonstrated robust effects on dopamine (DA) neuron function and survival. A post-translational processing model of the human GDNF proprotein theorizes the formation of smaller, amidated peptide(s) from the proregion that exhibit neurobiological function, including an 11-amino-acid peptide named dopamine neuron stimulating peptide-11 (DNSP-11). A single treatment of DNSP-11 was delivered to the substantia nigra in the rat to investigate effects on DA-neuron function. Four weeks after treatment, potassium (K+) and D-amphetamine evoked DA release were studied in the striatum using microdialysis. There were no significant changes in DA-release after DNSP-11 treatment determined by microdialysis. Dopamine release was further examined in discrete regions of the striatum using high-speed chronoamperometry at 1-, 2-, and 4-weeks after DNSP-11 treatment. Two weeks after DNSP-11 treatment, potassium-evoked DA release was increased in specific subregions of the striatum. However, spontaneous locomotor activity was unchanged by DNSP-11 treatment. In addition, we show that a single treatment of DNSP-11 in the MN9D dopaminergic neuronal cell line results in phosphorylation of ERK1/2, which suggests a novel cellular mechanism responsible for increases in DA function.

Evaluation of the physical and in vitro protective activity of three synthetic peptides derived from the pro- and mature GDNF sequence.

Recently, a small 11-amino acid amidated peptide, dopamine neuron stimulating peptide-11 (DNSP-11), was shown to exert neurotrophic-like actions on primary dopaminergic neurons and in parkinsonian rat models. This suggests smaller neurotrophic-like molecules may be deliverable and modifiable for therapeutic use. Here we evaluate the molecular and cellular protection properties of DNSP-11 and two other amidated-peptides, a 5-mer (DNSP-5) and a 17-mer (DNSP-17), hypothesized to be endoproteolytically processed from the pro- and mature glial cell line-derived neurotrophic factor (GDNF) protein sequence, respectively. Far-UV circular dichroism spectra show that the three DNSPs are soluble and act independently in vitro. Reverse phase HPLC and mass spectrometry analysis show that the three peptides are stable for one month at a variety of storage and experimental conditions. To gain insight into their biodistribution properties in the brain, we used affinity chromatography to show that DNSP-17 binds heparin equally as tight as GDNF, whereas DNSP-5 and DNSP-11 do not bind heparin, which should facilitate their delivery in vivo. Finally, we present data showing that DNSP-11 provides dose-dependent protection of HEK-293 cells from staurosporine and 3-nitropropionate (3-NP) cytotoxicity, thereby supporting its broad mitochondrial-protective properties.

Dopamine neuron stimulating actions of a GDNF propeptide.

BACKGROUND: Neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF), have shown great promise for protection and restoration of damaged or dying dopamine neurons in animal models and in some Parkinson's disease (PD) clinical trials. However, the delivery of neurotrophic factors to the brain is difficult due to their large size and poor bio-distribution. In addition, developing more efficacious trophic factors is hampered by the difficulty of synthesis and structural modification. Small molecules with neurotrophic actions that are easy to synthesize and modify to improve bioavailability are needed. METHODS AND FINDINGS: Here we present the neurobiological actions of dopamine neuron stimulating peptide-11 (DNSP-11), an 11-mer peptide from the proGDNF domain. In vitro, DNSP-11 supports the survival of fetal mesencephalic neurons, increasing both the number of surviving cells and neuritic outgrowth. In MN9D cells, DNSP-11 protects against dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA)-induced cell death, significantly decreasing TUNEL-positive cells and levels of caspase-3 activity. In vivo, a single injection of DNSP-11 into the normal adult rat substantia nigra is taken up rapidly into neurons and increases resting levels of dopamine and its metabolites for up to 28 days. Of particular note, DNSP-11 significantly improves apomorphine-induced rotational behavior, and increases dopamine and dopamine metabolite tissue levels in the substantia nigra in a rat model of PD. Unlike GDNF, DNSP-11 was found to block staurosporine- and gramicidin-induced cytotoxicity in nutrient-deprived dopaminergic B65 cells, and its neuroprotective effects included preventing the release of cytochrome c from mitochondria. CONCLUSIONS: Collectively, these data support that DNSP-11 exhibits potent neurotrophic actions analogous to GDNF, making it a viable candidate for a PD therapeutic. However, it likely signals through pathways that do not directly involve the GFRalpha1 receptor.

