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Activation of TLR3 and its adaptor TICAM-1 increases miR-21 levels in extracellular vesicles released from human cells
Biochemical and Biophysical Research Communications xxx (2018) 1e7
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Activation of TLR3 and its adaptor TICAM-1 increases miR-21 levels in extracellular vesicles released from human cells Yoshimi Fukushima a, Masaaki Okamoto a, Kana Ishikawa a, Takahisa Kouwaki a, Hirotake Tsukamoto a, Hiroyuki Oshiumi a, b, * a
Department of Immunology, Graduate School of Medical Sciences, Faculty of Life Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto, 860-8556, Japan JST PRESTO, 1-1-1 Honjo, Kumamoto, 860-8556, Japan
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 April 2018 Accepted 17 April 2018 Available online xxx
Pattern-recognition receptors (PRRs) recognizes viral RNAs and trigger the innate immune responses. Toll-like receptor 3 (TLR3), a PRR, recognizes viral double-stranded RNA (dsRNA) in endolysosomes, whereas cytoplasmic dsRNA is sensed by another PRR, MDA5. TLR3 and MDA5 utilize TICAM-1 and MAVS, respectively, to trigger the signal for inducing innate immune responses. Extracellular vesicles (EVs) include the exosomes and microvesicles; an accumulating body of evidence has shown that EVs delivers functional RNA, such as microRNAs (miRNAs), to other cells and thus mediate intercellular communications. Therefore, EVs carrying miRNAs affect innate immune responses in macrophages and dendritic cells. However, the mechanism underlying the regulation of miRNA levels in EVs remains unclear. To elucidate the mechanism, we sought to reveal the pathway that control miRNA expression levels in EVs. Here, we found that TLR3 stimulation increased miR-21 levels in EVs released from various types of human cells. Ectopic expression of the TLR3 adaptor, TICAM-1, increased miR-21 levels in EVs but not intracellular miR-21 levels, suggesting that TICAM-1 augmented sorting of miR-21 to EVs. In contrast, the MDA5 adaptor, MAVS, did not increase miR-21 levels in EVs. The siRNA for TICAM-1 reduced EV miR21 levels after stimulation of TLR3. Collectively, our data indicate a novel role of the TLR3-TICAM-1 pathway in controlling miR-21 levels in EVs. © 2018 Elsevier Inc. All rights reserved.
Keywords: Extracellular vesicles Innate immunity microRNA TLR3
1. Introduction In the innate immune system, pattern recognition receptors (PRRs) recognizes pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), and several other chemical substances [1,2]. Toll-like receptors (TLRs) and RIG-I-like receptors are PRRs and recognize viral nucleic acids as PAMPs [3,4]. Toll-like receptor 3 (TLR3) binds viral doublestranded RNA (dsRNA) and triggers the signal for inducing the innate immune response via TICAM-1 also called TRIF [5,6]. Singlestranded RNA is sensed by TLR7, which utilizes MyD88 adaptor to trigger the signal [7,8]. These TLRs bind to viral RNA within endolysosomes, whereas cytoplasmic viral RNAs are recognized by RIG-
* Corresponding author. Department of Immunology, Graduate School of Medical Sciences, Faculty of Life Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto, 860-8556, Japan. E-mail address: [email protected] (H. Oshiumi).
I-like receptors, including RIG-I and MDA5, whose adaptor is MAVS (also called IPS-1, VISA, and Cardif) [3]. Alum is an aluminum salt and is widely used an adjuvant for vaccines. Alum is internalized into cells and activates Nod-like receptors (NLR) [9e11]. Recent study reported that alum induces cell death, thereby releasing cellular DNA, which functions as a DAMP and is recognized by TLR9 [12]. Extracellular vesicles (EVs) include the exosomes and microvesicles [13]. The exosomes are released from multivesicular bodies and contain functional RNAs, such as mRNA and microRNA (miRNA). An accumulating body of evidence has shown that the exosomes deliver functional RNAs to recipient cells and thus mediate inter-cellular communications [14,15]. Unlike the exosomes, the microvesicles are released from the plasma membrane. Microvesicles also deliver functional RNAs to other cells, but the mechanism underlying this is still unclear [14]. Recent studies have shown that EVs deliver miRNAs to dendritic cells and macrophages and control the innate immune responses [13,15e17].
https://doi.org/10.1016/j.bbrc.2018.04.146 0006-291X/© 2018 Elsevier Inc. All rights reserved.
Please cite this article in press as: Y. Fukushima, et al., Activation of TLR3 and its adaptor TICAM-1 increases miR-21 levels in extracellular vesicles released from human cells, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.04.146
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Several mechanisms for sorting of miRNAs into the exosomes have been reported. miRNAs that contain the EXO motif are efficiently sorted into the exosomes via hnRNPA2B1 [18], and another motif, the hEXO motif, is recognized by hnRNP-Q, leading to sorting to the exosomes [19]. In addition to hnRNPA2B1 and hnRNP-Q, other proteins are also involved in miRNA sorting; for example, YBX1, an RNA binding protein, is also involved in sorting miRNAs into EVs [20]. Recently, we found that hepatitis B virus (HBV) increased miR21 and miR-29a levels in EVs released from HBV-infected cells, as compared to those in resting cells [16]. miR-21 and miR-29a are known to target IL-12, and thus EVs released from HBV-infected cells could regulate IL-12p35 and IL-12p40 expression [21,22]. HBV is recognized by several PRRs [16]. Therefore, we reasoned that the activation of PRRs would increase miR-21 and miR-29a levels in EVs. Here, we investigated miR-21 and miR-29a levels in EVs after stimulation of PRRs and found that a TLR3 ligand, polyI:C, can increase miR-21 levels in EVs.
