Medicine, Faculty of
Medicine, Department of
DSpace
UBCV
Chu, Tony Lok Heng
Master of Science - MSc
University of British Columbia
Inactivation of T cells is a widely used strategy for immunosuppression. Halofuginone (HF) is an antiprotozoal agent for treating parasites in veterinary medicine, and has been demonstrated to inhibit collagen type 1 synthesis, T helper 17 cell differentiations and cytokine production in activated T cells. The present study was designed to examine its actions against T cell proliferation, and in a combined therapy with conventional immunosuppressant rapamycin. To examine the anti-proliferative ability of HF, T cell proliferation in cultured murine splenocytes, cell apoptosis, and cell cycle analysis as well as autophagy markers were investigated. The expression of cleaved Poly ADP ribose polymerase and DNA fragmentation were further to characterize apoptosis. Here, I showed that addition of HF in naïve splenocyte cultures significantly suppressed cell proliferation in response to antigen- or anti-CD3 antibody or in activated T cell cultures in response to Interleukin (IL)-2 in a dose-dependent manner with activation of apoptosis, and cell cycle arrest at S phase. Further studies showed that proline supplement in cell culture medium significantly prevented HF-mediated suppression of T cell proliferation by reducing cell apoptosis and proliferation arrest. In addition, HF synergistically enhanced the anti-proliferative ability of rapamycin, which was correlated with increased induction of apoptosis in T cell cultures. My data suggests that HF interferes proline incorporation in protein synthesis machinery resulting in apoptosis and autophagy-induced cell cycle arrest via amino acid starvation response in T cells. The data of this study suggest that HF may be a potential adjuvant that can enhance anti-proliferative activity of rapamycin in preventing T cell proliferation.
THE MECHANISM OF ANTI-PROLIFERATION OF HALOFUGINONE IN T CELLS. by Tony Lok Heng Chu B.Sc., The University of British Columbia, 2009 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Experimental Medicine) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) June 2013 © Tony Lok Heng Chu, 2013 ii Abstract Inactivation of T cells is a widely used strategy for immunosuppression. Halofuginone (HF) is an antiprotozoal agent for treating parasites in veterinary medicine, and has been demonstrated to inhibit collagen type 1 synthesis, T helper 17 cell differentiations and cytokine production in activated T cells. The present study was designed to examine its actions against T cell proliferation, and in a combined therapy with conventional immunosuppressant rapamycin. To examine the anti-proliferative ability of HF, T cell proliferation in cultured murine splenocytes, cell apoptosis, and cell cycle analysis as well as autophagy markers were investigated. The expression of cleaved Poly ADP ribose polymerase and DNA fragmentation were further to characterize apoptosis. Here, I showed that addition of HF in naïve splenocyte cultures significantly suppressed cell proliferation in response to antigen- or anti-CD3 antibody or in activated T cell cultures in response to Interleukin (IL)-2 in a dose-dependent manner with activation of apoptosis, and cell cycle arrest at S phase. Further studies showed that proline supplement in cell culture medium significantly prevented HF-mediated suppression of T cell proliferation by reducing cell apoptosis and proliferation arrest. In addition, HF synergistically enhanced the anti-proliferative ability of rapamycin, which was correlated with increased induction of apoptosis in T cell cultures. My data suggests that HF interferes proline incorporation in protein synthesis machinery resulting in apoptosis and autophagy-induced cell cycle arrest via amino acid starvation response in T cells. The data of this study suggest that HF may be a potential adjuvant that can enhance anti-proliferative activity of rapamycin in preventing T cell proliferation. iii Preface This thesis is based on my own work. The work was completed under the supervision of Drs. Caigan Du and Christopher YC Nguan. I, Tony LH Chu, was responsible for experiments presented in this thesis. Proline concentration was measured using HPLC assisted by Hans Adomat (Vancouver Prostate Centre, Vancouver, BC). Identification of protein peptides in HF treated samples using LC-mass spectrometry was assisted by Dr. Bingyun Sun (Department of Chemistry, Simon Fraser University, Vancouver, BC) and Hans Adomat. I received training awards from the CIHR for Health Research Strategic Training Program in Transplantation Research, and the BC Proteomics Network Training programs. iv Table of Contents Abstract .......................................................................................................................................... ii Preface ........................................................................................................................................... iii Table of Contents ......................................................................................................................... iv List of Tables ............................................................................................................................... vii List of Figures ............................................................................................................................. viii List of Diagrams .............................................................................................................................x Acknowledgements ...................................................................................................................... xi Chapter 1: Introduction ...............................................................................................................1 1.1 Background of Autoimmune Diseases ............................................................................ 1 1.2 Autoimmune Diseases – Incidence, Mortality, Cost ...................................................... 2 1.3 The Immune System and the Role of Immune Cells in Autoimmune Diseases ............. 2 1.3.1 Innate System .............................................................................................................. 5 1.3.1.1 Leukocytes .......................................................................................................... 6 1.3.2 Adaptive System ....................................................................................................... 13 1.3.2.1 T Cell Migration ............................................................................................... 14 1.3.2.2 T Cell Activation – MHC/Peptide Complex ..................................................... 15 1.3.2.3 T Cell Signaling – T Cell Receptor................................................................... 16 1.3.2.4 T Cell Activation – Co-stimulation................................................................... 19 1.3.2.5 T Cell Differentiation ........................................................................................ 19 1.3.2.6 B Cell Activation .............................................................................................. 23 1.4 Immune Tolerance ........................................................................................................ 27 1.5 Drug Development and Current Immunosuppressant................................................... 28 v 1.6 HF, Rapamycin and Cyclosporin A .............................................................................. 31 1.7 Objectives of This Study ............................................................................................... 32 1.8 HF, Apoptosis and Cell Cycle Arrest ........................................................................... 34 1.8.1 What Is Apoptosis? ................................................................................................... 34 1.8.2 What Is Cell Cycle? .................................................................................................. 40 1.9 HF and Protein Synthesis .............................................................................................. 41 Chapter 2: Material and Method ..............................................................................................46 2.1 Cells and Reagents ........................................................................................................ 46 2.2 Preparation of T Cell Receptor-Stimulated Naïve T Cells ........................................... 47 2.3 Preparation of IL-2 Stimulated Activated T Cells ........................................................ 47 2.4 Determination of Cell Proliferation and Growth .......................................................... 48 2.5 Determination of Apoptosis by A Flow Cytometry ...................................................... 49 2.6 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) .................................... 49 2.7 Enzyme-Linked Immunosorbent Assay (ELISA) ......................................................... 50 2.8 Determination of Cell Cycle Arrest by A Flow Cytometry. ......................................... 51 2.9 Proline Measurement .................................................................................................... 51 2.10 Western Blot ................................................................................................................. 52 2.11 TUNEL Assay ............................................................................................................... 53 2.12 Determination of Synergistic Relationship Between HF and Rapamycine .................. 54 2.13 Statistical Analysis ........................................................................................................ 55 Chapter 3: Results.......................................................................................................................56 3.1 HF Induced Anti-proliferation In T Cells. .................................................................... 56 vi 3.1.1 The Effect of HF on T Cell Proliferative Response to the Stimulation of T Cell Receptor-CD3 Complex or IL-2 Receptor ............................................................................ 56 3.1.2 The Effect of HF on Anti-CD3 Antibody-Stimulated IL-2 Production in Naïve Splenocytes ........................................................................................................................... 58 3.1.3 The Effect of HF on IL-2 Receptor Signaling in Activated Splenocytes ................. 59 3.1.4 The Effect of HF on Cell Apoptosis in Both Naïve and Activated T Cells .............. 60 3.1.5 The Effects of HF in Cell Cycle Progress in Naïve T Cells ..................................... 64 3.1.6 The Effect of HF on Proline Uptake in Anti-CD3 Antibody-Stimulated Splenocytes…………………………………………………………………………………65 3.2 Proline Rescued HF Anti-proliferation ......................................................................... 66 3.2.1 The Effect of Additional Proline in Culture Medium on HF-induced T Cell Proliferative Suppression and Apoptosis .............................................................................. 66 3.2.2 The Effects of Proline in Cell Cycle Progress in HF-treated T Cells ....................... 71 3.3 HF Synergistically Enhanced RAPA Anti-proliferation in T Cells. ............................. 73 3.3.1 HF Enhanced Anti-proliferation of Rapamycin on T Cell Receptor-Stimulated T Cell Proliferation ................................................................................................................... 73 3.3.2 HF Enhanced Anti-prolifeation of Rapamycin on Allo-antigenic Stimulated T Cell Proliferation .......................................................................................................................... 76 3.3.3 HF Increased the Induction of Apoptosis in Rapamycin-treated T Cell Cultures .... 77 Chapter 4: Discussion .................................................................................................................80 Chapter 5: Conclusion ................................................................................................................89 Bibliography .................................................................................................................................91 Appendix A. ................................................................................................................................111 vii List of Tables Table 1.1 Important surface molecules of immune cells. ............................................................... 4 Table 3.1 The computed values of Loewe's Additivity between HF and RAPA or CsA. ........... 76 viii List of Figures Figure 1.1 Chemical structure of HF (up). Dichroa febrifuga (Chang Shan) (bottom). ............. 32 Figure 3.1 HF induces anti-proliferation in response to T cell receptor complex and IL-2R..... 57 Figure 3.2 HF doesn’t attenuate IL-2 production in anti-CD3 stimulated T cells as shown in PCR assay and ELISA assay......................................................................................................... 59 Figure 3.3 HF exerted no effect on IL-2 dependent phosphorylation of STAT5 signaling in activated T cells. ........................................................................................................................... 60 Figure 3.4 HF induced apoptosis in T cell receptor stimulated T cell proliferation. ................... 62 Figure 3.5 HF induced apoptosis in IL-2 stimulated T cell proliferation. ................................... 63 Figure 3.6 HF induced cell cycle arrest at S phase. Naive T cells were stimulated with anti-CD3 antibody in the presence or absence of HF. .................................................................................. 64 Figure 3.7 HF blocks proline uptake in anti-CD3 antibody-stimulated T cell cultures. .............. 65 Figure 3.8 The excess amount of proline inhibited anti-T cell proliferative activity of HF. ....... 67 Figure 3.9 The excess amount of proline rescued HF-induced apoptosis in T cells. .................. 68 Figure 3.10 The excess amount of proline inhibited HF-dependent PARP cleavage in splenocyte cultures. ......................................................................................................................................... 69 Figure 3.11 The excess amount of proline inhibited HF-dependent DNA fragmentation in splenocyte cultures. ....................................................................................................................... 70 Figure 3.12 The excess amount of proline inhibited HF induced cell cycle arrest in splenocyte cultures. ......................................................................................................................................... 72 Figure 3.13 Rapamycin (RAPA) induced anti-proliferation in T cells. ....................................... 74 Figure 3.14 The effect of HF + Rapamycin / HF + Cyclosporin A in T cell proliferation. .......... 75 ix Figure 3.15 HF enhanced anti-proliferation in RAPA treated T cell proliferation in response to allo-antigenic stimulation.............................................................................................................. 77 Figure 3.16 HF increased apoptosis in Rapamycin treated T cells. ............................................. 79 Figure 5.1 A proposed model of HF activity. ............................................................................... 90 Supplementary Figure A.1 Proline reduces the expression of HF-induced cleavage of caspase 3. ..................................................................................................................................................... 111 Supplementary Figure A.2 HF induces LC3 I/II conversion in splenocyte cultures. ................ 112 Supplementary Figure A.3 3-MA increases anti-proliferation in HF-treated T cell cultures. ... 113 Supplementary Figure A.4 HF induces up-regulation of CD25 (IL-2 α chain) in T cells. ........ 114 Supplementary Figure A.5 Proline reduces HF-induced up-regulation of CD25 (IL-2 α chain) in T cells. ......................................................................................................................................... 115 Supplementary Figure A.6 Identification of proyl-tRNA syntethase in HF-treated T cell cultures via drug affinity responsive target stability (DARTS). ............................................................... 118 Supplementary Figure A.7 CsA suppresses TCR stimulated T cell proliferation with IC50 at 9-10 nM. .............................................................................................................................................. 119 x List of Diagrams Diagram 1.1 Innate and adaptive immune response. ...................................................................... 4 Diagram 1.2 NK cells and Cytotoxic T cells (CTL). ..................................................................... 8 Diagram 1.3 The classical pathway of complement system. ........................................................ 12 Diagram 1.4 Cell mediated immunity and humoral immunity. .................................................... 13 Diagram 1.5 T cell activation....................................................................................................... 15 Diagram 1.6 TCR signaling induced IL-2 production. ................................................................. 18 Diagram 1.7 T cell subtypes. ........................................................................................................ 22 Diagram 1.8 T cell-dependent B cell activation. .......................................................................... 26 Diagram 1.9 Pathway to p53-dependent apoptosis. ...................................................................... 37 Diagram 1.10 The physiology of protein synthesis. .................................................................... 43 xi Acknowledgements I am deeply grateful to Dr. Caigan Du for having enabled me to be a graduate student in his laboratory. I wish to thank Dr. Caigan Du for his patience, understanding and kindness, and for all that has taught me about immunology, and most importantly, the way that a scientist thinks. Dr. Du has provided great supervision and guidance whenever I designed an experiment. It is an honor to be a part of Du’s Lab. I would like to thank Quinong Guan for her indispensable assistance during all my experiments. Thank you, Quinong, for always being there to resolve my frustration in performing experiments as well as interpretation of data. I thank Drs. Ken Harder and Christopher Ong for their help and advice. My thesis work was supported by training awards from the CIHR for Health Research Strategic Training Program in Transplantation Research (2011-2013), the Faulty of Medicine Graduate at the University of British Columbia (2012), and the BC Proteomics Network Training programs (2012). 