miR-223-3p Inhibits Antigen Endocytosis and Presentation and Promotes the Tolerogenic Potential of Dendritic Cells through Targeting Mannose Receptor Signaling and Rhob

Background The role of miR-223-3p in dendritic cells (DCs) is unknown. This study is aimed at investigating the effect of miR-223-3p on the antigen uptake and presentation capacities of DCs and the underlying molecular mechanism. Methods FITC-OVA antigen uptake and cell surface markers in bone marrow-derived DCs (BMDCs) were analyzed by flow cytometry. BMDCs were transfected with the miR-223-3p mimic or inhibitor. Cytokine levels were determined by ELISA. CD4+ T cell differentiation was determined by mixed lymphocyte culture assay. Results OVA treatment significantly downregulated miR-223-3p in BMDCs. The miR-223-3p mimic significantly inhibited OVA-induced antigen uptake and surface expression of MHC-II on BMDCs (P < 0.01). The miR-223-3p mimic increased TGF-β1 production in OVA-treated DCs (P < 0.01). Mixed lymphocyte reaction showed that the miR-223-3p mimic significantly promoted Treg cell differentiation. In addition, the miR-223-3p mimic significantly upregulated CD103 in DCs, indicating the promotion of tolerogenic DCs. The miR-223-3p mimic downregulated Rhob protein in OVA-induced DCs. Rhob knockdown significantly suppressed the ability of FITC-OVA endocytosis (P < 0.01) and surface MHC-II molecule expression (P < 0.01) in BMDCs, promoting promoted Treg cell differentiation. Mannose receptor (MR) knockdown significantly upregulated miR-223-3p, downregulated Rhob protein in OVA-treated DCs, inhibited the FITC-OVA endocytosis and surface MHC-II expression in BMDCs, and promoted Treg cell differentiation (all P < 0.01). Conclusion These data suggest that miR-223-3p has an inhibitory effect on the antigen uptake and presentation capacities of BMDCs and promotes Treg cell differentiation, which is, at least partially, through targeting MR signaling and Rhob.


Introduction
Dendritic cells (DCs) are the most potent antigen-presenting cells for inducing primary adaptive responses and maintaining self-tolerance [1]. DCs can uptake foreign antigens which were degraded into smaller peptides and subsequently loaded onto major histocompatibility complex (MHC) on the surface, presenting peptide fragments for recognition by antigen-specific T cells [2,3]. Class I MHC (MHC-I) is recognized by cytotoxic CD8+ T cells, while Class II MHC (MHC-II) is recognized by helper CD4+ T cells [4]. During antigen-specific T cell activation, DCs can produce cytokines, such as IL-1β, to facilitate the antigen-specific T cell proliferation and differentiation into one of the T cell phenotypes [5]. In addition to CD4 helper and CD8 cytotoxic T cells, DCs are capable of inducing regulatory T (Treg) cell differentiation, which plays a role in controlling the immune tolerance and homeostasis [6,7].
miRNAs are a class of small (20-25 nucleotides in length), single-stranded, non-coding, RNA molecules that exert function via binding mRNA targets, leading to their degradation or translational suppression [8]. miRNAs have been shown to be involved in a variety of important biological functions, such as cell development, apoptosis, signal transduction, and pathogenic conditions [8,9]. It has been predicted that up to 60% of human genes may be controlled by miRNAs [10]. Increasing evidence shows that miRNAs can be important regulators of immune responses, such as miR-146a [11], miR-29 [12], and miR-155 [13]. miRNAs have also been shown to be involved in DC development and DC function regulation, including the production of cytokines, differentiation, and homeostasis via affecting specific targets [14]. For instance, Naqvi et al. have demonstrated that overexpression of miR-24, miR-30b, and miR-142-3p attenuates uptake and processing of soluble antigen ovalbumin (OVA) in DCs [15]. Our preliminary highthroughput sequencing results showed that OVA treatment results in altered expression of miR-223-3p in DCs, suggesting miR-223-3p may take part in regulating DC function.
miR-223-3p is involved in innate immune responses by regulating myeloid differentiation and granulocyte functions [16,17]. miR-223-3p has been shown to be involved in regulating the differentiation and function of DCs [18][19][20]. Although the function of miR-223-3p in adaptive immune responses has been demonstrated, its role in antigen uptake and presentation is unknown. Therefore, the purpose of this study was to investigate the effect of miR-223-3p on the antigen uptake and presentation capacities of BMDCs and the underlying molecular mechanism.

