Forkhead box O1 (FOXO1) controls the migratory response of Toll-like receptor (TLR3)-stimulated human mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) can potently regulate the functions of immune cells and are being investigated for the management of inflammatory diseases. Toll-like receptor 3 (TLR3)-stimulated human MSCs (hMSCs) exhibit increased migration and chemotaxis within and toward damaged tissues. However, the regulatory mechanisms underlying these migratory activities are unclear. Therefore, we analyzed the migration capability and gene expression profiles of TLR3-stimulated hMSCs using RNA-Seq, wound healing, and transwell cell migration assay. Along with increased cell migration, the TLR3 stimulation also increased the expression of cytokines, chemokines, and cell migration-related genes. The promoter regions of the latter showed an enrichment of putative motifs for binding the transcription factors forkhead box O1 (FOXO1), FOXO3, NF-κB (NF-κB1), and RELA proto-oncogene and NF-κB subunit. Of note, FOXO1 inhibition by the FOXO1-selective inhibitor AS1842856 significantly reduced both migration and the expression of migration-related genes. In summary, our results indicate that TLR3 stimulation induces hMSC migration through the expression of FOXO1-activated genes.

One of the most important features in the therapeutic applications of MSCs is the homing of transplanted MSCs into inflammation sites within damaged tissues (4,27). Transplanted MSCs can migrate to injured sites and promote the repair process through their immunomodulatory activities (4,28). Migrated MSCs release proinflammatory or anti-inflammatory factors and regulate immune cells (16, 29 -33). Conversely, chemokines and cytokines of various origins, including stromal cell-derived factor-1␣ (34 -36), hepatocyte growth factor (37), and chemokine (C-C motif) ligand 2 (CCL2) (27,38), induce migration of MSCs. Also, activation of TLR3 stimulates the secretion of immune modulators and soluble factors that lead to immunosuppressive responses (2,25). Several studies have suggested that stimulation of TLR3 regulates migration properties and immunomodulatory factors, including indoleamine 2,3-dioxygenase (IDO), prostaglandin E 2 , and transforming growth factor ␤ (TGF␤) (2,26,39). However, the mechanism of the TLR3-activated migration of hMSCs is unknown. Therefore, we investigated whether TLR3-stimulated hMSCs contribute to the pathway in response to hMSC migration using gene expression profiling.
In this study, we performed RNA-Seq for gene expression profiling of hMSCs treated with a TLR3 ligand (poly(I:C), polyinosinic:polycytidylic acid) compared with unstimulated hMSCs (control hMSCs). We analyzed differentially expressed genes and validated the RNA-seq data using quantitative realtime PCR (qRT-PCR). Our results show that TLR3-stimulated hMSCs express inflammatory-and migration response-related genes, thus revealing the molecular effects of TLR3 activation. Additionally, our results show that the TLR3-stimulated hMSCs increased cell migration through the activation of forkhead box protein O1 (FOXO1). Together, these results strengthen the molecular foundation for the clinical utilization of the cell migration abilities of hMSCs.

Characterization of TLR3-stimulated hMSCs
To study the effects of TLR3 stimulation on hMSCs, we incubated them with poly(I:C) for 4 h. Nonstimulated hMSCs (control hMSCs) and TLR3-stimulated cells (TLR3-stimulated hMSCs) exhibited a similar spindle-shaped fibroblastic morphology (Fig. 1A). Flow cytometry expression analysis of the typical MSC-related surface antigens showed that both the control hMSCs and TLR3-stimulated hMSCs were positive for CD29, CD44, CD73, and CD105 and negative for the hematopoietic lineage markers CD31, CD34, CD45, and HLA-DR after 6 passages (Fig. 1B). The viability of these cells was not appreciably affected when treating them with various concentrations of poly(I:C) (1 to 20 g/ml) for 4 h (Fig. 1C). These data indicate that TLR3-stimulated hMSCs maintain the typical characteristics of hMSCs.

IDO gene expression and chemokine expression in TLR3-stimulated hMSCs
To determine the appropriate time points, we performed mRNA and protein expression analysis in hMSCs treated for 0.5 to 24 h with poly(I:C) at 10 g/ml. The poly(I:C) treatment induced the expression of IDO (Fig. 1D) as well as chemokine (C-C motif) ligand 5 (CCL5) and chemokine (C-X-C motif) ligand 10 (CXCL10) (Fig. 1, E and F). Although the time profiles differed, induction was in all cases maximal or near-maximal at 4 h. Based on these results, we used 10 g/ml concentration and the 4-h time point as the standard condition for TLR3 stimulation of hMSCs.

