Toll-like receptor 9 ligands enhance mesenchymal stem cell invasion and expression of matrix metalloprotease-13

doi:10.1016/j.yexcr.2010.05.024

Experimental Cell Research

Volume 316, Issue 16, 1 October 2010, Pages 2676-2682

https://doi.org/10.1016/j.yexcr.2010.05.024 Get rights and content

Abstract

Human mesenchymal stem cells (hMSCs) are multipotent cells that are found in the bone marrow. Inflammation and tissue damage mobilize MSCs and induce their migration towards the damaged site through mechanisms that are not well defined. Toll-like receptor-9 (TLR9) is a cellular receptor for microbial and vertebrate DNA. Stimulation of TLR9 induces inflammatory and invasive responses in TLR9-expressing cells. We studied here the expression of TLR9 in human MSCs and the effects of synthetic TLR9-agonists on their invasion. Constitutive expression of TLR9 was detected in human MSCs but the expression was suppressed when MSCs were induced to differentiate into osteoblasts. Using standard invasion assays and a novel organotypic culture model based on human myoma tissue, we discovered that stimulation with the TLR9 agonistic, CpG oligonucleotides increased the invasion capacity of undifferentiated MSCs. Simultaneously, an increase in MMP-13 synthesis and activity was detected in the CpG-activated MSCs. Addition of anti-MMP-13 antibody significantly diminished the CpG-induced hMSC invasion. We conclude that treatment with TLR9-ligands increases MSC invasiveness, and this process is at least partially MMP-13-mediated.

Introduction

Human mesenchymal stem cells (MSCs) are multipotent cells that can be found in many tissues, and most commonly they are isolated and cultured from bone marrow. Depending on the surrounding conditions, MSCs can further differentiate into osteoblasts, chondroblasts or adipocytes, and also into other cell lineages [1]. Physiological stress factors, such as inflammatory processes or tissue injuries, initiate cytokine cascades that can mobilize and attract mesenchymal stem cells to begin the tissue regeneration process [2]. During this process, MSCs are mobilized from the bone marrow and they migrate to the wounded or damaged sites. The mechanisms that control MSC homing into regenerating tissues are not clear but several different cytokines might be involved [3], [4]. Also malignancies cause secretion of inflammatory cytokines, and several reports from the recent years have demonstrated that MSCs home towards tumor tissues as well [5], [6], [7], [8]. The exact mechanisms how the mesenchymal stem cells are recruited, and the molecules that can enhance their capacities to cross tissue barriers and migrate towards the injured site or tumors, are, however, poorly understood.

It has been suggested that human MSC migration is increased through toll-like receptor (TLR) activation [9]. Toll-like receptors are a family of transmembrane proteins that were initially discovered to recognize pathogen-associated molecular patterns. Their activation triggers the innate immune system that leads to expression of various proinflammatory cytokines (reviewed in [10]). TLR9 is a cellular receptor for microbial and vertebrate DNA [10]. In addition to the inflammatory effects, stimulation of TLR9 with bacterial DNA or its synthetic analog (CpG-DNA sequence containing synthetic oligonucleotides) can induce cell migration and invasion, which are mediated via TLR9. For example TLR9 activation has been shown to increase invasion of the breast, brain and prostate cancer cells [11], [12], [13]. Furthermore, this invasion in cancer cells was shown to be mediated via matrix metalloprotease-13 (MMP-13) activation [11]. MMP activity, on the other hand, has been associated with tissue destruction during cancer invasion [14], [15].

The aim of this study was to investigate TLR9 expression in human MSCs, to evaluate the effects of TLR9-ligands on MSC invasion capacity, and to elucidate the mechanisms involved, in particular whether MMPs are involved in this process.