Materials and Methods
2.1. Culture and Transfection of Mouse DCs. BALB/C mice (6-8 weeks) were purchased from Guangdong Medical Experimental Animal Center (Guangzhou, China) and bred in specific pathogen-free conditions. Bone marrow-derived DCs (BMDCs) were isolated from BALB/C mice as previously described [21] with some modifications. Briefly, bone marrow progenitors were cultured in RPMI 1640 (HyClone, USA) supplemented with 10% FBS (Gibco, USA), 10 ng/ml GM-CSF (PeproTech, London, UK), and 10 ng/ml IL-4 (PeproTech, London, UK). Immature BMDCs were harvested at 5-6 days after differentiation. BMDC medium contains glutamine, but no antibiotics. DCs were positively selected using CD11c magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). All animal experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Scientific Investigation Board of Sun Yat-Sen University (Guangzhou, China).

2.2.
Quantitative RT-PCR Detection. Total RNA was extracted from DCs using TRIzol reagent (Invitrogen) according to the manufacturer's protocol and was reversely transcripted to cDNA with specific stem-loop primers by using a reverse transcription kit (Tiangen Biotech Co., Ltd., China). Quantitative PCR analysis of miRNAs was performed using an SYBR qPCR kit (Toyobo, Japan) and the ABI Prism 7500 Sequence Detection System (Applied Biosystems, Carlsbad, CA, USA). The relative expression level of miRNAs was normalized to the internal control (U6) using the 2 -ΔΔCt cycle threshold method. The primers for miR-223-3p and U6 were purchased from Guangzhou RiboBio Co., Ltd. (Guangzhou, China).

miRNA Mimic and
Inhibitor. The sequences of the miR-223-3p mimic/inhibitor and their controls are shown in Table 1. All of the oligonucleotides were synthesized and purified with high-performance liquid chromatography by Shanghai GenePharma (Shanghai, China). For miRNA mimic and inhibitor transfection, 2 × 10 6 cells/well were seeded onto a 6-well plate. Cells were transfected with the mimic and inhibitor or the negative control of the mimic and inhibitor using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's protocol. The final concentration of Lipofectamine 2000 was 10 μl/250 ml.

Statistical Analysis.
For all experiments, the data was presented as mean ± SD of three independent experiments (n = 3 for each group) and was compared between two groups using a paired t-test. For comparison among more than two groups, one-way ANOVA with post hoc Tukey's HSD test was performed for multiple comparisons. All analyses were performed with SPSS v16.0 (SPSS, Chicago, IL, USA). A P < 0:05 was considered statistically significant.

OVA Treatment Downregulated miR-223-3p in BMDCs.
Immature DCs from mouse bone marrow can be induced to mature in vitro via OVA stimulation, which was characterized by elevated surface expression of MHC-II, CD80, CD86, and CD40 (Figures 1(a) and 1(b)). Our preliminary highthroughput sequencing results showed that OVA treatment induces a decrease in the miR-223-3p level in DCs (unpublished data); therefore, the miR-223-3p expression kinetics in OVA-treated DCs was determined by qPCR analysis. As shown in Figure 1(c), OVA treatment significantly downregulated miR-223-3p in DCs from 0 min to 24 h in a time-dependent manner (P < 0:01).

