Modulation of TLR Expression and Activation in Mesenchymal Stromal Cells Leads to a Proinflammatory Phenotype

This information is current as Raphaëlle Romieu-Mourez, Moïra François, Marie-Noëlle of September 24, 2021. Boivin, Manaf Bouchentouf, David E. Spaner and Jacques Galipeau J Immunol 2009; 182:7963-7973; ; doi: 10.4049/jimmunol.0803864

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Supplementary http://www.jimmunol.org/content/suppl/2009/06/02/182.12.7963.DC1 Material http://www.jimmunol.org/ References This article cites 38 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/182/12/7963.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Cytokine Modulation of TLR Expression and Activation in Mesenchymal Stromal Cells Leads to a Proinflammatory Phenotype1

Raphae¨lle Romieu-Mourez,2* Moïra Franc¸ois,2* Marie-Noe¨lle Boivin,* Manaf Bouchentouf,* David E. Spaner,† and Jacques Galipeau3*

Bone marrow-derived mesenchymal stromal cells (MSC) possess an immune plasticity manifested by either an immunosuppressive or, when activated with IFN-␥, an APC phenotype. Herein, TLR expression by MSC and their immune regulatory role were investigated. We observed that human MSC and macrophages expressed TLR3 and TLR4 at comparable levels and TLR- mediated activation of MSC resulted in the production of inflammatory mediators such as IL-1␤, IL-6, IL-8/CXCL8, and CCL5.

IFN-␣ or IFN-␥ priming up-regulated production of these inflammatory mediators and expression of IFNB, inducible NO syn- Downloaded from thase (iNOS), and TRAIL upon TLR activation in MSC and macrophages, but failed to induce IL-12 and TNF-␣ production in MSC. Nonetheless, TLR activation in MSC resulted in the formation of an inflammatory site attracting innate immune cells, as evaluated by human neutrophil chemotaxis assays and by the analysis of immune effectors retrieved from Matrigel-embedded MSC injected into mice after in vitro preactivation with and/or TLR ligands. Hence, TLR-activated MSC are capable of recruiting immune inflammatory cells. In addition, IFN priming combined with TLR activation may increase immune responses induced by Ag-presenting MSC through presentation of Ag in an inflammatory context, a mechanism that could be applied in a http://www.jimmunol.org/ -based vaccine. The Journal of Immunology, 2009, 182: 7963–7973.

one marrow-derived mesenchymal stromal cells (MSC)4 However, in response to IFN-␥ stimulation, MSC acquire MHC are mesenchymal progenitors thought to give rise to cells class II expression (4) and are capable of Ag presentation (5–7) B that constitute the hematopoietic microenvironment. and to induce a T lymphocyte-mediated immune response in vivo MSC can be facilely isolated and expanded from the adherent cell (5). This suggests that MSC possess an immune plasticity with a fraction of bone marrow aspirates and serve as precursors for the default-suppressive phenotype and, when appropriately activated, generation of a variety of mesodermal tissues, including bone, car- an option to stimulate an immune response. This immune plasticity by guest on September 24, 2021 tilage, and muscle. In addition to their mesenchymal plasticity, appears akin to that observed with dendritic cells (DC), which in MSC are able to regulate the in a manner that the absence of maturation can induce T cell tolerance (8). depends on their state of activation (1). Resting MSC are described DC maturation can be achieved by activation of TLR. Twelve as strongly immunosuppressive, especially for the repression of TLR have been identified in mammals and are expressed prefer- allogeneic immune responses and autoimmune diseases (2, 3). entially by immune cells. TLR are pattern recognition receptors that recognize lipids, carbohydrates, peptides, or nucleic acids spe- cifically expressed by various pathogens. On the one hand, TLR *Department of Medicine and Oncology, Sir Mortimer B. Davis Jewish General Hos- pital & Lady Davis Institute for Medical Research, McGill University, Montreal, activation critically initiates the inflammatory and subsequent Quebec, Canada; and †Division of Molecular and Cellular Biology, Sunnybrook Re- adaptive immune responses. In macrophages and DC, activation search Institute, Sunnybrook Health Sciences Centre, Toronto, Canada with TLR ligands can result in an increase in MHC class I- and Received for publication November 18, 2008. Accepted for publication April 16, 2009. II-mediated Ag processing, expression of costimulatory molecules, and the production of inflammatory mediators. Among TLR-in- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance duced cytokines, biologically active IL-12p75, which is a polypep- with 18 U.S.C. Section 1734 solely to indicate this fact. tide composed of the IL-12p35 and IL-12/IL-23p40 chains, is a 1 This work was supported by a Terry Fox Foundation New Frontiers Program Project key factor in the induction of Th1 and CTL cells, production of Grant and by the Canadian Institute for Health Research Operating Grant MOP- 15017. M.F. is a PhD candidate at McGill University and is the recipient of a Cana- opsonic Abs, and activation of macrophages and NK cells that dian Institute for Health Research Studentship and J.G. is a Fonds de Recherche´en secrete high levels of IFN-␥ (9). On the other hand, the expression Sante´du Que´bec Chercheur Senior. and response to TLR is modified in the context of an immune 2 R.R.-M. and M.F. contributed equally to this study. response. For instance, stimulation with individual TLR induces 3 Address correspondence and reprint requests to Dr. Jacques Galipeau, Depart- the production of an excess of IL-12p40 in monocyte-derived DC ment of Medicine and Oncology, Sir Mortimer B. Davis Jewish General Hospital & Lady Davis Institute for Medical Research, McGill University, 3755 Cote and macrophages (10). Optimal production of IL-12p35 and active Sainte-Catherine Road, Montreal, Quebec, Canada H3T 1E2. E-mail address: IL-12p75 is observed in response to TLR ligands after priming [email protected] with IFN-␥ (11) or IFN-␣ (12), costimulation with CD40L (13), or 4 Abbreviations used in this paper: MSC, mesenchymal stromal cell; DC, dendritic to a specific combination of TLR ligands (12, 14). These combined cell; poly(I:C), polyinosinic:polycytidylic acid; mMSC, mouse MSC; hMSC, human MSC; rh, recombinant human; rm, recombinant mouse; yo, year old; PS, penicillin stimulations result in the activation of different signaling pathways and streptomycin; IRF, IFN regulatory factor; iNOS, inducible NO synthase; NOS, and factors that are particularly effective for the ad- NO synthase. ditive or synergistic up-regulation of IL-12 (15). In addition, in- Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 flammatory cytokines can regulate the expression of TLR, such as www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803864 7964 TLR-MEDIATED ACTIVATION IN MSC

