Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Immunol Invest. Author manuscript; available in PMC 2012 Feb 27.
Published in final edited form as:
PMCID: PMC3287069

Toll-like receptor expression and activation in astroglia: differential regulation by HIV-1 Tat, gp120, and morphine


In this study we determine whether morphine alone or in combination with HIV-1 Tat or gp120 affects the expression of Toll-like receptors (TLRs) by astrocytes and to assess whether TLRs expressed by astrocytes function in the release of inflammatory mediators in vitro. TLR profiling by immunofluorescence microscopy, flow cytometry, in-cell westerns, and RT-PCR showed that subpopulations of astrocytes possessed TLR 2, TLR3, TLR4, and TLR9 antigenicity. Exposure to HIV-1 Tat, gp120, and/or morphine significantly altered the proportion of TLR-immunopositive and/or TLR expression by astroglia in a TLR-specific manner. Subsets of astroglia displayed significant increases in TLR2 with reciprocal decreases in TLR9 expression in response to Tat or gp120 ± morphine treatment. TLR9 expression was also significantly decreased by morphine alone. Exposing astrocytes to the TLR agonists LTA (TLR2), poly I:C (TLR3), LPS (TLR4) and unmethylated CpG ODN (TLR9) resulted in increased secretion of MCP-1/CCL2 and elevations in reactive oxygen species. TLR3 and TLR4 stimulation increased the secretion of TNF-α, IL-6, and RANTES/CCL5, while activation of TLR2 caused a significant increase in nitric oxide levels. The results suggest that HIV-1 proteins and/or opioid abuse disrupt the innate immune response of the central nervous system (CNS) which may lead to increased pathogenicity.

Keywords: innate immunity, opioid abuse, cytokines, free radicals, HIV-1 proteins, glia, neuroplasticity


Alteration of the innate immune response and the corresponding release of inflammatory molecules are pivotal in the progression of many central nervous system (CNS) diseases including neuroAIDS. The inflammatory process is characterized by microglia-mediated superoxide production, nitric oxide (NO) release, and the release of pro-inflammatory cytokines and chemokines by both activated microglia and astrocytes (Akira and Kishimoto, 1992; Becher et al., 2000; Fuller et al., 2009; McGeer and McGeer, 2004). Toll like Receptors (TLRs), a major family of the pattern-recognition receptors (PRRs) that recognize conserved viral, bacterial, and fungal particles (Bsibsi et al., 2002; Iwasaki and Medzhitov, 2010; Medzhitov and Janeway, 1999; Takeda et al., 2003) are expressed in glia (microglia, astrocytes, and oligodendrocytes) and neurons (Bsibsi et al., 2006; Carpentier et al., 2005; Jack et al., 2005; Laflamme and Rivest, 2001; Lafon et al., 2006; McKimmie and Fazakerley, 2005; Rivieccio et al., 2006). The presence of CNS pathogens activate TLRs, whose signals are amplified (Akira et al., 2003; Jack et al., 2006; Lafon et al., 2006; Zekki et al., 2002) through MyD88-dependent and/or Toll/Interleukin-1 receptor (TIR) domain-containing adapter-inducing interferon-β (TRIF)-dependent pathways (Bafica et al., 2004; Doyle et al., 2002; Yamamoto et al., 2003).

Drug abuse with injectable opioids, such as heroin, is a major cause of the spread of human immunodeficiency virus type-1 (HIV-1) (Hauser et al., 2007; Nath et al., 2002; Sheng et al., 1997). In addition to disease propagation, histopathologic evidence suggests that chronic opioid abuse intrinsically enhances the severity of CNS complications of HIV-1 by almost four-fold (Bell et al., 1998), which is noteworthy since 15–40% of non-substance abusing AIDS patients are estimated to display significant HIV encephalitis (HIVE). Morphine, acting through μ-opioid receptors (MORs) on astrocytes, can potentiate Tat-induced increases in intracellular Ca2+, NF-κB activation, and in the release of IL-6, RANTES, and MCP-1 (El-Hage et al., 2005, 2006a, 2006b, 2008a), which directly affects macrophage recruitment and activation (Takeda et al., 2003). In independent research utilizing CCR2 or RANTES (CCL5) knockout mice, we showed that deletion of either gene alone is sufficient to reduce Tat ± morphine-induced astrogliosis and microgliosis, suggesting that astroglia contribute to the intensification of HIV-1 neuropathogenesis in opioid abusers (El-Hage et al., 2008a, 2008b; Gendelman et al., 1997; Kaul et al., 2001).

Until recently, only limited studies have evaluated the role of TLR-pathogen interactions in regulating HIV-associated disease. HIV-1 may interact directly with specific TLRs (Akira et al., 2003; Bsibsi et al., 2006; Salaria et al., 2007), or indirectly by affecting the TLR response of the host to other co-infecting pathogens, while HIV-1 uses the host defense to its advantage to infect immune cells and alter the immune response (Dong and Benveniste, 2001). In addition to the highly conserved structural motifs, known as pathogen-associated molecular patterns (PAMPs) that bind TLRs, several endogenous molecules reportedly activate the innate immune system in a TLR-dependent manner (Kariko et al., 2004; Tsan and Gao, 2004). Recent studies have suggested that opioid antagonists can activate glia through direct interactions with TLR4 and that this receptor serves overlapping pain functions with opioids (Hutchinson et al., 2007, 2008), inferring an antinociceptive role for TLR4 or the overlap of opioid and TLR4 signaling pathways.

Collectively, the established role of TLRs in innate immunity and in the response to HIV-1, combined with reported interactions of TLRs with opioids and opioid pain pathways, prompted us to explore the possible interactive effects of opioids and HIV-1 proteins on TLR expression and evaluated the consequences of TLR activation on free radical production and cytokine release by astrocytes. The findings confirm the presence of functional TLR expression by astrocytes and further implicate astrocytes as contributing to CNS immunity and inflammation. We found that exposure to morphine, HIV-1 Tat and/or gp120 is associated with changes in TLR expression, which may partially explain how opioids meditate the response of the CNS to the effects of HIV-1 and perhaps other pathogens.



Morphine sulfate was obtained from the National Institute on Drug Abuse (NIDA, Drug Supply System, Bethesda, MD). Tat1–72 recombinant protein was obtained from Phil Ray (University of Kentucky) and recombinant X4-tropic gp120 HIVIIIB was obtained from Immunodiagnostics, Inc. (Woburn, MA). The following ligands were used: TLR2 ligand: lipoteichoic acid (LTA; 0.1–100 μg/ml; InvivoGen, San Diego, CA); TLR3 ligand: polyinosinic–polycytidylic acid (Poly I:C; 0.1–25 μg/ml; Sigma–Aldrich, St. Louis, MO); TLR4 ligand: lipopolysaccharide (LPS; 1–25 μg/ml; Sigma); TLR5 ligand: recombinant flagellin (1–10 μg/ml; InvivoGen); and TLR9 ligand: unmethylated CpG containing oligonucleotides (ODN; 0.5–5 μM; InvivoGen). The specific concentrations of TLR ligands used in these studies were based on previous in vitro studies from others (Ashkar et al., 2003, 2004; Gill et al., 2006; MacDonald et al., 2007). Antibodies directed against the TLRs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and anti-GFAP was purchased from Chemicon (Ballerica, MA). Goat anti-mouse IRDye 800CW and goat anti-rabbit IRDye 680 were purchased from LI-COR Biosciences (Lincoln, NE), and goat anti-mouse TRITC and goat anti-rabbit FITC were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The specific concentrations of the primary and secondary antibodies used in these studies were based on manufacturer recommendations.

