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From mesenchymal stem cell therapy to discovery of drug therapy for systemic sclerosis

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From mesenchymal stem cell therapy to discovery of drug therapy for systemic sclerosis

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Content FROM MESENCHYMAL STEM CELL THERAPY TO DISCOVERY OF DRUG THERAPY FOR SYSTEMIC SCLEROSIS by Chider Chen A Dissertation Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (CRANIO-FACIAL BIOLOGY) May 2014 Copyright 2014 Chider Chen ii Acknowledgements I would like to express my deepest gratitude to my PhD advisor and dissertation chair, Dr. Songtao Shi for his continuous support and guidance throughout my learning process in USC, especially for the concept development, project management and proposal/manuscript preparation. I will be always grateful for the ubiquitous role of Dr. Shi in all aspects of my graduate program training and future academic career. I would like to extend my gratitude to my Graduate Committee, Dr. Yang Chai, Dr. Cheng-Ming Chuong, Dr. Qi-Long Ying, Dr. Michael Paine, and Dr. Anh Le. Your encouragement and challenges from different aspects throughout my reserch projects always guide me to focus on the main streams of the concepts. I would also like to thank Dr. Kentaro Akiyama, Dr. Xingtian Xu and Dr. Alireza Moshaverinia for the scientific collaborations, technical support, and knowledgeable discussion through all my projects at CCMB. Thank you for your time for the help and for the experience of teamwork. I am also grateful to our lab members for the opportunities of scientific collaborations and all the help during my time at CCMB. I have learned so many concepts and attitude toward doing science from them. Special thanks for Dr. Chia-lin Chen, who provides me her endless support and motivation to finish my acdamic training, as well as future career development. iii Finally, many thanks are to my family in Taiwan for their uncoditional love, support and encouragement, which provides me the environment to get this far. iv Table of Contents Acknowledgements ii List of Tables vi List of Figures vii List of Symbols & Abbreviations x Abstract xiii Chapter 1: Introduction 1 1.1 Mesenchymal Stem Cells 1 1.2 Immunomodulation of Mesenchymal Stem Cells 3 1.3 Tissue Regeneration of Mesenchymal Stem Cells 5 1.4 Systemic Sclerosis 8 1.5 Summary of the Project 9 Chapter 2: Mesenchymal Stem Cell-Induced Immunoregulation Involves Fas Ligand/ Fas- Mediated T Cell Apoptosis Is Governed by Telomerase. 11 2.1 Introduction 11 2.2 Material and Methods 13 2.2.1 Animals and Antibodies 13 2.2.2 Pripary Cells Isolation 15 2.2.3 Cell proliferation assay 16 2.2.4 Flow cytometry analysis 16 2.2.5 In vitro osteogenic differentiation 17 2.2.6 In vitro adipogenic differentiation 18 2.2.7 Western blot analysis 18 2.2.8 Real-time polymerase chain reaction 19 2.2.9 Co-culture of T cells with BMMSCs 19 2.2.10 T cell migration assay 19 2.2.11 In vitro CD4 + CD25 + Foxp3 + Treg cell induction 20 2.2.12 Overexpression of Fas ligand, Fas and MCP-1 21 2.2.13 RNAi and chemical treatments 22 2.2.14 Allogenic BMMSC transplantation into Disease Animal Models 23 2.2.15 Allogenic MSC transplantation into systemic sclerosis patients 24 2.2.16 ELISA assays 24 2.2.17 FASL + BMMSCs isolation 26 2.2.18 Luciferase reporter assay 26 2.2.19 Chromatin Immunoprecipitation Assays 27 2.2.20 Statistical analysis 27 v 2.3 Results 30 2.3.1 Fas ligand (FasL) in BMMSCs induces T cell apoptosis 30 2.3.2 FasL is required for BMMSC-based therapies in animal models 42 2.3.3 Fas is required for BMMSC-mediated therapy by recruitment of T cells 49 2.3.4 MSCT induced T cell apoptosis and Treg upregulation in SSc patients 58 2.3.5 TERT is associated with BMMSC-mediated immunomodulation 61 2.3.6 TERT is required for BMMSC-mediated therapy in animal models 66 2.3.7 TERT promotes FasL expression through Wnt/ -catenin pathway 68 2.3.8 TERT serves as a transcriptional modulator for FasL expression 70 2.3.9 Aspirin pretreatment activates TERT expression 75 2.4 Discussion 83 Chapter 3: Inhibition of mTOR Ameliorates Osteopenia Phenotype in Systemic Sclerosis by Regulating Mesenchymal Stem Cell Lineage Differentiation. 89 3.1 Introduction 89 3.2 Material and Methods 90 3.2.1 Animals and antibodies 90 3.2.2 MicroCT analysis 90 3.2.3 Histology 91 3.2.4 ELISA assays 92 3.2.5 In vivo Oil red O staining 92 3.2.6 In vivo BMMSC implantation assay 92 3.2.7 RNAi and chemical reagent treatments 93 3.2.8 Flow cytometric analysis of T H 2 cells 94 3.2.9 Immunofluorescent microscopy 94 3.2.10 Luciferase reporter assay 94 3.2.11 Chromatin Immunoprecipitation Assays 95 3.2.12 In vivo Rapamycin treatment 96 3.3 Results 96 3.3.1 Fbn1 deficiency alters BMMSC lineage differentiation 96 3.3.2 Fbn1 modulates MSC lineage differentiation via IL4R /mTOR signaling 101 3.3.3 IL4R  is activated by the TGF  pathway in Fbn1 +/- BMMSCs 108 3.3.4 mTOR cKO or rapamycin treatment ameliorates osteopenia in SSc mice 111 3.3.5 Activation of IL4R/mTOR signaling in SSc patients causes osteopenia 118 3.4 Discussion 120 Chapter 4: Conclusions 124 Bibliography 127 vi List of Tables Table 1. Primers Used in Chapter 2. 28 Table 2. SSc Patient Information. 59 vii List of Figures Figure 1.1 Isolation of mesenchymal stem cells. 2 Figure 1.2 Possible sources of mesenchymal stem cells for tissue engineering. 2 Figure 1.3 A summary of the immunomodulatory property of MSCs. 4 Figure 1.4 Proinflammatory T Cells Govern MSC-Based Bone Regeneration. 7 Figure 2.1 BMMSCs induce T cell apoptosis via Fas ligand (FasL). 34 Figure 2.2 FasL is required for BMMSC-induced T cell apoptosis and upregulation of CD4 + CD25 + Foxp3 + regulatory T cells (Tregs). 36 Figure S2.1 Fas Ligand (FasL) plays an important role in BMMSC-based immunotherapy. 39 Figure S2.2 Immunomodulation property of syngenic mouse BMMSC and human BMMSC transplantation. 40 Figure S2.3 Apoptosis of transplanted BMMSCs in peripheral blood and bone marrow. 41 Figure 2.3. FasL is required for BMMSC-mediated amelioration of systemic sclerosis (SSc) phenotypes. 45 Figure 2.4. FasL plays a critical role in BMMSC-mediated immune therapy for Dextran sulfate sodium (DSS)-induced experimental colitis. 46 Figure S2.4. FasL is required for BMMSC-mediated amelioration of skin phenotype in systemic sclerosis (SSc) mice. 47 Figure S2.5. Tregs are required in BMMSC-mediated immune therapy for DSS-induced experimental colitis. 48 Figure 2.5. Fas plays an essential role in BMMSC-mediated CD3 + T cell apoptosis and up regulation of Tregs via regulating monocyte chemotactic protein 1 (MCP-1) secretion. 52 Figure 2.6. MCP-1 plays an important role in T cell recruitment. 53 viii Figure S2.6. Fas is required for ameliorating disease phenotype in induced experimental colitis and systemic sclerosis (SSc). 55 Figure S2.7. Fas and MCP-1 regulate BMMSC-mediated B cell, NK cell, and immature dendritic cell (iDC) migration in vitro. 57 Figure 2.7. Allogenic MSC transplantation induces CD3 + T cell apoptosis and Treg upregulation in systemic sclerosis (SSc) patients. 60 Figure 2.8 TERT contributes to BMMSC-mediated immunomodulation. 63 Figure S2.8 Characterization of BMMSCs from TERT -/- mice. 65 Figure S2.9 BMMSCs from 6-month-old mice (6M-BMMSCs) have reduced immunomodulatory property. 65 Figure 2.9 TERT is required for BMMSC-mediated amelioration of disease phenotype in systemic sclerosis mice. 67 Figure 2.10 TERT serves as a transcriptional modulator to regulate FasL expression in BMMSCs. 72 Figure S2.10 FasL but not immunomodulatory factors mediates immunomodulation of BMMSCs. 74 Figure S2.11 FasL is required for BMMSCs to induce T cell apoptosis in vitro. 74 Figure 2.11 Aspirin pretreatment increases immunomodulation of BMMSCs through TERT activation. 78 Figure S2.12 Aspirin pretreatment increaseed inmmunomodulation of BMMSCs through TERT activation. 79 Figure 2.12 Aspirin-pretreated BMMSCs show increased capacity to ameliorate systemic sclerosis phenotypes. 80 Figure S2.13 Aspirin treatment failed to ameliorate systemic sclerosis phenotypes. 81 Figure 3.1 Fbn1-deficient (Fbn1 +/- ) mice exhibit osteopenia phenotype. 98 Figure 3.2 Fbn1 deficiency enhances adipogenic differentiation of BMMSCs. 100 ix Figure S3.1. Western blot showed that efficacy of Fbn1 siRNA in BMMSCs. 100 Figure 3.3 Fbn1 deficiency-induced activation of IL4R /mTOR signaling regulates osteogenic/adipogenic lineage differentiation of BMMSCs. 104 Figure S3.2 T H 2 infiltration in Fbn1 +/- mice. 105 Figure S3.3 Effecacy of shRNA. 105 Figure S3.4 Rapamycin treatment inhibited mTOR downstream signaling. 106 Figure S3.5 The efficacy of P70s6k by siRNA. 106 Figure S3.6 mTOR signaling governs osteogenic/adipogenic lineage differentiation of BMMSCs. 106 Figure 3.4 TGFβ enhances IL4Rα expression in Fbn +/- BMMSCs. 110 Figure 3.5 Conditional knockout of mTOR in BMMSC/osteoblastic lineage ameliorates osteopenia phenotype by rescuing impaired osteogenic/adipogenic differentiation. 114 Figure 3.6 Rapamycin treatment ameliorates osteopenia phenotype by rescuing impaired osteogenic/adipogenic differentiation of BMMSCs in Fbn1 +/- mice. 115 Figure S3.7 Rapamycin treatment ameliorated skin fibrosis phenotype in Fbn1 +/- mice. 116 Figure S3.8 Rapamycin treatment ameliorated autoimmune index in Fbn1 +/- mice. 117 Figure S3.9 Schematic diagram. 117 Figure 3.7 IL4R /mTOR signaling is activated in systemic sclerosis patients 119 x List of Symbols & Abbreviations BMMSCs bone marrow mesenchymal stem cells NK cells natural killer cells GvHD graft versus host disease SLE systemic lupus erythematosus IBD intestinal and bowel disease FASL Fas ligand FBN1 fibrillin-1 SSc systemic sclerosis/scleroderma TGF  transforming growth factor beta MCP-1 monocyte chemotactic protein 1 Tregs regulatory T cells TERT telomerase reverse transcriptase IL4Rα Interleukin-4 receptor α mTOR the mammalian target of rapamycin IL4 Interleukin-4 CFU-F colony-forming unit–fibroblasts Oct4 octamer-4 SSEA4 stage-specific embryonic antigen-4 IL10 interleukin 10 NO nitric oxide xi IDO indoleamine 2,3-dioxygenase PGE2 prostaglandin E2 Th17 T helper 17 cells nude immunocompromised mice MHC histocompatibility complex IL2 interleukin-2 LTBP-1 latent transforming growth factor-β binding protein-1 T H 2 type 2 helper T cells STAT6 signal transducer and activator of transcription-6 HSC hematopoietic stem cell ANCs all nucleated cells -MEM alpha minimum essential medium FBS  fetal bovine serum DMEM dulbecco's modified eagle’s medium MNCs mononuclear cells CFSE carboxyfluorescein diacetate N-succinimidyl ester RT-PCR real-time polymerse chain reaction DSS dextran sulfate sodium ANA antinuclear antibody dsDNA anti-double strand DNA HD hypodermal DAI disease activity index xii CXCL-10 C-X-C motif chemokine 10 TIMP-1 tissue inhibitor of matrix metalloprotease-1 TRAP tartrate-resistant acid phosphate ELISA enzyme-linked immunosorbent assay HA/TCP hydroxyapatite/tricalcium phosphate sRANKL soluble receptor activator of nuclear factor κB ligand OPG osteoprotegerin PPAR 2 peroxisome proliferator-activated receptor gamma 2 LPL lipoprotein lipase xiii Abstract Bone marrow mesenchymal stem cells (BMMSCs) are non-hematopoietic multipotent stem cells capable of differentiating into both mesenchymal and non-mesenchymal cell types. In addition to generate bone structure to replace damaged and diseased tissues, preclinical and clinical studies have shown that BMMSCs display profound immunomodulatory functions via inhibiting the proliferation and function of several major immune cells such as T lymphocytes, B lymphocytes, natural killer (NK), and dendritic cells. Thus, systemic infusion of BMMSCs has been used to treat a variety of diseases, including acute graft-versus-host-disease (GVHD), ameliorating HSC engraftment, systemic lupus erythematosus (SLE), intestinal and bowel disease (IBD), and sepsis. However, the detailed mechanism in which BMMSCs regulate immune function is not fully understood. In the first part of Chapter 2 of this study, we show that systemic infusion of BMMSCs induces a transient T cell apoptosis via the Fas Ligand (FasL)-mediated Fas pathway and ameliorates diseased phenotypes in fibrillin-1 (FBN1) mutated systemic sclerosis (SSc) and dextran sulfate sodium-induced experimental colitis mice. The therapeutic mechanism of BMMSC infusion is associated with phagocytosis of apoptotic T cell debris, leading to a high level of macrophage-mediated transforming growth factor beta (TGF ) production and a subsequent immune tolerance. Importantly, we provided clinical evidence to show that MSC infusion in SSc patients resulted in a T cell apoptosis xiv and up-regulation of Tregs. Additionally, we revealed that Fas null BMMSCs, with normal FasL function, failed to induce T cell apoptosis and offer therapeutic effect for SS and colitis mice. Mechanistic study showed that Fas governed monocyte chemotactic protein 1 (MCP-1) secretion in BMMSCs, which plays a crucial role in the recruitment of T cells to BMMSCs for FasL-mediated apoptosis. In summary, BMMSCs use Fas to control MCP-1 secretion for the recruitment of T cells and subsequently use FasL to induce activated T cell apoptosis. Macrophages take debris of apoptotic T cells to release a high level of TGF , leading to up-regulation of regulatory T cells (Tregs) and, ultimately, immune tolerance for immunotherapies. This study uncovers the role of Fas and FasL in BMMSC-based immune therapies, which may serve as a basis to develop novel strategies for improving cell-based therapies. The significance of this study is to identify a novel mechanism of BMMSC-associated immunomodulation and immune therapy. Also, Fas and FasL collaboratively induce immune tolerance suggests a potential new mechanism that receptor/ligand coupled to execute therapeutic effect in cell-based treatment. This study covered experimental evidences for stem cell biology, molecular mechanism of BMMSC associated immunomodulation, and stem cell-based immunotherapies. In second part of Chapter 2 of this study, we showed for the first time that telomerase activity is required for maintaining the immunomodulatory properties of MSCs. Telomerase deficient MSCs lose their capacity to inhibit T-cells, activate Foxp3-positive xv Tregs, and ameliorate disease phenotype in systemic sclerosis mice, which can be rescued by overexpression of telomerase reverse transcriptase (TERT). Mechanistically, TERT combined with -catenin and BRG1 to form a complex to bind to FasL promoter and upregulate FasL expression. Upregulated FasL expression can elevate MSC immunomodulation function, as shown in our recent publication (Akiyama et al., 2012). When MSCs were treated with aspirin, their immunomodulation was significantly improved due to elevated telomerase activity and the number of MSCs required to treat systemic sclerosis mice was markedly reduced. This study has uncovered the role of telomerase in MSC-based immunotherapies and the mechanism by which TERT binds to the promoter region to upregulate FasL expression. In fact, this is the first study to link telomerase activity to immunomodulatory therapies. Overall, therefore, this study has brought forth experimental evidence for stem cell biology, the molecular mechanism(s) underlying MSC-associated immunotherapies, and pathway-guided drug therapy. In Chapter 3, we reveal that Fbn1 +/- SSc mice show osteopenia phenotype with decreased osteogenic differentiation and increased adipogenic differentiation of bone marrow MSCs by the activation of Interleukin-4 receptor α (IL4Rα)/mTOR (the mammalian target of rapamycin) signaling. We further determine that mTOR signaling blocks osteogenic differentiation via the P70S6K/RUNX2 pathway, while it elevates adipogenic differentiation via P70S6K/PPARγ2 pathway. Since significantly elevated xvi levels of Interleukin-4 (IL4) and TGF  were observed in Fbn1 +/- SSc mice, we reveal that upregulation of the IL4Rα in Fbn1 +/- MSCs is governed by TGFβ/SMAD3/SP1 signaling via SP1 biding to the Il4rα promoter. Either knockdown of IL4Rα or inhibition of mTOR can rescue Fbn1 +/- MSC function by increasing osteogenesis and reducing adipogenesis. Additionally, we showed that conditional knockout of mTOR in MSCs/osteoblasts could ameliorate osteopenia phenotype in Fbn1 +/- mice by rescuing impaired osteogenic/adipogenic differentiation. To translate our findings to potential clinical applications, we used rapamycin treatment to inhibit mTOR signaling, thereby rescuing osteopenia phenotype in Fbn1 +/- SSc mice by rescuing osteo/adipo-lineage differentiation in MSCs. This result strongly suggests that rapamycin treatment may provide an anabolic therapy for systemic sclerosis. In summary, this study establishes the FBN1/TGFβ/SP1/IL-4Rα/mTOR cascade as a key determinant of MSC lineage selection, a finding which may serve as a basis for the development of novel therapies to treat SSc. 1 Chapter 1: Introduction 1.1 Mesenchymal Stem Cells Bone marrow mesenchymal stem cells are hierarchical postnatal stem/progenitor cells capable of self-renewing and differentiating into osteoblasts, chondrocytes, adipocytes, and neural cells (Friedenstein et al., 1974; Prockop, 1997). BMMSCs are thought to be derived from the bone marrow stromal compartment, initially appearing as adherent, single colony clusters (colony-forming unit–fibroblasts [CFU-F]), and subsequently proliferating on culture dishes (Friedenstein, 1980). To date, the CFU-F assay has been considered to be one of the gold standards for determining the incidence of clonogenic BMMSC (Clarke and McCann, 1989; Friedenstein et al., 1970) (Figure 1.1). BMMSCs express a unique surface molecule profile, including expression of STRO-1, CD29, CD73, CD90, CD105, CD146, Octamer-4 (Oct4), and stage-specific embryonic antigen-4 (SSEA4) (Gang et al., 2007; Greco et al., 2007). It is generally believed that BMMSCs are negative for hematopoietic cell markers such as CD14 and CD34 (Conget and Mingull, 1999; Covas et al., 2008). MSCs can be derived from a variety of tissues, including bone marrow, amniotic fluid, adipose and dental tissues, etc (Figure 1.2). Each type of tissue-specific MSC possesses advantage and disadvantage biological characteristics. MSC-based regeneration was associated with following functional properties: (1) their capacity to differentiate into 2 several cell lineages; (2) their ability to secrete soluble factors which regulate crucial biological functions, such as proliferation and differentiation over a broad spectrum of target cells; and (3) their ability to home to damaged tissues. Based on these properties MSCs are being exploited worldwide for a wide range of potential clinical applications (Keating , 2012; Wu et al., 2007; Battiwalla and Hematti, 2012). Figure 1.1 Isolation of mesenchymal stem cells. Whole bone marrow cells are isolated and seeded on plastic culture dish for primary culture. After 2 days, non-attached cells are removed, and adhesion cells are continued culture for 14 days. CFU-F can be passed to further applications. Figure 1.2 Possible sources of mesenchymal stem cells for tissue engineering (adapted from Liu et al., 2012). 3 BMMSCs have been widely used for tissue engineering (Kwan et al., 2002; Panetta et al., 2009; Liu et al., 2011). Recently, a growing body of evidence has indicated that BMMSCs produce a variety of cytokines and display profound immunomodulatory properties (Nauta and Fibbe, 2007; Uccelli et al., 2007, 2008), perhaps by inhibiting the proliferation and function of several major immune cells such as natural killer cells, dendritic cells, T and B lymphocytes (Nauta and Fibbe, 2007; Uccelli et al., 2007, 2008; Aggarwal and Pittenger, 2005). These unique properties make BMMSCs of great interest for clinical applications in the treatment of different immune disorders (Nauta and Fibbe, 2007; Bernardo et al., 2009; Le Blanc et al., 2004; Chen et al., 2006; Sun et al., 2009). 1.2 Immunomodulation of Mesenchymal Stem Cells Mesenchymal stem cells display profound immunomodulatory properties by inhibiting proliferation and function of several major immune cells, such as dendritic cells, T and B lymphocytes, and NK cells (Nauta and Fibbe, 2007; Uccelli et al., 2007, 2008; Aggarwal and Pittenger, 2005) (Figure 1.3). These unique properties have prompted researchers to investigate mechanisms by which MSCs ameliorate a variety of immune disorders (Nauta and Fibbe, 2007; Bernardo et al., 2009). In fact, MSC-based therapy has been successfully applied in various human diseases, including GvHD, SLE, rheumatoid arthritis, autoimmune encephalomyelitis, IBD, and multiple sclerosis (Aggarwal and Pittenger, 2005; Le Blanc et al., 2004; Chen et al., 2006; Polchert et al., 2008; Sun et al., 4 2009; Augello et al., 2007; Parekkadan et al., 2008; Zappia et al., 2005; González et al., 2009; Liang et al., 2009). The immunosuppressive properties of MSCs are associated with the production of cytokines, such as interleukin 10 (IL10), nitric oxide (NO), indoleamine 2,3-dioxygenase (IDO), prostaglandin (PG) E2, and TSG-6 (Batten et al., 2006; Zhang et al., 2010; Ren et al., 2008, Sato et al., 2007; Meisel et al., 2004; Aggarwal and Pittenger, 2005; Choi et al., 2011; Roddy et al., 2011). In addition, MSC-induced immune tolerance involves upregulation of CD4 + CD25 + Foxp3 + Tregs and downregulation of proinflammatory T helper 17 (Th17) cells (Sun et al., 2009; González et al., 2009; Park et al., 2011). However, the detailed mechanism of MSC-based immunotherapy is not fully understood. Figure 1.3 A summary of the immunomodulatory property of MSCs (adapted from Liu et al., 2012). 5 1.3 Tissue Regeneration of Mesenchymal Stem Cells BMMSCs are nonhematopoietic multipotent stem cells capable of differentiating into both mesenchymal and nonmesenchymal cell types, including osteoblasts, adipocytes and chondrocytes depending on the nature of the environmental signals that they receive (Bianco et al., 2001; Owen and Friedenstein, 1988; Pittenger et al., 1999; Prockop, 1997; Caplan, 1991; Friedenstein et al., 1970, 1974). MSCs are generally considered to be poorly immunogenic. Immunological characterization of MSCs revealed that they showed only intermediate expression levels of major histocompatibility complex (MHC) classI and no, or very low, expression of MHC class II antigen and co-stimulatory molecules CD40, CD80 and CD86 (Le Blanc, 2003; Zhang et al., 2009; Majumdar et al., 2003). Expression of MHC class I prevented MSCs from behaving like NK cells, whereas the absence of co-stimulatory molecules causes a state of anergy in T cells (Ryan et al., 2005; Nauta et al., 2007). Several studies indicated neither differentiated nor undifferentiated MSCs elicit proliferation of allogeneic lymphocytes (Li et al., 2005; Le Blanc, 2003; Djouad et al., 2003). Mesenchymal stem cells have already shown promise as an alternative therapeutic option for bone tissue engineering, with several important advantages over bone grafts (Caplan, 1991; Friedenstein et al., 1970). To date, a variety of preclinical and clinical studies have shown that exogenously added BMMSCs can give rise to the generation of new bone and bone-associated tissues to replace damaged and diseased tissues by 6 assisting the regenerative capacities of endogenous cells in the defect (Caplan, 1991; Garcia-Gomez et al., 2010; Tasso et al., 2010; Bueno and Glowacki, 2009). It was recently reported that the host immune system, especially T lymphocytes, could affect MSC-mediated bone regeneration (Liu et al., 2011). When BMMSCs were subcutaneously implanted with hydroxyapatite tricalcium phosphate as a carrier, autologous MSCs failed to regenerate bone in C57BL6 (wild type) mice. However, both human and mouse BMMSCs can form bone and bone-associated hematopoietic marrow components in immunocompromised (nude) mice. These data suggest that the recipient immune system may play an inhibitory role in regulating MSC-based tissue regeneration. Further study confirmed that Pan T, CD4 + or CD4 + CD25 − T cells infusion totally blocked MSC-mediated bone formation and that CD8 + T cells partially blocked MSC-mediated bone formation in nude mice. However, administration of CD4 + CD25 + Foxp3 + regulatory T cells had no inhibitory effect on MSC-mediated bone formation (Liu et al., 2011). It has been reported that interleukin-2 (IL2)–activated NK cells and CD3/CD28-stimulated activated T cells can induce MSC apoptosis through the FAS–FAS ligand (FASL) pathway (Yamaza et al., 2008; Kogianni et al., 2004). In addition, T cells can induce MSC and osteoblast apoptosis through the FAS–FASL pathway, as observed in some models of bone-related diseases (Hess and Engelmann, 1996; Li et al., 2011; Ahuja et al., 2003; Schrum et al., 2003) (Figure 1.4). The contribution of MSCs to tissue repair is also mediated by secretion of paracrine 7 factors with angiogenic and antiapoptotic properties (Rüster et al., 2006; Kinnaird et al., 2004, 2004). These paracrine factors not only attract endothelial cells and macrophages, but they are also likely to stimulate resident stem/progenitor cells to facilitate the process for tissue repair (Chen et al., 2008; Nakanishi et al., 2008). Since the recipient immune system plays a critical role in MSC-based tissue engineering, it is reasonable to assume that MSC-based tissue regeneration could be improved by modulating recipient T cells. On the other hand, manipulation of recipient T cells may provide a therapeutic avenue for autoimmune diseases, which prompt us to use immune suppression drugs, such as Aspirin and Rapamycin for disease treatment. Figure 1.4 Proinflammatory T Cells Govern MSC-Based Bone Regeneration. MSC infusion promotes bone repair. This function is regulated by IFN-g and TNF-a produced by CD4+T cells. IFN-g exerts its inhibition on osteogenetic differentiation of MSCs through Fas to downregulate Runx2 expression and upregulate Smad6 expression. When both IFN-g and TNF-a are present, MSCs undergo apoptosis (adapted from Shi et al., 2012). 8 1.4 Systemic Sclerosis Autoimmune disease is the failure of the immune system to recognize its own organs or tissues, which allows an abnormal immune response in the organism. The common characters of autoimmune disease are hyperactivity of T and B lymphocytes, multi- organ injuries, and production of anti-nuclear antibodies. Despite improved immunosuppressive medication, most of patients continue to suffer significant morbidity, especially in treatment-refractory patients, due to lack of effective therapeutic approach and limited knowledge of immune system. Fibrillin-1, a major structural component of microfibrils in extracellular matrix, plays an important role in organ development and tissue homeostasis (Zhang et al., 1995; Sakai et al., 1986). Recent studies show that latent transforming growth factor-β binding protein-1 (LTBP-1) interacts with FBN1 through its pro-domains (Ramirez et al, 2010; Charbonneau et al., 2004). Thus, Fbn1 deficiency increases the level of activated TGF  in the intercellular microenvironment. Clinically, Fbn1 gene mutation is linked to several human diseases, including systemic sclerosis/scleroderma, Marfan’s syndrome, ectopic lentis, and the dominant form of Weill-Marchesani syndrome (Charbonneau et al., 2004; Lee et al., 1991). These diseases are usually characterized by connective tissue fibrosis and skeletal disorders, but it is unclear whether Fbn1 deficiency contributes to bone disorders in these diseases. 