IFN-␣ known to increase expression of TLR3 in human primary Flow cytometry analysis DC, macrophages (16), or endothelial cells (17). The following Abs specific for human molecules were used for flow cytometry Observations of TLR expression and function on MSC revealed analysis: biotin-coupled anti-CD90 (clone 5E10), CD45 (clone HI30), and that mouse MSC (mMSC) expressed all TLR mRNA, with the TLR4 (clone HTA125); FITC-conjugated anti-CD64 (clone 10.1), CD105 exception of TLR9. A TLR2 ligand, inhibited mMSC differentia- (clone 8E11; Chemicon International) and CD64 (clone 10.1); PE-conjugated anti-␤ -microglobulin (clone TU¨ 99), CD31 (clone WM-59), CD73 (clone tion while sparing their ability to suppress allogeneic T cell acti- 2 AD2), CD80 (clone L307.4), TLR3 (clone TLR3.7; eBioscience), and TLR4 vation (18). Recently, the protective effect of i.v. injected synge- (clone HTA125; eBioscience); allophycocyanin-conjugated anti-CD4 (clone neic mMSC was reported against rapid septicemia induced by RPA-T4), CD34 (clone 581), and CD44 (clone G44-26); and isotypic controls. cecal ligation and puncture (19). In this model, mMSC located in Mouse specific Abs were: FITC-conjugated anti-CD11c (clone HL3), PE-con- the lungs where they recruited macrophages and reprogrammed jugated anti-CD117 (clone 2B8), CD14 (clone M⌽P9), and NK1.1 (clone PK136), PerCP-Cy5.5-conjugated anti-Ly-6G/6C (clone RB6-8C5), and allo- them for increased IL-10 release, an effect mediated by the com- phycocyanin-conjugated anti-CD3␧ (clone 145-2C11). Cell permeabilization bined production of NO by LPS-activated mMSC and macro- was performed using the Perm/Wash buffer. Flow cytometry analysis was phages. As for human MSC (hMSC), one study showed that hMSC performed on 20,000 events using a FACSCalibur cytometer and data were expressed only TLR3 and TLR4 and exposure to TLR ligands analyzed using CellQuest software. Except where indicated, Abs, reagents, and decreased their ability to suppress allogeneic T cell proliferation, apparatus were from BD Biosciences. an effect that correlated with the down-regulation of Notch ligand Jagged-1 expression on hMSC (20). The present work focused on Real-time RT-PCR assessing TLR expression and activation in MSC compared with Primer sequences (5Ј–3Ј forward and reverse, respectively) specific for “professional APC” such as primary macrophages for the induc- human mRNA were: TLR1, GCACCCCTACAAAAGGAATCTG and Downloaded from tion of an inflammatory state, as well as regulation of these prop- GGCAAAATGGAAGATGCTAGTCA (107 bp); TLR2, CTGGTAGTT erties by IFN-␣ or IFN-␥ priming. GTGGGTTGAAGCA and GATTGGAGGATTCTTCCTTGGA (102 bp); TLR3, TTAAAGAGTTTTCTCCAGGGTGTTTT and AATGCTTGT Materials and Methods GTTTGCTAATTCCAA (124 bp); TLR4, CCCCTTCTCAACCAAGA ACCT and ATTGTCTGGATTTCACACCTGGAT (120 bp); TLR5, TG Reagents CTAGGACAACGAGGATCATG and GAGGTTGCAGAAACGATAA

