Cross-talk between microglia and neurons regulates HIV latency.

Despite effective antiretroviral therapy (ART), HIV-associated neurocognitive disorders (HAND) are found in nearly one-third of patients. Using a cellular co-culture system including neurons and human microglia infected with HIV (hμglia/HIV), we investigated the hypothesis that HIV-dependent neurological degeneration results from the periodic emergence of HIV from latency within microglial cells in response to neuronal damage or inflammatory signals. When a clonal hμglia/HIV population (HC69) expressing HIV, or HIV infected human primary and iPSC-derived microglial cells, were cultured for a short-term (24 h) with healthy neurons, HIV was silenced. The neuron-dependent induction of latency in HC69 cells was recapitulated using induced pluripotent stem cell (iPSC)-derived GABAergic cortical (iCort) and dopaminergic (iDopaNer), but not motor (iMotorNer), neurons. By contrast, damaged neurons induce HIV expression in latently infected microglial cells. After 48-72 h co-culture, low levels of HIV expression appear to damage neurons, which further enhances HIV expression. There was a marked reduction in intact dendrites staining for microtubule associated protein 2 (MAP2) in the neurons exposed to HIV-expressing microglial cells, indicating extensive dendritic pruning. To model neurotoxicity induced by methamphetamine (METH), we treated cells with nM levels of METH and suboptimal levels of poly (I:C), a TLR3 agonist that mimics the effects of the circulating bacterial rRNA found in HIV infected patients. This combination of agents potently induced HIV expression, with the METH effect mediated by the σ1 receptor (σ1R). In co-cultures of HC69 cells with iCort neurons, the combination of METH and poly(I:C) induced HIV expression and dendritic damage beyond levels seen using either agent alone, Thus, our results demonstrate that the cross-talk between healthy neurons and microglia modulates HIV expression, while HIV expression impairs this intrinsic molecular mechanism resulting in the excessive and uncontrolled stimulation of microglia-mediated neurotoxicity.

Introduction

Approximately 30% of the HIV-infected individuals on combination anti-retroviral treatments (cART) display symptoms of cognitive impairment and central nervous system (CNS) pathology, a syndrome known as HIV-associated neurocognitive disorders (HAND) [1-5]. While the incidence of outright HIV-associated dementia (HAD), which is due to the neurological damage induced by actively replicating HIV, has sharply declined due to effective cART, the prevalence of the milder HAND conditions remains high [5, 6], and is expected to increase further as the HIV infected population ages [7]. In the US, 10–15% of HIV patients acknowledge METH use [8], which exacerbates the effects of HIV infection in the CNS [9, 10] due to a combination of neurotoxic effects and the enhancement of HIV replication in microglia [11-14]. There is compelling evidence that HIV exacerbates age-associated cognitive decline and diminishes neuropsychological performance across multiple cognitive domains [7, 15].

HIV-1 replication in the CNS is initiated from invading monocytes and CD4+ T cells, and then spreads to microglial cells and, arguably, astrocytes within the brain parenchyma [16-27]. Definitive evidence that HIV replicates in myeloid lineage cells within the CNS comes from the observation that HAD patients harbor macrophage-tropic HIV-1 variants that grow selectively in the CNS [21, 28–31]. A consequence of HIV-1 replication in longer-lived cell types in the brain, including microglia, is that virus is depleted more slowly in the cerebrospinal fluid (CSF) than virus in the peripheral circulation after the initiation of therapy [32]. Minimal viral replication still persists in the CNS [33, 34], especially in microglia and perivascular macrophages [35], in part because not all anti-HIV drugs are able to cross the blood-brain barrier with high efficiency. However, latent infections of microglial cells and astrocytes also appear to contribute to the long-term persistence of HIV in the CNS [36-44].

Microglia constitute the first barrier of the innate immune response in the CNS [45]. They constantly survey the brain parenchyma to detect physiological changes and then migrate to regions of damage, where they become activated [46]. Normally, the activation of microglia in response to inflammatory stimuli is characterized by a transition from a resting state (M0 cells) to an activated proinflammatory phenotype (M1 cells). It is generally believed that over-activated microglia exacerbate neuronal injury through the synthesis and secretion of proinflammatory and cytotoxic factors [47, 48]. Therefore, in NeuroHIV, microglia-mediated neuronal injury appears to result from excitotoxicity [49, 50], which disrupts the intrinsic molecular mechanisms that control ion homeostasis and energy production in neurons [51-53].

HIV-1 neuropathology arises because of the combined neurotoxic effects of the viral proteins and exaggerated inflammatory responses by microglial cells. The HIV proteins Tat [54-67], gp120 [66, 68–71], Vpr [72, 73], and Nef [74-78] can directly induce neuronal damage. Additionally, microglia can contribute to neurodegeneration through the release of cytokines and toxins that damage neurons and astrocytes [79-81]. The natural control mechanisms preventing over-activation of microglial cells are likely to be impaired as a consequence of HIV infection. HIV-infected microglia respond vigorously to proinflammatory signals and produce an excess of cytokines [37, 39]. Importantly, neuronal dysfunction does not correlate with the number of HIV-infected cells or viral antigens in CNS [82, 83], but rather with elevated inflammatory cytokine levels. Elevated TNF-α mRNA levels in microglia and astrocytes [82, 84] and high levels of IL-1β and TNF-α are seen in the CNS of patients with HAD [85, 86]. Similarly, increased IL-6 and IL-8 levels are found in the brains of HIV-1 infected patients [4, 87]. Immune-activated HIV-infected, brain-infiltrating macrophages and resident microglia release high levels of neurotoxic cytokines such as TNF-α and IL-1β [88].

Co-culture systems have been used extensively to study neuron-glia interactions and, because of the ability to measure the contribution of each cell type individually, are highly valuable tools to understand neuronal-microglial cross-talk [89-96]. Here, we developed a series of co-culture systems to study the impact of HIV expression in infected microglial cells on their interactions with neurons. Using LUHMES-derived neurons, which have been extensively use to study neurodegenerative processes because of their dopaminergic-like features [97], and iPSC-derived primary neurons, in co-culture with hμglia/HIV as a platform to study the effect of neuronal and microglial interactions on the regulation of HIV expression in the CNS, we found unexpectedly that healthy neurons induce HIV silencing in microglia and prevent spontaneous HIV reactivation in latent hμglia/HIV cells. By contrast, damaged neurons increased HIV expression in microglial cells. In addition, we found that METH, which can induce HIV expression in hμglia/HIV cells in a σ1R-dependent manner, sensitizes microglial cells to proinflammatory agents and exacerbates neurodegeneration in the neuronal-microglial co-cultures. We conclude that HIV expression disrupts the normal interplay between microglia and neurons and thereby induces neurodegeneration.

Results

Establishment of a co-culture system between LUHMES-derived neurons and μglia/HIV cells

We have shown previously that HIV readily establishes latency in immortalized human microglial cells [36-39]. In order to understand how neuronal and microglial cross-talk regulates HIV latency in the brain, we first established a co-culture system between LUHMES-derived neurons and human microglia infected with HIV reporter viruses (). Undifferentiated Lund human mesencephalic (LUHMES) cells, obtained from ATCC (CRL-2927) and originally developed and characterized at Lund University (Lund, Sweden) [98, 99], carrying a vector expressing red fluorescent protein (RFP) to allow cell visualization were kindly provided by Dr. Stefan Schildknecht (Konstanz, Germany) [100]. The cells were expanded in neuronal growth medium (NGM) for 4 days. After 4-days, NGM was replaced by a modified neuronal differentiation medium (mNDM), to inhibit the expansion of neuronal precursors and induce differentiation into dopaminergic neurons [99]. After differentiation for 1 day, immortalized human microglial cells (hμglia) infected with an HIV vector carrying a green fluorescence protein (GFP) reporter (e.g. HC69 cells) [36, 37, 39], were plated on top of the neurons, typically at a ratio of 3 microglia per 25 neurons (). The co-cultures could be maintained for up to 96 h and HIV expression and neuronal damage quantitatively and qualitatively evaluated using molecular, biochemical, flow cytometric, and morphologic endpoints.

Establishment of a neurons-hμglia/HIV co-culture system.

The time line is indicated by the large black arrow, with the corresponding day number shown in red. Undifferentiated, neuron precursor cells LUHMES/RFP are plated and allowed to expand for 2 days prior to transferring to the experimental wells at 500,000 neurons per well of a six-well plate at day 0. After 2 days, the neuronal differentiation is initiated by adding modified neuronal differentiation medium (mNDM). After differentiation for 1 day, 60,000 immortalized human microglial cells (hμglia/HIV) carrying a single round HIV construct with a GFP reporter (diagrammed, bottom left) are added. The viability of the neurons and the expression levels of HIV are monitored for 3 days.

A critical technical step in developing the co-culture system was to develop a culture medium that maximized survival for both the neurons and the microglia (). We found that a low concentration of fetal bovine serum (FBS 0.2%), was optimal to maintain microglial viability, but higher concentrations of FBS were toxic for the neurons. To further preserve microglial viability, the FBS was enriched with 1X insulin-transferrin-sodium selenite supplement in the final mNDM formulation.

Neurons silence HIV in a density-dependent manner

We have previously shown that HC69 cells undergo spontaneous HIV reactivation due to autocrine expression of TNF-α [39]. HIV reactivation could be potently repressed by addition of the glucocorticoid receptor agonist dexamethasone (DEXA), which blocked both HIV and proinflammatory cytokine production [39]. To determine the effect of neurons on spontaneous HIV expression, a mixed population of HC69 (hμglia/HIV cells) containing a combination of both GFP+ and GFP- cells, were co-cultured with LUHMES-derived neurons, as described above. Cultures were established at three different neuronal densities: 100 neurons per mm2, (a ratio 10:6 of neurons:HC69 microglial cells), 300 neurons per mm2 (25:6, neurons:HC69), and 600 neurons per mm2 (50:6, neurons:HC69). HIV expression was then evaluated after 24 h, using microglia cultured without neurons as a control. Flow cytometry and fluorescence microscopy () analyses demonstrated that HIV expression declines as the ratio of neurons:microglia is increased. The impact of neurons on HIV expression was evaluated over a time course ranging from 0 to 36 h (). At 36 h, the percent of HIV expression (Y-axis) was reduced significantly from ~45% in HC69 cells cultured without neurons (red line and squares) to ~25% when 100 neurons per mm2 were present (a 10:6 ratio). At higher ratios there was a further reduction to ~8%. There was no significant neurotoxicity in these co-cultures, with neuron viability remaining above ~90%, as measured by the resazurin assay ().

LUHMES-derived neurons inhibit HIV expression.

60,000 HC69 (hμglia/HIV) cells were plated in the absence or presence of increasing densities of LUHMES-derived neurons (Cells/mm2). The level of HIV expression was evaluated after 24 h by flow cytometry and fluorescence microscopy. (A) Flow cytometry profiles from representative single cultures (top) and microscopy (bottom). In the histograms GFP+ cells are indicated in bright green. Microglia were identified by phase contrast microscopy and are outlined by the white contours on the micrographs. (B) Progressive inhibition of HIV expression (Y-axis) at increasing neuronal densities during a time-course of 36 h (X-axis). The error bars represent the SD of n = 3 independent experiments. (C) Resazurin assay to evaluate neuronal viability. The resazurin reduction values (Y-axis) were normalized to the control culture of neurons alone. Each colored symbol represents one experiment. There was no statistically significant (N.S.) differences in cell viability in these experiments.

The gating strategy used to measure GFP expression in the microglial cells (CD14+ cells) by flow cytometry, and avoid counting neurons, is shown in . Gating on hμglia in the FSC vs. SCC scatter plot resulted in ~100% CD14+ cells and can be used instead of staining for CD14.

