miR-221 and miR-155 regulate human dendritic cell development, apoptosis, and IL-12 production through targeting of p27^kip1, KPC1, and SOCS-1

Dendritic cells (DCs) are the most potent antigen-presenting cells in the immune system with the unique capacity to uptake and efficiently present antigens to naive T lymphocytes.¹  DCs can also interact with B cells²  and natural killer (NK) cells³  and thus bridge the innate and adaptive immunity. DCs develop directly from myeloid progenitors in the bone marrow (BM) and circulating blood monocytes, ensuring that self versus non–self-discrimination is maintained in the steady state and during immunologic challenges.⁴  Through a series of differentiation events, progenitor cells develop into immature (imDCs) and then into mature DCs (mDCs). The developmental process ends with apoptotic cell death.⁵  During this process, genes that encode critical proteins involved in DC differentiation, maturation, and function are programmatically expressed.^6,7  Integrated genomic and proteomic analysis suggests that many of these proteins are regulated at a post-transcriptional level.⁸  However, the underlying molecular mechanism is unclear.

MicroRNAs (miRNAs) have recently emerged as a major class of gene expression regulators linked to most biologic functions, such as cell proliferation, differentiation, apoptosis, and tumorigenesis.^9,10  miRNAs are single-stranded RNA molecules approximately 19 to 24 nucleotides in length and post-transcriptionally regulate gene expression by forming imperfect base pairing with sequences in the 3′-untranslated region (3′-UTR) of target mRNAs. This interaction prevents protein accumulation by repressing translation or by inducing mRNA degradation.¹¹  In general, miRNAs do not completely shut down gene expression of their targets but rather fine-tune their expression. Although more than 1000 human miRNAs have been annotated in the miRBase database (Version 16), their functions in the immune system, especially in DC development and function, have just begun to be explored.

The most studied miRNA in DCs is miR-155. miR-155^−/− mice are defective in B- and T-cell immunity, but the role of miR-155 in antigen presentation by DCs remains controversial.^12,13  miR-155 is known to modulate the interleukin-1 signaling pathway in activated human monocyte-derived DCs.¹⁴  miR-155 also targets DC-specific intracellular adhesion molecule-3 grabbing nonintegrin and reduces its pathogen binding ability on maturation.¹⁵  miR-34a and miR-21 are mildly increased during human monocyte differentiation into imDCs, and they coordinately regulate DC differentiation by targeting JAG1 and WNT1.¹⁶  Although miRNA profiling in monocyte-derived DCs has been reported,^16,17  a systematic analysis of the miRNA profile during monocyte differentiation into imDCs and mDCs has not been performed. Moreover, few studies about miRNA regulation in DC apoptosis and function have been reported.

Here, we systematically analyzed miRNA signatures during human monocyte differentiation into imDCs and mDCs. We found that p27^kip1, a key cell cycle inhibitor, was differentially expressed during DC development and maturation. DC apoptosis was at least regulated by miR-221 and miR-155 through targeting the 3′-UTRs of p27^kip1 and Kip1 ubiquitination-promoting complex 1 (KPC1), respectively. miR-155 was also found to regulate IL-12p70 production by targeting suppressor of cytokine signaling 1 (SOCS-1). Collectively, these data indicate the regulatory role of miR-221 and miR-155 in human DC development, apoptosis, and interleukin-12 (IL-12) production.