Differentially expressed genes of TLR3-stimulated hMSCs
To analyze the gene expression pattern in response to TLR3 stimulation, three independent samples (biological triplicates) of the entire transcriptomes of the control 7F3915 hMSCs and TLR3-stimulated 7F3915 hMSCs were profiled using RNA-Seq. A total of 130 genes were differentially expressed, with 120 up-regulated and 10 down-regulated genes in the TLR3-stimulated versus control hMSCs. Of these differentially expressed genes (DEGs), the top 50 up-regulated genes are listed in Fig.  2A. Interestingly, most of the top 50 genes were related to interferons and inflammation, namely, an interferon-induced protein with tetratricopeptide repeats 2 (IFIT2), CXCL8, IFIT3, interleukin 1 ␤ (IL1␤), and 2Ј-5Ј-oligoadenylate synthetase-like (OASL) ( Fig. 2A and Table S1). Next, we performed functional annotation analysis for Gene Ontology (GO) based on the biological processes (BP) using DAVID Informatics Resources. The up-regulated genes in the TLR3-stimulated hMSCs were determined to be involved in several BPs such as response to viruses, inflammation, and anti-apoptosis (Fig. 2B).
To identify the degree to which the 7F3915 hMSCs genes that we detected were up-regulated in 7F3674 and 127756 hMSCs, we further constructed RNA-seq dataset to compare the up-regulated genes in 7F3915 hMSCs with that of 7F3674 and 127756 hMSCs. We compared the identified molecular signatures of the most highly up-regulated genes that we found to be expressed in 7F3915 hMSCs to those of 7F3674 and 127756 hMSCs. Of the top 30 up-regulated genes in TLR3-stimulated 7F3915 hMSCs, 7F3674 and 127756 hMSCs shared 20 genes following TLR3 stimulation, suggesting substantial similarities in their transcriptional regulation among different donors hMSCs (Fig. S1, A and B). The following inflammation-related genes were up-regulated all three different donors (7F3915, 7F3674, and 127756 hMSCs): cytokines/chemokines (CCL2, CXCL1, CXCL8, and CXCL8, etc.) and interferon-related (IFIT1, IFIT2, IFIT3, IFI44, OASL, MX1, etc.). However, we found that most of the positive regulators of inflammationrelated genes were more highly induced in 7F3915 hMSCs than in 7F3674 and 127756 hMSCs following TLR3 stimulation. We also observed 7F3674 and 127756 hMSCs also had similarities in their physiological functions according to DAVID GO anal-FOXO1 controls hMSCs migration to TLR3 stimulation ysis, such as response to viruses and immune response (Fig.  S1C).

Immune cells (BV-2 microglial cell lines) do not acquire the hMSCs transcriptional signature
To identify the degree to which the 7F3915 hMSCs genes that we detected were up-regulated in immune cell types, we analyzed our published RNA-seq dataset (40) to compare the up-regulated genes in TLR3-stimulated BV-2 microglial cell lines with that of 7F3915 hMSCs. Using Venn diagrams, and heat maps of BV-2 microglial cell lines and hMSCs transcriptome profiles, we found that hMSCs differed in their transcriptomes from BV-2 microglial cell lines (Fig. S2, A and C). We compared the identified molecular signatures of the top 100 up-regulated genes (fold-change Ն 1.5, and p Յ 0.01) between hMSCs and BV-2 microglial cell lines. In hMSCs, 71 genes were up-regulated that were not common to the BV-2 microglial cell lines. In contrast, in BV-2 microglial cell lines, also 71 genes were up-regulated that were not common to the TLR3-stimu-lated hMSCs (Fig. S2A). The unique gene sets for hMSCs and BV-2 microglial cell lines are presented in Fig. S2C. However, TLR3-stimulated hMSCs and BV-2 microglial cell lines also displayed similarities in their transcriptomes. The following inflammation-related genes were up-regulated only in hMSCs: cytokines/chemokines (CCL2, CCL20, CXCL1, CXCL3, CXCL6, CXCL8, IL7R, IL32, etc.) and interferon-related (GBP1, GBP4, IFI44, IFI44L, OASL, OAS1, OAS3, among others) (Fig. S2C). Thus, hMSCs and BV-2 microglial cell lines maintain their molecular signature during inflammatory responses.

