(Title of the Thesis)*

NEU1 SIALIDASE AND MATRIX METALLOPROTEINASE-9 CROSS-TALK IS ESSENTIAL FOR TOLL-LIKE RECEPTOR ACTIVATION AND CELLULAR SIGNALING

Samar Abdulkhalek

A thesis submitted to the Graduate Program in Microbiology and Immunology in

the Department of Biomedical and Molecular Sciences

In conformity with the requirements for

the degree of Doctor of Philosophy

Queen’s University

Kingston, Ontario, Canada

(April, 2013)

The molecular mechanism(s) by which Toll-like receptors become activated are not well understood. For the majority of TLR receptors, dimerization is a prerequisite to facilitate

MyD88-TLR complex formation and subsequent cellular signaling to activate NF-κB.

However, the parameters controlling interactions between the receptors and their ligands still remain poorly defined. Previous reports have identified that neuraminidase-1

(NEU1) is an important intermediate in the initial process of TLR ligand induced receptor activation and subsequent cell function. What we do not yet understand is how NEU1 is activated following TLR ligand binding. In this thesis, the findings disclose a receptor signaling paradigm involving a process of receptor ligand-induced GPCR-signaling via neuromedin-B (NMBR) Gα-proteins, matrix metalloproteinase-9 (MMP-9) activation, and the induction of Neu1 activation. Central to this process is that NEU1–MMP-9-

NMBR complex is associated with TLR-4 receptors on the cell surface of naive primary macrophages and TLR-expressing cell lines. Ligand binding to the receptor initiate

GPCR-signaling via GPCR Gα subunit proteins and MMP-9 activation to induce NEU1.

Activated NEU1 targets and hydrolyzes sialyl α-2-3-linked to β-galactosyl residues at the ectodomain of TLRs, enabling the removal of steric hindrance to receptor association, activation of receptors and cellular signaling. Furthermore, a novel glycosylation model is uncovered for the activation of nucleic acid sensing intracellular TLR-7 and TLR-9 receptors. It discloses an identical signaling paradigm as described for the cell-surface

TLRs. NEU1 and MMP9 cross-talk in alliance with neuromedin-B receptors tethered to

TLR-7 and -9 receptors at the ectodomain is essential for ligand activation of the TLRs ii and pro-inflammatory responses. However, the mechanism(s) behind this GPCR and

TLR cross-talk has not been fully defined. Here, GPCR agonists mediate GPCR- signaling via membrane Gα subunit proteins to induce NEU1 and MMP-9 cross-talk at the TLR ectodomain on the cell surface. This molecular organizational GPCR signaling platform is proposed to be an initial processing stage for GPCR agonist-induced transactivation of TLRs and subsequent cellular signaling. Collectively, these novel findings radically redefine the current dogma(s) governing the mechanism(s) of the interaction of TLRs and their ligands, which may provide important pioneering approaches to disease intervention strategies.

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Co-Authorship

Chapter 2. Neu1 Sialidase and Matrix Metalloproteinase-9 Cross-talk Is Essential for Toll-like Receptor Activation and Cellular Signaling

Schammim Ray Amith, Susan L. Franchuk, Preethi Jayanth, Merry Guo, Trisha Finlay, Alanna Gilmour, Christina Guzzo, Katrina Gee, Rudi Beyaert§¶, and Myron R. Szewczuk All co-authors gave permission to publish the data in the Journal of Biological Chemistry 2011;286(42): 36532–36549.

‡Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, Ontario K7L 3N6, Canada §Department for Molecular Biology, Ghent University, Technologiepark 927, B-9052 wijnaarde, Belgium ¶Unit for Molecular Signal Transduction in Inflammation, Department for Molecular Biomedical Research, VIB, 9052 Gent, Belgium

Chapter 3. G-protein coupled receptor agonists mediate Neu1 sialidase and matrix metalloproteinase-9 cross-talk to induce transactivation of TOLL-like receptors and cellular signaling

Merry Guo, Schammim Ray Amith, Preethi Jayanth and Myron R. Szewczuk All co-authors gave permission to publish the data in Cellular Signalling 24 (2012) 2035– 2042.

Chapter 4. NEU1 Sialidase, Matrix Metalloproteinase-9 and Neuromedin B Receptor Cross-talk Controls Endosomal TOLL-like Receptor-7 and -9 Responses

Myron R. Szewczuk Co-author gave permission to publish the data in Cellular Signaling under peer-review.

Acknowledgements

First, I thank God the most gracious and most merciful for blessing me with guidance, strength and patience to complete this work.

I would like to express my sincere thanks to my supervisor Dr. Szewczuk for his support, and mentorship through these years. His humor made the lab a friendly environment to spend the long hours in. His encouragement made this work possible.

I would like to extend my gratitude to my committee members: Dr. Gee for her thoughtful comments on my research and valuable suggestions; Dr. Basta for broadening my knowledge on various aspects of science and history; and Dr. Tayade for his support and guidance throughout my PhD journey.

I am grateful to all members of the previous microbiology and immunology department and the present Department of Biomedical and Molecular Sciences, particularly Dr.

Finnen, Dr. Geletu, Jenny, Jerry, Jackie and Diane for their assistance and cheerful chats.

I am thankful to the past and present members of the Szewczuk lab, and to my colleagues in the graduate program for their good companionship and for a lot of fun. I particularly acknowledge Farah, Alanna, Ila, Christina, Masany and Yan.

I am in debt to all those who made my stay in Kingston a memorable one, especially Dr.

Fatma and Rola, you have been wonderful neighbors and amazing friends.

I will always be grateful to my previous mentors in the faculty of medicine and health sciences in UAE university specially Dr. Lukic and Dr. Ramadi. Their good mentorship and sincere advices encouraged me to continue in the field of immunology. Their belief in my capabilities was vital for directing me to pursue my graduate studies.

In terms of funding, I am thankful to the Ontario Graduate Scholarship for supporting me and to the Canadian Institute of Health Research (CIHR) for granting me the Frederick

Banting and Charles Best Canada doctoral award.

To my dearest family, your valuable support is beyond measure. To my dearest mother, sisters and brothers, I feel so lucky for having you in my life. You always had great faith that I could be successful in whatever I do and therefore I dedicate this thesis to you.

Table of Contents

Abstract ...... ii Co-Authorship ...... iv Acknowledgements ...... v List of Figures ...... xi List of abbreviations ...... xiii Chapter 1 Introduction and Literature Review ...... 1 1.1 Overview of the immune system ...... 1 1.2 Toll-like Receptors...... 2 1.2.1 Molecular basis of TLR structure and ligand recognition ...... 4 1.2.2 TLR Signaling ...... 7 1.2.3 TLR Glycosylation ...... 9 1.3 Mammalian Sialidases ...... 10 1.3.1 NEU1 sialidase ...... 12 1.3.2 The role of NEU1 in modulating immune responses and inflammation ...... 13 1.4 Matrix metalloproteinases ...... 14 1.4.1 Role of MMP-9 in inflammation ...... 16 1.5 G protein-coupled receptors (GPCRs) ...... 17 1.5.1 GPCR crosstalk with TLRs ...... 20 1.6 Rationale and Hypothesis ...... 22 1.6.1 Overall hypothesis ...... 24 1.6.2 Aims of this study ...... 24 1.7 References: ...... 25 Chapter 2 Neu1 Sialidase and Matrix Metalloproteinase-9 Cross-talk Is Essential for Toll-like Receptor Activation and Cellular Signaling ...... 38 2.1 Abstract ...... 39 2.2 Introduction ...... 40 2.3 Experimental Procedures ...... 41 2.3.1 Cell Lines ...... 41 2.3.2 Mouse Models ...... 42 2.3.3 Primary Mouse Bone Marrow Macrophage Cells ...... 43 2.3.4 Silencing MMP9 mRNA Using Lentivirus MMP9 shRNA ...... 43 2.3.5 Silencing MMP9 mRNA Using MMP9 siRNA ...... 44

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2.3.6 Ligands and Enzymes ...... 45 2.3.7 Inhibitors ...... 46 2.3.8 Sialidase Assay ...... 47 2.3.9 OmniMMPTM Assay...... 47 2.3.10 Flow Cytometry of Cell Surface TLR4, CD14, and MMP9 ...... 48 2.3.11 Cell Surface Biotinylation and Western Blot of Immunoprecipitated Biotinylated Proteins ...... 48 2.3.12 Immunocytochemistry of NFκB and IKBα ...... 49 2.3.13 NFκB-dependent SEAP Assay...... 50 2.3.14 Co-immunoprecipitation ...... 50 2.3.15 MMP9 Colocalization with TLR4 ...... 51 2.3.16 MMP9 Colocalization with Neu1 ...... 52 2.3.17 Statistics ...... 53 2.4 Results ...... 53 2.4.1 Tamiflu, Pertussis Toxin, and Galardin Block Neu1 Activity Associated with LPS Binding to TLR4 in Live HEK-TLR4/MD2 Cells ...... 53 2.4.2 Matrix Metalloproteinase Activity Is Associated with Zymosan A (TLR2 Agonist), poly(I:C) (TLR3 Agonist), and LPS (TLR4 Agonist) Treatment of Live BMC-2 Cells ...... 58

2.4.3 Gαi-sensitive Pertussis Toxin, MMP Inhibitor Galardin, and Tamiflu Block TLR4 Ligand LPS and TLR2 Ligand Zymosan A-induced NFκB Activation...... 60 2.4.4 LPS-induced Phosphorylation of NFκB ...... 62 2.4.5 Silencing MMP9 mRNA Using Lentivirus MMP9 shRNA Knockdown ...... 68 2.4.6 Cell Surface Expression of MMP9 ...... 72 2.4.7 Interference MMP9 Using siRNA Knockdown ...... 75 2.4.8 Neu1 and MMP9 Cross-talk Is Essential for LPS-induced TLR4 Activation ...... 78 2.5 Discussion ...... 84 2.6 References ...... 93 Chapter 3 G-protein coupled receptor agonists mediate Neu1 sialidase and matrix metalloproteinase-9 cross-talk to induce transactivation of TOLL-like receptors and cellular signaling ...... 101 3.1 Abstract ...... 102 3.2 Introduction ...... 102 3.3 Materials and Methods ...... 104 3.3.1 Reagents ...... 104 viii

3.3.2 Inhibitors ...... 105 3.3.3 Antibodies ...... 106 3.3.4 Cell lines ...... 106 3.3.5 NFκB-dependent secretory alkaline phosphatase (SEAP) assay ...... 107 3.3.6 Silencing MMP9 mRNA using MMP9 siRNA ...... 107 3.3.7 Mouse Models ...... 108 3.3.8 Primary mouse bone marrow macrophage cells ...... 108 3.3.9 Sialidase activity in live cells ...... 109 3.3.10 Immunocytochemistry of NFκB ...... 109 3.3.11 Co-immunoprecipitation ...... 110 3.3.12 Statistics ...... 111 3.4 Results ...... 111 3.4.1 GPCR agonist induce sialidase activity in live BMC-2, BMA and RAW- blue macrophage cells and primary wild-type mouse macrophage cells but not in Neu1-deﬁcient cells ...... 111 3.4.2 Inhibitory effect of neuraminidase inhibitors Tamiﬂu and DANA (2-deoxy-2,3- dehydro-N-acetylneuraminic acid) on bombesin induced sialidase activity in live macrophage cells ...... 114 3.4.3 Bombesin induces NFκB activation in BMC-2 and RAW-blue macrophage cells .... 117 3.4.4 Isoforms of neuromedin B receptor co-immunoprecitate with TLR4 receptors in cell lysates from naïve and LPS stimulated BMA macrophage cells ...... 118 3.4.5 Silencing MMP9 using siRNA knockdown ...... 120 3.4.6 Bombesin-mediated NFκB activation in RAW-blue cells involves the TLR4 adaptor molecule MyD88 ...... 122 3.5 Discussion ...... 124 3.6 References ...... 130 Chapter 4 NEU1 Sialidase, Matrix Metalloproteinase-9 and Neuromedin B Receptor Cross-talk Controls Endosomal TOLL-like Receptor-7 and -9 Responses ...... 136 4.1 Abstract ...... 137 4.2 Introduction ...... 137 4.3 Materials and methods ...... 139 4.3.1 Cell lines ...... 139 4.3.2 Silencing Neu1, MMP9 and Neuromedin-B (NMBR) mRNA using siRNA ...... 140 4.3.3 Ligands ...... 141 ix

4.3.4 Inhibitors ...... 141 4.3.5 NF-κB-dependent secretory alkaline phosphatase (SEAP) assay ...... 141 4.3.6 Co-immunoprecipitation ...... 142 4.3.7 Neu1 or MMP9 colocalization with TLR-7 and -9 ...... 143 4.3.8 NMBR colocalization with Rab7 ...... 144 4.3.9 Bio-Plex cytokine microarray profiles in the cell culture supernatants using the multiplex bead-based assay ...... 144 4.3.10 Statistics ...... 145 4.4 Results ...... 145 4.4.1 NEU1 and MMP-9 are tethered to naive full-length and cleaved TLR7 receptors ..... 145 4.4.2 The effect of Tamiflu, MMP9i, BIM- 23127 and BIM- 46174 on Imiquimod-induced phosphorylation of NF-κB ...... 149 4.4.3 NEU1, MMP-9 and neuromedin B (NMBR) regulate Imiquimid-induced phosphorylation of NFκB...... 152 4.4.4 Tamiflu, MMP9i and neuromedin B receptor (NMBR, BB1) antagonist BIM-23127 attenuate imiquimod-induced TLR7 recruitment of adaptor molecule MyD88 in RAW-blue cells ...... 152 4.4.5 Neu1 and MMP-9 form a complex with TLR9 receptor ...... 154 4.4.6 Neu1 and MMP-9 cross-talk is essential for TLR9 receptor activation ...... 156 4.4.7 Tamiflu and MMP9i significantly blocked Imiquimod and CpG induced SEAP ...... 158 4.4.8 Isoforms of neuromedin B receptor co-immunoprecipitate with TLR-7 and -9 receptors in cell lysates from naive and ligand stimulated RAW-blue macrophage cells ...... 161 4.4.9 Tamiflu, MMP9i and the heterotrimeric G-protein complex antagonist, BIM-46174, abrogates pro-inflammatory cytokines ...... 164 4.5 Discussion ...... 166 4.6 References ...... 170 Chapter 5 General Discussion and Perspectives ...... 176 5.1 Disease intervention possibilities (mouse models) ...... 183 5.2 References ...... 187

List of Figures

Figure 1.1: Ligand-Induced TLR Dimers...... 6 Figure 1.2: TLR signaling pathways...... 8 Figure 1.3: G-Protein Coupled Receptor Signaling Pathways...... 19

Figure 2.1: LPS-induced sialidase activity is blocked by specific GPCR Gαi subunit and MMP inhibitors in live HEK-TLR4/MD2 cells...... 56 Figure 2.2: Zymosan A, poly(I:C), and LPS induce MMP activity in live BMC-2 cells, which is inhibited by galardin...... 59 Figure 2.3: LPS-induced NFκB activation and IκB degradation ...... 61 Figure 2.4: Endotoxin LPS induces phosphorylated NFκBp65 Ser(P)529 (pNFκB) in BMC-2 and BMA macrophage cells...... 64 Figure 2.5: MMP9i significantly inhibits LPS-induced sialidase activity in live RAW-Blue macrophage cells in a dose-dependent manner...... 67 Figure 2.6: Analysis of silencing MMP9 mRNA using lentiviral MMP9 shRNA...... 71 Figure 2.7: Analysis of MMP9 expression on the cell surface of live human monocytic CD14- THP1 and BMA cells...... 74 Figure 2.8: MMP9 siRNA transfection of RAW-Blue macrophage cells...... 77 Figure 2.9: MMP9 colocalizes and coimmunoprecipitates with TLR4...... 80 Figure 2.10: MMP9 colocalizes and coimmunoprecipitate with Neu1...... 83 Figure 3.1: Sialidase activity is associated with GPCR agonist treatments of live macrophage cells and primary bone marrow (BM) macrophage cells...... 113 Figure 3.2: Bombesin-induced sialidase and NFκB activities are blocked by Tamiﬂu and DANA in live RAW-blue and BMC-2 macrophage cells respectively...... 116 Figure 3.3: Neuromedin B receptor (NMBR) co-immunoprecipitates with TLR4 and MMP9. . 119 Figure 3.4: GPCR agonist-induced sialidase activity in live WT and siRNA MMP9 KD BMA macrophage cells ...... 121 Figure 3.5: MyD88 homodimizeration inhibitor blocks LPS-induced NFκB-dependent secretory alkaline phosphatase (SEAP) activity in RAW-blue macrophage cells...... 123 Figure 4.1: TLR7 co-immunoprecipitates with NEU1 and MMP9 ...... 147 Figure 4.2: NEU1 and MMP9 colocalize with TLR7 ...... 148 Figure 4.3: Western blot analysis of Imiquimod- and CpG ODN-induced phosphorylated NF-κB in RAW-blue whole cell lysates...... 151

Figure 4.4: MMP9i, Tamiflu and BIM23127 attenuate MyD88 recruitment...... 153 Figure 4.5: NEU1 and MMP9 are tethered to TLR9...... 155 Figure 4.6: Western blot analysis of CpG ODN-induced phosphorylated NF-κB in whole cell lysates...... 157 Figure 4.7: Tamiflu and MMP9i inhibit NF-κB-dependent SEAP activity...... 160 Figure 4.8: NMBR regulates TLR7 and TLR9 ...... 163 Figure 4.9: Bio-Plex cytokine microarray profiles in the cell culture supernatants...... 165 Figure 5.1: Model of TLR4 signaling platform...... 179

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List of abbreviations

4-MUNANA 2’(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid BM Bone marrow CD14 Cluster of differentiation 14 CLR C-type lectin receptor CpG-ODN CpG oligodeoxinucleotide Dpa N-3-(2,4-dinitrophenyl)-L-α,β-diaminopropionyl EBP Elastin-binding protein ECD Ectodomain ECM extracellular matrix EMH Extramedullary hematopoiesis ER Endoplasmic reticulum FRET Fluorescent resonance energy transfer GDP Guanosine diphosphate GPCR G protein-coupled receptor GRPR Gastrin-releasing peptide receptor GTP Guanosine triphosphate HER2 Human Epidermal Growth Factor Receptor 2

IC50 50% inhibitory concentration ICAM Intercellular adhesion molecule IGF Insulin-like growth factor IL Interleukin iNOS Induced nitric oxide synthase IFN Interferon IRAK IL-1 receptor-associated kinase KO Knock out LAMP Lysosomal- associated membrane protein LBP LPS-binding protein

xiii

LPS Lipopolysacharide LRR Leucine rich repeat LTA Lipoteichoic acid MAPK Mitogen-activated protein kinase MCA (7-methoxycoumarin-4-yl)acetyl MCP1 Monocyte chemotactic protein-1 MD-2 Myeloid differentiation factor-2 MHC II Major histocompatibility complex class II MMP Matrix metalloproteinase MMP3i MMP-3 inhibitor MMP9i MMP-9 inhibitor MyD88 Myeloid differentiation factor 88 NEU1-4 Neuraminidase 1-4 (sialidases) NFκB Nuclear factor kappa B NGF Nerve growth factor NLR NOD-like receptor NMBR Neuromedin B receptor NOD Nucleotide-binding oligomerization domain PAMP Pathogen associated molecular pattern PAR Proteinase-activated receptor PDGF Platelet derived growth factor PIPZ Piperazine Poly IC Polyinosinic-polycytidylic acid PPCA Protective protein/cathepsin A PRAT4A Protein-associated with TLR4A PRR Pattern recognition receptors PTX Pertussis toxin RIG Retinoic acid-inducible gene RLR RIG-I-like receptor

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RTK Receptor tyrosine kinase SEAP Secreted embryonic alkaline phosphatase siRNA Small interfering RNA Tamiflu Oseltamivir phosphate TGFβ Transforming growth factor β TIMP Tissue inhibitor of metalloproteinases TIR Toll-interleukin-1 receptor TIRAP TIR-associated protein TLR Toll like receptor TNF Tumor necrosis factor TRAM TRIF-related adaptor molecule TRIF TIR domain containing adaptor protein-inducing IFN-β

Chapter 1

Introduction and Literature Review

1.1 Overview of the immune system Immune systems in vertebrates are broadly divided into two branches: innate and adaptive. The innate immune system is immediately activated in response to invading pathogens and is therefore considered the first line of defense against microbial attacks.

In addition, the innate immune system mounts an effective response against infectious agents through the initiation of adaptive immunity, which is temporally delayed but long- lasting, and has immunological memory.

The innate immune system is genetically programmed to detect infection using a limited number of germ-line encoded receptors that recognize invariant features unique to the invading microbe known as pathogen-associated molecular patterns (PAMPs) (1).

PAMP receptors, known as pattern recognition receptors (PRRs), are expressed on the cell surface, in intracellular compartments, or secreted into the blood stream and tissue fluids. Several classes of PRRs have been discovered, which include Toll-like receptors

(TLRs), C-type lectin receptors (CLRs), RIG-I-like receptors (RLRs), NOD-like receptors (NLRs), and DNA receptors (cytosolic sensors for DNA) (2,3). On the other hand, the adaptive immune system uses antigen receptors that are generated de novo in each organism and appear to have infinite specificity (4,5). The combination of these two

mechanisms of pathogen recognition makes the mammalian immune system responses highly efficacious.

1.2 Toll-like Receptors Toll-like receptors (TLRs) represent the main family of PRRs. They play a key role in the initiation of the innate immune response and link innate to adaptive immunity

(1). TLRs are mainly expressed in tissues involved in immune function and those exposed to the external environment, such as the lung and skin. Since their discovery as mammalian homologues of the fruit fly Toll protein in 1997, the TLR family has significantly influenced the field of innate immunity research (6,7). The roles of TLRs in the pathogenesis of cancers, infections, autoimmune disorders, and allergic diseases have been a topic of rigorous study.

To date, there are 10 and 12 functional TLRs that have been reported in humans and mice, respectively. Interestingly, some primitive species such as the echinoderms, nematodes, and arthropods utilize TLRs for immune recognition, which indicate an early evolutionary role for these receptors (8). PAMPs recognized by TLRs include lipids, lipoproteins, proteins, and nucleic acids derived from a wide range of bacteria, viruses, parasites, and fungi (9). In addition to microbial products, TLRs recognize endogenous, altered host-derived ligands (10,11). The TLR family includes receptors residing both at the cell surface (TLRs -1, -2, -4, -5 and -6) and intracellularly (TLRs -3, -7, -8 and -9).

The proper cellular localization of TLRs is necessary for ligand accessibility, the downstream signal transduction and the maintenance of tolerance to self-molecules such

as nucleic acids (9). Cell surface TLRs recognize mainly microbial membrane components such as lipopolysaccharide (TLR4), lipopeptides (TLR2 associated with

TLR1 or TLR6), and bacterial flagellin (TLR5). On the other hand, intracellular TLRs are involved in recognition of nucleic acids. TLR9 recognizes microbial DNA; TLR7 and

TLR8 detect ssRNA while TLR3 recognizes dsRNA (12).

Intracellular TLRs have been shown to traffic from the ER through the Golgi to take up residence in the endolysosomes prior to stimulation (13,14). Surprisingly, following arrival into the endolysosome, TLR7 and TLR9 have been found to be proteolytically cleaved by multiple lysosomal proteases, including cathepsins and asparagine endopeptidase, to generate functional receptors. Hence, the activation of these receptors is limited to these intracellular compartments (14,15). In a more recent work, Qi et al (16) documented that TLR3 in HEK293T cells is proteolytically processed within

LRR-12 on the receptor ectodomain; however, unlike TLR -7 or -9, both the cleaved and full length TLR3 were able to bind the synthetic ligand, polyinosinic-polycytidylic acid

(poly IC) and induce cellular signaling (16). When receptor cleavage was inhibited by treating cells with cathepsin inhibitor, TLR3-poly IC-induced signaling was not affected; however this treatment resulted in accumulation of TLR3 in the recycling endosomes and lysosomes. This finding indicates that TLR3 cleavage may have an effect on the receptor’s trafficking to specific endosomal compartments. Toscano et al (2013) further confirmed TLR3 cleavage and intriguingly hypothesized a cleaved/associated model of

he receptor in which the LRR12 of TLR3 ectodomain is cleaved but the C and N terminal fragments do not detach (17).

1.2.1 Molecular basis of TLR structure and ligand recognition TLRs are type I transmembrane proteins that consist of three main domains: a leucine rich ectodomain, a transmembrane domain, and a cytoplasmic signaling domain.

The ectodomain contains 19-25 tandem copies of the “xLxxLxLxx” motif which forms a horseshoe structure and is involved in ligand recognition by TLRs. The ectodomains

(ECDs) of the 10 human TLRs vary in leucine rich repeat (LRR) numbers, the amino acid composition within these repeats, and the extent of N-linked glycosylation (18). The signaling domains of TLRs are known as Toll-interleukin-1 receptor (TIR) domains because they share homology with the signaling domains of interleukin-1 receptor (IL-

1R) family members (19). Most TLRs form homodimers in order to initiate signaling, while TLR2 forms a heterodimer with TLR1 to recognize triacyl lipopeptides or with

TLR6 in order to recognize diacyl lipopeptides. TLR4 forms a homodimer in complex with lipopolysacharide (LPS) and myeloid differentiation factor-2 (MD2). Additional proteins such as LPS-binding Protein (LBP) and cluster of differentiation 14 (CD14), a glycophosphatidylinositol-anchored membrane associated protein, are involved in LPS binding. CD14 binds LBP and delivers LPS to the TLR4-MD2 complex (20). Recently,

CD14 was also shown to be associated with TLR7 and TLR9, and to engage with their ligands promoting the uptake of nucleic acids (21).

To date, five crystallographic structures of the TLR ECDs and their ligand complexes have been reported (TLR-1, -2, -3, -4, and -6). The structures of TLR1-TLR2- lipoprotein, TLR3-dsRNA complex, as well as the model of TLR4-MD2-LPS complex, all acquire an “m” shaped dimeric architecture, suggesting that all other TLRs undergo similar dimerization upon ligand binding (Figure 1.1) (22-24).

Using homology modeling, Wei et al. (2009) generated structural models of cleaved ligand-binding domains of TLR-7, -8, and -9. Based on comparison of the structures, they identified potential ligand-binding sites as well as possible configurations of the receptor–ligand complexes (26). The final structures of the ectodomains of TLR-7,

-8 and -9 reveal a large, arc-shaped assembly consisting of 11 canonical LRRs and two terminal LRRs, which assumed a right-handed solenoid structure. Similar to other TLRs, the human TLR-7, -8 and -9 are glycosylated. The NCBI protein database provides seven and six predicted N-linked glycosylation sites for the cleaved forms of TLR-7, -8 and -9, respectively. Moreover, the glycans were shown to be nonfunctional for ligand binding

(22-24,27).

The mechanism by which TLR-3, -7 and -9 translocate from the ER to the endosome is not fully understood. A recent study using forward genetic mutagenesis indicated that a single point mutation in the gene encoding UNC93B, a highly conserved

ER resident protein, led to a complete deficiency in signaling of TLR-3, -7 and -9 (28).

UNC93B interacts with these TLRs and regulates their trafficking from the ER to the endolysosome. A second ER-resident protein that has been involved in TLR-1, -2, -4, -7

Figure 1.1: Ligand-Induced TLR Dimers. The ‘‘m’’-shaped TLR dimers induced by binding of agonistic ligands. The crystal structures of (A) TLR1-TLR2-Pam3CSK4 and (B) TLR3- dsRNA are colored gray, green, red, pink, and purple, respectively. (C) Shows a model of the TLR4-MD-2-LPS complex. TLR4, MD-2 and LPS are colored light blue, orange, and black, respectively. Adapted from Mi Sun Jun and Jie-Oh Lee, 2008 (25).

and -9 trafficking is protein-associated with TLR4A (PRAT4A) (29). TLR-7 and -9 mobilization was also reported to be controlled by gp96, a member of the heat shock protein 90 family (30).

1.2.2 TLR Signaling The accepted mechanism of TLR signaling involves the interaction of a ligand with the receptor’s ECD. The ligand binding to the receptor either induces receptor dimer formation or changes the conformation of a pre-existing dimer in such a way that it brings two intracellular TIR domains of the TLRs together. It is the resulting association of the two TIR domains that initiates the intracellular signaling pathway starting with the recruitment of adaptor proteins (25). To date, at least four different TLR adaptor proteins have been identified including the myeloid differentiation factor 88 (MyD88), TIR domain containing adaptor protein-inducing IFN-β (TRIF), TIR-associated protein

(TIRAP), and TRIF-related adaptor molecule (TRAM) (19). All TLRs with the exception of TLR3 initiate MyD88-dependent signaling. TLR3 and TLR4 recruit TRIF and initiate

TRIF-dependent signaling. Upon binding to the dimerized TIR domains, the adaptor proteins recruit downstream IL-1 receptor-associated kinases IRAK4, IRAK1, and

IRAK2. This binding in turn, leads to the activation of various transcription factors such as NFκB and IRF3/7, and MAP kinases to induce the production of pro-inflammatory cytokines and type-1 interferons (31) (Figure 1.2).

Figure 1.2: TLR signaling pathways. All TLRs, except TLR3, initiate the MyD88-dependent pathway whereas TLR3 initiates the TRIF-dependent pathway. TLR4 activates both MyD88-dependent and TRIF-dependent pathways. MyD88 recruits the IRAK family of proteins and TRAF6. In turn, TRAF6 activates TAK1. The activated TAK1 activates the IKK complex, which activates NFκB and also activates the MAPK pathway. TRIF interacts with TRAF6 which leads to NFκB activation and the transcription of proinflammatory cytokines; TRIF also interacts with TRAF3 to activate IRF3 or IRF7 transcription factors to induce the expression of type 1 interferons. Adapted from Kumar et al, 2009 (32).

Although the signaling pathways of TLRs are well characterized, the parameters controlling interactions between TLRs and their ligands have remained poorly defined until now. Upon ligand binding TLRs undergo conformational change that juxtaposes the

TIR domains providing interface for binding of adaptor proteins; and receptor glycosylation may be involved in this conformational change.

1.2.3 TLR Glycosylation Studies have shown that glycosylation of both secreted and cell membrane bound receptors is crucial for their transport and function. For instance, partial glycosylation is an essential requirement for the processing and/or ligand-binding of the receptor tyrosine kinase (RTK) insulin receptor (33) and epidermal growth factor receptor (34,35).

Additionally, glycosylation is critical for nerve growth factor (NGF) TrkA receptors localization to the cell surface; also, it prevents ligand-independent receptor autophosphorylation (36).

TLR3 glycosylation was shown to contribute significantly to the overall mass of the ectodomain (approximately 35%). Sun et al. (2006) have shown inhibition of TLR3 ligand-induced NFκB activation in cells treated with glycosylation inhibitors (37).

Additionally, they reported that mutations in two different glycosylation sites impaired

TLR3 function without altering receptor expression. The TLR4 ectodomain has 9 N- linked glycosylation sites, while MD2 contains 2 glycosylation sites as suggested by their sequence analysis. Studies with TLR4 and MD2 mutants lacking N-glycosylation sites have shown reduced LPS-induced IL8 production and decrease in the activation of c-Jun

kinase (38) . They have also revealed that mutations in TLR4 glycosylation sites reduced the cell surface expression of TLR4. These findings highlight the importance of glycosylation in maintaining the functional integrity of the LPS receptor complex. A more recent study demonstrated 75% and 83% decrease in LPS-mediated NF-κB reporter activation following mutagenesis of double and triple TLR4 glycosylation sites, respectively (39). In the same study, the authors also reported that glycan structures on

TLR4, MD2 and CD14 are sialylated as shown by lectin blot and the addition of exogenous neuraminidase which removes the sialic acid from the receptor’s glycan structure enhanced NFκB activity.

