ROLE OF IKAPPABZETA AND PYRIN AS MODULATORS OF MACROPHAGE INNATE IMMUNE FUNCTION

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

By

Sudarshan Seshadri, M.S

* * * * *

The Ohio State University 2008

Dissertation Committee:

Dr. Mark Wewers, Advisor Approved by Dr. Susheela Tridandapani

Dr. Scott Walsh

Dr. Daren Knoell

Advisor Biophysics Graduate Program

ABSTRACT

Innate immunity is the first line of defense against the pathogens mounted by the

host. The host response mediated by innate immunity is quick and takes place within the first few hours after the pathogen invasion. Proper functioning of innate immunity is

required for mounting the adaptive immune response. All lower order organisms,

animals and plants rely on innate immunity as their prime mode of defense. However,

studies on innate immunity have been very limited so far.

Innate immune responses are initiated by three main receptors, toll like receptors, nucleotide oligomerization domain-like receptors and RIG-like receptors. These

receptors get activated upon pathogen recognition and turn on several proinflammatory

pathways. The present study concentrated on two proinflammatory pathways, the

signalosome and the inflammasome pathway. The signalosome pathway leads to the

production of the pro-inflammatory cytokines that are involved in host defense and also

regulates the expression of proteins that are involved in host cell survival. IL-1β is one

such cytokine dependent on signalosome pathway for its production. However, the

produced IL-1β lacks biological activity and it needs to be processed to mature biologically active IL-1β. This process of converting the proIL-1β to mature form

requires a cysteine protease known as caspase-1. The mechanism by which caspase-1

gets activated is very complex and is mediated by the formation of multi-protein complex

ii called the inflammasome. I am interested in studying the function of two proteins MAIL and pyrin which are involved in signalosome and inflammasome activation respectively.

Chapter 1 provides summary of the recent literature about signalosome and inflammasome. Chapter 2 describes the analysis of the role of MAIL in IL-6 production.

In chapter 3, the role of pyrin in inflammasome mediated caspase-1 activation is elucidated. Chapter 4 describes with experiments designed to ascertain the role of pyrin mutations in caspase-1 activation. Chapter 5 describes some important experiments which have a potential to be developed into a new projects and some preliminary results which are significant. It also describes some hypotheses which can be tested to elucidate the function of pyrin and pyrin mutants.

Chapter 2 describes the role of human MAIL in the production of IL-6. MAIL

(IκBzeta) is a recently described homologue of IκBα that is rapidly induced by lipopolysaccharide (LPS) in monocytes. MAIL regulates the transcription of a number of inflammatory genes including IL-6 in the mouse. Although the role of IL-6 is well established in cancer and sepsis, the regulation of IL-6 in human monocytes and macrophages is poorly understood. Since we noted that monocytes are potent producers of IL-6, whereas their descendant macrophages are not, we proposed the hypothesis that

MAIL regulation is key to the IL-6 differences between monocytes and macrophages.

MAIL expression was suppressed with differentiation of monocytes to macrophages. In agreement with this finding, monocytes produced seven times more IL-6 compared to iii

macrophages. Furthermore, suppression of MAIL by small interfering RNA decreased

the production of IL-6 significantly up to 5 fold, whereas the production and release of

another pro-inflammatory cytokine, IL-8, was not affected. To test the role of MAIL in

NLR pathway, we used intracellular ligands which stimulate the NOD-like receptor Nod1

and Nod2. The results show that MAIL is important for Nod2 mediated IL-6 production.

Our data suggests that MAIL is a key regulator of IL-6 production in monocytes and

plays an important role in endotoxin-induced inflammation.

The activation of the signalosome pathway is sufficient for the production of

cytokines proIL-1β, proIL-18, proIL-33, IL-6, IL-8, IL-12 and TNFα. However, for

production of biologically active IL-1β, IL-18 and IL-33 needs caspase-1 mediated

processing. The inflammasome a multiprotein complex involved in activation of

caspase-1. IL-1β is a proinflammatory cytokine involved in host defense and host

homoeostasis. Dysregulation in IL-1β synthesis and production leads to many auto

inflammatory diseases like familial Mediterranean fever (FMF).

FMF is caused by mutations in MEFV gene which codes for a protein called

pyrin. The patients with mutations in pyrin have recurrent fever and inflammation. Pyrin interacts with proteins involved in the assembly of inflammasome namely, ASC, caspase-

1, IL-1β, and NALP3 and is involved in regulation of inflammasome activity. The exact role of pyrin in the regulation of inflammasome assembly is poorly understood and a

iv subject of controversy. Both activating and suppressing roles of pyrin have been proposed.

Chapter 3 describes the analysis of the role of pyrin in caspase-1 activation. The results show that pyrin levels in monocyte correlated positively with IL-1β processing and release. We found that macrophages were deficient in activating caspase-1 and also the expression level of pyrin. Suppression of pyrin decreased IL-1β processing and release in THP-1 cells and in monocytes. Thus it proves that in the endotoxin model, pyrin tends to augment IL-1β processing and release.

The role of pyrin in caspase-1 activation is controversial, so is the role of pyrin mutation in IL-1β processing and release. In chapter 4, the role of the two common mutations, E148Q and M694V in caspase-1 activation is elucidated. The results indicate that both these mutant forms of pyrin do not differ from wild type in co-localization and interaction with ASC. Also, the mutations in pyrin did not affect IL-1β processing and release when pyrin or the mutant forms of pyrin were co-expressed with the proteins of the inflammasome in HEK293 cells.

In chapter 5, some of the recent data regarding the role of MAIL in regulation of expression of COX-2, an enzyme required for the synthesis of prostaglandins is presented. I have put forth some hypotheses regarding function of pyrin which can be tested to understand the function of pyrin and the role of mutations in causing FMF. I have also presented some data regarding the release of inflammasome proteins by

v

monocytes. Contrary to the belief that caspase-1 activation is required for secretion of

the inflammasome proteins, I have presented some data that the key inflammasome

adaptor protein ASC is released in monocytes that does not require caspase-1 activation.

Thus, MAIL and pyrin are involved in regulating two distinct pathways the

signalosome pathway and the inflammasome pathway which are important for innate

immune defense. Understanding the role of MAIL and pyrin in monocytes will be

helpful in understanding mechanisms of inflammation and may open new avenues for

therapeutic interventions in inflammation and inflammation induced disease, such as cancer.

vi

Dedicated to my family

vii

ACKNOWLEDGMENTS

I would like to thank my advisor, Dr. Mark D. Wewers, for his support and guidance throughout my graduate education. His ideas and suggestions were important and crucial for the success of the research presented in this dissertation. I am lucky to have a good mentor as Dr. Wewers. I am sure; it will be hard for me to find a good mentor like him. The time that I have spent at Ohio State doing research in the lab of Dr.

Wewers is very instrumental in my career.

I am grateful to my committee members Dr. Susheela Tridandapani, Dr. Scott

Walsh, and Dr. Daren Knoell for their valuable suggestions in my research and in preparation of the thesis. I am also thankful to Dr. Thomas Clanton for serving in the committee for my candidacy examination.

I am indebted to the current and past members of Wewers lab, Dr. Mark Hall, Dr.

Matthew Exline, Dr. Mikhail Gavrilin, Dr. Anasuya Sarkar, Dr. Hee-Jung Kim, Judy

Hart, Michelle Duncan, Srabani Mitra, Jennifer Parker-Barnes, Jennifer Hollyfield and

Freweine Berhe for their valuable suggestions and untiring help with my projects. I appreciate Dr. Gavrilin’s help with my projects. My sincere thanks to Jennifer Hollyfield for proof reading the entire dissertation. I am thankful to my fellow graduate students in

Wewers Lab, Raquel Raices and Yashaswini Kannan for the stimulating discussions and help throughout the research. viii

I extend my special thanks to Susan Hauser (Biophysics Program) for all her help

during the graduate studies. I also thank the Division of Pulmonary and Critical Care, and

the administrative members, Tim Mazik, Thomasina Bowder-Long and Susan Meder for

all their help during the course of the study.

Words are not enough when it comes to acknowledge the support that my wife

Rakhi Rajan and my daughter Meena H. Seshadri have given for me to finish up my research projects. It is their support and encouragement that has led me to complete my projects successfully. I would like to thank my parents in law, Rajasekharan Nair and

Leelamony Rajasekharan for their encouragement and support throughout my doctoral studies. Finally, I would like to thank my parents, S.Seshadri and Rama Seshadri, and my brothers Arun Seshadri and Arvind Seshadri, who provided me with the strong foundation for all the achievements in my life. It is all their support and encouragement that has made me to achieve my goals.

This research was supported by the funds available to Dr. Mark D. Wewers from

the ‘National Institute of Health’ and from the Thematic Pre- Doctoral Grant from The

Heart and Lung Research institute which was awarded to me.

ix

VITA

November, 26, 1976...... Born- Chennai, India

1994-1998 ...... B.Sc. Agriculture, Annamalai University, Chidambaram, India

1998-2000 M.Sc. Biotechnology, Tamil Nadu Agricultural University, Coimbatore, India

2000-2001 ...... Junior Research Fellow, SPIC Science Foundation, Chennai, India

2001-2002 Teaching Assistant/Graduate Student, Northern Illinois University, De Kalb,

USA

2002- Present .Graduate Research Associate, The Ohio State University, Columbus, USA

PUBLICATIONS

1. Seshadri S, Duncan MD, Hart JM, Gavrilin MA, Wewers MD. (2007) Pyrin levels in human monocytes and monocyte-derived macrophages regulate IL-1beta processing and release. J Immunol. 179(2):1274-81.

FIELDS OF STUDY

Major Field: Biophysics

x

TABLE OF CONTENTS

Page

Abstract...... ii

Dedication...... vii

Acknowledgments...... viii

Vita ...... x

List of Tables ...... xx

List of Figures...... xxi

Abbreviations...... xxiv

Chapters:

1. Introduction

1.1 Innate Immunity...... 1

1.2 Toll Like Receptors...... 3

1.2.1 Structure of TLRs ...... 4

1.2.2 Ligand recognition by TLRs ...... 5

1.2.3 TLR signaling pathways ...... 7

xi

1.2.3.1 MyD88 signaling pathway/ The signalosome pathway...... 9

1.2.3.2 MyD88 independent signaling pathway ...... 10

1.3 NOD like receptors ...... 12

1.3.1 NLRs in health and disease...... 14

1.3.2 Domain structure of NLR proteins ...... 16

1.3.3 NOD signaling pathway in activation of NF-κB ...... 18

1.4 NF-κB pathway...... 20

1.5 Rel/NF-κB proteins...... 21

1.6 IκB proteins ...... 23

1.7 MAIL/IκBζ ...... 26

1.7.1 Identification ...... 26

1.7.2 Gene and protein structure...... 27

1.7.3 Expression and localization ...... 30

1.7.4 Mechanism of MAIL induction ...... 32

1.7.5 MAIL interacting proteins ...... 35

1.7.6 Targeted gene disruption of MAIL ...... 35

xii

1.7.7 Functions of MAIL ...... 37

1.7.7.1 Role of MAIL in NF-κB activation ...... 37

1.7.7.2 Role of MAIL in the production of cytokines ...... 39

1.7.7.3 Role of MAIL in the production of anti microbial proteins ...... 40

1.7.7.4 Role of MAIL in apoptosis ...... 41

1.7.7.5 Role of MAIL in adaptive immunity ...... 41

1.8 IL-1β, Caspase-1 and the inflammasome ...... 42

1.8.1 IL-1β and inflammation ...... 42

1.8.2 Caspase-1/ ICE (IL-1β converting enzyme) ...... 43

1.8.3 Inflammasome and its regulation...... 44

1.8.4 Types of inflammasome and their stimuli ...... 47

1.8.4.1 NALP1/ NLRP1 inflammasome ...... 47

1.8.4.2 NALP3/ NLRP3 inflammasome ...... 47

1.8.4.3 IPAF/NLRC4 inflammasome ...... 49

1.8.5 ASC and Pyrin – Regulators of inflammasome activity ...... 50

1.8.6 ASC- The caspase-1 activator ...... 50

xiii

1.8.7 Pyrin- The cause for familial Mediterranean fever ...... 52

1.8.7.1 Domain structure of pyrin protein ...... 53

1.8.7.2 Expression and co-localization ...... 55

1.8.7.3 Pyrin interacting proteins ...... 55

1.8.7.4 Functions of pyrin ...... 57

1.8.7.4.1 Role of pyrin in cytoskeletal remodeling...... 57

1.8.7.4.2 Role of pyrin in inflammation...... 57

1.8.7.4.3 Role of pyrin in apoptosis...... 60

2. MAIL- A key upstream regulator of monocyte IL-6 production

2.1 Introduction ...... 62

2.2 Materials and Methods ...... 65

2.2.1 Cell culture and transfection ...... 65

2.2.2 Small interfering RNA...... 66

2.2.3 ELISA ...... 66

2.2.4 Reagents and antibodies...... 66

2.2.5 Real Time PCR ...... 66

xiv

2.2.6 Plasmids ...... 67

2.2.7 Preparation of cell lysates and western blotting ...... 67

2.2.8 Statistical Analysis...... 67

2.3 Results ...... 68

2.3.1 Overexpression, purification and generation of MAIL antibody .... 68

2.3.2 Monocytes express both MAIL-L and MAIL-S mRNA upon LPS

stimulation ...... 68

2.3.3 MAIL is transiently induced in monocytes upon LPS stimulation.. 70

2.3.4 Monocytes express more MAIL and secrete more IL-6 compared to

macrophages ...... 72

2.3.5 EGFP-MAIL translocates to nucleus ...... 74

2.3.6 Knockdown of MAIL suppresses LPS induced IL-6 production in

monocytes ...... 74

2.3.7 Knockdown of MAIL suppresses NOD ligand induced IL-6

production ...... 76

2.4 Discussion...... 81

xv

3. Pyrin levels in monocytes and monocyte derived macrophages regulate IL-1β processing and release

3.1 Introduction...... 85

3.2 Materials and Methods...... 87

3.2.1 Cell culture and transfection ...... 87

3.2.2 Expression plasmids...... 88

3.2.3 Generation of pyrin antibody ...... 88

3.2.4 Antibodies...... 89

3.2.5 Quantitative polymerase chain reaction...... 89

3.2.6 Caspase-1 activity assay ...... 89

3.2.7 ELISA ...... 90

3.2.8 Preparation of cell lysates and Immunoblots...... 90

3.2.9 Small interfering RNA...... 90

3.2.10 Statistical Analysis...... 91

3.3 Results ...... 91

3.3.1 Overexpression and purification of pyrin and generation of pyrin

antibody...... 91 xvi

3.3.2 Testing the specificity of pyrin antibody ...... 92

3.3.3 Monocytes and monocyte derived macrophages differ in their ability

to process IL-1β ...... 94

3.3.4 Pyrin expression is suppressed in monocyte derived macrophages

compared to monocytes ...... 97

3.3.5 Pyrin increases caspase-1 activity in a dose dependent manner...... 97

3.3.6 Pyrin induces IL-1β release in a dose dependent manner in HEK293

cells ...... 99

3.3.7 Pyrin overexpression in THP-1 cells ...... 101

3.3.8 Suppression of pyrin decreased IL-1β release in THP-1 cells...... 101

3.3.9 Knockdown of pyrin suppresses IL-1β release in peripheral blood

monocytes ...... 105

3.4 Discussion...... 108

4. Familial Mediterranean fever is not due to enhanced casspase-1 activity

4.1 Introduction...... 114

4.2 Materials and Methods...... 117

xvii

4.2.1 Cell culture and transfection ...... 117

4.2.2 Plasmids and constructs ...... 117

4.2.3 Fluorescence Microscopy ...... 118

4.2.4 MBP pull down Assay ...... 118

4.3 Results and Discussion ...... 120

4.3.1 Pyrin or mutants form of pyrin co-localize with ASC...... 120

4.3.2 Pyrin or mutant forms of pyrin interact with ASC ...... 121

4.3.3 Pyrin or mutant forms of pyrin have no difference in IL-1β

processing and release...... 124

4.4 Conclusions...... 124

5. Conclusion and Future Perspectives

5.1 MAIL is a regulator of COX-2 expression ...... 129

5.1.1 Monocytes express COX-2 upon LPS stimulation ...... 131

5.1.2 COX-2 is induced by LPS, IL-1β and not by TNFα in monocytes 131

5.1.3 Knockdown of MAIL suppresses the expression of COX-2 ...... 133

5.2 Role of pyrin in macrophage innate immunity- The unsolved questions ..... 135

5.2.1 Do pyrin over expressing macrophages release IL-1β ...... 136 xviii

5.2.2 Does pyrin interact with other proteins of the inflammasome ...... 136

5.2.3 Role of mutations of pyrin in phagocytosis ...... 137

5.2.4 Is B30.2 domain a pathogen recognition receptor ...... 138

5.3 Secretion of inflammasome proteins ...... 141

5.3.1 Monocytes secrete ASC ...... 142

5.3.2 ASC is released from monocytes treated with LPS ...... 143

5.4 Conclusions...... 147

List of References ...... 149

xix

LIST OF TABLES

Table Page

1.1 List of diseases associated with NLRs...... 17

1.2 List of ligands for NLRs...... 51

xx

LIST OF FIGURES

Figure Page

1.1 Ligand recognition by TLRs...... 8

1.2 MyD88 dependent and independent pathways...... 11

1.3 NOD like receptor proteins and their domain structures...... 13

1.4 NOD signaling pathway...... 19

1.5 The NF-κB and IκB protein family protein family...... 24

1.6 MAIL gene and protein structure...... 29

1.7 Mechanism of MAIL action...... 38

1.8 Mechanism of IL-1β production and processing ...... 45

1.9 NALP1 inflammasome ...... 48

1.10 Domain structure of pyrin protein...... 54

1.11 Pyrin hypotheses...... 59

2.1 Overexpression and purification of MAIL protein and testing MAIL antibody .. 69

2.2 Monocytes express both MAIL-L and MAIL-S mRNA upon LPS stimulation... 71

xxi

2.3 MAIL is transiently induced in monocytes upon LPS stimulation...... 73

2.4 Monocytes express more MAIL and IL-6 compared to macrophages...... 75

2.5 MAIL translocates to nucleus and forms speckles ...... 77

2.6 Knockdown of MAIL suppresses LPS induced IL-6 production...... 79

2.7 Knockdown of MAIL suppresses NOD ligand induced IL-6 production...... 80

3.1 Purification of pyrin protein...... 93

3.2 Testing the specificity of pyrin antibody...... 95

3.3 Monocytes process and release IL-1β better than monocyte derived macrophages

...... 96

3.4 Expression of pyrin is decreased in macrophages compared to monocytes...... 98

3.5 Pyrin increases caspase-1 activity and IL-1β processing and release in HEK293

cells...... 100

3.6 Low dose ASC also supports pyrin induced IL-1β processing and release...... 102

3.7 Pyrin overexpression does not suppress IL-1β processing and release in THP-1

cells ...... 104

3.8 Knockdown of pyrin suppresses IL-1β processing and release in THP-1 cells.. 106

xxii

3.9 Knockdown of pyrin suppresses IL-1β processing and release in peripheral blood

monocytes ...... 107

4.1 Disease associated mutations in pyrin and constructs used in this study...... 119

4.2 Wild type or mutant forms of pyrin do not differ in co-localization with ASC. 122

4.3 Interaction of ASC with wild type or mutants of pyrin...... 123

4.4 Pyrin and mutant forms of pyrin do not differ in IL-1β processing and release in

HEK293 overexpression system...... 125

5.1 COX-2 is expressed in human monocytes upon LPS stimulation...... 132

5.2 MAIL suppressed monocytes have decreased COX-2 expression...... 134

5.3 Francisella binds pyrin...... 140

5.4 ASC is released from monocytes...... 144

5.5 ASC release is independent of caspase-1 activation...... 146

xxiii

ABBREVIATIONS

α alpha

β beta

γ gamma

˚C degree centigrade

NaCl Sodium chloride

DTT Dithiothreitol

IPTG Isopropyl β-D-1-thiogalactopyranoside

EDTA Ethylene diamine tetra-acetic acid

SDS-PAGE Sodium dodecyl sulphate – polyacrylamide gel electrophoresis

ATP Adenosine Tri Phosphate m milli

M molar

μ micro

% percentage

DNA Deoxyribo Nucleic Acid

OD Optical Density

L liter

BSA Bovine Serum Albumin xxiv bp base pair s second(s) min minute(s) h hour(s) nM nanomolar

MDP Muramyl di-peptide iE-DAP γ-D-Glu-m-da amino pimelic acid

LPS Lipopolysaccharide

NOD Nucleotide oligomerization domain pyd pyrin domain

CARD Caspase activation and recruitment domain

ASC Apoptotic speck like protein containing a CARD

IL-1R IL-1 Receptor

TNFR TNFα receptor

IFN Interferon

MHC Major histocompatability complex.

xxv

CHAPTER 1

INTRODUCTION

1.1 Innate Immunity

Innate immunity is the first line of defense mounted by the host against invading pathogens. Innate immunity is an evolutionarily conserved host defense mechanism present in all multi-cellular organisms. It was first described by Metchnikoff more than a century ago when he discovered that microorganisms can be phagocytosed by cells he called macrophages. The field of innate immunity has been under studied and under appreciated because of the discoveries of antibodies, B, and T cells. With the recent work of the sequencing genomes of many multi-cellular organisms, like sea urchins and the discovery of intracellular pathogen sensors have led us to think that innate immunity

consists of a complicated network of effector mechanisms which protects the organisms

until the adaptive immunity can mount its response. Knowledge about innate immunity

blossomed in 1989 when Janeway proposed that innate immune receptors are encoded by

germline cells and these are responsible for the detection of pathogens.