In control experiments, to rule-out the possibility that the observed decreased in GFP expression in HC69 cells exposed to neurons was due to microglia-specific cell death or toxicity, we measured both the growth rate and the level of toxicity of the microglial population in the presence and absence of neurons. After 24 h co-culture, there was no statistically significant difference in cell growth or toxicity level (Y-axis) between microglia cultured in the presence of neurons and control cultures at any of the three neuronal densities used (X-axis) ().

iPSC-derived GABAergic cortical and dopaminergic neurons induce HIV silencing

To demonstrate that primary neurons are also capable of silencing HIV, HC69 cells were co-cultured with iPSC-derived GABAergic cortical (iCort), dopaminergic (iDopaNer), or motor (iMotorNer) neurons at a ratio of 50 neurons:6 microglia (, which was the proportion of LUHMES cells to microglia that caused the greatest suppression of HIV expression. As shown in , the cortical and dopaminergic neurons, but not motor neurons, attenuated HIV expression in HC69 cells, as shown by both fluorescence microscopy () and flow cytometry (). HIV expression in HC69 cells was reduced from 52.34 ± 6.09% to 16.36 ± 3.44% (p < 10−7), after co-culture with iPSC-derived cortical neurons. Similarly, dopaminergic neurons decreased HIV expression in HC69 cells to 26.34 ± 8.84% (p < 10−7). HIV expression was not significantly inhibited by motor neurons ().

iPSC-derived neurons repress HIV expression.

(A) 60,000 hμglia/HIV HC69 cells were plated in the presence of 0.5 x 106 LUHMES-derived neurons (as positive control) or 0.5 x 106 iPSC-derived GABAergic cortical (iCort), dopaminergic (iDopaNer) or motor neurons (iMotorNer). HIV expression was evaluated after 24 h by fluorescence microscopy. Microglia identified by phase contrast microscopy are outlined by the white contours. (B) Flow cytometric analysis of microglial cell GFP expression. The p-values of pair-sample, Student’s t-tests comparing the microglial cells cultured alone or in the presence of neurons are shown. Individual independent experiments are color coded (n = number of independent samples). N.S.: non-significant. (C) Identification of differentiation of iPSC-derived neurons. Super-imposed images of DAPI stained nuclei (blue) and Alexa-Fluor 488 stained neuronal antigens (green) are shown. Neurons were stained with antibodies against GAD65/67, DAT, and AchE and Alexa Fluor 488-conjugated anti-rabbit secondary antibody.

To verify the differentiation of the neurons, iPSC-derived neurons were analyzed by immunocytochemical detection using phenotype-specific antibodies against GABAergic, dopaminergic, and cholinergic neuronal markers, respectively, glutamate decarboxylase (GAD65/67), dopaminergic transporter (DAT), and acetylcholinesterase (AchE) (). The pan-neuronal marker beta-TUJ [101, 102] (), dendritic marker MAP2 [103] (), and neuronal receptor marker CXCR3 [104] () were used as positive controls. As expected, the three iPSC cell lineages, as well as LUHMES-derived neurons, stained positive for these proteins. The microglial marker CD11b/c [105-107] was used as a negative control and, as expected, all the neurons were CD11b/c-negative (). LUHMES-derived neurons stained positive for all three phenotype-specific markers GAD65/67 (), DAT (), and AchE (). iPSC-derived GABAergic cortical neurons strongly expressed GAD65/67, a specific marker of inhibitory GABAergic neurons [108], and only expressed DAT or AchE at low levels ( and ). iPSC-derived dopaminergic neurons strongly expressed DAT, a dopaminergic neuron-specific marker [109, 110], and had reduced levels of AchE, and very low levels of GAD65/67 ( and ). iPSC-derived motor neurons were positive for AchE [111-113] and practically negative for DAT or GAD65/67 ( and ). The relative level of expression of these neuronal markers are summarized in .

Only neurons are able to induce efficient HIV silencing in microglial cells

In order to address the specificity of neurons in inducing HIV latency in infected microglial cells, HC69 cells were co-cultured in the absence or presence of two non-neuronal cell lines, 293T cells and primary human foreskin fibroblasts (HFF) (). After co-culture for 24 h, 293 T cells and HFF failed to significantly decrease HIV expression in HC69 cells, as measured by flow cytometry (profiles (). Quantitation of three similar experiments () showed that a slight decrease in HIV expression induced by HFF cells was not significant. Similarly, there was no significant toxicity detected in these co-cultures (), as measured by the resazurin assay. DEXA, a known inducer of HIV latency in microglial cells, was used as a positive control [39].

Similarly, neither iCort nor LUHMES neuronal cells were able to significantly reduce HIV expression in THP-1/HIV (A3) cells, a monocytic cell line [37], or Jurkat/HIV (2D10), a T-cell line [114] (). The ERK kinase inhibitor U0126, which has been shown to block HIV expression [115], was used as positive control in these experiments.

Human iPSC-derived neurons can induce HIV latency in infected human primary microglia

In order to verify that primary neuronal cells are also able to induce HIV latency in infected primary microglial cells, we co-cultured both HIV-infected human primary microglial cells and iPSC-derived microglial cells with either primary neurons or iPSC-derived GABAergic cortical neurons. The primary and iPSC-derived microglial cells were infected with VSVG-HIV-GFP viruses using the protocols we established for immortalized human microglial cells [36-39]. To confirm latency in the primary microglial cells, the cells were cultured in presence DEXA, to induce HIV silencing, and reactivated with TNF-α, to induce any silenced proviruses in the culture (). Both primary microglial (MG) () and iPSC-induced microglial (iMG) () cells can be readily infected with VSVG-HIV-GFP viruses. Approximately 40% of the MG cells and 30% of the iMG cells expressed HIV (GFP) at 72 h post infection (hpi). After treatment with DEXA for 24 h, virtually none of the infected MG or iMG cells expressed HIV. Treatment of the infected cells by TNF-α for 16 h increased the proportion of GFP+ cells in the culture by 25 to 50%.

Human primary and iPSC-derived microglia infected with VSVG-HIV-GFP can establish latent infections.

(A) Human primary microglial (MG) cells. (B) iPSC-derived microglial (iMG cells). Each cell type was infected with VSVG-HIV-GFP viruses for 72 h and GFP-expressing cells visualized by fluorescence microscopy. The infected cells were then either treated with DEXA (1 μM) for 24 h or with TNF-α (200 pg/mL) for 16 h and the proportion of GFP-expressing cells measured by fluorescence microscopy.

The availability of HIV-infected primary MG and iMG cells permitted us to perform co-culture experiments in an entirely primary cell context using human primary neurons (Primary HN) and iCort neurons (). The cells were cultured at a ratio of 50 neurons:6 microglia. Because of the limited numbers of cells that were available this experiment was performed semi-quantitatively using fluorescent microscopy. Nonetheless it is evident that Primary neurons were able to reduce HIV expression in both primary MG () and iMG cells () by more than 90%. The iCort neurons were able to reduce HIV expression both primary MG () and iMG cells () by more than 50%.

Primary neurons silence HIV expression in primary microglia.

(A) Human primary microglial (MG) cells. (B) iPSC-derived microglial (iMG cells). For each cell type, 60 x 103 cells were plated in the absence or presence of 0.5 x 106 human primary neurons (Primary HN) or iPSC-derived GABAergic cortical neurons (iCort). HIV expression was evaluated after 24 h by fluorescence microscopy. Microglia identified by phase contrast microscopy are outlined by the white contours. Healthy neurons prevent spontaneous HIV reactivation in GFP- cells.

We have reported previously that HIV expression increases spontaneously in cultured hμglia/HIV cells due to autocrine production of TNF-α, potentially a consequence of the lack of neuronal inhibitory signals [39]. Because neurons were able to inhibit HIV expression in HC69 cells, at least in the short-term (Figs ), we investigated whether neurons would also prevent spontaneous viral reactivation. A population of GFP- cells were prepared by cell sorting from the mixed HC69 cell population, and co-cultured in the absence or presence of LUHMES-derived neurons. Viral transcription was then evaluated using flow cytometry and fluorescence microscopy ().

Neurons prevent HIV emergence from latency.

(A) Flow cytometry profiles of representative single cultures. hμglia/HIV HC69 cells were sorted into a GFP- cell population and cultured in the presence or absence of neurons. GFP expression was measured after 24 h or 72 h. (B) Microscopy of HC69 cells unexposed or exposed to neurons for 24 or 72 h. Microglial cells are outlined by a white dashed-line. (C) Quantitation of GFP expression. (D) Resazurin assay to evaluate neuronal viability. The resazurin reduction values (Y-axis) plotted are referenced to the control culture (neurons only), set at 100%. MPP+ was used a positive control for resazurin reduction. For both (C) and (D), the p-values of pair-sample t-tests of multiple experiments (n = number of independent samples) comparing the unexposed vs. the exposed cells are shown. N.S.: non-significant. Individual experimental series are color-coded.

In the absence of neurons, there was a 13% increase in GFP+ cells (from ~9% to ~22%) after 72 h, indicating the emergence of HIV from latency. In the presence of neurons, viral rebound was inhibited and GFP levels remained at ~8% as seen by the representative flow cytometry profiles () and microscopy (). Quantitation of multiple experiments () showed that in the absence of neurons, HIV expression increased, on average, from 7.73 ± 2.59% at 24 h to 26.40 ± 6.79% (p < 10−3) at 72 h, whereas in the presence of neurons, HIV expression remained at basal levels and did not vary significantly (N.S.). Neuronal viability, as measured using the resazurin reduction method, was unaffected after 24 h of co-culture with HC69 cells. There was a significant decrease in neuronal viability to 54.00 ± 10.20% (p < 10−3) at 72 h (). For comparison, the neuronal toxin MPP+ decreased neuronal viability to 28.40 ± 11.89% (p < 10−3). The increased neuronal toxicity at 72 h () maybe due to the partial inhibition of HIV expression, which may be sufficient to induce neuronal damage.

In order to rule-out the possibility that the observed block to HIV spontaneous reactivation in HC69 cells exposed to neurons was due to microglia-specific cell death or toxicity, we measured both the growth rate and the level of toxicity of the microglia population at 24 and 72 h. At both time-points, no statistically significant (N.S.) difference was observed in cell growth or toxicity level (Y-axis) between microglia cultured in the absence of neurons and microglia cultured in the presence of neurons (X-axis) ().

Neuronal damage induces HIV expression

In the previous experiments, we showed that both LHUMES-derived and primary neurons can block HIV expression in microglial cells. To evaluate whether neuronal damage could reverse this repressive signal, HC69 cells sorted into GFP− and GFP+ subpopulations and co-cultured for 24 h with either healthy neurons or neurons damaged by treatment with 0.05% trypsin [36] (). Flow cytometry analysis of GFP expression in CD14+ cells from a representative experiment showed that, as expected, co-culture with healthy neurons slightly decreased HIV expression in the GFP− cells from ~15% to ~10% (). By contrast, co-culture with damaged neurons had the opposite effect and increased GFP expression to ~19%. The healthy neurons strongly decreased HIV expression in GFP+ cells (from ~78% to ~34%), whereas damaged neurons slightly increased it (from 78% to ~90%). Quantitation of multiple independent experiments () indicated that healthy neurons reduced HIV expression in GFP− cells from 13.99 ± 3.14% to 11.05 ± 2.50% (p < 10−3), whereas damaged neurons increased it up to 23.28 ± 1.68%; p < 0.05). In contrast, healthy neurons decreased HIV expression in GFP+ cells (from 77.03 ± 9.84% to 44.99 ± 8.98% (p = 0.003)), whereas damaged neurons had no effect (88.11 ± 7.52%; N.S.).

Effect of healthy neurons vs. damaged neurons on HIV expression.

(A) Representative flow cytometry profiles. hμglia/HIV HC69 cells were sorted into GFP- and GFP+ cells. Each population was expanded for 48 h prior to collection and co-cultured with either healthy neurons or damaged neurons at a ratio of 50:6. GFP (X-axis) and CD14 (Y-axis) expression, and the percentage of GFP+ cells that were CD14+ is shown. Isotype controls for the anti-CD14 antibody were performed for both the GFP+ and GFP- populations (left). (B) Quantitation of GFP expression. 60 x 103 HC69 cells were co-cultured in the presence of an increasing number of healthy (H) neurons in the absence damaged (H) neurons (X-axis) for 24 h prior to measuring GFP expression (Y-axis) (left). In parallel experiments, microglial cells were co-cultured with increasing numbers of healthy (H) neurons in the presence of 500 x 103 damaged (D) neurons (right). (C) Quantitation of GFP expression. 60 x 103 HC69 cells were co-cultured in the presence of 500 x 103 total neurons at the indicated ratios of damaged (D) to healthy (H) neurons (X-axis) for 24 h prior to measuring GFP expression (Y-axis). Diamonds of similar color represent an individual experimental series. (n = number of individual samples). The p-values of paired-sample t-tests comparing the unexposed vs. exposed cells are shown. N.S.; non-significant.

