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1 THE ROLE OF SUPPRESSOR OF CYTOKINE SIGNALING 1 (SOCS-1) MIMETIC AND ANTAGONIST PEPTIDES IN HOST DEFENSE By REA DABELIC A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT O F THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010
2 2010 Rea Dabelic
3 To Angie and Claudio Dabelic, for encouraging my curiosity and indulging my desire for learning
4 ACKNOWLEDGMENTS I would like to thank m y mentor, Dr. Howard M. Johnson, for taking me into his lab, for his financial and moral support, for the long hours of beneficial discussions, and for helping me think in technicolor. I would also like to thank the members of my graduate committee, Drs. J oseph Larkin, Janet Yamamoto, Peter Kima, and Marta Wayne for their patience, helpful suggestions, and time. This research would not have been completed without the aid of the members of the Johnson lab. Lindsey Jager has provided all kinds of aid that wou ld require many pages of thanks, so I would like to thank her profusely. I would also like to thank Dr. Lilian Waiboci for teaching me many of the assays and skills needed to perform all of this research. Dr. Iqbal Ahmed has been especially helpful with hi s molecular biology expertise and his humorous outlook on life. I would like to thank Tim Johnson for making sure the lab is always stocked and that we have all of the supplies we need. I would also like to thank Mohammad Haider for synthesizing the peptid es used in this study, and Ezra Noon -Song and James Martin for their helpful suggestions and assistance. I would also like to thank the members of the Larkin lab, Erin Collins, Ken Lau, and Patrick Benitez, and the rest of the graduate students in the department for their assistance and support. I would like to thank Dr. Shanmugam, Janet Lyles, and Mary Ann Soncrant for making my graduate studies run as smoothly as possible. I wish to thank the staff at the fiscal office, Joni Teerlink, Renee Patterson, Chr is Gough, Trisha Norris, and Anna Studstill for their assistance with supplies and transportation. I would also like to thank Kathy Pons and Sheila Yarber for keeping everything neat and clean.
5 I would like to profusely thank my parents, Angie and Claudio Dabelic, for all of the sacrifices that they have made to provide a chance for a better life for my sister and me. I would like to thank my sister, Zrinka Dabelic, for many late night phone calls and trips to Gainesville for moral support and pet -sitting s ervices. I also wish to thank Justin Reed for his words of encouragement and support in difficult times. Finally, I would like to thank Enzio Dabelic for his patience, unconditional love and understanding.
6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...................................................................................................... 4 LIST OF TABLES ................................................................................................................ 8 LIST OF FIGURES .............................................................................................................. 9 LIST OF ABBREVIATIONS .............................................................................................. 10 ABSTRACT ........................................................................................................................ 14 CHAPTER 1 INTRODUCTION ........................................................................................................ 16 Signaling Through the Janus Kinase (JAK)/ Signal Transducers and Activators of Transcription (STAT) Pathway ............................................................................ 16 The JAK Family of Kinases .................................................................................. 16 The STAT Family ................................................................................................. 19 Interferons ................................................................................................................... 22 The Interferon Family ........................................................................................... 22 Interferon Signaling Through the JAK/STAT Pathway ....................................... 23 Regulation of Cytokine Signaling ............................................................................... 24 Suppressors of Cytokine Signaling (SOCS) .............................................................. 24 SOCS Structur e and Function ............................................................................. 25 The Physiological Role of SOCS -1 ..................................................................... 26 The Role of SOCS 1 in Innate Immunity ............................................................. 27 The Role of SOCS 1 in Infection ......................................................................... 28 Specificity of Cytoki ne Signaling ................................................................................ 29 Small Molecule Mimetic and Antagonist Peptides ..................................................... 31 SOCS-1 Mimetic Peptides ................................................................................... 31 SOCS-1 Antagonist Peptide ................................................................................ 33 IFN Mimetic Peptide ........................................................................................... 34 2 MATERIALS AND METHODS ................................................................................... 42 Peptide Synthesis ....................................................................................................... 42 Cell Culture and Virus ................................................................................................. 42 Mice ............................................................................................................................. 42 Binding Assays ........................................................................................................... 43 Toxicity Studies ........................................................................................................... 44 Western Blot Analysis ................................................................................................. 44 Macrophage Activation ............................................................................................... 44 Interferon ELISA ...................................................................................................... 45 Titration of Virus .......................................................................................................... 45
7 Antiviral Assays ........................................................................................................... 46 Statistical Analysis ...................................................................................................... 46 3 CHARACTERIZATION OF THE SOCS 1 MIMETIC PEPTIDES .............................. 48 SOCS-1 Mimetics Recognize the Autophosphorylation Sites of Different JAKs ...... 48 SOCS-1 Mimetics are Not Cytotoxic .......................................................................... 49 SOCS-1 Mimetics Inh ibit STAT Phosphorylation ...................................................... 49 The Effect of SOCS -1 Mimetics on Macrophage Activation ..................................... 50 The Effects of SOCS1 -KIR on SOCS -1/ Mice .......................................................... 51 4 CHARACTERIZATION OF THE SOCS 1 ANTAGONIST PEPTIDE ........................ 61 The SOCS1 Antagonistic Activity of pJAK2(10011013) ......................................... 61 The SOCS 1 Antagonist Increases STAT1 Phosphorylation .................................... 61 The SOCS 1 Antagonist Increases Endogenous IFN Levels ................................. 61 The Effects of the SOCS 1 Antagonist on Macrophage Activation .......................... 62 The SOCS 1 Antagonist is Not Cytotoxic .................................................................. 63 The SOCS 1 Antagonist Possesses Antiviral Activity Against a Picornavirus ......... 63 5 DISCUSSION .............................................................................................................. 74 LIST OF REFERENCES ................................................................................................... 82 BIOGRAPHICAL SKETCH ................................................................................................ 91
8 LIST OF TABLES Table page 1 -1 Ligands that signal using Janus kinases (JAKs) and signal transducers and activators of transcription (STATs). ....................................................................... 36 1 -2 Phenotypes of JAK knockout mice. ....................................................................... 39 1 -3 Phenotypes of STAT knockout mice. .................................................................... 39 1 -4 Cytokines regulated by suppr essors of cytokine signaling (SOCS). .................... 39 2 -1 Peptides used in this study. ................................................................................... 47 4 -1 Yield reduction of herpes simplex virus 1. ............................................................. 71
9 LIST O F FIGURES Figure page 1 -1 Model of the Janus kinase (JAK) and signal transducers and activators of transcription (STAT) signaling pathway ................................................................. 37 1 -2 The JAK homology domains .................................................................................. 38 1 -3 Structure of the suppressor of cytokine signaling (SOCS) proteins ..................... 40 1 -4 Mechanisms for the regulatory activity of SOCS 1 on Toll like receptors (TLRs) ..................................................................................................................... 41 3 -1 Autophosp horylation sites of the JAKs .................................................................. 53 3 -2 SOCS-1 mimetics bind to the autophosphorylation site peptides of the JAKs .... 54 3 -3 SOCS-1 mimetic peptides are not cytotoxic .......................................................... 55 3 -4 SOCS-1 mimetic peptides inhibit STAT phosphorylat ion ..................................... 56 3 -5 SOCS-1 mimetic peptides inhibit lipopolysac charide (LPS) induced macrophage activation ........................................................................................... 57 3 -6 SOCS-1 mimetic peptides regulate TLR4 signaling ............................................. 58 3 -7 SOCS1 -KIR inhibits MyD88 adapter like (MAL) protein degradation ................... 59 3 -8 SOCS1 -KIR prolongs survival of SOCS -1/ mice ................................................. 60 4 -1 The SOCS 1 antagonist peptide increases STAT1 phosphorylation ................... 67 4 -2 The SOCS 1 antagonist increases endogenous interferon levels .................... 68 4 -3 The SOCS 1 antagonist increases macrophage activation .................................. 69 4 -4 The SOCS 1 antagonist is not cytotoxic ................................................................ 70 4 -5 The SOCS 1 antagonist has antiviral activity against encephalomyocarditis v irus (EMCV) .......................................................................................................... 72 4 -6 The SOCS 1 antagonist protects mice from lethal EMCV infection ..................... 73
10 LIST OF ABBREVIATION S AIDS acquired immune deficiency syndrome BLAST basic local alignment search tool Btk Brutons tyrosine kinase CIS cytokine inducible Src homology 2 containing protein CLC cardiotropinlike cytokine CNTF ciliary neurotrophic factor CPE cytopathic effect CT -1 cardiotrophin 1 CTL cytotoxic T cell DC dendritic cell DMSO dime thyl sulfoxide dsDNA double stranded DNA EAE experimental allergic encephalomyelitis EGFR epidermal growth factor receptor ELISA enzyme -linked immunosorbent assay ELISPOT enzyme -linked immunosorbent spot EMCV encephalomyocarditis virus EPO erythropoietin ESS extended SH2 sequence G -CSF granulocyte colony -stimulating factor GAF interferon activated factor GAS interferon activated sequence GFP green fluorescent protein GM -CSF granulocyte macrophage colony -stimulating factor
11 HPLC high performance liquid chromatography HSV-1 herpes simplex virus 1 IFN interferon IFNAR interferon receptor IFNGR interferon receptor IGF insulin -like growth factor IL interleukin i.p. intraperitoneal IRAK IL -1 receptor associated kinase IRF interferon regulatory factor ISG in terferon-stimulated gene ISGF interferon-stimulated gene factor ISRE interferon-stimulated response element JAB Janus kinase binding protein JAK Janus kinase JH JAK homology KIR kinase inhibitory region LIF leukemia inhibitory factor LPS lipopolysaccharide MAL MyD88 adapter -like MHC major histocompatibility complex moi multiplicity of infection NF B nuclear factor B NO nitric oxide OSM oncostatin M
12 PAMP pathogen associated molecular pattern pfu plaque forming unit PIAS protein inhibitors of activated sign al transducers and activators of transcription Poly I:C polyriboinosinic:polyribocytidylic acid RBX2 really interesting new gene (RING) box 2 SCID severe combined immunodeficiency SH2 Src homology 2 SHP SH2 -containing tyrosine phosphatase siRNA small inter fering RNA SOCS suppressor of cytokine signaling SPF specific pathogen free STAT signal transducer and activator of transcription TAK1 transforming growth factor activated kinase 1 TGF transforming growth factor TH1 T helper 1 TH2 T helper 2 Tkip tyro sine kinase inhibitor peptide TLR Toll -like receptor TNF tumor necrosis factor TPO thrombopoietin TRAF tumor necrosis factor receptor associated factor TRAM translocating chainassociating membrane protein TRIF Toll/interleukin 1 domain-containing adapt er -inducing interferon TRP2 tyrosine -related protein 2
13 TSLP thymic stromal lymphopoietin TYK tyrosine kinase VSV vesicular stomatitis virus
14 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy THE ROLE OF SUPPRESSOR OF CYTOKINE SIGNALING 1 (SOCS-1) MIMETIC AND ANTAGONIST PEPTIDES IN HOST DEFENSE By Rea Dabelic May 2010 Chair: Howard M. Johnson Major: Microbiology and Cell Science Suppressors of cytokine signaling (SOCS) are negative regulators of both innate and adaptive immunity via inhibition of signaling by cytokines and Toll -like receptors. In this study, we report on the development of SOCS 1 mimetic peptides SOCS1 -KIR and Tkip, and a SOCS -1 antagonist peptide pJAK2(1001 1013). The mimetic peptides imitate SOCS 1 by binding to the autophosphorylation sites of all of the Janus kinases (JAKs) and by inhibiting the activation of several signal transducers and activators of transcription (STATs). Moreover, they also directly inhibit Toll like receptor (TLR) 4 signaling by inactivating the MyD88 adapter -like protein (MAL). Mice deficient in SOCS-1 die on average by 15 days of age, but when administered SOCS1-KIR they are able to survive to weaning. The SOCS 1 antagonist peptide inhibits the replication of vaccinia virus, herpes simplex virus 1 (HSV 1), and encephalomyocarditis virus (EMCV) in cell culture, suggesting that it possesses broad antiviral activity. In addition, p JAK2(1001 1013) increases the intracellular level of IFN and STAT1 phosphorylation, which may play a role in the antagonist antiviral effect at the cellular level. Antibody neutralization suggests that the endogenous IFN may act intracellularly. pJAK2(10 01 1013) also synergizes with an IFN mimetic peptide,
15 IFN (95 132), to exert a multiplicative antiviral effect in vitro and in vivo In addition to its antiviral properties, pJAK2(1001 1013) enhances TLR3 and TLR4 activation. The SOCS-1 antagonist present s a novel approach to enhancement of host defense against viruses, while the SOCS 1 mimetics present a way to control inflammation and may be potential therapeutics for autoimmune diseases such as multiple sclerosis.
