Abstract Dendritic cells (DCs) play a critical role in triggering anti-tumor immune responses. Their intracellular p38 signaling is of great importance in controlling DC activity. In this study, we identified microRNA-22 (miR-22) as a microRNA inhibiting p38 protein expression by directly binding to the 3’ untranslated region (3’UTR) of its mRNA. The p38 down-regulation further interfered with the synthesis of DC-derived IL-6 and the differentiation of DC-driven Th17 cells. Moreover, overexpression of miR-22 in DCs impaired their tumor-suppressing ability while miR-22 inhibitor could reverse this phenomenon and improve the curative effect of DC-based immunotherapy. Thus, our results highlight a suppressive role for miR-22 in the process of DC-invoked anti-tumor immunity and that blocking this microRNA provides a new strategy for generating potent DC vaccines for patients with cancer.

Citation: Liang X, Liu Y, Mei S, Zhang M, Xin J, Zhang Y, et al. (2015) MicroRNA-22 Impairs Anti-Tumor Ability of Dendritic Cells by Targeting p38. PLoS ONE 10(3): e0121510. https://doi.org/10.1371/journal.pone.0121510 Academic Editor: Fabrizio Mattei, Istituto Superiore di Sanità, ITALY Received: August 21, 2014; Accepted: February 12, 2015; Published: March 31, 2015 Copyright: © 2015 Liang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: This research was supported by NSFC grants 31470876, 91029736, 91442111 and ISF-NSFC program 31461143010; a Ministry of Science and Technology grant (863 program, 2008AA02Z129); the National Key Scientific Program (2011CB964902); the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT13023) and State Key Laboratory of Medicinal Chemical Biology. Competing interests: The authors have declared that no competing interests exist.

Introduction Dendritic cells (DCs) are the most effective antigen presenting cells (APCs) with exclusive function of activating naïve T cells through presenting antigens to them [1]. They express high levels of MHCII molecules to exhibit antigens efficiently, and then activate CD8+ and CD4+ T cells. Besides that, DCs can also interact with natural killer (NK) cells and B cells to forge a bridge between innate and adaptive immune systems [2,3]. Thus, they have been considered as the primary activator of immune response and are closely involved in inflammation, autoimmune disease, transplantation immune response and so on. For a long time, researchers have been focusing on their ability to induce the reactions of T cells and B cells. However, in recent years, the anti-tumor function of DCs has been attracting more and more attention [4]. DCs play an important role in anti-tumor immune responses, while on the other hand, tumor cells can reciprocally secrete some soluble factors, including TGF-β, IL-10, etc, to disrupt the differentiation of DCs and their ability to activate immune responses, to fight back, which may be the crucial barrier holding back tumor treatment [5,6]. These tumor-derived factors interrupt the regular function of DCs by activating several intracellular signals, such as MAPK, JAK/STAT and NF-κB pathways. It has been recently reported that the dysfunction of DCs caused by tumor cells is accompanied by excessive activation of MAPK signaling pathways [7]. Thus, studying MAPK signals can lay a foundation for directly or indirectly mitigating tumor cells’ damage on DCs. As an important member of MAPK family, p38 plays a role in regulating various cell activities and is considered to be the joint center of signal transduction. Regulating the expression and function of p38, therefore, can be an effective method to improve DC-related tumor treatment. MicroRNAs, as small non-coding RNAs, widely distributed in various species are able to elaborately regulate expression of genes related to various physiological and pathological processes including immunity responses [8]. As to DCs, miRNAs are indispensable in regulation of their development, differentiation and functions. This may be seen via the actions of let-7i, miR-142-3p, miR-146a, the miR-148 family, miR-155, and miR-155* in regulating cytokine production in response to DC activation, and as an inherent characteristic of DCs via constitutive miR-146a expression [9]. Since DCs help to orchestrate immune responses by secreting appropriate cytokines and influencing CD4+ T cell subset differentiation [9], miRNAs may offer the foundation for modifying them to improve immune responses against tumors. MicroRNA-22 (miR-22), originally isolated from HeLa cell line, has been found to be ubiquitously expressed in various tissues [10–12]. Evolutionary clustering suggests that miR-22 is highly conserved in vertebrate evolution, indicating its functional importance in vertebrate species. It has been deduced from the statistical analysis of 3’ untranslated regions (3’UTRs) in transcriptome that miR-22 participates in the regulation of many target genes [13]. Here, we have found that miR-22 could be expressed in dendritic cells and proved that miR-22 can impair the tumor-suppressing function of DCs and directly bind to the 3’UTR of p38 mRNA to down-regulate p38 protein. The decreased expression level further interferes with the synthesis of DC-derived IL-6 and the differentiation of DC-driven Th17 cells.