Cell culture

Astroglial-enriched cultures were prepared using one- to four-day-old ICR mice (Charles River Laboratories, Charles River, MA) as previously described (Stiene-Martin et al., 1998). Briefly, mice were anesthetized and euthanized according to NIH and IACUC guidelines. Striata were aseptically isolated, minced in media, incubated with trypsin/DNAse (37°C, 30 min), triturated through a series of decreasing bore pipettes and filtered sequentially through 135 μm and 35 μm pore nitex filters. Cells for each experiment were pooled from the striata of two to four mice. Growth medium favoring astroglial enrichment consisted of DMEM (Dulbecco’s Modified Eagle’s Medium, Gibco, Grand Island, NY). DMEM was supplemented with glucose (27 mM), Na2HCO3 (6 mM), 10% (v/v) fetal bovine serum (FBS; JRH Biosciences, Lenexa, KS or Hyclone, Logan, UT) and penicillin/streptomycin (50 U/50 μg). Cells were grown for 10–14 days until they reached 80–90% confluency at 37°C, 5% CO2. To obtain purified astrocytes, glial cells were incubated for 90 min in 10 mM L-leucine methyl ester (LME) (Sigma-Aldrich). After addition of LME, cultures were visually inspected to ensure maximal microglial lysis with minimal toxicity to astrocytes. Astrocytes were then washed and resuspended in glial culture medium. Purity of astrocyte cultures was determined by the percentage of GFAP-immunofluorescent cells by flow cytometry and was routinely ≥ 95%.

Experimental treatments

Recombinant Tat1–72 was produced and purified as described previously (El-Hage et al., 2005). Briefly, the tat gene encoding the first 72 amino acids of HIV-1BRU (obtained from Dr. Richard Gaynor, through the NIH AIDS repository) was inserted into an Escherichia coli Pin Point Xa-2 vector (Promega, Madison, WI). Biotinylated Tat was purified on a column of soft release avidin resin, cleaved from the fusion protein using factor Xa, eluted and desalted using a PD10 column and treated with Detoxi-Gel (Pierce, Rockford, IL). A reticulocyte amoebocyte lysate assay was used to insure the absence of trace amounts of endotoxin (Associates of Cape Cod, Inc. East Falmouth, MA). Cells were continuously exposed to untreated medium (control) or medium containing morphine sulfate (500 nM), Tat1–72 (100 nM), or gp120 (500 pM) alone or in combination or alternatively with escalating concentrations of TLR2, 3, 4, or 9 ligands LTA (0.1, 1, 10, and 100 μg/mL), Poly I:C (5, 10, and 25 μg/mL), LPS (1, 5, 10, and 25 μg/mL), or ODN (0.5, 1, 2.5 and 5 μM), respectively at various time points. The drug and viral protein concentrations used were based on previously published values.


Cells were treated with media alone or media containing morphine or HIV-1 proteins alone or in combination for 30 min, 6 h or 12 h. For semi-quantitative RT-PCR, total RNA was isolated from treated cells using GenElute Mammalian Total RNA kit (Sigma). cDNA was synthesized from 1 μg of total RNA using the RETROscript kit from Ambion (Austin, TX). PCR was performed using Hot Master Taq DNA Polymerase and buffers from 5-prime (Gaithersburg, MD) and primer sets for TLR2: 3′-AAG TGA AGA GTC AGG TGA TGG ATG TCG, 5′-GCA GAA TCA ATA CAA TAG AGG GAG ACG; TLR3: 3′-TCT GGA AAC GCG CAA ACC, 5′-GCC GTT GGA CTC TAA ATT CAA GAT; TLR4: 3′-CAA GTT TAG AGA ATC TGG TGG CTG TGG, 5′-TGA AAG GCT TGG TCT TGA ATG AAG TCA; TLR9: 3′-CCA CAC CAA TGC CTT TCA GAA, 5′-TGG CTT CTG ACA GCG TTG AAG. RT-PCR was performed using the PTC-200 (MJ Research). TLR mRNA was normalized to β-actin.

In-cell Westerns

Cells were grown on 24-well plates and treated with morphine or HIV proteins alone or in combination for 30 min, 6 h, or 12 h. Subsequently, media was removed and cells were washed with cold PBS, fixed in 4% paraformaldehyde, permeabilized with PBS containing 0.1% Triton-X 100, 0.1% BSA and blocked in 1.5% goat serum in PBS for 1 h. Simultaneous incubation of primary antibodies towards each TLR with anti-β-actin antibody was performed at 4°C overnight in blocking buffer. Reaction products for each TLR (visualized with IRDye 680LT; LI-COR Biosciences, Lincoln, NE) were normalized to β-actin (visualized with IRDye 800CW; LI-COR) using appropriate secondary antibodies. Fluorescence measurements were performed on the LI-COR Odyssey system.


Cells were grown on coverslips and treated with media alone or media with LTA, Poly I:C, LPS, recombinant flagellin, or ODN for 12 h. A prior report indicated that pre-exposure ot TLR ligands could upregulate TLR antigenicity and hence their detection in astrocytes (Salaria et al., 2007). Cells were fixed in 4% paraformaldehyde, permeabilized in PBS containing 0.1% Triton-X 100 / 0.1% BSA and blocked in 1.5% goat serum in PBS for 1 h. Cells were incubated with anti-TLR and anti-GFAP antibodies overnight at 4 °C in blocking solution followed by 1 h incubation with goat anti-mouse TRITC and goat anti-rabbit FITC at room temperature. Nuclei were stained with 1 μg/ml Hoechst 33258 in PBS for 20 min. Fluorescently labeled cells were visualized using a Zeiss AxioObserver Z.1 microscope, AxioVision software and MRm digital camera. Fluorescent images were deconvolved using AutoQuant X software (Media Cybernetics; Bethesda, MD).

Cytokine release assay

Cells were treated with media alone or media with Tat or increasing concentrations of TLR agonists for 30 min, 6 h or 12 h on 24-well plates. Conditioned medium from treated cells was collected post-treatment, after which cytokines were assayed using ELISAs for IL-6, TNF-α, RANTES and MCP-1 (R&D Systems; Minneapolis, MN) according to the manufacturer’s instructions. Tetramethylbenzidine substrate (BD Pharmingen, San Diego, CA) was added for color development and microplates were read at 450 nm using a Victor 3 plate reader (PerkinElmer, Inc; Waltham, MA) immediately after terminating the reaction. Cytokine levels were determined based on a standard curve with known amounts of cytokine.

Reactive oxygen species (ROS) assay

Intracellular ROS production was measured using dihydrofluorescein diacetate (DCFH-DA, Invitrogen) for cells treated with media alone, media with Tat, or increasing concentrations of TLR agonists. Cells grown in 48-well plates were treated with 10 μM of cell-permeant dye for 45 min at 37°C in Hank’s balanced salt solution (HBSS), followed by a wash with HBSS and exposure to Tat and TLR2, TLR3, TLR4 or TLR9 ligands for 90 min. ROS production was measured using a Victor 3 plate reader (PerkinElmer, Inc., Waltham, MA) at λex = 488 nm and λem = 525 nm.

Nitric oxide production assay

Cells were treated with media alone, media with Tat, or increasing concentrations of TLR ligands for 24 or 48 h. At the indicated times, the supernatant was collected and the concentration of nitrite (the oxidized metabolite of NO) was assessed using the Griess reaction (Griess, 1879) (Promega, Madison) according to the manufacturer’s instructions. Briefly, conditioned medium from treated astrocytes was added to 96-well plates along with known standards made with sodium nitrite solution. Equal volumes (50 μL) of conditioned media, sulfanilamide solution and N-1-napthylethylenediamine dihydrochloride (NED) solution were mixed and incubated at room temperature for 10–15 min, after which the absorbance at 540 nm was measured with a Victor 3 plate reader. The concentration of NO in samples was calculated based on the standard curve using known concentrations of nitrite.

