N-Acetylcysteine Reverses Antiretroviral-Mediated Microglial Activation by Attenuating Autophagy-Lysosomal Dysfunction

Abstract

Successful suppression of viral replication by combined antiretroviral therapy (cART) in HIV-1 infected individuals is paradoxically also accompanied by an increased prevalence of HIV-associated neurocognitive disorders (HAND) in these individuals. HAND is characterized by a state of chronic oxidative stress and inflammation. Microglia are extremely sensitive to a plethora of stimuli, including viral proteins and cART. The current study aimed to assess the effects of cART-mediated oxidative stress on the induction of inflammatory responses in microglia. In the present study, we chose a combination of three commonly used antiretroviral drugs-tenofovir disoproxil fumarate, emtricitabine, and dolutegravir. We demonstrated that exposure of microglia to the chosen cART cocktail induced generation of reactive oxygen species, subsequently leading to lysosomal dysfunction and dysregulated autophagy, ultimately resulting in the activation of microglia. Intriguingly, the potent antioxidant, N-acetylcysteine, reversed the damaging effects of cART. These in vitro findings were further corroborated in vivo wherein cART-treated HIV transgenic (Tg) rats demonstrated increased microglial activation, exaggerated lysosome impairment, and dysregulated autophagy in the prefrontal cortices compared with HIV Tg rats not exposed to cART. Similar to in vitro findings, the treatment of HIV Tg rats with N-acetylcysteine also mitigated the deleterious effects of cART. Taken together, our findings suggest that oxidative stress-mediated lysosomal dysfunction plays a critical role in the pathogenesis of HAND in drug-treated HIV-infected individuals and that antioxidant-mediated mitigation of oxidative stress could thus be considered as an adjunctive therapeutic strategy for ameliorating/dampening some of the neurological complications of HAND.

Keywords: N-acetylcysteine; autophagy; combined antiretroviral therapy; lysosome; microglial activation; neuroinflammation.

Figure 1

Combined antiretroviral therapy (cART)-mediated reactive oxygen species (ROS) generation in rat primary microglial cells (rPMs). (A) Representative graph showing increased generation of ROS in rPMs exposed to cART at varying time points. (B) Representative fluorescent-microscopic image showing cART-mediated ROS generation at 3 and 24 h (Scale bar: 10 μm). (C) Representative graph showing effect of ROS scavengers N-acetylcysteine (NAC), 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL) or (2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (mitoTEMPO) on cART-mediated upregulation of ROS. rPMS was treated with ROS scavengers for 1 h, followed by exposure of cells to cART for an additional 24 h. Tert-butyl hydroperoxide (TBHP) was used as a positive control for ROS generation. Data are from three independent experiments and are represented as means ± SEM using a one-way analysis of variance followed by a Bonferroni (Dunn) comparison of groups. *P < 0.05 vs. control; ^#P < 0.05 vs. cART.

Figure 2

N-acetylcysteine (NAC) reverses combined antiretroviral therapy (cART)-mediated lysosomal dysfunction in rat primary microglial cells (rPMs). rPMs were seeded into six-well plates and subject to various treatments for 24 h. Protein homogenates were prepared for the detection of indicated molecules. (A,B) Representative Western blots showing treatment of microglia with NAC-reversed cART-mediated downregulation of lysosomal-associated membrane protein 2 (LAMP2) (A) and mature cathepsin D (mCTSD) expression levels (B). mCTSD is the mature form of pro-cathepsin (pCTSD). Maturation of cathepsins and their activity is dependent on the acidity of the lysosomes (low pH). (C–E) Representative bar graph showing NAC-mediated protection of lysosomal membrane permeabilization (LMP) (C), CTSD activity (D), and lysosomal pH (E) in the presence of cART. (F) Representative fluorescent-microscopic image showing the cART-mediated increase in LAMP2 and galectin 3 (GAL3) colocalization (Scale bar: 5 μm). (G) Representative bar graph showing quantitative values of LAMP2 and GAL3 colocalization. A minimum of 50 randomly chosen cells for each experimental group were analyzed. For all Western blots, ACTB served as a protein loading control. Data are from three independent experiments and are represented as means ± SEM and were analyzed using one-way ANOVA. *P < 0.05 vs. control; ^#P < 0.05 vs. cART.

