Introduction
The prevalence of HIV-associated neurocognitive disorders (HAND) is on the rise because of longer survival rates resulting from antiretroviral therapy. These effects are more predominant in HIV-1-infected opioid-dependent individuals [
1‐
4]. Furthermore, due to their immunosuppressive state, the incidence of systemic co-infection with opportunistic pathogens is also significantly higher in these patients. Frequent and repeated systemic infection in various neurodegenerative diseases, such as Alzheimer’s and Parkinson’s disease, results in unregulated activation of proinflammatory cytokines in the central nervous system (CNS) leading to progressive decline in cognitive function [
5,
6]. Despite a significant body of literature documenting the association of recurrent systemic infection with increased neurocognitive deficits, its role in the prevalence of HAND in opioid-dependent individuals infected with HIV-1 has not been delineated.
Peripheral leukocyte migration into the CNS contributes to pathogenesis of inflammatory neurologic responses during systemic infection [
7,
8] through induction of chemokines and their receptors. In the current study, we explored the contribution of specific chemokine receptors, i.e., CXCR4 and CCR5, in differential leukocyte trafficking into the CNS of mice following chronic morphine, HIV-1 Transactivator of transcription (Tat) protein, and/or co-infection with
Streptococcus pneumoniae. CCR5 and CXCR4 are the predominant co-receptors that interact with CD4+ T cells for HIV-1 entry into host cells [
9]. Additionally, various independent studies have shown that μ-opioid receptor (MOR) agonists, including morphine, exacerbate the expression of CXCR4 and CCR5 on peripheral immune cells, as well as on microglial cells, thereby increasing HIV-1 infectivity [
10‐
14].
Toll-like receptors (TLRs) on various immune cells recognize conserved motifs expressed by pathogens and play an important role in recruiting leukocytes to the sites of infection [
15‐
17]. Unregulated TLR activation has been implicated in HIV-1 pathogenesis [
18,
19]. We also previously showed that TLR activation on microglial cells results in significant neuronal apoptosis in a HIV-1 Tat model with co-infection of
S. pneumoniae in opioid-dependent mice [
20]. In the present study, we expanded our previous observations to further investigate the role of TLR-mediated CNS leukocyte trafficking as a contributing mechanism for HAND following chronic exposure to morphine in a co-infection murine model.
We demonstrated, in a murine model, morphine treatment in the context of HIV-1 Tat and S. pneumoniae orchestrate the migration of T cells through induction of chemokine ligands CXCL12 and CCL5. Furthermore, we attribute a specific role for TLR2 and TLR4 in the chemokine-mediated leukocyte trafficking into the CNS. In summary, secondary bacterial co-infection in a murine model of HIV-1 infection and drug abuse exacerbated peripheral immune cell trafficking, thus disrupting neuroimmune homeostasis, thereby contributing to HAND.
Materials and methods
Experimental animal
Experiments were conducted on 8- to 12-week-old male mice. Wild-type B6CBAF1 (wt), μ-opioid receptor knockout (MORKO), FVB/N luciferase transgenic, and Toll-like receptor 2 and 4 knockout (TLR2KO and TLR4KO) mice were obtained from Jackson Laboratory (Bar Harbor, ME) and maintained in pathogen-free animal housing facilities with a constant temperature (22 ± 1 °C) and humidity (50 %) and with a regulated 12-h light/dark cycle. Animals were housed three mice per cage and given standard food and tap water ad libitum. Animal studies were approved by the Institutional Animal Care and Use Committee at the University of Minnesota. All procedures are in agreement with the guidelines set forth by the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals.
Treatment
Mice were treated according to the procedure explained in our previously published paper with slight modification [
20]. Briefly, mice anesthetized with isoflurane (Halocarbon Products Corp., River Edge, NJ) were subcutaneously implanted either with placebo or with a slow-release morphine (25 mg, NIDA, Rockville, MD) pellet (blood levels are 0.9–1.6 μg/ml at steady state) and then injected intravenously (i.v.) with recombinant full-length HIV-1 Tat protein (82 aa, Immunodiagnostic Systems Inc., Gaithersburg, MD) with a dose of 10 μg/kg [
20‐
22]. To model an opportunistic infection in these mice, animals were inoculated intraperitoneally (i.p.) with
S. pneumoniae serotype 3 strain (American Type Culture Collection, ATTC 6303, Manassas, VA) with a dose of 1 × 10
3 colony-forming units (CFUs) in phosphate-buffered saline (PBS; 0.01 M, 500 μl) 24 h following morphine treatment. Five days following infection, animals were sacrificed and tissues were harvested for ex vivo analysis.