Previously our laboratory established the importance of the removal of -2,3 sialyl residues linked to the β-galactosyl of Trk by sialidase for receptor dimerization and subsequent activation (40). Further work on TLR4 identified a similar mechanism that leads to receptor activation following the hydrolysis of the -2,3-linked sialyl residues from the TLR4 ectodomain by NEU1 sialidase (41). These studies uncover the essential role of glycosylation and sialylation of TLRs as well as the role of sialidases in the receptor activation.

1.3 Mammalian Sialidases Sialic acids are a family of acidic monosaccharides that form α-glycosidic linkages with the terminal galactose residues on glycoconjugates. Sialoglycoconjugates are abundantly distributed on the cell surfaces of animals and bacteria as well as on the lysosomal and endosomal inner membrane surfaces (42). Sialic acids participate in many

biological processes including masking and recognition of antigens during cell differentiation, interaction, migration and metastasis (43). Hydrolytic cleavage of sialic acid residues linked to glycoconjugates is catalyzed by sialidases also known as neuraminidases (44).

To date four distinct mammalian sialidases (NEU1 to NEU4) have been cloned, purified and classified according to their subcellular localization and substrate specificities (45). NEU1 is mainly lysosomal and preferentially cleaves glycoproteins, also it can be translocated to the plasma membrane (46,47). It is highly expressed in all tissues mainly in the kidney, pancreas, skeletal muscle, liver, lungs, placenta and brain

(46). NEU2 is predominantly localized in the cytosol and was the first, and so far the only mammalian sialidase whose crystal structure was characterized (48). NEU3 is surface- associated and it is best recognized for cleaving sialic acid expressed on glycolipids

(gangliosides) (49-51). The last identified sialidase is NEU4 which was cloned from murine brain and has similarities to NEU3 (52). There is evidence for a human sialidase

(NEU4) localized to the mitochondrial (53) or lysosomal lumen (54). NEU1 shows higher sequence identity to bacterial sialidases than to the NEU2, NEU3, and NEU4 sialidases. The latter three enzymes appear to be more similar to each other (42–44%) than to NEU1 (<28%), this may indicate that their genes have evolved through “recent” gene duplication events within the vertebrate lineage (55). Sialidases play an essential role in the regulation of cell proliferation/differentiation, clearance of plasma proteins,

control of cell adhesion, metabolism of gangliosides and glycoproteins, developmental modeling of myelin and modification of receptors (55).

1.3.1 NEU1 sialidase Lysosomal (NEU1) sialidase is a glycoprotein enzyme that is only active as a part of a molecular multi-enzymatic complex that contains β-galactosidase and protective protein/ cathepsin A (PPCA) (56). On the cells surface NEU1 and PPCA associate with elastin-binding protein (EBP), which is an inactive, spliced form of β-galactosidase (57).

It has been reported that PPCA is required for NEU1 to keep its active and stable conformation (58,59). Bonten et al (2009) have also shown that in the absence of PPCA,

NEU1 self-associates into chain-like oligomers and stays inactive, while the addition of

PPCA expressed in insect cells was able to reverse the self-association (58). Genetic deficiency of PPCA leads to secondary deficiencies in NEU1 and β-galactosidase, which results in galactosialidosis, a lysosomal storage disorder (60). Sialidosis is another genetic metabolic disorder caused by deficiencies in NEU1 (59,61). Sialidosis patients suffer from hepatosplenomegaly and neurodegenerative symptoms (62). Neu1 knockout

(KO) mice exhibit degenerative effects similar to the human condition and develop a marked, age-dependent splenomegaly characterized by elevated numbers of hematopoietic progenitors, consistent with splenic extramedullary hematopoiesis (EMH)

(63).

1.3.2 The role of NEU1 in modulating immune responses and inflammation In addition to its role in the catabolism of glycoproteins, NEU1 regulates various important cellular phenomena by modulating cell signaling through desialylation of various cell receptors. For example, NEU1 was shown to desialylate both platelet derived growth factor (PDGF) and insulin-like growth factor (IGF)-1 receptors, which leads to reduction of the mitogenic effects of PDGF and IGF on smooth muscle cells (64). NEU1 also plays a role in modulating immune responses. Nan et al (2007) have shown that activation of lymphocytes by exposure to anti-CD3 and anti-CD28 IgG resulted in a nine fold increase in NEU1-specific activity (65). This NEU1 sialidase activity in activated T lymphocytes contributed to cell surface desialylation and to production of interferon-γ

(65). Treatment of freshly isolated monocytes with an exogenous bacterial neuraminidase enhanced their responsiveness to LPS and their production of inflammatory cytokines

(66) suggesting that the hydrolysis of sialic acids on both lymphocytes and monocytes by the sialidase enzyme influences their activation.

During the differentiation of freshly isolated human monocytes into macrophages, endogenous sialidase activity showed a 12-fold increase that was consistent with significant NEU1-mRNA and protein level increases (67). Liang et al (2006) have also reported similar findings in THP1-derived macrophages (68). The authors have also shown that NEU1 and PPCA are targeted to the lysosome and then are sorted to the

LAMP-2 negative, MHC II-positive vesicles that merge with the plasma membrane.

Transfecting THP1 cells with NEU1 small interfering RNA (siRNA) or treating them with anti-NEU1 antibodies significantly reduced the sialidase activity, as well as the 13

ability of the cells to engulf bacteria and to produce cytokines. Similarly, macrophages from NEU1-deficient mice were reported to have increased cell surface sialylation in addition to compromised phagocytosis. When these macrophages were treated with exogenous NEU1 they showed reduction in the cell surface sialylation and restoration of their phagocytosis ability (69).

More recent work from our lab by Amith et al (2010) indicated that NEU1 sialidase activity is required for the removal of steric hindrance to TLR4 receptor dimerization and the induction of NFκB activation (70). When NEU1 sialidase activity was blocked by Tamiflu, a neuraminidase inhibitor, or anti-NEU1 antibodies, this also blocked the NFκB activity, which suggests an essential role for NEU1 sialidase in TLR4 activation.

Collectively these data suggest that the upregulation of the NEU1 expression and activity is important for the primary function of macrophages and establish the link between NEU1 and the cellular immune responses; however, the mechanism that leads to the ligand-induced NEU1 sialidase activity was not defined. In this thesis, we identified a potential role of matrix metalloproteinases in activating NEU1that was not previously observed.

1.4 Matrix metalloproteinases Matrix metalloproteinases (MMPs) are a large family of zinc-dependent proteases responsible for degrading and rebuilding multiple components of the extracellular matrix

(ECM) such as collagen, elastin, gelatin, hyaluronan and proteoglycan (71). The family

of MMPs is composed of 28 endopeptidases that are subdivided based on their distinct and yet overlapping, substrate specificity (72). This includes the collagenases (MMP -1, -

8 and -13), the gelatinases (MMP -2 and -9), the stromelysins (MMP -3, -10 and -11), the matrilysins (MMP -7 and -26), and the final group is composed of membrane-associated

MMPs, known as membrane type MMPs (MT-MMP -14, -15, -16, -17, -24 and -25) (73).

MMP activity is tightly controlled by several endogenous inhibitors, of which the most thoroughly studied inhibitors are the tissue inhibitor of metalloproteinases (TIMPs), which bind to the MMPs and reversibly block MMP activity (73).

An increasing number of secreted MMPs are now recognized to at least transiently localize to the cell surface, most often in association with adhesion receptors or cell surface proteoglycans. For example, MMP-1 associates with integrins (74), MMP-

2 interacts with αvβ3-integrin (75), MMP-7 binds cell surface heparan sulphate proteoglycans (76) and MMP-9 binds several cell surface receptors including CD44 (77) and ICAM-1 (78).

In addition to their ECM substrates, MMPs also cleave cell surface molecules and other pericellular, non-matrix proteins, thereby regulating cellular processes including proliferation, angiogenesis, migration, host defense, cancer invasion, and metastasis

(79,80). MMPs have been shown to cause the release of precursor forms of growth factors, such as latent transforming growth factor β (TGF-β), activating them to bind their respective receptors on target cells (81). MMPs have also been shown to cleave the ectodomain of HER2 (a transmembrane tyrosine kinase receptor) enhancing the

receptor’s signaling activity, the case of which is seen in cancerous cells (82). These data indicate that MMPs influence physiological processes beyond simply ECM homeostasis.

1.4.1 Role of MMP-9 in inflammation MMP-9, also named gelatinase B, is able to cleave denatured collagen, fibronectin and elastin (73). MMP-9 expression on polymorphonuclear cells has been shown to increase by 10 fold when a proinflammatory mediator was used to activate these cells

(83). Also, enhanced MMP-9 expression was detected in murine bone marrow-derived mast cells following stimulation with LPS or peptitoglycan (84). In an attempt to study the role of MMPs in cases with sepsis, MMP-9 but not MMP-2 was secreted into the blood after infusion of bacterial LPS in healthy human volunteers (85). This scenario indicates that MMP-9 may contribute to the pathogenesis of sepsis via its pro- inflammatory effects on the vasculature. In another study, patients admitted to the intensive care unit with severe sepsis were reported to have significantly higher levels of

MMP-9 as well as interleukin 6 (IL-6) compared to healthy subjects (86); however,

MMP-2 levels were not different in the septic patients compared to healthy subjects.

These results support an association between markers of inflammation and MMP-9.

Moreover, MMP-9 deficient mice were found to be resistant to endotoxic shock when challenged with LPS (87). Activation of TLR 9 by its ligand CpG oligodeoxinucleotide

(CpG ODN) was reported to regulate the expression of MMP-9 in murine macrophage cells through AKt-TNF-mediated mechanism (88). Although there is a wealth of

information referring to the expression of MMP-9 in response to TLR stimulation, the role of MMP-9 in TLR activation was not studied.

Previous findings from our lab demonstrated that MMP-9 mediated TrkA receptor activation upon nerve growth factor (NGF) stimulation through NEU1 Activity (89).

Additionally, they found that MMP-9 forms a complex with the TrkA receptor on the cell surface. Here, we speculate a similar role played by MMP-9 in TLR activation. Given its proteolytic enzyme activity with elastase substrate specificity, MMP-9 is a good candidate to target the tripartite complex of NEU1/PPCA/EBP already associated with surface TLRs by cleaving the EBP from the NEU1 complex. MMP-9 can also degrade elastin, which produces elastin peptides. These peptides can then bind to the EBP and initiate NEU1 activity as previously reported (90).

How would MMP-9 become activated upon ligand binding to TLRs? A plausible answer would be that it becomes activated by a G protein coupled receptor (GPCR) Gα- signaling process. Indeed, GPCRs were involved in the transactivation of several receptors, a process that involves matrix metalloproteinases. For example, MMP-9 or

MMP-2 activities were required for gonadotropin-releasing hormone receptor-mediated epidermal growth factor receptor (EGFR) transactivation, and inhibition of the activities of these gelatinases suppressed EGFR phosphorylation (91).

1.5 G protein-coupled receptors (GPCRs) GPCRs represent the largest family of cell-surface signaling receptors with more than 800 genes in the human genome coding for these proteins (92). GPCRs respond to

signals of vast array of stimuli both internal, within the body, such as hormones and neurotransmitters and external such as light, odors and others. They are composed of a seven-transmembrane region, an extracellular amino-terminus involved in ligand recognition, and intracellular carboxyl-terminal domain that is important for internalization or desensitization of the receptor. Usually GPCRs are coupled to heterotrimeric G proteins consisting of an α,β, and γ-subunits (93). The Gα subunits are divided into four main categories: Gαi, Gαs, Gαq and Gα12/13, and they possess intrinsic

GTPase activity. The Gβγ subunit is noncovalently bound to the Gα subunit in its inactive guanosine diphosphate (GDP)-bound state. When the receptor is activated upon agonist- binding, this induces conformational changes in the extracellular domain that is transmitted to the G protein associated with the intracellular domain. This change dissociates the Gα subunit from the dimer βγ subunits and allows the exchange of guanosine triphosphate (GTP) for GDP on the Gα subunit. Active GTP-bound Gα subunit activates several second messenger pathways (94) Figure 1.3.

G-Protein Coupled Receptor

Figure 1.3: G-Protein Coupled Receptor Signaling Pathways. G-protein coupled receptors (GPCR) share a common structural architecture composed of extracellular N-terminal domain, a 7-transmembrane domain and an intracellular C- terminal domain. Intracellularly a G-protein, which consists of α, β, and γ-subunits, is connected with the receptor. Binding of an agonist to the receptor induces a conformational change that converts the receptor to its active state. This leads to the exchange of G-protein-bound GDP for GTP, after which the G-protein is disconnected from the receptor and the α-subunit dissociates from the βγ-dimer. The α-subunit can subsequently activate several second messenger pathways. Adapted from Veulens and Rodríguez, Biotecnología Aplicada 2009;26:24-33

1.5.1 GPCR crosstalk with TLRs Receptor crosstalk in the innate immune system is important to coordinate microorganism-sensing signals. Evidence for the interaction between TLRs and GPCRs came initially from the effect of TLRs on GPCR activity. Activation of TLRs has been shown to regulate GPCR responsiveness by modulating the regulators of G-protein signaling (RGS) proteins (95). RGS proteins have GTPase-activating activity and they enhance Gα-GTP hydrolysis, thereby negatively regulating G protein activity (96). After

LPS stimulation, RGS1 and RGS2 mRNAs were downregulated in murine bone marrow- derived macrophages as well as in the murine macrophage cell line J774. The ligand for

TLR2/6 heteromer and that for intracellular TLR9 also modulated RGS1 and RGS2 in a similar way to TLR4 ligand (LPS) (95).

It has been shown that pertussis toxin, which inhibits the coupling of the GPCR to the Gαi proteins, inhibited LPS-induced TNFα production in murine J774 and human

THP-1 cell lines. Similarly, pertussis toxin blocked TNFα production induced by the

Gram-positive bacterium Staphylococcus aureus (SA) that activates TLR2 (97). These results suggest that at least one Gi protein serves as a common post-receptor-signaling protein for both TLR4 and TLR2 ligands. In a follow up study, Fan et al (2007) have confirmed the involvement of Gα proteins in TLR4 and TLR2 signaling by demonstrating that genetic deletion of Gαi2 or Gαi1/3 in mice significantly reduced LPS and SA-induced TNFα production in peritoneal macrophages compared to wild type mice

(98).

Conversely, it is well known that GPCR agonists can induce transactivation of TLR receptors independent of TLR ligands, but the mechanism(s) behind GPCR agonist- induced transactivation of TLRs in the absence of TLR ligands is unknown.

CD14 is a glycosylphosphatidylinositol (GPI)-anchored protein which associates with TLR4 in the lipid membrane raft as part of the LPS-receptor complex with MD2. It was reported that CD14 physically interacts with and it co-immunoprecipitated with G protein subunits in human monocyte cells lysate (99). In addition, heterotrimeric G proteins were shown to have a specific regulatory function in CD14-associated LPS- induced mitogen-activated protein kinase (MAPK) activation and cytokine production in human monocytes.

Proteinase-activated receptor 2 (PAR2) is a member of the GPCR family that is expressed in the respiratory tract as well as in epithelial cells, monocytes and macrophages suggesting its participation in innate immune responses. In their study,

Rallabhandi et al (2008) demonstrated that PAR2 is physically associated with TLR4 in

HEK293T transfected cells and the co-expression of TLR4 and PAR2 enhanced NFκB signaling (100). To further confirm this novel cooperation model between GPCR and

TLR, they showed that TLR4-/- mice-derived macrophages exhibited a decrease in IL1β in response to a PAR2 agonistic peptide compared to wild type mice-derived

- - macrophages. Interestingly, macrophages from PAR2 / mice exhibited a down-regulation of induced nitric oxide synthase (iNOS) production after LPS stimulation.

Bombesin-like receptors such as gastrin-releasing peptide receptor (GRPR), neuromedin B receptor (NMBR) and the orphan bombesin receptor subtype 3 are members of the GPCR family that are involved in the activation of neutrophils and macrophages by chemokines (101). In a recent study, GRPR antagonist significantly reduced TLR-4 mRNA levels in RAW 264.7 murine macrophage cells after LPS stimulation, subsequently reducing NFκB activity (102). In the same study, lung tissue from rats treated with GRPR antagonist peptide after induced sepsis showed a significant decrease in TLR4 mRNA expression compared with untreated control animals. Also, animals treated with GRPR antagonist after induced sepsis demonstrated lower serum levels of both interleukin 6 (IL6) and the chemokine MCP1 (102).

Collectively, these studies strongly suggest a cross-talk between GPCRs and

TLRs whereby both receptors induce signaling that influences each other’s activity. Such intracellular interaction possibly includes transactivation pathways that modulate immune responses of which the molecular signaling platform is still to be elucidated.

1.6 Rationale and Hypothesis Since the discovery of TLRs sixteen years ago, much progress has been made in the field of their biology. Today, we know the specificities of TLRs for their microbial ligands. The structure of some of these receptors in complex with their ligands has been identified, which suggests the need for accessory proteins such as MD2 in the case of

TLR4. It has been discovered that some receptors are proteolytically processed in order to function properly, which means that a portion of the ectodomain is dispensable to these

receptors. The major components of the signaling pathways downstream of TLRs have been identified and different layers of complexity and regulation have been defined. Yet many questions remain unanswered. The important question is how ligand binding induces receptor activation and signaling? For the majority of TLRs, receptor dimerization is a prerequisite to facilitate MyD88-TLR complex formation and subsequent cellular signaling to activate NFκB. Ligand binding to TLR4 involves dimerization of the receptor chains, which in turn causes protein conformational changes in the receptor leading to the association of two receptor TIR domains (103). Fluorescent resonance energy transfer (FRET) microscopy revealed that the TIR-domains of TLR9 undergo substantial positional change following ligand binding to the receptor ectodomain (104). Therefore, it is quite reasonable to predict similar conformational changes taking place for the rest of TLRs upon ligand binding and that proper conformation of receptors in the dimer complex is an essential requirement for receptor activation in response to the specific ligands. However, the parameters controlling the interaction between the receptors and their ligands leading to the conformational change and dimerization remain to be explored.

Previous work by Amith et al (2009) from our lab identified NEU1 as an essential sialidase involved in cell surface TLRs activation (105). They demonstrated that NEU1 is already in complex with TLR2, -3 and -4 and it becomes activated upon ligand binding to these receptors. Activated NEU1 targets and cleaves the sialic acid residues bound to the

α-2,3 β-galactosides on the TLR ectodomain, the hydrolysis of which facilitates receptor

dimerization as reported in a follow-up study (70). A logical extension of this work would be to identify the mechanism that induces NEU1 activity in response to TLR- ligand stimulation. An insight related to this study came from the work by Jayanth et al

(2010) who reported a similar role of NEU1 in the activation of the neurotrophin receptor

TrkA, which they identified MMP-9 as an enzyme that triggers TrkA receptor phosphorylation by inducing NEU1 sialidase activity (89). In this thesis, therefore, we predict that a similar role for MMP-9 in the activation of TLR may exist. Also, we investigated further to identify a novel role for GPCR in the TLR signaling paradigm.

Since studies on TLR-deficient mice indicated a significant role played by these innate receptors in multiple pathologic conditions, our findings may provide an important pioneering approach to disease intervention strategies.

1.6.1 Overall hypothesis GPCR regulates Toll-like receptor activation and signaling through NEU1 sialidase and matrix metalloproteinase 9 crosstalk.

1.6.2 Aims of this study

1- To identify the mechanism of LPS-induced sialidase activity that mediates cell

surface TLR signaling

2- To investigate the mechanism of TLR4 transactivation by GPCR

3- To determine if intracellular TLR activation is regulated by NEU1-MMP-9

crosstalk similar to cell surface TLR.

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Chapter 2

Neu1 Sialidase and Matrix Metalloproteinase-9 Cross-talk Is Essential

for Toll-like Receptor Activation and Cellular Signaling

2.1 Abstract The signaling pathways of mammalian Toll-like receptors (TLRs) are well characterized, but the precise mechanism(s) by which TLRs are activated upon ligand binding remains poorly defined. Recently, we reported a novel membrane sialidase-controlling mechanism that depends on ligand binding to its TLR to induce mammalian neuraminidase-1 (Neu1) activity, to influence receptor desialylation, and subsequently to induce TLR receptor activation and the production of nitric oxide and pro-inflammatory cytokines in dendritic and macrophage cells. The α-2,3-sialyl residue of TLR was identified as the specific target for hydrolysis by Neu1. Here, we report a membrane signaling paradigm initiated by endotoxin lipopolysaccharide (LPS) binding to TLR4 to potentiate G protein-coupled receptor (GPCR) signaling via membrane Gαi subunit proteins and matrix metalloproteinase-9 (MMP9) activation to induce Neu1. Central to this process is that a Neu1-MMP9 complex is bound to TLR4 on the cell surface of naive macrophage cells. Specific inhibition of MMP9 and GPCR Gαi-signaling proteins blocks

LPS-induced Neu1 activity and NFκB activation. Silencing MMP9 mRNA using lentivirus MMP9 shRNA transduction or siRNA transfection of macrophage cells and

MMP9 knock-out primary macrophage cells significantly reduced Neu1 activity and

NFκB activation associated with LPS-treated cells. These findings uncover a molecular organizational signaling platform of a novel Neu1 and MMP9 cross-talk in alliance with

TLR4 on the cell surface that is essential for ligand activation of TLRs and subsequent cellular signaling.

2.2 Introduction The mammalian Toll-like receptors (TLRs) are one of the families of sensor receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of microbial infections for innate immune cells; they play important roles in the pathophysiology of infectious, inflammatory, and autoimmune diseases. Thus, the intensity and duration of TLR responses with these diseases must be tightly controlled. It follows that the structural integrity of TLR receptors, their ligand interactions, and their signaling components are important for our understanding of subsequent immunological responses.

Although the signaling pathways of TLR sensors are well characterized, the parameters controlling interactions between TLRs and their ligands have remained poorly defined until now. We have recently identified a novel paradigm of TLR activation by its natural ligand, which has not been observed previously (1). This paradigm suggests that ligand- induced TLR activation is tightly controlled by Neu1 activation. The data indicate that

Neu1 is already in complex with either TLR2, -3, or -4 receptors and is induced upon ligand binding to their respective receptors. In addition, activated Neu1 specifically hydrolyzes a-2,3- sialyl residues linked to β-galactosides, which are distant from ligand binding. This desialylation process is proposed to remove steric hindrance to TLR4 dimerization, MyD88-TLR4 complex recruitment, NFκB activation, and proinflammatory cell responses (1, 2). What we do not yet understand is how Neu1 is activated following TLR ligand binding. We know that the neuraminidase inhibitor,

Tamiflu (oseltamivir phosphate), specifically inhibits TLR ligand-induced Neu1 activity 40

on the cell surface of macrophage and dendritic cells and subsequently blocks TLR ligand-induced NFκB activation, nitric oxide (NO) production, and proinflammatory cytokines (1).

An insight into the mechanism of TLR ligand-induced Neu1 activity came from our recent report on Neu1 and matrix metalloproteinase-9 (MMP9) cross-talk in regulating nerve growth factor (NGF) TrkA receptors (3). The report disclosed a receptor signaling paradigm involving an NGF-induced G protein-coupled receptor (GPCR)-signaling process via Gαi proteins and MMP9 activation in inducing Neu1. This tripartite complex of G proteins, MMP9, and Neu1 forms an alliance with TrkA at the ectodomain on the cell surface. Active Neu1 in complex with TrkA hydrolyzes α-2,3-sialyl residues on the receptors, enabling the removal of steric hindrance to receptor association and allowing subsequent dimerization, activation, and cellular signaling.

This report describes the key players involved in the rapid propagation of a TLR ligand- induced Neu1 activity in TLR- expressing cells. Neu1 and MMP9 in complex with TLR receptors at the ectodomain form a molecular organizational signaling platform on the cell surface that is essential for ligand activation of TLR receptors and cellular signaling.

2.3 Experimental Procedures

2.3.1 Cell Lines Mouse BMC-2 and BMA macrophage cells (4) and mouse DC2.4 dendritic cells (5) were obtained from Dr. Ken L. Rock (University of Massachusetts Medical School, Worcester,

MA). Stable HEK-TLR cells were obtained by calcium phosphate transfection of a

pCDNA3 expression vector for a specific chimeric TLR with an in-frame COOH- terminal YFP and selection in 0.4 µg/ml G418. The HEK-TLR4/MD2 cell line was generated by additional co-transfection of an expression plasmid for human MD2. All cells were grown at 37 °C in 5% CO2 in culture medium containing Dulbecco’s modified

Eagle’s medium (DMEM) (Invitrogen) supplemented with 10% fetal calf serum (FCS)

(HyClone, Logan, UT).

THP-1 cells, a promonocytic cell line, were transfected with a plasmid containing CD14 cDNA sequences (CD14-THP1) as described elsewhere (6). CD14-THP1 cells were cultured in Iscove’s modified Dulbecco’s medium (Sigma) supplemented with 10% FCS and selection in 100 µg/ml G418.

RAW-BlueTM cells (Mouse Macrophage Reporter Cell Line, InvivoGen, San Diego, CA) derived from RAW 264.7 macrophages were grown in culture medium containing Zeocin as the selectable marker. They stably expressed a secreted embryonic alkaline phosphatase (SEAP) gene inducible by NFκB and AP-1 transcription factors. Upon stimulation, RAW-BlueTM cells activated NFκB and/or AP-1, leading to the secretion of

SEAP, which is detectable and measurable using QUANTI-BlueTM, a SEAP detection medium (InvivoGen). RAW-BlueTM Cells are resistant to ZeocinTM and G418 in the conditioned medium.

2.3.2 Mouse Models Wild-type (WT) and MMP9 knock-out (KO) C57Bl/6 and 129 mice were obtained from

Dr. V. Wee Yong (University of Calgary). Genotyping of the MMP9 KO mice revealed

that the active site (88-kDa) domain was deleted but that there appeared to be a pro-

MMP9 (105-kDa) protein fragment that was still made. Gelatin zymography showed that there is no MMP9 activity in extracts from these mice (V. Wee Yong, personal communication).

2.3.3 Primary Mouse Bone Marrow Macrophage Cells Bone marrow (BM) cells were flushed from femurs and tibias of mice with sterile Tris- buffered saline (TBS) solution. The cell suspension was centrifuged for 3 min at 900 rpm, and the cell pellet was resuspended in red cell lysis buffer for 5 min. The remaining cells were washed once with sterile TBS and then resuspended in RPMI conditioned medium supplemented with 10% FCS and 20% (v/v) L929 cell supernatant as a source of monocyte colony- stimulating factor (M-CSF) according to Alatery and Basta (7) and 1X

L-glutamine/penicillin/streptomycin (Sigma-Aldrich) in sterile solution. The primary BM macrophages were grown on 12-mm circular glass slides in RPMI conditioned medium for 7– 8 days in a humidified incubator at 37 °C and 5% CO2. By day 7, these primary macrophage cells are more than 95% positive for macrophage marker F4/80 molecule as detected by flow cytometry (7).

2.3.4 Silencing MMP9 mRNA Using Lentivirus MMP9 shRNA MMP9 shRNA (mouse) transduction-ready lentiviral particles (Santa Cruz

Biotechnology, Inc., Santa Cruz, CA) contain three target-specific constructs that encode

19 –25-nt (plus hairpin) shRNA designed to knock down gene expression. Briefly, BMA macrophage cells were cultured in 6-well tissue culture plates in DMEM containing 10% 43

FCS and 5 µg/ml plasmocin. After 24 h, medium was discarded, and 2 ml of 5 µg/ml

Polybrene medium was added to the cells followed by MMP9 shRNA lentiviral particles at a multiplicity of infection of 6. The plate was mixed, centrifuged at 2500 rpm for 90 min, and incubated at 37 °C in a 5% CO2 humidified incubator for 24 h. The cells were washed and recultured in medium for an additional 2 days. On day 5, the medium was replaced with selection medium containing optimal 2 µg/ml puromycin as predetermined in a cell viability assay. Selection medium was added every 40 h to expand puromycin resistance MMP9 shRNA-transduced BMA cell clones.

2.3.5 Silencing MMP9 mRNA Using MMP9 siRNA MMP9 siRNA (mouse) duplex components were obtained from Santa Cruz

Biotechnology, Inc. (sc-29401) containing a pool of three target-specific 20 –25-nt siRNAs designed to knock down gene expression. RAW-Blue cells were plated in a 6- well plate at 5 X 105 cells/well and incubated at 37 °C for 24 h. In serum-free medium, 10

µl of 40 µM siRNA duplex in a total volume of 250µl of medium was mixed with 10 µl of Lipofectamine 2000 (Invitrogen) for 20 min at room temperature. During the 20-min incubation, medium was aspirated from the wells containing the cells and replaced with

500 µl of fresh serum-free medium containing siRNA duplex together with

Lipofectamine 2000 complexes to each well and gently mixed by rocking the plate for 4h at 37 °C in a humidified 5% CO2 incubator. To each well was added 1.5 ml of serum-free medium to further incubate the cells at 37 °C in a humidified 5% CO2 incubator for 24 h.

After 24 h, the process of siRNA duplex with Lipofectamine 2000 complexes was

repeated on these same transfected cells. On day 4 of incubation, the serum-free medium was replaced with medium containing 10% FCS and further incubated at 37 °C in a humidified 5% CO2 incubator for 24 h. The transfection efficiency of 90% was determined using fluorescein-conjugated control siRNAs (Santa Cruz Biotechnology,

Inc.) and counting the proportion of labeled cells using fluorescence microscopy (Zeiss

Imager M2). On day 5 following the double siRNA transfection of the RAW-Blue cells, they were subjected to TLR ligand-induced sialidase activity using live WT and MMP9 siRNA RAW-Blue cells.

2.3.6 Ligands and Enzymes TLR4 ligand lipopolysaccharide (LPS; at the indicated concentrations, from Serratia marcescens and purified by phenol extraction; Sigma-Aldrich) and TLR2 ligands zymosan A (from Saccharomyces cerevisiae; Sigma-Al- drich), killed Mycobacterium butyricum (5 µg/ml; Difco), and lipoteichoic acid (LTA; 1 µg/ml; Invitrogen) were used at a predetermined optimal dosage. TLR3 ligand polyinosinic-polycytidylic acid

(poly(I:C); Sigma-Aldrich) was used at the indicated concentrations.

Purified neuraminidase (from Clostridium perfringens; specific activity of 1 unit/1.0 mmol of N-acetylneuraminic acid/ min; Sigma-Aldrich) specifically targets and hydrolyzes α-2,3-, α-2,6-, and α-2,8-sialic acids. Elastase (aqueous suspension from porcine pancreas; Sigma-Aldrich) is a serine protease that hydrolyzes elastin.

2.3.7 Inhibitors Tamiflu (pure oseltamivir phosphate, Hoffmann-La Roche Ltd., Mississauga, Ontario;

Lot number S00060168) was used at the indicated concentrations. Pertussis toxin (PTX; from Bordatella pertussis, in buffered aqueous glycerol solution; Sigma-Aldrich) catalyzes the ADP-ribosylation of the α subunits of the heterotrimeric G proteins Gi, Go, and Gt. This prevents the G protein heterotrimers from interacting with receptors, thus blocking their coupling and activation. Galardin (GM6001; Calbiochem-EMD Chemicals

Inc., Darmstadt, Germany) is a potent, cell-permeable, broad-spectrum hydroxamic acid inhibitor of matrix metalloproteinases (MMPs) (IC50 = 400 pM for MMP1; IC50 = 500 pM for MMP2; IC50 = 27 nM for MMP3; IC50 = 100 pM for MMP8; and IC50 =200 pM for MMP9). Piperazine (PIPZ; MMPII inhibitor; Calbiochem-EMD Chemicals Inc.) is a potent, reversible, broad-range inhibitor of matrix metalloproteinases. PIPZ inhibits

MMP1 (IC50 = 24 nM), MMP3 (IC50 = 18.4 nM), MMP7 (IC50 = 30 nM), and MMP9

(IC50 = 2.7 nM).