Recombinant human (rh) and mouse (rm) IFN-␣, IFN-␥, and TNF-␣ were AAGG (114 bp); TLR6, AGGCCCTGCCCATCTGTAA and GCAA http://www.jimmunol.org/ from Invitrogen, rhIFN-␣2b was from Schering ( A), and rhGM-CSF TTGGCAGCAAATCTAATTT (100 bp); TLR7, GCTATTGGGCCCAT was a generous gift from Cangen. Specific TLR activation was achieved CTCAAG and TCCACATTGGAAACACCATTTTT (112 bp); TLR8, using Pam3CSK4 (InvivoGen), polyinosinic-polycytidylic acid (poly(I:C); TCAGTGTTAGGGAACATCAGCAA and AACATGTTTTCCTTTTT Sigma-Aldrich) for TLR3, LPS from Escherichia coli O26:B6 (Sigma- AGTCTCCTTTC (134 bp); TLR9, GGGAGCTACTAGGCTGGTATA Aldrich) for TLR4, and gardiquimod (InvivoGen) for TLR7. Human CCL5 AAAATC and GCTACAGGGAAGGATGCTTCAC (103 bp); TLR10, and TNF-␣, mouse IL-6, IL-12p75, and TNF-␣ were detected by ELISA TTTACTCTGGGACGACCTTTTCC and ATAAGCCTTACCACCAAA kits from R&D Systems and human IL-6 and IL-12p75 from eBioscience. AGTCACA (100 bp); IL-8, CTGTTAAATCTGGCAACCCTAGTCT and Alternatively, levels of human IL-6, IL-1␤, IL-8, TNF-␣, and IL-12 were CAAGGCACAGTGGAACAAGGA (64 bp); IL-12A, ATGCTCCA measured by cytometric bead array (BD Biosciences). GAAGGCCAGACA and CCTCCACTGTGCTGGTTTTATCT (100 bp); IL-12B, TCATCAGGGACATCATCAAACC and CAGGGAGAAGTAG Cell isolation and culture GAATGTGGAGTA (131 bp); iNOS/NOS2A, GGCTCCTTCAAAGAG by guest on September 24, 2021 GCAAA and CATCTCCCGTCAGTTGGTAGGT (100 bp); and 18S hMSC from donors 240L, 5066R, and 5068L (24- year-old (yo) male, 22 rRNA, TTACCAAAAGTGGCCCACTA and GAAAGATGGTGAAC yo female, and 24 yo male, respectively) were given to us by D. J. Prockop TATGCC (200 bp). Human primers specific for CCL5, TNF-␣, TRAIL, (Tulane University, New Orleans, LA). hMSC from donors 52F, 291, 292, IFN-␤, and IFN-␣2 mRNA were as previously described (22). Primer se- 293, 294, and 302 (52 yo female, 73 yo male, 85 yo female, 73 yo female, quences (5Ј–3Ј forward and reverse, respectively) specific for mouse 69 yo female, and 48 yo male, respectively) were established from bone mRNA were: Cxcl1/KC, CACCTCAAGAACATCCAGAGCTT and GT marrow aspirates from patients undergoing hip surgery replacement at the GGCTATGACTTCGGTTTGG (53 bp); Cxcl2/Mip-2, CCCTGGTTCA Montreal Jewish General Hospital, as previously described (5). hMSC cul- GAAAATCATCCA and GCTCCTCCTTTCCAGGTCAGT (48 bp); ␣ ture medium was -MEM with 2 mM L-glutamine, 16.5% FBS, and 100 Ccl2, GTCTGTGCTGACCCCAAGAAG and TGGTTCCGATCCAG U/ml penicillin and streptomycin (PS). All tissue culture reagents were GTTTTTA (61 bp); Ccl3, CTCCCAGCCAGGTGTCATTTT and TTG from Wisent Technologies. hMSC were expanded for up to four passages 2 GAGTCAGCGCAGATCTG (59 bp); Ccl4, AGCCCTGATGCTTCT by plating cells every 7 days at 100 cells/cm . Karyotype analyses of CACTGA and GGGCCAGGAAATCTGAACGT (59 bp); Ccl5, hMSC from donors 240L, 5066R, 5068L, 292, and 293 were established on TCCAATCTTGCAGTCGTGTTTG and TCTGGGTTGGCACACACTTG 20 metaphasic cells and were shown to be normal in the absence of con- (58 bp); Ccl7, GCTGCTTTCAGCATCCAAGTG and GCAGCAT sistent numerical or structural anomalies (D. J. Prockop, per- GTGGATGCATTG (59 bp); Ccl8, CCAGACCAAGCAGGGTATGTC sonal communication, and data not shown). mMSC were obtained from and CATGTACTCACTGACCCACTTCTGT (43 bp); and Ccl9, CGGA C57BL/6 mice as described elsewhere (21). MSC were tested for the ab- ϩ ϩ ϩ GAGTTCAGAGATGCATTG and TTGTTTGTAGGTCCGTGGTTGT sence of CD31 endothelial cells and CD45 or CD34 hematopoietic (62 bp). Quantitative RT-PCR assays were performed in duplicate on an cells, expression of CD44, CD73, CD90, and CD105, and capacity to dif- Applied Biosystems 7500 Fast Real-Time PCR system thermal cycler and ferentiate into adipocytes and osteocytes as described previously (21). Un- SYBR Green Mastermix (Applied Biosystems) as described elsewhere (5– less indicated otherwise, hMSC and mMSC were cultured at 100 cells/cm2 2 7). Data were analyzed by the comparative threshold cycle method, based or 500 cells/cm , respectively, for 4 days before activation. PBMC (four on the relative expression of target mRNA vs 18S rRNA levels as a donors: MA-A, MA-B, MAL, and 692) were isolated from fresh apheresis reference, using the SDS version 1.3.1 Relative Quantification Software. with informed consent from healthy donors. Human monocyte-derived Expression levels of TLR mRNA were also determined using GAPDH or macrophage-enriched cell preparations were obtained by plating PBMC at ␤-actin mRNA levels as alternate references and similar results were ob- ϫ 6 2 10 cells/ml in IDMEM supplemented with 2% human serum A/B, 100 tained (data not shown). Specificity of PCR amplification was tested by U/ml PS, and 500 U/ml rhGM-CSF for 5–6 days before harvesting adher- melting curves and agarose gel analysis. The absence of genomic DNA ent cells. Flow cytometry analysis performed as described in Ref. 22 dem- contamination was demonstrated routinely by analysis of PCR performed Ͼ ϩ ϩ onstrated that 95% adherent cells were CD14 and/or CD64 . U937 with total RNA and with each of the primer sets. (ATCC CRL-1593.2) and Mono-mac 6 cells are human monocyte and macrophage-like immortalized cells, respectively, and were cultured in RPMI 1640 with 2 mM L-glutamine, 10% FBS, and 100 U/ml PS. Primary Immunoblot analysis HUVECs were obtained from Cambrex. Mouse peritoneal macrophages were exuded from C57BL/6 female retired breeder mice (Charles Rivers Immunoblot analyses were performed as described previously (5–7). Laboratories) by sterile lavage and plated in RPMI 1640, 10% FBS, and Primary Abs were specific for: c-Rel (N), RelA/p65 (C-20), ␣-tubulin 100 U/ml PS for 16 h before extensive washing to remove nonadherent (TU-02), IFN regulatory factor (IRF) 1 (C-60) (all from Santa Cruz cells. Biotechnology). The Journal of Immunology 7965 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 1. TLR3 and TLR4 expression in hMSC. A, hMSC characterization (donor 293). Top panel, Cells were analyzed by flow cytometry for surface expression of CD31, CD34, CD44, CD45, CD73, CD90, and CD105. The gray line represents the isotype control and the black line represents the specific Ab. Bottom panel, Cells were tested for their capacity to differentiate into bone and fat in the presence of osteocyte and adipocyte differentiation medium (original magnification, ϫ50 for nontreated cells and ϫ100 for differentiated cells). B, TLR1 to TLR10 mRNA levels. Total DNase-treated RNA from hMSC (donors 240L, 5068L, 5066R, and 52F), PBMC (donors MA-A and MA-B), and human monocytic immortalized cell lines U937 and Mono-mac 6 were prepared and processed for quantitative RT-PCR analysis of TLR1 to TLR10 expression. Relative quanti- fication (RQ) was calculated by normalizing the expression of target to reference 18S rRNA levels. Normalized values were compared between samples displaying detectable target gene expression. The minimal normalized detectable target gene expression level was given Not ,ء .the value of 1, and other values show fold increase expression compared with this minimal level. Shown are the means of triplicates Ϯ SDs detectable expression. Normalization to GAPDH and ␤-actin mRNA levels gave similar expression profiles (data not shown). C, TLR3 expression. hMSC (donors 240L and 293) and primary human macrophages (hM␾, donor 692) were analyzed for surface expression of Fc␥R1/CD64 and intracellular expression of TLR3 by flow cytometry as in A. D, Effect of cell permeabilization on TLR3 staining in hMSC (donor 240L) and U937 cells as analyzed by flow cytometry as in A.

Growth response to TNF-␣, IFN-␣, or IFN-␥ formazan product was measured on an ELX800 microplate reader (Bio- Tek Instruments). hMSC were seeded in 96-well plates at 300 cells/cm2 in complete medium. Three days later, cells were treated or not with TNF-␣ (1–10 ng/ml), IFN-␣ Neutrophil chemotaxis assay (100–10 000 U/ml), or IFN-␥ (10–300 U/ml) for 4 days before performing a MTT assay using the Cell Titer96 reagent (Promega) to measure cell hMSC were untreated or primed with 30 U/ml IFN-␥ for 18 h and activated viability. Conversion of tetrazolium compound to a 490-nm absorbing with 100 ng/ml LPS for 2 h, extensively washed to remove IFN-␥ and LPS, 7966 TLR-MEDIATED ACTIVATION IN MSC

and supernatants (conditioned medium) were harvested 48 h later. Neutro- phils were enriched from heparinized peripheral blood from healthy vol- unteers by Ficoll density gradient centrifugation (room temperature, 400 ϫ g, 45 min) and erythrocytes were removed by hypotonic shock in 168 mM ␮ ϫ NH4Cl, 10 mM KHCO3, and 100 M EDTA. Cells were washed at 120 g for 10 min with the break off to remove platelets. Human neutrophils were then isolated by negative selection using a human neutrophil enrich- ment (StemCell Technology). The 24-well cell invasion assay (Milli- pore) was used to examine the migration of neutrophils according to the manufacturer’s protocol. Briefly, this modified Boyden chamber is com- posed of a well (lower compartment) containing an insert (upper compart- ment) that is separated by an 8-␮m pore size polycarbonate membrane. Before cell seeding, Transwell permeable supports and cells were prein- cubated for 30 min and 1 h, respectively, in serum- free medium. Neutro- phils (4 ϫ 104 cells in 0.5 ml of serum-free medium) were loaded into the upper chamber of the Transwell. Lower chambers were loaded with hMSC- conditioned medium in the presence or absence of anti-IL-8-neutralizing Ab (3 ␮g/ml; R&D Systems) or isotype control Ab (goat IgG). Three hours later, cells remaining on the upper surface of the insert (nonmigrated cells) were removed gently. The inserts were then removed, placed into a sterile 24-well plate containing a cell detachment solution, and incubated for 30 min at 37°C. Dislodged cells were treated with a lysis buffer and incubated

