Methamphetamine functions as a novel CD4+ T-cell activator via the sigma-1 receptor to enhance HIV-1 infection

Methamphetamine (Meth) exacerbates HIV-1 pathobiology by increasing virus transmission and replication and accelerating clinical progression to AIDS. Meth has been shown to alter the expression of HIV-1 co-receptors and impair intrinsic resistance mechanisms of immune cells. However, the exact molecular mechanisms involved in augmenting HIV-1 replication in T-cells are still not yet clear. Here, we demonstrate that pretreatment with Meth of CD4+ T-cells enhanced HIV-1 replication. We observed upregulation of CD4+ T-cell activation markers and enhanced expression of miR-34c-5p and miR-155 in these cells. Further, we noted activation of the sigma-1 receptor and enhanced intracellular Ca2+ concentration and cAMP release in CD4+ T-cells upon Meth treatment, which resulted in increased phosphorylation and nuclear translocation of transcription factors NFκB, CREB, and NFAT1. Increased gene expression of IL-4 and IL-10 was also observed in Meth treated CD4+ T-cells. Moreover, proteasomal degradation of Ago1 occurred upon Meth treatment, further substantiating the drug as an activator of T-cells. Taken together, these findings show a previously unreported mechanism whereby Meth functions as a novel T-cell activator via the sigma-1 signaling pathway, enhancing replication of HIV-1 with expression of miR-34c-5p, and transcriptional activation of NFκB, CREB and NFAT1.


Meth pretreatment enhanced HIV-1 replication in CD4 + T-cells. Previous studies in in vitro models
have demonstrated that Meth enhances HIV-1 replication in T-cells, DCs, macrophages and neural progenitor cells [11][12][13][14] . The significance of these results is supported by an epidemiological study, which demonstrated increased viral loads in Meth using HIV-1 infected individuals compared with non-users who were infected 28 . However, the effects of Meth on HIV-1 replication in CD4 + T-cells are controversial, as Mantri et al. reported inhibition of HIV-1 replication in Meth treated CD4 + T-cells 15 . Here, we first examined HIV-1 BaL replication in PHA/IL-2 stimulated CD4 + T-cells (hereafter referred to as CD4 + T-cells) after either pretreating cells with 50 µM or 100 µM Meth for 24 hours and then infecting cells with HIV-1, or treating the cells with HIV-1 and Meth simultaneously. We analyzed HIV-1 p24 titer at different time points in the cell supernatants and observed significant increases in HIV-1 p24 titer in cells pretreated with 50 µM or 100 µM of Meth on days 1, 2 and 3 ( Fig. 1A). We confirmed these results by estimating the intracellular HIV-1 p24 by flow cytometry, which showed a significant increase in HIV-1 p24 in cells pretreated with Meth compared to control cells (Fig. 1B). It has been shown that naïve CD4 + T-cells or resting T-cells are refractory to HIV-1 infection 29 . Notably, unstimulated CD4 + T-cells (without PHA/IL-2) exhibited a naïve phenotype, characterized by low expression of CD4 + T-cell activation markers CD69 and CD25, as well as higher expression of naïve CD4 + T-cell marker CD45RA 30 . Thus we next analyzed the effects of Meth pretreatment on HIV-1 replication in unstimulated CD4 + T-cells; we observed enhanced HIV-1 replication in these cells compared to untreated cells (Fig. 1C). Intriguingly, we did not observe any significant change in the HIV-1 p24 titer when CD4 + T-cells were treated with Meth and HIV-1 simultaneously (Fig. 1D). These results indicate that Meth may alter cellular conditions favorable for HIV-1 infection in both stimulated and unstimulated CD4 + T-cells. 2+ ] in neuronal cells 30 . To further explore the molecular mechanisms involved in Meth mediated effects on CD4 + T-cells, we analyzed the release of second messengers Ca 2+ and cAMP after treating the cells with the drug. We observed significant increases in intracellular calcium levels upon Meth treatment; the maximum concentration was observed at 1 minute. This result suggests that Meth may have released the calcium from intracellular stores ( Fig. 2A,B). In addition, Meth has been shown to modulate cytokine secretion by regulating a cAMP/PKA/CREB signaling pathway in microglial cells 31 . We observed significant increases in cAMP at 30 minutes after Meth treatment indicating that Meth can activate cAMP mediated signaling pathways in CD4 + T-cells (Fig. 2C). Notably, pathways involving cAMP and transcription factors such as CREB have been associated with T-cell activation 32 . Western blot analysis also revealed increased phosphorylation of Akt, Src and ERK1/2 upon Meth treatment in these cells (Fig. 2D).

