• No results found

Baculovirus Transduction of Mesenchymal Stem Cells Triggers the Toll-Like Receptor 3 Pathway

N/A
N/A
Protected

Academic year: 2019

Share "Baculovirus Transduction of Mesenchymal Stem Cells Triggers the Toll-Like Receptor 3 Pathway"

Copied!
9
0
0

Loading.... (view fulltext now)

Full text

(1)

0022-538X/09/$08.00⫹0 doi:10.1128/JVI.01250-09

Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Baculovirus Transduction of Mesenchymal Stem Cells Triggers the

Toll-Like Receptor 3 Pathway

Guan-Yu Chen,

1

‡ Hsiao-Chiao Shiah,

1

‡ Hung-Ju Su,

2

Chi-Yuan Chen,

1

Yung-Jen Chuang,

3

Wen-Hsin Lo,

1

Jie-Len Huang,

1

Ching-Kuang Chuang,

1

Shiaw-Min Hwang,

4

* and Yu-Chen Hu

1

*

Department of Chemical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan1; HTP Transcriptomics and

Proteomics Department, Molecular Biomedical Technology Division, Biomedical Engineering Research Lab,

Industrial Technology Research Institute, Hsinchu 310, Taiwan2; Institute of Bioinformatics and

Structural Biology, National Tsing Hua University, Hsinchu 300, Taiwan3; and

Bioresource Collection and Research Center, Food Industry Research and

Development Institute, Hsinchu 300, Taiwan4

Received 16 June 2009/Accepted 26 July 2009

Human mesenchymal stem cells (hMSCs) can be genetically modified with viral vectors and hold promise as a cell source for regenerative medicine, yet how hMSCs respond to viral vector transduction remains poorly under-stood, leaving the safety concerns unaddressed. Here, we explored the responses of hMSCs against an emerging DNA viral vector, baculovirus (BV), and discovered that BV transduction perturbed the transcription of 816 genes associated with five signaling pathways. Surprisingly, Toll-like receptor-3 (TLR3), a receptor that generally recog-nizes double-stranded RNA, was apparently upregulated by BV transduction, as confirmed by microarray, PCR array, flow cytometry, and confocal microscopy. Cytokine array data showed that BV transduction triggered robust secretion of interleukin-6 (IL-6) and IL-8 but not of other inflammatory cytokines and beta interferon (IFN-). BV transduction activated the signaling molecules (e.g., Toll/interleukin-1 receptor domain-containing adaptor-induc-ing IFN-, NF-B, and IFN regulatory factor 3) downstream of TLR3, while silencing theTLR3gene with small interfering RNA considerably abolished cytokine expression and promoted cell migration. These data demonstrate, for the first time, that a DNA viral vector can activate the TLR3 pathway in hMSCs and lead to a cytokine expression profile distinct from that in immune cells. These findings underscore the importance of evaluating whether the TLR3 signaling cascade plays roles in the immune response provoked by other DNA vectors (e.g., adenovirus). Nonetheless, BV transduction barely disturbed surface marker expression and induced only transient and mild cytokine responses, thereby easing the safety concerns of using BV for hMSCs engineering.

Toll-like receptors (TLRs) are pattern recognition receptors that recognize a variety of pathogen-associated molecular pat-terns and are essential for activating innate immunity and potentiating adaptive immunity against pathogens (for a re-view, see references 2, 15, and 23). To date, 11 TLRs have been identified in humans (2). For example, TLR2 recognizes bac-terial lipoproteins and peptidoglycans, TLR3 recognizes virus-derived double-stranded RNA (dsRNA) and a synthetic dsRNA analogue poly(I:C) (polyriboinosinic-polyribocytidylic acid), TLR4 recognizes lipopolysaccharides, and TLR9 recog-nizes the unmethylated CpG DNA motifs. Upon the engage-ment of cognate ligands, TLRs are activated and recruit Toll/ IL-1 receptor-containing adaptor molecules such as myeloid differentiating factor 88 (MyD88) and Toll/interleukin-1 recep-tor domain-containing adaptor-inducing beta interferon

(TRIF). Among the TLRs, the TLR3 pathway is unique in that its signaling cascade begins by recruiting TRIF (2, 15, 33). TRIF can signal through interferon regulatory factor 3 (IRF-3) phosphorylation, leading to downstream beta interferon (IFN-␤) expression. TRIF also can orchestrate with TRAF6 and RIP1, leading to NF-␬B activation and subsequent expres-sion of cytokines and chemokines such as interleukin-1 (IL-1), IL-6, IL-8, IL-12, MCP-1 (CCL2), RANTES (CCL5), and MIP-2 (CXCL2).

The baculovirus (BV)Autographa californicamultiple nucleo-polyhedrovirus is a DNA virus that infects insects as its natural hosts and that has been developed as a biological insecticide. However, BV also efficiently transduces a broad range of mam-malian cells in which BV neither replicates nor is toxic. Also, recombinant virus construction, propagation, and handling can be performed readily in biosafety level 1 facilities. These at-tributes have inspired the development of BV vectors for in vitro and in vivo gene delivery (6, 28), cartilage tissue engi-neering (3), development of cell-based assays, delivery of vac-cine immunogens, production of viral vectors, and cancer ther-apy (for a review, see references 14 and 17). Furthermore, BV transduces human mesenchymal stem cells (hMSCs) derived from bone marrow at efficiencies greater than 80% (12) and accelerates osteogenesis of hMSCs in vitro and in vivo when expressing an osteogenic growth factor (4). hMSCs are capable of differentiating into multiple cell types (e.g., chondrocytes,

* Corresponding author. Mailing address for S.-M. Hwang: Biore-source Collection and Research Center, Food Industry Research and Development Institute, Hsinchu 300, Taiwan. Phone: 886 3 522 3191, ext. 576. Fax: 886 3 521 4016. E-mail: hsm@firdi.org.tw. Mailing address for Y.-C. Hu: Department of Chemical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan. Phone: 886 3 571 8245. Fax: 886 3 571 5408. E-mail: yuchen@che.nthu.edu.tw.

