A central role for glial CCR5 in directing the neuropathological interactions of HIV-1 Tat and opiates.

A central role for glial CCR5 in directing the neuropathological interactions of HIV-1 Tat and opiates.

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Abstract

Background

The collective cognitive and motor deficits known as HIV-associated neurocognitive disorders (HAND) remain high even among HIV+ individuals whose antiretroviral therapy is optimized. HAND is worsened in the context of opiate abuse. The mechanism of exacerbation remains unclear but likely involves chronic immune activation of glial cells resulting from persistent, low-level exposure to the virus and viral proteins. We tested whether signaling through C-C chemokine receptor type 5 (CCR5) contributes to neurotoxic interactions between HIV-1 transactivator of transcription (Tat) and opiates and explored potential mechanisms.

Methods

Neuronal survival was tracked in neuronal and glial co-cultures over 72 h of treatment with HIV-1 Tat ± morphine using cells from CCR5-deficient and wild-type mice exposed to the CCR5 antagonist maraviroc or exogenously-added BDNF (analyzed by repeated measures ANOVA). Intracellular calcium changes in response to Tat ± morphine ± maraviroc were assessed by ratiometric Fura-2 imaging (analyzed by repeated measures ANOVA). Release of brain-derived neurotrophic factor (BDNF) and its precursor proBDNF from CCR5-deficient and wild-type glia was measured by ELISA (analyzed by two-way ANOVA). Levels of CCR5 and μ-opioid receptor (MOR) were measured by immunoblotting (analyzed by Student’s t test).

Results

HIV-1 Tat induces neurotoxicity, which is greatly exacerbated by morphine in wild-type cultures expressing CCR5. Loss of CCR5 from glia (but not neurons) eliminated neurotoxicity due to Tat and morphine interactions. Unexpectedly, when CCR5 was lost from glia, morphine appeared to entirely protect neurons from Tat-induced toxicity. Maraviroc pre-treatment similarly eliminated neurotoxicity and attenuated neuronal increases in [Ca2+]i caused by Tat ± morphine. proBDNF/BDNF ratios were increased in conditioned media from Tat ± morphine-treated wild-type glia compared to CCR5-deficient glia. Exogenous BDNF treatments mimicked the pro-survival effect of glial CCR5 deficiency against Tat ± morphine.

Conclusions

Our results suggest a critical role for glial CCR5 in mediating neurotoxic effects of HIV-1 Tat and morphine interactions on neurons. A shift in the proBDNF/BDNF ratio that favors neurotrophic support may occur when glial CCR5 signaling is blocked. Some neuroprotection occurred only in the presence of morphine, suggesting that loss of CCR5 may fundamentally change signaling through the MOR in glia.

Keywords: Human immunodeficiency virus, Morphine, C-C chemokine receptor 5, Maraviroc, Brain-derived neurotrophic factor, NeuroHIV

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Background

Human immunodeficiency virus type 1 (HIV-1) remains a global epidemic [1]. Despite significant antiretroviral suppression of HIV-1 propagation in the periphery, limited penetration of combination antiretroviral therapy (cART) drugs through the blood-brain barrier [2, 3] as well as the early viral integration into the host genome cultivates a reservoir in which low levels of viral replication can be sustained in the central nervous system (CNS) [4–6]. Thus, HIV-1+ individuals are especially vulnerable to CNS injury, which afflicts as many as 50% of this population [7–9]. The neurological consequences of HIV-1 infection are known collectively as HIV-associated neurocognitive disorders (HAND). HAND presents as a spectrum of deficits ranging from mild or asymptomatic cognitive disorders to severe, HIV-associated dementia and includes a variety of cognitive, behavioral, and/or motor symptoms [10]. Postmortem findings in HIV-infected individuals, even those effectively treated with cART, often include signs of prominent CNS inflammation, such as increased numbers and/or activation of microglia and astroglia, perivascular inflammation, and leukocytic infiltration resulting in marked neuronal degeneration [11–13]. Notably, neuronal injury and alterations in signaling are not accompanied by direct viral infection of neurons [14–16]. Instead, microglia and macrophages are the major source of productive viral infection in the CNS [17–19]. Small numbers of astrocytes are infected and can produce toxic proteins that injure bystander neurons, but they have not been reliably shown to produce virus [20, 21]. These combined findings highlight the importance of glial impact on neurons, which is normally critical in maintaining proper neuronal activity and survival and suggests a mode of indirect injury that is a consequence of the innate CNS immune response to HIV.

