1 AGONISTS AND ANTAGONISTS OF IFN GAMMA SIGNALING By JAMES MARTIN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2010
2 2010 James Patrick Martin
3 To the mice
4 ACKNOWLEDGMENTS I thank all of those who worked alongside me at the Johnson lab, E. N. Noon Song, R. Dabelic, L. Jager, L. Waiboci, M. Mujtaba, M. Haider, C. M. Ahmed, and especially H. M Johnson.
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 6 LIST OF FIGURES ................................ ................................ ................................ .......... 7 ABSTRACT ................................ ................................ ................................ ..................... 8 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 10 2 MATERIALS AND METHODS ................................ ................................ ................ 17 3 RESULTS ................................ ................................ ................................ ............... 22 The Gamma Interferon (IFN 132) Prevents Encephalomyocarditis Virus Infection both in Tissue Culture and in Mice ........... 22 IFN Mimetic as a Therapeutic for Lethal Vaccinia Virus Infection: Possible Effects on Innate and Adaptive Immune Responses ................................ ........... 24 Both the Suppressor of Cytokine Signaling 1 (SOCS 1) Kinase Inhibitory Region and SOCS 1 Mimetic Bind to JAK2 Autop hosphorylation Site: Implications for the Development of a SOCS 1 Antagonist ................................ 26 Enhancement of Antiviral Immunity by Small Molecule Antagonist of Suppressor of Cytokine Signaling ................................ ................................ ........................... 30 4 DISCUSSION ................................ ................................ ................................ ......... 34 REFERENCES ................................ ................................ ................................ .............. 54 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 63
6 LIST OF TABLES Table page 3 1 The amino acid sequences of synthetic peptides used in this study ................... 46
7 LIST OF FIGURES Figure page 3 1 B8R neutralizes IFN 132) antiviral activity. ........................... 47 3 2 Protection of mice from EMC virus challenge by the IFN 132) peptide in the presence of B8R protein. ................................ ................................ .............. 48 3 3 Adjuvant effect of IFN .............................. 49 3 4 SOCS1 KIR and Tkip inhibit IFN induced macrophage activation ................... 50 3 5 Both SOCS1 KIR and Tkip inhibit proliferation of murine splenocytes. .............. 51 3 6 pJAK2(1001 1013) exerts an adjuvant effect at bo th cellular and humoral ................................ ................................ ................................ ............... 52 3 7 pJAK2(1001 1013) enhances macrophage activation via Toll like receptors.. ... 53
8 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science AGONISTS AND ANTAGONISTS OF IFN GAMMA SIGNALING By James Mart in December 2010 Howard M. Johnson Major: Microbiology and Cell Science We ha ve designed a series of peptide agonists and antagonists of IFN and JAK/STAT signal transduction based upon an alternative understanding of the classical model for this pathw ay. We have demonstrated previously that the C terminal gamma interferon (IFN 132)] and contains the requisite IFN tissue culture IFN 132) prevented EMC virus induced lethality in mice in a dose dependent manner compared to controls. IFN (95 132) also protected mice against lethal vaccinia virus infection with the virulence factor B8R, capable of rescuing 100% of animals two days into infection. B8R protein is a homologue for the extracellular interferon gamma receptor (IFNGR) encoded by poxviruses like vaccinia virus as a defense mechanism to neutralize host IFN IFN (95 32) bypasses B8R because its mode of action is int racellular, and synthesized with a lipophilic attachment, penetrates the cell plasma membrane in lieu of interacting with IFNGR extracellularly. The mimetic also possessed adjuvant effects which boosted humoral and cellular immunity to vaccinia virus. Supp ressor of cytokine signaling (SOCS) 1 protein modulates signaling by IFN by binding to the autophosphorylation site of JAK2 and by targeting bound
9 JAK2 to the proteosome for degradation. We have developed two small tyrosine kinase inhibitor peptides, Tkip and SOCS1 KIR, that are SOCS mimetics. Both are based on the kina se inhibitory region (KIR) of SOCS 1 and bind the autophosphorylation site of JAK2, JAK2(1001 1013) though not in precisely the same way. Tkip and SOCS1 KIR inhibited STAT1 phosphorylation, antagonizing IFN induced biological activity, including its imp act as an adjuvant, demonstrated here by the Tkip/SOCS1 KIR suppression of APC activation and Ag spe The fact that SOCS1 KIR binds to pJAK2(1001 1013) suggests that the JAK2 peptide could function as an antagonist of SOCS 1. T hus it was found that pJAK2(1001 1013) enhanced suboptimal IFN activity, blocked SOCS 1 induced inhibition of APCs, and enhanced IFN activation site promoter activity. Additionally, pJAK2(1001 1013) protected mice against lethal vaccinia and EMC virus infection. pJAK2(1001 1013) increased the intracellular level of the constitutive IFN which may play a role in the IFN agonist effect at the cellular level, and also synergizes with IFNs as per IFN mimetic to exert a multiplicative antiviral effect at the level of transcription. pJAK2(1001 1013) also exhibits adjuvant effects on humoral and cellular immunity in several direct and indirect ways including through the toll like receptors on APCs. These peptides present novel, effective approaches to ei ther promote, as with the IFN agonist peptides, or inhibit, via the IFN antagonist peptides, innate and adaptive host defenses.
10 CHAPTER 1 INTRODUCTION IFN signaling is one of the major systems coordinating the development, strength, and character of innate and adaptive imm une responses Its many biological effects include, but are not limited to, the induction of a number of antiviral proteins, up regulation of major histocompatibility complex antigen expression, and involvement in B cell maturation and immunoglobulin isot ype switching (1, 2, 3) IFN is also strongly correlated with the promotion of a T helper (Th)1 response, for which it has roles in the suppression of Th2 and Th17 specializations, activation and homing of natural killer cells, CD8+ T cells, and mononuclear macrophages, and the secretion of specific cytokine profiles, especially inflammatory ones marked by TNF and itself (4, 5, 6) The classical model of signaling for IFN contends that its effects are exerted solely through interactions with the extracellular domain of its r eceptor consisting of an subunit, IFNGR 1, and a subunit, IFNGR 2 (7). Receptor crosslinking results in the activation of receptor associated tyrosine kinases of the Janus kinase family, JAK1 and JAK2, leading to phosphorylation and dimerization of t he STAT1 transcription factors, which then dissociate from the receptor cytoplasmic domain and translocate to the nucleus. (8) This view ascribes no further role to the ligand or the receptor in the signaling process. (9) Further, there is the implicit as sumption that the phosphorylated STAT1 homodimer possesses an intrinsic nuclear localization sequence (NLS) that is responsible for its nuclear translocation. (8) However, based on current knowledge there is a potential paradox, as STATs like STAT1 do no t contain a polycationic or functional NLS (10).