9 As an established SSc mouse model, Fbn1 mutant tight-skin mice (B6.Cg-Fbn1 Tsk /J; Fbn1 +/- ) show significantly reduced femoral bone mineral density and altered trabecular microarchitecture (Barisic-Dujmovic, 2007). Fbn1 +/- mice represent an autoimmune connective tissue disorder characterized by type 2 helper T cells (T H 2) infiltration and vascular damage (Gabrielli et al., 2009). Interleukin-4, a key T H 2 cytokine, plays a critical role in the regulation of fibrotic tissue deposition through the signal transducer and activator of transcription-6 (STAT6) pathway (Wynn, 2004). Although the mechanism that results in an elevated level of IL4 in Fbn1 +/- mice is unknown, downregulation of the Il4 gene in Fbn1 +/- mice can rescue the pathogenesis in fibrotic diseases, suggesting that IL4 signaling is associated with fibrotic phenotype in SSc (Kodera et al., 2002). However, it is unknown whether IL4 signaling contributes to the osteoporotic phenotype in SSc mice. 1.5 Summary of the Project In this project, Systemic Sclerosis is used as an autoimmune disease model to answer questions: 1) the detailed mechanism of MSC-based immunotherapy; 2) the role of Telomerase in MSC-based immunotherapy; 3) the relationship between Fbn1 deficiency and bone disorder; 4) the mechanism contributes to the osteoporotic phenotype in SSc mice and patients. In chapter 2, we show that MSC-induced T cell apoptosis through FAS/FASL signaling is required for MSC-mediated therapeutic effects in SSc mice. Importantly, FAS controlled 10 chemokines releasing regulates immune cell migration, which is necessary for MSC- induced immune cell apoptosis. Secondly, we observe that TERT serves as a transcription modulator to regulate FASL expression, which governs immunomodulatory properties of BMMSCs. Interestingly, Aspirin increases immunomodulation of BMMSCs through TERT activation. In chapter 3, we show that Fbn1 regulates BMMSC osteogenic/adipogenic lineage selection via IL4Rα/mTOR signaling, leading to osteoporosis phenotype in Systemic Sclerosis mouse model and human patients. Blockage of the mTOR cascade by rapamycin, an anticancer and immune suppressive drug, ameliorates the osteopenia phenotype in Fbn1 +/- SSc mice. Importantly, conditional knockdown of mTOR or knockout of IL4Rα in Fbn1 +/- SSc mice rescues osteoporotic phenotype. Taken together, this study provides a systemic mechanism to improve safety and efficacy of MSC-based immune therapy in the first section, and identify a therapeutic FDA approved drug for Systemic Sclerosis treatment in the second. Therefore, this study has brought forth experimental evidence for stem cell biology, the molecular mechanism(s) underlying MSC-associated immunotherapies, and pathway-guided drug therapy. 11 Chapter 2: Mesenchymal Stem Cell-Induced Immunoregulation Involves Fas Ligand/Fas- Mediated T Cell Apoptosis which Is Governed by Telomerase. 2.1 Introduction Bone marrow mesenchymal stem cells are hierarchical postnatal stem cells capable of undergoing self-renewal and multipotent differentiation into osteoblasts, chondrocytes, myelosupportive stroma and adipocytes (Friedenstein et al, 1974; Prockop, 1997). BMMSCs are considered to be progenitors of osteoblasts with the capacity to regenerate bone and marrow components in vivo. These findings have led to extensive studies using BMMSCs for orthopaedic tissue engineering applications (Kwan et al, 2008; Panetta et al, 2009). Recently, a growing body of evidence has indicated that BMMSCs produce a variety of cytokines that display profound immunomodulatory properties by inhibiting the proliferation and function of several major types of immune cells, such as natural killer cells, dendritic cells, and both T and B lymphocytes (Aggarwal & Pittenger, 2005; Nauta & Fibbe, 2007; Uccelli et al, 2007; 2008). These unique properties make BMMSCs a plausible resource for the clinical treatment of immune disorders. To date, systemic infusion of BMMSCs has been successfully used for treating a variety of human diseases, including GvHD, as well as ameliorating hematopoietic stem cell (HSC) engraftment, SLE, diabetes, rheumatoid arthritis, autoimmune encephalomyelitis, 12 periodontitis, inflammatory bowel disease, sepsis, and systemic sclerosis (Chen et al, 2006; Le Blanc et al, 2004; Liang et al, 2009; 2012; Liu et al, 2013; Scuderi et al, 2012; Sun et al, 2009). A variety of factors, including TGF , IL10, PGE2, NO, IDO, and FAS/FASL, have been identified as potential regulators of BMMSC-based immunomodulation (Aggarwal & Pittenger, 2005; Akiyama et al, 2012; Batten et al, 2006; Meisel et al, 2004; Park et al, 2011; Ren et al, 2008; Sato et al, 2007; Zhang et al, 2008). However, the precise mechanisms underlying the immunomodulatory properties of BMMSCs remain to be elucidated. Telomerase reverse transcriptase is a nucleoprotein that functions to preserve chromosomal integrity and quell p53-dependent DNA damage, as well as perform DNA repair activity at telomere ends. In the absence of telomerase, continued cell division results in telomere shortening and p53 activation (Maser et al, 2002; Smogorzewska & De Lange, 2004). It has been reported that telomerase plays important roles in stem cell self-renewal and stem cell-based tissue regeneration (Liu et al, 2011; Yamaza et al, 2008), and is highly expressed in prospectively isolated BMMSCs from aspirates of human bone marrow (Gronthos et al, 2003). However, the role of TERT in regulating BMMSC-mediated immunomodulation has never been examined considering that TERT is rapidly down regulated in human BMMSCs during ex vivo expansion (Shi et al, 2002). 13 2.2 Material and Methods 2.2.1 Animals and Antibodies Female C57BL/6J (BL6), B6.129S-Tert tm1Yjc /J (TERT -/- ), B6.Cg-Fbn1 Tsk /J (Tsk/ + ), C57BL/6- Tg(TcraTcrb)1100Mjb/J (OT1TCRTG), B6Smn.C3-Fasl gld /J (BL6 gld), C3MRL-Fas lpr /J (C3H lpr), and B6.129S4-Ccl2 tm1Rol /J mice lines were purchased from the Jackson Lab. Female immunocompromised mice (Beige nude/nude XIDIII) were purchased from Harlan. gld and lpr strain have spontaneous mutation in FasL (Fasl gld ) and Fas (Fas lpr ), respectively, with no other spontaneous mutation. To maintain the TERT -/- strain and generate TERT +/+ (WT) mice, heterozygous (TERT +/- ) pairs were intercrossed. Also, TERT -/- mice were intercrossed to produce telomerase deficient fourth generation (G4) mice. Due to the embryonic lethality of a homozygous Fbn1 mutation, heterozygous Tsk/ + mice were intercrossed to generate WT and heterozygous Tsk/ + mice for the SS disease model. Aged-matched female littermates were used as controls in the present study. All animal experiments were performed under the institutionally approved protocols for the use of animal research (USC #10941, 11141 and 11327). Anti-mouse-CD4-PerCP, CD8-FITC, CD25-APC, CD11b-PE, CD34-FITC, CD45-APC, CD73- PE, CD90.2-PE, CD105-PE, CD117-PE, Sca-1-PE, CD3 , CD28, anti-human- CD73-PE, CD90- PE, CD105-PE, CD146-PE, CD34-PE and CD45-PE antibodies were purchased from BD Bioscience. Anti-mouse-CD178 (FasL)-PE, CD3-APC, Foxp3-PE, IL17-PE, anti-human-CD3- 14 APC, CD4-APC, IFN -APC, CD25-APC and Foxp3-PE antibodies were purchased from eBioscience. Anti-mouse IgG, Fas and Fas-ligand antibodies were purchased from Santa Cruz Biosciences. MCP-1 antibodies were purchased from Cell Signaling. Anti-rat-IgG- Rhodamine antibody was purchased from Southern Biotech. Anti-rat IgG-AlexaFluoro 488 antibody was purchased from Invitrogen. Anti- -actin antibody was purchased from Sigma. Anti-SSEA4, active -catenin and total -catenin monoclonal antibodies were purchased from Millipore. Anti-TERT, FasL and total -catenin (ChIP grade) polyclonal antibodies were purchased from Santa Cruz Biosciences. Anti-BRG1 antibody was purchased from Cell Signaling. Anti- -Actin antibody was purchased from Sigma. For Immunohistochemistry staining and TUNEL staining, femurs at 24 hours after BMMSC injection were harvested and used for paraffin embedded sections. For co- cultured sample, culture supernatant was removed and fixed by 1% paraformaldehyde at 4 o C overnight. The samples were blocked with serum matched to secondary antibodies, incubated with the CD3-specific antibodies (eBioscience, 1:400) 30min at room temperature, and stained using VECTASTAIN Elite ABC Kit (UNIVERSAL) and ImmPACT VIP Peroxidase Substrate Kit (VECTOR), according to the manufacturers’ instructions. For TUNEL staining, an apoptosis detection kit (Millipore) was used in accordance with the manufacturer's instructions, followed by TRAP staining and counterstaining with H&E. 15 2.2.2 Primary Cells Isolation For mouse BMMSC isolation, the single suspension of bone marrow-derived all nucleated cells (ANCs) from femurs and tibias were seeded at a density of 15x10 6 in 100 mm culture dishes (Corning) under 37 o C at 5% CO 2 condition. Non-adherent cells were removed after 48 hours and attached cells were maintained for 16 days in Alpha Minimum Essential Medium ( -MEM, Invitrogen) supplemented with 20% fetal bovine serum (FBS, Equitech-Bio, Inc.), 2 mM L-glutamine, 55 μM 2-mercaptoethanol, 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). Colonies forming attached cells were passed once for further experimental use. Flow cytometric analysis showed that 0.95% of BMMSCs was positive for CD34 + CD117 + antibody staining. For colony-forming unit-fibroblastic assays, 1x10 6 ANCs from bone marrow were seeded into 60 mm culture dishes. After 16 days, the cultures were washed by PBS and stained with 1% toluidine blue solution with 2% paraformaldehyde. Clusters with more than 50 cells were counted as colonies under microscopy. For isolation of mouse B cells, NK cells, immature Dendritic cells/macrophages, after removing red blood cells using ACK lycing buffer, mouse splenocytes were incubated with anti-mouse CD19-PE, CD49b-FITC and CD11c-FITC antibodies for 30 min, followed by a magnetic separation using anti-PE or anti-FITC micro beads (Milteny biotech) according to manufacturer’s instructions. 16 For T cell culture, complete medium containing Dulbecco's Modified Eagle’s Medium (DMEM, Lonza) with 10% heat-inactivated FBS, 50 M 2-mercaptoethanol, 10 mM HEPES, 1 mM sodium pyruvate (Sigma), 1% non-essential amino acid (Cambrex), 2 mM L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin. 2.2.3 Cell proliferation assay Mouse BMMSCs (10 x 10 3 /well) were seeded on 2-well chamber slides (Nunc) and cultured for 2-3 days. The cultures were incubated with BrdU solution (1:100) (Invitrogen) for 20 hours, and stained with a BrdU staining kit (Invitrogen) according to the manufacturer’s instructions. The samples were then stained with hematoxylin. BrdU-positive and total cell numbers were counted in ten images per subject. The number of BrdU-positive cells was indicated as a percentage of the total cell number. The BrdU assay was repeated on three independent samples for each experimental group. 2.2.4 Flow cytometry analysis Whole peripheral blood was stained with anti-CD45, anti-CD3, anti-CD4, and CD8a antibodies and treated with BD FACS™ Lysing Solution (BD Bioscience) to get mononuclear cells (MNCs). The apoptotic T cells were detected by staining with CD3 antibody, followed by Annexin-V Apoptosis Detection Kit I (BD Pharmingen). 17 For fluorescent labeling of cells, BMMSCs or T cells were incubated with Carboxyfluorescein diacetate N-succinimidyl ester (CFSE, SIGMA) for 15 min or PKH-26 (Invitrogen) for 5min, according to manufacturer’s instructions. For Foxp3 intercellular staining, T cells were stained with anti-CD4, CD8a, and CD25 antibodies (1 g each) for 30 min on ice. Next, cells were stained with anti-Foxp3 antibody using Foxp3 staining buffer kit (eBioscience). For IL17 staining, T cells were stained with anti-CD4 antibody and then stained with anti-IL17antibody using Foxp3 staining buffer kit. All samples were analyzed with FACS calibur (BD Bioscience). For BMMSC surface molecules analysis, WT BMMSCs or TERT -/- BMMSCs (0.2x10 6 ) were incubated with 1 g of PE-conjugated antibodies or isotype-matched control IgGs (Southern Biotech) at 4 o C for 30 min. After washing with PBS with 2% FBS and 2% paraformaldehyde fixation, samples were analyzed by FACS Calibur flow cytometry with CellQuest software. 2.2.5 In vitro osteogenic differentiation BMMSCs and TERT -/- BMMSCs were cultured under osteogenic culture conditions in medium containing 2 mM -glycerophosphate (Sigma), 100 μM L-ascorbic acid 2- phosphate and 10 nM dexamethasone (Sigma). After 4 weeks of induction, the cultures were either stained with alizarin red for mineralized nodule formation or lysed for RNA isolation to identify osteogenic gene expression. 18 2.2.6 In vitro adipogenic differentiation For adipogenic induction, 500 nM isobutylmethylxanthine (Sigma-Aldrich), 60 μM indomethacin (Sigma-Aldrich), 500 nM hydrocortisone (Sigma-Aldrich), 10 μg/ml insulin (Sigma-Aldrich), and 100 nM L-ascorbic acid phosphate were added into the growth medium. After 7 days, the cultured cells were stained with Oil Red-O (Sigma-Aldrich), and positive cells were quantified under microscopy and shown as a percentage of the total cells. 2.2.7 Western blot analysis Cells were lysed in M-PER mammalian protein extraction reagent (Thermo) with protease and phosphatase inhibitors (Roche), and proteins were quantified using a protein concentration assay (Bio-Rad Laboratories). Nuclear protein was obtained using NE-PER nuclear and cytoplasmic extraction reagent (Thermo). Twenty μg of total or nuclear proteins were separated by SDS-PAGE and transferred to 0.2 μm nitrocellulose membrane. The membranes were blocked with 5% non-fat dry milk and 0.1% Tween-20 for 1 hour, followed by incubation overnight with the primary antibodies diluted in blocking solution according to manufacturer’s instructions. The membranes were then incubated with primary antibodies overnight, followed by 1 h incubation in HRP- conjugated secondary antibody diluted at 1:10,000 in blocking solution. Immunoreactive proteins were detected using SuperSignal® West Pico Chemiluminescent Substrate 19 (Thermo) and BioMax film (Kodak). For TERT experiments, SuperSignal West Femto Chemiluminescent Substrate (Thermo) was used. The intensity of bands was measured by using NIH ImageJ software and normalized to β-Actin. 2.2.8 Real-time polymerase chain reaction Total RNA was isolated from the cultures using SV total RNA isolation kit (Promega) and digested with DNase I, following the manufacturer’s protocols. The cDNA was synthesized from 100 ng of total RNA using Superscript III (Invitrogen). Real-time polymerse chain reaction (RT-PCR) was performed using gene-specific primers (Table 1) and Cybergreen supermix (BioRad). RT-PCR was repeated in 3 independent samples. 2.2.9 Co-culture of T cells with BMMSCs BMMSCs (0.2x10 6 ) were seeded on a 24-well culture plate (Corning) and incubated 24 hours. The prestimulated T cells were directly loaded onto BMMSCs and co-cultured for 2 days. In some experiments, anti-Fas ligand neutralizing antibody (BD) or caspase 3, 8 or 9 inhibitors (R&D systems) were added in the co-culture. Apoptotic T cells were detected as described above. 2.2.10 T cell migration assay For T cell migration assay, a transwell system was used. PKH26-stained BMMSCs 20 (0.2x10 6 ) were seeded on the lower chamber of a 12-well culture plate (Corning) with transwell and incubated 24 hours. The prestimulated T cells with anti-CD3 and anti- CD28 antibodies for 48 hours were loaded onto the upper chamber of transwell and co- cultured for 48 hours, followed by observation under a fluorescent microscope. Green- labeled cell number was counted and normalized by red-labeled number of MSCs in five representative images. For immunofluorescent microscopy, the macrophages or BMMSCs were cultured on 4- well chamber slides (Nunc) (2x10 3 /well) and then fixed with 4% paraformaldehyde. The chamber slides were incubated with primary antibodies including anti-CD11b antibody (1:400, BD), anti-CD90.2 (1:400, BD) and anti-FasL (1:200, SantaCruz) at 4 o C for overnight followed by treatment with Rhodamine-conjugated secondary antibody (1:400, Southern biotech) or AlexaFluoro 488- conjugated secondary antibody (1:200, Invitrogen) for 30min at room temperature. Finally, slides were mounted with Vectashield mounting medium (Vector Laboratories). 2.2.11 In vitro CD4 + CD25 + Foxp3 + Treg cell induction To avoid natural Treg (nTreg) population in this inductive experiment, CD4 + CD25 - T- lymphocytes (1x10 6 /well) isolated from splenocytes using a CD4 + CD25 + regulatory T-cell Isolation kit (Miltenyi Biotec) were prestimulated with plate-bounded anti-CD3  antibody (3 g/ml) and soluble anti-CD28 antibody (2 g/ml) for 2 days. The activated T- 21 lymphocytes were loaded on a culture of 0.2x10 6 WT BMMSCs or TERT -/- BMMSCs with recombinant human TGF  (2 ng/ml) (R&D Systems) and recombinant mouse IL2 (2 ng/ml) (R&D Systems). After 3 days, cells in suspension were collected and stained with anti-CD4-PerCP, CD8 -FITC, and CD25-APC antibodies (1 g each) for 30 min on ice under dark conditions, followed by anti-Foxp3-PE antibody staining using a Foxp3 staining buffer kit (eBioscience) for cell fixation and permeabilization. The cells were analyzed by the FACS Calibur flow cytometer with CellQuest software. 2.2.12 Overexpression of Fas ligand, Fas, TERT and MCP-1 For FASL overexpression, 293T cells for lentivirus production were seeded in a 10 cm culture dish (Corning) until 80% confluence. Plasmids with proper proportion, FasL and TERT gene expression vector: psPAX:pCMV-VSV-G (all from Addgene) =5:3:2, were mixed in opti-MEM (Invitrogen) with Lipofectamin LTX (Invitrogen) according to the protocol of the manufacturer. EGFP expression plasmid (Addgene) was used as control. The supernatant was collected 24h and 48h after transfection and filtered through a 0.45μm filter to remove cell debris. For infection, the supernatant containing lentivirus was added into target cell culture in the presence of 4μg/ml polybrene (SIGMA), and the transgene expression was validated by GFP under microscopic observation. To generate Fas and MCP-1 overexpression vectors, a pCMV6-AC-GFP TrueORF mammalian expression vector system (Origene) was used. Fas and MCP-1 cDNA clones 22 generated from C57BL/6J strain mice were purchased from Open Biosystems (Huntsville) and amplified by PCR with Sgf I and Mlu I restriction cutting sites. The PCR products were directly subcloned into pCR-Blunt II-TOPO vector using Zero Blunt ® TOPO ® PCR Cloning Kit (Invitrogen). After sequencing, Fas and MCP-1 cDNAs with SgfI/MluI sites were subcloned into pCMV6-AC-GFP expression vector. All constructs were verified by sequencing before transfection into cells. After construction, lprBMMSCs were transfected with cDNAs using LIPOFECTAMINE PLUS reagent (LIFE TECHNOLOGIES), according to manufacturer’s instructions for 48 hours. 2.2.13 RNAi and chemical treatments BMMSCs (0.5x10 6 ) were seeded in a 6-well culture plate and treated with fasl, fas, tert, -catenin siRNAs (Santa Cruz), or the vehicle siRNA control (sc-36869) with lipofectamine reagent (Invitrogen), according to the manufacturers’ instructions. After transfection, cells were either used for protein extraction for Western immunoblotting or for in vitro co-culture with T- lymphocytes. For chemical reagent treatments, serum- starved BMMSCs were treated with 10 M -catenin activator (CHIRON 99021; Chiron Corporation) for 24 hours. For Western immunoblotting, BMMSCs were cultured in growth medium with drugs, and protein was extracted using M-PER mammalian protein extraction reagent. For differentiation induction, BMMSCs were cultured under inductive conditions in the presence of drugs (added every 3 days) for 3 weeks, followed by staining and gene expression analysis. 50 ug/mL aspirin was added to MSCs at 50% 23 confluence for 3 days. Aspirin-treated cells were harvested and directly used for further experiments. To analyze the effect of this treatment, cell lysates were harvested at 0, 1, 3, 5, 7, and 14 days post-treatment. In some experiments, after removal of aspirin at day 3, MSCs were cultured for 2 weeks. A TRAP-ELISA assay was performed to detect telomerase activity. For depletion of Phagocytes, clodronate-liposome (Encapsula Nano- Science, LLC) was injected (200 l/ mouse) into mice i.p. as described previously (Perruche et al. 2008). PBS-liposome was used as control. For depletion of Tregs in DSS-induced experimental colitis mice, anti-CD25 antibody (250 g/mouse, biolegend) was administrated intraperitoneally after 3 days of DDS induction. 2.2.14 Allogenic BMMSC transplantation into Disease Animal Models For colitis mice, acute colitis was induced by administering 3% (w/v) dextran sulfate sodium (DSS, molecular mass 36,000 –50,000 Da; MP Biochemicals) through drinking water, which was fed ad libitum for 10 days (Zhang et al., 2010). Passage one BMMSCs, gldBMMSCs or lprBMMSCs were infused (1x10 6 cells) into disease model mice (n=6) intravenously at day 3 after feeding DSS water. In control group, mice received PBS 24 (n=6). All mice were harvested at day 10 after feeding DSS water and analyzed. Induced colitis was evaluated as previously described (Alex et al., 2009). For SSc mice, passage one BMMSCs, gldBMMSCs or lprBMMSCs were infused (1x10 6 cells) into SSc mice intravenously at 8 weeks of age (n=6). In control group, SSc mice received PBS (n=5). All mice were sacrificed at 12 weeks of age for further analysis. The protein concentration in urine was measured using Bio-Rad Protein Assay (Bio-Rad). 2.2.15 Allogenic MSC transplantation into systemic sclerosis patients MSCs from umbilical cord were sorted out and expanded, following a previous report (Liang et al., 2009). Expanded MSCs were intravenously infused into the SSc recipients (1x10 6 /kg body weight). The trial was conducted in compliance with current Good Clinical Practice standards and in accordance with the principles set forth under the Declaration of Helsinki, 1989. This protocol was approved by the IRB of the Drum Tower Hospital of Nanjing, University Medical School, China. Informed consent was obtained from each patient. 2.2.16 ELISA assays Peripheral blood samples were collected from mice using micro-hematocrit tubes with heparin (VWR) and centrifuged at 1000g for 10 min to get serum samples. TGF  25 (eBioscience), mouse ANA, anti-dsDNA IgG and anti-dsDNA IgM (Alpha Diagnosis), human ANA (EUROIMMUN), mouse MCP-1, human MCP-1 (eBioscience) and creatinine (R&D Systems) levels were measured using a commercially available kit according to manufacturer’s instructions. The results were averaged in each group. The intra-group differences were calculated between the mean values. BMMSCs (0.5x10 6 /well) were seeded in 6-well culture plates with or without RNAi or chemicals at the indicated concentrations. For the telomerase activity assay, a TeloTAGGG Telomerase PCR ELISA kit (Roche) was used with cell lysates. For chemokine production assays, the supernatant samples from each culture were collected and measured according to manufacturers’ instructions, using a Total Nitric Oxide and Nitrate/Nitrite Parameter Assay kit, Prostaglandin E2 Parameter Assay Kit, and Mouse IL10 Quantikine ELISA Kit (R&D Systems). For cytokine array analysis, culture supernatants from BMMSC or lprBMMSC were analyzed using Mouse Cytokine Array Panel A Array Kit (R&D Systems) according to manufacturer’s instructions. The results were scanned and analyzed using Image J software to calculate blot intensity. Cytokine array was repeated in 2 independent samples. 26 2.2.17 FASL + BMMSCs isolation For FASL + BMMSCs isolation, culture expanded BMMSCs were harvested and stained with CD178 (FasL)-PE antibody (1 g antibody for 0.2 x10 6 BMMSCs staining) at 4 o C for 30 min. After washing by PBS with 2% FBS, samples were sorted by BD FACSAriaII and analyzed by FACS Calibur flow cytometry. After sorting, the FasL + BMMSC population showed greater than 95% expressing FasL, but the FasL - BMMSC population showed only 0.3% expressing FasL. 2.2.18 Luciferase reporter assay fasl-luciferase promoter reporter constructs were generated by PCR using Pfu polymerase and mouse genomic DNA as a template. Primers containing upstream XhoI and HindIII downstream restriction sites were used to generate fasl promoter fragments (Table 1). Restriction digested PCR products were subcloned into a pGL3-Basic vector (Promega). Point mutants were introduced into the reporter by the Pfu/DpnI method. All clones were confirmed by sequencing on both strands. BMMSCs cultured in 6-well plates were co-transfected with 2 μg luciferase reporter and 100 ng Renilla luciferase expression vector to control for transfection efficiency. Forty-eight hours after transfection, cells were lysed in 1x passive lysis buffer, and luciferase activity was measured using the Dual-Glo Luciferase System (Promega) with luminometer (Turner Biosystems). 27 2.2.19 Chromatin Immunoprecipitation Assays BMMSCs grown in 10 cm cell culture dishes were fixed for 10 min at room temperature by addition of 1% paraformaldehyde to the growth medium. Cells were washed twice in cold PBS supplemented with complete protease inhibitor cocktail and gently scraped from the plate. Cell lysis and chromatin immunoprecipitation were performed using the ChIP Assay Kit (Millipore). For chromatin fragmentation, cells were sonicated using a Branson Sonifier 450 on power setting 4 in 30 s bursts with 1 min cooling on ice for a total sonication time of 4 min. For immunoprecipitation, 1:100 dilutions of TERT or - catenin polyclonal antibodies were used to capture protein-DNA complexes, and non- specific serum IgG was used as a negative control. All resulting precipitated DNA samples were quantified with real-time PCR and expressed as a percentage of input DNA. The binding site was detected at 1,801 bp upstream of the TBE transcription start site, and the region surrounding the binding site was used for amplification (Table 1). For immunoprecipitation-Western blotting, nuclear proteins were extracted, followed by immunoprecipitation with TERT or -catenin polyclonal antibodies. Captured protein- protein complexes were further analyzed by Western blotting. 2.2.20 Statistical analysis Comparisons between two groups were analyzed by independent two-tailed Student’s t- tests, and comparisons between more than two groups were analyzed by one-way ANOVA. P values less than 0.05 were considered statistically significant. 28 Gene Name Sequence Application Mouse runx2 sense 5’-CCGCACGACAACCGCACCAT-3’ RT-PCR antisense 5’-CGCTCCGGCCCACAAATCTC-3’ RT-PCR Mouse ocn sense 5’-AAGCAGGAGGGCAATAAGGT-3’ RT-PCR antisense 5’-AGCTGCTGTGACATCCATAC-3’ RT-PCR Mouse mppar 2 sense 5’-GCTGTTATGGGTGAAACTCTG-3’ RT-PCR antisense 5’-ATAAGGTGGAGATGCAGGTTC-3’ RT-PCR Mouse lpl sense 5’-GGGCTCTGCCTGAGTTGTAG-3’ RT-PCR antisense 5’-AGAAATTTCGAAGGCCTGGT-3’ RT-PCR Mouse tert sense 5’-GGATTGCCACTGGCTCCG-3’ and, RT-PCR antisense 5’-TGCCTGACCTCCTCTTGTGAC-3’ RT-PCR Mouse gapdh sense 5’-CACCATGGAGAAGGCCGGGG-3’ RT-PCR antisense 5’-GACGGACACATTGGGGGTAG-3’ RT-PCR Human fasl sense 5’-CTCTTGAGCAGTCAGCAACAGG-3’, RT-PCR antisense ; 5’-ATGGCAGCTGGTGAGTCAGG-3’ RT-PCR Human fas sense 5’-TGATGTCAGTCACTTGGGCATTAAC-3’ RT-PCR antisense 5’-CAACAACCATGCTGGGCATC-3’ RT-PCR Human gapdh sense 5’-TGGTGAAGACGCCAGTGGA-3’ RT-PCR antisense 5’-GCACCGTCAAGGCTGAGAAC-3’ RT-PCR fasl promoter 1.1 kb sense 5’-CTCGAGTGTGCTGTGTGATGGTTAAGGCAC-3’ Cloning antisense 5’-AAGCTTAGCAAGTCCCTACTCCCACG-3’ Cloning 29 fasl promoter 2 kb sense 5’-CTCGAGATGGCACTACCAAACTCCAACCCA-3’ Cloning antisense 5’-AAGCTTAGCAAGTCCCTACTCCCACG-3’ Cloning fasl promoter sense 5’-TGTGATTGGTGGACAGTAGGGTGT ‑ 3´ ChIP assay fasl promoter antisense 5’- TGCTCTCCCTGTACCAGATGAGTCTT-3’ ChIP assay Table 1. PCR Primer Used in Chapter 2. 