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The inflammatory control valve: Modulation of responses to LPS and IFN-gamma by the RON receptor tyrosine kinase in vitro and in vivo

Dissertation
Author: Caleph B. Wilson
Abstract:
We have shown previously that activation of the receptor d-origine nantais (RON) receptor tyrosine kinase by macrophage stimulating protein (MSP) inhibits macrophage production of nitric oxide (NO) induced by interferon-gamma (IFN-γ) and lipopolysaccharide (LPS), in vitro, through the inhibition of inducible nitric oxide synthase (iNOS) expression. RON-/- mice exhibit elevated delayed-type hypersensitivity (DTH) responses and increased susceptibility to endotoxic shock in vivo. Here, we demonstrate that treatment of primary peritoneal macrophages with MSP followed by IFN-γ and LPS inhibits the production of interleukin-12 (IL-12) through the inhibition of IL-12p40 expression. IL-10 also inhibits IL-12p40 expression as part of a negative feedback response through the induction of signal transducer and activator of transcription 3 (STAT3) phosphorylation and suppressor of cytokine signaling (SOCS) gene expression, we have shown that the inhibition of IL-12 by MSP occurs independently of IL-10. LPS-induced IL-12 production by macrophages initiates the production of IFN-γ by NK, NKT and memory T cells during an innate immune response which forms a positive feedback loop by enhancing the production of IL-12 by activated macrophages. Our data demonstrate that splenocytes from RON knockout mice express elevated levels of IL-12p40 within three hours following LPS administration when compared with control animals. Furthermore, we observe enhanced IFN-γ levels in the serum of these animals at six and twelve hours following injection of endotoxin. By crossing RON knockout mice with IFN-γR knockout animals, we show that the enhanced susceptibility of RON knockout mice to endotoxemia is mediated, at least in part, through IFN-γ. Taken together, these data suggest that RON regulates IFN-γ production in vivo through its ability to regulate IL-12p40 expression in response to LPS, and that the de-regulation of IFN-γ production contributes to the increased sensitivity of RON knockout mice to septic shock. IFN-γ secreted during the innate immune response supports the development of Th1 cells by up-regulating major histo-compatibility complex (MHC) class II expression and subsequent antigen presentation. Furthermore, enhanced IL-12 secretion by macrophages in response to IFN-γ results in a further elevation of IFN-γ production during the acquired phase of the immune response by promoting Th1 differentiation. Data described herein demonstrate that, in addition to regulating IFN-γ production in vivo, MSP inhibits IFN-γ-mediated responses in macrophages including expression of class II trans -activator (CIITA), resulting in decreased MHC class II expression. Additionally, MSP stimulation of primary peritoneal macrophages induces the expression of SOCS1 and 3, and reduces cell surface expression of the IFN-γ receptor (IFN-γR) in a proteasome-dependent manner. However, unlike IL-10 which also induces SOCS expression in macrophages, MSP does not result in the up-regulation of STAT3 phosphorylation in these cells. These data indicate that MSP activation of the RON receptor negatively regulates IFN-γ-induced gene expression in macrophages via a signaling pathway that is distinct from IL-10 and STAT3. In the initial stages of an immune response, LPS induces the production of IFN-β by activated macrophages via a MyD88-independent signaling pathway which feeds back to cooperate with LPS in the induction of a subset of LPS-responsive genes, which are also dependent on IFN-induced STAT1 phosphorylation. This occurs 2-4 hours following LPS stimulation resulting in a delayed induction of the expression of this subset of genes, suggesting that this pathway may be a physiologically relevant target of RON signaling. Here we demonstrate that MSP inhibits LPS-induced IL-12p40 production, even in the absence of IFN-γ priming. However we find that MSP specifically inhibits IFN-γ-induced STAT1 tyrosine phosphorylation, but not LPS-induced STAT1 activation. Furthermore, we demonstrate that LPS-induced IRF3 phosphorylation and IFN-β expression are not diminished in the presence of MSP. Based on our previous studies demonstrating that RON inhibits LPS-induced NFκB activation, we conclude that RON targets TLR4 signaling via the MyD88-dependent, but not the MyD88-independent, pathway.