To further investigate the effect of damaged neurons in modulating HIV expression in infected microglia, we systematically varied the ratio of healthy and damaged neurons. As shown in and , increasing the proportion of healthy neurons resulted in the progressive silencing of HIV and decreased from 44.14 ± 1.82% to a minimum of 11.19 ± 0.87%. By contrast when 0.6 x 105 HC69 cells were co-cultured in the presence of 0.5 x 106 damaged LUHMES-derived neurons, HIV expression increased from 44.14 ± 1.82% to 65.94 ± 4.06% (p = 0.0011). The inductive effect of the damaged neurons was ameliorated by the addition of healthy neurons. For example, when the co-cultures included 0.5 x 103 damaged and 0.3 x 106 healthy neurons, HIV expression decreased to 46.19 ± 0.41%. Similarly, when the co-cultures included 0.5 x 103 damaged and 0.5 x 106 healthy neurons, HIV expression decreased to 29.15 ± 7.88%.

In a complementary set of experiments (), 0.6 x 105 HC69 cells were co-cultured for 24 h with different ratios of damaged (D) and healthy (H) neurons at a fixed concentration of 0.5 x 106 neurons. In the presence of 0.5 x 106 damaged neurons, HIV expression increased from 34.31 ± 4.55% to 59.65 ± 9.37% (p = 0.026), consistent with the previous results. As the proportion of healthy neurons in the co-culture increased and the number of damaged neurons decreased, HIV was progressively silenced. For example, at a ratio of 3:2 damaged to healthy neurons, HIV expression remained unchanged (32.76 ± 3.82%) compared to the control microglial cells (Day 0). At a ratio of 2:3 damaged to healthy neurons, HIV expression significantly decreased 24.97 ± 4.66% (p = 0.040). At a 1:4 ratio, HIV expression decreased to 13.85 ± 2.31% (p = 0.027), which was close in value to 10.99 ± 2.06% (p = 0.025) seen in the absence of damaged neurons. Thus, damaged neurons present microglial activation signals that enhanced HIV expression.

HIV expression rebounds in longer-term cultures with neurons

We next examined the effect of neurons on HIV expression in HC69 cells over a culture period of 4 days. A co-culture of LUHMES-derived neurons and GFP+ HC69 cells was established, and HIV expression and neuronal viability were monitored after 24, 72, and 96 h by microscopy and flow cytometry. As described above (Figs ), neurons induced HIV to enter latency in a significant population of HC69 HIV-expressing cells after 24 h, as demonstrated by reductions in the number of green, GFP+ cells (). At 24 h, neuronal viability, as measured by resazurin reduction, was comparable to time zero (; ~100%). After 72 h, the number of GFP+ cells increased from ~30% to ~50% (), and this correlated with a reduction of neuronal viability (resazurin ~65%) and the relative proportion of live neurons counted (down to ~50%). By 96 h, the proportion of GFP+ cells increased reaching ~60% (), while resazurin reduced the proportion of neurons to ~45% and percent of live neurons to ~30% ().

HIV expression rebounds in microglia exposed to neurons.

(A) Super-imposed phase contrast and fluorescence images of HC69 GFP+ cells exposed to neurons for 24, 72, or 96 h. Microglial cells are outlined by the white contours. (B) Quantitation of GFP expression (red), relative resazurin reduction (blue), and relative live neuron counts (purple), Y-axis, vs. Time, X-axis. Error bars: standard deviation (n = 3).

HIV expression in microglial cells induces neuronal damage

In order to demonstrate that the neuronal damage in the longer-term co-culture experiments was, at least in part, the result of the HIV expression, LUHMES-derived neurons were co-cultured (10:1 ratio) in the absence or presence of either C20 cells (the uninfected parental cell line) or HC20 cells (a mixed population of C20 cells infected with the HIV reporter) [36, 37]. After 96 h, neurons in the co-cultures were detected by immunocytochemistry with either anti-beta-TUJ antibody or anti-MAP2 antibody (Red), cultures were then stained with DAPI (Blue) (). The green (GFP) cells are HC20 cells induced to express HIV. In the cultures with HC20 cells, there was relatively little reduction in the number of beta-TUJ positive cells compared to control C20 cells, indicating preservation of axons. By contrast, there was a marked reduction in intact dendrites as assessed by reductions in microtubule associated protein 2 (MAP2) immunoreactivity in neurons exposed to HIV-infected microglial cells, indicating extensive dendritic pruning.

Activated human microglia/HIV induce neuronal damage.

LUHMES-derived neurons were co-cultured with hμglia clone C20 or mixed population of cells that had been infected with HIV (HC20). Top: The neurons or co-cultures were stained with anti-beta-TUJ antibody (Red). Bottom: Neurons were co-cultured with C20 or HC20 cells and stained with anti-MAP2 (Red). Green: GFP expression in activated HC20 cells. Red: Alexa Fluor 488 antibodies were used as secondary antibodies. Blue: DAPI stained cell nuclei.

Western blot analyses were also conducted to assess the extent of neuronal damage (). In these experiments, LUHMES-derived neurons were co-cultured in the absence or presence of C20 cells or HC69 cells, a clonal derivative of HC20 cells [36]. To maximize HIV expression, the HC69 cells were pre-treated with TNF-α or poly (I:C), a TLR3 agonist that mimics the effects of circulating bacterial rRNA and potently induces HIV by activation of IRF3 [37]. High levels of expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA receptor) [116-118], increased the phosphorylation of MAP2 (p-MAP2) [119, 120] and decreased phosphorylated synapsin I (p-synapsin) [121], which are both associated with neurodegeneration. Analysis of representative western blots showed no statistically significant (N.S.) difference between the effect of pre-activated C20 and pre-activated HC69 cells (), despite apparent increases in the expression of AMPA receptors in the presence of pre-activated microglia (). p(S136)-MAP2 levels increased more strongly with both TNF-α- and poly (I:C)-pre-activated HC69 than with similarly activated C20 cells (). p(S136)-MAP2 levels also showed significantly greater induction with TNF-α-pre-activated HC69 cells than with TNF-α-pre-activated C20 cells (p = 0.027). Similarly, poly (I:C)-pre-activated HC69 showed higher p(S136)-MAP2 levels than poly (I:C)-pre-activated C20 (p = 0.020). In contrast, p-synapsin levels (), were significantly lower in neurons exposed to TNF-α-pre-activated HC69 compared to C20 cells (p < 0.0001), but not altered when comparing neurons exposed to poly (I:C)-pre-activated HC69 or C20 cells (N.S.). Interestingly, based on the resazurin reduction method (), TNF-α- or poly (I:C)-pre-activated HC69 cells induced significantly more neuronal damage than TNF-α-pre-activated C20 cells (p = 0.048 and p = 0.030, respectively).

We also observed extensive neuronal damage following HIV induction when LUHMES-derived neurons were co-cultured (10:1 ratio) with rat-derived microglia CHME-5 cells [36]. Co-culture experiments were performed using either uninfected CHME-5 cells, cells that were latently-infected with the HIV reporter, or latently infected cells that were induced by treatment with TNF-α (100 ng/mL) (). After 48 h, the co-cultures were immunostained with anti-MAP2 antibody (Red) and the nuclei were visualized with DAPI (Blue). Green (GFP) cells are activated CHME-5/HIV. When neurons were co-cultured with uninfected CHME-5 cells, activation with TNF-α induced relatively little neuronal injury, as evidence by the reduced number of MAP2 immunoreactive neuronal dendrites. However, when neurons were co-cultured with CHME-5/HIV cells, addition of TNF-α greatly exacerbated neuronal damage, as evidenced by marked dendritic pruning compared to CHME-5 cells in the presence of TNF-α.

A similar effect was observed when we measured the effect of TNF-α-pre-activated CHME-5 and CHME-5/HIV cells on neuronal viability by measuring the relative number of healthy neurons visually and manually counted in time-lapse experiments. CHME-5/HIV cells, which are a rat cell model for HIV latency [36], decreased neuronal viability from 100% to ~35% (pink), compared to the co-cultured with CHME-5 cells (down to ~75%; blue) or neurons alone (black), where viability remained above 90% for the duration of the experiment ().

METH induces HIV expression in hμglia/HIV cells in a σ1R-dependent manner

We have shown previously that high concentrations of METH induce HIV reactivation in rat CHME-5/HIV cells [13]. To test the ability of METH to reactivate HIV in hμglia/HIV cells, HC69 cells were incubated with 50 to 500 μM METH (). These concentrations are in excess of the 100 nM to 10 μM METH levels seen in chronic drug abusers [122], but were chosen to evaluate the maximal level of METH that did not induce cytotoxicity. At the high METH concentrations, over a 96 h time course there was a progressive increase in HIV expression, as demonstrated by the levels of GFP+ cells in the population ().

METH-mediated reactivation of HIV.

(A) Effect of METH on HIV reactivation. (B) Cell viability. HC69 cells were incubated for 1, 2, 3, 4 or 5 days (X-axis) with indicated concentrations of METH prior to flow cytometry analysis and PI exclusion cell viability assay (Y-axis). Error bars represent standard deviations of three or more experiments. (C) σ1R mediates METH effect on HIV reactivation. HC69 cells were either untreated (Control) or treated with BD1047, rimcazole, or SM-21 prior to exposure to 300 μM of METH (+ METH) or untreated (- METH). Diamonds of similar color represent an individual experimental series. (n = number of individual samples). The p-values of pair-sample t-tests comparing the unexposed vs. the exposed cells are shown. N.S.: non-significant. (D) METH sensitizes hμglia for poly (I:C)-mediated HIV reactivation at low doses. HC69 cells were treated with increasing concentrations of METH (Control; 0, 1, 5, 50, 100 and 500 nM, and 1, 5, 10, 50 and 100 μM; X-axis, log scale) for 72 h prior to exposure to poly (I:C) (50 ng/mL). Parallel experiments were performed in the presence of BD1047 (10 μM). Error bars represent standard deviation of three or more experiments.

HIV was not reactivated during the first two days at any of the tested drug concentrations but became evident after 3 days at METH concentrations above 100 μM (). At 50 μM (blue circles and line), there was no effect on HIV reactivation () or cell viability as assessed by propidium iodide (PI) exclusion () compared to untreated controls during the course of the 5 days (0 μM; red squares and line). At 100 μM (yellow triangles and line), HIV reactivation was higher than in controls from day 3 (), but viability was somewhat compromised compared to untreated cells (; ~ 91% vs. ~ 86% at day 3, ~ 89% vs. 81% at day 4, and ~ 93% vs. 73% at day 5). At 300 μM and 500 μM METH, HIV reactivation was maximal () but cell viability was compromised. Flow cytometry histograms for cells treated with 300 μM METH from a similar experiment are shown in . No activation was observed after 24 h (from ~7% to ~4% GFP+) (), compared to the untreated controls; however, by 96 h, ~32% of the cells became GFP+ () compared to ~ 9% in the control cells (). In control experiments, potent proviral reactivation was seen at each time point using TNF-α (at 100 pg/mL; ~ 90%) () or poly(I:C) (at 100 ng/mL; ~68%) ().

Several studies have suggested that METH exhibits significant affinity for the σ1R. For example, BD1047, a specific inhibitor of the endoplasmic membrane-bound σ1R, was found to reduce neuronal injury in METH-exposed hippocampus [123]. More recently, it has been reported that σ1R antagonists attenuated METH-induced hyperactivity and neurotoxicity [124], and that σ1R is involved in METH-mediated microglial activation [125]. HC69 cells were exposed to METH at 300 μM, the dose found to be more effective at inducing HIV reactivation with low cell toxicity (), in either the absence or presence of BD1047 (10 μM), a σ1R antagonist [126], rimcazole (10 μM), a σ1R and σ2R antagonist [127], and SM-21 maleate (10 μM), a σ2R antagonist [128]. As shown in , BD1047, which by itself had no effect on HIV expression compared to controls, inhibited METH-mediated HIV reactivation from 19.44 ± 1.22% down to 5.66 ± 0.89% (p = 0.02). Rimcazole did not significantly reduce HIV expression and SM-21 maleate had no apparent effect.