cultured in serum-free medium during stimulation. EVs were isolated from cell culture media by using Total Exosome Isolation Reagent (from cell culture media) (Thermo Fisher Scientific), according to the manufacturer's instructions. 2.4. miRNA expression Total RNAs from cells and EVs was isolated using TRIzol (Thermo Fisher Scientific). The reverse transcription (RT) reaction for miRNAs generation was performed using miR-X miRNA First-Strand Synthesis kit (Clontech). The SYBR Green Real-Time PCR Master Mix (ABI) was used for qPCR reaction using the Step One Real Time PCR system (ABI). All the steps were performed as per the manufactures' instructions. The miRNA levels were normalized to U6 RNA expression levels. Primers used for PCR are; miR-21: TAG CTT ATC AGA CTG ATG TTG A, and miR-29a: TAG CAC CAT CTG AAA TCG GTT A. 2.5. ELISA
2. Materials and methods 2.1. Ethics statement All the animal studies were conducted in accordance with the Guidelines for Animal Experimentation of the Japanese Association for Laboratory Animal Science, and were approved by the Animal Care and Use Committe of Kumamoto University (A29-027, J27232). 2.2. Cells, plasmids, and reagents THP-1 cells (obtained from the Japanese Collection of Research Bioresources (JCRB) cell bank) were cultured in RPMI-1640 medium with 5% fetal calf serum (FCS). The cells were differentiated into macrophages with phorbol 12-myristate 13-acetate (PMA; 60 ng/ ml) for 16 h. A549 and HEK293 cells (provided by T. Seya, at Hokkaido University) were cultured in D-MEM (high Glc) and in DMEM (low Glc) with 10% FCS, respectively. FCS was heat-inactivated at 56 C for 30 min. HeLa cells, which were kindly gifted by T. Fujita (Kyoto University), were cultured in Eagle -MEM medium with 10% FCS. siRNAs for human TICAM-1 and the negative control were purchased from Thermo Fisher Scientific. PolyI:C and CL097 were purchased from GE Healthcare and Invivogen, respectively. Imject Alum, which contains an aqueous solution of aluminum hydroxide (40 mg/ml) and magnesium hydroxide (40 mg/ml), was purchased from Thermo Fisher Scientific. pEF-BOS/TICAM-1 and pEF-BOS/ MAVS, which carry human TICAM-1 or MAVS full-length ORF, have been described previously [6,23]. The plasmids and siRNA were transfected into cells with Lipofectamine 2000 and Lipofectamine RNAiMax transfection reagents (Thermo Fisher Scientific), respectively, according to the manufacture's instruction. 2.3. EV analysis C57BL/6 mice were purchased from KYUDO COMPANY (Tosu). PolyI: C (50 mg/head) was intraperitoneally injected into the mice when they were 8e10 weeks of age. Sera were obtained from blood samples taken from the tail veins, and EVs were extracted from sera by using Total Exosome Isolation Reagent (from serum) (Thermo Fisher Scientific), according to the manufacturer's instructions. To determine miRNA levels in EVs released from cultured cells, the cells were seeded onto a 24-well plate. They were then washed four times the next day with PBS and subsequently cultured in serumfree medium for 1 or 2 days; subsequently, they were stimulated. To avoid the contamination of EVs derived from FCS, the cells were
An ELISA kit for mouse IFN-b was purchased from PBL Biomedical Laboratories. The blood samples were obtained from mouse tail veins, and sera were subsequently prepared. Serum IFNb protein levels were determined according to the manufacture's instruction by using the ELISA kit. 2.6. Statistical analysis All the statistical analysis was performed using the Prism 7 software (Ver. 7; GraphPad Software, Inc.) and Excel. The t-test was used to investigate the significance of differences between control and target samples. 3. Results 3.1. PolyI:C increased miR-21 levels in EVs released from the human cell lines Alum is an aluminum adjuvant used for various types of vaccines and is recognized by NALP3, a PRR [10]. First, we investigated the effect of alum on the miR-21 and miR-29a levels in EVs. THP-1 macrophages were stimulated with alum for 24 h, and then the EVs were collected from cell culture medium. We determined miRNA levels by RT-qPCR, following which they were normalized to U6 RNA levels. Interestingly, miR-21 and miR-29a levels in EVs were increased on stimulation with alum (Fig. 1A). Second, we investigated the effect of other adjuvants, such as polyI:C, a dsRNA analog, and CL097, which are recognized by TLR3 and TLR7, respectively [6,24]. THP-1 macrophages were stimulated with CL097 and polyI:C for 24 h, and then EVs were collected from cell culture medium. We simultaneously extracted total RNA from THP-1 macrophages to determine intracellular miRNA levels. Interestingly, miR-21 levels in EVs were increased on stimulation with CL097 and polyI:C, whereas miR-29a levels did not (Fig. 1B). In contrast to the findings for levels in EVs, CL097 and polyI:C did not increase intracellular miR-21 and miR-29a levels (Fig. 1C), suggesting that CL097 and polyI:C promotes the sorting of miR-21 into EVs but do not promote miR-21 transcription. These data are consistent with our hypothesis that PRRs can increase miR-21 and miR-29a levels in EVs. Next, we investigated the miRNA expression in EVs released from other types of cells. A549 is a cell line derived from lung epithelial cells. When A549 cells were stimulated with alum, the miR-21 levels in EVs did not increase (Fig. 2A). Stimulation of HeLa and HEK293 cells also did not increase miR-21 levels in the EVs (Fig. 2B and C), suggesting that alum increases miR-21 levels in EVs
Please cite this article in press as: Y. Fukushima, et al., Activation of TLR3 and its adaptor TICAM-1 increases miR-21 levels in extracellular vesicles released from human cells, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.04.146
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Fig. 1. polyI:C increased miR-21 levels in EVs released from THP-1 macrophages. (A) Alum was added to THP-1 macrophages (final conc. 0.4 mg/ml) cultured in serum-free medium. At 24 h after stimulation, EVs were collected from cell culture, and miR-21 and miR-29a levels were determined by RT-qPCR and normalized to U6 RNA levels. Error bars represents standard deviation (n ¼ 3). (B, C) THP-1 macrophages cultured in serum free medium were stimulated with CL097 (final conc., 5 mg/ml) and polyI:C (final conc., 50 mg/ml) for 24 h. EVs were collected from cell culture. Total RNA was extracted from EVs (B) and THP-1 macrophages (C), and miR-21 and miR-29a levels were determined by RT-qPCR and normalized to U6 RNA levels. Error bars represents standard deviation (n ¼ 3).
Please cite this article in press as: Y. Fukushima, et al., Activation of TLR3 and its adaptor TICAM-1 increases miR-21 levels in extracellular vesicles released from human cells, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.04.146
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Fig. 2. polyI:C increases EV miR-21 levels released from various types of cells. A549 (A), HEK293 (B), and HeLa (C) cells were cultured in medium without serum and were subsequently stimulated with alum (final conc., 0.4 mg/ml), CL097, (final conc., 5 mg/ml), and polyI:C (final conc., 50 mg/ml) for 24 h. EVs were collected from each cell culture supernatant. Total RNA was extracted from the EVs and cultured cells that were collected, and miR-21 and miR-29a levels were determined by RT-qPCR and normalized to U6 RNA levels (n ¼ 3).
in a cell type-specific manner. In contrast, miR-21 levels were increased in EVs released from A549, HeLa, and HEK293 cells stimulated with polyI:C (Fig. 2A and C), suggesting that polyI:C can increase miR-21 levels in EVs released from various types of cells. miR-29 levels in EVs was increased on stimulation of HEK293 and HeLa cells with polyI:C, whereas those in EVs released from A549 cells did not (Fig. 2A and C), suggesting that polyI:C increases miR-29 levels in EVs in a cell type-specific manner. Unlike polyI:C, CL097 did not increase miR-21 and miR-29a levels in EVs released from A549, HEK293, and HeLa cells (Fig. 2A and C). Collectively, these data indicate that PRRs increase miR-29a levels in EVs in a cell type-specific manner.