‡ G.-Y.C. and H.-C.S. contributed equally to this work.

† Supplemental material for this article may be found at http://jvi .asm.org/.

Published ahead of print on 5 August 2009.

10548

on November 8, 2019 by guest

http://jvi.asm.org/

(2)

osteoblasts, and endothelial cells) and possess immunosup-pressive and immunomodulatory properties (32). Therefore, hMSC-based cell therapy has captured growing attention in regenerative medicine and has advanced to various phases of clinical trials for the treatment of damaged myocardium, knee injuries, graft-versus-host disease, and Crohn’s disease (22). hMSCs also serve as a gene delivery carrier for the treatment of cancer, osteogenesis imperfecta (13), and various neurological disorders (27). As such, the efficient BV transduction of hMSCs offers a new, attractive option for hMSC engineering.

Despite the potential of hMSCs for cell and gene therapy, whether the genetic modification provokes undesired cellular responses has yet to be explored. The lack of safety evaluation will hamper future clinical applications of genetically modified hMSCs. Therefore, the overriding objective of this study was to assess how hMSCs respond to BV transduction.

MATERIALS AND METHODS

BV and hMSCs.A recombinant BV harboring no mammalian transgene cas-sette was used for transduction. The virus was amplified and harvested, and titers were determined by an end point dilution assay based on virus infectivity in insect cells (25). Bone marrow-derived hMSCs were obtained from Cambrex Co.,

selected, enriched, cultured in alpha minimal essential medium (␣-MEM)

con-taining 10% fetal bovine serum (HyClone) as described previously (11), and expanded to passage 11 for all experiments.

The virus transduction was performed on six-well plates as described previ-ously (21) with minor modifications. Depending on the multiplicity of infection

(MOI), a certain volume of virus supernatant was mixed with NaHCO3-deficient

␣-MEM to adjust the final volume to 500␮l (per well). The transduction was

initiated by adding the virus mixture to the cells and was continued by gently shaking the plates on a rocking plate for 4 h at 25 to 27°C. For mock transduc-tion, the cells were incubated under the same conditions with a solution

consist-ing of 400␮l of NaHCO3-deficient␣-MEM and 100␮l of fresh TNM-FH

medium. After being washed, the cells were replenished with 2 ml of␣-MEM

containing 10% fetal bovine serum for culture.

For UV inactivation, the virus was exposed to short-wavelength UV radiation

at a distance of 5 cm for 30 min on ice (1.6⫻104

mJ/cm2

) as described previously

(1). For RNase treatment, the virus (per 100␮l) was incubated with 2␮l of

RNase A (⬎70 Kunitz units/mg of protein; Sigma) (35) or 2␮l of TNM-FH

medium for 1 h at 37°C as described previously.

Microarray and PCR array.Total RNA was extracted with an RNeasy Mini Kit (Qiagen) for cDNA synthesis using a Reverse Transcriptase 1st-Strand cDNA Synthesis Kit (Epicentre Biotechnologies). Labeled cRNA was prepared by using an Amino Allyl MessageAmpII aRNA amplification kit (Ambion). The

cRNA (10␮g) was fragmented and then hybridized on a Phalanx Human One

Array (HOA 4.3; Phalanx Biotech Group, Inc.) as described previously (9). Each microarray contains 32,050 oligonucleotide probes that include 30,968 human gene probes for transcription expression profiling and 1,082 experimental control probes. Detailed descriptions of the gene array list are available from http://www .phalanx.com.tw/tech_support/gene_lists.html. Arrays were scanned by a DNA Microarray Scanner (Agilent Technologies), and the fluorescence intensities were extracted by GenePix Pro, version 6.0 (Molecular Devices). The raw data

were preprocessed by log2transformation and global LOWESS normalization.

The preprocessed data were then analyzed by the Limma package of R software. The significantly changed genes, as determined by one-way analysis of variance,

were defined as the genes with relative changes in expression of⬎2-fold or

⬍0.5-fold and adjustedP values of less than 0.05. To identify which known

pathways were affected by BV transduction, the significantly changed genes were analyzed by a web-based tool, Pathway-Express (7), which is freely available as part of Onto-Tools (http://vortex.cs.wayne.edu).

Alternatively, the total RNA was reverse transcribed to cDNA using a Molo-ney murine leukemia virus Reverse Transcriptase 1st-Strand cDNA Synthesis Kit (Epicentre Biotechnologies). The cDNA was analyzed using the a human TLR

pathway-focused RT2

Profiler PCR Array (SABiosciences) following the manu-facturer’s instructions.

Flow cytometry.To characterize surface marker expression, hMSCs were la-beled with different antibodies. Anti-CD14-fluorescein isothiocyanate (FITC), anti-CD19-FITC, and anti-CD105-FITC were purchased from Miltenyi Biotec

(MACS); CD29-phycoerythrin (PE), CD73-PE, CD44-FITC, anti-CD90-FITC, anti-human leukocyte antigen class I (HLA-I)-PE, and anti-HLA-II-PE were purchased from BD Biosciences; anti-CD45-FITC and anti-CD34-PE were purchased from Chemicon. After being labeled, hMSCs were detached and analyzed with a flow cytometer (FACSCalibur; BD Biosciences). To characterize TLR expression, transduced and mock-transduced cells were fixed and perme-abilized with 4% formaldehyde and 0.5% Tween-20. After a washing step, the cells were incubated with different primary antibodies (1:150 dilution) for 1 h at 4°C in the dark. Mouse immunoglobulin G (IgG) primary antibodies were pur-chased from Abcam and were specific for human TLR2 (ab9100), TLR3 (ab12085), TLR4 (ab30667), and TLR9 (ab17236). After a washing step, the cells were incubated with Alexa Fluor 488-conjugated, goat anti-mouse IgG (Invitro-gen) for 1 h at 4°C in the dark. After a washing step, the cells were collected for flow cytometry analyses. As controls, the cells were incubated for 30 min with 500

␮l of NaHCO3-deficient␣-MEM containing 10␮g/ml poly(I:C) (Sigma), 10

ng/ml lipopolysaccharide (Sigma), or 1␮M CpG oligonucleotide (ODN 2216 and

ODN 2006; Invitrogen) and then analyzed for TLR expression in a similar fashion.