Network analysis of the altered genes in TLR3-stimulated hMSCs
Next, we used IPA software to identify the network of genes and related pathways that represent the interacting genes in TLR3-stimulated 7F3915 hMSCs. Networks 1 and 2 were among the networks of DEGs after 4 h of poly(I:C) treatment (Fig. 3, A and B). The genes in network 1 are known to be

FOXO1 controls hMSCs migration to TLR3 stimulation
involved in cellular movement, immune cell trafficking, and inflammatory response; these genes formed a direct or indirect network with the NF-B complex (Fig. 3A). The genes in network 2 are known to be involved in antimicrobial response, inflammatory response, and dermatological diseases and conditions; these genes formed a direct or indirect network with interferon regulatory factor 1 (IRF1) or the phosphatidylinositol 3-kinase family (Fig. 3B). The top 10 canonical pathways that include DEGs in the TLR3-stimulated hMSCs are listed in Fig.  3C. Interestingly, the genes in canonical pathways related to inflammation encode pattern recognition receptors for bacteria and viruses, interferon signaling, and activation of IRF by the cytosolic pattern recognition receptor. To further characterize the TLR3-stimulated hMSCs, we determined the molecular and cellular functions of the up-regulated and down-regulated genes in TLR3-stimulated hMSCs. We found that the top 6 of these functions are related to cellular movement, immunological disease, etc. (Fig. 3D).

Differential expression of transcription factors in TLR3-stimulated hMSCs
To better understand regulation, we focused on the transcription factors (TFs) among the DEGs in TLR3-stimulated 7F3915 hMSCs. Up-regulated TFs notably included the NF-B family, i.e. NF-B1, NF-B2, NF-BIA, NF-BIE, NF-BIZ, REL, and RELB (Fig. 4A), as confirmed by qRT-PCR (Fig. 4E). Next, we used IPA software to identify the target genes that were directly or indirectly regulated by TFs in TLR3-stimulated hMSCs. Interestingly, we found that cytokines, chemokines, and inflammation-related genes, including CXCL8, CXCL10, IL6, and IRF1, were regulated by TFs, including NF-B1 and FOXO1 (Fig. 4B). Next, we used DAVID 6.8 software to identify

FOXO1 controls hMSCs migration to TLR3 stimulation
functionally annotated processes of the FOXO1-and NF-B1regulated genes. We observed an enrichment of FOXO1 target genes associated with protein oligomerization and response to interleukin-1 (Fig. 4C). NF-B1-regulated genes were determined to be related to the chemokine-mediated signaling pathway and chemotaxis (Fig. 4D). The FOXO1 and NF-B1-regulated transcripts were determined to be associated with cytokine-mediated responses and immune responses. We analyzed the expression of several FOXO genes using real-time PCR and immunocytochemistry (Figs. 4F and 6B). Although FOXO1 was significantly up-regulated, the expression of FOXO3 and FOXO4 did not change after poly(I:C) treatment ( Fig. 4F and Fig. S3, A and B). We also analyzed the expression of FOXO1, FOXO3, and FOXO4 in 7F3674 and 127756 hMSCs ( Fig. S4A and S5A). The MSCs from both donors showed upregulated FOXO1 gene expression upon poly(I:C) treatment.

Up-regulation of cell migration-related genes in TLR3-stimulated hMSCs
In the therapeutic application of stem cells, their migration is important for wound healing effects. Therefore, after functional analysis of the up-and down-regulated genes in TLR3-stimulated 7F3915 hMSCs, we focused on cellular movement, except for genes associated with the inflammatory response and infectious disease-related functions. Using IPA software, we found that 42 genes, including cytokine/chemokine genes, were associated with cell migration responses (Fig. 5A). The IPA software further revealed that the cell migration responserelated genes are regulated by NF-B1 and FOXO1 (Fig. 5B). Using the Pscan software tool, we analyzed TF motifs to identify cis-regulatory elements within the promoter regions in cell migration response-related genes. The binding sites of NF-B1, RELA, FOXO1, and FOXO3 were significantly enriched in TLR3-stimulated hMSCs (Fig. 5C). Again, FOXO1 was found to be associated with the cell migration response.

