Identification of Differentially Expressed Long Non-coding RNAs in Polarized Macrophages

Abstract

Macrophages display remarkable plasticity, with the ability to undergo dynamic transition between classically and alternatively activated phenotypes. Long non-coding RNAs (lncRNAs) are more than 200 nucleotides in length and play roles in various biological pathways. However, the role of lncRNAs in regulating macrophage polarization has yet to be explored. In this study, lncRNAs expression profiles were determined in human monocyte-derived macrophages (MDMs) incubated in conditions causing activation toward M(IFN-γ + LPS) or M(IL-4) phenotypes. Compared with primary MDMs, 9343 lncRNAs and 5903 mRNAs were deregulated in M(IFN-γ + LPS) group (fold change ≥ 2.0, P < 0.05), 4592 lncRNAs and 3122 mRNAs were deregulated in M(IL-4) group. RT-qPCR results were generally consistent with the microarray data. Furthermore, we found that TCONS_00019715 is expressed at a higher level in M(IFN-γ + LPS) macrophages than in M(IL-4) macrophages. TCONS_00019715 expression was decreased when M(IFN-γ + LPS) converted to M(IL-4) whereas increased when M(IL-4) converted to M(IFN-γ + LPS). Knockdown of TCONS_00019715 following the activation of THP-1 cellls using IFN-γ and LPS diminished the expression of M(IFN-γ + LPS) markers, and elevated the expression of M(IL-4) markers. These data show a significantly altered lncRNA and mRNA expression profile in macrophages exposure to different activating conditions. Dysregulation of some of these lncRNAs may play important roles in regulating macrophage polarization.

Figure 1. Identifiation of ex vivo-programmed M(IFN-γ + LPS) and M(IL-4) macrophages.

MDMs were cultured in the presence of IFN-γ (20 ng/mL) plus LPS (100 ng/mL) or IL-4 (20 ng/mL). (A) Polarization-specific biomarkers were analyzed by RT-qPCR assays using RNA collected from MDMs at 18 hours post-treatment. (B–H) TNF-α (B), IL-6 (C), IL-12 (D), IL-10 (E), CCL17 (F), CCL18 (G) and CCL22 (H) in the supernatant were assayed by ELISA. Data are representative of three separate experiments, and show the means ± SEM. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001.

Figure 2. LncRNA and mRNA microarray expression data from MDMs incubated in distinct polarizing conditions.

Scatter plots showing the variation in lncRNAs (A) and mRNAs (B) expression between the M(−), M(IFN-γ + LPS) and M(IL-4) macrophages. The values of the X and Y axes in the scatter plot are averaged normalized values in each group (log2-scaled). Hierarchical clustering results of lncRNAs (C) and mRNAs (D) expression profiles among three groups. “Red” indicated high relative expression and “green” indicated low relative expression. One ANOVA test was used for statistical analysis. LncRNA or mRNA with expression fold change >2 and with FDR adjusted P value <0.05 was considered statistically significant.

Figure 3. Confirmation of lncRNAs expression by RT-qPCR.

Individual RT-qPCR assays were performed using samples from six additional MDM treated with polarizing conditions for 18 hours. After normalization to GAPDH expression, data were presented as mean ± SEM and obtained average expression value for each lncRNA was used for statistics. One ANOVA test for three groups or student’s t test for two groups was used for statistical analysis. Six lncRNAs were differentially expressed between three groups. ^*Significant difference between M(−) group and M(IFN-γ + LPS) group, as well as between M(−) group and M(IL-4) group. ^#Significant difference between M(IFN-γ + LPS) group and M(IL-4) group. P value <0.05 was considered statistically significant. Error bars in graphs referred to standard deviation. Each reaction was run three separate times, with technical triplicates in each reaction.

Figure 4. Differential expression of TCONS_00019715 during macrophage polarization.

TCONS_00019715 was assessed by RT-qPCR and normalized to GAPDH in THP-1 macrophages after 18 hours of stimulation with IFN-γ (20 ng/mL) plus LPS (100 ng/mL) (A) or GM-CSF (20 ng/mL) (B). (C) TCONS_00019715 levels in macrophages following M(IFN-γ + LPS)-to-M(IL-4) re-polarization by IL-4 (20 ng/mL) for 18 hours. (D) TCONS_00019715 levels in macrophages following M(IL-4)-to-M(IFN-γ + LPS) re-polarization by IFN-γ (20 ng/mL) plus LPS (100 ng/mL) for 18 hours.

Figure 5. Knockdown of TCONS_00019715 promotes transition of M(IFN-γ + LPS) macrophages to the M(IL-4) phenotype and diminishes the expression of M(IFN-γ + LPS) phenotypes in M(IFN-γ + LPS) macrophages.

Re-polarization of M(IFN-γ + LPS) macrophages to M(IL-4) macrophages by depleting TCONS_00019715 (A), correlating with reduction of TNF-α (B), IL-6 (C), IL-12 (D) and elevation of IL-10 (E), CCL17 (F), CCL18 (G) and CCL22 (H). Macrophages were transfected with an si-TCONS_00019715 (si-19715) or a control oligonucleotide (si-NC) and then stimulated with LPS and IFN-γ for M(IFN-γ + LPS) polarization (I), correlating with reduction of TNF-α (J), IL-6 (K), IL-12 (L) and elevation of IL-10 (M), CCL17 (N), CCL18 (O) and CCL22 (P). Data are representative of three separate experiments, and show the means ± SEM. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001.