1 Chapter 1: Introduction This chapter illustrates the rationale of this study by looking at the background of autoimmune diseases, the immune system, and current immunosuppressant development. 1.1 Background of Autoimmune Diseases Autoimmune diseases or autoimmune disorders are defined as “any disorder in which loss of function or destruction of normal tissue arises from humoral or cellular immune responses to the body’s own tissue constituents; may be systemic or organ specific[1]. The conception of autoimmune disease was first described in 1904 by Julius Donath and Karl Landsteiner [2]. In 1940s, encephalomyelitis was found to be one of complications of Pasteur’s rabies vaccine [2]. Thus, early immunologists started to study the effects of immunization of animals [2]. In 1950s, a T cell mediated autoimmune response in causing neural damage became widely accepted [3]. Adjuvant-based immunization that induced autoimmune disease in thyroid gland, testis and other tissues was developed. The development of clonal selection theory in 1957 [4] further solidified the idea of autoimmunity. This theory stated that the existence of self-reactive T cells was due to somatic mutation, and these self-reactive lymphocytes were the mediators of immunopathology. The innovation of fluorescence microscopy further enhanced immunologists’ understanding of autoimmune disease. The fluorescence microscopy was able to identify the deposited immunoglobins as well as antibodies in patients’ serum [5]. In 1965, autoimmunity was widely accepted in the general public to identify as one of the important causes of human pathology [6]. 2 1.2 Autoimmune Diseases – Incidence, Mortality, Cost More than 100 of diseases are classified under the category of autoimmune disease that includes bacterial/viral infection, Addison’s disease, anaphylaxis, arthritis, myasthenia gravis, and pernicious anemia, acute/chronic graft rejection, Type 1 diabetes, and multiple sclerosis [7]. The autoimmune diseases can be life-threatening under certain conditions, and can be classified by corresponding type of hypersensitivity: I, II, III or IV [7]. Researchers in the United States of America (USA) have reviewed more than 300,000 death certificates from 26 states collected from mid 1980s to the late 1990s. 50,000 of cases are certified as systemic autoimmune disease [7]. According to the American Autoimmune Related Diseases Association (AARDA), 50 million Americans suffer all kinds of autoimmune diseases, and 75% of these cases occurred in women [8]. The autoimmune diseases are the 4 th leading cause of disability in women in 2004 (The National Women’s Health Centre, 2004). There is a rising trend in incidence of autoimmune diseases [8]. The autoimmune diseases become a heavy financial burden on many health care systems around the world; the National Institutes of Allergy and Infectious diseases (NIAID) has estimated that the annual treatment cost of autoimmune disease is more than $100 billion in USA [9, 10]. 1.3 The Immune System and the Role of Immune Cells in Autoimmune Diseases There are a variety of immunosuppressants available for modulating the severity of autoimmune diseases in patients, but most of them have severe adverse side effects, such as nephrotoxicity [11]. Thus novel effective immunosuppressive drugs with more specific against the autoimmune response are needed. To develop such immunosuppressive strategies, an in- depth understanding of the immune system is required. 3 The immune system is a collection of biological processes and components within an organism that protects against pathogens that may cause harm to the host. The immune system is consisted of innate system and adaptive system (Diagram 1.1). When a pathogen successfully breaches skin barrier, the innate immune system is the second barrier which gives an immediate and non-specific response. The innate system is initiated when pathogens are identified by pattern recognition receptors (PRR) on leukocytes of innate system. These surface molecules can recognize components from wide broad groups of microorganism [12]. They can also identify damaged or injured cells [13]. However, this system does not yield a long lasting immunity against a pathogen. Here, Table 1.1 reveals the important surface molecules on immune cells. These molecules are frequently mentioned in the passages because they are essential in mediating immune responses. 4 Surface Molecules Cell Types Functions MHC I Major Histocompatibility Class I Nucleated Cells Present peptides to CD8 T cells MHC II Major Histocompatibility Class II Phagocytes, B cells Present peptides to CD4 T cells TCR T cell receptors T cells Recognize peptide:MHC complex BCR B cell receptors B cells Interact with antigens Table 1.1 Important surface molecules of immune cells. This table describes the roles of MHC I/II, TCR and BCR. Diagram 1.1 Innate and adaptive immune response. This flow chart simply illustrates how innate system and adaptive system are inter-connected to each other in protecting host from pathogens. APCs activate T cells which differentiate into different subtypes of T cells with immune functions. 5 1.3.1 Innate System Inflammation is one of the first responses of innate system to infection [14]. Inflammation is characterized as redness, swelling, heat, and pain. Such symptoms are caused by increased blood flow due to the release of eicosanoids and various cytokines at the site of injury [15, 16]. The goal of inflammation is to recruit leukocytes to the site of injury to encounter intruders. Leukocytes of innate system include phagocytes (macrophages, neutrophils, dendritic cells), mast cells, eosinophils, basophils and natural killer (NK) cells. Phagocytes eliminate the pathogens via engulfing or phagocytosis. In this process, pathogens are internalized and killed by secreting reactive oxygen species and hydrolytic enzymes in phagosome [17]. Other immune cells have unique roles in clearing pathogens. A leukocyte is recruited to the site of injury by binding to adhesion molecules on endothelial cells, so that leukocytes can be exited from a blood vessel once inflammatory response is triggered. The secretion of chemokines (CXCL8, CCL2) at the site of injury attracts more leukocytes binding to the endothelium. Leukocytes migrate through the endothelium to the site of injury by extravasation mediated by the integrins on leukocytes. If the innate system is evaded, the adaptive system will be triggered to act by activating T cells. Such system is antigen-specific and requires the recognition of specific pathogens in antigen presentation by MHC (major histocompatibility complex) on antigen presenting cells (APCs) [17]. 6 1.3.1.1 Leukocytes Phagocytes Macrophages, dendritic cells, and neutrophiles are important components of the innate immune system. Macrophages are phagocytes that are found in different parts of body tissues. Monocytes are immature form of macrophages in the blood, and differentiate into macrophages once they migrate into the tissues. Dendritic cells function as APCs presenting processed peptides to T cells. Immature dendritic cells migrate to the tissue from the bloodstream. Dendritic cells are both phagocytic, and macopinocytic referring as ingestion of extracellular fluid from surrounding. Once they recognize a pathogen, they become mature effector cells with increased expression of co-stimulatory molecules (which will be described in detail), and migrate to lymph nodes for subsequent immune synapse with T cells to elicit adaptive immune response. Neutrophils are another phagocytic cell, and also release anti-microbial proteins and cytokines to encounter infection. Eosinophils are believed to respond to parasitic infections. Mast Cells Mast cells are resident cells in many types of tissues. Following activation, these cells induce vascular permeability by releasing histamine, lipid inflammatory mediators such as prostaglandin D2 and leukotriene C4, and cytokines such as tumor necrotic factor-α (TNF-α), via antibody bound to Fc receptors specific for IgE and IgG [17]. Mast cells are also capable of expulsing pathogens out by triggering muscle contraction [18]. 7 Natural Killer Cells Natural killer cells (NK cells) in the innate immune system are found to be homologues in combating infection as CD8 + cytotoxic T lymphocyte (CTL). NK cells exert quick responses to infected cells as well as tumor development. NK cells can recognize stressed cells without the involvement of antibody and MHC complex. Thus, this permits a much faster immune reaction [19]. NK cells release perforin, granzymes to eliminate infected cells. Perforin forms pores in cell membrane of target cells, so that granzymes can travel to inside the cells where they induce apoptosis or osmotic cell lysis [17, 20]. Infected cells and pathogens can be combated by antibody-dependent cell mediated cytotoxicity (ADCC) which will be revealed in later section. NK cells can be activated by cytokine including IL-12, IL-15, IL-18, IL-2 and CCL5 [21]. NK cells possess activating receptors and inhibitory receptors implicated in “missing self model” and “induced self model” (Diagram 1.2). The activating receptors activate NK cells leading the release of interferon (IFN)-gamma as well as other cytokines and activation of cytotoxic activities. The inhibitory receptors identify infected cells by recognizing low level of MHC I on infected cells. Detailed description of MHC molecules will be revealed in the following section. Intracellular pathogens are evolved to down-regulate MHC I expression[22], resulting in NK cell activation. There are three kinds of interferons (IFN); interferon-alpha (IFN-alpha), interferon-beta (IFN-beta), and interferon-gamma (IFN-gamma). Interferon enhances expression of MHC class I molecules so that CTLs and NK cells can kill the infected cells by recognizing the viral peptide. 8 Diagram 1.2 NK cells and Cytotoxic T cells (CTL). NK cells are activated to kill infected cells with the release of perforin, granzymes, granulysin as implicated in induced self model and missing self theories. The induced self model indicates that activating receptors on NK cells recognize stress related ligands on the surface of infected cell. In missing self theory, NK cells attack infected cells showing reduced expression of MHC I molecules recognized by inhibitory receptors. Activated CTL recognizes the antigen : MHC I complex, and kills infected cells via the release of perforin, granulysin and granzyme. Acute Phase Response Macrophages release many different pro-inflammatory cytokines, such as IL-1beta, IL-6, IL-12, TNF-alpha and IL-8 in response to different pathogens. Chemokines are the cytokines released by variety of cells such as infected/damaged cells, epithelium and endotheilium at site of inflammation in response to infection. These chemokines act as chemoattractants for leukocytes, recruiting monocytes, neutrophils to the sites of infection. Neutrophils are the first 9 wave of cells to travel to inflammatory sites from the bloodstream. TNF-alpha, IL-1 and IL-6 are important to trigger this acute phase response. They act on hepatocytes resulting in a change in protein synthesis in which the production of acute phase protein increases [23]. One of these acute phase proteins, C-reactive protein binds to the phosphocholine of bacterial and fungal cell wall leading to the activation of complement system by binding to C1q (which will be revealed in the later sections). The second acute phase protein is mannose-binding lectin (MBL) which also triggers complement system. However, MBL also acts as an opsonin for monocytes. The pulmonary surfactant proteins (SP)-A and D are another class of acute phase protein with opsonizing properties [23]. Together, acute phase response provides with a quick immune response to a broad range of pathogens [23]. Complement System The complement system enhances antibodies and phagocytic cells to clear pathogens (Diagram 1.3). Complement system is part of innate immune system but it can be triggered by adaptive immune system as well [17, 20]. The complement system is composed of plasma proteins synthesized by the liver, and these proteins travel in the bloodstream as inactive precursors. They become active in cascade upon cleavage. There are three biochemical pathways to activate the complement system: the classical complement pathway, the alternative complement pathway, and the lectin pathway [17, 20]. The classical complement pathway requires antigen:antibody complexes for activation, whereas the alternative and lectin pathways can be activated by C3 hydrolysis or antigens the absence of antibodies [17, 20]. In all three pathways, c3-convertase activates component C3 by inducing cleavage, creating C3a and C3b. This initiates cascade of cleavage events. C3b binds to the surface of pathogens allowing 10 internalization by phagocytic cells via opsonization. The classical pathway is triggered by activation of the C1-complex. The C1-complex is composed of 1 molecule of C1q, molecules of C1r and 2 molecules of C1s. This complex forms when C1q binds to IgM or IgG in a complex with antigens. Alternatively, this complex is also formed when C1a binds directly to the pathogen. This binding leads to conformational changes in C1q molecules leading to the activation of C1r molecules which is a serine protease. They cleave C1s which is another serine protease. Then, C4 is cleaved by C1s generating C4a and C4b. C4b binds covalently to the pathogen surface. C4b then binds C2 which is cleaved by C1s to C2a and C2b forming the C4b2b complex. This complex is an active C3 convertase cleaving C3 to C3a and C3b which also binds to the microbial surface. The C3 convertase can cleave a large number of C3 molecules generating C3b molecules in which they can coat the pathogen surface. C3a is an inflammatory mediator inducing inflammatory response. The C3 convertase enzyme may bind covalently with C3b forming the C4 convertase which cleaves C5 to 5a and C5b. The C5b then recruits and assembles C6, C7, C8 and multiple C9 molecules to assemble membrane attack complex. This complex creates a hole or pore in the membrane that can kill or damage the pathogens or infected cells [17, 20]. Alternative Pathway The alternative pathway does not require pathogen binding antibodies. This pathway is mediated by spontaneous C3 hydrolysis due to the breakdown of the internal thioester bond since C3 is relatively unstable in aqueous environment [17, 20]. 11 Lectin Pathway The lectin pathway functions similar to the classical pathway. However, the opsonin, mannose-binding lectin (MBL), and ficolins are involved instead of C1q. MBL binds to mannose resides on the pathogen surface and activates the lectin pathway. Then, the MBL- associated serine proteases, MASP-1 and MASP-2, are activated which in turn convert C4 into C4a and C4b triggering the downstream cascade as in the classical pathway. MBL also acts as an opsonin for monocytes. Ficolins are found to be homologouse MBL in activating MASP [17, 20]. Lastly, activation of complement system is found to be involved in the pathogenesis of autoimmune disease including systemic small vessel vasculitis, cryoglobulinemic vaculitis, Henoch-Schonlein purpura/IgA nephropathy, and anti-neutrophil crytoplasmic autoantibody (ANCA)-associated vasculitides (AAV) [24]. 12 Diagram 1.3 The classical pathway of complement system. This pathway begins with antibody bound pathogen activates C1 complex. This complex cleaves C4 forming C4b resulting the formation of C3 convertase by binding to C2b. The C3 convertase cleaves C3 molecules forming C3b molecules. C3b can initiate opsonization by coating the pathogens. Alternatively, C3b molecules bind to C3 convertase, and cleave C5 to C5b. C5b molecules trigger the formation of membrane attack complex with the recruitment of C6, C7, C8, C9. 13 1.3.2 Adaptive System The adaptive system of the immune system is consisted of two major braches; cell- mediated immunity and humoral immunity (Diagram 1.4). In cell mediated immunity, it involves the activation of cytotoxic T cells (CTL), macrophages, NK cells and release of cytokines tailored to an antigen. Such immunity is mediated by T cells, whereas humoral immunity involves the release of antibodies mediated by B cells [17]. Diagram 1.4 Cell mediated immunity and humoral immunity. This is a diagram illustrated the interactions between these two branches of adaptive immune system. 14 1.3.2.1 T Cell Migration T cells enter the circulating bloodstream after their development in the thymus. They travel between the bloodstream and lymphoid organ for the purpose of immunosurvillience. These mature T cells are identified as naïve T cells. In lymphoid organs, naïve T cells sample MHC:peptide complexes on APCs. The pathogen-specific naïve T cells will be activated. T cells that do not respond to their specific antigens will travel via the medulla of the lymph node and back to the bloodstream via the efferent lymphatic. These cells will continue their duty of immunosurvillience. Activated T cells that recognize their specific antigen on the surface of a dendritic cell are activated to leave the lymphoid organ, and differentiate to armed effector cells. These armed effector T cells migrate to the sites of infection. Similarly, chemokines activate T cell integrins like other leukocyte homing in the migration to sites of infection. 15 1.3.2.2 T Cell Activation – MHC/Peptide Complex Diagram 1.5 T cell activation. T cell receptors recognize antigen in a MHC complex on an antigen presenting cell (APC), such as a macrophage, B cell or dendritic cell. Then, T cell will be activated in the presence of co- stimulatory molecules, and subsequently, secretes cytokines promoting proliferation and differentiation of memory cells and armed effector T cells included CD8 cytotoxic T cells and CD4 T helper cells. CD8 cytotoxic T cells kill infected cells that display peptides of pathogens bound to MHC class I molecules at the cell surface. T helper cells are differentiated to enhance the immune response by recruiting other immune cells. Together, T cell activation is one the main mediator of both cell mediated immunity and humoral immunity [25]. This image is released into public domain by National Institute of Allergy and Infectious Diseases, US Federal Government. 