Pascale Louis-Plence

Mesenchymal Stem Cells Inhibit the Differentiation of Dendritic Cells Through an Interleukin‐6‐Dependent Mechanism

Mesenchymal stem cells (MSC) are of particular interest for their potential clinical use in tissue engineering as well as for their capacity to reduce the incidence and severity of graft‐versus‐host disease in allogeneic transplantation. We have previously shown that MSC‐mediated immune suppression acts via the secretion of soluble factor(s) induced upon stimulation. The aim of this study was to identify the molecule(s) involved and the underlying mechanism(s). We show that murine MSC secrete high levels of interleukin (IL)‐6 and vascular endothelial growth factor, which are directly correlated to the inhibition of T‐cell proliferation. The T‐cell activation is partially restored upon addition of a neutralizing anti‐IL‐6 antibody or the prostaglandin E2 inhibitor indomethacin. Interestingly, no indoleamine 2,3‐dioxygenase activity was detected in our conditions. Instead, we show that MSC reduce the expression of major histocompatibility complex class II, CD40, and CD86 costimulatory molecules on mature dendritic cells (DC), which was responsible for a decrease in T‐cell proliferation. Moreover, we show that the differentiation of bone marrow progenitors into DC cultured with conditioned supernatants from MSC was partly inhibited through the secretion of IL‐6. Altogether, these data suggest that IL‐6 is involved in the immunoregulatory mechanism mediated by MSC through a partial inhibition of DC differentiation but is probably not the main mechanism.

Transcriptional profiles discriminate bone marrow-derived and synovium-derived mesenchymal stem cells

Previous studies have reported that mesenchymal stem cells (MSC) may be isolated from the synovial membrane by the same protocol as that used for synovial fibroblast cultivation, suggesting that MSC correspond to a subset of the adherent cell population, as MSC from the stromal compartment of the bone marrow (BM). The aims of the present study were, first, to better characterize the MSC derived from the synovial membrane and, second, to compare systematically, in parallel, the MSC-containing cell populations isolated from BM and those derived from the synovium, using quantitative assays. Fluorescent-activated cell sorting analysis revealed that both populations were negative for CD14, CD34 and CD45 expression and that both displayed equal levels of CD44, CD73, CD90 and CD105, a phenotype currently known to be characteristic of BM-MSC. Comparable with BM-MSC, such MSC-like cells isolated from the synovial membrane were shown for the first time to suppress the T-cell response in a mixed lymphocyte reaction, and to express the enzyme indoleamine 2,3-dioxygenase activity to the same extent as BM-MSC, which is a possible mediator of this suppressive activity. Using quantitative RT-PCR these data show that MSC-like cells from the synovium and BM may be induced to chondrogenic differentiation and, to a lesser extent, to osteogenic differentiation, but the osteogenic capacities of the synovium-derived MSC were significantly reduced based on the expression of the markers tested (collagen type II and aggrecan or alkaline phosphatase and osteocalcin, respectively). Transcription profiles, determined with the Atlas Human Cytokine/Receptor Array, revealed discrimination between the MSC-like cells from the synovial membrane and the BM-MSC by 46 of 268 genes. In particular, activin A was shown to be one major upregulated factor, highly secreted by BM-MSC. Whether this reflects a different cellular phenotype, a different amount of MSC in the synovium-derived population compared with BM-MSC adherent cell populations or the impact of a different microenvironment remains to be determined. In conclusion, although the BM-derived and synovium-derived MSC shared similar phenotypic and functional properties, both their differentiation capacities and transcriptional profiles permit one to discriminate the cell populations according to their tissue origin.

Microenvironmental changes during differentiation of mesenchymal stem cells towards chondrocytes

Chondrogenesis is a process involving stem-cell differentiation through the coordinated effects of growth/differentiation factors and extracellular matrix (ECM) components. Recently, mesenchymal stem cells (MSCs) were found within the cartilage, which constitutes a specific niche composed of ECM proteins with unique features. Therefore, we hypothesized that the induction of MSC differentiation towards chondrocytes might be induced and/or influenced by molecules from the microenvironment. Using microarray analysis, we previously identified genes that are regulated during MSC differentiation towards chondrocytes. In this study, we wanted to precisely assess the differential expression of genes associated with the microenvironment using a large-scale real-time PCR assay, according to the simultaneous detection of up to 384 mRNAs in one sample. Chondrogenesis of bone-marrow-derived human MSCs was induced by culture in micropellet for various periods of time. Total RNA was extracted and submitted to quantitative RT-PCR. We identified molecules already known to be involved in attachment and cell migration, including syndecans, glypicans, gelsolin, decorin, fibronectin, and type II, IX and XI collagens. Importantly, we detected the expression of molecules that were not previously associated with MSCs or chondrocytes, namely metalloproteases (MMP-7 and MMP-28), molecules of the connective tissue growth factor (CTGF); cef10/cyr61 and nov (CCN) family (CCN3 and CCN4), chemokines and their receptors chemokine CXC motif ligand (CXCL1), Fms-related tyrosine kinase 3 ligand (FlT3L), chemokine CC motif receptor (CCR3 and CCR4), molecules with A Disintegrin And Metalloproteinase domain (ADAM8, ADAM9, ADAM19, ADAM23, A Disintegrin And Metalloproteinase with thrombospondin type 1 motif ADAMTS-4 and ADAMTS-5), cadherins (4 and 13) and integrins (α4, α7 and β5). Our data suggest that crosstalk between ECM components of the microenvironment and MSCs within the cartilage is responsible for the differentiation of MSCs into chondrocytes.