Bioluminescence imaging and bacterial translocation
Morphine- or placebo-treated mice were inoculated intraperitoneally with luciferase-tagged
S. pneumoniae serotype 3 (Xen10, Xenogen Corporation, Alameda, CA) at a dose of 1 × 10
3 CFUs/500 μl PBS in the presence or absence of HIV-1 Tat protein. At day 5, in live animals, bacterial clearance and dissemination into the CNS were imaged using Xenogen’s IVIS CCD camera system [
23]. Total photon emission from selected and defined areas within the images of each mouse was quantified as photons/s/cm
2 using Living Image (Xenogen) and Igor (WaveMetrics, Lake Oswego, OR) image analysis software.
Luciferase activity
To measure luciferase activity in brain tissues, we used a luminometer (TD-20/20, Turner Designs Inc., Sunnyvale, CA). Animals were placed under gas anesthesia using isoflurane (2–2.5 %) and cardially perfused with ice-cold PBS (0.01 M). After perfusion, animals were sacrificed and PBS-perfused brains were aseptically removed and homogenized in lysis buffer (Promega Corporation, Madison, WI). After centrifugation at 10,000 g for 10 min, the supernatant of the brain lysate was collected and mixed with the substrate luciferin (1:4; Promega Corporation); luminescence was measured for 15 s by the luminometer. The total protein concentration of the brain homogenates was determined by a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific Inc., Rockford, IL) to normalize luciferase activity.
Adoptive transfer
Translocation of immune cells into the CNS of animals treated with or without morphine, HIV-1 Tat protein, and
S. pneumoniae was examined using adoptive transfer experiment. HIV-1 Tat protein (10 μg/kg) [
20,
22] was given i.v
. in age- and sex-matched donors, FVB/N luciferase transgenic mice (luciferase expression driven by the β-actin promoter; Xenogen Corporation, Alameda, CA) and recipient mice (B6CBAF1), after subcutaneous morphine (25 mg) and placebo pellet implantation. Twenty-four hours post pellet implantation,
S. pneumoniae (1 × 10
3 CFUs/500 μl) was given i.p. The spleens were aseptically removed from FVB/N luciferase transgenic mice at day 4 and splenocytes were adoptively transferred into wild-type mice as described elsewhere [
24]. Briefly, the harvested spleens were homogenized and red blood cells were lysed using ammonium chloride lysing reagent. Mixed luciferase-positive immunocytes (1 × 10
7 cells per mouse) were transferred via tail vein injection into the recipient mice at day 4. After 24 h, luciferase substrate (D-luciferin, 150 μg, Gold Biotechnology, St. Louis, MO) was administered by i.p. injection, mice were imaged using Xenogen’s IVIS CCD camera system, and data were acquired using a 5-min exposure window. The total photon emission of each mouse was quantified as described in the “Bioluminescence imaging and bacterial translocation” section. Further, luciferase activity in brain tissues was measured as described in the “Luciferase activity” section.
Immunohistochemistry
Prefrontal cortex (PFC) brain regions perfused with ice-cold PBS were divided into three different tissue sections and snap frozen in liquid nitrogen for different experiments, confocal imaging, RT-PCR, and Western blot. In HIV/AIDS infection, the PFC is especially important; it undergoes disease-associated changes in synaptic tone that produce abnormal neurocognitive phenotypes which strongly resemble many of the behavioral anomalies that occur in HAND [
25‐
27]. For immunohistochemistry experiments, brain coronal cryostat (Leica Microsystems Inc., IL) sections (5 μm) were prepared from groups of snap-frozen brain tissue. For intracellular immunostaining, cryostat tissue sections were fixed with 4 % paraformaldehyde, then permeabilized with 0.2 % PBS-Triton X, and blocked with PBS-BSA (5 %) [
25‐
27] for 1 h at room temperature. Slides were washed with PBS-Tween (PBS-T; 0.02 %) and subsequently stained with required antibodies.