vi TABLE OF CONTENTS LIST OF FIGURES.....................................................................................................viii LIST OF ABBREVIATIONS…………………………………………………… ......x ACKNOWLEDGEMENTS.........................................................................................xiv Chapter 1 Literature Review.......................................................................................1 1.1 Macrophages...................................................................................................1 1.2 Interferon-gamma/Interferon-gamma receptor (IFN-/IFN-R).....................3 1.3 Toll-like Receptor 4 (TLR4)...........................................................................12 1.4 RON/STK Receptor Tyrosine Kinase and Its Ligand MSP...........................16 1.5 The RON Receptor and Macrophage Activation............................................19 Chapter 2 The RON Receptor Tyrosine Kinase Regulates the Production of IL-12 by Macrophages Stimulated with LPS and IFN-................................................22 2.1 Introduction.....................................................................................................22 2.2 Materials and Methods...................................................................................24 2.2.1 Cells and Animals.................................................................................24 2.2.2 Reagents and Antibodies......................................................................25 2.2.3 RNA Extraction and RT-PCR...............................................................25 2.2.4 Western Blot Analysis...........................................................................26 2.2.5 Determination of Cytokine Concentrations..........................................27 2.2.6 Endotoxin Challenges...........................................................................27 2.3 Results.............................................................................................................28 2.3.1 IL-12 production in response to IFN-  and LPS stimulation is inhibited by MSP....................................................................................28 2.3.2 Suppression of IL-12 production by MSP/RON is independent of IL-10.......................................................................................................29 2.3.3 RON Regulates IL-12p40 Expression and IFN-  Production in vivo Following LPS Administration...............................................................29 2.3.4 Enhanced Susceptibility to Septic Shock of RON -/- Mice is Mediated by IFN- ..................................................................................................31 2.4 Discussion.......................................................................................................38 Chapter 3 MHC Class II Expression is Regulated by MSP/RON by Inhibition of IFN-/IFN-R Signal Transduction in Primary Peritoneal Macrophages In Vitro......................................................................................................................41 3.1 Introduction.....................................................................................................41 3.2 Materials and Methods...................................................................................43 3.2.1 Cells and Animals.................................................................................43

vii 3.2.2 Reagents and Antibodies......................................................................43 3.2.3 Flow Cytometry....................................................................................44 3.2.4 RNA Extraction and RT-PCR...............................................................44 3.2.5 Western Blot Analysis...........................................................................45 3.3 Results.............................................................................................................46 3.3.1 MSP inhibits IFN-  induced MHC class II surface expression and the transcriptional regulator CIITA.......................................................46 3.3.2 STAT1 activation by IFN- is suppressed by MSP...............................47 3.3.3 Exposure to MSP down-regulates IFN- R surface expression............48 3.3.4 MSP up-regulates Expression of SOCS1 and 3; however, STAT3 is not activated by MSP/RON....................................................................48 3.4 Discussion.......................................................................................................56 Chapter 4 MSP/RON Preferentially Inhibits the MyD88-dependent Pathway of TLR4.....................................................................................................................60 4.1 Introduction.....................................................................................................60 4.2 Materials and Methods...................................................................................62 4.2.1 Cells and Animals.................................................................................62 4.2.2 Reagents and Antibodies......................................................................63 4.2.3 RNA Extraction and RT-PCR...............................................................63 4.2.4 Western Blot Analysis...........................................................................64 4.2.5 Determination of Cytokine Concentrations..........................................65 4.3 Results.............................................................................................................65 4.3.1 Four hours of MSP exposure is adequate to prevent production of IL-12p40 by LPS alone or in combination with IFN- ...........................65 4.3.2 MSP does not prevent up-regulation of IFN- or IRF3 activation in response to LPS stimulation...................................................................66 4.3.3 Activation of STAT1 by LPS alone is not inhibited by MSP.................66 4.4 Discussion.......................................................................................................73 Chapter 5 Conclusion..................................................................................................76 References....................................................................................................................83

viii LIST OF FIGURES Figure 1-1: Macrophage Development.......................................................................5 Figure 1-2: Type I and II IFN Receptor Activation....................................................6 Figure 1-3: TLR4 Signal Transduction.......................................................................7 Figure 1-4: IFN- positively feeds forward to enhance IL-12 and iNOS induction by LPS..................................................................................................................8 Figure 2-1: MSP inhibits IL-12 production by primary peritoneal macrophages.......32 Figure 2-2: Inhibition of IL-12 by MSP is independent of IL-10................................33 Figure 2-3: IL-10 production is not induced by MSP.................................................34 Figure 2-4: RON -/- mice express more IL-12p40 in vivo after expose to LPS............35 Figure 2-5: IFN- production in RON -/- in response to LPS is induced at higher levels than in WT..................................................................................................36 Figure 2-6: RON -/- mice are more susceptible to LPS exposure than WT mice and IFN-R signaling plays a role in increased susceptibility.....................................37 Figure 3-1: MSP inhibits IFN- induced up-regulation of MHC Class II surface expression.............................................................................................................50 Figure 3-2: CIITA expression is reduced by MSP.......................................................51 Figure 3-3: Stimulation of the RON receptor tyrosine kinase with MSP inhibits IFN- induced STAT1 phosphorylation...............................................................52 Figure 3-4: MSP reduces IFN-R surface expression..................................................53 Figure 3-5: MSP up-regulates SOCS1 and 3 transcripts.............................................54 Figure 3-6: MSP induces moderate STAT3 tyrosine phosphorylation and RON -/-

have constant ability to activate STAT3 as WT...................................................55 Figure 4-1: Exposure to MSP as early as 4 hours inhibits IL-12p40 production by LPS with or without IFN- priming......................................................................68 Figure 4-2: The docking site tyrosines are required for ligand-dependent and - independent inhibition of IL-12p40 by RON…………………………………...69

ix Figure 4-3: MSP does not prevent the expression of IFN-........................................70 Figure 4-4: Activation of IRF3 is not inhibited by MSP.............................................71 Figure 4-5: MSP/RON does not inhibit STAT1 activation induced by LPS...............72 Figure 5-1: The RON Receptor Serves as an Inflammatory Control Valve................82