MMP3 inhibitor (MMP3i; stromelysin-1 inhibitor; Calbiochem-EMD Chemicals Inc.) inhibits MMP3 (IC50 = 5 nM). MMP9 inhibitor (MMP9i; Calbiochem-EMD Chemicals

Inc.) is a cell-permeable, potent, selective, and reversible MMP9 inhibitor (IC50 = 5 nM).

It inhibits MMP1 (IC50 = 1.05 µM) and MMP13 (IC50 = 113 nM) only at much higher concentrations.

2.3.8 Sialidase Assay TLR-expressing cells were grown on 12-mm circular glass slides in culture medium containing DMEM supplemented with 10% FCS. After removing medium, 2.04 mM 2'-(4-methylumbelliferyl)-a-D-N-acetylneuraminic acid (4-MUNANA) substrate

(Sigma-Aldrich) in Tris-buffered saline, pH 7.4, was added to each slide alone (control), with a predetermined dose of specific ligand, or in combination with ligand and inhibitor at the indicated doses as previously described (8). The substrate is hydrolyzed by sialidase to give free 4-methylumbelliferone, which has a fluorescence emission at 450 nm (blue color) following an excitation at 365 nm. Fluorescent images were taken after

2–3 min using epifluorescent microscopy (Zeiss Imager M2, X40 objective).

2.3.9 OmniMMPTM Assay TLR-expressing cells were grown on 12-mm circular glass slides in culture medium as described above. After removing medium, 0.91 mM OmniMMPTM (Mca- Pro-Leu-Gly-

Leu-Dpa-Ala-Arg-NH2•AcOH fluorogenic substrate (BIOMOL International Inc.,

Plymouth Meeting, PA) in dimethyl sulfoxide (DMSO)/Tris-buffered saline, pH 7.4, was added to each well alone (control), with a predetermined dose of specific ligand or in combination with ligand and 125 nM galardin. When OmniMMPTM is hydrolyzed by

MMP, it has a fluorescence emission at 393 nm following excitation at 328 nm.

Fluorescent images were taken after 1–3 min using epifluorescent microscopy (X40 objective).

2.3.10 Flow Cytometry of Cell Surface TLR4, CD14, and MMP9 Cells were grown in 25-cm2 flasks at 90% confluence. For cell surface staining, live cells in serum-free cold phosphate-buffered saline (PBS) containing 0.1% azide were stained with either fluorescein isothiocyanate (FITC)-conjugated mouse anti- TLR4 antibody, R- phycoerythrin-conjugated anti-CD14 antibody, or rabbit anti-MMP9 (H-129, Santa Cruz

Biotechnology, Inc.) for 15–20 min on ice, washed, followed by Alexa Fluor488 F(ab')2 goat anti-rabbit IgG for the primary anti-MMP9. After washing with cold PBS buffer containing azide, the cells were prepared for flow cytometry analysis. 40,000 cells were acquired on a Beckman Coulter (Miami, FL) Epics XL-MCL flow cytometer and analyzed with Expo32 ADC software (Beckman Coulter). For overlay histograms, control cells treated with Alexa Fluor488-conjugated goat anti-rabbit IgG alone are represented by the gray-filled histogram. Cells treated with anti-MMP9 antibody together with Alexa Fluor488 secondary antibody are depicted by the unfilled histogram with the black line. The mean fluorescence for each histogram is indicated for 80% gated cells.

2.3.11 Cell Surface Biotinylation and Western Blot of Immunoprecipitated Biotinylated Proteins BMA macrophage cells (WT) and BMA MMP9 shRNA KD (MMP9 KD) cells were suspended in ice-cold 1X PBS and washed twice. To 25x 106/ml cells was added 80 µl of freshly prepared 10 mM solution of sulfo-NHS- SS-biotin (Thermo Scientific, Pierce) for

30 min and mixing at room temperature. The cells were washed three times with 1X Tris- buffered saline to quench non-reacted biotinylation reagent. Cells were pelleted and lysed in lysis buffer (50 mM Tris, pH 8, 150 mM NaCl, 1% Nonidet P-40, 0.2 mg/ml leupeptin, 48

1% (3-mercaptoethanol, and 1 mM phenylmethanesulfonyl fluoride (PMSF)). For immunoprecipitation, MMP9 and TLR4 in cell lysates from BMA and MMP9 shRNA

KD cells were immunoprecipitated with 0.4, 1, or 4 µg of goat anti-MMP9, 2 µg of rat anti-TLR4 antibodies, or no antibody for 24 h. Following immunoprecipitation, complexes were isolated using protein G magnetic beads, washed three times in buffer

(10 mM Tris, pH 8, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 1% Triton X-100, and

0.2 mM sodium orthovanadate), and resolved by 8% gel electrophoresis (SDS-PAGE).

Proteins were transferred to polyvinylidene fluoride (PVDF) transfer membrane blot. The blots were probed with streptavidin-horseradish peroxidase (HRP) followed by Western

Lightning Chemiluminescence Reagent Plus. The chemiluminescence reaction was analyzed with x-ray film.

2.3.12 Immunocytochemistry of NFκB and IKBα TLR-expressing cells were pretreated with pure Tamiflu, galardin, or PTX at the indicated concentrations for 30 min followed by a pre-determined dose of specific ligand for 45 min. Cells were fixed, permeabilized, and immunostained with rabbit anti-

NFκBp65 (Rockland) or rabbit anti-IKBa (Rockland) antibodies followed by Alexa

Fluor594 goat anti-rabbit IgG. Stained cells were visualized by epifluorescence microscopy using a x40 objective. Quantitative analysis was done by assessing the density of cell staining corrected for background in each panel using Corel Photo Paint

8.0 software. Each bar in the figures represents the mean corrected density of staining ±

S.E. for all cells (n) within the respective images. p values represent significant

differences at 95% confidence using Dunnett’s multiple comparison test compared with control (Ctrl) in each group.

2.3.13 NFκB-dependent SEAP Assay Briefly, a cell suspension of 550,000 cells/ml in fresh growth medium was prepared, and

180 µl of cell suspension (~100,000 cells) were added to each well of a Falcon flat bottom 96-well plate (BD Biosciences). After different incubation times, 1.5 µg/ml LPS was added to each well together with different concentrations of either MMP9i, Tamiflu, or caffeic acid phenethyl ester (a known inhibitor of NFκB). The plates were incubated at

37 °C in a 5% CO2 incubator for 18 –24 h. A QUANTI-BlueTM (InvivoGen) solution, which is a detection medium developed to determine the activity of any alkaline phosphatase present in a biological sample, was prepared following the manufacturer’s instructions. Briefly, 180 µl of resuspended QUANTI-Blue solution were added to each well of a flat bottom 96-well plate, followed by 20 µl of supernatant from stimulated

RAW-Blue cells. The plate was incubated for 30 min to 3 h at 37 °C, and the SEAP levels were determined using a spectrophotometer at 620 – 655 nm. Each experiment was performed in triplicates.

2.3.14 Co-immunoprecipitation BMA macrophage cells were left cultured in medium or in medium containing 5 µg/ml

LPS for the indicated time intervals. Cells (1 X 107 cells) were pelleted and lysed in lysis buffer (50 mM Tris, pH 8, 150 mM NaCl, 1% Nonidet P-40, 0.2 mg/ml leupeptin, 1% (3- mercaptoethanol, and1 mM PMSF). For immunoprecipitation, Neu1, MMP9, and TLR4 50

in cell lysates from BMA cells were immunoprecipitated with 1.0 µg of rabbit anti-Neu1,

1.0 µg of rabbit anti-MMP9, or 2 µg of rat anti-TLR4 antibodies for 24 h. Following immunoprecipitation, complexes were isolated using protein A or G magnetic beads, washed three times in buffer (10 mM Tris, pH 8, 1 mM EDTA, 1 mM EGTA, 150 mM

NaCl, 1% Triton X-100, and 0.2 mM sodium orthovanadate), and resolved by 8% gel electrophoresis (SDS-PAGE). Proteins were transferred to a PVDF transfer membrane blot. The blots were probed for either MMP9 (78 or 84 kDa active) with anti-MMP9 (H-

129, Santa Cruz Biotechnology, Inc.), Neu1 (45.5 kDa) with anti-Neu1 (H-300, Santa

Cruz Biotechnology), or TLR4 (88 kDa) with anti-TLR4 (HTS510, Santa Cruz

Biotechnology, Inc.) followed by HRP-conjugated secondary IgG antibodies or Clean-

Blot IP Detection Reagent for immunoprecipitation/Western blots (Pierce, Thermo Fisher

Scientific) and Western Lightning Chemiluminescence Reagent Plus. The chemiluminescence reaction was analyzed with x-ray film. Sample concentration for gel loading was determined by the Bradford reagent (Sigma-Aldrich).

2.3.15 MMP9 Colocalization with TLR4 BMA macrophage cells were cultured in DMEM medium with 10% FCS. Cells were treated with 5 µg/ml LPS for 5, 15, 30, and 45 min or left untreated as controls. Cells were fixed, permeabilized, and immunostained with rat anti-mouse TLR4 (HTS510,

Santa Cruz Biotechnology, Inc.) and rabbit anti-mouse MMP9 (H-129, Santa Cruz

Biotechnology, Inc.) followed by Alexa Fluor594 goat anti-rabbit IgG or Alexa Fluor488 rabbit anti-rat IgG. Stained cells were visualized using a confocal inverted microscope

(Leica TCS SP2 MP inverted confocal microscope) with a x100 oil objective. Images were captured using a z-stage of 8 –10 images/cell at 0.5-mm steps and were processed using ImageJ version 1.38x software (National Institutes of Health, Bethesda, MD). To calculate the amount of colocalization in the selected images, the Pearson correlation coefficient was measured and expressed as a percentage using ImageJ version 1.38x software.

2.3.16 MMP9 Colocalization with Neu1 HEK-TLR4/MD2 cells were cultured in DMEM with 10% FCS and 100 µg/ml G418 selection reagent. Cells were treated with 5 µg/ml LPS for 5, 15, 30, and 45 min or left untreated as controls. Cells were fixed, non-permeabilized, and immunostained with rabbit anti-Neu1 (H-300, Santa Cruz Biotechnology, Inc.) and goat anti-MMP9 (C-20,

Santa Cruz Biotechnology, Inc.) followed by Alexa Fluor568 rabbit anti-goat IgG or

Alexa Fluor488 donkey anti- rabbit IgG. Stained cells were visualized using a confocal inverted microscope (Leica TCS SP2 MP inverted confocal microscope) with a x100 oil objective. Images were captured using a z-stage of 8 –10 images/cell at 0.5-mm steps and were processed using ImageJ version 1.38x software. To calculate the amount of colocalization in the selected images, the Pearson correlation coefficient was measured and expressed as a percentage using ImageJ version 1.38x software.

2.3.17 Statistics Comparisons between two groups were made by one-way analysis of variance at 95% confidence using Student’s unpaired t test and Bonferroni’s multiple comparison test or

Dunnett’s multiple comparison test for comparisons among more than two groups.

2.4 Results

2.4.1 Tamiflu, Pertussis Toxin, and Galardin Block Neu1 Activity Associated with LPS Binding to TLR4 in Live HEK-TLR4/MD2 Cells Reports have suggested that GPCRs (9, 10) and the specific induction of MMP (11, 12) play important roles in regulating TLR-mediated macrophage function. Other studies have demonstrated that PAR2 (proteinase-activated receptor-2), GPCR, and TLR4 are physically associated and that co-expression of TLR4 and PAR2 enhances NFκB signaling (13). The TLR4-associated CD14 protein has been shown to co- immunoprecipitate with G protein subunits (14), and CD14 can associate with TLR4 in lipid membrane rafts (15). Therefore, it is possible that there might be a Neu1 connection with GPCR signaling and MMPs in alliance with TLR4 as described previously for NGF

TrkA receptors (3). It is also known that an elastin receptor complex, a tripartite of elastin-binding protein (EBP) (16, 17) complexed to Neu1 and cathepsin A (18) is able to transduce signals through the catalytic activity of its Neu1 subunit (19). Accordingly, we propose that MMPs with metallo-elastase activity are required to remove EBP complexed to Neu1 and cathepsin A to activate Neu1. Furthermore, it is well known that agonist-bound GPCRs have been shown to activate numerous MMPs (20), including

MMP3 (21) and MMP2 and -9 (22, 23), as well as members of the ADAM family of metalloproteases: ADAM10, ADAM15, and ADAM17 (24, 25). The precise molecular mechanism(s) underlying GPCR- mediated MMP activation still remains unknown.

To test whether GPCR-mediated MMP activation plays a role in Neu1 activation associated with TLR ligand-stimulated macrophages, we initially asked whether galardin

(GM6001), a broad specific inhibitor of MMP1, -2, -3, -8, and -9, and PTX, a specific inhibitor of Gi2 and Gi3 (α subunits) of G protein subtypes, would have an inhibitory effect on Neu1 activity associated with LPS-induced live HEK-TLR4/MD2 cells. Here, we used a recently developed assay to detect sialidase activity on the surface of viable cells (1, 3, 8, 26, 27). This sialidase activity is revealed in the periphery surrounding the cells using a fluorogenic sialidase-specific substrate, 4-MUNANA, whose cleavage product 4-methylumbelliferone fluoresces at 450 nm. The data in Figure 2.1A clearly show this to be the case. The neuraminidase inhibitor Tamiflu (250 µg/ml), pertussis toxin (33.3 ng/ml), and galardin (125 nM) blocked the sialidase activity associated with

LPS-treated live HEK-TLR4/ MD2 cells compared with the LPS-positive control. The mean fluorescence surrounding the cells for each of the images was measured using

ImageJ software (Figure 2.1A). These results are consistent with our previous report for these compounds in inhibiting NGF-induced Neu1 activity in live TrkA-expressing cells

(3). As predicted, they suggest that GPCR Gαi-sensitive proteins and MMP(s) play a role in the sialidase activity associated with LPS-stimulated live HEK- TLR4/MD2 cells.

Figure 2.1: LPS-induced sialidase activity is blocked by specific GPCR Gαi subunit and MMP inhibitors in live HEK-TLR4/MD2 cells. Cells were incubated on 12-mm circular glass slides in conditioned medium for 24 h at 37 °C in a humidified incubator. After removal of medium, 0.2mM 4-MUNANA (4-MU) substrate in Tris- buffered saline, pH 7.4, was added with mounting medium to cells alone (Control), with 5 µg/ml LPS, or (A) with LPS in combination with 250 µg/ml Tamiflu, 500 nM galardin, or 25 ng/ml PTX. Fluorescent images were taken at 1-min intervals using epifluorescent microscopy ( x40 objective). The mean fluorescence surrounding the cells for each of the images was measured using ImageJ software. The data are a representation of one of six independent experiments showing similar results. Error bars, S.E. (B) LPS-induced sialidase activity in live BMC-2 macrophages is inhibited by galardin and piperazine in a dose-dependent manner. After removing medium, 0.2 mM 4-MUNANA substrate in Tris-buffered saline, pH 7.4, was added to cells alone (CTRL), with 5 µg/ml LPS, or with LPS in combination with galardin (GM6001) or piperazine (PIPZ, MMP inhibitor II) at the indicated concentrations. Fluorescent images were taken at 1 min after adding substrate using epifluorescent microscopy (x40 objective). The IC50 of each compound was determined by plotting the decrease in sialidase activity against the log of the agent concentration. The data are a representation of one of three independent experiments showing similar results. (C) MMP9i but not MMP3i blocks LPS-induced sialidase activity in BMC-2 macrophages. Images are as described in A. Data analyses are as described in B. The data are a representation of one of three independent experiments showing similar results.

Partial data taken from S.R. Amith’s PhD dissertation, Queen’s University, Kingston, Ontario 2009

Next, we tested whether MMP inhibitors would inhibit Neu1 activity associated with

LPS-treated live BMC-2 macrophage cells. As expected, LPS-induced sialidase activity was blocked by the MMP inhibitors galardin and PIPZ at 0.7 and 2 µM, respectively

(Figure 2.1B), but also in a dose-dependent manner. To further elucidate the inhibitory capacity of galardin and piperazine, the 50% inhibitory concentration (IC50) of each of the compounds was determined by plotting the decrease in sialidase activity against the log of the agent concentration. As shown in Figure 2.1B, galardin and piperazine inhibited Neu1 activity associated with LPS-treated live BMC-2 cells with an IC50 of 26 and 79 pM, respectively.

The 67-kDa EBP, identical to the spliced variant of β-galactosidase, acts as a recyclable chaperone that facilitates secretion of tropoelastin (17). This EBP also forms a cell surface-targeted molecular complex with protective protein cathepsin A and Neu1 in the lysosome (18). The evidence indicates that Neu1 activity is a prerequisite for the subsequent release of tropoelastin (16). Accordingly, MMPs with metallo-elastase activity are required to remove EBP (16, 17) forming a complex to Neu1 and cathepsin A

(18) to activate Neu1. We hypothesize that LPS binding to TLR4 receptors on the cell surface potentiates GPCR signaling via GPCR Gαi subunit proteins, leading to MMP9 activation. To determine whether MMP9 is associated with LPS-treated live macrophage cells, we used the MMP9i as well as MMP3i for their inhibitory effects on LPS-induced sialidase activity in live BMC-2 cells. The data in Figure 2.1C clearly show that MMP9i

(IC50 = 0.032 µg/ml) but not MMP3i (IC50 > 1000 µg/ml) blocked the sialidase activity associated with LPS-treated live BMC-2 macrophage cells.

2.4.2 Matrix Metalloproteinase Activity Is Associated with Zymosan A (TLR2 Agonist), poly(I:C) (TLR3 Agonist), and LPS (TLR4 Agonist) Treatment of Live BMC-2 Cells To confirm that MMP activity is associated with TLR ligand treatment of live BMC-2 macrophage cells, we used OmniMMPTM fluorogenic substrate (Mca-Pro-Leu-Gly-Leu-

Dpa-Ala-Arg-NH2•AcOH) in the live cell assay to detect MMP activity. The cells were allowed to adhere on 12-mm circular glass slides in medium containing 10% fetal calf serum for 24 h at 37 °C. After removing medium, 0.91 mM OmniMMPTM fluorogenic substrate was added to live cells alone (control) and in combination with either TLR2 ligand zymosan A, TLR3 ligand poly(I:C), or TLR4 ligand LPS or in combination with

LPS and 125nM galardin (GM6001). The OmniMMPTM substrate is hydrolyzed by

MMPs at the Gly-Leu sequence, releasing the Mca group. The Dpa group quenches the

Mca fluorescence. Mca has a fluorescence emission at 393 nm following excitation at

328nm. Fluorescent images were taken at 2 min after adding OmniMMPTM substrate using epi-fluorescent microscopy (x40 objective). The data in Figure 2.2 A show this to be the case. Zymosan A, poly(I:C), and LPS induced MMP activity on the cell surface of live BMC-2 macrophage cells after 2 min. Galardin significantly inhibited the MMP activity associated with LPS-treated live cells.

Figure 2.2: Zymosan A, poly(I:C), and LPS induce MMP activity in live BMC-2 cells, which is inhibited by galardin. (A) BMC-2 macrophage cells were allowed to adhere on 12-mm circular glass slides in medium containing 10% fetal calf serum for 24 h at 37 °C in a humidified incubator. After removing medium, 0.91 mM OmniMMPTM fluorogenic substrate (Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg- NH2•AcOH) in 20 µg/ml DMSO was added to cells alone (Control) or in combination with either 66.7 µg/ml zymosan A, 6.7 µg/ml poly (I:C), 1 µg/ml LPS, or LPS in combination with 125 nM galardin (GM6001). The OmniMMPTM substrate is hydrolyzed by MMP enzymes. Mca fluorescence is quenched by the Dpa group. MMP cleaves Gly-Leu of the OmniMMPTM substrate, releasing the Mca residue. Mca has a fluorescence emission at 393 nm following excitation at 328 nm. Fluorescent images were taken at 1 min after adding substrate using epifluorescent microscopy (x40 objective). The mean fluorescence for each of the images was measured using ImageJ software. The data are a representation of one of three independent experiments showing similar results. Error bars, S.E. (B) exogenous elastase induces sialidase activity in DC2.4 dendritic cells. Cells were grown on 12-mm circular glass slides in medium containing 10% fetal calf serum for 24 h at 37 °C in a humidified incubator. After removing medium, 2.04 mM 4-MUNANA substrate was added to each well alone (Control) or with 100 µg/ml pure elastase. Fluorescent images were taken at 1 min after adding substrate using epifluorescent microscopy (x40 objective). A positive control sialidase (from C. perfringens; specific activity of 1 unit/1.0 mmol of N-acetylneuraminic acid/min) or pure elastase. (C) was added to 2.04 mM 4-MUNANA substrate alone. Fluorescent images were taken at 1 min after adding substrate using epifluorescent microscopy (x40 objective). Partial data taken from S.R. Amith’s PhD dissertation, Queen’s University, Kingston, Ontario 2009. 59

Because an MMP with elastase activity may be required to cleave EBP from the elastin receptor complex of Neu1-cathepsin A-EBP to activate Neu1, we asked whether treating live cells with an exogenous elastase would induce sialidase activity in the absence of TLR ligands. Indeed, this was the case, as seen in Figure 2.2 B. When purified elastase was added to live DC2.4 dendritic cells in the presence of 4-MUNANA, sialidase activity was observed. This elastase-induced sialidase activity was not observed when the enzyme was added to substrate in the absence of cells in comparison with the exogenous

α-2,3-neuraminidase from Streptococcus pneumoniae as a positive control (Figure 2.2 C).

2.4.3 Gαi-sensitive Pertussis Toxin, MMP Inhibitor Galardin, and Tamiflu Block TLR4 Ligand LPS and TLR2 Ligand Zymosan A-induced NFκB Activation

If GPCR signaling via membrane Gαi proteins and MMP activation are important for

TLR signaling, inhibitors against these proteins should block LPS- or zymosan A- induced NFκB activation in TLR-expressing cells. Immunocytochemistry analyses shown here demonstrate that PTX, galardin, and Tamiflu significantly blocked LPS-induced

NFκBp65 activation in DC2.4 dendritic cells (Figure 2.3 A) and BMC-2 macrophage cells (Figure 2.3 B). The inhibitors blocked zymosan A-induced NFκBp65 activation in

HEK-TLR2 (Figure 2.3 C) cells as well as LPS-induced NFκBp65 activation in HEK-

TLR4/MD2 (Figure 2.3 D) with a loss of IκBα within 15 min. The anti-NFκBp65 (Rel A) antibody used here detects activated and non-activated NFκB. In these experiments, we also used anti-IκBα antibody in conjunction with anti-NFκBp65 (Rel A) antibody as an internal control for NFκB activation. IκBα binds to the p65 subunit, preventing nuclear

Figure 2.3: LPS-induced NFκB activation and IκB degradation (A) DC2.4 cells were pretreated with 200 µM Tamiflu, 500 nM galardin, 100 ng/ml PTX for 30 min followed by 3 µg/ml LPS for 15 min. Cells were fixed, permeabilized, and immunostained with rabbit anti-NFκBp65 or rabbit anti-IκBα followed by Alexa Fluor594 rabbit anti-goat IgG. Stained cells were visualized by epifluorescence microscopy using a x40 objective. Approx- imately 95% of LPS-treated cells immunostained with NFκBp65 had nuclear staining. Quantitative analysis was done by assessing the density of cell staining corrected for background in each panel using Corel Photo Paint 8.0 software. Each bar in the graphs represents the mean corrected density of staining ± S.E. (error bars) for all cells within the respective images. The control group was immunostained with only Alexa Fluor594 secondary goat anti-rabbit IgG. p values represent significant differences at 95% confidence using the Dunnett multiple comparison test compared with LPS-treated cells. The data are a representation of one of three independent experiments showing similar results. (B) LPS-induced NFκB activation and IκB degradation in BMC-2 macrophage cells. Cells were cultured and treated as described in A. (C) zymosan A-induced NFκB activation and IκB degradation in HEK-TLR2 cells. Cells were pretreated with 200 µM Tamiflu or 500 nM galardin for 30 min followed by 66.7 µg/ml zymosan A for 15 min. Cells were fixed, permeabilized, and immunostained as described in A. (D) LPS- induced NFκB activation and IκB degradation in HEK-TLR4/MD2 cells. Cells were cultured and treated as described in A. Partial data taken from S.R. Amith’s PhD dissertation, Queen’s University, Kingston, Ontario 2009.

localization and DNA binding. Activation of NFκB releases IκBα bound to p65 subunit, and it becomes degraded. Collectively, cells treated with their respective ligands showed a strong immunofluorescent signal for NFκB that was localized to the nucleus with a concomitant loss of IκB, whereas in the presence of Tamiflu, PTX, and galardin, the

NFκB signal was significantly weak or not present with no loss of IκB.

2.4.4 LPS-induced Phosphorylation of NFκB The inhibitory effect of MMP9i on LPS-induced NFκB phosphorylation was also examined in BMC-2 macrophage cells. Optimal activation of NFκB requires phosphorylation in the transactivation domain of p65. This transactivation domain of p65 subunit is responsible for the interaction with the inhibitor IκB and contains the phosphorylation sites. A phospho-specific polyclonal antibody against the human

NFκBp65 Ser(P)529 (pNFκB) that has minimal reactivity with non-phosphorylated p65 was used here. This phospho-specific antibody reacts with a peptide sequence

(PNGLLpSGDEDFC) corresponding to a region near phosphoserine 529 of the human

NFκBp65 (Rel A) protein. The data in Figure 2.4 A show that MMP9i inhibited LPS- induced pNFκB in a dose-dependent manner in these cells to the no ligand control levels compared with the LPS-positive control.

Figure 2.4: Endotoxin LPS induces phosphorylated NFκBp65 Ser(P)529 (pNFκB) in BMC-2 and BMA macrophage cells. (A) Cells were cultured on circular glass slides in 24-well tissue culture plates in medium containing 10% fetal calf serum for 24 h at 37 °C in a humidified incubator. Cells were pretreated with the indicated concentrations of MMP9i, in combination with 10 µg/ml LPS for 15 min or left untreated (no ligand). Cells were fixed, permeabilized, and immunostained with phospho-specific polyclonal rabbit antibody against the human NFκBp65 Ser(P)529 (pNFκB), followed by Alexa Fluor594 goat anti-rabbit IgG. Stained cells were visualized by epifluorescence microscopy using a x40 objective. Quantitative analysis was done by assessing the density of cell staining corrected for background in each panel using Corel Photo Paint 8.0 software. Each bar in the graphs represents the mean corrected density of staining ± S.E. (error bars) for all cells within the respective images. The data are a representation of one of five independent experiments showing similar results. (B) Western blot analyses of phosphorylated NFκB (Ser(P)311) in nuclear lysates. BMA macrophage cells were pretreated with 100 µg/ml MMP9i or 200 µM Tamiflu for 30 min followed by 5 µg/ml LPS. Nuclear lysates from the cells were separated by SDS-PAGE, and the blot was probed with phospho-specific polyclonal rabbit antibody against NFκBp65 Ser(P)311 with minimal reactivity with non-phosphorylated p65. Specific NFκBp65 Ser(P)311 blocking peptide was added to the anti-NFκBp65 Ser(P)311 antibody in probing the blot. MCM2 (highly conserved minichromosome maintenance complex protein-2) was used as an internal control protein for loading of the nuclear lysate. Cytoplasmic cell lysates from the same samples were separated by SDS-PAGE, and the blot was probed with anti-IκB antibody. Specific IκB blocking peptide was added to the anti-IκB antibody in probing the blot. (β-actin was used as an internal control protein for loading of the cytoplasmic cell lysate. Quantitative analysis was done by assessing the density of a band corrected for background in each lane using Corel Photo Paint 8.0 software. Each bar in the graphs represents the mean ratio of NFκBp65 Ser(P)311 to MCM2 or mean ratio of IκB to (3-actin of band density ± S.E. (error bars) for 5–10 replicate measurements. The data are a representation of one of three independent experiments showing similar results. (C) Western blot analyses of IκB in cell lysates. BMC-2 macrophage cells were pretreated with 200 µM Tamiflu for 30 min followed by 5 µg/ml LPS. Cell lysates from the cells were separated by SDS-PAGE and the blot probed with anti-IκB antibody. (β-actin was used as an internal control protein for loading of the cytoplasmic cell lysate. Quantitative analysis was done by assessing the density of a band corrected for background in each lane using Corel Photo Paint 8.0 software. Each bar in the graphs represents the mean ratio of IκB to β-actin of band density ± S.E. (error bars) for 5–10 replicate measurements. The data are a representation of one of three independent experiments showing similar results.

To further support these immunocytochemistry results, we also performed Western blots to detect phosphorylated NFκB (pNFκB-S311) in the nuclear cell extracts as well as IκB in the cytoplasmic cell lysates from untreated medium control, LPS-treated for 15 min, and LPS-stimulated in combination with MMP9i (195.5 µM) or Tamiflu (200 µM) in

BMA macrophage cells. The data in Figure 2.4 B clearly show this to be the case. Cells treated with MMP9i and Tamiflu inhibited LPS-induced pNFκB in the nuclear lysates compared with the LPS-treated control and the DNA replication licensing factor MCM2.

MCM2 is a protein encoded by the MCM2 gene and is one of the highly conserved minichromosome maintenance (MCM) proteins that are involved in the initiation of eukaryotic genome replication. The specificity of the anti-pNFκB-S311 antibody was confirmed using its specific pNFκB-S311 blocking peptide (Figure 2.4 B). In addition,

Western blot analyses of the BMA (Figure 2.4 B) and BMC-2 (Figure 2.4 C) cell lysates for IκBα revealed a diminution of IκBα expression in LPS-treated cells, which was reversed by prior treatment with MMP9i and Tamiflu.

To confirm MMP9 linkage with LPS-induced NFκB activation, we performed the NFκB- dependent SEAP assay. RAW- Blue cells are murine macrophages that stably express a

SEAP gene inducible by NFκB and AP-1 transcription factors, leading to the secretion of

SEAP. Secreted SEAP is detectable and measurable using QUANTI-Blue substrate.

Here, we used MMP9i as well as MMP3i to determine their inhibitory effects on LPS- induced sialidase activity in live RAW-Blue cells. The data in Figure 2.5 A and B, clearly

Figure 2.5: MMP9i significantly inhibits LPS-induced sialidase activity in live RAW-Blue macrophage cells in a dose-dependent manner. (A) LPS-induced sialidase activity in RAW-Blue cells was measured as described in the legend to Figure 2.1A. Fluorescent images were taken at 2 min after adding 0.318 mM 4-MUNANA substrate together with LPS and the indicated MMP inhibitors (MMP9i and MMP3i) using epifluorescent microscopy (Zeiss Imager M2, X40 objective). The mean fluorescence surrounding the cells for each of the images was measured using ImageJ Software. The data are a representation of one of three independent experiments showing similar results. (B) the 50% inhibition concentration (IC50) for MMP9i and MMP3i on sialidase activity induced by LPS in live RAW-Blue cells. Cells were treated with 5 µg/ml LPS in the presence or absence of different concentrations of the indicated inhibitors and 0.318 mM 4-MUNANA substrate using epifluorescent microscopy (X40 objective) as described in A. The IC50 of MMP9i compound was determined by plotting the decrease in sialidase activity against the log of the agent concentration. There was no inhibitory effect of MMP3i at 125 µg/ml. (C) Tamiflu and MMP9i inhibit NFκB- dependent SEAP activity. SEAP reporter-expressing RAW-Blue cells were treated with different doses of Tamiflu, MMP inhibitor, or caffeic acid phenethyl ester (CAPE; a known inhibitor of NFKB) for 24 h, and SEAP activity in the culture medium was assessed using Quanti-blue substrate. Results are representative of three experiments. Relative SEAP activity was calculated as fold increase of each compound (SEAP activity in medium from treated cells minus no cell background over SEAP activity in medium from untreated cells minus background). The IC50 of each compound was determined by plotting the decrease in SEAP activity against the log of the agent concentration. The data are a representation of one of three independent experiments showing similar results. (D) flow cytometry analysis of MMP9 expression on the cell surface of live RAW-Blue cells. Histograms show staining with rabbit anti-MMP9 antibody after incubation on ice for 15 min followed by Alexa Fluor488-conjugated F(ab')2 secondary goat anti-rabbit IgG for an additional 15 min on ice. Control cells were stained with Alexa Fluor488-conjugated F(ab')2 secondary antibody for 15 min on ice or untreated cells (auto). Cells were analyzed by Beckman Coulter Epics XL-MCL flow cytometry and Expo32 ADC software (Beckman Coulter). Overlay histograms are displayed. Live untreated cells (auto) are represented by a gray-filled histogram. Control Alexa Fluor488 secondary antibody-treated live cells are represented by the unfilled gray dashed line. Live cells stained with antibody against MMP9 are depicted by the unfilled histogram with the black line. The mean channel fluorescence (MCF) for each histogram is indicated for 40,000 acquired cells (80% gated). The data are a representation of one of three independent experiments showing similar results.

show that MMP9i (IC50 = 2 µM) but not MMP3i (IC50 > 316 mM) significantly blocked the sialidase activity associated with LPS-treated live RAW-Blue cells.