miR-223-3p Suppressed OVA Endocytosis and OVA-Mediated Surface Expression of MHC-II Molecules on
BMDCs. The changes in the miR-223-3p level led us to consider whether it participates in regulating the biological function of DCs. To investigate the role of miR-223-3p in DCs, the miR-223-3p mimic and miR-223-3p inhibitor were adopted. RT-PCR showed that miR-223-3p mimic transfection significantly elevated miR-223-3p expression in DCs, whereas miR-223-3p inhibitor transfection significantly reduced miR-223-3p expression as compared with the control group ( Supplementary Fig. S1A), suggesting a high efficiency of the miR-223-3p mimic and inhibitor. To determine if miR-223-3p regulates the antigen endocytosis capacity of BMDCs, the cells were transfected with the miR-223-3p inhibitor/mimic before FITC-OVA incubation. As shown in Figures 2(a) and 2(b), miR-223-3p inhibition significantly enhanced endocytic uptake of FITC-OVA in BMDCs as compared with the inhibitor control group (P < 0:01). By contrast, the miR-223-3p mimic markedly inhibited the endocytic activity of BMDCs (P < 0:01, Figures 2(c) and 2(d)).

miR-223-3p
Increased TGF-β1 Production in OVA-Treated DCs and Promoted Treg Cell Differentiation. The production of inflammatory cytokines and the upregulation of cell surface molecules, including MHC and costimulatory molecules in DCs, are essential for the priming of naive T cells [22]. To investigate if miR-223-3p plays a role in OVA-induced cytokine production in DC and T cell differentiation, miR-223-3p mimic or inhibitor transfections were performed. As shown in Figure 4(a), the miR-223-3p mimic remarkably increased the production of TGF-β1 in OVA-treated DCs, whereas the miR-223-3p inhibitor significantly reduced TGF-β1 production (both P < 0:01). However, miR-223-3p did not affect the secretion of IL-10, IL-12, and IL-6 (Figures 4(b)-4(d)).
It is known that TGF-β1 plays an important role in Treg cell differentiation [19]. To investigate if miR-223-3p has an effect on the DC-mediated T cell differentiation, mixed lymphocyte reaction was performed. The miR-223-3p mimic significantly increased the ratio of Foxp3+ CD4+ T cells as compared with the control group (P < 0:01, Figure 5(a)) but did not affect the ratio of T-bet+, GATA-3+, and IL-17A+ CD4+ T cells ( Figure 5(a)). Accordingly, miR-223-3p inhibition exhibited an opposite effect on the differentiation and polarization of T cells (P < 0:01, Figure 5(b)). These data suggested that miR-223-3p participated in the regulation of DC-mediated regulatory T cell polarization. The effect of miR-223-3p on CD103 (a tolerogenic marker) expression in OVA-treated DCs was investigated. As shown in Figures 5(e) and 5(f), the miR-223-3p mimic significantly upregulated CD103 in DCs, suggesting that miR-223-3p promoted tolerogenic DCs.

miR-223-3p Downregulated Rhob Protein Expression to
Inhibit Antigen Endocytosis and Presentation of BMDCs, Followed by Treg Cell Differentiation. It has been shown that Rhob plays a key role in antigen endocytosis and presentation in BMDCs [23]. In addition, Rhob is a target of miR-223-3p [24]. Hence, we investigated if Rhob takes part in the antigen endocytosis and presentation in BMDCs. Western blot demonstrated that the miR-223-3p mimic decreased Rhob expression  Journal of Immunology Research in OVA-induced DCs but did not affect the other members of the Rho family, including CDC42, Rhoa, and Rac-1 (Figure 6(a)). By contrast, the miR-223-3p inhibitor upregulated Rhob protein in OVA-treated DCs (Figure 6(b)). Next, siRNA-Rhob was used for knockdown experiments. As shown in Supplementary Fig. S1B, siRNA-Rhob (50nM) transfection effectively reduced the level of Rhob expression levels following OVA treatment. siRNA-Rhob knockdown significantly suppressed the ability of FITC-OVA endocytosis (P < 0:01, Figure 6(c)) and surface MHC-II molecule expression (P < 0:01, Figure 6(d)) in BMDCs. Rhob knockdown significantly promoted the preferential differentiation of Foxp3+ CD4+ T cells as compared with the control group (P < 0:01, Figure 6(e)) but did not affect T-bet+ cells, GATA-3+ cells, and IL-17A+ CD4+ T cells. These data suggested that miR-223-3p may target Rhob to regulate the antigen endocytosis and presentation in BMDCs and subsequent Treg responses.