Flow cytometry

Astrocytes were washed in PBS/0.1% BSA, and labeling was done in permeabilization buffer (PBS/0.1% BSA/0.1% Triton X-100) for detection of intracellular protein expression. TLR fluorescence associated with astrocytes was measured using a FACSCanto II (BD Biosciences) flow cytometer. TLR fluorescence intensity was assessed after gating on approximately 10,000 GFAP-positive astrocytes. Astrocyte autofluorescence was compensated by setting the phycoerythrin (PE) detector voltage to a minimum level that discriminates between autofluorescence and specific labeling in both negative and positive controls. Isotype control antibodies were used to define settings in histogram plot analyses. TLR expression is presented as a proportion of GFAP positive astrocytes.

Statistical analysis

All results are reported as mean ± SEM of at least 3 (typically 4–6) independent experiments. Data were analyzed using one-way analysis of variance (ANOVA) followed by appropriate post hoc tests. Resulting p values less than 0.05 were considered significant. Statistica Software (StatSoft; Tulsa, OK) was used for data analysis.


Localization of TLRs in individual GFAP-expressing astrocytes reveals phenotypically distinct subpopulations

The expression of TLRs by astrocytes has previously been described both in vivo and in vitro (Bowman et al., 2003; Hauser et al., 2007; McKimmie and Fazakerley, 2005; Park et al., 2006). However, these reports disagree greatly regarding whether a particular TLR is expressed by astrocytes, which perhaps results from differences in the brain region examined, the source of tissues, culture conditions, and/or the purity of astrocytes in different experiments. Specifically, astrocytes cultured from mouse and human brain tissues can display multiple TLR types (i.e., TLR1, TLR2, TLR3, TLR4, TLR5 and TLR9). TLR3, which recognizes dsRNA (Bsibsi et al., 2006; Carpentier et al., 2005; Jack et al., 2005; Lafon et al., 2006; McKimmie and Fazakerley, 2005; Rivieccio et al., 2006), suggesting that astrocytes may be particularly important for anti-viral responses in the CNS. Many viral types have dsRNA genomes or produce dsRNA during their replication cycles. Representative immunofluorescence images derived from a number of experiments from cultured astrocytes showed that subsets of astrocytes can display TLR2, TLR3, TLR4, and TLR9 immunoreactivity (Figs. 12) with diminished levels seen for TLR5 (data not shown). When astrocytes were challenged with cognate ligands for TLR 2, TLR3, TLR4, and TLR9 for 12 h, respectively, there appeared to minor changes in TLR4 and TLR9 immunofluorescent intensity, while TLR2 and TLR3 antigenicity was slightly more pronounced than untreated controls (Fig. 1). This differed from a prior report in human astrocytes indicating that pre-exposure to its cognate ligand dramatically upregulated TLR4 antigenicity in human astrocytes (Salaria et al., 2007). The labeling pattern for TLR2 and TLR3 was punctate, although TLR2 showed a more perinuclear localization within the cytoplasm. By contrast, TLR4 and TLR9 antigenicities were more broadly distributed within the cytoplasm. In addition, antigenicity for a particular TLR was not uniformly present on individual astrocytes. Flow cytometric analysis was used to better quantify the relative level of expression of each TLR in astrocytes. As expected, our flow cytometry data (Fig. 2C–D) correlated with the immunofluorescence findings suggesting that TLR2 (39 ± 2%), TLR3 (59 ± 1%), TLR4 (25 ± 5%), and TLR9 (21 ± 2%) are heterogeneously expressed by astrocytes. Next we determined the extent to which different TLR types might be co-expressed on individual cells. Cells were double-labeled with antibodies to TLR2 and TLR9, TLR3 and TLR9 or TLR4 and TLR9, counterstained with Hoechst 33258, and analyzed (Fig. 2E). TLR9 was readily co-localized with TLR2, TLR3 or TLR4 in subsets of astrocytes (Fig. 2E). Despite the apparent overlap between TLR9 and the other TLRs assessed, not all permutations were performed and it remains uncertain whether other TLRs are co-expressed by astrocytes. All together, these results strongly suggest that phenotypically distinct subsets of astrocytes differ in the expression of one or more TLRs.

Figure 1
Cellular localization of TLR immunoreactivity in astrocytes in the presence and absence of cognate agonists for TLR2 (LTA), TLR3 (Poly(I:C)), TLR4 (LPS), and TLR9 (ODN). TLR antigenicity was co-localized with GFAP immunofluorescence in astrocytes. TLR2, ...
Figure 2
Flow cytometric analysis of TLR immunofluorescence in astrocyte-enriched cultures. A) A plot of forward scatter (FSC-A) vs. GFAP (A). B) A histogram showing the relative differences in TLR2, TLR3, TLR4, and TLR9 fluorescence intensity associated with ...

Analysis of TLR levels in astrocytes treated with morphine and HIV-1 proteins alone or in combination

We examined Tat, gp120 and morphine’s effects on innate immunity and analyzed whether particular astrocytic TLRs might respond to exposure of morphine and HIV-1 proteins. TLR expression levels were quantitatively assessed by In-cell western analysis (Fig. 3A–B). In-cell westerns are a semi-quantitative near-infrared, immunofluorescent assay (LI-COR) to assess the relative abundance of two different proteins within cells in the same culture dish. Importantly, a protein of interest is quantified by normalization to a second protein standard. Astrocytes were treated with morphine, HIV-1 Tat, or gp120 alone or in combination for 30 min, 6 h, or 12 h, TLR fluorescence was normalized to β-actin, and reported as a percent of control levels (Fig. 3A–B). These results showed that 6 h and 12 h exposure to Tat alone or in combination with morphine caused an increase in TLR2 protein levels. Although the effect of the combined treatment of Tat and morphine was less than with Tat alone, it was significantly higher than either control or morphine treatment alone. Likewise, gp120 caused an increase in TLR2 expression above levels seen in control and morphine treated groups, although only a minimal increase in TLR2 expression was detected with combined gp120 and morphine treatment. Conversely, exposure to Tat, morphine, or gp120 alone or in combination significantly suppressed the effect of TLR9 protein levels after 12 h treatment. There were no significant changes in TLR3 and TLR4 transcript levels in astrocytes exposed to Tat, gp120, morphine or a combination thereof (data not shown).

Figure 3
Effects of morphine, HIV-1 Tat, and gp120 on the expression of TLR2 and TLR9 protein and mRNA in astrocyte cultures. A–B) Astrocyte-enriched cultures were treated with morphine, Tat, and gp120 alone or in combination for 30 min, 6 h and 12 h. ...