Estrogen receptor alpha inhibits the estrogen-mediated suppression of HIV transcription in astrocytes: implications for estrogen neuroprotection in HIV dementia.

Many human immunodeficiency virus (HIV) proteins including Tat are produced by HIV-infected astrocytes and secreted into the brain resulting in extensive neuronal damage that contributes to the pathogenesis of HIV dementia. The neuroprotective hormone 17beta-estradiol (E2) is known to negatively regulate the HIV transcriptional promoter in human fetal astrocytes (SVGA cell line) in a Tat-dependent manner. In the present study we extended our investigation in HIV-infected SVGA cells and found a reduction in HIV p24 levels following E2 treatment in comparison to control. Although many E2-mediated events occur through estrogen receptor alpha (ERalpha), we found low levels of ERalpha mRNA and failed to detect ERalpha protein in SVGA cells. Paradoxically, when ERalpha was overexpressed the E2-mediated decrease in Tat transactivation of the promotor was prevented. To determine whether ERalpha expression is altered in the human brain following HIV infection, postmortum hippocampal tissue was obtained from cognitively normal HIV- and HIV+ patients, patients diagnosed with either mild cognitive/motor disorder (MCMD) or HIV-associated dementia (HAD). Immunohistochemistry and quantitative real-time PCR (qRT-PCR) for ERalpha and glial fibrillary acidic protein (GFAP) showed that ERalpha mRNA levels were not significantly different between groups, while GFAP increased in the hippocampus in the HIV+ compared to the HIV- group and was decreased in the MCMD and HAD subgroups compared to HIV+ controls. Notably the ratio of ERalpha-positive reactive astrocytes to total reactive astrocytes increased and significantly correlated with the severity of cognitive impairment following HIV infection. The data suggest that E2 would have the most dramatic effect in reducing HIV transcription early in the disease process when the subpopulation of astrocytes expressing ERalpha is low.

NADPH oxidase drives cytokine and neurotoxin release from microglia and macrophages in response to HIV-Tat.

Previous reports have shown that the human immunodeficiency virus (HIV) regulatory protein Tat has both pro-oxidant and pro-inflammatory properties, suggesting that Tat might contribute to the neurological complications of HIV. However, the intracellular mechanisms whereby Tat triggers free radical production and inflammation, and the relationship between Tat-induced free radicals and inflammatory reactions, are still subject to debate. The present study was undertaken to evaluate the specific effects of Tat on NADPH oxidase in microglia and macrophages, and to determine the specific role of NADPH oxidase in Tat-induced cytokine/chemokine release and neurotoxicity. Application of Tat to microglia or macrophages caused dose- and time-dependent increases in superoxide formation that were prevented by both pharmacologic NADPH oxidase inhibitors and by specific decoy peptides (gp91ds). Furthermore, inhibition of NADPH oxidase attenuated Tat-induced release of interleukin-6 (IL-6), tumor necrosis factor alpha (TNF), and monocyte chemoattractant protein 1 (MCP-1), and decreased microglial-mediated neurotoxicity. Finally, macrophages derived from NADPH oxidase-deficient mice displayed reduced superoxide production, released lower levels of cytokines/chemokines, and induced less neurotoxicity in response to Tat compared to wild-type macrophages. Together, these data describe a specific and biologically significant signaling component of the macrophage/microglial response to Tat, and suggest the neuropathology associated with HIV infection might originate in part with Tat-induced activation of NADPH oxidase.