1

Now we know that the innate immune receptors are germ-line encoded receptors

and are known as the pathogen recognition receptors (PRRs). Pathogen recognition by

receptors is present both in plants and animals (1,2). These PRRs recognize highly

conserved structures on pathogens known as Pathogen Associated Molecular Patterns

(PAMPs) (3). The PAMPs are produced abundantly by the pathogens and not by the host

and are required for pathogen survival. Thus, the immune system can distinguish

between self and non-self and mounts a defensive response against the invading

pathogens. The sensing of PAMPs by PRRs leads to the expression of genes involved in

host defense. Such genes are involved in the production of various cytokines and

chemokines which leads to inflammation and recruitment of phagocytes. The production

of various defense molecules such as antimicrobial peptides is also mediated by this

pathway. All these processes help in host defense by regulating the infection caused by

the pathogens.

The discovery of the Drosphila melanogaster protein, Toll, in 1996 paved a way

in understanding the innate immune system (4). dToll, which is involved in dorso-ventral

patterning in the embryo of the fly, is also involved in the immune response to the fungus

Aspergillus fumigatus. It was shown that mutants of dToll were susceptible to the

infection of A. fumigatus and failed to produce the antifungal peptide drosomycin (4). A parallel discovery was made in humans which identified human toll protein now known

as TLR4 (5). It was shown that overexpression of the constitutive active form of TLR4

lead to increased production of pro-inflammatory cytokines and up regulation of co-

2

stimulatory molecules. Mice having mutations in the Tlr4 gene were unresponsive to

LPS and were also susceptible to endotoxin induced sepsis (6,7).

Work from many laboratories over the past few years have identified three major

classes of receptors which are involved in innate immunity. These are the Toll-like receptors (TLRs), NOD-like receptors (NLRs) and retinoid acid-inducible gene I (RIG-I)-

like receptors (RLRs)(8-10). TLRs are located in the cell membrane and endosomal compartments, and are involved in the recognition of extracellular pathogens. NLRs and

RLRs are present in the cytosol and recognize intracellular pathogens and viruses respectively. The presence of extracellular and intracellular receptors indicates that the defense response mediated by host is against both the extracellular and also against the intracellular pathogens which escape the extracelluar defense mechanism. Upon recognition these receptors turn on the host defense pathway such as NF-κB and MAP kinase which secrete inflammatory mediators that mount the immune response against the invading pathogens. Innate immunity is very ancient and is the first line of defense of

prime importance to the lower order organism which does not have the adaptive immunity. It is fascinating to know that sequencing the genome of the sea urchin has revealed around 222 TLRs and 203 NLRs (11,12). This indicates that innate immunity is far more complex and a lot of work needs to be done to understand and appreciate the complexity of this ancient form of defense.

1.2 Toll like receptors (TLRs)

TLRs are the major cell surface receptors which initiate an inflammatory response when they recognize the PAMPs. TLRs are found to be either associated with the plasma

3

membrane or they may be localized in cellular endosomal compartments. It has not even been a decade since the first discovery of TLR4 in humans, but we know much more about the TLRs. There are a total of 13 toll like receptors in mammals (10 in humans and

13 in mice) (13). TLR 1-9 are well conserved between humans and mouse. TLR 10 is expressed only in human, whereas the TLR11 is expressed only in mouse. Not much is known about the expression pattern, ligands and localization of TLR10, 12 and 13. All

TLRs recognize different ligands and activate distinct defense pathways that help the host to defend against the pathogens. Unlike the drosophila toll pathway, where pathogen recognition is mediated by protein upstream of Toll, mammalian TLRs are thought to be

mediated by direct binding of ligand to the extracellular domain (ECD) of the TLR. The

direct binding of TLR to the ligand has been shown in the case of TLR3 and its ligand,

dsRNA by extensive site directional mutation analysis (14). So far, there is no structural

evidence of TLR complexed with the ligand. Recently it was demonstrated that ligand- induced conformational change activates TLR9. This study revealed that the ligand, upon recognition by the receptor, leads to the receptor dimerization. This dimerization of the receptor leads to the change in conformation of the TIR domain of the TLR. This change in conformation helps in recruitment of adaptor molecules like MyD88 and Mal which leads to the activation of the Toll signaling pathway (15).

1.2.1 Structure of TLRs

TLRs are type I transmembrane receptors that have N-terminal leucine rich

repeats (LRR), a transmembrane region, and a C-terminal cytoplasmic domain which

shares a homology to type I IL-1 receptor. The C-terminal domain is known as Toll/IL-1

4 receptor (TIR) domain and is involved in signaling when the pathogens are recognized by the leucine rich repeats (16). The structure of TLR3 ecto domain has been solved recently. Structural analysis reveals that TLR3-ECD is a large horse shoe shaped solenoid containing 23 LRRs (17,18). The presence of a large concave surface at the ecto domain of TLR3 indicates that this may be useful in pathogen recognition and binding.

The ECD domain of TLR3 has two sides which differ in their glycosylation content. The side which is sugar free is thought to be the ligand recognition region of the TLR3.

Based on the structure of TLR3 ectodomain and also from the other LRR repeat containing proteins, it is known that each LRR forms a loop, all the loops are juxtaposed to form a solenoid like structure. The concave structure of the coil which is formed of large parallel β sheets and the convex structure are formed by α strand. Of all the TLR- ligand complex structures solved so far, the concave structure is involved in recognition of ligands. LRR of TLRs have insertions present at position 10 and 15 of the LRR, which is thought to play a role in the diversity of pathogen recognition (17). Signaling through the TLR is mediated by receptor dimerization.

1.2.2 Ligand recognition by TLRs

TLR2 deficient mice are hyporesponsive to Gram-positive bacterial products and

Staphylococcus aureus peptidoglycan (19). Now we know that TLR2 can function alone or functions by forming heterodimers with TLR1 and TLR6. TLR1/TLR2 heterodimer recognizes triacylated proteins which includes lipoproteins from Mycobacteria and

Meningococci (19). TLR2/TLR6 heterodimers recognizes diacylated proteins (20). It has been shown that TLR2 can function independently of TLR1 and TLR6. TLR2 can

5

recognize lipoteichoic acid, Mycobacterial lipoarabinomanan and atypical LPS from

Legionella (21). TLR2 and TLR2/TLR6 also recognizes fungal products such as yeast dectin and zymosan respectively (22)

TLR3 is present in the cytosolic endosome and is responsible for detection of

dsRNA from the viruses (23) (24). TLR3 knockout was hyporesponsive to dsRNA,

polyinosine-deoxycytidylic acid [poly(I:C)] and are susceptible to mouse cytomegalovirus infection (25). TLR4 was the first Toll identified in human and the mutations of which leads to hypo-responsiveness to lipopolysaccharides (LPS). Tlr4

knockout animals are hypo-responsive to LPS and susceptible to Gram-negative induced

bacterial infections (6). LPS is present in the cell wall of Gram-negative bacteria and is

the most well studied and most potent activator of the immune system. TLR4 receptor

recognizes the lipidA protein present in the LPS. LPS forms a complex with the LPS

binding protein present in the serum and this converts the oligomeric micelles of LPS to

the monomeric form which is then delivered to the glycosyl phosphotidylinositol (GPI)

anchored membrane protein CD14. CD14 concentrates LPS and then delivers it to the

TLR4/MD2 complex which triggers the intracellular signaling. TLR4 also recognizes

viral proteins from respiratory synctial virus (26). C3H/HeJ mouse which carry the

mutation in Tlr4 gene are susceptible to RSV infection (27). TLR5 is responsible for

recognition of flagellin which is a component of bacterial flagella involved in bacterial motility (28). TLR5 recognizes monomeric flagellin and the TLR5 recognition site is masked in the filamentous flagella (29). Human TLR7 and TLR8, but not murine TLR8, recognizes synthetic imidazoquinoline-like molecules, imiquimod (R-837), and

6

resiquimod (R-848), which are used as potent anti-viral compounds (30,31). These compounds are structurally similar to nucleic acids. TLR7 deficient mice are not capable of recognizing these compounds. TLR7 and TLR8 recognize guanosine and uridine rich single stranded RNA derived from viruses. Human nucleotides are highly modified and are different than the viral nucleotides and thus are not recognized by TLR7 and TLR8

receptors. TLR9 recognizes bacterial DNA containing unmethylated CpG motifs and it

has been shown that TLR9 deficient mice are not responsive to CpG motifs (32). CpG

motifs are very rare in humans and the cytosines of these motifs are highly methylated so

they are not recognized by the host immune system. Human TLR11 is not functional as

it has a stop codon. TLR11 knockouts experiments have revealed that mice deficient of

TLR11 are susceptible to uropathogenic infection (33). In accordance to this data it has been shown that TLR11 is highly expressed in kidney and bladder. The ligand for

TLR11 has not been discovered yet. Proteinase K treatment of uropathogenic bacteria destroys the recognition by TLR11 indicating that the TLR11 recognizes a protein ligand.

It is not clear how these 10 receptors recognize a plethora of PAMPs and trigger a specialized type of host defense in response to these pathogens. Figure 1.1 shows different TLRs and their respective ligands recognized by them.

1.2.3 TLR signaling pathways

The pathways starting from recognition of PAMPs to the production of pro-

inflammatory cytokines has been the topic of extensive research for the past few years.

There are two TLR signaling pathways. The first is the MyD88 dependent signaling

pathway and the second is MyD88 independent signaling pathway.

7

Figure 1.1: Ligand recognition by TLRs (Adapted from West et al. 2006.(34)). Various TLRs and their ligands. Ligands generated by the type of organism are indicated at the top. The pathways activated by the particular set of ligands are indicated at the bottom.

8

1.2.3.1 MyD88 signaling pathway/ the signalosome pathway

PAMPs binding to the LRR of the TLRs facilitate the dimerization/

oligomerization of the receptors. This receptor induced dimerization/oligomerization

causes a change in conformation of Toll-IL-1 receptor (TIR) domain present in the C-

terminus of the TLR receptor. This change of conformation helps in the recruitment of

adaptor protein myeloid differentiation primary response gene (88) (MyD88). MyD88

was first described to mediate IL-1R mediated signaling. Now we know that MyD88 is

an essential adaptor for both TLR and IL-1R pathways (35). The importance of TIR

domain is known from the C3H/HeJ mice which have a mutation in the TIR domain

which results in hyporesponsiveness to LPS. This is due to the inability of the TIR domain to interact with the adaptor MyD88. The adaptor molecule MyD88 has an N- terminal death domain and a C-terminal TIR domain. It interacts with the TLR receptor through the TIR-TIR domain interactions. Production of proinflammatory cytokines is dramatically decreased in macrophages and fibroblasts cells from MyD88 deficient mice on stimulation with IL-1β and TLR ligands. MyD88 recruits down stream signaling protein serine/threonine IL-1 receptor-associated kinase-4 (IRAK-4) through the homophilic death domain interactions. The complex of TLR/MyD88/IRAK-4 recruits and phosphorylates IRAK-1. The kinase function of IRAK-1 gets activated which autophosphorylates itself. IRAK-1 then interacts with TNFR associated factor-6 (TRAF-

6) causing the oligomerization and activation of TRAF-6. TRAF-6 is a ubiquitin E3 ligase which functions with Ubc13 and the Ubc like protein Uev1a to catalyze the synthesis of polyubiquitin chains on the Lys63 residue on other proteins and itself. The

9

ubiquitinated TRAF-6 binds to TAB2 and activates TAB2 associated kinase-1 (TAK-1).

Activated TAK-1 phosphorylates and activates the β subunit of the IKK complex, the

IKK complex in turn phosphorylate IκBα. The phosphorylated IκBα is then ubiquitinated

and gets degraded by the 26S proteosome leading to the translocation of NF-κB proteins

to the nucleus. The translocated NF-κB then turns on the pro-inflammatory genes which

are responsible for the host defense. TAK-1 is also important in activating other

pathways including p38 MAP kinase and JNK. Regulation of IκBα is tightly regulated

by a kinase signaling cascade. The pathway which senses PAMPs and mediates NF-κB

signaling is called the signalosome pathway. MyD88 is also essential in the production

of IFNα and IFN inducible genes by the activation of the IRF family transcription factors

(Figure 1.2).

1.2.3.2 MyD88 independent signaling pathway

MyD88 deficient cells were able to activate NF-κB and MAP kinase pathway

upon stimulation with TLR3 and TLR4 ligands (36). This indicated that there may be an

alternate pathway involved in the activation of NF-κB and the MAP kinase pathway. This

led to the identification of another adaptor protein known as TIR domain containing

adaptor inducing IFNβ (TRIF) /TIR containing adaptor molecule-1 (TICAM-1) (37).

TRIF mediates IRF3 activation through the association of TRAF family associated NF-

κB activator (TANK) binding Kinase-1 (TBK-1). TRIF also binds to receptor interacting protein-1 (RIP-1) through its C-terminal RIP homotypic interaction motif. The association of RIP is involved in TLR3 induced NF-κB activity. TRAM is another adaptor which is only utilized by the TLR4 receptor. TRAM serves as an adaptor

10

Figure 1.2: MyD88 dependent and independent pathways. (Adapted from Uematsu and Akira. 2006 (10)) When a ligand is recognized by the TLRs it recruits the TIR containing adapters MyD88 and TIRAP. MyD88 and TIRAP subsequently recruit IRAK4 and IRAK-1. IRAK-1 then activates TRAF-6, leading to activation of TAK-1. TAK-1 activates IKK complex, subsequently the IKK complex phosphorylates IκBα. IκBα is degraded which leads to the translocation of NF-κB to the nucleus. In MyD88 independent pathway leading to the activation of NF-κB, TLR3 recruits TRIF (for TLR4, TRAM is required for TRIF recruitment). TRIF then interacts to TRAF-6 and RIP-1. RIP-1 activates the IKK complex, which subsequently phosphorylates IκBα. MyD88 independent pathway also leads to the production of IFN-β and IFN-inducible genes. TLR3 recruits TRIF, TRIF then activates TBK-1. TBK-1 comprises of inducible kinases IKKi kinases. IKKi kinases directly phosphorylate IRF-3 which leads to the translocation of IRF-3 and expression of IFN-β and IFN-β inducible genes.

11

molecule which recruits TRIF to the TLR4 receptor. Thus both the MyD88 dependent

and MyD88 independent pathway work in concert to fight against the pathogens.

1.3NOD like receptors (NLRs)

The pathogen recognition and activation of the toll pathway leading to host

defense is explained in many circumstances of pathogen invasion but not in all forms of

infection. The observation that only an invasive form of Shigella led to the activation of

NF-κB, led to hypothesize that there are a class of intracellular receptors differing from

TLR in detection and activation of host defense (38). These bacteria were later shown to

be sensed by Nod1 (Card4), a new class of receptor having nuclear oligomerization

domain and leucine rich repeat (NOD-LRR) (38). These receptors are now known as

NOD like receptors (NLRs). Mammalian NLRs are a recently identified class of proteins

which are thought to be intracellular PRRs that recognize intracellular PAMPs. Figure

1.3 shows the various NLRs present in humans.

NLRs are very similar to the plant R proteins (NBS-LRR) which are involved in

plant defense against infection. These plant R proteins are both transmembrane and

cytosolic proteins. On detecting pathogens by the LRR they elicit the hypersensitivity

response such as isolation of lesion, formation of reactive oxygen species, production of anti-pathogen molecules and cell death (39-41). The current thinking is that NLRs are in a folded conformation in which the LRRs and NOD domain are bound together. When

LRRs are activated by the PAMPs, the NLR molecule undergoes conformational change that recruits adapters, like receptor interacting protein kinase2 (RIP2), which then activate NF-κB. There are about 20 different NLR proteins found in humans. The

12

Figure 1.3: NOD like receptor proteins and their domain structure. (Adapted from Inohara et al. 2005. (9)). All NLRs have a N-terminal variable domain namely pyrin, CARD, BIR, AD and X. X domain of Nod3 and Nod9 have no homology to known protein. All NLR have a central nucleotide oligomerization domain (NOD domain) and C-terminal leucine rich repeats (LRR). The LRRs are variable among the Nod proteins. APAF1 has a C-terminal WD40 repeats instead of LRRs.

13

expression of each NLRs vary upon cell types and are involved in sensing, of not only

PAMPs but are also involved in sensing cell associated danger signals. Nod1 and Nod2

were the first intracellular receptors to be described in pathogen sensing. Nod1 and Nod2

sense distinct sub structures from the bacterial cell wall. Nod1 senses meso-

diaminopimelic acid (meso-DAP) found in the Gram-negative bacteria and Nod2 senses

muramyl-di-peptide (MDP) which is found in both Gram-positive and Gram-negative

bacteria (42).