We reasoned that in HIV-infected individuals who utilize METH, they are also invariably exposed to chronically increased levels of microbial products, such as LPS and bacterial rRNA, and proinflammatory cytokines due to damage to the gut permeability barrier [129, 130]. To mimic these conditions, we evaluated the impact of METH on HIV expression in microglial cells in the presence of two pro-inflammatory agents, TNF-α and poly (I:C). As shown in , pre-treatment by 100 μM METH for 72 h sensitized HC69 cells to HIV reactivation in response to sub-saturating concentrations of poly(I:C) (50 ng/mL), but not for sub-saturating concentrations TNF-α (20 pg/mL). In this experiment, METH combined with poly(I:C) reactivated ~77% of the cells, compared to ~38% of the cells after treatment by poly(I:C) alone (p = 0.01).

Because of the synergy observed between METH and poly (I:C), we performed another similar experiments using a broad range of METH concentrations spanning the exposure doses seen in drug abusers (1 nM to 100 μM) in the absence or presence of 50 ng/mL of poly (I:C). As a control for the impact of METH on the cells, parallel samples were treated with BD1047. As shown in , even at nM concentrations, METH (red squares and line) sensitized HC69 cells for poly (I:C)-mediated HIV reactivation (yellow triangles and line; from ~28% to ~29% at 1 nM, ~31% at 5 nM, ~32% at 50 nM, 34% at 100 nM, and 35% at 500 nM). At higher METH doses, the sensitization effect was more pronounced (to ~35% at 1 μM, ~40% at 5 μM, ~44% at 10 μM, ~52% at 50 μM, and 74 at 100 μM). As expected, the inhibitor BD1047 (10 μM) abrogated the effect of METH ().

METH induces HIV expression in neuronal mixed-glial co-cultures

We hypothesized that METH-induced neurodegeneration is exacerbated due to a combination of its direct activation effect on HIV expression and indirect effects due to neuronal damage. To test our hypothesis, HC69-iCort neuronal co-cultures, as described above (), were exposed to METH 100 nM, a dose shown to slightly synergized with poly(I:C) for HIV reactivation in HC69 cells (), in the absence or presence of poly(I:C) 50 ng/mL for 96 h prior to evaluating GFP expression and toxicity. In the presence of METH, HIV reactivated in a significant proportion of HC69 cells (). GFP expression increased from 4.27 ± 1.88% to 10.22 ± 2.06%, p < 0.001 (), whereas toxicity decreased from 100% to 77.60 ± 5.01%, p = 0.016 (). In the absence of neurons (), we did not observe this effect suggesting that METH-mediated neuronal damage, evidenced by the increased dendritic fragmentation and toxicity (), induces HIV emergence from latency. Poly (I:C), by itself, also increased the number of HIV-expressing HC69 cells in co-cultures with GFP expression increasing up to 26.04 ± 4.60% (), while increasing dendritic fragmentation () and neurotoxicity with only 61.79 ± 10.30% surviving (). This result suggests that the neuronal toxicity induced by poly(I:C) is directly related to release of HIV and pro-inflammatory agents from the induced microglial cells. The combination of METH and poly(I:C) further significantly induced HIV expression and dendritic damage compared to cells treated with poly(I:C) alone (). The combination of METH and poly(I:C) increased GFP expression up to 33.61 ± 6.83% (, p = 0.03) and decreased resazurin reduction to 53.16 ± 8.73% (, p = 0.019).

Exposure of co-cultures to combinations of METH- and Poly (I:C) induce extensive neuronal damage.

(A) Microscopy of HC69 cells co-cultured with iCort neurons for 96 h in the presence and absence of 100 nM METH and 50 ng/ml Poly (I:C). Top: Phase contrast image. Middle: GFP positive microglial cells. Bottom: Neurons were stained with anti-MAP2 (Red). Green: GFP expression in activated HC69 cells. Red: Alexa Fluor 488 antibodies were used as secondary antibodies. Blue: DAPI stained cell nuclei. (B) Induction of GFP expression in HC69 cells co-cultured with iCort neurons for 96 h and then measured by flow cytometry. Cells were treated with 100 nM METH or 50 ng/ml Poly (I:C) or a combination of 100 nM METH or 50 ng/ml Poly (I:C). Error bars represent standard deviations of three experiments. (C) Resazurin assay to evaluate neuronal viability. The resazurin reduction values (Y-axis) plotted are referenced to the control culture (untreated HC69 cells), set at 100%. The p-values of pair-sample t-tests of multiple experiments comparing the control vs. the METH exposed cells and Poly (I:C) treated cells vs. the METH plus Poly (I:C) treated cells are shown. Error bars represent standard deviation of three experiments.

We also established a co-culture between primary mouse neurons and microglial cells carrying the HIV reporter (HC69) or uninfected control (C20) cells. HC69 cells treated with 300 μM METH for 3 days displayed increased HIV expression when co-cultured with neurons compared to cultures not exposed to METH. As shown in , when neurons were co-cultured with HC69 cells, METH exposure induced reductions in dendritic diameter and there was evidence of increased dendritic fragmentation compared to C20 or HC69 cells in the absence of METH ().

Finally, in similar experiments using LUHMES-derived neurons co-cultured with either C20 or HC69 cells (), we used the resazurin method to assess neuronal viability after treatment of microglial cells with a variety of inflammatory or anti-inflammatory agents. Reactivation of HIV by TNF-α (20 pg/mL) or poly(I:C) (50 ng/mL) decreased neuronal viability by 15–25% in C20 cell co-cultures. In combination with METH, neuronal viability was further decreased (by ~ 30%) (). These neurotoxic effects were significantly enhanced when HC69 cells were used in the co-cultures. Neuronal viability was decreased down to ~ 60% after activation by TNF-α or poly(I:C) and, reduced to ~ 50% when METH was combined with TNF-α (similar to METH alone) or to ~ 30% with poly(I:C) (). In control experiments assessing neuronal damage in the absence of microglia, toxicity was slightly less than that observed in the co-cultures with C20 cells (). By contrast, the neurotoxin MPP+ reduced neuronal viability to ~ 25%.

In comparison, dexamethasone (DEXA), which we have shown can potently repress HIV expression in microglial cells [39], was neuroprotective (). We attribute DEXA’s neuroprotective effects to its ability to restrict HIV reactivation and thereby limits HIV-induced bystander neuronal injury [39].

In a parallel experiment, METH (300 μM) reduced neuronal viability by about 30% in C20 cell co-cultures (p = 0.025), compared to losses of about 50% in the HC69 cell co-cultures (p = 0.005) (). However, the selective σ1R antagonist BD1047, which by itself had no apparent effect on neuronal viability, largely negated the neurotoxic effects of METH in C20 and HC69 cell co-cultures (). This finding indicates that METH acts through specific σ1Rs to reactivate HIV and trigger neurotoxicity in neuronal-microglial co-cultures.

We conclude that METH can exacerbate the neuronal damage seen in the presence of HIV-infected microglial cells through a combination of direct neurotoxic effects that activate the HIV transcription in infected microglial cells and by potentiating HIV reactivation responses to pro-inflammatory agents.

Discussion

Microglial dysregulation by HIV results in synaptodendritic injury and neuronal losses

HAND remains a significant clinical problem as it manifests in approximately 30–50% of cART-treated patients, despite peripheral viral suppression [5]. Pathophysiologically, HAND is driven by the sustained, low-level production of HIV proteins and proinflammatory toxins in microglia that result in synaptodendritic injury, reductions in CNS connectivity with accompanying neurobehavioral and cognitive declines [131-134]. The poor CNS penetration and consequently reduced effectiveness of certain ART drugs creates an environment where HIV replication is favored and has also been linked to the development of HAND [135].

In normal situations, activation of microglia in response to inflammatory stimuli is characterized by a transition from a resting state (M0 or M2 cells) to more activated cells (M1 cells). Although Ransohoff has argued that this model is an oversimplification [136], we believe it remains a useful broad classification of glial activation states. In contrast to other cells of the immune system, strict limitations on the activity of stimulated microglial cells are imposed to avoid an exaggerated response during infection and injuries. These mechanisms result both in the production of anti-inflammatory cytokines and inhibitory proteins, and attenuated production of pro-inflammatory cytokines through finely coordinated cell signaling and transcriptional programs such as the Nurr1/CoREST transrepression pathway [137, 138]. It is generally believed that over-activated microglia (constitutive M1 cells) exacerbate neuronal injury through the synthesis and secretion of cytotoxic factors, which increase excitotoxic synaptic transmission and damage healthy neurons [47, 48]. Therefore, microglia-mediated neurotoxicity, appears to be the result of excessive and uncontrolled stimulation [49, 50], and/or impaired functionality of intrinsic molecular mechanisms [51-53], which are likely to be further impaired as a consequence of HIV infection.

Neuronal-microglial communication is now realized to be far more dynamic than previously thought, and neurons play key roles in regulating microglial responses [139, 140]. For example, neurons regulate microglia through the CX3CL1 (expressed by neurons)/CX3CR1 (expressed in microglia) axis [141-145], and through the neuronal surface proteins CD200, CD47 and CD22, which bind to their cognate receptors CD200R, CD172 and CD45, respectively, on microglia [146].

Importantly, neuronal dysfunction in HAND does not correlate with the number of HIV-infected cells or viral antigens in CNS [82, 83], but rather with elevated inflammatory cytokine levels. These observations have led to some investigators to minimize the importance of viral persistence as part of the etiology of NeuroHIV [82, 84–86, 88].

Microglial-neuronal crosstalk regulates HIV latency

In-vitro neuron-glia co-culture strategies have been widely used to gain an in-depth understanding of regulatory interactions between neurons and microglia [90-96]. Here, we describe a co-culture method between primarily LUHMES-derived neurons and immortalized human microglia infected with an HIV reporter construct. To confirm these observations in the most realistic cell systems available, we have also co-cultured primary or iPSC-derived neurons with primary or iPSC-derived microglia.

Using these systems, we have observed, unexpectedly, that neurons can silence HIV expression in infected microglia and prevent spontaneous reactivation of latent virus. Interestingly, iPSC-derived GABAergic cortical and dopaminergic neurons, but not motor neurons, were capable of mediating HIV silencing, recapitulating the effect of LUHMES-derived neurons.

One plausible explanation for the neuronal specificity regulating HIV silencing is that dopaminergic and GABAergic cortical neurons, but not cholinergic motor neurons, are able to synthesize and release anti-inflammatory compounds such as glucocorticoids (or neurosteroids). Glucocorticoids (GC) play a key role in countervailing inflammation in the CNS and can protect the brain against excessive innate immune responsiveness [39, 147–152]. The hypothalamic-pituitary-adrenal (HPA) axis rapidly increases the release of circulating glucocorticoids in response to various cytokines such as TNF-α and IL-1β. Consequently, by binding to the glucocorticoid receptor (GR), GCs and their analogues such as dexamethasone (DEXA) repress transcription of the inflammatory genes by blocking recruitment of NF-κB and AP-1 to their promoters [39, 153]. Consistent with these mechanisms, activation of the GR by DEXA in hμglia/HIV cells promotes HIV latency because it reduces microglial pro-inflammatory responses, whereas down regulation of the GC receptor by shRNA leads to HIV reactivation [39]. Other possible explanations include the loss of essential neuronal proteins that promote microglial inactivation. Further studies are warranted to determine the level of expression of C200, CD47, CD22, and CX3CL1 on GABAergic cortical, dopaminergic and motor neurons, which could further explain the selectivity of GABAergic cortical and dopaminergic neurons to silencing HIV.

While healthy neurons can promote HIV silencing, damaged neurons reactivate microglial cells and enhance HIV transcription, resulting in further neuronal deterioration and the increased expression of damage-associated molecular patterns (DAMPs). Therefore, we hypothesize that the “off” signals that keep microglia in check [146] also inhibit viral expression in infected microglia. Thus, neuronal DAMPs can trigger low, persistent levels of viral expression/reactivation resulting in sustained cycles of microglial activation, inflammation, HIV reactivation, and the production of more DAMPS from injured bystander neurons.

In our co-culture experiments, we have observed that in after several days in culture with neurons HIV-expressing infected microglia induce excessive neuronal damage due to initiation of a vicious cycle of enhanced neuronal damage leading to enhanced microglial activation. After an initial period when the healthy neurons can repress HIV reactivation, a vicious cycle becomes established where damaged neurons induce HIV expression, leading to further neuronal damage. The results presented here, and our previous work, support the hypothesis that HIV expression in infected microglia leads to an enhanced response to inflammation and periodic cycles of neuronal damage [36, 37, 39] (). One implication of this hypothesis for the etiology of HAND is that multiple pro-inflammatory episodes that are damaging to neurons lead to increased HIV expression and creation of a constitutively M1 state for microglial cells in the brain.

