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

Abstract

In this study, we aimed to 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.

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, TLR3, TLR4, or TLR9 was co-localized in subsets of GFAP-positive astrocytes. Although TLR activation is reported to reveal some TLR types, we found only modest qualitative differences in the pattern and intensity of TLR immunofluorescence at 12 h post-treatment. Arrows indicate TLR immunoreactivity.

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 astrocytes (B); isotype controls (dashed line), TLR2 (red line), TLR3 (blue line), TLR4 (green line), TLR9 (purple line) and GFAP (black line). C–D) Data are plotted to show the intensity of the phycoerythrin (PE) fluorescent signal in isotype-control and GFAP to indicate negative and positive staining, respectively (PE was used for TLRs 2–4). E) TLR9 could be co-localized with TLR2, TLR3, or TLR4; the extent to which other TLRs are co-expressed in the same cell was not explored. Cell nuclei were counterstained with Hoechst 33258. Approximately 10,000 events were analyzed per treatment condition in each experiment and size discrimination was used as a crude method for viability determination. Representative histograms of three independent experiments are shown; values indicate the mean percentages of GFAP-immunopositive astrocytes ± SEM in which fluorescein isothiocyanate (FITC) labeled TLRs could be co-detected.

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. Cells were fixed, permeabilized and labeled with antibodies to TLR2, TLR3, TLR4 or TLR9 (IRDye 680LT), and normalized against β-actin (IRDye 800CW), and quantitated by In-cell western analysis (LI-COR) (A–B). C–D) After 12 h treatment with morphine, Tat, and/or gp120, total RNA was extracted, RT-PCR followed by gel electrophoresis was performed, TLR mRNA levels were quantitated densitometrically, and normalized to β-actin. A–D) Collectively, TLR3 or TLR4 expression was unaffected by exposure to Tat, gp120, morphine or combinations thereof (data not shown), while TLR2 (A) and TLR9 (B) protein or transcript levels (C–D), respectively, showed reciprocal increases and decreases in response to most treatments (*p < 0.05 vs. controls); protein and mRNA determinations are the mean ± SEM of 4 independent experiments (*p < 0.05 vs. controls).

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 (50 ± 2) increased the proportion of TLR2-immunopositive astrocytes compared to vehicle-treated controls (33 ± 2) (p < 0.05; one-way ANOVA; Duncan’s post hoc test), while the proportion of TLR9-immunofluorescent astroglia was unaffected by these treatments at 12 h. Approximately 10,000 events were analyzed per treatment condition in each experiment. Representative dot plots are shown (x-axis, PE; y-axis, FITC) on the x-axis); values are the mean percentage of TL2 (red) or TLR9 (green) positive astrocytes ± SEM from 3 independent experiments.

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 (A), IL-6 (B), RANTES (C), and/or MCP-1 (D) by astrocytes as determined by ELISA. Values represent the mean ± SEM of 4 independent experiments (*p < 0.05 vs. control).

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 affect on the accumulation and/or release of nitrites by astrocytes. Nitrite levels were assessed using the Griess reaction; HIV-1 Tat was included as a positive control; values represent the mean ± SEM of 4 independent experiments (*p < 0.05 vs. control).

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) (A), TLR3 (Poly(I:C)) (B), TLR4 (LPS) (C), or TLR9 (ODN) (D) agonists, and measuring DCF fluorescence after 90 min. treatment. Values represent the mean ± SEM of 4 independent experiments (*p < 0.05 vs. control).