Cytokine 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 http://www.jimmunol.org/content/182/12/7963 Downloaded from 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 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 24, 2021 • No Triage! 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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 cytokines 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/ cell-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 immune system 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 transcription 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