We further determined whether changes in protein levels correlated with that of mRNA. RNA was extracted from astrocytes treated with morphine and HIV-1 Tat or gp120 proteins alone or in combination for up to12 h and TLR expression was analyzed by RT-PCR. mRNA extracted from viral protein-treated astrocytes alone or in combination with morphine showed significant increases in levels of TLR2 (Fig. 3C). Conversely, after 12 h treatment with HIV-1 proteins or morphine alone or in combination, TLR9 mRNA levels were significantly below control level (Fig. 3D). There were no changes in TLR3 or TLR4 expression in astrocytes exposed to Tat, gp120, morphine or a combination thereof (data not shown). We further explored the possibility that viral protein or opioid drug exposure might change the proportion of TLR2 or TLR9-immunopositive astrocytes using flow cytometry (Fig. 4). Approximately 10,000 events were collected for each sample and size discrimination was used as a crude method for viability determination; one representative histogram of three independent experiments is shown (Fig. 4). Flow cytometric data showed a significant increase in the proportion of TLR2 immunopositive astrocytes following exposure to Tat (42 ± 1), gp120 (46 ± 3), or combined morphine and Tat (50 ± 2) when compared to vehicle-treated controls (33 ± 2) (p < 0.05; one-way ANOVA; Duncan’s post hoc test). By contrast, although the proportion of astrocytes expressing TLR9 tended to decrease following Tat (19 ± 3), gp120 (20 ± 4), morphine (16 ± 1), morphine + Tat (16 ± 1), or morphine + gp120 (20 ± 4), none of the treatments caused a significant decline compared to vehicle-treated controls (27 ± 4). The above numbers represent the average percentage of three independent studies. All together, (i) these results show that morphine ± HIV-1 proteins can influence the expression of TLR on astrocytes, and that (ii) the expression of a particular TLR by astrocytes may be plastic and modifiable by extracellular cues or environmental factors.

Figure 4
Effects of morphine, HIV-1 Tat and/or gp120 on the proportion of TLR2 and TLR9 immunofluorescent astrocytes at 12 h following continuous exposure. Dot plots show that exposure to Tat (42 ± 1), gp120 (46 ± 3), or combined morphine and Tat ...

Induction of inflammatory cytokines and chemokines by astrocytes following TLR treatment

We next examined the functional role of TLR activation in the production of proinflammatory cytokines and chemokines by astrocytes. This included the activation of TLR2, TLR3, TLR4 and TLR9 by specific agonists and comparing the effects of the agonist to that of media alone (negative control) or media with Tat (positive control). Following 30 min, 6 h, or 12 h of ligand treatment, conditioned medium from astrocytes was collected and assayed for the cytokines; IL-6 and TNF-α, and the chemokines; RANTES and MCP-1. Similar profiles of cytokine production were seen at both 6 h (not shown) and 12 h time points (Fig. 5), although the overall cytokine accumulation within the astrocyte-conditioned medium was higher at 12 h compared to 6 h. While a range of concentrations was assessed for each TLR ligand, the response was robust for each, even at the lowest concentrations studied, suggesting there is a very low threshold for PAMP detection by astrocytes. For this reason, one representative concentration of each ligand is shown for LTA (10 μg/mL), Poly I:C (10 μg/mL), LPS (1.0 μg/mL) and ODN (5.0 μM). In comparison to vehicle-treated controls, stimulation of astrocytes with Poly I:C and LPS led to increased levels of TNF-α, IL-6, RANTES and MCP-1 (Fig. 5C–D). Treatment with LTA caused a significant increase of MCP-1 alone (Fig. 5D), while ODN only caused increases in TNF-α and MCP-1 production as compared to control treatment (Fig. 5A, D). In comparing TLR2, TLR3, TLR4, and TLR9 stimulation with Tat-mediated activation of astrocytes, TLR4 signaling was observed to induce the strongest proinflammatory response, characterized by secretion of TNF-α, IL-6, RANTES and MCP-1 (Fig. 5A–D). These results suggest that TLR agonists activate astrocytes and trigger an innate immune response that is not homogenous but rather tailored according to a particular environmental signal.

Figure 5
TLR agonists induce cytokine/chemokine secretion by astrocytes at 12 h following exposure. A–D) HIV-1 Tat (positive control) and TLR2 (LTA), TLR3 (Poly(I:C)), TLR4 (LPS), and TLR9 (ODN) agonists differentially increased the release of TNF-α ...

Nitrite accumulation in astrocytes is largely unaffected by TLR agonists

NO release is involved in many biological functions such as normal and aberrant signaling (Lander et al., 1995). TLR ligands can induce NO production as part of an antiviral response in infected cells (McKimmie and Fazakerley, 2005). Therefore, we evaluated the consequences of TLR-induced activation by examining nitrite formation in astrocytes (Fig. 6). NO production was measured by the Griess assay, which is based on the accumulation of nitrite as the end product of NO metabolism. We previously found that treatment with gp120 or morphine alone or in combination had no significant effect on NO production and therefore their actions were not pursued further in the present study. After 24 h of exposure, Tat or LTA treatment caused significant accumulation of nitrite in astrocyte cultures, while cultures treatment with other TLR ligands had undetectable amounts of nitrite after 24 h of treatment. In astrocyte cultures, nitrite production was only seen following exposure to 1.0 μg/ml LTA (Fig. 6A). Thus, the results show that the TLR2 alone appears to be coupled to NO production.

Figure 6
Effects of TLR agonists on NO production by astrocytes at 24 h following exposure. A–D) With the exception of the lowest concentration of the TLR2 agonist LTA (A), TLR3 (Poly(I:C)) (B), TLR4 (LPS) (C), and TLR9 (ODN) (D) agonist exposure had no ...

TLR agonists induce ROS formation in astrocytes

ROS can control the production of the innate proinflammatory immune response through the activation of redox-sensitive signal transduction pathways such as MAPK, AP-1 and through TLR4 via their effects on NF-κB activation (Matsuzawa et al., 2005; Rao, 2001; Ryan et al., 2004; Suzuki et al., 1997). Since the TLR ligands were able to induce secretion of cytokines and chemokines in astrocytes, we evaluated the consequences of TLR agonist-induced activation of astrocytes by intracellular ROS formation. Furthermore, increases in ROS are reported to be downstream of TLR activation (Matsuzawa et al., 2005; Mustafa et al., 2009; Ryan et al., 2004). We previously found that treatment with gp120 and morphine alone or in combination had no significant effect on ROS production in astrocytes, and their actions were not examined in the present study. Compared to vehicle-treated control cells, exposure to Tat and as little as 1 μg/mL of LTA, 5 μg/mL of LPS or poly (I:C), or 1.0 μM ODN significantly increased the level of oxyradicals in astrocytes (Fig. 7).

Figure 7
TLR agonists stimulate ROS formation in astrocytes. A–D) Intracellular ROS production was measured by treating cells with DCF prior to treatment with media alone (control), HIV-1 Tat (positive control), or increasing concentrations of TLR2 (LTA) ...


The present findings show (i) that individual TLRs are not uniformly expressed in astrocytes and (ii) that the expression of a particular TLR may change dynamically with exposure to pathogenic insults or drug abuse. In addition, although TLR expression levels are reportedly low in astrocytes and some controversy whether astrocytes express TLRs at all (Bowman et al., 2003; Carpentier et al., 2007, 2008; El-Hage et al., 2006a; Farina et al., 2005, 2007; Hauser et al., 2007; McKimmie and Fazakerley, 2005; Rivieccio et al., 2006), our results suggest that TLR expression by astrocytes can be highly dynamic and modifiable by extracellular cues. Moreover, the ability of cognate PAMP ligands to induce ROS and cytokine production indicates the TLR receptors on astrocytes are functional. Our findings corroborate recent data indicating that TLR ligands (i.e., LTA, Poly I:C and LPS), can upregulate cytokine and chemokine expression in cultured murine and rat astrocytes (Akira et al., 2000; Borysiewicz et al., 2009; De Miranda et al., 2009). Moreover, the sheer number of astrocytes in the brain and the fact that subsets of astrocytes appear to express functional TLRs, dramatically underscores the potential importance of astroglia in CNS immunity. Clearly, additional studies are needed to determine the extent to which these in vitro findings mimic patterns present in vivo and to ascertain the exact role of astroglial TLRs in neuroimmune function. However, if disrupted TLR signaling can indeed be established as a potent driving force for opioid drug-HIVE comorbid disease progression, then understanding the origins of aberrant TLR signaling may be of therapeutic benefit.