In vitro human monocyte differentiation into DCs was performed as described with modifications.¹⁸  Human peripheral blood mononuclear cells were isolated through Ficoll-Hypaque (Mediatech Cellgro) density gradient centrifugation from buffy coats purchased from Memorial Blood Centers or blood from healthy donors after informed consent following the Declaration of Helsinki according to the University of Minnesota Institutional Review Board-approved study. Monocytes were purified from peripheral blood mononuclear cells using anti-CD14 microbeads (Miltenyi Biotec). Purified monocytes (> 95% purity) were cultured at 37°C in 6-well plates (1 × 10⁶ cells per well) in 3 mL of serum-free AIM V medium (Invitrogen) containing human granulocyte-macrophage colony-stimulating factor (GM-CSF, 100 ng/mL, Bayer HealthCare Pharmaceuticals) and human interleukin-4 (IL-4, 20 ng/mL, R&D Systems). A total of 1 mL of fresh medium with GM-CSF and IL-4 was added to the cell cultures at day 3. Old medium was replaced by 3 mL fresh medium with GM-CSF and IL-4 at day 5. DCs were matured with IL-1β (10 ng/mL, R&D Systems), IL-6 (10 ng/mL, PeproTech), tumor necrosis factor-α (TNF-α, 10 ng/mL, R&D Systems), and prostaglandin E₂ (PGE₂, 1 μg/mL, Sigma-Aldrich) on day 6, and DCs were harvested on day 8 or the indicated time.

Murine DCs were generated from BM cell culture in the presence of GM-CSF as described with modifications.¹⁹  Mice were maintained at the University of Minnesota Animal Facility in accordance with institutional guidelines. BM was collected from femurs and tibias of 8- to 12-week-old C57BL/6 mice (wild-type [WT]) and bic/mir-155^−/− mice (The Jackson Laboratory). Erythrocytes were lysed, passed through a nylon mesh, and cells washed and resuspended in RPMI 1640 medium (Invitrogen) supplemented with penicillin (50 U/mL), streptomycin (50 μg/mL), L-glutamine (2mM), 2-mercaptoethanol (50μM), and 10% heat-inactivated fetal bovine serum (HyClone). At day 0, BM cells were seeded at 2 × 10⁶ cells per 100-mm dish (Fisher Scientific) in 10 mL medium containing recombinant murine GM-CSF (20 ng/mL, R&D Systems). Three days later, another 10 mL medium containing murine GM-CSF (20 ng/mL) was added into the dish. For the induction of DC maturation, lipopolysaccaride (LPS, 1 μg/mL, catalog no. L2654, Sigma-Aldrich) was added to cell cultures for 24 hours at day 6. In some experiments, imDCs were further purified from suspension cells at day 6 using anti-CD11c microbeads (Miltenyi Biotec). LPS was then added for the indicated time.

Total RNA was isolated from purified monocytes, imDCs at day 6, and mDCs at day 8 using Trizol (Invitrogen) and subjected to custom miRNA microarray analyses.²⁰  Data were analyzed and normalized as described.²¹  The microarray data have been deposited in the GEO public database under accession number GSE21708.

Cells were washed with ice-cold phosphate-buffered saline twice and lysed with TRIZOL reagent to isolate total RNA, which was followed by DNase digestion. Single-strand cDNA was synthesized using the Ncode miRNA First-Strand cDNA Synthesis kit (Invitrogen). miRNA-specific primers were designed following the kit instruction and synthesized at the University of Minnesota DNA Core Facility. U6 and 5S RNA was used as endogenous controls. Primer sequences are detailed in supplemental Table 1 (available on the Blood Web site; see the Supplemental Materials link at the top of the online article). Comparative real-time polymerase chain reaction (PCR) using SYBR Green SuperMix (Invitrogen) was performed in a 96-well plate and run in a 7500 Real-Time PCR System (Applied Biosystems) at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 57°C for 1 minute. Each sample was analyzed in duplicate or triplicate. The level of miRNA expression was measured using threshold cycle (C_t) according to the ΔΔC_t method.²²  To normalize the relative abundance of miRNAs, 5S RNA was used as an endogenous control.

Antibodies and Fc blocking reagents were purchased from BD Biosciences or eBioscience. Flow cytometric analysis was carried out on a FACSCalibur (BD Biosciences) and analyzed using FlowJo software Version 7.2.2 (TreeStar).