11 We have proposed an alternative model for JAK/STAT signal transduction in which the ligand, receptor and auxiliary proteins are more involved It has been known for some time that IFN translocates to the nucleus of receptor expressing cells with kinetics as rapid as those for the activation and nuclear translocation of the transcription factor STAT1 that it activates ( 1 1 1 2 ). More recently, nuclear translocation of IFN has been shown to be driven by a n NLS in its C terminus (1 3, 14) Mutations of the IFN NLS destroy its biological activity, which can be restored by reconstitution with the NLS from T Ag of SV40 virus (1 4 1 5 ) The T Ag NLS is known to localize to the nucleus in a IMP / 1/Ran dependent f ashion. Excess T Ag NLS peptide inhibits IFN NLS dependent nuclear import, suggesting that IFN NLS mediates nuclear import through the same pathw ay (1 3 ). Results from immunoprecipitation experiments, which detected endocytosed IFN bound to IMP 5 (NPI 1) in cells actively transporting IFN to the nucleus, are c onsistent w ith this conclusion (16) Subsequent experiments showed that the receptor IFNGR1, of the hetero oligomeric receptor also translocates to the nucleus in IFN treated cells, while the I FNGR2 is not translocated ( 1 4 1 2 1 7 ). IFN binds to a soluble form of its receptor in which its N terminus interacts with the extracellular domain of the receptor, while the C terminus from residues 95 through 132 binds instead to the membrane proxima l region of the cytoplasmic d omain of the receptor (15, 1 8 19, 20) for IFNGR1 (7, 2 1 ) Insights from this alternative model led to the design of a short peptide mimetic of IFN w hich could demonstrate the intracellular role we suspected IFN fulfilled. The mimetic corresponds to a section of the C terminus [ IFN (95 132)] that interacts with
12 the intracellular domain of IFNGR1 and includes the polycationic NLS motif, 126 R KRKRSK ( 1 1 22). A lipophilic attachment, p almitate, was added to enable cell penetration ( 1 5 23). This mimetic proved capable of forming a complex with STAT1 along with IFNGR1 in the cytoplasm and provided the NLS s ignaling for nuclear transport (22). Signific antly, IFN (95 132) possessed agonist activity including upregulation of MHC class II in macrophages and protection against viral infection without toxicity, on macrophages similar to that of full length IFN (14, 1 9 2 0, 23). Like IFN initial findi ngs indicated the mimetic had a broad spectrum of effectiveness against viruses including large dsDNA viruses like HSV 1, a herpes family virus that replicates in the nucleus, and vaccinia virus (VV), a pox family virus that replicates in the cytoplasm, an d encephalomyocarditis, a small s sRNA member of picornavaridae (24, 25, 26). Viruses have developed a variety of mechanisms to antagonize the antiviral apparatus of IFN Poxviruses are particularly astute at thwarting the IFN system. These are large double stranded viruses that replicate in the cytoplasm of the cell. The variola strain of the poxviruses is responsible for smallpox, which historically has been the ca usative agent of pandemics that have resulted in considerable loss of human life until the unprecedented campaign of global immunization (27) E radication of smallpox was not however the permanent exit of pox viridae from the global stage, as evidenced by recurre nt outbreaks of monkeypox in Af rica and recently in the United States (2 8 ) The assembly of poxviruses in the cytoplasm of infected cells is complex, involving the generation of four types of infectious virus particles (27). Attachment, internalizat ion, and disassembling of poxviruses is followed by initiation of three waves of mRNA
13 receptors. Decoy receptors for both type I and type II IFNs are produced during early protein synthesis in poxvirus infected cells. An important virulence factor of poxviruses is the B8R protein, which is a homolog of the extracellular domain of the IFN receptor and can therefore bind to intact IFN and prevent its interaction with the r eceptor (29). The rodent picornovirus, encephalomyocarditis virus (EMCV), has an extremely wide r ange of hosts including humans (30, 31, 32). Instances of human infection with EMCV have manifested as generalized febrile illness, but the virus has also been isolated from patients with more severe illnesses, such as encephalitis, meningitis, and cardiomyopathy (31, 33). In mice, EMCV infection is lethal (32, 34, 35). We characterized the antiviral effects of the IFN (95 132), with and without the NLS region, and evaluated the therapeutic activity of the IFN mimetic peptide in vivo in the presence and absence of B8R protein in models of lethal EMCV and VV infection in mice (25, 26). The peptide mimetics act intr acellularly to activate the JAK/STAT signaling apparatus and do not recognize the IFN receptor extracellular domain and therefore bypass interactions wi th virulence factors like B8R (23). A family of proteins called suppressors of cytokine signaling (SO CS) negative ly regulates JAK/STAT signaling (36, 37, 38) These inducible proteins share domains of homology that characterize the SOCS family. SOCS proteins are also negative regulators of signaling by other cytokines, growth factors, and hormones (39, 40 41) 1 to SOCS 7 and cytokine inducible SH2 protein. SOCS 1 is of particular interest, because it is a
14 negative regulator of JAK2 as well as several other cytokines and hormone receptor syste ms, including epidermal growth factor receptor (EGFR) (42 ). We designed a short 12 mer peptide, WLVFFVIFYFFR, which antagonizes IFN signal transduction by binding to the autophosphorylation site of JAK2, resulting in inhibition of its autophosphorylation as well as its phosphorylation of IFNGR subunit IFNGR 1 (43) Tkip 1013 containing the phosphotyrosine residue (pY1007) in the activation loop of JAK2 similar to SOCS 1 KIR. From the SOCS1 kina se inhibitory region (KIR) we also designed another peptide corresponding to residues 53 DTHFRTFRSHSDYRRI near its N terminus, which we called SOCS1 KIR and showed that it has properties similar to Tkip (44) Tkip and SOCS1 KIR inhibit JAK2 phosphorylation activity and activation of STAT1 IL 6 activity is similarly suppressed by Tkip and SOCS1 KIR via inhibition of JAK2 phosphorylation of STAT3 (41, 45). The proliferation of prostate cancer cells, which depend on activation of the oncogene STAT3, were similarly blocked by Tkip inhibition of JAK2. Hence, these peptide antagonist of IFN signaling appear to have anti region (KIR) of SOCS 1 interacts with the aut ophos phorylation site of JAK2 (44, 46 ) We have tes ted Tkip in a murine model of multiple sclerosis (MS) called experimental allergic encephalomyelitis (EAE) (46) In the EAE model, immunization of mice with CNS antigens such as myelin basic protein (MBP), proteolysis protein, and myelin oligondendrocyte g lycoprotein results in tail and limb paralysis due to (47, 48) It was widely felt that Th 1 cells driven by the cytokine interleukin 12 (IL 12) were primarily responsible for the CNS
15 pathology of MS (49) Inflammatory cytokines that employ the JAK/STAT pathway such as IFN TNF and IL 2 were under this model implicated in the pathogenesis of both EAE and MS (50 54). More recently, IL 17 producing CD4+ T (Th17) cells have supplanted Th1 cells as the pr imary cause of MS (49, 55 58) During our investigation into the binding specificity of SOCS 1 KIR and Tkip we created another peptide, pJAK2(1001 1013). p JAK2(1001 101 3) correspond ed to the activation loop of JAK2, and we demonstrated that it blocked SOC S 1 activity in cells (44) The ability of pJAK2(1001 1013) to function as an antagonist of SOCS1 was reflected by its ability to function as an agonist of IFN type I and type II signaling through the JAK/STAT 1013) enhances suboptimal IFN activity, blocks SOCS 1 induced inhibition of STAT3 activation, enhances IFN activation site (GAS) promoter activity, and enh ances Ag It would be a great benefit for patients to receive the bonus of an adjuvant effect, and therefore long lasting immunity, as a side effect from a therapeutic already being used. The adjuvant effects of IFN are well known, and have been shown in various animals ( 59 64 ). Its upregulatio n of MHC, and modulations of APCs, as well as stimulation of B and CD8+T cells and effect on toll like receptors ( TLR ) s have long since been identified with its performance as an adjuvant in both c ellular and humoral arms The ability to enhance immune mem ory is desirable as it plays an important role in effective vaccines. This is especially true in scenarios where causative agents, or their components, present as weak antigens to the immune system. Such is often the case with tumors, which, in the absen ce of an adjuvant, are very difficult to mount any response against. Recently, tuberculosis patients that have resistance to standard
16 chemotherapy, have experie nced a beneficial effect of IFN when it is used as adjuvant in treatment (64). As IFN (95 132 ) has previously shown similarity to the effects expressed by native IFN it followed that its mimetic and its agonist, pJAK2, could also boost adjuvancy and were examined in a model with strong antigens, as with VV, and with weak ones, as with bovine se rum albumin (BSA). These were quantified both by splenocyte proliferation and antibody production after a delayed second exposure. Also, because JAK2 is a link in the Toll like receptor 3 (TLR3) signaling cascade, which serves both innate and adaptive ar ms, we tested to see whether this peptide had an adjuvant effect independent of Tcells. Contrastingly, we show that the JAK2 antagonism of SOCS1 KIR, as well as Tkip, inhibit IFN induced macrophage activation, and some of the underlining mechanisms associated with the formation of immune memory.
