Methamphetamine enhancement of HIV-1 gp120-mediated NLRP3 inflammasome activation and resultant proinflammatory responses in rat microglial cultures

Abstract

Background: Human Immunodeficiency Virus type 1 (HIV-1)-associated neurocognitive disorders (HAND) remain prevalent in HIV-1-infected individuals despite the evident success of combined antiretroviral therapy (cART). The mechanisms under HAND prevalence in the cART era remain perplexing. Ample evidence indicates that HIV-1 envelope glycoprotein protein 120 (gp120), a potent neurotoxin, plays a pivotal role in the HAND pathogenesis. Methamphetamine (Meth) abuse exacerbates HAND. How Meth exacerbates HAND is not fully understood. This study was to test the hypothesis that Meth exacerbates HAND by enhancing gp120-mediated proinflammatory responses in the brain, worsening the pathogenesis of HAND.

Methods: Experiments were carried out on primary microglial cultures prepared from neonatal SD rats. The purity of microglia was determined by staining with anti-CD11b. Meth and gp120 were applied to microglial cultures. Microglial activation was revealed by immunostaining and Iba-1 expression. The protein expression levels of Pro-IL-1β, Il-1β, Iba-1, iNOS, NLRP3, GSDMD and GSDMD-N were detected by western blotting analyses. The levels of proinflammatory cytokine and NO production in the microglia culture supernatants were assayed by ELISA and Griess reagent systems, respectively. NLRP3 activation was uncovered by fluorescent microscopy images displaying NLRP3 puncta labeled by anti-NLRP3 antibody. NLRP3 co-localization with caspase-1 was labeled with antibodies. One-way ANOVA with post hoc Tukey's multiple comparison tests was employed for statistical analyses.

Results: Meth enhanced gp120-induced microglia activation revealed by immunostaining and Iba-1 expression, and potentiated gp120-mediated NLRP3 expression, IL-1β processing and release assayed by immunoblot and ELISA. Meth also augmented the co-localization of NLRP3 and caspase-1, increased the numbers of NLRP3 puncta and ROS production, elevated levels of iNOS expression and NO production, and enhanced levels of cleaved gasderminD (GSDMD-N, an executor of pyroptosis) in gp120-primed microglia. The Meth-associated effects were attenuated or blocked by MCC950, an NLRP3 inhibitor, or Mito-TEMPO, a mitochondrial superoxide scavenger, indicating the involvement of mitochondria in Meth enhancement of NLRP3 inflammasome activation in gp120-primed microglia.

Conclusions: These results suggest that Meth enhanced gp120-associated microglial NLRP3 activation and resultant proinflammatory responses via mitochondria-dependent signaling.

Keywords: HIV-1gp120; Methamphetamine; NLRP3 inflammasome; microglia; neuroinflammation.

Figure 1

Meth potentiation of HIV-1 gp120-induced IL-1βprocessing and release. Data illustrated in three columns were obtained from rat microglial cultures treated with Meth (left) and gp120 (central), each alone at different concentrations, respectively, or with one concentration of gp120 (500 pM) and different concentrations of Meth (right). The upper row displayed western blot results of the expression levels of pro-IL-1β and IL-1β when microglia were treated with Meth (A) and gp120 (D) alone or gp120 500 pM with different concentrations of Meth (G). Bar graphs in the middle row were corresponding densitometry quantitation of pro-IL-1β, IL-1β on western blots shown in the upper row. The lower row showed the levels of IL-1β detected by ELISA from culture supernatants of microglia under different experimental conditions as indicated. *p<0.05, **p<0.01, ***p<0.001 vs. untreated controls (E, F) or vs. gp120 alone controls (H, I). Data represent mean ± SD derived from three independent experiments in triplicate.