Section snippets

Cell culture

Human bone marrow-derived mesenchymal stem cells were harvested and cultured as described previously [16]. The human bone marrow cells used in this particular study were obtained from patients who were operated for hip fracture or osteoarthritis. The ethical committee of Oulu University Hospital has approved the study protocol and the patients gave their written consent for participation in the study. MSCs were separated by virtue of their adherence to plastic, and after two days of initial

Undifferentiated human MSCs express TLR9

Previous reports of TLR9 expression in human MSC have been contradictory. For example it has been shown that human mesenchymal stem cells express TLR9 [20], but the TLR9 expression is lower compared to the other TLRs [10]. However, in a report by Pevsner-Fischer et al. [21], mouse MSCs were found to express TLR1-8, but not TLR9. Therefore, we first aimed to verify TLR9 expression by several lines of human MSCs. We observed that all the studied MSC cell lines expressed TLR9. The expression was

Acknowledgments

The expert technical assistance of Minna Savilampi, Sanna Juntunen, Eeva-Maija Kiljander, Maija-Leena Lehtonen and Merja Tyynismaa is gratefully acknowledged. Markku Santala is thanked for the surgical collection of myoma tissues. Ahti Niinimaa is acknowledged for statistical help and advice. This study was partially funded by grants from the Academy of Finland (TS, PN and PK), the Finnish Cancer Foundation (KSS and TS), Finnish Cultural Foundation (PN), Finnish Dental Society Apollonia (TS and