17 CHAPTER 2 MATERIALS AND METHOD S Cell culture, virus, B8R protein, and interferons from the American Ty minimal essential medium (JHR Biosciences, Lenexa, KS) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 /ml streptomycin (complete medium) at 37C in a 5% CO2 atmosphere incubator. Raw 264.7 were maintained in RPMI 1640 medium (SAFC Biosciences) supplemented with 10% FBS (HyClone), 100 U/ml penicillin and 100 U/ml streptomycin (complete medium). EMCV was obtained from the American Type Culture Collection and stored at 70 C until use. Vaccinia virus Western Reserve was a g i f t from Dr. Richard Condit (University of Florida, Gainesville, FL). The B8R protein was a kind gift from Tilahun Yilma (University of California, Davis). Rat and mouse IFN gamma (both at 10 7 U/mg) were purchased from PBL Biomedical Laboratories (Piscataway, NJ) and kept at 70C until use. Peptide synthesis All peptides [ IFN (95 132), IFN (95 125), IFN (95 106), IFNGR1(253 287), Tkip, SOCS1 KIR, pJAK2(1001 1013) ] were synthesized with an Applied Bios ystems 9050 automated peptide synthesizer (Foster City, CA) using 8 19, 79) IFN (95 132) peptide was synthesized without the cysteine residue at the C terminal end (residue no. 133 ). The addition of a lipophilic group (palmitoyl lysine) to the N termini of the synthetic peptides was performed as the last step by using a semi high performance liqui d chromatography. Peptides were dissolved in deionized water
18 and used for experimentation. All peptide solutions were negative for endotoxins as determined by a Limulus amebocyte lysate test (E toxate kit; Sigma, St. Louis, MO). Antiviral assays Antiviral assays were performed by using a cytopathic effect reduction assay with EMCV. Murine L929 cells (6 x 10 4 cell/well) were plated in a 96 well IFN IFN (95 132 ) pJAK2(1001 1013), derivatives, and control peptides were incubated for various times (7 to 24 h) at 37C. In othe r experiments, B8R protein (33 /ml) was preincubated for 2 h with rat IFN and IFN mimetic and control peptides. EMC virus (200 PFU/well) was added to the plate and incubated for 1 h, after which plates were washed and media added. Cells were incubate d for at least 24 h and then stained with 0.1% crystal violet. Unbound crystal violet was aspirated, and the plates were thoroughly rinsed with deionized water, blotted, and allowed to air dry. Plates were scanner (UMAX Technologies, Dallas, TX) and analyzed using Image J 1.29 software (Na tional Institutes of Health, Be thesda, MD) to assess cell survival. Percentages of cell survival were determined by comparing survival for the experimental treatment groups with that for the virus only control group. Macrophage activity. Murine macrophage cells, Raw 264.7, were seeded on 24 well plates at a concentration of 3 x 10 5 cells/well (300 l /well) and allowed to adhere. Varying concentrations of lipo peptides, Tkip, SOCS1 KIR, or mu IFN IFN (95 106), were then added to the wells and the cells incubated for2hat 37C in a 5% CO2 incubator. Varying concentrations of IFN were then added an d the cells were incubated for an additional 72 h at 37C in a 5% CO2 incubator, after which
19 s upernatants were transferred to fresh tubes and assayed for nitrite levels as a measure tructions (Alexis Biochemicals). To test for synergy between Tkip and SOCS1 KIR, the cells were incubated in the presence of IFN and varying concentrations of peptides as described above and also in the presence of both lipo Tkip and lipo SOCS1 KIR or lipo Tkip and lipo Mu IFN (95 106) and were collected after 48 h and tested for NO production as described above. MBP roliferation assay SJL/J mice were immunized with bovine myelin basic protein (MBP) as previously describe d (46) and spleens were extracted and homogenized into a single cell suspension. Splenocytes (1 x 10 5 cells/well) were incubated with medium, MBP (5 0 /ml), lipo Tkip, lipo SOCS1 KIR, or lipo MuIFNGR (253 287) for 48 h at 37C in 5% CO2. The cultures were pulsed with [ 3 H]thymidine (1.0 cell harvester. Cell associated scintillation counter, and data are reported as counts per minute. In vivo studies of mice One year old or younger (C57BL/6) or (SJL/J) mice were purchased from Jackson Laboratories and cared for at the Animal Resourc e Center of the University of Florida (Gainesville, FL). The Institutional Animal Care and Use Committee (IACUC) of the University of Florida approved all protocols prior to any study initiation. For EMCV experiments, female C57BL/6 mice were pretreated by intraperitoneal injection using a tuberculin syringe for 3 or 6 days with rat or mouse IFN and IFN (95 132) and control peptides at various concentrations (100 to 200 /mouse) every day. In some studies, B8R protein (250 /ml, diluted from crude stock
20 preparation) was preincubated with the rat IFN (200 U/mouse) and peptide (100 /mou se) injection cocktails prior to intraperitoneal administration. On the last day of treatment, mice were challenged with 50 PFU of EMC virus. The numbers of surviving mice were recorded starting on the day of EMC virus challenge (day 0) and are presented a s percent survival. Ten mice per treatment group were used in all mouse studies. For Vaccinia experiments, female C57BL/6 mice (6 8 wk old) were used Peptides dissolved in PBS in a volume of 100 were administered i.p. Vaccinia was administere For intranasal administration, vaccinia virus was taken in a volume of 10 were delivered in each of the nostrils of a lightly anesthetized mouse. After infection, mice were observed daily for signs of disease, euthanized and counted as dead. Measurement of anti vaccinia or anti BSA Ab response by ELISA Microtiter plates were coated w ith 10 6 inactivated vaccinia virus (900,000 J/cm 2 for 5 min in a DNA cross linker), or 0.5 of binding buffer (carbonate bicarbonate, pH 9.6) overnight at 4C. Plates were blocked for 2h at room temperature with PBS containing 5% FBS. Mouse sera (n = 5) were serially diluted in PBS containing 0.1% Tween 20 (wash buffer); 0.1 ml of the diluted serum was added to each well. The plate was incubated for 2h at room temperature and washed three times with wash buffer. Pero x idase conjugated goat anti mouse IgM (micro chain 1/2000 in a volume of 0.1 ml was added to each well, incubated for 1 h, and washed
21 Phenylened iamine (Pierce) in a volume of 0.1 ml was added and incubated for 15 min. The reaction was of 3 N HCl. The OD490 was determined using a microtiter plate reader. Measurement of vaccinia virus or BSA specific cellular res ponse by proliferation assay. Spleens from naive or pre inoculated mice at times indicated were homogenized to single cell suspension. Splenocytes (10 5 cells/well) were incubated with medium alone or medium containing UV inactivated vaccinia virus or BS A depending on the experiment, for 96 h. The cultures were then pulsed with [ 3 H]thymidine (1 Ci/well; Amersham Biosciences, Piscataway, NJ) for 8 h before harvesting onto associated radioactivity was counted using a scintillation counter. Sti mulation index refers to the in corporation in splenocytes cultured with test Ag divided by incorporation in splenocytes cultured with medium alone
22 CHAPTER 3 RESULTS The Gamma Interferon (IFN ) Mimetic Peptide IFN (95 132 ) Prevents Encephal omyocarditis Virus Infection both in Tissue Culture and in Mice We have demonstrated previously that the C peptide consisting of residues 95 to 132 [IFN 132)], which contains the crucial IFN sequence (NLS), has antiviral activity in tissue culture. Here we model of lethal viral infection with the encephalomyocarditis virus (EMCV). Deletion of the NLS region from the IFN 132) resulted in loss of antiviral activity. However, the NLS region does not have antiviral activity in itself. Replacing the NLS region of IFN 132) with the NLS region of the simian virus 40 large T ant igen retains the antiviral activity in tissue culture. IFN 132) prevented EMCV induced lethality in mice in a dose dependent manner compared to controls. Mice treated with IFN 132) had no or low EMCV titers in their internal organs, whereas contr ol mice had consistently high viral titers, especially in the heart tissues. Injection of B8R protein, which is encoded by poxviruses as a defense mechanism to neutralize host IFN not inhibit IFN 132) protection against a lethal dose of EMCV, w hereas mice treated with rat IFN mimetic peptide IFN 132) prevents EMCV infection in vivo and in vitro and may have potential against other lethal viruses, such as the smallpox virus, which encodes the B8R protein. Antiviral activity of IFN 132) peptide in the presence of the B8R protein in tissue culture and in v ivo Since we have shown previously that IFN 95 132 ) binds to the cytoplasmic domain of the IFN receptor and thus tr iggers signal
23 trans duction events associated with IFN this mimetic peptide was evaluated in the presence o f the B8R protein, which is pro duced by poxviruses for neutralization of IFN activity. The B8R protein is a homolog of the IFN receptor extrace llular domain; therefore, IFN 95 132 ) should retain its antiviral activity in the presence of this virulence factor of poxviruses. To demonstrate this in tissue culture, murine L929 cells were well plate, a fter which rat IFN IFN 95 132 ), and IFN 95 125), which were all preincu bated for 2 h with B8R protein, were added to the plate. Rat IFN was used here instead of mouse IFN due to the fact that B8R does not bind to mouse IFN but does bind to rat IFN ( 65 ). Further more, rat IFN has activity on mouse cells, as shown previously (65 ). Aft er overnight incubation, EMCV was added, and the cells were washed and reincubated in media for 24 h, followed by determination of EMCV cyto pathic effects. As shown in Fig. 3 1 A, IFN had antiviral activity against EMCV at all concentrations (100 U/ml, 33 U/ml, and 11 U/ml) used, but in the presence of B8R, IFN antiviral activity was lost at 33 U/ml and 11 U/ml. In contrast, the antiviral activities of IFN (9 5 132) were similar in the pres ence or absence of B8R protein, as denoted by almost 100% cell viability at the 11 peptide concentration used (Fig. 3 1 A and B). The control peptide IFN 95 125) did not have any antiviral activity in either case. Thus, B8R protein neutralize d IFN antiviral activity but not IFN (95 132) a ntiviral activity against EMCV in tissue culture. Based on the above described tissue culture study, the anti viral activity of IFN (95 132 ) was assessed in the presence of B8R protein in the mouse model o f lethal EMCV infection. C57BL/6 mice were pretr eated for 3 days with PBS, IFN (95 132 ) (100 /day), or rat IFN (200 U/day).