TLR activation can affect HIV-1 replication in vitro (Equils et al., 2001, 2003; Laflamme and Rivest, 2001; Liu et al., 2006; Scheller et al., 2004; Schlaepfer et al., 2004, 2006; Sundstrom et al., 2004), although the underlying mechanisms are not completely understood. Therefore, defects in normal TLR activation might be expected to compromise antimicrobial defenses and thereby modify disease progression. In the present study, we found enhanced TLR2 mRNA and protein levels in astrocytes treated with HIV-1 Tat, alone or in combination with morphine. Alternatively, gp120 by itself increased TLR2 protein levels at 6 h and 12 h; however in this instance, concurrent morphine exposure negated the gp120-ilicited increases in TLR2. The recent demonstration that NF-κB and TNF-α can regulate TLR2 expression in astrocytes through autocrine/paracrine feedback may provide an explanation for the increased expression of TLR2 (Phulwani et al., 2008), since both Tat and gp120 can elevate NF-κB-dependent cytokine expression (El-Hage et al., 2008b). TLR2 upregulation may contribute to the pathophysiology of transient cerebral ischemia (Ziegler et al., 2007). Outside the CNS, Heggelund et al. (2004a), showed increased surface expression of TLR2 on monocytes of HIV-infected patients and enhanced TNF-α release in response to TLR2 ligation, while others have shown levels of soluble TLR2 disrupted in HIV-1 infection (Heggelund et al., 2004b). Lastly, our findings differed somewhat from another study by Salaria et al. (2007), who found that gp120 increased TLR4 expression by human astrocytes. The reasons for the discrepancy are uncertain but may result from (i) species differences, (ii) astroglial heterogeneity among different brain regions (Fitting et al., 2010), or (iii) inherent differences in the effects of X4, R5, or dual-tropic gp120 variants. X4 and R5-tropic variants are reported to possess unique neurotoxic profiles (Kaul et al., 2007; Bachis et al., 2010; Medders et al., 2010), and each tropic variant differentially affects TLR function. Based on the present findings X4-tropic gp120IIIB, it is uncertain whether R5 or dual tropic gp120 variants might yield different results.

Unlike TLR2, TLR9 transcript levels decreased in astrocytes treated with Tat, gp120, and/or morphine. Likewise, both in-cell western and cytometric analyses showed corresponding declines in TLR9 proteins at 12 h after treatment. Notably, the proportion of astrocytes expressing TLR9 appeared to be less compared to TLR2 and TLR3 (Fig. 1B). The dynamic nature of TLR9 expression may partially explain why studies of TLR9 expression in astroglia have been controversial (Bowman et al., 2003). TLR9 participates in a coordinated response with TLR3 and TLR7/8 to suppress viral infections in the CNS (Mishra et al., 2006; Suh et al., 2009). Other investigators report aberrant TLR9 signaling in HIV-infected innate immune cells (Martinelli et al., 2007; Martinson et al., 2007), while polymorphisms in TLR9 affect HIV-1 progression clinically (Bochud et al., 2007). Then again, increased TLR3 expression was found in the lymph nodes of simian immunodeficiency virus (SIV)-infected macaques (Sanghavi and Reinhart, 2005), and Tat exposure was found to reduce TLR4 expression in cholangiocytes (O’Hara et al., 2009), suggesting that the virus or specific viral proteins can intrinsically affect TLR function—as seen in the present study. Alternatively, morphine can enhance LPS-mediated increases in vascular permeability suggesting additional modes of interaction with TLR signaling pathways (Liu et al., 2004). Collectively, HIV-1 infection may disrupt the innate immune response by systematically altering the function of a subset of TLRs, which appear to include an increase in deleterious TLR2 signaling coordinated with a loss in beneficial antiviral functions through reductions in TLR9 expression.

Even though morphine’s effects were more subtle than either viral protein, this should not discount morphine’s interactions with the TLR system. In the current studies, morphine alone caused a significant reduction in TLR9, while morphine attenuated gp120-induced increases in TLR2 transcript and protein levels. In combination, morphine and gp120 caused marked reductions in TLR9 protein levels at 6 h that were not seen with either substance alone. Thus, the immunosuppressive effect of morphine appears may be in part linked to alterations in TLR signaling. Wang et al. (2008) showed that morphine, in the presence of S. pneumoniae infection, inhibits NF-κB gene transcription through actions on TLR9 signaling in alveolar macrophages. Similarly, in human peripheral blood mononuclear cells (Bonnet et al. 2008), MOR activation mediates morphine-induced TNF-α and IL-6 inhibition in TLR2-stimulated monocytes and has been shown to suppress cytokine production by the TLR2 agonist, peptidoglycan (PGN). TLR2 mediates morphine-induced apoptosis in HEK293 cells in a MyD88-dependent manner (Li et al., 2009). Thus, morphine may potentially act at several direct and indirect levels to modify TLR expression astrocytes and to modify the host immune response.

Induction of neuropathic pain by opioid agonists was reversed using opioid receptor inhibitors, naloxone and naltrexone, through blockade of TLR4 activation in glia (Hutchinson et al., 2007; Watkins et al., 2009), suggesting TLR4 is an important mediator of functions outside of strict host-pathogen recognition and defense. Certainly, the upregulation of proinflammatory target genes encoding cytokines, chemokines and the production of ROS, as seen with activation of TLR 2, TLR3, TLR4, and TLR9 in the present study, are essential for pathogen elimination; however, these molecules can also mediate neuronal cell death (Brosnan et al., 1994; Chao et al., 1992). We hypothesize that the production of ROS, resulting from the activation of TLRs, may affect nociception in vivo as well. Alternatively, stimulation of astrocytes with TLR ligands can trigger the release of cytokines and downregulation of the glutamate transporter gene, excitatory amino acid transporter-1 (EAAT1 or GLAST), thereby limiting the ability of astrocytes to uptake excess glutamate (Bal-Price and Brown, 2001; Scumpia et al., 2005). The inability to sequester excess glutamate may enhance host defenses, but alternatively may contribute to excitotoxic insults in the CNS. Unfortunately, a trade-off for a heightened inflammatory response can be increased excitotoxicity and neuronal injury. Therefore, TLR stimulation on astrocytes has the ability to not only affect innate immunity, but astrocyte performance in general, which could have significant consequences for brain function.


We thank Drs. Seth M. Dever and Krista Scoggins for editing the manuscript. This work was supported by grants AD Williams (NE), and R03 DA026744-01 (NE), P01 DA19298, (KH), K02 DA027374 (KH), and T32 DA007027 (EP) from the NIH/NIDA. Flow cytometry was supported in part by NIH grant P30CA16059.


The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.