Cytokine measurement.Conditioned medium from the hMSC cultures was analyzed using a fluorescence bead immunoassay (Bender MedSystems) that detects 11 cytokines simultaneously. IL-6 and IL-8 were also measured using Module Sets of enzyme-linked immunosorbent assays (ELISAs) (Bender Med-systems).

Immunofluorescence labeling/confocal microscopy.The cells were fixed and permeabilized as described above, followed by extensive washing and primary antibody staining (1:150 dilution) for 1 h at 4°C in the dark. The primary antibodies were specific for human TLR3, IRF-3 (ab50772; Abcam), or

phos-phorylated NF-␬B (3033; Cell Signaling Technology). After a washing step, the

cells were incubated with the goat anti-mouse (for TLR3 and IRF-3) or goat

anti-rabbit (for pNF-␬B) antibody conjugated with Alexa Fluor 488 (Invitrogen)

for 1 h at 4°C in the dark. After a washing step, the cells were stained with 4,6-diamidino-2-phenylindole (DAPI; Vector Labs) and visualized with a confo-cal microscope (Nikon TE2000 equipped with the confoconfo-cal upgrade laser kit).

Western blotting.The cytoplasmic and nuclear proteins were separately ex-tracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce) supplemented with the Halt protease and phosphatase inhibitor cocktails (Pierce), followed by Western blotting. The primary antibodies (1:1,000 dilution) were specific for TRIF (4596; Cell Signaling Technology), phosphorylated IRF-3

(4947; Cell Signaling Technology), and phosphorylated NF-␬B or␤-actin

(A-2066; Sigma). The secondary antibody was species-specific horseradish peroxi-dase-conjugated IgG (1:5,000 dilution; Amersham Biosciences). The images were developed using an enhanced chemiluminescence kit (Amersham Bio-sciences).

siRNA knockdown of TLR3.To knock down TLR3, hMSCs were nucleofected

with 5␮g of a control small interfering RNA (siRNA) plasmid (InvivoGen) or a

plasmid expressing the TLR3 siRNA (psiTLR3; InvivoGen) using a Human MSC Nucleofector Kit (Amaxa Biosystems). At 48 h posttransfection, hMSCs were transduced with BV or treated with poly(I:C) as described above. The spent medium was collected at 24 h posttransduction (hpt) for ELISA, and the cells were trypsinized for migration assays.

Transwell migration assay.The trypsinized hMSCs (5⫻104

) were loaded

onto the upper chamber of the transwell inserts with 8-␮m-pore-size membrane

filters (BD Biosciences), while 500␮l of␣-MEM was loaded in the bottom

chamber. After 4 h of incubation, the upper sides of the filters were carefully washed, and nonmigrated cells were removed with a cotton-tipped applicator. The cells migrating to the lower sides were labeled with DAPI-containing mount-ing medium and counted under a fluorescence microscope. At least 10 fields

(magnification,⫻200) in two filters were counted for each sample, and the data

are expressed as the average number of migrated cells/field.

Statistical analysis.All data represent the mean⫾standard deviation or mean values of at least three independent culture experiments. The data were

statis-tically analyzed by one-way analysis of variance. APvalue of⬍0.05 was

consid-ered significant.

Microarray data accession number.Array data sets were deposited in the NCBI Gene Omnibus Express database under the accession number GSE15810.

RESULTS

Expression of hMSC surface markers.To examine whether BV transduction altered surface characteristics, hMSCs were mock transduced or transduced with a BV carrying no mam-malian gene cassette at an MOI of 100, followed by

immuno-VOL. 83, 2009 BACULOVIRUS TRIGGERS TLR3 PATHWAY IN hMSCs 10549

on November 8, 2019 by guest

http://jvi.asm.org/

(3)

fluorescence labeling and flow cytometry analyses at 24 h hpt. In agreement with the surface marker profiles in normal hMSCs (18, 31), the mock-transduced hMSCs expressed CD29, CD44, CD73, CD90, CD105, and HLA-I but were neg-ative for CD14, CD19, CD34, CD45, and HLA-II (Fig. 1). BV transduction did not apparently alter the surface expression profile, except that CD73 expression was slightly diminished while HLA-I expression was elevated.

BV transduction-upregulated genes associated with the TLR signaling pathway.To explore the global responses of hMSCs to BV transduction, hMSCs were treated as described in the legend of Fig. 1 and subjected to microarray analysis at 24 hpt. Of the 30,968 human genes on the microarray, we identified 548 upregulated (⬎2-fold) and 268 downregulated (⬍0.5-fold) known genes after BV transduction compared with the mock transduction control (see Tables S1 and S2 in the supplemental material). Pathway analysis using the Pathway-Express tool demonstrated five signaling pathways that were profoundly disturbed: cell adhesion molecules, TLR, Jak-STAT, apopto-sis, and antigen processing and presentation (see Tables S3 to S8 in the supplemental material). Since the activation of the TLR pathway is essential for initiating innate immunity and can trigger the other four pathways, we focused on the TLR pathway in subsequent experiments and analyses.

The microarray data revealed significant upregulation of

TLR1,TLR2, andTLR3but not of otherTLRgenes (Table 1). Certain genes encoding the TLR signaling molecules (e.g., MyD88 and IRAK2), downstream cytokines (e.g., IL-6 and IL-8), and other genes downstream of the TLR3 pathway (e.g.,

RSAD2, INDO, and PTGS2) were also significantly

upregu-lated. To confirm the data, transcription was also quantified by using the RT2 Profiler PCR Array, which detects 84 genes

involved in the TLR pathway (includingTLR1toTLR10). In accord with the microarray data, the PCR array revealed the upregulation of such genes as TLR3, MyD88, IRAK2, IL-6,

IL-8, andPTGS2 (Table 1). In contrast, the PCR array de-tected upregulation of neitherTLR1nor TLR2but revealed the upregulation of other genes involved in the TLR pathway (e.g.,NFKBIA,TRIF, andTRAF6). The discrepancy between the microarray and PCR array data sets probably arose from the relatively weak stimulation of these genes by BV transduc-tion.