Role of FOXO1 in the migratory response of TLR3-stimulated hMSCs
The foregoing bioinformatics analysis suggested an important role for FOXO1 in the migration response. To obtain direct experimental support for this notion, we treated the cells with the FOXO1 selective inhibitor AS1842856 (5-amino-7-(cyclohexylamino)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3carboxylic acid) (41, 42) and determined its effects on the FOXO1 controls hMSCs migration to TLR3 stimulation migratory response. Using the WST1 assay, we first determined the highest nontoxic concentration of AS1842856. Viability was not affected at lower doses (1 and 5 M) but was slightly decreased at higher doses (10 and 20 M) (Fig. 5D). Therefore, 5 M was used in the following.
First, we measured the expression of selected cell migrationrelated genes (Fig. 5E). We found that AS1842856 significantly down-regulated the expression of key cell migration-related genes, including CCL2, CCL5, CXCL1, CXCL8, CXCL10, ICAM1, IFIT2, IL6, MMP1, MMP3, NF-B1, NF-B2, SOD2, TNFSF10, and VCAM1 (Fig. 5E). By contrast, some of the genes regulated by NF-B (ATF3, HMOX1, IFITM1, and PTGS2) did not show altered expression (Fig. S6). We also analyzed the expression of cell migration-related genes in MSCs from 7F3674 and 127756 hMSCs. In both cell isolates, inhibition of FOXO1 regulated the expression of cell migration-related genes (Figs. S4B and S5B). Next, we investigated whether FOXO1 depletion affects key cell migration-related genes. hMSCs were treated with three different siRNA (si5257, si5258, and si5259), and si5258 led to significant depletion of FOXO1 expression compared with the negative control group (scrambled siRNA) (Fig. S7A). Furthermore, none of these FOXO1 siRNAs were able to inhibit positive control GAPDH (Fig. S7A). We also used positive control siRNA (GAPDH siRNA) to evaluate the methods of transfection and found that the positive control siRNA also had significant depletion of GAPDH, but not FOXO1 (Fig. S7B). Therefore, we used si5258 in the subsequent experiments. Using siRNA analysis we found that key cell migration-related genes were increased after poly(I:C) treatment and FOXO1 depletion cells led to significant reduction in key cell migration-related genes (Fig. 5F). However, some of the migration-related genes, including CXCL1, NF-B1, NF-B2, and SOD2 did not show low expression levels (data not shown).
Furthermore, we performed chromatin immunoprecipitation (ChIP) experiments to determine FOXO1 occupancy in target gene-promoter regions. FOXO1 directly bound to CCL2, CXCL8, and ICAM1 promoter regions (Fig. 5G). These findings suggest that FOXO1 might be involved in the regulation of cell migration in TLR3-stimulated hMSCs.

FOXO1 controls hMSCs migration to TLR3 stimulation
Second, we performed a wound-healing assay with control cells, TLR3-stimulated hMSCs, AS1842856-treated unstimulated cells, and AS1842856-treated TLR3-stimulated cells. These cells, including AS1842856-treated TLR3-stimulated cells, displayed similar spindle-shaped fibroblastic morphology (Fig. 6A). No morphological effects were visible in the TLR3stimulated cells treated with AS1842856. To determine the efficiency of AS1842856, we analyzed FOXO1 mRNA and protein levels using qRT-PCR and immunocytochemistry for 10 and 30 min. Immunofluorescence experiments revealed that AS1842856 treatment prevented the increased translocation of FOXO1 in TLR3-stimulated hMSCs (Fig. 6B). Furthermore, the qRT-PCR results revealed that in the TLR3-stimulated hMSCs, AS1842856 significantly down-regulated the expression of FOXO1 but not FOXO3 and FOXO4 (Fig. 6C). In addition, we performed a wound-healing assay to investigate the migration effects in TLR3-stimulated cells. Poly(I:C) treatment significantly increased the number of hMSCs that migrated into a wound field for 24 h. AS1842856 treatment in TLR3-stimulated hMSCs significantly decreased the number of cells that

FOXO1 controls hMSCs migration to TLR3 stimulation
migrated into a wound field compared with the control cells (Fig. 6D). We also performed a transwell migration assay to determine hMSCs migration in response to the AS1842856. We found that AS1842856 significantly suppressed the migration of 7F3674 and 127756 hMSCs toward TLR3 stimulation (Fig.  S8, A and B). Taken together, these data strongly suggest that FOXO1 is involved in the regulation of cell migration responses in TLR3-stimulated hMSCs.