x LIST OF ABBREVIATIONS Ab………………………...……………………………………………………….Antibody APC………………………………………………………………Antigen Presenting Cells ARGI…………………………………………………………………………….Arginase I ARGII…………………………………………………………………………..Arginase II BTK………………………………………………………..……Bruton’s Tyrosine Kinase CFU………………………………………………………………….Colony Forming Unit CFU-G…………………………………………………………………...CFU-Granulocyte CFU-GM……………………………………………….….CFU-Granulocyte/Macrophage CFU-M…………………………………………………………………..CFU-Macrophage CSF1…………………………………………………….…….Colony-stimulating factor 1 CSF1R…………………………………………...….Colony-stimulating factor 1 Receptor CIITA…………...…………………………………………………Class II Trans-activator COX2……………………………………………………………………Cyclooxygenase 2 ELISA……………………………………………...Enzyme-Linked Immunosorbet Assay ERK………………………………………………………..Extracellular Response Kinase FBS……………………………………………………………………Fetal Bovine Serum FITC……………………………………………………………Fluorescein Isothiocyanate GAB……………………………………………………………..GRB2 Associated Binder GAS………………………………………………………….Gamma Activating Sequence T cells……………………………………………………...……….gamma-delta T cells GAF…………………………………………………………gamma-IFN Activated Factor

xi GM-CSF……………………………Granulocyte Macrophage-Colony Stimulating Factor GRB2………..………………………...………..Growth-Factor-Receptor Bound Protein 2 HGF………………………………………………………….…Hepatocyte Growth Factor HGFL……................................................................……..Hepatocyte Growth Factor-Like ICSBP……………………………………Interferon Consensus Sequence Binding Protein IFN-………………………………………………………….…………..Interferon-alpha IFN-………………………………………………………………………..Interferon-beta IFN-…………………………………………………………………….Interferon-gamma IL-1……………………………………………………………………………Interleukin 1 IL-1RA…………………………………………………Interleukin 1 Receptor Antagonist IL-10………………………………………………………………………....Interleukin 10 IL-6…………………………………………………………………………....Interleukin 6 IL-12………………………………………………………………………....Interleukin 12 IL-2…………………………………………………………………………....Interleukin 2 iNOS…………………………………………………….Induciable Nitric Oxide Synthase IRF1……………………………………………………….…Interferon Response Factor 1 IRF2……………………………………………………….…Interferon Response Factor 2 IRF3………………………………………………………….Interferon Response Factor 3 JAK……………………………………………………………….Janus Activating Kinase JNK………………………………………………………………....Jun N-terminal Kinase LPS………………………………………………………………….…Lipopolysaccharide MAL………………………………………………………….……..MyD88 Adaptor-Like

xii MAPK………………………………………………….Mitogen Activated Protein Kinase M-CSF……………………………………………Macrophage Colony Stimulating Factor MEK…………………………………………..Mitogen Activated ERK-activating Kinase MHC……………………………………………….….Major Histocompatibility Complex mRON……………………………………………………………………….. murine RON MSP……………………………………………………...Macrophage Stimulating Protein MyD88………………………………Myeloid Differentiation Primary Response Gene 88 NCS……………………………………………………………….….Newborn Calf Serum NF-B……………………………………………………………..Nuclear Factor-kappa B NK cells………………………………………………………………...Natural Killer cells NO……………………………………………………………………………..Nitric Oxide TNF……………………………………………………..….Tumor Necrosis Factor alpha PBS……………………………………………………………..Phosphate Buffered Saline PCR……………………………………………………………Polymerase Chain Reaction PI3K…………………………………………………………Phosphatidylinsitol-3-Kinase PIAS………………………………………………....Protein Inhibitor of Activated STAT PP1……………………………………………………………….... Protein Phosphatase-1 RT-PCR……………………………………………………….Reverse Transcription-PCR RON…………………………………………………...……..Recepteur d-Origine Nantais S…………………………………………………………………………………..….Serine SH…………………………………………………………………………...Src Homology SOCS1………………………………………………..Suppressor of Cytokine Signaling 1

xiii SOCS3…………………………………..……………Suppressor of Cytokine Signaling 3 STK……………………………………………...……Stem- cell Derived Tyrosine Kinase Th1………………………………………………………………………………T-helper 1 Th2……………………………………………………………………………....T-helper 2 Y……………………………………………………………………......…………Tyrosine vs………………………………………………………………………………....….Versus WT…………………………………………………………………………...….Wild-Type

xiv ACKNOWLEDGEMENTS Thank you, to my advisor Dr. Pamela A. Hankey for allowing me to learn and develop in her laboratory and for her mentorship and guidance. I am very grateful to Dr. Avery August, Dr. Margherita Cantorna, Dr. Wendy Hanna-Rose, Dr. Andrew Henderson and Dr. Robert Paulson for their service as my thesis committee and for their steadfast dedication to providing solid support. Successfully matriculating the Pathobiology program was a pleasurable process due to the interactions with the past and present members of the Hankey lab: Dr. Lisa Finklestein, Shihan He, Qingping Liu, Dr. Michael Lutz, Dr. Amy Morrison, Dr. Shuang Ni, Dr. Manujendra Ray, Daniel Sharda, Dr. Hami Teal, Dr. Xin Wei and Jie Xu, as well as, the graduate students, principal investigators, research personnel and administrative staff of the Department of Veterinary and Biomedical Sciences.