Mannose Receptor-Mediated Antigen Endocytosis and Presentation
Were Involved in miR-223-3p Modulation. The previous study demonstrated that human alveolar macrophages phagocytose Pneumocystis organisms predominantly mediated by CDC42 and Rhob activation and mannose receptors (MRs) [25]. Therefore, we investigated if MR is involved in the mechanism of miR-223-3p-regulated antigen endocytosis and presentation in DCs. To determine the effect of miR-223-3p on MR expression, the protein levels of MR in DCs treated with OVA and miR-223-3p mimic or inhibitor were determined by Western blot. As shown in Figure 7(a), the miR-223-3p mimic markedly inhibited MR expression,        Supplementary Fig. S1B, siRNA-MR (50 nM) transfection effectively reduced endogenous MR expression levels. siRNA-MR knockdown significantly upregulated miR-223-3p from 15 min to 24 h (all P < 0:01, Figure 7(a)) and downregulated Rhob protein in OVA-treated DCs (Figure 7(b)). In addition, MR knockdown significantly inhibited the ability of FITC-OVA endocytosis (P < 0:01, Figure 7(c)) and surface MHC-II expression (P < 0:01, Figure 7(c)) as compared with the control group. Correspondingly, MR knockdown significantly promoted the preferential differentiation of Foxp3+ CD4+ T cells as compared with the control group but significantly decreased the ratio of T-bet+ cells, GATA-3+ cells, and IL-17A+ CD4+ T cells (Figure 7(e)).

Discussion
miR-223-3p has been shown to be involved in regulating the differentiation and function of DCs [18][19][20]. Bros et al. have demonstrated that mmu-miR-223-3p induces a protolerogenic state in BMDCs by attenuating the expression of its mRNA targets to control DC activation [18]. Ifergan et al. have reported that miR-223-3p regulates myeloid DC-(mDC-) induced activation of pathologic Th17 responses during autoimmune inflammation [20]. Zhou et al. have revealed that miR-223-3p regulates the differentiation and function of intestinal DCs and macrophages by targeting C/EBPβ [19]. In this study, we investigated the effect of miR-223-3p on the antigen uptake and presentation capacities of BMDCs and the underlying molecular mechanism. The results showed that OVA treatment significantly Mixed lymphocyte reaction showed that the miR-223-3p mimic significantly increased the ratio of OVA-induced Foxp3+ CD4+ cells, suggesting the promotion of Treg cell differentiation. In addition, the miR-223-3p mimic significantly upregulated CD103 in DCs, indicating the promotion of tolerogenic DCs. The miR-223-3p mimic decreased Rhob expression in OVAinduced DCs. Rhob knockdown significantly suppressed OVA-induced endocytosis and MHC-II molecule expression in BMDCs, promoting the preferential differentiation of Foxp3+ CD4+ T cells. MR knockdown significantly upregulated miR-223-3p from 15 min to 24 h, downregulated Rhob protein in OVA-treated DCs, inhibited the OVA-induced endocytosis and MHC-II expression, and promoted the preferential differentiation of Foxp3+ CD4+ T cells. Taken together, these data suggested that miR-223-3p inhibited MR-mediated antigen uptake and presentation capacities of BMDCs and promoted Treg cell differentiation via targeting MR signaling and Rhob. A schematic representation of the mechanism of miR-223-3p on regulating the antigen uptake and presentation capacities of BMDCs is shown in Figure 8.
Our results found that OVA treatment downregulated miR-223-3p, and miR-223-3p inhibited OVA antigen uptake and presentation in BMDCs, suggesting that miR-223-3p plays an inhibitory role in the DCs. To the best of our knowledge, this is the first study reporting an inhibitory effect of miR-223-3p on the antigen endocytosis and presentation capacities of DCs. Our findings are in line with Naqvi et al.'s in vitro observations that miR-24, miR-30b, and miR-142-3p interfere with the uptake and processing of OVA antigen in DCs [15]. These results indicate an inhibitory function of these miRNAs in mediating antigen internalization and presentation. It is worth investigating if there is a correlation among the molecular mechanisms of the inhibitory function of these miRNAs within DCs.
It is well known that the antigen endocytosis and presentation in DCs are essential for the direct interaction of DCs with naive T lymphocytes and initialization of adaptive immune responses [26]. Meanwhile, DCs are also the key players in maintaining immune tolerance, which is mainly demonstrated by inducing Treg cell differentiation [27]. Since we found that miR-223-3p inhibited antigen endocytosis and presentation in DCs, we then investigated if miR-223-3p affects the tolerogenic potential of DCs. Notably, the mixed lymphocyte reaction showed that the miR-223-3p mimic significantly promoted Treg cell differentiation. Furthermore, our ELISA results showed that immature DCs secreted large amounts of TGF-β1, while the secretion was gradually decreased during DC maturation. However, miR-223-3p mimic treatment induced elevated TGF-β1 secretion and promoted the differentiation of Treg cells. The inhibition of miR-223-3p exhibited the opposite biological effect. It is known that DC-induced Treg cell differentiation is largely mediated by TGF-β [28]. Therefore, these results indicated that miR-223-3p mimic treatment may inhibit antigen endocytosis and antigen presentation but promote the immune