30 2.3 Results 2.3.1 Fas ligand (FasL) in BMMSCs induces T cell apoptosis Since BMMSCs express FasL and activated T cells express elevated levels of Fas (Mazar et al., 2009; Figures S2.1A-2.1D), we hypothesized that FasL-mediated Fas signaling might play a critical role in BMMSC-based immunomodulation. To test this hypothesis, BMMSCs from C57BL6 mice and FasL-mutated B6Smn.C3-Fasl gld /J mice (gldBMMSC), along with FasL transfected gldBMMSCs (FasL + gldBMMSC) were injected into normal C57BL6 mice (Figure 2.1A). Peripheral blood and bone marrow samples were collected at 0, 1.5, 6, 24, and 72 hours after BMMSC transplantation (Figure 2.1A). Allogenic BMMSC infusion reduced the number of CD3 + T cells and increased the number of apoptotic CD3 + T cells in peripheral blood and bone marrow, starting at 1.5 hours, reaching the peak at 6 hours and lasting until 72 hours post-transplantation (Figures 2.1B-2.1E). In order to compare syngenic and allogenic BMMSCs, we showed that BMMSCs derived from a littermate were the same as allogenic BMMSCs in their capacity to induce T cell apoptosis (Figures S2.2A-2.2G). Meanwhile, infusion of FasL -/- gldBMMSCs failed to reduce the number of CD3 + T cells or elevate the number of apoptotic CD3 + T cells in peripheral blood and bone marrow (Figures 2.1B-2.1E). However, overexpression of FasL in gldBMMSCs by lentiviral transfection (Figure S2.1N) partially rescued the capacity to reduce the number of CD3 + T cells and elevate the 31 number of apoptotic CD3 + T cells in peripheral blood, bone marrow, spleen, and lymph node (Figures 2.1B-2.1E; S2.1P-2.1S). BMMSC infusion also resulted in reducing the number of both CD4 + and CD8 + T cells with correspondingly increased number of apoptotic CD4 + and CD8 + T cells in peripheral blood (Figures S2.1E and 2.1F). Interestingly, BMMSC transplantation induced CD4 + T cell apoptosis and Treg upregulation in OT1 TCR TG mice. However, the percentage of CD8 + T cells, which react with OVA-MHC class I antigen, was unchanged after BMMSC transplantation, indicating that transplanted BMMSCs need to be recognized as antigen to initiate CD8 + T cell apoptosis (Figures S2.1T-2.1AA). TUNEL staining confirmed that BMMSC infusion elevated the number of apoptotic T cells in bone marrow (Figure 2.1F). We next verified that BMMSC-induced T cell death was caused by apoptosis based on the in vitro blockage of BMMSC-induced CD3 + T cell apoptosis by neutralizing FasL antibody and caspase 3, 8, and 9 inhibitors (Figures 2.1G-2.1I). FasL neutralizing antibody injection could partially block BMMSC-induced CD3 + T cell apoptosis, upregulation of Tregs, and downregulation of Th17 cells in peripheral blood and bone marrow (Figure S2.1G-2.1M). These data indicate that BMMSCs are capable of inducing T cell apoptosis through the FasL/Fas signaling pathway (Figure 2.1J). Although BMMSCs failed to induce naïve T cell apoptosis in the co-culture system (data not shown), they were able to induce activated T cell apoptosis in vitro (Figures 2.1G and 2.1I). 32 In order to confirm the role of FasL in BMMSC-mediated T cell apoptosis in vivo, we used siRNA to knockdown FasL expression in BMMSCs (Figure S2.3A) and infused FasL knockdown BMMSCs to C57BL6 mice. As expected, infusion of FasL knockdown BMMSCs (FasL siRNA BMMSCs) failed to reduce the number of CD3 + T cells or induce CD3 + T cell apoptosis in peripheral blood and bone marrow (Figures 2.2A-2.2D). Moreover, infusion of FasL knockdown BMMSCs failed to elevate Foxp3 + regulatory T cell levels in peripheral blood (Figure 2.2E). This study confirms that FasL is required for BMMSC- induced T cell apoptosis and Treg upregulation. Since apoptotic T cells trigger TGF  production by macrophages and upregulate Tregs, which leads to immune tolerance in vivo (Perruche et al., 2008), we examined whether BMMSC-induced T cell apoptosis could also promote the upregulation of Tregs. We found that systemic infusion of BMMSCs did, in fact, elevate Treg levels in peripheral blood at 24 and 72 hours post-transplantation (Figures 2.2F and S2.2H-2.2M), along with elevated TGF  level and reduced Th17 cell level in peripheral blood (Figures 2.2G and S2.1O). Co-transplantation of BMMSCs and pan T cells resulted in T cell apoptosis at 1.5 and 6 hours post-transplantation. On the other hand, FasL -/- gldBMMSC infusion failed to upregulate the levels of either Tregs or TGF  (Figures 2.2F and 2.2G), suggesting that FasL-mediated T cell apoptosis plays a critical role in Treg upregulation. Indeed, overexpression of FasL in FasL -/- gldBMMSCs rescued BMMSC-induced Treg upregulation and TGF  production at 24 hours post-transplantation (Figures 2.2F and 2.2G). 33 To examine the mechanism by which BMMSC infusion resulted in TGF  upregulation in peripheral blood, we used fluorescence analysis to confirm that macrophages engulfed apoptotic T cells in vivo (Perruche et al., 2008; Figure 2.2H). Then we measured the number of CD11b + macrophages in spleen cells and found that the number was significantly increased in the BMMSC infusion group (Figure 2.2I). In contrast, treatment with macrophage inhibitor clodronate liposomes significantly reduced the number of CD11b + macrophages in spleen cells (Figure 2.2I) and blocked BMMSC infusion-induced upregulation of TGF  and Tregs (Figures 2.2J and 2.2K). These data suggest that T cell apoptosis, as induced by BMMSC infusion, activates macrophages to produce the TGF  that results in Treg upregulation (Figure 2.2L). We next asked whether apoptosis of infused BMMSCs also triggers macrophages to produce TGF  to upregulate Tregs. CFSE-labeled BMMSCs, gldBMMSCs and FasL knockdown BMMSCs were infused into C57BL6 mice. At 1.5 hours post-infusion, all CFSE + cells were detected and reached a peak in peripheral blood and bone marrow, after which the cell number was gradually decreased, becoming undetectable at 24 hours post-infusion (Figures S2.3C and 2.3D). In contrast, CFSE + apoptotic cells reached a peak at 6 hours post-infusion and became undetectable at 24 hours post-infusion in peripheral blood and bone marrow (Figures S2.3E and 2.3F). The apoptosis of transplanted BMMSCs was also observed by immunofluoresent analysis (Figure S2.3B). Although apoptosis of the infused FasL-deficient BMMSCs was observed, no 34 corresponding upregulation of TGF  or Tregs was observed in peripheral blood (Figures 2.2E, 2.2F, and 2.2G). These data suggest that T cell, not BMMSC, apoptosis is required for Treg upregulation (Figure 2.2L). Figure 2.1 BMMSCs induce T cell apoptosis via Fas ligand (FasL). (A) Schema of BMMSC transplantation procedure. 1x10 6 BMMSCs (n=5), FasL -/- gldBMMSCs (n=4) or FasL- transfected gldBMMSCs (FasL + gldBMMSCs, n=4) were infused into C57BL6 mice through the tail vein. All groups were sacrificed at indicated time points for sample collection. Zero hour represented that mice were immediately sacrificed after BMMSC injection. (B-E) BMMSC transplantation (BMMSC) induced transient reduction in the number of CD3 + T cells and increased AnnexinV + 7AAD + double positive apoptotic CD3 + T cells in peripheral blood mononuclear cells (PBMNCs; B, C) and bone marrow mononuclear cells (BMMNCs; D, E) at indicated time points, while FasL -/- BMMSCs from gld mice (gldBMMSCs) failed to reduce CD3 + T cells or elevate CD3 + T cell apoptosis in peripheral 35 blood (B, C) and bone marrow (D, E). FasL-transfected gldBMMSC transplantation (FasL + gldBMMSC) partially rescued the capacity to reduce the number of CD3 + T cells and induce CD3 + T cell apoptosis in peripheral blood (B, C) and bone marrow (D, E). *P<0.05; **P<0.01; ***P<0.001 vs. gldBMMSC, # P<0.05; ### P<0.001 vs. FasL + gldBMMSC, $ P<0.05; $$$ P<0.001 vs. gldBMMSC. (F) When BMMSCs were infused into mice, TUNEL and immunohistochemistry staining showed that TUNEL-positive apoptotic cell (brown, white arrow) number in CD3-positive T cells (purple, yellow arrowhead) was higher in the BMMSC-injected group compared to the control group in bone marrow. (G) When BMMSCs were co-cultured with T cells, BMMSC-induced AnnexinV + 7AAD + double positive apoptotic T cells were significantly blocked by anti-FasL neutralizing antibody (1mg/mL) compared to IgG antibody control group. (H) TUNEL and immunohistochemistry staining showed that TUNEL-positive apoptotic T cells (brown, white arrow) were observed in CD3 + T cells (purple, yellow arrowhead) when co- cultured with BMMSCs in vitro. In the presence of anti-FasL neutralizing antibody (FasL Ab), TUNEL-positive cell percentage was significantly less than the untreated control group. (I) In addition, the number of BMMSC-induced AnnexinV + 7AAD + double positive apoptotic T cells was significantly blocked by caspase 3, 8, and 9 inhibitor treatments. The results were representative of three independent experiments. (J) Schematic diagram indicating that BMMSCs induce T cell apoptosis. (*P<0.05; **P<0.01; ***P<0.001. The bar graph represents mean±SD). 36 Figure 2.2 FasL is required for BMMSC-induced T cell apoptosis and upregulation of CD4 + CD25 + Foxp3 + regulatory T cells (Tregs). (A, B) BMMSC transplantation (BMMSC, n=5) induced a transient reduction in the number of CD3 + T cells (A) and elevation of AnnexinV + 7AAD + double positive apoptotic CD3 + cells (B) in peripheral blood. Transplantation of FasL knockdown BMMSC (FasL siRNA BMMSC, n=3) failed to reduce CD3 + T cells (A) or increase the number of CD3 + apoptotic T cells (B) in peripheral blood. (C, D) BMMSC transplantation (BMMSC, n=5) showed a transient reduction of CD3 + T cells (C) and elevation of AnnexinV + 7AAD + double positive apoptotic CD3 + T cells (D) in bone marrow. Transplantation of FasL knockdown BMMSCs (FasL siRNA BMMSC, n=3) failed to reduce CD3 + T cells (C) or elevate CD3 + apoptotic T cells (D) in bone marrow. (E) BMMSC, but not FasL knockdown BMMSC, transplantation significantly upregulated levels of Tregs at 24 and 72 hours after transplantation in C57BL6 mice. (F) BMMSC transplantation resulted in a significant upregulation of Tregs when compared to the gldBMMSC transplantation group at 24 and 72 hours post-transplantation. FasL- transfected gldBMMSC transplantation (FasL + gldBMMSC) partially rescued BMMSC- induced upregulation of Tregs. (G) TGF  level in peripheral blood was significantly increased in both BMMSC and FasL + gldBMMSC groups at 24 hours post-transplantation. FasL -/- gldBMMSC transplantation failed to upregulate TGF  level. (H) Apoptotic pan T cells were engulfed by macrophages in vivo. Green indicates T cells, and red indicates 37 CD11b + macrophages. Bar=50 m. (I) BMMSC transplantation group increased the number of CD11b + cells in peripheral blood when compared to the control group (C57BL6). Depletion of macrophages by clodronate liposome treatment showed the effectiveness in reducing CD11b + cells in the BMMSC transplantation group (BMMSC+clodronate), as assessed by flow cytometric analysis. (J) TGF  level was significantly increased in peripheral blood after BMMSC transplantation. Clodronate liposome treatment blocked BMMSC-induced upregulation of TGF  (BMMSC+clodronate). (K) BMMSC transplantation upregulated the level of Tregs in peripheral blood compared to the control group (C57BL6). Clodronate liposome treatment inhibited BMMSC-induced Treg upregulation (BMMSC+clodronate). (L) Schematic diagram indicating that BMMSC-induced T cell apoptosis resulted in immune tolerance as evidenced by upregulation of Tregs. The results were representative of three independent experiments. (*P<0.05, **P<0.01, ***P<0.001. The bar graph represents mean±SD). 38 39 Figure S2.1 Fas Ligand (FasL) plays an important role in BMMSC-based immunotherapy (related to Figure 1). (A, B) Western blot analysis showed that moue BMMSCs (mBMMSC) and human BMMSCs (hBMMSC) express FasL. CD8 + T cells were used as positive control. (C) Immunocytostaining showed that mBMMSC co-expressed FasL (green: middle column) with mesenchymal stem cell surface marker CD73 (red; upper row) or CD90 (red; lower row). (Bar=50 m). (D) Western blot showed that T cells which were activated by anti CD3 antibody (3 g/mL) and anti CD28 antibody (2 g/mL) treatment expressed a higher level of Fas than naïve T cells. (E) BMMSC transplantation induced a transient reduction in CD4 + and CD8 + T cell number in peripheral blood. (F) The percentage of AnnexinV + 7AAD + double positive apoptotic cells was elevated in both CD4 + and CD8 + T cells after BMMSC transplantation (**P<0.01, ***P<0.005, vs. 0h after BMMSC transplantation in CD4 + T cell group, ##P<0.01, ###P<0.005 vs. 0h after BMMSC transplantation in CD8 + T cell group. The bar graph represents mean±SD). (G) Schema of BMMSC and anti-Fas Ligand neutralizing antibody (FasLnAb) transplantation in C57BL6 mice. (H, I) BMMSC transplantation, along with FasLnAb injection, showed a significant blockage of BMMSC-induced reduction of CD3 + T cell number (H) and elevation of apoptotic CD3 + T cells (I) in peripheral blood. (J, K) BMMSC transplantation, along with FasLnAb injection, failed to reduce the number of CD3 + T cells (J) and induce CD3 + T cell apoptosis (K) in bone marrow. (L) BMMSC transplantation, along with FasLnAb injection, showed lower level of Tregs compared to the BMMSC transplantation group at 72 hours post-transplantation in peripheral blood. (M) BMMSC transplantation, along with FasLnAb injection, showed significant inhibition of BMMSC-induced reduction of Th17 cells in peripheral blood. (N) Flow cytometric analysis showed that transfection of FasL into gldBMMSCs could significantly elevate the expression level of FasL. (O) BMMSC transplantation showed downregulated levels of Th17 cells from 6 to 72 hours post- transplantation, while gldBMMSC failed to reduce the number of Th17 cells in peripheral blood. (P, Q) BMMSC transplantation significantly reduced the number of CD3 + T cells (P) and induced CD3 + T cell apoptosis (Q) at 1.5 hours and 6 hours post- transplantation in spleen. (R, S) BMMSC transplantation induced a transient reduction of the number of CD3 + T cells (R) and elevation of apoptotic CD3 + T cells (S) in Lymph node. (T) Schema of BMMSC transplantation in OT1TCRTG mice. (U, V) BMMSC transplantation showed upregulation of CD4 + T cell apoptosis in peripheral blood (U) and bone marrow (V). (W, X) BMMSC transplantation showed no upregulation of CD8 + T cell apoptosis in peripheral blood (W) and bone marrow (X). (Y) BMMSC transplantation in OT1TCRTG mice showed upregulation of Tregs at 24 hours and 72 hours post- 40 transplantation. (Z) BMMSC transplantation in OT1TCRTG mice showed reduction of Th17 cell level from 24 hours to 72 hours post-transplantation in peripheral blood. (AA) CD8 + T cell in OT1TCRTG mice showed no alteration in BMMSC transplantation group. (*P<0.05, **P<0.01, ***P<0.005. The bar graph represents mean±SD). Figure S2.2 Immunomodulation property of syngenic mouse BMMSC and human BMMSC transplantation (related to Figure 1). (A) Schema of syngenic and allogenic BMMSC transplantation in C57BL6 mice. (B, C) Both syngenic and allogenic BMMSC transplantation showed similar effect in reducing the number of CD3 + T cells (B) and inducing CD3 + T cell apoptosis (C) in peripheral blood. (D, E) Both syngenic and allogenic BMMSC transplantation reduced the number of CD3 + T cells (D) and induced CD3 + T cell apoptosis (E) in bone marrow. (F, G) Both syngenic and allogenic BMMSC transplantation upregulated levels of Tregs (F) and downregulated levels of Th17 cells (G) in peripheral blood, while allogenic BMMSC transplantation showed a more significant reduction of Th17 cells compared to syngenic BMMSCs at 24 and 72 hours post-transplantation. (H) Flow cytometric analysis showed culture expanded human BMMSCs (hMSCs) express the stem cell markers CD73, CD90, CD105, CD146, and Stro1, but they are negative for the hematopoietic markers CD34 and CD45. Isotopic IgGs were 41 used as a negative control. (I) Schema of human BMMSCs (hMSC) transplantation in C57BL6 mice. (J, K) hMSC infusion induced CD3 + T cell apoptosis in peripheral blood (J) and bone marrow (K) in C57BL6 mice. (L, M) hMSC infusion induced upregulation of Tregs (L) and downregulation of Th17 cells (M) in peripheral blood. (*P<0.05, **P<0.01, ***P<0.005. The bar graph represents mean±SD). Figure S2.3 Apoptosis of transplanted BMMSCs in peripheral blood and bone marrow (related to Figure 2). (A) Western blot showed efficacy of FasL siRNA. (B) Immunofluorescent analysis showed that Annexin + /7AAD + double positive apoptotic cells, including transplanted GFP + BMMSCs (white arrowhead) and recipient cells (orange arrow) at 6 hours post-transplantation in peripheral blood (upper row) and bone marrow (lower row). Bar=50 m. (C-F) Carboxyfluorescein diacetate N-succinimidyl ester (CFSE)-labeled control BMMSCs, FasL -/- gldBMMSCs and FasL siRNA BMMSCs were transplanted into C57BL6 mice. Peripheral blood and bone marrow samples were collected at indicated time points for cytometric analysis. The number of CFSE-positive transplanted BMMSCs reached a peak at 1.5 hours post-transplantation in peripheral blood (C) and bone marrow (D) and then reduced to undetectable level at 24 hours post-transplantation. The number of AnnexinV + 7AAD + double positive apoptotic BMMSCs reached a peak at 6 hours post-transplantation in peripheral blood (E) and bone marrow (F) and then reduced to an undetectable level at 24 hours post- transplantation. (The bar graph represents mean±SD). 42 2.3.2 FasL is required for BMMSC-based therapies in Animal Models To further study the therapeutic mechanism of BMMSC transplantation, two mouse models, genetic tight-skin (Tsk/ + ) systemic sclerosis and inductive experimental colitis, were used to evaluate the therapeutic effect of BMMSC transplantation. Allogenic normal BMMSCs or gldBMMSCs (1x10 6 ) were systemically transplanted into Tsk/ + systemic sclerosis mice (Green et al., 1976) at 8 weeks of age, and samples were harvested at 12 weeks of age for further evaluation (Figure 2.3A). The BMMSC- transplanted group showed significant reduction in the number of CD3 + T cells and corresponding elevation in the number of apoptotic CD3 + T cells in peripheral blood from 6 to 72 hours post-transplantation (Figures 2.3B and 2.3C). On the other hand, FasL -/- gldBMMSC transplantation failed to induce CD3 + T cell apoptosis (Figures 2.3B and 2.3C). Tsk/ + mice showed an increase in the levels of antinuclear antibody (ANA), anti-double strand DNA (dsDNA) IgG and IgM antibodies, and creatinine in serum, along with an increase in the level of urine proteins (Figures 2.3D-2.3H). Normal BMMSC, but not FasL - /- gldBMMSC, transplantation significantly reduced the levels of ANA, dsDNA IgG and IgM, as well as serum creatinine and urine protein levels (Figures 2.3D-2.3H). Moreover, BMMSC transplantation rescued decreased level of Tregs and increased level of Th17 cells in Tsk/ + mice (Figures 2.3I, 2.3J, and S2.4B). As expected, gldBMMSC 43 transplantation failed to regulate the levels of Tregs and Th17 cells in Tsk/ + mice (Figures 2.3I and 2.3J). Histological analysis also showed that skin hypodermal (HD) thickness was significantly increased in Tsk/ + mice (Figure 2.3K). After BMMSC transplantation, HD thickness was reduced to a level equal to that of the control group, whereas gldBMMSC failed to reduce HD thickness (Figure 2.3K). Additionally, the tightness of skin, as measured by grabbed distance, was significantly improved in the BMMSC, but not the gldBMMSC, transplantation group (Figure S2.4A). The induced experimental colitis model was generated as previously described (Alex et al., 2009; Zhang et al., 2010). Allogenic normal BMMSCs or FasL -/- gldBMMSCs (1x10 6 ) were systemically transplanted into experimental colitis mice at day 3 post 3% dextran sulfate sodium induction (Zhang et al., 2010; Figure 2.4A). Normal BMMSC transplantation reduced the number of CD3 + T cells and elevated the number of AnnexinV + 7AAD + double positive apoptotic CD3 + T cells in peripheral blood starting at 1.5 hours and lasting to 72 hours after transplantation (Figures 2.4B and 2.4C). However, the gldBMMSC transplantation group showed no difference from the colitis group in terms of numbers of CD3 + T cells and apoptotic CD3 + T cells (Figures 2.4B and 2.4C). The body weight of mice with induced colitis was significantly reduced compared to control C57BL6 mice from day 5 to 10 post-DSS induction (Figure 2.4D). After normal BMMSC, but not gldBMMSC, transplantation, the body weight was partially restored at day 10 post-DSS induction. The disease activity index (DAI), including body weight loss, 44 diarrhea, and bleeding, was significantly elevated in the colitis mice compared to the control mice. After BMMSC transplantation, the DAI score was decreased, while gldBMMSCs failed to reduce the DAI score (Figure 2.4E). Both decreased Tregs and elevated Th17 cells were observed in the colitis mice from day 7 to 10 post-DSS induction (Figures 2.4F and 2.4G). BMMSC, but not gldBMMSC, transplantation significantly upregulated Tregs and downregulated Th17 cells (Figures 2.4F and 2.4G). Furthermore, colon tissue from each group was analyzed (Figure 2.4H). Both the absence of epithelial layer and infiltration of inflammatory cells were observed in the colitis and gldBMMSC transplantation groups. BMMSC transplantation recovered epithelial structure and eliminated inflammatory cells in colitis mice. Histological activity index (Alex et al., 2009) confirmed that BMMSC transplantation reduced the DAI, while gldBMMSCs failed to improve the DAI (Figure 2.4H). The data therefore suggest that BMMSC-induced T cell apoptosis with Treg upregulation might offer a potential treatment for the colitis (Figure 2.4I). Moreover, upregulation of Tregs was required in ameliorating disease phenotype in DSS-induced colitis model (Figures S2.5A-2.5F). 45 Figure 2.3. FasL is required for BMMSC-mediated amelioration of systemic sclerosis (SSc) phenotypes. (A) Schema showing how BMMSC transplantation ameliorates SSc phenotype. (B, C) BMMSC transplantation (n=6) showed a significantly reduced number of CD3 + T cells (B) and increased number of AnnexinV + 7AAD + double positive apoptotic CD3 + T cells (C) in SSc mice as assessed by flow cytometric analysis. However, FasL -/- gldBMMSC (n=6) failed to reduce the number of CD3 + T cells (B) or elevate the number of apoptotic CD3 + T cells (C). (D-F) Tsk/ + SSc mice showed elevated levels of antinuclear antibody (ANA, D) and anti-double strand DNA antibodies IgG (E) and IgM (F) when compared to control C57BL6 mice. BMMSC transplantation reduced the levels of ANA (D) and anti-double strand DNA antibodies IgG (E) and IgM (F). In contrast, FasL -/- gldBMMSC transplantation failed to reduce the levels of antinuclear antibody (ANA, D) or anti-double strand DNA IgG (E) and IgM (F) antibodies. (G) Creatinine level in serum was significantly increased in Tsk/ + mice. After BMMSC transplantation, creatinine level was significantly decreased to the level observed in C57BL6 mice. However, gldBMMSC transplantation failed to reduce the creatinine level. (H) The concentration of urine protein was significantly increased in Tsk/ + mice. BMMSC transplantation reduced urine protein to the control level. gldBMMSC transplantation failed to reduce urine protein levels in Tsk/ + mice. (I) Treg level was significantly decreased in Tsk/ + mice compared to C57BL6 mice. After BMMSC transplantation, Treg levels were significantly elevated, whereas gldBMMSC transplantation failed to increase Treg levels in Tsk/ + mice. (J) 46 CD4 + IL17 + Th17 cells were significantly increased in Tsk/ + mice compared to C57BL6 mice. Elevated Th17 level was significantly reduced in the BMMSC transplantation group, while gldBMMSC transplantation failed to reduce the Th17 level in Tsk/ + mice. (K) Hyperdermal thickness was significantly increased in Tsk/ + mice (Tsk/ + , n=5) compared to control mice (C57BL6, n=5). BMMSC, but not FasL -/- gldBMMSC, transplantation reduced hyperdermal thickness. (*P<0.05, **P<0.01, ***P<0.001. The bar graph represents mean±SD). Figure 2.4. FasL plays a critical role in BMMSC-mediated immune therapy for Dextran sulfate sodium (DSS)-induced experimental colitis. (A) Schema showing BMMSC transplantation in DSS-induced experimental colitis mice. (B, C) BMMSC transplantation (n=6) showed a significantly reduced number of CD3 + T cells at 24 hours post- transplantation (B) and increased number of AnnexinV + 7AAD + double positive apoptotic CD3 + T cells at 24-72 hours post-transplantation (C) in colitis mice as assessed by flow cytometric analysis. However, FasL -/- gldBMMSC (n=6) failed to reduce the number of CD3 + T cells (B) or elevate the number of apoptotic CD3 + T cells (C). (D) Colitis mice (colitis, n=5), BMMSC transplanted group, and gldBMMSC showed significantly reduced body weight from 5 to 10 days after DSS induction. The BMMSC transplantation group showed inhibition of body weight loss compared to the colitis and gldBMMSC 47 transplantation groups at 10 days after DSS induction. (E) Disease activity index (DAI) was significantly increased in colitis mice compared to C57BL6 mice from 5 days to 10 days after DSS induction. BMMSC transplantation significantly reduced DAI score, but it was still higher than that observed in C57BL6 mice. FasL -/- gldBMMSC transplantation failed to reduce DAI score at all time points. (F) Treg level was significantly reduced in colitis mice compared to C57BL6 mice at 7days after DSS induction. BMMSC, but not FasL -/- gldBMMSC, transplantation upregulated the Treg levels in colitis mice. (G) Th17 cell level was significantly elevated in colitis mice compared to C57BL6 mice at 7 days after DSS induction. BMMSC, but not FasL -/- gldBMMSC, transplantation reduced the levels of Th17 cells in colitis mice from 7 to 10 days after DSS induction. (H) Hematoxylin and eosin staining showed the infiltration of inflammatory cells (blue arrows) in colon with destruction of epithelial layer (yellow triangles) in colitis mice. BMMSC, but not FasL -/- gldBMMSC, transplantation rescued disease phenotype in colon and reduced histological activity index. (I) Schematic diagram of BMMSC transplantation for immunotherapies. (Bar= 200 m; *P<0.05, **P<0.01, ***P<0.001. The bar graph represents mean±SD). Figure S2.4. FasL is required for BMMSC-mediated amelioration of skin phenotype in systemic sclerosis (SSc) mice (related to Figure 3). (A) Systemic sclerosis mouse model (Tsk/ + ) showed tight skin phenotype compared to control C57BL6 mice. BMMSC, but not FasL -/- gldBMMSC, transplantation significantly improved skin phenotype in terms of grabbed skin distance. (B) BMMSC transplantation maintained spleen Treg level as observed in control mice at 2 month post-transplantation. (*P<0.05, **P<0.01, The bar graph represents mean±SD). 