with the CyQuant GR dye for 15 min at room temperature. The mixture Downloaded from was transferred to a 96-well plate and fluorescence was read at 480 nm. Migrated cell number was determined by running a fluorescent cell dose curve using 103,5ϫ 103,104,2ϫ 104, and 4 ϫ 104 neutrophils. Immune effector infiltration analysis mMSC (3 ϫ 106 cells) were untreated or primed with 30 U/ml IFN-␥ for 18 h and activated with 1 ␮g/ml LPS for 2 h, extensively washed to remove http://www.jimmunol.org/ IFN-␥ and LPS, mixed to 300 ␮l of Matrigel (BD Biosciences) at 4°C, and injected s.c. in mice. After 2 days, implants were surgically removed and incubated at 37°C with a solution of 1.6 mg/ml collagenase type I (Sigma- Aldrich) and 200 ␮g/ml DNase I (Sigma-Aldrich) until complete dissolu- tion of the Matrigel. The number of cells with a diameter larger than 7 ␮m (to exclude erythrocytes) was determined by automatic counting using a Z2 Coulter Particle Counter and Size analysis (Beckman Coulter) and cells were analyzed for the presence of Ly-6G/6Cϩ, NK1.1ϩ, CD11cϩ, CD117ϩ, CD14ϩ,orCD3ϩ cells by flow cytometry. Statistical analysis by guest on September 24, 2021 Otherwise indicated, experiments with hMSC were repeated at least twice using two different donors in each, giving a total of four independent data. Two groups were compared with the two-tailed unpaired Student t test using Prism software (GraphPad). Three groups and more were compared with the one-way ANOVA test and Dunnett’s multiple comparison or Bon- ferroni post tests.

Results Constitutive TLR expression pattern in hMSC To analyze the constitutive expression pattern of TLR in hMSC, adherent cell fractions from bone marrow aspirates performed in normal donors were selected for expression of CD44, CD73, CD90, and CD105, osteogenic and adipogenic differentiation ca- pacity, and the absence of CD31ϩ endothelial cells and CD45ϩ or CD34ϩ hematopoietic cells, (Fig. 1A). RNA were prepared from hMSC (four donors), unfractioned PBMC (two donors) that con- tain a mixture of immune cells expressing various TLR, and the monocytic immortalized cell lines U937 and Mono-mac 6. Real-time FIGURE 2. hMSC activated with TLR3 or TLR4 ligands produce inflam- RT-PCR analyses of TLR1 to TLR10 mRNA levels demonstrated matory cytokines or . A, Dose response to poly(I:C) or LPS. hMSC ␾ that hMSC expressed only TLR3 and TLR4 at levels comparable to (donor 294) or primary human macrophages (hM , donor 692) were stimu- ␮ hematopoietic cells (Fig. 1B). TLR5-, TLR6-, and TLR9-encoding lated with poly(I:C) (0.02–20 g/ml) or LPS (1–1000 ng/ml) for 48 h before analysis of IL-6 and CCL5 levels in supernatants by ELISA. Shows are means mRNA were detected in all hMSC donors. Since expression levels ,␤Not done. B, Poly(I:C) or LPS-induced IL-6, IL-1 ,ء .of triplicates Ϯ SDs were low compared with fresh PBMC, the functional responses to and IL-8 production. hMSC (donors 292 and 240L, plated at 500 cells/cm2 for corresponding ligands were not investigated. Cell culture density 2 days) were treated with poly(I:C) (20 ␮g/ml) or LPS (1 ␮g/ml) for 24 h. 2 (tested from 100 to 2000 cells/cm ) had no effect on the basal TLR Cytokine levels in supernatants were then measured by flow cytometry using expression in hMSC (data not shown). a cytometric bead array. C, Poly(I:C)-induced IL-8 mRNA expression. hMSC Flow cytometry analysis of TLR3 and TLR4 expression was (donor 294, plated at 500 cells/cm2 for 2 days), PBMC (donor MAL), and the next performed. Two commercially available Ab clones directed 293T human embryonic cell line were treated with poly(I:C) (20 ␮g/ml) or against TLR4 were tested but did not allow the detection of sig- LPS (1 ␮g/ml) for 20 h and processed for analysis of IL-8 expression as nificant expression of TLR4 in hMSC as well as Fc␥R1/CD64ϩ detailed in Fig. 1B legend. RQ, Relative quantification. The Journal of Immunology 7967

primary macrophages or U937 cells (data not shown). Expression of TLR4 was hence tested for responsiveness to TLR ligands (see below). Analysis of TLR3 expression demonstrated that hMSC homogenously expressed TLR3 at levels comparable to macro- phages (Fig. 1C). Previous studies demonstrated that TLR3 is found at the endosomal membrane and an internalization of exog- enously added dsRNA is necessary for TLR3 signaling in DC (23). In a human lung fibroblast cell line, however, TLR3 was observed at the cell surface (23). In this study, in both hMSC and U937 cells, detection of TLR3 expression was enhanced by cell permeabiliza- tion and intracellular staining (Fig. 1D), suggesting similar and intracellular localization of TLR3 in hMSC and macrophages.

Human MSC produce IL-1␤, IL-6, IL-8, and CCL5, but not IL-12p75 in response to TLR3 or TLR4 ligands The presence of functional TLR in hMSC was assessed by incu- bating cells with specific TLR3 or TLR4 ligands, respectively,

poly(I:C) (a synthetic dsRNA), or LPS, and measuring production Downloaded from of early inflammatory mediators such as IL-1␤, IL-6, IL-8/ CXCL8, and ␤ CCL5, as well as the Th1-inducing cytokine IL-12p75. As previously described (3), we observed that hMSC constitutively produced high levels of IL-6 (Ͼ10 ng/48 h/106 cells; Fig. 2, A and B). In response to stimulation with LPS ␮

