MicroRNA-146a and microRNA-146b regulate human dendritic cell apoptosis and cytokine production by targeting TRAF6 and IRAK1 proteins

Abstract

We have previously reported 27 differentially expressed microRNAs (miRNAs) during human monocyte differentiation into immature dendritic cells (imDCs) and mature DCs (mDCs). However, their roles in DC differentiation and function remain largely elusive. Here, we report that microRNA (miR)-146a and miR-146b modulate DC apoptosis and cytokine production. Expression of miR-146a and miR-146b was significantly increased upon monocyte differentiation into imDCs and mDCs. Silencing of miR-146a and/or miR-146b in imDCs and mDCs significantly prevented DC apoptosis, whereas overexpressing miR-146a and/or miR-146b increased DC apoptosis. miR-146a and miR-146b expression in imDCs and mDCs was inversely correlated with TRAF6 and IRAK1 expression. Furthermore, siRNA silencing of TRAF6 and/or IRAK1 in imDCs and mDCs enhanced DC apoptosis. By contrast, lentivirus overexpression of TRAF6 and/or IRAK1 promoted DC survival. Moreover, silencing of miR-146a and miR-146b expression had little effect on DC maturation but enhanced IL-12p70, IL-6, and TNF-α production as well as IFN-γ production by IL-12p70-mediated activation of natural killer cells, whereas miR-146a and miR-146b overexpression in mDCs reduced cytokine production. Silencing of miR-146a and miR-146b in DCs also down-regulated NF-κB inhibitor IκBα and increased Bcl-2 expression. Our results identify a new negative feedback mechanism involving the miR-146a/b-TRAF6/IRAK1-NF-κB axis in promoting DC apoptosis.

Keywords: Apoptosis; Cytokine; Dendritic Cell; Differentiation; IL-1 Receptor-associated Kinases (IRAK); MicroRNA (miRNA); NF-kappa B (NF-κB); TNF Receptor-associated Factor (TRAF); miR-146.

FIGURE 1.

miR-146 expression during monocyte differentiation into imDCs and mDCs. A, miR-146a and miR-146b expression in human monocytes (Mo) at day (D) 0; imDCs at days 2, 4, and 6; and mDCs at day 8 was analyzed by real-time PCR analysis. B, copy numbers of miR-146a and miR-146b. C, effect of the individual component in the maturation mixture on miR-146a and miR-146b expression. imDCs at day 6 were treated for 2 days with maturation mixture (MC) consisting of 10 ng/ml IL-1β, IL-6, and TNF-α and 1 μg/ml PGE₂, from which the individual cytokine was excluded. The relative expression of miR-146a and miR-146b was quantified by real-time PCR and normalized to mDCs that had been treated with the full maturation mixture. Data shown are mean percentages ± S.E. of four (A), two (B), or three (C) independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.005. w/o, without.

FIGURE 2.

miR-146 modulates DC apoptosis. imDCs at day 4 were transfected with miR-146a and/or miR-146b and scrambled control (Ctrl) inhibitors (A–C) or mimics (E–G) at a final concentration of 30 nm for 40 h. A Cy3-labeled negative control was used to monitor the transfection efficiency (close to 80%) of these miRNA inhibitors or mimics. A and E, the relative expression of miR-146a and miR-146b was quantified by real-time PCR. C and G, the percentages of annexin V⁺ cells (including annexin V⁺/propidium iodide⁻ (PI) and annexin V⁺/propidium iodide⁺) were determined as in B or F. imDCs at day 6 were transfected with miR-146a and/or miR-146b and scrambled control inhibitors (D) or mimics (H), followed by the maturation mixture for 40 h, and cells were then analyzed for apoptosis as described above. Caspase-3/7 activity was measured with the Caspase-Glo 3/7 assay after transfection of miR-146a and/or miR-146b and scrambled control inhibitors or mimics for 40 h in imDCs (I) and mDCs (J). Data shown are mean percentages ± S.E. of eight (C), four (A and E), six (D, G, and H), or two (I and J) independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.005 (by paired t test).

FIGURE 3.

Ectopic expression of miR-146 affects TRAF6 and IRAK1 expression. TRAF6 and IRAK1 expression in human monocytes (Mo) at day (D) 0; imDCs at days 2, 4, and 6; and mDCs at day 8 was analyzed by real-time PCR analysis (A) and Western blotting (B). There was a significant difference in TRAF6 protein (but not mRNA) expression between days 6 and 8 (p < 0.05). There was a significant difference in both IRAK1 mRNA and protein expression between days 6 and 8 (p < 0.05). imDCs at day 4 were transfected with miR-146a and/or miR-146b and scrambled control (Ctrl) inhibitors (C) or mimics (D) at a final concentration of 30 nm. Forty h later, cells were analyzed for TRAF6 and IRAK1 expression by Western blotting. Data shown are mean percentages ± S.E. and are representative of four (A and B) or three (C and D) independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.005.

FIGURE 4.