[image:3.585.139.450.68.409.2]

BV transduction of hMSCs triggered IL-6 and IL-8 produc-tion.To screen the BV-induced cytokines at the protein level, the conditioned medium collected at 24 hpt was analyzed by a multiplex cytokine array which simultaneously detects 11 cyto-kines (Fig. 2A and B). Compared with the mock transduction control, BV transduction did not significantly (P⬎0.05) elicit

FIG. 1. Expression of hMSC surface markers. Cells were mock transduced (black solid lines) or transduced with BV at an MOI of 100 (pink lines) and subjected to immunofluorescence labeling/flow cytometry analyses at 24 hpt.

on November 8, 2019 by guest

http://jvi.asm.org/

(4)

the secretion of IFN-␥, tumor necrosis factor alpha (TNF-␣), TNF-␤, IL-1␤, IL-2, IL-4, IL-5, IL-10, and IL-12 but provoked high-level secretion of IL-6 (⬇3,722 pg/ml) and IL-8 (⬇1,334 pg/ml). Such induction was dose dependent as IL-6 and IL-8 expression increased with elevating MOIs (Fig. 2C and D). To confirm the result and examine the kinetics, the protein con-centrations were measured again by ELISA at 24, 48, and 96 hpt. The results shown in Fig. 2E and F demonstrate that the expression of both IL-6 and IL-8 peaked at 24 hpt and fell to background levels at 96 hpt, indicating a transient cytokine response. It is also noteworthy that BV transduction did not provoke the secretion of antiviral IFN-␣(5) and IFN-␤(see Table S9 in the supplemental material).

Whether cytokine induction required infectious BV was ex-plored by inactivating the BV with UV prior to transduction. The ELISA data (Fig. 3A and B) showed that UV inactivation significantly (P ⬍ 0.05) abolished the BV-induced IL-6 and IL-8 secretion, indicating the essential role of the live virus. Since IL-6 and IL-8 can be elicited by dsRNA as a result of TLR3 activation (18, 31), the virus solutions were treated with RNase or TNM-FH medium prior to transduction. The results shown in Fig. 3C and D show that RNase treatment retarded secretion of neither IL-6 nor IL-8 after BV transduction, thus ruling out a role for RNA. These data collectively confirmed that BV itself provoked the cytokine response.

BV transduction of hMSCs triggered the TLR3 pathway.To examine the induction of TLR3 and other TLRs at the protein level, hMSCs were transduced with BV or treated with differ-ent TLR agonists and were subjected to immunofluorescence labeling/flow cytometry analyses (Fig. 4A). Compared with the mock transduction control, BV transduction led to the emer-gence of a peak when cells were labeled with the TLR3 anti-body. Such a peak shift was due to receptor activation, inter-nalization, and degradation (31) and was similarly observed for the sample treated with the TLR3 ligand, poly(I:C). However, BV transduction did not apparently provoke TLR2, TLR4, or TLR9.

TLR3 activation was further visualized by confocal mi-croscopy (Fig. 4B). TLR3 expression was diffuse in the cy-toplasm of the untreated hMSCs but was more focused along the edge of the BV-transduced cells, which was like-wise observed in the poly(I:C)-treated hMSCs (Fig. 4B) (31). The results shown in Fig. 4, in conjunction with the microarray and PCR array data, concretely attested to TLR3 activation by BV transduction.

In immune cells, TLR3 activation induces TRIF expression and results in the nuclear translocation of phosphorylated IRF-3 and NF-␬B (16). Western blot analyses of hMSCs (Fig. 5A) demonstrated that both BV transduction and poly(I:C) treatment stimulated a gradual increase in TRIF expression for 4 h and accumulation of phosphorylated IRF-3 and NF-␬B in the nucleus. The nuclear trafficking of IRF-3 and NF-␬B was further confirmed by confocal microscopy (Fig. 5B), which illustrated the absence of IRF-3 and NF-␬B in the nuclei of untreated hMSCs and the presence of IRF-3 and NF-␬B in the nuclei after BV transduction and poly(I:C) treatment.

TLR3 knockdown diminished BV-induced cytokine secre-tion and promoted migrasecre-tion.To correlate TLR3 activation and cytokine secretion, cells were nucleofected with a plasmid expressing the control siRNA or psiTLR3. After 48 h of cul-ture, the cells were mock transduced, transduced, or treated with poly(I:C). As depicted in Fig. 6A and B, psiTLR3 treat-ment of hMSCs considerably abrogated poly(I:C)-induced IL-6 and IL-8 secretion, confirming the TLR3 knockdown by psiTLR3. Accordingly, TLR3 silencing by psiTLR3 treatment significantly (P⬍0.05) attenuated BV-induced IL-6 and IL-8 secretion.

Additionally, we examined the effect of TLR3 knockdown on BV-induced migration by the transwell migration assay. The results shown in Fig. 6C indicate that the migration of cells treated with the control siRNA was remarkably impeded by both poly(I:C) treatment and BV transduction, but psiTLR3 treatment significantly (P⬍0.05) ameliorated the migration of poly(I:C)-treated and BV-transduced hMSCs.