(1–1000 ng/ml) or poly(I:C) (0.02–20 g/ml), hMSC from donors http://www.jimmunol.org/ 292 (Fig. 2A) and 240L (data not shown) produced elevated levels of IL-6 compared with macrophages and comparable amounts of CCL5. The fold induction in the increase of IL-6 production in response to poly(I:C) or LPS was nevertheless similar in both cell types. Although doses as low as 0.02 ␮g/ml poly(I:C) or 1 ng/ml LPS induced secretion of CCL5 in hMSC, optimal responses for IL-6 production were observed at 20 ␮g/ml poly(I:C) or 1 ␮g/ml LPS (Fig. 2A). We hence used the latter doses for subsequent anal-

yses. Response study in two other hMSC donors demonstrated that by guest on September 24, 2021 poly(I:C) or LPS activation induced production of inflammatory IL-1␤ and IL-6 cytokines and IL-8 chemokine (Fig. 2B). In sharp contrast, levels of IL-12p75 were below detection in supernatants of resting or TLR ligand-activated hMSC, as tested by flow cy- tometry-microbead array or ELISA (data not shown). In addition, activation of hMSC with Pam3CSK4, a TLR1 and TLR2 ligand, did not up-regulate IL-6 or CCL5 production by opposition to macrophages (data not shown), confirming the specificity of the TLR expression and responsiveness in hMSC. TLR3 is not the sole receptor specific for dsRNA, as most mam- malian cells express RIG-I-like receptors (e.g., RIG-I, MDA5, and LGP2). Although TLR3 binds to endosomal dsRNA, RIG-I-like receptors are cytosolic RNA helicases able to sense dsRNA result- ing from or electroporation of poly(I:C), leading to the production of inflammatory mediators in most cell types (24–26). Hence, we compared the response to poly(I:C) between 293T cells, which express RIG-I but not TLR3 or TLR4, and hMSC. Poly(I:C) or LPS added to 293T cell cultures did not in- crease expression of IL-8, in contrast to hMSC and PBMC (Fig. 2C), suggesting that the response to poly(I:C) overlaid to cell cul- tures is mediated by TLR3 in hMSC. FIGURE 3. Effect of TNF-␣, IFN-␣, and IFN-␥ on TLR expression in These data support the hypothesis that the constitutive expres- hMSC. A, TLR mRNA levels. hMSC were activated with 3 ng/ml TNF-␣, sion of TLR4 and TLR3 in hMSC resulted in a response to their 1 000 U/ml IFN-␣, or 100 U/ml IFN-␥ for 6 h and then processed for specific ligand that was predominantly characterized by secretion quantification of TLR expression as detailed in Fig. 1B legend. RQ, Rela- of inflammatory cytokines and chemokines, with the exception of tive quantification. B, TLR3 protein expression. hMSC (donor 240L) were IL-12p75. treated with 1 000 U/ml IFN-␣ or 100 U/ml IFN-␥ or not (Ϫ) for 48 h and Exposure to TNF-␣, IFN-␣, or IFN-␥ in hMSC regulates the analyzed for TLR3 expression by flow cytometry, as detailed in Fig. 1C expression of TLR in hMSC legend. B7H1 expression was tested as an additional control for IFN stimulation. To study the TLR response in the context of an immune or in- flammatory response, we investigated the effects of exposure to 7968 TLR-MEDIATED ACTIVATION IN MSC Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 4. Combination of TLR3 or TLR4 activation with IFN-␣ or IFN-␥ priming increases hMSC inflammatory response. hMSC (donors 292, 240L, and 302) were cultured at 500 cells/cm2 for 4 days. PBMC and primary macrophages (hM␾) were obtained from blood aphereses (donors MAL and 692). A, Expression of inflammatory genes. Cells were primed or not with IFN-␣ (2 000 U/ml) for 18 h and treated or not with poly(I:C) (20 ␮g/ml) or LPS (1 ␮g/ml) for 6 h. Total DNase-treated RNA was prepared and processed for quantitative RT-PCR analysis of IL-12A, IL-12B, TNFA, CCL5, IFNB, TRAIL, and iNOS expression, as described in Fig. 1B legend. RQ, Relative quantification. B, Cells were primed or not with IFN-␥ (30 U/ml) for 18 h, treated or not with poly(I:C) (20 ␮g/ml) or LPS (1 ␮g/ml), and processed as in A. C, Inflammatory cytokine and chemokine production. Cells were primed or not with IFN-␥ (30 U/ml) for 18 h, treated or not with poly(I:C) (20 ␮g/ml) or LPS (1 ␮g/ml) for 48 h, and processed for quantification of IL-12p75, TNF-␣, IL-6, IL-8, and CCL5 levels by ELISA. D, Neutrophil chemotaxis. hMSC donors (292, 294, and 302) were activated or not with IFN-␥ (30 U/ml) for 18 h and LPS (100 ng/ml) for 2 h and extensively washed to remove IFN-␥ and LPS. Conditioned medium (CM) from the three hMSC donors (nontreated or IFN-␥ ϩ LPS-activated) were harvested 48 h later and pooled in equal volumes. Human neutrophils were isolated from peripheral blood and were placed in the upper compartment of a Transwell filter system. Conditioned medium was added in triplicate to the bottom wells in the absence or presence of anti-human IL-8 or isotype control (goat IgG). Neutrophils migration was quantified 4 h later. Results are shown as the total number of migrated cells .(p Ͻ 0.01 ,ءء p Ͻ 0.05 and ,ء ;means Ϯ SEM) The Journal of Immunology 7969

TNF-␣, IFN-␣, or IFN-␥ on hMSC growth and TLR expression. IFN-␥ induced a marginal but statistically significant growth in- hibition (Ͻ20% in OD values at all tested doses, p values Ͻ0.05) in two hMSC donors, as detected by the measure of mitochondrial activity in an MTT assay (supplemental Fig. 15). At all tested doses, TNF-␣ significantly inhibited growth in only one donor (35–40% decrease in OD values, p values Ͻ0.001) and IFN-␣ in both donors (25–49% decrease in OD values, p values Ͻ0.001). Automatic cell counting performed on hMSC from four donors cultured with 3 ng/ml TNF-␣, 1000 U/ml IFN-␣, or 100 U/ml IFN-␥ for 48 h showed 5–20% decrease in cell numbers (data not shown). Thus, hMSC displayed moderate to medium growth re- tardation when cultured with TNF-␣, IFN-␣, or IFN-␥. Exposure to TNF-␣, IFN-␣, or IFN-␥ had no significant effects FIGURE 5. c-Rel and IRF-1 expression in hMSC. A, Basal c-Rel ex- on expression levels of TLR1, TLR4, TLR5, TLR8, TLR9, and pression. Samples of WCE from hMSC (donors 291, 292, and 240L, plated TLR10 mRNA in hMSC, as tested by real-time RT-PCR after 6 h at 2000 cells/cm2 for 3 days) and primary human macrophages (hM␾, (data not shown) or 24 h (Fig. 3A and data not shown). In contrast, donor MAL) were run on 4–20% SDS-PAGE gels and subsequently sub- TNF-␣ up-regulated expression of TLR2 and TLR7, IFN-␣ jected to immunoblot analysis of RelA (p65), c-Rel, or ␣-tubulin (control) strongly increased the expression of TLR3, and IFN-␥ increased expression. B, IFN-␣- or IFN-␥-induced IRF-1 expression. hMSC (donor Downloaded from the expression of TLR2 and TLR3 (Fig. 3A). Up-regulation of 240L, cultured as in A) were treated or not with IFN-␣ (2000 U/ml) or TLR3 expression by IFN-␣ and IFN-␥ in hMSC was also observed IFN-␥ (30 U/ml) for either 24 or 48 h. Samples of WCE were run on by intracellular staining (Fig. 3B). Importantly, in three tested 4–20% SDS-PAGE gels and subsequently subjected to immunoblot anal- ␣ hMSC donors (240L, 5068L, and 293) quantification of IFN-␣ or ysis of IRF-1 or -tubulin (control) expression. IFN-␥ effects on TLR3 expression assayed by RT-PCR could not