16 An adaptive immune response is initiated with the recognition of MHC/peptide complexes by a T cell receptor (TCR) as shown in Diagram 1.5 [25]. MHC is a cell surface molecule with high diversity. Such diversity is created by genetic recombination. The molecules present peptides to TCR. Thus, T cell activation is dependent on the MHC. High polymorphism of MHC molecules offers evolutionary benefit to encounter a wide spectrum of pathogens in a dynamic environment. MHC molecules have peptide binding groove. The amino acid sequences at the groove determine the specificity of peptides presenting to TCR. T cell activation can only be triggered when there is a complex of MHC molecules and foreign peptides via recognition by TCR. 1.3.2.3 T Cell Signaling – T Cell Receptor TCRs are associated on the T cell surface with the CD3 complex. Most T cells possess TCR composed of alpha and beta chain. In generating antigen specific TCRs, the alpha chain is manipulated by VJ recombination, whereas the beta chain is generated by V(D)J recombination. Such recombination processes involve random joining gene segments at VDJ region. Similarly, the gamma chain involves VJ recombination, and delta chain is generated by VDJ recombination. However, gamma delta T cells are a small subset of T cells with TCRs composed of gamma and delta chain. These T cells are more abundant in the gut mucosa; however they are believed in playing a role in recognizing lipid antigens [26]. Engagement of TCR induces phosphorylation of TCR associated proteins such as the zeta chain as well as a variety of adapter proteins. The phosphorylation of TCR leads to the activation of several biochemical pathways including the calcineurin pathway, the protein kinase C (PKC) pathway and the Ras- and Rac-mitogen activated protein (MAP) kinase pathways. Upon activation, 17 NFAT (Nuclear factor of activated T-cells) , a transcription factor shown to be important in the immune response, binds to regulatory sequences and increases the transcription of genes for several cytokines including IL-2 [27, 28] (Diagram 1.6). Such signal is also essential to activate activator protein (AP)-1 and NF-B that further increase IL-2 gene transcription [27]. IL-2 signaling prevents T cell anergy which is termed as an absence of immune response to foreign peptides [29]. Anergy is a state of T cells which lacks of reaction to foreign pathogens, as well as involves peripheral lymphocyte tolerance, especially, to self-antigen. Thus, anergy is important in preventing self-destruction [30]. IL-2 is the cytokine produced by activated T cells, and it drives proliferation of differentiation of other stimulated T cells. In the presence of co- stimulatory signal, it triggers T cells to enter G1 phase. It also promotes the production of IL-2 and IL-2 receptor (IL-2R, CD25). IL-2 resumes the responsive activity of T cells to antigenic stimulation by permitting cell cycle progression from G1 to S phase resulting DNA synthesis for subsequent clonal expansion [31, 32]. Thus, IL-2 serves as a pro-survival factor promoting the generation of armed effector T cells. 18 Diagram 1.6 TCR signaling induced IL-2 production. T cell receptors recognize antigen in a MHC complex on an antigen presenting cell (APC), such as a macrophage, B cell or dendritic cell. This signal pathway leads to activation of calcineurin which dephosphorylates NFAT. Consequently, NFAT translocates to the nucleus for transcription of IL-2 gene [28, 33]. This diagram is obtained from Crabtree et al., 2002 published on Cell (2002) with permission. 19 1.3.2.4 T Cell Activation – Co-stimulation T cell activation occurs via the recognition of MHC-peptide complex on APC by TCR. However, CD28 co-stimulation on T cell is required to give full activation giving an effective adaptive immune response. In the lack of CD28 co-stimulation, TCR signaling induces anergy [34, 35]. Activated APCs express B7 molecules which bind to CD28 for co-stimulation. Therefore, only specific T cells are activated to trigger immune response. CD40 ligand (CD40L) is expressed when a naïve T cell is activated. CD40 on APCs will bind to CD40L. This interaction transmits activating signals to the T cells and also activates the APCs to express B7 for further proliferation [17, 20](Diagram 1.4). 1.3.2.5 T Cell Differentiation Upon activation, naïve T cells resume their cell cycle, and they are differentiated into different subsets depending on the distinct cytokine profiles during inflammatory response. CD4 + T cells can be differentiated into T helper 1/2 cells, regulatory T cells as well as T helper 17 cells. MHC class I presents peptides to CD8 + T cells whereas MHC calss II presents peptides to CD4 + T cells. Mosmann and Coffman as well as Bottomly and her colleagues identified different subpopulations of CD4 T cells [36, 37]. CD4 and CD8 molecules are used as markers to define subsets of T cells. CD4 classifies as the helper T cells whereas CD8 classifies as the cytotoxic T cells. Naive CD8 T cells developing in the thymus are designed to become cytotoxic T cells (CTL). In CD4 + T cells, they can differentiate into either T helper cells 1 (Th1) in the presence of IL-12 mediated by the transcriptional activity of STAT1, STAT4 and T-Bet, or T helper cells 2 (Th2) upon activation in the presence of IL-4 mediated by the transcriptional activity of STAT6 and GATA3. These two subgroups of CD4 T cells differ in cytokine 20 productions as well as their cellular functions. The activation of Th1 cells leads to cell mediated immunity and production of IgG which is opsonizing antibody. They also secrete IL-2, interferon-γ (IFN-γ), and tumor necrosis factor (TNF). Th1 cells activate macrophages which destroy intracellular micro-organisms. Conversely, activation of Th2 cells triggers humoral immunity such as IgM, IgA, IgE production by driving B cells to differentiate. Th2 cells secrete IL-4, IL-5, and IL-13 which activate eosinophils and stimulate production of IgE antibody. These two responses become one side polarized once either one is initiated. Another population of CD4 + T cells is identified as T helper 17 cells (Th17) in 2003 [38]. Th17 is differentiated in the presence of transforming growth factor β1 (TGF-β1), IL-6 and IL-21. Th17 cells are found to pro-inflammatory with the secretion of IL-17. In addition, similar to Th 1, Th 17 cells are involved in cell-mediated immunity. Th 17 cells were identified to have broad implication in manifesting autoimmune disease. IL-17 functions to recruit and activate neutrophils [39]. Regulatory T cells (T reg cells) are another subset of T cell. This subpopulation of T cells regulates the immune system by maintaining tolerance to self-antigens. During development, T reg cells are selected when they receive intermediate signals in interaction with self-peptide MHC complex. T reg cells modulate the immune response by secreting immunosuppressive cytokines TGF-β and IL-10 mediated by the activation of transcription factor FoxP3 [40]. CD4+ T cells are required to activate naïve CD8 + T cells during priming [17, 20]. In this approach, both CD4 + and CD8 + T cells will respond to the same antigen. CTLs kill target cells by releasing lytic granules. Firstly, effector cytotoxic T cells bind to target cells via LFA-1 and ICAM-1 or ICAM-2 interaction. Such binding enable cytotoxic T cells to seek the presence of specific MHC:peptide complexes on the surface of target cell. Cytotoxic T cells possess lytic granules which are modified lysosomes which contain three classes of cytotoxic molecules. Perforin 21 polymerizes at the membranes of target cell resulting the formation of transmembrane pores. Another class of lytic granules is granzymes, a serine proteases, which serves as digestive enzymes trypsin and chymotrypsin. Granzymes are proteases but they cannot cleave DNA; however, they enable to cleave and activate apoptosis (described in detail in following passage) via caspase proteolytic cascasde. In turn, the cascade activates lead to DNA fragmentation. Granulysin is the third class, and this cytotoxic protein can induce apoptosis as well in target cells [17, 20]. Effector CTLs also bear Fas ligand (CD178) which is membrane-associated TNF- related molecule triggering apoptosis in target cells by binding to the receptor protein Fas (CD95) on the surface [17, 20]. T cell subtypes are shown on Diagram 1.7 [41]. 22 Diagram 1.7 T cell subtypes. CD4+ T cells differentiate into different subsets in response to cytokine profiles. IFN-γ and IL- 12 promotes the differentiation of T helper 1 cells (Th1 cells) mediated by the transcription factors of STAT1, STAT4 as well as T-bet, whereas T helper 2 cells (Th2 cells) will be differentiated in the presence of IL-2 mediated by STAT6 and GATA3. IL-23 and IL-6 promotes the differentiation of T helper 17 (Th 17 cells) cells mediated by transcription factors of RORγt and STAT3. T regulatory cells (T reg cells) are mediated by FoxP3 transcription factor [41]. This diagram is obtained from Turner et al., 2010 published on Kidney International (2010) with permission. 23 1.3.2.6 B Cell Activation B cells mediate the humoral immunity of the adaptive immune system. Immature B cells are produced in the bone marrow, and these immature B cells travel to secondary lymphoid tissues. B cell development is involved in several stages. In each stage, there is a change at antibody loci via VDJ rearrangement. An antibody is composed of two identical light (L) and two identical heavy (H) chains. The specificity of an antibody is illustrated by the “V” (variable) region and the “C” (constant) region. Pro-B cells are the earliest B lineage cells where the rearrangement of heavy chain locus takes place (Diagram 1.4). In the heavy chain “V” region, there are three segments; V, D and J. These segments recombine randomly (VDJ) to produce a unique viable domain in the immunoglobulin of each individual B cell. Similar rearrangements are also identified for “V” region of light chain, but only V and J segments are involved. And, such rearrangements initiate in the stage of development of pre-B cells. When the light chain gene arrangement is established, immature B cell is developed with expression of IgM molecule on the cell surface. When the B cell fails in any one step of the maturation process, it will die by apoptosis or clonal deletion [42]. After the selection process, the survived B cells differentiate into mature B cells as know as naïve B cells with the expression of IgD and IgM on the surface. B cells are continuously produced in bone marrow. B cell mediated immune response to foreign antigen requires the presence of an APC in combination with a B cell (Diagram 1.8 [20]). When an APC presents an antigen to a B cell, the B cell is signaled to proliferate and produce antibodies which are specific for that pathogen, and differentiated into B memory cells specific for the same antigen. If the antibodies bind to antigens on bacteria or parasites, it signals phagocytes to kill them via phagocytosis or ADCC. In ADCC, infected cells or pathogens are oponsied with antibodies which can be recognized by phagocytes or other immune 24 cells. Antibodies bind to antigen in which it can be recognized by NK cells through CD16 receptors expressing on cell surface. Such interaction activates NK cells resulting in the release of cytolytic granules and subsequently cell apoptosis as mentioned above [21]. Another important function of antibodies is to initiate the complement system. When antibodies bind to cells or bacteria, complement proteins in serum bind to the immobilized antibodies and destroy the bacteria by creating pores in them. Antibodies can also signal NK cells to kill viral or bacterial-infected cells (Diagram 1.1, 1.3, 1.4). Naïve B cells can be activated in a T cell dependent or T cell independent manner. T Cell-Dependent Activation CD4 T helper (Th) cells are activated MHC:peptide complex on APC. These Th cells produce cytokines resulting in the proliferation and differentiation to effector and memory T cells. Cytokines released by T helper cells activate B cells for production of antibodies which recognize the same foreign antigen (Diagram 1.8; [17, 20]). These cytokines trigger B cell proliferation and differentiation into plasma cells. The production of antigen-specific antibodies by B cells requires an armed CD4 T helper cells via the differentiation that recognizes the same antigen binding to the same antigen presented on B cells [20]. In T cell dependent activation, the first signal comes from antigen cross-linking the B cell receptor, and at the same time, B cells internalize pathogen and present the processed peptides to the activated Th cells to the same pathogen. The second signal comes from co-stimulation provided by these activated Th cells. Here, BCRs possess two important functions. The first one is to transducer signal upon binding to Ag. The second one is to initiate the internalization of Ag for subsequent presentation to T helper cells. Antibody production with isotype switching to IgG, IgA, IgE is termed as class 25 switch recombination. Particular B cell will usually no longer make the earlier isotypes, IgM or IgD when the switch recombination is occurred [43-45]. In conclusion, we understand that T cells are the major mediator in modulating elevated inflammatory response in many kinds of autoimmune diseases [2, 3]. Also, activation of T cells also enhances the ability of innate system in eliminating pathogens (Diagram 1.4). T Cell-Independent Activation Many antigens can activate B cells in T cell independent manner. There are two types of T cell independent activation; Type 1 and Type 2. In Type 1 activation, a B cell binds to an antigen and receives secondary activation by toll like receptors (TLRs). Type 2 activation occurs when the antigen with multiple repeating subunits which can simultaneously cross-link with high numbers of B cell receptors to fully activate B cells in the absence of T helper cells [46]. 26 Diagram 1.8 T cell-dependent B cell activation. B cell cross-links to antigen, and momentarily processes and presents the same antigen to the antigen-specific primed Th2 cell which secretes cytokines. Signals from bound antigen and Th2 cell promote B cell proliferation and differentiation into plasma cells as well as memory cells in the presence of cytokines. Production of IgG, IgA and IgE occur in reponse to the specific antigens via isotype switching. Pathogens that are bound with these antibodies will be eliminated by neutralization, opsonization via phagocytosis by macrophages, or activation of complement cascade [17, 20]. This diagram was released into public domain in which it is granted any user the right to use this work for any purpose. 27 1.4 Immune Tolerance Immune tolerance is a process that the immune system does not combat an antigen [47]. It can be either “self tolerance”, in which the body does not trigger an immune response to self- antigens, or “Ag-specific tolerance”, in which tolerance to external antigen can be created by manipulating the immune system. Genetic defects in these processes lead to Autoimmune polyendocrine syndrome type 1, immunodysregulation polyendocrinopahty enteropathy X-linked syndrome, and type 1 diabetes [48]. Central tolerance occurs during lymphocyte development in the thymus and bone marrow. Immature T and B cells which recognize self antigens are deleted [17, 20, 49]. Central tolerance involved positive selection and negative selection. In positive selection, naïve T cells that have sufficient affinity for self peptide-MHC presented by thymic APCs molecules are selected. Other naïve T cells that have no affinity to self-peptide: MHC complex will be deleted by activating apoptosis. In negative selection, T cells interact self peptide:MHC molecules with a very high affinity resulting the activation of anergy or development to T regulatory cells (T reg cells) [17, 20, 49]. Negative selection is found in B cell development. Peripheral tolerance is immunological tolerance developed to counteract the self- reactive T and B cells in periphery after development. Activation of anergy and activation induced cell death are involved in modulating these harmful lymphocytes [17, 20, 50]. 28 Ag-Specific Tolerance Induction of self-antigen or alloantigen-specific tolerance is one therapeutic approach in treating autoimmunity and preventing graft rejection, respectively. Strategies of antigen specific therapeutic tolerance induction include administration of high or low doses of autoantigen or its T cell epitope(s), antibody to TCR BV chain, TCR antagonist peptide vaccination, and DNA- encoding self-peptide. For non antigen-specific approaches, they are administration of soluble cytokine receptor or antibodies to cytokine, gene therapy by delivering regulatory protein to the site of inflammation. Antibodies reactive with specific V region families can be used to identify restricted TCR variable region usage in autoimmune disease and other immune-mediated disease [51-54]. In transplantation, the survival of cardiac allograft was prolonged by oral tolerance to immunodominant MHC donor derived synthetic class I peptide [55]. Transient antibody therapy targeted the adhesion molecule LFA-1 (CD11a) inducing donor-specific tolerance to pancreatic islet allografts in multiple donor-recipient strain combination [56]. All of these further suggest that T cell is the ultimate target in prevent manifestation of immune response in autoimmunity and graft rejection. 1.5 Drug Development and Current Immunosuppressant The discovery of small compounds which interact with disease causing molecules often yields promising therapeutic effect [57]. Thus, it is ideal to develop a small immunosuppressive agent to target a T cell proliferation-specific endogenous molecule. Therefore, theoretically, this small immunosuppressive compound gives high potency and high specificity. However, today, immunosuppressive drugs come along with extreme toxicity. The non-specificity of these drug 29 compounds impairs normal functions of other cell types. Thus, many immunosuppressants do not offer benefits in a chronic usage [58]. Cyclosporin A Cyclosporin A (CsA) is a calcineurin inhibitor, and commonly used in immunosuppressive therapy. It binds to cyclophilin as a complex inhibiting calcineurin which has a role in transcription of IL-2. During T cell activation, intracellular calcium is increased in which calcineurin is activated. Calcineurin is then able to dephosphorylates NFAT which translocates to nucleus freely for the subsequent transcription. Thus, CsA suppress cytokine production by binding to cycophilin as a complex [59]. CsA enhances the expression of TGF- that inhibits IL-2 production and the generation of cytotoxic T cells. However, CsA is highly toxic to the kidneys, and it furthers worsen the pathological conditions of any pre-existing kidney diseases. Also, TGF- has a promoting role in tumor growth. Rapamycin (mTOR Inhibitor) Rapamycin (RAPA) is another common immunosuppressant. RAPA binds to the cytosolin protein FK-binding protein 12 (FKBP12). The rapamycin-FKBP12 complex inhibits mTOR (mammalian target of rapamycin) pathway by directly binding to the mTOR complex 1 (mTORC1). The mTOR pathway has a huge implication in cell proliferation, cell growth, cell motility, cell survival, protein synthesis and transcription [60, 61]. However, RAPA is in association with the risk for decreased renal function. Lung toxicity is also associated with RAPA therapy [62]. 