Finally, I am blessed to have a lifetime of love, faith and support from family and friends. A special, thank you, to my parents, Bennie L. Sledge, Jr. and Mary L. Wilson, for giving me life; to my grandmother, Luvenia S. Goss, for her unconditional love, encouragement and wisdom; my wife, Dr. Melanie Ragin, for being there every step of the way; and to all members of my family that have always looked out for me.

Chapter 1

Literature Review 1.1 Macrophages “Big eaters”, better known as macrophages, are a diverse lineage of phagocytic cells of hematopoetic origin. In the late 19 th century Ilia Metchnikokk performed two experiments in starfish larva and Daphnia. In these studies he observed phagocyte migration, attachment and microbial elimination by what became known as macrophages. The earliest sources of macrophages during development are the yolk sac and fetal liver [1]. In the yolk sac, cells with macrophage morphology were identified as macrophages by the expression of markers such as cFMS/ colony-stimulating factor 1 receptor (CSF1R), CD11b and the mannose receptor (MR) [2]. As development progresses the fetal liver increasingly becomes the source of hematopoesis, in general, and macrophages, in particular [3, 4].

Hematopoietic development of macrophages in the adult bone marrow begins with a common hematopoietic stem cell (HSC) which develops into the myeloid lineage and differentiates into a CFU-GM, CFU-M, monoblast, pro-monocyte, monocyte and macrophage after exposure to growth factors. Colony-stimulating factor 1 (CSF1) has

2 been show to be critical in macrophage development in mice, as well as tissue remodeling and organogenesis during fetal/neonatal

life [5, 6]. The absence of CSF1 in osteopetrotic (op/op) mice, results in defects in macrophage development, differentiation and proliferation, resulting in osteopetrosis due to the lack of osteoclastic bone resorption [7, 8]. Additionally, granulocyte-macrophage CSF (GM-CSF) assists in the maintenance and population regulation of macrophages. During bacterial infection, GM-SF is produced and promotes increased hematopoiesis and macrophage development. GM- CSF -/- mice do not have defects in macrophage development; however, they do exhibit defects in macrophage mediated responses [9].

Tissue resident macrophages are primarily derived from circulating monocytes that migrate to tissues and become “resident” phagocytes. Migration of this population from circulating monocytes is mediated by soluble CX3C-chemokine ligand or CXC- chemokine ligand 12 [10]. Recognition of a plethora of endogenous and exogenous ligands, and the ability to respond appropriately, is critical to macrophage function in homeostasis (removal of cellular debris/ senescent cells and tissue remodeling/repair after injury or infection) as well as host defense in innate and acquired immunity. The inability to appropriately control these responses can result in autoimmunity, inflammation, and other immunopathologies. Tissue resident populations of macrophages express CD14, CD16, F4/80 and high amounts of MHC class II [11, 12]. Kupffer cells of the liver, alveolar macrophages of the lungs, microglia, Langerhans cells of the epidermis and peritoneal macrophages are all examples of tissue resident populations of macrophages. Additionally, tissue resident macrophages are also found

3 within the lamina propria of the gut and the interstitium of organs such as the heart, pancreas, and kidney.

Macrophages exposed to IFN- with or without microbial constituents, TNF and/or GM-CSF are considered to be classically activated. In response, these cells produce substantial amounts of IL-12, reactive oxygen species, NO, IL-1, TNF, IL-6 and IL-23 [13]. The resulting cytokine profile supports activation of NK cells and multiple T lymphocyte populations. IL-1 and TNF produced during endotoxin exposure are associated with septic shock, antigen specific immune responses, hepatitis and pancreatitis, and suppression of IFN- production by IL-6 has been linked to the shift of Th1 to Th2 in acquired immunity [14-16]. 1.2 Interferon-gamma/Interferon-gamma receptor (IFN-/IFN-R) Interferon-gamma (IFN-) and its receptor (IFN-R) have been shown to be very close to the crux of many immunological activities in the nearly 50 years since it was discovered [Reviewed in [17-19]] In the family of IFNs, named for their ability to exhibit viral interference [20, 21], there are two known classes, types I and II. The former class, type I, includes a wide range of IFNs, some of which are species specific. IFNs - and - the most widely studied of the type I IFNs, are notably recognized for their potent anti-viral activity. Both are expressed within a wide range of cell types in response to viral infection. IFN- also plays a major role in innate immunity by promoting antibacterial activities following induction by pathogen-associated molecular

4 patterns (PAMPs) of bacteria. Up-regulation of IFN-/ activ ates a positive-feedback loop resulting in amplification of IFN-/expression [22-25]. Expression of type II IFN, IFN-is induced by a wide range of stimuli, yet production of IFN- is limited in cell type. Natural killer (NK) cells, gamma-delta T cells (T cells), NKT cells, CD8 +

memory T cells and activated T-helper 1 (Th1) cells release IFN- in response to interleukin 12 (IL-12) stimulation [26-29]. Dendritic cells (DC) and macrophages have also been reported to produce IFN- [30-32]. Although IFN- has antiviral activities, it primarily promotes immunity to unicellular pathogens by activating macrophages and other cells of the myeloid lineage [Reviewed in [17]].