Materials and Methods Mice, cell lines and murine bone marrow derived dendritic cells Four- to six-wk-old female C57BL/6 mice (Beijing Animal Center) were maintained in a specific pathogen-free animal facility for at least 1 wk prior to use. The animal experiments were performed in accordance with institutional guidelines and the study was approved by the ethics committee of Nankai University. The following cell lines were purchased from American Type Culture Collection: murine monocyte/macrophage RAW264.7, murine melanoma B16 and human embryonic kidney 293T. 293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Hyclone) at 37°C in a humidified 5% CO 2 atmosphere. RAW264.7 and B16 cells were maintained in the same condition except that the basic culture medium was RMPI 1640 rather than DMEM. Murine bone marrow derived dendritic cells (BMDCs) were generated from C57BL/6 mice. Mice were sacrificed and their bone marrow cells were collected by removing the femurs and tibia, cutting off each end, and flushing out the bone marrow with PBS using a syringe. The pooled cells were harvested by centrifugation at 600 ×g for 10 min and the erythrocytes contained were removed by incubation with Ammonium Chloride lysing buffer for 5 min at room temperature. The cells were then washed in RMPI 1640 medium supplemented with 10% fetal bovine serum. BMDCs were obtained by culturing these bone marrow cells for 5–6 days in 500 U/mL GM-CSF (R&D Systems). The percentage of CD11c+ dendritic cells (DCs) in the final cell population was 70–80% by fluorescence activated cell sorting (FACS) analysis. Surface and intracellular staining Single cells were prepared from the removed mouse inguinal lymph nodes and 106 cells/well were cultured in 24-well plates. They were re-stimulated for 6 h with 50 ng/mL PMA, 1 μg/mL Ionomycin and 0.66 μL/mL GolgiStop Protein Transport Inhibitor (BD Pharimingen). Then surface and intracellular staining was performed using the mouse Th1/Th2/Th17 phenotyping Kit (BD Pharmingen) according to the manufacture’s instructions. Stained cells were analyzed with CellQuest software on an FACSCalibur (BD Biosciences). Prediction of microRNA-binding sites The predicted microRNA binding sites were downloaded from TargetScan 5.1 Mouse (http://www.targetscan.org/mmu_61/) [14]. Real-time RT-PCR Total RNA was isolated using TRIzol Reagent (Invitrogen). 1 μg of RNA was reverse transcribed with the M-MLV Reverse Transcription System (Invitrogen) according to the manufacture’s instructions. PCR was performed using the SYBR Green Real-time RT-PCR Master Mix plus (TOYOBO) as described by the manufacture. PCR primers used here were as follows. GAPDH: 5′-TGCACCACCAACTGCTTAG-3′ (sense), 5′-GATGCAGGGATGATGTTC-3′ (antisense); p38: 5′-ACAAACCAAGTCATCAAGG-3′ (sense), 5′-ATCAGAAGGAACCACACT-3′ (antisense); IL6: 5′-AACGATGATGCACTTGCAGA-3′ (sense), 5′-GAGCATTGGAAATTGGGGTA-3′ (antisense). Amplification was performed by denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 30 sec, 58°C for 30 sec and 72°C for 30 sec. GAPDH was performed on each experimental sample as an endogenous control. The real-time RT-PCR was carried out in a Bio-rad IQ 5 Multicolor real-time RT-PCR system and their software was used to calculate the cycle threshold of each reaction. All reactions were run in triplicate. Transfection RNA mimics, inhibitor and negative control oligonucleotide were purchased from RiboBio (Guangzhou, China). Cells were transfected with the indicated oligonucleotides (100 nM) using the Entranster-R system (Engreen Biosystem) according to the manufacturer’s instructions. Western blotting (WB) Cells were lysed in lysis buffer (Beyotime) containing complete protease mixture (Sigma-Aldrich). After centrifugation, the lysates were boiled in SDS loading buffer and resolved