Morphine and HIV-Tat increase microglial-free radical production and oxidative stress: possible role in cytokine regulation.

Opiate abuse alters the progression of human immunodeficiency virus and may increase the risk of neuroAIDS. As neuroAIDS is associated with altered microglial reactivity, the combined effects of human immunodeficiency virus-Tat and morphine were determined in cultured microglia. Specifically, experiments determined the effects of Tat and morphine on microglial-free radical production and oxidative stress, and on cytokine release. Data show that combined Tat and morphine cause early and synergistic increases in reactive oxygen species, with concomitant increases in protein oxidation. Furthermore, combined Tat and morphine, but not Tat or morphine alone, cause reversible decreases in proteasome activity. The effects of morphine on free radical production and oxidative stress are prevented by pre-treatment with naloxone, illustrating the important role of opioid receptor activation in these phenomena. While Tat is well known to induce cytokine release from cultured microglia, morphine decreases Tat-induced release of the cytokines tumor necrosis factor-alpha and interleukin-6, as well as the chemokine monocyte chemoattractant protein-1 (MCP-1). Finally, experiments using the reversible proteasome inhibitor MG115 show that temporary, non-cytotoxic decreases in proteasome activity increase protein oxidation and decrease tumor necrosis factor-alpha, interleukin-6, and MCP-1 release from microglia. Taken together, these data suggest that oxidative stress and proteasome inhibition may be involved in the immunomodulatory properties of opioid receptor activation in microglia.

Morphine causes rapid increases in glial activation and neuronal injury in the striatum of inducible HIV-1 Tat transgenic mice.

HIV encephalitis (HIVE) is accompanied by brain inflammation, leukocyte infiltration, and glial activation, and HIV patients who abuse opiates are more likely to develop HIVE. To better understand how opiates could alter HIV-related brain inflammation, the expression of astrocyte (GFAP immunoreactivity) and macrophage/microglial (F4/80 or Mac1 immunoreactivity) markers in the striatum, and the percentage of 3-nitrotyrosine (3-NT) positive macrophages/microglia, was determined following a 2-day exposure to morphine (5 mg/kg/day via time-release, subcutaneous implant) and doxycycline in GFAP-driven, doxycycline-inducible HIV-1 Tat transgenic mice. Data show that both morphine and Tat induction via doxycycline increased astrocyte activation, with significant additive increases achieved with combined morphine and doxycycline exposure. By contrast, combined Tat induction and morphine exposure, but neither manipulation alone, significantly increased the proportion of macrophages/microglia present in the striatum of transgenic mice, although morphine exposure was necessary to elevate 3-NT co-detection in Mac1-positive macrophages/microglia. Finally, Tat induction increased the percentage of neurons expressing active caspase-3, and this was even more significantly elevated by co-administration of morphine. In spite of elevations in caspase-3, neuronal TUNEL reactivity was unchanged in all groups, even after 10 days of Tat induction. Importantly, co-administration of naltrexone completely antagonized the effects of morphine. These findings indicate that morphine rapidly and significantly increases the activation of astrocytes and macrophages/microglia in the brains of inducible Tat transgenic mice, supporting the theory that early inflammatory changes in glia could underlie the development of HIVE in opiate-abusing AIDS patients.

Cell-specific actions of HIV-Tat and morphine on opioid receptor expression in glia.