5 Figure 1-1

Figure 1-1: Macrophage Development. Macrophages are present at multiple stages of maturation of mammals from embryo to adulthood. Primitive macrophages are found in the yolk sac during embryo genesis. As a fetus HSC in the fetal liver progress from CFU-GM → CFU-M → monoblast → pro- monocyte → macrophages. In adult bone marrow macrophage development evolves with comparable stages as in the fetal liver with a divergence of monocytes circulating in the bloodstream into tissues where the terminally differentiate into tissue residen t

macrophage after reaching adult tissues. Image adapted from [2] and reprinted with permission.

6 Figure 1-2

Figure 1-2: Type I and II IFN Receptor Activation. Upon binding of ligand, IFN receptor–associated JAKs are activated and phosphorylate receptor chains on tyrosine. Cytoplasmic STATs bind to the phosphorylated receptors vi a their SH2 domains. JAKs associated with the type I IFN receptor (IFNAR) the n phosphorylate STAT1 and STAT2 on tyrosine, causing the formation of predominantly STAT1/STAT2 hetero-dimers. IFN-γR–associated JAKs phosphorylate STAT1, leading to the formation of STAT1 homo-dimers. STAT dimers translocate to the cell nucleus. Thereafter, STAT1/STAT2 hetero-dimers associate with a third protein, IRF9, and bin d the ISRE, whereas STAT1 homo-dimers or GAFs activate gene expression by binding the GAS. Image adapted from [33] and reprinted with permission.

7 Figure 1-3

Figure 1-3: TLR4 Signal Transduction. The MyD88-dependent pathway is initiated by binding of the TIR domain of Mal to the TIR domain of TLR4, which has dimerized after ligand binding. Mal recruits MyD88, which binds IRAK4 and -1. IRAK1 is phosphorylated by itself and IRAK4 and leaves the membrane to activate TRAF6. After TRAF6 is ubiquitinated, it interacts with TAB2 to activate TAK1. TAK1 activates the IKK complex and IB is phosphorylated, ubiquitinated and degraded allowing NFB to translocate to the nucleus to produce proinflammatory cytokines. TAK1 also activates MKK6 which in turn activates JNK and p38 leading to AP-1 activation and the production of proinflammatory cytokines. TRAF6 can also activate IRF5. The MyD88-independent pathway is activated by binding o f

TLR4 and TRAM at the cell membrane. TBK-1 is activated, thus leading to the activation of IRF3, a transcription factor that translocates to the nucleus to produce IFN-inducible genes. Adapted from [34] and reprinted with permission.

8 Figure 1-4

Figure 1-4: IFN- positively feeds forward to enhance IL-12 and iNOS induction by LPS. An alternate pathway of TL4 signaling cascade independent of MyD88 is guided by IRF- 3, which induces the expression of IFN- as part of a positive feed by mechanism tha t

results in IL-12 and iNOS transcription.

9 IFN-γ exists as a 143 amino acid homo-dim er that is intercalated, and differs from type I IFNs due to the lack of disulfide bonds and C-terminal helical configuration [35- 37]. Functional IFN-Rs exist as linked hetero-dimers of IFN-R1 and IFN-R2 prior to IFN- ligation [38, 39]. Addition of ligand to the receptor complex results in the formation of a symmetrical quatrimer of IFN-R1 and IFN-R2 with IFN- binding sites located on IFN-R1. Several tyrosine (Y) residues allow for adequate signal transduction upon phosphorylation by the constitutively associated Janus-kinases (JAKs) 1 and 2, which cross phosphorylate both -receptors. JAK2, phosphorylates tyrosine 457 (TYR457) on IFN-R1, the binding site of the Src-homology 2 (SH2) containing signaling transducer and activator of transcription 1 (STAT1) [40-44]. Post docking of STAT1 to IFN-R1, TYR701 of STAT1 is phosphorylated, leading to receptor disassociation and homo-dimerization of STAT1 [42, 45]. Activated STAT1 homo- dimers translocate to the nucleus and bind the gamma activated site (GAS) element (TTNCNNNAA), resulting in the primary IFN- response.

The type I and II IFN signaling pathways consist of common components, yet result in the activation of distinctive sets of genes [Reviewed in [46]]. Both classes of IFNs incorporate the JAK family of tyrosine kinases associated with their respective receptors. IFN-R1 and IFN-R2 constitutively associate with tyrosine kinase 2 (TYK2) and JAK1, respectively [47, 48]. Although both type I and II IFN receptors utilize STAT1 as a transcription factor, IFN-R2 binds STAT2 via its SH2 domain which hetero-dimerizes with STAT1 upon phosphorylation [45, 49]. This hetero-dimer

10 associates with interferon-stimulated gene factor 3- (ISGF3)/p48 forming ISGF3/interferon response factor (IRF)9 which then translocates to the nucleus [50, 51]. DNA binding of this complex occurs at the interferon sequence response element (ISRE) site (AGTTTCNNTTTCNC/T) and promotes gene transcription (Figure 1-2).