  • Akira S, Kishimoto T. IL-6 and NF-IL-6 in acute phase response and viral infection. Immunol Rev. 1992;313:47–50. [PubMed]
  • Akira S, Hoshino K, Kaisho T. The role of Toll-like receptors and MyD88 in innate immune responses. J Endotoxin Res. 2000;6:383–387. [PubMed]
  • Akira S, Yamamoto M, Takeda K. Role of adapters in Toll-like receptor signalling. Biochem Soc Trans. 2003;31:637–642. [PubMed]
  • Ashkar AA, Bauer S, Mitchell WJ, Vieira J, Rosenthal KL. Local delivery of CpG oligodeoxynucleotides induces rapid changes in the genital mucosa and inhibits replication, but not entry, of herpes simplex virus type 2. J Virol. 2003;77:8948–8956. [PMC free article] [PubMed]
  • Ashkar AA, Yao XD, Gill N, Sajic D, Patrick AJ, Rosenthal KL. Toll-like receptor (TLR)-3, but not TLR4, agonist protects against genital herpes infection in the absence of inflammation seen with CpG DNA. J Infect Dis. 2004;190:1841–1849. [PubMed]
  • Bachis A, Cruz MI, Mocchetti I. M-tropic HIV envelope protein gp120 exhibits a different neuropathological profile than T-tropic gp120 in rat striatum. Eur J Neurosci. 2010;32:570–578. [PMC free article] [PubMed]
  • Bafica A, Scanga CA, Schito M, Chaussabel D, Sher A. Influence of coinfecting pathogens on HIV expression: evidence for a role of Toll-like receptors. J Immunol. 2004;172:7229–7234. [PubMed]
  • Bal-Price A, Brown GC. Inflammatory neurodegeneration mediated by nitric oxide from activated glia-inhibiting neuronal respiration, causing glutamate release and excitotoxicity. J Neurosci. 2001;21:6480–6491. [PubMed]
  • Becher B, Prat A, Antel JP. Brain-immune connection: immuno-regulatory properties of CNS-resident cells. Glia. 2000;29:293–304. [PubMed]
  • Bell JE, Brettle RP, Chiswick A, Simmonds P. HIV encephalitis, proviral load and dementia in drug users and homosexuals with AIDS. Effect of neocortical involvement. Brain. 1998;121:2043–2052. [PubMed]
  • Bochud PY, Hersberger M, Taffe P, Bochud M, Stein CM, Rodrigues SD, Calandra T, Francioli P, Telenti A, Speck RF, Aderem A. Polymorphisms in Toll-like receptor 9 influence the clinical course of HIV-1 infection. AIDS. 2007;21:441–446. [PubMed]
  • Bonnet MP, Beloeil H, Benhamou D, Mazoit JX, Asehnoune K. The mu opioid receptor mediates morphine-induced tumor necrosis factor and interleukin-6 inhibition in toll-like receptor 2-stimulated monocytes. Anesth Analg. 2008;106:1142–1149. [PubMed]
  • Borysiewicz E, Fil D, Konat GW. Rho proteins are negative regulators of TLR2, TLR3, and TLR4 signaling in astrocytes. J Neuroscie Res. 2009;87:1565–1572. [PubMed]
  • Bowman CC, Rasley A, Tranguch SL, Marriott I. Cultured astrocytes express toll-like receptors for bacterial products. Glia. 2003;43:281–291. [PubMed]
  • Brosnan CF, Battistini L, Raine CS, Dickson DW, Casadevall A, Lee SC. Reactive nitrogen intermediates in human neuropathology: An overview. Dev Neurosci. 1994;16:152–161. [PubMed]
  • Bsibsi M, Ravid R, Gveric D, van Noort JM. Broad expression of Toll-like receptors in the human central nervous system. J Neuropathol Exp Neurol. 2002;61:1013–1021. [PubMed]
  • Bsibsi M, Persoon-Deen C, Verwer RW, Meeuwsen S, Ravid R, van Noort JM. Toll-like receptor 3 on adult human astrocytes triggers production of neuroprotective mediators. Glia. 2006;53:688–695. [PubMed]
  • Carpentier PA, Begolka WS, Olson JK, Elhofy A, Karpus WJ, Miller SD. Differential activation of astrocytes by innate and adaptive immune stimuli. Glia. 2005;49:360–374. [PubMed]
  • Carpentier PA, Williams BR, Miller SD. Distinct roles of protein kinase R and toll-like receptor 3 in the activation of astrocytes by viral stimuli. Glia. 2007;55:239–252. [PubMed]
  • Carpentier PA, Duncan DS, Miller SD. Glial toll-like receptor signaling in central nervous system infection and autoimmunity. Brain Behav Immun. 2008;22:140–147. [PMC free article] [PubMed]
  • Chao CC, Hu S, Molitor TW, Shaskan EG, Peterson PK. Activated microglia mediate neuronal cell injury via a nitric oxide mechanism. J Immunol. 1992;149:2736–2741. [PubMed]
  • De Miranda J, Yaddanapudi K, Hornig M, Lipkin WI. Astrocytes recognize intracellular polyinosinic-polycytidylic acid via MDA-5. The FASEB Journal. 2009;23:1064–1071. [PMC free article] [PubMed]
  • Dong Y, Benveniste EN. Immune function of astrocytes. Glia. 2001;36:180–190. [PubMed]
  • Doyle S, Vaidya S, O’Connell R, Dadgostar H, Dempsey P, Wu T, Rao G, Sun R, Haberland M, Modlin R, Cheng G. IRF3 mediates a TLR3/TLR4-specific antiviral gene program. Immunity. 2002;17:251–263. [PubMed]
  • El-Hage N, Gurwell JA, Singh IN, Knapp PE, Nath A, Hauser KF. Synergistic increase in intracellular Ca2+, and the release of MCP-1, RANTES and IL-6 by astrocytes treated with opiates and HIV-1 Tat. Glia. 2005;50:91–106. [PMC free article] [PubMed]
  • El-Hage N, Wu G, Wang J, Ambati J, Knapp PE, Bruce-Keller A, Hauser KF. HIV Tat1–72aa and Opiate-induced changes in astrocytes promote chemotaxis of microglia through the expression of MCP-1 and alternative chemokines. Glia. 2006a;53:132–146. [PMC free article] [PubMed]
  • El-Hage N, Wu G, Ambati J, Bruce-Keller A, Knapp PE, Hauser KF. CCR2 mediates increases in glial activation caused by exposure to HIV-1 Tat and opiates. J Neuroimmunol. 2006b;178:9–16. [PMC free article] [PubMed]
  • El-Hage N, Bruce-Keller AJ, Knapp PE, Hauser KF. CCL5/RANTES gene deletion attenuates opioid-induced increases in glial CCL2/MCP-1 immunoreactivity and activation in HIV-1 Tat-exposed mice. J Neuroimmune Pharmacol. 2008a;3:275–285. [PMC free article] [PubMed]
  • El-Hage N, Bruce-Keller AJ, Yakovleva T, Bazov I, Bakalkin G, Knapp PE, Hauser KF. Morphine exacerbates HIV-1 Tat-induced cytokine production in astrocytes through convergent effects on [Ca2+]i, NF-kappaB trafficking and transcription. PLoS One. 2008b;3:e4093. [PMC free article] [PubMed]
  • Equils O, Faure E, Thomas L, Bulut Y, Trushin S, Arditi M. Bacterial lipopolysaccharide activates HIV long terminal repeat through Toll-like receptor 4. J Immunol. 2001;166:2342–2347. [PubMed]
  • Equils O, Schito ML, Karahashi H, Madak Z, Yarali A, Michelsen KS, Sher A, Arditi M. Toll-like receptor 2 (TLR2) and TLR9 signaling results in HIV-long terminal repeat trans-activation and HIV replication in HIV-1 transgenic mouse spleen cells: implications of simultaneous activation of TLRs on HIV replication. J Immunol. 2003;170:5159–5164. [PubMed]
  • Farina C, Krumbholz M, Giese T, Hartmann G, Aloisi F, Meinl E. Preferential expression and function of Toll-like receptor 3 in human astrocytes. J Neuroimmunol. 2005;159:12–19. [PubMed]