by 12% acrylamide gel electrophoresis in the presence of SDS (SDS-PAGE), then transferred to a PVDF membrane (Millipore) and probed with 1:1000 dilution of a rabbit anti-p38 or anti-beta-actin polyclonal antibody (Santa Cruz Biotechnology), followed by 1:1000 dilution of a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Promega). Then the polypeptides were revealed using ECL reagent (Amersham Biosciences). All of the figures illustrating Western blotting analyses are representative of at least three independent experiments. Enzyme linked immunosorbent assay (ELISA) After stimulation with LPS at a concentration of 1 μg/mL for 24 h, cell supernatants of different groups were collected and analyzed using the Quantikine ELISA Kit for IL-6 (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. Luciferase reporter assay To get the recombinant luciferase mRNAs, the DNA of the 3’ untranslated region (3’UTR) of mouse p38 mRNA or its mutant, changing the 8nt binding site for microRNA-22 (miR-22), was amplified by PCR using modified primers (sense: 5'-AACTCGAGCGAGTCCTCTCCTAGGACTA-3', and antisense: 5'-TTGCGGCCGCACACAAAGCTTAAATATG-3' for both the wild-type and mutant 3’UTRs. As for the mutant one, 2 additional primers were required to generate the mutant site from the original wild-type template. One was paired with the aforementioned sense primer: 5'-TCAGTGTCATCGACGGGGGGGGTGGGG-3', and the other was used together with the antisense one: 5'-CCACCCCCCCCGTCGATGACACTGAATC-3'. Then the 2 kinds of products were mixed to perform an overlapping PCR experiment and the final product was the expected mutant 3’UTR.), digested with Xho I and Not I. Then each of them was cloned into the vector siCHECK-2, downstream of a luciferase CDS, respectively. The validity of these constructs was verified by sequencing. 293T cells were seeded in 24-well plates and cotransfected with 1 of the 2 constructs combined with dsRNA control or dsRNA mimics of miR-22 using Lipofectamine 2000 Reagent (Invitrogen). After 24 h of incubation, the cells were collected for application in the Luciferase Reporter System (Promega, Madison, WI) following the manufacturer’s instructions. All the luciferase reporter assays were repeated 3 times within each experiment. Tumor challenge model B16 melanoma cells were maintained as mentioned above and injected s.c. into C57BL/6 mice to form solid tumors. 106 of these cells were administrated to each mouse. Then the mice were monitored for tumor growth every 2 days. When solid tumors formed and their volumes could be measured by calipers 12 days after inoculation, mice were randomly divided into several groups to receive intratumoral DC injection. Differentially treated DCs were administrated to different groups respectively at a number of 106 per mouse every 7 days. In the meantime, analgesic steps were also performed to minimize the suffering of the mice by a subcutaneous Buprenorphine (Temgesic, 0.05 mg/kg) treatment every 12 h, and hence their condition was also monitored at this frequency from then on. Humane endpoints were introduced into the study and mice were euthanized by cervical dislocation method when their smallest tumor volume reached to 3000 mm3. Tumor size was measured in two dimensions by calipers and determined by the following formula: Width2×Length×π/6, where Width was the lesser value of the two dimensions. However, as to the survival study, the criterion to implement euthanasia to a mouse was that it exhibited a moribund state, and the period between tumor inoculation and this endpoint was collected as its survival time. A mouse would be conceived as being moribund if it exhibited the characteristics of severe mobility loss, hunched back, piloerection, ruffled fur and weight loss, and then it would be sacrificed humanely.