Disruption of microglial cell-neuron communication drives HIV replication in the brain and leads to neuronal degeneration.

Healthy neurons suppress HIV expression in microglia. Although the mechanisms are not fully understood, HIV silencing correlates with establishment of a resting (M0) state. This is likely to be mediated by glucocorticoids (GC) and the fractalkine (CX3CL1) system. Inflammatory cytokines such as TNF-α and IL-1β, or TLR agonists such as LPS and microbial metabolites, including rRNA fragments, activate microglia (M1) and induce HIV transcription. The production of inflammatory cytokines and HIV proteins then leads to further neuronal damage. METH, acting through the σ1R receptor on microglia and DAT on neurons, works in concert with the pro-inflammatory agents to further disrupt normal cell physiology and enhance HIV transcription. Thus, inflammation can initiate a vicious cycle of neuronal damage/microglial cell activation leading to HIV reactivation.

Primary human microglia are efficiently infected by replication competent R5 HIV (AD8gNef-GFP), and primary macaque microglia with replication competent SIV 17E-Fr particles [36, 154]. Unfortunately these cells loose the CD4 receptor upon passaging making it technically impossible to obtain sufficient susceptible primary microglia to perform studies of the type shown here. Therefore, for most of our studies we have relied on the HC69 clonal population of immortalized human microglial cells bearing an HIV construct (hμglia/HIV) [36, 37, 39] as a proxy. HC69 bears an HIV construct that lacks Gag, Pol and other accessory viral proteins. We have also previously shown that the proviral integration site of the HC69 cells was sequenced and located within the host genome, demonstrating that HC69 is a single integrant and, as expected from the extensive studies characterizing HIV proviral integration sites, the provirus was located in the introns of host genes [39]. There is extensive evidence that these reporters, which we used extensively to study HIV transcription in T-cell systems [155-157] and microglial cells [36-39] accurately reflect the transcriptional state of the virus since GFP expression is strictly dependent upon Tat activation in this system. We therefore believe that the neuronal silencing we have observed is the result of blocking of signaling pathways that activate HIV expression. It is also important to note that even microglial cells carrying defective proviruses can induce extensive neuronal damage once they become activated. The degree of neurotoxicity is likely to increase in the context of replication competent viruses.

METH exacerbates neuronal damage and enhances HIV reactivation

METH dependence is one of the most common co-morbid conditions among the HIV-infected population [158], and several studies clearly demonstrate that HIV patients who abuse METH have exacerbated neurocognitive impairments [159, 160]. Acute use of METH can lead to psychological and behavioral abnormalities [161-164], while the deleterious effects of chronic METH use on neurons have been well documented in rodents, non-human primates [165-167], and humans [168]. Strong clinical evidence indicates that METH-induced immune activation in the CNS in combination with HIV effects increase neuronal injury in individuals with both risk factors [124, 125, 169, 170].

It is now widely recognized that microglia-mediated inflammation is critical in METH-induced neurotoxicity [171-174], and that microgliosis is the result of METH abuse that persists long after abstinence [175, 176]. Acute exposure of hμglia/HIV cells to high concentrations of METH does not induce immediate viral expression, as seen when latently infected cells are exposed to pro-inflammatory molecules such as TNF-α and poly (I:C). However, a significant level of HIV reactivation is observed after 96 h, suggesting that indirect autocrine mechanisms have been activated. Experiments performed with physiological concentrations of METH in drug abusers (for example at 100 nM; [122]) indicate that this low concentration is enough to induce HIV expression in microglia co-cultured with neurons as well as neuronal damage. Under this condition, inflammation, represented by activation of TLR3 on microglia, we found further increased HIV expression and exacerbation of neuronal damage.

We also demonstrated that METH acts through a specific receptor, σ1R, to reactivate HIV and trigger neurotoxicity in neuronal-microglial co-cultures. The selective σ1R antagonist BD1047 not only fully ameliorated METH-induced HIV reactivation, it also significantly reduced bystander neurotoxicity at 24 h. By contrast, the selective σ2R antagonist, SM-21 failed to restrict METH-induced increases in HIV expression. This finding indicates that METH acts through specific σ1R to reactivate HIV and trigger neurotoxicity, and strongly suggests σ1R is a key molecular site of METH and HIV comorbid interactions in neuroHIV. Since METH can act on both microglia and neurons through different receptors, a wide number of different molecular mechanisms can potentially play a role in regulating neuron-microglia communication in the context of METH abuse. Further experiments investigating these mechanisms are warranted, and they should include evaluation of the ON/OFF signaling through knock-out/knock-down strategies. Nonetheless, based on our findings and the work of others on the impact of METH on HIV replication [10, 14], it seems clear that chronic METH abuse enhances HIV-induced neuro-inflammation both through direct effects on σ1R-expressing microglia, and through indirect effects due to the induction of neuronal damage, leading to enhanced neurodegeneration, and an exacerbation of disease progression.

Conclusions

Although systemic inflammation and antiretroviral and other drug toxicities are likely to contribute to the development of HAND, substantial evidence indicates that microglia, together with perivascular macrophages, are the main cellular reservoirs for HIV within the CNS parenchyma, and also contribute to HIV-related neuropathology and neurologic disorders. We have developed a reliable co-culture method to study the interactions between neurons and microglia and how inter-cellular signaling can regulate HIV latency in microglial cells. Our key observations are: First, that neurons can suppress viral expression, which likely involves repression of HIV through the GR. Second, that HIV-expressing infected microglia induce excessive neuronal damage, due to initiation of a vicious cycle of enhanced neuronal damage leading to enhanced microglial activation. Third, σ1Rs modulate the deleterious effects of METH on HIV reactivation and neurotoxicity. These results support the hypothesis that neurological complications of HAND result from the periodic emergence of HIV from latency within microglia in response to neuronal damage or inflammatory signals.

Bidirectional, interdependent cross-talk between neurons and microglia is therefore crucial in driving HIV infectivity in the CNS, and we believe that interrogating these signals is needed to identify basic mechanisms underlying the development of HAND. The recognition that HIV expression disrupts the normal interplay between microglia and neurons (i.e. the M0 to M1 transition cycle) and thereby exacerbates neurodegeneration should lead to the development of therapeutic anti-inflammatory strategies. Additionally, we believe that the screens of receptor agonists and antagonists will help identify potentially novel therapeutic strategies to improve the clinical management of HAND, especially in HIV-infected individuals who abuse METH.

Materials and methods

Establishment of co-culture between LUHMES-derived neurons and hμglia/HIV

Primary human microglia are efficiently infected by replication competent R5 HIV (AD8gNef-GFP), and primary macaque microglia with replication competent SIV 17E-Fr particles [36]. However, due to the scarcity of primary microglia, for most of our studies here, we have use the HC69 clonal population of immortalized human microglial cells bearing an HIV construct (hμglia/HIV) [36, 37, 39] as a proxy for infected primary microglia. HC69 bears an HIV construct that lacks Gag, Pol and other accessory viral proteins. We have previously shown that the proviral integration site of the HC69 cells was sequenced and located within the host genome, demonstrating that HC69 is a single integrant and, as expected from the extensive studies characterizing HIV proviral integration sites, the provirus was located in the introns of host genes [39].

Undifferentiated, neuron precursor cells LUHMES/RFP, developed [100] and kindly provided by Dr. Stefan Schildknecht at the laboratory of Dr. Marcel Leist (Konstanz, Germany), contain a lentiviral construct bearing the sequence of red fluorescent protein (RFP) that allows visualization of neurite growth and its disturbance by toxicants. These cells were plated and allowed to expand for two days prior to transferring to the experimental wells at 500,000 neurons per well of a 6-well plate, or another quantity specified in the Figure Legends, for differentiation into dopaminergic neurons. One day after the neuronal differentiation process started, 60,000 immortalized human microglial cells bearing an HIV construct (Proviral HIV Structure) (hμglia/HIV; [36, 37, 39, 177]) were added. After 2 days, the classical dopaminergic markers are still in the induction phase, therefore LUHMES-derived neurons are still not considered full dopaminergic cells. After 3 days, these cells can then be considered maturing neurons, since they start producing classical markers of dopaminergic neurons, reaching their full mature status by day 6 [100, 178]. For the next 3 to 4 days, assays were performed to evaluate the viability of the neurons. All co-cultures with CHME-5 cells lasted only 48 h.

Where indicated, primary or iPSC-derived neurons were also used in co-culture with hμglia/HIV HC69, primary or iPSC-derived microglial cells. Primary human neurons (HN) and microglia were obtained from ScienCell Research Laboratories (CA), whereas human iPSC-derived microglia (Tempo-iMG), cortical (Tempo-iCort), dopaminergic (Tempo-iDopaNer), and motor (Tempo-iMotorNer) neurons were obtained from Tempo Bioscience (CA). These cells were plated, allowed to differentiate and maintained in culture on mouse laminin-coated plates or on Matrigel matrix (Corning), as directed by the manufacturer.

Neuronal, microglia, and co-culture media

For expansion of undifferentiated, neuron precursors cells LUHMES/RFP [100], we used DMEM-F12 (ScienCell, Cat. #09411) supplemented with 1X N2 supplement (ThermoFisher Scientific, Cat. #17502048), 40 ng/mL bFGF (ThermoFisher Scientific, Cat. #13256029), 1% penicillin streptomycin (Gibco, Cat. #15070–063), and 250 ng/mL amphotericin B (Gibco, Cat. #15290018). For differentiation of LUHMES/RFP into dopaminergic neurons, DMEM-F12 was supplemented with 1 mM dibutyryl cAMP (StemCell Technologies, Cat. #73886), 1 μg/ml tetracycline (Sigma-Aldrich, Cat. #87128), 2 ng/mL GDNF (Sigma-Aldrich, Cat. #SRP3309), 1% P/S, and 250 ng/mL amphotericin B. As recommended by the manufacturer, primary human neurons were grown in Neuronal Medium (NM, ScienCell Cat. #1521), primary human microglia in Microglia Medium (MM, ScienCell Cat. #1901), iPSC-derived microglia in DMEM/F-12 (LifeTech) supplemented with 1X N2 supplement (LifeTech), 0.5X NEAA (LifeTech), 2 mM L-Glutamine or 1X GlutaMax (LifeTech), 100 ng/mL GM-CSF (Peprotech), and 50 ng/mL IL-34 (Peprotech), iPSC-derived cortical neurons were grown in neurobasal-based medium supplemented with 1X B27 (LifeTech), 1X essential amino acids (LifeTech), 1X GlutaMax (LifeTech), and 10 μM all-trans-retinoic acid (Sigma Aldrich), iPSC-derived dopaminergic neurons also in neurobasal-based medium supplemented in 1X B27 (LifeTech), ascorbic acid (0.2 μM, Sigma Aldrich), cAMP (5 μM, Sigma Aldrich), and TGF-β3, recombinant human BDNF and GDNF (all at 10 ng/ml, Peprotech), and iPSC-derived motor neurons in DMEM/F12:neurobasal medium (50:50), supplemented with 1X B27 (LifeTech), 1X N2 (LifeTech), GlutaMax (0.5 mM (LifeTech), cAMP (5 μM, Sigma Aldrich), and recombinant human SHH, BDNF, GDNF, and CNTF (all at 10 ng/ml, Peprotech).

Unless otherwise noted, microglial cells (hμglia C20 and hμglia/HIV HC69; [36, 37, 39]) were cultured, as previously reported [39], in BrainPhys medium (StemCell Technologies, Cat. #05790) containing 1X N2 supplement, 1X penicillin streptomycin, 100 μg/mL normocin (InvivoGen, Cat. #ant-nr-1), 25 mM glutamine (Gibco, Cat. #25030081), 1% fetal bovine serum (FBS; Gibco, Cat. #10438026), and 1 μM DEXA (freshly added to the cell culture) (Sigma-Aldrich, Cat. #D4902). For the co-culture of microglia with neurons, the specific neuronal medium was used supplemented 1X insulin-transferrin-sodium selenite media supplement (Sigma-Aldrich, Cat. #I1884-1VL) and 0.2% FBS. Where indicated, METH, poly (I:C), alone or in combination were added to the HC69/primary HN co-cultures prior to collection of supernatant, and fixation for 15 min in 4% paraformaldehyde. MAP-2 was detected using rabbit anti-MAP-2 primary antibodies (1:500 dilution; Chemicon/EMD Millipore, Billerica, MA) followed by secondary goat anti-rabbit antibodies conjugated to Alexa Fluor-594 (red). summarizes the composition of these formulations.