Immature Dendritic Cells Suppress Collagen-Induced Arthritis by In Vivo Expansion of CD49b+ Regulatory T Cells

Dendritic cells (DCs) are specialized APCs with an important role in the initiation and regulation of immune responses. Immature DCs (iDCs) reportedly mediate tolerance in the absence of maturation/inflammatory stimuli, presumably by the induction of regulatory T cells. In this study, we show for the first time that repetitive iDC injections trigger the expansion of a novel regulatory population with high immunomodulatory properties, able to protect mice from collagen-induced arthritis. These regulatory T cells are characterized by the expression of the CD49b molecule and correspond to a CD4+ α-galactosylceramide/CD1d-nonrestricted T cell population producing IL-10. Adoptive transfer of <105 TCRβ+CD49b+ cells isolated from the liver of iDCs-vaccinated mice, conferred a complete protection against arthritis. This protection was associated with an attenuation of the B and T cell response associated with a local secretion of IL-10. Thus, together these data demonstrate that iDCs can expand and activate a novel regulatory population of CD49b+ T cells, with high immunosuppressive potential able to mediate protection against a systemic autoimmune disease.

Immunomodulatory Dendritic Cells Inhibit Th1 Responses and Arthritis via Different Mechanisms

Dendritic cells (DCs) are professional APCs which have the unique ability to present both foreign and self-Ags to T cells and steer the outcome of immune responses. Because of these characteristics, DCs are attractive vehicles for the delivery of therapeutic vaccines. Fully matured DCs are relatively well-defined and even used in clinical trials in cancer. DCs also have the potential to influence the outcome of autoimmunity by modulating the underlying autoimmune response. To gain a better appreciation of the abilities and mechanisms by which immunomodulatory DCs influence the outcome of T cell responses, we studied several immunomodulatory DCs (TNF-, IL-10-, or dexamethasone-stimulated bone marrow-derived DCs) side by side for their ability to modulate T cell responses and autoimmune diseases. Our data show that these differentially modulated DCs display a different composition of molecules involved in T cell activation. Although, all DC subsets analyzed were able to inhibit the induction of collagen-induced arthritis, the modulation of the underlying immune response was different. Vaccination with TNF- or IL-10-modulated DCs altered the Th1/Th2 balance as evidenced by the induction of IL-5- and IL-10-secreting T cells and the concomitant reduction of the IgG2a-IgG1 ratio against the immunizing Ag. In contrast, DCs modulated with dexamethasone did not affect the ratio of IL-5-producing vs IFN-γ-producing T cells and tended to affect the Ab response in a nonspecific manner. These data indicate that distinct mechanisms can be used by distinct DC subsets to change the outcome of autoimmunity.

Interleukin‐6 Receptor Blockade Enhances CD39+ Regulatory T Cell Development in Rheumatoid Arthritis and in Experimental Arthritis

The rationale for blocking interleukin‐6 (IL‐6) in rheumatoid arthritis (RA) lies chiefly in the proinflammatory effect of this cytokine. Few studies have evaluated the consequences of anti–IL‐6 receptor (IL‐6R) antibody treatment on Treg cells. This study was undertaken to elucidate the mechanism of action of anti–IL‐6R antibody treatment by studying the effects on Treg cells in an experimental arthritis model and in patients with RA.

A3.10 IL-6 Receptor Blockade Enhances CD39+ Regulatory T-Cell Development in Rheumatoid Arthritis and in Experimental Arthritis