Cells were harvested at the indicated time points, washed, labeled with annexin V-allophycocyanin (APC; eBioscience) for 30 minutes on ice, and subsequently stained with propidium iodide (PI; 1 μg/mL, Invitrogen). Annexin V/PI staining was analyzed by flow cytometry. Sub-G₀ analysis of cell apoptosis was also used. Briefly, cells were washed with 1mM ethylenediaminetetraacetic acid/phosphate-buffered saline and fixed with 90% ethanol for at least 30 minutes at −20°C. The cells were then washed, and cellular DNA was stained with RNase (50 μg/mL, Sigma-Aldrich) and PI (10 μg/mL) for 45 minutes at room temperature. Fluorescence intensity was quantified by flow cytometry.

An miR-155 expression cassette containing the human miR-155 hairpin sequence and flanking regions was cloned from human genomic DNA isolated from mDCs using the primer set in supplemental Table 1 and inserted into a bidirectional lentiviral expression vector²³  downstream of EF1α promoter (LV-miR-155WT). The mutant miR-155 expression lentiviral construct (LV-miR-155MUT) was generated based on LV-miR-155WT sequence with 8 nucleotide mutations in the miR-155 seeding region (see Figure 2C). For construction of luciferase reporter plasmids, 3′-UTRs of the respective mRNAs (KPC1, CD115, and SOCS-1) were cloned into pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega) after amplification of genomic DNA from human mDCs using the primer sets in supplemental Table 1. Mutated 3′-UTRs of respective mRNAs were amplified with the specific primers in which 4 nucleotides at the miRNA seed regions were changed.

NIH3T3 cells (2 × 10⁵ cells per well) were plated in 24-well plates with Dulbecco modified Eagle medium (Invitrogen) containing 10% fetal bovine serum one day before transfection. pmirGLO Dual-Luciferase miRNA Target Expression Vectors (20 ng per well) containing either WT or mutated (MUT) KPC1, CD115, or SOCS-1 3′-UTR, and LV-miR-155WT or LV-miR-155MUT plasmids (800 ng per well) were cotransfected into NIH3T3 cells in triplicate using Lipofectamine 2000 (Invitrogen). Cells were harvested 24 hours later, and luciferase activity was assessed using Dual-Luciferase Reporter Assay System (Promega).

Pre-miR miRNA precursors for hsa-miR-155, hsa-miR-198, and a negative control (Negative Control #1) were purchased from Ambion. Fluorescein isothiocyanate-labeled miRCURY LNA knockdown probes for hsa-miR-221, hsa-miR-155, hsa-miR-198, and a scramble-miR control were purchased from Exigon. To overexpress or silence miRNAs, imDCs (2.5 × 10⁵ per well) in 24-well plates were transfected with miRNA precursors or miRNA knockdown probes and respective controls at the indicated concentrations for the indicated time using Lipofectamine RNAiMAX (Invitrogen).

DCs were harvested, washed, and lysed in 20mM 3-(N-morpholino) propane sulfonic acid buffer containing 0.5% Nonidet P-40, 2mM ethyleneglycoltetraacetic acid, 5mM ethylenediaminetetraacetic acid, 1% Triton X-100, and one complete Protease Inhibitor Cocktail tablet (Roche Diagnostics) in a 10-mL volume. Membranes were stripped with stripping buffer (0.2M glycine-HCl, pH 2.5, 0.05% Tween-20, 100mM 2-mercaptoethanol) to probe the expression of several specific proteins within one experiment. Each protein sample was separated under reducing conditions on a 4% to 12% polyacrylamide sodium dodecyl sulfate gel and transferred onto polyvinylidene fluoride membranes. Proteins were detected with the primary antibody, mouse-antihuman p27^kip1 (BD Biosciences), rabbit-antihuman KPC1,²⁴  rabbit-antihuman SOCS-1 (Santa Cruz Biotechnology), or goat-antihuman γ-tubulin (Santa Cruz Biotechnology) and horseradish peroxidase-conjugated secondary antibodies (Pierce Chemical). Protein quantification was performed using National Institutes of Health Image J software (www.rsb.info.nih.gov/ij).

