Integration of Molecular Inflammatory Interactome Analyses Reveals Dynamics of Circulating Cytokines and Extracellular Vesicle Long Non-Coding RNAs and mRNAs in Heroin Addicts During Acute and Protracted Withdrawal

Abstract

Heroin addiction and withdrawal influence multiple physiological functions, including immune responses, but the mechanism remains largely elusive. The objective of this study was to investigate the molecular inflammatory interactome, particularly the cytokines and transcriptome regulatory network in heroin addicts undergoing withdrawal, compared to healthy controls (HCs). Twenty-seven cytokines were simultaneously assessed in 41 heroin addicts, including 20 at the acute withdrawal (AW) stage and 21 at the protracted withdrawal (PW) stage, and 38 age- and gender-matched HCs. Disturbed T-helper(T_h)1/T_h2, T_h1/T_h17, and T_h2/T_h17 balances, characterized by reduced interleukin (IL)-2, elevated IL-4, IL-10, and IL-17A, but normal TNF-α, were present in the AW subjects. These imbalances were mostly restored to the baseline at the PW stage. However, the cytokines TNF-α, IL-2, IL-7, IL-10, and IL-17A remained dysregulated. This study also profiled exosomal long non-coding RNA (lncRNA) and mRNA in the plasma of heroin addicts, constructed co-expression gene regulation networks, and identified lncRNA-mRNA-pathway pairs specifically associated with alterations in cytokine profiles and T_h1/T_h2/T_h17 imbalances. Altogether, a large amount of cytokine and exosomal lncRNA/mRNA expression profiling data relating to heroin withdrawal was obtained, providing a useful experimental and theoretical basis for further understanding of the pathogenic mechanisms of withdrawal symptoms in heroin addicts.

Keywords: cytokine; exosome; heroin; non-coding RNA; withdrawal.

Figure 1

Alterations in cytokine profiles in heroin addicts during withdrawal. (A–J) The levels of various cytokines in the plasma samples of healthy controls, heroin addicts from AW and PW groups was investigated, including IL-4 (A), Eotaxin (B), G-CSF (C), MCP-1 (D), VEGF (E), IL-2 (F), IL-7 (G), IL-10 (H), IL-17A (I), TNF-α (J). (K–M) Th1/Th2/Th17/Treg balance in heroin addicts across two withdrawal stages (AW and PW). The ratio of T_h1/T_h2, T_h1/T_reg (K) and T_h1/T_h17 (L) was significantly decreased in AW stage, and recovered in PW stage. The ratio of T_h2/T_h17 and T_reg/T_h17 (M) was significantly increased in AW stage, and recovered in PW stage. Each dot represents one patient in each group. Values of all significant correlations (p < 0.05) are given with degree of significance indicated (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001). All intergroup comparisons by Mann-Whitney U-test.

Figure 2

Identification and functional enrichment analysis of differentially expressed Ex-mRNA. (A, D, G) The volcano plot and MA plot (A, D) and hierarchical clustering heatmap (G) of differentially expressed exosome mRNAs in Heroin AW versus HC (P < 0.05, fold change > 1.5). (B, E, H) The volcano plot and MA plot (B, E) and hierarchical clustering heatmap (H) of differentially expressed exosome mRNAs in Heroin PW versus HC (P < 0.05, fold change > 1.5). (C, F, I) The volcano plot and MA plot (C, F) and hierarchical clustering heatmap (I) of differentially expressed exosome mRNAs in Heroin AW versus Heroin PW (P < 0.05, fold change > 1.5). (J–M) Top enrichment of KEGG pathways related to differentially expressed exosome mRNAs in Heroin AW versus HC (J, K) and in Heroin PW versus HC (L, M).

Figure 3

Identification and functional enrichment analysis of differentially expressed exosome lncRNAs. (A, D, G) The volcano plot and MA plot (A, D) and hierarchical clustering heatmap (G) of differentially expressed exosome lncRNAs in Heroin AW versus HC (P < 0.05, fold change > 1.5). (B, E, H) The volcano plot and MA plot (B, E) and hierarchical clustering heatmap (H) of differentially expressed exosome lncRNAs in Heroin PW versus HC (P < 0.05, fold change > 1.5). (C, F, I) The volcano plot and MA plot (C, F) and hierarchical clustering heatmap (I) of differentially expressed exosome lncRNAs in Heroin AW versus Heroin PW (P < 0.05, fold change > 1.5). (J, M) Top enrichment of KEGG pathways related to differentially expressed exosome lncRNAs in Heroin AW versus HC (J, K) and in Heroin PW versus HC (L, M).

Figure 4

WGCNA of plasma exosome samples uncovered dynamic changes of exosome mRNAs and lncRNAs signatures during substance withdrawal. (A) Hierarchical cluster dendrogram using WGCNA analyzing RNA sequencing data. A total of 5 modules were identified after 0.25 threshold merging. (B) Module trait correlation analysis revealed that 5 key modules were significantly correlated with Heroin AW and Heroin PW. (C) The eigengene dendrogram and heatmap showed the relevancies of cytokines and identified RNA-seq modules. TNF-a, IL-2, IL-4, IL-10 and IL-17A were selected. (D) The correlation and connectivity of the turquoise module and TNF-a (r = 0.48, p = 2.6E-69). (E) The hub-gene network of turquoise module. The network of Top 50 lncRNAs/mRNAs (Rankings related to turquoise module) were showed. (F) The correlation and connectivity of the red module and IL-4 (r = 0.35, p = 1.1E-10). (G) The hub-gene network of red module. The network of Top 50 lncRNAs/mRNAs (Rankings related to red module) were showed. (H) The correlation and connectivity of the blue module and IL-10 (r = 0.59, p = 1.1E-45). (I) The hub-gene network of blue module. The network of Top 50 lncRNAs/mRNAs (Rankings related to blue module) were showed.

Figure 5

Identification of immune-related lncRNA-mRNA-pathway network. Circos plot showing the top-ranked lncRNA–mRNA-pathway pairs in heroin AW and PW stages. Red, blue (AW stage) and turquoise (green, PW stage) colors represent the corresponding modules and stages.