16 CHAPTER 1 INTRODUCTION Signaling Thr ough the Janus Kinase (JAK)/Signal Transducers and Activators of Transcription (STAT) Pathway The ability of cells to respond to distinct stimuli from the environment is the key to the complexity of multicellular organisms. Cytokines and their receptors ar e the major players in this response (Borden et al., 2007). Many hormones and cytokines use the Janus kinase (JAK) family of kinases and the signal transducers and activators of transcription (STAT) family to initiate intracellular signaling ( Table 1 1 ). A few examples are interferons (IFNs), some interleukins (ILs), erythropoietin (EPO), growth hormone, insulin, leptin, and lactin (OSullivan et al., 2007). The current model for signaling through the JAK/STAT pathway (illustrated in Figure 11 ) involves a cytokine binding to its receptor(s), which activates the JAKs that are associated with their receptor subunits via JAK binding sites located proximal to the membrane (Miura et al., 1994; VanderKuur et al., 1994; DaSilva et al., 1994). The JAKs then phosphorylate the cytoplasmic domain of their receptors, creating docking sites for the Src homology 2 (SH2) domain of STATs. Once the STATs are recruited to the receptor, they are phosphorylated on key tyrosine and serine residues by JAKs and other kinases. This activation of the STATs allows them to form homoor hetero dimers that subsequently translocate to the nucleus to bind specific sequences on the genomic DNA to activate gene expression (reviewed in OShea et al., 2002). The JAK Family of Kinases The JAK family of cytoplasmic kinases consists of four members in the mammalian system: JAK1, JAK2, JAK3, and tyrosine kinase 2 (TYK2). They are differentially activated in response to various cytokines, and are expressed in many
17 types of tissue with the exceptio n of JAK3, which is limited to cells of the hematopoietic system (Ihle, 1995). The JAKs are large tyrosine kinases (120 140 kDa) that are associated with the cytoplasmic regions of cytokine receptor subunits (Haan et al., 2006). They consist of seven highl y conserved JAK homology (JH) domains (illustrated in Figure 1 -2 ) (Wilks et al., 1991). The JH1 domain, located at the C -terminus, is a classical kinase domain that is required for kinase activity. It contains the activation loop that becomes phosphorylated at a critical tyrosine residue (Y), located around position 1000 in all four JAKs, which activates the kinase. This autophosphorylation site is critical for the activity of JAKs as well as for downstream signaling events (Feng et al. 1997). Phosphorylat ion of this tyrosine residue causes a conformational change in the activation loop, allowing substrate access to binding sites in the catalytic groove (Yasukawa et al. 1999). Comparison of the amino acid sequence of the autophosphorylation site of JAK2 (1 001LPQDKEYYKVKEP) showed 100% sequence identity among mammalian species including human, mouse, rat, and pig, as determined using the basic local alignment search tool (BLAST, http://www.ncbi.nlm.nih.gov/b last/ ). Among all four JAK kinases, the protein sequences of the autophosphorylation sites are very similar, highlighting the importance of the autophosphorylation site in JAK function. Preceding the JH1 domain N -terminally is the JH2 domain, a pseudokin ase domain thought to be involved in the autoregulatory activity of JAKs (reviewed in Haan et al., 2006). It has a classical kinase domain fold but lacks the residues required for catalytic activity and for nucleotide binding. There is an SH2 region spanni ng the JH3
18 and JH4 domains. The JH3 JH7 domains comprise the rest of the N terminus, and are involved in cytokine receptor binding. The JAK kinases are linked to signaling by cytokines of the hematopoietic system, including interleukins, colony -stimulatin g factors, interferons, erythropoietin, and thrombopoietin (reviewed in Khwaja, 2006). The majority of these cytokines bind to a family of transmembrane receptors, either monomeric or heterodimeric, that share structural features. The heterodimeric recepto rs share a common signaling subunit and a unique ligandbinding chain (reviewed in Rane and Reddy, 2002). These can further be grouped into receptors which share the common -chain (granulocyte macrophage colony -stimulating factor, IL -3, IL -5), the gp130 s ubunit (IL6, leukemia inhibitory factor, oncostatin M, IL11), or the common -chain (IL2, IL 4, IL 7, IL 9, IL13, IL15). The single chain and heterodimeric group together make up the type I cytokine receptors, which are characterized by the presence o f a WSXWS motif, fibronectin type III domains in the extracellular part of the receptor, and by conserved Box1/Box2 regions in the membrane proximal cytoplasmic domain (Khwaja, 2006). The type II cytokine receptors include the interferon and IL10 receptor s, and they lack the WSXWS motif but do have the Box1/Box2 region (Khwaja, 2006). Signaling via these cytokine receptors is initiated by ligand binding, which induces the dimerization or a conformational change of receptor subunits. The JAKs are constituti vely associated with the receptor subunits via their FERM domain and the receptor Box1 domain, thereby conferring the functional equivalent of a receptor tyrosine kinase, as these receptors lack this quality (Behrmann et al., 2004). Receptor oligomerization brings the associated JAKs to close proximity, allowing their autoor trans phosphorylation and activation.
19 The JAKs have a multitude of physiological roles, including regulating the cell surface expression of their associated receptors (Ragimbeau et al., 2003), and the recycling and degradation of their associated receptors (Gauzzi et al., 1997). Their uncontrolled activation has been implicated in numerous hematological malignancies, immunodeficiency syndromes, oncogenesis, myeloid proliferative disor ders, and cardiovascular diseases (reviewed in Sandberg et al., 2004; Khwaja, 2006). Gene targeting studies of the JAK kinases in mice show distinct phenotypes. ( Table 1 -2 ) (reviewed in Igaz et al. 2001). JAK1 knockout mice die perinatally due to profound defects in lymphoid development (Rodig et al., 1998), while JAK2 knockout mice die in the embryonic period due to a lack of erythropoiesis (Parganas et al., 1998). This correlates with the role of JAK2 in erythropoietin (EPO) and thrombopoietin (TPO) sign aling. Mice deficient in JAK3 are viable, owing to the limited expression of JAK3. They exhibit the murine severe combined immunodeficiency (SCID) phenotype that affects B and T -cell populations. TYK2 knockout mice are phenotypically normal but have been shown to have exercise intolerance compared to their littermates, suggesting a mitochondrial respiration disregulation (Potla et al. 2006). The STAT Family STAT proteins are a family of latent cytoplasmic transcription factors that, when phosphorylated on a tyrosine residue by a member of the JAK family, dimerize via their SH2 domains and translocate to the nucleus where they activate gene transcription (reviewed in Darnell, 1997). There are seven known mammalian STAT family members: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6. Each STAT is composed of five domains, including a four -helix bundle transactivation domain, a central -barrel DNA binding domain, a helical linker domain, an SH2 domain, and an
20 effector domain. The helical linker domain f orms a bridge between the DNA binding and SH2 domains. The C terminal transactivation domain, involved in communication with transcription complexes, has a conserved serine residue (not found in STAT2 or STAT6) that, when phosphorylated, regulates STAT transcriptional activity (Kovarik et al., 2001). The effector domain is involved in regulatory function of the STATs and may be responsible for their nuclear export. The N -terminal region of the STATs is highly conserved and contributes to the stability of ST AT DNA binding, thereby increasing the transcriptional activity (reviewed in Imada and Leonard, 2000). STATs bind to receptors via the interaction of their SH2 domains with their appropriate receptor docking site. Phosphorylation of a single tyrosine residue located around amino acid residue 700 in each of the STATs is required for their activation. The proteins responsible for the phosphorylation can be cytokine receptors with intrinsic tyrosine kinase activity, or receptors that are associated with JAKs. Once they are phosphorylated, the STATs form homoor heterodimers. This dimerization is required for DNA -binding activity, as monomers are not capable of binding DNA. The STATs that form heterodimers are STAT1:STAT2 and STAT1:STAT3, while the ones that f orm homodimers are STAT1, STAT3, STAT4, STAT5, and STAT6 (reviewed in Darnell, 1997). The physiological role of the STATs has been widely investigated in a wide variety of experimental models, but due to the complexity of the interactions between and among the various JAKs and STATs, the most clear -cut results were determined using STAT knockout mice (reviewed in Khwaja, 2006). Table 1 -3 summarizes their phenotypes. STAT1 deficient mice have no innate immune response to either bacterial
21 or viral infection, but if kept in a pathogen-free environment they are phenotypically normal and capable of reproducing (Meraz et al., 1996). Knocking out the STAT4 gene resulted in mice deficient in T helper 1 (TH1) cell function. Likewise, STAT6 null mice had deficient T h elper 2 (TH2) cell function. This is supported by findings that STAT4 is activated in response to IL12, a cytokine that drives T cells to the TH1 phenotype (Kaplan et al., 1996), and that STAT6 is activated in response to IL-4, which promotes a TH2 phenot ype (Shimoda et al., 1996). Male mice deficient in STAT5A are phenotypically normal, while the female mice are not able to develop normal breast tissue and cannot lactate. Deficiency in STAT5B causes male mice to grow slowly, and their serum levels of live r -produced proteins are more characteristic of female mice. This sexual dimorphism is due to the activation of STAT5A and STAT5B by growth hormone (Udy et al., 1997). In addition, STAT5A and STAT5B deficiency in CD4+ T cells results in a significant reduct ion of CD4+CD25+Foxp3+ T cells in the thymus and periphery (Burchill et al., 2007; Yao et al., 2007). Further, humans with mutations in the STAT5A/B genes display immune disregulation that is associated with decreased CD25 and Foxp3 expression (Cohen et al ., 2006). Finally, the IL -12R chain -dependent activation of STAT5 is necessary for the development and homeostasis of T regulatory cells (Burchill et al., 2007). STAT3 is expressed in the visceral endoderm, whose function is required for gastrulation. In accord with these findings, STAT3 knockout mice die before reaching the gastrulation phase. STAT2 deficiency is also embryonically lethal, but the exact stage has not been determined (Darnell, 1997).
22 Interferons Interferons (IFNs) were first described over 50 years ago as agents that interfered with viral infection (Lindenmann et al., 1957). Since then, much work has been done on elucidating the wide variety of biological effects of the IFNs. This includes control over cell growth and tumors, as well as reg ulating both the innate and adaptive arms of the immune response (reviewed in Bonjardim et al., 2009). Interferons are now described as a family of secreted autocrine and paracrine proteins that serve to stimulate intracellular and extracellular networks t hat regulate resistance to viral infections, modulate normal and tumor cell survival and death, and enhance innate and adaptive immune responses (Borden et al., 2007). The Interferon Family There are three types of IFNs (reviewed in Bonjardim et al., 200 9). The type I IFNs include IFN (which in humans includes 13 members), , and These IFNs all have antiviral activity, share a high degree of homology in terms of protein sequence, and do not have introns. They signal through the type I IFN receptor subunits, IFN receptor 1 and 2 (IFNAR1 and IFNAR2). Type II IFNs have only one member, IFN It has antiviral activity like the type I IFNs, but also has an immunomodulatory function. IFN signals through the IFN receptor (IFNGR) subunits 1 and 2 (IFNGR1 and IFNGR2). A newly recognized IFN family is the type III IFNs, or IFN s, which are also known as IL -28A, IL-28B, and IL29. They signal through the IL 10 receptor and IL28 receptor chains to control cell proliferation and exert their antiv iral activity. Unlike the other IFNs, however, the type III IFNs contain introns (Kotenko et al., 2003).