3.2. miR-21 levels in EVs circulating in the blood did not increase on stimulation with polyI:C EVs are abundant in the blood. Considering that polyI:C stimulation increased miR-21 levels in EVs from various types of cells, we reasoned that polyI:C injection would increase miR-21 levels in EVs circulating in the blood. To test this hypothesis, we used a mouse animal model. PolyI:C was intraperitoneally injected into mice. At 24 h after injection, sera were obtained from blood samples taken from mouse tail veins, and EVs were isolated from these sera. miR21 and miR-29a levels in EVs were determined by RT-qPCR. We
confirmed that polyI:C stimulation transiently increased the type I IFN levels in mouse sera by using ELISA (Fig. 3A). Unexpectedly, polyI:C injection did not increase miR-21 and miR-29a levels in EVs circulating in the blood (Fig. 3B). These data suggest that polyI:C injection is not sufficient for increasing the systemic EV miR-21 levels, although it can increase systemic IFN-b protein level.
3.3. TICAM-1 increased miR-21 levels in EVs Next, we focused on the mechanism underlying the increase in miR-21 levels in EVs, because it was increased on polyI:C stimulation in various types of cells. PolyI:C is recognized by TLR3 in the endolyosomes [25], whereas transfected polyI:C localized in the cytoplasm is recognized by MDA5 [26]. TLR3 and MDA5 trigger the signal via TICAM-1 and MAVS, respectively [6,26]. To determine the pathway responsible for the increase in miRNA levels in EVs after polyI:C stimulation, TICAM-1- and MAVS-expressing vectors were transfected into cells, and miR-21 and miR-29a levels in EVs and cells were determined by RT-qPCR. Over-expression of TICAM-1 and MAVS leads to auto-activation of their signalings [6,27]. We found that TICAM-1 overexpression increased miR-21 and miR-29a levels in EVs, whereas MAVS overexpression did not (Fig. 4A). In contrast to the findings for expression in EVs, TICAM-1 and MAVS overexpression did not increase the intracellular miR-21 and miR-
Please cite this article in press as: Y. Fukushima, et al., Activation of TLR3 and its adaptor TICAM-1 increases miR-21 levels in extracellular vesicles released from human cells, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.04.146
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Fig. 3. PolyI:C injection does not affect miR-21 and miR-29a levels in EVs circulating in the blood. (A) 50 mg of polyI:C was intraperitoneally injected into C57BL/6 mice. To measure serum IFN-b protein levels, samples were collected at the indicated time points. IFN-b protein levels were determined by ELISA (n ¼ 3). (B) 50 mg of polyI:C or PBS (mock) was intraperitoneally injected into C57BL/6 mice (n ¼ 5 or 6). Sera were obtained from mice 24 h after stimulation, and EVs were subsequently extracted from each serum sample. miR-21 and miR-29a levels were determined by RT-qPCR and normalized to U6 RNA levels.
Fig. 4. TICAM-1 increased miR-21 levels in EVs. (A, B) THP-1 macrophages were cultured in serum-free medium and were transfected with empty, TICAM-1-, and MAVS-expressing vectors for 24 h. EVs were collected from the cell culture supernatant. Total RNA was extracted from EVs and cultured cells. miR-21 and miR-29a levels in EVs (A) and cells (B) were determined by RT-qPCR and normalized to U6 RNA levels (n ¼ 3, t-test, *p < 0.05). (C) siRNA for negative control or TICAM-1 was transfected into HeLa cells for 2 days. Cells were stimulated with 50 mg of polyI:C for 24 h. EVs were collected from cell culture sup., and total RNA was extracted. miR-21 levels in EVs were determined by RT-qPCR and normalized to U6 RNA levels. The fold change in expression was calculated by dividing the value for each sample with that for the mock-stimulated sample (n ¼ 3, t-test, *p < 0.05). (D) siRNA for negative control (NC), hnRNPA2B1, YBX1, and hnRNP-Q were transfected into THP-1 macrophages for 2 days. Cells were stimulated with 50 mg of polyI:C for 24 h, and EVs were subsequently collected from the cell culture supernatant. miR-21 levels in EVs were determined by RT-qPCR and normalized to U6 RNA levels. The fold change in expression was calculated by dividing the value for each sample with that for NC sample (n ¼ 3, t-test, *p < 0.05).
29a levels (Fig. 4B). These data suggest that TICAM-1 can increase the sorting of miR-21 into EVs. To further confirm the involvement of TICAM-1 in miR-21
expression in EVs, we performed knockdown study using siRNA for TICAM-1. siRNA for TICAM-1 or negative control was transfected into A549 cells for 2 days. Cells were subsequently stimulated with
Please cite this article in press as: Y. Fukushima, et al., Activation of TLR3 and its adaptor TICAM-1 increases miR-21 levels in extracellular vesicles released from human cells, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.04.146
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polyI:C, and EVs were collected at 1 day after stimulation. The siRNA for TICAM-1 decreased EV miR-21 levels after polyI:C stimulation (Fig. 4C). Collectively, our data indicate that TLR3 stimulation triggers the signal for increasing EV miR-21 levels via TICAM-1. Sorting of miRNAs into EVs depend on RNA-binding proteins, such as hnRNPA2B1, hnRNP-Q, and YBX1 [18e20]. We investigated whether those RNA binding proteins are involved in the TICAM-1mediated increase in miR-21 levels in EVs. The siRNA for YBX1 decreased miR-21 levels in EVs from TICAM-1 overexpressing cells, whereas that for hnRNP-Q did not (Fig. 4D). Although the siRNA for hnRNPA2B1 decreased EV miR-21 levels, the decrease was not statistically significant. These data are consistent with our hypothesis that TICAM-1 promotes the sorting of miR-21 into EVs.
miR-21 is reported to regulate the IL-12 expression. EVs delivers miRNAs into the cytoplasm of recipient cells, and the miRNAs subsequently repress target gene expression [14,16,21]. Thus, it is expected that TLR3 would promotes sorting of miR-21 into EVs, which results in the downregulation of IL-12 in recipient cells. TLR3 itself is known to increase IL-12p40 production [31]. Therefore, miR-21 levels in EVs are expected to be involved in a negative feedback loop for attenuating inflammation. Considering that polyI:C administration did not increase miR-21 levels in EVs circulating in the blood, the EVs released from TLR3-stimulated cells would affect only cells in local tissue. Further studies are required to completely elucidate the physiological significance of EV miR-21 released from TLR3-stimulated cells.