30 Mycophenolate Mofetil (MMF) MMF is a reversible inhibitor of inosine monophosphate dehydrogenase (IMPDH). IMPDH is an enzyme involved synthesis of purines and catalyzes the formation of guanosine nucleotides from inosine. Depletion of guanosine nucleotides by MMP suppresses T cell proliferation. It does not affect cytokine production as well as antigen recognition. However, MMF can inhibit antibody production by suppressing B cell proliferation. MMF can also down- regulates the expression of adhesion molecules on T cells. Therefore, the interaction between T cells and endothelium is weakened. However, MMF in immunosuppressive therapy also increases the risk of developing lymphoma and other infections. OKT3 Muromonab-CD3 (OKT3) is another immunosuppressant drug which primarily targets T cell proliferation [63]. It is a monoclonal antibody targeted at the CD3 receptor. However, upon binding, OKT3 induces T cell activation with cytokine production [64], but, subsequently, it induces apoptosis in these T cells [65]. Thus, the early cytokine release induced by OKT3 brings along with various adverse effects, so called cytokine release syndrome (CRS). The CRS gives skin reactions, fatigue, fever, chills, myalgia, headaches, nausea and diarrhea [66]. Lethal conditions caused by this antibody therapy include cardiac arrest [67], apnea , and non- cardiogenic pulmonary edema [68]. Therefore, an ideal immunosuppressant is always in demand in which it gives high specificity but low toxicity. 31 1.6 HF, Rapamycin and Cyclosporin A Graft transplant is one of the biggest medical breakthroughs which rescue thousands of patients’ lives with end stage organ failure. However, seeking perfect matches in HLA typing is nearly impossible. Thus, the discovery of immunosuppressive drugs makes transplantation possible by preventing graft rejection. Nevertheless, these immunosuppressive drugs do not offer long term protection of the graft as in decades [58], because they also come along with extreme toxicity resulting in graft damage as well as multiple graft failure. Physicians tend to adjust dosage to maintain the drug level falling within therapeutic range. Alternatively, adjunctive agent is added in combined drug therapy to reduce the toxicity of standard protocol. It is common to have mTOR inhibitor, rapamycin (RAPA) in conjunction with calcineurin inhibitors, such as Cyclosporin A (CsA) and/or mycophenolate mofetil (MMF) in immunosuppressive therapy. In this study, we investigated the anti-proliferative ability of HF in combination with CsA and RAPA which are common drug candidate in standard protocol. RAPA is associated with the risk for decreased renal function. CsA is highly toxic to the kidneys, and can further worsen the pathological conditions of any preexisting kidney diseases. RAPA is the backbone medication in many cocktail treatments for post-operative protocol for treating acute or chronic rejection. Thus, it is a great interest to investigate the therapeutic effect of the combined therapy of RAPA and HF in preventing T cell proliferation. In the present study, the potential of HF as an adjuvant in combination with RAPA was investigated by examining the effects of HF with RAPA on T cell proliferation. 32 1.7 Objectives of This Study Throughout an extensive understanding on immunology and development of current immunosuppressant, targeting T cell proliferation is one of ultimate goal in developing a novel immunosuppressant with high specificity and low toxicity. Halofuginone (HF) as shown in Figure 1. is a synthetic halogenated derivative of febrifugine, a natural quinazolinone alkaloid that can be found in the Chinese herb Dichroa febrifuga (Chang Shan) as shown in Figure 1.1 (bottom; obtained from Daniel Mosguin with permission [69]) [70], and has been used for treating parasite infection in veterinary medicine, such as in dairy calves to prevent cryptosporidiosis [71-73] and in poultry against coccidiosis [74, 75]. Many experimental studies have revealed that HF is a collagen type 1 synthesis inhibitor or an anti-fibrotic agent; HF inhibits TGF-β-mediated collagen type 1 synthesis in fibroblasts [76-78] and treatment with HF Figure 1.1 Chemical structure of HF (up). Dichroa febrifuga (Chang Shan) (bottom). This picture of Dichroa febrifuga was obtained from Daniel Mosguin [69] with permission. 33 prevents dermal fibroblast , liver fibrosis [79], extracellular matrix deposition in diabetic nephropathy [80], and capsular fibrosis[81]. Interestingly, It has also been demonstrated that in vitro HF inhibits human Th 17 differentiation [82] and cytokine production (i.e. TNF-, IFN-, IL-4, IL-13 and TGF-) in activated T cells [83], and treatment with HF reduces the severity of experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis [82], and delayed-type hypersensitivity (HTH) response [83]. These studies suggest the potential of HF for immunosuppression and anti-fibrosis. In this present study, we investigated the potential of HF in suppressing T cell proliferation via caspase 3 mediated apoptosis as well as autophgic cell cycle arrest in a proline dependent manner. Also, the synergistic relationship between HF and RAPA in preventing T cell proliferation is reported as well. 34 1.8 HF, Apoptosis and Cell Cycle Arrest HF has reported to suppress cell growth in the cultures of fibroblasts and several cell lines recently in association with activation of apoptosis, more probably via amino acid starvation response (AAR) [82, 83]. Apoptosis is one of the most fundamental biological processes in eukaryotes, in which an individual cell dies through the activation of an intrinsic suicidal mechanism. The morphological and physiological events of the apoptotic process include phosphatidylserine (PS) externalization, the loss of mitochondria membrane potential, the activation of caspases, PARP (poly (ADP-ribose) polymerase) cleavage, nuclear DNA fragmentation, and cellular shrinkage. Induction of apoptosis as well as proliferation arrest is an ultimate therapeutic target for developing immunosuppressants and anti-cancer agents. Here, in the following chapters, we reported the anti-proliferative ability of HF in suppressing T cell proliferation in response to either the stimulation of TCR complex or IL-2 receptor (IL-R). 1.8.1 What Is Apoptosis? Apoptosis is a programmed cell death [84]. Apoptosis is important in maintaining tissue homeostasis involving cell removal and recycling [85]. For instance, the differentiation of fingers and toes during developmental stage of human embryo involves apoptosis. Thus, the digits can be separated. Apoptosis produces cell fragments called apoptotic bodies, which can be engulfed by phagocytic cells without spreading the content to surrounding cells and causing damage while necrosis induces inflammation [86]. Thus, in contrast to necrosis, which is a form of traumatic cell death that results from acute cellular injury, apoptosis offers advantages for all organisms’ survival? However, excessive apoptosis leads to atrophy, whereas low level of apoptosis promotes uncontrolled cell proliferation or cancer. Mutation causing failure in 35 apoptosis often leads to the development, progression or metastasis of tumor [87]. Apoptosis undergoes three phases; activation, execution, and death. Activation Phase Intrinsic Pathway Apoptosis can be initiated by either an intrinsic pathway or extrinsic pathway. The intrinsic pathway occurs in response to the signal originating from the inside of the cells. When cells are exposed to stress from the inside, this triggers the intrinsic pathway. The causes of that include heat, radiation, nutrient depletion, viral infection, hypoxia and increased calcium concentration [88, 89]. A damaged cell can produce intracellular apoptotic signals. The p53 protein is a sensor of cellular stress activating the intrinsic pathway. The DNA checkpoints proteins, ATM (Ataxia elangiectasia mutated protein), and Chk2 (checkpoints factor 2) directly phosphorylate and stabilize p53. They also suppress MDM2 (Mouse double minute 2 homolog) activity that inactivates p53. Once activated, p53 initiates apoptosis by activating transcription of pro-apoptotic Bcl2 family members but repressing expressions of anti-apoptotic Bcl2 proteins and cellular inhibitor of apoptosis proteins (cIAPs). Other p53 targets include BAX, Noxa, PUMA (p53 up-regulated modulator of apoptosis) and BID. Protein p53 may transcribe other genes including PTEN (phosphatase and tensin homolog), Perp, p53AIP1 (p53 regulated apoptosis inducing protein 1), and genes that lead to increase in reactive oxygen species (ROS). These genes are related to the activation of apoptosis. The increased level of ROS produces oxidative damage to all mitochondrial components [90]. Intrinsic induced apoptosis converges the suicidal signal at mitochondria resulting increased mitochondrial permeability (MMP) and subsequent cytochrome c release. Once 36 cytochrome c is released it binds with apoptotic protease activating factor -1 (Apaf-1) and ATP which then bind to pro-caspase 9 to create a protein complex known as an apoptosome. The apoptosme cleaves the pro-caspase 9 to its acive form of caspase 9, which in turn activates the effector caspase 3 [91, 92]. Caspase is a group of cysteine proteases involving the execution of apoptosis, especially mediated by caspase 3. However, the release of cytochrome c is regulated by Bcl2 family proteins. Bcl2L (Bcl2-Like), BclXL (Bcl2 related protein long isoform) and other anti-apoptotic Bcl2 family members reside in the outer mitochondrial membrane and prevent cytochrome c release. Conversely, activated BAX (released from 14-3-3), BID and BIM translocate to mitochondria to induce apoptosis either by forming pores in mitochondria directly or by binding via BH3 domains to Bcl2. Thus, MMP is a pivotal event in the progression of apoptosis [93]. Mitochondrial proteins known as SMACs (small mitochondria-derived activator of caspases) are released into the cytosol when MMP increases. SMAC binds to the inhibitor of apoptosis proteins (IAPs) and deactivates them. IAP also normally suppresses the activity of caspases, thereby arrests apoptosis [94]. The pathway inducing apoptosis is illustrated in part in Diagram 1.9 [19]. 37 Diagram 1.9 Pathway to p53-dependent apoptosis. This is a schematic diagram of the p53 induced apoptosis with activation of BAX, PUMA and APAF-1. Nuclear p53 induces the expression of PUMA which releases inactive form of p53 residing in cytoplasm. Cytosolic p53 which is in active form induces Bax oligomerization and mitochondrial translocation. Bax oligomerization antagonizes the anti-apoptotic roles of Bcl-2. These lead to disruption of mitochondrial membranes with subsequent release of cytochrome c. Consequently, cytochrome c binds to Apaf1 leading to the formation of apoptosome as in a complex with caspase-9. Apoptosome in turn triggers the execution stage of apoptosis by activating caspase 3 [19]. 38 Extrinsic Pathway The extrinsic pathway occurs when cells expose stress from the environment include toxins, hormone, nitric oxide or granzyme secreted by cytotoxic T cells [95, 96]. These death molecules can transmit the signal via receptor-mediated pathways. These death receptors activate caspase molecules. These receptors belong to the super family of TNFR (tumor necrosis factor receptor) with a cys-rich extracellular domain and a homologous intracellular domain known as the death domain. Adapter molecules like FADD (Fas-associated via death domain), TRADD (Tumor necrosis factor receptor-1-associated death domain) or Daxx contain death domains so that they can interact with the receptor and transmit the apoptotic signal to the death machinery. The well-studied receptors inducing apoptosis include Fas, TNRF1[97]. FasL acts as ligand for Fas. It leads to oligomerization at the receptor upon binding. The binding between FADD, Fas and pro-caspase 8 forms a complex known as the DISC (death inducing signaling complex). Pro-caspase 8 is then drives itself activation via self-cleavage. Active caspase 8 subsequently cleave caspase 3 and 7 which are the execution stage of apoptosis [98]. In TNFR1 mediated apoptosis, different death domain containing proteins can form distinct complex (complex 1) when the receptor is bound and activated. The complex is composed of TRADD, TRAF2, CIAP1 (cellular inhibitor of apoptosis 1) and the kinase RIP1 (receptor interacting protein 1) assemble at the plasma membrane which recruit IKK (I kappaB kinase) leading to NF-B activation and the subsequent cell survival. The second complex (complex 2) is formed when TRADD complex dissociates from the receptor and recruits FADD and pro-casapse8. The balance of complex 1 and complex 2 is determined by FLIP expression. Adequate FLIP expression inhibits caspase 8 in complex 2. Thus, complex 2 can mediate cell 39 death when complex 1-induced NF-B activation is insufficient [99]. BAD (Bcl2-antagonist of cell death) is a proapoptotic member of Bcl2 family which can be activated by Akt pathway [100]. Execution Phase and Death Phase Both extrinsic and intrinsic pathways lead to cleavage of caspase proteins that plays a role in executing the apoptotic signals. Initiator caspases (i.e. caspase 8, 9) as mentioned above are activated by binding to specific oligomeric adaptor protein, followed by the activation of effector caspases (i.e. caspase 3, 7) by these active initiator caspases through proteolytic cleavage. These active effector caspases then degrade intracellular proteins as well as organelles to execute the apoptotic cell death. Therefore, the expression of caspase 3, 7 is the final execution step in mediating apoptotic pathway resulting nuclear fragmentation [101]. DNA fragmentation associated with apoptosis is induced by the DNA fragmentation factor (DFF). DFF is composed of two protein subunits, a 40 kDa caspase activated nuclease (DFF40/CAD), and its 45 kDa inhibior (DFF45/ICAD). The cleavage of DFF45/ICAD by caspase 3 releases DFF40/CAD from its inhibitor leading to the induction of nuclease activity, nuclear condensation and DNA fragmentation [102]. The activated caspase 3 and/or caspase 7 cleave poly-(ADP-ribose) (PARP) to an 89-kDa fragment leading to induction of DNA fragmentation resulting in cell death. PARP is a nuclear DNA binding protein, and it involves DNA repair. PARP inhibits the pro-apoptotic Ca 2+ /Mg 2+ endonuclease, and protects cells against apoptosis [103]. 40 1.8.2 What Is Cell Cycle? Cell cycle is series of intracellular events that a cell produces daughter cells via duplication and replication [104]. In eukaryotes, the cell cycle is consisted of interphase and mitosis. The interphase is that the cell grows and prepares for mitosis with duplication of DNA material, whereas mitosis (M) phase where the cell splits itself into distinct cells called daughter cells. The interphase is divided into four distinct phases: G0/G1, S, M and G2 phases. For the proper progression, each phase has to be completed prior the activation of the subsequent phase. Therefore, suppression of significant biosynthesis in any of distinct phases can prevent cells from undergoing cell cycle progression. In G0 phase or quiescence state, cells are in the state of resting in which they are temporarily not dividing stopped dividing. In G1 phase, activated cells grow in size, and prepare for DNA synthesis in S phase. DNA replication takes place in S phase, and the chromosomes will be duplicated before reaching G2 phase. G2 is the phase between S phase and M phase. In G2 phase, the cell will expand continuingly for subsequent dividing processes taken place in M phase. The G2 checkpoint mechanisms regulate the proper production of microtubules which are required for mitosis. Proper cell cycle progression regulated by various control mechanisms determines the survival of a cell. Cyclin-dependent kinases (cdks) are the important mediators for cell cycle progression by monitoring the initiation, progression, and completion of cell cycle events, so that the cell cycle progression can be maintained in an ordered and directional manner. The activity of the cdks is determined by their interactions with cyclins and cdk inhibitors via phosphorylation and by ubiquitin-mediated proteolysis. Thus, suppression of the activities of cdks becomes therapeutic targets in treating various cancers by inducing cell cycle arrest. Many anti-tumor agents as well as immunosuppressant act in the cell cycle machinery [105]. 41 Accumulating evidences indicate that HF is a potential immunosuppressant in inducing apoptosis in activated T cells as well as various cell lines [83, 106]. HF links to activation of amino acid starvation response (AAR) [82], and such response leads to induction of cell cycle arrest[107]. In this chapter, we investigate the pro-apoptotic activity of HF in suppressing antigenic proliferation of T cells in response to TCR and IL-2 signaling in association with induction of cell cycle arrest. In our experiments, we found that HF suppressed antigenic T cell proliferation via apoptosis as well as cell cycle arrest in a dose response manner via AAR. 1.9 HF and Protein Synthesis Very recently, HF is demonstrated to inhibit protein synthesis via binding to proyl-tRNA synethetase in vitro [108]. Proyl-tRNA synthetase is one of aminoacyl-tRNA synthetases. This enzyme catalyzes the reaction in charging tRNA with proline which is important in the elongation process during protein synthesis. Proline is a source of glutamate, arginine, and tricarboxylin acid cycle intermediates, so proline is essential for protein synthesis. This amino acid also involves a metabolic cycle that shuttles redox equivalents between mitochondria and cytosol [109]. Proline containing regions of some tertiary protein structure are important in protein to protein interactions as well as the processes of recognition and phosphorylation [110]. Increasing evidences suggest that HF activity is regulated by proline as evident in our study as well as previous studies. The proline supplement is able to remove the suppressive activity of HF on protein synthesis in vitro [108]. Blocking protein synthesis is one target in developing antibiotics. Some antibiotics such as anisomycin, cycloheximde or tetracycline function to suppress protein synthesis. In this chapter, we investigated the potential of HF in blocking protein synthesis resulting apoptosis demonstrated with the addition of the proline supplement in 42 culture medium. Thus, this present study uncovers the mechanism of action of HF at least in part in immunosuppression. 43 The Physiology of Protein Synthesis Diagram 1.10 The physiology of protein synthesis. Translation refers as the synthesis of proteins. Translation is carried in four phases: activation, initiation, elongation, and termination. In activation, the correct amino acid is joined to the correct transfer RNA molecule (tRNA). The amino acid is joined byits carboxyl group to the 3’OH of the tRNA by an ester bond. When the tRNA has an amino acid linked to it, it is termed “charged”. Initiation involves the small subunit of the ribosome binding to 5’ end of mRNA with the help of initiation factors (IF), other proteins that assist the process. Elongation initiates when the next aminoacyl-tRNA (charged tRNA) binds to the ribosome with GTP and an elongation factor. Termination of the synthesized protein chain occurs when the A site of the ribosome binds to a stop codon (UAA, UAG, or UGA). Then, releasing factor can recognize nonsense codons releasing the polypeptide chain [111]. This diagram was obtained from Khade et al., 2010 published on Transfer RNA with permission. The synthesis of proteins is known as translation. In translation, mRNA is decoded to produce corresponded proteins specified by the trinucleotide genetic codes appeared on mRNA. Translation occurs in the cytoplasm, where the ribosomes are located. Ribosome is a protein making machinery that is composed of a small and large subunit. There are four phases in translation: activation, initiation, elongation, and termination [112, 113] ( Diagram 1.10; [111]). 