imDCs (2.5 × 10⁵ per well) in 24-well plates were transfected with hsa-miR-155 precursors and a negative control at a final concentration of 40nM using Lipofectamine RNAiMAX. Six hours later, DCs were stimulated with the maturation cocktail for 12 hours. Old medium was then removed, and transfected DCs were cocultured with NK cells (5 × 10⁵ cells in 0.5 mL AIM V medium), which were isolated from human peripheral blood mononuclear cells using an NK cell negative isolation kit (Miltenyi Biotec). Cells were cocultured for 24 hours, and the supernatant was harvested for an enzyme-linked immunosorbent assay (ELISA) assay.

Cell culture supernatants were recovered at the indicated times and evaluated in triplicate for IL-12p70 and interferon-γ (IFN-γ) levels using ELISA kits (eBioscience).

For non-normally distributed data, the nonparametric Wilcoxon signed rank test was used in the evaluation of the statistical differences between groups. Two-sample t test was used when the data were approximately normally distributed. For repeated measurement outcomes, a linear mixed model with random subject effect to evaluate miRNA groups and time effects was used (means reported are least square means). An interaction term was introduced in the initial model and removed if it was insignificant. Time was treated as a discrete variable. Groups with values of P ≤ .05 were considered to be statistically significant.

To systematically investigate miRNA expression profiles during human monocyte differentiation into imDCs and mDCs, monocytes from 2 healthy donors were differentiated into imDCs and matured with IL-1β, IL-6, TNF-α, and PGE₂, commonly used in DC clinical trials.²⁵  Total RNA from human monocytes, imDCs, and mDCs was analyzed using a custom miRNA microarray platform.²⁰  Supplemental Figure 1 shows that the difference in miRNA expression between monocytes versus imDCs and monocytes versus mDCs appeared more dramatic than the difference between imDCs versus mDCs, suggesting that miRNA identity between imDCs and mDCs is similar.

Fifty-eight miRNAs whose expression increased more than 1.5-fold or decreased more than 2-fold in comparisons between monocytes and DC maturation subtypes were selected for validation by real-time PCR. Table 1 details the fold change and P values of the differentially expressed miRNAs. The expression levels of 15 miRNAs were decreased more than 2-fold during monocyte differentiation into DCs, and the expression levels of 12 miRNAs were increased more than 2-fold at the imDC and/or mDC stages. Interestingly, all of the miRNAs that were down-regulated at the imDC stage remained at the same level for the mDC stage. Both miRNA-106b-25 (miR-106b, miR-93, and miR-25) and -15/16 (miR-15a and miR-16) clusters were down-regulated during monocyte differentiation into imDCs and mDCs. miRNA-155 was dramatically up-regulated on DC maturation, although it was only moderately increased in imDCs. miR-198 expression was nearly undetectable in monocytes and imDCs but was clearly increased on DC maturation, whereas conversely miRNA-221 was first up-regulated on monocyte differentiation into imDCs and then down-regulated during DC maturation. These results demonstrate that there are at least 4 types of miRNA signatures during human monocyte development into imDCs and mDCs, suggesting that they may play a pivotal role in DC development, maturation, and function.