Discussion
In addition to their differentiation potentials, the immunomodulatory properties of MSCs create exciting possibilities for therapies that aim to repair damaged tissues (12,30). MSCs inhibit the proliferation of immune cells, including T cells, macrophages, natural killer cells, and dendritic cells (4,7,28). MSCs are widely investigated for their use in the treatment of inflammatory diseases, such as graft versus host disease, collagen-induced arthritis, experimental autoimmune encephalomyelitis, type 1 diabetes mellitus disease, and inflammatory bowel disease (43).
MSCs can be isolated and expanded ex vivo before being administered to patients (14, 44 -46). In many studies, researchers that used the expansion phase for pretreatments hoped to enhance therapeutic effects such as immunomodulation, transdifferentiation, and cell migration (11,(47)(48)(49). In this respect, TLRs are promising targets, because they are known to modulate immune responses in inflammation-associated conditions, such as infection and tissue injury (28,33,50). Of the various TLRs, the present study has focused on TLR3. Activation of TLR3 is known to induce the expression of cytokines and chemokines that repress T cell proliferation, exert various immunosuppressive effects, and stimulate cell migration (2,24,51). In agreement with this notion, TLR3-stimulated MSCs (poly(I:C)-treated MSCs) exhibit enhanced immunosuppressive properties. For example, they inhibit T cell proliferation and reduce intestinal inflammation in dextran sulfate sodiuminduced acute colitis (52). Poly(I:C) treatment also improves the immunosuppression effects of MSCs in septic mice (53) and ameliorates myocardial fibrosis, reduces apoptosis, and enhances cardiomyocyte regeneration in hamsters (54). Pre-