Mice with targeted disruptions of IFN-, the IFN-R or STAT1 are defective in their response to pathogens and pathogen particulates. Targeted deletions in IFN- prevents MHC Class II surface expression, splenocyte proliferation, and NK cell mediated killing [52]. Lymphocytic choriomeningitis virus (LCMV) induces increased footpad swelling in IFN-R1 -/- [53]. STAT1 -/- mice produce reduced levels of nitric oxide (NO) in response to both type I and II IFNs [54]. These mice are also more susceptible to Listera monocytogenes, vesicular stomatitis virus (VSV) and murine herpes virus (mHSV) [54, 55]. These observations are attributed to the fact that STAT1 -/-

cells do not up-regulate IRF1 [54, 55]. Additionally, many of the phenotypes exhibited by STAT1 -/- mice have also been reported in IFN- -/- and IFN-R1 -/- animals, including reduced levels of NO and increased susceptiblity to Listeria monocytogenes.

The primary response of cells to IFN-/IFN-R1 signaling promotes transcription implementing binding of the DNA GAS element by STAT1. Most GAS responsive genes are forward acting transcription factors, while others are antagonists of IFN-. The family of IRFs comprise the secondary response of cells to IFN- [56, 57]. IRF1 and IRF2 exhibit 75% homology and have antagonistic effects [58]. This class of

11 transcrip tion factors interacts with the ISRE. IRF1 drives the expression of pro- inflammatory genes including inducible NO synthase (iNOS)/NOS2, IL-12p40 and cycloxygenase 2 (COX2), as well as the MHC class II master transcriptional regulator, class II trans-activator (CIITA). IRF2 binds to the same relative site of the ISRE and inhibits binding of IRF1 to the site, thus inhibiting the transcription of IRF1 responsive genes [58-60]. IRF1 -/- mice exhibit defects in type I IFN gene induction [61, 62]. IL-12 production in IRF1 -/- mice is reduced post IL-12 treatment due to defects in IFN- gene induction [63]. CIITA mRNA expression is also negatively affected by the absence of IRF1 -/- [64].

The IFN consensus binding protein (ICSBP)/IRF8 is related to (ISGF3)/p48 and induced by IFN- [65-67]. Interactions between ICSBP, PU.1 and IRF1 result in cooperative DNA binding and activation of gene transcription [68]. In addition to serving as a GAS element gene activator, ICSBP combines with IRF2 to suppress ISRE- mediated transcription [69, 70].

While IFN- up-regulates gene expression, in order to mediate immunity, it also activates genes that serve to control cellular responses. Suppressor of cytokine signaling (SOCS) and protein inhibitor of activated STAT (PIAS) are two of the most studied negative regulators of cytokine signal transduction and gene activation. SOCS proteins inhibit cytokine signaling and are up-regulated by cytokine receptor activation [71-76]. SOCS proteins are characterized by a conserved carboxyl-terminal SOCS box and an

12 SH2 domain. Interruption of the JAK/STAT pathway is the main function of SOCS proteins. SOCS1 binds activated cytokine receptors via its SH2 domain, which inhibits recruitment of STAT proteins and subsequent signal transduction [77, 78]. Cytokine gene induction is also regulated by SOCS1 through the control of cytokine receptor degradation [79-81].

Phosphorylated STAT proteins are also bound by PIAS preventing them from binding DNA and thus preventing gene expression [82-84]. PIAS has also been reported to possibly target STAT proteins for proteosomal degradation [85, 86]. In addition, dephosphorylation of cytokine receptors, associated JAK kinases and both cytoplasmic and nuclear STATs by receptor and non-receptor tyrosine phosphatases including SHP1, SHP2 and CD45 plays a critical role in the regulation of cytokine signaling [77, 87-89]. 1.3 Toll-like Receptor 4 (TLR4) During an immune challenge from intracellular pathogens, the highly evolutionarily conserved pattern recognition receptors (PRRs) on the surface of tissues at the site of infection bind pathogen associated molecular patterns (PAMPs) of pathogens such as lipopolysaccharide (LPS) from Gram-negative bacteria, flagellin, double-stranded RNA (dsRNA), peptidoglycan, CpG DNA and lipotechoic acid (LTA) from Gram- positive bacteria [Reviewed in [90] and [91]]. A subclass of the PRRs were first identified as Toll in Drosophila melanogaster for conferring antimicrobial defense to fungus infection [92-97]. Drosophila melanogaster’s Toll structure consists of an extra-

13 cellular domain of leucine-rich repeats with a cytoplasmic domain that is homologous to the mammalian interleukin 1 receptor (IL1R), referred to as the Toll/IL-1R (TIR) domain. Signal transduction through Toll in Drosophila melanogaster results in the nuclear translocation of Dorsal, a Rel transcription family member [Reviewed in [98]].

TLR4, which was originally known as human Toll (hToll), was the first mammalian homolog of Drosophila melanogaster Toll identified [99, 100]. Subsequently, the mammalian receptors were coined as Toll-like receptors (TLR) by Rock et al [100]. Interestingly, a constitutively active form of TLR4 was shown to activate NFB and induce pro-inflammatory mediators, thus creating a link between TLRs and immunity [99].