To identify the cellular or acellular mode of S. pneumoniae trafficking into the CNS, confocal microscopy was used. Mouse anti-CD45, anti-CD3, and anti-Ly6C (BD Biosciences; dilution 1:500) and rabbit anti-S. pneumoniae (Serotec; dilution 1:500) were used as primary antibodies. Alexa Fluor 488-conjugated anti-mouse IgG (Invitrogen; dilution 1:1000) and Rhodamine Red goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories; dilution 1:1000) were used as secondary antibodies.
Chemokine ligands (CXCL12 and CCL5) and their co-localization with glia, astrocytes, and microglia were also observed using confocal microscopy. Mouse anti-CCL5 and mouse anti-CXCL12 were used as primary antibodies (eBioscience; dilution 1:200), and Alexa Fluor 488-conjugated anti-mouse IgG (Invitrogen; dilution 1:1000) was used as a secondary antibody. To identify glia, Alexa Fluor 555-conjugated glial fibrillary acidic protein antibody (GFAP; Cell Signaling Technology; dilution 1:1000) was used as an astrocyte marker and rabbit anti-ionized calcium-binding adapter molecule 1 (IBA-1; Wako, CA; dilution 1:200) was used as a microglia marker. Rhodamine Red goat anti-rabbit IgG was used as a secondary antibody for microglia detection (Jackson ImmunoResearch Laboratories).
After staining with specific antibodies, tissues were washed with PBS-T and mounted using ProLong Gold antifade reagent with 4′,6′-diamidino-2-phenylindole (DAPI; nuclei marker; Invitrogen; dilution 1:5000) and dried overnight in the dark. Immunofluorescence images were obtained on a Nikon Eclipse Ti confocal microscope using a ×60 oil immersion objective. Sectioning was performed on a minimum of five random sections from each of six individual mice per experiment.
Isolation of brain leukocytes and flow cytometry
For immunophenotyping of brain-infiltrated leukocytes, we used flow cytometry. At the end of the treatment period, mice were lethally anesthetized using CO2 asphyxiation and the brains were removed after being cardially perfused with ice-cold PBS as described in the “Luciferase activity” section. Brain tissue was cut into small pieces and enzymatically digested using collagenase (Liberase, Roche) and single-cell suspension separated by conventional 40/70 Percoll centrifugation. Mononuclear single cells were incubated with purified rat anti-mouse CD16/CD32 (2.4G2) (BD Biosciences) for 10 min at 4 °C to reduce the nonspecific binding to the Fcγ receptor. Cells were subsequently incubated for 30 min at 4 °C with monoclonal antibodies (10 μg/ml) ACPCy7-CD45 (30-F11), PECy7-CD11b (M1/70), PerCP-CD3 (145-2C11), and phycoerythrin-conjugated Ly6C (AL-21) (all from BD Biosciences).
To detect chemokine receptors on CNS immune cells, we used biotinylated CCR5 (C34-3448) and CXCR4 (2B11/CXCR4) antibodies (10 μg/ml) as primary antibodies; the secondary antibody was streptavidin conjugated with PerCP/PE. To stain TLRs on immune cells, we used Alexa Fluor 488-conjugated anti-mouse TLR4 (UT41) and FITC-TLR2 (6C2) (eBioscience; dilution 1:200). The cells were washed twice and resuspended in 200 μl PBS with 0.1 % sodium azide. Dead cells were excluded by TROPO3 (Invitrogen) staining, and specific isotype controls (BD Biosciences) were used in parallel. Cells (10,000 events) were acquired using a FACSCanto II cytometer, and data were analyzed using Diva software (BD Biosciences).