44 Activation and Initiation Phase In activation, the carboxyl group of specific amino acid is bound to the 3’ OH of corresponded tRNA via an ester bond. The linked tRNA is also known as charged tRNA. The codon on mRNA is recognized by the anticodon on a tRNA molecule. In the phase of initation, the initiator tRNA bound with the amino acid methionine is first tRNA incorporated into the small ribosomal subunit with the help of translation initiation factors. The charged initiator tRNA binds to P site of the small ribosome subunit. Then, 5’ end of an mRNA is recognized and captured by the loaded ribosome subunit. When AUG (start codon) is recognized by charged initiator tRNA, initiation factors are dissociated where the large ribosomal subunit assemble to form the full active form of ribosome. Now, the addition of the next charged tRNA is set to incorporate along the chain [113]. Elongation Phase Elongation involves the next charged tRNA binds to ribosome at A site in the presence of GTP and elongation factor. The free amino group of the amino acid linked to the tRNA at the A site joins to the carboxyl end of the growing polypeptide chain dissociated from the tRNA at the P site. Protein synthesis is catalyzed by a peptidyl transferase in the ribosome. The peptidyl transferase reaction involves in shifting the large subunit relative to the small subunit holding the mRNA. This moves tRNAs into the E and P sites of the large subunits from P and A sites in the small subunit. Then charged tRNA at E site with the amino acid joining to the polypeptide chain dissociates from the ribosome. In next step, the small subunit moves along the mRNA molecule, and resumes its original position relative to the large subunit. The cycle repeats until a stop codon is encountered [113]. 45 Termination Phase Termination of protein synthesis is triggered when the A site of the ribosome reaches a stop codon (UAA, UAG or UGA) on mRNA. Only releasing factor can recognize the stop codon resulting in the release of polypeptide chain. The releasing factor at A site catalyzes the addition of a water molecule to the peptidyl tRNA at P site. This reaction releases the polypeptide chain from tRNA molecule into the cytoplasm. The two subunits of the ribosome separate when synthesis of the protein is finished. The newly synthesized polypeptide chains can be folded to native secondary and tertiary structures [113]. In this study, we investigated whether the pro-apoptotic activity of HF initiated via suppression of protein synthesis by uncovering the potential role of proline in reversing the pro-apoptotic activity of HF. Our results indicated that the addition proline was demonstrated to resume the proliferation in HF treated T cells. 46 Chapter 2: Material and Method This chapter illustrates the detailed procedures of all experiments performed in this study, as well as the detailed information of each reagent used in this study. 2.1 Cells and Reagents In this thesis work, a single cell suspension of splenocytes was used to study anti- proliferation of HF. This cell suspension was prepared by gently smashing the spleens of mice with phosphate buffered saline (PBS) in a Cell strainer (BD – Canada, Mississauga, ON, Canada), followed by removal of erythrocytes using lysis buffer (0.15M NH4Cl, 1.0 mM KHCO3, 0.1 mM EDTA, pH 6.8). After washed with PBS again, splenocytes were resuspended in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 100 U/mL penicillin/streptomycin (complete RPMI medium), and grown at 37°C in a humidified atmosphere of 5% CO2. HF was purchased from the Toronto Research Chemicals Inc. (Catalogue number: H102500, Toronto, ON, Canada). Anti-mouse anti-CD3 antibody was purified from anti-mouse CD3 hybridoma ascite [114]. Recombinant mouse IL-2 (rmIL-2) was purchased from BD Pharmingen (San Diego, CA, USA). L-Proline (proline) was obtained from the Life Technologies Inc. (Burlington, ON, Canada). 3-methyladenine (3-MA) was obtained from Sigma-Aldrich Canada (Catalog #: M9281). RAPA was purchased from Cayman Chemical Company (Catalogue number: 13346, Ann Arbor, Michigan, USA). CsA was purchased from Novartis Pharmaceutical Canada (Catalogue number: 486200, Quebec, Canada). 47 2.2 Preparation of T Cell Receptor-Stimulated Naïve T Cells Naïve T cells were activated by either anti-CD3 antibody or alloantigens in one way mixed lymphocyte reaction (MLR). Anti-CD3 antibody stimulated T cell cultures were naïve splenocytes, 2 105 cells in 100 µL of RPMI complete medium per well in 96-well microplates, incubated with anti-CD3 antibody (2 µg/mL) for 48 hrs. This time point was able to demonstrate apoptotic events induced by HF. MLR was employed to explore the effects of HF on antigenic stimulated T cell proliferation. MLR was a mixed culture of splenocytes (2 x 10 5 /well) from BALB/c mice (H-2 d haplotype) with mitomycin C (Sigma-Aldrich Canada, Oakville, ON, Canada)-pretreated splenocytes (1 x 10 5 /well) from C57BL/6j (B6, H-2 b haplotype) mice in 96- well U-bottom microculture plates (Corning Inc, Corning, NY). The splenocytes from B6 mice as allogeneic stimulators were prepared by pretreated the cells (1 × 10 6 cells/ml) with 50 μg/ml mitomycin C at 37°C for 30 min, followed by extensively washed with PBS. The controls for basal levels of responder proliferation (splenocytes from BALB/c mice) were the cultures without stimulator cells. Cultures were maintained in RPMI-1640 complete medium for 48 hrs at 37°C in 5% CO2. The T cell proliferation both in anti-CD3 antibody stimulated cultures and MLR as well as IL-2 stimulated cultures was quantitatively measured using methylthiazol tetrazolium (MTT) assay as described below. 2.3 Preparation of IL-2 Stimulated Activated T Cells In order to examine HF anti-proliferation in IL-2 dependent T proliferation, splenocytes were harvested from naïve B6 mice and were cultured in the presence of 4 µg/mL of Concanavalin (Con) A (Sigma-Aldrich Canada) in complete RPMI 1640 medium at 37 o C in 5% CO2. After 3 days of incubation, cells were harvested and maintained in RPMI 1640 medium 48 containing low serum (0.5% FBS) for 18 hrs in order to synchronize T cells to G1 phase of the cell cycle. The purpose of synchronization is to have all the cells yielding the same cellular response toward stimulation and treatment. These cells were then activated by exogenous IL-2 for 48 hours. This time point was able to demonstrate apoptotic events induced by HF. The resting T cells were used as activated T cells after dead cells were removed by a gradient centrifugation. 2.4 Determination of Cell Proliferation and Growth Cell proliferation or an increase in viable cell number in cultured splenocytes or activated T cells was measured using MTT assay. In brief, 10 µL of 0.5 mg/mL of 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich Canada) were added to each well (100 µL of cell culture) and incubated at 37 o C for 4 hrs. The mitochondria of MTT treated living cells reduce MTT into insoluble, dark purple formazan crystals. Thus, active mitochondrial activity determines the viability of cells. The formazan crystals in viable cells were then dissolved in 100 µL/well of dimethyl sulfoxide (DMSO, Sigma-Aldrich Canada). The absorbance of the color in each well was quantified as absorbance unit (AU) at 560 nm wavelength using ELx808 Ultra Microplate Reader (BioTek, Winooski, VT, USA). The percentage of inhibition in cell proliferation in drug-treated cultures against nondrug-treated control was calculated as follows: Inhibition (%) = (Drug-treated - Control)/Control ×100%. However, one limitation of MTT assay is that it cannot reflect the mechanisms causing anti- proliferation. Thus, determination of apoptosis by flow cytometry was performed in this study. 49 2.5 Determination of Apoptosis by A Flow Cytometry Apoptosis of splenocytes or T cells in cultures was measured by fluorescence-activated cell sorter (FACS) analysis following the manufacturer’s protocol (BD Biosciences, Mississauga, ON, Canada), in which Annexin-V conjugated with phycoerythrin (Annexin-V-PE) staining was for early apoptosis and 7 amino-actinomycin D (7-AAD) for late apoptosis. Annexin-V is a phospholipid binding protein that binds to PS. In normal cells, PS is located at the cytosolic side of the plasma membrane, whereas early apoptotic cells allow PS translocating to extracellular membrane. 7AAD binds strongly with DNA molecules with a high affinity for GC-regions [115]. Annexin V bond cytosolic PS paired with 7AAD are able to detect fragmented cells in late apoptosis. Briefly, splenocytes or T cells (1  106 cells/well in 1 mL of complete RPMI medium) in 24-well plates were treated with the indicated concentrations of HF in complete RPMI medium for 48 hrs, followed by stained with Annexin-PE and 7-AAD for 15 min in dark. This time point was able to demonstrate apoptotic events induced by HF. The proportions of cell population undergoing early apoptosis and/or late apoptosis were quantified by flow cytometry and analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA). 2.6 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) IL-2 is one of the important cytokines promoting T cell growth [116]. It is interesting to investigate the effects of HF on IL-2 production. The levels of IL-2 transcript were determined using RT-PCR kits following the provided protocol (Invitrogen, Carlsbad, CA, USA). Briefly, splenocytes (1  106 cells/well) in 24-well plates were stimulated with 2 µg/mL anti-CD3 antibody in the presence or absence of HF for 48 hrs (to be consistent with the time points studying HF anti-proliferation), followed by total RNA extraction using TRIzol Reagent 50 (Invitrogen), and 5 µg of total RNA was used for cDNA synthesis in reverse transcription, followed by PCR amplification with appropriate cycle number and annealing temperature using specific primers (IL-2: sense, 5’-AAC CTG AAA CTC CCC AGG AT, and antisense, 5’-TCC ACC ACA GTT GCT GAC TC; internal control glyseraldehyde-3-phosphate dehydrogenase [GAPDH]: sense, 5’-ATC ACT GCC ACC CAG AAG ACT G, and antisense, 5’-CCC TGT TGC TGT AGC CGT ATT C). All the PCR products were visualized in 1% agarose in tris- acetate-EDTA (TAE) buffer containing 0.5 µg/ml of ethidium bromide, and the expression levels (the size and density of the band) of IL-2 transcripts were measured using a densitometry, and were presented as ratio units (RU) after normalized with GAPDH transcript from the same RT product. 2.7 Enzyme-Linked Immunosorbent Assay (ELISA) Secreted levels of IL-2 in culture supernatants were measured using Mouse IL-2 ELISA kits according to the manufacturer's instruction (eBioscience, San Diego, CA, USA). Briefly, splenocytes (1  106 cells/well) in 24-well plates were stimulated with 2 µg/mL anti-CD3 antibody in the presence or absence of HF for 48 hrs (to be consistent with the time points studying HF anti-proliferation), and the culture supernatants were collected by centrifugation. The total amount of secreted IL-2 at each indicated concentrations of HF was normalized by its number of total viable cells. Data were presented as the amount of total IL-2 protein per million cells. 51 2.8 Determination of Cell Cycle Arrest by A Flow Cytometry. Cell cycle sorting of splenocytes or T cells in cultures was measured by FACS analysis. In brief, naïve T cells (1x10 6 cells/ml in 1 mL of complete RMPI medium) in 24 well plates were stimulated with 2 µg/mL anti-CD3 antibody, and treated with in the indicated concentrations of HF. After 24 hrs, cells were centrifuged, and subsequently, supernatant was discarded. The time point as 24 hrs was employed instead of using 48 hrs because it was believed that cell cycle arrest was induced prior the activation of apoptosis in HF treated T cell cultures. Cell pellets were re-suspended in ice-cold 70% ethanol and stored at 4ºc for 4 hrs or more. Fixed cells were then washed with PBS at room temperature. Washed cells were incubated at 37ºc in 50µl of PBS and RNaseA at final concentration of 2 mg/ml for 30 minutes, followed by DNA staining with propidium iodide (PI) in 50µl of PBS at final concentration of 0.2 mg/ml for 30 min at 37ºc. Stained cells were treated with 250 µl of PBS followed by flow cytometric analysis. However, cell cycle analysis via PI staining by a flow cytomtry cannot accurately determine whether proliferation arrest occurs at S phase or S/G2 phase transition. Thus, the use of BrdU staining assay in future study can further determine the anti-proliferative ability of HF specifically in S phase arrest by measuring DNA synthesis in HF-treated T cells. 2.9 Proline Measurement The concentrations of proline in culture supernatants were quantitatively measured using a high-performance liquid chromatography (HPLC) as described previously [117]. In brief, 1  10 6 cells/well in 1 mL of complete RPMI medium in 24-well plates were activated by anti-CD3 antibody in the absence or presence of HF. After 6 or 24 hrs of treatment, 50 µL of culture supernatant sample was mixed with 150 µL of acetonitrile, followed by the incubation with 52 coupling reagent (TEA/distilled water/phenyl isothiocyanate/methanol: vol/vol ratio of 1:1:1:7) for 20 min at room temperature. The mixture solution was dried under vacuum at room temperature using a Centrivap Centrifugal Vacuum Concentrator (Labconco, Kansas City, MO, USA). The dried samples were re-dissolved in 100 µL of 10 mM ammonium acetate, and pelleted the undissolved particles by centrifugation. The proline-pheny isothiocyanate in the supernatants was then quantified by Waters Acquity HPLC compared with proline standard (10 µg/mL in water). The concentration of proline in culture supernatant was normalized by the number of viable cells in each sample. Proline measurement was performed at earlier time points because it was believed that intracellular proline depletion was the pro-apoptotic signal. 2.10 Western Blot The cleaved form of poly ADP ribose polymerase (PARP) (markers of apoptosis), caspase 3, phosphorylated form STAT5 (intracellular signaling of IL-2) as well as light chain 3 (LC3) I/II were examined by Western blot analysis. Briefly, splenocytes or T cells (10  106 cells/well) in 6-well plates were stimulated with 2 µg/mL of anti-CD3 antibody or rmIL-2 (1 ng/mL) in the presence or absence of HF. After indicated periods of treatment, the total protein was extracted by a brief sonication of cell suspension in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS) containing protease inhibitor cocktail (Roche, Mannheim, Germany). The protein content in each sample was determined by the Bradford assay using the Bio-Rad dye reagent compared to bovine serum albumin (BSA) as standards. The proteins in extracts were denatured by boiling with Laemmli sample buffer for 5 min. Equal amount of protein (100 to 150 µg) from each sample was fractioned by15% or 7% SDS-polyacrylamide gel electrophrosis (SDS-PAGE) and transferred to 53 nitrocellulose membranes (Bio-Rad Lab, Hercules, CA, USA), that were then blocked with 5% skim milk in TBST (10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.1% Tween-20) for 1 hr, and then probed with anti-cleaved PARP (Cell Signaling Technology) or anti-cleaved caspase 3 (Cell Signaling Technology) or anti-phospho-STAT5 (anti-p-STAT5) antibody (Cell Signaling Technology) or LC3 I/II (Cell Signaling Technology) in TBST containing 2.5% of milk at 4 o C overnight. The membranes were then washed with TBST and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies or alexa fluor 680 secondary antibodies for 60 min. Proteins on the blot were visualized by an enhanced chemiluminescence assay (ECL, Amersham Pharmacia Biotech, Buckinghamshire, England). Alternatively, proteins on blots were scanned and visualized by Odyssey infrared imaging system (LI-COR, USA). The blots of cleaved PARP, LC3 I/II were re-probed with anti-β-actin IgG antibody (Sigma-Aldrich Canada) to confirm equal protein loading in each sample, or of p-STAT5 with anti-STAT5 (Cell Signaling Technology) to show the total STAT5 in each sample. The expression levels were measured using a densitometry, and were presented as RU after normalized with β-actin or STAT5 from the same samples. 2.11 TUNEL Assay The TUNEL (terminal deoxynucleotidyl transferasemediated dUTP-biotin nick end- labeling) assay was performed for the detection of DNA fragmentation. In brief, splenocytes (1  106 cells/well) in 24-well plates were stimulated with 2 µg/mL anti-CD3 antibody in the presence or absence of HF for 24 hrs. This time point was able to demonstrate apoptotic events induced by HF. These cells were fixed in 1% paraformaldehyde and then treated with ice-cold 70% ethanol. Apoptotic cells with DNA fragmentation were labeled at the terminal end of 54 nucleic acids by FITC-tagged dUTPs (eBioscience). The intensity of fluorescent of cell population with DNA fragmentation was quantified by a flow cytometry and analyzed using FlowJo software (Tree Star Inc.). 2.12 Determination of Synergistic Relationship Between HF and Rapamycine To determine the synergistic/antagonistic relationship between HF and conventional immunosuppressants (i.e. RAPA and CsA), Loewe’s equation was employed [118]. As the equation stated (Table 3.1), D1 and D2 are the concentrations of drugs to result in χ % inhibition of each respective drug alone, d1 and d2 are concentration of each drug in the mixture that yield χ % inhibition. The Loewe additivity model is derived from the use of isobologram approach which is a graphical tool to assess drug interaction. Isobologram is manipulated by experimental dose data which are used to draw a contour. Such contour is a function of concentrations of both drugs, and the chosen inhibition level. Loewe’s additivity (D1/D2 = 1) gives contours that are straight lines connecting the concentrations of two drugs in isobologram. Synergy will be identified when the data contour falls below the additivity line, whereas antagonism occurs when the data contour falls above the additivity line. Thus, as Loewe additivity model implied, the computed value of > 1 indicates the antagonistic relationship between agents while the value of < 1 indicates synergistic relationship. The values of IC50 of HF (Fig 3.14) or/and RAPA (Fig 3.13) or/and CsA (Fig 3.14 and Suppl Fig 6) collected from the calculated inhibition rate from MTT assay were plugged into the equation. 