48 Figure S2.5. Tregs are required in BMMSC-mediated immune therapy for DSS-induced experimental colitis (related to Figure 4). (A) Schema of BMMSC transplantation with blockage of Treg using anti-CD25 antibody in DSS-induced colitis mice. (B) Colitis mice (colitis, n=5), BMMSC-treated colitis mice (n=6), and BMMSC-treated colitis mice with anti-CD25 antibody injection (BMMSC+antiCD25ab, n=5) showed reduced body weight from 5 to 10 days after DSS induction. BMMSC transplantation, but not BMMSC transplantation along with anti CD25ab injection, could partially inhibit colitis-induced body weight loss at 10 days after DSS induction. (C) Disease Activity Index (DAI) was significantly increased in colitis mice compared to C57BL6 mice from 5 to 10 days after DSS induction. BMMSC transplantation significantly reduced the DAI score compared to colitis model, but it was still higher than that observed in C57BL6 mice. The BMMSC+antiCD25ab group failed to reduce the DAI score at all observed time points. (D) Treg level was significantly reduced in colitis mice compared to C57BL6 mice at 7days after DSS induction. The BMMSC transplantation group showed upregulation of Treg levels in colitis mice. The BMMSC+antiCD25ab group showed reduced Treg level at all time points. (E) Th17 cell level was significantly elevated in colitis mice compared to C57BL6 mice at 7 days after DSS induction. The BMMSC transplantation reduced the levels of Th17 cells in colitis mice from 7 to 10 days after DSS induction. The BMMSC+antiCD25ab group showed lower level of Th17 cells compared to colitis group, but still higher than the BMMSC group at 10 days post-DDS induction. (F) Hematoxylin and eosin staining showed the infiltration of inflammatory cells (blue arrows) in colon with destruction of epithelial layer (yellow triangles) in colitis mice. The BMMSC transplantation group showed rescued disease phenotype in colon and histological activity index, while the BMMSC+antiCD25ab group failed to reduce disease phenotype at 10 days after DSS induction. (Bar= 200 m; *P<0.05, **P<0.01, ***P<0.001. The bar graph represents mean±SD). 49 2.3.3 Fas is required for BMMSC-mediated therapy by recruitment of T cells In addition to the production of FasL, BMMSCs also express Fas (Figure S2.6A). To examine whether Fas plays a role in BMMSC-based immunotherapies, we infused Fas -/- BMMSCs, derived from C3MRL-Fas lpr /J mice (lprBMMSCs), to C57BL6 mice and found that Fas -/- lprBMMSCs failed to reduce the number of CD3 + T cells or elevate the number of apoptotic CD3 + T cells in peripheral blood and bone marrow (Figures 2.5A- 2.5D). In addition, we revealed that lprBMMSC transplantation failed to upregulate the levels of Tregs and TGF  and downregulate Th17 cell level in peripheral blood (Figures 2.5E, 2.5F, and S2.6X). Moreover, Fas knockdown BMMSCs using siRNA showed the same effect as observed in Fas null lprBMMSC (Figure S2.6Y-2.6EE). When transplanted into DSS-induced colitis mice, lprBMMSCs neither provided therapeutic effects on body weight, disease activity index, histological activity index nor balanced the levels of Tregs and Th17 cells (Figures S2.6B-2.6G). In addition, lprBMMSC transplantation failed to treat Tsk/ + SSc mice, showing no rescue of the levels of ANA, anti-dsDNA antibodies IgG and IgM antibodies, creatinine, urine protein, Grabbed distance, Tregs, or Th17 cells (Figures S2.6H-2.6Q). Taken together, these data suggest that Fas -/- lprBMMSCs, like FasL -/- gldBMMSCs, were unable to ameliorate disease phenotypes in SS and colitis mouse models. 50 Next, we investigated the underlying mechanisms by which lprBMMSC transplantation failed to treat the diseases. We showed that lprBMMSCs expressed a normal level of FasL by Western blot analysis (Figure S2.6R) and induced CD3 + T cell apoptosis in a co- culture system (Figure 2.5G). This was blocked by anti-FasL neutralizing antibody (Figure 2.5G), suggesting that the failure to induce in vivo T cell apoptosis by lprBMMSCs does not result from the lack of expression of functional FasL. We therefore hypothesized that Fas expression affects the BMMSC immunomodulatory property via a non-FasL-related mechanism, such as regulating the recruitment of T cells. To test this, we used an in vitro transwell co-culture system to show that activated T cells migrate to BMMSCs to initiate cell-cell contact (Figure 2.5H). However, lprBMMSCs showed a significantly reduced capacity to recruit activated T cells in the co-culture system when compared to control BMMSCs (Figures 2.5H and 2.5I). We then used a cytokine array analysis to determine that lprBMMSCs express a low level of MCP-1, a member of the C-C motif chemokine family and a T cell chemoattractant cytokine (Carr et al. 1994; Figure S2.6S). Interestingly, overexpression of MCP-1 in lprBMMSCs partially rescued their capacity to recruit T cells (Figures 2.5H-2.5J). Overexpression of Fas in lprBMMSCs showed that the secretion level of multiple cytokine was restored (Figures S2.6S and S2.6U) and fully rescued their capacity to recruit T cells (Figures 2.5H, 2.5I, 2.5K). However, the expression level of MCP-1 protein in lprBMMSCs was higher than that in control BMMSCs, and overexpression of Fas reduced MCP-1 cytoplasm protein level in lprBMMSCs (Figure 2.5L), indicating that Fas regulates MCP-1 secretion, but not expression. Next, we examined MCP-1 level in the culture supernatant, and we found 51 that the MCP-1 level in lprBMMSCs was significantly lower than BMMSCs (Figure 2.5M). Overexpression of MCP-1 and Fas in lprBMMSCs rescued MCP-1 levels in culture supernatant (Figure 2.5M). We next confirmed that Fas regulated MCP-1 secretion using the siRNA knockdown approach (Figure S2.6T). Downregulation of Fas expression in BMMSCs resulted in a reduced MCP-1 secretion (Figure 2.5N), with a corresponding reduction in the capacity to recruit activated T cells in the co-culture system (Figures 2.5O and 2.5P). In order to confirm that MCP-1 contributes to BMMSC-based immunoregulation, we isolated BMMSCs from MCP-1 mutant B6.129S4-Ccl2 tm1Rol /J mice and showed that MCP-1 -/- BMMSCs were defective in reducing the number of CD3 + T cells or elevating apoptotic CD3 + T cells in C57BL6 mice when compared to control BMMSCs (Figures 2.6A and 2.6B). Also, MCP-1 -/- BMMSCs failed to upregulate the levels of Tregs and TGF  within 72 hours post-transplantation (Figures 2.6C and 2.6D). The deficiency of inducing T cell apoptosis and Treg upregulation by MCP-1 -/- BMMSCs was not associated with FasL function (Figure 2.6E). When MCP-1 -/- BMMSCs were co-cultured with activated T cells in a transwell culture system, the number of T cells migrating to BMMSCs was significantly reduced compared to control BMMSCs (Figure 2.6F). Also, Fas and MCP-1 play an important role in attracting B cells, NK cells, and immature dendritic cells (iDCs) in an in vitro culture system (Figure S2.7A-2.7C). These data indicate that MCP-1 secretion regulates BMMSC-induced T cell migration (Figure 2.6G). Moreover, we showed that Fas also regulated the secretion of other cytokines, such as C- 52 X-C motif chemokine 10 (CXCL-10) and tissue inhibitor of matrix metalloprotease-1 (TIMP-1) (Figures S2.6V and 2.6W). Figure 2.5. Fas plays an essential role in BMMSC-mediated CD3 + T cell apoptosis and up-regulation of Tregs via regulating monocyte chemotactic protein 1 (MCP-1) secretion. (A-D) BMMSC transplantation (BMMSC) induced transient reduction in the number of CD3 + T cells and increase in the number of AnnexinV + 7AAD + double positive apoptotic CD3 + T cells in peripheral blood mononuclear cells (PBMNCs; A, B) and bone marrow mononuclear cells (BMMNCs, n=5; C, D) at indicated time points, while Fas -/- BMMSC from lpr mice (lprBMMSC, n=5) failed to reduce the number of CD3 + T cells or increase the number of CD3 + apoptotic T cells in peripheral blood (A, B) and bone marrow (C, D). (E, F) lprBMMSC transplantation failed to elevate Treg levels (E) and TGF  (F) in C57BL6 mice compared to the BMMSC transplantation group at indicated time points. (G) lprBMMSC induced activated T cell apoptosis in a BMMSC/T cell in vitro co-cultured system, which was blocked by anti-FasL neutralizing antibody (1 g/mL). (H- K) Activated T cells (green) migrate to BMMSCs (red) in a transwell co-culture system (H). lprBMMSCs showed a significantly reduced capacity to induce activated T cell migration (I), which could be partially rescued by overexpression of MCP-1 (J) and totally 53 rescued by overexpression of Fas (K) in lprBMMSCs. The results were representative of three independent experiments. (L) Quantitative RT-PCR analysis showed no significant difference between BMMSC and lprBMMSC in terms of MCP-1 expression level. However, overexpression of MCP-1 and Fas in lprBMMSC significantly elevated gene expression level of MCP-1. (M) Western blot showed that lprBMMSCs express a higher cytoplasm level of MCP-1 than BMMSC. Overexpression of Fas in lprBMMSC reduced the expression level of MCP-1 in cytoplasm. (N) ELISA analysis showed that MCP-1 secretion in culture supernatant was significantly reduced in lprBMMSCs compared to BMMSC. Overexpression of MCP-1 and Fas in lprBMMSCs significantly elevated MCP-1 secretion in culture supernatant. (O) ELISA data showed that knockdown Fas expression using siRNA resulted in reduction of MCP-1 level in culture medium compared to control siRNA group. (P-Q) Fas siRNA-treated BMMSCs (Q) showed reduced T cell migration in transwell co-culture system compared to control siRNA group (P). (*P<0.05, **P<0.01, ***P<0.001. The bar graph represents mean±SD). Figure 2.6. MCP-1 plays an important role in T cell recruitment. (A) MCP-1 -/- BMMSC transplantation showed a slightly reduced number of CD3 + T cells in peripheral blood, but the level of reduction was significantly less than that of the BMMSC transplantation group. (B) AnnexinV + 7AAD + double positive apoptotic CD3 + T cell percentage was slightly increased in the MCP-1 -/- BMMSC transplant group. (C) Treg level was slightly increased in the MCP-1 -/- BMMSC-transplanted group at 72 hours post-transplantation, but significantly lower than the BMMSC transplantation group. (D) TGF-  level in serum was slightly increased in the MCP-1 -/- BMMSC-transplanted group at 72 hours after transplantation compared to 0 hour, but the elevation level was lower than the BMMSC transplantation group. (E) When T cells were stimulated with CD3 and CD28 antibody and co-cultured with BMMSC or MCP-1 -/- BMMSC in a transwell culture system, the 54 number of migrated T cells was significantly higher in the BMMSC group (E) than the MCP-1 -/- BMMSC group. (F) Schematic diagram showing the mechanism of BMMSC- induced immunotherapies. (**P<0.01, ***P<0.005; The bar graph represents mean±SD). 55 Figure S2.6. Fas is required for ameliorating disease phenotype in induced experimental colitis and systemic sclerosis (SSc) (related to Figure 5). (A) Western blot analysis showed that mouse BMMSCs express Fas. CD8 + T cells were used as a positive control. (B) Schema of BMMSC transplantation in experimental colitis mice. (C) lprBMMSC transplantation failed to inhibit body weight loss in colitis mice. (D) Increased disease activity index in colitis mice was not reduced in the lprBMMSC transplantation 56 group. (E) Histological analysis of colon showed no remarkable difference between experimental colitis mice and lprBMMSC transplantation group. Bar=200 m. (F) lprBMMSC transplantation failed to upregulate Treg level in experimental colitis mice. (G) Increased Th17 level in experimental colitis mice was not reduced in the lprBMMSC transplantation group. (H) Schema of BMMSC transplantation in Tsk/ + mice. (I) Increased ANA level in SSc (Tsk/ + ) mice was not reduced in the lprBMMSC transplantation group. (J, K) The levels of Anti-dsDNA were not reduced in lprBMMSC treated Tsk/ + mice (IgG: J, IgM; K). (L) Increased creatinine level in Tsk/ + mice was not reduced in the lprBMMSC transplantation group. (M) lprBMMSC failed to reduce urine protein level in Tsk/ + mice. (N) Bent vertebra and skin tightness, as indicated by grabbed distance in Tsk/ + mice, were not improved in the lprBMMSC transplantation group. (O) The reduced Treg level in Tsk/ + mice was not upregulated in lprBMMSC transplantation group. (P) lprBMMSC transplantation failed to reduce Th17 level in Tsk/ + mice. (Q) lprBMMSC transplantation failed to reduce hypodermal thickness in Tsk/ + mice. (R) Western blot analysis showed that Fas -/- lprBMMSCs express FasL at the same level as observed in BMMSCs. (S) Cytokine array analysis showed that BMMSCs express a higher level of MCP-1 than lprBMMSCs in the culture supernatant. After Fas overexpression in Fas -/- lprBMMSC (Fas + lprBMMSC) by cDNA transfection, the secretion level of multiple cytokines/chemokines was restored to the level observed in BMMSCs. (T) Western blot analysis showed efficacy of Fas siRNA in BMMSCs. (U) Flow cytometric analysis showed that transfection of Fas into lprBMMSCs could significantly elevated the expression level of Fas. (V-W) ELISA analysis showed that Fas -/- lprBMMSCs and Fas knockdown BMMSCs (Fas siRNA BMMSC) had a significantly reduced level of CXCL-10 (V) and TIMP-1 (W) in the culture supernatant compared to BMMSCs or control siRNA group. (X) BMMSC transplantation showed downregulated levels of Th17 cells from 6 to 72 hours post- transplantation, while lprBMMSCs failed to reduce the number of Th17 cells in peripheral blood. (Y) Schema of Fas knockdown BMMSC transplantation in C57BL6 mice. (Z, AA) Fas knockdown BMMSCs using siRNA (Fas siRNA BMMSC) showed a significantly reduced capacity to reduce the number of CD3 + T cells (Z) and induce CD3 + T cell apoptosis (AA) in peripheral blood. (BB, CC) Fas siRNA BMMSCs showed reduced capacity to reduce the number of CD3 + T cells (BB) and induce CD3 + T cell apoptosis (CC) when compared to the BMMSC transplantation group in bone marrow. (DD) Fas siRNA BMMSCs failed to upregulate Tregs compared to the BMMSC group in peripheral blood. (EE) Fas siRNA BMMSC failed to significantly reduce Th17 cell compared to BMMSC group in peripheral blood. (*P<0.05, **P<0.01, ***P<0.005, The bar graph represents mean±SD). 57 Figure S2.7. Fas and MCP-1 regulate BMMSC-mediated B cell, NK cell, and immature dendritic cell (iDC) migration in vitro (related to Figure 6). (A-C) When B cells, NK cells, and iDCs were co-cultured with BMMSCs, Fas -/- lprBMMSCs, Fas knockdown BMMSCs using siRNA (Fas siRNA BMMSC), or MCP-1 -/- BMMSCs in a transwell culture system, the number of migrated B cells (A), NK cells (B), and iDCs (C) was significantly higher in the BMMSC group. (**P 58 2.3.4 MSCT induced T cell apoptosis and Treg upregulation in SSc patients Based on the above results in experimental animal models, we conducted a pilot clinical investigation to assess whether T cell apoptosis and Treg upregulation occurred in SSc patients treated with MSCT. Five patients (4 females and 1 male, Table 2), ranging in age from 44 to 61 years old (average 51.2 7.8 years old) and having SSs for a duration of 48- 480 months (average 163.2 182.1 months) were enrolled for allogenic MSCT (Sun et al., 2009; Liang et al., 2009) and peripheral blood was collected at indicated time points (Figure 2.7A). Allogenic MSC transplantation induced a significant reduced number of CD3 + T cells and upregulated number of AnnexinV-positive apoptotic CD3 + T cells at 6 hours post-MSCT, followed by a decrease in CD3 + T cell number and apoptotic rate to baseline level by 72 hours (Figure 2.7B and 2.7C). A reduced number of CD4 + T cells was also observed at 6 hours post-MSCT (Figure 2.7D). Importantly, the frequency of Tregs in peripheral blood was significantly upregulated at 72 hours post-MSCT (Figure 2.7E), along with elevated level of TGF  (Figure 2.7F). Assessment of Modified Rodnan Skin Score (MRSS) and Health Assessment Questionnaire (HAQ-DI) indicated that MSCT provided optimal treatment for SSc patients at follow-up period (Figure 2.7G and 2.7H). Furthermore, reduced level of ANA was observed in SSc patients at the 12-month follow-up period (Figure 2.7J). Interestingly, the MSC derived from SSc patients (SSMSC) showed a deficiency in FasL and Fas expression when compared to MSC derived from 59 healthy donors (MSC) (Figures 2.7K and 2.7M). SSMSCs showed a reduced capacity to induce T cell apoptosis (Figure 2.7L) and secrete MCP-1 (Figure 2.7N) by the reduced expression levels of FasL and Fas. In addition, we found that MSCT significantly improved skin ulcers in a patient (Figure 2.7I). These early clinical data demonstrate safety and efficacy of MSCT in SSc patients and improvement of disease activities at post-allogenic MSCT. However, the long-term effects of MSCT on SSc patients will require further investigation. Table 2. SSc Patient Information. 60 Figure 2.7. Allogenic MSC transplantation induces CD3 + T cell apoptosis and Treg upregulation in systemic sclerosis (SSc) patients. (A) Schema of MSC transplantation in SSc patients. (B) Flow cytometric analysis showed reduced number of CD3 + T cells from 2 to 72 hours post-transplantation. (C) AnnexinV + -positive apoptotic CD3 + T cells were significantly increased at 6 hours after MSC transplantation. (D) Flow cytometric analysis showed reduced number of CD4 + T cells from 2 to 72 hours post-transplantation. (E) Treg levels in peripheral blood were significantly increased at 72 hours after allogenic MSC transplantation. (F) Serum level of TGF  was significantly increased in MSC transplantation group at 72 hours post-transplantation. (G, H) Modified Rodnan Skin Score (MRSS, G) and Health assessment Questionnaire disease activity index (HAQ-DI, H) were significantly reduced after allogenic MSC transplantation. (I) Representative images of skin ulcers prior to MSC transplantation (pre-MSC) and at 6 months post- transplantation (post-MSC). (J) The reduced ANA level was maintained at 12 months after MSC transplantation. (K) Real-time PCR analysis showed significantly decreased FasL expression in SSc patient MSCs (SSMSC) compared to MSC from healthy donor (MSC). (L) SSMSC showed a significantly decreased capacity to induce T cell apoptosis compared to normal MSC in vitro. (M) SSMSC showed a reduced expression of Fas by real-time PCR analysis. (N) MCP-1 secretion level in SSMSC was significantly lower than that in MSC culture supernatant. (*P<0.05, **P<0.01, ***P<0.005; The bar graph represents mean±SD). 61 2.3.5 TERT is associated with BMMSC-mediated immunomodulation To address whether TERT is important in regulating BMMSC-mediated immunomodulation, we isolated BMMSCs from TERT null mice, B6.129S-Tert tm1Yjc /J (TERT -/- ), and found that the number of single colony clusters (colony-forming unit fibroblasts, CFU-F) was significantly reduced in TERT -/- BMMSCs (Figure 2.8A). To examine the proliferative capacity of TERT -/- BMMSCs, we performed a BrdU-labeling assay to show that TERT -/- BMMSCs have a reduced proliferative rate compared to TERT +/+ age-matched littermate (wild-type) BMMSCs (Figure 2.8B). Flow cytometric analysis showed generally lower expression of mesenchymal stem cell surface molecules, including CD90, CD105, Sca1, and SSEA4, in TERT -/- BMMSCs, while the hematopoietic lineage markers CD34 and CD45 were absent in TERT -/- BMMSCs, similar to observations of BMMSCs derived from wild type mice (WT BMMSCs) (Figure 2.8C and 2.8D). We observed that TERT -/- BMMSCs exhibited decreased osteogenic differentiation and increased adipogenic differentiation potential, as indicated by alizarin red staining of mineralized nodule formation and Oil red O staining of lipid-containing adipocytes, respectively. As expected, gene expression analysis revealed downregulated expression of the osteogenic genes runt-related transcription factor 2 (runx2) and osteocalcin (ocn), along with upregulated expression of the adipogenic genes peroxisome proliferator- 62 activated receptor gamma 2 (ppar γ2) and lipoprotein lipase (lpl) in TERT -/- BMMSCs (Figures S2.8E-2.8H). Next, we used a BMMSC/T-cell co-culture system to show that TERT -/- BMMSCs had significantly reduced capacity to induce AnnexinV + 7AAD - and AnnexinV + 7AAD + double positive apoptotic T-cells and upregulate CD4 + CD25 + Foxp3 + regulatory T-cells (Tregs), when compared to the WT BMMSCs (Figures 2.8E and 2.8F). Western blot analysis confirmed the absence of telomerase activity and TERT expression in TERT -/- BMMSCs, as assessed by a telomeric repeat amplification protocol (TRAP)-ELISA assay and Western blot, respectively (Figures S2.8A and 2.8B). In addition, we performed quantitative PCR (qPCR) to examine the RNA level of TERT from passage-0 to passage-10 to further confirm our Western blot data. TERT expression is maintained at a certain level from P0 to P2 of WT BMMSCs, which were used in this study. However, the expression level of TERT was significantly decreased in passage 5 and undetectable by qPCR in passage 10. On the other hand, TERT expression was undetectable in TERT -/- BMMSCs from p0 to p10 (Figure S2.8A). Moreover, siRNA-mediated knockdown of TERT expression in BMMSCs showed that TERT expression levels and telomerase activity were markedly decreased in TERT knockdown BMMSCs compared to the scrambled siRNA treated BMMSCs (Figures S2.8C and 2.8D). TERT knockdown BMMSCs also showed a significantly decreased capacity to induce T-cell apoptosis and upregulate Tregs when compared to the WT BMMSCs (Figures 2.8G and 2.8H). Previous studies have reported 63 that aged BMMSCs exhibit decreased proliferation and differentiation potential (Bonab et al, 2006). We found that BMMSCs from 6-month-old mice (6M-BMMSCs) had downregulated levels of TERT and a reduced capacity to induce T-cell apoptosis and upregulate Tregs when compared to BMMSCs from 1-month-old mice (Figures S2.9A- 2.9C). Thus, we have used TERT -/- , TERT knockdown and BMMSCs from mice of different ages to demonstrate the key role telomerase plays in governing BMMSC-based immunomodulation. Figure 2.8 TERT contributes to BMMSC-mediated immunomodulation. (A) TERT -/- BMMSCs showed reduced single colony-forming ability. (Student's t-test, n=5 in each group, **p < 0.01). Error bars present the s.d. of the mean values. (B) BrdU labeling was performed to show reduced proliferative capacity of TERT -/- BMMSCs (Student's t-test, n=5 in each group, ***p < 0.005). Error bars present the s.d. of the mean values. (C-D) 64 FACS analysis showed downregulated expression levels of BMMSC surface markers in TERT -/- BMMSCs (Student's t-test, n=5 in each group, *p < 0.005). Error bars present the s.d. of the mean values. (E) In vitro coculture system showed a significantly decreased capacity of TERT -/- BMMSCs to induce AnnexinV + 7AAD - and AnnexinV + 7AAD + double positive apoptotic T cells compared to regular (WT) BMMSCs (One-way Anova, Bonferroni, n=5 in each group, ***p < 0.005). Error bars present the s.d. of the mean values. (F) Treg induction assay indicated a significantly decreased capacity of TERT -/- BMMSCs to upregulate Treg levels in comparison with WT BMMSCs (One-way Anova, Bonferroni, n=5 in each group, ***p < 0.005, *p < 0.05). Error bars present the s.d. of the mean values.. (G) In vitro coculture system showed a decreased capacity of TERT knockdown BMMSCs by siRNA to induce AnnexinV + 7AAD - and AnnexinV + 7AAD + double positive apoptotic T cells BMMSCs (One-way Anova, Bonferroni, n=5 in each group, ***p < 0.005). Error bars present the s.d. of the mean values. (H) Treg induction assay was performed to show a significantly decreased capacity of TERT knockdown BMMSCs to upregulate the level of Tregs in comparison with WT BMMSCs (One-way Anova, Bonferroni, n=5 in each group, ***p < 0.005, *p < 0.05). Error bars present the s.d. of the mean values. Vehicle: scrambled siRNA-treated BMMSCs. 65 Figure S2.8 Characterization of BMMSCs from TERT -/- mice. (A) Western blot showed no TERT expression in TERT -/- BMMSCs. (B) TRAP-ELISA analysis showed that TERT -/- BMMSCs had no telomerase activity. (C) TERT siRNA significantly knocked down TERT expression. (D) Telomerase activity was significantly decreased in TERT siRNA-treated BMMSCs. (E-F) TERT -/- BMMSCs showed downregulation of the osteogenic genes runx2 and ocn (E) and bone nodule formation (F). (G-H) In vitro adipogenesis evaluation showed that TERT -/- BMMSCs had elevated expression of pparg and lpl (G), and Oil red-O staining showed that TERT -/- BMMSCs had increased capacity to differentiate to adipocytes (H). Error bars present the s.d. from three independent experiments (***p<0.005, **p<0.01, *p<0.05). Figure S2.9 BMMSCs from 6-month-old mice (6M-BMMSCs) have reduced immunomodulatory property. (A) TERT and FASL expression levels were significantly decreased in 6M-BMMSCs compared to regular BMMSCs by Western blot analysis. (B) BMMSC-induced T cell apoptosis in an in vitro co-culture system was dramatically decreased in 6M-BMMSCs. (C) In vitro Treg induction by 6M-BMMSCs was markedly decreased. Error bars present the s.d. from three independent experiments (***p<0.005). 66 2.3.6 TERT is required for BMMSC-mediated therapy in Animal Models Recently, immunomodulatory properties were identified as an important characteristic of BMMSCs, which has led to their systemic infusion to treat a variety of immune diseases (Aggarwal & Pittenger, 2005; Nauta & Fibbe, 2007; Uccelli et al, 2007; 2008). Therefore, in order to assess the therapeutic effect of TERT -/- BMMSCs, we infused either regular BMMSCs (WT; from TERT +/+ littermates) or TERT -/- BMMSCs into B6.Cg- Fbn1 Tsk /J (Tsk/ + ) systemic sclerosis (SSc) mice at 8 weeks of age and analyzed treatment response at 12 weeks of age (Figure 2.9A). Flow cytometric analysis revealed that WT BMMSC transplantation (MSCT) significantly upregulated the number of Tregs and downregulated the number of CD4 + IL17 + IFN  - T helper 17 (Th17) cells in comparison to the untreated group, while TERT -/- MSCT failed to either upregulate Treg levels or downregulate the level of Th17 cells in SSc mice (Figures 2.9B and 2.9C). Furthermore, SSc mice showed a significant increase in the levels of antinuclear antibody (ANA) and anti-double strand DNA (dsDNA) IgG and IgM antibodies in serum. WT MSCT, but not TERT -/- MSCT, showed significant reduction in the levels of ANA, and dsDNA IgG and IgM in SSc mice (Figures 2.9D-2.9F). Additionally, the tightness of skin, as measured by grabbed distance, was significantly improved in the WT MSCT group, but not the TERT -/- MSCT group (Figure 2.9G). Histological analysis also confirmed that skin hypodermal thickness was significantly increased in SSc mice. After WT MSCT, hypodermal thickness 67 was reduced to a level equal to that of the control group, whereas TERT -/- MSCT failed to reduce hypodermal thickness (Figure 2.9H). These data indicate that TERT -/- MSCT failed to offer effective treatment for SSc mice. Figure 2.9 TERT is required for BMMSC-mediated amelioration of disease phenotype in systemic sclerosis mice. (A) Schema showing BMMSC transplantation (MSCT) for treating systemic sclerosis (SSc) tight skin (Tsk/ + ) mice. (B) FACS analysis showed that the Treg level was significantly decreased in Tsk/ + mice compared to Fbn1 +/+ control littermates. After MSCT, the Treg level was significantly elevated, whereas TERT -/- MSCT failed to upregulate the Treg level in Tsk/ + mice. (C) FACS analysis showed that CD4 + IL17 + Th17 cells were significantly increased in Tsk/ + mice compared to control littermates. WT MSCT, but not TERT -/- MSCT, was able to significantly reduce the Th17 level in Tsk/ + mice. (D-F) ELISA assays showed that Tsk/ + mice had elevated levels of anti- double strand DNA antibodies IgG (D), IgM (E) and antinuclear antibody (ANA, F) when compared to control littermates. WT MSCT reduced the levels of anti-double strand DNA antibodies IgG (d), IgM (e) and ANA (f). In contrast, TERT -/- MSCT failed to reduce the levels of anti-double strand DNA antibodies IgG (d), IgM (e) and ANA (f). (G) Tsk/ + mice showed a tight skin phenotype. Grabbed skin distance measurement showed that WT BMMSC, but not TERT -/- BMMSC, transplantation significantly improved the tight skin phenotype. (H) Histological examination identified that hyperdermal thickness was significantly increased in Tsk/ + mice compared to control littermates. WT BMMSC, but not TERT -/- BMMSC, transplantation improved hyperdermal thickness in Tsk/ + mice. Scale Bar, 100 m. D: Dermal, M: Muscle, and HD: Hyperdermal. Error bars represent the s.d. from the mean values (One-way Anova, Bonferroni, n=6 in each group, ***p < 0.005, **p < 0.01, *p < 0.05). 