RT-PCR
Total RNA was extracted from frozen perfused brain tissue using TRIzol reagent (Life Technologies, Carlsbad, CA). RNA was reverse-transcribed using random hexamers from the TaqMan Reverse Transcription Reagents and RT Reaction Mix (Applied Biosystems, Inc., Foster City, CA) in a total volume of 40 μl at 25 °C for 10 min, at 42 °C for 30 min, and at 94 °C for 5 min on a PCR machine (Mastercycler gradient, Eppendorf, Germany). The resulting cDNA was used as a template for RT-PCR. Specific oligonucleotide primer sequences used in the amplification of chemokine ligand genes were CCL5 and CXCL12 (Integrated DNA Technologies, Inc, Coralville, IA). Details of primer sequences are given in Table
1. Real-time PCR was performed using SYBR Green Master Mix (Applied Biosystems) on the ABI Prism 7500 sequence detection system (Applied Biosystems). 18S rRNA served as an internal control in relative RT
-PCR
. The relative levels of chemokine ligands in the brain homogenate were quantified using the ΔCt-ΔCt method and were expressed as the percentage-fold change.
Table 1
Primers used for quantitative real-time RT-PCR
CCL5 | CCC TCA CCA TCA TCC TCA CT | TCC TTC GAG TGA CAA ACA CG |
CXCL12 | TCA GTG GCT GAC CTC CTC TT | TTT CAG CCA GCA GTT TCC TT |
18S | TTG ACG GAA GGG CAC CAC CAG | CTC CTT AAT GTC ACG CAC GAT TTC |
Western blot
Semi-quantitative estimation of CCL5 and CXCL12 chemokine ligand levels in the mouse brain was done using Western blot. Proteins were harvested from frozen brain tissues using RIPA cell lysis buffer (Biotech Corporation). The protein concentrations were measured by using BCA protein assay kit. Equal aliquots of total SDS-soluble proteins (100 μg) were resolved to 15 % discontinuous SDS-PAGE. The transferred nitrocellulose membranes were blocked with 5 % fat-free milk in PBS-T (blocking buffer) at room temperature for 1 h and then incubated with primary antibodies (mouse anti-CCL5 and rabbit anti-CXCL12 or anti-β-actin antibody (eBioscience; 1:1000 dilution) at 4 °C overnight. After incubating with primary antibodies, the membranes were washed with PBS-T three times. Then the membranes were incubated for 1 h with IRDye 800CW-conjugated goat anti-rabbit IgG and IRDye 680-conjugated goat anti-mouse IgG secondary antibodies (LI-COR Biosciences, Lincoln, NE) diluted in blocking buffer. The blots were then washed three times with PBS-T and rinsed with PBS. Proteins were visualized by scanning the membrane on Odyssey Infrared Imaging System (LI-COR Biosciences) with both 700- and 800-nm channels. ImageJ software was used to quantify the band intensity. The protein levels of CCL5 and CXCL12 in the CNS were represented by the ratios of optical densities in their bands normalized against β-actin.
Statistical analysis
SPSS 16.0 statistical software was used for statistical analysis. Data were collected from three independent experiments and expressed as mean ± SEM. Statistical significance was calculated by one-way ANOVA followed by Bonferroni’s multiple-comparison test among different groups. For correlation analysis, the Spearman rank order correlation coefficient was used. Results were considered significant at P ≤ 0.05.
Discussion
Opioid use and abuse accelerates HIV-1-associated neurocognitive disorders (HAND) [
36,
37]. Peripheral leukocyte recruitment into the CNS has been implicated in neuropathogenesis associated with neuroAIDS [
3,
8,
20,
32,
33,
38,
39]. However, the detailed mechanism underlying this process is unclear. The exacerbated induction of proinflammatory mediators from activated leukocytes into the CNS through repetitive episodes of systemic infection contributes to neurocognitive deficits often observed in neurodegenerative diseases, such as Alzheimer’s and Parkinson’s [
5,
40,
41].
In the current study, we demonstrate that chronic administration of morphine either independently or together with HIV-1 Tat protein resulted in persistent bacterial infection in mice. This finding is consistent with our earlier findings where opiate abuse resulted in decreased bacterial clearance and increased susceptibility to opportunistic infection [
42]. In addition, in our current study, we also delineated the specific roles of morphine and HIV-1 Tat on differential TLR expressions that contribute to distinct leukocyte recruitment into the CNS following systemic bacterial infection. We also demonstrate a Trojan horse mechanism for bacterial dissemination across the blood-brain barrier into the CNS by monocytes.
