Morphine treatment of human monocyte-derived macrophages induces differential miRNA and protein expression: impact on inflammation and oxidative stress in the central nervous system

Abstract

HIV-1-infected opiate abusers often exhibit an accelerated form of HIV-1-associated dementia and enhanced neurological dysfunction. Productive HIV-1 infection of microglia and perivascular macrophages and the resultant secretion of neurotoxic molecules by these cells contribute to this phenomenon. In order to understand the role of morphine in this process, we performed a genome-wide association study at the micro RNA (miRNA) and protein levels in human monocyte-derived macrophages (h-mdms). A total of 26 differentially expressed miRNA were identified (P < 0.01), of which hsa-miR-15b and hsa-miR-181b had the greatest increase and decrease in expression levels, respectively. Computational analysis predicted fibroblast growth factor-2 (FGF-2) as the strongest target gene for hsa-miR15b. Of note, we observed a decrease in FGF-2 protein expression in response to morphine. Both hsa-miR-15b and hsa-miR-181b have several predicted gene targets involved in inflammation and T-cell activation pathways. In this context, we observed induction of MCP-2 and IL-6 by morphine. Moreover, proteomic analysis revealed the induction of mitochondrial superoxide dismutase in response to morphine treatment. HIV-1 infection did not induce mitochondrial superoxide dismutase. Collectively, these observations demonstrate that morphine induces inflammation and oxidative stress in h-mdms thereby contributing to expansion of HIV-1 CNS reservoir expansion and disease progression. Of note, differentially expressed miRNAs (hsa-miR-15b and 181-b) may have a potential role in regulating these processes.

Fig. 1

Morphine treatment of h-mdms induces differential miRNA expression. miRNA isolated from morphine-treated and control h-mdms were labeled with Cy5 or Cy3, respectively. A: Labeled miRNA were hybridized simultaneously to a miRNA array (Sanger Version 12.0) to detect morphine-induced differential miRNA expression. The majority of the differentially expressed miRNAs had <0.5 fold change in expression level as compared to control. The outer lines along the median indicate a 0.5 fold change in expression levels. B: A total of 26 different miRNAs with P-values ≤ 0.01 were identified to exhibit differential expression. The heat-map of these miRNAs is depicted on the right-side. Green signal on the heat-map indicates a decrease in miRNA expression and red signal indicates an increase in miRNA expression level as compared to control (range: -2.0 to +2.0). C: Eight of these 26 miRNAs had a log₂ ratio ≥ 0.05 and P-values 0.01.

Fig. 2

Morphine treatment of h-mdms decreases secretion of FGF-2. Cell-free culture supernatants were concentrated to equivalent volumes (25×) on ultra-filtration membranes (cut-off: 3,000 kDa). Concentrated cell-free culture supernatants from uninfected and HIV-1 YU-2-infected h-mdms at 5dpi were either treated with 0.1μM morphine or 0.1μM morphine and 1.0μM naloxone for a duration of 24h. These concentrated cell-free supernatants along with the same from untreated controls were subjected to Western blot analysis. Image-quantitation of the blot suggests FGF-2 secretion was decreased to 85% and 66% in response to morphine treatment and morphine treatment during HIV-1 infection, respectively. Signal intensities depicted were normalized with protein concentration and compared as % of control.

Fig. 3

Morphine-induced secretion of inflammatory cytokines and chemokines. A: Cell-free culture supernatants from morphine-treated, HIV-1 JR-FL-infected, HIV-1 JR-FL-infected plus morphine-treated or control h-mdms were subjected to an inflammatory chemokine and cytokine antibody array analysis. B: Quantitation of signal intensities revealed that morphine treatment and HIV-1 infection of h-mdms increased secretion of MCP-2 and IL-6. A cumulative effect was not observed when HIV-1-infected h-mdms were treated with morphine. Increase in PDGF-BB secretion was not statistically significant. No induction of TNF-α was observed in this array analysis.

Fig. 4

Morphine treatment of h-mdms induces differential protein expression. Protein extracts from control h-mdms, morphine-treated and HIV-1 YU-2-infected h-mdms were subjected to 2D gel electrophoresis. Proteins were resolved in first dimension by isoelectric focusing at pI 4-7 and 6-9. Signal intensities of resolved protein spots from morphine-treated and HIV-1 YU-2-infected h-mdms was compared to the same from control h-mdms. The 3D plot on the top row shows change in protein expression from within the square in the pI 4-7 gel electrophoresis. Both morphine and HIV-1 infection decrease intensity of spot 1. In addition, spot 2 was induced by morphine treatment and spot 3 was induced in response to HIV-1 YU-2 infection. The 3D plot in the bottom row shows changes in protein expression from within the square in the pI 6-9 gel electrophoresis. Spot 9 was induced only in response to morphine treatment.

Fig. 5

Mitochondrial superoxide dismutase was induced by morphine treatment of h-mdms. Differentially-expressed protein spots were identified by MALDI-TOF MS analysis. A: Majority of these protein spots were suppressed as a result of HIV-1 YU-2 infection. Identity of 6 of the 15 spots was successfully confirmed by MALDI-TOF MS analysis. B. Only 1 of these 6 spots was specifically induced by morphine and was identified as mitochondrial superoxide dismutase.

Fig. 6

Mechanisms through which morphine contributes to expansion of HIV-1 reservoir in CNS. Interaction of morphine with the μ-opioid receptor is known to trigger GPCR signaling pathways that can regulate miRNA expression, pro-inflammatory response and oxidative stress. Each of these biochemical responses induced by morphine may lead to expansion of the HIV-1 viral reservoir in the CNS and explain the accelerated disease progression in opioid abusing cohorts.