The NFκB-dependent SEAP assay shown here clearly demonstrates that MMP9i (IC50 =

0.1 µM) and Tamiflu (IC50 = 2 µM) significantly blocked SEAP activity compared with caffeic acid phenethyl ester, a known inhibitor of NFKB, associated with LPS-treated live

RAW-Blue macrophage cells (Figure 2.5 C). The immunolocalization of MMP9 on the cell surface of these cells was also confirmed by flow cytometry immunostained with anti-MMP9 antibodies followed by Alexa Fluor596-conjugated F(ab')2 secondary antibody. The data shown in Figure 2.5 D clearly indicate that 40,000 acquired live, untreated cells showed significant immunostaining for MMP9 expression on the cell surface of RAW-Blue cells. These findings are consistent with our previous report showing that Tamiflu blocks TLR ligand-induced NFκB activation, LPS-induced nitric oxide (NO) production, and LPS-induced proinflammatory IL-6 and TNFα cytokines (1).

In addition, primary BM macrophages from Neu1-deficient mice do not respond to TLR ligand-induced NFκB activation or NO production (1). We have also shown from EMSA analysis that the nuclear extracts of LPS-treated HEK-TLR4/MD2 cells contained specific NFκB DNA binding activity that was abrogated with Tamiflu (2).

2.4.5 Silencing MMP9 mRNA Using Lentivirus MMP9 shRNA Knockdown BMA macrophage cells were treated with MMP9 shRNA transduction-ready lentiviral particles containing three target-specific constructs that encode 19 –25-nt (plus hairpin) shRNA designed to knock down MMP9 gene expression. MMP9 shRNA-transduced

BMA cell clones (MMP9 KD) were expanded under the selection of puromycin. Western blot analyses of whole cell lysates from two separate preparations of parental BMA (WT) and BMA (MMP9 KD) cells revealed differential knockdowns of three different MMP9 isoforms (Figure 2.6 A). Lentiviral MMP9 shRNA transduction of BMA cells revealed a significant knockdown of the proactive 105-kDa (~50%) and the active 65-kDa (>90%) isoforms of MMP9 but a marginal knockdown of the active 78- or 84-kDa (~28%)

MMP9 isoforms. We also performed Western blots to detect phosphorylated NFκB

(pNFκB-S311) in the cytoplasmic cell extracts from parental (WT) and MMP9 KD BMA cells. There was a ~30% reduction in LPS-induced pNFκB-S311 in cell lysates from

MMP9 KD cells compared with the LPS-stimulated WT cells, (β-actin, and specific pNFκB-S311 blocking peptide (Figure 2.6 B). The WT BMA cells pretreated with

MMP9i (195.5 µM) or Tamiflu (200 µM) followed by LPS stimulation showed a similar reduction in LPS-induced pNFκB-S311. We also tested whether lentiviral MMP9 shRNA transduction of BMA cells had an effect on Neu1 activity in live cells. The data shown in

Figure 2.6 C revealed a significant reduction in sialidase activity associated with LPS- stimulated live BMA MMP9 KD cells compared with the WT BMA cells.

Figure 2.6: Analysis of silencing MMP9 mRNA using lentiviral MMP9 shRNA. (A) MMP9 shRNA (mouse) transduction-ready lentiviral particles (multiplicity of infection = 6) contain three target-specific constructs that encode 19 –25-nt (plus hairpin) shRNA designed to knock down MMP9 gene expression (MMP9 KD). WT and MMP9 KD BMA macrophage cells were cultured in DMEM conditioned selection medium containing 10% FCS, 5 µg/ml plasmocin, and optimal 2 µg/ml puromycin. Cell lysates from untreated cells were separated by SDS-PAGE, and the blot was probed with anti-MMP9 antibody. Quantitative analysis was done by assessing the density of a band corrected for background in each lane using Corel Photo Paint 8.0 software. Each bar in the graphs represents the mean of band density ± S.E. (error bars) for 5–10 replicate measurements. (B) Western blot analysis of LPS-induced phosphorylated NFκB (Ser(P)311) in cytoplasmic cell lysates. WT macrophage cells were pretreated with 100 µg/ml MMP9i or 200 µM Tamiflu for 30 min followed by 5 µg/ml LPS. MMP9 KD BMA cells were treated with 5 µg/ml LPS or left untreated as medium control. Cell lysates from the WT and MMP9 KD BMA cells were separated by SDS-PAGE, and the blot was probed with phospho-specific polyclonal rabbit antibody against NFκBp65 Ser(P)311 with minimal reactivity with non-phosphorylated p65. Specific NFκBp65 Ser(P)311 blocking peptide was added to the anti-NFκBp65 Ser(P)311 antibody in probing the blot. β-actin was used as an internal control protein for loading of the cytoplasmic cell lysate. Quantitative analysis was done by assessing the density of a band corrected for background in each lane using Corel Photo Paint 8.0 software. Each bar in the graphs represents the mean ratio of NFKBp65 Ser(P)311 to β-actin band density ± S.E. (error bars) for 5–10 replicate measurements. The data are a representation of one of three independent experiments showing similar results. (C) LPS-induced sialidase activity in live WT and MMP9 KD BMA macrophage cells. After removing medium, 0.2 mM 4-MUNANA substrate in Tris- buffered saline, pH 7.4, was added to cells alone (control) or with 5 µg/ml LPS. Fluorescent images were taken at 1 min after adding substrate using epifluorescent microscopy (X40 objective). The mean fluorescence surrounding the cells for each of the images was measured using ImageJ software. The data show two separate experiments showing similar results.

2.4.6 Cell Surface Expression of MMP9 Because most studies describing the expression of MMP9 on the cell surface use growth factors or phorbol esters, we questioned whether MMP9 is generally expressed on the cell surface of live, untreated, and LPS-treated CD14-THP-1 human monocytic cells and

BMA macrophage cells. The immunolocalization of MMP9 on the cell surface of these live cells was confirmed by flow cytometry immunostained with anti-MMP9 antibodies followed by Alexa Fluor596-conjugated F(ab')2 secondary antibody. The data shown in

Figure 2.7 A clearly indicate that 40,000 acquired live, untreated cells showed significant immunostaining for MMP9 expression on the cell surface of CD14-THP-1 cells. In addition, LPS treatment of live CD14-THP-1 cells for 5, 15, 30, and 45 min did not have any significant effects on the expression of TLR4 on the cell surface (Figure 2.7 B) compared with the untreated control cells. LPS treatment of live BMA macrophage cells

(see Figure 2.9 B) for 5 min also did not have any effects on the expression of MMP9 on the cell surface compared with the untreated controls.

Figure 2.7: Analysis of MMP9 expression on the cell surface of live human monocytic CD14-THP1 and BMA cells. (A) Flow cytometry histograms show staining with FITC-conjugated anti-TLR4 antibody, R- phycoerythrin (R-PE)-conjugated anti-CD14 antibody, or rabbit anti-MMP9 antibodies after incubation on ice for 15 min followed by Alexa Fluor488-conjugated F(ab')2 secondary goat anti- rabbit IgG for an additional 15 min on ice. Control cells were stained with Alexa Fluor488- conjugated F(ab')2 secondary antibody for 15 min on ice or untreated cells (Auto). Cells were analyzed by Beckman Coulter Epics XL-MCL flow cytometry and Expo32 ADC software (Beckman Coulter). Overlay histograms are displayed. Live untreated cells are represented by a gray-filled histogram. Control Alexa Fluor488 secondary antibody-treated live cells are represented by the unfilled gray dashed line. Live cells stained with antibody against TLR4, CD14, or MMP9 are depicted by the unfilled histogram with the black line. The mean channel fluorescence (MCF) for each histogram is indicated for 40,000 acquired cells (80% gated). (B) flow cytometry analysis of TLR4 expressed on the cell surface of live human monocytic CD14- THP1 cells following LPS treatment for 5, 15, 30, and 45 min as described in A. Overlay histograms are displayed. Live untreated cells (Auto) are represented by a gray-filled histogram. Live cells stained with FITC- conjugated anti-TLR4 are depicted by the unfilled histogram with the black line. Live cells treated with LPS and stained with FITC-conjugated anti-TLR4 are depicted by the unfilled histogram with the gray line. The mean channel fluorescence (MCF) for each histogram is indicated for 40,000 acquired cells (80% gated). (C) immunoprecipitation of MMP9 and Western blot analysis of biotinylated cell surface of WT and shRNA MMP9 KD BMA cells. Cells were left untreated as medium control. Cells were pelleted and lysed in lysis buffer, and the protein lysates were immunoprecipitated with the indicated amount (µg) of anti- MMP9 antibody for 24 h. Immunocomplexes were isolated using protein G magnetic beads and resolved by SDS-PAGE, and the blot was probed with streptavidin-HRP followed by Western Lightning Chemiluminescence Reagent Plus. The data are a representation of one of three independent experiments showing similar results. Error bars, S.E. (D) immunoprecipitation of TLR4 and Western blot analysis of biotinylated cell surface of WT and shRNA MMP9 KD BMA cells. Cells were left untreated as medium control. Cells were pelleted and lysed in lysis buffer, and the protein lysates were immunoprecipitated with anti-TLR4 antibody for 24 h. The same protein lysates were immunoprecipitated with anti-IgG isotype control antibodies. Immunocomplexes were isolated using protein G magnetic beads and resolved by SDS-PAGE, and the blot was probed with streptavidin-HRP followed by Western Lightning Chemiluminescence Reagent Plus. The data are a representation of one of three independent experiments showing similar results.

To further confirm the expression of MMP9 on the cell surface, we performed a biotinylation of the cell surface of live WT and shMMP9 KD BMA cells, immunoprecipitated MMP9 and TLR4 in the cell lysates with specific antibodies, and probed the blots with streptavidin-HRP and Western Lightning Chemiluminescence

Reagent Plus. The data shown in Figure 2.7 C revealed a predominant cell surface expression of the active 88-kDa MMP9 isoform followed by the active 65-kDa MMP9 isoform. The BMA MMP9 KD cells revealed a ~15–20% reduction in the expression of

65- and 88-kDa MMP9 isoforms on the cell surface. As expected, the proactive 105-kDa

MMP9 zymogen was not expressed on the cell surface of either WT or MMP9 KD BMA cells (Figure 2.7 C). In addition, BMA MMP9 KD cells expressed similar amounts of

TLR4 on the cell surface like the WT BMA cells (Figure 2.7 D).

2.4.7 Interference MMP9 Using siRNA Knockdown To further confirm the role of MMP9 in LPS-induced TLR4 activation, we transfected

RAW-Blue macrophage cells with MMP9 siRNA duplexes containing a pool of three target-specific 20–25-nt siRNAs using Lipofectamine 2000 reagent. Western blot analyses of whole cell lysates from WT and siRNA MMP9 KD RAW-Blue cells revealed a complete knockdown of the active 88-kDa isoform of MMP9 compared with the WT results, but there appeared to be a proactive MMP9 (105-kDa) protein fragment that is still present (Figure 2.8 A). Western blot analyses of the WT and siRNA MMP9 KD

RAW-Blue cell lysates revealed a complete deletion of the 50- and 65-kDa pNFκB-S311 protein levels in the cell lysates from LPS-treated siRNA MMP9 KD cells compared with

Figure 2.8: MMP9 siRNA transfection of RAW-Blue macrophage cells. (A) Cells were doubly transfected with siRNA MMP9 using Lipofectamine 2000. The cells were lysed, the lysates from untreated WT and siRNA MMP9 KD cells were separated by SDS-PAGE, and the blot was probed with anti-MMP9 antibody. β-actin was used as an internal control protein for loading of the cytoplasmic cell lysate. The data are a representation of one of three independent experiments showing similar results. (B) Western blot analysis of LPS-induced phosphorylated NFκB (Ser(P)311) in cytoplasmic cell lysates. WT and siRNA MMP9 KD RAW-Blue macrophage cells were stimulated with 5 µg/ml LPS for 45 min or left untreated as medium control. Cell lysates from the WT and siRNA MMP9 KD cells were separated by SDS-PAGE, and the blot was probed with phospho-specific polyclonal rabbit antibody against NFκBp65 Ser(P)311. β-actin was used as an internal control protein for loading of the cytoplasmic cell lysate. The data are a representation of one of three independent experiments showing similar results. (C) LPS-induced sialidase activity in live WT and siRNA MMP9 KD RAW-Blue macrophage cells. After removing medium, 0.2 mM 4-MUNANA substrate in Tris-buffered saline, pH 7.4, was added to cells alone (no ligand control) or with 5 µg/ml LPS. Fluorescent images were taken at 1 min after adding substrate using epifluorescent microscopy (X40 objective). The mean fluorescence surrounding the cells for each of the images was measured using ImageJ software. The data are a representation of one of five independent experiments showing similar results. Error bars, S.E. (D) TLR2 ligands LTA and killed M. butyricum (Myco) and TLR4 ligand LPS induced sialidase activity in live primary BM macrophage cells from WT and MMP9 KO mice. Primary BM macrophage cells obtained from normal, WT and MMP9 KO C57Bl/6 and 129 mice were cultured in conditioned medium supplemented with 20% (v/v) M-CSF, 10% FCS, and penicillin/streptomycin/glutamine for 7– 8 days on circular glass slides in 24-well tissue culture plates. After removing medium, 0.2 mM 4- MUNANA substrate in Tris-buffered saline, pH 7.4, was added to cells alone (no ligand control) or with 5 µg/ml LPS, 1 µg/ml of LTA, or 10 µg/ml M. butyricum. Fluorescent images were taken at 1 min after adding substrate using epifluorescent microscopy (X40 objective). The mean fluorescence surrounding the cells for each of the images was measured using ImageJ software.

the WT controls and β-actin (Figure 2.8 B). In addition, Neu1 sialidase activity associated with TLR4 ligand LPS- treated live siRNA MMP9 KD cells was completely inhibited to the background levels of no ligand controls and compared with the LPS-treated WT cells as positive controls (Figure 2.8 C).

In addition, we used primary bone marrow macrophages derived from WT and

MMP9 KO C57Bl/6 and 129 mice. After 7 days in culture with conditioned medium containing M-CSF, the primary macrophage cells were stimulated with the TLR2 ligands

LTA and killed M. butyricum (Myco in Figure 2.8 D) as well as the TLR4 ligand LPS to induce sialidase activity. Preliminary data (not shown) indicated that the proactive 105- kDa MMP9 isoform is present in the cell lysates from the WT and MMP9 KO primary macrophages, which is consistent with the siRNA MMP9 results with the RAW-Blue cells. As expected, the sialidase activity associated with TLR ligand-treated MMP9 KO primary macrophages was completely reduced to the background no ligand controls and compared with the WT primary macrophages as positive controls (Figure 2.8 D).

2.4.8 Neu1 and MMP9 Cross-talk Is Essential for LPS-induced TLR4 Activation If MMP9 is expressed on the cell surface of TLR-expressing cells, we asked whether

MMP9 would colocalize with TLR4 receptors. Confocal microscopy revealed the cell surface colocalization of TLR4 and MMP9 in naive and LPS- treated BMA macrophage cells (Figure 2.9 A). In addition, there were no significant reductions of MMP9 and

TLR4 colocalizations in these cells treated with LPS for 5 min (28% overlay), 15 min

(47% overlay), 30 min (36% overlay), and 45 min (31% overlay) compared with the

Figure 2.9: MMP9 colocalizes and coimmunoprecipitates with TLR4. (A) BMA macrophage cells were treated with 5 µg/ml LPS for 5, 15, 30, and 45 min or left untreated as controls. Cells were fixed, non-permeabilized, and immunostained with rat anti- mouse TLR4 and rabbit anti-mouse MMP9 followed by Alexa Fluor594 goat anti-rabbit IgG or Alexa Fluor488 rabbit anti-rat IgG. Stained cells were visualized using a confocal inverted microscope (Leica TCS SP2 MP inverted confocal microscope) with a X100 oil objective. Images were captured using a z-stage of 8 –10 images/cell at 0.5-mm steps and were processed using ImageJ version 1.38x software. To calculate the amount of colocalization in the selected images, the Pearson correlation coefficient was measured and expressed as a percentage using ImageJ version 1.38x software. (B) flow cytometry analysis of MMP9 expressed on the cell surface of live BMA macrophage cells following LPS treatment for 5 min as described in the legend to Figure 2.7 A. MMP9 co-immunoprecipitates with TLR4 (C) and conversely, TLR4 co- immunoprecipitates with MMP9 (D) BMA macrophage cells are left cultured in medium or in medium containing 5 µg/ml LPS. Cells (1 x 107 cells) are pelleted and lysed in lysis buffer. MMP9 and TLR4 in cell lysates from BMA cells are immunoprecipitated with 1.0 µg of rabbit anti-MMP9 or 2 µg of rat anti-TLR4 antibodies for 24 h. Following immunoprecipitation, complexes are isolated using protein A or G magnetic beads and resolved by 8% gel electrophoresis (SDS-PAGE). The blots are probed for TLR4 (88 kDa) with anti-TLR4 or MMP9 (78 or 84 kDa) with anti-MMP9 antibodies followed by Clean-Blot IP Detection Reagent for immunoprecipitation/Western blots and Western Lightning Chemiluminescence Reagent Plus. The chemiluminescence reaction was analyzed with x-ray film. Sample concentration for gel loading was determined by Bradford assay. Quantitative analysis was done by assessing the density of TLR4 or MMP9 bands corrected for background in each lane using Corel Photo Paint 8.0 software. Each bar in the graphs represents the mean ratio corrected density of TLR4 over the MMP9 band for 6 – 8 replicate measurements within each lane. The data are a representation of one of five independent experiments showing similar results. IP, immunoprecipitation.

untreated cells (45% overlay). These data provide strong evidence for MMP9 colocalization with TLR4 receptors in naive macrophage cells. Surprisingly, LPS treatment of these cells did not have any effect on the colocalization of TLR4 and MMP9 as well as MMP9 expression on the cell surface of live BMA cells (Figure 2.9 B). Co- immunoprecipitation experiments using cell lysates from BMA cells further demonstrated that MMP9 forms a complex with TLR4 receptors in naive and LPS-treated cells for 5 min and for 5– 45 min (Figure 2.9 C). Conversely, TLR4 co- immunoprecipitated with MMP9 in the cell lysates of BMA cells (Figure 2.9 D). In support of the confocal colocalization data, there were no reductions in the ratios of

TLR4 to MMP9 following LPS treatment. Collectively, the additional intracellular and cell surface colocalization of TLR4 and MMP9 validated the predicted alliance between

TLR4 and MMP9 on the cell surface.

If Neu1 and MMP9 cross-talk is localized to the cell surface in regulating TLR receptor activation, they should be associated with each other in alliance with TLR receptors. To test this hypothesis, we performed confocal microscopic colocalization and co-immunoprecipitation experiments using BMA cells. The data shown in Figure 2.10 A-

C, validated the predicted association of Neu1 with MMP9. Confocal microscopy revealed the cell surface colocalization of Neu1 and MMP9 in naive and LPS-treated

BMA cells (Figure 2.10 A). Surprisingly, there was a significant reduction of Neu1 and

MMP9 colocalization in these cells treated with LPS for 5 min (30% overlay), 15 min

(24% overlay), 30 min (18% overlay), and 45 min (1% overlay) compared with the

Figure 2.10: MMP9 colocalizes and coimmunoprecipitate with Neu1. (A) BMA macrophage cells were treated with 5 µg/ml LPS for 5, 15, 30, and 45 min or left untreated as controls. Cells were fixed, non-permeabilized, and immunostained with rabbit anti- Neu1 and goat anti-MMP9 followed by Alexa Fluor568 rabbit anti-goat IgG or Alexa Fluor488 donkey anti-rabbit IgG. Stained cells were visualized using a confocal inverted microscope (Leica TCS SP2 MP inverted confocal microscope) with a X100 oil objective. Images were captured using a z-stage of 8 –10 images/cell at 0.5-mm steps and were processed using ImageJ version 1.38x software. To calculate the amount of colocalization in the selected images, the Pearson correlation coefficient was measured and expressed as a percentage using ImageJ version 1.38x software. The data are a representation of one of three independent experiments showing similar results. (B) and (C) BMA macrophage cells were left cultured in medium or in medium containing 5 µg/ml LPS for the indicated time intervals. Cells (1 X 107 cells) were pelleted and lysed in lysis buffer. MMP9 and Neu1 in cell lysates from BMA cells were immunoprecipitated with 1.0 µg of goat anti-MMP9 or 1 µg of rabbit anti-Neu1 antibodies for 24 h. Following immunoprecipitation, complexes were isolated using protein A or G magnetic beads, washed 3 times in buffer, and resolved by 8% SDS-PAGE. The blots were probed for MMP9 (78 or 84 kDa) with anti-MMP9 or Neu1 (45.5 kDa) with anti-Neu1 antibodies followed by Clean-Blot IP Detection Reagent for immunoprecipitation/Western blots and Western Lightning Chemiluminescence Reagent Plus. The chemiluminescence reaction was analyzed with x-ray film. Sample concentration for gel loading was determined by Bradford assay. Quantitative analysis was done by assessing the density of Neu1 or MMP9 bands corrected for background in each lane using Corel Photo Paint 8.0 software. Each bar in the graphs represents the mean ratio corrected density of the Neu1 band over the MMP9 band for 6 – 8 replicate measurements within each lane. The data are a representation of one of three independent experiments showing similar results. Error bars, S.E. IP, immunoprecipitation.

untreated control cells (38% overlay). As predicted, MMP9 co-immunoprecipitated with

Neu1 in cell lysates from naive BMA cells (Figure 2.10 B), and conversely, Neu1 co- immunoprecipitated with MMP9 in cell lysates from naive BMA cells (Figure 2.10 C).

As expected, there was a clear diminution of MMP9 co-immunoprecipitation with Neu1 and conversely with Neu1 co-immunoprecipitation with MMP9 in the cell lysates from

BMA cells after 5– 45-min treatment with LPS (Figure 2.10 B and C). These data further validated that Neu1 forms a complex with MMP9 on the cell surface of naive cells, but it is lost over time with LPS treatment. Collectively, the additional intracellular and cell surface colocalization of Neu1 and MMP9 validated the predicted cross-talk between

Neu1 and MMP9 in alliance with TLR4 receptors. These results indicate that Neu1 in alliance with GPCR-signaling Gαi subunit proteins and MMP9 is expressed on the cell surface of TLR- expressing cells. This tripartite alliance would make Neu1 readily available to be induced by TLR ligand binding to the receptor, enabling removal of steric hindrance for receptor association.

2.5 Discussion The molecular mechanism(s) by which Toll-like receptors become activated are not well understood. For the majority of TLR receptors, dimerization is a prerequisite to facilitate MyD88-TLR complex formation and subsequent cellular signaling to activate

NFκB. However, the parameters controlling interactions between the receptors and their ligands still remain poorly defined. We previously reported that Neu1 is an important intermediate in the initial process of TLR ligand-induced receptor activation and

subsequent cell function (1, 2, 26). The data indicated an initial rapid activation of Neu1 activity that was induced by ligand binding to the receptor. Central to this process is that

Neu1, and not the other three mammalian sialidases, forms a complex with either TLR2, -

3, or -4 receptors in naive TLR-expressing cells or primary macrophage cells. The findings in this report provide evidence for an unprecedented membrane signaling paradigm initiated by TLR ligands binding to TLR receptors. The results indicate that the interaction of TLR ligands with their receptors initiates the potentiation of GPCR signaling via membrane Gαi subunit proteins and MMP9 activation to induce Neu1 activity. Neu1 in alliance with GPCR-signaling Gαi subunit proteins and MMP9 is expressed on the cell surface of TLR-expressing cells. This tripartite alliance makes Neu1 readily available to be induced by TLR ligands binding to their receptors. How Neu1 is rapidly induced by MMP9 and Gαi proteins still remains unknown. It can be speculated that TLR ligand binding to its receptor on the cell surface initiates GPCR signaling via

GPCR Gαi subunit proteins to activate MMP. It is well known that agonist-bound GPCRs have been shown to activate numerous MMPs (20), including MMP3 (21) and MMP2 and -9 (22, 23), as well as members of the ADAM family of metalloproteases: ADAM10,

ADAM15, and ADAM17 (24, 25). However, the precise molecular mechanism(s) underlying GPCR-mediated MMP activation is also unclear. The paradigm could involve a conformational change following TLR ligand binding. For instance, it has been reported that the binding of DNA containing CpG to TLR9 receptors leads to substantial conformational changes in the TLR9 ectodomain (28 –30). Perhaps a similar TLR ligand-

induced conformational change(s) in TLR ectodomain might in turn induce Neu1 activity. We speculate that TLR-ligand binding to its receptor induces a conformational change in the ectodomain to initiate the GPCR-signaling process and MMP9 activation in inducing Neu1. Active Neu1 in complex with TLR hydrolyzes α-2,3-sialyl residues, enabling the removal of steric hindrance to receptor association for TLR activation and cellular signaling.

The TLR signaling model in our study would also predict that MMP9 would be suppressed prior to TLR ligand binding to the receptor. The data in this report support this prediction. It is well known that MMP activity is blocked by general inhibitors, including α2-macroglobulin, which are present in the plasma and tissue fluids, as well as by more specific inhibitors, such as tissue inhibitors of metalloproteinases (TIMP) (31,

32). There are four human TIMPs that have been identified. They are either anchored in the extracellular membrane or secreted extracellularly. These TIMPs bind MMPs tightly and noncovalently. Several other proteins have also been described as novel MMP inhibitors, and some of these contain domains that are homologous to the TIMP- inhibitory domains. For example, tissue factor pathway inhibitor-2 (TFPI2) is a serine protease inhibitor that can function as an MMP inhibitor (33). The pro-collagen COOH- terminal proteinase enhancer releases a COOH-terminal fragment that is similar to the inhibitor domain of TIMPs, and this fragment possesses significant MMP-inhibitory activity (34). RECK (reversion-inducing cysteine-rich protein with Kazal motifs) is another cell surface MMP inhibitor that is a key regulator of extracellular membrane

integrity and angiogenesis (31, 35). The RECK protein contains serine protease inhibitor- like domains and is associated with the cell membrane through a COOH-terminal glycosyl-phosphatidylinositol modification (31). It has been reported that the glycosylation of asparagine sites had no role in the cell surface localization of RECK as a glycosylphosphatidylinositol- anchored protein, but more interestingly, the glycosylation of the RECK Asn297 residue was involved in the suppression of MMP9 secretion, whereas the Asn352 residue was necessary to inhibit MMP2 activation (36).

Interestingly, RECK suppression of tumor cell invasion was reversed by inhibiting glycosylation at the Asn86, Asn297, and Asn352 residues of RECK. Thus, it is reasonable to expect that RECK or any other tissue inhibitors of MMP might play a role in the suppression of MMP9 in complex with TLR receptors. As predicted in the model,

MMP9 in complex with TLR becomes activated upon TLR ligand binding to the receptor. It is possible that this MMP9 activation may involve GPCR signaling.

Surprisingly, galardin and piperazine were found to be highly potent (IC50 of 26 and 79 pM, respectively) in inhibiting Neu1 activity induced by TLR ligand treatment of live macrophage cells when compared with a specific inhibitor of MMP9 (IC50 = 0.1 µM) or MMP3i (IC50 > 3160 µM). The reason(s) for this inhibitory potency of these broad range inhibitors of MMPs on Neu1 activity is unknown. However, it may be due to a unique orientation of Neu1 with the molecular multienzymatic complex that contains β- galactosidase, cathepsin A, and EBP, the complex of which would be associated with

MMP9 within the ectodomain of TLR receptors (Figure 2.10 A). Galardin is a broad

inhibitor of MMP1 (interstitial collagenase), MMP2 (72-kDa type IV collagenase or gelatinase A), MMP3 (stromelysin-1), MMP8 (neutrophil collagenase), and MMP9, whereas piperazine inhibits MMP1, -3, -7, and -9. MMP2 has been shown to interact with

TIMP2 and TIMP4 (37), and galardin inhibition of MMP2 may provide an additive potency for it to keep MMP9 inactive. The MMP3 enzyme degrades collagen types II,

III, IV, IX, and X, proteoglycans, fibronectin, laminin, and elastin. In addition, MMP3 can also activate other MMPs, such as MMP1, MMP7, and MMP9, rendering MMP3 crucial in connective tissue remodeling (38). The findings in this report indicate that a specific inhibition of MMP3 had no effect on Neu1 activity associated with TLR ligand activation of receptors. In addition, silencing MMP9 mRNA using lentivirus MMP9 shRNA transduction of BMA cells or short interference MMP9 RNA transfection of

RAW-Blue cells as well as using primary bone marrow macrophages from MMP9 knockout mice validated the results obtained with the MMP inhibitors at the genetic level.

In this report, the TLR signaling paradigm on the cell surface would also predict that TLR receptors are in alliance with a functional GPCR signaling complex. The data in this and our other reports (1–3) show that ligand binding to either of these receptors induces Neu1 activity within 1 min and that this activity is blocked by Gαi-sensitive pertussis toxin. The rapidity of the ligand-induced Neu1 activity as mediated by the ligand-bound receptor suggests that glycosylated receptors like NGF TrkA, brain-derived neurotrophic factor (BDNF) TrkB, and Toll-like receptors (1, 2) form a functional signaling complex with Gαi proteins of GPCRs. In support of this hypothesis, others have

provided important evidence to show that the cross-talk between GPCR and TLR signaling pathways and the GPCR signaling molecules may have uncharacterized functions in macrophage cells (9). In addition, TLR receptor signaling has been shown to alter the expression of the regulator of G protein signaling proteins in dendritic cells (39).

Other reports have shown that sphingosine 1-phosphate S1P1 and S1P3 expression was induced by LPS in human gingival epithelial cells, and this elevated expression enhanced the influence of S1P in its cooperation with TLR4 to increase cytokine production (40).

The S1P receptors (S1P1 to -5) are a family of GPCRs with a high affinity for sphingosine 1-phosphate, a serum-borne bioactive lipid associated with diverse biological activities, such as inflammation and healing (40). Furthermore, the relationship between

GPCR signaling and TLR has been shown for (a) CC chemokine ligand-2 synergizing with the non-chemokine G protein-coupled receptor ligand formyl methionyl leucyl phenylalanine in monocyte chemotaxis (41), (b) complement C1q expression in macrophages requiring β-arrestin 2 where β-arrestins (ARRB1 and ARRB2) regulate

GPCR-dependent and -independent signaling pathways (42), (c) leukotriene B4 (LTB4) receptor BTL1 by reducing SOCS1 inhibition of MyD88 expression in mouse macrophages (43), and (d) GPCR-derived cAMP signaling influencing TLR responses in primary macrophages through peptide disruptors of protein kinase A anchoring protein

(AKAP10) involving prostaglandin E2 (44).