23 Interferon Signaling Through the JAK/STAT Pathway All IFNs are helical cytokines that signal through the JAK/STAT pathway. The type I IFNs use IFNAR associated JAK1 and TYK2 to activate STAT1 and STAT2, which in combination with IFN -regulatory factor (IRF) 9 form the heterotrimeric transcription factor IFN -stimulated gene factor (ISGF) 3. ISGF3 then translocates to the nucleus to bind to IFN -stimulated response elements (ISREs) in the DNA to promote gene expression. IFN uses IFNGR associated JAK1 and JAK2 to activate STAT1, which dimerizes with itself to form the transcriptional regulator IFN activated factor (GAF). The GAF recognizes elements in the DNA termed the IFN activated sequences (GAS) and binds to them to initiate gene activation. The type III IFNs use JAK1 to activate STAT1, STAT2, and ISGF 3 to turn on the expression of IFN -stimulated genes (ISGs) that are also turned on upon type I IFN s timulation (reviewed in Bonjardim et al., 2009). As previously mentioned, the current model of JAK/STAT signaling shows the cytokine -stimulated receptor activation, followed by auto or transphosphorylation of the receptor associated JAKs, which also phosphorylate the receptor subunits to create docking sites for the SH2 regions of the STATs, causing them to form homoor heterodimers and translocate to the nucleus, where they activate gene expression. The IFN signaling pathway is illustrated using this sam e model, where the only function of the cytokines, receptors, and JAKs is to turn on the relevant STATs so they can activate gene expression. Recent studies have shown that, in the case of type II IFNs, the IFNGR1 and IFNGR2 receptor subunits come together upon stimulation with IFN IFN moves to the cytoplasm, followed by activation of JAK1, JAK2, and STAT1. The
24 IFNGR1 receptor subunit, IFN and STAT1 homodimer are subsequently translocated to the nucleus as a complex (Subramaniam et al., 2001; Ahmed et al., 2003; Johnson, 2004). Regulation of Cytokine Signaling Cytokine signaling has to be tightly regulated, as unregulated signaling could result in unwanted inflammation and/or autoimmune diseases that are harmful to the organism. There are multiple regul atory pathways to contain cytokine signaling, including activation of tyrosine phosphatases, receptor internalization, proteasomal degradation of signaling adapter molecules, soluble receptor agonists (usually employed by viruses), and specific inhibitors of cytokine signaling (Croker et al., 2008). These inhibitors are important not only for keeping the immune response in check, but also for the differentiation of the cells that are the responders of the immune system (Yoshimura et al., 2007). There are c urrently three known classes of regulators of the JAK/STAT pathway. They are the protein inhibitors of activated STATs (PIAS), the SH2-containing tyrosine phosphatases (SHPs), and the suppressors of cytokine signaling (SOCS). PIAS proteins regulate transcr iption through multiple mechanisms, one of which is blocking the DNA binding ability of transcription factors (Shuai, 2006). SHPs are phosphatases that dephosphorylate activated phosphotyrosine -containing proteins, such as JAKs and STATs (Lorenz, 2009). SO CS can regulate the JAK/STAT pathway in multiple ways, which are discussed in detail below. Suppressors of Cytokine Signaling (SOCS) The discovery of the SOCS proteins was published concurrently by three groups: as a JAK binding protein (JAB) (Endo et al., 1997), as a suppressor of IL-6 signaling
25 (Starr et al., 1997), and based on sequence homology with the STAT3 SH2 domain (Naka et al., 1997). The SOCS proteins have been shown to regulate over 30 different cytokines, including IL 6, leukemia inhibitory fac tor (LIF), IL -10, growth hormone, IFN and most recently IL17 and IL23 ( Table 1 4 ) (reviewed in Croker et al., 2008) SOCS Structure and Function There are currently eight members of the SOCS family: SOCS -1 through 7 and cytokine inducible SH2-containing protein (CIS). They each have a central SH2 domain, an N terminal domain of variable length and sequence, and a C -terminal SOCS box motif ( Figure 1 -3 ). The SOCS box interacts with elongins B and C, cullin-5, and RING box -2 (RBX2), which recruits the E2 ubiquitin transferase, allowing the SOCS proteins to act as E3 ubiquitin ligases, which mediate the proteasomal degradation of proteins that are associated with them (Kamura et al., 2004). The structure of the SOCS box of SOCS-3 in complex with elongins B an d C has recently been solved (Babon et al., 2008), as has the partial structure of SOCS 3 in complex with a peptide from the IL-6 receptor gp130 subunit (Bergamin et al., 2006). These studies showed that there is a great deal of disorder in the SOCS box of SOCS3 when it is not bound to a ligand, and that it is this flexibility that is a key feature of the interaction between SOCS -3 and its various ligands. SOCS-1 and SOCS -3 (and possibly SOCS -5 [Croker et al., 2008]) have an N -terminal kinase inhibitory re gion (KIR) that is important for their inhibitory effects on kinases such as JAK2 (Waiboci et al., 2007; Ahmed et al., 2009). The KIR consists of a 12 amino acid region that interacts specifically with the activation loop of JAK2 to inhibit its kinase acti vity (Waiboci et al., 2007; Ahmed et al., 2009). The importance of this
26 region in the binding of JAKs has been debated, with most of the binding being ascribed to the SH2 region. The SH2 region of the SOCS -1 and SOCS 3 proteins is preceded N terminally by a short sequence termed the extended SH2 sequence (ESS), and it has been shown to be critical in the binding of phosphotyrosine residues. It forms a 15 -residue helix that has been shown to directly contact the phosphotyrosine-binding loop and determines its orientation, providing structural integrity to the SOCS molecule (Sasaki et al., 1999; Babon et al., 2006). The central SH2 region of the SOCS proteins binds to phosphorylated tyrosine residues found on many proteins, including the IFNGR1 and IFNAR1 re ceptor subunits in the case of SOCS -1 (Fenner et al., 2006; Qing et al., 2005), and the gp130 (Schmitz et al., 2000) and IL12R 2 receptor subunits in the case of SOCS -3 (Yamamoto et al., 2003). The regulation of receptor phosphorylation by SOCS would allow for the suppressive effect on cytokine signaling even in the presence of low amounts of SOCS proteins (Yoshimura et al., 2007). The Physiological Role of SOCS 1 SOCS-1 is necessary for survival, as SOCS 1 knockout mice die as neonates due to low body wei ght, multi organ inflammation, necrosis of the liver, and monocytic infiltration of the pancreas, lung, and heart. In addition, their thymuses are severely reduced in size and they have a deficiency in mature B and T lymphocytes (Starr et al., 1997; Naka et al., 1997). The pathology in the SOCS -1 knockout mice is similar to that observed in wildtype mice treated with excess IFN This is complemented with the observation that SOCS-1/ -IFN / mice did not show the lethal phenotype of the SOCS -1/ mice. In
27 addition, SOCS -1/ mice treated from birth with neutralizing antibodies to IFN for three weeks remained viable and healthy while their untreated counterparts succumbed to disease (Alexander et al., 1999). These observations led to the conclusion that SO CS 1 is a key regulator of IFN signaling. However, the SOCS 1/ -IFN / mice eventually died by 6 months of age due to inflammation and polycystic kidneys, which would suggest that the regulatory abilities of SOCS -1 are not limited to IFN (Metcalf et al. 2002). The regulation of IFN by SOCS 1 was confirmed by injecting IFN into SOCS -1/ mice and looking for STAT1 phosphorylation levels. In wild-type mice, phosphorylated STAT1 was found in the liver 15 minutes after treatment, and it declined 2 hours a fter treatment. In the SOCS 1/ mice, phosphorylation of STAT1 was evident 8 hours after treatment, suggesting continuous, unregulated IFN signaling (Brysha et al., 2001). It has additionally been shown that SOCS 1 is also a regulator of type I IFN signaling. Using neutralizing antibodies to IFN and IFN and mice deficient in IFN IFNAR1 or IFNAR2, Fenner et al. (2006) demonstrated that SOCS 1 deficiency amplified type I IFN antiviral actions independently of IFN The Role of SOCS 1 in Innate Immunity Toll -like receptors (TLRs) are key players in the immune response to invading pathogens. There are over 10 recognized TLRs in humans and mice. They recognize pathogen associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS) or double -strande d DNA (dsDNA). These signals must be tightly regulated to avoid excessive inflammation and damage to the host (reviewed in Carpenter and ONeill, 2009). TLR ligands such as LPS and CpG -containing DNA are potent inducers of SOCS-1 (reviewed in Yoshimura et al., 2007).
28 Mice deficient in SOCS -1 are hyper responsive to LPS and hypersensitive to LPS induced lethality (Nakagawa et al., 2002). In vivo treatment with LPS results in a strong production of IFN IL -12, and nitric oxide (NO) (Kinjyo et al., 2002). Sev eral mechanisms for the regulatory activity of SOCS 1 on TLRs have been proposed and a few are shown in Figure 1-4 One mechanism is through SOCS -1 binding to p65 of nuclear factor B (NF B) and mediating its proteasomal degradation via the SOCS box (Ryo et al., 2003). SOCS -1 has been shown to bind to tyrosine phosphorylated MyD88 adapter like (MAL) protein and induce its proteasomal degradation (Mansell et al., 2006). MAL is an adapter protein that is used by MyD88dependent TLR signaling pathways, specif ically, those activated by TLR2 and TLR4 stimulation. After stimulation by TLR ligands, the MyD88 mediated signaling pathway leads to activation of NF B and induction of IFN and other inflammatory gene products. The induction of IFN leads to activation of JAK/STAT signaling, which is also regulated by SOCS -1 (Fenner et al., 2006). TLR activation is extremely important in pathogen clearance, but excessive activation can lead to harmful pathogenesis to the host. It is therefore important to be able to regulate overactive signaling in order to prevent fatal responses to systemic infection. The Role of SOCS 1 in Infection SOCS-1 is an important mediator of IFN signaling. As such, it contributes to the balance of beneficial antiviral and detrimental pro inflam matory effects of IFN signaling. It has been shown that SOCS 1/ mice are resistant to viral infection (Fenner et al., 2006). These mice responded to type I IFNs for a longer amount of time to clear virus more efficiently and therefore survive a lethal vi ral infection. In addition, the expression
29 of a dominant negative form of SOCS 1 in cardiac myocytes increased their resistance to enteroviral infection (Yasukawa, 2003). These data collectively indicate that low SOCS-1 protein levels are important for hos t defense against viral infection through their regulation of type I and II IFNs. SOCS-1/ mice are not only resistant to viral infection, but certain parasitic infections as well. SOCS 1 is directly induced by Toxoplasma gondii parasites, and is involved in IFN inhibition in infected cells (Zimmermann et al., 2006). The induction of SOCS-1 is therefore most likely a strategy to evade the immune response. Specificity of Cytokine Signaling With only four receptor associated JAK kinases and seven STATs being involved in the signaling of over 40 different cytokines and hormones, how is specificity of gene activation achieved? Further, we still do not understand the exact interactions between the JAKs and their receptors, and between JAKs and STATs, in terms o f protein structure. The exact sequence of events from ligand binding to the receptor, to the phosphorylation of the JAKs, to the recruitment and subsequent nuclear translocation of STATs, is still not clear. We do know that the JAK mediated phosphorylation of the receptor subunits creates docking sites on the receptor for the SH2 domain of the relevant STAT, and that this recruitment is followed by phosphorylation of critical tyrosine and serine residues on the STAT proteins. From here, the events are yet to be clearly understood. There are several theories on what happens after the STATs are recruited to the receptors. The classical model of cytokine signaling has the STATs forming homoor heterodimers and then translocating to the nucleus by themselves in order to activate