HIV-1 infection and injection drug use are interlinked epidemics, due in large part to needle sharing and increased risky sexual behavior. Because heroin (diacetyl morphine) is widely abused and its active metabolite, morphine, is an opiate prescribed for pain syndromes experienced by HIV patients, we and others are interested in neurological interactions of HIV-1 and heroin/morphine. The comorbid effects of HIV and opiates are not trivial. HIV+ individuals who also abuse opiates demonstrate more severe neuropathology than those who do not, and these findings can translate to exacerbated and accelerated HAND [22–25]. The actions of morphine in this context occur primarily through the activation of μ-opioid receptors (MORs) expressed on glial cells. MOR activation results in the potentiation of HIV-induced release of pro-inflammatory factors (e.g., TNFα, IL-1β, IL-6, CCL5, and CCL2), as well as oxidative and nitrosative stress, mitochondrial dysregulation, elevated intracellular calcium levels, and excess extracellular glutamate (via restriction of astroglial glutamate uptake), all of which promote neurotoxicity [26–30]. Dysregulated release of inflammatory factors upon chronic exposure to viral proteins may recruit and activate more immune cells, propagating a cycle of increasing inflammation with significant downstream neuronal consequences.

One receptor vulnerable to the aforementioned dysregulation by HIV infection is C-C chemokine receptor 5 (CCR5). CCR5 is widely expressed on T lymphocytes, macrophages, microglia, and dendritic cells and plays a critical role in inducing migration of immune cells to sites of infection and injury in response to elevated levels of certain C-C-chemokine ligands (MIP-1α/CCL3, MIP-1β/CCL4, CCL5/RANTES) [31]. CCR5 and its ligands are upregulated during HIV infection, leading to excess activation of CCR5-expressing cells, including CNS microglia and astrocytes [32, 33]. A homozygous deletion of 32 base pairs in the CCR5 gene prevents its expression on the cell surface and confers improved but not absolute immunity to infection with R5-tropic strains of HIV-1 [34]. Individuals carrying the allele show slowed disease progression upon infection with HIV and less cognitive impairment [35–38]. Furthermore, maraviroc, a CCR5 antagonist with relatively high CNS penetrance [39], reduces microglial activation in the simian immunodeficiency virus-infected model to uninfected control levels and reduces the expression of several pro-inflammatory factors [40]. cART regimens that are supplemented with maraviroc improve the neurocognitive status of HIV+ patients and reduce CSF levels of TNFα [41, 42]. Morphine can alter CCR5 expression by monocytes and activated T cells, contributing to increased viral entry and replication, and excess CNS immune activation [43, 44]. We previously demonstrated that loss of CCL5, a CCR5 ligand, prevented widespread glial activation and reduced levels of another inflammatory ligand (CCL2), suggesting CCL5 may be an upstream activating signal that promotes the expansion of downstream pro-inflammatory responses [45]. The present studies investigate whether interrupting CCR5 signaling may protect neurons against the comorbid effects of HIV-1 and opiate exposure, apart from any effect of blocking HIV entry. We demonstrate using mixed glial-neuronal co-cultures that morphine potentiates Tat-induced neuronal death and that a loss of CCR5 expression on glial cells rescues neurons from such enhanced neurotoxicity. Surprisingly, morphine completely protected against Tat neurotoxicity in cultures with CCR5-null glia even though Tat by itself was still toxic. Levels of brain-derived neurotrophic factor (BDNF) are reduced in HIV+ individuals and by glycoprotein 120 (gp120), an HIV-1 envelope protein with neurotoxic properties [46, 47]. We found that the ratio of neurotrophic BDNF to its neurotoxic precursor (proBDNF) was altered in CCR5-null glia exposed to Tat and morphine co-treatment such that the environment favored neuronal support. BDNF also rescued neurons from Tat + morphine neurotoxicity in a manner similar to the loss of glial CCR5 expression. Overall, we postulate that CCL5/CCR5 signaling is a point of convergence for opiate-Tat interactions within the inflammatory milieu of the HIV-infected CNS. Blocking CCR5 appears to enhance neuroprotection, perhaps by increasing BDNF-related neuroprotection. Inactivation or loss of CCR5 may also change heterologous interactions between MOR and CCR5 related to toxicity and protection.

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Methods

Experiments were conducted in compliance with procedures reviewed and approved by the Virginia Commonwealth University Institutional Animal Care and Use Committee.