4. Discussion
Acknowledgement
In our previous study, we found that EVs released from HBVinfected hepatocytes contained higher levels of miR-21 than those from resting cells, and regulated IL-12 expression [16]. In the current study, we found that TLR3 stimulation increased miR-21 levels in EVs released from human cells and that this increase required the TICAM-1 adaptor molecule. Moreover, the TICAM-1mediated increase in EV miR-21 levels depended on YBX1, which is known to be involved in sorting of miRNAs into exosomes [20]. On TLR3 stimulation, miR-29a levels increased in HEK293 and HeLa cells but not in A549 cells and THP-1 macrophages. Therefore, sorting of miR-29a appears to be regulated not only by TLR3 signaling but also by other unknown mechanisms. Other PAMPs also increase miR-21 and/or miR-29a levels in EVs. For instance, alum increased miR-21 and miR-29a levels in EVs. Alum is recognized by NALP3, resulting in the activation of the inflammasome [10]. Thus, it is expected that the inflammasome would be involved in the increase in miR-29a levels in EVs. In contrast, CL097, a ligand for TLR7, barely increased miR-21 and miR-29a levels. Thus, the mechanism underlying the increase in levels seems to be complicated, and further studies are required to clarify it. miRNAs that contain the EXO-motif and hEXO motif are sorted into the exosomes via hnRNPA2B1 and hnRNP-Q, respectively. Several other proteins are required for sorting of miRNAs into EVs, although the motif or features of miRNAs that are sorted by those proteins are remain unclear. The YBX1 is previously identified as a protein required for miR-224 sorting into the exosomes in HEK293T cells [20]. A subsequent study reported that YBX1 is involved in the sorting of non-coding RNAs, such as tRNAs, Y RNAs, and vault RNAs as well as miRNAs [28]. Considering that the siRNA for YBX1 decreased the TICAM-1-mediated increase in miR-21 levels in EVs, it is expected that TICAM-1 would regulate the YBX1 activity and that TICM-1 signaling might be required for sorting of other types of RNAs into EVs. EVs are released from various types of cells, and consequently, blood contains high amount of EVs [13]. PolyI:C administration increases type I IFN protein levels in the blood. Because polyI:C increased miR-21 levels in EVs released from several types of cells, we expected that it would increase miR-21 levels in the EVs circulating in blood. However, we found that polyI:C administration did not increase miR-21 levels in those EVs. PolyI:C is known to activate not only TLR3 but also MDA5 in vivo, and MDA5 plays a major role in type I IFN production in vivo [26]. Our in vitro overexpression study showed that over-expression of MAVS, which is the sole adaptor for MDA5, did not increase miR-21 levels in EVs. MDA5 is ubiquitously expressed in various type of cells, and its expression is increased by stimulation [26,29]. In contrast, TLR3 exhibits cell type-specific expression [30]. Therefore, it is possible that polyI:C administration stimulated only the MDA5 pathway and was not sufficient to systemically increase miR-21 levels in EVs.
We thank all of our laboratory members for the helpful discussions and technical assistance. This work was supported in part by Grants-in-Aid from the following: Ministry of Education, Science and Technology (15K08517); Japan Agency for Medical Research and Development (A-163); PRESTO JST (09801362); Tokyo Metropolitan Government (09802207); the Takeda Science Foundation; the Japanese Association for Complement Research; the Uehara Memorial Foundation; the Daiichi Sankyo Foundation of Life Science; and the Waksman Foundation of Japan. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.04.146. References [1] K. Newton, V.M. Dixit, Signaling in innate immunity and inflammation, Cold Spring Harb Perspect Biol 4 (2012). [2] V.E. Schijns, E.C. Lavelle, Trends in vaccine adjuvants, Expert Rev. Vaccines 10 (2011) 539e550. [3] Y.M. Loo, M. Gale Jr., Immune signaling by RIG-I-like receptors, Immunity 34 (2011) 680e692. [4] T. Kawai, S. Akira, Toll-like receptors and their crosstalk with other innate receptors in infection and immunity, Immunity 34 (2011) 637e650. [5] M. Yamamoto, S. Sato, H. Hemmi, K. Hoshino, T. Kaisho, H. Sanjo, O. Takeuchi, M. Sugiyama, M. Okabe, K. Takeda, S. Akira, Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway, Science 301 (2003) 640e643. [6] H. Oshiumi, M. Matsumoto, K. Funami, T. Akazawa, T. Seya, TICAM-1, an adaptor molecule that participates in Toll-like receptor 3-mediated interferon-beta induction, Nat. Immunol. 4 (2003) 161e167. [7] S.S. Diebold, T. Kaisho, H. Hemmi, S. Akira, C. Reis e Sousa, Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA, Science 303 (2004) 1529e1531. [8] H. Hemmi, T. Kaisho, O. Takeuchi, S. Sato, H. Sanjo, K. Hoshino, T. Horiuchi, H. Tomizawa, K. Takeda, S. Akira, Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway, Nat. Immunol. 3 (2002) 196e200. [9] S.L. Demento, S.C. Eisenbarth, H.G. Foellmer, C. Platt, M.J. Caplan, W. Mark Saltzman, I. Mellman, M. Ledizet, E. Fikrig, R.A. Flavell, T.M. Fahmy, Inflammasome-activating nanoparticles as modular systems for optimizing vaccine efficacy, Vaccine 27 (2009) 3013e3021. [10] V. Hornung, F. Bauernfeind, A. Halle, E.O. Samstad, H. Kono, K.L. Rock, K.A. Fitzgerald, E. Latz, Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization, Nat. Immunol. 9 (2008) 847e856. [11] E. Kuroda, K.J. Ishii, S. Uematsu, K. Ohata, C. Coban, S. Akira, K. Aritake, Y. Urade, Y. Morimoto, Silica crystals and aluminum salts regulate the production of prostaglandin in macrophages via NALP3 inflammasomeindependent mechanisms, Immunity 34 (2011) 514e526. [12] T. Marichal, K. Ohata, D. Bedoret, C. Mesnil, C. Sabatel, K. Kobiyama, P. Lekeux, C. Coban, S. Akira, K.J. Ishii, F. Bureau, C.J. Desmet, DNA released from dying host cells mediates aluminum adjuvant activity, Nat. Med. 17 (2011) 996e1002. [13] M. Colombo, G. Raposo, C. Thery, Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles, Annu. Rev. Cell Dev. Biol. 30 (2014) 255e289. [14] T. Kouwaki, M. Okamoto, H. Tsukamoto, Y. Fukushima, H. Oshiumi,