Meth enhanced intracellular calcium and cAMP levels in CD4 + T-cells. Meth has been shown to induce increased intracellular calcium concentration [Ca
Meth induces increased phosphorylation and nuclear translocation of CREB, NFAT1 and NFκB in CD4 + T-cells. We next analyzed the effects of Meth on the downstream cellular transcription factors, CREB, nuclear factor of activated T-cells (NFAT1), and NFκB, which are associated with T-cell immune activation 32 . We first examined the phosphorylation status and levels of expression of these molecules after treating CD4 + T-cells with Meth at various time points. Our analysis revealed increased phosphorylation of CREB and the p65 subunit of NFκB. However, total expression of these molecules was unaltered (Fig. 3A). Next, we analyzed the effects of Meth on their expression in nuclear and cytoplasmic fractions. We observed increased nuclear translocation of p-NFκB, p-CREB and NFAT1 in Meth treated cells compared to untreated cells, with levels of p-NFκB and NFAT1 significantly reduced in the cytoplasmic fractions (Fig. 3B). We performed confocal microscopy to confirm these results and observed increased nuclear translocation of p-NFκB, p-CREB and NFAT1 in Meth treated cells compared to control cells (Fig. 3C). These results indicate that Meth induced translocation of p-NFκB, p-CREB and NFAT1 from the cytoplasm to the nucleus in CD4 + T-cells. NFAT1 has been shown to regulate the expression of cytokines IL-4 and IL-10 during T-cell activation 33,34 . qRT-PCR analysis revealed increased levels of mRNA of both IL-4 and IL-10 in Meth treated cells compared to Meth mediated its effect through activating the sigma-1 receptor in CD4 + T-cells. Meth has been shown to exert its effects by activating dopamine receptors and/or sigma-1 receptors in the brain 18,19 . Hence, we analyzed effects of Meth on these receptors in CD4 + T-cells. All four dopamine receptors (D1DR, D2DR, D3DR, and D4DR) and the sigma-1 receptor were expressed constitutively, and we observed significant increased expression only of the sigma-1 receptor after Meth treatment (Fig. 4A). These observations indicated that Meth may act via the sigma-1 receptor in CD4 + T-cells (Fig. 4A,B). To confirm its functional role, we treated the cells with sigma-1 receptor specific inhibitor (Dihydrobromide, BD1047), then with Meth, and analyzed the activation of downstream signaling molecules by Western blot analysis. The sigma-1 receptor inhibitor significantly blocked the phosphorylation of NFκB, ERK and Src kinases (Fig. 4C). These results indicate that Meth induces the activation of transcription factors in CD4 + T-cells by activating the sigma-1 receptor. Next, to confirm the role of sigma-1 receptor in Meth mediated enhanced HIV-1 replication in CD4 + T-cells, we employed the sigma-1 receptor inhibitor in both unstimulated and stimulated cells. We found that this inhibitor significantly abrogated Meth mediated enhanced HIV-1 replication in both the stimulated and unstimulated cell types (Fig. 4D). These results confirm that sigma-1 receptor signaling is needed for Meth mediated enhanced HIV-1 replication in CD4 + T-cells.