CCR5-null mice

Transgenic mice in which there has been a loss in CCR5 expression were obtained from Jackson Labs (Bar Harbor, ME) and maintained as homozygous breeding trios. Briefly, the insertion of a neomycin resistance gene to replace the single coding exon has resulted in the constitutive loss of CCR5 expression. To confirm the loss of CCR5, tail snips and harvested glial cells were digested and DNA was isolated as per the instructions of the manufacturer (KAPA Mouse Genotyping; KAPA Biosystems; Wilmington, MA). Primer sets designed to identify the neomycin resistance gene as well as the CCR5 coding exon were obtained from Jackson Labs. Polymerase chain reaction was carried out to confirm the presence of the neomycin resistance gene and the absence of the CCR5 sequence (Fig. 1a). Because the knockout is global, CCR5-deficient glia or neurons are reconstituted into co-cultures with wild-type neurons or glia, respectively, to study effects of mutations in a single cell type. Mice of the C57Bl6/J background strain were used as wild-type controls. The CCR5-null mice displayed no overt signs of illness or problems during development, and litters occur in a similar frequency and size as the C57Bl6/J strain.

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Fig. 3

Neurotoxic effects of HIV-1 Tat and morphine are reversed by loss of glial CCR5. a In C57Bl6/J wild-type co-cultures, Tat is neurotoxic (*p = 0.001 vs control), and co-exposure to morphine enhanced Tat-induced toxicity over a 72-h period (**p < 0.001 vs control, p < 0.05 vs Tat). This interaction was blocked by pretreatment with naloxone, a broad-spectrum opioid receptor antagonist. Naloxone or morphine by themselves had no effect on neuronal survival (n = 4–8). bd To explore the role of CCR5 in mediating neurotoxic interactions between Tat and morphine, co-cultures in which glia, neurons, or both were deficient in CCR5 were established. b In co-cultures where glia are CCR5-null but neurons are wild-type, exposure to Tat by itself still led to significant neurotoxicity (*p < 0.001 vs control); however, the morphine-enhanced neurotoxicity seen in wild-type cultures was eliminated. In fact, morphine co-treatment entirely abolished Tat toxic effects, restoring neuronal survival to control levels. Pre-treatment with naloxone re-established Tat toxicity, suggesting that actions at the μ-opioid receptor mediate this neuroprotection (n = 4–8). c In co-cultures where neurons are CCR5-null but glia are wild-type, the survival curves are similar to wild-type co-cultures (n = 5). d In co-cultures between CCR5-deficient glia and neurons, the survival curves are similar to co-cultures where only glia were CCR5-deficient (n = 5). Overall, the results from the CCR5-deficient co-cultures suggest an important role for glial CCR5 in the neurotoxic interactions of HIV-1 Tat and opiates that act at the MOR

Constitutive CCR5 loss affects neuronal survival differently than short-term CCR5 blockade

Long-term, constitutive knockout of CCR5 might result in compensatory changes during development that alter neuronal sensitivity to Tat or morphine. To explore this hypothesis, we used a paradigm where the length of CCR5 blockade was controlled using the CCR5 antagonist maraviroc (Fig. 4). Here we show that a relatively long-term, 2-week incubation with maraviroc (LT-MVC) mimicked the effects on neuron survival seen in co-cultures with CCR5-deficient glia. That is, morphine–Tat interactions that enhance neurotoxicity were negated, and morphine additionally protected completely against Tat neurotoxicity. However, shorter-term exposure to maraviroc, starting immediately before Tat and morphine were co-administered (ST-MVC), had much more limited effects. Short-term maraviroc treatment reduced the interactive effects of Tat and morphine; however, it did not reduce the neurotoxicity of Tat itself irrespective of whether morphine was present.

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Fig. 4

Constitutive CCR5 loss affects neuron survival differently than short-term CCR5 blockade. Maraviroc was applied to the co-culture to compare the effects of a CCR5 antagonist to a genetic knockout. Maraviroc was applied in two different paradigms that permitted us to manipulate the time period of CCR5 loss. The first was a short-term pre-treatment immediately before adding Tat and/or morphine (ST-MVC; n = 4); in this paradigm, maraviroc was on the cultures for a period of 72 h, during the time of Tat and morphine treatments. The second was a longer-term exposure starting 3 days after glia were plated and continuing for the entire 2-week duration of the experiment with replacement of the media every 48 h (LT-MVC; n = 4). The Tat + morphine + LT-MVC survival curve matched that of cultures with CCR5-deficient glia. The Tat + morphine + ST-MVC eliminated the morphine–Tat interaction and only showed a trend towards eliminating Tat toxicity (p = 0.08 vs control). This set of studies suggests that compensatory effects occur over time with CCR5, which dramatically alter morphine–Tat interaction and neurotoxicity

Maraviroc protects against acute increases in neuronal [Ca2+]i

Disruptions in calcium homeostasis are a common response to neurotoxic signals. As an indicator of how maraviroc affected neuronal function, we performed ratiometric imaging with Fura-2 to assess changes in the [Ca2+]i level of individual neurons over a 60-min period of treatment with Tat ± morphine ± maraviroc. Tat ± morphine treatments significantly increased [Ca2+]i by 15 min, and this was maintained for the duration of the trial (Fig. 5). Importantly, even at this early time point, co-exposure to maraviroc blocked the changes, suggesting that reduced CCR5 signaling had the effect of maintaining normal [Ca2+]i levels and stabilizing neuronal function.