1.3.1 NLRs in health and disease

Genetic variations in many of the genes of the NLR family are associated with

development of genetic diseases. Polymorphisms in Nod1 gene are associated with

development of asthma, atopic eczema and increased IgE concentrations in the serum

(43). As the microbial exposure at early childhood prevents the occurrence of asthma, it

is thought that polymorphisms in Nod1 may play a role in bacterial recognition and thus

Th2 polarization and IgE concentration. Mutations in Nod2 gene causes Crohn’s disease

which is characterized by abnormal T cell responses in intestinal epithelium. R702W,

G908R, and L1007insC, the three most common mutations found in or near the LRR of

Nod2 gene, are associated with Crohn’s disease (44,45). This is explained by the fact that

the mutation harboring cell lines or primary monocytes from patients have reduced ability

to respond and produce cytokines in response to MDP (46,47). However, in

macrophages from L1007insC transgenic mice expressing a mutant form of Nod2 there

was increased IL-1β production (48). An increase in IL-1β secretion was not observed

when monocytes were isolated from the patients having the Nod2 mutations (49). This

14 demonstrates that Nod2 mutations have increased inflammation which may be due to reduced bacterial clearance and may lead to uncontrolled infection and enhanced bacterial response by other PRRs independent of Nod2. It has also been shown that mutations may lead to decreased α defensin production by Paneth cells and impaired

TLR2 induced inflammation (6,50,51). Mutations in the NOD region of Nod2 gene are associated with Blau syndrome, an autosomal dominant trait characterized by arthritis, uveitis and skin rashes (44,52-56). Unlike Crohn’s disease, which is mediated by loss of function of Nod2 gene, Blau syndrome is thought to be due to enhanced function of Nod2 protein leading to hyper-inflammatory response. Mutations in CIAS1 genes coding for the protein cryopyrin are associated with many dominant auto-inflammatory disorders such as familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome

(MWS) and neonatal onset multisystem inflammatory disease (NOMID) (57-59).

Patients with these diseases have mutations in the NOD region of cryopyrin. Mutations in cryopyrin are gain of function mutations which lead to enhanced NF-κB and pro- inflammatory caspase activation. Recently, it has been shown that mutations in NALP12 gene causes hereditary periodic syndrome. These mutations lead to deleterious effect on

NF-κB production (60). Mutations in CIITA are known to cause bare lymphocyte syndrome, a condition in which there in absence of expression MHC class II molecules which leads to defective antigen presentation (61,62). The diseases associated with different NLR proteins are listed in Table 1.1.

It has been subjected to research for many years that both Nod1 and Nod2 are involved in the production of antimicrobial proteins which limit the growth of infection.

15

Recently, it was shown that Nod1 and epidermal growth factors are involved in the

production of beta defensins during Helicobacterium pylori infection (63). Nod1 is also

involved in detecting Campylobacter jejuni infection and production of antimicrobial proteins to combat C. jejuni infection. Nod1 and RIP2 are necessary for production of chemokine and anti microbial proteins in mesothelial cells (64). Over expression of

Nod2 in HEK293 cells resulted in increase in hBD2 production whereas this was not seen when the mutant form of Nod2 was expressed. NOD induced production of anti- microbial proteins in mucosa is governed by caspase-12 through RIP2 (65,66). However, the role of NOD proteins in the production of anti-microbial proteins is not well understood yet.

1.3.2 Domain structure of NLR proteins

NLR proteins have three distinct domains. The N-terminal effector binding

domains are either a CARD, pyrin or baculoviral inhibitory repeat (BIR) domain. The N- terminal effector binding domains are involved in protein-protein interactions. The centrally located NOD or NACHT domain binds to ATP and is involved in oligomerization. The C-terminal LRRs are involved in sensing the ligands.

Nod1 and Nod2 were the first to be identified and most studied NLRs. Nod1 is

expressed in epithelial cells and many other tissues whereas Nod2 is expressed only in

monocytes (68). Nod1 is present constitutively in intestinal epithelial cells and it is up

regulated upon IFNγ treatment (69). It has been shown by different groups that Nod1 or

Nod2 synergizes with TLR ligands for the production of may pro-inflammatory cytokines

like IL-6, IL-1β, IL-12 and IL-8 (70-72).

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Auto inflammatory Gene Mutated Disease Inheritance Allelic Variants R260W, V198M, D303N,

Muckle Wells A352V, A439T, G569R Dominant syndrome Familial cold R260W, V198M, L305P, NALP3 autoinflammatory Dominant L353P, A439V, E627G syndrome Chronic infantile R260W, D303N, Q306L, neurological Dominant F309S, H358R, T436N, cutaneous and F573S, M662T articular syndrome

Nod2 Crohn’s Disease L1007fsinsC, G908R, Recessive R702W

Nod2 Blau Syndrome Dominant R334W, R334Q, L469F Δ24(IVSDS G-A), E381ter,

Bare lymphocyte W688ter, 991del, Δ1027, CIITA Recessive syndrome Δ28 (IVSDS G-A), L469P

M694V, M694I, M680I, familial V726A, E167D, (E148Q), Mediterranean fever Pyrin Recessive P369S, R408Q, R653H,

E148V, T267I, F479L,

K695R, A744S, R761H

Table 1.1: Diseases Associated with NLRs (Adapted from Inohara and Nunez. 2003. (67))

17

1.3.3 NOD signaling pathway in activation of NF-κB

It has been shown recently that NLR are involved in sensing intracellular pathogens and mediate apoptotic and pro-inflammatory signaling. It is now known that

NOD1 detects γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP) and NOD2 detects muramyl-di-peptide (MDP). Upon sensing the intracellular pathogens the NOD proteins get activated by an unknown mechanism which then recruits RIP2/CARDIAK through

CARD-CARD interactions (68). Recent studies have indicated that Nod-2 and Nod1 translocates RIP2 to the membrane and this membrane association is involve in Nod1 and

Nod2 function, i.e., NF-κB activation (73,74). Upon recruitment of RIP2, RIP2 gets poly-ubiquitinated and this polyubiquitination is necessary for the function of RIP2

(75,76). RIP2 is also involved in K63 ubiquitination of NEMO/IKKγ, which then phosphorylates IκB leading to the ubiquitination and degradation of IκBα. This leads to the activation of NF-κB (Figure 1.4). Earlier it was believed that RIP2 is an essential adaptor which links the inflammasome pathway to the signalosome pathway and is essential for NF-κB activation (77). This is based on the fact that RIP2 binds both caspase-1 and IKKγ and is involved in both the TLR and NLR induced NF-κB pathway

(78). But recent research shows that RIP2 is dispensable for the TLR pathway but may be required for NOD induced NF-κB pathway (79,80). There are other NLRs which sense pathogens and trigger the host defense by the activation of caspase-1. These NLRs containing multi-protein complexes are known as the inflammasomes and are involved in activation of caspase-1. The Inflammasome will be discussed in detail later.

18

Figure 1.4: NOD Signaling Pathway. Products of peptidoglycan iE-DAP and MDP are recognized by Nod1 and Nod2 respectively. Upon recognition both Nod1 and Nod2 recruit RIP2. RIP2 gets polyubiquitinated and then interacts with the γ subunit of the IKK complex. Since RIP2 does not require its own kinase function to promote NF-κB activation, the role of its binding to IKK is still unclear. IKK subsequently phosphorylates IκBα. IκBα gets ubiquitinated upon phosphorylation. The ubiquitinated IκBα gets degraded through the proteosome pathway. Upon degradation of IκBα, NF-κB translocates to nucleus.

19

1.4 NF-κB pathway

NF-κB is one of the important inducible transcription factors that helps the

organisms to cope with environmental changes. NF-κB is an evolutionarily conserved

transcription factor involved in a variety of cellular functions. It is involved in processes

like regulation of the immune system, survival, development and apoptosis. A proper regulation of NF-κB is required for normal health and homoeostasis (81). Dysregulation

of NF-κB leads to many diseases like hereditary periodic fever and cancer (82).

Understanding how NF-κB functions in with such diverse environmental stimuli to produce the necessary outcomes has been the subject for research for many years.

Regulation of host immune response in response to pathogens is one of the functions of

NF-κB among many other functions known so far.

NF-κB plays a critical role in the function of both the innate and adaptive

immune system in response to a pathogen infection. It helps in the transcription of many genes that are involved in production of cytokines, chemokines, adhesion molecules, acute phase proteins and inducible effector enzymes.

There are five mammalian NF-κB proteins namely RelA (p65), RelB, c-Rel,

p50/p105 and p52/p100 (83). In non-stimulated cells, the NF-κB proteins exist in the

cytoplasm as homo- or heterodimers bound to IκB family proteins. All NF-κB proteins

have rel homology domain (RHD) at the N-terminus. There are two pathways involved

in NF-κB activation. In the classical pathway, upon stimulation, the β subunit of the IκB

kinase (IKK) gets activated which in turn phosphorylates the IκB proteins on two N-

terminal serine residues. In the alternate pathway, IKKα gets activated which leads to the

20

phosphorylation of p100 (84). The phosphorylated IκB proteins are recognized by the

ubiquitin ligase machinery and are poly-ubiquitinated. The ubiquitin ligase identifies the

phosphorylated E3 sequence (DS*GXXS*) on the IκB proteins (85,86). The poly-

ubiquitinated proteins are degraded (IκB) or processed (p100) by the proteosome

machinery to expose the nuclear localization signal and to free the NF-κB which

translocates to the nucleus to activate the transcription of the target genes. NF-κB binds

to the κB sites of the promoter and/or enhancer regions of the genes which have the

consensus sequence 5’-GGGRNNYYCC-3’ (where N is any base, R is purine and Y is

pyrimidine). The activated NF-κB is then down regulated by feedback mechanism in

which the newly synthesized IκBα binds to the NF-κB and exports it to the cytosol.

1.5 Rel/NF-κB proteins

NF-κB proteins were first purified by using the κB site specific affinity column.

This led to the identification of two proteins p65 and p50. Subsequent cloning of p50 led to the identification of other NF-κB proteins. There are five NF-κB proteins identified so far.

NF-κB/Rel proteins are characterized by the presence of RHD in the N-terminus

of the molecule. The 300 amino acids conserved domain is known as the Rel homology

domain (RHD). The RHD is located at the N-terminus of these proteins. The RHD helps

in DNA binding, dimerization, and interacting with the IκB family proteins. Two of the

NF-κB proteins p105 and p100 need to be processed for them to function. p105

undergoes constitutive processing to yield p50 by limited proteolysis (87,88). The

glycine rich region (GRR) present between amino acids 376 and 404 is required for the

21

processing of p105 (89). The presence of precursor p105 in multiple heterodimeric and

homodimeric forms regulates the processing to p50. It has been speculated that the

partial processing may be carried out by an ubiquitin ligase other than SCFβTrCP. It has been reported that p105 can also undergo inducible degradation by IKKβ-dependent and

IKKα-independent phosphorylation (81). This process is similar to that of IκBα degradation by the ubiquitin dependent and proteosome mediated degradation.

Polyubiquitination of p105 involves multiple lysine residues located at the N-terminus region of the protein and this process is dependent on the presence of acidic regions between residues 445 and 453. The processing of p100 is similar to p105 and IκBα.

P100 undergoes very minimal constitutive processing. Upon stimulation NF-κB inducing kinase (NIK) phosphorylates and activates IKKα (90). NIK also acts as a scaffolding molecule which brings IKKα and p100 together. IKKα then phosphorylates p100 which leads to the recruitment of SCFβTrCP ubiquitin ligase. Polyubiquitinated p100 is then partially processed by the 26S proteosome machinery. Like p105 the presence of GRR is required for this partial processing of p100 to p52. The active DNA binding form of NF-

κB is a dimer. Almost all possible homo- and heterodimers of the Rel family have been identified. Not all dimers of Rel proteins are transcriptionally active. RelA, RelB and c-

Rel have the C-terminal transactivation domain and are transcriptionally active. On the other hand, p52 and p50 lack the C-terminal transactivation domain and are considered to be transcription repressors. Homo- and heterodimers of p50 and p52 have been shown to suppress gene expression by competing with the transcriptionally efficient forms of NF-

κB. Both p52 and p50 homodimers have been shown to associate with Bcl-3, a proto-

22

oncoprotein and a member of IκB family. This interaction of Bcl-3 leads to activation of

transcription by two mechanism. First, Bcl-3 can displace the DNA bound p50

homodimers so that the transactivating heterodimer can activate the transcription.

Second, the Bcl-3 can interact with the p50 and p52 homodimers to activate the transcription.

The function of all these NF-κB protein is modified by the binding of IκB

proteins in the cytoplasm. The following section will deal about the various IκB proteins.

1.6 IκB proteins

There are seven IκB family members known so far which includes the newly

identified members, IκB ζ and IκBNS (91). All these proteins namely IκBα, IκBβ, IκBε,

IκBγ, p105, p100 and Bcl-3, are characterized by the presence of 5-7 ankyrin repeats

present at the C-terminus of these proteins. Precursor molecules of p50 and p52 namely

p105 and p100 respectively have ankyrin repeats at the C-terminal end (Figure 1.5).

These proteins inhibit NF-κB activity prior to their processing. The ankyrin repeats are

around 30 residues long and assemble to cylindrical structures which bind to the RHD/

dimerization domain and inhibit NF-κB activity (83). The NF-κB protein (p65/p50 or

p65/c-Rel) was crystallized with IκBα and IκBβ as a complex (92,93). The structural data

revealed that IκBα protein masked only the nuclear localization signal (NLS) of p65 but

not of p50. The accessible NLS of p50 directed the NF-κB/IκBα to translocate to the

nucleus. However, the presence of nuclear export sequence (NES) in IκBα tranlocates it

23

Figure 1.5: The NF-κB and IκB protein family (Adapted from Hayden and Ghosh. 2008. (91)) Members of the NF-κB and IκB protein family are shown. RHD, Rel homology domain, TAD, transcriptional activation domain, DD, death domain, Glycine rich region is indicated by green round rectangle and the ankyrin repeats are indicated by yellow semi-cylinder.

24

back to cytoplasm. The presence of NES and the NLS of p50 helps in the shuttling of

IκBα/NF-κB molecule in and out of the nucleus. This shuttling makes the IκBα/NF-κB localization to both nucleus and cytoplasmic, but it is cytoplasmic at steady state. Upon stimulation, the IκBα is phosphorylated, ubiquitinated, and degraded. The degradation of

IκBα exposes the NLS of p65 and this leads to the nuclear localization of p65. However,

NF-κB/IκBβ masks both the NLS of p50 and p65 which makes it locate only to the cytoplasm.

IκB proteins are less understood compared to their counterparts Rel/NF-κB proteins. IκBα is well studied IκB protein (81,83). The lack of availability of IκBβ knockout mouse makes it difficult to understand the precise role of IκBβ. However, it has been shown that IκBβ plays a role in NF-κB regulation by binding to NF-κB and preventing its nuclear localization. The role of Bcl-3 has been shown to be both activating and repressing. Earlier studies have shown that Bcl-3 binds the p50 and p52 homodimers and removes their repressing effects. Later studies have shown that Bcl-3 based on its state of phosphorylation can bind p50 and p52 homo dimers and promotes transcription. Bcl-3 knockout mice lack the humoral response against multiple pathogens. Bcl-3 knockout also lack spleen germinal centers and fail to generate antigen specific responses. IκBε inhibits the activity of NF-κB (p65/p50 and p50/c-Rel) by interacting with various NF-κB proteins namely p65, p50 and c-Rel. The degradation of

IκBε occurs similarly to IκBα but with slower kinetics. IκBε has nuclear and cytoplasmic shuttling similar to that of IκBα but at slower kinetics and therefore IκBε containing complexes are mainly cytoplasmic. It is believed that IκBε regulates the later stages of

25

p65/c-Rel complexes as the induction of IκBε is dependent on NF-κB activation. IκBγ is

the C-terminal region of the mouse p105 (94,95). The functional significance of are IκBγ

mRNA has been questioned recently. Finally, IκBζ (MAIL) was identified by subtractive

hybridization as the gene product which is highly induced by LPS and IL-1β by two

independent groups (96,97). We identified it in our yeast 2- hybrid systems as a target

protein which interacted with the 28 kDa IL-1β protein. The details of MAIL will be

explained in detail in the coming session.

1.7 MAIL/ IκBζ

MAIL (Molecule containing ankyrin repeats induced by LPS), INAP (IL-1 inducible nuclear ankyrin repeat protein) or IκBζ is a recently identified member of the

IκB protein family. The C-terminal region of MAIL contains six ankyrin repeats and thus

it is classified as the member of IκB family of proteins.

1.7.1 Identification

MAIL was identified recently by five independent groups including our group. Kitamura

et al. 2000 first reported the identification of MAIL (96). They identified MAIL from a

differential display of the genes which were up regulated by LPS in the mouse brain.

They studied 1500 genes which were responsive to LPS in the mouse brain and picked 11

of these genes which were upregulated upon LPS injection intraperitonially. They

identified and named MAIL as a 728 amino acids containing protein with 6 ankyrin

repeats at the C-terminus. There were two set of clones in their library which lacked -63

to +286. They labeled this MAIL-S, a short form of MAIL. Haruta et al. 2001 identified

MAIL by subtractive hybridization as a novel gene induced when OP9 stromal cells were

26

stimulated with IL-1α (97). They named this gene as INAP (IL-1 inducible nuclear

ankyrin repeat protein). Yamazaki et al. (2001) identified MAIL by subtractive

hbridization of LPS treated mouse macrophage cell line RAW264.7 (98). They cloned

this protein which contained 629 amino acids residues having 6 ankyrin repeats at the C-

terminus of the molecule. As this protein was very similar to the proteins of IκB family

they named it IκBζ. We, Parker-Barnes et al. (unpublished data), identified MAIL-S from LPS stimulated monocyte cDNA library as a protein interacting with 28 kDa IL-1β.

This protein is similar to MAIL-S and is an alternatively spliced form which lacks exon

3. Totzke et al. 2006 identified MAIL by suppressive subtractive hybridization as a gene involved in apoptosis of HeLa cells (99). When they treated HeLa cells D98 (TNF

sensitive) and H21 (TNF-Resistant) with TNF and cycloheximide for 1 hour, MAIL was

one of the genes identified as a differentially induced gene. Northern blot analysis

revealed an mRNA size of 3.5 kb. The full length cDNA is 1857 bp encoding a protein of

618 amino acids harboring 6 ankyrin repeats at the C-terminus. This protein showed a

30% homology to the other IκB proteins.

1.7.2 Gene and protein structure

MAIL gene is located in chromosome 3p12-q12. The MAIL gene spans around

30 kb. The gene contains 14 exons. Exon 1 and 2 are located around 22kb upstream of exon 3. There are two major forms of MAIL reported so far. MAIL-L encodes mRNA of

4.3 kb and MAIL-S which is an alternate spliced form of MAIL-L encodes a mRNA of

3.5 kb. Exon 1 and 2 of MAIL-S are not translated. MAIL-L cDNA is 2187 bp and

encodes a protein of 728 amino acid residues. MAIL-S lacks the first 99 residues present

27 in MAIL-L. There has been a third alternative splice form reported recently (Figure

1.6A) (100). This form of MAIL designated as IκBζ (D) or MAIL-D lacks amino acids

188-456 in the exon 7 region. However, the protein product for MAIL-D has not been shown so far. The N-terminal of MAIL is not similar to any known protein.

