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. 2022 Sep 12;19(1):225.
doi: 10.1186/s12974-022-02586-9.

Chronic delta-9-tetrahydrocannabinol (THC) treatment counteracts SIV-induced modulation of proinflammatory microRNA cargo in basal ganglia-derived extracellular vesicles

Hussein Kaddour  1   2 , Marina McDew-White  3 , Miguel M Madeira  1 , Malik A Tranquille  1 , Stella E Tsirka  1 , Mahesh Mohan  4 , Chioma M Okeoma  5   6
Affiliations

Chronic delta-9-tetrahydrocannabinol (THC) treatment counteracts SIV-induced modulation of proinflammatory microRNA cargo in basal ganglia-derived extracellular vesicles

Hussein Kaddour et al. J Neuroinflammation. .

Abstract

Background: Early invasion of the central nervous system (CNS) by human immunodeficiency virus (HIV) (Gray et al. in Brain Pathol 6:1-15, 1996; An et al. in Ann Neurol 40:611-6172, 1996), results in neuroinflammation, potentially through extracellular vesicles (EVs) and their micro RNAs (miRNA) cargoes (Sharma et al. in FASEB J 32:5174-5185, 2018; Hu et al. in Cell Death Dis 3:e381, 2012). Although the basal ganglia (BG) is a major target and reservoir of HIV in the CNS (Chaganti et al. in Aids 33:1843-1852, 2019; Mintzopoulos et al. in Magn Reson Med 81:2896-2904, 2019), whether BG produces EVs and the effect of HIV and/or the phytocannabinoid-delta-9-tetrahydrocannabinol (THC) on BG-EVs and HIV neuropathogenesis remain unknown.

Methods: We used the simian immunodeficiency virus (SIV) model of HIV and THC treatment in rhesus macaques (Molina et al. in AIDS Res Hum Retroviruses 27:585-592, 2011) to demonstrate for the first time that BG contains EVs (BG-EVs), and that BG-EVs cargo and function are modulated by SIV and THC. We also used primary astrocytes from the brains of wild type (WT) and CX3CR1+/GFP mice to investigate the significance of BG-EVs in CNS cells.

Results: Significant changes in BG-EV-associated miRNA specific to SIV infection and THC treatment were observed. BG-EVs from SIV-infected rhesus macaques (SIV EVs) contained 11 significantly downregulated miRNAs. Remarkably, intervention with THC led to significant upregulation of 37 miRNAs in BG-EVs (SIV-THC EVs). Most of these miRNAs are predicted to regulate pathways related to inflammation/immune regulation, TLR signaling, Neurotrophin TRK receptor signaling, and cell death/response. BG-EVs activated WT and CX3CR1+/GFP astrocytes and altered the expression of CD40, TNFα, MMP-2, and MMP-2 gene products in primary mouse astrocytes in an EV and CX3CR1 dependent manners.

Conclusions: Our findings reveal a role for BG-EVs as a vehicle with potential to disseminate HIV- and THC-induced changes within the CNS.

Keywords: Astrocytes; Basal ganglia; CX3CR1; Extracellular vesicles; Neuroinflammation; THC.

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Conflict of interest statement

The authors report no biomedical financial interests or potential conflicts of interest.