Background and Objectives Studies have demonstrated the clinical efficacy of tocilizumab, a humanised anti-IL-6 receptor (R) antibody (Ab), in patients with rheumatoid arthritis (RA). The rational for blocking IL-6 in this disease mainly lays on the pro-inflammatory role of this cytokine in the disease. However, only few works have studied the consequences of anti-IL-6R treatment on Tregs cells and mainly focuses on their frequency. Our objective was to elucidate anti-IL-6R mode of action on Tregs in RA patients treated with tocilizumab and in a RA model. Methods Mice with collagen-induced arthritis (CIA) were treated at day 0 by MR16–1 (a rat anti-mouse IL-6 receptor monoclonal Ab provided by Chugai Pharmaceutical Co. LTD, Japan) and the evolution of CD4+ FoxP3+ Tregs during arthritis course was assessed at key time points (day 8–18–28 and 42 after CIA induction) by studying their number, frequency and phenotype (expression of GITR, ICOS, Helios, CD62L, CTLA-4 and CD39) in lymph nodes (LN), thymus and spleen by flow cytometry. Numerical analysis of Th17 and Th1 cells was also performed by flow cytometry. Twenty patients with severe and active RA were recruited and treated with 8 mg/kg of tocilizumab monthly. Peripheral blood was recovered at day 0, as well as 1 and 3 month, and Th17and Tregs were analysed by flow cytometry. Results Clinical and histological evaluation of arthritides in mice treated with anti-mouse IL-6R mAb showed, as expected, a less severe disease as compared to control Ig treated mice. Th17 frequency was unchanged, but Tregs frequency was enhanced in the LN of MR16–1 treated mice. In the thymus, we observed an enhanced frequency of Tregs CD4+CD8−FoxP3+. Tregs phenotype was also modified in treated mice, with an increased frequency of CD39+ Tregs (LN and spleen), suggesting an enhanced ATP hydrolysis immunosuppressive activity of Tregs. In RA patients, Th17 frequencies were not modified by tocilizumab therapy and did not differ between responders and non-responders. Interestingly, CD39+ Treg cell among CD4+ cells frequencies were significantly higher in responders than in non-responders after 3 months of tocilizumab therapy. Conclusions Tregs, but not Th17, are modified by anti-IL-6R treatment in both CIA and RA. These results support a beneficial effect in RA of treatments responsible for CD39+ Tregs enhancement and emphasise the relevance of the monitoring cell populations after cytokine blockade used to treat arthritis.

Antitumoral Activity and Osteogenic Potential of Mesenchymal Stem Cells Expressing the Urokinase-Type Plasminogen Antagonist Amino-Terminal Fragment in a Murine Model of Osteolytic Tumor

Prostate cancer metastasis to bone results in mixed osteolytic and osteoblastic lesions associated with high morbidity, and there is mounting evidence that the urokinase‐type plasminogen system is causatively involved in the progression of prostate cancer. Adult mesenchymal stem cells (MSCs) are promising tools for cell‐mediated gene therapy with the advantage of osteogenic potential, a critical issue in the case of osteolytic metastases. In this study, we evaluated the therapeutic use of engineered murine MSCs for in vivo delivery of the urokinase‐type plasminogen antagonist amino‐terminal fragment (hATF) to impair osteolytic prostate cancer cell progression in bone and to repair bone lesions. Bioluminescence imaging revealed that both primary MSCs and the MSC line C3H10T1/2 (C3) expressing hATF (MSC‐hATF) significantly inhibited intratibial PC‐3 Luciferase (Luc) growth following coinjection in SCID mice. Furthermore, microcomputed tomography imaging of vascular network clearly demonstrated a significant decrease in tumor‐associated angiogenesis and a protection from tumor‐induced osteolysis in MSC‐hATF‐treated mice. Importantly, the osteogenic potential of MSC‐hATF cells was unaffected, and an area of new bone formation was evidenced in 60% of animals. Together, these data support the concept of MSC‐based therapy of tumor osteolysis disease, indicating that MSCs may combine properties of vehicle for angiostatic agent with osteogenic potential.

The control of dendritic cell maturation by pH-sensitive polyion complex micelles.

Double-hydrophilic block copolymer micelles were designed as vectors for ex vivo dendritic cell engineering to improve the delivery of therapeutic molecules in such immune cells. Polymethacrylic acid-b-polyethylene oxide (PMAA(2100)-b-POE(5000))/poly-L-lysine micelles were optimised and showed a hydrodynamic diameter of 30 nm with a peculiar core organised with hydrogen bonds as well as hydrophobic domains. The micelles proved high stability in physiological conditions (pH and ionic strength) and were also able to disassemble under acidic conditions mimicking acidic endolysosomes. The efficient endocytosis of the optimised micelles tested on bone marrow-derived dendritic cells was monitored by fluorescence-activated cell sorting and microscopy analysis. Finally, the micelle biocompatibility permitted a complete control of the dendritic cell-maturation process widening the therapeutical potential of such engineered dendritic cells for cancer vaccines as well as for immunomodulation in autoimmune diseases.

A comparative study on intra-articular versus systemic gene electrotransfer in experimental arthritis

Electric pulse mediated gene transfer has been applied successfully in vivo for increasing naked DNA administration in various tissues. To achieve non‐viral gene transfer into arthritic joint tissue, we investigated the use of electrotransfer (ET). Because anti‐inflammatory cytokine strategies have proven efficient in experimental models of arthritis, we compared the therapeutic efficiency of local versus systemic delivery of the interleukin‐10 (IL‐10) using in vivo ET.