Figure 2

Meth augments gp120-primed microglia activation. (A) immunofluorescence labeling of Iba-1 expression in microglia under different experimental conditions as indicated. Note that an enhanced Iba-1 expression was detected in microglia treated with gp120+Meth. (B) Western blot detection of Iba-1 protein expression and an increased level of Iba-1 expression was observed with the treatment of gp120+Meth. (C) Image J densitometry analyses of Iba-1 protein expression in western blots. **p<0.01 One-way ANOVA followed by Dunnett’s comparisons test. Scale bar equals 100μM.

Figure 3

Meth-induced co-localization of NLRP3 and caspase-1 in gp120-primmed microglia. Immunofluorescence images were taken from microglial cultures treated with gp120 (middle row) and gp120+Meth (bottom row) after staining separately with primary antibodies against NLRP3 (red) and caspase-1 (green). The upper row was untreated controls. Note that Meth induced co-localization of NLRP3 and caspase-1 as shown in merged images. Blue in color was a DAPI stain. Images were taken at 60 magnification.

Figure 4

Meth triggers NLRP3 activation in gp120-primed microglia. Panel A: Representative fluorescence microscopy images display NLRP3 puncta (red) labeled by anti-NLRP3 antibody (left column) in different experimental conditions as indicated. Cell nuclei were stained with DAPI (blue, middle column). The right column showed merged images. Panel B: Percentage of microglia containing NLRP3 puncta as estimated by microscopic scoring. Panels C and D: Western blot detection of NLRP3 and corresponding densitometric analysis of NLRP3 from different treatment conditions. Note the synergic effects of Meth and gp120 on the increase of NLRP3 puncta positive cells and NLRP3 expression levels and their significant blockade by MCC 950, an NLRP3 inflammasome activation blocker, or by Mito-TEMPO, a mitochondria-targeted antioxidant. ***p<0.001, ^###p<0.001, **p<0.01 One-way ANOVA followed by Tukey’s multiple comparisons tests. Abbreviations used in figure: U (untreated), G (gp120), M (Meth), MC (MCC950), MT (Mito-TEMPO).

Figure 5

Meth increased proinflammatory cytokine production in gp120-primmed microglia. ELISA analyses of the levels of proinflammatory cytokines in the culture supernatants recovered from microglia with experimental treatments as indicated. Increased production levels of IL-1β (A), TNF-α(B), IL-6 (C) and IL-18 (D) were detected gp120-primed, Meth-treated microglia. Pretreatment of microglia with NLRP3 inhibitor MCC950 or mitochondrial superoxide scavenger Mito-TEMPO significantly decreased the levels of cytokine production. ***p<0.001, ^###p<0.001.

Figure 6

Meth and HIV-1 gp120 synergistically magnify total ROS production. Panel A: Combined stimulation of Meth and gp120 synergistically activated total ROS production. Panel B: Quantified results were displayed in a bar graph. All images were captured at 40X original magnification. ***p<0.001.

Figure 7

Meth enhancement of iNOS expression and NO production in gp120-primmed microglia. Panel A showed Meth-induced enhancement of NO production in culture supernatants recovered from gp120-primed microglia. Panels B and C exhibited an increase of iNOS expression detected using microglial lysate *p<0.1, ***p<0.001 vs gp120 only treated control.

Figure 8

Meth-induced microglial pyroptosis. Panel A was a representative western blot result showing that microglia cells underwent pyroptosis after treatment with gp120, as detected by increased N-terminal GSDMD (GSDMD-N), which was further augmented by Meth treatment. The bar graph in Panel B showed average expression levels of GSDMD and GSDMD-N. Note the augment effect of Meth on gp120-mediated pyroptosis. Data in Panel B represent three independent experiments and each in triplicate. **p<0.01, ***p<0.001 vs untreated control,

Figure 9

Potential mechanisms for Meth enhancement of NLRP3 inflammasome activation in gp120-primed microglia in a two signal model. Gp120 works as priming (first signal) and induces upregulation of transcription of pro-IL-1βand NLRP3. Meth helps in processing (the second signal) for the NLRP3 inflammasome activation via mitochondria-associated ROS signaling.