Meth enhanced expression of miR-34c-5p and miR-155 and induced abnormal activation of CD4 + T-cells. Previous studies have shown that miR-34c-5p can activate naïve CD4 + T-cells by TCR stimulation and thereby make cells more susceptible to HIV-1 infection 35 . In addition, miR-155 has been shown to be upregulated in activated T-cells 35 . Since we observed increased HIV-1 infectivity after Meth pretreatment, we examined whether Meth treatment altered the expression of these two miRNAs in unstimulated CD4 + T-cells, thus driving them toward an activated state. Interestingly, we found that the expression of both miR-34c-5p and miR-155 increased in unstimulated CD4 + T-cells by more than 3 fold and 1.5 fold respectively after drug treatment (Fig. 5A). The increase we observed in these two miRNAs, which have been associated with T-cell activation, further indicates that Meth-treatment facilitates CD4 + T-cell activation. Next, we analyzed expression of miR-34c-5p expression in PHA/IL-2 stimulated CD4 + T-cells upon treating with Meth. We also found enhanced expression of this miRNA in drug treated activated cells compared to control cells (Fig. 5B). These results indicate that Meth induced increased expression of miR-34c-5p may further predispose CD4 + T-cells to HIV-1 replication. To explore a possible mechanism for increased miR-34c-5p and miR-155 expression, we tested their abundance with or without the sigma-1 receptor inhibitor; this also allowed us to analyze the role of Meth mediated activation of the sigma-1 receptor. We observed that the increased expression of these two miRNAs due to Meth was significantly abrogated in the presence of the inhibitor (Fig. 5C). These results indicate that Meth induces the expression of miR-34c-5p and miR-155 via activation of the sigma-1 receptor. Next, to analyze the effects of Meth mediated activation of signaling molecules and alteration of miRNA expression on T-cell activation, we tested the expression levels of CD4 + T-cell activation markers by flow cytometry after treating unstimulated CD4 + T-cells with Meth at different time points. Drug treatment modestly increased CD45RO expression (1.3 fold) and HLA-DR (1.5 fold). Intriguingly, we observed significant increases in the expression of other CD4 + T-cell activation markers such as CD69 (>4.5 fold) and CD25 (>2.5 fold). Naïve T-cells have a CD45RA + phenotype, which we found to be decreased in unstimulated CD4 + T-cells by 40% on day 3 of Meth treatment (Fig. 6A). Thus, taken together, Meth treatment can elevate the expression of miRs-34c-5p and 155 which have been shown to increase CD4 + T-cell activation, and may make the cells more susceptible to HIV-1 infection. We then analyzed the role of Meth mediated activation of the sigma-1 receptor in enhanced expression of T-cell activation markers in unstimulated CD4 + T-cells using sigma-1 receptor inhibitor. We observed that drug mediated increased expression of CD69 and HLA-DR was significantly abrogated in the presence of the sigma-1 receptor inhibitor (Fig. 6B). These studies further indicate that Meth induces a novel pathway of CD4 + T-cell activation by triggering sigma-1 receptor signaling.

Meth induced degradation of Ago1 and altered structural integrity of P-bodies. A recent study
has revealed that T-cell activation induces global post-transcriptional miRNA down-regulation and proteasomal  25,37 . Hence, we analyzed the effects of Meth treatment on Ago1 expression in CD4 + T-cells. We found that Meth treatment significantly inhibited Ago1 expression within 24 hours after treatment, which is consistent with T-cell activation (Fig. 7A). Further, we analyzed the mechanisms involved in Meth mediated inhibition of Ago1 expression; we found increased ubiquitination of Ago1 in drug treated cells compared to untreated cells (Fig. 7B). Moreover, the level of Ago1 mRNA was unaltered in Meth treated cells (data not shown). These results indicate that Meth treatment may induce proteasomal degradation of Ago1 in CD4 + T-cells as a result of induced activation. In addition, we also found that mRNA expression levels of Drosha and Dicer were unaltered in Meth treated CD4 + T-cells (data not shown).