In conjunction with the TLR dimerization process, we would also predict that the

TLR receptors need to undergo conformational changes following ligand binding, which

allow proper orientation of the ectodomains of TLR for receptor-receptor association (45,

46). In another study, we reported the importance of the involvement of α-2,3-sialyl residues linked to β-galactosides in receptor activation, which was further emphasized by exogenous α2,3-sialyl-specific neuraminidases (2,47). Primary bone marrow macrophages derived from Neu1-deficient mice treated with a purified recombinant neuraminidase (C. perfringens) or recombinant Trypanosoma cruzi trans-sialidase (TS) but not the mutant TSΔD98E-induced phosphorylation of TrkA (3) and the activation of

NFκB (2). These results are consistent with our other reports (1–3, 8, 27) supporting the glycosylation model in corroborating the importance of sialyl α-2,3-linked β-galactosyl residues of Trk and TLR in the initial stages of ligand-induced receptor activation. For mammalian sialidases, we have shown that Neu1 desialylation of α-2,3-sialyl residues of

TLR receptors enables receptor dimerization (2). The report showed that TLR ligand- induced NFκB responses were not observed in TLR-deficient HEK293 cells but were reestablished in HEK293 cells stably transfected with TLR4/MD2 and were significantly inhibited by α-2,3-sialyl-specific Maackia amurensis (MAL-2) lectin, α-2,3- sialyl specific galectin-1, and neuraminidase inhibitor Tamiflu but not by α-2,6-sialyl specific

Sambucus nigra lectin. Also, Tamiflu inhibited LPS-induced sialidase activity in live

BMC-2 macrophage cells with an IC50 of 1.2 µM compared with an IC50 of 1015 µM for its hydrolytic metabolite oseltamivir carboxylate (1). Tamiflu blockage of LPS-induced

Neu1 activity was not affected in BMC-2 cells pretreated with the anticarboxylesterase agent clopidogrel. The other neuraminidase inhibitors oseltamivir carboxylate and

zanamivir had limited inhibitory effect on Neu1 activity associated with LPS-treated live

TLR4- expressing cells (1). The reason(s) for this inhibitory potency of Tamiflu on Neu1 activity is unknown. Possibly, it may be due to a unique orientation of Neu1 with the molecular multienzymatic complex that contains β-galactosidase and cathepsin A (48) and EBP (16), the complex of which would be associated within the ectodomain of TLR receptors. Stomatos and co-workers (50) have also shown that Neu1 on the cell surface is tightly associated with a subunit of cathepsin A, and the resulting complex influences cell surface sialic acid in activated cells and the production of IFNγ. Neu1-deficient mice produced markedly less IgE and IgG1 antibodies following immunization with protein antigens, which may be the result of their failure to produce IL-4 cytokine (51). In other studies, Neu1 was found to negatively regulate lysosomal exocytosis in hematopoietic cells, where it processes the sialic acids on the lysosomal membrane protein LAMP-1

(52). On the cell surface, Seyrantepe et al. (53) have reported that Neu1 regulates phagocytosis in macrophages and dendritic cells through the desialylation of surface receptors, including Fc receptors for immunoglobulin G (FcγR). The present report discloses another functional role for Neu1. It suggests a Neu1 and MMP9 cross-talk in alliance with GPCR signaling through Gαi proteins and TLR receptors on the cell surface to mediate receptor activation.

In conclusion, the data presented in this report signify a novel role of Neu1 as an intermediate in the initial process of ligand-induced TLR receptor activation and subsequent cellular signaling. The premise is that Neu1 forms a complex with glyco-

sylated TLR receptors within the ectodomain as reported previously (1). Second, Neu1 may be a requisite intermediate in regulating TLR activation following ligand binding to the receptor. Third, Neu1 activated by ligand binding to the receptor predicts a rapid removal of α-2,3-sialyl residues linked to β-galactosides on TLR ectodomain to generate a functional receptor (2). Although there are four identified mammalian sialidases classified according to their subcellular localization (54), the sialidases classified as cytosolic (Neu2), plasma membrane-bound (Neu3) (54 –58), and Neu4 (49, 59) are not involved in the sialidase activity associated with ligand-treated live TLR-expressing cells and primary BM macrophages (1). Fourth, the potentiation of GPCR signaling via membrane targeting of Gαi subunit proteins and MMP9 activation by ligand binding to

TLRs in this report and to Trk receptors (3) is involved in the activation process of Neu1 sialidase on the cell surface. Using confocal microscopy on permeabilized naive and endotoxin LPS-treated cells and co-immunoprecipitation experiments, the additional intracellular and cell surface colocalization of Neu1, MMP9, and TLR4 validated the predicted association of MMP9 with Neu1 in alliance with TLR receptors (Figures 2.9 and 2.10). Taken all together, these findings uncover a molecular organizational signaling platform of a Neu1 and MMP9 cross-talk in alliance with TLR receptors on the cell surface that is essential for ligand-induced TLR activation and cellular signaling.

2.6 References 1. Amith, S. R., Jayanth, P., Franchuk, S., Siddiqui, S., Seyrantepe, V., Gee, K., Basta, S., Beyaert, R., Pshezhetsky, A. V., and Szewczuk, M. R. (2009) Dependence of pathogen molecule-induced toll-like receptor activation and cell function on Neu1 sialidase. Glycoconj J 26, 1197-1212 2. Amith, S. R., Jayanth, P., Franchuk, S., Finlay, T., Seyrantepe, V., Beyaert, R., Pshezhetsky, A. V., and Szewczuk, M. R. (2010) Neu1 desialylation of sialyl alpha-2,3-linked beta-galactosyl residues of TOLL-like receptor 4 is essential for receptor activation and cellular signaling. Cell Signal 22, 314-324 3. Jayanth, P., Amith, S. R., Gee, K., and Szewczuk, M. R. (2010) Neu1 sialidase and matrix metalloproteinase-9 cross-talk is essential for neurotrophin activation of Trk receptors and cellular signaling. Cell Signal 22, 1193-1205 4. Kovacsovics-Bankowski, M., and Rock, K. L. (1994) Presentation of exogenous antigens by macrophages: analysis of major histocompatibility complex class I and II presentation and regulation by cytokines. Eur.J.Immunol. 24, 2421-2428 5. Shen, Z., Reznikoff, G., Dranoff, G., and Rock, K. L. (1997) Cloned dendritic cells can present exogenous antigens on both MHC class I and class II molecules. J.Immunol. 158, 2723-2730 6. Ma, W., Lim, W., Gee, K., Aucoin, S., Nandan, D., Kozlowski, M., Diaz-Mitoma, F., and Kumar, A. (2001) The p38 Mitogen-activated Kinase Pathway Regulates the Human Interleukin-10 Promoter via the Activation of Sp1 Transcription Factor in Lipopolysaccharide-stimulated Human Macrophages. Journal of Biological Chemistry 276, 13664-13674 7. Alatery, A., and Basta, S. (2008) An efficient culture method for generating large quantities of mature mouse splenic macrophages. J Immunol Methods 338, 47-57 8. Woronowicz, A., Amith, S. R., De Vusser, K., Laroy, W., Contreras, R., Basta, S., and Szewczuk, M. R. (2007) Dependence of neurotrophic factor activation of Trk tyrosine kinase receptors on cellular sialidase. Glycobiology 17, 10-24

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24. Gooz, M., Gooz, P., Luttrell, L. M., and Raymond, J. R. (2006) 5-HT2A Receptor induces ERK phosphorylation and proliferation through ADAM-17 Tumor Necrosis Factor-{alpha}-converting Enzyme (TACE) activation and Heparin- bound Epidermal Growth Factor-like Growth Factor (HB-EGF) shedding in mesangial Cells. J. Biol. Chem. 281, 21004-21012 25. Prenzel, N., Zwick, E., Daub, H., Leserer, M., Abraham, R., Wallasch, C., and Ullrich, A. (1999) EGF receptor transactivation by G-protein-coupled receptors requires metalloproteinase cleavage of proHB-EGF. Nature 402, 884-888 26. Takahashi, M., Tsuda, T., Ikeda, Y., Honke, K., and Taniguchi, N. (2004) Role of N-glycans in growth factor signaling. Glycoconj J 20, 207-212 27. Finlay, T. M., Jayanth, P., Amith, S. R., Gilmour, A., Guzzo, C., Gee, K., Beyaert, R., and Szewczuk, M. R. (2010) Thymoquinone from nutraceutical black cumin oil activates Neu4 sialidase in live macrophage, dendritic, and normal and type I sialidosis human fibroblast cells via GPCR Galphai proteins and matrix metalloproteinase-9. Glycoconj J 27, 329-348 28. Ewald, S. E., Lee, B. L., Lau, L., Wickliffe, K. E., Shi, G. P., Chapman, H. A., and Barton, G. M. (2008) The ectodomain of Toll-like receptor 9 is cleaved to generate a functional receptor. Nature 456, 658-662 29. Latz, E., Verma, A., Visintin, A., Gong, M., Sirois, C. M., Klein, D. C., Monks, B. G., McKnight, C. J., Lamphier, M. S., Duprex, W. P., Espevik, T., and Golenbock, D. T. (2007) Ligand-induced conformational changes allosterically activate Toll-like receptor 9. Nat Immunol 8, 772-779 30. Haas, T., Metzger, J., Schmitz, F., Heit, A., Muller, T., Latz, E., and Wagner, H. (2008) The DNA sugar backbone 2' deoxyribose determines toll-like receptor 9 activation. Immunity 28, 315-323 31. Takahashi, C., Sheng, Z., Horan, T. P., Kitayama, H., Maki, M., Hitomi, K., Kitaura, Y., Takai, S., Sasahara, R. M., Horimoto, A., Ikawa, Y., Ratzkin, B. J., Arakawa, T., and Noda, M. (1998) Regulation of matrix metalloproteinase-9 and

inhibition of tumor invasion by the membrane-anchored glycoprotein RECK. Proceedings of the National Academy of Sciences of the United States of America 95, 13221-13226 32. Brew, K., Dinakarpandian, D., and Nagase, H. (2000) Tissue inhibitors of metalloproteinases: evolution, structure and function. Biochimica et biophysica acta 1477, 267-283 33. Izumi, H., Takahashi, C., Oh, J., and Noda, M. (2000) Tissue factor pathway inhibitor-2 suppresses the production of active matrix metalloproteinase-2 and is down-regulated in cells harboring activated ras oncogenes. FEBS letters 481, 31- 36 34. Mott, J. D., Thomas, C. L., Rosenbach, M. T., Takahara, K., Greenspan, D. S., and Banda, M. J. (2000) Post-translational proteolytic processing of procollagen C-terminal proteinase enhancer releases a metalloproteinase inhibitor. The Journal of biological chemistry 275, 1384-1390 35. Oh, J., Takahashi, R., Kondo, S., Mizoguchi, A., Adachi, E., Sasahara, R. M., Nishimura, S., Imamura, Y., Kitayama, H., Alexander, D. B., Ide, C., Horan, T. P., Arakawa, T., Yoshida, H., Nishikawa, S., Itoh, Y., Seiki, M., Itohara, S., Takahashi, C., and Noda, M. (2001) The membrane-anchored MMP inhibitor RECK is a key regulator of extracellular matrix integrity and angiogenesis. Cell 107, 789-800 36. Simizu, S., Takagi, S., Tamura, Y., and Osada, H. (2005) RECK-mediated suppression of tumor cell invasion is regulated by glycosylation in human tumor cell lines. Cancer Research 65, 7455-7461 37. Kai, H. S., Butler, G. S., Morrison, C. J., King, A. E., Pelman, G. R., and Overall, C. M. (2002) Utilization of a novel recombinant myoglobin fusion protein expression system to characterize the tissue inhibitor of metalloproteinase (TIMP)-4 and TIMP-2 C-terminal domain and tails by mutagenesis. The

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prostaglandin E2 modulation of toll-like receptor signaling in alveolar macrophages. J Biol Chem 45. Bell, J. K., Askins, J., Hall, P. R., Davies, D. R., and Segal, D. M. (2006) The dsRNA binding site of human Toll-like receptor 3. Proc.Natl.Acad.Sci.U.S.A 103, 8792-8797 46. Bell, J. K., Botos, I., Hall, P. R., Askins, J., Shiloach, J., Segal, D. M., and Davies, D. R. (2005) The molecular structure of the Toll-like receptor 3 ligand-binding domain. Proc.Natl.Acad.Sci.U.S.A 102, 10976-10980 47. Woronowicz, A., De Vusser, K., Laroy, W., Contreras, R., Meakin, S. O., Ross, G. M., and Szewczuk, M. R. (2004) Trypanosome trans-sialidase targets TrkA tyrosine kinase receptor and induces receptor internalization and activation. Glycobiology 14, 987-998 48. Lukong, K. E., Elsliger, M. A., Chang, Y., Richard, C., Thomas, G., Carey, W., Tylki-Szymanska, A., Czartoryska, B., Buchholz, T., Criado, G. R., Palmeri, S., and Pshezhetsky, A. V. (2000) Characterization of the sialidase molecular defects in sialidosis patients suggests the structural organization of the lysosomal multienzyme complex. Hum Mol Genet 9, 1075-1085 49. Seyrantepe, V., Landry, K., Trudel, S., Hassan, J. A., Morales, C. R., and Pshezhetsky, A. V. (2004) Neu4, a novel human lysosomal lumen sialidase, confers normal phenotype to sialidosis and galactosialidosis cells. J Biol Chem 279, 37021-37029 50. Nan, X., Carubelli, I., and Stamatos, N. M. (2007) Sialidase expression in activated human T lymphocytes influences production of IFN-gamma. J Leukoc Biol 81, 284-296 51. Chen, X. P., Enioutina, E. Y., and Daynes, R. A. (1997) The control of IL-4 gene expression in activated murine T lymphocytes: a novel role for neu-1 sialidase. J Immunol 158, 3070-3080

52. Yogalingam, G., Bonten, E. J., van de Vlekkert, D., Hu, H., Moshiach, S., Connell, S. A., and d'Azzo, A. (2008) Neuraminidase 1 is a negative regulator of lysosomal exocytosis. Dev Cell 15, 74-86 53. Seyrantepe, V., Iannello, A., Liang, F., Kanshin, E., Jayanth, P., Samarani, S., Szewczuk, M. R., Ahmad, A., and Pshezhetsky, A. V. (2010) Regulation of phagocytosis in macrophages by neuraminidase 1. J Biol Chem 285, 206-215 54. Miyagi, T., Wada, T., Yamaguchi, K., Hata, K., and Shiozaki, K. (2008) Plasma Membrane-associated sialidase as a crucial regulator of transmembrane signalling. J Biochem 144, 279-285 55. Kato, K., Shiga, K., Yamaguchi, K., Hata, K., Kobayashi, T., Miyazaki, K., Saijo, S., and Miyagi, T. (2006) Plasma-membrane-associated sialidase (NEU3) differentially regulates integrin-mediated cell proliferation through laminin- and fibronectin-derived signalling. Biochem.J. 394, 647-656 56. Valaperta, R., Chigorno, V., Basso, L., Prinetti, A., Bresciani, R., Preti, A., Miyagi, T., and Sonnino, S. (2006) Plasma membrane production of ceramide from ganglioside GM3 in human fibroblasts. FASEB J. 20, 1227-1229 57. Papini, N., Anastasia, L., Tringali, C., Croci, G., Bresciani, R., Yamaguchi, K., Miyagi, T., Preti, A., Prinetti, A., Prioni, S., Sonnino, S., Tettamanti, G., Venerando, B., and Monti, E. (2004) The plasma membrane-associated sialidase MmNEU3 modifies the ganglioside pattern of adjacent cells supporting its involvement in cell-to-cell interactions. J.Biol.Chem. 279, 16989-16995 58. Sasaki, A., Hata, K., Suzuki, S., Sawada, M., Wada, T., Yamaguchi, K., Obinata, M., Tateno, H., Suzuki, H., and Miyagi, T. (2003) Overexpression of plasma membrane-associated sialidase attenuates insulin signaling in transgenic mice. J.Biol.Chem. 278, 27896-27902 59. Yamaguchi, K., Hata, K., Koseki, K., Shiozaki, K., Akita, H., Wada, T., Moriya, S., and Miyagi, T. (2005) Evidence for mitochondrial localization of a novel human sialidase (NEU4). Biochem J 390, 85-93

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Chapter 3

G-protein coupled receptor agonists mediate Neu1 sialidase and matrix metalloproteinase-9 cross-talk to induce transactivation of TOLL-like

receptors and cellular signaling

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3.1 Abstract The mechanism(s) behind GPCR transactivation of TLR receptors independent of TLR ligands is unknown. Here, GPCR agonists bombesin, bradykinin, lysophosphatidic acid

(LPA), cholesterol, angiotensin-1 and ‐2, but not thrombin induce Neu1 activity in live macrophage cell lines and primary bone marrow macrophage cells from wild-type (WT) mice but not from Neu1-deficient mice. Using immunocytochemistry and NFκB- dependent secretory alkaline phosphatase (SEAP) analyses, bombesin induced NFκB activation in BMC-2 and RAW-blue macrophage cells, which was inhibited by MyD88 homodimerization inhibitor, Tamiflu, galardin, piperazine and MMP-9 inhibitor.

Bombesin receptor, neuromedin B (NMBR), forms a complex with TLR4 and MMP9.

Silencing MMP9 mRNA using siRNA transfection of RAW-blue macrophage cells markedly reduced Neu1 activity associated with bombesin-, bradykinin- and LPA-treated cells relative to the untreated controls. These findings uncover a molecular organizational

GPCR signaling platform to potentiate Neu1 and MMP-9 cross-talk on the cell surface that is essential for the transactivation of TLR receptors and subsequent cellular signaling.

3.2 Introduction G-protein coupled receptors (GPCRs) exhibit key regulatory functions in innate and acquired immunity. In macrophage and dendritic cells, GPCRs have been shown to regulate diverse cell functions including cell–cell interactions, survival, chemotaxis and activation (1–3). There is now evidence for a cross-talk between GPCR and TOLL-like

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receptor (TLR) signaling pathways (2,3), but the mechanism(s) behind this cross-talk has not been fully deﬁned.

An insight into the mechanism(s) of GPCR agonist-induced transactivation of TLRs in the absence of TLR ligands came from our recent reports on the role of Neu1 sialidase and matrix metalloproteinase-9 (MMP-9) cross-talk in regulating TLR (4) and Trk (5) receptors. The ﬁndings disclosed a receptor signaling paradigm involving a process of receptor ligand-induced GPCR-signaling via Gαi-proteins, MMP-9 activation, and the induction of Neu1 activation. Central to this process was that Neu1–MMP-9 complex is tethered to TLR-4 receptors on the cell surface of naïve primary macrophages and TLR- expressing cell lines. This signaling paradigm proposes that ligand binding to the receptor on the cell surface induces a conformational change of the receptor to initiate GPCR- signaling via GPCR Gα subunit proteins and MMP-9 activation to induce Neu1.

Activated Neu1 tethered to TLR on the cell surface targets and hydrolyzes sialyl α-2-3- linked β-galactosyl residues at the ectodomain of TLR receptors (4). Taken together, these ﬁndings predict a prerequisite desialyation of TLR receptors caused by activated

Neu1 enabling the removal of a steric hindrance to receptor association with subsequent activation of TLR receptors and cellular signaling.

This report describes the key players involved in the GPCR agonist-induced transactivation of TLR receptors in the absence of TLR ligands. They radically redeﬁne the current dogma(s) governing the mechanism of the cross-talk between GPCR and TLR activation and cellular signaling, which may provide important pioneering approaches to

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disease intervention strategies. Here, GPCR agonists mediate GPCR-signaling via membrane Gα subunit proteins to induce Neu1 and MMP-9 cross-talk at the TLR's ectodomain on the cell surface. This molecular organizational GPCR signaling platform is proposed to be an initial processing stage for GPCR agonist-induced transactivation of

TLRs and subsequent downstream NFκB signaling.

3.3 Materials and Methods

3.3.1 Reagents TLR4 ligand, lipopolysaccharide (LPS from Serratia marcescens was puriﬁed by phenol extraction, Sigma) or LPS-EK from E. coli K12 (Invivogen, San Diego, CA) was used at indicated optimal dosage. TLR3 ligand polyinosinic–polycytidylic acid (polyI:C, 20

μg/ml; Sigma) and TLR2 ligand, killed Mycobacterium butyricum (MYCO, 5 μg/ml,

DIFCO) were used at the indicated optimal dosage. Bombesin acetate salt hydrate, bradykinin acetate salt, angiotensin I and II human acetate salt, oleoyl-L-α- lysophosphatidic acid sodium salt (LPA), and cholesterol (all purchased from Sigma-

Aldrich) in Tris buffered saline were used at predetermined optimal dosage.

The sialidase substrate, 2′-(4-methylumbelliferyl)-α-D-N-acetyl neuraminic acid (98% pure, 4-MUNANA, Biosynth International Inc., Itasca, IL, USA) was used at the optimal concentration of 0.318 mM for the live cell sialidase assay as previously described (6–8).

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3.3.2 Inhibitors Tamiﬂu (99% pure oseltamivir phosphate, Hoffmann-La Roche Ltd., Mississauga,

Ontario, Lot # BS00060168) and DANA (2-deoxy- 2,3-dehydro-D-N-acetylneuraminic acid) (Sigma-Aldrich) were used at indicated concentrations. Pertussis toxin (PTX, from

Bordetella pertussis, in buffered aqueous glycerol solution, Sigma-Aldrich) catalyzes the

ADP-ribosylation of the α subunits of the heterotrimeric G proteins Gi, Go, and Gt. This prevents the G protein heterotrimers from interacting with receptors, thus blocking their coupling and activation. Galardin (GM6001; N-[(2R)-2-(Hydroxamidocarbonylmethyl)-

4-methylpentanoyl]-L-tryptophan methylamide; Calbiochem-EMD Chemicals Inc.,

Darmstadt, Germany) is a potent, cell-permeable, broad-spectrum hydroxamic acid inhibitor of matrix metalloproteinases (MMPs). Piperazine (PIPZ, a MMPII inhibitor, N-

Hydroxy-1,3-di-(4-methoxybenzenesulphonyl)-5,5-dimethyl-[1,3]-piperazine-2- carboxamide; Calbiochem-EMD Chemicals Inc.) is a potent, reversible, broad-range inhibitor of MMP-1 (IC50 = 24 nM), MMP-3 (IC50 = 18.4 nM), MMP-7 (IC50 =30 nM), and MMP-9 (IC50 = 2.7 nM).

MMP-3 inhibitor (MMP-3i, Stromelysin-1 Inhibitor, Calbiochem-EMD Chemicals Inc.) inhibits MMP-3 (IC50 = 5 nM). MMP-9 inhibitor (MMP-9i, Calbiochem-EMD Chemicals

Inc.) is a cell-permeable, potent, selective, and reversible inhibitor (IC50 = 5 nM). It also inhibits MMP-1 (IC50 =1.05 μM) and MMP-13 (IC50 = 113 nM) only at much higher concentrations.

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MyD88 homo-dimerization inhibitor peptide (Imgenex, San Diego, CA; IMG-2005-1) contains a sequence from the MyD88 TIR homo-dimerization domain to which MyD88 monomers bind to the inhibitor peptide and block MyD88 homo-dimerization. The corresponding control peptide does not contain the MyD88 homodimerization sequence.

The compounds were used according to Imgenex protocol.

3.3.3 Antibodies Rabbit anti-human Neu1 IgG and rabbit polyclonal anti-MMP9, were all acquired from

Santa Cruz Biotechnology, Santa Cruz, CA, USA. AlexaFluor-labeled secondary antibodies included F(ab′)2 goat anti-rabbit AlexaFluor 488 (Molecular Probes, Eugene,

OR, USA), F(ab′)2 goat anti-rabbit AlexaFluor 594 (Molecular Probes), goat anti-mouse

AlexaFluor 594 (Invitrogen, Corp.) were used at predetermined optimal concentrations in these studies. Horse radish peroxidase-labeled goat anti-rabbit antibody was obtained from Santa Cruz Biotechnology.

3.3.4 Cell lines BMC-2 and BMA macrophage cells (9,10) were obtained from Dr. Ken L. Rock,

University of Massachusetts Medical School, Worcester, MA. All cell lines were grown at 37 °C in 5% CO2 in culture media containing 1 × Dulbecco's Modiﬁed Eagle Medium

(DMEM, Gibco, Rockville, MD) supplemented with 10% fetal calf serum (FCS)

(HyClone, Logan, Utah, USA).

RAW-Blue™ cells (mouse macrophage reporter cell line, Invivogen, San Diego, CA) derived from RAW 264.7 macrophages were grown in culture medium under the 106

selection of zeocin. The cells stably express a secreted embryonic alkaline phosphatase

(SEAP) gene inducible by NF-κB and AP-1 transcription factors. Upon stimulation,

RAW-Blue™ cells activate NF-κB and/or AP-1 leading to the secretion of SEAP which is detectable and measurable when using QUANTI-Blue™, a SEAP detection medium

(Invivogen). RAW-Blue™ cells are resistant to Zeocin™ and G418.

3.3.5 NFκB-dependent secretory alkaline phosphatase (SEAP) assay Briefly, a cell suspension of 550,000 RAW-blue cells/mL in fresh growth medium was prepared, and 180μL of cell suspension (∼100,000 cells) was added to each well of a Falcon flat-bottom 96-well plate (Becton Dickinson). After different incubation times, 1.5 μg/mL of LPS was added to each well together with either 250 μM Tamiflu or

100 μg/mL piperazine. The plates were incubated at 37 °C in a 5% CO2 incubator for

18–24 h. A QUANTI-Blue™ solution which is a detection medium developed to determine the activity of any alkaline phosphatase present in a biological sample was prepared following the manufacturer's instructions. Brieﬂy, 180 μL of QUANTI-Blue solution was added to each well of a ﬂat-bottom 96-well plate, followed by 20 μL of supernatant from stimulated RAW-blue cells. The plate was incubated for 30 min to 3 h at 37°C and the SEAP levels were determined using a spectrophotometer at 620–655 nm.

Each experiment was performed in triplicate.

3.3.6 Silencing MMP9 mRNA using MMP9 siRNA MMP9 siRNA (mouse) duplex components were obtained from Santa Cruz

Biotechnology, Inc., containing a pool of three target-speciﬁc 20– 25-nt siRNAs designed 107

to knock down gene expression. RAW-blue or BMA cells were plated in a 6-well plate at

5 × 105 cells/well and incubated at 37 °C for 24 h. In serum-free medium, 10 μL of 40

μM siRNA duplex in a total volume of 250 μL of medium was mixed with 10 μL of

Lipofectamine 2000 (Invitrogen) for 20 min at room temperature and further described in detail elsewhere (4).

3.3.7 Mouse Models Wild-type (WT), Neu1–CathA KD (Neu1 deﬁcient and cathepsin A deﬁcient) and CathA

KD (normal Neu1 sialidase bound to inactive cathepsin A Ser190Ala mutant) mice were obtained from Dr. Alexey Pshezhetsky's laboratory. Neu1–CathA KD mice have a hypomorphic cathepsin A phenotype with a secondary ~ 90% reduction of the Neu1 activity (11). CathA KD mice have an inactive cathepsin A with a S190A point mutation

(11).

3.3.8 Primary mouse bone marrow macrophage cells Bone marrow (BM) cells were ﬂushed from femurs and tibias of mice with sterile Tris- buffered saline (TBS) solution. The cell suspension was centrifuged for 3 min at 900 rpm, and the cell pellet resuspended in red cell lysis buffer for 5 min. The remaining cells were washed once with sterile TBS, and then resuspended in 1 × RPMI conditioned medium supplemented with 10% FCS and 20% (v/v) of L929 cell supernatant as a source of monocyte colony-stimulating factor according to Alatery and Basta (12) and 1× L- glutamine–penicillin– streptomycin (Sigma-Aldrich) in sterile solution. The primary BM macrophages were grown on 12 mm circular glass slides in 1 × RPMI conditioned 108

medium for 7–8 days in a humidified incubator at 37 °C and 5% CO2. By day 7, these primary macrophage cells were more than 95% positive for macrophage marker F4/80 molecule as detected by ﬂow cytometry (12).

3.3.9 Sialidase activity in live cells Primary BM macrophages, TLR-expressing cells were grown on 12 mm circular glass slides in conditioned medium as described above. After removing medium, 0.318 mM 4-

MUNANA substrate in TBS pH7.4 was added to each well alone (control), with predetermined dose of LPS (3–5 μg/mL), or in combination of LPS and 200–500 μM

Tamiflu as previously described (4,6,7). The substrate is hydrolyzed by sialidase to give free 4-methylumbelliferone which has a fluorescence emission at 450 nm (blue color) following an excitation at 365 nm. Fluorescent images were taken after 1–2 min using epi-fluorescent microscopy (40X objective).

3.3.10 Immunocytochemistry of NFκB TLR-expressing cells were pretreated with pure Tamiflu, galardin or PTX at indicated concentrations for 30 min followed with predetermined dose of specific ligand for 45 min. Cells were fixed, permeabilized, and immunostained with rabbit anti-NFκB p65

(Rockland, Gibertsville, PA), or rabbit anti-IκBα (Rockland) antibodies followed with

Alexa Fluor594 goat anti-rabbit IgG. Stained cells were visualized by epi-ﬂuorescence microscopy using a 40X objective. Quantitative analysis was done by assessing the density of cell staining corrected for background in each panel using Corel Photo Paint

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8.0 software. Each bar in the ﬁgures represents the mean corrected density of staining ±

S.E. for all cells (n) within the respective images.

3.3.11 Co-immunoprecipitation BMA macrophage cells were left cultured in media or in media containing 5 μg/mL LPS for the indicated time intervals. Cells (1 × 107 cells) were pelleted and lysed in lysis buffer (50 mM Tris, pH 8, 150 mM NaCl, 1% NP-40, 0.2 mg/mL leupeptin, 1% β- mercaptoethanol, and 1 mM phenylmethanesulfonyl ﬂuoride (PMSF). For immunoprecipitation, NMBR, TLR4 and MMP9 in cell lysates from BMA cells were immunoprecipitated with 1.0 μg of rabbit anti-NMBR, 2 μg of rat anti-TLR4 or rabbit anti-MMP9 antibodies for 24 h. Following immunoprecipitation, complexes were isolated using protein A or G magnetic beads, washed 3 × in buffer (10 mM Tris, pH8, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 1% Triton X-100 and 0.2 mM sodium orthovanadate) and resolved by 8% gel electrophoresis (SDS-PAGE). Proteins were transferred to polyvinylidene ﬂuoride (PVDF) transfer membrane blot. The blots were probed for either NMBR with anti-NMBR (M-52, Santa Cruz Bio- technology, Inc., CA),

TLR4 (88 kDa) with anti-TLR4 (HTS510, Santa Cruz Biotech) or MMP9 (88 kDa) with anti-MMP9 (H-129, Santa Cruz Biotech) followed by HRP conjugated secondary IgG antibodies or Clean-Blot IP Detection Reagent for IP/Western blots (Pierce Biotech- nology, Thermo Fisher Scientiﬁc, Rockford, IL) and Western Lightning

Chemiluminescence Reagent Plus. The chemiluminescence reaction was analyzed with

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X-ray ﬁlm. Sample concentration for gel loading was determined by the Bradford reagent

(Sigma-Aldrich).

3.3.12 Statistics Comparisons between two groups were made by one-way analysis of variance at 95% conﬁdence using Student's unpaired t test and Bonferroni's multiple comparison test or

Dunnett's multiple comparison test for comparisons among more than two groups.

3.4 Results

3.4.1 GPCR agonist induce sialidase activity in live BMC-2, BMA and RAW- blue macrophage cells and primary wild-type mouse macrophage cells but not in Neu1- deﬁcient cells We recently reported a receptor signaling paradigm involving a process of receptor ligand-induced GPCR-signaling via Gαi-proteins, MMP-9 activation, and the induction of Neu1 activation (4). Central to this process was that Neu1–MMP-9 complex is tethered at the ectodomain of TLR-4 receptors on the cell surface of naïve primary macrophages and TLR-expressing cell lines. Furthermore, we reported a similar signaling paradigm for

Trk receptors (5). Collectively, it was proposed that ligand binding to its receptor may induce allosteric conformational change(s) in the receptor, which in turn potentiates

GPCR-signaling and MMP-9 activation to induce Neu1 sialidase.