The reference for the culture of the other cell lines used as control, 293T cells, primary human foreskin fibroblasts, THP-1/HIV (A3) cells, and Jurkat/HIV (2D10) cells, are given in the corresponding figure legend.

To obtain damaged neuronal cells, cultured LUHMES-derived neurons were treated with 0.05% trypsin, followed by 1–3 min vortex, and cell death was verified by propidium iodide staining, as previously described [36]; at least 90% of cells were positive.

Chemicals & reagents

To drive HIV emergence from latency and/or HIV expression increase, we used TNF-α (Invitrogen, Cat. #PHC3015), poly(I:C) (InvivoGen, #tlrl-pic), and METH (Sigma-Aldrich, Cat. #51-57-0) alone or in combination at the concentrations stated in the corresponding Figures and/or Figure Legends. To study the involvement of the σ receptors in METH-mediated HIV reactivation, BD1047 (Tocris, Cat. #0956), rimcazole (Tocris, Cat. #1497), and SM21 (Tocris, Cat. #0751) were used at the concentrations specified in the Figure Legends.

SDS-PAGE/Western blot analysis

For determining the total expression of AMPA and phosphorylation of MAP2 and synapsin in LUHMES/RFP-derived neurons untreated or treated with TNF-α (10 pg/mL) or poly(I:C) (10 ng/mL) unexposed or not to C20 or HC69 cells untreated or treated with similar amount of TNF-α or poly (I:C), whole cell extracts (WCE) were prepared from 5 X 105 neurons collected by gentle shaking and added to RIPA buffer (25 mM Tris, pH 7–8, 150 mM Na, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100). Protein concentration in WCE was measured by Bradford assay, and protein solutions were subjected to SDS-PAGE/Western blot using anti-glutamate receptor 1 (AMPA subtype) antibody [EPR5479] (Abcam, Cat. #ab109450), anti-MAP2 (phospho S136) antibody [EPR2361] (Abcam, Cat. #ab96378), and anti-synapsin I (Abcam, Cat. #phospho S9) antibody (Abcam, Cat. #ab194778). β-tubulin III (beta-TUJ) is a microtubule element of the tubulin family found exclusively in neurons [101, 102], therefore, we used anti-beta-TUJ (Abcam, Cat. #ab18207) as loading control. These primary antibodies were bound by the appropriate IRDye 800CW secondary antibody, and the membranes were scanned and analyzed using the Odyssey Infrared Imaging System (LI-COR Biosciences, NE).

Flow cytometry and microscopy

As previously [36, 37, 39, 177], quantitation of GFP-expressing cells was carried out by fluorescence-activated cell sorting (FACS or flow cytometry) analysis using the LSRFortessa instrument for cell sorting, the FACSDiva software (BD, NJ) for data collection, and the WinList 3D software for data analysis, gating neurons separated from the microglia. To confirm microglia identity, we used anti-CD14-PE conjugated antibody (eBioscience, Cat. #12–0149), since we have shown that the CD14 receptor, a marker of monocytes, macrophages and dendritic cells [179], is present in the totality of the hμglia cells [36].

For phase contrast and fluorescence microscopy, we used the services of CWRU Visual Sciences Research Center core (grant #p30-15411373). In brief, a Leica DMI6000 Widefield Microscope with a QImaging EXI Aqua camera was used with a 1000 ms exposure time for FITC (488) to capture GFP fluorescence from HIV-expressing hμglia/HIV cells, and a 100 ms exposure time for the TxRed (546) to capture RFP fluorescence from neurons.

For neuronal cells immunostaining, we used anti-beta-TUJ (D71G9), XP rabbit (Cell Signaling Technology, #5568), anti-MAP2 rabbit (Cell Signaling Technology, #4542), anti-CXCR3 rabbit (Novus Biologicals, #NB10056404), anti-CD11b/c (Novus Biologicals, #NB11040766), anti-GAD 65/67 rabbit (Millipore, #AB1511), anti-dopamine transporter (DAT) (Millipore, #MAB369), and anti-acetylcholinesterase monoclonal (AchE) (Thermo, #MA3-042) antibodies. As secondary antibodies, Alexa Fluor 488 anti-rabbit or 594 anti-mouse were used.

Neuronal viability assay

For viability of neuronal cells, we performed the in-vitro toxicology assay, based on resazurin reduction, following the instructions of the supplier (Sigma-Aldrich, Cat. #TOX8). Briefly, resazurin dye was added at a final concentration of 10% (v/v) to each well and incubated with the cells for ~2 hours, before transferring 100 μL of each well’s supernatant to individual wells of a 96-well flat-bottom plate, and measuring absorbance (endpoint) at 600 nm and 690 nm (final absorbance = absorbance at 600- absorbance at 690). Healthy cells are able to reduce the dark blue resazurin dye, turning the solution to a lighter pink color. The lighter the dye the healthier and more viable the cells are, since they undergo more metabolic activity, which reduces the dye to a lighter pink color and, therefore, absorbance is lower. Quantitation was then calculated in percent using untreated neurons as reference. We also used healthy cell counting per field as endpoint for cell neuronal viability.

Optimal co-culture medium.

LUHMES-derived neurons (red) and microglial cells, C20 (blue) and HC69 (green), were independently cultured in the presence of the indicated medium formulations (X-axis) and cell viability (Y-axis) was measured by the resazurin method.

(TIF)

Click here for additional data file.

Microglia growth and viability is not affected in co-culture with neurons in a short-term.

(A) Growth rate. 60,000 hμglia/HIV HC69 cells were plated in the presence of increasing density of LUMHES-derived neurons (X-axis). After 24 h (short-term), neurons were killed with 0.25% trypsin for 30 seconds, and washed away with PBS prior to further trypsinization for 5 minutes to recover microglial cells. Cells were counted (Y-axis). (B) PI exclusion assay for measuring viability (Y-axis; right panel). N.S.: not significant.

(TIF)

Click here for additional data file.

Flow cytometry gating strategy for measuring CD14- and GFP-expressing hμglia/HIV cells.

Flow cytometry profiles representing single cultures. The distinct populations of HC69 (μglia) and neuronal cells are indicated on the far-left flow cytometry profiles. Top flow cytometry profiles represent cells bound to isotype control; bottom profiles represent cells bound to anti-CD14 antibody. Anti-CD14 bound population is shown on the Y-axis, and GFP-expressing cells are shown on the X-axis in the CD14 vs. GFP graphs. The population of CD14-expressing cells is shown in orange and the populations of GFP-expressing cells are shown in green.

(TIF)

Click here for additional data file.

beta-TUJ immunochemistry.

LUHMES- and iPSC-derived neurons were stained with antibody against beta-TUJ (green). Alexa Fluor 488 anti-rabbit was used as secondary antibody. DAPI (blue) indicates nuclear staining.

(TIF)

Click here for additional data file.

MAP2 immunochemistry.

LUHMES- and iPSC-derived neurons were stained with antibody against MAP2 (green). Alexa Fluor 488 anti-rabbit was used as secondary antibody. DAPI (blue) indicates nuclear staining.

(TIF)

Click here for additional data file.

CXCR3 immunochemistry.

LUHMES- and iPSC-derived neurons were stained with antibody against CXCR3 (green). Alexa Fluor 488 anti-rabbit was used as secondary antibody. DAPI (blue) indicates nuclear staining.

(TIF)

Click here for additional data file.

CD11b/c immunochemistry.

LUHMES- and iPSC-derived neurons were stained with antibody against CD11b/c. Alexa Fluor 488 anti-rabbit was used as secondary antibody. DAPI (blue) indicates nuclear staining.

(TIF)

Click here for additional data file.

GAD65/67 immunochemistry.

LUHMES- and iPSC-derived neurons were stained with antibody against GAD65/67 (green). Alexa Fluor 488 anti-rabbit was used as secondary antibody. DAPI (blue) indicates nuclear staining.

(TIF)

Click here for additional data file.

DAT immunochemistry.

LUHMES- and iPSC-derived neurons were stained with antibody against DAT (green). Alexa Fluor 488 anti-rabbit was used as secondary antibody. DAPI (blue) indicates nuclear staining.

(TIF)

Click here for additional data file.

AchE immunochemistry.

LUHMES- and iPSC-derived neurons were stained with antibody against AchE (red or green). Alexa Fluor 488 anti-rabbit (green) or Alexa Fluor 594 anti-mouse was used as secondary antibodies. DAPI (blue) indicates nuclear staining.

(TIF)

Click here for additional data file.

293T cells and human foreskin fibroblasts failed to induce HIV latency in HC69 cells.

(A) 60,000 hμglia/HIV HC69 cells were plated in the absence or presence of 0.5 x 106 293T cells or human foreskin fibroblasts (HFF), both grown in DMEM/10% FBS, or DEXA (positive control). The co-culture medium was the immortalized microglia medium (Table 2). HIV expression was evaluated after 24 h by flow cytometry. Flow cytometry profiles representing single cultures indicate the proportion of the CD11b/c-expressing cells (total microglia; Y-axis) that expresses GFP (X-axis). (B). Flow cytometric analysis quantification of microglial cell GFP expression in three similar experiments. The p-values of pair-sample, Student’s t-tests comparing the microglial cells cultured alone or in the presence of cells are shown. Individual independent experiments are color coded (n = number of independent samples). N.S.: non-significant. (C) PI exclusion assay to evaluate co-culture viability. Viability values (Y-axis) were normalized to the control culture of HC69 cells alone. Each colored symbol represents one experiment.

Table 2

Medium composition for the growth and maintenance of LUHMES and microglial cells, and the co-culture between LUHMES-derived neurons and microglia.

Cells	Microglia or Neuronal Growth Medium (NGM)	Neuronal Differentiation Medium (NDM)	Modified Neuronal Differentiation Medium (mNDM) or Co-culture Medium
Immortalized Microglia	BrainPhys (w/o phenol)1x N21x P/S100 μg/mL normocin2.5 mM glutamine0.2%x FBS1 μM dexamethasone	None	None
Primary microglia	Microglia Medium (MM, ScienCell Cat. #1901)	None	None
iPSC-derived microglia	DMEM/F-12 (LifeTech) 1X N2 supplement0.5X NEAA2 mM L-Glutamine100 ng/mL GM-CSF50 ng/mL IL-34	None	None
LUHMES	DMEM-F121% N2 supplement40 ng/mL bFGF1% Pen/Strep250 ng/mL amphotericin B	DMEM-F121 mM dibutyryl cAMP1 μg/mL tetracycline2 ng/mL GDNF1% P/S250 ng/mL amphotericin B	None
Primary human neurons	None	Neuronal Medium (NM, ScienCell Cat. #1521)
iPSC-derived cortical neurons	None	Neurobasal1X B271X non-essential amino acids1X GlutaMax10 μM all-trans-retinoic acid	None
iPSC-derived dopaminergic neurons	Neurobasal Medium1X B270.2 μM ascorbic acid5 μM cAMP10 ng/mL TGFbeta310 ng/mL rhBDNF10 ng/mL rhGDNF10 μM cytosine arabinofuranoside	Neurobasal Medium1X B270.2 μM ascorbic acid5 μM cAMP10 ng/mL TGFbeta310 ng/mL rhBDNF,10 ng/mL rhGDNF	None
iPSC-derived motor neurons	None	50:50 DMEM/F12: Neurobasal Medium1X B271X N20.5 mM GlutaMax5 μM cAMP10 ng/mL rhSHH10 ng/mL rhBDNF10 ng/mL rhGDNF10 ng/mL rhCNTF	None
Immortalized microglia-LUHMES-derived neuronal co-culture	None	None	NMDInsulin-transferrin-sodium selenite (Sigma-Aldrich I1884-1VL)0.2% FBS
Primary or iPSC-derived microglia-primary or iPSC-derived neurons	None	None	1:1 microglia medium/neuronal medium

(TIF)

Click here for additional data file.

Neurons failed to induce HIV latency in THP-1/HIV and Jurkat/HIV cells.

60,000 THP-1/HIV (A3) and Jurkat/HIV (2D10) cells, grown as previously described [37], were plated in the absence or presence of 0.5 x 106 iCort or LUHMES-derived neurons, or U0126 (positive control). NDM (Table 2) was co-culture medium. HIV expression was evaluated after 24 h by fluorescence microscopy (A) and flow cytometry. (B). Flow cytometric analysis of GFP expression in three similar experiments: the p-values of pair-sample, Student’s t-tests comparing the A3 and 2D10 cells cultured alone or in the presence of neurons are shown. Individual independent experiments are color-coded (n = number of independent samples). N.S.: non-significant.