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Activation of TLR3 and its adaptor TICAM-1 increases miR-21 levels in extracellular vesicles released from human cells Yoshimi Fukushima a, Masaaki Okamoto a, Kana Ishikawa a, Takahisa Kouwaki a, Hirotake Tsukamoto a, Hiroyuki Oshiumi a, b, * a
Department of Immunology, Graduate School of Medical Sciences, Faculty of Life Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto, 860-8556, Japan JST PRESTO, 1-1-1 Honjo, Kumamoto, 860-8556, Japan
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 April 2018 Accepted 17 April 2018 Available online xxx
Pattern-recognition receptors (PRRs) recognizes viral RNAs and trigger the innate immune responses. Toll-like receptor 3 (TLR3), a PRR, recognizes viral double-stranded RNA (dsRNA) in endolysosomes, whereas cytoplasmic dsRNA is sensed by another PRR, MDA5. TLR3 and MDA5 utilize TICAM-1 and MAVS, respectively, to trigger the signal for inducing innate immune responses. Extracellular vesicles (EVs) include the exosomes and microvesicles; an accumulating body of evidence has shown that EVs delivers functional RNA, such as microRNAs (miRNAs), to other cells and thus mediate intercellular communications. Therefore, EVs carrying miRNAs affect innate immune responses in macrophages and dendritic cells. However, the mechanism underlying the regulation of miRNA levels in EVs remains unclear. To elucidate the mechanism, we sought to reveal the pathway that control miRNA expression levels in EVs. Here, we found that TLR3 stimulation increased miR-21 levels in EVs released from various types of human cells. Ectopic expression of the TLR3 adaptor, TICAM-1, increased miR-21 levels in EVs but not intracellular miR-21 levels, suggesting that TICAM-1 augmented sorting of miR-21 to EVs. In contrast, the MDA5 adaptor, MAVS, did not increase miR-21 levels in EVs. The siRNA for TICAM-1 reduced EV miR21 levels after stimulation of TLR3. Collectively, our data indicate a novel role of the TLR3-TICAM-1 pathway in controlling miR-21 levels in EVs. © 2018 Elsevier Inc. All rights reserved.
Keywords: Extracellular vesicles Innate immunity microRNA TLR3
1. Introduction In the innate immune system, pattern recognition receptors (PRRs) recognizes pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), and several other chemical substances [1,2]. Toll-like receptors (TLRs) and RIG-I-like receptors are PRRs and recognize viral nucleic acids as PAMPs [3,4]. Toll-like receptor 3 (TLR3) binds viral doublestranded RNA (dsRNA) and triggers the signal for inducing the innate immune response via TICAM-1 also called TRIF [5,6]. Singlestranded RNA is sensed by TLR7, which utilizes MyD88 adaptor to trigger the signal [7,8]. These TLRs bind to viral RNA within endolysosomes, whereas cytoplasmic viral RNAs are recognized by RIG-
* Corresponding author. Department of Immunology, Graduate School of Medical Sciences, Faculty of Life Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto, 860-8556, Japan. E-mail address: [email protected] (H. Oshiumi).
I-like receptors, including RIG-I and MDA5, whose adaptor is MAVS (also called IPS-1, VISA, and Cardif) [3]. Alum is an aluminum salt and is widely used an adjuvant for vaccines. Alum is internalized into cells and activates Nod-like receptors (NLR) [9e11]. Recent study reported that alum induces cell death, thereby releasing cellular DNA, which functions as a DAMP and is recognized by TLR9 [12]. Extracellular vesicles (EVs) include the exosomes and microvesicles [13]. The exosomes are released from multivesicular bodies and contain functional RNAs, such as mRNA and microRNA (miRNA). An accumulating body of evidence has shown that the exosomes deliver functional RNAs to recipient cells and thus mediate inter-cellular communications [14,15]. Unlike the exosomes, the microvesicles are released from the plasma membrane. Microvesicles also deliver functional RNAs to other cells, but the mechanism underlying this is still unclear [14]. Recent studies have shown that EVs deliver miRNAs to dendritic cells and macrophages and control the innate immune responses [13,15e17].
https://doi.org/10.1016/j.bbrc.2018.04.146 0006-291X/© 2018 Elsevier Inc. All rights reserved.
Please cite this article in press as: Y. Fukushima, et al., Activation of TLR3 and its adaptor TICAM-1 increases miR-21 levels in extracellular vesicles released from human cells, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.04.146
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Several mechanisms for sorting of miRNAs into the exosomes have been reported. miRNAs that contain the EXO motif are efficiently sorted into the exosomes via hnRNPA2B1 [18], and another motif, the hEXO motif, is recognized by hnRNP-Q, leading to sorting to the exosomes [19]. In addition to hnRNPA2B1 and hnRNP-Q, other proteins are also involved in miRNA sorting; for example, YBX1, an RNA binding protein, is also involved in sorting miRNAs into EVs [20]. Recently, we found that hepatitis B virus (HBV) increased miR21 and miR-29a levels in EVs released from HBV-infected cells, as compared to those in resting cells [16]. miR-21 and miR-29a are known to target IL-12, and thus EVs released from HBV-infected cells could regulate IL-12p35 and IL-12p40 expression [21,22]. HBV is recognized by several PRRs [16]. Therefore, we reasoned that the activation of PRRs would increase miR-21 and miR-29a levels in EVs. Here, we investigated miR-21 and miR-29a levels in EVs after stimulation of PRRs and found that a TLR3 ligand, polyI:C, can increase miR-21 levels in EVs.