The gross structure of MAIL protein contains an N-terminal potential PEST region, a nuclear localization sequence (NLS), a Transcriptional Activation Domain

(TAD) located in the middle region and 6 ankyrin repeats located at the C-terminal end of the protein (Figure 1.6B). PEST find program predicts a potential PEST sequence spanning amino acids 184-203 (MAIL-L) and 84-103 (MAIL-S). Unlike other IκB protein family members who have the PEST sequence at the C-terminal end of the protein, MAIL has PEST sequence at the N-terminus of the protein. The PEST sequences are enriched with amino acids proline, glutamic acid, serine and threonine and function in rapid turn over of proteins. However the role of the PEST sequence in MAIL has not been tested so far. There is a bi-partite NLS sequence spanning amino acids 163-

178 in MAIL-L (100). The amino acid sequence of NLS was determined using indirect immunofluorescence studies. It was found that K163, R164, K177 and R178 of MAIL-L is indispensable for nuclear localization, mutating these residues affected the nuclear localization dramatically when expressed in isolation. The ankyrin region of MAIL was localized uniformly in the cytoplasm. The region after the NLS and preceding the ankyrin repeats was of unknown function. Recently, Motoyama et al. 2005 found the transcriptional activity of the amino acids spanning the region 329-403 of mouse MAIL-

L (100). They found this activity by generating a series of deletion constructs of MAIL-L

28

Figure 1.6: MAIL Gene and Protein Structure (Adapted from Muta. 2006. (101)) A) Location of MAIL gene intron exon in human genome. The numbers depict each exon which encodes the MAIL protein. The exon information of MAIL-L and MAIL-S are depicted below. Exon 3 is spliced out in MAIL-S. B) Domain information of MAIL protein. Nuclear localization signal (NLS), Transactivation domain (TAD) and the box at C-terminal end represent the 6 ankyrin repeats.

29

fused with GAL4 protein and analyzing the role of this fusion protein in a GAL4 reporter assay in HEK293 cells. However, when full length MAIL-L was fused to GAL4 protein there was no detectable transcriptional activity. The C-terminal ankyrin region was

attributed to the suppression in the transcriptional activity. This suppression was over

come by transfecting p50 protein which binds to the ankyrin repeats of MAIL-L through

the RHD. Ankyrin repeats are found in around 400 proteins having diverse function.

Like the other IκB proteins, MAIL has six ankyrin repeats, with each repeat having

around 30 amino acids. It has been shown in the case of IκB protein and MAIL that the

ankyrin repeats function is to bind the RHD and inhibit gene trans-activation. IκB

members inhibit the localization of NF-κB members to the nucleus and prevent the

activation of NF-κB. MAIL, as it is, localized in the nucleus prevents the activation by

binding to NF-κB in the nucleus. The precise mechanism of how MAIL functions to

inhibit NF-κB activation is subjected to further research.

1.7.3 Expression and localization

MAIL-L is the predominant form of MAIL expressed upon LPS stimulation

(102,103). Mouse MAIL-L has been shown to express in lung, lymph node, liver, heart,

eyes and kidney upon stimulation with LPS and IL-1β (96,97). However there are

conflicting reports on the expression of mouse MAIL-L in spleen and brain upon LPS

stimulation (96,98). It has been shown by in situ hybridization and immunohistochemical

analysis that MAIL mRNA is expressed in B lymphocytes of white pulp of the spleen and

cortical lymphoid follicle of lymph nodes in the LPS injected mice (102). Mail expression was also seen in F4/80 positive macrophages in these organs. MAIL

30

expression was faintly detected in T-lymphocytes, fibroblasts and endothelial cells.

Northern and Western blot analysis revealed MAIL-L as the major form of MAIL in the

B-lymphocyte and macrophages (102). There are conflicting reports about the expression

of MAIL in the skin. Ueta et al. 2005 detected no MAIL expression in the skin of mice by real time PCR (104). However, Oonuma et al. 2007 have detected constitutive MAIL

expression in the skin of mice (105). Expression of MAIL has been shown to be

constitutive in skin epidermis but not in the dermis (105). This expression of human

MAIL has been shown to be expressed in different tissues such as lung, placenta, liver,

heart, spleen, kidney, skeletal muscle and in peripheral blood leukocytes. The expression

was highest in lung and peripheral blood leukocytes and low in skeletal muscle, spleen,

kidney and heart (99).

It has been shown that MAIL when transfected translocates to the nucleus (96,97).

Both MAIL-L and MAIL-S have been shown to translocate to nucleus and form a

granulated or a speckled pattern (96-98). The NLS present at the N-terminal of MAIL is

responsible for nuclear localization, as N-terminal deletion of MAIL failed to localize to

nucleus. It will be interesting to see whether MAIL can shuttle to nucleus and cytoplasm

like IκBα. However, as there are no NES reported so far, it is unlikely for this to happen.

Immuno-fluorescent microscopy indicates that MAIL forms speckles or dot-like

structures in the nucleus. Recently it was shown that MAIL co-localizes with nuclear co-

repressor SMRT and HDAC5 in matrix associated deacetylated nuclear bodies (99). It is

possible that MAIL may regulate transcription by modulating HDAC5 in the nucleus.

31

1.7.4 Mechanism of MAIL induction

MAIL is an inducible gene whose expression is induced upon stimulation. MAIL

expression is constitutive in primary keratinocytes (105). MAIL-L has been shown to be induced by ligands that trigger Toll/IL-1 pathway namely LPS (TLR4), Bacterial Lipo

Protein (BLP) (TLR1/TLR2), flagellin (TLR5), peptidoglycan (PGN)( TLR2), MALP-2

(TLR6/TLR2), R848 (TLR7), CpG (TLR9) and IL-1β. However the ligands that do not induce the MyD88 dependent Toll/IL-1 pathway like TLR3 and TNF do not induce

MAIL expression. TLR3 signaling pathway utilizes TRIF/TICAM1 as an adapter molecule and not MyD88. It has been shown that MyD88 is required for MAIL expression as macrophages from MyD88 knockout animal do not express MAIL upon stimulation with various TLR ligands and IL-1β (106). On the other had it has been shown that overexpression of MyD88, TRAF6 but not TRAF2 increases the expression of MAIL. TNF pathway utilizes TRAF2 and not TRAF6 to activate NF-κB and MAP kinase pathway. This data agrees with the previous finding that MAIL expression is up regulated by LPS, IL-1β and not by TNF (98). MAIL expression was inhibited by NF-κB inhibitors namely PDTC, BAY 11-7082, acetylsalicylic acids and proteosome inhibitors

MG132 and lactacystin (98,107). Overexpression of components of NF-κB namely p65 and p50 induced IκBα expression but not of MAIL expression. This indicates that there is a second stimulus/signal derived from the MyD88 pathway which is essential for the expression of MAIL (108). The promoter region upstream of the MAIL gene is highly conserved in mouse and humans. It was also shown that NF-κB actively binds to this region of the MAIL promoter upon LPS stimulation in RAW264.7 mouse macrophages.

32

There are four NF-κB sites present in the promoter of MAIL gene (107). Three of these

NF-κB binding sites are highly conserved between mouse and human. Mutational and

deletion analysis revealed that the region -229 to -220 is very important in LPS induced

MAIL promoter activation. Apart from NF-κB activation in MAIL promoter activity,

CREB may also be involved in the expression of MAIL. This was demonstrated by

overexpression of dominant negative form of CREB and the use of deletion constructs of

CREB binding region present in the promoter region of the MAIL gene. Use of dominant

negative form of CREB or the use of deletion constructs of CREB in MAIL promoter

decreased the activity by 50 percent. Overexpression of MAIL-L or MAIL-S also

decreased the MAIL promoter activity. This suggests that there might be a feedback

regulation of MAIL. However the work by Yamazaki et al. 2005 identified -359 to

initiation site of the MAIL promoter to be the important region which is responsible for

LPS responsiveness (103). They identified three NF-κB binding sites κB1(-256 to -247),

κB2 (-218 to -209) and κB3(-83 to -74) in the promoter region of the MAIL gene. The two NF-κB sites κB1 and κB2 were highly conserved in humans and mouse. They showed that the promoter region of MAIL having these three NF-κB sites was more LPS responsive than the traditional 4X-κB sites. Mutational analysis of each of the κB sites revealed that κB1 and κB3 sites were dispensable for LPS responsiveness; however the second NF-κB site (κB2) was indispensable for LPS responsiveness in RAW264.7 cells.

Mutation of the κB2 site completely abolished LPS mediated promoter activation in

RAW264.7 cells. Analyzing the promoter region of MAIL yielded no elements that explained the IL-1β and LPS specific expression of MAIL. Stimulation of NIH3T3 cells

33 with the three stimuli LPS, IL-1β, and TNFα stimulated the activation of MAIL promoter activity and the κB2 site was important for this activation. In order to find the difference between LPS, IL-1β and TNFα in the induction of MAIL, whether the differences observed were at transcriptional or post transcription level, nuclear run on analysis were performed. TNFα induced the transcription of MAIL gene in NIH3T3 cells similar to the induction by IL-1β and LPS. Nuclear run on analysis and NF-κB promoter analysis revealed that TNFα can indeed induce the expression of MAIL mRNA, and there is no difference in transcriptional regulation of MAIL gene. This revealed that MAIL gene may be regulated at the level of post-transcription. Analyzing the stability of MAIL mRNA indicated that IL-1β and LPS induced a stable MAIL mRNA whereas TNFα induced MAIL mRNA was not very stable. This was attributed to a cis-element present in

765 bp N-terminal region in the ORF of the MAIL gene. However the work from the same group has shown that a cis-element present in the 3’ untranslated leader sequence present in the MAIL mRNA governs the stimulus specific expression of the MAIL mRNA. A 165 nucleotide fragment present at the 3’ untranslated region of MAIL mRNA is critical for stimulus specific induction. The apparent differences in the finding may be attributed to the methods used to identify the destabilizing fragment. In the first method, actual mRNA was measured after actinomycin D treatment and in the second method the mRNA of luciferase was fused with MAIL and the protein product was used as a marker to check the stability of the transcripts and stimulus specific induction. The role of TNFα in the induction of MAIL is subjected to further research as there are conflicting reports about the role of TNFα in MAIL induction (98,99,102). Recently it

34

was also shown that MAIL is induced upon cross linking of surface Ig complex on B

cells (109). Stimulation of FCγRIIB which is an inhibitory receptor suppressed the expression of MAIL. The expression of MAIL upon stimulation of B cell receptor is NF-

κB mediated. Use of NF-κB inhibitors during stimulation suppressed the expression of

MAIL.

1.7.5 MAIL interacting proteins

Little is known about the binding partners of MAIL. There are conflicting reports

about binding of MAIL to NF-κB members. Haruta et al. 2001 when they first reported

MAIL detected no binding of MAIL to NF-κB members (p65, p50 or p52), C/EBPβ and

c-Fos/c-Jun. Since MAIL is a close homolog to Bcl3, they also tried immuno-

precipitating MAIL with Bcl3 binding partners, namely RXR and AP-1. They did not

detect any interactions of MAIL to any of these proteins. Subsequent research has

identified p50 as MAIL binding partner. The discrepancy in the finding of the two

groups is not clear but this may be attributed to the use of different systems. It was shown

by different groups that MAIL binds preferentially to p50 but not to p65 (106). However

recent data shows that human MAIL can bind to p65 also (99). We identified MAIL as a

binding partner to 28 kDa IL-1β in our yeast two hybrid system. We have proved in

systems other than yeast that MAIL indeed binds to IL-1β. But we do not know the

functional significance of this interaction.

1.7.6 Targeted gene disruption of MAIL

Two groups generated mice which have targeted disruption of the MAIL gene

(106), (110). One targeted the first 5 exons and the other had replaced a 2.0 kb fragment

35 replacing a central portion of the MAIL gene. The ratio of wild type (+/+): heterozygous

(+/-): homozygous (-/-) homozygous was (1:1.7:0.1) and did not maintain a Mendelian type of inheritance, indicating that most of the knockout animals died before birth. The animals which survived were normal and asymptomatic for 4-8 weeks after birth. After that all the mice developed scaling and thickening of the periocular skin and thickening of the eyelids.

The symptoms became worse with age progression. All the mice at the age of 10 weeks developed allergic/ atopic dermatitis with acanthosis, parakeratosis, hyperkeratosis, and lichenoid changes (110). The affected tissue was infiltrated with mononuclear cells, neutrophils, lymphoid cells, melanophages, mast cells and eosinophils. Many chemokines which are associated to AD like TARC, eotaxin and

CCR3 were expressed in the skin of the affected animals. The serum concentrations of

IgE were around 2.4 times higher in MAIL (-/-) mice compared to the wild type mice.

Also, MAIL (-/-) splenocytes showed a defect in proliferation when they were stimulated with LPS but not with anti CD40, IL-4 and anti IgM. MAIL (-/-) animals showed spontaneous ocular inflammation with infiltration of inflammatory cells like

CD45R/B220+ and CD4+ cells. MAIL (-/-) animals had a heavy lymphocyte infiltration in their conjunctivae which led to the loss of goblet cells. The symptoms exhibited by the

MAIL (-/-) mice is similar to the Stevens-Johnson syndrome in human where there is a complete loss of goblet cells (110).

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1.7.7 Functions of MAIL

MAIL was first identified and reported by Kitamura et al. 2000. They over expressed MAIL and found that MAIL potentiated LPS induced IL-6 production. Since then, many groups have been involved in understanding the mechanism of action and function of MAIL. Now, based on the knock out mouse model, we know that MAIL acts as a transcriptional regulator and is involved in production of many cytokines. Also other than regulating transcription MAIL is also involved in NF-κB activation and apoptosis.

Figure 1.7 shows the mechanism of action of MAIL.

1.7.7.1 Role of MAIL in NF-κB activation

It has been shown that MAIL acts as both a positive and negative regulator of NF-

κB activity. Members of the IκB family of proteins act as a negative regulator of NF-κB activity by binding to the RHD of NF-κB/Rel proteins and thereby preventing their localization to the nucleus. MAIL, unlike the other IκB proteins is localized in the nucleus and it regulates the NF-κB activity by inhibiting the NF-κB proteins to bind

DNA. Recombinant MAIL inhibited the p65/p50 binding to DNA in vitro. In

RAW264.7 cell transfection experiments, overexpressing different forms of MAIL, (full length, MAIL-N (amino acids 1-314), MAIL-C (amino acids 315-629)), all the three forms MAIL inhibited NF-κB activity. Full length and MAIL-N localized in nucleus and inhibited NF-κB by inhibiting DNA binding. Whereas MAIL-C was more effective in inhibiting NF-κB, was cytoplasmic. MAIL-C inhibited NF-κB by inhibiting the translocation of NF-κB. NF-κB activity induced by LPS, different cytokines (IL-1β and

TNFα) and overexpression of NIK, p65/p50 was suppressed by overexpressing MAIL.

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Figure 1.7: Mechanism of MAIL action. (Adapted from Muta. 2006. (101)) TLR stimulation activates the NF-κB pathway which results in degradation of IκBα and translocation of NF-κB to the nucleus. Translocated NF-κB turns on different sets of genes including that of MAIL. Synthesized MAIL goes to the nucleus binds to the promoter elements of MAIL responsive gene with the help of p50. This leads to expression of MAIL dependent genes A) like IL-6, GM-CSF, G-CSF and IL-12. MAIL is also involved in suppression of expression of certain genes B) However, the mechanism by which MAIL suppresses the expression is not yet known.

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Now, it is well agreed by many groups that the C-terminal of MAIL inhibits NF-κB

activity. On the other hand, when human MAIL-N was over expressed in HEK293 cells, there was no observable suppression of NF-κB activity when induced by TNFα. MAIL

has also been shown to induce transcriptional activation. HEK293 cells transfected with different fragments of MAIL fused with GAL4 DNA binding domain showed transcriptional activity measured by GAL4 reporter plasmid. The transcription activity was attributed to amino aids 329-403 (103). Full length MAIL did not have any transcriptional activity. However when full length MAIL was expressed with either p65 or p50 the transcriptional activity was enhanced. This indicated that the C-terminal region of MAIL might have an inhibitory role in transcriptional activity of MAIL.

1.7.7.2 Role of MAIL in the production of cytokines

Macrophages from a MAIL knockout animal failed to produce IL-6 in response to

Toll ligands namely LPS (TLR4), CpG (TLR9), R-848 (TLR7), PGN (TLR2) MALP-1

(TLR) and BLP (TLR). Mouse embryonic fibroblasts cells from MAIL knockout

animals were deficient in producing IL-6 in response to IL-1β but not to TNFα (106). It

was also shown that MAIL specifically interacted with p50 subunit of the NF-κB.

Transfection of MAIL increased the Il6 but not Elam1 promoter activity in RAW264.7 cells. Chromatin immunoprecipitation assay revealed that both MAIL and p50 subunit of the NF-κB bound to the κB site of the Il6 promoter. In accordance with the above data, macrophages from NF-κB deficient mouse had significantly low IL-6 but not TNFα

production. MAIL deficient macrophages were also deficient in production of GM-CSF,

IL-12p40, G-CSF, C/EBPδ and endothelin upon LPS stimulation (106).

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Microarray analysis comparing the wild type and MAIL knockout macrophages upon LPS stimulation revealed an array of genes that were affected by the absence of

MAIL. Among the genes that were affected by MAIL deficiency were IL-18, GMCSF,

G-CSF, TSP1, caspase-4, and CCL3 (106). It is to note that MAIL knockout macrophages produced more serum TNFα upon LPS stimulation compared to wild type mice. However, there was no difference in level of IL-6 in the serum between MAIL knockout or wild type animals upon LPS injection. The difference in IL-6 production could be restored when the animals were given anti-TNFα antibody along with LPS. This was attributed to excess production of TNFα in MAIL knockout animals. It is not clear whether MAIL might regulate the expression of TNFα in macrophages. But in the microarray data TNFα was not in the list of genes that were regulated by MAIL. It will be interesting to test whether MAIL might regulate the expression of TNFα.

1.7.7.3 Role of MAIL in the production of anti microbial proteins

The role of MAIL in the production of Neutrophil Gelatinase Associated

Lipocalin (NGAL) and human beta defensin2 (hBD2) was elucidated recently (111).

Macrophages from MAIL knockout animal showed low levels mRNA expression of

NGAL and hBD2. NGAL is siderophore which binds iron and thereby having a bacteriostatic effect. NGAL is produced by epithelial cells upon stimulation with IL-1β.

Suppression of MAIL in A549 cells resulted in suppression of NGAL expression. It was also shown that MAIL also regulated the expression of hBD2 in A549 cells indicating the importance of MAIL in the lung epithelium against pathogen defense. MAIL also regulates the production of hBD2 upon stimulation with IL-17 (112).

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1.7.7.4 Role of MAIL in apoptosis

NF-κB pathway increases the expression of many genes that are anti-apoptotic and are involved in cell survival. As MAIL inhibits NF-κB activity it may be involved in regulation of apoptosis. Overexpression of MAIL in HeLa cells make them susceptible to

TNFα induced apoptosis (98). Totzke et al. 2006 isolated MAIL by a differential screening technique. MAIL was one among the various genes that modulates the sensitivity of cancer cells (HeLa) to TNFα mediated apoptosis. Suppression of MAIL in

HeLa D98 cells by using stably transfected siRNA decreased TNFα and anti-CD95 mediated apoptosis. Similarly, overexpression of MAIL by itself but not its homolog

IκBα in HT-1080 cells rendered them more sensitive apoptosis (99). This finding is in agreement with the anti-apoptotic role of NF-κB and MAIL might make the cells susceptible to apoptosis by inhibiting the activation of NF-κB.