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Fig. 5

Maraviroc reduces Tat-mediated increases in [Ca2+]i. Intracellular calcium levels were assessed in neuron-glia co-cultures by ratiometric imaging of Fura-2. A series of images were taken every 15 min for 1 h to track the response of individual neurons. Initial [Ca2+]i measurements were taken prior to any treatment at the 0-min time point. Tat and/or morphine treatments were applied 10 min prior to the second reading (marked by arrow). There were significant effects for both time (p = 0.001) and treatment (p = 0.009) when assessed by repeated measures ANOVA. Treatment with Tat or Tat + morphine (marked by asterisk) led to significant increases in [Ca2+]i, as indicated by increased F340/F380 ratios. Pre-treatment with maraviroc blocked the Tat + morphine-induced increase (p = 0.008; Duncan’s post hoc test) as well as the Tat-mediated response (p = 0.054). Morphine and maraviroc alone did not significantly alter [Ca2+]i. Results are presented as percent of the control F340/F380 ratios for each concurrent time point (n = 3 independent experiments)

BDNF protects against Tat toxicity and HIV-1 Tat and morphine interactions

BDNF was applied to co-cultures to see if it would promote the survival of striatal neurons co-exposed to Tat + morphine. Co-cultures of wild-type neurons and glia were treated with BDNF concurrently with combined Tat and morphine for 72 h. Time-lapse analysis demonstrated that exogenous BDNF was partially protective against the neurotoxic effects of Tat alone, which was not the case for CCR5 deficiency. However, similar to CCR5 deficiency, BDNF reversed the combined neurotoxic effects of Tat + morphine (Fig. 6). BDNF alone at this concentration did not increase the survival of neurons in untreated, wild-type cultures; survival of both was over 90%

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Fig. 6

Exogenous BDNF protects against Tat + morphine treatment. Wild-type, mixed glial-neuronal co-cultures were treated with BDNF in conjunction with Tat or Tat + morphine co-treatment (represented by dotted survival curves). Tat alone was toxic compared to no treatment (*p < 0.05), and Tat + morphine co-treatment was significantly more toxic than Tat treatment alone (**p < 0.0001). BDNF applied for 72 h was entirely protective against Tat and morphine co-exposure, reducing toxicity to control levels (n = 4). BDNF was partially protective against Tat alone. Survival of neurons treated with Tat + BDNF was not significantly different from either controls or cultures treated with Tat alone (#) (n = 4)

Loss of glial CCR5 expression produces a shift in proBDNF/BDNF levels

Based on our prior studies demonstrating significant but reversible reduction in glial production of mBDNF after exposure to HIV-infected supernatant ± morphine [48], as well as other studies where HIV-1 gp120 altered BDNF processing [47], we analyzed levels of both mBDNF and its precursor, proBDNF, which binds p75NTR to activate cell death pathways. We also compared changes in their ratios. Wild-type and CCR5-deficient glial cultures were treated with Tat (Fig. 7a), morphine (Fig. 7b), or concurrent Tat and morphine (Fig. 7c) and harvested at 6- and 24-h time points for protein analysis. After Tat treatment, mBDNF levels measured by ELISA were unchanged from levels in media in untreated control cultures at both 6 h and 24 h (Fig. 7a (i, iv)). Tat by itself significantly reduced proBDNF in wild-type cultures versus control cultures at 6 h, with a strong trend towards reduction in both wild-type and CCR5-deficient cultures at 24 h (Fig. 7a (ii, v)). Morphine by itself reduced only proBDNF and only in CCR5-deficient cultures (Fig. 7b (ii, v)). The combination of Tat and morphine showed a strong trend to reduce mBDNF in wild-type cultures at 6 h (Fig. 7c (i)) and was the only treatment to affect mBDNF. The ratio of proBDNF to mBDNF has been used as one index of relative neurotrophic support [47]. CCR5 deficiency strongly reduced this ratio by over twofold at 6 h in cells treated with Tat and morphine (Fig. 7c (iii)), and the protection of neurons in the CCR5-deficient glial environment may reflect the relative increase in mBDNF. A similar trend noted at 24 h was noted (p = 0.17) (Fig. 7c (vi)). The only other significant change in this ratio was a much smaller, but still significant, decrease in CCR5-deficient cultures treated with Tat (Fig. 7a (vi)).

 

 

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Jawahar Raina

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