Figures

Fig. 1
Fig. 1
Physicochemical characterization of BG-EVs. A Absorbance at 280 nm of PPLC-isolated collagenase-digested BG eluates from VEH, SIV and THC/SIV groups (n = 4/group). B 3D-surface plot featuring the EV-turbidimetric signature. C Contour representation highlighting the non-membranous molecular condensates (MCs) whose UV-peak blue-shifted to 262 nm. D R2 index detected in fractions 7–16, confirming the presence of EVs in these fractions, whereas fractions 19–28 contained the MCs. E, F PPLC-derived particle size (E) and concentration (F). G Zeta (ζ)-potential of BG-EVs as measured by NTA (ZetaView). H Quantification of EV-associated total protein as measured by the Bradford assay (top) and assessment of the protein weight per EV particle (Bottom). N = 4 per group. Error bars represent standard error of the mean. No statistical differences were noted
Fig. 2
Fig. 2
TEM–EDX elemental analysis of BG structures. A Representative TEM images of CD9-immunolabeled EVs from BG tissues, from the three RM groups. The black spots on the structures correspond to 6-nm-gold-nanoparticles-labeled goat anti-mouse secondary antibody. B Total of 25 TEM images were analyzed for size and counted for the immunogold label per vesicle and the means were reported per group. Error bar represent standard error of the mean. The number above the bars represent the number of vesicles analyzed per group. C Representative images of MC from BG tissues. Seen are dense, sharp-edged and membraneless structures of primary particle size of ~ 10–20 nm that aggregate into large structures up to ~ 500 nm. D Corresponding EDX spectra of the MC structures revealing presence of SPONCH (Sulphur, S; Phosphorus, P; Oxygen, O; Nitrogen, N; Carbon, C; and H, Hydrogen) elements, as a signature of biomolecules. Despite the strong signal of copper, Cu and chromium, Cr which constitute the main material of the specimen holder, and the fact that H is beyond the EDX detection limit and C may also be a specimen artifact, persistent traces of SPON, sulfur, phosphorus, oxygen, and nitrogen, were detected, supporting a ribonucleo-proteinaceous nature of the MC structures
Fig. 3
Fig. 3
Changes in miRNA expression in BG-EVs from chronically SIV-infected rhesus macaques administered vehicle (VEH) or THC. A–C Volcano plots showing the relationship between fold-change and statistical significance of differentially expressed miRNAs in A VEH/SIV RMs relative to uninfected control, B THC/SIV relative to VEH/SIV RMs, and C THC/SIV relative to control RMs. The blue and red vertical lines correspond to 1.5-fold down and up, respectively, and the horizontal black lines represent p < 0.05. The miRNAs of interest are denoted with arrows. D Heatmap of significant pathways predicted by DIANA-miRPath (v.3) of the human counterparts of the 11 SIV-downregulated miRs, showing inflammation and immune regulation pathway cluster (red arrows), a TLR signaling pathway cluster (orange arrows), in addition to Neurotrophin TRK receptor signaling pathway and response to stress (green arrows), as well as cell death and viral process (black arrows), among others. E Raw counts of selected neuroregulatory miRNAs, downregulated in the VEH/SIV group. F TaqMan PCR validation using mml-let-7a-5p and mml-let-7c-5p specific TaqMan microRNA stem-loop RT-qPCR assays. Statistical differences were assessed by a two-way ANOVA test with Tukey’s correction (n = 3). *p < 0.05
Fig. 4
Fig. 4
BG-EVs are taken up by astrocytes: we incubated 10,000 astrocytes from CX3CR1+/GFP mice with increasing concentrations (0, 12.5, 25, 50, 100 and 200 µg/mL) of SytoSELECT labeled BG-EVs in the presence of NucBlue (a live cell stain, 30 µL/mL) and seeded in a glass-bottom 96 well plate 24-h prior to experiment. Kinetics imaging (every 3 h) was recorded over the course of 18 h using automated Lionheart FX software [18] (Biotek) after which total GFP (ex/em) and Nucblue (ex/em) intensity were recorded using a plate reader (Synergy H1 Biotek). Astrocytes were assessed for viability using Cell Titer Glow assay (Promega). A Representative 10 × images at 18 h timepoint after addition of BG-EVs. B Total gfp and nucblue intensity as measured by the plate reader. E Viability of astrocytes treated with BG-EVs. Ordinary one-way ANOVA multiple comparison test (Tukey’s test) was used to assess statistical differences. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, non-significant. Error bars represent Standard Deviation
Fig. 5
Fig. 5
BG-EVs elevate Glial Fibrillary Acidic Protein (GFAP) levels: 10,000 primary mouse astrocytes per well were seeded in a glass-bottom 96 well plate 24-h prior to experiment. Cells were treated with vehicle PBS or with 100 µg/mL pooled BG-EVs (n = 4, 25 µg/pool from each group) for 24 h, in triplicate wells and 9 field of views. Cells were then fixed, immuno-stained for GFAP, and imaged using an automated scope (Lionheart FX, Biotek). A, C Representative 10 × images for A WT and C CX3CR1 + GFP + astrocytes. Scale bar = 50 µm. B, D Mean fluorescence intensity (MFI) of GFAP calculated using single-cell analysis for B WT and D CX3CR1 + GFP + astrocytes. Ordinary one-way ANOVA multiple comparison test (Dunnett’s test) was used to assess statistical differences in B and D. *p < 0.05; ***p < 0.001; ****p < 0.0001. Error bars represent S.E.M. of 600–1000 cells per treatment. E Graph depicting genotype-dependent differential response to the treatments between WT and CX3CR1 + GFP + astrocytes. Unpaired T test with Welch’s correction was used to assess statistical differences. **p < 0.01; ***p < 0.001; ****p < 0.0001. Error bars represent S.E.M. of 600–1000 cells per treatment
Fig. 6
Fig. 6
BG-EVs mediate distinct changes in the transcriptome of astrocytes: 200,000 primary mouse astrocytes per well were seeded in a 12 well-plate 24-h prior to experiment. Cells were treated with vehicle PBS or with 100 µg/mL pooled BG-EVs (n = 4, 25 µg/pool from each group) and incubated for 24 h. Cellular RNA was extracted, and gene expression was assessed by RT-qPCR. A–D Differential analysis of A CD40, B TNFα, C MMP2, and D MMP9. Top graph is WT and bottom graph is CX3CR1 + GFP + (TG) astrocytes. E–H Graphs depicting the differential response to the treatments between WT and CX3CR1 + GFP + (TG) astrocytes. Ordinary one-way ANOVA multiple comparison test (Dunnett’s test) was used to assess statistical differences in A–D. Unpaired T test with Welch’s correction was used to assess statistical differences between SIV and THC/SIV groups in panels A–H. Error bars represent S.E.M. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant. I The levels of untreated astrocyte CX3CR1 mRNA expression relative to normalization against GAPDH. The RT-qPCR products representative of mRNA levels were normalized to GAPDH signal and shown on the bar, while the amplicons separated on agarose gel is the inset. J Protein–protein interaction (PPI) network of the altered genes in astrocytes
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