68 2.3.7 TERT promotes FasL expression through Wnt/ -catenin pathway In order to learn how telomerase activity contributes to BMMSC-mediated immunomodulation, we examined the levels of BMMSC-associated immunomodulatory factors, including IL10, PGE2, and FasL, in TERT -/- and TERT knockdown BMMSCs. ELISA analysis showed that IL10 and PGE2 were not significantly altered in either TERT null or knockdown BMMSCs (Figures S2.10A and 2.10B). However, Western blot analysis indicated that the FasL expression level was markedly decreased in both TERT null and knockdown BMMSCs (Figures 2.10A and 2.10B). To further confirm that FasL is required for BMMSC-mediated immunosuppression, we isolated FasL mutant BMMSCs from B6Smn.C3-Fasl gld /J mice (gldBMMSC) and examined their immunomodulatory properties in an in vitro coculture system. The capacity of gldBMMSCs to induce T cell apoptosis was significantly decreased when compared to WT BMMSCs (Figure S2.10C). These findings are supported by previous studies, which showed that FasL plays a crucial role in BMMSC-based immunomodulation (Akiyama et al, 2012). In the present study, we isolated FasL + and FasL - subpopulations of BMMSCs by fluorescence cell sorting (Figure S2.11A) and used a T-cell co-culture system to show that FasL + BMMSCs had an increased capacity to induce both AnnexinV + 7AAD - and AnnexinV + 7AAD + double positive apoptotic T-cells when compared to WT BMMSCs, while FasL - BMMSCs lost this 69 immunomodulatory function in the in vitro co-culture system, confirming that FasL expression affects the immunomodulatory properties of BMMSCs (Figure S2.11B). It has been reported that TERT is able to act as a cofactor to modulate transcriptional responses by regulating the Wnt signaling pathway and is also able to execute a stem cell activation program by interacting with the chromatin-remodeling protein BRG1 (Park et al, 2009). We next examined the expression levels of Wnt/ -catenin and BRG1 in TERT-deficient BMMSCs. Western blot analysis showed that the expression level of active -catenin (non-phosphorylated), but not BRG1, was markedly decreased in both TERT null and knockdown BMMSCs (Figures 2.10A and 2.10B). -catenin activator (CHIRON 99021) treatment could significantly elevate expression levels of activated - catenin and FasL, but not TERT, in BMMSCs. FasL knockdown by siRNA in -catenin activator-treated BMMSCs significantly diminished the FasL expression level, but not that of TERT or activated -catenin (Figure 2.10C). Co-culture of BMMSCs and T-cells indicated that -catenin activator treatment could significantly elevate the capacity of BMMSCs to induce both AnnexinV + 7AAD - and AnnexinV + 7AAD + double positive apoptotic T cells when compared to the untreated group, but that such elevation could be abrogated by FasL siRNA treatment (Figure 2.10D). TRAP-ELISA assays also showed that -catenin activator treatment failed to affect telomerase activity when compared to WT BMMSCs (Figure 2.10E). Downregulation of -catenin expression in BMMSCs resulted in decreased FasL expression levels, as evaluated by Western blot assay, 70 confirming that Wnt/ -catenin signaling regulated FasL (Figure 2.10F). Flow cytometric analysis showed that -catenin knockdown BMMSCs have a reduced capacity to induce AnnexinV + 7AAD - and AnnexinV + 7AAD + double positive apoptotic T cells (Figure 2.10G). Interestingly, overexpression of tert in TERT -/- BMMSCs (TERT TF) rescued the expression levels of TERT, active -catenin, and FasL, as well as the capacity to induce T cell apoptosis, while fasl overexpression (FASL TF) only elevated FasL expression, but also rescued the capacity to induce T-cell apoptosis (Figures 2.10H and 2.10I). This experimental evidence suggests that TERT serves as an upstream activator of Wnt/ - catenin signaling to regulate FasL expression, which, in turn, regulates the immunomodulatory properties of BMMSCs. 2.3.8 TERT serves as a transcriptional modulator for FasL expression To examine whether -catenin directly controls FasL expression at the transcriptional level, we used PROMO search tools (http://alggen.lsi.upc.es/) to examine the fasl promoter sequence. We found two possible transcription factor candidate binding sites, TCF/LEF1 binding element (TBE) and nuclear factor kappa B (NF B), both closely matching the consensus targets. We therefore generated 1.1kb (only NF B targets) and 2kb (both TBE and NF B targets) fasl promoter reporter constructs in which the defined region of the fasl promoter and flanking region were placed upstream of a reporter 71 gene encoding firefly luciferase (Figure 2.10J). Luciferase reporter analysis demonstrated that the 2kb construct, but not the 1.1kb construct, showed markedly higher promoter activity in both normal BMMSCs (WT) and tert overexpressed BMMSCs (TERT TF) compared to TERT null BMMSCs (TERT -/- ), suggesting that the TBE transcriptional element may contribute to FasL expression. When TERT -/- BMMSCs were transfected with a reporter vector, the luciferase assay showed significantly decreased promoter activity. Introduction of a TBE-mutated reporter vector markedly diminished the expression of the fasl-luciferase reporter, suggesting a direct initiation of fasl expression by Wnt/ -catenin cascades (Figure 2.10J). We next determined whether - catenin directly binds to the fasl promoter in BMMSCs. Using chromatin immunoprecipitation (ChIP)-qPCR, the TBE binding consensus sequence within the promoter region was examined to determine its ability to recruit -catenin. Unexpectedly, the -catenin-bound DNA at the candidate site was significantly enriched in both normal and TERT -/- BMMSCs (Figure 2.10K). These findings prompted us to examine whether TERT contributes to this transcriptional process. ChIP-qPCR analysis demonstrated that TERT-bound DNA at the candidate site was enriched in normal BMMSCs, but not TERT -/- BMMSCs, indicating that TERT acts as a cofactor with -catenin to drive FasL expression (Figure 2.10K). To further confirm the role of TERT and - catenin in binding to the fasl promoter, immunoprecipitation of nuclear protein by either -catenin or TERT antibodies was performed. The results showed that TERT, - catenin, and BRG1 formed a complex in normal BMMSCs, while only -catenin and BRG1 72 formed a complex in TERT -/- BMMSCs (Figure 2.10L). Together, these findings indicate that TERT, together with -catenin, serves as a transcriptional regulator of FasL expression (Figure 2.10M). Figure 2.10 TERT serves as a transcriptional modulator to regulate FasL expression in BMMSCs. (A and B) Western blot analysis showed decreased levels of FasL and active - catenin, but not BRG1, in TERT -/- BMMSCs (A) and tert knockdown BMMSCs by siRNA (B) compared to TERT +/+ (WT) BMMSCs. (C) -catenin activator (Chir, 10 M) treatment elevated levels of active -catenin and FasL in WT BMMSCs. fasl knockdown BMMSCs by siRNA showed a decreased level of FasL expression, but not active -catenin. (D) In vitro coculture system showed -catenin activator (Chir)-treated BMMSCs had increased capacity to induce AnnexinV + 7AAD - and AnnexinV + 7AAD + double positive apoptotic T cells compared to control group. fasl siRNA treatment could reduce Chir-elevated T cell apoptosis in the co-culture system. (E) Telomerase activity in Chir-treated BMMSCs showed no significant difference from the untreated group. 293T cells were used as a positive control, and heat-inactivated (H.I.) samples were used as a negative control. (F) 73 Western blot analysis showed decreased expression levels of -catenin and FasL in - catenin knockdown BMMSCs by siRNA. (G) -catenin knockdown BMMSCs by siRNA showed decreased capacity to induce AnnexinV + 7AAD - and AnnexinV + 7AAD + double positive apoptotic T cells compared to the control siRNA group. (H) Western blot showed that TERT -/- BMMSCs decreased expression levels of TERT, active -catenin, and FasL. Tert transfection (TERT TF) rescued the expression levels of TERT, active -catenin, and FasL, assessed by Western blot, while fasl transfection (FASL TF) only rescued FasL expression, but not that of TERT or -catenin, in TERT -/- BMMSCs. (I) In vitro coculture system showed a decreased capacity of TERT -/- BMMSCs to induce AnnexinV + 7AAD - and AnnexinV + 7AAD + double positive apoptotic T cells when compared to the control group, whereas transfection of both tert and fasl rescued the capacity to induce AnnexinV + 7AAD - and AnnexinV + 7AAD + double positive apoptotic T cells. (J) fasl promoter-luciferase fusions were examined in WT, TERT -/- and TERT TF BMMSCs. Promoter activity was expressed as relative light units (RLU) normalized to the activity of co-transfected Renilla luciferase. The activity of 2kb promoter-luciferase fusion was significantly elevated compared to 1.1kb fusion in WT BMMSCs and TERT TF BMMSCs when compared to TERT -/- BMMSCs. The activity of TBE-specific site-mutated promoters was markedly decreased in WT BMMSCs and TERT TF BMMSCs. (K) Chromatin immunoprecipitation (ChIP)-qPCR assay showed enrichment of direct association of - catenin on the fasl promoter in WT and TERT -/- BMMSCs, while the enrichment of direct association of TERT on the fasl promoter was only found in WT BMMSCs. (L) ChIP- Western blot assays showed direct association of TERT, -catenin and BRG1 on the fasl promoter in WT BMMSCs, but only direct association of -catenin and BRG1 on the fasl promoter in TERT -/- BMMSCs. (M) Schematic diagram indicates that TERT, as a transcriptional modulator in a complex with -catenin and BRG1, mediates FasL expression in BMMSC-induced immunoregulation. Vehicle: scrambled siRNA-treated BMMSCs. Error bars represent the s.d. from the mean values (One-way Anova, Bonferroni, n=3 in each group, ***p < 0.005, *p < 0.05). 74 Figure S2.10 FasL but not immunomodulatory factors mediates immunomodulation of BMMSCs. Knockout of TERT or knockdown of TERT expression by siRNA in BMMSCs failed to affect expression levels of Interleukin-10 (IL10) (A) and prostaglandin E2 (PGE2) (B). (C) FasL null BMMSC isolated from gld mouse (gldBMMSC) confirmed that FasL is essential for BMMSC-mediated immunomodulation. Error bars present the s.d. from six independent experiments. Figure S2.11 FasL is required for BMMSCs to induce T cell apoptosis in vitro. (A) FasL expression level before and after flow cytometric sorting of FasL in BMMSCs. (B) FasL + BMMSCs showed increased capacity to induce AnnexinV + 7AAD + double positive apoptotic T cells, while FasL - BMMSCs showed decreased capacity to induce T cell apoptosis when compared to nonsorted BMMSCs in an in vitro co-culture system. Error bars present the s.d. from six independent experiments (***p<0.005). 75 2.3.9 Aspirin pretreatment activates TERT expression Previously, we reported that aspirin-pretreated BMMSCs (Asp-BMMSCs) showed increased telomerase activity and improved bone regeneration (Yamaza et al, 2008). Thus, we examined whether aspirin-induced telomerase activity could improve BMMSC- based immunoregulation. A TRAP-ELISA assay showed that telomerase activity was significantly increased at 3 days after aspirin pretreatment and that it maintained this elevated level for more than 7 days (Figure 2.11A). To examine whether upregulated TERT activity by aspirin is specific to BMMSCs, naïve T cells from splenocytes were isolated and pretreated with aspirin for 3 days. TRAP-ELISA assays showed that telomerase activity was elevated in the BMMSC group, but not in the naïve T cell group (Figure S2.12A). We found that Asp-BMMSCs expressed higher levels of TERT, active - catenin, and FasL and showed elevated capacity to induce AnnexinV + 7AAD - and AnnexinV + 7AAD + double positive apoptotic T cells when compared to the untreated group (Figures 2.11B and 2.11C). Knockdown TERT expression in Asp-BMMSCs by siRNA significantly decreased the expression levels of TERT and FasL and the capacity to induce T-cell apoptosis, suggesting that aspirin elevated the immunomodulatory capacity of BMMSCs through TERT activation (Figures 2.11D and 2.11E). These data suggest that aspirin pretreatment increases telomerase activity and elevates the immunomodulation capacity of mouse BMMSCs. To further extend these findings to clinical application, human BMMSCs were isolated and pretreated with aspirin. We verified that Asp- 76 BMMSCs from human bone marrow showed increased telomerase activity using a TRAP- ELISA assay; they also showed increased TERT expression levels, as evaluated by Western blot analysis, when compared to the non-pretreated group (Figure S2.12B). To confirm that elevated telomerase activity was related to the immunomodulatory properties of Asp-BMMSCs, BMMSC-T cell co-culture experiments were performed to show that Asp-BMMSCs induced increased T cell apoptosis when compared to the BMMSC group (Figure S2.12B). In addition, cytogenetic analysis of Asp-BMMSCs showed no karyotype alterations, suggesting that in vitro aspirin pretreatment (50 g/ml) may be a safe approach to improve BMMSC immunomodulatory properties (Figure S2.12C). Treatment with 0.2x10 6 BMMSCs (positive control group in our study) is considered a standard dosage to elicit a therapeutic response. Therefore, we infused 10% of that amount (0.02x10 6 of either Asp-BMMSCs or BMMSCs) into Tsk/ + SSc mice at 8 weeks of age to examine whether aspirin pretreatment could reduce the dosage of BMMSCs in immunotherapy (Figure 2.12A). When we analyzed treatment responses at 12 weeks of age, we found that a 10% dose of Asp-BMMSCs, but not 10% untreated BMMSCs, was capable of elevating Treg levels equal to that of the positive control group (Figure 2.12B). Flow cytometric analysis further revealed that the 10% Asp-BMMSCs group showed a greater efficacy in reducing the number of Th17 cells, levels of ANA and dsDNA IgG and IgM antibodies in peripheral blood, when compared to the 10% untreated BMMSCs group (Figures 2.12C-2.12F). In addition, the tightness of skin, as measured by grabbed 77 distance, was significantly improved in the 10% Asp-BMMSCs group, but not the 10% BMMSCs group in Tsk/ + mice (Figure 2.12G). Histological analysis also confirmed that skin hypodermal thickness was significantly reduced to a level equal to that of the control group in the 10% dose of Asp-BMMSCs group, whereas the 10% dose of untreated BMMSCs group failed to reduce hypodermal thickness (Figure 2.12H). Moreover, the 10% Asp-BMMSCs group showed a therapeutic effect similar to that observed in the positive control. To confirm that aspirin-elevated telomerase activity contributes to BMMSC-mediated immune therapy, we infused TERT -/- BMMSCs, with or without aspirin pretreatment, into Tsk/ + mice and found that they failed to rescue the disease phenotypes, as indicated by no significant changes in terms of the levels of Tregs, Th17, ANA, and dsDNA IgG and IgM antibodies in peripheral blood, tightness of skin, and histological skin hypodermal thickness when compared to the untreated WT BMMSC infusion group (Figure S2.13A-2.13H). In addition, infusion of aspirin alone also failed to rescue the disease phenotypes (Figure S2.13A-2.13H). These data lead us to hypothesize that the number of BMMSCs used for immunotherapy could be dramatically reduced if BMMSCs are first treated with a telomerase activator, such as aspirin. 78 Figure 2.11 Aspirin pretreatment increases immunomodulation of BMMSCs through TERT activation. (A) TRAP-ELISA assays showed that Aspirin-pretreated (50 g/mL) BMMSCs exhibited increased telomerase activity from day 3 to day 7 compared to the untreated group. 293T cells were used as a positive control, and heat-inactivated (H.I.) samples were used as negative control. (B) Western blot analysis showed elevated levels of TERT, FasL, and active -catenin expression in aspirin-pretreated BMMSCs. (C) In vitro coculture system showed that Aspirin-pretreated BMMSCs exhibited increased capacity to induce AnnexinV + 7AAD - and AnnexinV + 7AAD + double positive apoptotic T cells compared to the untreated group. (D) Western blot analysis showed that aspirin pretreatment elevated levels of TERT and FasL expression in BMMSCs, which could be blocked by TERT siRNA treatment. (E) In vitro coculture system showed that aspirin- pretreated BMMSCs exhibited increased capacity to induce AnnexinV + 7AAD - and AnnexinV + 7AAD + double positive apoptotic T cells, which could be diminished by TERT siRNA treatment. Vehicle: scrambled siRNA-treated BMMSCs. Error bars represent the s.d. from the mean values (One-way Anova, Bonferroni, n=3 in each group, ***p < 0.005). 79 Figure S2.12 Aspirin pretreatment increaseed inmmunomodulation of BMMSCs through TERT activation. (A) Aspirin treatment showed specific increased telomerase activity in BMMSC, but not naïve T cells. (B) Aspirin-treated human BMMSCs showed increased telomerase activity, assessed by TRAP-ELISA telomere length assay. Western blot analysis showed no increased TERT expression in Aspirin-treated human BMMSCs. In an in vitro coculture system, aspirin-treated human BMMSCs showed increased capacity in inducing AnnexinV + 7AAD - and AnnexinV + 7AAD + double positive apoptotic T cells. (C) there is no karyotype alteration after aspirin treatment compared to untreated group. Error bars present the s.d. from three independent experiments (***p<0.005, *p<0.05). 80 Figure 2.12 Aspirin-pretreated BMMSCs show increased capacity to ameliorate systemic sclerosis phenotypes. (A) Schema showing procedure for using aspirin- pretreated BMMSC transplantation (Asp-MSCT) to treat systemic sclerosis (SSc) tight skin (Tsk/ + ) mice. (B) FACS analysis showed that the Treg level was significantly decreased in Tsk/ + mice compared to control littermates. After MSCT, the Treg level was significantly elevated. Using ten times less BMMSCs during MSCT (Low-BMMSC) failed to increase the Treg level, while Low-Asp-BMMSC significantly elevated the Treg level in Tsk/ + mice. (C) FACS analysis showed that CD4 + IL17 + Th17 cells were significantly increased in Tsk/ + mice compared to control littermates. After MSCT, the Th17 level was significantly reduced. Low-BMMSC failed to diminish Th17 level, while Low-Asp-BMMSC significantly reduced the Th17 level in Tsk/ + mice. (D-F) ELISA assays showed that Tsk/ + mice had elevated levels of antinuclear antibody (ANA, D) and anti-double strand DNA antibodies IgG (E) and IgM (F) when compared to control littermates. MSCT reduced the levels of ANA (d) and anti-double strand DNA antibodies IgG (e) and IgM (f). Low- BMMSC failed to diminish the levels of ANA (D) and anti-double strand DNA antibodies IgG (E) and IgM (F), while Low-Asp-BMMSC significantly reduced the levels of ANA (D) and anti-double strand DNA antibodies IgG (E) and IgM (F) in Tsk/ + mice. (G) Tsk/ + mice showed tight skin phenotype compared to control littermates. MSCT significantly 81 improved skin phenotype in terms of grabbed skin distance in Tsk/ + mice. Grabbed skin distance measurement showed that Low-BMMSC failed to ameliorate tight skin phenotypes, while Low-Asp-BMMSC significantly improved skin phenotypes in Tsk/ + mice. (H) Hyperdermal thickness was significantly increased in Tsk/ + mice compared to control littermates. MSCT improved hyperdermal thickness in Tsk/ + mice. Histological examination identified that Low-BMMSC failed to diminish hyperdermal thickness, while Low-Asp-BMMSC significantly reduced hyperdermal thickness in Tsk/ + mice. Scale Bar, 100 m. D: Dermal, M: Muscle, and HD: Hyperdermal. Error bars represent the s.d. from the mean values (One-way Anova, Bonferroni, n=6 in each group, ***p < 0.005, **p < 0.01, *p < 0.05). Figure S2.13 Aspirin treatment failed to ameliorate systemic sclerosis phenotypes. (A) Schema showed procedure of using TERT -/- BMMSCs, aspirin-pretreated TERT -/- BMMSCs (TERT -/- -Asp), or aspirin alone (Asp) to treat systemic sclerosis (SSc) tight skin (Tsk/ + ) mice. (B) Flow sytometric analysis showed that Treg level was significantly decreased in Tsk/ + mice compared to control littermates. After MSCT, Treg level was significantly elevated, whereas TERT -/- MSCT failed to upregulate Treg level in Tsk/ + mice. TERT -/- -Asp, along with Asp group, also failed to upregulate Treg levels in Tsk/ + mice. (C) Flow 82 cytometric analysis showed that CD4 + IL17 + Th17 cells were significantly increased in Tsk/ + mice compared to control littermates. MSCT, but not TERT -/- MSCT, was able to significantly reduce Th17 level in Tsk/ + mice. TERT -/- -Asp, along with Asp group, also failed to reduce Th17 levels in Tsk/ + mice. (D-F) ELISA assays showed that Tsk/ + mice had elevated levels of anti-double strand DNA antibodies IgG (D), IgM (E) and antinuclear antibody (ANA, F) when compared to control littermates. MSCT reduced the levels of anti-double strand DNA antibodies IgG (D), IgM (E) and ANA (F). In contrast, TERT -/- MSCT failed to reduce the levels of anti-double strand DNA antibodies IgG (D), IgM (E) and ANA (F). TERT -/- -Asp, along with Asp group, also failed to reduce the levels of anti- double strand DNA antibodies IgG (D), IgM (E) and ANA (F). (G) Tsk/ + mice showed tight skin phenotype. Grabbed skin distance measurement showed that BMMSC, but not TERT -/- MSCT, significantly improved tight skin phenotype. TERT -/- -Asp, along with Asp group, also failed to improved tight skin phenotype. (H) Histological examination identified that hyperdermal thickness was significantly increased in Tsk/ + mice compared to control littermates. BMMSC, but not TERT -/- MSCT improved hyperdermal thickness in Tsk/ + mice. TERT -/- -Asp, along with Asp group, also failed to reduce hyperdermal thickness in Tsk/ + mice. D: Dermal, M: Muscle, and HD: Hyperdermal. Error bars represent the s.d. of the mean values (n=6 in each group, ***p<0.005, **p<0.01, *p<0.05). 83 2.4 Discussion The FasL/Fas-mediated cell death pathway represents typical apoptotic signaling in many cell types (Hohlbaum et al., 2000; Pluchino et al., 2005; Zhang et al., 2008). MSCs derived from bone marrow (BMMSCs) express FasL and induce tumor cell apoptosis in vitro (Mazar et al., 2009). However, it is unknown that whether BMMSCs induce T cell apoptosis via Fas/FasL pathway leading to immune tolerance. Therefore, we transplanted BMMSCs into C57BL6 mice and demonstrated that BMMSCs, but not FasL- deficient BMMSCs, induced transient T cell apoptosis. Furthermore, we found a reduced number of T cells in multiple organs, including peripheral blood, bone marrow, spleen, and lymph node. It appears that alteration of T cell number, owing to T cell redistribution, is not supported by the experimental evidence. Since CD3 antibody- induced T cell apoptosis resulted in immune tolerance (Chatenoud et al., 1994 and 1997), we confirm here that BMMSC-induced T cell apoptosis upregulates Tregs via high levels of macrophage-released TGF  (Kleinclauss et al., 2006; Perruche et al., 2008). Although transplanted FasL -/- gldBMMSCs and FasL knockdown BMMSCs undergo apoptosis in vivo, they failed to induce upregulation of Tregs. This evidence further confirms that T cell, but not transplanted BMMSC, apoptosis is required for inductive upregulation of Tregs (Perruche et al., 2008). Despite the expression of functional FasL by Fas -/- lprBMMSCs, they failed to induce T cell apoptosis and upregulate Tregs in vivo. Mechanistically, Fas controls 84 chemoattractant cytokine MCP-1 secretion in BMMSCs. Decreased MCP-1 secretion from lprBMMSC results in the failure to recruit activated T cells to BMMSCs (Carr et al., 1994; Xu et al., 1996) and, hence, infusion of Fas -/- lprBMMSCs failed to induce T cell apoptosis in vivo. However, when lprBMMSCs were directly co-cultured with CD3 + T cells, they could induce T cell apoptosis, suggesting that lprBMMSC may not able to initiate cell-cell contact with T cells in vivo. Moreover, Fas -/- lprBMMSCs show a higher cytoplasm level of MCP-1 than control BMMSCs, suggesting that Fas regulates MCP-1 secretion, but not MCP-1 production. When MCP-1 -/- BMMSCs were systemically transplanted into C57BL6 mice, CD3 + T cell apoptosis and Treg upregulation were significantly reduced compared to control BMMSC group, suggesting that MCP-1 is one of the factors regulating MSC-based immune tolerance. It was reported that BMMSCs could inhibit CD4/Th17 T cells with MCP-1 paracrine conversion from agonist to antagonist (Rafei et al., 2009). Here we showed that MCP-1 helped to recruit T cells to up-regulate Tregs. Prospectively, it will be important to dissect the mechanism by which Fas regulates MCP-1 secretion. In addition to MCP-1 secretion, Fas also regulates multiple cytokine and chemokine secretions in BMMSCs, which may extensively affect the recruitment of T cells. Therefore, MCP-1 may only represent one of those chemokines contributing to BMMSC-mediated T cell recruitment. Significantly, our primary clinical investigation showed that MSC infusion induced CD3 + T cell apoptosis and Treg upregulation in allogenic MSC-infused SSc patients. In our 1-12 85 month follow-up period, we did not find any clinical sign of side effects, including cardiovascular and pulmonary insufficiencies, infection, malignancy, or metabolic disturbances, suggesting the safety of the MSC therapy in SSc patients. The therapeutic effects of allogenic MSC transplantation were significant as shown by the reduction of MRSS, HAQDI, in addition to improved quality of life. Furthermore, we demonstrated that MSC transplantation dramatically improved treatment-refractory skin ulcers. Thus, we have uncovered a previously unrecognized BMMSC-mediated therapeutic mechanism by which BMMSCs use Fas to regulate MCP-1 secretion for T cell recruitment and subsequently use FasL to induce T cell apoptosis. Macrophages subsequently take the debris of apoptotic T cells to release a high level of TGF , leading to upregulation of Tregs and, ultimately, immune tolerance for immunotherapies. Collaborative execution of therapeutic effect between Fas and FasL may therefore represent a new functional role of receptor/ligand in cell-based therapies. BMMSCs exhibit immunomodulatory functions by mediating the proliferation, migration and function of several major types of immune cells, and systemic infusion of BMMSCs has been shown to yield therapeutic benefits for a variety of immune disorders (Aggarwal & Pittenger, 2005; Chen et al, 2006; Le Blanc et al, 2004; Liang et al, 2009; 2012; Liu et al, 2013; Nauta & Fibbe, 2007; Scuderi et al, 2012; Sun et al, 2009; Uccelli et al, 2007; 2008). However, the stem cell properties of BMMSCs in maintaining immunomodulatory function are poorly understood. Specifically, it is yet to be 86 determined whether the unique gene-driven functional commonality of stem cells, including BMMSCs, plays a role in regulating immune response at therapeutic levels. Previous reports have demonstrated that telomerase reverse transcriptase (TERT) plays a key role in progenitor cell survival and stem cell self-renewal and that it controls telomere maintenance to ensure chromosome stability (Gronthos et al, 2003; Liu et al, 2011; Maser & DePinho, 2002; Shi et al, 2002; Smogorzewska & De Lange, 2004; Yamaza et al, 2008). In the present study, we found that expression of MSC surface markers, including CD90, CD105, Sca1 and SSEA4, was significantly reduced in TERT -/- BMMSCs. CD90 may function as an activator for stem cell differentiation (Chen et al, 1999). CD105 is important for BMMSC adhesion and angiogenesis (Duff et al, 2003). Sca1 and SSEA4 are general stem cell markers for BMMSCs (Battula et al, 2009; Gang et al, 2007). TERT is highly expressed in newly isolated BMMSCs from aspirates of bone marrow (Gronthos et al, 2003) and is rapidly downregulated in BMMSCs during ex vivo expansion (Shi et al, 2002). TERT may contribute to the maintenance of stem cell properties of BMMSCs. Clinically, a correlation between successful immunosuppressive therapy and the dosage of donor stem cells has been well documented (Liang et al, 2010; Wang et al, 2012). In this study, we reveal a novel mechanism by which telomerase governs the immunomodulatory properties of BMMSCs via upregulation of the FasL-induced apoptotic pathway. Further, activation of telomerase activity in BMMSCs by a pharmacological approach, such as in vitro aspirin treatment, can markedly improve 87 BMMSC-based immunomodulation and reduce the number of BMMSCs used for immunotherapy in SSc mice. By a mechanistic study, we reveal that telomerase- enhanced FasL production is associated with Wnt/ -catenin signaling, in which a TERT/ -catenin/BRG1 complex directly binds to the fasl promoter to drive gene expression at the transcriptional level. This study provides experimental evidence that links telomerase activity to BMMSC-based immunomodulation and demonstrates the potential to improve BMMSC-based clinical therapies with reduced cell dosage. It has been reported that aspirin promotes osteogenesis and bone regeneration (Liu et al, 2011; Yamaza et al, 2008), but inhibits proliferation (Deng et al, 2009; Wang et al, 2006) in BMMSCs. Activation of Wnt/β-catenin by aspirin treatment may contribute to elevated osteogenesis (Yamaza et al, 2008); however, the detailed mechanism is not clear. Moreover, it is well known that TERT can regulate the Wnt/β-catenin pathway (Choi et al, 2008; Park et al, 2009). In this study, we showed that TERT associates with β- catenin to form a transcriptional complex to control the expression of FasL, thereby affecting BMMSC-mediated immunomodulation. Aspirin is a widely used anti- inflammatory drug, and treatment using a 50 g/ml dosage appears to have no negative effect on BMMSCs. It is therefore reasonable to continue examining the efficacy of using aspirin-treated BMMSCs for clinical therapies. Taken together, this translational study substantially extends current knowledge about BMMSC-based immunotherapy and provides a new strategy for improving it. We also 88 reveal a novel mechanism by which TERT is, for the first time, linked to BMMSC- mediated immunomodulation. 