55 2.13 Statistical Analysis Data were presented as mean  standard derivation (SD) of separate experiments (at least three experiments if results were consistent). Statistical analysis of difference between groups was performed by t-test (two-tailed distribution) or analysis of variance (ANOVA) as indicated in the text. A p value of  0.05 was considered statistically significant. 56 Chapter 3: Results This chapter presents data collected all experiments performed in this study. 3.1 HF Induced Anti-proliferation In T Cells. This section presents data revealing the anti-proliferative ability of HF in T cell proliferation in response to either TCR or IL-2 stimulation. 3.1.1 The Effect of HF on T Cell Proliferative Response to the Stimulation of T Cell Receptor-CD3 Complex or IL-2 Receptor Naïve T cells were activated by the signal from T cell receptor (TCR)-CD3 complex for the initiation of T cell immune response, whereas activated T cells were expanded by the stimulation with IL-2, a common cytokine for T cell growth. The anti-T cell proliferation of HF was tested in both naïve and activated T cells. As shown in Fig. 3.1A, addition of HF to anti- CD3 antibody-stimulated splenocytes suppressed T cell proliferation in a dose-dependent manner, indicated by the inhibition of 14.91  8.31% in cultures with 0.625 nM of HF, 28.86  1.28% with 1.25 nM of HF, 57.92  2.30% with 2.5 nM of HF and 85.14  0.60% with 5 nM of HF (IC50, 2 nM; P < 0.0001, one-way ANOVA, n = 3). Similar inhibition of HF was seen in MLR (Fig. 3.1B) that showed 12.51  6.66% of inhibition with 0.625 nM of HF, 39.15  7.3% with 1.25 nM of HF, 49.97  5.46% with 2.5 nM of HF and 63.71  1.73% with 5 nM of HF (IC50, 2.5 nM; P < 0.0001, one-way ANOVA, n = 5). The anti-T cell proliferation of HF was also tested in activated T cells in response to IL-2 stimulation. As shown in Fig. 3.1C, HF inhibited IL-2-mediated cell proliferation in activated T cells but was less effective compared to those in 57 naïve T cells, indicated by the inhibition of 18.28  13.72% in cultures with 5 nM of HF, 36.21  25.17% with 10 nM of HF, 65.74  12.2% with 20 nM of HF and 81.9  18.65% with 80 nM of HF (IC50, 16 nM; P < 0.0001, one-way ANOVA, n = 5). Figure 3.1A Figure 3.1B Figure 3.1C Figure 3.1 HF induces anti-proliferation in response to T cell receptor complex and IL-2R. T cell proliferation in response to the stimulation of TCR complex or IL-2R was measured by MTT assay. (A) Anti-CD3 antibody-stimulated naïve T cells. Naïve splenocytes were stimulated with anti-CD3 antibody in the presence or absence of HF for 48 hrs. Data are 58 presented as mean ± SD of three separate experiments. (B) MLR. A mixed culture of splenocytes from BALB/c mice and B6 mice was incubated in the presence or absence of HF for 48 hrs. Data are presented as mean ± SD of five separate experiments. (C) IL-2 stimulated activated T cells. Activated T cells were stimulated with IL-2 in the presence or absence of HF for 48 hrs. Data are presented as mean ± SD of five separate experiments. 3.1.2 The Effect of HF on Anti-CD3 Antibody-Stimulated IL-2 Production in Naïve Splenocytes IL-2 is a key cytokine for the promotion of T cell growth after activation by antigen-TCR stimulation [116], and HF has been reported to inhibit pro-inflammatory cytokine (i.e. IFN- TNF-) production via blocking NF-κB translocation and p38 MAPK phosphorylation in peripheral blood T cell [83]. The expression of both IL-2 mRNA and protein in anti-CD3 antibody-stimulated naïve splenocytes in the presence of HF was examined. As shown in Fig. 3.2 (left), IL-2 mRNA was significantly up-regulated by the stimulation with anti-CD3 antibody in the absence of HF (RU: 0.7) as compared to the basal level (RU: 0.5) in cultures without the antibody stimulation, but was not significantly affected by the addition of HF, indicated by the RU value of 0.79 in cultures with 0.625 nM of HF, 0.82 with 1.25 nM of HF, 0.63 with 2.5 nM of HF and 0.61 with 5 nM of HF. The same results were seen in the measurement of secreted IL- 2 protein in culture supernatants (Fig. 3.2. (right)); the level of IL-2 production per million viable cells was not significantly attenuated by the treatment with HF. These data suggest that HF does not attenuate IL-2 production in its effect on T cell growth following activation. 59 Figure 3.2 HF doesn’t attenuate IL-2 production in anti-CD3 stimulated T cells as shown in PCR assay and ELISA assay. Naïve splenocytes were stimulated with anti-CD3 antibody in the presence or absence of HF for 48 hrs. (Left) The levels of IL-2 mRNA in HF-treated or untreated cultures were determined using RT-PCR assay. Data are a representative of three separate experiments. (Right) The secreted IL-2 in HF-treated or untreated cultures was measured by ELISA assay. 3.1.3 The Effect of HF on IL-2 Receptor Signaling in Activated Splenocytes We next explored the biological effect of HF on T cells in response to IL-2 by examining the expression level of phosphorylated STAT5 (p-STAT5) via Western Blot analysis. The JAK/STAT5 pathway is one of the important pathways mediating T cell proliferation in response to IL-2 signaling [116, 119-121]. As shown in Fig. 3.3, the expression of p-STAT5 in IL-2 stimulated cultures was not significantly affected by the presence of HF; p-STAT5 was upregulated by the stimulation with IL-2 in the absence of HF (RU: 0.51) as compared to the basal level (RU: 0.06). The addition of HF did not change the expression of p-STAT5 significantly, indicated by the RU value of in cultures with HF, 0.51 with 10 nM of HF, 0.36 with 20 nM of HF, 0.55 with 40 nM of HF, and 0.60 with 80 nM of HF (P = 0.408, one-way ANOVA), while the expressions of total STAT5 were not different in all of samples. These data indicate that HF did not affect IL-2-mediated JAK/STAT5 signaling cascade in T cells. 60 Figure 3.3 HF exerted no effect on IL-2 dependent phosphorylation of STAT5 signaling in activated T cells. Activated T cells were stimulated with IL-2 in the presence or absence of HF for 48 hrs. The phosphorylation of STAT5 in protein extracts was analyzed using Western blot. Data are a representative of two separate experiments. 3.1.4 The Effect of HF on Cell Apoptosis in Both Naïve and Activated T Cells Earlier studies have showed that HF induces apoptosis in acute promyelocytic leukemia (APL) cell lines [76, 106] as well as in activated T cells [83]. Thus, it is possible that HF exerts its inhibitory activity through induction of apoptosis. As shown by FACS analysis in Fig. 3.4A- B, HF induced cell apoptosis, stained positively by 7-AAD and/or annexin V, in anti-CD3 antibody-stimulated splenocytes in a dose dependent manner, reflected by an increase in the percentage of apoptotic cells from 6.41  2.44% in cultures without HF to 8.92  1.85% in cultures with 0.625 nM of HF, 9.88  3.06% with 1.25 nM of HF, 19.12  2.65% with 2.5 nM of 61 HF and 34.97  9.98% with 5 nM of HF (P = 0.0002, one-way ANOVA, n = 3), or by a decrease in the percentage of viable cells (double-negative stained cells) from 82.77  7.5% in HF- untreated cultures to 72.34  10.35% in cultures with 0.625 nM of HF, 70.84  7.52% with 1.25 nM of HF, 56.79  6.35% with 2.5 nM of HF and 48.7  9.16% with 5 nM of HF (P = 0.0038, one-way ANOVA, n = 3). In IL-2-stimulated activated T cells, HF also induced dose-dependent cell apoptosis (Fig. 3.5C-D), indicated by an increase in cell apoptosis from 17.61  8.0 in untreated controls to 23.63  0.55% in cultures with 20 nM of HF, 31.55  1.2% with 40 nM of HF and 51.3  0.42% with 80 nM of HF (P = 0.0012, one-way ANOVA, n = 2), or by a decrease in viable cells from 79.8  8.63% in untreated controls to 74.15  0.64% in cultures with 20 nM of HF, 65.85  1.34% with 40 nM of HF and 41.3  0.57% with 80 nM of HF (P = 0.0009, one- way ANOVA, n = 2). Taken together, these data suggest that the anti-proliferative activity of HF in T cell cultures is associated with its induction of apoptosis. 62 Figure 3.4 HF induced apoptosis in T cell receptor stimulated T cell proliferation. Cell apoptosis was measured by FACS analysis with Annexin-V and 7-AAD staining. (A-B) Naïve splenocytes were stimulated with anti-CD3 antibody in the presence or absence of HF for 48 hrs. Data are presented as a typical FACS graph of cells labeled by Annexin-V-PE and/or 7- AAD in each group (A), or as mean ± SD of apoptotic cells (Annexin-V stained in right upper and lower quadrants) or viable cells (double negative in left and lower quadrant) of three separate experiments (B). 63 Figure 3.5 HF induced apoptosis in IL-2 stimulated T cell proliferation. Activated T cells were stimulated with IL-2 in the presence or absence of HF for 48 hrs. Data are presented as a typical FACS graph of cells labeled by Annexin-V-PE and/or 7-AAD in each group (C), or as mean ± SD of apoptotic cells (Annexin-V stained in right upper and lower quadrants) or viable cells (double negative in left and lower quadrant) of two separate experiments (D). 64 3.1.5 The Effects of HF in Cell Cycle Progress in Naïve T Cells Cell cycle arrest is one of biological features during the deprivation of nutrients [122]. By using a flow cytometry, we reported that HF induced cell cycle arrest in a dose response manner in anti-CD3 stimulated splenocytes, as reflected by propidium iodide (PI) staining. HF induced accumulation of proportion of cells at S phase indicating a cell cycle arrest occurring at S phase (Figure 3.6). As Figure 3.6 shown, the percentage of S phase was increased from 12 ± 0.127% in control cultures to 19.2 ± 0.679% in HF treated cultures with 5 nM. The cultures with 0.625 nM of HF yielded 13.2 ± 0.06% of S phase accumulation. This indicated that the induction of cell cycle arrest by HF was in a dose response manner. (p < 0.05 using one way ANOVA. n = 2) Figure 3.6 HF induced cell cycle arrest at S phase. Naive T cells were stimulated with anti-CD3 antibody in the presence or absence of HF. Data are presented as a typical FACS graph of cells labeled by propidium iodide (PI) in each group (left), or as mean ± SD of percentage of cells accumulated at S phase (right). 65 3.1.6 The Effect of HF on Proline Uptake in Anti-CD3 Antibody-Stimulated Splenocytes It has been reported that HF activates AAR in its inhibition of Th17 differentiation [82], and in a cell-free system it inhibits glutamyl-prolyl-tRNA synthetase, which can be reversed by the addition of exogenous proline [108]. To verify if HF suppressed T cell proliferation by the same action, the effect of HF on proline uptake was examined in T cell cultures. As shown in Fig. 3.7, activation of T cells by anti-CD3 antibody in naïve splenocyte cultures significantly depleted proline in culture medium (6 hrs: 0.95  0.071 µg/mL; 24 hrs: 0.53  0.042 µg/mL) as compared to the basal levels (6 hrs: 1.245  0.078 µg/mL; 24 hrs: 1.035  0.092 µg/mL) in non- stimulated cultures (P = 0.0015, two-way ANOVA, n = 2). And treatment with 5 nM HF significantly blocked proline uptake or depletion, indicated by an increase in proline Figure 3.1 HF blocks proline uptake in anti-CD3 antibody-stimulated T cell cultures. Naïve splenocytes were stimulated with anti-CD3 antibody in the presence or absence of 5 nM HF. The levels of proline in culture medium were measured using HPLC after 6 hrs or 24 hrs of incubation. Data are presented as mean ± SD of two separate experiments. 66 concentration (6 hrs: 1.425  0.134 µg/mL; 24 hrs: 0.95  0.071 µg/mL) in anti-CD3 antibody- stimulated splenocyte cultures in the presence of HF as compared to those in anti-CD3 antibody- stimulated cultures without addition of HF (P = 0.0018, two-way ANOVA, n = 2), and also by no significant difference of proline concentrations in between non-stimulated cultures and anti- CD3 antibody-stimulated cultures in the presence of HF (P = 0.5263, two-way ANOVA, n = 2). 3.2 Proline Rescued HF Anti-proliferation This section presents data demonstrating that proline was able to rescue the anti- proliferative ability of HF in T cell proliferation in response to either TCR or IL-2 stimulation. 3.2.1 The Effect of Additional Proline in Culture Medium on HF-induced T Cell Proliferative Suppression and Apoptosis To verify if the inhibition of proline uptake caused HF-mediated T cell proliferative suppression and apoptosis, the effect of proline supplement on T cell proliferation and apoptosis in HF-treated cultures was examined. Similar to the results shown in Fig. 3.1.1a-c, HF inhibited T cell proliferation in anti-CD3 antibody-stimulated splenocytes in normal complete RPMI medium (Fig. 3.8A), but the inhibitory activity of HF was significantly reduced by the addition of 1 mM proline in the culture medium (Fig. 3.8A), indicated by the inhibition of 25.46  8.62% in cultures without HF, 19.54  6.54 in cultures with 0.625 nM of HF, 13.16  13.67% with 1.25 nM of HF, 18.83  9.82% with 2.5 nM of HF and 27.64  6.14% with 5 nM of HF (P < 0.0001, Medium vs. Medium + Proline, two-way ANOVA, n = 15). The same effect of additional proline in culture medium on the inhibitory activity of HF in IL-2 stimulated activated T cell cultures was seen (Fig. 3.8B). HF inhibited IL-2-stimulated T cell proliferation in activated T cells in 67 normal culture medium, the same as shown in Fig. 3.1C, but its inhibition was significantly reduced by the addition of 1 mM of proline (P < 0.0001, Medium vs. Medium + Proline, two- way ANOVA, n = 5). Figure 3.8 The excess amount of proline inhibited anti-T cell proliferative activity of HF. The effect of proline supplement in T cell cultures on HF-mediated suppression of cell growth was examined by MTT assay. (A) Naïve splenocyte in normal culture medium (Medium) or medium containing additional 1 mM proline (Medium + proline) were stimulated with anti-CD3 antibody in the presence or absence of HF for 48 hrs. Data are presented as mean ± SEM (Standard error of the mean) of fifteen separate experiments. (B) Activated T cells in normal culture medium (Medium) or medium containing additional 1mM proline (Medium + proline) were stimulated with IL-2 in the presence or absence of HF for 48 hrs. Data are presented as mean ± SEM of five separate experiments. FACS analysis demonstrated that HF induced cell apoptosis or reduced cell viability in both anti-CD3 antibody-stimulated naïve splenocytes and IL-2-stimulated activated T cells in normal culture medium (Fig. 3.9) as compared to those in untreated controls (Medium groups), which were the same as shown in Fig. 3.4 and Fig. 3.5. However, the pro-apoptotic activity of HF was significantly inhibited by the addition of proline in the culture medium, indicated by a decrease in apoptosis from 28.52  2.14% in HF group to 16.9  1.7% in HF + Proline group (P = 0.0005, t-test, n = 3) in anti-CD3 antibody-stimulated naïve splenocytes (Fig. 3.9A), or from 68 44.4  0.71% in HF group to 20.15  1.06% in HF + Proline group in IL-2-stimulated activated T cells (P < 0.0001, t-test, n = 3) (Fig. 3.9B). Figure 3.9 The excess amount of proline rescued HF-induced apoptosis in T cells. Apoptotic cells were determined by FACS analysis, and were presented by the number of Annexin-V stained cell population. (A) Naïve splenocytes in normal culture medium were stimulated with anti-CD3 antibody in the absence (Medium) or presence of 5 nM HF (HF), or in the medium containing additional 1 mM proline were stimulated with anti-CD3 antibody in the absence of (Medium + proline) or presence 5 nM HF (HF + Proline) for 48 hrs. Data are presented as mean ± SD of three separate experiments. (B) Activated T cells in normal culture medium were stimulated with IL-2 in the absence (Medium) or presence of 16 nM HF (HF), or in the medium containing additional 1 mM proline were stimulated with IL-2 in the absence of (Medium + proline) or presence 16 nM HF (HF + Proline) for 48 hrs. Data are presented as mean ± SD of three separate experiments. To further confirm these results, the cleaved form of PARP and DNA fragmentation as markers of apoptosis in these cultures were examined. As shown in Fig. 3.10, the level of PARP in HF-treated cultures (HF group) were significantly induced as compared to those in controls (Medium and Medium + Proline groups), but was declined to the control levels by the addition of proline in culture medium (HF + Proline group). 69 TUNEL assay was performed to determine the DNA fragmentation in these T cells. As shown in Fig. 3.11, HF induced DNA fragmentation, as indicated by labeled dUTPs, in anti-CD3 antibody-stimulated splenocytes, reflected by an increase in the percentage of DNA fragmentation from 35.1  8.2% in cultures without HF to 58.4  9.5% in cultures with 5 nM of HF. The level of DNA fragmentation was significantly reduced by the addition of proline in the culture medium from 58.52  9.5% in HF group to 31.2  6.8% in HF + Proline group (P < 0.0001, t-test, n = 3). Taken together, these data suggest that HF is a competitive inhibitor of proline uptake that is required for T cells proliferation, and blocking proline uptake by HF induces T cell apoptosis. Figure 3.2 The excess amount of proline inhibited HF-dependent PARP cleavage in splenocyte cultures. Naïve splenocytes in normal culture medium were stimulated with anti-CD3 antibody in the absence (Medium) or presence of 5 nM HF (HF), or in the medium containing additional 1 mM proline were stimulated with anti-CD3 antibody in the absence of (Medium + proline) or presence 5 nM HF (HF + Proline) for 48 hrs. Un-treated splenocytes were used as a basal control. The cleaved form of PARP in protein extracts of these samples was determined by Western blot analysis. Data are a representative of three separate experiments. 70 Figure 3.11 The excess amount of proline inhibited HF-dependent DNA fragmentation in splenocyte cultures. Naïve splenocytes in normal culture medium were stimulated with anti-CD3 antibody in the absence (Medium) or presence of 5 nM HF (HF), or in the medium containing additional 1 mM proline were stimulated with anti-CD3 antibody in the absence of (Medium + proline) or presence 5 nM HF (HF + Proline) for 48 hrs. The DNA fragmentation was determined by TUNEL assay. (A) A typical FACS graph of cells labeled by FITC-dUTP in each group. (B) Data are presented as mean ± SD of two separate experiments. 71 3.2.2 The Effects of Proline in Cell Cycle Progress in HF-treated T Cells Cell cycle arrest is one of biological features during the deprivation of nutrients [122]. By using a flow cytometry, we reported that HF induced cell cycle arrest in a dose response manner in anti-CD3 stimulated splenocytes. HF induced accumulation of proportion of cells at S phase indicating a cell cycle arrest occurring at S phase (Figure 3.12 (up)). As Figure 3.5 (bottom) shown, the percentage of S phase was increased from 12 ± 0.127% in control cultures (Medium group) to 20.1 ± 3.4% in HF treated cultures with 5 nM (HF group). The cultures with proline and HF reduced the percentage of S phase accumulation to 13.4±3.7%. This indicated that proline reversed the induction of cell cycle arrest by HF. (HF vs. HF + Proline; P = 0.018, t-test, n = 2) 72 Figure 3.12 The excess amount of proline inhibited HF induced cell cycle arrest in splenocyte cultures. Naïve splenocytes in normal culture medium were stimulated with anti-CD3 antibody in the absence (Medium) or presence of 5 nM HF (HF), or in the medium containing additional 1 mM proline were stimulated with anti-CD3 antibody in the absence of (Medium + proline) or presence 5 nM HF (HF + Proline). The cell cycle analysis was determined by FACS. (up) A typical FACS graph of cells labeled by propidium iodide (PI) in each group. (bottom) Data are presented as mean ± SD of two separate experiments. 