1.7.7.5 Role of MAIL in adaptive immunity

Not much work has been done to elucidate the role of MAIL in adaptive

immunity. It was shown by in situ hybridization and immuno histochemical analysis that

MAIL mRNA is expressed in B lymphocytes of white pulp of the spleen and cortical

lymphoid follicle of lymph nodes in the LPS injected mice (102). LPS, but not IL-4,

induced splenocytes from MAIL knockout animal were defective in proliferation. This

indicates that MAIL may play an important role in governing the adaptive immunity of

the host. Recent work has shown that MAIL can be induced by the activation of B cell

receptor with F(ab’)2 fragment. This was dependent on NF-κB activation. However, when an intact IgG (which co-crosslinks FCγRIIB) was used this resulted in suppression

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of MAIL expression. Macrophages from MAIL knockout animals are defective in

production of IL-12p40 upon stimulation with toll ligands (106). As secreted by

macrophages play an important role in triggering the onset of adaptive immunity, we can speculate that MAIL has an important role in normal function of adaptive immunity. It would be very interesting to test whether MAIL knockout animals are defective in adaptive immunity.

1.8 IL-1β, caspase-1 and the inflammasome

1.8.1 IL-1β and inflammation

IL-1β is a pro-inflammatory cytokine which plays an important role in innate host

defense and host homeostasis (113,114). It is produced mainly by activated monocytes

and macrophages. IL-1β affects many cell types and therefore has a diverse effect on

host homeostasis. IL-1β functions in systemic and local responses to infection, injury,

and immunological challenges and is the primary cause of inflammation. Dysregulation of IL-1β is involved in many inflammatory diseases such as which are characterized by recurrent fevers, leukocytosis, and increase of acute phase proteins. It is an endogenous pyrogen, injecting a small dose of IL-1β results in fever, thrombocytosis, acute phase proteins, and neutrophilia. Elevated level of IL-1β is observed in chronic inflammatory diseases such as arthritis, systemic lupus erythematous, and septic shock (115).

Although we know more about the biological effects of IL-1β, not much is known

about the processing and secretion of IL-1β. IL-1β is synthesized by monocytes very

rapidly upon stimulation with bacterial products which activate the TLRs or by host

inflammatory mediators. NF-κB pathway is essential for the production of IL-1β. ProIL-

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1β is a leaderless peptide containing protein of mass 31 kDa. Following synthesis, the

proIL-1β is present primarily in the cytosol until it is processed by a cysteine protease

known as caspase-1 or IL-1β converting enzyme (ICE) and secreted out of the cell

Recently, the role of active caspase-1 in secretion of proteins with leaderless peptides

have been described (116). It was shown that leaderless proteins need the activity of

caspase-1 for their secretion. Binding of these proteins either directly or indirectly to

caspase-1 facilitates their secretion.

1.8.2. Caspase-1/ICE (IL-1β Converting Enzyme)

Caspases are family of cysteine protease that are involved in two diverse function

in the cell namely apoptosis and inflammation. Caspases recognize a tetra-peptide

sequence and cleave the peptide bond C-terminal to the aspartic acid residue. Caspases

can be broadly classified into inflammatory caspases and apoptotic caspases based on

their function. In mammals there are five inflammatory caspases caspase-1, caspase-4,

caspase-5, caspase-11 and caspase-12. Caspase-11 is present in mouse and not in

humans. Humans have a non-functional form of caspase-12 (117,118). The

inflammatory caspases as the name suggests are involved in inflammation. Of the known

inflammatory caspases, caspase-1 is well studied. Caspase-1 cleaves proIL-1β to the

mature form and it is also involved in cleaving proIL-18 and may also be involved in the processing of IL-33 (119-123). Caspase-1 cleaves proIL-1β at two places, aspartic acid

27 and aspartic acid 116. In vitro and in silico approaches indicate the aspartic acid 27 cleavage occurs first and then the cleavage at position 116 (124). The mechanism of processing and release of IL-1β is shown in Figure 1.8. The requirement of caspase-1 in

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IL-1β processing and release was revealed by generation of caspase-1 knock out animal.

These mice are impaired in processing of IL-1β and are resistant to lethal effects of

endotoxic shock and also in infection with live E.coli (125,126). Caspase-1 belongs to the ICE/CED-3 family. Most of the proteins of this family cause apoptosis on overexpression (127). Based on this, caspase-1 is thought to be involved both in inflammation and apoptosis. The role of caspase-1 in cell survival has been recently elucidated (128). Like other caspases, caspase-1 is expressed in monocytes and macrophages as an inactive zymogen precursor of 45 kDa. The precursor form is cleaved into the P10 and P20 heterodimer, which forms the mature protein. Similar to other initiator caspases, which require the formation of multi-protein complexes for activation, caspase-1 requires a multiprotein complex called inflammasome. The idea behind the requirement of multi-protein complex is derived from the earlier studies involving the apoptosome which is required for the proteolytic processing of caspase-9 (129). The multiprotein complex helps in bringing the initiator caspases into close proximity and this leads to the proteolytic processing of caspases. The multiprotein complex called the inflammasome activates caspase-1 by induced proximity model. The complex is different based on the cell types and the nature of the PAMPs (130-136).

1.8.3 Inflammasome and its regulation

The proteins that form the scaffold of the multiprotein complex have three

domains which help in activation of caspases. First, they should have a protein-protein

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Figure 1.8: Mechanism of IL-1β production and processing. When endotoxin is sensed by TLRs here TLR4 leads to activation of NF-κB pathway through IKK complex. NF-κB activation leads to the production of proIL-1β. ProIL-1β is cleaved by caspase-1 leading to the formation of mature IL-1β. Mature IL-1β is secreted outside the cell by an unknown mechanism which causes the inflammation.

45

interaction domain or also called an effector domain which is usually a domain from the

death domain family. The N-terminal effector domain is capable of interacting with the

initiator caspases. Second, an oligomerization domain that helps to form dimers and

oligomers and also a domain which would regulate the activity of the protein. The NLR proteins have all these characteristics of the scaffolding protein. They have the N- terminal pyrin/CARD domain, a central nucleotide binding and oligomerization domain

(NOD) and C-terminal LRR domain. The C-terminal LRR domain restricts the activity of

the NLRs by directly binding to the NOD domain. The recognition of pathogen or

danger signals by the LRRs, brings about a change in conformation of the scaffolding

molecule which later binds adaptor molecules and the caspases to activate the

oligomerization and proteolytic processing of the caspases. NLRs are ideal candidates

for the formation of the inflammasome. The inflammasome was first characterized and

described based on biochemical experiments in THP-1 cells (130). NALP1 (NLRP1)

was the first central scaffolding protein for an inflammasome to be described. In the

NALP1 inflammasome, NALP1 is the scaffolding molecule which has both a pyrin

domain at the N-terminus and CARD domain at the C-terminus, a central NOD domain followed by LRR. Upon activation the NALP1 binds to the adaptor molecule apoptotic speck like protein containing a CARD (ASC) through the pyrin-pyrin domain interaction.

The ASC recruits caspase-1 through the CARD-CARD interaction. The C-terminus of the

NALP1 recruits caspase-5 through CARD-CARD interaction (Figure 1.9). This recruitment of all the molecules brings about activation of both caspase-1 and caspase-5

46

by induced proximity model. Now we know that there are at least three inflammasomes in different cell types and based on diverse stimuli.

1.8.4 Types of inflammasome and their stimuli

1.8.4.1 NALP1/NLRP1 inflammasome

NALP1, the first inflammasome identified, was based on biochemical studies in which

the inflammasome formation was triggered by activating cell free lysates at 30 ºC. Now

we know that NALP1b inflammasome senses the anthrax lethal B toxin (LT) and activates caspase-1(137). Recent studies have shown that K+ efflux and proteosome

activity is required for NALP1 activation in response to LT. Use of K+ channel blocker

or a proteosome inhibitor blocked the activation of caspase-1 thereby caspase-1 induced

necrosis (138,139).

1.8.4.2 NALP3/NLRP3 inflammasome

NALP3 inflammasome is the most characterized inflammasome. Mutations of

NALP3 are associated with inflammatory diseases such as MWS, NOMID and CINCA.

The NALP3 inflammasome consists of NALP3 as the scaffolding molecule, ASC as the

adaptor which binds to the caspase-1 through CARD-CARD interaction and the protein

cardinal but there is no caspase-5 (133,140). The NALP3 inflammasome senses a variety

of PAMPS and danger associated molecular patterns (DAMPs). NALP3 recognizes

MDP, Listeria monocytogenes, Staphylococcus aureus, gout, and pseudogout crystals,

bacterial RNA, antiviral compounds R837 and R848, UVB, ATP, and viruses and

bacterial or mammalian DNA (132-136,141-143). It is quite intriguing to think that an

47

Figure 1.9: NALP1 inflammasome. NALP1 inflammasome was the first inflammasome described by Martinon et al. 2002. Inflammasome a multi protein complex is formed by unknown mechanism. The proteins of the inflammasome assemble though the homotypic protein-protein interactions. NALP1 inflammasome consists of NALP1 as NLR, ASC as the activating adaptor, caspase-1 and caspase-5. Upon assembly caspase-1 and caspase-5 gets processed by induced proximity model.

48 intracellular PRR recognizes a wide variety of PAMPs and DAMPs unlike the TLRs which are not known to have such diversity in recognition.

1.8.4.3 IPAF/NLRC4 inflammasome

IPAF or NLRC4 is another NLR which is homologous to APAF-1. APAF-1 is a key molecule involved in the activation of caspase-9. Based on overexpression experiments IPAF has been implicated in caspase-1 activation and apoptosis (144,145).

IPAF binds directly to caspase-1 through CARD-CARD interactions and therefore there is no need for the activation adaptor, ASC. Recent studies with IPAF deficient mice support a role of IPAF in Salmonella mediated caspase-1 activation (146). It was known from the IPAF knock out studies that IPAF is essential for Salmonella induced caspase-1 activation. However the ligand for IPAF was not known, until recently when two groups demonstrated that flagellin can be recognized by IPAF. This recognition was independent of TLR5 and required type III secretion system (147,148). Now we know that IPAF is also required for sensing the intracellular bacteria Pseudomanas aeruginosa.

It is not clear whether the sensing of Pseudomonas is mediated through recognition of flagellin by IPAF (149-151).

NAIP5, another NLR protein, in combination with IPAF has been shown to activate caspase-1 in response to Legionella pneumophila. Genetic screening of mouse identified NAIP as the gene which makes the mouse susceptible to Legionella pneumophila (152,153).

So far, these are the three inflammasome which have been identified. Table 1.2 describes the types of inflammasome characterized and their ligands. There are many

49

reports of other inflammasome proteins in caspase-1 activation solely based on the

overexpression models. Since there are around 23 NLRs more work needs to be done to

elucidate the functions of other NLR proteins.

1.8.5 ASC and Pyrin – Regulators of inflammasome activity

The Death Domain super family consists of death domain (DD), death effector

domain (DED), caspase recruitment domain (CARD) and a pyrin domain (PYD). The

proteins of death domain super family have six helices that are arranged in classical death

fold. Proteins containing death folds play important roles in apoptosis and inflammation

(154-156). Proteins having these death domains are involved in caspase activation by the

formation of multi-protein complexes such as apoptosome, inflammasome, and

piddosome by homotypic protein-protein interaction domains (157). Mutations in the

number of PYD and CARD domain containing proteins have been linked to many

autoinflammatory diseases (48,57,60,158).

1.8.5.1 ASC- The caspase-1 activator

ASC was initially identified in HL-60 cells as a detergent insoluble protein that

forms specks when these cells were undergoing apoptosis upon retinoic acid treatment

(159). ASC is a bipartite protein consisting of two interaction domains, an N-terminal pyrin domain and a C-terminal CARD domain. A number of reports document a proapoptotic role for ASC (162). ASC also co-localizes with BAX to the mitochondria to induce apoptosis (163). However, ASC knockout animals did not show any signs of defect in apoptosis (146). ASC is also a target for methylation-induced gene silencing.

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NLR Elicitor Reference NALP1b Lethal Factor from Anthrax (137)

Microbial Motifs MDP (133) NALP3 Bacterial RNA (141) Bacterial DNA (136) Imidazoquinoline Compounds (135) Live Bacteria S. aureus (134) L. monocytogenes (134) Microbial Toxins Aerolysin (A.hydrophila) (128) Maitotoxin (Marine dinoflagellates) (134) Nigericin (Streptomyces hygroscopicus) (134) Danger Associated host components ATP, NAD+ (134) Mammalian DNA (136) Uric acid crystals (132) Skin Irritants (160) UVB (142,143) IPAF Microbial Motifs Cytosolic Flagellin (147),(148) Live Bacteria S. typhimurium (134) P. aeruginosa (150,151)

Microbial Motifs Naip5 Flagellin (152) Live Bacteria

L. pneumophila (153)

Table 1.2: Types of inflammasomes and their respective ligands. (Adapted from Petrilli et al. 2007. (161)

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ASC gene is methylated in many breast tumors and other cancers implying a role in

tumorigenesis (164,165). ASC expression is up regulated by inflammation and apoptosis

in neutrophils (166). ASC activates caspase-1 by the formation of the inflammasome

(130,167,168). In this context, macrophages from ASC knockout are unable to process

and release IL-1β and IL-18 (146). However, in IPAF inflammasome that recognizes

Salmonella typhimurium, there is no requirement of ASC. This is because IPAF can

directly bind to caspase-1 through CARD-CARD interactions and this binding leads to

the activation of caspase-1. ASC also interacts with other proteins such as pyrin,

caspase-8, proteins of the NOD family (NALP1, NALP3 and IPAF) to induce NFκB

activation and apoptosis (162,169-172). Recently it has been demonstrated that ASC is

involved in a supra molecule complex called the pyroptosome which cleaves caspase-1 in

the absence of an NLR protein and thereby leading to apoptosis (173).

1.8.6 Pyrin – The cause for familial Mediterranean fever

Pyrin was identified by positional cloning, mutations of which caused familial

Mediterranean fever (FMF) (174,175). FMF is an autosomal recessive disease

characterized by spontaneous bouts of inflammation with no underlying cause of

infection (158). Pyrin belongs to a family of proteins that have the death domain fold.

Pyrin has been shown to play a role in apoptosis and inflammation. This is based on the idea that pyrin interacts with ASC though the pyrin-pyrin domain interactions. As ASC

is involved in both apoptosis and inflammation, pyrin is thought to be involved in these

two processes. In contrast to the role of ASC to promote apoptosis, pyrin has been shown

to suppress ASC induced apoptosis (176,177). Similar to role of pyrin in suppression of

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apoptosis by binding to ASC, pyrin is thought to be a negative regulator of caspase-1

activation and thereby IL-1β induced inflammation. However, recent data suggests that

this idea of pyrin suppressing ASC induced caspase-1 activation may not be true. As of

now, the role of pyrin in caspase-1 activation is controversial and is subjected to area of

intense research (169).

1.8.6.1 Domain structure of pyrin protein

Pyrin is an 86 kDa protein expressed in myeloid cells and is upregulated during myeloid

differentiation (178). The pyrin protein is divided into four major domains. The N-

terminus (residues 1-92) encodes a pyrin domain (PYD), the B-box Zinc Finger motif

(residues 370-417), the coiled-coil region and the C-terminal B30.2/ rfp/ SPRY like domain (residues 598-774) (Figure 1.10 ). Pyrin domain is a member of death fold super family and is found in many proteins, which are involved in inflammation and apoptosis.

The B-box Zn finger motif and the coiled coiled domain are involved in protein-protein interactions. The C-terminal B30.2 domain is present in more than 700 mammalian proteins. It is present in proteins that are involved in diverse functions such as cytokine signaling, RNA metabolism, intracellular Calcium release, differentiation and immunity to viruses (179-181). The C-terminal domain of pyrin resembles a transcription factor, but experiments failed to show that native pyrin localizes in the nucleus and had transcriptional activity (178). The structure of B30.2 domain was recently solved by three different groups. B30.2 domain folds like a carbohydrate binding domain. This implies that the function of B30.2 domain in innate immunity may be due to the carbohydrate

recognition capability of the B30.2 domain (69,182,183).

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Figure 1.10: Domain structure of pyrin protein. (Adapted from Yu et al. 2007. (131)) Domain information of pyrin protein. Various domains and their interacting proteins are indicated.

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1.8.6.2 Expression and co-localization

Pyrin is predominantly expressed in granulocytes, monocytes and fibroblasts but

not in macrophages (184,185). Proinflammatory agents such as IFNγ, TNFα and LPS

stimulated the expression of pyrin. The expression of pyrin mediated by anti

inflammatory cytokines like IL-10 and IL-4, is not clear as the same group reported

conflicting data (184,186). Pyrin co-localization is not completely understood yet. Pyrin

is predominantly nuclear in synovial fibroblasts, neutrophils, and dendritic cells, but is

cytoplasmic in monocytes. The co-localization of pyrin may depend on the existence of

alternate splice forms and its interacting partner (187,188). Pyrin co-localizes with actin and it interacts with microtubules (189). The co-localization and interaction of pyrin with the cytoskeleton proteins may indicate that pyrin has a role to play in cytoskeleton remodeling and therefore the phagocytosis of microorganisms.

1.8.6.3 Pyrin interacting proteins

There are only few proteins that are known to interact with pyrin. Pyrin interacts with proteins which are involved in a variety of functions. To determine the proteins interacting with the C-terminal domain, Chen et al. 2000 did a yeast 2-hybrid of B30.2

domain of pyrin as bait. They found that pyrin interacted with a putative golgi transporter

(178). The functional significance of this interaction is yet to be ascertained. Richards et

al. 2001 found ASC to be pyrin’s interacting protein by yeast 2-hybrid assay (176).

Pyrin-ASC interaction was mediated by PYD domain present in both these proteins.

They demonstrated that pyrin was able to suppress ASC mediated apoptosis in HeLa

cells.

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Papin et al. 2004 showed 14.3.3τ and 14.3.3ε as pyrin binding partners by yeast 2-

hybrid. This pyrin-14.3.3 interaction modulated the localization of pyrin in the cell

(190). Pyrin-14.3.3 interaction was shown to be required for cytoplasmic localization of

pyrin. Pyrin was shown to interact with 14.3.3τ and 14.3.3ε which was mediated by exon

2. A alternate spliced form of pyrin which lacks exon 2, was unable to interact with pyrin

and this alternatively spliced form of pyrin localized to nucleus. This interaction was

also dependent on the phosphorylation state of pyrin. Pyrin undergoes phosphorylation at

3 serine residues located in exon 2 namely S208, S209 and S242. They could no longer

observe this interaction when the serine residues were modified to alanine. Recently, two

groups have shown that pyrin interacts with caspase-1 thereby modulating caspase-1

function (191,192). Pyrin-caspase-1 interaction was mediated by the B30.2 domain of

pyrin. This interaction was shown to suppress the activation of caspase-1 and thereby IL-

1β processing and release. Papin et al. 2007 demonstrated that B30.2 domain of pyrin

interacted with other proteins of the inflammasome namely, caspase-1, NALP3 and

proIL-1β (191). Recently Balci-Peynircioglu et al. 2008 demonsrated that pyrin is also

involved in interacting with a proapoptotic protein siva-1 (177). This interaction of pyrin

with siva-1 modulates recruitment of siva-1 to the ASC specks and thereby apoptosis

induced by oxidative stress. Thus we can see that pyrin interacts with a variety of proteins involved in diverse functions. Understanding the pyrin interacting proteins and their functions may lead to identification of mechanism involved in FMF disease.

