Suppression of HIV-TAT and cocaine-induced neurotoxicity and inflammation by cell penetrable itaconate esters

Abstract

HIV-associated neurological disorder (HAND) is a serious complication of HIV infection marked by neurotoxicity induced by viral proteins like Tat. Substance abuse exacerbates neurocognitive impairment in people living with HIV. There is an urgent need for therapeutic strategies to combat HAND comorbid with Cocaine Use Disorder (CUD). Our analysis of HIV and cocaine-induced transcriptomes in primary cortical cultures revealed significant overexpression of the macrophage-specific gene aconitate decarboxylase 1 (Acod1). The ACOD1 protein converts the tricarboxylic acid intermediate cis-aconitate into itaconate during the activation of inflammation. Itaconate then facilitates cytokine production and activates anti-inflammatory transcription factors, shielding macrophages from infection-induced cell death. However, the immunometabolic function of itaconate was unexplored in HIV and cocaine-exposed microglia. We assessed the potential of 4-octyl-itaconate (4OI), a cell-penetrable ester form of itaconate known for its anti-inflammatory properties. When primary cortical cultures exposed to Tat and cocaine were treated with 4OI, microglial cell number increased and the morphological altercations induced by Tat and cocaine were reversed. Microglial cells also appeared more ramified, resembling the quiescent microglia. 4OI treatment inhibited secretion of the proinflammatory cytokines IL-1α, IL-1β, IL-6, and MIP1-α induced by Tat and cocaine. Transcriptome profiling determined that Nrf2 target genes were significantly activated in Tat and 4OI treated cultures relative to Tat alone. Further, genes associated with cytoskeleton dynamics in inflammatory microglia were downregulated by 4OI treatment. Together, the results strongly suggest 4-octyl-itaconate holds promise as a potential candidate for therapeutic development to treat HAND coupled with CUD comorbidities.

Keywords: Acod 1; HAND; HIV; Itaconate; Microglia; Neuroinflammation; Tat.

Fig. 1

Acod1 expression in Tat and/or cocaine-treated primary neuronal cultures, and neuroprotective effects of 4OI. a Acod1 expression was assessed in primary cortical cultures treated with cocaine (25 μM) alone for 24 h, Tat (6 ng/mL) alone for 48 h, or Tat for 48 h followed by the addition of cocaine for 24 h. Acod1 levels were quantified using RNA-seq and presented as CPM read counts. b qPCR analysis of the same experimental samples measured Acod1 expression fold change, normalized to ACTB. Statistical analysis was performed using the Mann–Whitney/Wilcoxon Rank-Sum test, followed by Bonferroni’s post hoc test. c Boxplots illustrate the dose-dependent effect of 4OI (30, 60, 120, or 250 μM) on Tat (60 ng/mL) and cocaine (25 μM) treated cultures. Non-treated control cultures and cultures treated with the highest concentration of 4OI (250 μM) are shown on the left. Each data point represents an image containing 300–450 cells. Statistical significance was determined using the Mann–Whitney-Wilcoxon test, followed by Bonferroni post hoc test (ns: not significant; *: p < 0.05; **: p < 0.01; ***: p < 0.001). d Immunofluorescent staining of cultures treated with Tat (60 ng/mL) with or without 4OI (250 μM) for 48 h followed by cocaine (25 μM) for 24 h, fixed with 4% PFA. Dead/apoptotic cells were detected using the CellEvent Caspase 3/7 Assay Kit (Green: Caspase 3/7; Red: Ethidium Bromide). Scale bar: 100 μm

Fig. 2

Effects of 4OI treatment on microglia morphology in Tat and/or cocaine-treated primary neuronal cultures. Immunofluorescent staining of primary cortical cultures: a Cells were treated with Tat alone (6 ng/ml) or Tat combined with 4OI (60 or 250 μM) for 48 h, followed by cocaine (25 μM) for additional 24 h. Cells were fixed with 4% PFA. Staining: Iba1 (green, microglia marker), MAP2 (red, neuronal marker), DAPI (blue, nuclei). Scale bar: 100 μm. b Cells were subjected to the same treatment. Staining: CD11b/c (magenta, activated microglia marker), GFAP (green, astrocyte marker), MAP2 (red, neuronal marker), DAPI (blue, nuclei)

Fig. 3

Effects of Tat and cocaine on microglia cell morphology in primary neuronal cultures. a Proportion of Iba1 positive microglial cells in cultures treated with Tat (6 ng/mL) and/or 4OI (60 or 250 µM) for 48 h, with or without an additional 24 h of cocaine (25 µM). Tat-Coc vs Tat-Coc-4OI (60 µM) yielded not significant p-adj value = 0.126. Tat-Coc vs Tat-Coc-4OI (250 µM) yielded not significant p-adj value = 0.114. b Length of microglia processes and c microglia cell body size in the same cultures. The intensity of the blue color indicates increasing 4OI concentration (60, 250 µM). The box covers 50% of the data in each condition, and the line inside indicates the median value. The Mann–Whitney-Wilcoxon test was conducted to calculate the statistical significance, followed by Bonferroni post hoc test (ns, not significant; *, p < 0.05; **, p < 0.01; ***, P < 0.001). d 3D projection of microglia morphology affected by 4OI and/or Tat treatment. e 3D projection of microglia morphology affected by 4OI and/or Tat-Cocaine treatment (x: Body size; y: Percentage; z: Process length)

Fig. 4

Genome-wide effects of 4OI on primary neuronal cultures treated with Tat and/or cocaine. RNA-seq transcriptomics of primary cortical cultures treated with Tat (6 ng/mL) and/or cocaine (25 μM) with/without 4OI (250 μM). Volcano plot comparing differential gene expression levels of a Tat treated samples vs Control, b Tat-Cocaine treated samples vs Control, c and cocaine treated samples vs Control. Volcano plot comparing the differential gene expression between d 4OI vs Control e Tat-4OI vs Tat, and f Tat-Cocaine-4OI vs Tat-Cocaine conditions. Scatter plot illustrating the effect of 4OI on transcriptomic fold changes of select differentially expressed genes (FDR < 0.05), and how they correlate between Tat-4OI vs Tat against g 4OI vs Control and h Tat-Cocaine-4OI vs Tat-Cocaine conditions. The colors correspond with the listed gene set pathway, with green representing select anti-inflammatory and magenta representing select pro-inflammatory pathways. Heatmap showing gene expression levels for i phosphodiesterase-associated genes, j purinergic receptor-associated genes, k phagocytic cytoskeleton-associated genes, l interleukin superfamily genes, and m tumor necrosis factor family genes

Fig. 5

Effects of 4OI on cytokine secretion profile in primary neuronal cultures treated with Tat and/or cocaine. Bar graphs showing the levels of cytokine secretion followed by Tat 6 ng/mL (A) or 60 ng/ml (B) and Tat-Cocaine (25 μM), with or without 4OI (250 μM). The cytokine levels were determined by the 27-plex chemokine/cytokine array thrice from each sample of collected cell culture. Bars represent chemokine/cytokine concentrations in the medium (pg/ml) ± SD

Fig. 6

Possible model of 4OI-mediated neuroprotection. The observed neuronal damage and Acod1 overexpression resulting from Tat and cocaine treatment are significantly attenuated upon 4OI administration. This potential neuroprotection might stem from 4OI's negative feedback inhibition of Acod1