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Patients with immune deficiencies from cancers and associated treatments represent a growing population within the intensive care unit with increased risk of morbidity and mortality from sepsis. Mesenchymal stromal cells (MSCs) are an integral part of the hematopoietic niche and express toll-like receptors, making them candidate cells to sense and translate pathogenic signals into an innate immune response. In this study, we demonstrate that MSCs administered therapeutically in a murine model of radiation-associated neutropenia have dual actions to confer a survival benefit in Pseudomonas aeruginosa pneumo-sepsis that is not from improved bacterial clearance. First, MSCs augment the neutrophil response to infection, an effect that is enhanced when MSCs are preconditioned with CpG oligodeoxynucleotide, a toll-like receptor 9 agonist. Using cytometry by time of flight, we identified proliferating neutrophils (Ly6G^lowKi-67⁺) as the main expanded cell population within the bone marrow. Further analysis revealed that CpG-MSCs expand a lineage restricted progenitor population (Lin⁻Sca1⁺C-kit⁺CD150⁻CD48⁺) in the bone marrow, which corresponded to a doubling in the myeloid proliferation and differentiation potential in response to infection compared with control. Despite increased neutrophils, no reduction in organ bacterial count was observed between experimental groups. However, the second effect exerted by CpG-MSCs is to attenuate organ damage, particularly in the lungs. Neutrophils obtained from irradiated mice and cocultured with CpG-MSCs had decreased neutrophil extracellular trap formation, which was associated with decreased citrullinated H3 staining in the lungs of mice given CpG-MSCs in vivo. Thus, this preclinical study provides evidence for the therapeutic potential of MSCs in neutropenic sepsis.
TLR-induced immunomodulatory cytokine expression by human gingival stem/progenitor cells
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The differential expression of immunosuppressive or promotive molecules was reported to be controlled by two main factors; namely the MSCs’ tissues of origin and the TLR ligands activating them [6]. TLR activation in MSCs has been reported to promote a receptor internalization and to initiate the intracellular pathways of NFκβ, MAPK, and AKT [9,36,37], as well as to affect different functions of MSCs [5,38,39]. Studies on TLR-induced immunomodulation of MSCs presented inconsistent results.
During therapeutic application, mesenchymal stem cells (MSCs) may interact with their environment via their expressed toll-like-receptors (TLRs) leading to pro- or anti-inflammatory immune responses. The present study aimed to describe the gingival margin-derived stem/progenitor cells’ (G-MSCs) TLR-induced immune regulatory response to specific TLR agonists.
Gingival cells were obtained, immunomagnetically sorted via anti-STRO-1 antibodies and seeded out to achieve colony forming units (CFUs). G-MSCs were investigated for stem cell characteristics and TLR expression. Specific TLR agonists were applied and m-RNA expression of pro- and anti-inflammatory factors was analyzed via real-time polymerase chain reaction.
G-MSCs showed all characteristics of stem/progenitor cells. All TLR agonists induced pro-inflammatory cytokines, except for the TLR3 agonist, which significantly promoted the anti-inflammatory response. (p ⩽ 0.05, Wilcoxon-Signed-Ranks-Test).
TLR-induced immunomodulation by G-MSCs could impact their therapeutic potential in vivo. Two distinctive pro-inflammatory and an anti-inflammatory TLR-induced phenotypes of G-MSCs become noticeable in this study.
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During inflammation, the ligation of TLR9 attracts MSCs into injured tissues. Nurmenniemi et al. [98] stated that invasion and migration of BM-MSCs may be mediated by increasing synthesis and activation of MMP-13. In agreement with their study, Merrell et al. [99] reported that the CpG-ODN-primed TLR9-expressing breast cell increased the activation of MMP-13.
Understanding the role of toll-like receptors (TLRs) in the immunomodulation potential, differentiation, migration, and survival of mesenchymal stem cells (MSCs) is absolutely vital to fully exploiting their MSC-based therapeutic potential. Furthermore, through recognition of exogenous or endogenous ligands produced upon injury, TLRs have been linked to allograft rejection and maintenance of chronic inflammatory diseases, including Crohn’s disease, rheumatoid arthritis. Characterizing the effect of TLRs in biological control of MSCs fate and function could improve our knowledge about the MSC-based cell therapy and immunotherapy. In this paper, we outline the impacts of TLR activation and mechanisms on MSCs immunomodulatory functions, differentiation, migration, and survivability. Moreover, we indicate that the expression patterns of TLRs in MSCs from different sources.
TLR4- and TLR9-dependent effects on cytokines, cell viability, and invasion in human bladder cancer cells
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In contrast, we observed strong induction of cell invasion when employing CpG-A and CpG-B in UMUC3 and RT112 cells. This observation is in accordance with CpG-ODN studies performed in glioma [22], breast cancer cells [18,20], prostate cancer cells [19], and mesenchymal stem cells [29]. Noteworthy, CpG-ODN-induced invasion was shown to be independent of the conventional MyD88 pathway in breast cancer cells [18] and in prostate cancer cells [19].
Adjuvant immunotherapy of bladder cancer by instillation of bacillus Calmette-Guérin (BCG) is highly recommended within certain groups of non–muscle-invasive stages but only partially effective. Toll-like receptors (TLRs) TLR4 and TLR9 likely mediate BCG effects by triggering innate systemic immune cell responses. In addition, TLR4 and TLR9 expressed in bladder cancer cells may contribute to the outcome of BCG treatment. Here, we studied the expression and function of TLR4 and TLR9 in human bladder cancer cell lines.