73 3.3 HF Synergistically Enhanced RAPA Anti-proliferation in T Cells. This section presents data demonstrating that the interaction of HF with RAPA in suppressing T cell proliferation. 3.3.1 HF Enhanced Anti-proliferation of Rapamycin on T Cell Receptor-Stimulated T Cell Proliferation RAPA inhibits T cell proliferation by attenuating responses to IL-2 signaling [123, 124], whereas HF is demonstrated to exhibit its anti-proliferation in T cell cultures by inducing apoptosis. As demonstrated in the Fig. 3.13, the treatment with RAPA alone yielded a significant suppression in TCR-stimulated T cell proliferation in a dose response manner, as reflected by 16.4±10.6% with 0.1 nM of RAPA, 53.1±5.8% with 1 nM of RAPA, 65.6±6.2% with 10 nM of RAPA, 69.1±3.6% with 100 nM of RAPA. The anti-proliferative activity of RAPA was significantly enhanced by the presence of HF (2.5 nM) as shown in Fig 3.14 (up); 68.6±5.2% at 0.1nM of RAPA, 90.5±0.3% at 1nM of RAPA, 90.9±0.4% with 10nM of RAPA, 91.0±0.4% at 100nM of RAPA. Comparing with those treated with RAPA alone, there were significant differences between two treatments (RAPA vs, RAPA + HF) at all doses (p < 0.001, t-test, n=2) in suppressing T proliferation. 74 As demonstrated in the Fig. 3.14 (bottom), the inhibitory activity of combination treatment of CsA and HF (2.5 nM) was reflected by 29.2±6.6% at 1.25 nM of CsA, 35.1±5.5% at 2.5nM of CsA, 47.5±2.8% at 5nM of CsA, 65.6±2.8% with 10nM of CsA. As comparing with the treatment of CsA alone, there is significant differences at the doses of 1.25nM and 2.5nM of CsA (p < 0.05, t-test, n=2), whereas there is no significant differences at the doses of 5 nM and 10nM of CsA (p = 0.06, t-test, n=2; p = 0.15, t-test, n=2, respectively) in suppressing T proliferation. The Loewe’s Additivity was applied to determine the synergistic or antagonistic relationship between HF and RAPA or HF and CsA (Table 3.1). The computed value between HF and RAPA at IC50 is 0.18 which was less than 1, whereas the computed value between HF and CsA at IC50 is 2.2. The IC50 dosage of CsA was found to be 10 nM (Suppl Fig. 6). This indicates that HF and RAPA are synergistic to each other in preventing T cell proliferation while HF and CsA are antagonistic to each other. Figure 3.3 Rapamycin (RAPA) induced anti-proliferation in T cells. Naïve splenocytes were stimulated with anti-CD3 antibody in the presence or absence of RAPA for 48 hrs. Data are presented as mean ± SD of five separate experiments. 75 Figure 3.14 The effect of HF + Rapamycin / HF + Cyclosporin A in T cell proliferation. The combination of HF + Rapamycin yielded higher inhibitory activity in an additive effect. Naïve splenocytes were stimulated with anti-CD3 antibody in the presence or absence of RAPA or CsA and/or HF for 48 hrs. Data are presented as mean ± SD of three separate experiments. 76 Drug Combination: Loewe’s Additivity Value: at IC50 HF + RAPA 0.18 HF + CsA 2.12 Table 3.1 The computed values of Loewe's Additivity between HF and RAPA or CsA. The combination treatment of RAPA with HF yielded the value of 0.18 indicating the existence of synergistic relationship. The combination treatment of CsA with HF yielded the value of 2.12 indicating the existence of antagonistic relationship. 3.3.2 HF Enhanced Anti-prolifeation of Rapamycin on Allo-antigenic Stimulated T Cell Proliferation As demonstrated in the Fig. 3.15, the treatment of RAPA (1nM) alone yielded 36.1.3±9.1% in allo-antigenic stimulated T cell proliferation in MLR. In the combination treatment of RAPA and HF, the inhibitory activity was demonstrated by 44.1±5.1% with 0.625nM of HF, 51.7±6.5% with 1.25nM of HF, 59.3±3.9% with 2.5nM of HF, 55.3±11.1% with 5nM of HF. The inhibitory pattern is in a dose response manner (p = 0.0008, two way ANOVA, n=5). 77 Figure 3.15 HF enhanced anti-proliferation in RAPA treated T cell proliferation in response to allo-antigenic stimulation. MLR; a mixed culture of splenocytes from BALB/c mice and B6 mice was incubated in the presence or absence of HF for 48 hrs. Data are presented as mean ± SD of five separate experiments. 3.3.3 HF Increased the Induction of Apoptosis in Rapamycin-treated T Cell Cultures HF is demonstrated to induce apoptosis [83, 106] whereas RAPA is proven to induce cell cycle arrest in T cell proliferation [125]. Thus, it is possible that RAPA enhances the pro- apoptotic activity of HF. As shown by FACS analysis in Fig 3.16, RAPA did not lead to a high increase in apoptosis stained positively by 7-AAD and/or annexin V, in anti-CD3 antibody- stimulated splenocytes. The cultures with RAPA yielded 13.5±3.0% of apoptotic cells as compared to 9.5±1.4% of untreated control, with no significant differences (p = 0.233, t-test, n=2). HF and RAPA together yielded additive effect in inducing apoptosis, reflected by an increase from 13.5±3.0% in cultures without HF to 16.8±2.1% in cultures with 0.625 nM of HF, 22.1±2.6 with 1.25 nM of HF, 31.7±3.8% with 2.5 nM of HF, and 28.7±4.1 with 5 nM of HF (p = 0.0017, one-way ANOVA, n=2), or by a decrease in the percentage of viable cells (double- negative stained cells) from 79.1±0.4% in RAPA treated cultures without HF to70.5±1.9% in cultures with 0.625 nM of HF, 56.8±2.4% with 1.25 nM of HF, 45.5±1.1% with 2.5 nM of HF, 78 44.8±5.9% with 5 nM of HF (p < 0.0001, one-way ANOVA, n=2). Data are presented as a typical FACS graph of cells labeled by Annexin-V-PE and/or 7-AAD in each group (A), or as mean ± SD of apoptotic cells (Annexin-V stained in left upper and lower quadrants) or viable cells (double negative in right and lower quadrant) of two separate experiments (B), or mean ± SD of apoptotic cells (Annexin-V stained in left upper and lower quadrants) in HF + RAPA treated cultures compared to the one is seen in HF treated cultures collected from the data shown Figure 3.16 (C). Using the Loewe’s Additivity model, it demonstrates that there is a synergistic relationship in inducing apoptosis between RAPA and HF in T cell cultures with the value of 0.217. Taken together, these data show that the combination of HF and RAPA increased induction of apoptosis as compared to what is seen in RAPA treated cultures. 79 Figure 3.16 HF increased apoptosis in Rapamycin treated T cells. Cell apoptosis was measured by FACS analysis with Annexin-V and 7-AAD staining. (A-B) Naïve splenocytes were stimulated with anti-CD3 antibody in the presence or absence of RAPA and/or HF for 48 hrs. Data are presented as a typical FACS graph of cells labeled by Annexin- V-PE and/or 7-AAD in each group (A), or as mean ± SD of apoptotic cells (Annexin-V stained in right upper and lower quadrants) or viable cells (double negative in left and lower quadrant) of three separate experiments (B). Data are presented as mean ± SD of apoptotic cells (Annexin-V stained in right upper and lower quadrants) in the presence or absence of RAPA in splenocytes treated with HF (C). 80 Chapter 4: Discussion HF is an anti-protozoal drug for the treatment of Coccidia parasite infection in veterinary medicine. Recent studies have documented its potential for immunosuppression; it inhibits cytokine production from activated T cells [83] and Th17 cell differentiation by the induction of AAR in human peripheral blood mononuclear cell culture [82] and in a murine model of chronic graft-versus-host disease [126]. The present study for the first time shows that HF potently suppresses T cell proliferation in association with the induction of apoptosis as well as cell cycle arrest in the response to the stimulation of TCR-CD3 complex and IL-2R. In this study, the anti- proliferation of HF in T cell growth is examined by investigating induction of apoptosis and proliferation arrest. These two cellular pathways are important in maintaining tissue homeostasis by removing damaged or stressed cells. Also, these mechanisms are shared by many anti- proliferative agents in treating autoimmune diseases and tumor growth. Similar to the findings of this study, HF has been reported to suppress cell growth in cultures of fibroblasts [127, 128] and several tumor cell lines recently [106, 129, 130] which is also associated with the induction of cell apoptosis [106, 130, 131]. This study has clearly demonstrated that HF does not attenuate the IL-2 production or its associated intracellular signaling (Fig 3.2-3.3). Thus, growth factor withdrawal is not the cause of apoptosis by HF. Indeed, increasing evidences demonstrate that HF activates AAR pathway in its inhibition of Th17 differentiation in vitro [82] and its renal protection against ischemia-reperfusion injury in mice [128], and the supplement with amino acids in culture medium inhibits HF-activated AAR pathway which prevents the inhibition of Th17 differentiation [82]. Similarly, data of this study identifies that the proline levels in culture medium were significantly deprived following the stimulation of T cell proliferation with anti-CD-3 antibody, and proline deprivation or uptake in 81 culture media is prevented in the presence of HF (Fig. 3.7). However, the limitation of this proline measurement is that it cannot measure the intracellular proline level in HF treated T cells. Thus, the proline measurement presented in this study provides indirect evidence showing HF- induced proline depletion resulting activation AAR, and subsequently, the induction of apoptosis. Nonetheless, all these data may strongly suggest that HF may induce cell apoptosis in T cells by activating AAR via the induction of intraceullular proline depletion. Cell cycle arrest is a pro-survival mechanism in stressed cells, as proliferation arrested cells are permitted to gain some times to get repaired and recover from damage before resuming normal proliferation [132-134]. Some stress stimuli may activate genes associated with cell cycle arrest and DNA repair [135, 136]. The present study demonstrates that proliferation arrest occurred at S phase significantly in HF-treated T cell cultures, and such arrest was in a dose response manner (Fig 3.6). To further confirm the notion that HF induced cell cycle arrest, alternative experimental approach can be performed such as dUTP labeling assay. Alternatively, BrdU assay can be employed to demonstrate whether HF leads to suppression of DNA replication in S phase arrest. Extensive mitotic arrest can lead to the induction of apoptosis [137], and glucocorticoid treatment in lymphocyte proliferation induces cell cycle arrest followed by apoptosis [138]. Therefore, it is believed that HF may activate cell cycle arrest in encountering pro-apoptotic stress stimuli, and subsequently, the prolonged activation of cell cycle arrest may lead to apoptosis. Little is known about the mechanism of direct or indirect action of HF in modulating apoptosis and cell cycle arrest. Thus, it is unknown whether HF- induced S phase arrest and apoptosis are in parallel, independent pathways or sequential cascade events. Further experiments are required to examine the expressions of cell cycle phase specific cyclin such as CDK2, CDK4 and CDC2 as well as pro-apoptotic proteins such as Bcl family to 82 identify the link between apoptosis and cell cycle arrest. Nonetheless, the results presented in this study provide a lead about the mechanisms underlying HF anti-proliferation in T cell expansion. In this study, further experiments reveal that the proline supplement in culture medium reverses the anti-proliferative activity of HF in T cell cultures. Thus, the present study for the first time shows that HF potently suppresses T cell proliferation including induction of apoptosis and cell cycle arrest in the response to the stimulation of TCR-CD3 complex and IL-2R in a proline dependent manner. A recent study by Keller’s group has shown that in a cell-free system, HF competitively inhibits the activity of glutamyl-prolyl-tRNA synthetase (EPRS) by preventing the binding of its substrate proline [108], suggesting that HF may act as a competitive inhibitor of EPRS in vitro and in vivo. Interestingly, the present study was able to identify peptides of EPRS which might bind to HF in T cell cultures by performing drug affinity responsive target stability (DARTS) [139] followed by LC-mass spectrometry with the identified peptide sequence “AKVTSAVR” (Suppl. Table 1). Indeed, an early study has shown that supplement with proline in the diets mitigate the weakened skin of broiler chickens receiving HF treatment [140], and recently Keller et al (2012) have reported that of 10 EAAs (essential amino acids) and 7 NEAAs (non-essential amino acids) tested, only proline rescues HF cytotoxicity against cell growth in cultured mouse embryonic fibroblasts [108]. Intracellular starvation of any of essential amino acids (EAA) and non-essential amino acids (NEAA) activating AAR will result in accumulation of unaminoacylated (i.e., uncharged) tRNA that functions as an amino acid delivery molecule to the ribosome for protein synthesis. The uncharged tRNAs binds GCN2 (general control nonderepressible 2) kinase [141] that subsequently phosphorylates and inactivates the eukaryotic translation initiation factor 2α (eIF2α), an essential factor for protein 83 synthesis [142, 143], thereby, resulting in a reduction of protein synthesis. Inhibition of protein synthesis augments the effects of pro-apoptotic factors [144-146], so amino acid deprivation leads to apoptosis and oxidative stress [107, 147]. HF stimulates the phosphorylation of GCN2 and eIF2α in T cells [82]. And, the inhibition of eIF2α causes apoptosis in Jurkat T cells [148, 149], which is similar to the apoptosis in HF-treated T cells, characterized by an increase in the intensity of Annexin-V binding (Fig. 3.4 and 3.5), cleavage of PARP (Fig. 3.10), DNA fragmentation (Fig. 3.11), and caspase 3 (Suppl. Fig. 1), suggesting that HF induces T cell apoptosis probably by activation of this AAR pathway. Similarly, addition of proline in culture medium inactivates HF in the suppression of T cell proliferation or in the induction of casapse 3 dependent apoptosis (Fig 3.9, Suppl Fig. 1) as well as cell cycle arrest (Fig. 3.12). Together, this study further suggest that HF may induce cell apoptosis and proliferation arrest in T cells via the inhibition of EPRS; subsequently, blocking cellular proline incorporation or utilization. The data of this study also suggest that HF anti-proliferation leads to autophagy activation upon proline depletion via AAR in T cell cultures. AAR is demonstrated to activate autophagy [150-152]. Autophagy is a pro-survival strategy which is widely employed for a starving cell as well as an apoptotic cell. This allows cells to re-allocate nutrients degraded from self organelles and recyclable proteins for essential biological processes. During the autophagy, cells are instructed to construct a double membrane vesicle known as atuophagosome by catalyzing the scaffolding of Atg proteins to the pre-autophagosome membrane. Autophagosome acting as an eating machine engulfs aging organelles and recyclable proteins for extraction of nutrients. Starvation leads to the induction of autophagic cell cycle arrest [122]. The knockout of different autophagy genes has demonstrated to affect in maintenance of normal numbers of B cells, T cells [153, 154]. Thus, these evidences indicate that autophagic growth 84 suppression may serve as a pro-survival role by preventing damaged cells from proliferation with activation of genes associated with cell cycle arrest [132, 134, 155]. Similarly, this study demonstrates that HF induced cycle arrest at S phase (Figure 3.6) as well as LC3 I/II conversion (Suppl. Fig. 2). LC3 is microtubule protein, which normally presents in the cytoplasm. Cleaved LC3 forms LC3 I which is lapidated with phospholipid, phosphatidylethanolamine (PE) to form LC3 II in autophagosomes [156, 157]. All of these results indicate that anti-proliferative activity of HF is associated with cell cycle arrest in part which may serve as a pro-survival role in encountering the apoptotic stimuli induced by HF. 3-MA, the specific inhibitor of autophagy is known to target the class III PI3K and suppress autophagic vesicle formation [158, 159]. In this study, 3-MA is found to increase anti-proliferation induced by HF by nearly 20% (Suppl. Fig. 3). Taken together, these results provide further support to the notion that HF induces autophagy as a pro-survival mechanism upon intracellular starvation of proline via AAR resulting cell cycle arrest. However, this experiment did not explain whether apoptotic signal induced by HF activates autophagy, or they occurred in a synchronized manner in the same cell. Previous studies state that autophagy inducation may activate or delay the onset of apoptosis [160, 161]. Some finds these autophagy and apoptosis are mutually exclusive [162]. Thus, to further explore the inter-changing relationship between autophagy and apoptosis in HF anti-proliferation deserves further investigation. Eukaryotic initiation factor 2 (eIF2) as mentioned earlier is crucial in protein synthesis. It functions in the initiation of translation by mediating the binding of the initiaior Met-tRNA to the ribosome [163]. The activity of eIF2 is attenuated by phosphorylation at alpha subunit during AAR [164]. In addition to the attenuation of eIF2α -dependent protein synthesis, phosphorylation of eIF2α enhances the translation of a group of specific mRNAs, including 85 ATF4 (activating transcription factor 4), which contain initiating codons downstream of noncoding micro-open reading frames (ORFs) and are normally translated with low efficiency [165, 166]. Indeed, HF up-regulates ATF4 expression in T cells [82]. In this study, an increase in CD25 expression (Suppl Fig. 4) in anti-CD3 antibody-activated T cells in the presence of HF is evident, whereas the proline supplement in medium reduces the up-regulation of CD25 induced by HF (Suppl Fig. 5). Similarly, starvation of L-arginine leads to up-regulation of CD25, and production of IL-2 were found to be unaffected [167, 168]. Interestingly, HF-treated T cells also yielded normal expression of IL-2 as demonstrated in this study. This further suggests that HF-induced CD25 expression is one of the cellular responses to amino acid starvation. However, additional experiments are required to demonstrate the link between CD25 expression and proline depletion, as well as mechanisms underlying this linkage. Also, it may be a great interest to examine the change in CD25 expression in depletion of other amino acids. This helps to solidify the idea that AAR and CD25 up-regulation are related. Nonetheless, the data of this study contribute to the