89 Chapter 3: Inhibition of mTOR Ameliorates Osteopenia Phenotype in Systemic Sclerosis by Regulating Mesenchymal Stem Cell Lineage Differentiation. 3.1 Introduction Bone marrow mesenchymal stem cells constitute a population of self-renewal and multipotent cells that can differentiate into osteoblasts, adipocytes, fibroblasts, chondrocytes and non-mesenchymal cell types (Friedenstein et al., 1974; Prockop, 1997). BMMSCs are a promising cell source for bone regeneration and immunoregulatory therapies by interacting with several subsets of immune cells (Uccelli et al., 2007; Uccelli and Mancardi, 2010; Ren et al., 2008; Le Blanc et al., 2004; Sun et al., 2009). In response to stimulation from multiple environmental factors, BMMSCs can differentiate into different lineage cells, which are regulated at both transcriptional and translational levels (Shi and Gronthos, 2003; Shi et al., 2002). In the present study, we show that Fbn1 regulates BMMSC osteogenic/adipogenic lineage selection via IL4Rα/mTOR signaling. Blockage of the mTOR cascade by rapamycin, an anticancer and immune suppressive drug, ameliorates the osteopenia phenotype in Fbn1 +/- SSc mice. 90 3.2 Material and Methods 3.2.1 Animals and antibodies Female C57BL/6J, B6.Cg-Fbn1 Tsk /J, B6.Cg-Tg(Prrx1-cre)1Cjt/J, B6.Cg-Tg (Sp7-tTA, tetO- EGFP/cre)1Amc/J, and B6.129S4-Mtor tm1.2koz /J mice were purchased from Jackson Lab. Il4r -null (Il4r  -/- ) and mice with the floxed allele of Il4r  (Il4r  f/f ) were described previously (De’Broski et al., 2004) and kindly provided by Dr. Frank Brombacher. Female immunocompromised nude mice (Beige nu/nu XIDIII) were purchased from Harlan. All animal experiments were performed under the institutionally approved protocols for the use of animal research (University of Southern California protocol number 11141). 3.2.2 MicroCT analysis Femurs were harvested and analyzed by Inveon micro-CT system (Siemens AG, Germany). Cross sectional volumetric bone mineral density (BMD) was measured at right femur mid-diaphysis with a density phantom. Using 3-dimensional images, a region of interest in secondary spongiosa was manually drawn near the endocortical surface, and bone volume relative to tissue volume (BV/TV) was assessed as a cancellous bone morphometric parameter. 91 3.2.3 Histology To assess trabecular bone and bone marrow areas, femurs were fixed in 4% paraformaldehyde (Sigma-Aldrich) and then decalcified with 5 % EDTA (pH 7.4), followed by paraffin embedding. Paraffin sections (6 μm) were stained with hematoxylin and eosin (H&E) and analyzed by NIH ImageJ software. To perform Immunohistochemistry staining, the paraffin embedded sections were blocked with serum matched to secondary antibodies, incubated with the ALP or IL4 specific antibodies (Santa Cruz Biotechnology, Inc, 1:400) at 4 o C for overnight, and then stained using VECTASTAIN UNIVERSAL elite ABC kit and ImmPACT VIP Peroxidase Substrate kit (VECTOR) according to the manufacturer’s instruction. To quantify osteoclast activity, mature osteoclasts were determined by tartrate-resistant acid phosphate (TRAP)- positive cells on the bone surface. Deparaffinized sections were refixed with a mixture of 50% ethanol and 50% acetone for 10 min. TRAP staining solutions (1.6% naphthol AS- BI phosphate in N, N-dimethylformamide, 0.14% fast red-violet LB diazonium salt, 0.097% tartaric acid, and 0.04% MgCl 2 in 0.2 M sodium acetate buffer at pH 5.0) were freshly made. The sections were incubated in the solution for 10 min at 37 o C under shield and counterstained with toluidine blue. All reagents for TRAP staining were purchased from Sigma-Aldrich. 92 3.2.4 ELISA assays Peripheral blood serum and cell culture medium were collected, and sRANKL, OPG, IL4 and TGF  protein levels were analyzed by using mouse ELISA Ready-SET-GO kits (eBioscience), according to the manufacturer’s instructions. For measurement of anti- dsDNA antibodies and ANA, peripheral blood serum samples were collected from all experimental mice and analyzed by commercially available enzyme-linked immunosorbent assay (ELISA) kits (Alpha Diagnostics) according to the manufacturer’s instructions. 3.2.5 In vivo Oil red O staining To assess the adipose tissue in trabecular areas, femurs were fixed in 4% paraformaldehyde and decalcified with 5 % EDTA (pH 7.4), followed by cryosection. Sections were stained with Oil Red-O, and positive areas were quantified under microscopy and shown as a percentage of total areas. 3.2.6 In vivo BMMSC implantation assay Approximately 4.0x10 6 BMMSCs were mixed with hydroxyapatite/tricalcium phosphate (HA/TCP) ceramic powders (40 mg, Zimmer Inc.) and subcutaneously implanted into 8- week-old immunocompromised mice. At 8 weeks post-implantation, the transplants were harvested, fixed in 4% PFA, and decalcified with 5% EDTA (pH 7.4), followed by 93 paraffin embedding. The 6 m paraffin sections were stained with H&E chemical staining. Total bone volume per total volume was quantified by NIH ImageJ software. For IL4-treated in vivo BMMSC implantation assay, BMMSCs were mixed with HA/TCP ceramic powders, and Extracel-HP™ hydrogel (Glycosan Biosystems), containing 200 ng IL4, was covered on the surface of the implants for slow release of the cytokine. At eight weeks post-implantation, the implants were harvested and decalcified. Newly formed mineralized tissue areas were analyzed by paraffin section and H&E staining. 3.2.7 RNAi and chemical reagent treatments BMMSCs (0.5x10 6 ) were seeded to a 6-well culture plate and treated with Fbn1 siRNA (Santa Cruz), P70s6k siRNA (Santa Cruz), Il4r  shRNA (Santa Cruz), or Mtor shRNA (Addgene), according to the manufacturers’ instructions. After transfection, cells were either used for protein extraction for Western immunoblotting or for differentiation induction. For chemical reagent treatments, serum-starved MSCs were treated with 10 ng/mL rIL-4 (R&D Systems), 50 nM rapamycin (LC Laboratories), or 1 μg/mL TGF  neutralized antibody (R&D systems). For Western immunoblotting, MSCs were cultured under growth medium with drugs for 24 hours, and protein was extracted by using M- PER mammalian protein extraction reagent. For differentiation induction, MSCs were cultured under inductive conditions in the presence of drugs (added every 3 days) for 3 weeks, followed by staining and gene expression analysis. 94 3.2.8 Flow cytometric analysis of T H 2 cells To detect T H 2 percentage of CD4 + cells in bone marrow, bone marrow-derived single- suspension ANCs were incubated with anti-CD4-PerCP (BD Bioscience), followed by staining with anti-interferonγ-APC (eBioscience) and anti-IL4-PE (eBioscience) antibodies using an intracellular antigen staining kit (eBioscience). Cells were analyzed by FACS Calibur with CellQuest software (BD Bioscience). 3.2.9 Immunofluorescent microscopy BMMSCs were cultured on 4-well chamber slides (Nunc) (2x10 3 /well) and then fixed with 4% paraformaldehyde. The Chamber slides are incubated with primary anti CD73 antibody (1:400, BD) and anti IL4Rα (1:400, BD) at 4 o C for overnight. And then treated with Rhodamine-conjugated secondary antibody (1:400, Southern biotech) or Alexafluoro 488 conjugated secondary antibody (1:200, Invitrogen) for 30min at room temperature. Finally, slides were mounted with Vectashield mounting medium (Vector Laboratories). 3.2.10 Luciferase reporter assay Il4r -luciferase promoter reporter constructs were generated by PCR using Pfu polymerase and mouse genomic DNA as a template. Primers containing upstream XhoI and HindIII downstream restriction sites were used to generate Il4r  promoter fragment 95 (forward, 5’-CTCGAGGAATTCATGCTGCTTTCTCG-3’ and reverse, 5’- AAGCTTGGCTTTCCCACCGCCCGTT-3’). Restriction digested PCR products were subcloned into pGL3-Basic vector (Promega). Point mutants were introduced into the reporter by the Pfu/DpnI method. All clones were confirmed by sequencing on both strands. BMMSCs cultured in 6-well plates were co-transfected with 2 μg of luciferase reporter and 100 ng of Renilla luciferase expression vector to control for transfection efficiency. Forty-eight hours after transfection, cells were lysed in 1x passive lysis buffer, and luciferase activity was measured using the Dual-Glo Luciferase System (Promega) and a luminometer (Turner Biosystems). 3.2.11 Chromatin Immunoprecipitation Assays BMMSCs grown in 10 cm cell culture dishes were fixed for 10 min at room temperature by addition of 1% paraformaldehyde to the growth medium. Cells were washed twice in cold PBS supplemented with complete protease inhibitor cocktail and gently scraped from the plate. Cell lysis and chromatin immunoprecipitation were performed using the ChIP Assay Kit (Millipore). For chromatin fragmentation, cells were sonicated using a Branson Sonifier 450 on power setting 4 in 30 s bursts with 1 min cooling on ice for a total sonication time of 4 min. For immunoprecipitations, 1:100 dilution of SP1 antibody was used to capture protein-DNA complexes, and isotype-matched IgG was used as negative control. All resulting precipitated DNA samples were quantified with real-time PCR and expressed as the percentage of input DNA. 96 3.2.12 In vivo Rapamycin treatment Rapamycin (LC Laboratories) was in the vehicle containing 0.2% sodium carboxymethylcellulose (Sigma) and 0.25% polysorbate 80 (Sigma). Rapamycin with vehicle was intraperitoneally (IP) administered to Fbn1-deficient mice at a dose of 1.5 mg/kg/day for 14 and 28 days, respectively. The disease group and control mice were treated with vehicle only. Treatment was started at age 6 or 8 weeks, and all groups of mice were healthy. 3.3 Results 3.3.1 Fbn1 deficiency alters BMMSC lineage differentiation Since Fbn1 gene mutation leads to significant loss of bone volume and increase in bone marrow adipocytes in B6.Cg-Fbn1 Tsk /J (Fbn1 +/- ) mice 6 , we hypothesize that Fbn1 deficiency may reduce osteogenic differentiation of BMMSCs and elevate their adipogenic differentiation. To test this hypothesis, we confirmed that Fbn1 deficiency resulted in an osteopenia phenotype in Fbn1 +/- mice. MicroCT and histological analysis showed that bone mineral density (BMD), bone volume versus total volume (BV/TV), and distal femoral trabecular bone structure of Fbn1 +/- mice were markedly decreased compared to the control wild type (WT) mice (Figure 3.1A-3.1D). Histomorphometric analysis revealed that the numbers of both osteoblasts and osteoclasts in the femur of 97 Fbn1 +/- mice were significantly reduced in comparison to the WT group by alkaline phosphatase (ALP) immunohistochemical staining and tartrate-resistant acid phosphatase (TRAP) staining, respectively (Figure 3.1E-3.1F). The level of soluble receptor activator of nuclear factor κB ligand (sRANKL), but not osteoprotegerin (OPG), was significantly reduced, implying that the loss of bone volume in Fbn1 +/- mice may be mainly associated with an insufficient bone formation (Figure 3.1G-3.1H). To examine whether Fbn1 deficiency affects the stem cell properties of BMMSCs, we showed that the number of colony forming unit-fibroblasts (CFU-F) was significantly reduced in Fbn1 +/- BMMSCs when compared to the WT control (Figure 3.1I) and that the osteogenic differentiation capacity of Fbn1 +/- BMMSCs was decreased, as indicated by reduced mineralized nodule formation and reduced expression of the osteogenic regulators RUNX2, ALP, and OCN (Figure. 3.1J-3.1K). Using an established in vivo BMMSC implantation assay, in which 4×10 6 BMMSCs with carrier HA/TCP particles were subcutaneously implanted into immunocompromised mice, we showed that BMMSCs derived from Fbn1 +/- mice generated less bone structure than the WT group at 8 weeks post-implantation (Figure. 3.1L). To further confirm the role of Fbn1 in BMMSC differentiation, we used siRNA to silence Fbn1 gene expression in BMMSCs (Figure S3.1) and found a significantly reduced osteogenic differentiation, as assessed by alizarin red staining to show reduced mineralized nodule formation, immunoblotting analysis to show decreased expression of the osteogenic markers RUNX2, ALP, and OCN, and in vivo BMMSC implantation assay to show reduction in new bone formation (Figure 3.1M- 98 3.1O). In contrast, Oil red O staining showed that the number of adipocytes in Fbn1 +/- bone marrow was markedly increased when compared to the WT control group (Figure 3.2A). To confirm that Fbn1 deficiency results in elevated adipogenesis, Fbn1 +/- BMMSCs were found to exhibit a significantly elevated number of Oil red O-positive cells and upregulation of the adipogenic regulators peroxisome proliferator-activated receptor gamma 2 (PPAR 2) and lipoprotein lipase (LPL) under adipogenic culture conditions (Figure 3.2B-3.2C). Furthermore, we showed that Fbn1-silenced BMMSCs by siRNA had a significantly increased number of Oil red O-positive cells and upregulation of PPAR 2 and LPL (Figure 3.2D-3.2E). These data suggest that Fbn1 governs osteogenic/adipogenic lineage differentiation in BMMSCs. 99 Figure 3.1 Fbn1-deficient (Fbn1 +/- ) mice exhibit osteopenia phenotype. (a-c) MicroCT (a), bone mineral density (BMD) (b), and bone volume over total volume (BV/TV) (c) analysis showed a significant reduction in trabecular bone (TB) area in Fbn1 +/- mouse distal femurs. (d) H&E staining showed a decreased TB volume (yellow circled area) in distal femurs of Fbn1 +/- mice (n=15) compared to wild type (WT) group (n=10), Scale bar, 1 mm. (e) Immunohistochemical staining revealed a reduced number of ALP-positive osteoblasts in Fbn1 +/- mice when compared to WT group. (f) TRAP staining showed a reduced osteoclast number in distal femur trabecular bone (TB) and bone marrow (BM) in Fbn1 +/- mice. Arrowheads, TRAP + osteoclasts (purple cells). Scale bar, 25 m. (g and h) ELISA analysis showed a decreased level of serum sRANKL in Fbn1 +/- mice in comparison to WT control group (g). In contrast, serum OPG level was not significantly altered in Fbn1 +/- mice (h). (i) Fbn1 +/- BMMSCs showed a significant reduction in the number of colony-forming unit fibroblast (CFU-F) compared to the WT group, as shown by toluidine blue staining. (j and m) Alizarin red staining showed that Fbn1 +/- (j) and Fbn1 siRNA knockdown (m) BMMSCs had reduced capacity to form mineralized nodules when cultured under osteoinductive conditions. (k and n) Western blot analysis showed that Fbn1 +/- (k) and Fbn1 siRNA knockdown (n) BMMSCs expressed reduced levels of the osteogenic genes RUNX2, ALP and OCN. β-Actin was used as a protein loading control. (l and o) Subcutaneous implantation of BMMSCs in immunocompromised mice showed that bone (B), bone marrow (BM) and connective tissue (CT) were generated around HA/TCP (HA) at 8 weeks post-implantation. Fbn1 +/- (l) and Fbn1 siRNA knockdown (o) BMMSCs had decreased capacity to regenerate bone tissue compared to the control group (WT and Vehicle). Scale bar, 50 m. A semiquantitative analysis showed the amount of bone formation in BMMSC implants. Representative results from three independent experiments. *** P < 0.005; NS, not significant. 100 Figure 3.2 Fbn1 deficiency enhances adipogenic differentiation of BMMSCs. (a) Representative histological images of distal femurs showed a significantly increased number of adipocytes in Fbn1 +/- mouse bone marrow, as assessed by H&E and Oil red O staining. (b and c) Fbn1 +/- BMMSCs showed increased number of Oil red O-positive cells when cultured under adipoinductive conditions (b) and upregulation of the adipogenic genes PPAR2 and LPL, as assessed by Western blot (c). (d and e) Knockdown of Fbn1 by siRNA in BMMSCs showed increased number of Oil red O-positive cells when cultured under adipoinductive conditions (d) and upregulation of the adipogenic genes PPAR 2 and LPL, as assessed by Western blot (e). Scale bar, 50 m. * P < 0.05; ** P < 0.001. Figure S3.1. Western blot showed that efficacy of Fbn1 siRNA in BMMSCs. 101 3.3.2 Fbn1 modulates MSC lineage differentiation via IL4R /mTOR signaling Since previous studies showed that Fbn1 mutation induced T H 2 infiltration with a high level of IL4 7 , we used immunohistochemical staining to confirm a higher number of IL4- positive cells in Fbn1 +/- mouse bone marrow (Figure S3.2). Next, we examined whether activation of IL4 downstream signaling contributed to reduced osteogenesis and elevated adipogenesis in Fbn1 +/- mice. We used immunofluorescence staining to confirm that BMMSCs coexpressed the MSC marker CD73 with IL4R  (Figure 3.3A). Compared to control BMMSCs, Fbn1 +/- BMMSCs show upregulated expression of IL4R  (Figure 3.3B). Western blot analysis also showed that Fbn1 deficiency upregulated IL4R  downstream signaling Phosphoinositide 3-kinase (PI3K)-p110, phosphorylated Extracellular Signal- regulated Kinase1/2 (p-Erk1/2), phosphorylated Akt (p-Akt) and phosphorylated mTOR (p-mTOR) in Fbn +/- BMMSCs (Figure 3.3B). In order to verify that IL4Rα/mTOR signaling contributes to BMMSC osteo/adipo-lineage selection, we examined whether knockdown of the IL4Rα/mTOR cascade could rescue the altered lineage differentiation in Fbn1 +/- BMMSCs. Having confirmed that shRNAs could knock down Il4rα and Mtor (Figure S3.3A) and that rapamycin treatment could inhibit mTOR signaling in a dose-dependent manner (Figure S3.4), we showed that Il4r α and Mtor shRNAs, as well as rapamycin treatment, significantly improved osteogenic differentiation of Fbn1 +/- BMMSCs, as indicated by alizarin red staining to show increased mineralized nodule formation, 102 Western blot to show elevated expression of the RUNX2, ALP, and OCN, and in vivo BMMSC implantation assay to show increased new bone formation at 8 weeks post- implantation (Figure 3.3C-3.3E). Conversely, the elevated adipogenic differentiation in Fbn1 +/- BMMSCs was significantly reduced in the shRNAs or rapamycin treatment groups, as indicated by a decreased number of Oil red O-positive adipocytes and downregulation of the PPAR 2 and LPL (Figure 3.3F). We next revealed that mTOR regulated osteogenic differentiation of Fbn1 +/- BMMSCs through its downstream signaling phosphorylated P70 ribosomal S6 protein kinase (P70S6K). Western blot analysis showed that p-P70S6K expression level was increased in Fbn1 +/- BMMSCs (Figure 3.3G). When p-mTOR expression was knocked down by Il4r  and Mtor shRNAs, or rapamycin treatment, in Fbn1 +/- BMMSCs, a corresponding downregulation of p- mTOR and P70S6K was revealed by Western blot analysis (Figure S3.3B). Although knockdown of P70s6k expression by siRNA in Fbn1 +/- BMMSCs resulted in upregulation of RUNX2 expression (Figure 3.3H), the expression levels of mTOR was unaffected (Figure S3.5). In addition, the osteogenic differentiation was markedly increased in P70s6k knockdown BMMSCs, as indicated by elevated mineralized nodule formation (Figure 3.3I) and expression of the RUNX2, ALP, and OCN (Figure 3.3J). In contrast, the number of Oil red O-positive cells (Figure 3.3K) and the expression levels of the adipogenic genes (Figure 3.3L) were significantly downregulated, suggesting that mTOR regulates P70S6K to control RUNX2-mediated osteogenic differentiation and PPAR 2- mediated adipogenic differentiation. 103 To further confirm that the IL4/IL4R  pathway activates mTOR signaling, we showed that a long-term treatment of 100 ng/mL IL4 was capable of activating mTOR signaling in normal BMMSCs, as shown by upregulation of PI3K, p-Erk1/2, p-Akt, p-mTOR and p- P70S6K (Figure S3.6A). Furthermore, we showed that IL4 treatment-activated mTOR signaling inhibited osteogenic differentiation of normal BMMSCs, as indicated by reduced mineralized nodule formation, decreased expression of the RUNX2, ALP, and OCN, and reduced bone formation when subcutaneously implanted into immunocompromised mice (Figure S3.6B-3.6D), along with elevated adipogenic differentiation, as indicated by the increased number of Oil red O-positive cells and the expression of PPAR 2 and LPL (Figure S3.6E-3.6F). When treated with IL4, Fbn1 +/- BMMSCs exhibited a more significant increase in the expression of p-mTOR when compared to the WT control BMMSCs (Figure S3.6G). These data suggest that activation of the IL4/IL4R /mTOR/P70S6K cascade contributes to osteogenic deficiency of Fbn1 +/- BMMSCs and that blockade of mTOR signaling may be a promising therapeutic approach for rescuing osteogenic deficiency in Fbn1 +/- mice. 104 Figure 3.3 Fbn1 deficiency-induced activation of IL4R /mTOR signaling regulates osteogenic/adipogenic lineage differentiation of BMMSCs. (a) Immunofluorescence staining showed that BMMSCs coexpressed mesenchymal stem cell marker CD73 with IL4R . Scale bar, 100 m. (b) Western blot showed that Fbn1 +/- BMMSCs exhibited activation of mTOR signaling, including upregulation of IL4R , PI3K-p110, p-Erk1/2, p- Akt and p-mTOR when compared to the control group. (c) Alizarin red staining showed that Fbn1 +/- BMMSCs exhibited decreased capacity to form mineralized nodules when cultured under osteoinductive conditions and that such decrease was rescued by Il4r  and Mtor shRNAs, as well as rapamycin treatment when compared to scrambled shRNA control (sh-Scr)-treated Fbn1 +/- BMMSCs. (d) Accordingly, Western blot showed that Il4r  and Mtor shRNAs, as well as rapamycin treatment, elevated the expression of the osteogenic genes RUNX2, ALP and OCN in Fbn1 +/- BMMSCs when compared to sh-Scr- treated Fbn1 +/- BMMSCs. (e) H&E staining showed that Il4r  and Mtor shRNAs, as well as rapamycin treatment, improved Fbn1 +/- BMMSC-mediated bone (B) and bone marrow (BM) regeneration when subcutaneously implanted into immunocompromised mice using HA/TCP (HA) as a carrier. Scale bars, 50 μm. A semiquantitative analysis showed that the amounts of bone formation in shRNAs and rapamycin-treated Fbn1 +/- BMMSC implants were significantly increased. (f) Oil red O staining showed that shRNAs and rapamycin-treated Fbn1 +/- BMMSCs had a significantly decreased capacity to differentiate into adipocytes compared to sh-Scr-treated Fbn1 +/- BMMSCs when 105 cultured under adipoinductive conditions. Scale bar, 50 m. Accordingly, Western blot showed that shRNAs and rapamycin-treated BMMSCs had downregulation of the adipogenic genes PPAR 2 and LPL. (g) Western blot showed that Fbn1 +/- BMMSCs exhibited activation of p-P70S6K. (h) Knockdown of P70s6k by siRNA (si P70s6k), Fbn1 +/- BMMSCs showed elevated expression of RUNX2 by Western blot analysis. (i-l) Knockdown of P70s6k expression by siRNA (siP70s6k), normal BMMSCs showed increased mineralized nodule formation by alizarin red staining (i), elevated expression of RUNX2, ALP, and OCN by Western blot analysis (j), decreased Oil red O-positive cells, Scale bar, 50 m (k), and decreased expression of PPAR 2 and LPL by Western blot analysis (l). Figure S3.2 T H 2 infiltration in Fbn1 +/- mice. Immunohistochemical staining of distal femoral in Fbn1 +/- mice showed an elevated number of IL4-positive cells (arrows) in bone marrow (BM). The results were representative of five independent experiments. Scale bar, 25 μm. *** P < 0.005. Figure S3.3 Effecacy of shRNA. (a) Western blot analysis showed efficacy of Il4ra and Mtor shRNAs in Fbn1 +/- BMMSCs. (b) Western blot showed that shRNAs and rapamycin- treated Fbn1 +/- BMMSCs had decreased expression of p-mTOR and p-P70S6K. 106 Figure S3.4 Rapamycin treatment inhibited mTOR downstream signaling. Western blot showed that rapamycin treatment induced a dose-dependent inhibition of phosphrylation of mTOR downstream P70S6K in Fbn1 +/- BMMSCs. The results were representative of three independent experiments. Figure S3.5 The efficacy of P70s6k by siRNA. Western blot analysis showed that knockdown of P70s6k by siRNA in BMMSCs had no significantly effect on the expression of p-mTOR and mTOR. Figure S3.6 mTOR signaling governs osteogenic/adipogenic lineage differentiation of BMMSCs. (a) Western blot showed that IL4-treated BMMSCs exhibited activation of mTOR signaling, including upregulation of PI3K-p110, p-Erk1/2, p-Akt, p-mTOR and p- P70S6K when compared to the control group. (b) Alizarin Red staining showed that mTOR activation reduced BMMSC-mediated mineralized nodule formation when cultured under osteoinductive conditions. (c) Western blot analysis indicated that mTOR 107 activation resulted in a downregulation of the osteogenic genes RUNX2, ALP and OCN in BMMSCs. (d) H&E staining showed that mTOR-activated BMMSCs had decreased capacity to form new bone when subcutaneously implanted into immunocompromised mice. Scale bars, 50 μm. A semiquantitative analysis showed the amount of bone formation (B) in BMMSC implants. HA/TCP (HA) was used as carrier vehicle for BMMSC implantation. (e) Oil red O staining showed a significantly increased number of adipocytes in mTOR-activated BMMSCs compared to the control group. Scale bars, 50 μm. (f) Western blot analysis showed that mTOR-activated BMMSCs exhibited elevated expression of the adipogenic genes PPAR 2 and LPL. (g) When treated with IL4 in vitro, Fbn1 +/- BMMSCs showed a more significantly elevated expression of phosphorylated mTOR than control BMMSCs, as assessed by Western blot. *** P < 0.005. 