Morphine treatment results in attenuated bacterial killing following
S. pneumoniae infection. The regulatory mechanism associated with this outcome is either impaired TLR9-NF-κB signaling or blunted phagocytic properties of immune cells following exposure to morphine [
23,
43]. Compromised bacterial killing increased bacterial load and resulted in persistent systemic infection. Morphine induced inhibition of systemic bacterial clearance leading to increased bacterial burden following their dissemination into the CNS which was significantly abolished in the MORKO mice, suggesting that morphine-mediated augmentation of
S. pneumoniae infection was mediating through μ-opioid receptors. Furthermore, in wild-type animals, we observed higher bacterial dissemination into the CNS of mice treated with HIV-1 Tat alone. A possible mechanism for the increased bacterial dissemination with HIV-1 Tat-treated animals is significant disruption of blood-brain barrier integrity following HIV-1 Tat treatment, as reported earlier [
44].
Furthermore, recent studies by Olin et al. [
45] show profound effects on splenocyte infiltration into the CNS in an inflammation model in the presence of chronic morphine. Their finding is consistent with our present finding where we observed a significant infiltration of peripheral immune cells into the CNS of animals treated with morphine and/or HIV-1 Tat protein following systemic infection with
S. pneumoniae. The characterization of phenotypic markers on live immune cells revealed a differential influx of T lymphocytes (CD3+) and inflammatory monocytes (Ly6C+) into the CNS
. HIV-1 Tat alone resulted in CNS trafficking of both CD3+ T lymphocytes and Ly6C+ inflammatory monocytes. Surprisingly, systemic bacterial infection resulted in dramatic trafficking only of T lymphocytes. Although morphine treatment alone did not alter the trafficking of any immune cell, we observed robust and synergistic CNS trafficking of both T cells and inflammatory monocytes when morphine-treated animals were exposed to HIV-1 Tat and co-infected with
S. pneumoniae.
T lymphocytes are considered to be neuroprotective in CNS damage, but their deleterious role in myelin degradation in various autoimmune diseases has also been reported [
46,
47] through activation of inflammatory cytokines such as IFN-γ and IL-17. We previously reported that the synergistic effect of chronic morphine with
S. pneumoniae and HIV-1 Tat increased proinflammatory cytokine (TNF-α, IL-6, and MCP-1) synthesis in the CNS [
20]. We now demonstrate exacerbated T cell and inflammatory monocyte recruitment into the CNS following morphine,
S. pneumoniae, and HIV-1 Tat treatment, suggesting that migrated immune cells contribute to the inflammatory milieu, disrupting CNS hemostasis, eventually resulting in neuropathology.
The contributing role of chemokines and their receptors in potentiating leukocyte trafficking during infection is well documented. In the present study, we demonstrated that HIV-1 Tat alone resulted in increased CCR5 expression on T lymphocytes with their subsequent trafficking into the CNS. We further showed that systemic bacterial infection modulated the trafficking of both T lymphocytes and monocytes by activating the expression of both CXCR4 and CCR5. However, the activation of chemokine receptors alone is not sufficient for the migration of leukocytes. Rather, a highly regulated network of chemokine gradient was needed for directing cell trafficking. Exaggerated production in the microglia of the cognate ligands (CXCL12 and CCL5) for the corresponding chemokine receptors (CXCR4 and CCR5) provided chemical gradients for attracting peripheral leukocytes to the site of expression. Detailed analyses of our data revealed that morphine induced an increase in CXCL12 generation in the presence of systemic infection. However, the generation of CCL5 is predominantly a HIV-1 Tat effect. When all three insults are on board, a synergistically increased production of both chemokine ligands in the CNS resulted in a massive infiltration of activated peripheral immune cells into the brain. Excessive leukocyte recruitment with surplus chemokine production in the CNS has been shown to trigger neuronal injury and death via overactivation of p38 MAPK [
48].
