Profiles of MicroRNAs in Interleukin-27-Induced HIV-Resistant T Cells: Identification of a Novel Antiviral MicroRNA

Abstract

Objectives: Interleukin-27 (IL-27) is known as an anti-HIV cytokine. We have recently demonstrated that IL-27-pretreatment promotes phytohemagglutinin-stimulated CD4(+) T cells into HIV-1-resistant cells by inhibiting an uncoating step.

Purpose: To further characterize the function of the HIV resistant T cells, we investigated profiles of microRNA in the cells using microRNA sequencing (miRNA-seq) and assessed anti-HIV effect of the microRNAs.

Methods: Phytohemagglutinin-stimulated CD4(+) T cells were treated with or without IL-27 for 3 days. MicroRNA profiles were analyzed using miRNA-seq. To assess anti-HIV effect, T cells or macrophages were transfected with synthesized microRNA mimics and then infected with HIVNL4.3 or HIVAD8. Anti-HIV effect was monitored by a p24 antigen enzyme-linked immunosorbent assay kit. interferon (IFN)-α, IFN-β, or IFN-λ production was quantified using each subtype-specific enzyme-linked immunosorbent assay kit.

Results: A comparative analysis of microRNA profiles indicated that expression of known miRNAs was not significantly changed in IL-27-treated cells compared with untreated T cells; however, a total of 15 novel microRNAs (miRTC1 ∼ miRTC15) were identified. Anti-HIV assay using overexpression of each novel microRNA revealed that 10 nM miRTC14 (GenBank accession number: MF281439) remarkably suppressed HIV infection by (99.3 ± 0.27%, n = 9) in macrophages but not in T cells. The inhibition was associated through induction of >1000 pg/mL of IFN-αs and IFN-λ1.

Conclusion: We discovered a total of 15 novel microRNAs in T cells and characterized that miRTC14, one of the novel microRNAs, was a potent IFN-inducing anti-HIV miRNA, implicating that regulation of the expression of miRTC14 may be a potent therapeutic tool for not only HIV but also other virus infection.

FIGURE 1.

Comparison of baseline novel miRNA expression in T cells and MDMs. CD4(+) T cells and CD14(+) monocytes were isolated from peripheral blood mononuclear cells of 3 independent healthy donors. A, T cells were stimulated with phytohemagglutinin for 3 days and then cultured for additional 3 days without (Ctrl-Tc) or with 100 ng/mLIL-27 (27-Tc) in the presence of 20 units/mL of IL-2. B, Monocytes were differentiated into macrophages in the presence of 25 ng/mL M-CSF alone (MDMs) or 25 ng/mL M-CSF with 100 ng/mL IL-27 (I-Mac) for 7 days. Total RNA from Ctrl-Tc, 27-Tc, MDMs, and I-Mac were extracted, and the expression of each novel miRNA was quantified by real time qRT-PCR. Gene-specific probes were custom-made by Thermo Fisher Scientific. As an internal control, the small nuclear protein RNU44 probe was used. Base line of the expression of each miRNA was calculated by comparing the Ct values of each miRNA with the Ct value of RNU44 and then subtracting this from 40 (40-delta Ct). Results represent mean ± SE (n = 3) of 3 independent assays.

FIGURE 2.

Characterization of the anti-HIV effect of each novel miRNA in MDMs. A, MDMs were transfected with 10 nM miRNA using RNAimax lipofectamine for 72 hours and then infected with HIVAD8 (opened bars) or HIVLuc-V (closed bars) as described in the Materials and Methods. HIVAD8 and HIVLuc-V infected cells were culture for 14- and 2-days, respectively. HIV replication was monitored by a p24 antigen capture kit (PerkinElmer) and Luciferase was detected by the Bright-Glo (Promega). Each experiment was performed triplicate using cells from 3 independent donors. Results represent relative infection compared to HIV infection value in miRCtrl-transfected MDMs. Data indicate mean ± SE (n = 3). *P < 0.05, ***P < 0.001. B, MDMs were transfected with 0, 0.1, 1.0, 10.0, and 100.0 nM of miRCtrl or miRTC14 mimic for 72 hours, and total RNA was extracted, and change of miRTC14 expression was quantified using qRT-PCR, results indicate relative gene expression increase dose dependently in miRTC14 transfected cells. C, MDMs were transfected with 0, 0.1, 1.0, 10.0, and 100.0 nM miRCtrl or miRTC14 mimic for 72 hours and then infected with HIVLuc-V. HIV infection was monitored at 48 hours after infection by the luciferase activity using the Bright Glo. Results represent mean ± SD. ***P < 0.001.

FIGURE 3.

miRTC14 significantly induces type-I and type-III IFNs. A, Impact of RNA transfection lipid on the anti-HIV effect. MDMs were treated with a transfection lipid reagent, lipofectamine RNAiMAX, miRNA (10 nM miRCtrl or miRTC14), or miRNA with the lipid for 72 hours. The cells were then infected with HIVLuc-V. HIV-infection was monitored by the luciferase activity. The data are a representative result from 2 independent experiments, the results show mean ± SD ***P < 0.001. B, MDMs were transfected with 10 nM miRCtrl or miRTC14 and cultured in the absence or presence of 1 μg/mL of B18R for a total 72 hours. The transfected cells were infected with HIVLuc-V and then incubated for 48 hours. HIV infection was monitored by the luciferase activity. As a control, untreated cells or IFN-α (R&D systems)-treated cells were also cultured with or without the B18R. The data are a representative result from 2 independent experiments, data shown are mean ± SD (n = 3). ***P < 0.001. C, Quantitation of concentrations of IFNs in transfection supernatants. MDMs were transfected with 10 nM of miRCtrl or miRTC14 for 72 hours, and, then, cell-free supernatants were collected. Concentrations of IFN-α, β, and α were determined through ELISA kits. Detection limits of IFN-αs (all subtypes), β, and λs (all subtypes) were 1.25, 1.2, and 15.6 pg/mL, respectively. The box plots show data the results from 8 donors for IFN-αs and IFN-λs and 5 donors for IFN-β.

Figure 4.

miRTC14 induces the activation of multiple subtypes of IFN genes. MDMs were transfected with 10 nM miRCtrl or miRTC14 and cultured for 48 hours. Total RNAs were extracted from the cells, and real time qRT-PCR was performed using a gene-specific probe for each subtype of IFNs. Gene expression is presented as relative expression units compared with miRCtrl-transfection after normalization to GAPDH. Results represent ± SE from 3 independent experiments. The inserted table shows the corresponding P values of all the type of interferon gene expressions.