DISCUSSION

The present study primarily aimed to explore the hMSC response to BV transduction and to decipher the molecular pathway. We determined that most hMSC surface markers remained undisturbed after BV transduction (Fig. 1), suggest-ing that hMSC characteristics are retained. This response con-trasted markedly with the evident BV-induced upregulation of surface molecules (e.g., HLA-II) in dendritic cells (29) but was in line with the negligible perturbation of hMSC marker

ex-TABLE 1. Significantly changed genes associated with the TLR signaling pathway by BV transduction

Gene group and name

Relative change in expression

(n-fold) as determined by:a

Microarray PCR array

TLR genes

TLR1 2.6 ND

TLR2 6.6 ND

TLR3 33.4 1157.1

TLR signaling-associated molecules

MYD88 8.9 5.8

IRAK2 5.7 10.2

NFKBIA ND 9.4

TRIF ND 3.4

TRAF6 ND 1.8

Cytokines and chemokines

CXCL10 814.0 465.1

CCL5 231.8 NA

IL1B 34.6 95.4

IL1A 27.7 26.0

IL-6 13.7 27.0

IL12A 12.6 12.0

CXCL2 10.8 NA

IL-8 6.2 26.4

CCL2 4.4 NA

Other genes associated with TLR3

RSAD2 1220.6 NA

INDO 106.9 NA

PTGS2 5.2 19.3

SIGIRR 2.9 3.4

PELI1 2.7 3.0

a

The data represent the average values from three to five independent culture experiments. ND, not detectable; NA, not available in the PCR array.

VOL. 83, 2009 BACULOVIRUS TRIGGERS TLR3 PATHWAY IN hMSCs 10551

on November 8, 2019 by guest

http://jvi.asm.org/

[image:4.585.43.282.90.396.2]
(5)

pression (e.g., CD34 and CD105) after poly(I:C) treatment (18). BV transduction only slightly upregulated HLA-I, which is desirable since HLA-I is responsible for presenting endog-enously synthesized proteins to CD8⫹T cells. BV transduction also marginally downregulated CD73, but the physiological significance of this is unknown.

We identified 816 known genes that were significantly per-turbed by BV transduction. Among all TLR genes,TLR3 ex-pression showed the most pronounced upregulation. Concur-rent with the TLR3 pathway (see introduction), BV transduction upregulated not onlyTLR3 but its downstream genes such asTRIF, TRAF6,NFKB1A (encoding I␬B),IL-6,

IL-8, IL12A, CCL2, CCL5, and CXCL2 (Table 1; see also

[image:5.585.135.450.66.452.2]

Tables S1 and S2 in the supplemental material). At the protein level, BV elicited transient IL-6 and IL-8 production in a dose-dependent manner (Fig. 2 and 3), which concurred with the activation of TLR3 (Fig. 4) and its signaling molecules like TRIF, IRF-3, and NF-␬B (Fig. 5). Critically, silencing TLR3 expression considerably abolished BV-induced cytokine secre-tion and augmented hMSC migrasecre-tion (Fig. 6). These data

FIG. 2. Cytokine production by BV-transduced hMSCs. (A and B) Cytokine production at an MOI of 100. (C and D) IL-6 and IL-8 production at different MOIs. (E and F) IL-6 and IL-8 production at different times. The mock-transduced hMSCs served as the controls. Cytokine production was measured using a fluorescence bead immunoassay that detects 11 cytokines (A to D) or ELISA kits (E and F).

FIG. 3. Cytokine production required infectious BV. (A and B) IL-6 and IL-8 production by the hMSCs transduced with virus (MOI of 100) that was untreated (⫺) or treated (⫹) with UV light. (C and D) IL-6 and IL-8 production by the hMSCs transduced with virus (MOI of 100) that was pretreated with 2␮l of RNase A (⫹RNase) or 2␮l of TNM-FH (⫺RNase) for 1 h at 37°C. In parallel, cells were mock transduced and served as controls. All spent media were collected at 24 hpt for ELISAs.

on November 8, 2019 by guest

http://jvi.asm.org/

(6)

collectively confirmed the activation of the TLR3 signaling pathway by BV.

However, BV transduction provoked no secretion of IL-1␤, IFN-␥, IL-12, and TNF-␣ (Fig. 2A). These proteins were highly expressed by BV-transduced dendritic cells (1) but were not robustly secreted by the poly(I:C)-treated hMSCs (18, 31). Nor did we detect IFN-␤secretion from 0.25 to 24 h after BV transduction or poly(I:C) treatment (see Table S9 in the sup-plemental material). IFN-␤is the signature IFN induced after TLR3 activation in murine cells (2, 15), but its expression was not reported in studies that treated hMSCs with poly(I:C) (18, 31). In contrast, Opitz et al. recently showed that poly(I:C) treatment of hMSCs induced detectable IFN-␤secretion and a subsequent signaling loop (24). One key difference was the

[image:6.585.134.454.67.493.2]

poly(I:C) dose (50␮g/ml) these investigators used, which was markedly higher than amounts used in this (10 ␮g/ml) and other studies. As such, it appears that in hMSCs TLR3 ligation could elicit IFN-␤secretion but at a fairly low magnitude. This suggests that in hMSCs certain pathways downstream of IRF-3 might be lacking or blocked unless potently stimulated. In this study, the virus dose (MOI of 100) used is sufficient to trans-duce 80 to 90% of hMSCs (12) and intrans-duce ectopic bone for-mation in vivo when hMSCs express an osteogenic factor (4). Given that these IFNs and cytokines are pivotal in establishing the antiviral state and immune responses, the undetectable induction of these proteins at an MOI of 100 is instrumental for the safe use of BV-transduced hMSCs for tissue regener-ation.

FIG. 4. BV transduction of hMSCs triggered TLR3 activation. (A) hMSCs were mock transduced or transduced with BV. At 0.5 hpt the cells were subjected to fixation, permeabilization, immunostaining with anti-TLR antibodies, and flow cytometry analyses. The green and pink lines indicate the mock-transduced and transduced cells, respectively. As controls, cells were treated with TLR ligands for 30 min and subjected to the same analyses. The peak shift as indicated by the arrows demonstrated TLR3 activation after BV transduction and poly(I:C) treatment. (B) Cells were treated as described in panel A, labeled with anti-TLR3 antibody, counterstained with DAPI, and examined by confocal microscopy (Nikon TE2000 equipped with a confocal upgrade laser kit). Magnification,⫻1,000. LPS, lipopolysaccharide.