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1.8.7 Functions of pyrin

1.8.7.1 Role of pyrin in cytoskeletal remodeling

Mansfield et al. 2001 demonstrated that pyrin co-localized with microtubules and actin (189). They showed a direct interaction of pyrin with microtubules which was

mediated by the N-terminal region of pyrin protein. Pyrin co-localized with actin in

perinuclear filaments and also in peripheral lamellar ruffles. Supporting the role of pyrin

in cytoskeletal remodeling, it was shown by the same group that pyrin interacted with

PSTPIP1, a tyrosine phosphorylated protein involved in cytoskeletal organization (193).

The mutations of the gene coding for PSTPIP1 leads to an auto-inflammatory disorder

called pyogenic arthritis, pyoderma gangrenosum and acne. Pyrin interacted with

PSTPIP1 in a phosphorylation dependent manner. The two mutations of PSTPIP1 which

are associated with the disease were hyper-phosphorylated and they bound to pyrin more

than the wild type. Interestingly, monocytes isolated from patients having this disease

had more IL-1β secretion supporting the role of PSTPIP1- pyrin interaction in regulating inflammation. Interaction of pyrin with cytoskeletal proteins indicates that pyrin may regulate the inflammatory response at the level of leukocyte cytoskeletal remodeling.

This idea is supported by the fact that colchicine a microtubule polymerization inhibitor, is traditionally prescribed to patients having FMF to decrease the inflammatory response.

1.8.7.2 Role of pyrin in inflammation

Yeast 2-hybrid analysis of pyrin indicated that pyrin interacts with ASC through

the N-terminal pyrin domain. As ASC is involved in both NF-κB activation and caspase-

1 activation, we can speculate that pyrin may have role in ASC mediated caspase-1

57 activation and NF-κB activation (78,162,167,170,172). As mentioned earlier the role of pyrin in caspase-1 activation is controversial. Both activating and suppressing effect of pyrin has been reported. There are two hypotheses regarding the function of pyrin. One is the sequestration hypothesis, in which pyrin takes away ASC and caspase-1 which are needed for the inflammasome formation by sequestering ASC and caspase-1 molecules.

In support of sequestration hypothesis, it has been demonstrated that pyrin blocks IL-1β processing at two levels. Pyrin blocks the function of ASC by binding ASC through the

N-terminal pyrin domain and also blocks the caspase-1 activation by binding to procaspase-1 molecule. Thus, pyrin is believed to be a negative regulator of inflammation because it blocks caspase-1 activation and thereby IL-1β processing and release. In this context, pyrin deficient macrophages produce more IL-1β when stimulated with IL-4,

LPS or both compared to the wild type (186). In the pyrin mutant macrophages the pyrin and ASC interaction is blocked, ASC activates caspase-1 and this active caspase-1 cleaves the pro IL-1 β. The other hypothesis is the inflammasome hypothesis which indicates that pyrin might be an activator of caspase-1 by forming the pyrin inflammasome in which the B30.2 domain of pyrin may be involved in pathogen recognition (Figure 1.11). In support of inflammasome hypothesis Yu et al. 2006 demonstrated that overexpression of pyrin resulted in increased IL-1β processing and release. When pyrin was overexpressed in the presence of NALP3, they still observed an increase in IL-1β processing and release which was contrary to the belief that pyrin sequesters ASC from assembling the NALP3 inflammasome. Recently, Yu et al. also

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Figure 1.11: Pyrin Hypotheses. In sequestration Hypothesis pyrin is thought to be a negative regulator of caspase-1 activation by sequestering ASC, caspase-1, NALP3 and proIL-1β. This sequestration suppresses the activation of caspase-1 and thereby IL-1β induced inflammation. In inflammasome hypothesis pyrin is believed to be a positive regulator of caspase-1 activation by the assembly of pyrin inflammasome. B30.2 domain of pyrin may be a ligand recognition receptor. By the assembly of pyrin inflammasome, pyrin activates caspase-1 and thereby IL-1β induced inflammation.

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demonstrated that pyrin was involved in the activation of ASC-mediated apoptosome

along with PSTPIP1(131). The differences observed by different groups regarding the

function of pyrin in caspase-1 activation may be just due to the differences in the system

used to test the function of pyrin.

The role of pyrin in NF-κB activation is not clear yet. It is shown in overexpression systems that pyrin inhibits ASC induced NF-κB activation, when ASC is

over expressed with caspase-8, NALP3/ cryopyrin/ PYPAF1, or IPAF (176,177,194).

However recent report describes that pyrin has no role in NF-κB activation. In this study

ASC and caspase-1 was expressed in HEK293 cells at a physiological expression levels

seen in THP-1 cells. When they analyzed the ability of pyrin to activate NF-κB, they did

not observe NF-κB activation with overexpression of pyrin. However, in the same

setting cryopyrin did enhance NF-κB activation.

1.8.7.3 Role of pyrin in apoptosis

Pyrin is thought to suppress ASC mediated apoptosis. It is well known in

literature that ASC causes apoptosis upon overexpression and methylation induced

silencing of ASC expression is seen to occur in cancers (164). It was demonstrated by

different groups that pyrin reduces ASC induced speck formation and thereby ASC

induced apoptosis (162,176). Recently, it was shown that pyrin interacts with a pro-

apoptotic protein Siva-1 through B30.2 domain of pyrin. Pyrin modifies the apoptotic

response of Siva-1 induced by oxidative stress (177).

As there has been conflicting reports regarding function of pyrin in caspase-1 activation we wanted to determine the role of pyrin in caspase-1 activation and IL-1β

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processing and release. We noted that monocytes process and release IL-1β in response to TLR4 ligand LPS, whereas their decendents macrophages lack this ability. We hypothesized that pyrin may play a role in this difference in caspase-1 activation between monocytes and macrophages. The experiments and results depicted in Chapter III tests the role of pyrin in caspase-1 activation in monocytes and macrophages.

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CHAPTER 2

MAIL – A KEY UPSTREAM REGULATOR OF

MONOCYTE IL-6 PRODUCTION

2.1 Introduction

NF-κB is an important transcription factor involved in many processes including inflammation, cell growth and differentiation, immunity and apoptosis. NF-κB plays an

important role in host cell defense and host homeostasis (81,91). It is required for proper

functioning of both the innate and adaptive immune response. In response to diverse

stimuli, NF-κB pathway activates the production of many different proteins, which helps

the cell to cope up and adapt to the stimulus (81). The mammalian NF-κB proteins

include RelA, RelB, c-Rel, p50 and p52. These proteins bind to the target NF-κB site as

homo or hetero dimers and are involved in the activation or suppression of gene

expression. For example, RelA and p50 initiate the transcription of many different genes

whereas p50/p50 hetero dimers repress the expression of specific genes.

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NF-κB proteins are involved in diverse cell functions so their activity needs to be tightly regulated. Besides the post translational mechanisms which regulate the NF-κB

pathway, the function of NF-κB protein is regulated by IκB proteins (195). IκB proteins

are characterized by the presence of the C-terminal ankyrin containing repeats. IκB

proteins bind to the NF-κB proteins through the interaction of ankyrin and Rel homology

domain. This interaction is thought to mask the nuclear localization signal of NF-κB and

therefore tethers the NF-κB proteins to the cytoplasm (91).

Activation of the TLR pathway leads to phosphorylation and ubiquitination of the

IκBα. The ubiquitinated IκBα undergoes degradation through the proteosome pathway.

Proteosome mediated degradation of the ubiquitinated IκBα releases the NF-κB protein to

the nucleus. Upon translocation the NF-κB binds to the NF-κB sites on the promoter

region of different genes and this leads to the expression of many different genes

including the members of the IκB family of proteins.

MAIL (molecule containing ankyrin repeats induced by LPS), also called INAP

(IL-1 inducible nuclear ankyrin repeat protein) or IκBζ, is a newly described homolog of

IκB proteins (96-98). It is induced by TLR agonists and other pro-inflammatory

cytokines but not by TNFα (98,107,108). Unlike the other IκB proteins, MAIL has a

long N-terminal region which anchors a nuclear localization sequence and a

transactivation domain. The C-terminal region of MAIL harbors ankyrin repeats which

are thought to suppress NF-κB activity. However the role of MAIL in suppression of

NF-κB is not clearly understood yet. The transactivation domain of MAIL is involved in regulating transcriptional activation of many genes. It has been demonstrated that mouse

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MAIL binds to the p50 subunit of NF-κB and regulates transcriptional activation. In this

context, recent experiments from the MAIL knockout animals indicate that MAIL is a

transcriptional regulator involved in the production of many proinflammatory cytokines

and innate defense proteins. Macrophages from MAIL knockout animals are deficient in

the production IL-6 upon LPS stimulation (106). MAIL has also been implicated as an

important molecule in innate immunity. It has been shown recently that MAIL regulates

the expression anti microbial defense proteins namely h-beta defensin 2 (hBD2) and

neutrophil gelatinase associated lipocalin (NGAL) in epithelial cells upon stimulation

with IL-1β or IL-17 (111,112).

Although the role of mouse MAIL in regulating the expression of IL-6 is clearly demonstrated, the role of human MAIL in IL-6 production has not been elucidated yet.

Mouse MAIL and human MAIL differ in the N-terminal region which harbors the transactivation domain. Also, it has also been reported that mouse MAIL and human

MAIL differ in their ability to bind NF-κB proteins. Mouse MAIL binds only to p50 whereas the human MAIL binds both p65 and p50 (99,106). Differences also exist in

TNFα mediated MAIL induction in humans and mice. In contrast to human MAIL which is inducible by TNFα, mouse MAIL is not. Since, IL-6 plays an important role in many inflammatory diseases such as sepsis, heart attacks, stroke, and in many human cancers including hepatocarcinoma, multiple carcinoma, and ovarian cancer we wanted to elucidate the function of human MAIL in IL-6 production. To our knowledge, MAIL dependent regulation of IL-6 has not been reported in humans.

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In this study we show that human MAIL is comparable in function to mouse

MAIL for the production of LPS induced IL-6. We also show that macrophages unlike their precursor’s monocytes have less MAIL and therefore have reduced ability to produce IL-6 in response to LPS. Furthermore, we demonstrate that intracellular Nod2 ligand (muramyl di-peptide) synergizes with LPS for the production of IL-6 and this is response is dependent on the MAIL gene expression. Our data indicates that monocytes have robust MAIL dependent IL-6 production when compared to macrophages in response to TLR and NOD ligands. Thus, we show that MAIL is a key positive regulator of monocytes IL-6 production in response to extracellular and intracellular pathogens.

2.2 Materials and Methods

2.2.1 Cell culture and transfection

Human PBMCs were isolated by Histopaque density gradients from fresh source

leukocytes from the American Red Cross. Monocytes were isolated from PBMC by

CD14 positive selection. For culturing monocytes derived macrophages, PBMCs

(2x106/ml) were plated in Teflon containers and cultured in RPMI supplemented with

20% human AB serum for 5 days. Five day old macrophages were isolated with CD14 positive selection. Purified macrophages (2x106) were plated in RPMI supplemented with

5% FCS and 1% Pen Strep in a 12 well plate. Macrophages were stimulated with LPS

(1.0 μg/ml) for the time points indicated. HEK293 cells were grown in DMEM (Low

glucose) supplemented with 10% heat inactivated FBS and 1% PenStrep. Transfection of

plasmids into HEK293 and HeLa were done using Lipofectamine 2000 using

manufacturer’s recommendation (Invitrogen, Carlsbad, CA).

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2.2.2 Small interfering RNA

Control and MAIL siRNA (Dharmacon) was dissolved to 40 μM concentration

and 2 μl of siRNA was used to nucloefect 10x106 cells using the Amaxa Nucleofector kit.

The program used was Y-01. After the nucleofection the cells were suspended in the

media supplied with the nucleofection kit supplemented with L-glutamine and 25 ng/ml

of MCSF. The cells were plated in 12 well plate at a concentration of (2.5x106) cells per ml for 18 h. The cells were stimulated with LPS (1.0 μg/ml) for additional 6 h.

2.2.3 ELISA

ELISA for IL-6 was purchased from e-Bioscience (San Diego, CA). ELISA for

IL-1β, IL-8 and TNFα were developed in our laboratory and have been described

previously (196,197)

2.2.4 Reagents and antibodies

LPS was bought from sigma. MDP, iE-DAP and αDAP was bought from Bachem

(Torrence, CA). IL-1β was purchased from R&D (Minneapolis, MN). Antibodies were

purchased from IκBα (Millipore), actin (monoclonal clone C4, MP Biomedicals, Solon,

OH), antibody to IL-1β and MAIL was generated in our laboratory against recombinant

proteins expressed in bacteria.

2.2.5 Real Time PCR

Monocytes and macrophages were stimulated for the time points indicated and

total RNA was extracted using Trizol. Cells were lyzed in Trizol (Invitrogen, Carlsbad,

CA). Total RNA was extracted following the manufacturers recommendations. RNA

was quantified using spectrophotometer. Total RNA (0.5-1.0 μg) was used to convert to 66 cDNA using Thermoscript (Invitrogen, Carlsbad, CA). This cDNA was used for Real time PCR for analyzing MAIL gene expression with primers specific for MAIL. A detailed protocol has been described previously (198).

2.2.6 Plasmids

MAIL was cloned from a cDNA library of human monocytes stimulated with LPS for 2h into pEGFPN1 vector. All the sequences were sequenced to verify the integrity of the plasmids.

2.2.7 Preparation of cell lysates and Western blotting

Cells were lysed in plate by adding 100μl of lysis buffer (Tris 50 mM, NaCl 150 mM, Triton X 100 1%, EDTA 10 mM ) with inhibitor cocktail, CMK and 2 mM PMSF

(Sigma). The cells lysates were incubated in ice for 15 minutes. Cell lysates were centrifuged at 14,000Xg for 10 minutes and the supernatants were transferred into a new tube. Protein was quantified by Lowry’s assay (Bio-Rad) and equal protein (10-50μg) per lane was loaded in a 10% Gel. The separated proteins were transferred onto a nitrocellulose membrane. The membranes were blocked in 5% non fat milk. The membranes were blotted with appropriate antibody over night, and then blotted with appropriate secondary antibody followed by ECL substrate (GE healthcare).

2.2.8 Statistical analysis

Values are expressed as mean ± standard error of the mean. For comparisons standard paired t-test was used, with significance defined as a p value < 0.05.

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2.3 Results

2.3.1 Overexpression, purification and antibody production of MAIL

As there was no commercially available antibody for MAIL, we decided to over

express MAIL protein in bacteria. MAIL was cloned in pET28b with both N and C-

terminal histidine tags. This construct was over expressed in BL21DE3RIL strain of

bacteria. The over expressed protein was purified using Ni-NTA column. Figure 2.1A

shows the western blotting of the steps involved in recombinant MAIL purification

blotted with His tag antibody. We show that MAIL is purified to homogeneity by using

Ni-NTA column. This recombinant protein was used to immunize rabbits. To confirm

the specificity of the antibody, recombinant protein at two different concentrations were

used to probe with MAIL antibody or recombinant MAIL added to MAIL antibody. As shown in Figure 2.1B recombinant MAIL protein blocked the specificity of MAIL antibody. To further test the specificity of MAIL antibody, we used HEK293 cell lysates transfected with EGFP or EGFP-MAIL. These lysates were run on SDS-PAGE and probed with MAIL antibody. As shown in Figure 2.1C, MAIL antibody detected EGFP-

MAIL transfected in HEK293 cells at different concentrations. However, we did not detect the presence of MAIL when HEK293 cells were transfected with EGFP alone.

2.3.2 Monocytes express both MAIL-S and MAIL-L mRNA upon LPS stimulation

There are two alternate splice forms of MAIL that have been reported in literature

(Figure 2.2A). MAIL-L containing exon 3 and expresses a protein product of 78 kDa

and MAIL-S which lacks exon 3 expresses a protein product of 68 kDa. As there is not 68

Figure 2.1: Overexpression and purification of MAIL protein and testing MAIL antibody. A) MAIL was cloned in pET28b with a N and C-terminal His tag. The over expressed protein was purified using Ni-NTA column and analyzed by immunoblot with His tag antibody. B) Recombinant MAIL protein blocks MAIL antibody. 10 and 50 ng of rMAIL were blotted onto a membrane. The membrane was probed with MAIL antibody or MAIL antibody + rMAIL protein. C) MAIL antibody detects MAIL–EGFP protein from HEK293 cell lysates and rMAIL. HEK 293 cells were transfected with EGFP or increasing concentration of MAIL-EGFP. Cell lysates were subjected to immunoblot analysis after SDS-PAGE with MAIL antibody.

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much known about the splice variants expressed in monocytes, we wanted to check

whether monocytes expressed both forms of MAIL. We made use of PCR with primers

specific to each form of MAIL. As shown in the Figure 2.2B, we could amplify band of

appropriate size with primers specific for MAIL-L and MAIL-S from the cDNA samples

of monocytes stimulated with LPS. Both forms of MAIL are induced by LPS stimulation.

All these finding indicate that both MAIL-L and MAIL-S is express in monocytes upon

LPS stimulation.

2.3.2 MAIL is transiently induced in monocytes upon LPS stimulation

To determine the expression pattern of MAIL mRNA in monocytes, we made use

of the quantitative PCR. Monocytes were treated with LPS (1.0 μg/ml) for various time

points for a period of 24 h. Cells were lysed in TRIZOL for mRNA analysis and cell

lysates were used for analyzing MAIL protein expression by immunoblotting. Freshly

isolated monocytes had little or no MAIL mRNA. However, MAIL mRNA was induced

in monocytes at 30 min and the expression peaked at 2 h (Figure 2.3A). After 2 h there

was a gradual decrease in MAIL mRNA and was almost undetectable at 24 h. To

analyze whether MAIL mRNA data matched MAIL protein data we analyzed the cell

lysates by immunoblotting with antibody for MAIL and actin.

MAIL protein was absent in fresh monocytes and the protein expression increased on LPS stimulation peaking at 4 h (Figure 2.3B). There was no detectable protein at 24 h after LPS stimulation. Our antibody to MAIL reacted with a band around 82 kDa in LPS induced monocytes lysates indicating that MAIL-L is the predominant form seen in monocytes cell lysates. We did not see the expression of MAIL-S, indicating that MAIL-

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Figure 2.2: Monocytes express both MAIL-L and MAIL-S mRNA upon LPS stimulation. A) Exon information of MAIL-L and MAIL-S. B) Monocytes (2x106) were induced with 1.0 µg/ml LPS for the time points indicated and lysed in Trizol. Total RNA was extracted and 0.5 – 1.0 μg of RNA was converted to cDNA. 2 μl of cDNA was amplified with primers specific for MAIL-L and MAIL-S by PCR. M represents 1kb+ marker.

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S protein is unstable. Our data agrees with the previously reported data that in

RAW264.7 cells the predominant form of MAIL is MAIL-L.

As it has been shown previously that MAIL regulates IL-6 production in mouse

(96,100,106), we hypothesized that human MAIL might also regulate IL-6 production in

monocytes. So, we analyzed the mRNA expression of MAIL and IL-6 in monocytes at

time points indicated upon LPS stimulation by real time PCR. As shown in Figure 2.3A,

MAIL mRNA expressed very early peaking at 2 h after LPS stimulation and then

gradually decreasing for a period of 24 h. In contrast to MAIL mRNA which is early in expression, IL-6 mRNA was very late in expression peaking only at 8 h after LPS

stimulation (Figure 2.3C). This indicated that MAIL is an early inducible gene whereas

IL-6 is a late inducible gene in monocytes upon LPS stimulation.