(TIF)

Click here for additional data file.

Microglia growth and viability is not affected in co-culture with neurons.

(A) Growth rate. 60,000 hμglia/HIV HC69 cells were plated in the presence of 0.5 x 106 LUMHES-derived neurons (X-axis). After either 24 h or 72 h, neurons were killed with 0.25% trypsin for 30 seconds, and washed away with PBS prior to further trypsinization for 5 minutes to recover microglial cells. Cells were counted (Y-axis; left panel). (B) PI exclusion assay for measuring viability (Y-axis; right panel). N.S.: not significant.

(TIF)

Click here for additional data file.

Quantitation of the effect of healthy neurons vs. damaged neurons on HIV expression.

(A) hμglia/HIV HC69 cells were sorted into GFP- cells. The population was expanded for 48 h prior to collection and co-cultured with either healthy neurons or damaged neurons (X-axis) at a ratio of 50:6. (B) GFP+ cells. Quantitation of GFP expression (Y-axis). Diamonds of similar color represent an individual experimental series. (n = number of individual samples). The p-values of pair-sample t-tests comparing the unexposed vs. the exposed cells are shown. N.S.: non-significant.

(TIF)

Click here for additional data file.

HIV induces neuronal damage signals.

A. Western blot analysis. LUHMES-derived neurons were unexposed (-Microglia) or exposed to either C20 or HC69 cells in the absence or presence of either TNF-α or poly(I:C) for 48 h. Whole cell extracts were prepared from neurons and subjected to SDS-PAGE/Western blot analysis. Western blot membranes were blotted against anti-AMPA, anti-p-MAP2, and anti-p-synapsin antibodies, using anti-β-tubulin III as loading control. Approximate molecular weights are indicated in KDa. B. Quantitation of AMPA, p-MAP2, and p-synapsin band intensity. Numbers were plotted in Relative Intensity (Arbitrary Units; Y-axis) vs. experimental treatments (represented by a specific color; X-axis), graphs for each AMPA, p-MAP2, and p-synapsin. C. Resazurin reduction assay. The resazurin reduction values (Y-axis) plotted are referenced to the control culture (neurons only), set at 100%, next to the other experimental treatments (X-axis). For (B) and (C), the p-values of statistically significant pair-sample t-tests (at the 0.05 confidence level, where the difference of the sample means is significantly different from the test difference of zero) of three experiments (n = 3) comparing the neurons exposed to C20 and either TNF-α or poly(I:C) with the neurons exposed to HC69 and either TNF-α or poly (I:C), respectively. N.S. stands for non-significant. Similar geometric figures represent a unique experiment.

(TIF)

Click here for additional data file.

Activated rat CHME-5/HIV cells exacerbate neuronal damage.

A. LUHMES-derived neurons were co-cultured or not with CHME-5 or CHME-5/HIV cells for 48 h. TNF-α (100 ng/mL) was added or not, as indicated. The neurons or co-cultures were stained with anti-MAP2 antibody followed by anti-rabbit Alexa Fluor 594 antibody (red) and DAPI for nuclear visualization. Green (GFP) depicts activated CHME-5/HIV cells. Dendrites and microglia are indicated by the white lines. B. LUHMES-derived neurons were co-cultured with CHME-5 (blue squares) or CHME-5/HIV (red triangles) cells that had been pre-activated with 100 ng/mL of TNF-α. Neurons alone (black circles) were used as control. Time-lapse images were taken every four hours, at the indicated time points, from 0 to 48 h (X-axis). The number of healthy neurons was counted in every field the relative number of viable neurons quantified (Y-axis).

(TIF)

Click here for additional data file.

Flow cytometry profiles representing single cultures of HC69 cells were incubated for 24, 48 or 96 h. (A) Untreated. (B) 300 μM METH. (C) 100 pg/ml TNF-α. (D) 100 ng/mL poly (I:C). GFP+ cell populations were measured by flow cytometry and indicated in bright green.

(TIF)

Click here for additional data file.

METH sensitizes hμglia for poly (I:C)-mediated HIV reactivation.

HC69 cells were either untreated (- METH) or treated with METH 300 μM for 72 h prior to exposure to either TNF-α 20 pg/mL) or poly(I:C) (50 ng/mL) for another 24 h. Diamonds of similar color represent an individual experimental series. (n = number of individual samples). The p-values of pair-sample t-tests comparing the unexposed vs. the exposed cells are shown. N.S.: non-significant.

(TIF)

Click here for additional data file.

HIV exacerbates METH-mediated neuronal damage.

Human neuronal and glial mixed-cultures containing astrocytes (Advanced Biosci. Resources) were maintained for 17 days in vitro (DIV) in BrainPhys supplemented with insulin-transferrin-sodium selenite prior to co-culture with either C20 or HC69 cells in either the absence or presence of 300 μM METH for 72 h. Top: brightfield. Middle: Green fluorescence channel. Bottom: Green (GFP+ cells). Red (MAP2, neuronal dendrites). Blue (DAPI, all nuclei).

(TIF)

Click here for additional data file.

Effect of METH, TNF-α and poly(I:C) on neuronal damage.

LUHMES-derived neurons were either cultured alone (red) or co-cultured with either C20 (blue) or HC69 (green) cells in either the absence (control) or presence of (A) TNF-α, poly (I:C), METH, METH + TNF-α or METH + poly (I:C), or (B) METH, BD1047, METH + BD1047 or DEXA for 72 h (X-axis) prior to neuronal survival quantitation by the resazurin method (Y-axis). MPP+ was used as positive control for neuronal damage.

(TIF)

Click here for additional data file.

7 Jul 2019

Dear Prof. Karn,

Thank you very much for submitting your manuscript "Cross-talk between microglia and neurons regulates HIV latency" (PPATHOGENS-D-19-00977) for review by PLOS Pathogens. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the manuscript as it currently stands. These issues must be addressed before we would be willing to consider a revised version of your study. We cannot, of course, promise publication at that time.

We therefore ask you to modify the manuscript according to the reviewers' recommendations. Your revisions should address the specific points made by each reviewer; given the importance of the issues raised it will be necessary to send your manuscript back out for review.  Under such circumstances it will be important for the reviewers to feel the paper is very close to "publication-ready" with the second submission to consider acceptance.  One reviewer raised concerns about the form of the manuscript (with its 170+ references) which seems like a distraction to the science.  More importantly, the reviewers asked in a number of different ways to enhance our level of understanding of relevance of your model system.  This question is at the crux of how useful the observations are so it will be important to revise the manuscript (including new experiments) to address the concerns of the reviewers.

In addition, when you are ready to resubmit, please be prepared to provide the following:

(1) A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

(2) Two versions of the manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Additionally, to enhance the reproducibility of your results, PLOS recommends that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see http://journals.plos.org/plospathogens/s/submission-guidelines#loc-materials-and-methods

We hope to receive your revised manuscript within 60 days. If you anticipate any delay in its return, we ask that you let us know the expected resubmission date by replying to this email. Revised manuscripts received beyond 60 days may require evaluation and peer review similar to that applied to newly submitted manuscripts.

[LINK]

We are sorry that we cannot be more positive about your manuscript at this stage, but if you have any concerns or questions, please do not hesitate to contact us.

Sincerely,

Ronald Swanstrom

Associate Editor

PLOS Pathogens

Thomas Hope

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Grant McFadden

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-2556-3526

***********************

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: This is a very interesting manuscript that addresses an important and under researched topic. The findings are of considerable interest and raise important questions regarding the ability of neurons to exert control over HIV replication in the nervous system. The use of a novel microglial HIV reporter line is a strength that facilitates the ability to perform the experiments. However, the use of cell lines and a model expression system is also a limitation as it may not accurately reflect HIV production and release. The general design of the studies and the rationale are strong but are impacted by a number of weaknesses. Neural damage can influence microglial replication but growth and death of microglia is not addressed. Overall, the manuscript is not well focused and lacks clarity in many sections as outlined below.

Reviewer #2: The manuscript by Alvarez-Carbonell examines whether the cross talk between microglia and neurons using a co-culture model system, where a microglia cell line HC69 that has a HIV reporter system were co-cultured with different types of neurons including LUHMES/RFP neurons, differentiated cortical, dopaminergic and motor neurons as well as mouse primary neurons. The authors analyzed whether co-culture of neurons with HC69 cells altered the GFP expression in these cells under different conditions and in combination with METH. The author conclude that neurons induce HIV latency, but that dying neurons then reverses this phenotype. The question the authors are addressing is important, however, the authors only use one system (GFP expression of the lentiviral reporter) to address this and do not validate their studies with any other system

Reviewer #3: There is a great deal of interest is examining viral latency in the CNS, however, there are few tools for doing so. Alvarez-Carbonell et al have developed an in vitro system to examine how interactions between neurons and microglia influence HIV expression and neuronal damage. This system involves their previously developed immortalized microglia cell line that has been infected with a VSV-g pseudotyped reporter vector. This cell line now harbors a partial HIV genome and GFP under control of the HIV LTR. In this study they use this immortalized microglia cell line and neuronal cells lines to examine whether interactions between these cells influences viral latency. Based on experiments co-culturing these cells they observed that HIV expression in microglia is lower when healthy neurons are present, but HIV expression increases when damaged neurons are present. Further, their results suggest that neuron types differ in their ability to suppress HIV expression and that other substances can stimulate HIV expression including TNF-a and meth. Together, these results suggest that HIV latency in the CNS may be highly regulated by both local cells that suppress expression when healthy, increase expression when damaged and increased expression when the environment becomes inflammatory. These results suggest that CNS reservoirs may be ‘shocked’ by unique triggers that may not alter latency in CD4+ T cells.

Reviewer #4: - strong/credible previous work from this group establishing their model of immortalized brain microglial cells

- co-culture approach is a vaulable extension to the microglia-only work and, while a cell culture model with the limitations of that simplified system, allows for a window into interactions between microglia and neurons that has value

- co-culture of neurons and microglia has been standardly used in other models

-finding that damaged neurons slightly increased HIV expression in the microglia is interesting and plausible

-overall the finding that the condition of the neurons impacts HIV expression is novel and provocative, though there are some limitations in interpreting these data

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: Neuron viability is assessed but not microglial which is more important for interpretation of the results. The authors need to address the question of microglial growth/survival. The growth rate of the immortalized microglia may be sufficient to have an impact on the interpretation of the studies. Any differential growth or death could alter the % of GFP expressing cells.

Meth concentrations used in the study are very high - at least an order of magnitude higher than plasma concentrations in Meth abusers. Data from studies in the low µM range would provide more translatable conclusions.

Reviewer #2: 1) There is a lack of explanation of the system in the introduction or methods and the lack of a verification with any other type of system. The authors use GFP+ cells as the sole read-out of HIV latency. They also do not describe the system in this manuscript. In their previous paper (Garcia-Mesa et al. JNV 2017), the % of cells expressing GFP was very low (<5%). In this paper, the HC69 cells have varied expression of GFP in normal culture. Is this due to cell division (ie, do dividing cells have reduced GFP which then rebounds). The authors use GFP- and GFP+ sorted population and compare the differences between them without providing any information on what is regulating this GFP expression in the absence of any other stimuli.

2) Additionally, the “induced latency” is only at a single time point (24 hrs) of decreased # of cells expressing GFP to declare that neurons induce HIV latency. A more clear kinetics of GFP expression (6 hr, 12 hr, 24 hr, 36 hr) would provide better evidence of suppressed GFP production.

3) One of the strongest correlates with the loss of suppression is the loss of neuronal viability. Could it not simply be that the neurons are dying and therefore there are less neurons to suppress latency. The authors suggest strongly in the abstract that damage neurons increase HIV suppression, but they would have to add damaged neurons to a normal co-culture to show this.

4) The authors show a substantial drop in live neuron counts at 72 hrs in their co-culture system (Fig. 6). However, in the study to examine neuronal damage, there was little reduction in cell numbers (Fig. 7), with reduced dendrites being the main criteria for damaged neurons. This is a slightly different system, but a huge difference in outcomes that appears to be primarily glossed over. It should be discussed.