  • Farina C, Aloisi F, Meinl E. Astrocytes are active players in cerebral innate immunity. Trends Immunol. 2007;28:138–145. [PubMed]
  • Fitting S, Zou S, Chen W, Vo P, Hauser KF, Knapp PE. Regional Heterogeneity and diversity in cytokine and chemokine production by astroglia: differential responses to HIV-1 Tat, gp120 and morphine revealed by multiplex analysis. J Proteome Res. 2010;9:1795–1804. [PMC free article] [PubMed]
  • Fuller S, Steele M, Munch G. Activated astroglia during chronic inflammation in Alzheimer’s disease-Do they neglect their neurosupportive roles? Mutat Res. 2009 Sep 11; [Epub ahead of print] [PubMed]
  • Gendelman HE, Persidsky Y, Ghorpade A, Limoges J, Stins M, Fiala M, Morrisett R. The neuropathogenesis of the AIDS dementia complex. AIDS. 1997;11(Suppl A):S35–S45. [PubMed]
  • Gill N, Deacon PM, Lichty B, Mossman KL, Ashkar AA. Induction of innate immunity against herpes simplex virus type 2 infection via local delivery of Toll-like receptor ligands correlates with beta interferon production. J Virol. 2006;80:9943–9950. [PMC free article] [PubMed]
  • Griess P. Bemernkugen zu der Abhandlung der HH: Wesely und Benedikt “Ueber einige Azoverbindungen” Ber Dtsch Chem Ges. 1879;12:426–428.
  • Hauser KF, El-Hage N, Stiene-Martin A, Maragos WF, Nath A, Persidsky Y, Volsky DJ, Knapp PE. HIV-1 neuropathogenesis: glial mechanisms revealed through substance abuse. J Neurochem. 2007;100:567–586. [PMC free article] [PubMed]
  • Heggelund L, Muller F, Lien E, Yndestad A, Ueland T, Kristiansen KI, Espevik T, Aukrust P, Froland SS. Increased expression of toll-like receptor 2 on monocytes in HIV infection: possible roles in inflammation and viral replication. Clin Infect Dis. 2004a;39:264–269. [PubMed]
  • Heggelund L, Flo T, Berg K, Lien E, Mollnes TE, Ueland T, Aukrust P, Espevik T, Froland SS. Soluble toll-like receptor 2 in HIV infection: association with disease progression. AIDS. 2004b;18:2437–2439. [PubMed]
  • Hutchinson MR, Bland ST, Johnson KW, Rice KC, Maier SF, Watkins LR. Opioid-induced glial activation: Mechanisms of activation and implications for opioid analgesia, dependence, and reward. The Scientific world Journal. 2007;7:98–111. [PubMed]
  • Hutchinson MR, Zang Y, Brown K, Coats BD, Shridhar M, Sholar PW, Patel SJ, Crysdale NY, Harrison JA, Maier SF, Rice KC, Watkins LR. Non-stereoselective reversal of neuropathic pain by naloxone and naltrexone: involvement of toll-like receptor 4 (TLR4) Eur J Neuroscie. 2008;28:20–29. [PMC free article] [PubMed]
  • Iwasaki A, Medzhitov R. Regulation of adaptive immunity by the innate immune system. Science. 2010;327:291–295. [PMC free article] [PubMed]
  • Jack CS, Arbour N, Manusow J, Montgrain V, Blain M, McCrea E, Shapiro A, Antel JP. TLR signaling tailors innate immune responses in human microglia and astrocytes. J Immunol. 2005;175:4320–4330. [PubMed]
  • Kariko K, Weissman D, Welsh FA. Inhibition of toll-like receptor and cytokine signaling--a unifying theme in ischemic tolerance. J Cereb Blood Flow Metab. 2004;24:1288–1304. [PubMed]
  • Kaul M, Garden GA, Lipton SA. Pathways to neuronal injury and apoptosis in HIV-associated dementia. Nature. 2001;410:988–994. [PubMed]
  • Kaul M, Ma Q, Medders KE, Desai MK, Lipton SA. HIV-1 coreceptors CCR5 and CXCR4 both mediate neuronal cell death but CCR5 paradoxically can also contribute to protection. Cell Death Differ. 2007;14:296–305. [PubMed]
  • Laflamme N, Rivest S. Toll-like receptor 4: the missing link of the cerebral innate immune response triggered by circulating gram-negative bacterial cell wall components. FASEB J. 2001;15:155–163. [PubMed]
  • Lafon M, Megret F, Lafage M, Prehaud C. The innate immune facet of brain: human neurons express TLR-3 and sense viral dsRNA. Mol Neurosci. 2006;29:185–94. [PubMed]
  • Lander HM, Ogiste JS, Pearce SF, Levi R, Novogrodsky A. Nitric oxide-stimulated guanine nucleotide exchange on p21ras. J Biol Chem. 1995;270:7017–7020. [PubMed]
  • Li Y, Sun X, Zhang Y, Huang J, Hanley G, Ferslew KE, Peng Y, Yin D. Morphine promotes apoptosis via TLR2, and this is negatively regulated by beta-arrestin 2. Biochem Biophys Res Commun. 2009;378:857–861. [PubMed]
  • Liu HC, Anday JK, House SD, Chang SL. Dual effects of morphine on permeability and apoptosis of vascular endothelial cells: morphine potentiates lipopolysaccharide-induced permeability and apoptosis of vascular endothelial cells. J Neuroimmunol. 2004;146:13–21. [PubMed]
  • Liu X, Mosoian A, Li-Yun CT, Zerhouni-Layachi B, Snyder A, Jarvis GA, Klotman ME. Gonococcal lipooligosaccharide suppresses HIV infection in human primary macrophages through induction of innate immunity. J Infect Dis. 2006;194:751–759. [PubMed]
  • MacDonald EM, Savoy A, Gillgrass A, Fernandez S, Smieja M, Rosenthal KL, Ashkar AA, Kaushic C. Susceptibility of human female primary genital epithelial cells to herpes simplex virus, type-2 and the effect of TLR3 ligand and sex hormones on infection. Biol Reprod. 2007;77:1049–1059. [PubMed]
  • Martinelli E, Cicala C, Van RD, Goode DJ, Macleod K, Arthos J, Fauci AS. HIV-1 gp120 inhibits TLR9-mediated activation and IFN-α secretion in plasmacytoid dendritic cells. Proc Natl Acad Sci U S A. 2007;104:3396–3401. [PMC free article] [PubMed]
  • Martinson JA, Tenorio AR, Montoya CJ, Al-Harthi L, Gichinga CN, Krieg AM, Baum LL, Landay AL. Impact of class A, B and C CpG-oligodeoxynucleotides on in vitro activation of innate immune cells in human immunodeficiency virus-1 infected individuals. Immunol. 2007;120:526–535. [PMC free article] [PubMed]
  • Matsuzawa A, Saegusa K, Noguchi T, Sadamitsu C, Nishitoh H, Nagai S, Koyasu S, Matsumoto K, Takeda K, Ichijo H. ROS-dependent activation of the TRAF6-ASK1-p38 pathway is selectively required for TLR4-mediated innate immunity. Nat Immunol. 2005;6:587–592. [PubMed]
  • McGeer PL, McGeer EG. Inflammation and the degenerative diseases of aging. Ann N Y Acad Sci. 2004;1035:104–116. [PubMed]
  • McKimmie CS, Fazakerley JK. In response to pathogens, glial cells dynamically and differentially regulate Toll-like receptor gene expression. J Neuroimmunol. 2005;169:116–125. [PubMed]
  • Medders KE, Sejbuk NE, Maung R, Desai MK, Kaul M. Activation of p38 MAPK is required in monocytic and neuronal cells for HIV glycoprotein 120-induced neurotoxicity. J Immunol. 2010;185:4883–4895. [PMC free article] [PubMed]
  • Medzhitov R, Janeway CA. Innate immune induction of the adaptive immune response. Cold Spring Harb Symp Quant Biol. 1999;64:429–435. [PubMed]
  • Mishra BB, Mishra PK, Teale JM. Expression and distribution of Toll-like receptors in the brain during murine neurocysticercosis. J Neuroimmunol. 2006;181:46–56. [PMC free article] [PubMed]