24 The 200 U/day dose of rat IFN was administered in order to detect the neutral izing effect of the B8R protein. On the last day of treatment, mice were challenged with 50 PFU o f EMCV. The numbers of sur viving mice were recorded over time. As shown in Fig. 3 2 injection of rat IFN resulted in 20% survival of mice in response to EMC V challenge. In contrast, administration of B8R with rat IFN resulted in 0% of mice surviving, which was similar to results for PBS controls. Administration of IFN (95 132 ) in the presence or absence of B8R resulted in 40% and 30% survival, respectively 9 days after the EMCV challenge. Furthermore, there was a delay in the onset of lethality in IFN 132) and rat IFN treated groups compar ed to results for PBS and IFN /B8R treated groups. Thus, the IFN was effective against EMCV in the presence of B8R. I additionally contributed to the characterization of IFN injection, observation, non survival surgeries and euthanization of C57BL/6 mice which were challenged with EMCV. I conducted and assisted in various cy topathic effect reduction assays, and virus yield reduction and plaque assays all involving EMCV. For the above procedures which involved tissue I maintained murine L929 fibroblast cell lines. IFN Mimetic as a Therapeutic for Lethal Vaccinia Virus Infec tion: Possible Effects on Innate and Adaptive Immune Responses We have developed small peptide mimetics of IFN virulence factor B8R protein, which binds to intact IFN receptor extracellular domain. Thus, these peptides inhibit vaccinia virus (VV) repli cation i n cell culture where intact IFN is ineffective. We demonstrate here that the mouse IFN mimetic peptide, IFN 132), protects C57BL/6 mice against
25 overwhelming lethal VV infection. The mimetic peptide was synthesized with an attached lipophil ic group for penetration of cell plasma membrane. Injection of mimetic i.p. before and at the time of intranasal (10 6 PFU) or i.p. (10 7 PFU) challenge with virus resulted in complete protection at 200 of mimetic and 40 60% protection at 5 of mimetic. Initiation of treatment of mice with IFN resulted in complete protection against death, whereas initiation of treatment at 6 days postinfection resulted in 40% protection. Administration of mimetic by the oral route al so completely protected mice against the intranasal route of a lethal dose of VV challenge. In addition to its direct antiviral effect, the mimetic also possessed adjuvant effects in boosting humoral and cellular immunity to VV. The combination of antivira l and adjuvant effects by the IFN mimetic probably plays a role in its potent anti VV properties. These results suggest an effective therapeutic against ongoing, lethal poxvirus infections that taps into innate and adaptive host defenses. IFN m imetic IFN 132) possesses a djuvant ac tivity We were interested in determining whether the IFN mimetic possessed adjuvant activity that may have contributed to the anti VV immune response in protecting mice. Thus, mice were infected with a sub lethal dose of VV in the presence of mimetic or control peptide, and the cellular and humoral immune response to virus were monitored. Mice were injected i.p. with peptides at days 2, 1, and 0 relative to the intranasal challenge with 10 4 PFU of VV. Proliferation in the p resence of VV was determined with splenocytes at 4 wk postinfection. As shown in Fig. 3 3 A both lipo IFN 132) and nonlipo IFN 132) treated murine splenocytes showed greater proliferation in the presence of VV than those of the control peptide treated mice, with the lipophilic form being more
26 effective. An unrelated peptide from resid ues 253 to 287 from IFNGR1 with a lipophilic residue was used as a control peptide in this experiment. Thus, the mimetic in either a lipophilic state for cell penetration or in a nonlipophilic state enhanced the proliferation of splenocytes from mice infec ted with VV. Uptake of a nonlipophilic peptide suggests uptake required pinocytosis by antigen presenting cells ( APC ) s. Since proliferation for the nonlipophilic group corresponded closely to the lipophilic group, we can speculate that APCs may account fo r most of the proliferation effect, and additionally that this may be the route of mimetic action. VV is a potent Ag and thus may possess intrinsic adjuvant activity. This in turn could mask some of the adjuvancy of the mimetic. We thus determined the ad juvancy of the IFN mimetic against a soluble protein, BSA, focusing on the humoral response. As shown in Fig. 3 3 B lipo mimetic BSA IgG Ab by ELISA at weeks 2 through 4 when compared with the BSA treatment alone. Mice treated with lipo control peptide from residues 95 to 106 had Ab responses similar to that of BSA alone. The IFN mimetic thus possesses adjuvant effects against VV and BSA Ags. Both the Suppressor of Cytokine Signaling 1 (SOCS 1) K inase Inhibitory Region and SOCS 1 Mimetic Bind to JAK2 Autophosphorylation Site: Implications for the Development of a SOCS 1 Antagon ist Suppressor of cytokine signalin g (SOCS) 1 protein modulates signaling by IFN by binding to the autophosphorylation site of JAK2 and by targeting bound JAK2 to the proteosome for degradation. We have developed a small tyrosine kinase inhibitor peptide (Tkip) that is a SOCS 1 mimetic. T kip is compared in this study with the kinase inhibitory region (KIR) of SOCS 1 for JAK2 recognition, inhibition of kinase activity, and regulation of IFN induced biological activity. Tkip and a peptide corresponding to the
27 KIR of SOCS 1, 53 DTHFRTFRSHSDY RRI (SOCS1 KIR), both bound similarly to the autophosphorylation site of JAK2, JAK2(1001 1013). The peptides also bound to JAK2 peptide phosphorylated at Tyr1007, pJAK2(1001 1013). Dose response competitions suggest that Tkip and SOCS1 KIR similarly recogn ize the autophosphorylation site of JAK2, but probably not precisely the same way. Although Tkip inhibited JAK2 autophosphorylation as well as IFN induced STAT1 KIR, like SOCS 1, did not inhibit JAK2 autophosphorylation but inhib ited STAT1 Both Tkip and SOCS1 KIR inhibited IFN ophages and inhibited Ag specific splenocyte proliferation. The fact that SOCS1 KIR binds to pJAK2(1001 1013) suggests that the JAK2 peptide could function as an antagonist of SOCS 1. Thus, pJAK2(1001 1013) enhanced suboptimal IFN 1 induced inhibition of STAT3 pho sphorylation in IL 6 treated cells, enhanced IFN activation site promoter activity, and enhanced Ag SOCS 1 competed with SOCS1 KIR for pJAK2(1001 1013). Thus, the KIR region of SOCS 1 binds directly to the autophosphor ylation site of JAK2 and a peptide corresponding to this site can function as an antagonist of SOCS 1. T kip and SOCS KIR inhibit IFN induced a ctivation of m acrophages IFN plays an important role in activation of macrophages for innate host defense aga inst intracellular pathogens as well as serving to bridge the link between innate and adaptive imm une responses ( 1). Tkip and SOCS1 KIR were examined for their ability to block IFN ion of nitric oxide ( NO ) production using Griess reagent (Alexis Biochemicals). Lipo versions of the peptides were synthesized with palmitic acid for pen etration of the cell membrane
28 (66 ). Both Tkip and SOCS KIR, compared with control peptide, inhibited in duction of NO by various concentrations of IFN 3 4A. Dose response with varying concentrations of the peptides against IFN NO production by Tkip and SOCS1 KIR with Tkip being the more effect ive of the inhibitors as shown in Fig. 3 4B. Control peptide was relatively ineffective at inhibition, KIR inhibition. Tkip and SOCS1 KIR in combination (33 M each) were the most effective in inhibit ion of IFN the autophosphorylation site of JAK2 by the two peptides. Thus, Tkip and SOCS1 KIR both inhibited IFN re effective inhibitor. Tkip and SOCS1 KIR inhibit Ag roliferation We have previously shown that Tkip inhibits Ag vitro (2 5 ice immunized with bovine MBP. In this study, we compared Tkip and SOCS1 KIR for their relative ability to inhibit proliferation of MBP 10 5 cells/well) were incubated with MBP (50 /ml) in the presence of lipo Tkip, lipo SOCS1 KIR, or lipo control peptide for 48 h and proliferation was assessed by testing for [ 3 H] thymidine incorporation. As shown in Fig. 3 5, both Tkip and SOCS1 KIR inhibited MBP induced proliferation of splenocytes, while the control p eptide had a negligible effect on proliferation. Similar to inhibition of NO production by macrophages, Tkip was more effective than SOCS1 KIR in inhibition of MBP induced splenocyte proliferation with 84, 88, and 97% inhibition at 1.2, 3.7, and 11 M, respectively,