If Neu1 activity is associated with GPCR-signaling and MMP-9 activation in live TLR- expressing macrophage cells, we asked whether GPCR agonists binding to their respective GPCRs would directly induce sialidase activity in different macrophage cell

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lines and primary bone marrow derived mouse macrophage cells in the absence of TLR speciﬁc ligands. The data in Figure 3.1 are consistent with this hypothesis. GPCR agonist lysophosphatidic acid (LPA), cholesterol, angiotensin-1 and ‐2 and bombesin induced sialidase activity similar to the positive control results with TLR4 ligand lipopolysaccharide (LPS) in live BMC-2 (Figure 3.1 A), BMA (Figure 3.1 A) and RAW-blue

(Figure 3.1 B) macrophage cell lines, while thrombin did not. In addition, the sialidase activity was blocked by pertussis toxin using LPA, and by Tamiﬂu and galardin using bombesin in live BMC-2 macrophage cells (Figure 3.1 A). It is noteworthy that LPA is generated by macrophages, dendritic cells, mast cells and platelets, and that there are distinctive proﬁles of GPCRs for LPA, which are expressed by each type of immune cell

(13–15). Bradykinin also induced sialidase activity in live BMA macrophage cells, which was blocked by pertussis toxin (Figure 3.1 A). Also, bombesin, bradykinin, LPA, angiotensin-1 and ‐2 induced sialidase activity in live RAW-blue macrophage cells, which was inhibited by Tamiflu (Figure 3.1 B). Interestingly, these findings also propose a novel GPCR signaling paradigm that has a common uncharacterized property with broader specificities than expected.

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Figure 3.1: Sialidase activity is associated with GPCR agonist treatments of live macrophage cells and primary bone marrow (BM) macrophage cells. (A) BMC-2, BMA, (B) RAW-blue macrophage cells and (C) primary BM macrophage cells derived from wild-type (WT), Neu1–CathA KD (Neu1 deficient and cathepsin A deficient) and CathA KD (normal Neu1 sialidase bound to inactive cathepsin A Ser190Ala mutant) mice were allowed to adhere on 12 mm circular glass slides for 24 h at 37 °C in a humidified incubator. After removing the media, 0.318 mM 4-MUNANA substrate in Tris buffered saline pH 7.4 was added to cells alone (control) or with either 250 ng/mL bombesin, 50 U/mL thrombin, 200 μg/mL LPA, 100 μg/mL bradykinin, 160 μg/mL cholesterol, 200 μg/mL angiotensin-1 (angioten-1), 200 μg/mL angiotensin-2 (angioten-2), 5 μg/mL LPS, 20 μg/mL polyinosinic–polycytidylic acid (poly-IC) and 5 μg/mL killed Mycobacterium butyricum (MYCO) or in combination with the indicated agonist and 250 μg/mL Tamiflu, 50 μg/mL galardin or 25 ng/mL pertussis toxin (PTX). Fluorescent images were taken at 2 min after adding substrate using epi-fluorescent microscopy (40X objective). The mean fluorescence surrounding the cells for each of the images was measured using Image J Software. The data are a representation of one out of five independent experiments showing similar results

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To conﬁrm that Neu1 activity is associated with GPCR agonist-stimulation of live macrophage cells, we also used primary bone marrow macrophages derived from the hypomorphic cathepsin A mice with secondary ∼ 90% reduction of the Neu1 activity

(Neu1–CathA KD) (11). For controls, we used primary macrophages derived from wild- type (WT) mice and from cathepsin A KD mice (CathA KD, normal Neu1 sialidase bound to inactive cathepsin A S190A point mutation) (11). After 7 days in culture with conditioned medium containing monocyte colony-stimulating factor (7,12), the live primary macrophage cells were treated with either the TLR4 ligand LPS, bombesin, the

TLR3 ligand poly-IC or the TLR2 ligand killed M. butyricum (MYCO) to induce sialidase activity. The data in Figure 3.1 C indicate that neither LPS, MYCO nor bombesin induced sialidase activity in the live primary macrophage cells derived from these Neu1-deﬁcient mice, but that they did in cells from the WT and CathA KD mice.

These latter data provide additional evidence for Neu1 involvement in the bombesin- induced sialidase activity.

3.4.2 Inhibitory effect of neuraminidase inhibitors Tamiﬂu and DANA (2-deoxy-2,3- dehydro-N-acetylneuraminic acid) on bombesin induced sialidase activity in live macrophage cells Next, we asked whether the neuraminidase inhibitors, Tamiﬂu (oseltamivir phosphate) or

DANA (2-deoxy-2,3-dehydro-N-acetylneuraminic acid) would dose-dependently inhibit the sialidase activity associated with bombesin treatment of live macrophage cells. As shown in Figure 3.2 A, Tamiﬂu or DANA inhibited bombesin-induced sialidase activity in live RAW-blue macrophage cells with IC50 of 17 μM and > 344 μM, respectively. 114

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Figure 3.2: Bombesin-induced sialidase and NFκB activities are blocked by Tamiflu and DANA in live RAW-blue and BMC-2 macrophage cells respectively. (A) Cells were incubated on 12-mm circular glass slides in conditioned medium for 24 h at 37 °C in a humidified incubator. After removal of the medium, 0.2 mM 4-MUNANA substrate in Tris- buffered saline, pH 7.4 was added with mounting medium to cells alone (Control), with 250 ng/mL bombesin, or with bombesin in combination with indicated concentrations of Tamiflu or DANA. Fluorescent images were taken at 1-min intervals using Zeiss M2 epi-fluorescent microscopy (40X objective). The mean fluorescence surrounding the cells for each of the images was measured using ImageJ software. The IC50 of each compound was determined by plotting the decrease in sialidase activity against the log of the agent concentration. The data are a representation of one of six independent experiments showing similar results. (B) Immunocytochemistry of bombesin induced NFκB activation in BMC-2 macrophage cells. BMC- 2 macrophage cells were pretreated with 200 μM Tamiflu for 30 min followed with 3 μg/mL LPS or 250 μg/mL bombesin for 15 min. Cells were fixed, permeabilized, and immunostained with rabbit anti-NFκB p65 or rabbit anti-IκBα followed with Alexa Fluor594 rabbit anti-goat IgG. Stained cells were visualized by epi-fluorescence microscopy using a 40X objective. Control images had Alexa Fluor594 secondary but no primary antibody. Approximately 95% of LPS- and bombesin-treated cells immunostained with NFκBp65 had nuclear staining. The data are a representation of one out of three independent experiments showing similar results. Quantitative analysis was done by assessing the intensity of cell staining corrected for background (no cells) in each panel image using Corel Photo Paint 8.0 software. (C) Tamiflu, MMP inhibitor piperazine and MMP9i inhibit bombesin-induced NFκB-dependent secretory alkaline phosphatase (SEAP) activity in RAW-blue macrophage cells. SEAP reporter-expressing RAW-blue cells were treated with either 300 and 400 μg/mL Tamiflu, 200 μg/mL piperazine or 100 μg/mL MMP9i followed by 250 μg/mL bombesin for 24 h. SEAP activity in the culture medium was assessed using Quanti-blue substrate. Results are representative of three to four experiments. Relative SEAP activity was calculated as fold increase of each group [SEAP activity in medium from treated cells minus no cell background over SEAP activity in medium from untreated cells minus background]. p values represent significant differences at 99.9% confidence intervals using the Dunnett's multiple comparison test compared with bombesin-treated cells.

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Tamiflu is the ethyl ester pro-drug of the anti-influenza drug oseltamivir carboxylate, which is converted to this biologically active form in vivo (16). We have previously shown that oseltamivir carboxylate as well as other purified neuraminidase inhibitors such as DANA, BCX-1827 and zanamivir (4-guanidino-Neu5Ac2en) had a limited inhibition of LPS-induced sialidase activity in live BMC-2 cells (7).

3.4.3 Bombesin induces NFκB activation in BMC-2 and RAW-blue macrophage cells We reported previously that inhibition of TLR ligand-induced sialidase activity in TLR- expressing cells treated with speciﬁc lectins and/or Tamiﬂu results in reductions in NFκB activity (4,7,17). Here, bombesin-treated BMC-2 macrophage cells revealed a strong

NFκB (p65, RelA) immunofluorescence localized to the nuclei of these cells, similar to those findings with the LPS-treated cells (Figure 3.2 B). This immunofluorescence was not seen if cells were previously treated with Tamiflu and then stimulated with LPS

(Figure 3.2 B). The anti-NFκB antibody used here detects both active (localized in the nucleus) and inactive (localized in the cytoplasm) forms of NFκB, whereas the anti-IκBα antibody which detects the NFκB-inactivating IκBα is mostly localized in the cytoplasm.

Following NFκB phosphorylation and entry into the nucleus, IκBα becomes degraded. A strong IκBα signal is indicative of the presence of the inactive form of NFκB, and thus serves as a robust internal control for this assay.

To conﬁrm that bombesin induces NFκB activation in macrophage cells; we also performed the NFκB-dependent secretory alkaline phosphatase (SEAP) assay using

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RAW-blue reporter macrophage cell line. RAW-blue cells are murine macrophages that stably express a SEAP gene inducible by NF-κB and AP-1 transcription factors leading to the secretion of SEAP. Secreted SEAP is detected using QUANTI-Blue substrate. The data in Figure 3.2 C clearly show that bombesin treatment of live RAW-blue macrophage cells induced SEAP activity and that this activity was signiﬁcantly inhibited by Tamiﬂu

(400 μg/mL), the broad range MMP inhibitor piperazine (PipZ, 200 μg/mL) or MMP9 inhibitor (MMP9i, 100 μg/mL).

3.4.4 Isoforms of neuromedin B receptor co-immunoprecitate with TLR4 receptors in cell lysates from naïve and LPS stimulated BMA macrophage cells Since bombesin or LPS similarly induce Neu1 activity within a minute and subsequently activate NFκB in macrophages, we questioned whether the bombesin-like receptor, neuromedin B (NMBR) forms a complex with TLR4. Co-immunoprecipitation experiments using cell lysates from BMA macrophage cells showed that NMBR is tethered to TLR4 receptors in naïve and LPS treated cells (Figure 3.3 A). Collectively, the data validated the predicted alliance between TLR4 and NMBR in naïve macrophage cells.

Recently, we reported that MMP-9 is tethered to TLR4 receptors in both naïve and LPS- treated BMA cells (4). Conversely, TLR4 was found to be co-immunoprecipitated with

MMP9. Since both MMP9 and NMBR form a complex with TLR4 receptors, we asked whether or not they are associated with each other. Co-immunoprecipitation experiments using cell lysates from RAW-blue cells further demonstrated that NMBR 80 kDa isoform

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Figure 3.3: Neuromedin B receptor (NMBR) co-immunoprecipitates with TLR4 and MMP9. (A) BMA macrophage cells were left cultured in media or in media containing 5 μg/mL LPS for 30 min. Cells (1 × 107 cells) were pelleted and lysed in lysis buffer. NMBR and TLR4 receptors in the cell lysates from BMA cells were immunoprecipitated with 1 μg of goat anti-NMBR or 1 μg of rabbit anti-TLR4 antibodies for 24 h. Following immunoprecipitation, complexes were isolated using protein A or G magnetic beads, washed 3 × in buffer and resolved by 8% SDS- PAGE. The blots are probed for NMBR with anti-NMBR or TLR4 (88 kDa) with anti-TLR4 antibodies followed by Clean-Blot IP Detection Reagent for IP/Western blots and Western Lightning Chemiluminescence Reagent Plus. The chemiluminescence reaction was analyzed with X-ray ﬁlm. Sample concentration for gel loading was determined by Bradford assay. Quantitative analysis was done by assessing the density of NMBR or TLR4 bands corrected for background in each lane using Corel Photo Paint 8.0 software. Each bar in the ﬁgures represents the mean ratio corrected density of NNBR band over TLR4 for 6–8 replicate measurements within each lane. The data are a representation of one out of three independent experiments showing similar results. (B) RAW-blue macrophage cells were left cultured in media or in media containing 5 μg/mL LPS for 30 min. Cells (1 × 107 cells) were pelleted and lysed in lysis buffer. NMBR receptor and MMP9 in the cell lysates from RAW-blue cells were immunoprecipitated with 1 μg of goat anti- NMBR or 1 μg of rabbit anti-MMP9 antibodies for 24 h. 119

forms a complex with the active 88 kDa MMP9 isoform from naïve or LPS-stimulated cells (Figure 3.3 B). These data further validated that NMBR forms a complex with

MMP9 on the cell surface of naive cells.

3.4.5 Silencing MMP9 using siRNA knockdown To further conﬁrm the role of MMP9 in GPCR agonist-induced sialidase activity, we knocked down MMP9 in RAW-blue macrophage cells using siRNA MMP9 transfection.

Neu1 activity associated with addition of the TLR4 ligand LPS, bombesin or LPA in siRNA MMP9 knockdown (KD) cells was reduced to levels compared to wild-type

RAW-blue cells (Figure 3.4).

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Figure 3.4: GPCR agonist-induced sialidase activity in live WT and siRNA MMP9 KD BMA macrophage cells After removing medium, 0.2 mM 4-MUNANA substrate in Tris-buffered saline, pH 7.4, was added to cells alone (no ligand control) or with either 5 μg/ mL LPS, 250 μg/mL bombesin, 100 μg/mL bradykinin or 200 μg/mL LPA. Fluorescent images were taken at 2 min after adding substrate using Zeiss M2 epi-ﬂuorescent microscopy (40X objective). The mean ﬂuorescence surrounding the cells for each of the images was measured using Image J software. The data are a representation of one of three independent experiments showing similar results.

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3.4.6 Bombesin-mediated NFκB activation in RAW-blue cells involves the TLR4 adaptor molecule MyD88 Since bombesin induces NFκB in BMC-2 or RAW-blue macrophage cells (Figure 3.2 B and C), and its receptor NMBR is tethered to TLR4 (Figure 3.3 A), we assessed if bombesin-mediated NFκB activation in RAW-blue cells involves the TLR4 adaptor molecule MyD88. Our data clearly show that the MyD88 inhibitor peptide of homodimerization signiﬁcantly inhibited LPS (Figure 3.5 A) and bombesin (Figure 3.5 B) induced SEAP activity in live RAW-blue macrophage cells compared to the MyD88 control peptide. These results suggest that bombesin-mediated NFκB activation involves the TLR4 adaptor molecule MyD88. They also suggest that bombesin binding to NMBR receptor activates MMP9 which in turn induces Neu1 activity to hydrolyze α-2,3-sialyl residues of TLR-4 receptors. This effect is proposed to enable removal of steric hindrance to receptor association, MyD88 recruitment and subsequent NFκB activation.

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Figure 3.5: MyD88 homodimizeration inhibitor blocks LPS-induced NFκB-dependent secretory alkaline phosphatase (SEAP) activity in RAW-blue macrophage cells. (A) SEAP reporter-expressing RAW-blue cells were treated with either 5 μg/mL LPS or in combination with MyD88 control peptide or MyD88 homodimerization inhibitor for 48 h. SEAP activity in the culture medium was assessed using Quanti-blue substrate after 30 and 60 min. Relative SEAP activity was calculated as fold increase of each group [SEAP activity in medium from treated cells minus no cell background over SEAP activity in medium from untreated cells minus background]. Results are mean of three separate experiments. p values represent significant differences at 99.9% confidence intervals using the Dunnett's multiple comparison test compared with LPS-treated cells. (B) MyD88 homodimizeration inhibitor blocks bombesin-induced NFκB- dependent secretory alkaline phosphatase (SEAP) activity in RAW-blue macrophage cells. SEAP reporter-expressing RAW-blue cells were treated with either 250 μg/mL bombesin (bomb) or in combination with MyD88 control peptide or MyD88 homodimerization inhibitor for 48 h as described in (A). Results are mean of three separate experiments. p values represent significant differences at 99.9% confidence intervals using the Dunnett's multiple comparison test compared with bombesin-treated cells.

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3.5 Discussion Here, we report ﬁndings that clarify the elements involved in the GPCR agonist-induced transactivation of TLRs via metalloproteinase activation. Our ﬁndings have uncovered a molecular organizational GPCR signaling platform that potentiates Neu1 and MMP-9 cross-talk at the ectodomain of TLRs on the cell surface. This GPCR signaling platform is proposed to be the cellular signaling mechanism for the transactivation of TLR receptors by GPCR-ligands. It also predicts that TLR receptors are in alliance with a functional GPCR signaling complex.

The data in this report show that GPCR agonists such as bombesin, LPA, cholesterol, angiotensin-1 and ‐2, and bradykinin binding to their respective GPCR receptors can induce Neu1 activity within 1 min and that this activity is blocked by Gαi-sensitive pertussis toxin, neuraminidase inhibitor Tamiﬂu, broad range MMP inhibitors galardin and piperazine and MMP9 specific inhibitor, and siRNA knockdown of MMP9. The rapidity of the GPCR agonist-induced Neu1 activity suggests that glycosylated receptors like TLR receptors form a functional GPCR signaling complex. In support of this hypothesis, the bombesin-related neuromedin-B receptor was found to form a complex tethered to TLR4 receptors on the cell surface in BMA macrophage cells. These data are consistent with the reports describing a cross-talk between GPCR and TLR signaling pathways (2, 3).

In addition, our data indicate that the three different isoforms of NMBR are tethered to

TLR-4 receptors, but it is the 80 kDa isoform of NMBR which forms a complex with

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MMP-9, making it readily available to activate MMP-9 on the cell surface of naive macrophage cells. Unexpectedly, the 63 and 80 kDa isoforms of NMBR are eventually lost over time with LPS treatment. The reason(s) for these differential activities of the

NMBR isoforms is unknown. It is possible that the 115 kDa isoform of NMBR is a protein with ligand-stimulated protein–tyrosine kinase activity as reported elsewhere

(18). Perhaps, it might be indirectly involved in the signaling process to activate MMP-9.

These latter results suggest that the 115 kDa isoform of NMBR may have uncharacterized properties with broader substrate speciﬁcities than expected. The ﬁndings suggest that the molecular mechanism of GPCR agonist for the transactivation of TLR receptors in the absence of TLR ligands requires the GPCR signaling platform to be tethered to TLR receptors.

It is noteworthy that the activation of Neu1 requires the removal of elastin binding protein (EBP) from the Neu1/cathepsinA/EBP complex which is tethered to NGF TrkA

(5) and TLR (4,7,17) receptors on the cell surface. In the lysosome, Neu1 sialidase is associated with a serine carboxypeptidase (protective protein cathepsin A), β- galactosidase and N-acetyl-galactosamine-6-sulfate sulphatase (19). Cathepsin A is required for normal Neu1 enzymatic activity (20) and is sorted to the plasma membrane of differentiating monocytes similar to Neu1 (19). Cell surface Neu1–cathepsin A complex can also be associated with elastin-binding protein (EBP) forming the elastin receptor complex, where the catalytic activity of Neu1 facilitates elastic ﬁber assembly

(21) and signal transduction (22). The data in this and another report (7) indicate that

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Neu1 activity can be induced by TLR ligands and GPCR agonists in live primary macrophage cells derived from CathA KD (normal Neu1 with an inactive cathepsin A with a S190A point mutation) mice. These results suggest that the activation of Neu1 by

TLR ligands does not require an active cathepsin A.

For GPCR transactivation of TLR receptors, we propose that GPCR receptors need to be tethered to TLR receptors in order to activate MMP-9 already in complex with the elastin receptor complex containing Neu1. The ﬁndings in this report support this premise.

However, the precise molecular mechanism(s) underlying GPCR-mediated MMP activation still remains undetermined. Other reports have provided compelling evidence to indicate that a cross-talk between G protein signaling molecules and TLR receptors may have uncharacterized functions in macrophage signaling pathways (1–3). TLR receptor signaling pathways have been shown to alter the expression of the regulator of G protein signaling in dendritic cells (2,3). Others have shown that sphingosine 1-phosphate

S1P1 and S1P3 expressions were induced by LPS in human gingival epithelial cells

(HGEC), and that these elevated expressions enhanced the inﬂuence of S1P in its cooperation with TLR4 to increase cytokine production (23). The relationship between

GPCR-signaling and TLR has been shown for (a) CC chemokine ligand-2 synergizing with the non-chemokine G protein-coupled receptor ligand fMLP in monocyte chemotaxis (24), (b) complement C1q expression in macrophages requiring beta-arrestin

2 where beta-arrestins (ARRB1 and ARRB2) regulate G-protein coupled receptor

(GPCR) dependent- and independent-signaling pathways (25), (c) leukotriene B4

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(LTB4) receptor BTL1 by reducing SOCS1 inhibition of MyD88 expression in mouse macrophages (26) and (d) GPCR-derived cAMP signaling in inﬂuencing TLR responses in primary macrophages through peptide disruptors of A-kinase anchoring protein

(AKAP10) involving prostaglandin E2 (PGE2) (27). In addition, the ﬁndings in this present report provide another novel cross-talk between G protein signaling proteins and

TLR receptors. Using MyD88 homodimerization inhibitor peptide, the data indicate that

GPCR bombesin activation of NFκB in macrophage cells is initially mediated via this novel molecular GPCR organizational signaling platform in complex with the TLR receptor as previously proposed by us (4), but the subsequent bombesin-induced NFκB is mediated through the activation of TLR.

The involvement of heterotrimeric Gα proteins in TLR ligand-mediated receptor function has also been proposed in this report. Others have demonstrated that CD14, an integral component of the Gram-negative bacterial lipopolysaccharide receptor complex, along with TLR4 and MD2, was associated with Gi (inhibitory class) and Go (olfactory class) α subunits of G proteins (28). Heterotrimeric G proteins were shown to have a speciﬁc regulatory function in CD14-associated LPS-induced mitogen-activated protein kinase

(MAPK) activation and cytokine production in normal human monocytes (28). In support of a regulatory role for Gα proteins in LPS-induced TLR4 signaling, a signiﬁcant decrease in LPS-induced activation of c-Jun-N-terminal kinase (JNK) and p38 kinase was reported with a subsequent loss in the production of tumor necrosis factor-α (TNF-α) when THP-1 human monocyte cells were pretreated with Gαi-sensitive pertussis toxin

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(29). In addition, Fan et al. (30) have shown that Gαi proteins differentially regulate LPS- mediated signaling through TLR4, whereas Gram-positive Staphylococcus aureus- mediated signaling was through TLR2. Using the knockout mouse models of Gαi2−/−and

Gαi3−/− proteins, they have shown a signiﬁcant decrease in TLR ligand-mediated TNF-α and IL-10 production in peritoneal macrophages of the knockout mice compared to wild- type mice. Surprisingly, in splenocytes from the same knockouts, signiﬁcant increases in

TNF-α and IFN-γ production in response to TLR ligands were detected. These ﬁndings suggest a regulatory role for Gαi proteins in TLR signaling, which is potentially dependent on a cellular phenotype (31). In the murine RAW 264.7 macrophage cell line and primary murine macrophages, G protein dysregulation induced by wasp venom-derived peptide mastoparan caused a signiﬁcant inhibition in LPS-induced TLR4-, but not in

TLR2-mediated gene expression (32). However, the process(es) by which TLR-mediated cellular responses are regulated by Gαi proteins is not well understood. The data in the present report support a unique organizational GPCR signaling platform tethered to TLR receptors as the initial processing stage for GPCR agonist-induced transactivation of these receptors and subsequent downstream cellular signaling.

In conclusion, the data presented in this report signify a novel role of GPCR receptor signaling in the initial process of ligand-induced TLR receptor activation. The premise is that GPCR receptors form a complex with glycosylated TLR receptors at the ectodomain, as previously suggested (4,5). Secondly, Neu1 may be a requisite intermediate in regulating TLR activation following ligand binding to the receptor (7). Thirdly, activated

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Neu1 by ligand binding to the receptor predicts a rapid removal of α-2,3-sialyl residues linked to β-galactosides on TLR ectodomain to generate a functional receptor (17). The sialidases classiﬁed as cytosolic (Neu2), plasma membrane bound Neu3 (33–37) and

Neu4 (38,39) are not involved in the sialidase activity associated with ligand-treated live

TLR-expressing cells and primary BM macrophages (7). Fourthly, the potentiation of

GPCR-signaling via membrane Gαi subunit proteins induces MMP-9 activation either directly by GPCR ligands or indirectly in alliance with TLR (4) receptors. This GPCR- signaling is proposed to be involved in the activation of Neu1 in alliance with MMP-9 and TLR receptors on the cell surface. Using co-immunoprecipitation experiments, the additional cell surface co-localization of GPCR bombesin-related NMBR receptor with

TLR4 and MMP9 validates the predicted association of GPCR with TLR in alliance with

MMP9 and Neu1 in forming a tripartite complex with TLR receptors. Collectively, these

ﬁndings uncover a molecular organizational GPCR signaling platform to potentiate Neu1 and MMP-9 cross-talk on the cell surface that is essential for the transactivation of

TOLL-like receptors in the absence of TLR natural ligands and subsequent cellular signaling.

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alpha-2,3-linked beta-galactosyl residues of TOLL-like receptor 4 is essential for receptor activation and cellular signaling. Cell Signal 22, 314-324 18. Cirillo, D. M., Gaudino, G., Naldini, L., and Comoglio, P. M. (1986) Receptor for bombesin with associated tyrosine kinase activity. Mol Cell Biol 6, 4641-4649 19. Liang, F., Seyrantepe, V., Landry, K., Ahmad, R., Ahmad, A., Stamatos, N. M., and Pshezhetsky, A. V. (2006) Monocyte differentiation up-regulates the expression of the lysosomal sialidase, Neu1, and triggers its targeting to the plasma membrane via major histocompatibility complex class II-positive compartments. J Biol Chem 281, 27526-27538 20. van der Spoel, A., Bonten, E., and d'Azzo, A. (1998) Transport of human lysosomal neuraminidase to mature lysosomes requires protective protein/cathepsin A. EMBO J 17, 1588-1597 21. Hinek, A., Pshezhetsky, A. V., Von, I. M., and Starcher, B. (2006) Lysosomal sialidase (neuraminidase-1) is targeted to the cell surface in a multiprotein complex that facilitates elastic fiber assembly. J.Biol.Chem. 281, 3698-3710 22. Duca, L., Blanchevoye, C., Cantarelli, B., Ghoneim, C., Dedieu, S., Delacoux, F., Hornebeck, W., Hinek, A., Martiny, L., and Debelle, L. (2007) The elastin receptor complex transduces signals through the catalytic activity of its Neu-1 subunit. J Biol Chem 282, 12484-12491 23. Eskan, M. A., Rose, B. G., Benakanakere, M. R., Zeng, Q., Fujioka, D., Martin, M. H., Lee, M. J., and Kinane, D. F. (2008) TLR4 and S1P receptors cooperate to enhance inflammatory cytokine production in human gingival epithelial cells. Eur J Immunol 38, 1138-1147 24. Gouwy, M., Struyf, S., Verbeke, H., Put, W., Proost, P., Opdenakker, G., and Van Damme, J. (2009) CC chemokine ligand-2 synergizes with the nonchemokine G protein-coupled receptor ligand fMLP in monocyte chemotaxis, and it cooperates with the TLR ligand LPS via induction of CXCL8. J Leukoc Biol 86, 671-680

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25. Lattin, J. E., Greenwood, K. P., Daly, N. L., Kelly, G., Zidar, D. A., Clark, R. J., Thomas, W. G., Kellie, S., Craik, D. J., Hume, D. A., and Sweet, M. J. (2009) Beta-arrestin 2 is required for complement C1q expression in macrophages and constrains factor-independent survival. Mol Immunol 47, 340-347 26. Serezani, C. H., Lewis, C., Jancar, S., and Peters-Golden, M. (2011) Leukotriene B4 amplifies NF-kappaB activation in mouse macrophages by reducing SOCS1 inhibition of MyD88 expression. J Clin Invest 121, 671-682 27. Kim, S. H., Serezani, C. H., Okunishi, K., Zaslona, Z., Aronoff, D. M., and Peters-Golden, M. (2011) Distinct protein kinase a anchoring proteins direct prostaglandin E2 modulation of toll-like receptor signaling in alveolar macrophages. J Biol Chem 28. Solomon, K. R., Kurt-Jones, E. A., Saladino, R. A., Stack, A. M., Dunn, I. F., Ferretti, M., Golenbock, D., Fleisher, G. R., and Finberg, R. W. (1998) Heterotrimeric G proteins physically associated with the lipopolysaccharide receptor CD14 modulate both in vivo and in vitro responses to lipopolysaccharide. J Clin Invest 102, 2019-2027 29. Ferlito, M., Romanenko, O. G., Guyton, K., Ashton, S., Squadrito, F., Halushka, P. V., and Cook, J. A. (2002) Implication of Galpha i proteins and Src tyrosine kinases in endotoxin-induced signal transduction events and mediator production. J Endotoxin Res 8, 427-435 30. Fan, H., Zingarelli, B., Peck, O. M., Teti, G., Tempel, G. E., Halushka, P. V., Spicher, K., Boulay, G., Birnbaumer, L., and Cook, J. A. (2005) Lipopolysaccharide- and gram-positive bacteria-induced cellular inflammatory responses: role of heterotrimeric Galpha(i) proteins. Am.J.Physiol Cell Physiol 289, C293-C301 31. Fan, H. K., Zingarelli, B., Peck, O. M., Teti, G., Tempel, G. E., Halushka, P. V., and Cook, J. A. (2005) Lipopolysaccharide- and gram-positive bacteria-induced

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Chapter 4

NEU1 Sialidase, Matrix Metalloproteinase-9 and Neuromedin B

Receptor Cross-talk Controls Endosomal TOLL-like Receptor-7 and -9

Responses

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4.1 Abstract The precise mechanism(s) by which intracellular TOLL-like receptors (TLRs) become activated by their ligands remains unclear. Here, we report a molecular organizational G- protein coupled receptor (GPCR) signaling platform to potentiate a novel mammalian neuraminidase-1 (Neu1) and matrix metalloproteinase-9 (MMP9) cross-talk in alliance with neuromedin B GPCR receptor (NMBR), all of which form a tripartite complex with

TLR-7 and -9. siRNA silencing Neu1, MMP9 and NMBR in RAW-blue cells significantly reduced TLR7 imiquimod- and TLR9 CpG ODN-induced NF-κBpSer536 activity. Targeting this novel signaling platform attenuates MyD88 recruitment, ligand- induced NF-κB activity and significantly inhibits the production of pro-inflammatory

TNFα and MCP-1 cytokines. For the first time, a Neu1 and MMP9 cross-talk in alliance with neuromedin B GPCR is uncovered that controls nucleic acid-induced, intracellular

TLR activation and pro-inflammatory responses.

4.2 Introduction Dimerization of the extracellular domain of most mammalian Toll-like receptors (TLRs) is essential for their ligand induced activation. However, the mechanism(s) by which

TLRs become activated by this process is not well understood. For the majority of TLR receptors, dimerization is a prerequisite to facilitate MyD88/TLR complex formation and subsequent cellular signaling to activate NF-κB. However, the parameters controlling interactions between the receptors and their ligands remained poorly defined until now.

For the cell-surface TLRs, we have identified a novel molecular organizational G-protein

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coupled receptor (GPCR) signaling platform to potentiate Neu1 sialidase and matrix metalloproteinase-9 (MMP9) cross-talk in regulating lipopolysaccharide (LPS) induced

TLR4 receptors and cellular function (1-3). For intracellular TLRs, namely TLR-3, -7, -8, and -9, that are known to recognize nucleic acids (4), their subcellular compartmentalization and cellular distribution are maintained in their correct subcellular location by endoplasmic reticulum (ER) chaperone gp96 (5), protein associated with

TLR4 (PRATA/B) (6-8), and the ER membrane protein, UNC93B (9,10). However the mechanistic machinery driving ligand-induced intracellular TLR dimerization and subsequent receptor function is unknown. To gain an insight into the molecular mechanism of intracellular TLR7 triggering, Visintin and colleagues, using N-nitrose-

N9-ethyl urea (ENU)-induced mutations in mice, identified a crucial role for N- glycosylation for TLR7 receptor activation and function (11).