TLR4 and TLR9 messenger RNA and protein levels were determined by real-time reverse transcription polymerase chain reaction and Western blot. Selected cell lines were analyzed with respect to cytokine induction, proliferation, and cell invasion after addition of BCG, TLR4-specific agonist lipopolysaccharide (LPS), or TLR9 agonist (CpG-oligodeoxynucleotide [ODN]).
TLR4 and TLR9 were expressed quite heterogeneously in human bladder cancer cells. BCG caused induction of interleukin (IL)-6 or IL-8 in BFTC905 and T24 cells as representatives for TLR4-/TLR9-expressing cells. The study aimed to dissect TLR4- and TLR9-mediated effects. For functional analysis of TLR4 with LPS, we selected T24 and BFTC905 cells with high and undetectable TLR4 levels, respectively. For TLR9 analysis with CpG-ODN, we selected UMUC3 and RT112 cells with high and low TLR9 levels, respectively. Addition of LPS caused significant induction of TNFα and IL-6 messenger RNA in T24 cells but not in BFTC905 cells. Addition of CpG-ODN induced interferon ß (INFß), IL-8, tumor necrosis factor α (TNFα) and the angiogenic factors vascular endothelial growth factor-A and placental growth factor in UMUC3 cells; whereas in RT112 cells, induction of IL-8 and TNFα was noticed. Interestingly, addition of CpG-ODN significantly reduced cell viability and increased cell invasion in UMUC3 and RT112 cells.
Our findings demonstrate that bladder cancer cell lines express functional TLR4 and TLR9 with possible effects on cancer progression and outcome of BCG-based immunotherapy.
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Mesenchymal stromal cells (MSC) constitutively express low levels of human leukocyte antigen-G (HLA-G), which has been shown to contribute to their immunomodulatory and anti-inflammatory properties. Here, we hypothesized that overexpression of HLA-G on bone marrow-derived MSC would improve their immunomodulatory function, thus increasing their therapeutic potential. Therefore, we investigated which gene transfer system is best suited for delivering this molecule while maintaining its immunomodulatory effects. We performed a side-by-side comparison between three nonviral plasmid-based platforms (pmax-HLA-G1; MC-HLA-G1; pEP-HLA-G1) and a viral system (Lv-HLA-G1) using gene transfer parameters that yielded similar levels of HLA-G1-expressing MSC. Natural killer (NK) cell–mediated lysis assays and T cell proliferation assays showed that MSC modified with the HLA-G1 expressing viral vector had significantly lower susceptibility to NK-lysis and significantly reduced T cell proliferation when compared to nonmodified cells or MSC modified with plasmid. We also show that, in plasmid-modified MSC, an increase in Toll-like receptor (TLR)9 expression is the mechanism responsible for the abrogation of HLA-G1's immunomodulatory effect. Although MSC can be efficiently modified to overexpress HLA-G1 using viral and nonviral strategies, only viral-based delivery of HLA-G1 is suitable for improvement of MSC's immunomodulatory properties.
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In agreement with these prior observations, we did not detect proteins corresponding to the majority of transcripts determined to be absent. However, we identified proteins encoded by 24 of the 187 CD-coding transcripts, and in keeping with our positive identification, other groups also detected the expression of 11 of the 24 CD transcripts (CD39, CD104, CD108, CD220, CD228, CD246, CD271, CD275, CD289, CD309, and CD324) on hBM-MSC surface [7,10,48,49]. Apart from the sample preparation steps, the high capacity of the MS instruments used (i.e., resolution, sensitivity, and accuracy) was critical in identifying proteins, which otherwise could not be detected in previous works using alternative approaches.
A comprehensive analysis of the membrane proteome is essential to explain the biology of multipotent stromal cells and identify reliable protein biomarkers for the isolation as well as tracking of cells during differentiation and maturation. However, proteomic analysis of membrane proteins is challenging and they are noticeably under-represented in numerous proteomic studies. Here we introduce new approach, which includes high pressure-assisted membrane protein extraction, protein fractionation by gel-eluted liquid fraction entrapment electrophoresis (GELFREE), and combined use of liquid chromatography MALDI and ESI tandem mass spectrometry. This report presents the first comprehensive proteomic analysis of membrane proteome of undifferentiated and culture-expanded human bone marrow multipotent stromal cells (hBM-MSC) obtained from different human donors. Gene ontology mapping using the Ingenuity Pathway Analysis and DAVID programs revealed the largest membrane proteomic dataset for hBM-MSC reported to date. Collectively, the new workflow enabled us to identify at least two-fold more membrane proteins compared to published results on hBM-MSC. A total of 84 CDs were identified including 14 CDs identified for the first time. This dataset can serve as a basis for further exploration of self-renewal, differentiation and characterization of hBM-MSC.

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¹: These authors made equal contribution.

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Research ArticleToll-like receptor 9 ligands enhance mesenchymal stem cell invasion and expression of matrix metalloprotease-13

Abstract

Introduction

Section snippets

Cell culture

Undifferentiated human MSCs express TLR9

Acknowledgments

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Am. J. Pathol.

Blood

J. Biol. Chem.

Blood

Biochimie

Cancer Gen. Cytogen.

Stem cell plasticity and carcinogenesis

Neoplasma

Recent advances into the understanding of mesenchymal stem cell trafficking

Br. J. Haematol.