p27^kip1 is an inhibitor of cell cycle progression, exerting its effect through interaction with cyclin-dependent kinase complexes and arrestment of cells in G_0/1 phase. p27^kip1 is present at relatively high levels in quiescent cells and is down-regulated by mitogenic stimulation.^26,27  p27^kip1 plays a decisive role in nonproliferating cell types, such as eosinophils and DCs.^28,29  Because p27^kip1 is a direct target of miR-221 in human cancer cells and mast cells,^30-32  we investigated whether there was an inverse correlation between p27^kip1 and miR-221 expression during human monocyte differentiation into DCs. Human monocytes, differentiating DCs at days 2, 4, and 6, and mDCs were analyzed for expression of p27^kip1 and miR-221. As shown in Figure 1A, p27^kip1 protein was present at a high level in monocytes. On DC differentiation, p27^kip1 expression was progressively down-regulated with the lowest level at the imDC stage. On DC maturation, p27^kip1 expression was up-regulated to a level similar to that seen in monocytes. In contrast to p27^kip1 expression, miR-221 expression was increased during monocyte differentiation into imDCs and then reduced during DC maturation (Figure 1B; Table 1). miR-221 expression almost disappeared after 4-day maturation (data not shown). To determine whether miR-221 regulates p27^kip1 in DCs, imDCs were transfected with an miR-221 inhibitor. As shown in Figure 1C, silencing of miR-221 expression resulted in higher levels of p27^kip1 protein expression than scramble controls 6 and 12 hours after transfection, respectively. Consistent with the findings in other cell types,^33,34  silencing of miR-221 in imDCs at a time of higher (day 2) or lower (day 4) miR-221 expression levels resulted in modestly increased apoptosis compared with the scrambled control (eg, 16% vs 11% annexin V⁺ in fluorescein isothiocyanate⁺ cell populations on day 2 imDCs; Figure 1D; day 4 data not shown). Taken together, these data indicate that miR-221 expression was inversely correlated with the expression of p27^kip1, suggesting that p27^kip1 differential expression during DC development and maturation is at least partially regulated by miR-221.

In contrast to miR-221, miR-155 expression positively correlated with p27^kip1 expression. miRNA-155 expression was significantly up-regulated on DC maturation, whereas miR-155 expression in monocytes and during differentiation into imDCs at days 2, 4, and 6 was low (Figure 2A). Up-regulation of miR-155 in mDCs was mainly mediated by IL-1β, TNF-α, and PGE₂ but probably not IL-6 (Figure 2B). miR-155 is known to target multiple genes, such as PU.1 and SHIP1.^15,35  A recent report showed that miR-155 overexpression in the human monocytic leukemia THP-1 cell line depletes the G₂ population and accumulates a sub-G₁ population, suggesting that miR-155 is involved in cell apoptosis.³⁶  Using the miRGator miRNA target prediction program,³⁷  miR-155 was predicted to target human KPC1, which can recognize free p27^kip1 (Figure 2C). KPC1 associates with KPC2, and p27^kip1 is then polyubiquitylated. Deletion of KPC1 by miR-155 would result in the inhibition of p27^kip1 degradation.²⁴ 

To definitively validate KPC1 as a target of miR-155, the full-length KPC1 3′-UTR was cloned and inserted downstream of the luciferase gene in the pmirGLO expression vector (pGL-KPC1-WT). The 3′-UTR of KPC1 with a mutant sequence in the miR-155 predicted binding site was cloned and inserted into the pmirGLO vector as a negative control (pGL-KPC1-MUT). NIH3T3 cells were cotransfected with LV-miR-155WT (or LV-miR-155MUT) and pGL-KPC1-WT (or pGL-KPC1-MUT). Ectopic expression of miR-155 resulted in an appreciable repression of the luciferase reporter activity by targeting KPC1 WT but not the mutant 3′-UTR, whereas mutant miR-155 did not affect luciferase reporter activity with the intact or mutant KPC1 3′-UTR (Figure 2D). These results suggest that the KPC1 is a direct target of miR-155.

To further investigate whether miR-155 regulates p27^kip1 expression in DCs, imDCs at day 4 were transfected with miR-155 precursors or negative controls for 24 hours. Cell lysates were analyzed for the expression of KPC1 and p27^kip1 by Western blot. As shown in Figure 2E, KPC1 expression was reduced and p27^kip1 was increased after overexpression of miR-155 in imDCs. In addition, KPC1 expression was reduced along with miR-155 up-regulation during DC maturation (Figure 2F). Taken together, these results demonstrate that miR-155 indirectly regulates p27^kip1 expression by targeting KPC1.