30 gene expression (reviewed in Borden et al., 2007). Recent studies in our lab based on signaling by IFN have proposed another model that is currently being tested using other cytokines that signal through the JAK/STAT pathway (Ahmed et al., 2003). Binding of IFN to the IFNGR1 and IFNGR2 receptor subunits causes the activation of JAK1 and JAK2 and induces binding of STAT1 to IFNGR1. The binding of IFN additionally initiates the selective endocytosis of IFNGR1 and the associated proteins JAK2 and STAT1 homodimer. Interestingly, IFN is found bound to the cytoplasmic domain of the IFNGR1 subunit, allowing for its C terminal nuclear localization sequence (NLS, 126RKRKRSR) to be exposed and recognized by the nuclear importin machinery, al lowing the entire complex to be translocated to the nucleus, where the complex binds to the GAS region of the IFN promoter. This model ascribes roles to the ligand and receptor beyond that of signal transduction initiators. As such, replacing the basic re sidues of IFN NLS with alanines and expressing the mutant intracellulary, failed to induce nuclear translocation of IFNGR1 or STAT1, and led to a loss of IFN activities (Ahmed et al., 2003). Further, in cells expressing the IFN NLS mutant, STAT1 was not translocated to the nucleus, even though it was phosphorylated and activated (Ahmed et al., 2003), suggesting that an important function of IFN in this model is to act as a chaperone for the nuclear import of activated STAT1. Another theory involves the tissue -specific expression of cytokine receptors. Different tissues and cell types express distinct receptor combinations unique to their microenvironment, allowing the cells to integrate signals from multiple cytokine receptors. The complication here is that cytokine receptors preferentially associate with certain JAKs. For example, the IFNGR complex only associates with JAK1 and JAK2
31 (OShea et al., 2002). This raises several questions: with over 40 different cytokines using only four JAKs, and their onl y function is to phosphorylate tyrosine residues on the receptors and STATs, why are the JAKs not interchangeable? Does one cytokine activate the same genes in every cell type that is responsive to it? What role do negative regulators of cytokine signaling such as the SOCS proteins, play in the specificity of signaling? The complexity of the subject is increased when the interactions among the different pathways are taken into account. It has been shown that some functions of the STATs are redundant, i.e. if one STAT is knocked out, another STAT can compensate (Gil et al., 2001; Ramana et al., 2001). Additionally, SOCS proteins such as SOCS -1 and SOCS -3 are induced by STATs, and while they have similar mechanisms of action, they are activated in a cytokine-specific fashion (Murray, 2007). Further, certain cytokines can induce the expression of receptors, coreceptors, or adapter molecules during a priming phase (Bezbradica and Medzhitov, 2009), enabling the cell to respond to the cytokine for a prolonged p eriod. This, in turn, further upregulates the expression of the negative regulators of cytokine signaling, like SOCS, which are able to modify the output of activated STATs and therefore alter downstream gene activation. Although the specificity of signaling is being intensively studied, there is still a large gap of knowledge in this field. With some of the results presented in this study and additional experiments being performed, we hope to fill some of these gaps. Small Molecule Mimetic and Antagonist P eptides SOCS -1 Mimetic Peptides Inflammatory diseases in which cytokine signaling is hyperactive could be treated using SOCS 1 proteins or SOCS -1 like molecules (mimetics). Hyperactive proinflammatory cytokine signaling has been implicated in the pathogen esis of autoimmune
32 diseases such as multiple sclerosis, rheumatoid arthritis, and inflammatory diseases of the gastrointestinal tract. The current treatment options for inflammatory and autoimmune diseases are limited to the treatment of symptoms, not the actual disorder itself. Our group has designed two SOCS -1 mimetic peptides, SOCS1-KIR and tyrosine kinase inhibitor peptide (Tkip). The first peptide to be made, Tkip, was designed to be complementary to the autophosphorylation site of JAK2, which encompa sses residues 10011013 and contains the important phospho -tyrosine 1007 that is required for JAK2 kinase activity. As such, Tkip has been shown to bind to the JAK2 autophosphorylation site peptide, as well as inhibit JAK2 autophosphorylation and the JAK2-mediated phosphorylation of the IFNGR1 subunit. Tkip also inhibited the ability of IFN to upregulate major histocompatibility complex (MHC) I, and the ability to induce an antiviral state. In addition to its regulatory activity of IFN Tkip was shown to inhibit epidermal growth factor receptor (EGFR) autophosphorylation (Flowers et al., 2004). Tkip was also shown to protect mice from experimental allergic encephalomyelitis (EAE), a mouse model of multiple sclerosis (Mujtaba et al., 2005). These data colle ctively would suggest that Tkip is able to act as a SOCS 1 mimetic, and that it has the potential to be used as a therapeutic treatment in autoimmune diseases such as multiple sclerosis. The SOCS1-KIR peptide contains the 12-residue sequence that makes up the kinase inhibitory region of SOCS -1. It has been shown to bind to the autophosphorylation site peptide of JAK2 in a similar, but not identical, way to Tkip. SOCS1 -KIR was also shown to inhibit the IFN -induced STAT1 phosphorylation in a
33 manner similar t o Tkip, the IFN -induced activation of macrophages and antigenspecific splenocyte proliferation, and EGFR autophosphorylation, similar to Tkip (Waiboci et al., 2007). As for Tkip, these data suggest that SOCS1-KIR acts as a SOCS-1 mimetic. In this study, we will show that SOCS1 -KIR and Tkip are able to act as SOCS -1 mimetics. They are able to bind to and regulate the activity of all four JAKs, and they are able to decrease or inhibit the activation of STATs in response to cytokines that use the type II cyt okine receptors. In addition, the SOCS 1 mimetics are able to decrease macrophage activation in response to TLR ligands such as lipopolysaccharide (LPS). Further, SOCS1 -KIR is able to partially remedy the pathophysiology of SOCS -1 knockout mice and prolong their survival. SOCS -1 Antagonist Peptide The ability of SOCS1KIR to bind to the autophosphorylation site peptide of JAK2, pJAK2(1001 1013), would suggest that pJAK2(1001 1013) has the ability to act as a SOCS-1 antagonist. LNCaP cells overexpressing SO CS 1 showed a reduced level of IL -6 -induced STAT3 phosphorylation that was increased 2-fold when treated with pJAK2(1001 1013) peptide. In addition, pJAK2(1001 1013) was able to enhance IFN function at the level of gene activation, as well as the antiviral activity of IFN against encephalomyocarditis virus (EMCV) in mice (Waiboci et al., 2007). Therefore, pJAK2(1001 1013) can be classified as a SOCS -1 antagonist. This has implications for treatments of diseases and infections where an increase in IFN sign aling and decrease in SOCS -1 protein levels would be advantageous in order to clear an invading pathogen such as T. gondii which has been shown to induce SOCS 1 in order to evade the
34 immune response (Zimmermann et al., 2006), or viruses that have similar immune evasion strategies. In this study, we will show that pJAK2(1001 1013) is able to activate STAT1 in the absence of added cytokine stimulation, and that it is able to increase the activation of macrophages in response to TLR ligands that are encounter ed during bacterial or viral infection. In addition, pJAK2(1001 1013) is able to increase levels of endogenous IFN while simultaneously decreasing levels of SOCS 1. Further, pJAK2(1001 1013) can be used to protect cells and mice from infection by various viruses, suggesting that it can be used as a broad spectrum antiviral. IFN Mimetic Peptide With the studies performed in our lab on the signaling of IFN we have identified the C -terminal domain of IFN as having biological function, using a synthetic peptide approach (Ahmed et al., 2003). We made a peptide corresponding to a portion of the C -terminal portion of murine IFN spanning residues 95 132, IFN (95 132). It has been shown to bind to a soluble IFNGR1 chain that lacked the transmembrane domain (Jo hnson et al., 2004), suggesting a possible interaction with the cytoplasmic portion of the receptor subunit. This was confirmed by synthesizing overlapping peptides corresponding to the cytoplasmic portion of IFNGR1 and testing the binding ability of IFN (95 132) (Subramaniam et al., 2001). The IFN (95 132) peptide was further shown to have IFN -like biological activity, including upregulation of MHC class I expression, and reduction of vesicular stomatitis virus (VSV) titer (Szente and Johnson, 1994; Szent e et al., 1994). In addition, the IFN (95 132) peptide was able to form a complex with IFNGR1, STAT1, and the nuclear import machinery (Subramaniam et al., 2000).
35 Collectively, these data suggest that IFN (95 132) can function as an IFN mimetic. In this s tudy, we show that IFN (95 132) can synergize with pJAK2(1001 1013) to protect cells and mice from viral infection.
36 Table 1 1. Ligands that signal using Janus kinases (JAKs) and signal transducers and activators of transcription (STATs). Ligands JAKs ST ATs Erythropoietin JAK2 STAT5 Growth Hormone JAK2 STAT3, STAT5 Thrombopoietin JAK2 STAT5 IL 2 JAK1, JAK3 STAT3, STAT5 IL 4 JAK1, JAK3 STAT6 GM CSF JAK2 STAT5 IL 6 JAK1, JAK2, TYK2 STAT1, STAT3 LIF JAK1, JAK2, TYK2 STAT1, STAT3, STAT5 G CSF JAK1, J AK2, TYK2 STAT3 Leptin JAK2 STAT3, STAT5, STAT6 IL 12 JAK2, TYK2 STAT1, STAT3, STAT4, STAT5 IL 23 JAK2, TYK2 STAT1, STAT3, STAT4, STAT5 Type I IFNs JAK1, TYK2 STAT1, STAT2, STAT3 6 IFN JAK1, JAK2 STAT1 IL 10 JAK1, TYK2 STAT1, STAT3, STAT5 IL 11 JAK 1, JAK2, TYK2 STAT3 IL 3 JAK2 STAT3, STAT5 IL 5 JAK2 STAT1, STAT3, STAT5 Angiotensin JAK2, TYK2 STAT1, STAT2, STAT3 Serotonin JAK2 STAT3 Thrombin JAK2 STAT1, STAT3 IL 7 JAK1, JAK3 STAT3, STAT5 IL 9 JAK1, JAK3 STAT1, STAT3, STAT5 Insulin JAK1 STAT1, STAT5 Adapted with modifications from Schindler, 2002.
37 Figure 11. Model of the Janus kinase (JAK) and signal transducers and activators of transcription (STAT) signaling pathway. A ligand binds to its receptor(s), activating the JAKs that are associ ated with their receptor subunits. The JAKs then phosphorylate the cytoplasmic domain of their receptors, creating docking sites for the SH2 domain of STATs. Once the STATs are recruited to the receptor, they are phosphorylated on key tyrosine and serine r esidues by JAKs and other kinases. This activation of STATs allows them to form homo or hetero dimers that subsequently translocate to the nucleus to bind specific sequences on the genomic DNA to activate gene expression.
38 Figure 12. The JAK homology d omains. The JH1 domain is a classical kinase domain that contains Y1007 of JAK2, required for kinase activity. The JH2 domain is a pseudokinase domain. There is an SH2 region spanning the JH3 and JH4 domains. The JH3 JH7 domains comprise the rest of the N terminus, and are involved in cytokine receptor binding. Adapted with modifications from Imada and Leonard, 2000.
39 Table 1 2. Phenotypes of JAK knockout mice. Targeted Gene Phenotype JAK1 Die perinatally, small at birth, no nursing JAK2 Embryonic lethal, no erythropoiesis JAK3 SCID TYK2 Normal phenotype Adapted with modifications from Igaz et al., 2001. Table 1 3. Phenotypes of STAT knockout mice. Targeted Gene Phenotype STAT1 No innate response to viral or bacterial infection STAT2 Viable and fert ile, defective type I IFN functions STAT3 Early embryonic lethal STAT4 No T H 1 function STAT5A No breast development or lactation STAT5B No breast development or lactation STAT6 No T H 2 function Adapted with modification from Darnell, 1997. Table 1 4. Cytokines regulated by suppressors of cytokine signaling (SOCS). SOCS Regulates CIS Growth hormone, prolactin, IL 3 SOCS-1 IFN IFN IFN IL -2, IL3, IL4, IL 6, IL 7, IL 10, IL-12, IL 13, IL-15, IL 17, prolactin, erythropoietin, OSM, TSLP, TNF TPO LIF, LPS SOCS 2 Growth hormone SOCS 3 IL -1, TGF IL 6, IL 10, IL-11, IL 12, IL-17, IL 23, IL-27, G -CSF, leptin, LIF, OSM, CT 1, CNTF, CLC SOCS 4 EGFR SOCS 5 IL 4, EGFR SOCS 6 Insulin SOCS 7 Insulin, IGF Adapted with modifications from Croker et al., 2008.
40 Figure 13. Structure of the suppressor of cytokine signaling (SOCS) proteins. The function of the SOCS box is to recruit the ubiquitintransferase system. SOCS-1 and SOCS -3 contain a kinase inhibitory region (KIR) immediately upstream of the central SH2 domain, which inhibits the catalytic activity of JAKs by binding to the activation loop. Point mutations in this region completely abolish the suppressive effect of SOCS -1 and SOCS -3 on cytokine signaling. Adapted with modifications from Yoshim ura et al., 2007.
41 Figure 14. Mechanisms for the regulatory activity of SOCS -1 on Toll like receptors (TLRs ). Activation of TLR4 by LPS transmits signals through adapter proteins MyD88 MAL TRIF and TRAM NF is activated by TRAF6 and TAK1 through MyD88 and MAL, whereas IRF3 is activated by TRIF and TRAM. apidly induced through the TRIF/ IRF3 pathway and activates the JAK/STAT pathway. The JAK2/ STAT5 pathway has also been shown to be activated by LPS and is responsible for IL6 production. Phosphorylated MAL interacts with SOCS 1, which results in MAL inactivation, ubiquitination and subsequent degradation. SOCS -1 also binds to the p65 subunit of NF induces its degradation. Adapted with modi fications from Yoshimura et al., 2007.