HIV-1 patients who abuse opiate-based drugs, including heroin and morphine, are at a higher risk of developing HIV dementia. The effects of opiates are mediated predominantly through opioid receptors, which are expressed on glial cells. As HIV-1 infection in the CNS is restricted to glial cells, experiments were designed to measure the cell-specific effects of HIV Tat and morphine exposure on opioid receptor expression in both astrocytes and microglia. Specifically, the cell-type-specific pattern of mu opioid receptor (MOR), delta opioid receptor (DOR), and kappa opioid receptor (KOR) localization (surface vs. intracellular) and expression of opioid receptor mRNA were determined after exposure to morphine in the presence and the absence of Tat in primary cultured microglia and astrocytes. Data show that morphine treatment caused significantly decreased cell surface expression of opioid receptors in microglia but not in astrocytes. However, morphine treatment in the presence of Tat significantly increased intracellular expression of opioid receptors and prevented morphine-induced cell surface opioid receptor down-regulation in microglia. These findings document that cell surface opioid receptor expression is divergently regulated by morphine in microglia compared with in astrocytes, and further suggest that HIV-Tat could exacerbate opioid receptor signaling in microglia by increasing receptor expression and/or altering ligand-induced trafficking of opioid receptors.

Increased vulnerability of ApoE4 neurons to HIV proteins and opiates: protection by diosgenin and L-deprenyl.

Human immunodeficiency virus (HIV) infection continues to rise in drug-abusing populations and causes a dementing illness in a subset of individuals. Factors contributing to the development of dementia in this population remain unknown. We found that HIV-infected individuals with the E4 allele of Apolipoprotein E (ApoE) or history of intravenous drug abuse had increased oxidative stress in the CNS. In vitro studies showed that HIV proteins, gp120 and Tat, Tat + morphine but not tumor necrosis factor-alpha (TNF-alpha), caused increased neurotoxicity in human neuronal cultures with ApoE4 allele. Microarray analysis showed a differential alteration of transcripts involved in energy metabolism in cultures of ApoE3 and 4 neurons upon treatment with Tat + morphine. This was confirmed using assays of mitochondrial function and exposure of the neurons to Tat + morphine. Using this in vitro model, we screened a number of novel antioxidants and found that only L-deprenyl and diosgenin protected against the neurotoxicity of Tat + morphine. Furthermore, Tat-induced oxidative stress impaired morphine metabolism which could also be prevented by diosgenin. In conclusion, opiate abusers with HIV infection and the ApoE4 allele may be at increased risk of developing dementia. L-deprenyl and a plant estrogen, diosgenin, may have therapeutic potential in this population.

Gonadal steroids differentially modulate neurotoxicity of HIV and cocaine: testosterone and ICI 182,780 sensitive mechanism.

BACKGROUND: HIV Associated Dementia (HAD) is a common complication of human immunodeficiency virus (HIV) infection that erodes the quality of life for patients and burdens health care providers. Intravenous drug use is a major route of HIV transmission, and drug use is associated with increased HAD. Specific proteins released as a consequence of HIV infection (e.g., gp120, the HIV envelope protein and Tat, the nuclear transactivating protein) have been implicated in the pathogenesis of HAD. In primary cultures of human fetal brain tissue, subtoxic doses of gp120 and Tat are capable of interacting with a physiologically relevant dose of cocaine, to produce a significant synergistic neurotoxicity. Using this model system, the neuroprotective potential of gonadal steroids was investigated. RESULTS: 17beta-Estradiol (17beta-E2), but not 17alpha-estradiol (17alpha-E2), was protective against this combined neurotoxicity. Progesterone (PROG) afforded limited neuroprotection, as did dihydrotestosterone (DHT). The efficacy of 5alpha-testosterone (T)-mediated neuroprotection was robust, similar to that provided by 17beta-E2. In the presence of the specific estrogen receptor (ER) antagonist, ICI-182,780, T's neuroprotection was completely blocked. Thus, T acts through the ER to provide neuroprotection against HIV proteins and cocaine. Interestingly, cholesterol also demonstrated concentration-dependent neuroprotection, possibly attributable to cholesterol's serving as a steroid hormone precursor in neurons. CONCLUSION: Collectively, the present data indicate that cocaine has a robust interaction with the HIV proteins gp120 and Tat that produces severe neurotoxicity, and this toxicity can be blocked through pretreatment with ER agonists.