FOXO1 controls hMSCs migration to TLR3 stimulation
conditioning of human umbilical cord-derived MSC with poly(I:C) protects against mortality in trinitrobenzene sulfonate-induced colitis in mice (55). These examples illustrate that it is of medical interest to clarify the molecular mechanisms triggered by TLR3 stimulation in hMSCs.
Of the above list, CXCL1, CXCL8, and CCL2 are well-studied examples of chemokines and cytokines involved in migration and chemotaxis (56). For example, Pu et al. (57) reported that the up-regulation of CXCL1 and CXCL8 promotes chemotaxis and regulates adiponectin-induced chemotaxis in human jaw bone marrow MSCs. CXCL1 induces chemotaxis and stimulates wound repair in a scratch assay using bone marrowderived mesenchymal cells (BM-MCs). In addition, an anti-CXCL1 antibody decreased CXCL1-stimulated migration activity in BM-MCs (58). Furthermore, CCL2 promotes the migration of SG2 cells, a TGF␤-responsive MSC line (59). MSC-derived CCL2 increases the migration of immune cells such as monocytes, whereas an anti-CCL2 antibody counteracts their migration (60). CCL2 is also important for the mediation of rat BM-MSC migration toward gliomas (61). Altogether, these literature results are consistent with our data that show increased cell migration and chemotaxis in association with the induction of cytokines and chemokines in TLR3-stimulated hMSCs (Fig. 2, A-E).
TLR3 also activated inflammatory responses through the upregulation of infectious disease-and inflammatory responserelated genes. In IPA network analysis, the infectious disease network was highly enriched with interferon-related genes (IFI6, IFIH1, IFIT1, IFNAR2, IRF1, IRF7, ISG15, and MX1) (Fig.  3B) that are regulated by the NF-B family (Fig. 4B). TLR signaling leads to activation of downstream effectors, such as NF-B, MAPK, and IRFs. Among these, NF-B plays a key role in the induction of inflammatory cytokine genes and the induction of chemotaxis or cell migration (62,63). In our study, TLR3 stimulation induced the expression of the NF-B family, such as NF-B1, NF-B2, NF-BIA, NF-BIE, NF-BIZ, REL, and RELB (Fig. 4, A and E). Thus, in agreement with the literature, our data indicate that NF-B controls inflammatory response genes, chemokine-related pathways, chemotaxis, and cell migration in TLR3-stimulated hMSCs.
To further analyze transcriptional regulation downstream of TLR3 stimulation, we performed TF binding motif analysis. We found that the promoters of cell migration-related genes were enriched for putative binding sites of FOXO1, FOXO3, NF-B1, and RELA. IPA software then identified the NF-B1 and FOXO1 target genes that are involved in similar biological processes including immune response and cytokine-related pathways (Fig. 5B). In a previous study, it was shown that FOXO1 regulates immune responses by protecting hematopoietic stem cells against oxidative stress (64). FOXO1 induces cell migration through TGF␤1 in keratinocytes, and knockdown of FOXO1 inhibits keratinocyte migration (65). Taken together, these results suggest that FOXO1 and NF-B1 have an important role in TLR3-mediated cell migration as well as immune responses of hMSCs.
Among the DEGs identified by RNA-seq, the genes of adhesion molecules and matrix metallopeptidases (MMPs) were significantly up-regulated in TLR3-stimulated hMSCs. Adhesion molecules, such as ICAM1 and VCAM1, play a critical role in MSCs migration (66) and mediate TNF␣-stimulated MSCs migration (67). Likewise, MMPs play an essential role in cell migration (68). Ho et al. (69) reported that migration properties parallel the activation and inhibition of MMP1. In the present study, MMPs and adhesion molecules such as ICAM1, MMP1, MMP3, and VCAM1 were significantly up-regulated by TLR3 stimulation. Furthermore, the expression of cell migration-related cytokine and chemokine genes (CCL2, CCL5, CXCL1, CXCL8, and CXCL10), an interferon-related gene (IFIT2), NF-B family genes (NF-B1 and NF-B2), and TNF-related genes (TNFSF10) were significantly up-regulated in TLR3stimulated hMSCs. Importantly, treatment with AS1842856, a highly specific FOXO1 inhibitor, blunted these effects of TLR3 stimulation on gene expression (Fig. 5E) as well as the associated cell migration response. Interestingly, many of the studies researchers used AS1842856 for the specific inhibition of FOXO1, because they are known to affect only the FOXO1 transcription-activating domain (41,42,70). Nevertheless, further experimental validation using knockouts or overexpression models are warranted to determine the role of FOXO1 in MSCs. Taken together, our results demonstrate that FOXO1 is important for cell migration and the regulation of cell migration-related genes in TLR3-stimulated hMSCs.
Overall, our RNA-seq data reveal the transcriptional landscape of TLR3-stimulated hMSCs, reflecting the altered gene expression and modulation of cell responses by external stimulation. Based on these data, we have used gene expression profiling, gene clustering, pathway analysis, and TF motif analysis to generate information about the molecular mechanisms that underlie therapeutic effects such as immunomodulation and cell migration. Future analysis of histone modifications, TF binding, and noncoding RNA expression will provide further insights into the regulatory systems that control the therapeutically relevant cell migration-related gene expression in TLR3stimulated hMSCs.

Conclusion
Our comparative analysis of global transcriptomes revealed the altered expression of inflammation-and cell migration-related genes in TLR3-stimulated versus nonstimulated hMSCs. Bioinformatics approaches identified NF-B and FOXO1 as potential regulators of the migratory response of TLR3-stimu-FOXO1 controls hMSCs migration to TLR3 stimulation lated hMSCs. In support, direct inhibition of FOXO1 significantly down-regulated the expression of cell migration-related genes and cell migration responses. Together, these findings provide a better understanding of the molecular mechanisms that underlie the TLR3-mediated migration response of hMSCs.