108 3.3.3 IL4R  is activated by the TGF  pathway in Fbn1 +/- BMMSCs It was reported that FBN1 interacts with the latent transforming growth factor-β binding protein-1 (LTBP-1) to bind TGF  (Ramirez and sakai, 2010; Charbonneau et al., 2004). As such, Fbn1 deficiency causes an increased release of TGF  to the skin and lung (Holm et al., 2011; Gabrielli et al., 2009). We examined the level of TGF  in Fbn1 +/- mouse bone marrow and Fbn1 +/- BMMSC culture medium and found that both bone marrow and medium contained elevated levels of TGF , as assessed by ELISA assay (Figure 3.4A). Next, we used Western blot to confirm that TGF  downstream signaling p-SMAD3 and SMAD3-associated transcription factor SP1 21-22 were significantly increased in Fbn1 +/- BMMSCs compared to the control BMMSCs (Figure 3.4B). In order to further confirm that Fbn1 deficiency-mediated IL4R  expression occurs through TGF  release, we treated BMMSCs with recombinant TGF 1 or Fbn1 siRNA, followed by TGF  neutralizing antibody blocking. Western blot analysis revealed that both TGF 1 and Fbn1 siRNA treatment increased IL4R  levels, which could be blocked by TGF  neutralizing antibody treatment (Figure 3.4C-3.4D). To further confirm that TGFβ signaling activates IL4Rα expression, we used Smad2/3 siRNA to block canonical TGF  pathway and then examine the expression level of IL4Rα in Fbn1 +/- BMMSCs. Western blot analysis showed that IL4Rα was significantly decreased in Fbn1 +/- BMMSCs after Smad2/3 siRNA treatment (Figure 3.4E), indicating that TGFβ/SMAD3 signaling may directly regulate IL4Rα expression. In order to determine whether increased expression of IL4Rα in Fbn1 +/- 109 BMMSCs is attributed to the TGFβ/SMAD3 signaling, we generated an Il4r α promoter reporter construct in which the defined region of the Il4r α promoter and flanking region were placed upstream of a reporter gene encoding firefly luciferase. We used BIOBASE biological databases to search the Il4rα promoter sequence and found two candidate sites, which were closely matching with the SMAD3-associated SP1 transcription factors consensus targets (Figure 3.4F). We next demonstrated that Il4r α promoter activity was markedly induced in both Fbn1 +/- and TGFβ1-treated BMMSCs. When Fbn1 +/- BMMSCs were transfected with reporter vector, the luciferase assay showed a significantly increased Il4r α promoter activity. Introduction of SP1 mutated reporter vector markedly diminished the expression of the Il4r α-luciferase reporter in both Fbn1 +/- and TGFβ1- treated BMMSCs, indicating the direct initiation of IL4Rα expression by TGFβ-SMAD3- SP1 cascades (Figure 3.4F). We next determined whether SP1 regulates Il4rα promoter in BMMSCs. Using chromatin immunoprecipitation (ChIP), the SP1 binding consensus sequence within the promoter region was examined to confirm its ability to recruit SP1. As expected, SP1-bound DNA at the candidate site was significantly enriched in TGF β1- treated BMMSCs and Fbn1 +/- BMMSCs (Figure 3.4G), suggesting that TGFβ may act as an initiator to elevate IL4Rα expression. These results prompted us to examine whether TGFβ and IL4 synergistically activate the mTOR signaling in BMMSCs. We found that short-term TGFβ, but not IL4, treatment elevated the expression levels of IL4Rα in BMMSCs by Western blot analysis (Figure 3.4H). While TGFβ and IL4 treatments each elevated expression levels of p-mTOR in BMMSCs, combinatorial treatment of TGFβ and 110 IL4 resulted in a marked increase in p-mTOR expression (Figure 3.4H). These results indicate that the Fbn1 deficiency-induced TGFβ-SMAD3-SP1/IL4Rα cascade enhances mTOR signaling in Fbn1 +/- BMMSCs at the transcriptional level. Figure 3.4 TGFβ enhances IL4Rα expression in Fbn +/- BMMSCs. (a) ELISA assay showed an elevated level of TGFβ in Fbn1 +/- mouse serum and cultured medium of Fbn1 +/- BMMSCs when compared to the WT control groups. (b) Western blot showed upregulation of p-SMAD3 and SP-1 in Fbn1 +/- BMMSCs. (c) Western blot analysis showed that TGFβ treatment elevated IL4Rα expression in BMMSCs, which could be blocked by anti-TGFβ neutralizing antibody (TGFβ NAb). (d) Western blot analysis showed that Fbn1 knockdown by siRNA in BMMSCs elevated IL4Rα expression, which could be blocked by TGF β NAb. (e) Western blot showed that Smad2/3 siRNA treatment resulted in reduced expression of IL4Rα compared to vehicle-treated control group. (f) Il4rα promoter- luciferase fusions were examined in WT, TGFβ-treated and Fbn1 +/- BMMSCs. Promoter activity was expressed as relative light units (RLU) normalized to the activity of co- transfected Renilla luciferase. There was a significantly increased Il4ra promoter activity in TGFβ1-treated and Fbn1 +/- BMMSCs. However, transfection of SP1-specific site- mutated promoters resulted in markedly decreased Il4ra promoter activity in TGFβ1- treated and Fbn1 +/- BMMSCs. (g) Chromatin immunoprecipitation (ChIP)-qPCR assay showed enrichment of direct association of SP1 on Il4ra promoter in TGFβ1-treated and Fbn1 +/- BMMSCs compared to WT control. (h) Western blot showed that TGFβ1 treatment activated IL4Rα expression, but both TGFβ1 and IL4 treatment could elevate expression levels of p-mTOR. Combinatorial treatment with TGFβ1 and IL4 induced a more significant elevation of p-mTOR expression. *** P < 0.005. 111 3.3.4 mTOR cKO or rapamycin treatment ameliorates osteopenia in SSc mice Since IL4Rα/mTOR alters BMMSC lineage commitment, depletion of mTOR expression in BMMSC/osteoblastic lineage may serve as a key approach to recover Fbn1 deficiency- induced osteopenia. To achieve such a tissue-specific knockout, we generated SP7-Cre; Mtor f/+ ; Fbn1 +/- (TKO) conditional Mtor knockout mice (Figure 3.5A). Floxed Mtor littermates (Mtor f/+ ) were used as normal control and Mtor f/+ ; Fbn1 +/- (DKO) littermates were used as osteopenia model (Figure 3.5A). MicroCT analysis showed that TKO mice have significantly increased BMD compared to DKO mice (Figure 3.5B), suggesting a rescue of BMD in Fbn1 deficiency-induced osteopenia by Mtor conditional knockout. Histological analysis showed that trabecular bone volume in TKO mice was increased compared to the DKO mice (Figure 3.5C). The numbers of adipocytes in TKO mouse bone marrow were significantly reduced when compared to the DKO mice, as assessed by Oil red O staining (Figure 3.5C). To investigate the role of mTOR in the differentiation of Fbn1 deficient BMMSCs, we isolated BMMSCs from DKO mice and knocked down Mtor using an adenovirus expressing Cre with GFP (Ad-Cre-BMMSCs) or an adenovirus expressing GFP only (Ad-GFP-BMMSCs) as a control. Ad-Cre-BMMSCs showed significantly increased osteogenic differentiation by alizarin red staining indicating elevated mineralized nodule formation (Figure 3.5D), Western blot analysis indicating recovered expression of the mTOR/osteogenic cascade genes p-mTOR, RUNX2, ALP, and 112 OCN (Figure 3.5E), and in vivo implantation indicating elevated bone formation capacity when compared to the Ad-GFP-BMMSCs (Figure 3.5F). In contrast, Ad-Cre-BMMSCs showed a significant decrease in adipogenic differentiation compared to Ad-GFP- BMMSCs, as shown by the decreased number of Oil red O-positive cells and downregulation of the adipogenic genes PPARγ2 and LPL (Figure 3.5G-3.5H). Next, we examined whether rapamycin treatment rescued the osteopenia phenotype in Fbn1 +/- mice. Rapamycin was intraperitoneally administered to Fbn1 +/- mice at 8 or 6 weeks of age for 14 or 28 consecutive days, respectively. The samples were harvested at 10 weeks of age for further evaluation (Figure 3.6A). Histological and microCT analysis showed that rapamycin treatment elevated BMD and trabecular bone volume in Fbn1 +/- mice (Figure 3.6B). The number of adipocytes in bone marrow of rapamycin-treated Fbn1 +/- mice was significantly reduced when compared to untreated Fbn1 +/- mouse bone marrow, as assessed by Oil red O staining (Figure 3.6B). BMMSCs isolated from rapamycin-treated Fbn1 +/- mice showed significantly increased CFU-F number compared to the untreated group (Figure 3.6C). Osteogenic differentiation of BMMSCs from rapamycin-treated Fbn1 +/- mice was markedly improved when compared to the untreated Fbn1 +/- BMMSCs, as analyzed by alizarin red staining to show elevated mineralized nodule formation and Western blot analysis to show decreased expression of p-mTOR and P70S6K and increased expression of the RUNX2, ALP, and OCN (Figure 3.6D-3.6E). In contrast, BMMSCs isolated from rapamycin-treated Fbn1 +/- mice showed a 113 significant decrease in adipogenic differentiation compared to the untreated Fbn1 +/- BMMSCs, as shown by decreased number of Oil red O-positive cells and downregulation of the PPARγ2 and LPL (Figure 3.6F-3.6G). Besides skeletal deficiency, connective tissue fibrosis is another major systemic sclerosis phenotype observed in Fbn1 +/- mice. Histological analysis showed that skin hypodermal thickness was significantly increased in Fbn1 +/- mice. After rapamycin treatment, hypodermal thickness was reduced to a level equal to that observed in WT mice (Figure S3.7). In addition, Fbn1 +/- mice showed an increase in the levels of autoimmune markers anti-nuclear antibodies (ANA), anti-double strand DNA (dsDNA) IgG and IgM, and urine proteins. Rapamycin treatment significantly reduced the levels of ANA, dsDNA IgG and IgM, as well as urine protein levels (Figure S3.8), suggesting that rapamycin treatment is capable of rescuing osteoporotic and autoimmune phenotypes in the Fbn1 +/- SSc mice. These findings suggest that mTOR signaling may also contribute to connective tissue fibrosis and osteopenia phenotype in Fbn1 +/- mice (Figure S3.9). 114 Figure 3.5 Conditional knockout of mTOR in BMMSC/osteoblastic lineage ameliorates osteopenia phenotype by rescuing impaired osteogenic/adipogenic differentiation. (a) Experimental outline describing the use of floxed Mtor mice (Mtor f/+ ) crossed with Fbn1 +/- mice to generate Mtor f/+ ; Fbn1 +/- (DKO) mice. Then the DKO mice were crossed with Sp7-Cre;tTA-tet off mice to generate Sp7-Cre; Mtor f/+ ; Fbn1 +/- (TKO) conditional Mtor knockout mice. (b) MicroCT analysis showed that Mtor conditional knockout mice (TKO) have significantly increased BMD compared to DKO mice. Mtor f/+ mice were used as normal control. Scale bar, 1 mm. (c) H&E staining and Oil red O staining showed that TKO mice have significantly increased trabecular bone (TB) volume, but decreased the percentage of adipocytes in distal femur when compared to DKO mice. Mtor f/+ mice were used as normal control. Scale bar, 1mm for H&E staining and 50 m for Oil red O staining. (d) Alizarin red staining showed that mineralized nodule forming capacity was rescued in Adenovirus-Cre-treated BMMSCs derived from DKO mice (Ad-Cre) compared to Adenovirus-GFP-treated DKO group (Ad-GFP). (e) Western blot analysis showed that Ad-Cre-BMMSCs (Ad-Cre) had downregulated p-mTOR and upregulated expression of the RUNX2, ALP and OCN compared to Ad-GFP-BMMSCs from DKO mice (Ad-GFP). (f) When implanted into immunocompromised mice subcutaneously using HA/TCP (HA) as a carrier, Ad-Cre-BMMSCs showed significantly increased bone formation (B) compared 115 to Ad-GFP-BMMSCs from DKO mice. Scale bar, 50 m. (g) Oil red O staining showed a significantly decreased number of adipocytes in Ad-Cre-BMMSCs compared to Ad-GFP- BMMSCs from DKO mice when cultured under adipoinductive conditions. Quantitative analysis showed a significantly decreased amount of Oil red O + cells in Ad-Cre-BMMSCs. Scale bar, 50 m. (h) Western blot showed that Ad-Cre-BMMSCs had downregulated expression of the adipogenic genes PPAR 2 and LPL when compared to Ad-GFP- BMMSCs from DKO mice. *** P < 0.005. Figure 3.6 Rapamycin treatment ameliorates osteopenia phenotype by rescuing impaired osteogenic/adipogenic differentiation of BMMSCs in Fbn1 +/- mice. (a) Experimental outline describing the use of rapamycin to treat Fbn1 +/- mice. Rapamycin was given intraperitoneally for 14 or 28 consecutive days to 8- or 6-week-old Fbn1 +/- mice (n=6 per group). Tissues and samples were collected from 10-week-old mice. (b) MicroCT, H&E staining and Oil red O staining showed that rapamycin treatment for 14 or 28 days significantly increased BMD and trabecular bone (TB) volume, but decreased the percentage of adipocytes in distal femur of Fbn1 +/- mice. Scale bar, 1 mm for H&E staining and 50 m for Oil red O staining. (c) BMMSCs isolated from rapamycin-treated Fbn1 +/- mice showed increased CFU-F number compared to the untreated group by 116 toluidine blue staining. (d) Alizarin red staining showed that mineralized nodule forming capacity was rescued in BMMSCs derived from rapamycin-treated Fbn +/- mice compare to untreated Fbn +/- BMMSCs. (e) Western blot showed that BMMSCs from rapamycin- treated Fbn1 +/- mice had downregulated expression of p-mTOR and p-P70S6K, but upregulated expression of the RUNX2, ALP and OCN compared to untreated Fbn1 +/- BMMSCs. (f) Oil red O staining showed a significantly decreased number of adipocytes in BMMSCs derived from rapamycin-treated Fbn1 +/- mice compared to untreated Fbn1 +/- BMMSCs when cultured under adipoinductive conditions. Scale bar, 50 m. Quantitative analysis showed a significantly decreased amount of Oil red O positive cells in rapamycin-treated Fbn1 +/- mice. (g) Western blot showed that BMMSCs derived from rapamycin-treated Fbn1 +/- mice had downregulated expressions of the PPAR 2 and LPL. *** P < 0.005. Figure S3.7 Rapamycin treatment ameliorated skin fibrosis phenotype in Fbn1 +/- mice. Hypodermal thickness was significantly increased in Fbn1 +/- mice (n=5) compared to the WT group (C57BL6, n=5). Rapamycin treatment significantly reduced hypodermal thickness in Fbn1 +/- mice. The results were representative of three independent experiments. Abbreviations: D, Dermis; Ad, Adipose; PC, Panniculus Carnosus; HD, Hypodermis; M, Muscle. Scale bar, 50 μm. *** P < 0.005. 117 Figure S3.8 Rapamycin treatment ameliorated autoimmune index in Fbn1 +/- mice. ELISA analysis showed elevated levels of antinuclear antibody (ANA), anti-double strand DNA antibodies IgG and IgM, and urine protein in Fbn1 +/- mice (n=5) compared to the WT group (C57BL6, n=5). Rapamycin treatment for 14 or 28 days significantly reduced these autoimmune index. The results were representative of three independent experiments. *** P < 0.005. Figure S3.9 Schematic diagram shows that Fibrillin-1 (Fbn1) deficiency induces alteration of osteo/adipo-linage differentiation by activation of IL4/IL4R /PI3K/mTOR/P70S6K cascade in mesenchymal stem cells (MSCs), leading to a low-turnover osteoporotic phenotype in Fbn1 mutant mice. In addition, Fbn1 deficiency-induced elevation of TGF  activates SMAD3/SP1/IL4R  signaling to upregulate expression of IL4R . Rapamycin, a specific mTOR inhibitor, restores MSC/osteoblast function and rescues osteoporotic phenotype in Fbn1 +/- mice. 118 3.3.5 IL4R /mTOR signaling is activated in systemic sclerosis patients In order to confirm that IL4R /mTOR signaling contributes to systemic sclerosis, we conducted a pilot clinical investigation to assess whether IL4R /mTOR signaling is activated in systemic sclerosis (SSc) patients. Twenty patients diagnosed with SSc were enrolled for this study. Flow cytometric analysis showed that CD4 + IL4 + T H 2 cells were significantly increased in the peripheral blood of SSc patients when compared to the control group (Figure 3.7A). ELISA further confirmed that the serum IL4 levels were significantly elevated in SSc patients (Figure 3.7B). To examine whether IL4 induces downstream mTOR signaling, we showed that BMMSCs from SSc patients had upregulated expression of p-Raptor, p-Rictor, and p-mTOR (Figure 3.7C). Next, we showed that human recombinant IL4 treatment elevated expression of p-Raptor, p- Rictor, and p-mTOR, reduced in vitro mineralized nodule formation, and decreased expression of RUNX2, ALP, and OCN in human BMMSCs (Figure 3.7D-3.7F). Rapamycin treatment rescued IL4-induced osteogenic differentiation deficiency (Figure 3.7E-3.7F). Finally, we revealed that SSc patients had significantly elevated levels of serum TGF  and expression of IL4R  in BMMSCs (Figure 3.7G-3.7H), along with reduced levels of RANKL and OPG (Figure 3.7I-3.7J). 119 Figure 3.7 Systemic sclerosis patients showed highly activated IL4R /mTOR signaling. (a) Flow cytometric analysis showed elevated levels of T H 2 cells in peripheral blood of systemic sclerosis (SSc) patients. (b) ELISA assay showed increased levels of IL4 in the serum of SSc patients. (c) Western blot analysis showed upregulation of p-Raptor, p- Rictor, and p-mTOR in BMMSCs from SSc patients. (d) Western blot analysis showed that IL4 treatment activated p-Raptor, p-Rictor, and p-mTOR expression in human BMMSCs. (e) IL4 treatment reduced the capacity of human BMMSCs to form mineralized nodules, assessed by alizarin red staining, while rapamycin treatment rescued mineralized nodule formation in IL4-treated human BMMSCs. (f) IL4-treated human BMMSCs showed upregulation of p-mTOR and downregulation of RUNX2, ALP and OCN compared to untreated BMMSCs, while rapamycin treatment rescued IL4- induced expression of p-mTOR, RUNX2, ALP, and OCN in human BMMSCs. (g) SSc patients showed increased serum TGF  levels, assessed by ELISA. (h) Quantitative RT- PCR analysis showed that SSc patient BMMSCs had upregulated expression of Il4r . (i and j) ELISA showed that SSc patients had decreased serum levels of sRANKL (i) and OPG (j). 120 3.4 Discussion FBN1 is essential for the formation of elastic fibers or microfibrils that provide strength and flexibility to connective tissue. Normally, FBN1 is abundant in the connective tissue of the aorta, ligature of the human eye lens, bones and lungs. Mutation of the fibrillin gene causes systemic sclerosis and Marfan’s syndrome, in which bone and connective tissue disorders are observed. Recently, it was suggested that FBN1 and FBN2 might differentially regulate endogenous BMP and TGF  activity in osteoblasts to affect bone growth and development (Nistala et al., 2010). In this study, we reveal that Fbn1 deficiency results in an increased level of TGF  in bone marrow, which, in turn, initiates a cascade in which IL4R  is upregulated in BMMSCs via SMAD3/SP1 binding to Il4r  promoter, leading, as a consequence, to the activation of mTOR-P70S6K signaling to directly suppress osteogenesis via RUNX2 and promote adipogenesis via PPAR 2, respectively. Osteogenic differentiation deficiency in Fbn1 +/- BMMSCs may contribute to an osteoporotic phenotype, such as low bone mineral density or reduced trabecular bone structure, in Fbn1 +/- SSc mice. However, inhibition of mTOR signaling using rapamycin treatment appears to be a promising approach to rescue osteoporotic disorders in the SSc mice. It is known that mTOR signaling positively regulates expression of the adipogenic gene PPAR 2, which is critical for adipogenesis of BMMSCs (Kim and Chen, 2004; Yu et al., 121 2008; Zhang et al., 2009). However, controversial findings were reported regarding mTOR signaling in osteogenesis in that mTOR may be required for osteoblast proliferation, which is involved in several signaling pathways through interleukin-6 (IL6) (Takai et al., 2007, 2008; Kozawa et al., 2001). In contrast, rapamycin may either inhibit (Shoba and Lee, 2003) or stimulate (Lee et al., 2010; Martin et al., 2010) osteogenesis, depending on cell type or differentiation stages. Recently, it has been reported that bone matrix secretes insulin-like growth factor-1 (IGF-1) to recruit normal BMMSCs for bone remodeling by activating mTOR signaling (Xian et al., 2012), indicating that mTOR may contribute to the activation of different sets of biological regulatory controls under certain conditions. In this study, we reveal that elevated IL4R  expression leads to the activation of mTOR-P70S6K signaling in Fbn1 +/- BMMSCs, which directly inhibits the osteogenic gene RUNX2 and suppresses bone regeneration in vitro and in vivo. On the other hand, rapamycin represses mTOR/P70S6K signaling, which results in rescuing BMMSC deficiency and ameliorating the osteoporotic phenotype in Fbn1 +/- mice, implicating that IL4R /mTOR is, in fact, a major signaling pathway contributing to the osteopenia phenotype in Fbn1 +/- mice. Since Fbn1 -/- mice are embryonic lethal, we cannot examine whether Fbn1 null shows a phenotype similar to that observed in Fbn1- deficient conditions (Barisic-Dujmovic et al., 2007). Fbn1 +/- mice show T H 2 infiltration and high levels of IL4. Disruption of IL4 can rescue the fibrotic phenotype, suggesting that a high level of IL4 may also play a critical role in the 122 disease pathogenesis (Gabrielli et al., 2009; Wynn et al., 2004; Kodera et al., 2002). While overproduction of IL4 can induce osteoporotic phenotype in mice (Lewis et al., 1993), activation of IL4R  may inhibit osteoblast proliferation (Frost et al., 2001), indicating that IL4 may affect BMMSC function. Conversely, it was reported that local delivery of IL4, using a gene therapy approach, prevented bone erosion in arthritis animal models via abrogation of osteoclastogenesis (Lubberts et al., 2000; Woods et al., 2001; Saidenberg-Kermanac’h et al., 2004). These findings indicate that elevated IL4 caused by Fbn1 deficiency may contribute to a low osteoblast/osteoclast turnover rate, as observed in Fbn1 +/- mice. Our data suggest that IL4/IL4R  signaling activates mTOR signaling in Fbn1 +/- BMMSCs, which alters BMMSC osteogenic/adipogenic lineage differentiation, as a consequence of reduced osteogenesis and elevated adipogenesis in bone marrow. Rapamycin, a specific mTOR inhibitor, is a novel nonsteroidal anticancer and immunosuppressive drug (Abraham and Wiederrecht, 1996; Wang et al., 2003; Shaw et al., 2004). Several preclinical studies showed that rapamycin is effective in treating autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis (Yoon, 2009; Islander et al., 2011). These diseases are usually associated with bone disorders (Islander et al., 2011; Weinstein, 2010). Although efficacy of rapamycin treatment in SSc patients must be assessed in clinical studies, phase I clinical trials showed optimal safety for its use in treating scleroderma patients (Su et al., 2009). 123 Added experimental evidence showed that rapamycin treatment resulted in a significant improvement of skin phenotype with regulation of immune index, such as reducing the levels of IL4 and IL17 in Fbn1 +/- mice (Yoshizaki et al., 2010). These data suggest that the underlying mechanism of rapamycin-mediated treatment may be associated with immunomodulation, leading to the downregulation of T H 2 cells (Lee et al., 2010). Upregulation of osteogenesis and downregulation of adipogenesis in rapamycin-treated Fbn1 +/- mice via inhibition of mTOR signaling in BMMSCs may help to rebuild functional osteoblasts to improve niche microenvironment for immune cells and rebuild homeostasis for the immune system. Because of the extensive functional roles of rapamycin in immune, anticancer, and osteoporosis therapies, it is necessary to explore detailed mechanisms by which rapamycin may target several signaling pathways simultaneously. 124 Chapter 4: Conclusions Fibrillin-1 is a major structural component of microfibrils in extracellular matrix. FBN1 gene mutation is linked to several human diseases, including systemic sclerosis/scleroderma, Marfan’s syndrome, ectopic lentis, and the dominant form of Weill-Marchesani syndrome. These diseases are usually characterized by connective tissue fibrosis and skeletal disorders. Although it is known that FBN-1 deficiency-induced elevation of IL4 causes connective tissue fibrosis, it is unclear whether FBN1 deficiency contributes to bone disorders in these diseases. Mesenchymal stem cells are multipotent stem cells capable of differentiating into osteoblasts, adipocytes, chondrocytes, and other cell types. MSCs are considered as a promising cell source for regenerative medicine and immune therapies. A variety of preclinical and clinical studies have shown that MSCs can be used to generate bone structure to replace damaged and diseased tissues, as well as treat a variety of immune- related diseases, including GVHD, SLE, IBD, and sepsis. Recent studies suggest that MSC disorder may contribute to a variety of human diseases, such as osteoporosis and SLE. However, the detailed mechanism in which BMMSCs regulate immune function is not fully understood. In addition, it is unknown that whether signaling pathway alteration in MSCs is associated with disease phenotypes in these diseases. It is also unknown if rescuing the altered pathway would provide a fruitful direction for the development of novel therapies. 125 First of all, we show that in mice systemic infusion of BMMSCs induced transient T-cell apoptosis via the Fas ligand (FasL)-dependent Fas pathway and could ameliorate disease phenotypes in fibrillin-1 mutated systemic sclerosis (SSc) and dextran sulfate sodium- induced experimental colitis. FasL -/- BMMSCs did not induce T-cell apoptosis in recipients, and could not ameliorate SSc and colitis. Mechanistic analysis revealed that Fas-regulated MCP-1 secretion by BMMSCs recruited T-cells for FasL-mediated apoptosis. The apoptotic T-cells subsequently triggered macrophages to produce high levels of TGF  which in turn led to the upregulation of Tregs and, ultimately, to immune tolerance. These data therefore demonstrate a previously unrecognized mechanism underlying BMMSC-based immunotherapy involving coupling via Fas/FasL to induce T- cell apoptosis. Secondly, we show that telomerase-deficient BMMSCs lose their capacity to inhibit T cells and ameliorate the disease phenotype in systemic sclerosis mice. Restoration of TERT transfection in TERT -/- BMMSCs rescues their immunomodulatory functions. Mechanistically, we reveal that TERT, combined with -catenin and BRG1, serves as a transcriptional complex, which binds the Fas ligand (FasL) promoter to upregulate FasL expression, leading to an elevated immunomodulatory function. To test the translational value of these findings in the context of potential clinical therapy, we used aspirin treatment to upregulate telomerase activity in BMMSCs, and found a significant improvement in the immunomodulatory capacity of BMMSCs. Taken together, these 126 findings identify a previously unrecognized role of TERT in improving the immunomodulatory capacity of BMMSCs, suggesting that aspirin treatment is a practical approach to significantly reduce cell dosage in BMMSC-based immunotherapies. Connective tissue fibrosis in systemic sclerosis is attributed to FBN1 deficiency-induced elevation of interleukin-4, the mechanism underlying FBN1-deficiency-associated osteoporosis is not fully understood. In Chapter 3, we show that BMMSCs from FBN1- deficient SSc mice exhibit decreased osteogenic differentiation and increased adipogenic differentiation, which is regulated by IL4/IL4Rα-mediated activation of mTOR signaling to downregulate RUNX2 and upregulate PPAR 2, respectively, via P70S6K. Additionally, we reveal that activation of TGF /SMAD3/SP1 signaling results in enhancement of SP1 binding to the IL4R  promoter to synergistically activate mTOR pathway in Fbn1 +/- BMMSCs. Blockage of Mtor signaling by MSC/osteoblastic-specific knockout or rapamycin treatment rescue osteopenia phenotype in SSc mice by improving osteogenic differentiation of BMMSCs. 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Abstract (if available)