42 CHAPTER 2 MATERIALS AND METHOD S Peptide Synthesis The peptides used in this study were synthesized on an Applied Biosystems 431A automated peptide synthesizer (Applied Biosystems, Carlsbad, CA), using conventional f luorenylmethyloxycarbonyl (f -moc) chemistry, as previously described (Szente et al., 1994). To ensure cell penetration, a lipophilic palmitoyl lysine was added to the N -terminus of each peptide used for cell culture or animal studies, using a semi automated protocol (Thiam et al., 1999). The peptides were characterized by mass spectrometry, purified as needed by highperformance liquid chromatography (HPLC), and dissolved in dimethyl sulfoxide (DMSO) or phosphate buffered saline (PBS) (Sigma -Aldrich, St. Lo uis, MO). The sequences of the peptides used in this study are presented in Table 2-1. Cell Culture and Virus L929, WISH, RAW264.7, and HEL-30 cells were obtained from ATCC (Manassas, VA) and propagated on DMEM with 10% fetal bovine serum (L929, WISH), EME M with 10% calf serum (HEL30), or RPMI 1640 with 10% fetal bovine serum (RAW264.7) supplemented with penicillin/streptomycin. All cells were grown at 37 C in humidified atmosphere with 5% CO2. Encephalomyocarditis virus (EMCV) was grown and titrated on L9 29 cells, as described (Mujtaba et al., 2006). Herpes simplex virus 1 (HSV 1) was a kind gift from Dr. Nancy Bigley (Wright State University, Dayton, OH). Mice All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Florida. All mice were housed in standard
43 specific pathogen free (SPF) facilities. For viral infections, female C57BL/6 mice (6-8 weeks old) were purchased from Jackson Laboratories (Bar Harbor, ME). Peptides diluted in PBS in a volume o f 100 l were administered i.p. Fifty pfu of EMCV were diluted into PBS in a volume of 100 l and administered i.p. Mice were monitored twice per day for signs of disease, such as lethargy, ruffled hair, weight loss, and eye secretions. Moribund mice were euthanized. SOCS-1+/ mice were purchased from St. Jude Childrens Hospital (Memphis, TN). Peptides were diluted in PBS and 10 g of peptide per gram of body weight was administered daily i.p. in a volume of 30 l for neonates, and 100 l for adults. The m ice were weighed daily and monitored for signs of inflammation. Binding Assays Peptides (3 g) were bound to 96well plates in binding buffer (0.1 M sodium carbonate -sodium bicarbonate, pH 9.6). The wells were washed three times with wash buffer (0.9% NaCl and 0.05% Tween -20) and incubated in blocking buffer (2% gelatin and 0.05% Tween -20 in PBS) for 1 h at room temperature. Then, the wells were washed three times with wash buffer and incubated with various concentrations of biotinylated peptides for 1 h at room temperature. The wells were washed five times with wash buffer and bound biotinylated peptides were detected using HRP -conjugated neutravidin (Invitrogen, Carlsbad, CA) and o phenylenediamine in stable peroxidase buffer (Pierce Biochemicals, Rockford IL). The chromogenic reaction was stopped by the addition of 2 M H2SO4 (50 l) to each well. Absorbance was measured at 490 nm using a microplate reader (Bio-Tek, Winooski, VT).
44 Toxicity Studies Cell viability experiments were performed on murine L929 ce lls and splenocytes isolated from healthy SJL/J mice using CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI). Cells were plated out to confluence, and then incubated at 37 C with 5% CO2 with medium or various peptides. After 24 h, the Solution Reagent was added and the cells were incubated for 2 h at 37 C, and the absorbance was measured at 490 nm using a microplate reader (BioTek, Winooski, VT). Western Blot Analysis Cells were incubated with various peptides or cytokines. T he cells were washed in cold PBS and harvested in RIPA buffer containing protease and phosphatase inhibitor cocktails (Santa Cruz Biotechnology Inc., Santa Cruz, CA). Protein concentration was measured using a BCA kit (Pierce Biochemicals, Rockford, IL) an d lysates were resolved with SDS PAGE, transferred onto nitrocellulose membranes, and probed with various antibodies. Detection of proteins was accomplished using ECL Protein Detection Reagents (Amersham Biosciences, Piscataway, NJ). Macrophage Activation Murine macrophages (RAW264.7) were seeded on 24 well plates at a concentration of 3 x 105 cells/well and allowed to adhere. Varying concentrations of peptides were then added to the cells and incubated for 2 h at 37 C in 5% CO2. Purified lipopolysaccharide (LPS) at 2 g/ml, or poly I:C (Sigma -Aldrich, St. Louis, MO) at 0.1 g/ml were then added and incubated for an additional 48 h at 37 C. Supernatants were transferred into fresh tubes and assayed for nitrite levels as a measure of nitric
45 oxide production using Griess reagent according to manufacturers instructions (Alexis Biochemicals, Plymouth Meeting, PA). Interferon ELISA L929 cells were treated with peptides for 30 or 60 min and then lysed with RIPA lysis buffer containing protease inhibitor cocktail s (Sigma Aldrich, St. Louis, MO). The cell lysates were analyzed with a murine IFN ELISA kit (PBL Biomedical Laboratories, Piscataway, NJ), following manufacturers instructions. Briefly, cell lysates were plated onto plate strips for 1 h at room temperat ure. The strips were washed three times with wash buffer, and then incubated with the Antibody Solution for 1 h at room temperature. After washing the strips three times, they were incubated with the HRP Solution for 1 h at room temperature. The strips wer e washed three times and incubated with TMB Substrate Solution for 15 minutes at room temperature. The reaction was stopped by addition of Stop Solution, and the absorbance was measured at 450 nm with a standard plate reader (BioTek, Winooski, VT). Titrati on of Virus HEL -30 keratinocytes were seeded onto 6well plates at 2 x 104 cells/well and grown to confluence. Various peptides were added to the cells for 24 h, followed by removal of peptides and washing of cells with PBS. HSV -1 was then added to the cel ls at a multiplicity of infection (moi) of 0.1 for 1 h at 37 C. Then the virus was removed, the cells were washed, and fresh maintenance media was added. The cells were incubated for 2 days at 37 C in 5% CO2. The media were collected and the viral titer wa s determined using a standard plaque assay protocol.
46 Antiviral Assays Murine L929 cells were seeded in a 96well plate at a cell density of 6 x 104 cells/well, and grown to confluence. Various concentrations of peptides were added and incubated for 2 h, af ter which 200 pfu of EMCV was added and incubated for 1 h. Then, the virus was removed, the cells were washed with PBS, and fresh maintenance media was added. After 24 h of incubation at 37 C, 5% CO2, the cells were washed and stained with 0.1% crystal violet. Unbound crystal violet was removed and the plates were thoroughly rinsed with deionized water, blotted, and allowed to air dry. The plates were then scanned and analyzed using ImageJ 1.29 software (National Institutes of Health, Bethesda, MD) to asses s cell survival. Percentages of cell survival were determined by comparing survival for the experimental treatment groups with that for the virus only control group. Statistical Analysis Statistical differences between groups were determined by ANOVA with multiple comparisons. The Students t test was used for comparisons when only two parameters were evaluated. Binding and proliferation assays were analyzed using the MannWhitney U test. p values < 0.05 were considered significant. Mouse survival data are presented as KaplanMeier plots, and analyzed using the log-rank test. Experimental data were measured for statistical significance using the GraphPad Prism software from GraphPad Software, Inc., San Diego, CA.
47 Table 2 1. Peptides used in this study. Pep tide Name Sequence Tkip WLVFFVIFYFFR Tkip2A WLVFFVIAYFAR SOCS1 KIR 53 DTHFRTFRSHSDYRRI SOCS1 KIR2A 53 DTHFATFASHSDYRRI JAK1 autophosphorylation site 1028 IETDKE Y YTVKDD JAK2 autophosphorylation site 1001 LPQDKE Y YKVKEP JAK3 autophosphorylation site 974 LPL DKD Y YVVREP TYK2 autophosphorylation site 1048 VPEGHE Y YRVRED MAL(82 94) 82 WSKD Y DVCVCHSE MAL(154 166) 154 DPWCK Y QMLQALT pJAK2(1001 1013) 1001 LPQDKE Y YKVKEP JAK2(1001 1013)2A 1001 LPQDKEAAKVKEP IFN (95 132) 95 AKFEVNNPQVQRQAFNELIRVVHQLLPESSL IFN (95 125) 95 AKFEVNNPQVQRQAFNELIRVVHQ IFN (95 106) 95 AKFEVNNPQVQR IFNGR1(253 287) 253 TKKNSFKRKSIMLPKSLLSVVKSATLETKPESKYS Tyrosines shown in bold are phosphorylated.
48 CHAPTER 3 CHARACTERIZATION OF THE SOCS1 MIMETIC PEPTIDES SOCS -1 Mimetics Recognize the Autophosphorylation Sites of Different JAKs SOCS-1 has been reported to recognize and modulate the function of all four JAKs (Starr et al., 1997; Endo et al., 1997; Naka et al., 1997; Yoshimura et al., 2007). We were therefore interested in determining the ability of the SOCS 1 mimetics Tkip and SOCS1-KIR to bind to and modulate the kinase activity of JAK1, JAK2, JAK3, and TYK2. First, we plotted the hydropathic profiles of the JAK autophosphorylation sites. All four JAKs have similar protein sequences and therefore s imilar hydropathic profiles, suggesting similar structure ( Figure 31a ). Next, we synthesized peptides corresponding to the autophosphorylation sites of the four JAKs ( Figure 3-1b ) and compared them for binding to Tkip and SOCS1-KIR. As previously reported, both Tkip and SOCS1-KIR bound to the JAK2 autophosphorylation site ( Figure 3 -2a ). Tkip and SOCS1-KIR also specifically bound to the autophosphorylation site peptide of TYK2 ( Figure 3-2b ), a JAK that plays a key role in mediation of type I IFN signaling ( Leung et al., 1995). SOCS1 -KIR, but not Tkip, bound to the JAK1 and JAK3 autophosphorylation site peptides ( Figure 3-2c and d). This would suggest that the KIR region of SOCS -1 might be the key to SOCS -1 recognition of the JAKs. Phenylalanines at position s 56 and 59 of SOCS 1 have previously been identified as critical for SOCS -1 binding to JAK2 and for its function (Yasukawa et al., 1999). Accordingly, the bindings of Figure 32 also include SOCS1KIR with alanine substitutions at positions 56 and 59 (SOC S1 -KIR2A), as well as Tkip with alanine substitutions at positions 8 and 11 (Tkip2A), which correspond to possible sites of homology with SOCS1KIR (Flowers et al., 2004). SOCS1-KIR2A was unable to bind to