Experimental procedures
Cell culture and stimulation of hMSCs hBM-MSCs were derived from donors 7F3915 (21-year-old female), 7F3674 (22-year-old female), and 127756 (43-year-old male). These cells were purchased from Lonza (Walkersville, MD) and cultured in low glucose Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and penicillin (100 units/ml)/streptomycin (100 mg/ml) (Gibco). Most experiments were performed using donor 7F3915. The medium was replaced every 3-4 days. Experiments were performed using hBM-MSCs at passage 6. The cells were maintained in a humidified incubator with 95% air and 5% CO 2 atmosphere at 37°C, and FOXO1 inhibitor AS1842856 (41) was purchased from Merck Millipore, Billerica, MA. The cells were treated with the TLR3 agonist poly(I:C) (10 g/ml, Sigma), and AS1842856 for the specified concentrations under normal culture conditions. At 70 -80% confluence, the cells were treated for 4 h with fresh growth medium (control) or growth medium containing poly(I:C) (TLR3-stimulated MSCs)and AS1842856.

Chemokine measurements with enzyme-linked immunosorbent assay (ELISA)
Human MSCs were treated with poly(I:C) (10 g/ml) for 4 h. After treatment, the concentration of the inflammatory chemokines CCL5 and CXCL10 in cell culture supernatants were determined using human CCL5 and CXCL10 ELISA kits (KOMABIOTECH, Seoul, Korea) according to the manufacturer's instructions. The data represent three independent experiments (n ϭ 3).

Cell viability assay
The cell viability assay was performed using a tetrazolium salt colorimetric assay with PreMixWST-1 according to the manufacturer's instructions (Takara, Shiga, Japan). The cells were seeded at a density of 1 ϫ 10 3 per well in 96-well plates and incubated from 0 to 96 h. After this incubation, PreMixWST1 was added to each well, and the plate was incubated for an additional 4 h. Finally, the absorbance of the microplate at 450 nm was measured. The data represent three independent experiments (n ϭ 3).
To analyze the differentially expressed genes, FASTQ files from RNA-seq were clipped, trimmed of adapters, and reads of low-quality were removed using Trimmomatic (72). These FASTQ files were aligned using the STAR (version 2.5.1) aligner software (73) with the UCSC hg19 reference. We analyzed the differential gene expression using DESeq2 with the default parameters (74). Genes identified by RNA-seq that had an absolute log 2 -fold change larger than 0.7 (log 2 -fold change Ն 0.7 and log 2 -fold change Յ Ϫ0.7, p value Ͻ 0.01) were selected as being DEGs in TLR3-stimulated hMSCs. The acquired data were deposited in the Gene Expression Omnibus database under dataset accession number GSE97724.

Analysis of the networks and pathways
The RNA-seq dataset was mapped using Ingenuity's Knowledge Base IPA (Ingenuity System, Mountain View, CA) to analyze the networks and pathways. RNA-seq data were cutoff at the fold-change (Ն0.7 log 2 -fold change, p value Յ 0.01) in TLR3-stimulated hMSCs.

Functional annotation
To determine functional annotations, gene ontology analysis was performed using DAVID (Database for Annotation, Visualization, and Integrated Discovery) version 6.8 (75). The Gene Ontology was examined using a modified Fisher's exact p value in the DAVID program. Annotation categories with p values less than 0.001 were considered to be enriched.

Transcription factor-binding motif enrichment analysis
GenBank accession numbers were input into the Pscan software to analyze transcription factor binding motifs (76). The JASPAR 2016 database of TF binding sequences of the promoter regions ranged from 1000 bp (bp) downstream of the transcription start site to ϩ0 bp (77).

FOXO1 controls hMSCs migration to TLR3 stimulation Gene expression analysis using quantitative real-time RT-PCR
Total RNA was extracted from hMSCs using RNAiso Plus (Takara, Shiga, Japan) according to the manufacturer's instructions. The cDNA was generated by PrimeScript reverse transcriptase (Takara, Shiga, Japan). The synthesized cDNA was amplified using gene-specific primers ( Table 1). The primers were designed by the Primer Bank (http://pga.mgh.harvard-.edu/primerbank/index.html). 9 Real-time PCR was performed with SYBR Premix Ex Taq TM II (Takara, Shiga, Japan). Glyceraldehyde-3-phosphate dehydrogenase was used as the internal control. The data represent three independent experiments (n ϭ 3). After performing real-time PCR, the results were analyzed using the critical threshold (⌬C T ) and the comparative critical threshold (⌬⌬C T ) methods in the ABI 7500 software with the NormFinder and geNorm PLUS algorithms.