VOL. 83, 2009 BACULOVIRUS TRIGGERS TLR3 PATHWAY IN hMSCs 10553

on November 8, 2019 by guest

http://jvi.asm.org/

(7)

Among the cytokines investigated, we detected only IL-6 and IL-8. This is conceivable as they are constitutively ex-pressed by hMSCs (19) and are potently stimulated after poly(I:C) treatment (Fig. 6) (18, 31). IL-6 dictates the regula-tion of both inflammatory responses and hematopoiesis (26), and its overproduction relates to the pathology of autoimmune diseases and tissue remodeling. Conversely, IL-8 is present in the inflammatory milieu during tissue repair (34). The robust secretion of IL-6 and IL-8 thus suggests that BV transduction might impact hMSC differentiation and potentiate the inflam-matory response after transplantation. To date, the conse-quences of TLR3 activation and IL-6/8 expression on hMSCs remain elusive. It was reported that TLR3 activation in hMSCs promotes migration (31) and hampers immunosuppressive properties but interferes with neither the phenotype nor the differentiation potential (18). However, it was also shown that TLR3 activation enhances the immunosuppressive properties of hMSCs (24). The discrepancy likely stems from the

differ-ences in experimental procedures, poly(I:C) dose, and dura-tion of ligand treatment (24). For example, hMSCs have been incubated with poly(I:C) for 5 days (18) or 24 h (24) prior to evaluation of the immunosuppressive properties. In our hands, BV transduction of hMSCs did not impair long-term prolifer-ation (11), differentiprolifer-ation (12), and immunosuppressive prop-erties (5). The disparity in the immunosuppressive propprop-erties could arise from the differences in the protocols because the cells were exposed to BV for only 4 h, after which the virus was withdrawn. Also the BV-induced cytokine response was tran-sient, precipitously dropping after 24 hpt and vanishing at 96 hpt (Fig. 2E and F). As a result, unlike results of previous studies that continuously stimulated hMSCs with poly(I:C), BV transduction only transiently activated the TLR3-mediated responses, which accounts for the intangible adverse effects. Our data, however, suggest that hMSCs be transplanted after cytokine responses wane in order to circumvent the distur-bance of hMSC functions and elicitation of immune responses in vivo.

Our findings also raised an intriguing question: how did BV, a DNA virus, trigger the TLR3 pathway that is generally re-garded as a sensor of dsRNA? Since BV genes (e.g., immedi-ate-early geneie1) can be expressed at low levels in

[image:7.585.349.496.69.341.2]

mamma-FIG. 5. BV transduction activated the TLR3 signaling pathway. (A) Activation of TRIF, IRF-3, and NF-␬B. (B) Nuclear translocation of IRF-3 and NF-␬B. The nuclear and cytoplasmic proteins were separately extracted from the transduced hMSCs (MOI of 100) and analyzed by Western blotting using primary antibodies specific for human TRIF, phosphorylated IRF-3 (p-IRF-3), phosphorylated NF-␬B (p-NF-␬B), or␤-actin. Alternatively, cells were immunostained with antibodies specific for human IRF-3 or phosphorylated NF-␬B for confocal microscopy. For comparison, the cells were left untreated or were treated with poly(I:C) for 24 h for the same analyses. Magnifi-cation,⫻1,000.

FIG. 6. TLR3 silencing diminished BV-induced cytokine secretion and promoted migration. (A) IL-6 expression. (B) IL-8 expression. (C) Cell migration. hMSCs were nucleofected with a control siRNA plasmid or with psiTLR3, which expresses the TLR3 siRNA. After 48 h of culture, the cells were mock transduced, transduced with BV, or treated with poly(I:C). Twenty-four hours later, the medium was col-lected for ELISAs, and the trypsinized hMSCs (5⫻104) were loaded

onto the transwell inserts. The migrated cells were labeled with DAPI-containing mounting medium and counted under a fluorescence mi-croscope. Data are expressed as the average number of migrated cells/field.

on November 8, 2019 by guest

http://jvi.asm.org/

(8)

lian cells (20), the most likely explanation is that some BV genes were transcribed in hMSCs and that the RNA interme-diates were recognized by TLR3. However, the underlying mechanism(s) awaits further investigation. Also intriguing is that BV DNA activated the TLR9 pathway in mouse immune cells (1), yet only TLR3 activation was detected in hMSCs. Because hMSCs express high levels of TLR3 and TLR4 but low levels of TLR1, TLR2, TLR5, and TLR6 and negligible levels of TLR7 to TLR10 (18), the undetectable activation of TLR7 to TLR9 may be explained by the lack of viral DNA-sensing (TLR9) and single-stranded RNA-DNA-sensing (TLR7) re-ceptors.

In summary, hMSCs can be genetically engineered with var-ious viral vectors (8) and serve as a promising cell and gene therapy vehicle, yet little is known about how hMSCs respond to viral vector transduction. This study, for the first time, sys-tematically explored the cellular responses of hMSCs to vi-rus transduction at the molecular level. We revealed that BV transduction of hMSCs barely perturbed surface marker ex-pression even while altering the exex-pression of genes implicated in several pathways. We also provided the first evidence that a DNA viral vector can activate the TLR3 pathway in hMSCs, leading to a cytokine expression profile distinct from that in immune cells. Although TLR3 has been implicated in control-ling the infection of two DNA viruses (vaccinia virus [10] and mouse cytomegalovirus [30]), there was no direct evidence confirming the induction of the TLR3 pathway by a DNA virus until the recent discovery that Kaposi’s sarcoma-associated herpesvirus triggers the TLR3 pathway in human monocytes (35). Since DNA vectors including adenovirus, herpes simplex virus, and adeno-associated virus have been employed for ge-netically modifying hMSCs, our findings underscore the im-portance of evaluating whether these vectors also provoke the TLR3 signaling cascade and downstream immune responses. Our data also indicate that BV transduction elicits only mild and transient responses, thereby easing the safety concerns of using BV for hMSC engineering.