cultured in serum-free medium during stimulation. EVs were isolated from cell culture media by using Total Exosome Isolation Reagent (from cell culture media) (Thermo Fisher Scientific), according to the manufacturer's instructions. 2.4. miRNA expression Total RNAs from cells and EVs was isolated using TRIzol (Thermo Fisher Scientific). The reverse transcription (RT) reaction for miRNAs generation was performed using miR-X miRNA First-Strand Synthesis kit (Clontech). The SYBR Green Real-Time PCR Master Mix (ABI) was used for qPCR reaction using the Step One Real Time PCR system (ABI). All the steps were performed as per the manufactures' instructions. The miRNA levels were normalized to U6 RNA expression levels. Primers used for PCR are; miR-21: TAG CTT ATC AGA CTG ATG TTG A, and miR-29a: TAG CAC CAT CTG AAA TCG GTT A. 2.5. ELISA
2. Materials and methods 2.1. Ethics statement All the animal studies were conducted in accordance with the Guidelines for Animal Experimentation of the Japanese Association for Laboratory Animal Science, and were approved by the Animal Care and Use Committe of Kumamoto University (A29-027, J27232). 2.2. Cells, plasmids, and reagents THP-1 cells (obtained from the Japanese Collection of Research Bioresources (JCRB) cell bank) were cultured in RPMI-1640 medium with 5% fetal calf serum (FCS). The cells were differentiated into macrophages with phorbol 12-myristate 13-acetate (PMA; 60 ng/ ml) for 16 h. A549 and HEK293 cells (provided by T. Seya, at Hokkaido University) were cultured in D-MEM (high Glc) and in DMEM (low Glc) with 10% FCS, respectively. FCS was heat-inactivated at 56 C for 30 min. HeLa cells, which were kindly gifted by T. Fujita (Kyoto University), were cultured in Eagle -MEM medium with 10% FCS. siRNAs for human TICAM-1 and the negative control were purchased from Thermo Fisher Scientific. PolyI:C and CL097 were purchased from GE Healthcare and Invivogen, respectively. Imject Alum, which contains an aqueous solution of aluminum hydroxide (40 mg/ml) and magnesium hydroxide (40 mg/ml), was purchased from Thermo Fisher Scientific. pEF-BOS/TICAM-1 and pEF-BOS/ MAVS, which carry human TICAM-1 or MAVS full-length ORF, have been described previously [6,23]. The plasmids and siRNA were transfected into cells with Lipofectamine 2000 and Lipofectamine RNAiMax transfection reagents (Thermo Fisher Scientific), respectively, according to the manufacture's instruction. 2.3. EV analysis C57BL/6 mice were purchased from KYUDO COMPANY (Tosu). PolyI: C (50 mg/head) was intraperitoneally injected into the mice when they were 8e10 weeks of age. Sera were obtained from blood samples taken from the tail veins, and EVs were extracted from sera by using Total Exosome Isolation Reagent (from serum) (Thermo Fisher Scientific), according to the manufacturer's instructions. To determine miRNA levels in EVs released from cultured cells, the cells were seeded onto a 24-well plate. They were then washed four times the next day with PBS and subsequently cultured in serumfree medium for 1 or 2 days; subsequently, they were stimulated. To avoid the contamination of EVs derived from FCS, the cells were
An ELISA kit for mouse IFN-b was purchased from PBL Biomedical Laboratories. The blood samples were obtained from mouse tail veins, and sera were subsequently prepared. Serum IFNb protein levels were determined according to the manufacture's instruction by using the ELISA kit. 2.6. Statistical analysis All the statistical analysis was performed using the Prism 7 software (Ver. 7; GraphPad Software, Inc.) and Excel. The t-test was used to investigate the significance of differences between control and target samples. 3. Results 3.1. PolyI:C increased miR-21 levels in EVs released from the human cell lines Alum is an aluminum adjuvant used for various types of vaccines and is recognized by NALP3, a PRR [10]. First, we investigated the effect of alum on the miR-21 and miR-29a levels in EVs. THP-1 macrophages were stimulated with alum for 24 h, and then the EVs were collected from cell culture medium. We determined miRNA levels by RT-qPCR, following which they were normalized to U6 RNA levels. Interestingly, miR-21 and miR-29a levels in EVs were increased on stimulation with alum (Fig. 1A). Second, we investigated the effect of other adjuvants, such as polyI:C, a dsRNA analog, and CL097, which are recognized by TLR3 and TLR7, respectively [6,24]. THP-1 macrophages were stimulated with CL097 and polyI:C for 24 h, and then EVs were collected from cell culture medium. We simultaneously extracted total RNA from THP-1 macrophages to determine intracellular miRNA levels. Interestingly, miR-21 levels in EVs were increased on stimulation with CL097 and polyI:C, whereas miR-29a levels did not (Fig. 1B). In contrast to the findings for levels in EVs, CL097 and polyI:C did not increase intracellular miR-21 and miR-29a levels (Fig. 1C), suggesting that CL097 and polyI:C promotes the sorting of miR-21 into EVs but do not promote miR-21 transcription. These data are consistent with our hypothesis that PRRs can increase miR-21 and miR-29a levels in EVs. Next, we investigated the miRNA expression in EVs released from other types of cells. A549 is a cell line derived from lung epithelial cells. When A549 cells were stimulated with alum, the miR-21 levels in EVs did not increase (Fig. 2A). Stimulation of HeLa and HEK293 cells also did not increase miR-21 levels in the EVs (Fig. 2B and C), suggesting that alum increases miR-21 levels in EVs
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Fig. 1. polyI:C increased miR-21 levels in EVs released from THP-1 macrophages. (A) Alum was added to THP-1 macrophages (final conc. 0.4 mg/ml) cultured in serum-free medium. At 24 h after stimulation, EVs were collected from cell culture, and miR-21 and miR-29a levels were determined by RT-qPCR and normalized to U6 RNA levels. Error bars represents standard deviation (n ¼ 3). (B, C) THP-1 macrophages cultured in serum free medium were stimulated with CL097 (final conc., 5 mg/ml) and polyI:C (final conc., 50 mg/ml) for 24 h. EVs were collected from cell culture. Total RNA was extracted from EVs (B) and THP-1 macrophages (C), and miR-21 and miR-29a levels were determined by RT-qPCR and normalized to U6 RNA levels. Error bars represents standard deviation (n ¼ 3).
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Fig. 2. polyI:C increases EV miR-21 levels released from various types of cells. A549 (A), HEK293 (B), and HeLa (C) cells were cultured in medium without serum and were subsequently stimulated with alum (final conc., 0.4 mg/ml), CL097, (final conc., 5 mg/ml), and polyI:C (final conc., 50 mg/ml) for 24 h. EVs were collected from each cell culture supernatant. Total RNA was extracted from the EVs and cultured cells that were collected, and miR-21 and miR-29a levels were determined by RT-qPCR and normalized to U6 RNA levels (n ¼ 3).
in a cell type-specific manner. In contrast, miR-21 levels were increased in EVs released from A549, HeLa, and HEK293 cells stimulated with polyI:C (Fig. 2A and C), suggesting that polyI:C can increase miR-21 levels in EVs released from various types of cells. miR-29 levels in EVs was increased on stimulation of HEK293 and HeLa cells with polyI:C, whereas those in EVs released from A549 cells did not (Fig. 2A and C), suggesting that polyI:C increases miR-29 levels in EVs in a cell type-specific manner. Unlike polyI:C, CL097 did not increase miR-21 and miR-29a levels in EVs released from A549, HEK293, and HeLa cells (Fig. 2A and C). Collectively, these data indicate that PRRs increase miR-29a levels in EVs in a cell type-specific manner.