Abstract Bone marrow mesenchymal stem cells (BMMSCs) are non‐hematopoietic multipotent stem cells capable of differentiating into both mesenchymal and non‐mesenchymal cell types. In addition to generate bone structure to replace damaged and diseased tissues, preclinical and clinical studies have shown that BMMSCs display profound immunomodulatory functions via inhibiting the proliferation and function of several major immune cells such as T lymphocytes, B lymphocytes, natural killer (NK), and dendritic cells. Thus, systemic infusion of BMMSCs has been used to treat a variety of diseases, including acute graft-versus-host-disease (GVHD), ameliorating HSC engraftment, systemic lupus erythematosus (SLE), intestinal and bowel disease (IBD), and sepsis. However, the detailed mechanism in which BMMSCs regulate immune function is not fully understood. ❧ In the first part of Chapter 2 of this study, we show that systemic infusion of BMMSCs induces a transient T cell apoptosis via the Fas Ligand (FasL)‐mediated Fas pathway and ameliorates diseased phenotypes in fibrillin-1 (FBN1) mutated systemic sclerosis (SSc) and dextran sulfate sodium‐induced experimental colitis mice. The therapeutic mechanism of BMMSC infusion is associated with phagocytosis of apoptotic T cell debris, leading to a high level of macrophage‐mediated transforming growth factor beta (TGFβ) production and a subsequent immune tolerance. Importantly, we provided clinical evidence to show that MSC infusion in SSc patients resulted in a T cell apoptosis and up‐regulation of Tregs. Additionally, we revealed that Fas null BMMSCs, with normal FasL function, failed to induce T cell apoptosis and offer therapeutic effect for SS and colitis mice. Mechanistic study showed that Fas governed monocyte chemotactic protein 1 (MCP-1) secretion in BMMSCs, which plays a crucial role in the recruitment of T cells to BMMSCs for FasL‐mediated apoptosis. In summary, BMMSCs use Fas to control MCP-1 secretion for the recruitment of T cells and subsequently use FasL to induce activated T cell apoptosis. Macrophages take debris of apoptotic T cells to release a high level of TGFβ, leading to up‐regulation of regulatory T cells (Tregs) and, ultimately, immune tolerance for immunotherapies. ❧ This study uncovers the role of Fas and FasL in BMMSC‐based immune therapies, which may serve as a basis to develop novel strategies for improving cell‐based therapies. The significance of this study is to identify a novel mechanism of BMMSC‐associated immunomodulation and immune therapy. Also, Fas and FasL collaboratively induce immune tolerance suggests a potential new mechanism that receptor/ligand coupled to execute therapeutic effect in cell‐based treatment. This study covered experimental evidences for stem cell biology, molecular mechanism of BMMSC associated immunomodulation, and stem cell-based immunotherapies. ❧ In second part of Chapter 2 of this study, we showed for the first time that telomerase activity is required for maintaining the immunomodulatory properties of MSCs. Telomerase deficient MSCs lose their capacity to inhibit T‐cells, activate Foxp3‐positive Tregs, and ameliorate disease phenotype in systemic sclerosis mice, which can be rescued by overexpression of telomerase reverse tran¬scriptase (TERT). Mechanistically, TERT combined with β‐catenin and BRG1 to form a complex to bind to FasL promoter and upregulate FasL expression. Upregulated FasL expression can elevate MSC immunomodulation function, as shown in our recent publication (Akiyama et al., 2012). When MSCs were treated with aspirin, their immunomodulation was significantly improved due to elevated telomerase activity and the number of MSCs required to treat systemic sclerosis mice was markedly reduced. ❧ This study has uncovered the role of telomerase in MSC‐based immunotherapies and the mechanism by which TERT binds to the promoter region to upregulate FasL expression. In fact, this is the first study to link telomerase activity to immunomodulatory therapies. Overall, therefore, this study has brought forth experimental evidence for stem cell biology, the molecular mechanism(s) underlying MSC‐associated immunotherapies, and pathway‐guided drug therapy. ❧ In Chapter 3, we reveal that Fbn1+/- SSc mice show osteopenia phenotype with decreased osteogenic differentiation and increased adipogenic differentiation of bone marrow MSCs by the activation of Interleukin-4 receptor α (IL4Rα)/mTOR (the mammalian target of rapamycin) signaling. We further determine that mTOR signaling blocks osteogenic differentiation via the P70S6K/RUNX2 pathway, while it elevates adipogenic differentiation via P70S6K/PPARγ2 pathway. Since significantly elevated levels of Interleukin-4 (IL4) and TGFβ were observed in Fbn1+/- SSc mice, we reveal that upregulation of the IL4Rα in Fbn1+/- MSCs is governed by TGFβ/SMAD3/SP1 signaling via SP1 biding to the Il4rα promoter. Either knockdown of IL4Rα or inhibition of mTOR can rescue Fbn1+/- MSC function by increasing osteogenesis and reducing adipogenesis. Additionally, we showed that conditional knockout of mTOR in MSCs/osteoblasts could ameliorate osteopenia phenotype in Fbn1+/- mice by rescuing impaired osteogenic/adipogenic differentiation. ❧ To translate our findings to potential clinical applications, we used rapamycin treatment to inhibit mTOR signaling, thereby rescuing osteopenia phenotype in Fbn1+/- SSc mice by rescuing osteo/adipo‐lineage differentiation in MSCs. This result strongly suggests that rapamycin treatment may provide an anabolic therapy for systemic sclerosis. ❧ In summary, this study establishes the FBN1/TGFβ/SP1/IL-4Rα/mTOR cascade as a key determinant of MSC lineage selection, a finding which may serve as a basis for the development of novel therapies to treat SSc.

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Creator Chen, Chider (author)

Core Title From mesenchymal stem cell therapy to discovery of drug therapy for systemic sclerosis

School School of Dentistry

Degree Doctor of Philosophy

Degree Program Craniofacial Biology

Publication Date 03/31/2014

Defense Date 02/24/2014

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag immunomodulation,mesenchymal stem cell,OAI-PMH Harvest,osteopenia,rapamycin,systemic sclerosis,telomerase

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Shi, Songtao (committee chair), Chai, Yang (committee member), Chuong, Cheng-Ming (committee member), Le, Anh (committee member), Paine, Michael L. (committee member), Ying, Qi-Long (committee member)

Creator Email chiderch@usc.edu,mike0916kimo@yahoo.com.tw

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-370638

Unique identifier UC11295335

Identifier etd-ChenChider-2306.pdf (filename),usctheses-c3-370638 (legacy record id)

Legacy Identifier etd-ChenChider-2306.pdf

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Document Type Dissertation

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Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

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