Figure 3

N-acetylcysteine (NAC) reverses combined antiretroviral therapy (cART)-mediated autophagy dysregulation in rat primary microglial cells (rPMs). (A,B) Representative Western blots showing treatment of NAC-reversed cART-mediated upregulation of autophagy markers microtubule-associated protein 1 light chain 3 beta (MAP1LC3B) (A) and sequestosome 1 (SQSTM1) (B). (C,D) rPMs were seeded into 12-well plates followed by transfection of cells with the tandem fluorescent-tagged MAP1LC3B plasmid. Cells were then exposed to various treatments for an additional 24 h and fluorescent intensity assessed by confocal microscopy (Scale bar: 5 μm). A minimum of 50 randomly chosen cells for each experimental group were analyzed. For all Western blots, ACTB served as a protein loading control. Data are from three independent experiments and are expressed as means ± SEM and were analyzed using a one-way analysis of variance followed by a Bonferroni (Dunn) comparison of groups. *P < 0.05 vs. control; ^#P < 0.05 vs. cART.

Figure 4

N-acetylcysteine (NAC) reverses combined antiretroviral therapy (cART)-mediated activation of rat primary microglial cells (rPMs). rPMs were seeded into six-well plates and subject to various treatments for 12 h. (A–C) Representative bar graph demonstrating NAC-mediated abrogation of cART-induced messenger RNA expression of pro-inflammatory cytokines: interleukin 1 beta (Il1β), interleukin 6 (Il6), and tumor necrosis factor (Tnf). Data are from three independent experiments and are represented as means ± SEM and were analyzed using one-way ANOVA. *P < 0.05 vs. control; ^#P < 0.05 vs. cART.

Figure 5

N-acetylcysteine (NAC) reverses combined antiretroviral therapy (cART)-mediated lysosome impairment and autophagy dysregulation in vivo. HIV Tg rats received cART injection with or without NAC (200 mg/kg) treatment (n = 3/group, intraperitoneal, 3 weeks). Saline-injected HIV Tg rats were used as controls. Protein homogenates of prefrontal cortices were prepared to detect the levels of indicated molecules. (A,B) Representative Western blots showing treatment of NAC-reversed cART-mediated downregulation of both LAMP2 (A) and mCTSD (B) in the prefrontal cortices. (C) Representative bar graph showing NAC-reversed cART-mediated downregulation of CTSD activity. (D–F) Representative Western blots showing treatment of NAC-reversed cART-mediated upregulation of beclin 1 (BECN1) (D), microtubule-associated protein 1 light chain 3 beta (MAP1LC3B) (E), and sequestosome 1 (SQSTM1) (F) in the prefrontal cortices of HIV Tg rats. For all Western blots, ACTB served as a protein loading control. Data are from three independent experiments and are represented as means ± SEM and were analyzed using one-way ANOVA. *P < 0.05 vs. control; ^#P < 0.05 vs. cART.

Figure 6

N-acetylcysteine (NAC) reverses combined antiretroviral therapy (cART)-mediated microglial activation in vivo. (A,B) Representative Western blot and bar graph showing treatment of NAC-reversed cART-mediated upregulation of integrin subunit alpha M (ITGAM) in the prefrontal cortices of HIV Tg rats. (C) Representative fluorescent-microscopic image showing the fluorescent intensity of allograft inflammatory factor 1 (AIF1) and lysosome-associated membrane protein 2 (LAMP2) in the prefrontal cortices of HIV Tg rats receiving various treatments (Scale bar: 20 μm). A minimum of 50 randomly chosen cells for each experimental group were analyzed. Red boxes in control, NAC + cART, and NAC-alone groups represent the microglial cells with ramified cellular processes with LAMP2 colocalization, which is absent in the cART group. (D) Representative bar graph showing treatment of NAC-reversed cART-mediated downregulation of microglial process length. (E,F) Representative bar graphs showing treatment of NAC-reversed cART-mediated downregulation of LAMP2 staining and decreases colocalization of LAMP2 and AIF1 in microglia in the prefrontal cortex region. (G) Representative bar graph showing treatment of NAC-reversed cART-mediated upregulation of Il1β protein levels in the prefrontal cortex. Data are from three independent experiments and are represented as means ± SEM and were analyzed using one-way ANOVA. *P < 0.05 vs. control; ^#P < 0.05 vs. cART.

Figure 7

Schematic depicting the involvement of reactive oxygen species (ROS) in combined antiretroviral therapy (cART)-mediated autophagy–lysosomal dysfunction in microglia. Exposure of microglia to cART increases ROS generation, leading, in turn, lysosomal dysfunction and autophagy dysregulation, which ultimately led to microglial activation and increased expression of pro-inflammatory cytokines. ROS scavenger N-acetylcysteine (NAC) reversed these deleterious effects of cART.