Although the list of ligands to TLR4 continues to grow, endotoxin or LPS, is the most widely studied ligand and was identified as such due to the tempered response of C3H/HeJ and C57BL10/ScCr mice to endotoxin [101, 102]. Activation of TLR4 by LPS is not achieved by direct binding to the receptor in isolation. LPS-binding protein (LBP) circulates in the serum and presents LPS to TLR4 by binding to cluster of differentiation 14 (CD14), which is a glycosylphosphatidylinositol anchored molecule preferentially expressed in monocytes/macrophages and neutrophils [103-108]. Surface expression of CD14 is usually in close proximity to TLR4 thus presenting optimal conditions for recognition and initiation of signal transduction [109]. After CD14 links to LPS, MD2, associated with TLR4, confers ligation and activation of TLR4. Depletion of MD2 in

14 animal m odels, or targeted deletion of CD14 and TLR4 conveys resistance to endotoxin [110-117].

The TIR domain is the site of initiation of cytoplasmic signal transduction down- stream of TLR4; however two distinct pathways, the myeloid differentiation primary response gene 88 (MyD88) -dependent and –independent pathways, are engaged to activate LPS responsive genes. MyD88 is an important adaptor protein that was previously linked to IL-1R signal transduction [118, 119]. Prior to engagement of MyD88, the TLR4 TIR domain co-adaptor MyD88-adaptor like (MAL)/ TIR-domain- containing adaptor protein (TIRAP), which is associated with the cell membrane by attaching to phosphatidylinositol 4,5-bisphosphate (PIP2), assists in recruiting MyD88 to TLR4 [120]. As signal transduction begins, the TIR domain of MyD88 associates with the TIR domain of TLR4 leading to the activation of a series of kinases that results in the nuclear translocation of multiple transcription factors such as NFB, AP-1 and IRF5, as well as the induction of pro-inflammatory mediators including IL-1, IL-6, TNF and IL-8 [Reviewed in [34, 121-123]. Mice deficient in MyD88 are protected from endotoxin mediated death and vesicular stomatitis virus (VSV) infection [124, 125]. In response to LPS, MyD88 -/- mice fail to express IL-6, TNF and IL-1 [125].

MyD88-independent signaling incorporates TIR domain interactions between the TRIF-related adaptor molecule (TRAM) and TLR4. This pathway is responsible for inducing TIR-domain-containing adaptor inducing IFN- (TRIF), also know as TIR-

15 containing adaptor molecule 1 (TICAM1) [126, 127]. IRF3 is the major transcription factor activated by the MyD88-independent signal transduction pathway downstream of TLR4 (Figure 1-4), which converges with the MyD88-dependent pathway to up-regulate IFN- in an autocrine-paracrine loop [22-25, 128]. IFN- then positively feeds back to cooperate with LPS in the induction of a subset of LPS responsive genes, including iNOS and IL-12p40, that are dependent on IFN-induced STAT1 phosphorylation (Figure 1-4) [46, 129, 130].

The pro-inflammatory cytokines produced by exposure to LPS of bacterial pathogens also induce negative feedback genes, such as IL-10. IL-10 was first described due to its ability to inhibit synthesis of the Th1 cytokine IFN-[131, 132] In addition to inhibiting the induction of the pro-inflammatory cytokines IL-12, IL-6 and TNF [133] just to name a few, IL-10 also up-regulates inflammatory antagonist like IL-1 receptor antagonist (IL-1RA) and soluble tumor necrosis-factor receptor (sTNFR) in macrophages [134, 135]. Mice deficient in IL-10 develop inflammatory bowel disease (IBD) and exhibit increased immune responses to Listeria monocytogenes [136, 137]. IL-10 induces gene expression primarily through the activation of STAT3. Mice with tissue specific deletions of STAT3 in macrophages and neutrophils also develop spontaneous IBD due to the dysfunction of the anti-inflammatory actions of STAT3 [138].

SOCS1 expression is also induced in response to LPS mediated TLR4 activation. Phosphorylated MAL is prevented from mediating TLR4 MyD88-dependant signaling after binding of SOCS1 [139] . Phosphoinositide Kinase-3 (PI3K) is also up-regulated

16 and perturbs IL-12 transcription in DCs after exposure to LPS [140, 141]. Additionally, LPS treatment reduces the expression of TLR4 which is attributed to the development of endotoxin tolerance. 1.4 RON/STK Receptor Tyrosine Kinase and Its Ligand MSP The recepteur d-origine nantais (RON) receptor tyrosine kinase (RTK) is a member of the proto-oncogene c-MET family of receptor tyrosine kinases (RTKs) [142- 145]. Screening of a human keratinocyte cDNA library resulted in the isolation of RON while the murine homolog, stem cell-derived tyrosine kinase (STK), was isolated from murine hematopoietic stem cells in an unrelated study [142, 143]. RON exists as a disulfide-linked 190 kD hetero-dimer composed of a 40 kD extra-cellular -chain and a 150 kD trans-membrane -chain that is formed after cleavage of a single precursor [143, 146]. Although the N-terminal -chain and extra-cellular portion of the -chain forms the ligand binding domain of RON, only the -chain has kinase activity [146]. Additionally, the C-terminal tail of the -chain has tyrosine residues that serve as docking sites that mediate the binding of down-stream signal transduction molecules that contain SH2 domains [147].