Reviewer #3: Studying viral reservoirs in the CNS is a complicated proposition that likely involves the use of suboptimal in vitro systems or human samples. My primary concern with this study is that the model in question has deviated so much from in vivo biology as to be uninformative. In this study the authors examine interactions between two cell lines rather than primary cells. In their previous work the authors found that expression in these cell lines differs substantially from that of primary cells. In addition, both cell lines are infected with lentiviral vectors. The microglia are infected with a partial HIV reporter genome (see below) and the neurons are infected with a lentivirus reporter vector that is not/poorly described. The authors clearly show that expression of a partial HIV genome changes in culture conditions that include neurons. However, these experiments often lack the necessary controls to assess the cause of these changes. Is it due to interactions that are specific to microglia and neurons? Or is it due to the high density of cells in some culture conditions (see below) or interactions between microglia and any cells type (also see below). Further, would the effects be the same if the HIV reporter virus was intact? These types of questions should be addressed in order to assess whether this model captures features of the in vivo biology. Without these controls it is very difficult to assess whether these provocative observations represent specific interactions that likely occur in the CNS in vivo or are a product of the culture conditions.

1. The methods are insufficiently described. I am assuming that this study infects cells using a VSV-g pseudotyped vector, as in their previous work (PMC5294768), but that needs to be clarified. Infection with a VSV-g pseudotyped variant would not represent a true HIV Env-mediated infection. Did the authors choose this approach because it generates a higher rate of infection than infection with reporters pseudotyped with an actual HIV Env? I have three primary concerns with it. a) It suggests that HIV may infect microglia very inefficiently; raising questions about the importance of this event. b) If a very high percentage of cells are infected, we would expect that some cells are infected with multiple virions and may express more viral proteins than cells with a single integrated provirus. c) The vector used in this study doesn’t encode all of the viral proteins. This is unfortunate given that the study is examining cellular interactions that may be mediated by viral proteins. All of these points should be discussed.

2. Did the authors examine whether the results outlined in Figure 2 are specific to microglia culture with neurons? If I understand correctly, as the ratio of neurons to microglia increased, the total number of cells also increased. Is it possible that the reduction in GFP expression when the ratio of neurons to microglia is high could be due to there being a larger number of cells in the coculture?

3. Similar to the point above, do the authors examine whether the effect observed in Figure 4 is specific to neurons or influenced by the total density of cells in the culture? There are many ways to address this. It would be nice to try a couple of the following:

• Keep the total cell density constant across treatments (I don’t think that the authors did this).

• Repeat experiment with cells other than neurons

• Culture in a trans-well system where the density of microglia is constant but factors produced by neurons can reach the microglia culture or add spent media from neuron cultures to the microglia.

4. The authors should confirm that the neurons also mediate HIV expression in primary microglia. In a previous study (PMC5329090) the authors observed that their immortalized human microglia have gene expression patterns that differ substantially from that of primary microglia.

5. The authors should discuss the significance of the five types of neurons examined in Figure 3. Were they are terminally differentiated?

6. The author argue that neurons block HIV expression. They should also examine whether this is due to changes in global expression.

7. It would be helpful if the authors listed samples sizes. For example, does each flow plot in figure 8 represent a single culture?

8. What does including figures 7 and 9 accomplish? Summary figures would be more informative. I suggest moving 7 and 9 to the supplemental materials.

9. Figure 10 is very hypothetical. This hypothesis provides easily tested predictions that glucocorticoids can influence HIV expression. In the absence of additional evidence, I recommend that Figure 10 be moved to the supplemental materials.

10. Lines 593-595: “Substantial evidence indicates that microglia, together with perivascular macrophages, are the main cellular reservoirs for HIV within the CNS parenchyma, and hence responsible for HIV-related neuropathology and neurologic disorders.” This is a very strong statement suggesting that HAND is due to infection of macrophage/microglia. I would recommend softening this statement given that this mechanism has not been demonstrated in vivo and the fact that other mechanisms such as drug toxicities haven’t been ruled-out.

Reviewer #4: -HIV 'silencing' by increasing concentrations of neurons:microglia is a surprising findings and can't help but raise more questions than it answers. Why cortical and dopaminergic neurons but not motor neurons woudl selectively reduce HIV expression is unexplained/unexplored

- short term silencing and 'long term' considered 72 hoursin this cell culture system has questionable significance for human disease

- Methamphetamine inclusion in this study is not well explained/justified. Further experiments investigating the mechanisms of the neuronal effects on microglia and HIV expression would be more relevant to the current paper. I would consider removing the methamphetamine experiments from the paper and focusing on the huglia/neuronal interactions.

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: 1) Too much of the manuscript contains general information and reads like a review rather than a research paper. Along the same line, the reference list is exceptionally long. The authors should focus the manuscript by eliminating background material that is not directly relevant with a corresponding reduction in the number of references.

2) Resolution of the images in Fig 1 is not very good. Higher quality images would improve the ability to see what the cells actually look like.

3) The concentration of FBS needs to be clarified. It is listed as 0.1% 0.2% or 1% in different places.

4) The authors should define LUHMES derived neurons and add a justification for their use.

5) It would be useful to determine if the cellular interaction effects are specific to neurons. Addition of the microglia to other cells types would help to solidify the argument.

6) The role of neuronal damage in GFP expression is confusing. In Fig 4 there is substantial neuron death associated with decreased GFP expression whereas in Fig 5 & 6 neural damage/death is associated with increased GFP expression.

7) In Fig 7 it is difficult to tell what is circled. In addition, it appears that the HC69 cells were pre-activated to induce HIV expression. Under these circumstances it is unclear how the resulting toxicity can be attributed to HIV induction. A comparison of GFP+ vs GFP- HC69 cells without added cytokines would provide a cleaner comparison.

8) In the supplemental Figs, the cortical, dopamine and motor “neurons” do not look neuronal which raises questions about the adequacy of the differentiation. How do the authors know these are functional neurons? The addition of primary neurons to the experiments would add additional credibility.

9) It would be useful to know if the effect of the neurons on the microglia is contact dependent. Does neuronal conditioned medium have the same effect?

10) It is not clear why the paradigm shifted to use of CHME-5 cells. The authors should provide a rationale. Also in supplemental Fig 11, it appears that the neural damage is restricted to TNF treated cells not the HIV expressing cells. This again suggests that cytokine treatment and not HIV expression is the critical variable (which is already well established).

11) Are microglia added while neurons are in the differentiation medium? It is not clear when and how medium changes take place. (Neurobasal is misspelled in the Table)

12) Exposure to the different differentiation factors may confound the interpretation of the experiments. The culture conditions are very complex and it is not clear how the effects of growth factors and other components can be factored out.

13) Figure 10 is very speculative and not evidence based. The conclusions should be restricted to what is supported by data from the study.

14) How does the proposed involvement of glucocorticoids relate to this study?

15) Dexamethasone is in the microglial culture medium. Is this a confounding factor that needs to be addressed?

16) The conclusions that can be drawn are restricted by the nature of the model system. Addition of data on actual HIV production from infected cells would greatly strengthen the manuscript.

Reviewer #2: 1) The authors make statements of their system to indicate they are studying the effect of HIV infection (lines 104-105 “study the impact of HIV infection of microglia cells on their interaction with neurons” is just one example). Unless measurable virus is being produced and released, they are not studying the effect of virus infection on neurons. They are using a virus-reporter system, not virus production, which can have different effects on the cells. This needs to be put in context throughout the paper.

2) In Figure 2A, the GFP+ cells by themselves have cytoplasmic GFP expression, while cells cultured with neurons has what appears to be nuclear staining of GFP in the cells that are expressing it. However, the average GFP expression per positive cells seems similar (flow data). Does induction of latency affect GFP localization in the cell?

3) The authors should check with a statistician whether a One-way ANOVA would be the more correct statistical test for most of these studies.

4) Fig. 3C is not clear enough to show different cell populations

5) Box and whiskers are not needed on graphs when individual data points are given.

5) The discussion was basically a reiteration of the introduction until line 533.

Reviewer #3: Lines 467-470: “Pathophysiologically, HAND is thought to be driven the sustained, low-level production of HIV proteins and proinflammatory toxins that result in synaptodendritic injury, reductions in CNS connectivity with accompanying neurobehavioral and cognitive declines [123-126].” These are a couple of explanations, but may want to include others including drug toxicities (see PMC4937456).

Are the cells called μglia or mμglia?

Lines 59-62. There is an error in this sentence “In the US, 10-15% of HIV patients acknowledge METH use [8], exacerbates the effects of HIV infection in the CNS [9, 10] due to a combination of neurotoxic effects and the enhancement of HIV replication in microglia [11-14].”

Reviewer #4: (No Response)

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

1 Dec 2019

Dear Prof. Karn,

We are pleased to inform that your manuscript, "Cross-talk between microglia and neurons regulates HIV latency", has been editorially accepted for publication at PLOS Pathogens.

Before your manuscript can be formally accepted and sent to production, you will need to complete our formatting changes, which you will receive by email within a week. Please note that your manuscript will not be scheduled for publication until you have made the required changes.

IMPORTANT NOTES

(1) Please note, once your paper is accepted, an uncorrected proof of your manuscript will be published online ahead of the final version, unless you’ve already opted out via the online submission form. If, for any reason, you do not want an earlier version of your manuscript published online or are unsure if you have already indicated as such, please let the journal staff know immediately at plospathogens@plos.org.

(2) Copyediting and Proofreading: The corresponding author will receive a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript.

(3) Appropriate Figure Files: Please remove all name and figure # text from your figure files. Please also take this time to check that your figures are of high resolution, which will improve the readbility of your figures and help expedite your manuscript's publication. Please note that figures must have been originally created at 300dpi or higher. Do not manually increase the resolution of your files. For instructions on how to properly obtain high quality images, please review our Figure Guidelines, with examples at: http://journals.plos.org/plospathogens/s/figures.

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

(4) Striking Image: Please upload a striking still image to accompany your article if one is available (you can include a new image or an existing one from within your manuscript). Should your paper be accepted, this image will be considered for our monthly issue image and may also appear on our website to feature your article. Please upload this as a separate file, selecting "striking image" as the file type upon upload. Please also include a separate "Other" file with a caption, including credits and any potential copyright information. Please do not include the caption in the main article file. If your image is from someone other than yourself, please ensure that the artist has read and agreed to the terms and conditions of the Creative Commons Attribution License at http://journals.plos.org/plospathogens/s/content-license. Please note that PLOS cannot publish copyrighted images.

(5) Press Release or Related Media: If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team in advance at plospathogens@plos.org as soon as possible. We ask that you contact us within one week to plan ahead of our fast Production schedule. If you need to know your paper's publication date for related media purposes, you must coordinate with our press team, and your manuscript will remain under a strict press embargo until the publication date and time. This means an early version of your manuscript will not be published ahead of your final version.

(6)  PLOS requires an ORCID iD for all corresponding authors on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager.  To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field.  This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager

(7) Update your Profile Information: Now that your manuscript has been provisionally accepted, please log into Editorial Manager and update your profile, if needed. Go to https://www.editorialmanager.com/ppathogens, log in, and click on the "Update My Information" link at the top of the page. Please update your user information to ensure an efficient production and billing process.

(8) LaTeX users only: Our staff will ask you to upload a TEX file in addition to the PDF before the paper can be sent to typesetting, so please carefully review our Latex Guidelines http://journals.plos.org/plospathogens/s/latex in the meantime.

(9) If you have associated protocols in protocols.io, please ensure that you make them public before publication to guarantee immediate access to the methodological details.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Ronald Swanstrom

Associate Editor

PLOS Pathogens

Thomas Hope

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Grant McFadden

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-2556-3526

***********************************************************

Reviewer Comments (if any, and for reference):

19 Dec 2019

Dear Prof. Karn,

We are delighted to inform you that your manuscript, "Cross-talk between microglia and neurons regulates HIV latency," has been formally accepted for publication in PLOS Pathogens.

We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Pearls, Reviews, Opinions, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript, if you opted to have an early version of your article, will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Grant McFadden

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-2556-3526

Table 1

Level of expression of neuronal markers in different neuronal cell types.

Marker	LUHMES	Cortical	Dopaminergic	Motor
beta-TUJ	+++	++	+++	+++
MAP2	+++	+++	+++	+++
CXCR3	+	+++	+++	++
CD11b/c	-	-	-	-
GAD65/67	+++	+++	+	+
DAT	+++	+	+++	+
AchE	+++	+	++	+++