3.2. miR-21 levels in EVs circulating in the blood did not increase on stimulation with polyI:C EVs are abundant in the blood. Considering that polyI:C stimulation increased miR-21 levels in EVs from various types of cells, we reasoned that polyI:C injection would increase miR-21 levels in EVs circulating in the blood. To test this hypothesis, we used a mouse animal model. PolyI:C was intraperitoneally injected into mice. At 24 h after injection, sera were obtained from blood samples taken from mouse tail veins, and EVs were isolated from these sera. miR21 and miR-29a levels in EVs were determined by RT-qPCR. We
confirmed that polyI:C stimulation transiently increased the type I IFN levels in mouse sera by using ELISA (Fig. 3A). Unexpectedly, polyI:C injection did not increase miR-21 and miR-29a levels in EVs circulating in the blood (Fig. 3B). These data suggest that polyI:C injection is not sufficient for increasing the systemic EV miR-21 levels, although it can increase systemic IFN-b protein level.
3.3. TICAM-1 increased miR-21 levels in EVs Next, we focused on the mechanism underlying the increase in miR-21 levels in EVs, because it was increased on polyI:C stimulation in various types of cells. PolyI:C is recognized by TLR3 in the endolyosomes [25], whereas transfected polyI:C localized in the cytoplasm is recognized by MDA5 [26]. TLR3 and MDA5 trigger the signal via TICAM-1 and MAVS, respectively [6,26]. To determine the pathway responsible for the increase in miRNA levels in EVs after polyI:C stimulation, TICAM-1- and MAVS-expressing vectors were transfected into cells, and miR-21 and miR-29a levels in EVs and cells were determined by RT-qPCR. Over-expression of TICAM-1 and MAVS leads to auto-activation of their signalings [6,27]. We found that TICAM-1 overexpression increased miR-21 and miR-29a levels in EVs, whereas MAVS overexpression did not (Fig. 4A). In contrast to the findings for expression in EVs, TICAM-1 and MAVS overexpression did not increase the intracellular miR-21 and miR-
Please cite this article in press as: Y. Fukushima, et al., Activation of TLR3 and its adaptor TICAM-1 increases miR-21 levels in extracellular vesicles released from human cells, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.04.146
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Fig. 3. PolyI:C injection does not affect miR-21 and miR-29a levels in EVs circulating in the blood. (A) 50 mg of polyI:C was intraperitoneally injected into C57BL/6 mice. To measure serum IFN-b protein levels, samples were collected at the indicated time points. IFN-b protein levels were determined by ELISA (n ¼ 3). (B) 50 mg of polyI:C or PBS (mock) was intraperitoneally injected into C57BL/6 mice (n ¼ 5 or 6). Sera were obtained from mice 24 h after stimulation, and EVs were subsequently extracted from each serum sample. miR-21 and miR-29a levels were determined by RT-qPCR and normalized to U6 RNA levels.
Fig. 4. TICAM-1 increased miR-21 levels in EVs. (A, B) THP-1 macrophages were cultured in serum-free medium and were transfected with empty, TICAM-1-, and MAVS-expressing vectors for 24 h. EVs were collected from the cell culture supernatant. Total RNA was extracted from EVs and cultured cells. miR-21 and miR-29a levels in EVs (A) and cells (B) were determined by RT-qPCR and normalized to U6 RNA levels (n ¼ 3, t-test, *p < 0.05). (C) siRNA for negative control or TICAM-1 was transfected into HeLa cells for 2 days. Cells were stimulated with 50 mg of polyI:C for 24 h. EVs were collected from cell culture sup., and total RNA was extracted. miR-21 levels in EVs were determined by RT-qPCR and normalized to U6 RNA levels. The fold change in expression was calculated by dividing the value for each sample with that for the mock-stimulated sample (n ¼ 3, t-test, *p < 0.05). (D) siRNA for negative control (NC), hnRNPA2B1, YBX1, and hnRNP-Q were transfected into THP-1 macrophages for 2 days. Cells were stimulated with 50 mg of polyI:C for 24 h, and EVs were subsequently collected from the cell culture supernatant. miR-21 levels in EVs were determined by RT-qPCR and normalized to U6 RNA levels. The fold change in expression was calculated by dividing the value for each sample with that for NC sample (n ¼ 3, t-test, *p < 0.05).
29a levels (Fig. 4B). These data suggest that TICAM-1 can increase the sorting of miR-21 into EVs. To further confirm the involvement of TICAM-1 in miR-21
expression in EVs, we performed knockdown study using siRNA for TICAM-1. siRNA for TICAM-1 or negative control was transfected into A549 cells for 2 days. Cells were subsequently stimulated with
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polyI:C, and EVs were collected at 1 day after stimulation. The siRNA for TICAM-1 decreased EV miR-21 levels after polyI:C stimulation (Fig. 4C). Collectively, our data indicate that TLR3 stimulation triggers the signal for increasing EV miR-21 levels via TICAM-1. Sorting of miRNAs into EVs depend on RNA-binding proteins, such as hnRNPA2B1, hnRNP-Q, and YBX1 [18e20]. We investigated whether those RNA binding proteins are involved in the TICAM-1mediated increase in miR-21 levels in EVs. The siRNA for YBX1 decreased miR-21 levels in EVs from TICAM-1 overexpressing cells, whereas that for hnRNP-Q did not (Fig. 4D). Although the siRNA for hnRNPA2B1 decreased EV miR-21 levels, the decrease was not statistically significant. These data are consistent with our hypothesis that TICAM-1 promotes the sorting of miR-21 into EVs.
miR-21 is reported to regulate the IL-12 expression. EVs delivers miRNAs into the cytoplasm of recipient cells, and the miRNAs subsequently repress target gene expression [14,16,21]. Thus, it is expected that TLR3 would promotes sorting of miR-21 into EVs, which results in the downregulation of IL-12 in recipient cells. TLR3 itself is known to increase IL-12p40 production [31]. Therefore, miR-21 levels in EVs are expected to be involved in a negative feedback loop for attenuating inflammation. Considering that polyI:C administration did not increase miR-21 levels in EVs circulating in the blood, the EVs released from TLR3-stimulated cells would affect only cells in local tissue. Further studies are required to completely elucidate the physiological significance of EV miR-21 released from TLR3-stimulated cells.