ACKNOWLEDGMENTS

We acknowledge the support from the VTY Joint Research Pro-gram, Tsou’s Foundation (VGHUST98-P5-17), National Tsing Hua University Booster Program (98N2901E1), National Science Council (NSC 97-2627-B-007-014), Ministry of Economic Affairs (98-EC-17-A-17-R7-0525), and National Health Research Institutes (NHRI-EX97-9412EI), Taiwan.

We declare that we have no competing financial interests. G.-Y.C., H.-C.S., and Y.-C.H. designed the research; G.-Y.C., H.-C.S., C.-Y.C., S.-M.H., Y.-J.C., W.-H.L., C.-K.C., J.-L.H., and H.-J.S. per-formed the research; S.-M.H., H.-J.S. and Y.-J.C. contributed new re-agents or analytic tools; G.-Y.C., H.-C.S., and H.-J.S. analyzed data; and G.-Y.C., H.-C.S., S.-M. H., and Y.-C.H. wrote the paper.

REFERENCES

1.Abe, T., H. Hemmi, H. Miyamoto, K. Moriishi, S. Tamura, H. Takaku, S. Akira, and Y. Matsuura.2005. Involvement of the Toll-like receptor 9 signaling pathway in the induction of innate immunity by baculovirus. J.

Vi-rol.79:2847–2858.

2.Akira, S., S. Uematsu, and O. Takeuchi.2006. Pathogen recognition and

innate immunity. Cell124:783–801.

3.Chen, H.-C., L.-Y. Sung, W.-H. Lo, C.-K. Chuang, Y.-H. Wang, J.-L. Lin, and Y.-C. Hu.2008. Combination of baculovirus-mediated BMP-2 expression and rotating-shaft bioreactor culture synergistically enhances cartilage

for-mation. Gene Ther.15:309–317.

4.Chuang, C.-K., L.-Y. Sung, S.-M. Hwang, W.-H. Lo, H.-C. Chen, and Y.-C.

Hu.2007. Baculovirus as a new gene delivery vector for stem cells

engineer-ing and bone tissue engineerengineer-ing. Gene Ther.14:1417–1424.

5.Chuang, C.-K., T.-H. Wong, S.-M. Hwang, Y.-H. Chang, Y.-H. Chen, Y.-C. Chiu, S.-F. Huang, and Y.-C. Hu.2009. Baculovirus transduction of mesen-chymal stem cells: in vitro responses and in vivo immune responses after cell

transplantation. Mol. Ther.17:889–896.

6.Condreay, J. P., S. M. Witherspoon, W. C. Clay, and T. A. Kost.1999. Transient and stable gene expression in mammalian cells transduced with a

recombinant baculovirus vector. Proc. Natl. Acad. Sci. USA96:127–132.

7.Draghici, S., P. Khatri, A. L. Tarca, K. Amin, A. Done, C. Voichita, C. Georgescu, and R. Romero.2007. A systems biology approach for pathway

level analysis. Genome Res.17:1537–1545.

8.Evans, C. H., S. C. Ghivizzani, and P. D. Robbins.2009. Orthopedic gene

therapy in 2008. Mol. Ther.17:231–244.

9.Fong, S., M. Shoemaker, J. Cadaoas, A. Lo, W. Liao, M. Tagliaferri, I. Cohen, and E. Shtivelman.2008. Molecular mechanisms underlying selective

cytotoxic activity of BZL101, an extract ofScutellaria barbata, towards breast

cancer cells. Cancer Biol. Ther.7:577–586.

10.Harte, M. T., I. R. Haga, G. Maloney, P. Gray, P. C. Reading, N. W. Bartlett, G. L. Smith, A. Bowie, and L. A. O’Neill.2003. The poxvirus protein A52R targets Toll-like receptor signaling complexes to suppress host defense. J.

Exp. Med.197:343–351.

11.Ho, Y.-C., Y.-C. Chung, S.-M. Hwang, K.-C. Wang, and Y.-C. Hu.2005. Transgene expression and differentiation of baculovirus-transduced human

mesenchymal stem cells. J. Gene Med.7:860–868.

12.Ho, Y.-C., H.-P. Lee, S.-M. Hwang, W.-H. Lo, H.-C. Chen, C.-K. Chung, and Y.-C. Hu.2006. Baculovirus transduction of human mesenchymal stem cell-derived progenitor cells: variation of transgene expression with cellular

dif-ferentiation states. Gene Ther.13:1471–1479.

13.Horwitz, E. M., D. J. Prockop, L. A. Fitzpatrick, W. W. K. Koo, P. L. Gordon, M. Neel, M. Sussman, P. Orchard, J. C. Marx, R. E. Pyeritz, and M. K. Brenner.1999. Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat.

Med.5:309–313.

14.Hu, Y.-C.2008. Baculoviral vectors for gene delivery: a review. Curr. Gene

Ther.8:54–65.

15.Ishii, K. J., S. Koyama, A. Nakagawa, C. Coban, and S. Akira.2008. Host innate immune receptors and beyond: making sense of microbial infections.

Cell Host Microbe3:352–363.

16.Jiang, Z., T. W. Mak, G. Sen, and X. Li.2004. Toll-like receptor 3-mediated

activation of NF-␬B and IRF3 diverges at Toll-IL-1 receptor

domain-con-taining adapter inducing IFN-␤. Proc. Natl. Acad. Sci. USA101:3533–3538.

17.Kost, T. A., J. P. Condreay, and D. L. Jarvis.2005. Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat.

Biotech-nol.23:567–575.

18.Liotta, F., R. Angeli, L. Cosmi, L. Fili, C. Manuelli, F. Frosali, B. Mazz-inghi, L. Maggi, A. Pasini, V. Lisi, V. Santarlasci, L. Consoloni, M. L. Angelotti, P. Romagnani, P. Parronchi, M. Krampera, E. Maggi, S. Ro-magnani, and F. Annunziato.2008. Toll-like receptors 3 and 4 are ex-pressed by human bone marrow-derived mesenchymal stem cells and can inhibit their T-cell modulatory activity by impairing Notch signaling. Stem

Cells26:279–289.

19.Liu, C. H., and S. M. Hwang.2005. Cytokine interactions in mesenchymal

stem cells from cord blood. Cytokine32:270–279.