Because up-regulation of p27^kip1 is not only linked to cell cycle arrest, but also associated with apoptosis,^28,29  we wanted to determine whether overexpression of miR-155 could induce apoptosis in DCs. imDCs at day 4 were transfected with miR-155 precursors or negative controls. Indeed, overexpression of miR-155 in imDCs for 24 hours induced the typical characteristics of apoptosis as demonstrated by an increased sub-G₀ fraction (73% vs 11% in the control group; Figure 3A) and annexin V⁺PI⁺ populations (28% vs 4%; Figure 3B). Overexpression of miRNA-155 in mDCs also led to transition from apoptosis to necrosis (30% vs 16% of annexin V⁺PI⁺ populations; Figure 3C). To further confirm the relationship between miR-155 expression and apoptosis, BM-derived DCs were generated from WT and miR-155^−/− mice and stimulated with LPS. Up to day 4 after LPS maturation, there was no significant difference in DC apoptosis between WT and miR-155^−/− mice. However, on day 5, apoptotic populations were dramatically increased in DCs from WT versus miR-155^−/− mice (Figure 3D). Nearly all WT DCs died on day 6 after LPS stimulation, although most of miR-155^−/− DCs still survived for one additional week (data not shown). Consistently, mDCs from WT mice had higher level of p27^kip1 than that from miR-155^−/− mice (Figure 3E). These data indicate the regulatory role of the miR-155-KPC1-p27^kip1 pathway in DC apoptosis.

Because miR-155 and miR-198 were dramatically up-regulated in mDCs (Table 1), we hypothesized that these 2 miRNAs may influence DC maturation. To test this, DCs were transfected with miRNA-155 and miRNA-198 knockdown probes to silence endogenous cognate miRNAs. Analysis of DC surface molecules, including CD40, CD80, CD83, CD86, and human leukocyte antigen (HLA) class II, revealed no difference in expression compared with scramble-treated DCs before and after maturation (Figure 4A-B). To further confirm these results in mice, BM-derived imDCs were generated from miR-155^−/− and WT mice and matured with LPS. Flow cytometric analysis of major histocompatibility complex class II and costimulatory molecule levels in imDCs and mDCs indicated no differences in expression between miR-155^−/− and WT mice (Figure 4C-D). Taken together, these results indicate that miR-155 (and miR-198) has no impact on HLA and costimulatory molecule expression in DCs.

In contrast, CD115, a marker of terminal maturation of DCs, was found to be a target of miR-155 (Figure 5A). Luciferase reporter assays demonstrated that cotransfection of the intact CD115 3′-UTR with LV-miR-155WT resulted in a significant repression of the luciferase reporter activity. As a control, LV-miR-155MUT did not affect luciferase expression with the intact and mutant CD115 3′-UTR (Figure 5B). More importantly, forced overexpression of miR-155 in imDCs resulted in a decrease of CD115 expression on the cell surface (Figure 5C). Consistent with this, miR-155^−/− mDCs exhibited higher levels of CD115 than WT cells (Figure 5D). These results suggest that CD115 is a direct target of miR-155.

Next, we investigated whether miR-155 regulates cytokine production in DCs. We found that IL-12p70 (Figure 6A) and IL-10 (data not shown) production was significantly reduced after miRNA-155 silencing during DC maturation. Consistent with this, activated DCs from miR-155^−/− mice secreted less IL-12p70 than those from WT mice (supplemental Figure 2). IL-12p70 produced by DCs plays an important role in the development of IFN-γ-producing T cells and NK-cell activation.¹  Several studies have shown that IL-12 production in DCs was related to SOCS-1 expression.^38,39  During human monocyte differentiation into DCs, SOCS-1 expression at mRNA level was up-regulated and reached its highest level at maturation stage (Figure 6B). However, the expression of SOCS-1 at protein level was not elevated along with the mRNA level on DC maturation (Figure 6C), suggesting that SOCS-1 was post-transcriptionally regulated. SOCS-1 has been shown to be regulated by miR-155 in murine T regulatory cells.⁴⁰  We also confirmed that human SOCS-1 was a direct target of miRNA-155 in a luciferase report assay (supplemental Figure 3). Consistent with this, silencing of miR-155 in mDCs resulted in increased SOCS-1 expression at protein level (Figure 6D), whereas forced expression of miR-155 led to reduced SOCS-1 expression in imDCs (Figure 6E).