49 any of the JAKs, while Tkip2A showed reduced bindi ng to JAK2 and TYK2 as compared to Tkip. Tkip2A and SOCS1KIR2A did not bind to JAK1 or JAK3 autophosphorylation peptides. SOCS -1 Mimetics are Not Cytotoxic In order to assess possible toxicity of the SOCS 1 mimetic peptides, we incubated the peptides at various concentrations (3.7 to 100 M) for 24 hours with murine L929 cells ( Figure 33a ) and splenocytes ( Figure 33b ). The assay was repeated for 72 hours with similar results. None of the peptides tested showed toxicity as assessed in a cytopathic assay. Thus, any biological effects of the test peptides are not due to toxicity. SOCS -1 Mimetics Inhibit STAT Phosphorylation As a functional correlate of the binding of Tkip and SOCS1KIR to the autophosphorylation site peptides of the JAK kinases, we stimulat ed cells with IFN IFN or IL -10. The interaction of IFN with its receptor activates JAK1 and JAK2, which are responsible for the phosphorylation of STAT1 (Leaman et al., 1996). Both Tkip and SOCS1 -KIR inhibited IFN -induced phosphorylation of STAT1 in L929 fibroblast cells (Figure 34a ). Like other type I IFNs, IFN activates JAK1 and TYK2, which phosphorylate STAT1, STAT2, and STAT3 (Bazer et al., 1996). Tkip and SOCS1-KIR inhibited IFN -induced phosphorylation of STAT1 and STAT3 in L929 cells, and STA T2 in WISH cells ( Figure 3-4b ), while SOCS1 -KIR2A and Tkip2A failed to inhibit the phosphorylation of STAT1 in response to IFN (Figure 34c), supporting the previous findings that they were unable to bind to the relevant JAKs. IL10 also activates JAK1 a nd TYK2, which leads to phosphorylation of STAT1 and STAT3 (Finbloom and Winestock, 1995). Treatment of RAW264.7 macrophages with IL10 resulted in
50 activation of STAT1 and STAT3, both of which were inhibited by Tkip and SOCS1-KIR (Figure 34d). The Effect of SOCS -1 Mimetics on Macrophage Activation SOCS-1 has been shown to inhibit LPS -induced activation of macrophages by blocking signal transduction events that occur via TLR4 (Mansell et al., 2006). TLR4 signaling involves activation of the adapter protein MAL by Brutons tyrosine kinase (Btk), resulting in MAL-dependent p65 phosphorylation and the resultant activation of NF B (reviewed in Kobayashi et al., 2006). The induced SOCS -1 protein modulates this activation by binding to activated MAL, causing ubi quitination and then proteasomal degradation of MAL (Mansell et al., 2006). To determine if the SOCS 1 mimetic peptides could imitate the SOCS -1 inhibition of LPS -induced activation of these cells via TLR4, we treated murine RAW264.7 macrophages with Tkip and SOCS1-KIR, and determined the amount of nitric oxide produced. Both Tkip and SOCS1-KIR inhibited LPS -induced nitric oxide (NO) production ( Figure 35 ). Based on a similar hydropathic profile of a tyrosine kinase phosphorylation site on MAL by Btk to th at of pJAK2(1001 1013) ( Figure 3 -6a ), we synthesized a MAL(82 94) peptide and determined its ability to bind to SOCS1KIR. MAL(82 94) peptide showed a similar binding pattern to SOCS1 -KIR as that of pJAK2(1001 1013) ( Figure 36b). Another tyrosine kinase p hosphorylation site on MAL, MAL(154 166), had a hydropathic profile different from pJAK2(1001 1013) ( Figure 3 -6a ) and did not bind to SOCS1-KIR (Figure 3-6b ). We hypothesized that the binding of MAL(82 94) peptide to SOCS1KIR reflected the mechanism of SO CS -1 recognition of MAL in LPS -treated cells. Accordingly, we treated RAW264.7 macrophages with SOCS1-KIR and MAL(82 94) to determine
5 1 whether the peptides could compete with endogenous proteins for binding to their targets. LPS treatment of cells in the pr esence of control peptide showed essentially complete degradation of MAL by 60 minutes, while LPS treatment in the presence of SOCS1 -KIR completely blocked MAL degradation. Similarly, MAL(82 94) treatment also protected against MAL degradation ( Figure 3-7 ). The inhibition of LPS activation of macrophages by SOCS1 -KIR by binding to a critical functional site on MAL, yet blocking the SOCS 1 mediated degradation of MAL, suggests that inhibition of function can occur through the binding without the necessity fo r MAL degradation. The Effects of SOCS1-KIR on SOCS -1/ Mice SOCS-1 is essential for survival, as evidenced by the fact that SOCS -1/ mice die by three weeks of age due to severe inflammatory pathology (Starr et al., 1997; Naka et al., 1997). It was ther efore of interest to determine the effects of SOCS1 -KIR on the survival of SOCS -1/ mice. We first established breeding pairs of SOCS -1+/ mice to generate progeny that would include SOCS 1+/+, SOCS -1+/ -, and SOCS -1/ pups. When the pups were born, we injected them with 10 g per gram of body weight (measured immediately prior to injection) of SOCS1-KIR intraperitoneally (i.p.) every day. The pups were monitored daily for weight and signs of inflammation. The untreated SOCS -1/ pups died by day 17, while the SOCS1-KIR treated mice survived until day 22 ( Figure 38a ). SOCS-1/ mice have stunted growth as compared to their wild-type littermates, so one of the parameters that we measured was weight change. The SOCS1-KIR treated knockout pups had weights th at were similar to their wild-type littermates, up to a point (Figure 38b ). This would suggest that SOCS1 -KIR is able to at least partially remedy
52 the low body weight that is associated with the SOCS 1/ phenotype. Further studies are being done in colla boration with Dr. Larkins group, where the SOCS1-KIR peptide is being used in conjunction with adoptive transfer of CD4+CD25+ regulatory T cells to determine the survival outcome and reduction of inflammatory pathology of the SOCS 1 knockout mice.
53 Figu re 31. Autophosphorylation sites of the JAKs. a. A hydropathic plot of the autophosphorylation sites of the JAKs. b. A partial sequence alignment of the four JAKs, with the autophosphorylation sites outlined in red.
54 Figure 32. SOCS 1 mimetics bind to the autophosphorylation site peptides of the JAKs. Biotinylated pJAK2(1001 1013) ( a ), TYK2(1048 1060) ( b ), JAK1(1028 1040) (c ), or JAK3(974 986) ( d ), at the indicated concentrations, were added in triplicate to a 96well plate coated with binding buffer, IFN (95 106), SOCS1 -KIR, SOCS1 -KIR2A, Tkip, or Tkip2A, and binding assays were carried out as described in Chapter 2. Values represent the means ( SEM) of triplicate wells from three independent experiments. Stars (*) indicate statistically significant d ifferences when compared to SOCS1-KIR2A or Tkip2A, as determined by the Mann-Whitney U test.
55 Figure 33. SOCS 1 mimetic peptides are not cytotoxic. L929 cells ( a ) or spleen cells isolated from healthy SJL/J mice ( b ) were seeded onto 96well plates at 1 x 106 cells/ml. Peptides were added at various concentrations and the cells were incubated at 37 C at 5% CO2. After 24 h, Solution Reagent was added and the cells were incubated for 2 h at 37 C, and the absorbance was measured at 490 nm. Values represent the means ( SEM) of triplicate wells from three independent experiments.
56 Figure 34. SOCS 1 mimetic peptides inhibit STAT phosphorylation. a. SOCS1 -KIR and Tkip inhibit IFN -induced STAT1 activation. L929 cells were incubated with various peptides for 2 h at 37 C. Following a 2 h incubation in the presence or absence of IFN (4000 U/ml), the cells were washed and lysed. Whole cell extracts were resolved on 12% SDS -PAGE, transferred to nitrocellulose membranes, and the membranes were probed using pSTAT1 or STAT1 Ab. b. SOCS1 -KIR and Tkip inhibit IFN -induced STAT1, STAT2, and STAT3 activation. L929 cells were treated as in part a except 10,000 U/ml of IFN was used in lieu of IFN Membranes were probed with Abs to pSTAT1, pSTAT2, pSTAT3, STAT1, STAT2, or STAT3. c. SOCS1 -KIR2A and Tkip2A do not inhibit the phosphorylation of IFN -induced STAT1. L929 cells were treated as in part b and membranes were probed with pSTAT1 or STAT1 Abs. d. SOCS1 -KIR and Tkip inhibit IL 10induced STAT1 and STAT3 activation. RAW264.7 cells were treated as in part a except 10 ng/ml of IL10 was used in lieu of IFN Membranes were probed with Abs to pSTAT1, pSTAT3, STAT1, or STAT3. Data are representative of three independent experiments.
57 Figure 35. SOCS 1 mimetic peptides inhibit lipopolysaccharide ( LPS ) induced macrophage activation. RAW264.7 macrophages were incubated with varying concentrations of LPS alone or with various peptides at 24 M for 48 h at 37 C. Culture supernatants were collected and nitric oxide concentration was determined using Griess reagent. Values represent the means ( SEM) of triplicate wells from three independent experiments. Stars (*) indicate statistically significant differences when compared to IFN (95 106) treated cells, as determined by twoway ANOVA with Bonferroni post -tests.
58 Figure 36. SOCS 1 mimetic peptides regulate TLR4 signaling. a. Hydropathic profiles of the JAK2 autophosphorylation site pJAK2(1001 1013) and the tyrosine phosphorylation site peptides of MAL. pJAK2(1001 1013) and MAL(82 94) show similar hydropathic profiles, while MAL(154 166) has a different hydropathic profile. b. SOCS1 -KIR binds to MAL(82 94). Bindings were carried out as previously described with biotinylated MAL(82 94), MAL(154 166), and pJAK2(1001 1013). Val ues represent the means ( SEM) of triplicate wells from three independent experiments. Stars (*) indicate statistically significant differences (p < 0.05) when compared to binding buffer, as determined by the MannWhitney U test.
59 Figure 37. SOCS1 -KIR inhibits MyD88 adapter like (MAL) protein degradation. a. RAW264.7 macrophages were seeded onto 6well plates at 2.5 x 106 cells/ml and treated with LPS (1 g/ml) in the presence or absence of SOCS1 -KIR, MAL(82 94) or IFN (95 106) (control peptide) for the indicated times. Westerns were performed as previously described and probed with Ab to MAL. The media lane is a negative control for all treatments, which were carried out in one experiment. Data are representative of three independent experiments. Densit ometry is presented in part b. Values represent the means ( SEM) of western blot bands from three independent experiments. Stars (*) indicate statistically significant differences when compared to 60 min Control Peptide (12 M) treated cells, as determined by two way ANOVA with Bonferroni post -tests.
60 Figure 38. SOCS1 -KIR prolongs survival of SOCS 1/ mice. Pups were injected daily with 10 g per gram body weight of SOCS1-KIR, i.p., and monitored for survival ( a ) and weight change ( b ). There were stati stically significant differences between the survival of treated and untreated SOCS -1/ mice, as determined by the log-rank test. There were statistically significant differences between weights of untreated and treated SOCS 1/ mice, as determined by a two way ANOVA.