20.Liu, C. Y. Y., C. H. Wang, J. C. Wang, and Y. C. Chao.2007. Stimulation of baculovirus transcriptome expression in mammalian cells by baculoviral

transcriptional activators. J. Gen. Virol.88:2176–2184.

21.Lo, W.-H., S.-M. Hwang, C.-K. Chuang, C.-Y. Chen, and Y.-C. Hu.2009. Development of a hybrid baculoviral vector for sustained transgene

expres-sion. Mol. Ther.17:658–666.

22.Mack, G. S. 2009. Osiris seals billion-dollar deal with Genzyme for cell

therapy. Nat. Biotechnol.27:106–107.

23.O’Neill, L. A., and A. G. Bowie.2007. The family of five: TIR-domain-containing adaptors in Toll-like receptor signaling. Nat. Rev. Immunol.

7:353–364.

24.Opitz, C. A., U. M. Litzenburger, C. Lutz, T. V. Lanz, I. Tritschler, A. Koppel, E. Tolosa, M. Hoberg, J. Anderl, W. K. Aicher, M. Weller, W. Wick, and M. Platten.2009. Toll-Like receptor engagement enhances the immu-nosuppressive properties of human bone marrow-derived mesenchymal stem

cells by inducing indoleamine-2,3-dioxygenase-1 via interferon-␤and protein

kinase R. Stem Cells27:909–919.

25.O’Reilly, D., L. Miller, and V. Luckow.1992. Baculovirus expression vectors: a laboratory manual. W. H. Freeman and Co., New York, NY.

26.Pevsner-Fischer, M., V. Morad, M. Cohen-Sfady, L. Rousso-Noori, A. Zanin-Zhorov, S. Cohen, I. R. Cohen, and D. Zipori.2007. Toll-like receptors and

their ligands control mesenchymal stem cell functions. Blood109:1422–1432.

27.Phinney, D. G., and L. Isakova.2005. Plasticity and therapeutic potential of

mesenchymal stem cells in the nervous system. Curr. Pharm. Des.11:1255–

1265.

28.Sarkis, C., C. Serguera, S. Petres, D. Buchet, J. L. Ridet, L. Edelman, and J. Mallet.2000. Efficient transduction of neural cells in vitro and in vivo by a

baculovirus-derived vector. Proc. Natl. Acad. Sci. USA97:14638–14643.

29.Strauss, R., A. Huser, S. Ni, S. Tuve, N. Kiviat, P. S. Sow, C. Hofmann, and A. Lieber. 2007. Baculovirus-based vaccination vectors allow for efficient

VOL. 83, 2009 BACULOVIRUS TRIGGERS TLR3 PATHWAY IN hMSCs 10555

on November 8, 2019 by guest

http://jvi.asm.org/

(9)

induction of immune responses againstPlasmodium falciparum

circumsporo-zoite protein. Mol. Ther.15:193–202.

30.Tabeta, K., P. Georgel, E. Janssen, X. Du, K. Hoebe, K. Crozat, S. Mudd, L. Shamel, S. Sovath, J. Goode, L. Alexopoulou, R. A. Flavell, and B. Beutler.

2004. Toll-like receptors 9 and 3 as essential components of innate immune defense against mouse cytomegalovirus infection. Proc. Natl. Acad. Sci. USA

101:3516–3521.

31.Tomchuck, S. L., K. J. Zwezdaryk, S. B. Coffelt, R. S. Waterman, E. S. Danka, and A. B. Scandurro.2008. Toll-like receptors on human mesenchy-mal stem cells drive their migration and immunomodulating responses. Stem

Cells26:99–107.

32.Uccelli, A., L. Moretta, and V. Pistoia.2008. Mesenchymal stem cells in

health and disease. Nat. Rev. Immunol.8:726–736.

33.Vercammen, E., J. Staal, and R. Beyaert.2008. Sensing of viral infection and activation of innate immunity by Toll-like receptor 3. Clin. Microbiol. Rev.

21:13–25.

34.Wang, J. P., G. N. Bowen, C. Padden, A. Cerny, R. W. Finberg, P. E. Newburger, and E. A. Kurt-Jones.2008. Toll-like receptor-mediated

activa-tion of neutrophils by influenza A virus. Blood112:2028–2034.

35.West, J., and B. Damania.2008. Upregulation of the TLR3 pathway by Kaposi’s sarcoma-associated herpesvirus during primary infection. J. Virol.

82:5440–5449.

on November 8, 2019 by guest

http://jvi.asm.org/

Figure

FIG. 1. Expression of hMSC surface markers. Cells were mock transduced (black solid lines) or transduced with BV at an MOI of 100 (pinklines) and subjected to immunofluorescence labeling/flow cytometry analyses at 24 hpt.
TABLE 1. Significantly changed genes associated with the TLRsignaling pathway by BV transduction
FIG. 2. Cytokine production by BV-transduced hMSCs. (A and B) Cytokine production at an MOI of 100
FIG. 4. BV transduction of hMSCs triggered TLR3 activation. (A) hMSCs were mock transduced or transduced with BV
+2

References

Related documents

Aeromagnetic data covering an area of 1800km 2 were analysed to characterise the dimension of Mersing fault zone and the surrounding faults by conventional

Children can truly benefit learning a second language as long as they are exposed to the language and are given opportunities to use the language in the environment where the

Moreover, pressure from consumers has ensured that the minimum standards (w ith respect to food safety, animal w elfare, environmental stew ardship etc.) in general and for

The proposed hybrid technique for frequency domain identification of servo systems with friction incorporates a blend of GA and neural network.. The system identification

The purpose of this study was to investigate the capability of different imaging procedures such as US, MRI and CT of the recent generation to predict the nature (benign/ malignant)

The main effect (or first order) sensitivity index measures only the main effect contribution of each input parameter on the out- put variance. The interactions among the

Using these standardized guidelines to interpret AST data from a large collection of Abiotrophia and Granulicatella species isolated across the United States, we identified