understanding of modulating T cell response to proline depletion. Depletion of a single amino acid resulting suppression of protein synthesis leads to an increase in a program of gene expression under GCN4 control in yeast [169]. GCN4 is a primary regulator in response to amino acid starvation leading to the transcription of genes that are required for amino acid synthesis [170-172]. GCN4 belongs to a family of mammalian activator protein (AP)-1 [173]. Homology between the DNA binding sites for AP-1 and GCN4 suggested that AP-1 and GCN4 are related to each other [174]. AP-1 is a transcription factor dedicated to function in cell proliferation, differentiation and apoptosis. AP-1 is composed of the DNA binding proteins fos and jun. In mammalian cells, AP-1 family is activated by stress signals 86 [175-178]. Since GCN4 is activated in amino acid starvation in yeast, AP-1 is predicted to activate in the mammalian cells under the same conditions. Also, there is association between AP-1 and autophagy. The jun N-terminal kinase (JNK)/AP-1 pathway contributes to autophagic responses [179, 180]. Similarly, autophagy is found to be a protective role for tumor cells encountering anti-cancer therapy. Recent study implicates that increased AP-1 transcriptional activity is evident in association with tamoxifen resistant breast cancer [181]. These data reflect that AP-1 has some implications in autophagy activation. Thus, it is reasonable to speculate that AP-1 transcriptional activity is activated during proline depletion leading to activation of autophagy. AP-1 is also an essential transcription factor in mediating T cell development by inducing IL-2 gene expression [182-184]. Therefore, HF-induced up-regulation of CD25 and unaffected IL-2 production might be mediated by AP-1 transcriptional activity. However, further investigations are required to support this notion by looking at the expression level of AP-1 in the presence of HF. RAPA is known for the most commonly used immunosuppressant in post-operative medication in transplant patients. RAPA remains as the backbone of many protocols in preventing both acute and chronic rejection of transplants. However, RAPA does not offer a long term protection for the grafts. Thus, it is always a great interest to develop an adjuvant compound which can enhance the immunosuppressive ability of RAPA in treating graft rejection while reducing its toxicity. HF has been demonstrated its potential for immunosuppression [82, 108], as also supported by the data presented previously. Here, this study is the first literature showing the potential of HF as an adjuvant compound in synergistically enhancing the anti- proliferative ability of RAPA in TCR stimulated T cell proliferation. An increase in activation of 87 apoptosis in T cell cultures is evident with the combination of HF and RAPA as compared to the monotherapy of RAPA (Fig. 3.16). RAPA is known for targeting mammalian target of rapamycin (mTOR) pathway. mTOR is an integrator of signals that sense energy and nutrients [185]. It controls cell cycle and survival. mTOR exists in two complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), most commonly distinguished by their association with the companion proteins Raptor and Rictor [125]. These two existed forms of mTOR complex can be defined by the levels of sensitivity to RAPA. mTORC1 is sensitive to RAPA, whereas mTORC2 is resistant to RAPA [186]. The synergistic relationship between RAPA and HF is identified with the Loewe’s value of 0.18 (Table 3.1) in the present study, suggesting that HF is a potential adjuvant in strengthening the therapeutic effects of immunosuppressive protocol with RAPA in treating graft rejection. Such synergistic relationship may be attributed by the differential biological functions that induced by RAPA and HF separately inside the cells. HF is found to induce caspase 3 dependent apoptosis and cell cycle arrest at S phase as demonstrated in our unpublished data as well as in previous findings [82, 106], whereas RAPA is found to induce autophagy which is associated with induction of cell cycle arrest at the G1-S transition followed by increased apoptosis [187]. RAPA induces apoptosis in several cancer cell lines at micro-molar concentration [188-190]. Differential doses of RAPA yield differential biological functions. Low dosage of RAPA at nano-molar level leads to cell cycle arrest via suppression of S6 kinase which is a mTORC1 substrate [191]. Conversely, dosage at micro-molar level of RAPA leads to apoptosis via completed dissociation of mTORC1 and suppression of phosphorylation of eukaryotic initiation factor 4E-binding protein (4E-BP1) [125]. Similarly, this study clearly shows that RAPA at nano-molar level played a minor role in inducing apoptosis (Fig. 3.16). 88 Intriguingly, the combined treatment with HF increases proportion of apoptotic cells (Fig. 3.16C). This may indicate that the addition of HF promoted the pro-apoptotic activity of RAPA at nano-molar concentration. It has demonstrated a clear correlation between apoptosis and the suppression of 4E-BP1 phosphorylation at dosages of RAPA at micro-molar level [125] resulting in extreme toxicity to the patients. Conversely, lose doses of RAPA are associated with increased lifespan in patients, and these doses also play an important role in sensitizing cancer cells to other treatments [192- 197]. RAPA is also found to induce autophagy that is associated with induction of cell cycle arrest. RAPA is known to induce cell cycle arrest at the transition between G1 and S [125]. Data of this study suggest that the effect of HF might promote RAPA-treated T cell cultures more prone to apoptosis. Beclin-1, a key player of inducing autophagy, is found to have a central role in the induction of apoptosis upon cleavage by caspase 3 [198]. Beclin-1 is induced under the conditions of nutrient deprivation and RAPA treatment [199]. Thus, it is reasonable to speculate that caspase 3 cleavage activated by HF, subsequently cleaves Beclin-1 resulting the demolishment of RAPA induced autophagy. Thus, RAPA- treated T cells become less protected from apoptotic signal; therefore, increased induction of apoptosis is observable. However, additional experiments are necessary to support this notion. Taken together, HF is demonstrated as a potential adjuvant in strengthening RAPA immunosuppressive activity. 89 Chapter 5: Conclusion HF has been reported as an anti-fibrotic agent by inhibition of collagen I synthesis and interference of TGF- signaling with potential immunosuppressive activity. Our study further confirms that HF potently suppresses T cell proliferative response to the stimulation of TCR- CD3 complex or IL-2R, at least in part by inducing cell apoptosis with increased cleavage of PARP and DNA fragmentation. We also demonstrate that HF also prevents T cell proliferation via activation of cell cycle arrest at S phase. Here, we report that increased apoptotic cell proportions induced by HF could be reversed by the proline supplement in the medium, as also reflected by the reduction in the HF-induced expression of cleaved PARP as well as DNA fragmentation. The proline supplement also rescued HF-treated T cell cultures from cell cycle arrest. This present study demonstrates that HF induced proline depletion resulting in induction of autophagy via suppression of protein synthesis. These results may suggest that HF is a novel compound that can be designed for specifically targeting T cells as well as tissue fibrosis, which are the feathers of chronic rejection of organ transplants as well as of graft-versus-host disease. Also, we have identified a synergistic relationship between HF and RAPA in preventing T cell proliferation. Thus, HF may be a potential adjuvant for the RAPA-based immunosuppressive protocol. 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The protein content in each sample was determined by the Bradford assay using the Bio-Rad dye reagent compared to bovine serum albumin (BSA) as standards. The proteins in extracts were denatured by boiling with Laemmli sample buffer for 5 min. Equal amount of protein (100 to 150 µg) from each sample was fractioned by 7% SDS-polyacrylamide gel electrophrosis (SDS-PAGE) and transferred to nitrocellulose membranes (Bio-Rad Lab, Hercules, CA, USA), that were then blocked with 5% skim milk in TBST (10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.1% Tween-20) for 1 hr, and then probed with anti-caspase 3 antibody (Cell Signaling Technology) in TBST containing 2.5% of milk at 4 o C overnight. The membranes were then washed with TBST and incubated with alexa fluor 680 secondary antibodies for 60 min. Proteins on blots were scanned and visualized by Odyssey infrared imaging system (LI-COR, USA). The blots of cleaved caspase 3 were re-probed with anti- -actin IgG antibody (Sigma-Aldrich Canada) to confirm equal protein loading in each sample. The expression levels were measured using a -actin from the same samples. The image demonstrates a typical Western blot labeled with anti-caspase 3 antibody and anti- - actin IgG antibody. 112 Supplementary Figure A.2 HF induces LC3 I/II conversion in splenocyte cultures. Naïve splenocytes in normal culture medium were stimulated with anti-CD3 antibody in the absence (Medium) or presence of 5 nM HF (HF) were stimulated with anti-CD3 antibody for 24 hrs. Stimulated splenocytes in the absence of HF were used as a basal control. The expression of LC3 I/II in protein extracts of these samples was determined by Western blot analysis. Briefly, splenocytes or T cells (10  106 cells/well) in 6-well plates were stimulated with 2 µg/mL of anti-CD3 antibody in the presence or absence of HF. After indicated periods of treatment, the total protein was extracted by a brief sonication of cell suspension in RIPA buffer (50 mM Tris- HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS) containing protease inhibitor cocktail (Roche, Mannheim, Germany). The protein content in each sample was determined by the Bradford assay using the Bio-Rad dye reagent compared to bovine serum albumin (BSA) as standards. The proteins in extracts were denatured by boiling with Laemmli sample buffer for 5 min. Equal amount of protein (100 to 150 µg) from each sample was fractioned by15% SDS-polyacrylamide gel electrophrosis (SDS-PAGE) and transferred to nitrocellulose membranes (Bio-Rad Lab, Hercules, CA, USA), that were then blocked with 5% skim milk in TBST (10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.1% Tween-20) for 1 hr, and then probed with anti-LC3 I/II antibody (Cell Signaling Technology) in TBST containing 2.5% of milk at 4 o C overnight. The membranes were then washed with TBST and incubated with alexa fluor 680 secondary antibodies for 60 min. Proteins on blots were scanned and visualized by Odyssey infrared imaging system (LI-COR, USA). The blots were re-probed with anti-β-actin IgG antibody (Sigma-Aldrich Canada) to confirm equal protein loading in each sample. The expression levels were measured using a densitometry, and were presented as RU after normalized with β-actin from the same samples. The image demonstrates a typical Western blot labeled with anti-LC3 I/II antibody and anti-β-actin IgG antibody 113 Supplementary Figure A.3 3-MA increases anti-proliferation in HF-treated T cell cultures. The effect of 3-MA in T cell cultures on HF-mediated suppression of cell growth was examined by MTT assay. Naïve splenocytes in normal culture medium (HF), and the same cell cultures containing additional 1 µM 3-MA (HF + 3-MA) were stimulated with anti-CD3 antibody in the presence of HF for 48 hrs. After 48 hrs incubation, 10 µL of 0.5 mg/mL of 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich Canada) were added to each well (100 µL of cell culture) and incubated at 37 o C for 4 hrs. The mitochondria of MTT treated living cells reduce MTT into insoluble, dark purple formazan crystals. Thus, active mitochondrial activity determines the viability of cells. The formazan crystals in viable cells were then dissolved in 100 µL/well of dimethyl sulfoxide (DMSO, Sigma-Aldrich Canada). The absorbance of the color in each well was quantified as absorbance unit (AU) at 560 nm wavelength using ELx808 Ultra Microplate Reader (BioTek, Winooski, VT, USA). The percentage of inhibition in cell proliferation in drug-treated cultures against nondrug-treated control was calculated as follows: Inhibition (%) = (Drug-treated - Control)/Control ×100%. Data are presented as mean ± SD (Standard deviation of the mean) of five experiments. 114 Supplementary Figure A.4 HF induces up-regulation of CD25 (IL-2 α chain) in T cells. CD25 staining assay was performed for the measurement of CD25 expression level in T cells. In brief, splenocytes (1  106 cells/well) in 24-well plates were stimulated with 2 µg/mL anti-CD3 antibody in the presence or absence of HF for 24 hrs. These cells were stained with allophycocyanin (APC) conjugated anti-mouse CD25α antibody (eBioscience) in 1% PBS, and incubated in dark for 30 min followed by washing. The intensity of fluorescent of cell population was correlated with the expression level of CD25, which was quantified by flow cytometry and analyzed using FlowJo software (Tree Star Inc.). The figure demonstrates a typical FACS graph of cells labeled by APC conjugated anti-CD25 antibody in each group. 115 Supplementary Figure A.5 Proline reduces HF-induced up-regulation of CD25 (IL-2 α chain) in T cells. CD25 staining assay was performed for the measurement of CD25 expression level in T cells. In brief, splenocytes (1  106 cells/well) in 24-well plates were stimulated with 2 µg/mL anti-CD3 antibody in the presence or absence of 5 nM HF for 24 hrs. These cells were stained with allophycocyanin (APC) conjugated anti-mouse CD25 antibody (eBioscience) in 1% PBS, and incubated in dark for 30 min followed by washing. The intensity of fluorescent of cell population was correlated with the expression level of CD25, which was quantified by flow cytometry and analyzed using FlowJo software (Tree Star Inc.). The figure demonstrates a typical FACS graph of cells labeled by APC conjugated anti-CD25 antibody in each group. 116 Supplementary Figure A.6A Control HF LDMCKLLLR VQGDWIRER SPHIDNVR ELAEGEEQSEK KMLLIDADLR KMLLIDADLR QIGIWDAFNIEK LCQDLGPGAFR ALCDLGPWVK ANEAAESVGNK TVQQSTVDQLK AAPMVSEAPIVMDCK VAYSRISNLHK GSYADELVK ASAMYSGKGPLNDLR QESRTK TLNNDIMLIK TLSTSDDVEDRENEK ANEAAESVGNK AKVTSAVR MIKETMDK EIGHGELAR QYTAKSEGLAR ALTKDSDEIVPK SGQETIGGIDMIVR SGQETIGGIDMIVR NMAFVMLNR MNKIALVMDLR KESVASK TLESQITLEK ADADTMKAMR IGIPYDLMENK KPFKCIECGK DFVIQPTSEEVVTDIARQELK EALMTTVHATTATQK EFDETIIK AFEIAGYDK EVSESLHKR ENTKLYLNLLEMEYSGDLK IDSELMLYK ALTKDSDEIVPK AGAAPMETVDKR RMFGALGR NVSTGSAENEEK YGMPDLRPFFEADMR AAMVVEINGEFK QMLQLMVAK MLQIADTTFTSR AMREENGLR VMVEPRANMK ATEPYKCTFAELPEK GVTEGFR WILDNVDGARETAQSGR QIGIWDAFNIEK KEWTTT IGLQAVPSLASSQEKAK AAALLTYSSFCPPDDLADR ANTNRGPWVR AGMVAVDEK IESSLERFGDR MMVESPTQPNFWER DNEDDIQQSFSVNRSHK 117 Control HF VPEWSDVTKMGISK MELHYLAK DQCIVDGLTYNVNSSFTK EGKFILELK AFALKQAAMAFGK SMIEYIGGGDDK GADINNLVLRML ITGIA HIHAQK EHLVVQSK MKSSMAMIK MDPDRQADIAALAATLTTVER ANTNRGPWVR ASRSMPFSSK NGEILTIDTIPK NSPISRAK AEIIIK MATEQEFTR EGPTWHAKK AEAVETEVK ENIINLAK AEIIIK NGNMTSHDSYISPLIMENK MVIQDIFK AGYTEKQR FMTNKYK QHVVAAWR ISISTSGGSFR AGEEISRR EGNDLYAEMKESK HNTPQEISAMILQK ADGNHNTTK ARGMMVR IMMACLK DLTINAIAMDQDGKIYDPYGGQNDLENK MVVYEFPK ELLQGMYK AHMFCVNALAARDPIWAAR ASSSFLK KYGLENEVK NLFTWSIMINGYAKNNCFEK AIKAGVDSTK ADGNHNTTK HGRHAVR CAICYKAGHGQHFGVETCR TAKETK GATSTNGNPVDISGNGLTNGFPK AAEELGIDLGRR ADQCRSSPQEPSK DLTINAIAMDQDGKIYDPYGGQNDLENK DLDDKYFK GAETLTEQLTEKGYSAVSLSK MGYKDLPPGK IKPHDDAVEEGIRYK HSPQMEVSTVK CSGLMGAAPPGPPSPSAADTPAK APAVV ATFLDADGK LMANEWAK MLESIGMKDLR SKLALDESTMR NLFTWSIMINGYAKNNCFEK TGSELFK ASDALLKAQNDVMTMK 118 Control HF DQGEIK Supplementary Figure A.6B Supplementary Figure 6 Identification of proyl-tRNA syntethase in HF-treated T cell cultures via drug affinity responsive target stability (DARTS). (A) DARTS protocol was adapted from the publication in 2009 by Lomenick et al [200]. Splenic lymphocytes treated with HF after 30 min were washed with cold PBS and M-Per(Pierce, USA). Lysates were sonicated, and put into cellulose tubular membrane (Membrane Filtration Products, Inc., USA) for dialysis for overnight. Protein concentrations were determined by the Bradford assay using the Bio-Rad dye reagent, bovine serum albumin (BSA) was used as standard. Then, the protein samples were incubated with theromlysin at 70ºc for 10 min (according to manufacturer’s instruction). Equal amounts of protein were resolved by SDS- polyacrylamide gel electrophrosis (SDS-PAGE) on 10%. The gel was stained with comassie blue. Protein bands of interest were excised from the gel, and cut into small pieces. The gel pieces were then incubated in destaining solution (50% methanol, 10% acetic acid) and rotated for 30 min. Supernatant was removed. The samples were incubated in 100mM ammonium bicarbonate and rotated for 10 min, and supernatant was removed. 50% acetontrile was added and rotated for 10 min following by the vacuum drying in centrifugal evaporator. The samples were treated with 10mM dithiothreitol (DTT) at 65°C for 35 min. Supernatant was removed following by the addition of 55mM iodoacetamide (IAc) in dark for 30 min. After the removal of supernatant, the samples were incubated and rotated in acetonitrile for 10 min, and dried in centrifugal evaporator. Trypsin (10ng/ul) was added to the gel pieces, and incubated for overnight. The supernatant was removed for the subsequent peptide identification by LC mass spectrometry (Quattro Premier XE). (B) The protein identification of peptide fragments that were sequenced by LC/MS were further identified using National Center for Biotechnology Information (NCBI)-Basic Local Alignment Search Tools (Blast). Data was accessed on November 30 th , 2012. 119 Supplementary Figure A.7 CsA suppresses TCR stimulated T cell proliferation with IC50 at 9-10 nM. MTT assay was performed for the measurement of T cell proliferation. In brief, splenocytes (2  10 5 cells/well) in 96-well plates were stimulated with 2 µg/mL anti-CD3 antibody in the presence or absence of CsA (1.25 nM, 2.5 nM, 5 nM, 10 nM). At 48 hours, 10 µL of 0.5 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich Canada, ) were added to each well (100 µL of cell culture) and incubated at 37 o C for 4 hrs. The formazan crystals in viable cells were then dissolved in 100 µL/well of dimethyl sulfoxide (DMSO, Sigma- Aldrich Canada). The absorbance of the color in each well was quantified as absorbance unit (AU) at 560 nm wavelength using ELx808 Ultra Microplate Reader (BioTek, Winooski, VT, USA). The percentage of inhibition in cell proliferation in drug-treated cultures against nondrug- treated control was calculated as follows: Inhibition (%) = (Drug-treated - Control)/Control ×100%.
Thesis/Dissertation
eng
Experimental Medicine
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
The mechanism of anti-proliferation of Halofuginone in T cells.
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