The earliest expression of RON is observed in the embryonic trophoblast where it is involved in embryonic implantation [148, 149]. During murine embryonic development and in the adult animal, RON is widely expressed in the epithelium of the digestive tract [146, 150]. Moreover, RON is overexpressed in a number of malignant

17 epithelia l cancers including breast, pancreatic, ovarian and colon carcinomas [Reviewed in [151]]. RON is also expressed on hematopoietic progenitor cells and is down- regulated as these cells differentiate [152]. Circulating monocytes do not express RON; however when they migrate into tissues and differentiate into tissue resident macrophages RON expression is up-regulated [153]. Tissue resident macrophage populations that are RON positive include Kupffer cells, osteoclasts, resident peritoneal macrophages , Langerhan’s cells, mesangial cells, splenic marginal zone macrophages and microglia [153-157].

Full document contains 132 pages
Abstract: We have shown previously that activation of the receptor d-origine nantais (RON) receptor tyrosine kinase by macrophage stimulating protein (MSP) inhibits macrophage production of nitric oxide (NO) induced by interferon-gamma (IFN-γ) and lipopolysaccharide (LPS), in vitro, through the inhibition of inducible nitric oxide synthase (iNOS) expression. RON-/- mice exhibit elevated delayed-type hypersensitivity (DTH) responses and increased susceptibility to endotoxic shock in vivo. Here, we demonstrate that treatment of primary peritoneal macrophages with MSP followed by IFN-γ and LPS inhibits the production of interleukin-12 (IL-12) through the inhibition of IL-12p40 expression. IL-10 also inhibits IL-12p40 expression as part of a negative feedback response through the induction of signal transducer and activator of transcription 3 (STAT3) phosphorylation and suppressor of cytokine signaling (SOCS) gene expression, we have shown that the inhibition of IL-12 by MSP occurs independently of IL-10. LPS-induced IL-12 production by macrophages initiates the production of IFN-γ by NK, NKT and memory T cells during an innate immune response which forms a positive feedback loop by enhancing the production of IL-12 by activated macrophages. Our data demonstrate that splenocytes from RON knockout mice express elevated levels of IL-12p40 within three hours following LPS administration when compared with control animals. Furthermore, we observe enhanced IFN-γ levels in the serum of these animals at six and twelve hours following injection of endotoxin. By crossing RON knockout mice with IFN-γR knockout animals, we show that the enhanced susceptibility of RON knockout mice to endotoxemia is mediated, at least in part, through IFN-γ. Taken together, these data suggest that RON regulates IFN-γ production in vivo through its ability to regulate IL-12p40 expression in response to LPS, and that the de-regulation of IFN-γ production contributes to the increased sensitivity of RON knockout mice to septic shock. IFN-γ secreted during the innate immune response supports the development of Th1 cells by up-regulating major histo-compatibility complex (MHC) class II expression and subsequent antigen presentation. Furthermore, enhanced IL-12 secretion by macrophages in response to IFN-γ results in a further elevation of IFN-γ production during the acquired phase of the immune response by promoting Th1 differentiation. Data described herein demonstrate that, in addition to regulating IFN-γ production in vivo, MSP inhibits IFN-γ-mediated responses in macrophages including expression of class II trans -activator (CIITA), resulting in decreased MHC class II expression. Additionally, MSP stimulation of primary peritoneal macrophages induces the expression of SOCS1 and 3, and reduces cell surface expression of the IFN-γ receptor (IFN-γR) in a proteasome-dependent manner. However, unlike IL-10 which also induces SOCS expression in macrophages, MSP does not result in the up-regulation of STAT3 phosphorylation in these cells. These data indicate that MSP activation of the RON receptor negatively regulates IFN-γ-induced gene expression in macrophages via a signaling pathway that is distinct from IL-10 and STAT3. In the initial stages of an immune response, LPS induces the production of IFN-β by activated macrophages via a MyD88-independent signaling pathway which feeds back to cooperate with LPS in the induction of a subset of LPS-responsive genes, which are also dependent on IFN-induced STAT1 phosphorylation. This occurs 2-4 hours following LPS stimulation resulting in a delayed induction of the expression of this subset of genes, suggesting that this pathway may be a physiologically relevant target of RON signaling. Here we demonstrate that MSP inhibits LPS-induced IL-12p40 production, even in the absence of IFN-γ priming. However we find that MSP specifically inhibits IFN-γ-induced STAT1 tyrosine phosphorylation, but not LPS-induced STAT1 activation. Furthermore, we demonstrate that LPS-induced IRF3 phosphorylation and IFN-β expression are not diminished in the presence of MSP. Based on our previous studies demonstrating that RON inhibits LPS-induced NFκB activation, we conclude that RON targets TLR4 signaling via the MyD88-dependent, but not the MyD88-independent, pathway.