be paralleled to protein levels determined by flow cytometry. In the http://www.jimmunol.org/ case of hMSC donor 240L, a 16- or 350-fold increase in TLR3 strongly up-regulated expression of IL-12A in hMSC. Activation mRNA levels was observed upon IFN-␥ or IFN-␣ activation, but with IFN-␥ and poly(I:C) or LPS, however, did not further in- this corresponded to only a 1.3- and 1.4-fold increase in protein crease expression of IL-12A and did not induce expression of IL- levels, respectively. Overall, these results suggest that single treat- 12B, in clear contrast to macrophages. ments with TNF-␣, IFN-␣, or IFN-␥ resulted in up-regulation of ELISA analyses using cell supernatants harvested 48 h after the expression of TLR2, TLR3, or TLR7 in hMSC. stimulation confirmed that in macrophages high levels of TNF-␣ production was induced by LPS alone; while IL-12p75 secretion Combination of TLR3 or TLR4 activation with IFN-␣ or IFN-␥ was synergistically increased by IFN-␥ and LPS (Fig. 4C). In priming increases the inflammatory response in hMSC hMSC, production of TNF-␣ and IL-12p75 was detected upon dual by guest on September 24, 2021 ␥ To further address the regulation of the TLR response by cytokines stimulation with IFN- and poly(I:C) or to a lesser extent with ␥ ␣ in hMSC, we analyzed the effects of preexposure to IFN-␣ or IFN- and LPS. Consistent with gene expression analyses, TNF- IFN-␥ on TLR3 or TLR4 ligand-induced expression of a set of and IL-12p75 levels were at least 50- and 3- fold lower, respec- genes encoding cytokines, chemokines, and proinflammatory me- tively, in supernatants from hMSC compared with macrophages. diators, namely, IL-12A (encoding IL-12p35), IL-12B (encoding In contrast, a robust production of CCL5 was induced by LPS Ͻ IL-12p40), TNFA, CCL5/RANTES, IFNB, IFNA2, TRAIL, and alone in hMSC ( p values 0.001), which was increased at least ␥ ␥ NOS2A/iNOS. For this task, hMSC from donors 292, 240L, and two times by preexposure to IFN- ( p values of IFN- - or LPS- ␥ Ͻ 302 (Fig. 4) and 291 and 5068L (data not shown) as well as pri- treated vs IFN- plus LPS-treated hMSC 0.001; Fig. 4C). Sim- mary macrophages were stimulated with IFN-␣ or IFN-␥ for 18 h ilarly, production of IL-6 and IL-8 were increased by the dual ␥ before the addition of poly(I:C) or LPS for 6 h for analysis of gene stimulation of IFN- and LPS in hMSC and macrophages (Fig. expression by real-time RT-PCR or 48 h for protein secretion by 4C). Because IL-8 is a specific neutrophil-attracting chemokine, ELISA. we tested the capacity of activated hMSC to mediate neutrophil Treatment with IFN-␣ alone had no effect on expression levels recruitment. For this task, hMSC were untreated or primed with ␥ of IL-12A, IL-12B, TNFA, CCL5, IFNB, TRAIL, and iNOS (Fig. IFN- for 18 h and activated with LPS for 2 h, extensively washed ␥ 4A). Activation of hMSC with poly(I:C) or LPS increased expres- to remove IFN- and LPS, and supernatants (conditioned medium) sion of TNFA, CCL5, IFNB, and TRAIL ( p values Ͻ0.05). Ex- were harvested 48 h later. Neutrophil chemotaxis was tested in a pression of IL-12A and IL-12B was not detected in poly(I:C) or Transwell system with purified human blood neutrophils in the LPS-activated MSC, in contrast to macrophages. Pretreatment upper compartment and supernatants from hMSC in the bottom with IFN-␣ and poly(I:C) acted in synergy for the up-regulation of compartment. Neutrophils migrated in higher numbers toward ␥ IL-12A, TNFA, CCL5, IFNB, and iNOS but not of IL-12B mRNA compartments with conditioned medium from IFN- -primed LPS- levels in hMSC. activated hMSC compared with nontreated hMSC ( p value Ͻ When poly(I:C) or LPS was added to hMSC preexposed to 0.01), an effect that was significantly inhibited by the neutral- ␥ IFN-␥, a synergistic up-regulation of CCL5, TRAIL, and iNOS was ization of IL-8 in supernatants from IFN- -primed LPS-activated observed in hMSC ( p values Ͻ0.05) at levels comparable to or hMSC but not from nontreated cells (Fig. 4D). higher than macrophages (Fig. 4B). TNFA was also synergistically Overall, mRNA and protein expression analyses suggested that the ␣ up-regulated; however, its expression in hMSC was minimal com- association of TLR3 or TLR4 ligands with priming with IFN- or ␥ pared with macrophages. Of note, sole activation with IFN-␥ IFN- increased the production of inflammatory cytokines and che- mokines, such as IL-6, CCL5, and IL-8 in hMSC. The most striking difference between hMSC and macrophages, however, was the low 5 The online version of this article contains supplemental material. levels of production of IL-12p75 and TNF-␣ by the former. 7970 TLR-MEDIATED ACTIVATION IN MSC

FIGURE 6. In vivo inflammatory response induced by TLR-activated MSC. A, IL-12, TNF-␣, and IL-6 pro- duction by TLR3- or TLR4-activated mMSC. C67BL/6 mMSC or primary peritoneal macrophages (mM␾) were primed or not with IFN-␣ (2000 U/ml) or IFN-␥ (30 U/ml) for 18 h and treated or not with poly(I:C) (20 ␮g/ml) or LPS (1 ␮g/ml) for 48 h. Supernatants were then processed for quantification of IL-12p75, TNF-␣, Not ,ء .and IL-6 levels by ELISA done. B, Chemokine gene expression. mMSC were primed or not with Downloaded from IFN-␥ (30 U/ml) for 18 h and treated or not with LPS (100 ng/ml) for 4 h. Total DNase-treated RNA was pre- pared and processed for quantitative RT-PCR analysis of Cxcl1, Cxcl2, Ccl2, Ccl3, Ccl4, Ccl5, Ccl7, Ccl8,