29 compared with 61, 67, and 72% for SOCS1 KIR. Thus, both Tkip and SOCS1 KIR inhibited Ag induced splenocyte proliferation, which is consistent with SOCS 1 protein inhibition of A g ). I contributed to the Tkip SOCS1 KIR experiments by maintaining LNCaP and murine macrophage cells (RAW 267.4). I helped in peptide preparation and dilution, I cooperated with Dr. Mujtaba in the EAE experiments on SJL/J mice i n which we found SOCS1 KIR could protect mice from relapsing/remitting EAE. In this experiment I administered injections of the various peptide and control species, as well as MBP immunizations to induce disease. Throughout the experiment I assisted in t he supervision of mice, the progression of their disease, and some of the post mortem surgeries, such as splenectomies. I also assisted Dr. Mujtaba when we examined SOCS1 KIR in a role of prevention and reversal of lymphocyte infiltration into the CNS du ring EAE. Here, we harvested the brains from three individual mice, one nave, and t wo immunized with MBP The two MBP mice had been treated every other da y for 38 days post immunization, one with PBS (60 ), and the other with SOCS1 KIR2A (60 ). The t hree m ice were sacrificed, their brains removed to a 4% PFA in PBS and fixed overnight before being transferred to 70% ethanol. Brains w e re embedded in paraffin, sliced and stained with H&E for imaging. In addition to work directly associated with the Tk ip model, I helped train other members of our lab in animal handling, injection, anesthetization, blood sampling, organ harvesting, euthanasia and other often difficult, intimidating, and precarious
30 experimental procedures which revolve around live, willfu l animals during in vivo experimentation. Enhancement of Antiviral Immunity by Small Molecule Antagonist of Suppressor of Cytokine Signaling Suppressors of cytokine signaling are negative regulators of both innate and adaptive immunity via inhibition of si gnaling by cytokines such as type I and type II interferons ( IFN ) s. We have developed a small peptide antagonist of SOCS 1 that corresponds to the activation loop of JAK2. SOCS 1 inhibits both type I and type II IFN activities by binding to the kinase acti vation loop via the kinase inhibitory region of the SOCS. The antagonist, pJAK2(1001 1013), inhibited the replication of VV and EMCV in cell culture, suggesting that it possesses broad antiviral activity. In addition, pJAK2(1001 1013) protected mice agains t lethal VV and EMCV infection. pJAK2(1001 1013) increased the intracellular level of the constitutive IFN the antagonist antiviral effect at the cellular level. Ab neutralization suggests that constitutive IFN ( 1 9 ) pJAK2(1001 1013) also synergizes with IFNs as per IFN multiplicative antiviral effect at the level of transcription, the cell, and protection of mice against lethal viral infection. pJAK2(1001 1013) binds to the kinase inhibitory r egion of both SOCS 1 and SOCS 3 and blocks their inhibitory effects on the IFN promoter. In addition to a direct antiviral effect and synergism with IFN, the SOCS antagonist also exhibits adjuvant effects on humoral and cellular immunity as well as an enhancement of polyinosinic polycytidylic acid (poly I:C) activation of TLR3. The SOCS antagonist thus presents a novel and effective approach to enhancement of host defense against viruses.
3 1 pJAK2(1001 1013) exerts an adjuvant effect on t he i mmune system I n addition to its inhibitory effects on virus replication in cells and related to the potent anti VV response, we were interested in determining possible adjuvant effects of SOCS 1 antagonist on the immune response. Accordingly, C57BL/6 mice were immunized i.p. with 50 BSA, treated i.p. with 200 pJAK2(1001 1013) on days 2, 1, and 0, and then assessed for enhancement of cellular and humoral immune responses. BSA is a relatively weak Ag in mice. Four weeks postimmunization, splenocytes from the mice were stimulated in cell culture with 0.5 BSA. As shown in Fig. 3 6A, untreated mice or mice given PBS mounted a weak proliferation response. By comparison, mice treated with pJAK2 (1001 1013) had an ~8 fold greater proliferative response t o BSA. The humoral immune response as assessed by the serum IgG Ab response to BSA in the 1013) treated mice at 3 and 4 w ee k s postimmunization (Fig. 3 6B). The SOCS antagonist can also enhance the Ab re sponse to the T cell independent Ag LPS. This is shown in Fig. 3 6C, in which the Ab pJAK2(1001 1013). We previously showed that staphylococcal enterotoxin superantigens st aphylococcal enterotoxin A (SEA) and staphylococcal enterotoxin B (SEB) enhanced T cell dependent Ab production (68 ) SEA/SEB did not enhance the anti LPS response. SEA/SEB did enhance the Ab response to BSA, a T cell dependent Ag. Thus, the SOCS antagon ist has a direct effect on B cell function independent of T h elper cells. At the level of macrophage TLR function, RAW264.7 cells treated with pJAK2(1001 1013) produced a ~5 fold increase in NO production upon LPS stimulation (via TLR4) compared with a con trol peptide (Fig. 3 7A ). We also examined the effect of
32 pJAK2 (1001 1013) on TLR3 activation. Poly I:C is a synthetic dsRNA that activates macrophages and dendritic cell s via TLR3 (69 ). TLR3 thus plays an important role in the antiviral responses to HSV 1 which have a dsRNA stage in their replication (32, 33). SOCS 1 negatively regulates TLR signaling at several stages including signaling by type I IFNs and by NF transcription factor ( 45, 72, 73, 74 ). Given the importance of TLR3 in the innate immune response against viruses, we treated the macrophage cell line RAW264.7 with poly I:C and determined the enhancing effect of the SOCS 1 antagonist pJAK2(1001 1013) on NO production. As shown in Fig 3 7B poly I:C at 0.1 /ml had a negligible effect on NO production, which was increased >20 fold by 25 M pJAK2 (1001 1013). Alanine substituted JAK2(1001 1013)2A had a negligible effect on NO production. Thus, the SOCS 1 antagonist enhanced the poly I:C activation of TLR3. T he SOCS 1 effect would suggest that SOCS 1 induction has a regulatory effect on TLR3 activation and that the SOCS antagonist blocks this effect. These results demonstrate that pJAK2(1001 1013) has an adjuvant effect in terms of the cellular and humoral imm une responses as well as in macrophage activation. Thus, in addition to direct inhibition of virus replication, the antagonist also has an adjuvant effect on the immune response. I additionally contributed to many other levels of the pJAK2 inve stigations, performing numerous antiviral assays that assess ed the strength and toxicity of pJAK2 in L929 cell cultures. Here, cells treated with various doses of peptides including pJAK2 and 132) were challenged with EMCV or VV. I also re assessed EMCV and VV viral stocks for their infectivity I ran other experiments especially in animal models to assess peptide utility, including the IFN mimetic and Tkip The protective