Processing bodies (P-bodies) are important, discrete foci in the cytoplasm that play a key role in mRNA gene silencing and regulation of mRNA turnover 38,39 . Ago proteins and GW182 are the main components of P-bodies [38][39][40] . Since we observed decreased expression of Ago1 in Meth treated CD4 + T-cells, we next analyzed the structural integrity of P-bodies in Meth treated CD4 + T-cells. Confocal microscopic analysis and immunoprecipitation analysis showed decreased co-localization of Ago1 and GW182 in Meth treated cells compared to control cells (Fig. 7C,D). These results indicate that Meth may alter the structural integrity of P-bodies in CD4 + T-cells.

Discussion
Meth use is associated with a high risk of contracting HIV-1 infection, and its transmission [1][2][3][4][5] . Moreover, Meth enhances the viral load in HIV-1 positive, active drug using individuals by increasing viral replication in T-cells, monocytes and neural progenitor cells 7,11 . However, the molecular mechanisms involved in these Meth mediated effects in the immunopathogenesis of HIV-1 still are not fully elucidated. Here, we characterized novel molecular effects of Meth on CD4 + T-cells and their implications in HIV-1 replication.
Our experiments reveal that pretreatment of CD4 + T-cells with Meth significantly enhanced HIV-1 replication; however, there was no significant change in replication when cells were treated with HIV-1 and Meth simultaneously. A previous study by Skowronska demonstrated enhanced HIV-1 replication in neural progenitor cells when they were pretreated with Meth but not when they are exposed to HIV-1 and Meth together 14 . To address the mechanism of this effect, they point to Meth mediated NFκB/SP1 dependent activation of the HIV-1 LTR 14   However, in vivo the tissue microenvironment facilitates the activation of naïve T-cells and provides conditions favorable for productive HIV-1 infection [41][42][43] . Hence, CD4 + T-cell activation is considered to be a key factor that facilitates infection 44,45 . Moreover, expression of the T-cell activation markers CD25 and HLA-DR has been shown to correlate with enhanced HIV-1 infection 43 . When we analyzed cell activation markers in unstimulated CD4 + T-cells upon Meth treatment, we observed significant increases in CD25 and HLA-DR. We also observed increased expression of the activation markers CD69 and CD45RO, and a modest decline in the naïve CD4 + T-cell marker CD45RA. In addition, after Meth treatment of unstimulated CD4 + T-cells, we observed significant increases in the expression of miR-34c and miR-155. Transcriptional upregulation of miR-34c has been shown to occur during activation of CD4 + T-cells. Further, both of these miRNAs are reported to promote HIV-1 replication in CD4 + T-cells 35 .These findings indicate that Meth can act as an activator of CD4 + T-cells which could contribute to enhanced HIV-1 infection. Our finding corresponds to a clinical study by Massanella et al., where they observed enhanced CD4 + and CD8 + T-cell proliferation, and CD4 + T-cell activation and exhaustion in Meth using HIV-1-infected ART-suppressed individuals 16 .
Our study showed that Meth significantly increased intracellular [Ca 2+ ] and cAMP levels in CD4 + T-cells. Meth is known to induce increased cytosolic calcium concentration flux and cAMP release in neuronal cells 20,31 . Both of these pathways play major roles in T-cell signaling and activation, and are involved in downstream regulation of the transcription factors NFκB, CREB and NFAT1 32 . Interestingly, the NFκB/SP1 complex has been shown to mediate enhanced HIV-1 transcription in T-cells 14 . In addition, CREB has been shown to play a role in Meth addiction and activation of neuroinflammatory signaling pathways 21 . We observed increased phosphorylation Meth induced CD4 + T-cell activation via the sigma-1 receptor. Involvement of the sigma-1 receptor was also implicated in Meth mediated activation of astrocytes via Src, ERK/ mitogen-activated protein kinase, and CREB pathways 19 . Indeed, in our study we found Meth treatment similarly led to the activation of these downstream signaling pathways. Furthermore, enhanced nuclear translocation of CREB may have interacted with the promoter of the sigma-1 receptor resulting in the enhanced expression of the sigma-1 receptor in CD4 + T-cells.