Surprisingly, we found that chronic morphine, either alone or with HIV-1 Tat, did not result in the trafficking of any immune cells, even though it significantly increased the surface expression of CCR5 on T lymphocytes. A potential explanation lies in the heterodimerization and cross-desensitization of the chemokine CCR5 receptor with μ-opioid receptors in the presence of morphine, which can alter the efficiency of ligand binding and signaling, thereby contributing to decreased chemotaxis [
49]. Moreover, it has been previously shown that CD3+ T lymphocyte trafficking is mediated primarily through the CXCR4 chemokine receptor [
9]. In our study, morphine, primarily, in the presence of HIV-1 Tat downregulated both CXCR4 expression and the production of its cognate ligand, CXCL12, in the CNS, thereby explaining the decreased T cell trafficking into the CNS, although the expression of CCR5 and its cognate ligand was high.
Numerous studies including ours have suggested that morphine modulates various intracellular signaling elements of TLR pathways or directly binds to MD-2, thereby inducing TLR4 oligomerization and activating TLR4 signaling [
20,
50,
51]. We previously demonstrated that morphine potentiated TLR2 and TLR4 expression on microglial cells leading to significant induction of proinflammatory cytokines with a concurrent increase in reactive oxygen species, nitric oxide production, and caspase-3 activation, thereby contributing to neuronal damage [
20]. In the current study, we demonstrated that chronic morphine in the presence of HIV-1 Tat and
S. pneumoniae resulted in TLR2 activation on T lymphocytes (Fig.
7). However, we observed induction of both TLR2 and TLR4 on inflammatory monocytes. Activated TLRs induce upregulation of proinflammatory transcription factors (e.g., NF-κB, activator protein 1 (AP-1), signal transducer and activator of transcription (STAT)) which drives promoters of proinflammatory cytokines and chemokines that play a critical role in the pathophysiology of neuroAIDS [
18,
19,
52].
To the best of our knowledge, the present study is the first to clearly demonstrate the distinct role of TLRs in chemokine-induced peripheral leukocyte trafficking into the CNS of HIV-infected opioid-dependent animals particularly in the presence of systemic pneumonia infection. Data shows that chronic morphine treatment in the presence of HIV-1 Tat resulted in significant CNS trafficking of Ly6C+CCR5+ and Ly6C+CXCR4+ following co-infection with systemic
S. pneumoniae infection, an effect that was significantly attenuated in both TLR2KO and TLR4KO mice. Significantly less monocyte (the bacterial transporters) trafficking into the CNS results in reduced
S. pneumoniae dissemination into the CNS of TLR2KO and TLR4KO mice as reported previously [
20]. Further, the generation of cognate chemokine ligands CCL5 and CXCL12 is significantly attenuated in both TLR2KO and TLR4KO mice. HIV-1 Tat treatment activates TLR2 induction of CCL5 ligand and mediates the CNS trafficking of CD3+CCR5+ T lymphocyte. Till date, HIV-1 Tat protein is reported to activate TLR4 on monocytes which contribute to abnormal hyper-activation of the immune system via TNF-α production [
53]. The present investigation suggests that TLR2 activation by HIV-1 Tat on T lymphocytes might also play a significant role. Further, TLR2 signaling upregulates T cell infiltration and microglial expansion through the MyD88-dependent pathway [
35].
Taken together, our study shows that chronic morphine treatment results in significant upregulation of TLR2 and TLR4 expression, both on peripheral immune cells and on microglia. Subsequent activation of TLRs by HIV-1 Tat and/or S. pneumoniae in morphine-treated mice results in upregulation of chemokine receptor expression on peripheral immune cells, with a concurrent increase in their cognate ligand secretion by microglial cells. A combination of receptor and ligand expression in different compartments induces migration of peripherally activated peripheral leukocytes into the CNS. The resulting exacerbated neuroinflammatory responses might be a contributing factor in HIV-1-associated neuropathogenesis observed in the drug-abusing population that are HIV positive. These results also indicate that systemic infection with Gram-positive bacteria may potentiate neuropathogenesis in this population. Therapeutic interventions that target TLR activation could be targeted to mitigate neuroinflammation associated with neuroAIDS.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
RD performed the experiments. RD and SR designed the research, analyzed the data, and drafted the manuscript. Both authors read and approved the final manuscript.