33 abilities of the peptides were tested in VV in and sepsis mouse models I invest igated and q uantified the interference associated with DMSO which we used to dissolve relatively insolvent peptides 132), Tkip and related controls D MSO is a known anti inflammatory, and could compete with the inflammatory or anti inflamma tory natures of our peptides. I revealed the which c onsistently arose from supposedly inactive controls which were dissolved in DMSO, suggesting an alternative solution be used I also ran a number of assays and mouse experiments comparing the therapeutic effects of IFN ic, and the superantigens, SEA and SEB in an EMCV model which may have provided small insights
34 CHAPTER 4 DISCUSSION Interferon gamma is central to the development and character of the host defense, responsible for targeting a wide scope of i mmunologically relevant genes that range from the initiation of innate cellular programs, to the local recruitment and activation of leukocytes, to the selection and maturation of adaptive imm une effecter and memory cells (97). As a potent cytokine, IFN is subject to intensive positive and negative regulation from the host and also from pathogens. While control of IFN inflammation and pathology associated with I FN similarly be seen as one of the essential attributes of successful pathogens such as poxviruses, Mycobacterium and Leishmani a which, without virulence factors to antagonize IFN ownstream machinations (29, 75, 76) Poxviruses are highly adept at evading the host innate immune response due to their many evasion genes and their resultant protein products ( 77 ) There are, for example, greater than 18 proteins produced by poxviruses t hat interfere with different aspects of the host defense, including the actions of IFNs, TNF, chemokines, interleukins, and others. Vaccinia virus (VV) produces decoy receptors to deal with type I and type II IFNs. These homologues of IFNGR, and IFNAR ext racellular domain tie up the ligand in an effective dead end (77) Owing to its importance, the B8R gene is transcribed in the first wave of viral protein synthesis (29). We have engineered small, peptide mimetics of IFN terminus a mino acids 95 to 132 which possesses antiviral and agonist activity, and is
35 nontoxic to animals (23). These mimetics were developed contrary to the classical model of JAK/STAT signaling in which IFN interaction. Rathe r the design for a C terminal IFN logic of direct intracell ular signaling by IFN (10, 78). IFN are delivered to the nucleus of cells as a conglomerate wh e re IFN provides a cla ssical polycationic NLS for the nuclear importation of the group (14). Partial IFN mimetics demonstrate the importance of the NLS as a component in this complex molecule. An NLS peptide, IFN 132) a nd the truncated IFN mimetic where the NLC has been removed IFN 125), were equally without antiviral activity in viral assay s Replacement of the IFN 132) NLS region with the NLS of the SV40 large T antigen maintained the mimetics antiviral acti vity as assessed by its ability to protect cells from encephalomyocarditis virus (EMCV) in a cytopathic assay (25) This suggests that the NLS in IFN pathway utilized by the SV40 T antig en NLS for transportation into the nucleus (15, 16, 18). We have previously shown that IFN 132) introduced into the cytoplasm was that it provided the NLS signaling for nuclear transport like tha t of full length IFN Both mouse IFN 132) and human IFN 134) mimetics induced an antiviral state and up regulated MHC cla ss I molecules in cell culture (23). Intracellular signaling begins when IFN res idues 253 to 287 on the cytoplasmic domain of receptor subunit IFNGR1. This binding induces the tyrosine phosphorylation events catalyzed by JAK1 and JAK2
36 domain of IFNGR1 (9) It may also be the case that JAK1 and JAK2, which are known to be initially attached to IFNGR1, remain attached and are carried on the IFN the transcription of IFN nes such as the phosphorylation of histones and enhancement of promoter binding (81, 82, C.M. Ahmed, E.N. Noon Song, and H.M. Johnson, unpublished observations). Inside the nucleus, chromatin immunoprecipitations and reporter gene studies of IFN treated cells find the se ligand s site element on the DNA and indicate that they participate in mediated transcription (83). We have characterized IFN 132) fitted with the mem brane penetrating lipophilic group palmitate for its ability to counteract lethal viral disease in tissue cultures and C57BL/6 mice In a dose dependent manner IFN 132) protect ed L929 against EMCV challenge (25). In an animal model where EMCV had 100% lethality in controls, a three day prophylactic regimen of IFN 132) resulted in 100% survival of mice, equal to that obtained by IFN days postinfection showed little or no EMCV in the hearts, livers, and spleens of IFN 132) treated mice, opp osite what we found for control peptide and PBS treatment groups. Major organs assayed for EMCV 13 days after inoculation showed that IFN 132) treated mice had achieved total clearance of the virus (25) The prophylactic effect observed in these experiments is probably the consequence of IFN IFN mimetic preparing an antiviral state within cells and generally inducing immune
37 system activity, for example the enhancement of immune surveilla nce. An additional experiment was devised to exhibit IFN 132) intracellular mode of action wherein B8R virulence factor was combined with EMCV and added to cell cultures treated with IFN (95 132) activity was independent of the extracellular domain of IFNGR, it retained its protective effect where native IFN The strong antiviral activity IFN 132) against EMCV like IFN EMCV+B8R in contrast to IFN encouraged us to apply the mimetic against a poxvirus model in mice Treating a highly lethal VV infection with IFN 132) was completely protective compared with the 100% fatality of controls, even for mice who had therapy withheld two days after vira l ingress. Initiation of treatment as late as days 6 resulted in the recovery of 40% of the infected animals (26) By bypassing the virulence factor B8R, the IFN benign one. In a ddition to the direct antiviral properties, IFN 132 also had an adjuvant effect on both the humoral and cellular immune response. Mimetic enhanced the IgG antibody ( Ab ) response to both VV and BSA. Cellular immunity to VV was also enhanced as determine d by splenocyte proliferation. The adjuvancy of the mimetic was observed with or without the lipophilic group attached. Thus, it does not depend on mimetic penetration of the plasma membrane of nonphagocytic cells. Given that uptake of the mimetic by APCs does not depend on the lipophilic group, internalization by pinocytosis could play an important role in the enhanced of adaptive immune response s
38 We have been particularly interested in regulation by the suppressors of cytokine signaling (SOCS) proteins that modulate interferons, including IFN cytokines and growth factors, as they attempt to signal through the JAK/STAT pathway. SOCS 1 function is requisite for an survival past early infancy. Although SOCS 1 knockout mice appea r to be normal at birth, they exhibit stunted growth and die as neonates in their third week (84). These mice suffer a syndrome characterized by severe lymphopenia, activation of T lymphocytes, fatty degradation and necrosis of the ration of multiple organs, and high levels of constitutive IFN well as an abnormal sensitivity to it (41, 84, 85). IFN likely play s a central role in the pathology since SOCS 1 knockout mice ie nt in IFN neonates. Similar pathology and lethality is observed in normal neonates that are injected in excess with IFN SOCS 1 is transcribed alongside other IFN in a negative feedback loop (45, 72, 73, 74, 86). The dynamics of induction of S OCS 1 by IFN 1 attenuates IFN astrocytes with IFN 1 gene at ~90 min (87, 88) Low doses of IFN 1 mRNA that returned to baseline after 4 h, whereas high concentrations of IFN SOCS 1 mRNA up to 24 h. Thus, SOCS 1 suppression appears to be induced by the IFN 1 +/+ mice with IFN p eak ing at 2 h before dec lining (88). Although 1 a similar fashion, it persists for 8 h.