Discussion Dendritic cells (DCs) are the most important antigen-presenting cells (APCs) that bridge innate and adaptive immunity by triggering the activation and differentiation of naïve T cells. They express a repertoire of pattern recognition receptors (PRRs) that sense microbial pathogen products and endogenous ligands to initiate a signaling cascade that culminates in the activation of DCs and adaptive immunity [27]. Among the central pathways regulating this process, the MAPK pathways involving ERK, JNK and p38 play a critical role [28], and human interventions in them may play a part in enhancing the curative effect of DC vaccines for patients with cancer. In this study, we have focused on the p38 MAPK because of its importance in facilitating DC functions, especially in inducing the secreation of cytokine IL-6 and differentiation of Th17 cells. To intervene in this molecular target in DCs, we adopted a microRNA strategy and firstly screened the endogenous microRNAs related to p38. Based on the TargetScan Mouse prediction and actual experiments, we identified microRNA-22 (miR-22) as a negative regulator of p38 protein expression through directly binding to the 3’UTR of the mRNA. And as we expect, this endogenous microRNA exerts a negative influence on DC functions via silencing p38, which is consistent with the reports demonstrating the critical role of p38 in DC maturation and that p38 inhibition leads to the down-regulation of CD40, CD80 and CD86 markers, raising the risk of DC defunctionalization [29]. Overexpression of miR-22 mimics had an inhibitory effect on IL-6 mRNA and protein synthesis in DCs while miR-22 inhibitor had a reverse influence. This impact on IL-6 level further changed the Th17 cell proportion in tumor microenvironment. It is a significant phenomenon to DCs because initiating Th17 response is one of the most important functions of them. This process is initiated by the C-type lectin-like receptors (CLRs) coupling to signaling via the kinase Syk and the adaptor CARD9 [30]. The significant influences of miR-22 mimics and inhibitor on this DC function hinted the importance of this microRNA in DCs. Consistent with our result was the report that murine splenic DCs with p38 deletion showed a selectively lower IL-6 expression than those of wild-type mice after immunization in vivo or LPS stimulation in vitro. This impairment of IL-6 level further led to the weakened ability of these DCs to drive the expression of IL-17A and IL-23R in T cells because IL-6 was the most potent positive regulator of Th17 polarization [21]. Follow this axis, the inhibitory effect of miR-22 to p38 in DCs made miR-22 mimics a negative regulator and miR-22 inhibitor a positive regulator of both IL-6 expression and DC-induced Th17 differentiation as revealed in our study. This effect on IL-6 and Th17 partially contributed to another function of DCs influenced by miR-22, their anti-tumor ability, which is our primary research focus. It is well-known that a controversy has been existed for a long time around the question of whether IL-6 and IL-17A are anti-tumor or tumor promoting factors. This contradiction between the 2 standpoints is likely to have a close relationship with the various experimental conditions and a specific conclusion may only be suitable for its specific condition. As to the B16 tumor challenge model, which is adopted in this study, existing reports have concluded that both IL-6 and IL-17A function as anti-tumor factors and can enhance the immune response [31,32]. It proves the rationality of our results demonstrating the higher tumor clearance efficiency of miR-22 inhibited DCs with up-regulated IL-6 and Th17. MiR-22 was an endogenous tumor-promoting factor. It could be seen from the fact that miR-22 mimics transfected DCs exhibited a weaker therapy effect than the normal ones. The original tumor-suppressing ability of DCs was impaired under the influence of miR-22. This hinted that inhibition of endogenous miR-22 could be a strategy to release more active p38 and DC anti-tumor activity and miR-22 inhibitor could be served as a promising reagent to improve the performance of the existing DC-based therapeutic tumor vaccine. In summary, this study demonstrates that miR-22, as an endogenous microRNA of DCs, could suppress the translation of p38 mRNA and further down-regulate the expression of IL-6, which in turn interferes with Th17 cell development in tumor microenvironment. This could explain in part the negative effect of miR-22 mimics and positive influence of miR-22 inhibitor on the anti-tumor ability of DCs. These phenomena also suggest that miR-22 could be treated as a novel target of intervention and blocking miR-22 could be a promising strategy to improve the performance of DCs in immunotherapy. Of course, further preclinical studies are needed to test the applicability of this approach to prepare DC vaccines for patients with cancer.

Supporting Information S1 Materials and Methods. Supporting Information on ethics statement, mice and tumor challenge model. Detailed description on ethics statement, mice and tumor chanllenge model involved in this study. https://doi.org/10.1371/journal.pone.0121510.s001 (DOC)

Author Contributions Conceived and designed the experiments: RY. Performed the experiments: XL YL SM MZ JX YZ. Analyzed the data: XL YL. Contributed reagents/materials/analysis tools: XL YL SM MZ JX YZ. Wrote the paper: XL YL RY.