TRAF6 and IRAK1 modulate DC apoptosis. imDCs at day 4 were transfected with TRAF6 and/or IRAK1 siRNA and scrambled control (Ctrl) siRNA at a final concentration of 30 nm for 40 h. A, reduced expression of TRAF6 and/or IRAK1 was analyzed by Western blotting. B, all cells were analyzed for apoptosis by staining with FITC-annexin V and 7-aminoactinomycin D (7-AAD). C, the percentages of annexin V⁺ cells (including annexin V⁺/propidium iodide⁻ and annexin V⁺/propidium iodide⁺) were determined as in B. D, imDCs at day 6 were transfected with TRAF6 and/or IRAK1 siRNA and scrambled control siRNA, followed by the maturation mixture for 40 h, and cells were then analyzed for apoptosis as described above. E, schematic representation of the pEhfflucmCNsin lentiviral vector expressing human IRAK1 and TRAF6. LTR, long terminal repeat; mC, minimal core promoter element; EF1α, elongation factor 1α. imDCs at days 4 (G) and 6 (F and H) were transduced with the control lentiviral vector (Luc) and with the lentiviral vector (LV) expressing TRAF6 and/or IRAK1, and cells were then incubated for 48 h with (F and H) or without (G) the maturation mixture. The transduction efficiency was determined to be >80% NGFR⁺ by flow cytometry. Cells were analyzed for Western blotting (F) or apoptosis (G and H) by staining with FITC-annexin V and allophycocyanin-NGFR. NGFR⁺ cells were used only to determine the percentages of annexin V⁺ cells. High baseline apoptosis in the control lentiviral vector-transduced imDCs and mDCs was due to use of Polybrene for lentiviral transduction and serum-free AIM V medium for DC culture. Data shown are mean percentages ± S.E. and are representative of four (A–D) or two (F–H) independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.005 (by paired t test).

FIGURE 5.

miR-146 induces apoptosis via the NF-κB pathway in human DCs. imDCs at day 4 were transfected with miR-146a and/or miR-146b and scrambled control (Ctrl) inhibitors (A) or mimics (B) or with TRAF6 and/or IRAK1 siRNA and scrambled control siRNA (C) at a final concentration of 30 nm. Forty h later, cells were analyzed for IκB and Bcl-2 expression by Western blotting. D, imDCs at day 6 were transduced with the control lentiviral vector (Luc) and with the lentiviral vector (LV) expressing TRAF6 and/or IRAK1, and cells were then incubated for 48 h with the maturation mixture. Blots were quantified by densitometry and normalized to GAPDH. Data shown are representative of four (A–C) or two (D) independent experiments.

FIGURE 6.

miR-146 modulates cytokine production in mDCs. A, imDCs at day 5 were treated with the LPS + R848 or with poly(I:C) + maturation mixture (MC) in AIM V medium supplemented with human GM-CSF and IL-4 for 16 h. miR-146a and miR-146b expression in human monocytes (Mo) at day 0, imDCs at day 6, and mDCs by LPS + R848 or poly(I:C) + maturation mixture at day 6 was analyzed by real-time PCR analysis. imDCs at day 5 were transfected with miR-146a and/or miR-146b and scrambled control (Ctrl) inhibitors (B, D, and E) or mimics (C, F, and G). Six h later, cells were treated with LPS + R848 (B–D and F) or with poly(I:C) + maturation mixture (B–G) in AIM V medium supplemented with human GM-CSF and IL-4 for 16 h. Supernatants were harvested, and IL-12p70, IL-6, and TNF-α were measured by ELISA. Data shown are mean percentages ± S.E. of three (A), four (B and C), or two (D–G) independent experiments. There is a large variation between individual donors in C. *, p < 0.05; **, p < 0.01; ***, p < 0.005 (by paired t test).

FIGURE 7.

miR-146 modulates IFN-γ production by NK cells through increased IL-12p70 production by DCs. imDCs at day 6 were transfected with miR-146a and/or miR-146b and scrambled control (Ctrl) inhibitors. Six h later, cells were treated with the maturation mixture for another 12 h. After washing, DCs were co-cultured with NK cells at a 1:2 ratio for 24 h. Supernatants were harvested, and IFN-γ (A) and IL-12p70 (B) were measured by ELISA. Data shown are mean percentages ± S.E. of two independent experiments with two different donors. ***, p < 0.005.

FIGURE 8.

Working model for miR-146a/b-mediated regulation of DC functions. In human monocytes, miR-146a and miR-146b are expressed at low levels. During differentiation of monocytes into imDCs and mDCs, stimulation of TRAF6 and IRAK1 via various signals such as the TLR/IL-1 receptor and CD40 leads to subsequent activation of NF-κB, which up-regulates miR-146a and miR-146b expression. The increased miR-146a and miR-146b levels in turn suppress NF-κB activity through inhibition of TRAF6 and IRAK1 expression. This negative feedback loop regulates DC survival, maturation, and cytokine production.