39 SOC S 1 thus normalizes IFN while still permitting a lower amplitude of signaling more likely to be beneficial. The power to antagonize cytokines which depend on tyrosine kinases for signal trans d uction could have much therapeutic usefulness, especially in inflammatory disease where IFN 1 supplementation used in co njunction with other modulaters may be able to abolish certain proinflammatory activity, or to res tore equilibrium, as well as to affect long term development within the adaptive arm, as with in the T helper subsets that are sensitive to the presence of certain cytokines and interleukins. Given the critical importance of SOCS 1 modulation of IFN other cytokines employing JAK2, we have developed small peptide mimetics of SOCS 1, Tkip and SOCS1 KIR (43, 44) Tkip and SOCS1 KIR recognized the autophosphorylation site on JAK2, specifically residues 1001 1013 including the critical tyrosine at 1007 whi ch would be phosphorylated (44, 45). We showed that Tkip blocked JAK2 autophosphorylation as well as tyrosine p hosphorylation of substrates such as STAT We subsequently showed that Tkip blocked IL 6 induced activation of the STAT3 oncogene in LNCaP prostate cancer cells, which involved inhibition of JAK2 activation (90) These studies presented a proof of concept demonstration of a peptide mimetic of SOCS 1 that regulates JAK2 tyrosine kinase function. We chose EAE, the r odent model for MS, to test the potential of SOCS1 KIR and Tkip in an T cell mediated auto immune disease considered to involve cytokines which depend on JAK2 ( 46 ). SJL/J mice were immunized with MBP for induction of the relapsing/remitting form of EAE. T he SOCS 1 mimetic and KIR protected the mice
40 against relapses compared with control groups in which >70% of the mice relapsed after primary incidence of disease. Protection of mice correlated with lower MBP Ab titers in Tkip and SOCS1 KIR treated groups as well as suppression of MBP induced proliferation of splenocytes taken from EAE afflicted mice (44) Consistent with its JAK2 inhibit ory factor (44). Thus, Tkip and SOCS1 KIR as with SOCS 1, possess anti relapsing/remitting EAE. More recent findings have indicated the additional involvement of the Th17 subset after its isolation from MS patients ( 90 ) Mice with EAE produce IFN 17A when stimulated with IL 23. Pathogenic Th17 cells can be generated by a combination of IL 6, Il 23, and IL (91) Not only will SOCS1 KIR and Tkip downregulate the IFN these cells, but IL 23 also signals through JAK2 and is regu lated by SOCS1 KIR/Tkip itself ( L. Jager R. Dabelic, and H.M. Johnson u npublished results ). Treg cells essential to the bodies maintenance of self tolerance through their inhibitor effects on ef fecter cells are also relevant to dise a ses like MS and EAE defined by self re a ctive T cells ( 92 ). It was thought that Treg and Th17 effectors arose in a mutually exclusive fashion, depending on whether they were activated in the presence of TGF 6 (90) Even more recent studies show plasticity between T cell phenotypes involves cytokines that differ from those previously considered the central players (93). IL 6 is still considered to be involved in th e current formulas for Th17 generati on. Since Tkip and SOCS1 KIR can suppress IL 6 transduction, it suggests cytokine antagonists like SOCS mimetics may have influence over active T cell phenotypes.
41 Tkip and SOCS1 KIR were also significant in their ability to blunt IFN effect on cellular and humor immunity. Activated macrophages exhibit enhanced microbicidal behavior, as well as improved and expanded antigen presentation ( 1 ). IFN suppressive effect w hen exposed to SOCS1 KIR or Tkip. This was determined by the SOCS mimetics ability to inhibit nitric oxide (NO) production, a telltale indicator of activation. NO production was blocked in RAW macrophages, especially so when Tkip and SOCS1 KIR were admini stered in tandem, an effect which reflects their different means of recognizing JAK2. Another experiment focused on SOCS1 KIR and Tkip hindrance of Ag specific induction of splenocyte proliferation. The expansion of MBP sensitized splenocytes after stimul ation was significantly reduced by the SOCS mimetics, with Tkip being the more effective inhibitor. Regulation or control of the SOCS 1 modulatory arm of the immune system prov ides an approach to enhance host response s that would normally be suppressed or reduced. As we showed with the SOCS mimetics Tkip and SOCS1 KIR, the regulatory role of SOCS 1 extends to APCs and Ag presentation. Dendritic cells (DC)s are probably the most efficient cells at capturing, processing, and presentati ng Ags. In another stu dy, knockdown of DC SOCS 1 by siRNA led to more effective cancer vaccination ( 94 tyrosinerelated Ag 2 by DCs transfected with SOCS 1 siRNA protected mice against the well established B16 m elanoma tumor. The enhanced antitumor immunity was accompanied by enhanced tyrosine related Ag 2 assessed by IFN
42 regulation of Ag presentation by suppression of DC SO CS 1 showed promise for more effective tumor vaccines. Related to the SOCS 1 siRNA studies is 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 IFNA R1 receptor subunit (95). The fact that the KIR of SOCS 1 can bind directly to pJAK2(1001 1013) raised the possibility that pJAK2(1001 1013) might function as an antagonist of SOCS 1. Four initial experiments explored the potential of pJAK2(1001 1013) to a ntagonize SOCS 1 function. First, pJAK2(1001 1013) enhanced suboptimal IFN tivity in EMCV infection Second, prostate cancer cells transfected for constitutive production of SOCS 1 protein had reduced activation of STAT3 by IL 6 treatment. pJAK2(1001 101 3) reversed the SOCS 1 effect. Third, pJAK2(1001 1013) enhanced IFN luciferase rep orter gene via the GAS promoter ( 44 ) Fourth, pJAK2(1001 1013) enhanced Ag 1013) not only has the ability to neutralize SOCS1 which impedes the pathways that develop adaptive immunity, but also contributes to cellular and humoral immunity directly as an IFN agonist suggest it is a capable adjuvant This was further underlined by the apparant ra nge of different routes through which p JAK2 (1001 1013) could affect adaptive immunity Toll like receptors (TLRs) are key players in both the innate and adaptive arms of host defense and represent T cell independent means of effecting immunity. TLRs sig nal through transcription factors such as NF subject to SOCS1 regulation (73) pJAK2(1001 1013) working as an adjuvant could stimulate Ab production to LPS through TLR4 as observed by the downstream production of IgG by B cells in a mouse
43 model where antibodies to LPS were insignificantly produced when LPS was coupled to the typically strong adjuvant s staphylococcal enterotoxin s which require Tcells. TLR3 functions in viral immunity to detect dsRNAs specifically, a signature of the replicatio n of HSV 1, influenza, CMV and others. Treatment of the macrophages with poly I:C, synthesized dsRNA, in the presence of pJAK2(1001 enhancement of NO production suggesting they had been activated through TLR3 which trigger type I IFNs, which upregulate NO, hydrogen peroxide, MHC, etc. Th e diversity in ways which pJAK2(1001 1013) can amplify a signal directed at specific cells of adaptive immunity, as through T cells or APCs n ot only amplifies an immune response by increasing t he number of participating cells, but may also strengthen alternative routes to immune responses This could be critical i n a system that has be en compromised in one capacity or another, as for example case s of T cell depletion. Another mechanism by whi ch pJAK2(1001 1013) may exert direct antiviral effects has to do with a well recognized but not fully understood aspect of IFN function in which cells constitutively produce low levels of intracellular IFN interferons overlap in thei r signaling pathways and their genes ( 96 ). The signaling of one type of IFN will increase the frequency that mutual components are in circulation and therefore more readily available for use by other IFNs, as is the case with SOCS1 homodimers, ISGF3, and JAK2 (IFN OVERVIEW HUME) IFNs may also benefit from the expression of shared genes, and cooperate to synergize or antagonize certain functions, for example the induction of the antiviral state (overview). Low level IFN activity is thought to keep the with other IFNs ( 98 ). We found that pJAK2(1001 1013) treatment increased intracellular
44 levels of IFN 99 ). pJAK2(1001 1013) coupled with IFN 132) worked in syner gy, and cells that received them in combination required lower doses for complete protection against EMCV and VV Measurements made at the level of transcription demonstrated that the two IFN o boost GAS promoter activa tion The cooperative nature of the SOCS 1 antagonist and IFN appears to arise from the reduction of regulatory restraints imposed by SOCS 1 under normal physiological conditions as was indicated by reduced SOCS 1 levels in pJAK2(1001 1013) treated cells ( 99 ). The mechanism of this reduction is currently not known, but may be related to proteasomal degradation via the SOCS box of SOCS 1 ( 100 ). IFN not orious for immune evasion (64 ). One of the causitve agents of TB, M. bovis has recently been shown to cultivate immune tolerance by inducing SOCS 1 and SOCS 3 ( 75 ). The dual strength of pJAK2(1001 1013) as both an antagonizer of SOCS1 and an agonist of IFN e for treatment and vaccine adjuvancy. The studies presented here further contribute to the development and classification of IFN 1 (mimetics) and SOCS antagonist. My focus has primarily involved adjuvant properties of the IFN s and SOCS antagonists. I helped to show how the mimetic and the SOCS antagonists could significantly enhance immune memory when they are associated with specific antigens whether they be weak or strong. I also helped to establish the involvement that AP Cs had in conjunction with our peptides ability to elicit long term adaptive immunity. IFN agonists can activate APCs directly and indirectly by strengthening pathways like TLRs.