Because silencing of miR-155 resulted in decreased levels of IL-12p70 in mDCs, we predicted that overexpression of miR-155 in mDCs could enhance IL-12p70 secretion. To test this, imDCs at day 6 were transfected with miR-155, miR-198 precursors, or a negative control followed by incubation with the maturation cocktail 6 hours after transfection. Indeed, miR-155 overexpression in mDCs led to significantly high levels of IL-12p70 secretion at 8, 16, and 24 hours after transfection (P < .01), whereas overexpression of miR-198 in mDCs or negative control-transfected mDCs had little effect on IL-12p70 production (Figure 6F). Moreover, enhanced IL-12 production by overexpression of miR-155 was demonstrated using more potent IL-12 inducers, including LPS plus R484 and polyinosinic-polycytidylic acid plus the cocktail (supplemental Figure 4).

To investigate whether overexpression of miR-155 could enhance mDCs in activating NK cells, mDCs with overexpression of miR-155 were cocultured with autologous CD3⁻CD56⁺ NK cells. As shown in Figure 6G, NK cells cocultured with mDCs that were transfected with miR-155 precursors secreted significant higher levels of IFN-γ than control cultures (P < .01), suggesting that miRNA-155 overexpression can enhance the ability of mDCs to activate NK-cell production of IFN-γ.

In this study, we comprehensively analyzed miRNA profiles in human monocytes, imDCs, and mDCs and demonstrated the following novel observations. First, we identified 4 types of miRNA signatures during monocyte differentiation into imDCs and mDCs (Table 1). Second, we revealed the cooperatively regulatory role of miR-221 and miR-155 in human DC development and apoptosis through modulation of p27^kip1 expression (Figure 7). miR-221 directly targets p27^kip1, whereas miR-155 indirectly modulates p27^kip1 via targeting KPC1. Third, our data indicate that overexpression of miR-155 in mDCs led to higher levels of IL-12p70 secretion by DCs, enchancing IFN-γ production by NK cells (Figure 6).

Our study is the first to demonstrate the differential expression of p27^kip1 during human monocyte differentiation into imDCs and mDCs. Previous studies have shown that p27^kip1 levels are regulated by alterations of protein stability.⁴¹  The degradation of p27^Kip1 is regulated by 2 distinct mechanisms: translocation-coupled cytoplasmic ubiquitination by KPC and nuclear ubiquitination by Skp2.²⁴  The third new mechanism identified here is post-transcriptional regulation of p27^kip1 by miRNAs. p27^kip1 regulation by miR-221 has been reported in several types of cells, including cancer cells and mast cells.^30-32  Our current study confirmed this finding in human DCs (Figure 1). Furthermore, we have identified KPC1 as a novel target by miR-155 in human DCs, which indirectly regulate p27^kip1 (Figure 2). KPC, a ubiquitin ligase, consisting of KPC1 and KPC2, recognizes free p27^kip1 via the NH2-terminal region and then polyubiquitinate p27^kip1 for proteasome-mediated degradation by the COOH-terminal RING-finger domain of KPC1.²⁴  It appears that miR-155 functions like a tumor suppressor in DCs via its indirect effects on p27^kip1 stability, although it is oncogenic in other types of cells and tissues, including B-cell, myeloid cell, breast, lung, colon, pancreatic, and thyroid tissues.⁴²  It may be possible that the miR-155-KPC1-p27^kip1 pathway is defective in tumors.