2.3.4 Monocytes express more MAIL and secrete more IL-6 compared to

macrophages

Monocytes and macrophages are very important cells involved in the host innate

response. We have previously shown that monocytes and macrophages differ in their

ability to process IL-1β. Recently it has been shown that macrophage produced IL-6 is

involved in tumor metastasis, we were curious to know whether monocytes and

macrophages differed in the expression of MAIL and therefore the production of IL-6.

We isolated monocytes from blood and compared the LPS induced production of MAIL

and IL-6 with in vitro matured macrophages. To our surprise, we observed that

macrophages expressed less MAIL (Figure 2.4A) and therefore produced less IL-6.

Monocytes produced 7 fold more IL-6 compared to macrophages (81.7 ± 29.7 ng/ml Vs

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Figure 2.3: MAIL is transiently induced in monocytes upon LPS stimulation. A) Monocytes (2x106) were induced with 1.0 µg/ml LPS for the time points indicated. mRNA expression was analyzed by real time PCR. B) Cell lysates (10 µg) were separated by SDS-PAGE followed by Western blotting with MAIL antibody. The same membrane was used to reprobe with actin antibody. Shown is the data from one donor. Asterisk indicates a non specific protein. C) Monocytes (2x106) were induced with 1.0 µg/ml LPS for the time points indicated. mRNA expression was analyzed by real time PCR for MAIL (black) and IL-6 (red).

73

12.6 ± 6.8 ng/ml) (Figure 2.4 B). In contrast, TNFα production was not different between monocytes and macrophages (Figure 2.4 C).

2.3.5 EGFP-MAIL translocates to nucleus and forms a distinct speckled pattern

As mouse MAIL has been shown to co-localize to nucleus we hypothesized that human MAIL should also be a nuclear protein. To determine the cellular localization of

MAIL, EGFP–MAIL plasmid was transfected into HEK293 cells and the cells were examined by fluorescent microscopy. In the samples transfected with EGFP alone, GFP was localized through out the cell whereas in samples transfected with EGFP-MAIL

MAIL was seen only in the nucleus. In agreement with our finding, it has been shown by other groups that MAIL translocates to nucleus. In the nucleus, MAIL had a speckled pattern of staining (Figure 2.5).

2.3.6 Knockdown of MAIL suppresses LPS induced IL-6 production in monocytes

As the function of human MAIL in regulating IL-6 production has not yet been studied, we wanted to determine the function of MAIL in monocytes. To do this, expression of MAIL in human monocytes was knocked down using the small interference

RNA technology. Monocytes were nucleofected with MAIL or control siRNA.

Nucleofected cells were rested for 18 h and then stimulated with 1.0 μg/ml LPS for additional 6 h. Cell media supernatants were collected and analyzed for IL-6 and IL-8 by

ELISA. One set of samples were lyzed in Trizol for RNA isolation. Cell lysates were analyzed for the expression of MAIL, IκBα and actin by western blotting. 74

Figure 2.4: Monocytes express more MAIL and IL-6 compared to macrophages. A) Monocytes or macrophages (PBMC matured to macrophages) were stimulated with 1.0 μg/ml LPS for time points indicated. A) Cell lysates were analyzed by Western blot for MAIL. The same membrane was reprobed with IL-1β and actin. B) Cell culture media for monocytes (closed bars) and macrophages (open bars) were by ELISA for IL-6 and C) TNF.

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Upon analyzing the RNA samples we saw a significant decrease in the production of IL-6 with MAIL siRNA treated samples compared to the control siRNA treated samples (Figure 2.6A). In contrast to the decrease in expression of IL-6 mRNA between

MAIL treated and control siRNA treated monocytes, we did not see any significant change in IL-8 mRNA expression between control or MAIL siRNA treated samples.

There was no significant difference in the expression of MAIL between MAIL siRNA treated and the control siRNA treated samples. We attribute this to the late time (6 h after stimulation) at which we harvested the samples for the RNA isolation. Since there was no significant difference in MAIL mRNA expression between control siRNA and MAIL siRNA treated samples, we decided to analyze the expression of MAIL protein in cell lysates. There was about a 90 percent suppression of MAIL expression in MAIL siRNA treated samples compared to the control siRNA treated. Suppression of MAIL significantly decreased the IL-6 production. MAIL siRNA samples had 5 fold decrease in

IL-6 production compared to control siRNA treated samples (Figure 2.6B). However, the suppression of MAIL did not affect the production of IL-8. Also, in cell lysates, suppression of MAIL did not affect the expression of IκBα protein, a homolog of MAIL, indicating that the siRNA is target specific.

2.3.7 Knockdown of MAIL suppresses NOD ligand induced IL-6 production

Stimulation of the intracellular NOD like receptors turns on the NF-κB pathway which leads to the production of many pro-inflammatory genes including IL-1β, IL-6 and

IL-8 (199-202). Since MAIL is a NF-κB dependent protein, we hypothesized that

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Figure 2.5: MAIL translocates to nucleus and forms speckles. HEK293 cells were transfected with EGFP or with MAIL-EGFP. 24 h after transfection the cells were visualized with a fluorescent microscope.

77 suppression of MAIL will also suppress NOD ligand induced IL-6 production. We made use of two NOD ligands namely iE-DAP, a NOD1 agonist and MDP, a NOD2 agonist and we also used αDAP which was our control. Monocytes were treated with MAIL siRNA or the control siRNA. The MAIL or control siRNA treated samples were stimulated with LPS, iE-DAP and MDP individually, or in combination for 6 h. Cell media supernatants were harvested 6 h after stimulation and analyzed for IL-6. Cells were lysed and lysates were subjected to western blot analysis with antibodies for MAIL and actin.

As we have seen before, treatment of monocytes with MAIL siRNA decreased the expression of MAIL protein. In agreement with the data shown in figure 2.6, LPS induced IL-6 production was decreased in MAIL siRNA treated samples compared to control siRNA treated samples. Neither of the NOD ligands stimulated MAIL expression or IL-6 production when they were treated alone, indicating that they are not potent in stimulating the monocytes. When monocytes were treated with Nod1 stimulus iE-DAP in combination with LPS they did not show any synergistic effect on IL-6 production, indicating that monocytes do not respond to Nod1 stimulus. However, when monocytes were treated with LPS in combination with MDP, it had a synergistic effect on the production of MAIL and IL-6. MAIL siRNA treated samples had dramatically less IL-6 production compared to the control siRNA treated samples when they were stimulated with LPS or LPS in combination with MDP (Figure 2.7). This indicates that MAIL is an important regulator of IL-6 production in response to both TLR and NLR ligands.

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Figure 2.6: Knockdown of MAIL suppresses LPS induced IL-6 production. Monocytes (10x106) were nucleofected with control siRNA or MAIL siRNA. Cells were stimulated 18 h after nucleofection with 1.0 μg/ml LPS for additional 6 h. A) RNA expression for control siRNA treated (open bar) and MAIL siRNA treated (closed bar) was analyzed by real time PCR for MAIL, IL-6 and IL-8. B) IL-6 and IL-8 for control siRNA (open bar) and MAIL siRNA treated (closed bar) were analyzed in cell culture media. The lysates were analyzed by immunoblotting for MAIL and actin using the same membrane. The data represents mean ± SEM for 5 donors for RNA data and 4 donors for protein data.

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Figure 2.7: Knockdown of MAIL suppresses NOD ligand induced IL-6 production. Monocytes (10x106) were nucleofected with control siRNA or MAIL siRNA. Cells were stimulated 18 h after nucleofection for additional 6 h with 1.0 μg/ml LPS, 5.0 μg/ml NOD ligands (α DAP, MDP or iE-DAP) each of them alone or in combination with LPS IL-6 was analyzed in cell culture media. The lysates were analyzed by immunoblot with MAIL antibody the same membrane was reprobed with actin antibody. The data represents mean ± SEM for 3 donors.

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2.4 Discussion

Inducible transcriptional factors help organisms to adjust to the environmental changes. NF-κB is one of the important inducible transcription factors that help the

organism to cope with environmental changes. NF-κB is an evolutionarily conserved transcription factor involved in a variety of cellular functions. Proper regulation of NF-

κB is required for normal health and homoeostasis. Dysregulation of NF-κB leads to

many diseases. IκB proteins are natural inhibitors of the NF-κB proteins. They bind and

inhibit the function of NF-κB proteins. All these IκB proteins are characterized by the

presence of 5-7 ankyrin repeats present at the C-terminus of these proteins (81).

MAIL is a newly described homolog of IκB protein family having six ankyrin

repeat at the C-terminal region (96-98). Unlike the other IκB proteins, which are

constitutively expressed, MAIL is an inducible gene. So far, there are two major

alternatively spliced form of MAIL reported. In monocytes, we find that both MAIL-L

and MAIL-S mRNA is expressed. However, we did not observe any protein product as of

the size of MAIL-S. From our data we show that MAIL is an early inducible gene upon

LPS stimulation. Expression of MAIL is very transient. Based on this, we speculate that

like IκBα, MAIL may also undergo phosphorylation and ubiquitination leading to its

degradation by the proteosome machinery. It has been reported by other groups that

MAIL is an inducible gene and the expression of MAIL depends on the NF-κB pathway

(107).

The presence of ankyrin repeats at the C-terminus end of MAIL suggests that, like

other IκB proteins, it may be involved in suppression of NF-κB. It has been reported by

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other groups that MAIL suppresses NF-κB activity upon overexpression (98). However there is no direct evidence for the suppression of NF-κB activity by MAIL. In contrast to the suppressive ability, mouse MAIL has shown to be a transcriptional regulator of IL-6 production. IL-6 is an important cytokine thought to have both pro and anti- inflammatory effects (203). Its role in sepsis has been well documented (204). Recently the role of IL-6 in cancer has been reported by two different groups (205,206). Their finding supported the role of macrophage produced IL-6 in tumor progression and metastasis.

The role of MAIL in the production of innate defense proteins has been well

documented. MAIL acts as a transcriptional regulator by binding to p50 of NF-κB for the production of NGAL and hBD-2 (111). The role of MAIL in regulation of apoptosis has also been studied recently (99). Susceptibility of HeLa cells to TNF induced apoptosis was based on the level of MAIL expression. Cells that were suppressed in MAIL expression were less susceptible to TNF induced apoptosis. They also reported that human MAIL, unlike the mouse MAIL bound both p50 and p65 subunits of the NF-κB molecules (99). They hypothesized that there may be differences in production of IL-6 between mouse and human MAIL. Based on their finding, we wanted to test whether there were any difference in regulation of IL-6 production between mouse and human

MAIL.

Many groups have implicated that MAIL might be involved in the production of

IL-6 based on the overexpression model system. Yamamoto et al. 2004 reported that

macrophages from MAIL knockout animal are deficient in LPS induced IL-6 production

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(106). To our knowledge, we are the first to describe the role of human MAIL in the

production of IL-6. We show that MAIL is expressed by monocytes very early upon LPS stimulation and the production of IL-6 mRNA followed MAIL protein expression. We also show that monocytes express more MAIL and produced more IL-6 upon LPS stimulation. We have no explanation why there is a difference in the production of MAIL and therefore MAIL dependent IL-6 between monocytes and macrophages. But this may be attributed to the difference in the expression of transcription factors which may regulate the expression of MAIL and therefore MAIL-induced IL-6.

Since we have reported earlier that macrophages are deficient in production of IL-

1β and now we showed that macrophages produce less IL-6, it is tempting to speculate

that macrophages are quite stable in their reaction to various stimuli when compared to

their precursor monocytes, which are over reactive (207-209). This idea is supported by the fact that alveolar macrophages (similar to in vitro derived macrophages) which are present in lungs come across various molecules and bacteria and if they are very reactive

may lead to over active immune response.

Unlike other IκB proteins, MAIL translocates to nucleus with a speckled pattern

(96-99). Here in this study we show that like the mouse MAIL, human MAIL is also a

nuclear protein. It translocates to nucleus upon overexpression. Human MAIL formed a

speckled pattern in nucleus. As reported earlier, this speckled pattern may be due to the interaction of MAIL with nuclear deacetylases (99).

We also showed that MAIL is involved in the production of both TLR induced

(extracellular receptor) and NOD (intracellular receptor) induced IL-6. Our data suggests

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that MAIL, like its mouse counter part, is involved in production of IL-6. It will be of a

great interest to see other genes that are regulated by MAIL. Micro array analysis would help in analyzing the global expression of genes. It is intriguing to think, how MAIL could be involved in two opposite functions. MAIL induced IL-6 production helps in tumor progression and MAIL is also involved in apoptosis by an unknown mechanism

(99). We do not have an explanation for these two distinct functions of MAIL.

Since MAIL has been implicated in the production of human host innate immune

proteins we think that MAIL is a key player in regulating innate host defense. This also

is supported by the fact that MAIL knockout animals have atopic like dermatitis and eye

inflammation. It will be interesting to test whether these diseases are due to the lack of

host defense molecules and/or to lack of essential cytokine, namely IL-6. However, the

other function of MAIL i.e., suppression of NF-κB activity cannot be excludes. The

atopic dermatitis and ocular inflammation may also be due to the overexpression of

cytokines that MAIL might inhibit with it’s the C-terminal ankyrin region. Future studies should be directed in understanding the role of MAIL in two different functions namely, inflammation and cancer. To conclude, our data supports that, like the mouse counterpart human MAIL is involved in the production of IL-6 in monocytes.

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CHAPTER 3

1 PYRIN LEVELS IN HUMAN MONOCYTES AND

MONOCYTE DERIVED MACROPHAGES REGULATE IL-

1β PROCESSING AND RELEASE*

3.1 Introduction

Caspase-1 regulation is central to the innate host response. Caspase-1 activation

is required to generate the functional proinflammatory cytokines, IL-1β and IL-18

(119,121,210). It may also be required to process and activate a newly described

cytokine IL-33 (123). However, the activation of caspase-1 is a highly regulated event

that requires the assembly of several cytosolic proteins that are members of the NACHT-

LRR family (NLRs) (211-213). This protein assemblage termed the inflammasome, is likely to exist in varying formats depending upon cell type and instigating factors

* This work has been published in Journal of Immunology 179 (2), 1274-81. Copyright 2007 The American Association of Immunologists, Inc. 85

(53,130). In this context, one related protein, pyrin (marenostrin), has been postulated to

provide a regulatory function within inflammasomes (186). Pyrin is an 86 kDa protein

discovered as part of a genome wide search for the cause of familial Mediterranean fever

(174,175). Pyrin contains an amino terminal pyrin domain (PYD) which is a member of

the death domain family that promotes specific protein-protein interactions. The PYD of

pyrin is believed to cooperate with other PYD-containing proteins within the

inflammasome. Specifically, pyrin interacts with ASC, a 22 kDa adaptor protein that

contains a PYD linked to a caspase recruitment domain (CARD) (176). ASC knockout

animals lack caspase-1 activation (106,146). In agreement with the knockout data, ASC

overexpression readily induces caspase-1 activation in HEK293 cells via an induced

proximity mechanism that requires interaction between the CARD of caspase-1 and the

CARD of ASC (214). Thus, it is hypothesized that pyrin’s interaction with ASC

regulates the inflammasome structure and hence caspase-1 activation.

In this context, there is controversy concerning pyrin’s function in the regulation

of the inflammasome. Both an inhibiting and an activating function have been postulated

(169,186,192). However, the fact that pyrin mutations are associated with inflammatory

responses lends itself to the concept that these mutations confer loss of function and that

pyrin down regulates the inflammasome function and hence, caspase-1 activation and IL-

1β/IL-18 mediated innate responses. Given our long-standing interest in understanding differences in caspase-1 regulation between blood monocytes and tissue macrophages

(208,208,209,215) we elected to study the relative role of pyrin expression in these cells as it pertains to IL-1β processing and release. Although macrophages are derived from

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monocytes and contain abundant amounts of caspase-1, macrophages are limited in their

ability to process 31 kDa proIL-1β to the active 17 kDa form (207,209). Therefore we

sought to test the hypothesis that pyrin may be responsible for the differences in the

inflammasome function between fresh blood monocytes and monocyte-derived

macrophages. Our experiments examined the relative abundance of pyrin in monocytes

and macrophages and the effect of modulating pyrin levels on caspase-1 activation and

IL-1β processing and release.

3.2 Materials and Methods

3.2.1 Cell culture and transfection

Human peripheral blood monocytes (PBMC) were isolated from fresh source

leukocytes from the American Red Cross (Columbus, OH). PBMC were isolated by using Histopaque-1077 density gradient. Monocytes were isolated by CD14 positive

selection (Miltenyi Biotec, Auburn, CA). This method yields >95% pure monocytes as

confirmed by FACS. Monocytes were cultured in RPMI 1640 (Cambrex, East

Rutherford, New Jersey) supplemented with 5% FBS (endotoxin-free; HyClone, Logan,

UT) and with 1% penicillin and streptomycin solution. Monocyte derived macrophages were generated by plating 2x107/ml PBMC in RPMI 1640 without serum. The cells were

allowed to adhere for two hours. The non-adherent cells were washed with PBS and the

cells were cultured in RPMI 1640 supplemented with 5% FBS and with 1% penicillin and

streptomycin solution for 5 days. HEK293 (ATCC, Manassas, VA) cells were cultured in

DMEM supplemented with 10% FBS and 1% penicillin and streptomycin solution.

HEK293 cells were transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) as 87

per the manufacturer’s instructions. The total amount of DNA was kept constant in all

transfections using the appropriate empty vector. THP-1 (ATCC, Manassas, VA) cells

were cultured in RPMI 1640 supplemented with 10% FBS and 1% penicillin and

streptomycin solution. THP-1 cells (10x106) were nucleofected with 5 μg of plasmid or

1x106 THP-1 cells were transfected using Lipofectamine 2000 with 100 pmoles of siRNA

according to the manufacturer’s instructions (Amaxa, Cologne, Germany and Invitrogen,

Carlsbad, CA).

3.2.2 Expression plasmids

The complete open reading frame for pyrin was amplified from the cDNA of monocytes

stimulated with LPS and cloned into pcDNA3.1 Myc/His(-)B (Invitrogen, Carlsbad, CA)

with a C-terminal Myc/His tag. Plasmids encoding for proIL-1β, ASC and caspase-1 were cloned into pEGFPC2 (Clontech, Mountain View, CA) by amplifying from the cDNA of monocytes stimulated with LPS. The integrity of the plasmids was confirmed by sequencing. pEGFP-Pyrin plasmid was a kind gift from Deborah Gumucio (University of Michigan, Ann Arbor).

3.2.3 Generation of pyrin antibody

Pyrin was cloned into pMALC2X plasmid (New England Biolabs, Ipswich, MA)

with an N-terminal maltose binding protein tag. MBP-pyrin fusion protein was expressed

in E. coli strain BL21DE3RIL (Stratagene, La Jolla, CA). A major product of the column

eluate was the MBP with N-terminus of the pyrin protein. The N-terminus of the protein was confirmed by LC MS/MS mass spectrometry analysis (Campus Chemical

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Instrumentation Centre, The Ohio State University). A rabbit was immunized with the

purified fusion protein and the antisera recovered (Covance, Denver, PA).

3.2.4 Antibodies

Antibodies for Western blotting were from the following sources: ASC (rabbit

polyclonal generated in our laboratory and monoclonal clone 23-4 from MBL

International, Woburn, MA), caspase-1 (was a kind gift from Doug Miller, Merck

Research Labs), actin (monoclonal clone C4, MP Biomedicals, Solon, OH) and IL-1β

(rabbit polyclonal generated in our laboratory).