and Ccl9 as described in Fig. 1B leg- http://www.jimmunol.org/ .Not detectable expression ,ء .end Analysis on mouse spleen cells dem- onstrated expression of Ccl3 and Ccl4 genes (data not shown). RQ, Relative quantification. C, Immune infiltration induced by TLR-activated mMSC. mMSC were primed with IFN-␥ (30 U/ml) for 18 h and activated with LPS (1 ␮g/ml) for 3 h. After extensive washing, cells (3.106) were mixed to by guest on September 24, 2021 Matrigel and injected s.c. in C57BL/6 mice. After 2 days, implants were ex- cised and dissolved. Cells larger than 7 ␮m were automatically counted and analyzed for the presence of Ly6C/ 6Cϩ granulocytes, NK1.1ϩ NK cells, CD11cϩ DC cells, and CD3ϩ T cells by flow cytometry. Results were re- produced in two other sets of experi- ments comparing nontreated to IFN-␥ ϩ LPS-treated mMSC (data not shown). Data represent values ob- p Ͻ ,ء) tained with individual mice p Ͻ ,ءءء p Ͻ 0.01; and ,ءء ;0.05 0.001, n ϭ 5).

Basal and IFN-␣- or IFN-␥-regulated expression of c-Rel, RelA, complexes (27–29). Activation of ubiquitous RelA-p50 NF-␬B and IRF-1 in hMSC complexes, in contrast, was reported sufficient for expression of Given the lack of production of IL-12p75 by TLR-activated inflammatory cytokines such as IL-1 or IL-6 (29). In the present hMSC, we tested whether hMSC and macrophages differently ex- study, we observed that hMSC from three donors displayed basal pressed latent transcription factors that, when activated by TLR expression of RelA but almost undetectable expression of c-Rel signaling , are involved in the expression of IL-12A and compared with primary macrophages (Fig. 5A). IL-12B. Notably, it was shown in monocyte-derived DC or mac- IFN-␥-induced IRF-1 DNA binding was demonstrated to act in rophages that TLR-induced activation of IL-12A and IL-12B pro- synergy with c-Rel/p50 for optimal IL-12A activation in moters is critically dependent on the binding of c-Rel-p50 NF-␬B primary mouse macrophages (30). Likely, in hMSC, IRF-1 was The Journal of Immunology 7971 strongly up-regulated by 24 h after exposure to IFN-␥ and to a tion of hMSC with poly(I:C) or LPS resulted in the production of much lesser extent to IFN-␣ (Fig. 5B). In addition, we observed inflammatory mediators such as IL-1, IL-6, IL-8, and CCL5 that that c-Rel levels were unaffected by exposure for 24–48 h to was increased by IFN-␣ or IFN-␥ priming, resulting in the attrac- IFN-␥ or IFN-␣ (data not shown). Overall, these results suggest tion of innate immune cells in vivo. The most notable difference that the absence of basal expression of c-Rel in hMSC compared between hMSC and macrophages was the lack of production of with macrophages hindered the up-regulation of IL-12A and IL- IL-12 or TNF-␣ by the former. 12B mRNA by TLR ligands. In IFN-␥-stimulated hMSC, IRF-1 TLR engagement in macrophages or DC results in the in- was likely to contribute to the activation of IL-12A promoter, as creased production of inflammatory mediators, chemokines, and seen above (Fig. 4C); however, the absence of induction of IL-12B T- and B-activating cytokines such as IL-1␣, IL-1␤, IL-6, IL-8, may explain the low level production of active IL-12p75. IL-12, CCL5/RANTES, type I IFN, TNF-␣, antitumoral and antimicrobial molecules such as NOS2A/iNOS-induced NO or In vivo immune cell infiltration induced by TLR-activated MSC TRAIL, as well as Ag processing and presentation (31, 32). In Supernatants from hMSC activated with IFN-␥ and LPS contained this study, we observed that hMSC expressed high basal levels chemokines involved in the attraction of neutrophils (Fig. 4C)as of TLR3 and TLR4 and responded to their respective ligand by well as unfractioned PBMC (data not shown). To address the in the production of inflammatory cytokines IL-1␤ and IL-6 as vivo immune recruitment induced by MSC upon TLR activation, well as the chemokines IL-8 and CCL5. IFN-␥ priming in- we first characterized the response of mMSC to TLR ligands. In creased both TLR3- and TLR4-mediated inflammatory re- distinction to hMSC, mMSC were previously shown to express all sponses, as detected by the measure of TRAIL and iNOS mRNA

TLR, with the exception of TLR9 (18). In this study, we observed transcripts as well as of the production of IL-6, IL-8, and CCL5 Downloaded from that C57BL/6 mMSC exposed to poly(I:C) or LPS increased IL-6 (Fig. 4). IFN-␣ priming in hMSC and macrophages increased production, as detected by ELISA on supernatants of mMSC ac- expression of TLR3 and responsiveness to poly(I:C). By con- tivated for 48 h (Fig. 6A). IFN-␣ or IFN-␥ priming combined with trast, TNF-␣ priming up-regulated TLR7 expression in hMSC; TLR activation resulted in a 19- to 180-fold additional increase in however, hMSC remained unresponsive to guardiquidimod, a IL-6 production compared with single treatments ( p Ͻ 0.001). TLR7 ligand (data not shown).