MiRNAs are implicated in various biological processes including drug addiction and as modulators of post-transcriptional gene expression 23,46,47 . Recently, Liu et al. reported significant downregulation of Ago2, indicating that miRNA downregulation may be due to inhibition of Ago2 mediated splicing of precursor miRNA and not due to inhibition at the transcriptional level 23 . Consistent with this study, we observed proteasomal degradation of Ago1, that may have resulted in altered structural integrity of P-bodies, which play key roles in silencing of target mRNAs. Interestingly, prior studies have shown that during T-cell activation, Ago proteins undergo proteasome mediated degradation 36 . This further confirms Meth as a novel activator of CD4 + T-cells.
In sum, we elucidate several mechanisms through which Meth can enhance HIV-1 replication in CD4 + T-cells, and act as a novel activator of CD4 + T-cells through upregulation of miR-34c-5p and increased expression of several CD4 + T-cell activation markers. Consistent with this new insight into the drug as a T-cell activator, we found that Meth alone and in combination with HIV-1 infection induced extensive Ago1 degradation, which can result in loss of structural integrity of P-bodies and formation of miRNA induced silencing complexes, and has been linked to decreased anti-viral response and increased HIV-1 transcription 48 . We also show that Meth mediated activation of T-cells resulted in activation of the transcription factors NFκB, CREB and NFAT1, which may contribute to increased HIV-1 replication and production of inflammatory cytokines. Thus, multiple molecular mechanisms emanating from Meth as a T-cell activator likely contribute to enhanced HIV-1 pathogenesis in drug using individuals and provide insights into the development of novel therapeutic strategies to limit HIV-1 in these individuals.

Materials and Methods
Cells, HIV-1 and constructs. Buffy coats were obtained from the Blood Transfusion Service, Massachusetts General Hospital, Boston, MA, in compliance with the Beth Israel Deaconess Medical Center Committee on Clinical Investigations (CCI) protocol #2008-P-000418/5. Buffy coats were provided at this institution for research purposes; therefore, no informed consent was further needed. In addition, buffy coats were provided without identifiers. This study was approved by Beth Israel Deaconess Medical Center's CCI, Institutional Review Board, and Privacy Board appointed to review research involving human subjects. The experimental procedures were carried out in strict accordance with approved guidelines. Human peripheral blood mononuclear cells were isolated from buffy coats and CD4 + T cells were isolated using a negative selection kit respectively, per manufacturer's protocol (STEMCELL Technologies, Inc.). For PHA/IL-2 stimulated CD4 + T-cells, CD4 + T-cells were cultured in complete RPMI culture medium supplemented with PHA-L (1 μg/ml) and IL-2 (10 ng/ml) (PeproTech, Rocky Hill, NJ) at 2 × 10 6 cells/ml for 3 days. For unstimulated CD4 + T-cells, CD4 + T-cells were cultured in complete RPMI culture medium without PHA and IL-2. Purity of these T cells was analyzed using CD3 and CD4 staining and flow cytometry.

Antibodies and reagents. HIV-1 BaL was obtained from the NIH AIDS Research and Reference Reagent
Program, National Institute of Allergy and Infectious Diseases, NIH. The HIV-1 BaL strain has been shown to efficiently infect, via the CCR5 co-receptor, and replicate within CD4 + T-cells 49  Flow cytometric analyses. CD4 + T cells, isolated as aforementioned, were cultured in complete medium without PHA and IL-2 but were treated with or without 100 µM Meth for 3 days. Cells were harvested on days 0, 1 and 3, stained with the T-cell activation markers, and analyzed by flow cytometry. CD4 + T cells were stained with the marker antibodies conjugated with fluorophores or with their respective isotypes. The positively stained cells were gated based off the respective isotype.