12
Journal of Immunology Research     tolerance function of DCs. On the other hand, our results also revealed that the miR-223-3p mimic upregulated CD103 in DCs. CD103 is a tolerogenic marker [29]. It is known that intestinal CD103+ DCs induce Treg differentiation and intestinal mucosa homing to control tolerance by production of retinoic acid and TGF-β [30]. Taken together, these findings suggested that miR-223-3p promoted the tolerogenic potential of DCs. However, the detailed molecular mechanism requires further investigation.
Rhob has been shown as a target of miR-223-3p [24] and is implicated in antigen presentation of BMDCs [23]. In this study, the miR-223-3p mimic markedly downregulated Rhob protein, confirming that Rhob is a target of miR-223-3p in DCs. Moreover, we found that Rhob-silenced BMDCs exhibited similar behaviors with those with the miR-223-3p mimic, including suppressed antigen-presenting ability, increased TGF-β1 production, and elevated Treg cell differentiation. These data implied that miR-223-3p may exert its modulating function in BMDCs by targeting Rhob. It has been shown that MR is crucial for antigen uptake and antigen presentation by DCs [31]. In the current study, we observed the miR-223-3p mimic downregulated MR in DCs, implying MR is a target of miR-223-3p. Nevertheless, we also found that MR knockdown significantly upregulated miR-223-3p. These data may indicate that MR is a target of miR-223-3p, but MR signaling may feedback inhibit miR-223-3p expression. However, the detailed molecular mechanism remains to be further investigated. Our results showed that MR knockdown also downregulated Rhob protein in BMDCs, suppressed antigen-presenting ability, and promoted Treg cell differentiation, indicating that miR-223-3p may inhibit MR-mediated antigen internalization and presentation capacities of BMDCs ( Figure 8). However, more evidence is needed to support this notion.
There are still some limitations to this study. Although we found that miR-223-3p inhibited antigen-presenting function but promoted tolerogenic potential of DCs, the detailed molecular mechanism remains to be elucidated. In addition, these in vitro results should be further validated in an animal model. Although we found concordant results employing the miR-223-3p mimic, Rhob siRNA, and MR siRNA, the direct pathway has not been verified experimentally. For example, it remains to be determined if the miR-223-3p inhibitor reverses the effects of MR siRNA. All these limitations should be addressed in the following study.

Conclusions
In summary, our findings suggested that miR-223-3p has an inhibitory effect on the antigen uptake and presentation capacities of DCs and promotes Treg cell differentiation, which is, at least partially, through targeting MR signaling and Rhob. Our findings aid in understanding the modulating role of miR-223-3p in DC functions.

Data Availability
The data used to support the findings of this study are included within the article.

Conflicts of Interest
The authors declare no financial or commercial conflict of interest.