Real-time PCR analyses performed on mMSC primed or not for The most notable difference between MSC and macrophages http://www.jimmunol.org/ 18 h with IFN-␥ and/or activated with LPS for 4 h demonstrated was the low levels of IL-12B expression as well as the marginal that the dual treatment increased transcription of mRNA encoding production of IL-12p75 and TNF-␣ by the former upon TLR3 or the neutrophil-attracting mouse chemokines CXCL1 and CXCL2, TLR4 activation, even after IFN-␣ or IFN-␥ priming. TLR3 and as well as monocyte-macrophage or leukocyte chemokines such as TLR4 use MyD88-Toll/IL-1 receptor domain-containing adap- CCL2, CCL5, CCL7, CCL8, and CCL9, with the exception of tor protein and Toll/IL-1 receptor domain-containing adaptor CCR1 and/or CCR5-binding chemokines CCL3 and CCL4 (Fig. protein inducing IFN-␤ adaptor molecules, respectively, to ac- 6B). By contrast, secretion of TNF-␣ or IL-12 in activated mMSC tivate NF-␬B transcription factors (33). It was suggested that remained low compared with macrophages (Fig. 6A). the constitutive expression of the c-Rel NF-␬B subunit by DC In vivo inflammatory chemoattracting properties of MSC were and macrophages plays a determinant role for their capacity to by guest on September 24, 2021 assessed by injecting C57BL/6 mice with extensively washed and produce of IL-12 and to induce T cell cross-priming (27). By Matrigel-embedded mMSC. Two days later, plugs were excised, contrast, the ubiquitous p65/RelA NF-␬B subunit is able to ac- dissolved by collagenase, and cells larger than 7 ␮m, to exclude tivate IL-1, IL-6, and IL-8 promoters. In line with these obser- erythrocytes, were automatically counted. Increases of 114 Ϯ 30% vations, we report that hMSC displayed low basal levels of and 146 Ϯ 35% in cell numbers (not statistically significant) were c-Rel compared with macrophages, while expression of RelA observed in implants after injection of IFN-␥- or LPS-treated MSC was detected in both cell types. Further investigations are re- compared with nontreated MSC. Combined treatment with IFN-␥ quired to identify mechanisms explaining the lack of production priming and LPS resulted in an increase of 189 Ϯ 52% in cell of TNF-␣ by hMSC. It was observed that the human TNFA numbers ( p Ͻ 0.01; data not shown). In all plugs, infiltrating im- promoter contains p50/RelA binding sites but its activation mune cells consisted of Ly6C/6Cϩ granulocytes, i.e., neutrophils seems to rather depend on an enhancer complex containing and eosinophils, NK1.1ϩ NK cells, CD11cϩ DC cells, and CD3ϩ NFAT, Ets/Elk, Sp1, ATF-2-Jun, and the coactivator T cells as analyzed by flow cytometry (Fig. 6C). No CD14ϩ mono- CBP and p300 (34). cytes/macrophages and CD117ϩ mast cells were detected (data not Several mechanisms may account for the effect of IFN-␣ or shown). Implantation of LPS-activated mMSC, especially cells IFN-␥ on the tonic signaling through TLR. As reported in DC (9), primed with IFN-␥, resulted in a significant increase in granulo- we observed in hMSC that IFN-␥ alone induced the transcriptional cytes and NK cell infiltration (Fig. 6C). Only one of the mice activation of IL-12A. IFN-␥-induced IRF-1 DNA binding was injected with IFN-␥ plus LPS-treated mMSC displayed an increase shown to act in synergy with c-Rel/p50 for optimal IL-12A pro- in infiltrating CD3ϩ T cells (Fig. 6C). Immune cell infiltration was moter activation (30). This synergy was not clear when studies reduced by 5 days (data not shown), suggesting it correlated with were performed with IL-12B instead of IL-12A (30). In the present the transient production of inflammatory mediators by activated study, we observed that IFN-␥, and to a lesser extent with IFN-␣, mMSC. These reports support the notion that mMSC closely par- up-regulated levels of IRF-1 in hMSC. Among other factors pos- allel hMSC for their response to TLR3 or TLR4 activation, which sibly implicated in the IFN-␥ and TLR pathway cross-talk in MSC was boosted by IFN-␣ or IFN-␥ priming, resulting in an in vivo is IRF-8. IRF-8 expression is up-regulated by IFN-␥ and IRF-8 inflammation and recruitment of innate immune effectors. was described to bind to the mouse il-12b promoter (35), as well as to increase TLR signaling and activation of ERK and JNK by Discussion interacting with TNFR-associated factor 6 (36). Since TLR are main players in the induction of the innate and What are the physiological outcomes and possible therapeutic subsequent adaptive immune responses, we studied the effects of applications of in vitro pretreatments with TLR ligands combined TLR activation in MSC. We observed that hMSC and macro- or not with IFN priming in MSC? On the one hand, we observed phages expressed TLR3 and TLR4 at comparable levels. Activa- here that in vitro stimulation of mMSC with LPS before injection 7972 TLR-MEDIATED ACTIVATION IN MSC in Matrigel led to increased recruitment of NK and granulocytes. 2. Stagg, J., and J. Galipeau. 2007. Immune plasticity of bone marrow-derived mes- Infiltration was more noticeable when mMSC were primed with enchymal stromal cells. Handb. Exp. Pharmacol. 45–66. 3. Nasef, A., N. Ashammakhi, and L. Fouillard. 2008. Immunomodulatory effect of IFN-␥ before LPS activation. Supernatants from IFN-␥-primed mesenchymal stromal cells: possible mechanisms. Regen. Med. 3: 531–546. and LPS-activated hMSC induced human neutrophil Transwell mi- 4. Le Blanc, K., C. Tammik, K. Rosendahl, E. Zetterberg, and O. Ringden. 2003. gration. Correspondingly, ELISA or gene expression analyses re- HLA expression and immunologic properties of differentiated and undifferenti- ated mesenchymal stem cells. Exp. Hematol. 31: 890–896. ported that production of chemokines or expression of encoding 5. Stagg, J., S. Pommey, N. Eliopoulos, and J. Galipeau. 2006. -␥-stim- mRNAs was increased by the dual stimulation in hMSC and ulated marrow stromal cells: a new type of nonhematopoietic antigen-presenting mMSC. Among factors produced by TLR-activated MSC and cell. Blood 107: 2570–2577. 6. Chan, J. L., K. C. Tang, A. P. Patel, L. M. Bonilla, N. Pierobon, N. M. Ponzio, deemed for a role in chemoattraction of these innate immune ef- and P. Rameshwar. 2006. Antigen-presenting property of mesenchymal stem fectors are IL-8/CXCL8 in humans or CXCL1 and CXCL2 in cells occurs during a narrow window at low levels of interferon-␥. Blood 107: mice, and CCL2, CCL5, CCL7, CCL8, and CCL9 that are che- 4817–4824. 7. Romieu-Mourez, R., M. Francois, M. N. Boivin, J. Stagg, and J. Galipeau. 2007. mokines produced during inflammation and acting as essential Regulation of MHC class II expression and antigen processing in murine and chemotactic and activators for neutrophils (37) and monocytes, human mesenchymal stromal cells by IFN-␥, TGF-␤, and cell density. J. Immu- DC, NK, or some T cell subsets (38), respectively. In addition, nol. 179: 1549–1558. ␣ ␥ 8. Hawiger, D., K. Inaba, Y. Dorsett, M. Guo, K. Mahnke, M. Rivera, J. V. Ravetch, IFN- and IFN- stimulation was previously reported to up-reg- R. M. Steinman, and M. C. Nussenzweig. 2001. Dendritic cells induce peripheral ulate Ag- presenting functions in MSC (5–7); however, this up- T cell unresponsiveness under steady state conditions in vivo. J. Exp. Med. 194: regulation was unaffected by the dual stimulation with TLR li- 769–779. 9. Trinchieri, G. 2003. -12 and the regulation of innate resistance and gands (data not shown). Hence, these data suggest that in vitro adaptive immunity. Nat. Rev. Immunol. 3: 133–146. pharmacological modulation of TLR3- or TLR4-mediated activity 10. D’Andrea, A., M. Rengaraju, N. M. Valiante, J. Chehimi, M. Kubin, M. Aste, Downloaded from on MSC could be exploited as a means to further increase their S. H. Chan, M. Kobayashi, D. Young, E. Nickbarg, et al. 1992. Production of ␥ natural killer cell stimulatory factor () by peripheral blood mono- IFN- -up-regulated APC functions through the formation of an nuclear cells. J. Exp. Med. 176: 1387–1398. appropriate inflammatory milieu after immunization with MSC- 11. Ma, X., J. M. Chow, G. 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