61 CHAPTER 4 CHARACTERIZATION OF THE SOCS1 ANTAGONIST PEPTIDE The SOCS 1 Antagonistic Activity of pJAK2(10011013) As previously mentioned, SOCS1-KIR can bind to the autophosphorylation site peptide of JAK2, pJAK2(1001 1013) ( Figure 3-2 ). T his raised the possibility that pJAK2(1001 1013) can inhibit the function of endogenous SOCS -1 and thus enhance the various cytokine activities that are mediated by JAK2. Previous studies done in our lab have shown that the pJAK2(1001 1013) peptide can enhance the antiviral activity of IFN increase STAT3 activation in IL6 -treated cells that overexpress SOCS -1, enhance the GAS promoter activity of IFN and enhance antigen -specific splenocyte proliferation (Waiboci et al., 2007). In addition, it was shown that SOCS -1 protein competes with SOCS1KIR for binding to pJAK2(1001 1013) (Waiboci et al., 2007). This suggests that pJAK2(1001 1013) acts as a SOCS 1 antagonist. The SOCS 1 Antagonist Increases STAT1 Phosphorylation To determine if the SOCS -1 antagonis t had any effect on STAT1 activation, we treated L929 cells with pJAK2(1001 1013) and JAK2(1001 1013)2A (alanines substituted for tyrosines 1007 and 1008), and probed for phosphorylated STAT1 ( Figure 4 -1 ). Treatment with pJAK2(1001 1013) at 25 M increased pSTAT1 levels by more than two-fold over untreated cells, while JAK2(1001 1013)2A had minimal effects. The SOCS 1 Antagonist Increases Endogenous IFN Levels For efficient induction of an antiviral state, cells contain low levels of constitutive IFN (Taniguchi and Takaoka, 2002). A subtle increase in this low level of IFN plays an important role in a positive feedback loop to increase type I IFN production and induction of a potent antiviral state in cells (Takaoka et al., 2000). To determine if
62 pJAK2(10 01 1013) affected the level of constitutive IFN we treated L929 fibroblasts with pJAK2(1001 1013) and JAK2(1001 1013)2A. Cells treated with pJAK2(1001 1013) for 30 or 60 minutes showed an increase in IFN as determined by Western blot, while JAK2(1001 10 13)2A had little or no effect on IFN levels ( Figure 42a ). In comparison, IFN levels were not altered in the same cells ( Figure 42b). Western blots showed a decline in SOCS -1 protein levels in pJAK2(1001 1013) treated cells, while JAK2(1001 1013)2A had no discernable effect on SOCS 1 protein levels ( Figure 4 2c ). This decrease in SOCS 1 levels corresponded to the increase in IFN levels in the cells, and this would suggest that pJAK2(1001 1013) played a role in SOCS -1 degradation. To confirm the increase in IFN protein levels in L929 cells treated with pJAK2(1001 1013), we determined the intracellular IFN levels by ELISA. The cells treated with pJAK2(1001 1013) had an approximately two-fold increase in IFN levels as compared to untreated cells ( Figure 4 2d ). JAK2(1001 1013)2A -treated cells had IFN levels comparable to those of untreated cells. The Effects of the SOCS 1 Antagonist on Macrophage Activation SOCS-1 negatively regulates TLR signaling at several stages, including signaling by type I IFNs and by the transcription factor NF B. Since the SOCS -1 mimetic peptides are able to inhibit macrophage activation, we were interested to see if the SOCS-1 antagonist peptide would increase macrophage activation. We treated RAW264.7 macrophages with pJAK2(100 1 1013) and JAK2(1001 1013)2A and measured the amount of NO produced upon LPS stimulation. The pJAK2(1001 1013)
63 treated cells produced an approximately five-fold increase in NO, while the JAK2(1001 1013)2A peptide had a minimal effect ( Figure 4-3a ). We al so examined the effect of pJAK2(1001 1013) on TLR3 activation. TLR3 recognizes double -stranded RNA (dsRNA) and the synthetic dsRNA analog polyriboinosinic:polyribocytidylic acid (poly I:C) and induces type I IFN (Matsumoto and Seya, 2008). TLR3 plays an im portant role in the antiviral response to viruses that have a dsRNA stage in their life cycle, including HSV 1, influenza virus, cytomegalovirus, and respiratory syncytial virus (Vercammen et al., 2008). We treated RAW264.7 macrophages with pJAK2(1001 1013 ) and JAK2(1001 1013)2A, and determined the effects on poly I:C -induced NO production. The pJAK2(1001 1013) treatment increased NO production over 20-fold as compared to poly I:C alone, while JAK2(1001 1013)2A had a negligible effect ( Figure 4 3b). The SOC S1 Antagonist is Not Cytotoxic In order to assess possible toxicity of the SOCS 1 antagonist peptide, we incubated pJAK2(1001 1013) and JAK2(1001 1013)2A at various concentrations (3.7 to 25 M) with murine L929 cells ( Figure 44 ). None of the peptides te sted showed significant toxicity as assessed in a cytopathic assay. Thus, any biological effects of the test peptides are not due to toxicity. The SOCS 1 Antagonist Possesses Antiviral Activity Against a Picornavirus Encephalomyocarditis virus (EMCV) is a small single -stranded RNA picornavirus of the plus strand orientation with a wide host range. EMCV infection can cause myocarditis leading to arrhythmias, heart failure, and death (Robinson et al., 2009). During cardiac transplantation and valve replacement, infection by EMCV has been
64 implicated in the development of cardiomyopathy, which makes the development of effective therapies against this virus particularly important (Yajima and Knowlton, 2009). In mice, EMCV infection is lethal, but is quite suscept ible to IFN or an IFN mimetic (IFN (95 132)) treatment at early stages of infection (Mujtaba et al., 2006). The IFN mimetic is a small peptide that corresponds to the C -terminus of IFN and functions intracellularly. It has been shown to be an effective treatment against vaccinia virus (Ahmed et al., 2007) and herpes simplex virus 1 (HSV 1) (Frey et al., 2009). We reasoned that the SOCS 1 antagonist peptide would either induce or enhance an antiviral state by limiting the ability of SOCS 1 to modulate constitutive or added IFN antiviral activity. We have previously shown that pJAK2(1001 1013) antagonized the effect of SOCS 1 in HSV 1 infected keratinocytes (Frey et al., 2009). It reduced HSV 1 titers by two -fold as compared to untreated cells ( Table 4 1 ) (Frey et al., 2009). We were therefore interested to see whether pJAK2(1001 1013) would have an antiviral effect against EMCV. We treated L929 fibroblasts with pJAK2(1001 1013), JAK2(1001 1013)2A, IFN the control peptide IFN (95 125), or IFN (95 132) pri or to infection with 200 plaque forming units (pfu) of EMCV. Both IFN and IFN (95 132) as well as pJAK2(1001 1013) inhibited EMCV infection ( Figure 45a ). Specifically, pJAK2(1001 1013) and IFN reduced cytopathic effects (CPE) by approximately 50%, while IFN (95 132) was completely protective. The JAK2(1001 1013)2A peptide was only about 7% protective. Synergy between pJAK2(1001 1013) and IFN (95 132) was observed in treatments using suboptimal concentrations of both peptides, where 2 M of pJAK2(1001 10 13) and 5 M of IFN (95 132) combined completely protected L929
65 cells against EMCV infection, while separately the peptides at these concentrations showed 20% or less protection ( Figure 4-5b ). The increase of IFN in cells treated with pJAK2(1001 1013) rai ses the possibility that this IFN exerts its effects intracellularly and thus does not need to be secreted for subsequent interaction with the extracellular domain of the type I IFN receptor. To address this, we treated L929 cells with pJAK2(1001 1013) in the presence or absence of neutralizing antibodies to IFN prior to infection with 200 pfu of EMCV. Complete protection by pJAK2(1001 1013) was reduced to approximately 60% in the presence of a saturating level of anti -IFN antibody ( Figure 4 5c ). JAK2(100 1 1013)2A was not protective. These data suggest that some of the increased IFN exerted its effects intracellularly. Based on the antiviral effects of pJAK2(1001 1013) in tissue culture, we tested the therapeutic effects of the antagonist in a mouse model of lethal EMCV infection. C57BL/6 mice were treated i.p. with 50, 100, or 200 g of pJAK2(1001 1013) or 200 g of JAK2(1001 1013)2A every day beginning 2 days prior to challenge with 50 pfu of EMCV per mouse, and the mice were monitored daily for signs of infection. Mice treated with JAK2(1001 1013)2A all died by day 5 after EMCV challenge ( Figure 4-6a ). In contrast, mice treated with 100 and 200 g pJAK2(1001 1013) showed 80% and 60% survival, respectively. Treatment with 50 g of pJAK2(1001 1013) resulted in 20% survival. To assess synergistic effects between pJAK2(1001 1013) and IFN (95 132), mice were treated with suboptimal doses of both peptides every day beginning 2 days prior to challenge with 50 pfu of EMCV. Treatment with pJAK2(1001 1013) at 10 g and
66 IFN (95 132) at 2 g resulted in 80% survival of infected mice, while treatment with pJAK2(1001 1013) alone resulted in 40% survival, and IFN (95 132) alone resulted in 60% survival (Figure 4-6 b ).
67 Figure 41. The SOCS -1 antagonist peptide increase s STAT1 phosphorylation. L929 cells were seeded onto a 6 well plate at 1 x 106 cells/ml, grown to confluency, and incubated with pJAK2(1001 1013) or JAK2(1001 1013)2A for 1 h. Cells were lysed and whole cell lysates were resolved by 12% SDS -PAGE, proteins were transferred onto nitrocellulose membrane, and the membranes were probed with pSTAT1 and STAT1 antibodies. Data are representative of three independent experiments. Values represent the means ( SEM) of bands from three independent experiments. Stars ( *) indicate statistically significant differences when compared to untreated cells, as determined by oneway ANOVA with Bonferroni post -tests.
68 Figure 42. The SOCS -1 antagonist increases endogenous interferon levels. a c. L929 cells were seeded onto 6well plates at 1 x 106 cells/ml and grown to confluence. The cells were incubated with pJAK2(1001 1013) and JAK2(1001 1013)2A (JAK2m) for 30 or 60 minutes at 37 C. The cells were lysed and immunoblotted as previously described for IFN (a ), IFN ( b ), or SOCS -1 ( c ). Relative intensities are shown in numbers below each blot, and in a graph above all blots. Values represent the means ( SEM) of bands from three independent experiments. Stars (*) indicate statistically significant differences when compared to untreated cells, as determined by oneway ANOVA with Bonferroni post -tests. d. Intracellular IFN levels were determined for the cell lysates with an IFN ELISA kit, following manufacturers instructions. Values represent the means ( SEM) of triplicate we lls from three independent experiments. Stars (*) indicate statistically significant differences when compared to untreated cells, as determined by two way ANOVA with Bonferroni post -tests.
69 Figure 43. The SOCS -1 antagonist increases macrophage activat ion. pJAK2(1001 1013) increases LPS induced macrophage activation. RAW264.7 cells were seeded onto 24well plates at 5 x 106 cells/ml and grown overnight. They were incubated with pJAK2(1001 1013) (pJAK2) or JAK2(1001 1013)2A (JAK2m) at 24 M for 4 h, after which 2 g/ml of LPS ( a ) or 0.1 g/ml of poly I:C ( b ) was added and the cells were incubated for 48 h at 37 C. Supernatants were collected and nitric oxide concentration was determined using Griess reagent. Values represent the means ( SEM) of triplicat e wells from three independent experiments. Stars (*) indicate statistically significant differences when compared to untreated cells, as determined by twoway ANOVA with Bonferroni post -tests.
70 Figure 44. The SOCS -1 antagonist is not cytotoxic. L929 c ells were seeded onto 96well plates at 1 x 106 cells/ml. Peptides were added at various concentrations and the cells were incubated at 37 C at 5% CO2. After 24 h, Solution Reagent was added and the cells were incubated for 2 h at 37 C, and the absorbance was measured at 490 nm. Values represent the means ( SEM) of triplicate wells from three independent experiments.
71 Table 4 1. Yield reduction of herpes simplex virus 1. Treatment Virus Yield (pfu/ml) Fold Reduction Untreated 7 x 10 7 pJAK2(1001 1013) ( 25 M) 4 x 10 7 2 IFN (95 132) (25 M) 5 x 10 6 14 IFN (95 132) (50 M) 9 x 10 5 77 Frey et al., 2009.
72 Figure 45. The SOCS -1 antagonist has antiviral activity against encephalomyocarditis virus (EMCV). a. L929 cells were treated with IFN pJAK2(10 01 1013) (pJAK2), JAK2(1001 1013)2A (JAK2m), IFN (95 125), or IFN (95 132) for 2 h, after which 200 pfu of EMCV were added. After 1 h, virus was removed and fresh media was added followed by incubation for 24 h at 37 C. Cells were stained with crystal viol et and the plates were scanned and analyzed using ImageJ 1.29 software. b. Synergy between pJAK2(1001 -1013) and IFN (95 132) in inhibition of EMCV. L929 cells were treated as in part a, and pJAK2(1001 1013) at 2 M and IFN (95 132) at 5 M were incubated together as well. c. L929 cells were incubated with the peptides (24 M) in the presence or absence of 500 U/ml of neutralizing antibody to IFN for 2 h, after which the cells were infected with EMCV and processed as in part a Values represent the means ( SEM) of triplicate wells from three independent experiments. Stars (*) indicate statistically significant differences when compared to EMCV -infected cells, as determined by twoway ANOVA with Bonferroni post -tests.
73 Figure 46. The SOCS -1 antagonist protects mice from lethal EMCV infection. a. C57BL/6 mice (n = 5 per group) were injected daily i.p. beginning at day 2 with pJAK2(1001 1013) (pJAK2) at 50, 100, and 200 g and JAK2(1001 1013)2A (JAK2m) at 200 g. On day 0, 50 pfu of EMCV per mouse were in jected i.p. Survival data are presented as KaplanMeier plots. Survival curves were not found to be significantly different when compared to the PBS treated group, as determined by the log-rank statistical test. b. Synergy in protection of mice infected w ith EMCV as for part a using suboptimal levels of pJAK2(1001 1013) (pJAK2) (10 g) and IFN (95 132) (95 132) (2 g). Survival data are presented as KaplanMeier plots. Survival curves were not found to be significantly different when compared to the PBS t reated group, as determined by the log-rank statistical test.