This present report describes a novel glycosylation model in the activation of nucleic acid sensing, intracellular TLR-7 and TLR-9 receptors. It discloses an identical signaling paradigm as described for the cell-surface TLRs (1-3). Here, a Neu1 and MMP9 cross- talk in alliance with GPCR neuromedin-B receptors tethered to TLR-7 and -9 receptors at the ectodomain is essential for ligand activation of the TLRs and cellular signaling. Since

TLR9 receptors undergo ligand-induced conformational changes in their ectodomains

(12), we propose here that ligand binding to TLR-7 and -9 receptors initiates a conformational change to potentiate GPCR-signaling via Gα subunit proteins and MMP9 activation to induce Neu1. Activated Neu1 specifically hydrolyzes α-2,3-sialyl residues

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linked to β-galactosides at the ectodomain of the receptor, the structural perturbation of which facilitates MyD88/TLR recruitment and subsequent pro-inflammatory cell responses.

4.3 Materials and methods

4.3.1 Cell lines The HEK-TLR7-HA cells were obtained by stable transfection of HEK293XL cells with the pUNO-hTLR7-HA plasmid which expresses the human TLR7 gene fused at the 3'end to the influenza hemaglutinin (HA) (Invivogen, San Diego, CA) . The cells were grown at 37oC in 5% CO2 in culture media containing Dulbecco's Modified Eagle Medium

(DMEM) (Gibco, Rockville, MD) supplemented with 10% fetal calf serum (FCS)

(HyClone, Logan, Utah, USA) and selection in 100 μg/mL Normocin™.

RAW-Blue™ cells (Mouse Macrophage Reporter Cell Line, Invivogen, San Diego, CA) derived from RAW 264.7 macrophages were grown in culture medium containing Zeocin as the selectable marker. They stably express a secreted embryonic alkaline phosphatase

(SEAP) gene inducible by NF-kB and AP-1 transcription factors. Upon stimulation,

RAW-Blue™ cells activate NF-kB and/or AP-1 leading to the secretion of SEAP which is detectable and measurable using QUANTI-Blue™, a SEAP detection medium

(Invivogen). RAW-Blue™ Cells are resistant to Zeocin™ and G418 in the conditioned medium.

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RAW264.7 cells were obtained from Dr Andrew Craig (Queen’s University) and maintained in DMEM with 10% FCS and 2 mM L-glutamine at 37 °C in 5% CO2 humidified incubator.

4.3.2 Silencing Neu1, MMP9 and Neuromedin-B (NMBR) mRNA using siRNA Mouse NEU1, MMP9 and NMBR ON-TARGETplus SMART pool, were obtained from

Thermo Scientific Dharmacon each containing a mixture of four predesigned siRNAs targeting one gene. RAW-blue cells were plated in 6-well plate at 3x105 cells/well and incubated at 37oC for 24hr or until 50 -60% confluent. 250 µL OPTIMEM media

(Invitrogen) was pipetted into each of 2 tubes, 100 pmoles siRNA were added to one tube and 6µL Lipofectamine 2000 (Invitrogen) into the other and incubated for 5 minutes. The

Lipofectamine containing tube was transferred into the siRNA containing tube by gently mixing. The mixture was incubated for 20 min at room temperature. During the 20 min incubation, medium was aspirated from the wells containing the cells and replaced with

500 μL of fresh OPTIMEM media and the mixture containing siRNA duplex together with Lipofectamine 2000 complexes was added to each well incubating for 5-6 hrs at 37

°C in a humidified 5% CO2 incubator. At the end of transfection, the mixture was replaced with fresh complete media and plate was incubated for 24 hrs. The process of siRNA transfection was repeated on these same transfected cells. The transfection efficiency of 90% was determined using fluorescein conjugated control siRNAs (Santa

Cruz Biotech) and counting the proportion of labelled cells using fluorescence

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microscopy (Zeiss Imager M2). 72 hours post double siRNA transfection, RAW-blue cells were assayed for protein level to assess silencing.

4.3.3 Ligands TLR7 ligand, Imiquimod from BioVision was used at 10 or 20μg/mL. TLR9 ligand, CpG

ODN (1826) from InvivoGen was used at 10 or 20μg/mL.

4.3.4 Inhibitors Tamiflu (oseltamivir phosphate) was used at indicated concentrations. MMP-3 inhibitor

(MMP3i; Stromelysin-1 Inhibitor, Calbiochem-EMD Chemicals Inc.) inhibits MMP-3.

MMP9 inhibitor (MMP9i, Calbiochem-EMD Chemicals Inc.) a cell-permeable, potent, selective, and reversible MMP9 Inhibitor. BIM-46174 is a G-protein inhibitor kindly provided by IPSEN Innovation. BIM-23127 is a specific neuromedin B receptor inhibitor from TOCRIS bioscience.

4.3.5 NF-κB-dependent secretory alkaline phosphatase (SEAP) assay Briefly, a cell suspension of 1x106 cells/ml in fresh growth medium was prepared, and

100μL of the cell suspension (∼100,000 cells) were added to each well of a Falcon flat- bottom 96-well plate (Becton Dickinson). Different concentrations of either specific

MMP9 inhibitor (MMP9i), Tamiflu or BIM-46174 were added to each well 1 hour before ligand stimulation. The plates were incubated at 370C in a 5% CO2 incubator for 18–24 h. A QUANTI-BlueTM (InvivoGen) solution, which is detection medium developed to determine the activity of any alkaline phosphatase present in a biological sample, was prepared following the manufacturer instructions. Briefly, 160 μL of resuspended 141

QUANTI-Blue solution were added to each well of a flat-bottom 96-well plate, followed by 40 μL of supernatant from stimulated RAW-blue cells. The plate was incubated for 60 min at 370C and the SEAP levels were determined using a spectrophotometer at 620–655 nm. Each experiment was performed in triplicates.

4.3.6 Co-immunoprecipitation Macrophage cells were left cultured in media or in media containing 20 μg/mL

Imiquimod or CpG ODN for indicated time intervals. Cells (1x107 cells) were pelleted and lysed in lysis buffer (50mM Tris, pH 8, 150 mM NaCl, 1% NP-40, 0.2mg/ml) containing Halt Protease and Phosphatase Inhibitor cocktail (Thermo Scientific). For immunoprecipitation, Neu1, MMP-9, TLR7, TLR9 or NMBR in cell lysates from RAW- blue or HEKTLR7cells were immunopreciptated with 1.0 µg of either rabbit anti HA, rabbit anti-Neu1, rabbit anti-MMP-9, rabbit anti-TLR7, rabbit anti TLR9 or rabbit anti-

NMBR antibodies for 24 hrs. Following immunoprecipitation, complexes were isolated using protein A or G magnetic beads, washed 3x in buffer (10mM Tris, pH8, 1mM

EDTA, 1mM EGTA, 150mM NaCl, 1% Triton X-100 and 0.2mM sodium orthovanadate) and resolved on 8% gels by SDS-PAGE electrophoresis. Proteins were transferred to polyvinylidene fluoride (PVDF) transfer membrane blot. The blots were probed for either

MMP-9 with anti-MMP-9 (H-129, Santa Cruz Biotechnology, Inc. CA), Neu1 with anti-

Neu1 (H-300, Santa Cruz Biotech), TLR7 with anti-TLR7 (H-114, Santa Cruz Biotech),

TLR9 with anti TLR9 (H-100, Santa Cruz Biotechnology) or NMBR with anti-NMBR

(M-52, Santa Cruz Biotechnology) followed by HRP conjugated secondary IgG

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antibodies or Clean-Blot IP Detection Reagent for IP/Western blots (Pierce

Biotechnology, Thermo Fisher Scientific, Rockford, IL) and Western Lightning

Chemiluminescence Reagent Plus. The chemiluminescence reaction was analyzed with x- ray film. Sample concentration for gel loading was determined by the Bradford reagent

(Sigma-Aldrich Canada Ltd., Oakville, Ontario).

4.3.7 Neu1 or MMP9 colocalization with TLR-7 and -9 RAW-blue macrophage cells were cultured in DMEM medium with 10% FCS. Cells were treated with 10 µg/mL Imiqumod or 10 µg/mL CpG ODN for 30 min or left untreated as controls. Cells were fixed, permeabilized and immunostained with either rabbit anti-TLR7 (H-114, Santa Cruz Biotech) or rabbit anti-TLR9 (H-100, Santa Cruz

Biotech) and goat anti- MMP-9 (M-17, Santa Cruz Biotechnology) or goat anti-Neu1 (N-

20, Santa Cruz Biotechnology) followed with Alexa Fluor488 Donkey anti-rabbit IgG or

Alexa Fluor594 Donkey anti-goat IgG. Stained cells were visualized using a confocal inverted microscope (Leica TCS SP2 MP inverted Confocal Microscope) with a 100x oil objective. Images were captured using a z-stage of 8–10 images per cell at 0.5-mm steps and were processed using Image J 1.38x software (NIH, USA). To calculate the amount of colocalization in the selected images, the Pearson correlation coefficient was measured and expressed as a percentage using Image J 1.38x software.

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4.3.8 NMBR colocalization with Rab7 RAW-blue mouse macrophage cells were cultured in DMEM medium with 10% FCS.

Cells were fixed, permeabilized and immunostained with rabbit anti- NMBR (M-52,

Santa Cruz Biotechnology, Inc., California, USA) and mouse monoclonal anti-Rab7

(Abcam) followed with Alexa Fluor568 donkey anti-mouse IgG or Alexa Fluor488 donkey anti-rabbit IgG. Stained cells were visualized using a confocal inverted microscope (Leica TCS SP2 MP inverted Confocal Microscope) with a 100x oil objective. Images were captured using a z-stage of 8–10 images per cell at 0.5-mm steps and were processed using Image J 1.38x software (NIH, USA). To calculate the amount of colocalization in the selected images, the Pearson correlation coefficient was measured and expressed as a percentage using Image J 1.38x software.

4.3.9 Bio-Plex cytokine microarray profiles in the cell culture supernatants using the multiplex bead-based assay RAW-blue mouse macrophage cells were grown at 3x104 cells/well in flat-bottom 96-

o well plate at 37 C and 5% CO2 for 18- 24 hours. Media was discarded and 100 µL of fresh media alone or with inhibitors at indicated concentrations added to the wells, and the plate is incubated for 60 mins before adding the specific ligand. The plate was incubated for additional 24 hours. Supernatant was collected and the assay was carried out according to the manufacturer instructions. The cytokines in individual supernatant samples were measured using the Bio-Plex 200 System and quantified using standard curve for each cytokine.

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4.3.10 Statistics Comparisons between two groups were made by one-way ANOVA at 99% confidence using unpaired t-test and Bonferroni’s Multiple Comparison Test or Dunnett’s Multiple

Comparison Test for comparisons among more than two groups.

4.4 Results

4.4.1 NEU1 and MMP-9 are tethered to naive full-length and cleaved TLR7 receptors According to the trafficking and processing models of intracellular TLRs (4), TLR-3, -7,

-8, -9, and murine TLR13, are expressed in intracellular compartments such as the endoplasmic reticulum (ER), endosomes, multi-vesicular bodies and lysosomes.

However, ligand-induced TLR activation only occurs within acidified endolysosomal compartments (13, 14). In these compartments, the TLR7 as well as TLR9 ectodomains are proteolytically cleaved by proteases. The full-length TLR-7 and -9 are found only in the ER, whereas the cleaved, biologically active product is restricted to endolysosome.

Multiple proteases have been implicated in TLR9 cleavage, including various cathepsins and asparagine endopeptidase (13-18) as well as asparagine endopeptidase cleavage of

TLR7 (19).

For the cell-surface TLR4 receptors, we reported a receptor signaling platform involving a process of receptor ligand-induced Gαi-proteins, MMP9 activation and the induction of

Neu1 activation (2). To test whether this same signaling paradigm is involved with intracellular TLRs, we initially asked if Neu1 and MMP9 form a complex with TLR7

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receptors. Co-immunoprecipitation experiments using cell lysates from HEK-TLR7-HA cells demonstrated that both Neu1 and MMP9 are tethered to full-length (120 kDa) and cleaved (72 kDa) TLR7 receptors in naive and 30 min imiquimod stimulated cells (Fig.

4.1A and B). In addition, only Neu1 and Neu4 but not Neu-2 and -3 co- immunoprecipitated with full-length 120kDa TLR7 in the cell lysates of HEK-TLR7-HA cells (Fig. 4.1B). Recent reports have indicated that Neu4 is tethered to MMP9 but requires a special ligand to become activated (20, 21). To further confirm this, Neu1 and

MMP9 were co-immunoprecipitated with TLR7 in cell lysates from naïve and imiquimod-treated RAW-blue macrophage cells (Fig. 4.1C). Using fluorescence microscopy, MMP9 as well as Neu1 colocalized with TLR7 in naïve and imiquimod- treated RAW-blue macrophage cells (Fig. 4.2 A and B).

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Figure 4.1: TLR7 co-immunoprecipitates with NEU1 and MMP9 (A) HEK-TLR7-HA cells were left cultured in media or in media containing 10 μg/mL imiquimod for 30 min. Cells (1×107 cells) were pelleted and lysed in lysis buffer. Neu1, MMP9, TLR7-HA and TLR7 receptors in the cell lysates were immunoprecipitated with 1 μg of rabbit anti-Neu1, rabbit anti-MMP9 or 1 μg of rabbit anti-HA antibodies for 24 h. Following immunoprecipitation, complexes were isolated using protein A or G magnetic beads, washed 3× in buffer and resolved by 8% SDS-PAGE. The blots were probed for HA with anti-HA or TLR7 with anti-TLR7 antibodies followed by Clean-Blot IP Detection Reagent for IP/Western blots and Western Lightning Chemiluminescence Reagent Plus. The chemiluminescence reaction was analyzed with X-ray film. Sample concentration for gel loading was determined by Bradford assay. The data are a representation of one out of three independent experiments showing similar results. (B) Neu1 co-immunoprecipitates with TLR7. HEK-TLR7-HA cells were used as described in (A) the blot probed with antibodies against the indicated proteins. DMSO/PBS: cells were treated with DMSO in PBS before lysing to control for effect of Imiquimod diluent. (C) Neu1 and MMP9 co-immunoprecipitate with TLR7 in RAW blue cells. RAW blue cells lysates were prepared as described for HEK-TLR7 in (A).

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Figure 4.2: NEU1 and MMP9 colocalize with TLR7 RAW-blue macrophage cells were treated with 10 μg/mL imiquimod for 60min or left untreated as controls. Cells were fixed, permeabilized and immunostained with rabbit anti-TLR7 and goat anti NEU1 (A) or goat anti-MMP9 (B) followed with Alexa Fluor488 donkey anti-rabbit IgG or Alexa Fluor594 donkey anti-goat IgG. Stained cells were visualized using a Zeiss M2 imager with a 40x objective. To calculate the amount of colocalization in the selected images, the Pearson correlation coefficient was measured and expressed as a percentage using Image J 1.38x software.

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4.4.2 The effect of Tamiflu, MMP9i, BIM- 23127 and BIM- 46174 on Imiquimod- induced phosphorylation of NF-κB We also asked whether inhibitors of Neu1 and MMP9 would block phosphorylation of

NF-κB associated with imiquimod-treated RAW-bue cells. The data in Fig. 4.3A indicate a differential optimal activation of NF-κBpSer536 associated with imiquimod and CpG

ODN treated RAW-blue cells. Cells treated with imiquimod induced optimal NF-

κBpSer536 activity after 30 min followed by a concomitant decline at 45 and 60 min.

However, CpG ODN induced NF-κBpSer536 activity after 45 min with a marked decline at 60 min. Therefore, we chose these specific times for subsequent western blot experiments.

The inhibitory effect of Tamiflu, MMP9 inhibitor (MMP9i), BIM23127 (a specific antagonist of neuromedin B receptor, NMBR), and BIM-46174 (22) (a selective inhibitor of the whole heterotrimeric G-protein complex) on imiquimod-induced NF-κBpSer536 was examined in RAW-blue macrophage cells. The data in Fig. 4.3B show that these specific compounds inhibited imiquimod-induced NF-κBpSer536 in these cells comparable to the no ligand control levels and to the imiquimod positive control.

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Figure 4.3: Western blot analysis of Imiquimod- and CpG ODN-induced phosphorylated NF-κB in RAW-blue whole cell lysates. (A) RAW-blue macrophage cells stimulated with either 10 μg/ml imiquimod or 10 μg/ml CpG ODN for the indicated time or left untreated as medium control. Cell lysates were separated by SDS-PAGE, and the blot was probed with phospho-specific polyclonal rabbit antibody against NF-κB (Ser(P)536) with minimal reactivity with non-phosphorylated p65. β-Actin was used as an internal control protein for equal loading of the cytoplasmic cell lysate. Quantitative analysis was done by assessing the density of a band corrected for background in each lane using Corel Photo Paint 8.0 software. Each bar in the graphs represents the mean ratio of NF-κB (Ser(P)536) to β- actin band density±S.E. (error bars) for 5–10 replicate measurements. The data are a representation of one of three independent experiments showing similar results.

(B) RAW-blue macrophage cells were pretreated with 200 μM Tamiflu, 100 μg/ml MMP9i, 100μg/ml BIM-23127 or 20μM BIM-46174 for 30 min followed by 10μg/ml imiquimod. RAW- blue siRNA KD Neu1, KD MMP9 and KD NMBR cells were treated with 10μg/ml imiquimod. Cell lysates from the WT and KD cells were separated by SDS-PAGE, and the blot was probed with phospho-specific polyclonal rabbit antibody against NF-κBp65 Ser(P)536. Quantitative analysis was done by assessing the density of a band corrected for background in each lane using Corel Photo Paint 8.0 software. Each bar in the graphs represents the mean ratio of NF-κBp65 Ser(P)536 to β-actin of band density±S.E. (error bars) for 3 independent measurements. p values represent significant differences at 95% confidence using the Dunnett multiple comparison test compared with ligand-treated control cells. (C) RAW-blue cells were transfected with scrambled siRNA and stimulated with10μg/ml imiquimod. β-actin was used as internal control protein for equal loading of the cell lysate and scrambled siRNA transfected cells for transfection control. The data are a representation of one of three independent experiments showing similar results.

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4.4.3 NEU1, MMP-9 and neuromedin B (NMBR) regulate Imiquimid-induced phosphorylation of NFκB. At the genetic level, we transfected RAW-blue and the parental RAW264.7 macrophage cells with either Neu1, MMP9 or NMBR siRNA ON-TARGETplus SMART pool, each containing a mixture of four predesigned siRNAs targeting one gene using OPTIMEM media and Lipofectamine 2000 reagent. Using this transfection protocol and Western blot analyses of whole cell lysates from siRNA MMP9 KD RAW-blue cells, we previously reported a complete knockdown of the active 88 kDa isoform of MMP9 compared to the wild-type (WT), but the pro-active MMP9 (105kDa) protein fragment was still present

(2). Western blot analyses of the WT and siRNA Neu1, MMP9 and NMBR KD in RAW- blue cells revealed a complete reduction of 65kDa NF-κBpSer536 protein levels in the cell lysates from imiquimod-treated siRNA KD cells compared to the WT controls and

β-actin (Fig. 4.3B). Scrambled siRNA had no effect on NF-κBpSer536 levels (Fig. 4.3C).

4.4.4 Tamiflu, MMP9i and neuromedin B receptor (NMBR, BB1) antagonist BIM- 23127 attenuate imiquimod-induced TLR7 recruitment of adaptor molecule MyD88 in RAW-blue cells Since Tamiflu, MMP9i and NMBR antagonist BIM23127 inhibit imiquimod-induced

NF-κBpSer536 in RAW-blue macrophage cells (Fig. 3B), we assessed if they would inhibit imiquimod-mediated recruitment of the adaptor molecule MyD88 to TLR7. Our data clearly show this to be the case. MyD88 co-immunoprecipitates with the 65kDa cleaved TLR7 receptor following stimulation with imiquimod for 30 min, and this co-IP effect was reduced by Tamiflu, MMP9i and BIM23127 (Fig. 4.4). It is well known that

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Figure 4.4: MMP9i, Tamiflu and BIM23127 attenuate MyD88 recruitment. RAW-blue macrophage cells were pretreated with 200 μM Tamiflu, 100 μg/ml MMP9i, or 100 μg/ml BIM-23127 for 30 min followed by 10μg/ml imiquimod or left cultured in media. Cells (1x107 cells) are pelleted and lysed in lysis buffer. TLR7 in cell lysates from RAW-blue cells are immunopreciptated with 1.0 µg of rabbit anti-TLR7 antibodies for 24 hrs. Following immunoprecipitation, complexes are isolated using protein A or G magnetic beads, washed 3x in buffer and resolved by 8% SDS-PAGE. The blots are probed for MyD88 (33 kDa) with anti- MyD88 antibodies followed by Clean-Blot IP Detection Reagent for IP/Western blots and Western Lightning Chemiluminescence Reagent Plus. The chemiluminescence reaction was analyzed with x-ray film. Sample concentration for gel loading was determined by Bradford assay.

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both TLR-7 and -9 are completely dependent upon MyD88 in eliciting a signal (4), and our data are consistent with this model. However, the process (es) by which nucleic acid sensing TLRs undergo ligand-induced conformational changes to make them competent to signal is not well understood. For efficient TLR7 signaling, cleavage of the TLR7 receptor is required for ligand binding and subsequent association with MyD88. Our data here support another level of a unique organizational Neu1-MMP9-NMBR signaling platform tethered to TLR7 receptors as the final processing stage for imiquimod-induced activation of these receptors and subsequent recruitment of the adaptor molecule MyD88.

4.4.5 Neu1 and MMP-9 form a complex with TLR9 receptor If Neu1 and MMP9 complex is tethered to TLR7 receptors, we asked whether Neu1 and

MMP9 would also form a complex with intracellular TLR9 receptors. Fluorescence microscopy revealed the intracellular colocalization of Neu1 and MMP9 with TLR9 in naïve and CpG ODN-treated RAW-blue macrophage cells (Fig. 4.5A and B). Co- immunoprecipitation experiments using cell lysates from RAW-blue cells further demonstrated that Neu1 forms a complex with TLR9 receptors in naïve and CpG ODN treatment of cells for 30 min (Fig. 4.5C). In addition, the two active isoforms of MMP9

(65 and 80 kDa) were found to co-immunoprecipitate with TLR9 in the cell lysates of

RAW-blue cells (Fig. 4.5C).

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Figure 4.5: NEU1 and MMP9 are tethered to TLR9. (A) Neu1 and (B) MMP9 colocalizes with TLR9. RAW-blue macrophage cells were treated with 10 μg/mL CpG ODN for 60min or left untreated as controls. Cells were fixed, permeabilized and immunostained with rabbit anti- TLR9, goat anti-Neu1 or goat anti- MMP9 followed with Alexa Fluor488 donkey anti-rabbit IgG or Alexa Fluor594 donkey anti-goat IgG. Stained cells were visualized using a Zeiss M2 imager with a 40x objective. To calculate the amount of colocalization in the selected images, the Pearson correlation coefficient was measured and expressed as a percentage using Image J 1.38x software. (C) Neu1 and MMP9 co-immunoprecipitate with TLR9. RAW-blue macrophage cells are left cultured in media or in media containing 10 μg/mL imiquimod for the indicated time intervals. Cells (1x107 cells) are pelleted and lysed in lysis buffer. TLR9 in cell lysates from RAW-blue cells are immunopreciptated with 1.0µg of rabbit anti-TLR9 antibodies for 24 hrs. Following immunoprecipitation, complexes are isolated using protein A or G magnetic beads and resolved by 8% SDS-PAGE. The blots are probed for MMP9 and Neu1 with specific antibodies followed by Clean-Blot IP Detection Reagent for IP/Western blots and Western Lightning Chemiluminescence Reagent Plus. The chemiluminescence reaction was analyzed with x-ray film. Protein concentration for gel loading was determined by Bradford assay.

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4.4.6 Neu1 and MMP-9 cross-talk is essential for TLR9 receptor activation If Neu1 and MMP9 regulate TLR9 activation, inhibitors against these enzymes should block CpG ODN-induced phosphorylation of NFκB in RAW-blue cells. Cells treated with MMP9i and Tamiflu inhibited CpG-induced pNFκB in the cell lysates compared to the CpG-treated positive control and the β-actin (Fig. 4.6A). Live cells pretreated with

MMP3i or anti-Neu1 antibody followed with CpG stimulation showed no reduction of pNF-κB activity in the cell lysates. The anti-Neu1 antibody treatment of the live cells would neutralize the cell surface Neu1 activity, but not expected to internalize into the endosomal compartment to neutralize Neu1 activity associated with endosomal TLRs.

Western blot analyses of the WT and siRNA Neu1 KD, MMP9 KD and NMBR KD in

RAW264.7 cells revealed a significant reduction of 65kDa NF-κBpSer536 protein levels in the cell lysates prepared from CpG ODN-treated cells compared to the WT controls and β-actin (Fig. 4.6B). Scrambled siRNA had no significant effect on NF-κBpSer536 levels (Fig. 4.6B).

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Figure 4.6: Western blot analysis of CpG ODN-induced phosphorylated NF-κB in whole cell lysates. (A) Cells were pretreated with 200μM Tamiflu, 100μg/ml MMP9i, 100μg/ml MMP3i or 50μg/ml anti-Neu1 for 30 min followed by 10μg/ml CpG ODN or left untreated as medium control. Cell lysates were separated by SDS-PAGE, and the blot was probed with phospho-specific polyclonal rabbit antibody against NF-κB Ser(P)311 with minimal reactivity with non-phosphorylated NK- κB. β-actin was used as internal control proteins for equal loading of the cytoplasmic cell lysate. The data are a representation of one of three independent experiments showing similar results. (B) RAW264.7 macrophage cells and RAW264.7 siRNA KD Neu1, KD MMP9, KD NMBR and scrambled siRNA cells were treated with 10μg/ml CpG ODN for 45min. Cell lysates from the WT and KD cells were separated by SDS-PAGE, and the blot was probed with phospho-specific polyclonal rabbit antibody against NF-κBp65 Ser(P)536 with minimal reactivity with non- phosphorylated p65. Quantitative analysis was done by assessing the density of a band corrected for background in each lane using Corel Photo Paint 8.0 software. Each bar in the graphs represents the mean ratio of NF-κBp65 Ser(P)536 to β-actin of band density±S.E. (error bars) for 3 independent measurements. p values represent significant differences at 99.9% confidence using the Dunnett multiple comparison test compared with ligand-treated control cells.

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4.4.7 Tamiflu and MMP9i significantly blocked Imiquimod and CpG induced SEAP To further confirm Neu1 and MMP9 linkage with imiquimod-and CpG-induced NFκB activation, we also performed the NFκB-dependent SEAP assay. Secreted SEAP is detectable and measurable using QUANTI-Blue substrate. Here, we used Tamiflu, BIM-

46174, MMP9i as well as the specific inhibitor of MMP3 (MMP3i) to determine their inhibitory effects on imiquimod-or CpG-induced SEAP activity in live RAW-blue cells.

The NFκB-dependent SEAP assay shown here clearly demonstrates that MMP9i

(IC50=79μM), Tamiflu (IC50=240μM) and BIM-46174 (IC50=7.9μM) significantly blocked SEAP activity compared to a lack of inhibition with MMP3i associated with imiquimod treated live RAW-blue macrophage cells (Fig. 4.7A).

Similarly, the data in Fig. 4.7 B clearly show that MMP9i (IC50=199μM), Tamiflu

(IC50=398μM) and BIM-46174 (IC50=20μM) but not MMP3i significantly blocked the

NFκB-dependent SEAP activity associated with CpG treated live RAW-blue macrophage cells.

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Figure 4.7: Tamiflu and MMP9i inhibit NF-κB-dependent SEAP activity. SEAP reporter-expressing RAW-Blue cells were treated with different doses of Tamiflu, MMP9 inhibitor , BIM 46174 or MMP3 inhibitor and stimulated with Imiquimod (A) or CpG (B) for 24 h, and SEAP activity in the culture medium was assessed using Quanti-blue substrate. Results are representative of three experiments. Relative SEAP activity was calculated as fold change of each compound (SEAP activity in medium from treated cells minus no cell background over SEAP activity in medium from untreated cells minus background). The IC50 of each compound was determined by plotting the decrease in SEAP activity against the log of the agent concentration. The data are a representation of mean±S.E. (n=3 independent experiments).

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4.4.8 Isoforms of neuromedin B receptor co-immunoprecipitate with TLR-7 and -9 receptors in cell lysates from naive and ligand stimulated RAW-blue macrophage cells Since NMBR antagonist BIM-23127 and the heterotrimeric G-protein complex antagonist BIM-46174 markedly blocked NF-κB activation associated with imiquimod- and CpG ODN- stimulated RAW-blue cells (Fig. 4.3B and Fig. 4.7B) and MyD88 recruitment associated with imiquimod-stimulated cells (Fig. 4.4), we questioned whether

NMBR forms a complex with TLR-7 and -9 receptors. Co-immunoprecipitation experiments using cell lysates from RAW-blue macrophage cells showed that NMBR is tethered to TLR-9 (Fig. 4.8A) and TLR-7 (Fig. 4.8B) receptors in naïve and ligand treated cells. In addition, BIM-23127 and BIM-46174 significantly inhibited SEAP activity associated with CpG ODN (Fig. 4.8C) and imiquimod (Fig. 4.8D) stimulation of live RAW-blue cells. The results from the NFκB-dependent SEAP assay shown in Fig.

4.8E demonstrate that siRNA NMBR KD in RAW-blue cells significantly inhibited

SEAP activity associated with imiquimod stimulation compared to the WT cells.

Collectively, the data validated the predicted alliance between nucleic acid sensing TLR-

7 and -9 receptors and NMBR in naive and ligand stimulated macrophage cells. They also support recent evidence for a novel concept of intracellular GPCR signaling where it suggests new and intriguing scenarios for the functions of GPCRs in the endocytic compartments (23,24).

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Figure 4.8: NMBR regulates TLR7 and TLR9 Neuromedin B receptor (NMBR) co-immunoprecipitates with TLR9 (A) and TLR7 (B). RAW- blue macrophage cells were left cultured in media or in media containing 10 μg/mL CpG ODN or imiquimod for 30 min. Cells (1×107 cells) were pelleted and lysed in lysis buffer. NMBR and TLR-7 and -9 receptors in the cell lysates from cells were immunoprecipitated with 1 μg of goat anti-NMBR, 1 μg of rabbit anti-TLR7 or rabbit anti-TLR9 antibodies for 24 h. Following immunoprecipitation, complexes were isolated using protein A or G magnetic beads, washed 3× in buffer and resolved by 8% SDS-PAGE. The blots are probed for NMBR with rabbit anti- NMBR or TLR7 or TLR9 with respective antibodies followed by Clean-Blot IP Detection Reagent for IP/Western blots and Western Lightning Chemiluminescence Reagent Plus. The chemiluminescence reaction was analyzed with X-ray film. Sample concentration for gel loading was determined by Bradford assay. (B) BIM-23127 and (C) BIM-46174 inhibit NF-κB-dependent SEAP activity. SEAP reporter-expressing RAW-Blue cells were treated with 85μM BIM-23127 or 100μM BIM-46174 plus indicated ligand for 24 h, and SEAP activity in the culture medium was assessed using Quanti-blue substrate. Results are representative of three experiments. Relative SEAP activity was calculated as fold increase of each compound (SEAP activity in medium from treated cells minus no cell background over SEAP activity in medium from untreated cells minus background). The data are the mean±S.E. (error bars) of three independent experiments. p values represent significant differences at 99% confidence using the Dunnett multiple comparison test compared with ligand-treated control cells. (E) siRNA NMBR RAW- blue cells inhibit NF-κB-dependent SEAP activity. WT RAW-Blue and siRNA NMBR KD RAW-blue cells were treated with imiquimod for 24 h, and SEAP activity in the culture medium was assessed using Quanti-blue substrate. Results are representative of three experiments. (F) NMBR colocalizes with Rab7. RAW-blue macrophage cells were left untreated as controls. Cells were fixed, permeabilized and immunostained with rabbit anti-NMBR and mouse anti-Rab7 followed with Fluor568 donkey anti-mouse IgG or Alexa Fluor488 donkey anti-rabbit IgG. Stained cells were visualized using a Zeiss M2 imager with a 40x objective. To calculate the amount of colocalization in the selected images, the Pearson correlation coefficient was measured and expressed as a percentage using Image J 1.38x software.