  • Mustafa AK, Gadalla MM, Snyder SH. Signaling by gasotransmitters. Sci Signal. 2009;2:re2. [PMC free article] [PubMed]
  • Nath A, Hauser KF, Wojna V, Booze RM, Maragos W, Prendergast M, Cass W, Turchan JT. Molecular basis for interactions of HIV and drugs of abuse. J Acquir Immune Defic Syndr. 2002;31(Suppl 2):S62–S69. [PubMed]
  • O’Hara SP, Small AJ, Gajdos GB, Badley AD, Chen XM, Larusso NF. HIV-1 Tat protein suppresses cholangiocyte toll-like receptor 4 expression and defense against Cryptosporidium parvum. J Infect Dis. 2009;199:1195–1204. [PMC free article] [PubMed]
  • Park C, Lee S, Cho I-H, Lee H-K, Kim D, Choi S-Y, Oh S-B, Park K, Kim J-S, Lee S-J. TLR 3-mediated signal induces proinflammatory cytokine and chemokine gene expression in astrocytes: differential signaling mechanisms of TLR3-induced IP-10 and IL-8 gene expression. Glia. 2006;53:248–256. [PubMed]
  • Phulwani NK, Esen N, Syed MM, Kielian T. TLR2 expression in astrocytes is induced by TNF-alpha- and NF-kappa B-dependent pathways. J Immunol. 2008;181:3841–3849. [PMC free article] [PubMed]
  • Rao KM. MAP kinase activation in macrophages. J Leukoc Biol. 2001;69:3–10. [PubMed]
  • Rivieccio MA, Suh HS, Zhao Y, Zhao ML, Chin KC, Lee SC, Brosnan CF. TLR3 ligation activates an antiviral response in human fetal astrocytes: a role for viperin/cig5. J Immunol. 2006;177:4735–4741. [PubMed]
  • Ryan KA, Smith MF, Jr, Sanders MK, Ernst PB. Reactive oxygen and nitrogen species differentially regulate Toll-like receptor 4-mediated activation of NF-kappa B and interleukin-8 expression. Infect Immun. 2004;72:2123–2130. [PMC free article] [PubMed]
  • Salaria S, Badkoobehi H, Rockenstein E, Crews L, Chana G, Masliah E, Everall IP. and the HNRC Group. Toll-like receptor pathway gene expression is associated with human immunodeficiency virus–associated neurodegeneration. J NeuroVirol. 2007;13:496–503. [PubMed]
  • Sanghavi SK, Reinhart TA. Increased expression of TLR3 in lymph nodes during simian immunodeficiency virus infection: implications for inflammation and immunodeficiency. J Immunol. 2005;175:5314–5323. [PubMed]
  • Scheller C, Ullrich A, McPherson K, Hefele B, Knoferle J, Lamla S, Olbrich AR, Stocker H, Arasteh K, ter MV, Rethwilm A, Koutsilieri E, Dittmer U. CpG oligodeoxynucleotides activate HIV replication in latently infected human T cells. J Biol Chem. 2004;279:21897–21902. [PubMed]
  • Schlaepfer E, Audige A, von BB, Manolova V, Weber M, Joller H, Bachmann MF, Kundig TM, Speck RF. CpG oligodeoxynucleotides block human immunodeficiency virus type 1 replication in human lymphoid tissue infected ex vivo. J Virol. 2004;78:12344–12354. [PMC free article] [PubMed]
  • Schlaepfer E, Audige A, Joller H, Speck RF. TLR7/8 triggering exerts opposing effects in acute versus latent HIV infection. J Immunol. 2006;176:2888–2895. [PubMed]
  • Scumpia PO, Kelly KM, Reeves WH, Stevens BR. Double-stranded RNA signals antiviral and inflammatory programs and dysfunctional glutamate transport in TLR3-expressing astrocytes. Glia. 2005;52:153–162. [PubMed]
  • Sheng WS, Hu S, Gekker G, Zhu S, Peterson PK, Chao CC. Immunomodulatory role of opioids in the central nervous system. Arch Immunol Ther Exp (Warsz) 1997;45:359–366. [PubMed]
  • Stiene-Martin A, Zhou R, Hauser KF. Regional, developmental, and cell cycle-dependent differences in δ, κ, and μ opioid receptor expression among cultured mouse astrocytes. Glia. 1998;22:249–259. [PMC free article] [PubMed]
  • Suh HS, Brosnan CF, Lee SC. Toll-like receptors in CNS viral infections. Curr Top Microbiol Immunol. 2009;336:63–81. [PubMed]
  • Sundstrom JB, Little DM, Villinger F, Ellis JE, Ansari AA. Signaling through Toll-like receptors triggers HIV-1 replication in latently infected mast cells. J Immunol. 2004;172:4391–4401. [PubMed]
  • Suzuki YJ, Forman HJ, Sevanian A. Oxidants as stimulators of signal transduction. Free Radic Biol Med. 1997;22:269–285. [PubMed]
  • Takeda K, Kaisho T, Akira S. Toll-like receptors. Annu Rev Immunol. 2003;21:335–376. [PubMed]
  • Tsan MF, Gao B. Endogenous ligands of Toll-like receptors. J Leukoc Biol. 2004;76:514–519. [PubMed]
  • Wang J, Barke RA, Charboneau R, Schwendener R, Roy S. Morphine induces defects in early response of alveolar macrophages to Streptococcus pneumoniae by modulating TLR9-NF-kappa B signaling. J Immunol. 2008;180:3594–3600. [PubMed]
  • Watkins LR, Hutchinson MR, Rice KC, Maier SF. The “Toll” of Opioid-Induced Glial Activation: Improving the Clinical Efficacy of Opioids by Targeting Glia. Trends Pharmacol Sci. 2009;30(11):581–591. [PMC free article] [PubMed]
  • Yamamoto M, Sato S, Hemmi H, Hoshino K, Kaisho T, Sanjo H, Takeuchi O, Sugiyama M, Okabe M, Takeda K, Akira S. Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway. Science. 2003;301:640–643. [PubMed]
  • Zekki H, Feinstein DL, Rivest S. The clinical course of experimental autoimmune encephalomyelitis is associated with a profound and sustained transcriptional activation of the genes encoding toll-like receptor 2 and CD14 in the mouse CNS. Brain Pathol. 2002;12:308–319. [PubMed]
  • Ziegler G, Harhausen D, Schepers C, Hoffmann O, Rohr C, Prinz V, Konig J, Lehrach H, Nietfeld W, Trendelenburg G. TLR2 has a detrimental role in mouse transient focal cerebral ischemia. Biochem Biophys Res Commun. 2007;359:574–579. [PubMed]
PubReader format: click here to try


Save items

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


  • Compound
    PubChem chemical compound records that cite the current articles. These references are taken from those provided on submitted PubChem chemical substance records. Multiple substance records may contribute to the PubChem compound record.
  • Gene
    Gene records that cite the current articles. Citations in Gene are added manually by NCBI or imported from outside public resources.
  • GEO Profiles
    GEO Profiles
    Gene Expression Omnibus (GEO) Profiles of molecular abundance data. The current articles are references on the Gene record associated with the GEO profile.
  • HomoloGene
    HomoloGene clusters of homologous genes and sequences that cite the current articles. These are references on the Gene and sequence records in the HomoloGene entry.
  • MedGen
    Related information in MedGen
  • PubMed
    PubMed citations for these articles
  • Substance
    PubChem chemical substance records that cite the current articles. These references are taken from those provided on submitted PubChem chemical substance records.

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...