45 Through such pathways in APCs we have shown that our peptides can increas e splenocyte proliferation and B cell antibody production. In relation to this I was also involved in investigations as to how IFN could be suppressed by antagonists, specifically our synthetic SOCS 1 mimetics, Tkip a nd SOCS1 KIR. The SOCS 1 mimetics inhibit the activation of APCs directly and indirectly, limiting their ability to process antigens and transmit them to T and B lymphocytes as evidenced by their stunted proliferation despite the presence of strong antigens. As we have shown that IFN 132) synergizes with pJAK2, and also Tkip to some degree with SOCS1 KIR, we present here an effective dual approach to adjuvancy with potential for future therapeutic application.
46 Table 3 1 The amino acid sequences of synthetic peptides used in this study Peptide Sequence 132) 95 AKFEVNNPQVQRQAFNELIRVVHQLLPESSLRKRKRSR 125 95 AKFEVNNPQVQRQAFNELIRVVHQLLPESSL 132)SV40 95 AKFEVNNPQVQRQAFNELIRVVHQLLPESSLPKKRKV V40 T antigen PKKKRKV IFNgamm(126 132 126 RKRKRSR IFNGR(253 2 87 253 TKKNSFKRKSIMLPKSLLVVKSATLETKPESKYS SOCS1 KIR 53 DTHFRTFRSHSDYRRI Tkip WVLVFFVIFYFFR pJAK2(1001 1013 1001 LPQDKEYYKVKEP JAK2(1001 1013)2A 1001 LPQDKEAAKVKEP
47 Figure 3 1 B8R neutralizes IFN 132) antiviral activity. Murine L929 of IFN 132), and IFN 125) that were preincubated for 2 h with or without B8R (33 g/ml) were added to the plate. After 24 h of incubation, EMCV (EMCV) (200 PFU/m l) was added for1hof incubation and washed with media. Cells were then incubated with media for 24 h, after which wells were stained w ith crystal violet and washed. A ) Digital image of the plate. B ) The plate was scanned for cell viability assessment using Image J software (NIH). Percent cell viability is presented for 33 U/ml of IFN 11 M of IFN 132). Error bars indicate standard errors of the means.
48 Figure 3 2 Protection of mice from EMC virus challenge by the IFN 132 ) peptide in the p resence of B8R protein. C57BL/6 mice were pretreated for 3 days with PBS, IFN 132 ) (100 g/day), or rat IFN absence of the B8R protein (25 g). On the last day of treatment, mice were challenged with 50 PFU of EMC viru s. The numbers of surviving mice were recorded starting on the day of EMC virus challenge (day 0) and are presented as percent survival. Ten mice per treatment group were used, and representative data from one of two experiments are shown.
49 Figure 3 3. Adjuvant effect of IFN A ) Mice (n = 5) were infected intranasally with VV in the presence of lipo (L) IFN 132), IFN 132), control peptide, or PBS. Proliferation in the presence of ermined 48 h later by using Alamar blue dye (25). Statistical measurements using the Wilcoxon Mann Whitney rank sum test indicated p < 0.05 for mimetic vs control. B ) Mice (C57BL/6, n = 5) were immunized using BSA as an Ag in the presence of lipo IFN 132), control peptide, or PBS. On the weeks indicated, blood was drawn from mice and measured for the presence of BSA values represent the average with SD. p < 0.05 was obtained for the lipo mimetic vs the PBS treated gr oup.
50 Figure 3 4. SOCS1 KIR and Tkip inhibit IFN induced macrophage activation. A ) Inhibition of IFN induced NO production in macrophages. Murine macrophage cells, Raw 264.7, were incubated with varying concentrations of IFN e of either lipo Tkip, lipo SOCS1 KIR, or lipo control peptide MuIFN 106), all at 15 37C and 5% CO 2 atmosphere. Culture supernatants were collected and nitrite concentration determined using Griess reagent. SOCS1 K IR and Tkip, induced nitrite production. The inhibition of macrophage activation by Tkip and SOCS1 KIR two way ANOVA (p < 0.0001). B) Dose response inhibition of induction of NO and synergy between Tkip and SOCS KIR. Raw 264.7 cells were treated with IFN SOCS1 KIR, or control peptide (Mu IFN (253 287)) an d assayed for NO production as described above. The differences between Tkip and SOCS1 determined by two way ANOVA (p < 0.0001). To show synergy, Raw 264.7 cells were treated with IFN in the presence of varying concentrations of Tkip and SOCS1 KIR and screened for NO production as described above.
51 Figure 3 5 Both SOCS1 KIR and Tkip inhibit proliferation of murine splenocytes. Splenocytes (1 x 105cells/well) were obtained from MBP se nsitized SJL/J mice that had developed EAE and were in remission. The splenocytes were incubated with RPMI 1640 medium containing MBP (50 g/ml) and varying concentrations of lipo SOCS1 KIR, lipo Tkip, or lipo control peptide MuIFNGR1 (253 287) for 48 h. Cu ltures were then incubated with [3H]thymidine for 18 h before harvesting. Radioactivity was counted on a liquid scintillation counter and data reported as counts per minute above background (medium only). Both lipo SOCS1 KIR and lipo Tkip, but not the cont rol peptide, inhibited splenocyte proliferation in a dose dependent manner. The inhibition of proliferation by lipo SOCS1 KIR and lipo Tkip, by two way ANOVA (p < 0.0003). The da ta are representative of two independent experiments, each conducted in triplicate.
52 Figure 3 6 pJAK2(1001 1013) exerts an adjuvant effect at both cellular and humoral levels. A ) Splenocyte stimulation. Mice (n = 5) were pretreated i.p. on day 2, 1, and 0 with pJAK2(1001 1013), control peptide JAK2(1001 1013)2A, or PBS. On day 0, 50 g BSA was injected in mice in all groups, except the naive group. Four weeks later, isolated splenocytes (5 x 106/well) were seeded in quadruplicate and incubated with 0 .5 g BSA for 3 d with the addition of 1 Ci/well [3H] thymidine for the last 6 h, and its incorporation was measured. Data are representative of three individual experiments. B ) IgG production. Mice (n = 5) were treated as in A. Sera obtained in the weeks indicated were diluted (1:1000) and added to microtiter plates. IgG Abs were measured in an ELISA assay. C ) SOCS antagonist enhances T cell independent Ab production. Mice (C57BL6, n = 3) were injected i.p. with T cell independent Ag, LPS (50 each), or the T cell dependent Ag BSA (50 ). Some of the mice received SOCS antagonist (200 ), the control peptide (JAK2A) (200 ), or a combination of SEA/SEB (SAg, 25 each). A set of mice was also injected with BSA (50 ) and SAg. Two weeks later, mice w ere bled. Sera were tested for IgG to LPS or BSA by ELISA. The secondary Ab used was anti mouse IgG conjugated to HRP. After washing, substrate was added, and color was allowed to develop before reading absorbance at 490 nm. Comparison of LPS versus LPS an d SOCS antagonist by Student t test resulted in p < 0.01 at 1/100 dilution.
53 Figure 3 7. pJAK2 (1001 1013) enhances macrophage activation via Toll like receptors. A ) LPS stimulation. RAW264.7 cells (5 x 106/well) were seeded in triplicate and incubated overnight. The indicated amounts of pJAK2(1001 1013) or control peptide were added to the cells and incubated for 4 h, after which 2 /ml LPS was added, and the cells were incubated for 3 d. NO was measured by Griess reagent, and absorbance was read. B ) P oly I:C stimulation. Murine macrophages (RAW264.7) were incubated with lipophilic pJAK2(1001 1013), or control peptide for 2 h, followed by stimulation with poly I:C at 0.1 /ml for 72 h. Culture supernatants were collected and nitrite concentration deter mined using Griess reagent. *p < 0.001.
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63 BIOGRAPHICAL SKETCH James Martin is both a biologist and an artist. He has previously earned a bachelor s degree in microbiology and cell science from the University of Florida C ollege of Liberal A rts. He is also working on a novel and a series of paintings. This thesis marks hi s departure from the field of i mmunology he looks to continue his research in either the f ields of plant genetics or aging