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To confirm the location of NMBR in the endocytic compartment, we asked whether the small GTP binding protein Rab7 which has a role in the late endocytic pathway and lysosome biogenesis would colocalize with NMBR. The data in Fig. 4.8F show this to be the case. NMBR colocalizes with Rab7 associated with naïve RAW-blue cells.

4.4.9 Tamiflu, MMP9i and the heterotrimeric G-protein complex antagonist, BIM- 46174, abrogates pro-inflammatory cytokines To further confirm the molecular signaling platform of Neu1 and MMP9 cross-talk in alliance with NMBR in regulating nucleic acid sensing TLR-7 and -9 receptors, we assessed cytokine profiles in the tissue culture supernatants using the multiplex bead- based assays designed to quantitate multiple cytokines in a single sample. The Bio-Plex suspension array system incorporates a novel technology using color-coded beads, which permits the simultaneous detection of cytokines in a single well of a 96-well microplate.

The 96-well microplate-format Bio-Plex assays are optimized for the Bio-Plex suspension array system, which utilizes xMAP detection technology. Here, the chemokine (C-C motif) ligand 2 (CCL2) also referred to as monocyte chemotactic protein-1 (MCP-1) and TNFα were analyzed in tissue culture supernatants after 24 hr stimulation of RAW-blue macrophage cells with either imiquimod or CpG ODN.

Tamiflu, MMP9i and the heterotrimeric G-protein complex antagonist, BIM-46174 significantly and dose-dependently abrogated MCP-1 and TNFα secretion associated with imiquimod- and CpG ODN-stimulated RAW-blue cells after 24 hr (Fig. 4.9A and B).

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Figure 4.9: Bio-Plex cytokine microarray profiles in the cell culture supernatants. RAW-blue cells were cultured in 96-well microplate at 30,000 cells per well for 24 hr followed by pretreatment with Tamiflu, MMP9i and BIM-46174 at the indicated doses for 1 hr. The pretreated cells were stimulated for 24hrs with either 20μg/mL imiquimod (A) or CpG ODN (B). The Bio-Plex suspension array system was optimized using the xMAP detection technology. The quantitation of the level of TNFα and MCP-1 cytokines in a single well of tissue culture sample was assessed from a standard curve. Each bar in the graphs represents the mean±S.E. (error bars) three separate experiments. p values represent significant differences at 99.9% confidence intervals using the Dunnett's multiple comparison test compared with ligand-treated positive controls.

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4.5 Discussion The precise molecular mechanism(s) by which nucleic acid sensing TOLL-like receptors becomes activated is not well understood. For the majority of cell-surface TLR receptors, dimerization is a prerequisite to facilitate MyD88/TLR complex formation and subsequent cellular signaling to activate NF-κB. For intracellular TLR3 receptors, they also form dimers upon interaction of dsRNA. In contrast, the intracellular TLR8 (25) and

TLR9 (12, 26) appear to exist as preformed naïve signaling dimers. For TLR9, it is proposed that nucleic acids binding to two distinct binding sites (26, 27) in the ectodomain of the receptors induce conformational changes which enable subsequent signaling. For TLR7 however, the precise mechanism(s) of nucleic acid action on the receptors is unknown. In this report, we uncovered a mechanism of signaling by nucleic acid ligands binding to TLR-7 and -9, which is orchestrated by a novel Neu1 and MMP9 cross-talk in alliance with NMBR GPCR. This signaling paradigm was elucidated to be essential for subsequent nucleic acid activation of intracellular TLR7 and TLR9 receptors. In addition, targeting Neu1, MMP9 and the heterotrimeric G-protein complex tethered to both TLR-7 and -9 receptors in live RAW-blue macrophage cells significantly abrogates pro-inflammatory cytokines MCP-1 and TNFα. Indeed, other studies have provided evidence to show that CpG ODN induces TNFα and TNFR-II at the transcriptional level and that these molecular players are involved in MMP9 expression in the supernatants derived from murine RAW264.7 macrophage cell line by a TLR9 and

Akt-mediated mechanism (28, 29). This CpG ODN-induced MMP9 expression fits well within our novel Neu-1 and MMP9 cross-talk signaling paradigm for TLR9 receptors. 166

We previously reported that Neu1 is an important intermediate in the initial process of cell surface TLR ligand-induced receptor activation and subsequent cellular function (30, 31). The data also provided evidence for a physiological relevance of Neu1 in regulating LPS induced pro-inflammatory cytokines and nitric oxide production (31).

Collectively, pro-inflammatory IL-6 and TNFα cytokines and nitric oxide production associated with endotoxin LPS activation of cell surface TLR4 receptors were determined to be partially dependent on Neu1 in juxtaposition with MMP9 and GPCR signaling (31,

20). The data in this present report provide additional evidence to support an identical signaling platform in controlling nucleic acid-induced endosomal TLRs to produce TNFα and MCP-1 cytokines.

It has become evident that TLRs require additional proteins to be activated by their respective ligands. For an example, not only CD14 is associated with MyD88- dependent TLR4 receptors on the cell surface, but it also constitutively interacts with the

MyD88-dependent TLR7 and TLR9 receptors (32). It was found that CD14 was necessary for TLR7- and TLR9-dependent induction of pro-inflammatory cytokines in vitro and for TLR9-dependent innate immune responses in mice. In addition, the absence of CD14 led to a reduced nucleic acid uptake in macrophages. Using various types of vesicular stomatitis virus, the report showed that CD14 is dispensable for viral uptake but is required for the triggering of TLR-dependent cytokine responses (32). These findings suggested that CD14 has a dual role in nucleic acid–mediated TLR activation whereby it can promote the selective uptake of nucleic acids, and at the same time, it acts as a co-

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receptor for endosomal TLR activation. Interestingly, others have demonstrated other important roles for CD14 (33-35) especially those that involve the heterotrimeric G proteins.

In this report, the involvement of heterotrimeric G-protein complex in TLR ligand-mediated receptor function has also been shown for endosomal TLRs. The neuromedin B receptor antagonist BIM-23127 and the heterotrimeric G-protein complex antagonist BIM-46174 markedly blocked NF-κB activation and reduced MyD88 recruitment associated with imiquimod- and CpG ODN-stimulated RAW-blue cells.

Collectively, the data validated the predicted alliance between nucleic acid sensing TLR-

7 and -9 receptors and neuromedin B receptor localized in the late endocytic lysosomal pathway of naïve macrophage cells. This TLR signaling paradigm also predicts that endosomal TLR receptors are in alliance with a functional GPCR signaling complex. In support of this premise, our studies and others have provided important evidence to show that the crosstalk between GPCR and TLR signaling pathways and the GPCR signaling molecules may have uncharacterized functions in macrophage cells (36, 1-3).

Although it is not recognized, the diverse multiple actions of GPCRs regulating

TLRs and its translation to human disease has yet to be uncovered (1). Accordingly, the regulation and sorting of GPCRs by endocytic membrane trafficking and its potential implications is eloquently reviewed by Hanyaloglu and colleagues (37, 38) and Marchese et al. (39). Indeed, achieving a coordinated regulation of multiple receptor-mediated signaling is one of the mandates for GPCRs which represent the largest family of

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signaling receptors expressed in animals and respond to a wide range of stimuli. The evidence suggests an unprecedented degree of specificity and plasticity in the cellular regulation of mammalian GPCRs by endocytic membrane trafficking (37). The diverse physiological roles played by GPCRs, together with evidence for disordered GPCR signaling in various pathological conditions are indicated by (a) dysregulation of GPCR function (40), (b) the ‘loss of function’ mutation or the ‘gain of function’ mutation (41),

(c) constitutively active GPCRs especially those that cause syndromes of hyperfunction and/or tumors in humans including diseases involving infectious viral agents (40, 42) and

(d) the mutant GPCRs as a cause of human diseases (43). To date, over 600 inactivating and almost 100 activating mutations in GPCR have been identified, which are responsible for more than 30 different human diseases. The number of human disorders is expected to increase given the fact that over 160 GPCRs have been targeted in mice (43).

Collectively, the parameters controlling interactions between the TLRs and their ligands have remained poorly defined until now. This report proposes that Neu1 is an important intermediate player in the initial process of endosomal TLR ligand-induced receptor activation and subsequent cellular function. Central to this process is that Neu1 and MMP9 in alliance with the 63 kDa isoform of NMBR receptor are tethered to TLR-7 and -9 receptors in naïve TLR-expressing cells. This paradigm signifies an unprecedented molecular GPCR signaling platform of a Neu1 and MMP9 cross-talk in alliance with endosomal TLR-7 and -9 that is essential for nucleic acid activation of endosomal TLRs and subsequent cellular signaling.

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Morishita, Y., Miyazono, K., Kato, S., Saftig, P., and Takayanagi, H. (2008) Cathepsin K-dependent toll-like receptor 9 signaling revealed in experimental arthritis. Science 319, 624-627 16. Fukui, R., Saitoh, S., Matsumoto, F., Kozuka-Hata, H., Oyama, M., Tabeta, K., Beutler, B., and Miyake, K. (2009) Unc93B1 biases Toll-like receptor responses to nucleic acid in dendritic cells toward DNA- but against RNA-sensing. J Exp Med 206, 1339-1350 17. Matsumoto, F., Saitoh, S., Fukui, R., Kobayashi, T., Tanimura, N., Konno, K., Kusumoto, Y., Akashi-Takamura, S., and Miyake, K. (2008) Cathepsins are required for Toll-like receptor 9 responses. Biochem Biophys Res Commun 367, 693-699 18. Sepulveda, F. E., Maschalidi, S., Colisson, R., Heslop, L., Ghirelli, C., Sakka, E., Lennon-Dumenil, A. M., Amigorena, S., Cabanie, L., and Manoury, B. (2009) Critical role for asparagine endopeptidase in endocytic Toll-like receptor signaling in dendritic cells. Immunity 31, 737-748 19. Maschalidi, S., Hassler, S., Blanc, F., Sepulveda, F. E., Tohme, M., Chignard, M., van Endert, P., Si-Tahar, M., Descamps, D., and Manoury, B. (2012) Asparagine endopeptidase controls anti-influenza virus immune responses through TLR7 activation. PLoS pathogens 8, e1002841 20. Finlay, T. M., Abdulkhalek, S., Gilmour, A., Guzzo, C., Jayanth, P., Amith, S. R., Gee, K., Beyaert, R., and Szewczuk, M. R. (2010) Thymoquinone-induced Neu4 sialidase activates NFκB in macrophage cells and pro-inflammatory cytokines in vivo. Glycoconjugate Journal 27, 583-600 21. Finlay, T. M., Jayanth, P., Amith, S. R., Gilmour, A., Guzzo, C., Gee, K., Beyaert, R., and Szewczuk, M. R. (2010) Thymoquinone from nutraceutical black cumin oil activates Neu4 sialidase in live macrophage, dendritic, and normal and type I sialidosis human fibroblast cells via GPCR Galphai proteins and matrix metalloproteinase-9. Glycoconjugate Journal 27, 329-348

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22. Prevost, G. P., Lonchampt, M. O., Holbeck, S., Attoub, S., Zaharevitz, D., Alley, M., Wright, J., Brezak, M. C., Coulomb, H., Savola, A., Huchet, M., Chaumeron, S., Nguyen, Q. D., Forgez, P., Bruyneel, E., Bracke, M., Ferrandis, E., Roubert, P., Demarquay, D., Gespach, C., and Kasprzyk, P. G. (2006) Anticancer activity of BIM-46174, a new inhibitor of the heterotrimeric Galpha/Gbetagamma protein complex. Cancer Res 66, 9227-9234 23. Calebiro, D., Nikolaev, V. O., and Lohse, M. J. (2010) Imaging of persistent cAMP signaling by internalized G protein-coupled receptors. Journal of molecular endocrinology 45, 1-8 24. Calebiro, D., Nikolaev, V. O., Persani, L., and Lohse, M. J. (2010) Signaling by internalized G-protein-coupled receptors. Trends Pharmacol Sci 31, 221-228 25. Zhu, J., Brownlie, R., Liu, Q., Babiuk, L. A., Potter, A., and Mutwiri, G. K. (2009) Characterization of bovine Toll-like receptor 8: ligand specificity, signaling essential sites and dimerization. Mol Immunol 46, 978-990 26. Peter, M.E., Kubaranko, A.V., Weber, A.N., and Dalpke, A.H. (2009) Identification of an N-terminal recognition site in TLR9 that contributes to CpG-DNA-mediated receptor activation. J Immunol : 7690-7697. 27. Rutz, M., Metzger, J., Gellert, T., Luppa, P., Lipford, G. B., Wagner, H., and Bauer, S. (2004) Toll-like receptor 9 binds single-stranded CpG-DNA in a sequence- and pH-dependent manner. Eur J Immunol 34, 2541-2550 28. Lim, E. J., Lee, S. H., Lee, J. G., Chin, B. R., Bae, Y. S., Kim, J. R., Lee, C. H., and Baek, S. H. (2006) Activation of toll-like receptor-9 induces matrix metalloproteinase-9 expression through Akt and tumor necrosis factor-alpha signaling. FEBS Lett. 580, 4533-4538 29. Lim, E. J., Lee, S. H., Lee, J. G., Kim, J. R., Yun, S. S., Baek, S. H., and Lee, C. (2007) Toll-like receptor 9 dependent activation of MAPK and NF-kB is required for the CpG ODN-induced matrix metalloproteinase-9 expression. Exp.Mol.Med. 39, 239-245

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30. Amith, S.R., Jayanth, P., Franchuk, S., Finlay, T., Seyrantepe, V., Gee, K., Basta, S., Beyaert, R., Pshezhetsky, A.V., and Szewczuk, M.R. (2010) Neu1 desialylation of sialyl alpha-2,3-linked beta-galactosyl residues of Toll-like receptor 4 is essential for receptor activation and cellular signaling. Cell Signal 22, 314-324 31. Amith, S.R., Jayanth, P., Franchuk, S., Siddiqui, S., Seyrantepe, V., Gee, K., Basta, S., Beyaert, R., Pshezhetsky, A.V., and Szewczuk, M.R. (2009) Dependance of pathogen molecule-induced Toll-like receptor activation and cell function on Neu1 sialidase. Glycoconj J 26, 1197-1212 32. Baumann, C. L., Aspalter, I. M., Sharif, O., Pichlmair, A., Bluml, S., Grebien, F., Bruckner, M., Pasierbek, P., Aumayr, K., Planyavsky, M., Bennett, K. L., Colinge, J., Knapp, S., and Superti-Furga, G. (2010) CD14 is a coreceptor of Toll- like receptors 7 and 9. J Exp Med 207, 2689-2701 33. Solomon, K. R., Kurt-Jones, E. A., Saladino, R. A., Stack, A. M., Dunn, I. F., Ferretti, M., Golenbock, D., Fleisher, G. R., and Finberg, R. W. (1998) Heterotrimeric G proteins physically associated with the lipopolysaccharide receptor CD14 modulate both in vivo and in vitro responses to lipopolysaccharide. J Clin Invest 102, 2019-2027 34. Ferlito, M., Romanenko, O. G., Guyton, K., Ashton, S., Squadrito, F., Halushka, P. V., and Cook, J. A. (2002) Implication of Galpha i proteins and Src tyrosine kinases in endotoxin-induced signal transduction events and mediator production. J Endotoxin Res 8, 427-435 35. Fan, H., Zingarelli, B., Peck, O. M., Teti, G., Tempel, G. E., Halushka, P. V., Spicher, K., Boulay, G., Birnbaumer, L., and Cook, J. A. (2005) Lipopolysaccharide- and gram-positive bacteria-induced cellular inflammatory responses: role of heterotrimeric Galpha(i) proteins. Am.J.Physiol Cell Physiol 289, C293-C301 36. Lattin, J., Zidar, D. A., Schroder, K., Kellie, S., Hume, D. A., and Sweet, M. J. (2007) G-protein-coupled receptor expression, function, and signaling in macrophages. J.Leukoc.Biol. 82, 16-32

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37. Hanyaloglu, A. C., and von Zastrow, M. (2008) Regulation of GPCRs by endocytic membrane trafficking and its potential implications. Annu Rev Pharmacol Toxicol 48, 537-568 38. Jean-Alphonse, F., and Hanyaloglu, A. C. (2011) Regulation of GPCR signal networks via membrane trafficking. Molecular and cellular endocrinology 331, 205-214 39. Marchese, A., Paing, M. M., Temple, B. R., and Trejo, J. (2008) G protein- coupled receptor sorting to endosomes and lysosomes. Annu Rev Pharmacol Toxicol 48, 601-629 40. Arvanitakis, L., Geras-Raaka, E., and Gershengorn, M. C. (1998) Constitutively signaling G-protein-coupled receptors and human disease. Trends Endocrinol Metab 9, 27-31 41. Spiegel, A. M., and Weinstein, L. S. (2004) Inherited diseases involving g proteins and g protein-coupled receptors. Annual review of medicine 55, 27-39 42. Tao, Y. X. (2008) Constitutive activation of G protein-coupled receptors and diseases: insights into mechanisms of activation and therapeutics. Pharmacol Ther 120, 129-148 43. Schoneberg, T., Schulz, A., Biebermann, H., Hermsdorf, T., Rompler, H., and Sangkuhl, K. (2004) Mutant G-protein-coupled receptors as a cause of human diseases. Pharmacol Ther 104, 173-206

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Chapter 5

General Discussion and Perspectives

The mammalian TLRs are sensor receptors that recognize pathogen-associated molecular patterns. Not only are TLRs important sensors of microbial infections for innate immune cells, they also play important roles in the pathophysiology of inflammatory and autoimmune diseases. Thus, the intensity and duration of TLR responses with these diseases must be tightly controlled. Accordingly, characterizing of

TLRs structural integrity, their interaction with their ligand and their signaling components will help us understand their subsequent immunological responses.

Although the signaling pathways of TLR sensors are well characterized, the parameters controlling interactions between TLRs and their ligands have remained poorly defined until now. For the majority of TLR receptors, dimerization is a confirmed prerequisite to facilitate MyD88-TLR complex formation and subsequent cellular signaling to activate NFκB. Previous reports from our lab identified NEU1 to be an important intermediate in the initial process of TLR ligand induced receptor activation and subsequent cell function (1,2); however, the molecular mechanism(s) involved in

NEU1 activation remains to be uncovered. This thesis provides evidence for an unprecedented membrane signaling paradigm initiated by TLR ligands binding to their respective receptors. The results indicated that the interaction of TLR ligands with their receptors potentiates GPCR-signaling via membrane Gαi subunit proteins and MMP-9 activation to induce NEU1 activity. NEU1 together with GPCR-signaling Gαi subunit 176

proteins and MMP-9 form a complex with TLR receptor on the cell surface of TLR- expressing cells. This tripartite complex makes NEU1 readily available to be induced by

TLR ligands binding to their receptors. It has been shown that agonist-bound GPCRs activate numerous MMPs (3) including MMP-3 (4), MMP-2 and MMP-9 (5,6). However, the precise mechanism by which GPCR could activate each of the aforementioned MMPs is unclear until now and would be a potential future work. These findings suggest that following ligand binding the receptor undergoes a conformational change that activates a

GPCR most probably tethered to the TLR receptor complex. In support of our speculation, a substantial conformational change in the TLR9 ectodomain was previously reported following CpG-ODN ligand binding to its receptor (7). This induced- conformational change may be a common feature of all TLRs, that may then play a role in GPCR activation. Indeed, TLR4 was also reported to undergo a conformational change upon LPS binding (8). It is also possible that LPS binding to CD14 in the TLR4 receptor complex mediates the activation of GPCR through the Gα subunit that was reported to interact with CD14. While, in this thesis, I did not confirm CD14 involvement in this model, investigating the role of CD14 in the suggested signaling paradigm may lead to a wider understanding of the molecular mechanisms involved in TLR signaling.

It is known that GPCR activates MMP-9 which is a gelatinase enzyme with elastase activity. It is proposed that activated MMP-9 will most likely degrade the elastin binding protein (EBP) in the NEU1 complex. This in turn leads to NEU1 activation. Activated NEU1 hydrolyzes the terminal α-2,3 sialyl residues bound to the β-

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galactosides in the glycan chain on the TLR ectodomain. The hydrolysis of these sialic acid residues removes steric hindrance to receptor dimerization which then brings the

TIR domains into close proximity serving as a platform for adaptor protein binding and the initiation of TLR signaling. Figure 5.1 presents a model for this new signaling paradigm associated with TLR4 on the cell surface. Our data recognizes new players directly involved in TLR signaling: GPCRs, MMP-9and NEU1, which further enrich the available literature on the biology of these receptors and enzymes in addition to elucidating a novel mechanism by which TLRs are activated.

In Chapter 3 of this thesis, we investigated the role of GPCR in TLR activation, with a specific focus on the mechanism(s) behind GPCR agonist-induced transactivation of TLRs in the absence of TLR ligands. Our data show that GPCR agonists such as bombesin, lysophosphatidic acid (LPA), cholesterol, angiotensin-1 and -2, and bradykinin binding to their respective GPCR receptors can induce NEU1 activity within

1 minute and that this activity is blocked by Gαi-sensitive pertussis toxin, neuraminidase inhibitor Tamiflu, broad range MMP inhibitors galardin and piperazine, anti-NEU1 and anti-MMP9 antibodies, and siRNA knockdown of MMP9. The rapidity of the GPCR agonist-induced NEU1 activity suggests that glycosylated receptors like

TLR receptors form a functional GPCR signaling complex. In support of this hypothesis, we found that the bombesin-related neuromedin-B receptor forms a complex tethered to

TLR4 receptors on the cell surface in BMA macrophage cells. These data are consistent with the reports describing a cross-talk between GPCR and TOLL-like receptor (TLR)

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Figure 5.1: Model of TLR4 signaling platform. Upon TLR4 ligand (LPS) binding to the receptor complex consisting of CD14-MD2-TLR4 on the cell surface a conformational change in the TLR4 potentiates GPCR signaling through the Gαi protein which activates MMP-9. Activated MMP-9 with elastase activity degrades the elastin binding protein in the NEU1 complex (NEU1-PPCA-EBP) which initiates NEU1 activation. Activated NEU1 targets and hydrolyzes the α-2,3 sialyl residue on the TLR4 ectodomain which then removes steric hinderance to TLR4 receptor dimerization providing a platform for MyD88 recruitment to initiate a signaling cascade leading to NFκB activation and translocation into the nucleos to transcribe inflammatory responses.

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signaling pathways (9,10). In addition, we have shown that other GPCR-specific agonists binding to their respective receptors induce sialidase activity in live macrophage cells.

Using MyD88 homodimerization inhibitor peptide, we have reported that GPCR bombesin activation of NFκB in macrophage cells is initially mediated via a novel molecular GPCR organizational signalling platform in complex with the TLR receptor as previously proposed in chapter 2, but the subsequent bombesin-induced NFκB is actually mediated through the activation of TLR. The question for further research is how different GPCR agonists were able to induce sialidase activity? Is it possible that different GPCR receptors are associated with the same TLR? It is well known that

GPCRs form heterodimers to respond to vast array of ligands. Our results propose that there may be a common GPCR tethered to these receptors. The findings propose a novel

GPCR signaling platform at the receptor level that has a common uncharacterized property with broader specificities than expected. To test this proposition further investigations are still required. Another question raised by our observations that remains unanswered is why thrombin did not induce sialidase activity in our experimental settings? Thrombin activates the heterodimer PAR1 and PAR4 in human or PAR3 and

PAR4 in mouse cells. It is PAR4 that is capable of signaling while the other 2 PARs are co-receptors (11). It was reported that human monocytes as well as monocyte derived macrophages do not express PAR4 (12,13), which may be the case of murine macrophages used in our experiments. This offers a plausible explanation for not detecting sialidase activity upon BMC2 (mouse macrophages) cells stimulation with

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thrombin. However this explanation requires confirmation through testing the protein expression or the mRNA copies in the BMC2 cells. Another scenario would make use of the available literature pertaining to PAR4 being not involved in activating Gαi protein

(14). Our model involves the pertussis toxin sensitive Gαi protein in activating MMP-9 because pertussis toxin completely blocked sialidase activity associated with other GPCR agonists (LPA, bradykinin and bombesin). Thus, thrombin may not be able to induce sialidase activity even if BMC2 cells turn out to express PAR4. Our findings up to now have highlighted several areas that require further investigations.

Since several macrophage-expressed GPCRs have been identified and targeted with therapeutic intent in human diseases which involve infectious disease to cancer.

Studies about the key players involved in the GPCR agonist-induced transactivation of

TLR receptors in the absence of TLR ligands, or reciprocally, TLR ligands potentiate

GPCR-signaling to activate MMP-9 and NEU1 would radically redefine the current dogma(s) governing the mechanism of the cross-talk between GPCR and TLR activation and cellular signalling. Targeting the molecular GPCR signaling platform tethered to

TLR receptors may provide important pioneering approaches to disease intervention strategies.

In these studies, we have identified a new signaling platform essential for surface

TLRs activation. We further asked the question whether this novel signaling platform is common for all TLRS. Therefore in chapter 4 of this thesis, we investigated whether

NEU1, MMP-9 and GPCRs are required for endosomal TLR-7 and TLR-9 signaling. The

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data in these studies provide evidence to support an identical signaling platform in regulating ligand-induced endosomal TLRs. Nucleic acid binding to TLR7 and TLR9 initiates a NEU1 and MMP-9 crosstalk in alliance with neuromedin B GPCR (NMBR) which is essential for TLR activation. In addition, molecular targeting NEU1, MMP9 and the heterotrimeric G-protein complex tethered to both TLR-7 and -9 receptors in live

RAW-blue macrophage cells significantly abrogates pro-inflammatory cytokines MCP-1 and TNFα. Indeed, other studies have provided evidence to show that CpG ODN induces

TNFα and TNFR-II at the transcriptional level, and that these molecular players are involved in MMP-9 expression in the supernatants derived from murine RAW264.7 macrophage cell line by a TLR9 and Akt-mediated mechanism (15,16). This CpG ODN- induced MMP-9 expression fits well within our novel NEU1 and MMP-9 cross-talk signaling paradigm for TLR9 receptors. Previously CD14 was reported to co-localize with TLR-7 and TLR-9 in the endosome (17). Additionally, CD14 was essential for TLR-

7 and TLR-9-induction of proinflammatory cytokines in vitro and for TLR-9 dependent innate immune responses in mice. Interestingly, others demonstrated that CD14 physically associates with Gα proteins (18). In line with these studies, our results showed that NMBR (GPCR) coimmunoprecipitates with TLR-7 and TLR-9. Whether the interaction is directly or through the associated CD14 co-receptor will be a project for future investigations. Flourescence resonance energy transfer (FRET) techniques may be deployed in future experiments because of their ability to confirm protein-protein interaction. This may be used to support out findings that NEU1, MMP-9 and GPCR co-

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immunoprecipitate with endosomal TLRs. We have shown by using siRNA to knock down NEU1, MMP9 and NMBR that NFκB activation in response to TLR-7 and TLR-9 ligands was significantly reduced confirming the role of NEU1, MMP-9 and NMBR in the signaling pathway for endosomal TLRs. Our experiments were carried in RAW macrophage cell line and would be advantageous to confirm such findings in primary macrophages from NEU1, MMP-9 and NMBR deficient mice.

Our findings have set the foundations for future research to investigate when and where NEU1 and MMP-9 associate with TLRs and whether they are required for transporting TLRs from the ER to their signaling cellular compartments, or even for their expression. In this thesis we identified key molecules involved in TLR activation and signaling. We uncovered a novel signaling paradigm of GPCR, MMP-9 and NEU1 in alliance with TLRs both on the cell surface and intracellularly.

5.1 Disease intervention possibilities (mouse models) The immune system defends the host against invading pathogens, including bacteria and viruses. However, the immune responses must be rigorously controlled because over activation of the immune system may lead to damaging effects on the host in different ways. Several immune disorders result from exaggerated immune responses, including allergies, sepsis and autoimmune diseases. TLRs have been suggested to play a key role in sepsis and several autoimmune diseases. Therefore, our findings may open new avenues for potential therapeutic approaches in such disease conditions.

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Sepsis which is triggered by systemic LPS release into the blood stream, remains a leading cause of morbidity and mortality in critically ill patients (19). TLR4 recognizes

LPS and initiates a cascade of cytokines which includes IL1, TNFα and IL6. Amplified, overwhelming production of these mediators can lead to vasodilation and multiple organ failure (20) encountered in sepsis. The benefit of available treatments, such as the use of anti-TNFα or corticosteroids, is uncertain (21). Thus the need for new treatments to combat sepsis is quite important and therefore understanding how TLRs are activated to induce production of cytokines would be of great benefit. TLR antagonists such as anti-

TLR4 showed beneficial effect by improving survival rate in animal models (22). Our findings that Inhibitors of either NEU1, MMP9 or GPCR significantly reduced TNFα production in response to TLR stimulation in vitro may provide promising therapeutic intervention for the treatment of sepsis, and can be taken further and applied in vivo in an animal model of induced sepsis. Indeed, hypomorphic cathepsin A mice with a secondary

NEU1 deficiency respond poorly to LPS induced pro-inflammatory cytokines compared to the wild-type or hypomorphic cathepsin A with normal NEU1 mice (1).

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by chronic simulation of the innate immune system by endogenous nucleic acids released from apoptotic cells. These nucleic acids are detected by TLR7 and TLR9 expressed in the endosomal compartments which then provide the stimulus that leads to proliferation of autoreactive B-cells and production of autoantibodies characteristic of the autoimmune disease (23). TLR7 deficient mice did not induce the production of autoreactive

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antibodies, such as anti-double stranded DNA and anti-histone antibodies when injected with syngeneic late apoptotic thymocytes and compared to wild type mice (24). These findings highlight a role of TLR7 in the development of SLE. Other studies reported an up-regulation of TLR9 in B cells from lupus patients, which also indicates a role of TLR9 in enhancing the autoantibodies production (25). Multiple studies have indicated a role of

TLRs and their signaling pathways molecules to play a role in SLE which makes them excellent targets to combat the disease. For example, interferon α (IFNα) is downstream

TLR-signaling product, and it is a characteristic inflammatory cytokine elevated in SLE patients (26). Therefore, the use of monoclonal antibodies to target IFNα for the treatment of SLE is being used in clinical trials (27). In addition, TLR7 and TLR9 antagonists have also shown efficacy in mouse models of SLE. Hence our results which identified new molecules involved in the TLR signaling regulation will widely increase the number of potential therapeutic developments and may lead to a more tolerated treatment options for this complicated disorder. It would be interesting to use the available SLE mouse model such as MRL-lpr multigenic SLE-prone mouse model, in which SLE susceptibility correlates with mutations at several loci, as for human SLE

(28,29). These mice can be used to test the effect of neuraminidase, MMP9 or GPCR inhibitors in these animals, especially after we have reported the efficacy of these inhibitors in blocking TLR signaling in cell lines. Autoantibodies or IFN blood levels from treated mice can be compared with the untreated ones to monitor the efficacy of such inhibitors which is expected to further support our findings.

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Our research findings which increase our understanding of the TLR signaling regulation, can also offer new opportunities for providing more practical and needed clinical applications in targeting TLRs.

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