4. Discussion
Acknowledgement
In our previous study, we found that EVs released from HBVinfected hepatocytes contained higher levels of miR-21 than those from resting cells, and regulated IL-12 expression [16]. In the current study, we found that TLR3 stimulation increased miR-21 levels in EVs released from human cells and that this increase required the TICAM-1 adaptor molecule. Moreover, the TICAM-1mediated increase in EV miR-21 levels depended on YBX1, which is known to be involved in sorting of miRNAs into exosomes [20]. On TLR3 stimulation, miR-29a levels increased in HEK293 and HeLa cells but not in A549 cells and THP-1 macrophages. Therefore, sorting of miR-29a appears to be regulated not only by TLR3 signaling but also by other unknown mechanisms. Other PAMPs also increase miR-21 and/or miR-29a levels in EVs. For instance, alum increased miR-21 and miR-29a levels in EVs. Alum is recognized by NALP3, resulting in the activation of the inflammasome [10]. Thus, it is expected that the inflammasome would be involved in the increase in miR-29a levels in EVs. In contrast, CL097, a ligand for TLR7, barely increased miR-21 and miR-29a levels. Thus, the mechanism underlying the increase in levels seems to be complicated, and further studies are required to clarify it. miRNAs that contain the EXO-motif and hEXO motif are sorted into the exosomes via hnRNPA2B1 and hnRNP-Q, respectively. Several other proteins are required for sorting of miRNAs into EVs, although the motif or features of miRNAs that are sorted by those proteins are remain unclear. The YBX1 is previously identified as a protein required for miR-224 sorting into the exosomes in HEK293T cells [20]. A subsequent study reported that YBX1 is involved in the sorting of non-coding RNAs, such as tRNAs, Y RNAs, and vault RNAs as well as miRNAs [28]. Considering that the siRNA for YBX1 decreased the TICAM-1-mediated increase in miR-21 levels in EVs, it is expected that TICAM-1 would regulate the YBX1 activity and that TICM-1 signaling might be required for sorting of other types of RNAs into EVs. EVs are released from various types of cells, and consequently, blood contains high amount of EVs [13]. PolyI:C administration increases type I IFN protein levels in the blood. Because polyI:C increased miR-21 levels in EVs released from several types of cells, we expected that it would increase miR-21 levels in the EVs circulating in blood. However, we found that polyI:C administration did not increase miR-21 levels in those EVs. PolyI:C is known to activate not only TLR3 but also MDA5 in vivo, and MDA5 plays a major role in type I IFN production in vivo [26]. Our in vitro overexpression study showed that over-expression of MAVS, which is the sole adaptor for MDA5, did not increase miR-21 levels in EVs. MDA5 is ubiquitously expressed in various type of cells, and its expression is increased by stimulation [26,29]. In contrast, TLR3 exhibits cell type-specific expression [30]. Therefore, it is possible that polyI:C administration stimulated only the MDA5 pathway and was not sufficient to systemically increase miR-21 levels in EVs.
We thank all of our laboratory members for the helpful discussions and technical assistance. This work was supported in part by Grants-in-Aid from the following: Ministry of Education, Science and Technology (15K08517); Japan Agency for Medical Research and Development (A-163); PRESTO JST (09801362); Tokyo Metropolitan Government (09802207); the Takeda Science Foundation; the Japanese Association for Complement Research; the Uehara Memorial Foundation; the Daiichi Sankyo Foundation of Life Science; and the Waksman Foundation of Japan. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.04.146. References [1] K. Newton, V.M. Dixit, Signaling in innate immunity and inflammation, Cold Spring Harb Perspect Biol 4 (2012). [2] V.E. Schijns, E.C. Lavelle, Trends in vaccine adjuvants, Expert Rev. Vaccines 10 (2011) 539e550. [3] Y.M. Loo, M. Gale Jr., Immune signaling by RIG-I-like receptors, Immunity 34 (2011) 680e692. [4] T. Kawai, S. Akira, Toll-like receptors and their crosstalk with other innate receptors in infection and immunity, Immunity 34 (2011) 637e650. [5] M. Yamamoto, S. Sato, H. Hemmi, K. Hoshino, T. Kaisho, H. Sanjo, O. Takeuchi, M. Sugiyama, M. Okabe, K. Takeda, S. Akira, Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway, Science 301 (2003) 640e643. [6] H. Oshiumi, M. Matsumoto, K. Funami, T. Akazawa, T. Seya, TICAM-1, an adaptor molecule that participates in Toll-like receptor 3-mediated interferon-beta induction, Nat. Immunol. 4 (2003) 161e167. [7] S.S. Diebold, T. Kaisho, H. Hemmi, S. Akira, C. Reis e Sousa, Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA, Science 303 (2004) 1529e1531. [8] H. Hemmi, T. Kaisho, O. Takeuchi, S. Sato, H. Sanjo, K. Hoshino, T. Horiuchi, H. Tomizawa, K. Takeda, S. Akira, Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway, Nat. Immunol. 3 (2002) 196e200. [9] S.L. Demento, S.C. Eisenbarth, H.G. Foellmer, C. Platt, M.J. Caplan, W. Mark Saltzman, I. Mellman, M. Ledizet, E. Fikrig, R.A. Flavell, T.M. Fahmy, Inflammasome-activating nanoparticles as modular systems for optimizing vaccine efficacy, Vaccine 27 (2009) 3013e3021. [10] V. Hornung, F. Bauernfeind, A. Halle, E.O. Samstad, H. Kono, K.L. Rock, K.A. Fitzgerald, E. Latz, Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization, Nat. Immunol. 9 (2008) 847e856. [11] E. Kuroda, K.J. Ishii, S. Uematsu, K. Ohata, C. Coban, S. Akira, K. Aritake, Y. Urade, Y. Morimoto, Silica crystals and aluminum salts regulate the production of prostaglandin in macrophages via NALP3 inflammasomeindependent mechanisms, Immunity 34 (2011) 514e526. [12] T. Marichal, K. Ohata, D. Bedoret, C. Mesnil, C. Sabatel, K. Kobiyama, P. Lekeux, C. Coban, S. Akira, K.J. Ishii, F. Bureau, C.J. Desmet, DNA released from dying host cells mediates aluminum adjuvant activity, Nat. Med. 17 (2011) 996e1002. [13] M. Colombo, G. Raposo, C. Thery, Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles, Annu. Rev. Cell Dev. Biol. 30 (2014) 255e289. [14] T. Kouwaki, M. Okamoto, H. Tsukamoto, Y. Fukushima, H. Oshiumi,
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Please cite this article in press as: Y. Fukushima, et al., Activation of TLR3 and its adaptor TICAM-1 increases miR-21 levels in extracellular vesicles released from human cells, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.04.146