ChIP assay
ChIP assay was performed as previously described (63). Briefly, chromatin from 2 ϫ 10 7 cells was used for ChIP assay. The cells were immunoprecipitated with antibodies against FOXO1 (Abcam, Cambridge; ab39670) and normal rabbit IgG (Santa Cruz Biotechnology, Inc., Dallas, TX; number sc2025) used as a control with Dynabeads Protein A beads (Invitrogen). The immunoprecipitated DNA was analyzed by real-time quantitative PCR, and the resulting levels were normalized to the amounts of input DNA. The data represent three independent experiments (n ϭ 3). Primers used for ChIP-PCR are listed in Table 2.

In vitro wound-healing assay
The wound-healing assay was performed as previously described (63). Briefly, the cells were seeded onto Ibidi culture inserts (Ibidi, Martinsried, Germany) and incubated for 24 h. After incubation, the cells were treated with either poly(I:C) or FOXO1 inhibitor AS1842856 for 4 h. The insert was removed and incubated in growth medium for 24 h. The cells were imaged by an inverted microscope (Leica, Wetzlar, Germany).

Migration assay
Transwell migration assays were performed using Fluoroblock migration plates as described previously (78). Briefly, hMSCs were loaded at a density at 5 ϫ 10 4 cells with Calcein AM (Thermo fisher C3100MP), and washed prior to seeding hMSCs in the upper chamber of the tissue culture insert. After 24 h in culture, the hMSCs were stimulated for 4 h with poly(I:C) (10 g/ml) in the presence or absence of FOXO1 inhibitor AS1842856 (5 M). Poly(I:C) and AS1842856 were added to the lower chamber to stimulate migration. Then hMSCs were rinsed and the medium was replaced. After 24 h incubation, the fluorescence of migrating hMSCs were measured using an inverted fluorescence live cell imaging system (Leica, Wetzlar, Germany).

Immunocytochemistry
The cells were incubated on coverslips in 4-well plates. After 24 h, the cells were treated with either poly(I:C) or FOXO1 inhibitor, washed with PBS, and then fixed with 4% paraformaldehyde. Next, the cells were incubated with cold methanol for 5 min and blocking solution (5% BSA in PBS) for 1 h. The cells were then incubated with the primary antibody anti-rabbit FOXO1 (1:200, Abcam, Cambridge, UK) and the secondary antibody donkey anti-rabbit IgG (Jackson Laboratory, West Grove, PA). After incubation with the antibodies, the cells were washed with PBS, mounted with 4Ј,6-diamidino-2-phenylindole (DAPI) mounting solution (Vectashield, Vector Laboratories, Burlingame, CA), and imaged by microscopy (Zeiss, Oberkochen, Germany). The percentage of total fluorescence 9 Please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party hosted site.

FOXO1 controls hMSCs migration to TLR3 stimulation
intensity ratio of nuclear versus cytoplasmic area was determined and quantified with the Intensity Ratio Nuclei Cytoplasm macro from ImageJ software, using DAPI staining to define the nucleus area available from National Institutes of Health as described (79).

Knockdown of FOXO1 gene expression by siRNA treatment
Oligonucleotide specific for FOXO1 (ID numbers s5257, s5258, and s5259) was from Thermo Fisher Scientific. MSCs were transfected with FOXO1 siRNA constructs following the Silencer Select siRNA transfection protocol with nontargeting siRNAs (Ambion Applied Biosystems, Waltham, MA). FOXO1 siRNA was used at concentrations of 5 nM for 48 h using Dulbecco's modified Eagle's medium.

Western blotting
The extraction of protein from the cells was performed using RIPA buffer (1% Triton X-100 in 50 mM phosphate buffer (pH 7.4)) containing a complete EDTA-free protease inhibitor mixture (Roche Diagnostics, Basel, Switzerland). The extracted protein was separated on SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Schleicher and Schuell Bioscience, Inc.). The Western blot analysis was performed using anti-FOXO1 (Abcam, Milton, Cambridge, UK: ab39670 and anti-␤-actin (Sigma; A-5316) antibodies. ␤-Actin was used as an internal control.

Statistical analysis
Data were analyzed as the mean Ϯ S.D. Data were analyzed using the SPSS 17.0 program (SPSS Inc., Chicago, IL) using a one-way analysis of variance, followed by the Tukey's honestly significant difference post hoc test. p values Ͻ 0.05 were considered significant.