Briefly, cell surface staining was performed by washing cells in 0.5% BSA in 1X PBS followed by incubation with fluorescent antibodies. Cells were fixed in 10% formalin with 4% formaldehyde (Sigma Aldrich, St. Louis, MO) for 30 minutes before washing twice more with 0.5% BSA in 1X PBS. Cells were analyzed in 1X PBS solution. Intracellular p24 was analyzed by staining the cells using FITC-conjugated p24 GAG antibody and analyzed on BD LSRII (BD Biosciences, Franklin Lakes, NJ). For p24 intracellular staining, the cells were stained with anti-gag antibody conjugated to FITC or FITC isotype control. The FITC positive cell population was gated based off the isotype control. Intracellular staining was performed by first washing cells in 0.5% BSA in 1X PBS. Then, cells were fixed in 10% formalin with 4% formaldehyde (Sigma Aldrich, St. Louis, MO) for 30 minutes before washing twice with 0.5% BSA in 1X PBS. Cells were permeabilized in 1X BD FACS TM Permeabilizing Solution 2 (BD Biosciences, Franklin Lakes, NJ) followed by incubation with fluorescent antibodies. Cells were washed with 1X PBS, and analyzed in 1X PBS solution.
Western blotting and immunoprecipitation. Western blotting was performed as previously described 51 .
Briefly, uninfected and HIV-1 infected or untreated and Meth treated CD4 + T-cells (after incubation period) were collected in cell lysis buffer, protein lysates were separated on NuPAGE precast gels (Life Technologies Corp.), transferred to 0.45 μm nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA), and probed with appropriate primary antibodies followed by incubation with their respective secondary antibodies. Proteins were visualized with Western Lightning Plus ECL Substrate (PerkinElmer, Waltham, MA).
For immunoprecipitation assay, CD4 + T-cells were left untreated or treated with Meth (100 μM) and incubated for times indicated. Cells were lysed with cell lysis buffer (Cell Signaling Technology). Cell lysates were subjected to immunoprecipitation using Millipore PureProteome TM Protein A and Protein G Magnetic beads, which were used according to manufacturer's protocol (MilliporeSigma, Burlington, MA) and the immune-complexes were further processed by Western blotting 51 .
Confocal microscopy. CD4 + T-cells were cultured on chamber slides. They were starved for 2 hours, and then untreated or treated with Meth (100 μM) for 24 hours. They were fixed in 4% paraformaldehyde and blocked with 5% normal goat serum in PBS/0.1% Triton X-100 (1 hour). Cells were then incubated with primary antibodies overnight at 4 °C, washed thrice with PBS, and stained with AlexaFluor 488-labeled anti-rabbit IgG antibody and AlexaFluor 594-labeled anti-mouse IgG antibody (Molecular Probes ® ; Invitrogen) for 2 hours.
Subsequently, cells were washed thrice with PBS, and slides were mounted using Prolong Gold antifade with DAPI (4′,6-diamidino-2-phenylindole; Invitrogen). Slides were examined under a Zeiss 880 Meta confocal microscope (Carl Zeiss Microimaging, LLC, Thornwood, NY), and images were acquired using ZEN2 software (Carl Zeiss). according to the manufacturer's protocol. Live cell microscopy was performed and fluorescence was measured before and after addition of Meth. Pixel density of the fluorescence was determined using Image J software.
Quantitative RT-PCR. RNA was isolated from CD4 + T-cells using TRIzol ™ reagent according to the manufacturer's instructions (Thermo Fisher Scientific, Waltham, MA). DNase treatment was performed using TURBO DNA-free kit (Ambion RNA, Carlsbad, CA). 1ug of RNA was used to prepare cDNA using iSCRIPT cDNA synthesis kit (Bio-Rad, Hercules, CA). qRT-PCR was done in triplicate for each sample with SYBR green based PowerUp ™ SYBR ™ Green Master Mix (Thermo Fisher Scientific, Waltham, MA) using 100 ng cDNA. Gene expression was normalized to TATA-box binding protein (TBP) and fold change in expression was calculated using 2−ΔΔCt method. Specificity of the primer sets was confirmed by melting curve analysis. Primer sequences used were: miR-34c (RefSeq: Statistical Analysis. All experiments were performed in triplicate. Differences between untreated and Meth treated samples were calculated using a standard 2-tailed Student's t-test. P-values ≤ 0.05 were considered statistically significant. For comparisons involving both Meth and the Sigma-1 inhibitor, one way ANOVA was performed and p-values ≤ 0.05 were considered statistically significant.