74 CHAPTER 5 DISCUSSION Knocking out the SOCS 1 gene in mice results in neonatal death. The mice appear to be normal at birth, but exhibit stunted growth and die at approximately 3 weeks of age (Starr et al., 1997). The primary cause of the pathology that leads to death is unregulated or inadequate regulation of IFN activity, since SOCS 1 knockout mice that are deficient in IFN or IFN receptor do not die as neonates (Alexander et al., 1999). H ow SOCS 1 keeps the IFN system under control is complex. Structurally, optimal recognition of JAK2 by SOCS 1 involves a 12residue KIR region, a 12residue extended SH2 sequence (ESS), and a longer SH2 domain (Yasukawa et al., 1999). This study has shown that the KIR can recognize the autophosphorylation site of JAK2 independently of the ESS and SH2 regions, using the SOCS1-KIR peptide. SOCS1 -KIR can also recognize the autophosphorylation site of the other JAKs, and it can block the phosphorylation of vari ous STATs associated with type I and II IFN, as well as IL-10, signaling. The ability of the KIR region to inhibit IFN and other cytokine functions is of interest in assessing other functional domains of SOCS 1, particularly the SOCS box domain. SOCS 1, as well as the other seven SOCS proteins, contains a homology domain called the SOCS box that makes up the C -terminus of the protein (Yasukawa et al., 1999). The conserved SOCS box domain binds to elongins B and C, which form part of an E3 ubiquitin ligase complex that ubiquitinates proteins and targets them for proteasomal degradation (Kamura et al., 2004). Deleting only this domain from the SOCS-1 gene and comparing the mouse phenotype to that of mice lacking the entire SOCS-1 protein determined the role of the SOCS box in SOCS 1 function (Zhang et
75 al., 2001). The SOCS box knockout mice, although possessing increased responsiveness to IFN did not die by 3 weeks as the SOCS 1/ mice do. Rather, these mice had approximately 50% survival at 50 days, and approximately 20% survival as late as 90 days. Prolonged activation of STAT1 in hepatocytes was intermediate between that of wild -type and SOCS 1/ mice. This suggests that the protective effect of SOCS-1 in wild -type mice against unregulated IFN appears to be due to both KIR/SH2 binding to JAK2 and proteasomal degradation of JAK2 via activity of the SOCS box. We have shown in this study that both the SOCS1 KIR and Tkip peptides bound to JAK2 and TYK2, while only SOCS1KIR bound to JAK1 and JAK3. This is c onsistent with SOCS 1 recognition of all of the JAKs (reviewed in OSullivan et al., 2007). This study has also shown that the SOCS -1 mimetic peptides inhibit type I IFN -induced activation of STAT1, STAT2, and STAT3. IL -10 treatment of macrophages resulted in activation of STAT1 and STAT3, which were also inhibited by the SOCS -1 mimetic peptides. Additionally, the SOCS -1 mimetic peptides blocked IFN -induced STAT1 phosphorylation, as previously shown (Waiboci et al., 2007). The demonstration here that SOCS1 -KIR binds to all of the JAKs provides a direct correlate to SOCS -1 induction by and regulation of numerous cytokines, growth factors, and hormones that use the JAK/STAT signaling pathway. Using alanine substitutions for essential phenylalanines at positio ns 56 and 59 of SOCS-1, we showed that SOCS1 -KIR2A failed to recognize all four JAKs and did not block IFN induced activation of STAT1. Alanine substitutions at potential phenylalanine homology sites 8 and 11 of Tkip, Tkip2A, resulted in reduced binding t o JAK2 and TYK2 and loss of ability to inhibit IFN -induced activation of STAT1. This would suggest that
76 there is specificity involved in the recognition of the autophosphorylation sites of the JAKs by the SOCS-1 mimetic peptides, which is consistent with previously identified critical phenylalanine residues in KIR (Yasukawa et al., 1999). The inhibition of LPS -induced activation of macrophages by the SOCS -1 mimetic peptides demonstrates that they, like SOCS -1, can regulate TLR4 function (Mansell et al., 2 006). SOCS 1 regulates TLR4 at multiple sites in macrophage signaling, including MAL, p65 of NF B, an IRAK tyrosine kinase, or induced IFN autocrine activation of JAKs (Kobayashi et al., 2006). MAL is one of the adapter proteins that are involved in TLR4 signaling (Fitzgerald et al., 2001). Its activation leads in turn to activation of the transcription factor NF B. Enhanced binding of SOCS -1 to activated MAL results in its proteasomal degradation (Mansell et al., 2006). We identified a binding site on M AL for the KIR region of SOCS-1, which is consistent with SOCS1KIR peptide inhibition of TLR4 signaling. We also showed that SOCS1KIR competes with endogenous SOCS -1 protein for binding to MAL, and that SOCS1-KIR prevents the proteasomal degradation of M AL while inhibiting TLR4 signaling. This would suggest that SOCS1-KIR is able to block MAL function without the need for proteasomal degradation, since SOCS1 -KIR lacks the SOCS box. SOCS-1 acts intracellularly in the cells in which it is induced and this is most likely the key to the selective aspects of its regulation of various functions. The use of the SOCS-1 mimetic peptides as therapeutics for immunological or other disorders presents a potential challenge in inhibition of inflammatory cytokines such as IFN while minimally affecting anti inflammatory cytokines such as IL10, since the mimetics are
77 able to inhibit the actions of both. We have previously shown that Tkip has therapeutic effectiveness in the experimental allergic encephalomyelitis (EAE) model of multiple sclerosis under a given protocol of dosage and time of Tkip administration (Mujtaba et al., 2005). This demonstrates that these peptides should have therapeutic value in SOCS-1 based treatment of immunological diseases. Studies currently under way in our lab have shown that SOCS1 -KIR can also be used as a therapeutic for EAE, as treatment with SOCS1 -KIR lowered disease incidence and the resulting pathology to levels that are similar to those of naive mice (unpublished data). In addition to treatment of autoimmune diseases, SOCS1-KIR is able to prolong survival of SOCS -1/ mice and reduce the associated inflammation (unpublished data). Regulation of the SOCS -1 modulatory arm of the immune response provides an approach to enhancement of the response to infectious agents as well as to weak antigens that are the target of tumor vaccine studies. In this regard, the regulatory role of SOCS -1 extends to dendritic cells (DCs) and antigen presentation. Recently, it was shown that knockdown of DC SOCS1 by siRNA led to more effective cancer vaccination (Shen et al., 2004). Specifically, presentation of murine melanocyte differentiation antigen tyrosine-related protein 2 (TRP2) by DCs transfected with SOCS-1 siRNA protected C57BL/6 mice against the well established B16 melanoma tumor. Protection was not observed in DC vaccination where the siRNA was disrupted by GFP. The enhanced anti -tumor immunity was accompanied by enhanced TRP2specific cytotoxic T cells (CTLs) in protected mice as assessed by IF N ELISPOT and CTL responses. The authors concluded that regulation of antigen presentation by suppression of DC SOCS 1 showed promise for more effective tumor vaccines. The
78 SOCS-1 siRNA treatment also enhanced HIV -1 envelope specific CD8+ CTL responses, w hich suggests that suppression of SOCS -1 in DCs is of potentially general value for immune enhancement against AIDS (Song et al., 2006). These observations are related to the observation that SOCS -1/ mice are more resistant to viral infection than their wild -type counterparts, due to enhanced type I IFN activity involving the IFNAR1 subunit (Zimmerer et al., 2007). The development of the SOCS 1 antagonist pJAK2(1001 1013) was based on the observation that the KIR region of SOCS 1 binds directly to the autophosphorylation site peptide of JAK2, raising the possibility that the peptide could function as an antagonist of SOCS -1 (Waiboci et al., 2007). It was shown that pJAK2(1001 1013) enhanced suboptimal IFN activity, reversed the SOCS -1 induced reduced act ivation of STAT3 by IL -6 treatment, enhanced IFN activation of the luciferase reporter gene via the GAS promoter element, and enhanced antigen-specific splenocyte proliferation (Waiboci et al., 2007). The antiviral effects of the SOCS 1 antagonist appear to operate through direct effects on the cell as well as by indirect effects in mice by enhancement of both the cellular and humoral arms of the immune system. TLRs are key players in both the innate and adaptive arms of host defense. With respect to TLR3 and virus immunity, treatment of the macrophage cell line RAW264.7 with poly I:C in the presence of pJAK2(1001 1013) resulted in significant enhancement of NO production. Classically, poly I:C induces type I IFNs and these in turn activate myeloid cells to produce NO, reflecting the activation of these cells (Matsumoto and Seya, 2008). In addition,
79 pJAK2(1001 1013) enhanced NO production in response to LPS, an activator of TLR4, suggesting that pJAK2(1001 1013) may have antibacterial properties. A well reco gnized but not fully understood aspect of IFN function in cells is that most cells constitutively produce low levels of intracellular IFN that is thought to play a role in induction of an antiviral state in cells treated with type I and type II IFNs (Tani guchi and Takaoka, 2001). pJAK2(1001 1013) increased the level of intracellular IFN in cells where it induced the antiviral effect. Intracellular signaling by a nonsecretable form of type I IFN has been reported by several independent studies (Ahmed et al., 2001; Shin Ya et al., 2005). Both IFN and IFN were expressed in a nonsecretable form, and were able to induce an antiviral state without being secreted. Related to these findings, SOCS -1 deficiency has been shown to amplify type I IFN antiviral acti ons (Fenner et al., 2006). Additionally, type I IFNs activate STAT1, an effect that is increased by pJAK2(1001-1013) in vitro Mice deficient in STAT1 are phenotypically normal when kept in a specific pathogen free environment, but they are extremely susce ptible to viral and bacterial infection (Meraz et al., 1996). In keeping with the TLR findings of this study, it would be interesting to determine the effects of pJAK2(1001 1013) on the ability of STAT1/ mice to clear viral or bacterial infection. The S OCS 1 antagonist exerted a direct effect and synergized with IFN and IFN (95 132) to protect cells and mice from EMCV infection. These observations, along with the inhibition of HSV -1 replication in keratinocytes and protection against vaccinia virus, dem onstrate the broad antiviral activity of the SOCS -1 antagonist. The connection of the SOCS 1 antagonist with IFN appears to reduce the regulatory restraints imposed by SOCS 1 under normal physiological conditions as shown by SOCS -1 reduction in
80 cells treated with pJAK2(1001 1013). The mechanism of the reduction is not currently known, but may be related to proteasomal degradation via the SOCS box of SOCS -1. Targeting of SOCS -1 expression in cardiac myocytes by expression of a dominant negative SOCS -1 incre ased myocyte resistance to acute cardiac injury as well as reduced mortality in coxsackievirus -infected mice (Yasukawa, 2003). This transfection study with a generic approach to inhibition of SOCS -1 function is analogous to the SOCS-1 antagonist results pr esented here. These results provide an approach to targeting SOCS -1 with a flexible drug therapeutic potential. Induction of SOCS -1 in other viral infections such as influenza (Pothlichet et al., 2008) further suggests a role for the SOCS1 antagonist in m odulation of these infections. There has been great interest in developing small molecule agonists or inhibitors of various cytokines and cytokine regulators. This study presents both, in the form of SOCS-1 mimetics and antagonists. The SOCS 1 mimetics ar e able to emulate the biological activity of SOCS -1, and this property can be used to treat a variety of immunological diseases that have a disregulation in the JAK/STAT signaling pathway, as well as certain viral infections. We have recently revealed a novel endogenous antipoxviral activity of SOCS -1 (Ahmed et al., 2009). The SOCS 1 mimetics prevent vaccinia virus from hijacking the hosts replication machinery by blocking the activity of the ErbB1 tyrosine kinase, a kinase that is necessary for viral repl ication and release (Buller et al., 1988). Additionally, the SOCS 1 mimetics inhibited virus -induced activation of JAK2, similar to the inhibition of myxoma virus infection in rabbits by tyrophostin AG490, a JAK -specific inhibitor (Masters et al., 2001).
81 T he SOCS 1 antagonist pJAK2(1001 1013) is able to function as a broad spectrum antiviral, as it is effective against vaccinia virus, HSV -1, and EMCV. It would be interesting to test its effects on parasitic infections, as SOCS 1/ mice are resistant to not only viral infections, but certain parasitic infections as well (Zimmermann et al., 2006). Toxoplasma gondii parasites are able to block IFN signaling pathways via induction of SOCS 1, and this enables the parasite to evade the immune response and live a nd replicate happily in macrophages (Zimmermann et al., 2006). The ability of the parasite to interfere with IFN signaling was abolished when macrophages from mice deficient in SOCS 1 were infected with T. gondii suggesting that the parasite induces endo genous SOCS 1. Future studies will focus on further elucidating the complete mechanisms of action of the SOCS 1 mimetic and antagonist peptides, including looking closer into their physiological roles. The SOCS 1 mimetics will be used to treat SOCS -1 knoc kout mice and their effects on the inflammatory pathology, as well as their direct effects on the various cell types including B cells and T regulatory cells, will be determined. The SOCS-1 antagonist peptide will be used as a potential antiviral therapy f or influenza A virus. Its effects on virus replication and infection in vitro will be determined, followed by in vivo studies in a mouse model of influenza A infection.
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91 BIOGRAPHICAL SKETCH Rea Dabelic was born in Dubrovnik, Croatia, to Angie and Claudio Dabelic. She spent the first eight years of her life there, until the civil war broke out in early summer of 1991. Reas family was one of the lucky families to escape the war on the last airplane to leave the country. Their flight took them to th e Netherlands, where a loving family took them into their home. There, Rea spent the next year learning Dutch and adjusting to a new culture. In August of 1992, Rea and her family emigrated to Coral Springs, Florida and they made that their permanent home. Rea spent the next nine years of her life there and graduated from J.P. Taravella High School in 2001. Upon graduating, Rea enrolled in the University of Florida, where she earned her Bachelor of Science in integrative biology in 2005. Rea earned her Do ctor of Philosophy degree in microbiology and cell science from the University of Florida in May of 2010. She plans to pursue a career in immunological research.