There are many putative functions attributed to p27^kip1, including proliferation, differentiation, and apoptosis.⁴¹  Although the role of p27^kip1 in nonproliferating cells has not been well investigated, one study in DCs suggested that increased p27^kip1 expression (eg, recombinant adenovirus infection) promotes apoptosis.²⁹  Consistent with this finding, an increase of apoptosis was found in imDCs when miR-221 was silenced (Figure 1D) or miR-155 was overexpressed (Figure 3A-B), suggesting that a low level of p27^kip1 expression is necessary for imDC development. Overexpression of miR-155 in mDCs also led to an increase in apoptosis (Figure 3C). It was further confirmed that mDCs from miR-155^−/− mice survived longer than WT mDCs (Figure 3D). These results provide the first evidence that miRNAs are critical regulators in DC apoptosis.

DC apoptosis is physiopathologically essential in controlling tolerance and immunity.⁴³  The most convincing evidence demonstrating a central role for DCs in maintaining self-tolerance is that blocking DC apoptosis in mice leads to DC buildup and the development of systemic autoimmune diseases.⁴⁴  Consistent with this, DCs in autoimmune patients with mutations in caspase 10, an enzyme activated during apoptosis, are less apoptotic and hence accumulate.⁴⁵  In addition, it has been recently shown that uptake of apoptotic DCs induces immature DCs to secrete TGF-β, which in turn promotes the differentiation of naive T cells into regulatory T cells.⁴⁶  Furthermore, constitutive ablation of DCs breaks self-tolerance and results in spontaneous fetal autoimmunity.⁴⁷  Therefore, down-regulated miR-221 and up-regulated miRNA-155 expression during DC maturation by various stimuli, resulting in p27^kip1 accumulation in mDCs and subsequent DC apoptosis, could be physiologically important.

DCs are a major source of many cytokines, including IL-12, which is considered as a critical third signal for naive CD8 T-cell activation, strong effector cell function, and memory T-cell formation.⁴⁸  SOCS-1, an inducible negative feedback inhibitor of JAK/STAT signal pathway, functions like an antigen presentation attenuator in DCs through limiting IL-12 production.^38,39  In this study, we demonstrate that IL-12 production by human DCs is regulated by miR-155-mediated targeting of SOCS-1. Of note, the finding that SOCS-1 is a target of miR-155 has been previously reported in murine T regulatory cells and more recently in breast cancer cell lines and murine DCs.^13,40,42  Nevertheless, enhanced production of IL-12 by DCs or IFN-γ by NK cells via overexpression of miR-155 in mDCs suggests that miR-155 could be used as an adjuvant to improve DC vaccines.

In conclusion, we have identified differentially expressed miRNAs during human monocyte differentiation into imDCs and mDCs. More importantly, we have uncovered the important role of miR-221 and miR-155 in DC apoptosis and IL-12 production. Whether these miRNAs are critical regulators in human DC development and function in vivo remains to be investigated. Our finding that miRNAs are involved in regulation of DC survival and cytokine production may provide additional knowledge about miRNA regulation in DC biology and implicate modulation DCs using miRNAs for clinical applications.

The authors thank University of Minnesota Supercomputing Institute for microarray data acquisition. K.R. is a Directed Research undergraduate student at the University of Minnesota, College of Biologic Sciences.

This work was supported by startup funds from the University of Minnesota Masonic Cancer Center and Department of Pediatrics, the Children's Cancer Research Fund in Minneapolis, University of Minnesota Medical School Dean's Commitment, and in part by grants from the Alliance for Cancer Gene Therapy, the Gabrielle's Angel Foundation for Cancer Research, and the Sidney Kimmel Foundation for Cancer Research Kimmel Scholar Program (X. Zhou).

National Institutes of Health

Contribution: C.L. designed the research, performed the experiments, analyzed the data, and wrote the manuscript; X.H., X. Zhang, and K.R. performed the research; Q.C. performed statistical analyses; K.I.N. provided critical reagents and edited the paper; B.R.B discussed the work and edited the paper; Y.Z. discussed the work and wrote the paper; and X. Zhou oversaw the research and wrote the paper.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Xianzheng Zhou, Masonic Cancer Center, University of Minnesota, MMC 366, 420 Delaware St, Minneapolis, MN 55455; e-mail: zhoux058@umn.edu.