3.2.5 Quantitative polymerase chain reaction (qPCR)

Monocytes and monocyte-derived macrophages were lysed in Trizol (Invitrogen,

Carlsbad, CA) and mRNA was extracted and converted into cDNA using Thermoscript

RT-PCR systems (Invitrogen, Carlsbad, CA). The cDNA was used for qPCR using

primers specific for pyrin. The values obtained by qPCR were normalized based on two

housekeeping genes, CAP-1 and GAPDH, as previously described (198).

3.2.6 Caspase-1 activity assay

For caspase-1 activity measurements using a fluorescent substrate, 2x106 cells

were typically lysed in 100 µl of lysis buffer (50 mM HEPES pH 7.4, 100 mM NaCl,

0.1% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, 0.1 mM EDTA),

supplemented with complete protease inhibitor cocktail (Roche, Indianapolis, IN) for 20 min on ice, followed by centrifugation at 14,000 x g for 10 min at 4°C. Cell extracts (50

μl) were used in the assay. A detailed protocol has been described previously (196). 89

3.2.7 ELISA

Sandwich ELISAs developed in our laboratory were used to detect mature IL-1β,

IL-8 and TNFα as previously described (197,207,216).

3.2.8 Preparation of cell lysate and Immunoblots

Monocytes (2x106 cells) were lysed in cold lysis buffer (50 mM Tris-HCl pH 8.0,

150 mM NaCl, 2 mM EDTA, and 1% Triton X-100) supplemented with complete

protease inhibitor cocktail (Roche) and 1 mM PMSF on ice for 20 min. Cell debris and nuclei were removed by centrifugation at 14,000 rpm at 4°C for 10 min. The protein concentrations in the cell extracts were determined using Bio-Rad Dc protein Lowry

assay (Bio-Rad, Hercules, CA) and 10 μg of total protein per lane were resolved by 10%

SDS-PAGE gel and transferred to a nitrocellulose membrane. Nonspecific sites on the

nitrocellulose membrane were blocked with 5% nonfat dry milk (Carnation; Nestle,

Solon, OH) in 25 mM Tris-Cl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20 for 2 h at

room temperature. The membranes were probed with the appropriate primary antibodies.

Primary antibodies were followed by peroxidase-conjugated secondary antibodies as

indicated, and protein bands were visualized by chemiluminescence (GE Health Care,

Arlington Heights, IL). In some cases, membranes were reprobed with different

antibodies without stripping.

3.2.9 Small interfering RNA

siRNA for pyrin (cat # M-011081-00) and control siRNA were purchased from

Dharmacon RNA technologies (Lafayette, CO). THP-1 cells (1.0x106) were transfected 90

with 100 pmoles of siRNA using Lipofectamine 2000. After 3 days the transfected cells

were treated with 1.0 μg/ml LPS (E. coli strain 0127:B8, Westphal preparation, phenol

extraction; Difco, Detroit, MI) for 6 h and the cell culture media was analyzed for IL-1β and IL-8 by ELISA. Cell lysates were analyzed for pyrin expression by immunoblot.

Monocytes (10x106) were nucleofected with 500 pmoles of siRNA according to the

manufacturer’s instructions (Amaxa, Cologne, Germany). After 18 h of nucleofection, the

cells were stimulated with 1.0 µg/ml of LPS for an additional 6 h. Cell culture media was

collected and analyzed for IL-1β and IL-8 by ELISA. Pyrin suppression was confirmed

by Western blotting.

3.2.10 Statistical analysis

Values are expressed as mean ± standard error of the mean. For comparisons

standard paired t-test was used, with significance defined as a p value < 0.05.

3.3 Results

3.3.1 Overexpression, purification and antibody production of pyrin

There were no commercially available antibodies for pyrin, so we decided to over

express and purify pyrin protein to generate a poly clonal antibody for pyrin. Pyrin was cloned in pET28b with both N and C-terminal histidine tag. This construct was over expressed in BL21DE3RIL strain of bacteria. The over expressed protein was purified using Ni-NTA column. As we did not see any pyrin protein in the supernatants of pyrin purified with Ni affinity column, we attributed this to poor solubility and stability of pyrin protein. It has been reported by others that many of the death domain containing

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protein like pyrin, ASC and other NALP proteins are highly insoluble (217). So, we

decided to increase solubility of pyrin in solution by cloning pyrin with the maltose

binding protein (MBP) tag. MBP tag has been recommended to increase the solubility of

proteins which are highly insoluble (218,219). We decided to sub clone pyrin with an N-

terminal MBP tag. This MBP-pyrin was transformed in Rosetta (DE3). Upon reaching

the OD of 0.6, the cells were induced by 0.4 mM IPTG for 6-8 h. Cells were harvested

and cell lysates were loaded on to the amylose column. Figure 3.1A shows the

purification of MBP-pyrin using the amylose column. Upon analyzing the elutes of the

amylose column, we found an N-terminal piece of MBP-pyrin (size 70 kDa) getting

eluted in our fractions (Figure 3.1A). We could see very little of full length MBP-Pyrin

(Size 120 kDa). We thought that the 70 kDa piece of MBP-pyrin may be a degradation

product of full length pyrin protein. So we decided to analyze the 70 kDa of MBP-pyrin

by LC-Ms/Ms mass spectrometry analysis. Upon analysis we found that the 70 kDa

piece was indeed N-terminal portion of MBP-pyrin (Figure 3.1B). We used this

fragment of pyrin for generating a poly clonal antibody for pyrin.

3.3.2 Testing the specificity of pyrin antibody

To confirm the specificity of the antibody cell lysates from Monocytes and THP-1

lines with or without LPS stimulation, cell lysates from HEK293 transfected with empty

vector or pyrin were blotted and probed with pre immune serum or with immune serum

(Figure 3.2A). As expected, when the blots were probed with pre-immune serum there was no band of expected size of pyrin. On the other hand, when we used pyrin immune serum we got a band cross reacting at the expected size of pyrin. This indicated that the

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Figure 3.1: Purification of pyrin protein. A) Pyrin was cloned in pMALC2X with a N-terminal maltose binding tag in EcoRI and HindIII site. This plasmid was transformed in BL21DE3 and over expressed with 0.4 mM IPTG. Shown here are the steps involved in purification. Coomassie staining of the product in each step is shown. B) LC MS/MS Mass Spectrometry analysis of the fragment revealed that the N-Pyrin was a N-terminal fragment of pyrin.

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antibody was reacting to pyrin protein (Figure 3.2A). To reconfirm our finding, we used

excess pyrin protein to block the pyrin antibody. This was used for blotting the above

mentioned cell lysates. As shown in Figure 3.2B recombinant pyrin protein blocked the

pyrin antibody from detecting pyrin protein by immunoblot. This experiment reconfirmed

our earlier finding that pyrin antibody was specific for pyrin protein and could detect

pyrin protein in native cells such as monocytes and THP-1 cells.

3.3.3 Monocytes and monocyte derived macrophages differ in their ability

to process proIL-1β

To analyze the difference between monocytes and monocyte derived

macrophages in their ability to process IL-1β, monocytes and macrophages were

stimulated with LPS (1.0 μg/ml), and IL-1β and TNFα release was measured over a

period of 24 h. Cell lysates at 0, 8 and 24 h were analyzed for proIL-1β, pyrin, caspase-1

and ASC expression. Mature IL-β was measured in the cell culture media by ELISA.

While both monocytes and macrophages had similar proIL-1β, caspase-1 and ASC expression, monocytes released more IL-1β as compared to macrophages. (Figure 3.3A,

B). TNFα measured in the cell culture media (Figure 3.3C) indicated that the

macrophages were capable of responding to LPS stimulation. Thus, macrophages are

deficient in their ability to process and release IL-1β in response to LPS.

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Figure 3.2: Testing the specificity of pyrin antibody. A) Pyrin antibody detects pyrin transfected in HEK293 cells and native pyrin protein from THP-1 cells and monocytes. Lysates from HEK293 cells transfected with or without pyrin, lysates from monocytes and THP-1 with or without stimulation with LPS were blotted with Preimmune serum or antibody against pyrin. B) Recombinant pyrin protein blocks pyrin antibody. Lysates from HEK293 cells transfected with pyrin or cell lysates from THP-1 cells and monocytes blotted onto a membrane. The membrane was probed with pyrin antibody or pyrin antibody incubated with recombinant pyrin to block the antibody detection sites.

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Figure 3.3: Monocytes process and release IL-1β better than monocyte derived macrophages. A) Fresh human monocytes were compared to monocytes allowed to mature to macrophages for 5 days. Fresh monocytes and macrophages were stimulated with LPS (1.0 μg/ml) for 0, 8 and 24 h. Lysates were collected and analyzed for the presence of IL-1β, caspase-1, ASC and actin by immunoblot. D1 and D2 represent two different donors. B) Cell culture media from the monocyte (dark bar) and macrophage (open bar) samples from four donors were analyzed by ELISA specific for mature IL-1β. C) Cell culture media were also analyzed for TNFα from monocytes (dark bar) and macrophages (open bar) to confirm ability to sense the endotoxin. The data represents mean ± SEM of three donors.

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3.3.4 Pyrin expression is suppressed in monocyte derived macrophages

compared to monocytes

Since pyrin may be a regulator of caspase-1 activation and therefore IL-1β

processing and release, we hypothesized that monocytes may differ in pyrin levels when

compared to macrophages. First, we used qPCR to check the mRNA expression of pyrin

in monocytes and macrophages. Fresh monocytes had increased pyrin mRNA as

compared to macrophages (10.7±1.9 vs. 0.04±0.02 relative copy numbers, respectively).

Pyrin mRNA was induced approximately two-fold in monocytes but only slightly in

macrophages following LPS (1.0 μg/ml) treatment (Figure 3.4A).

To determine if the mRNA data matched protein production, we generated a

polyclonal antibody to the N-terminal portion of pyrin. We used immunoblots to detect

pyrin in HEK293 cells transfected with a pyrin expressing plasmid and in fresh or LPS

stimulated monocytes in order to demonstrate the specificity of the antisera (Figure

3.4B). As shown in Figure 3.4C monocytes expressed more pyrin as compared to

macrophages. Thus, monocyte and macrophage pyrin levels matched the IL-1β release

which correlates with our original findings that monocytes quickly lose their IL-1β

release capacity with time in culture (215).

3.3.5 Pyrin increases caspase-1 activity in a dose dependent manner

Our finding that monocytes express higher levels of pyrin than macrophages led us to speculate a role of pyrin as an activator of caspase-1. To test this hypothesis, we used the HEK293 transfection system in which plasmids encoding caspase-1 and ASC

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Figure 3.4: Expression of pyrin is decreased in macrophages compared to monocytes. A) Monocytes (2x106/ml) or macrophages were stimulated with LPS (1.0 μg/ml) for 0, 8 and 24 h and then lysed in Trizol. RNA was extracted and transcribed into cDNA for quantitative PCR. The mRNA levels were referenced to the mean of the two housekeeping genes CAP-1 and GAPDH and expressed as relative copy numbers. Monocyte (dark bars) and macrophage (open bars) mRNA for pyrin is shown for the time points as the mean ± SEM of three donors. B) Pyrin rabbit antiserum is specific for human pyrin. Lysates from HEK293 cells transfected with pcDNA or pyrin pcDNA (two different protein concentrations) were compared to monocytes cultured 4 h alone (NT) or with LPS (1.0 μg/ml). Shown is the immunoblot of the lysates. C) Pyrin is expressed in monocytes and its expression is dramatically decreased in macrophages. Monocytes and macrophages were stimulated with LPS (1.0 μg/ml) for 0, 8 or 24 h, then lysed and 10 μg of total protein electrophoresed in SDS-PAGE, transferred to nitrocellulose and blotted with anti-pyrin antibody. D1 and D2 represent donor 1 and donor 2 respectively. These are the same blots shown in Figure 1 with actin blotting used as the loading control.

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were cotransfected with or without increasing amounts of pyrin. Cell lysates were

subjected to caspase-1 activity assay 24 h after the transfection. Cells transfected with

caspase-1 alone had minimal caspase-1 activity, whereas, caspase activity was augmented

by the addition of ASC and further induced by pyrin in a dose dependent fashion (Figure

3.5A).

3.3.6 Pyrin induces IL-1β release in a dose dependent manner in HEK293 cells

To further test whether pyrin increased the processing and release of proIL-1β to

mature IL-1β, we used the HEK293 transfection system in which plasmids encoding

proIL-1β, procaspase-1 and ASC were cotransfected with or without pyrin (Figure 3.5B).

Released IL-1β was measured after 24 h later in the cell culture media. As shown when

pyrin was cotransfected with ASC (0.2 µg plasmid), caspase-1 was further activated and

increased amounts of processed IL-1β were released. This pyrin enhancement behaved

in a dose response fashion. Increasing pyrin expression was associated with diminished

intracellular proIL-1β and ASC protein expression (Figure 3.5B) presumably due to their

activation induced release although decreased solubility due to overexpression of these

proteins cannot be excluded.

In an effort to confirm the specificity of the pyrin overexpression findings we next

asked whether a lower dose of ASC would have the same effect on IL-1β release with

pyrin overexpression. Figure 3.6A shows that pyrin’s augmentation was present with

ASC expression using only 0.05 µg of ASC plasmid.

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Figure 3.5: Pyrin increases caspase-1 activity and IL-1β processing and release in HEK293 cells. A) Caspase-1 activity. HEK293 cells were transfected with plasmids encoding for caspase-1 (0.1 µg), ASC (0.2 µg) and increasing doses of pyrin (0.005, 0.2 and 0.4 µg). The total DNA was kept constant at 0.7 μg by using pcDNA vector control. Caspase-1 activity was measured from cell pellets using Ac-WEHD-AMC, and results are presented as two independent experiments. The cell lysates from one representative experiment were blotted for caspase-1, ASC and pyrin. B) IL-1β release. HEK293 cells were transfected with plasmids encoding for proIL-1β (0.15 µg), caspase-1 (0.05 µg), ASC (0.2 µg) and with or without pyrin (0.005, 0.05, 0.15 and 0.3 µg). Cell culture media were harvested 24 h after the transfection and IL-1β was measured by ELISA. The data is presented as the mean ± SEM of three independent experiments. The cell lysates from one representative experiment were blotted for pyrin. The same membrane was reprobed with proIL-1β and ASC.

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We were concerned that the pyrin overexpression might be inducing IL-1β release

in response to injury to the cells due to the nonspecific effect of over expressing a protein

in HEK293 cells. To test this hypothesis we next utilized the pEGFP-pyrin plasmid with

the pEGFP plasmid control. In this case, all experimental cells would be experiencing

protein overexpression. As shown in Figure 3.6B, pEGFP-pyrin also induced an

enhanced IL-1β release from HEK293 cells. However when pEGFP (without pyrin) was expressed with ASC, caspase-1 and proIL-1β, caspase-1 activation and IL-1β processing and release was not induced (Lane 3, Figure 3.6B).

3.3.7 Pyrin overexpression in THP-1 cells

To determine if pyrin is capable of regulating IL-1β production in response to a

physiologically relevant stimulus, we nucleofected THP-1 monocytic cells with pEGFP-

pyrin plasmid or the pEGFP plasmid control (Figure 3.7). Although after 6 h, the THP-1 cells expressed the EGFP-pyrin protein, LPS (10 μg/ml) stimulation for an additional 18 h did not induce a differential release of pyrin in response to the increased pyrin expression. The lack of an effect of pyrin overexpression here is unexplained but may be due to the fact that THP-1 cells already express high levels of pyrin. We therefore chose to test the suppression of pyrin in response to LPS.

3.3.8 Suppression of pyrin decreases IL-1β release in THP-1 cells

If pyrin is an activator of IL-1β processing and release, then pyrin knockdown

should inhibit IL-1β processing and release. Therefore, we transfected THP-1 cells using

Lipofectamine2000 with pyrin or control siRNA. Three days after transfection, the cells

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Figure 3.6: Low dose of ASC also supports pyrin induced IL-1β processing and release. IL-1β release was compared in response to two pyrin expression plasmids (pcDNA3.1Myc/His and pEGFPC2N1). A) pcDNA-pyrin. HEK293 cells were transfected with plasmids encoding for proIL-1β (0.15 µg), caspase-1 (0.05 µg), ASC (0.05 µg) and with or without pcDNA-pyrin (0.05, 0.10 and 0.45 µg). Cell culture media

102 were harvested 24 h after the transfection and IL-1β was measured in the cell culture media by ELISA. The data is presented as the mean ± SEM of three independent experiments. ND indicates cytokine levels not detectable by ELISA. The cell lysates from one representative experiment were blotted for pyrin. The same membrane was used to reprobe with ASC, caspase-1 and pro-IL-1β. The asterisk indicates a non specific band. B) pEGFP-pyrin. To confirm the specificity of pyrin transfection on IL-1β processing and release pEGFP-pyrin plasmid was also used. Transfection was carried out using the above protocol. The cell lysates from one representative experiment were blotted for pyrin, ASC, caspase-1 and pro-IL-1β. The asterisk indicates a non specific band. ND indicates cytokine levels not detectable by ELISA.

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Figure 3.7: Pyrin overexpression does not suppress IL-1β processing and release in THP-1 cells. THP-1 cells were nucleofected with plasmid encoding pyrin (EGFP-pyrin) or the plasmid control (EGFP). Six hours after nucleofection, the cells were stimulated with LPS (10 μg/ml) for 18 h. Cell culture media were analyzed for IL-1β by ELISA and lysates subjected to immunoblots with antibody for pyrin, IL-1β and actin. The IL-1β data is presented as the mean ± SEM of four independent experiments. The blots shown are from one representative experiment. The asterisk indicates endogenous pyrin.

104 were stimulated with LPS (1.0 μg/ml) for 6 h. IL-1β and IL-8 release was measured by

ELISA. In support of pyrin’s role as an activator of IL-1β, suppression of pyrin by siRNA significantly decreased IL-1β release in THP-1 cells, p = 0.05 (Figure 3.8). The

IL-1β was suppressed approximately 40 percent in THP-1 cells treated with pyrin siRNA as compared to the control siRNA treated cells. Pyrin siRNA decreased pyrin protein levels in lysates. However, suppression of pyrin had no effect on IL-8, a cytokine which does not require processing or inflammasome activation for its function. As a control for siRNA, ASC, another PYD-containing protein, was not affected by pyrin siRNA.

Importantly, the pyrin siRNA did not affect intracellular proIL-1β levels (i.e., the substrate for caspase-1 was not limited).

3.3.9 Knockdown of pyrin suppresses IL-1β release in peripheral blood monocytes

To further corroborate the THP-1 studies, we nucleofected human peripheral blood monocytes with control siRNA or pyrin siRNA in seven donors. Monocytes were allowed to recover for 18 h and were then stimulated with LPS (1.0 μg/ml) for an additional 6 h. Cell culture media were analyzed for IL-1β and IL-8 by ELISA. Pyrin siRNA suppressed intracellular pyrin levels and IL-1β release was also diminished

(208±64 vs. 62±21 pg/ml, p = 0.03) (Figure 3.9). Again suppression of pyrin did not affect IL-8.

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Figure 3.8: Knockdown of pyrin suppresses IL-1β release in THP-1 cells. THP-1 cells were transfected with control siRNA or pyrin siRNA and three days later stimulated with LPS (1.0 μg/ml) for 6 h. The cell culture media were analyzed for A) IL-1β and B) IL-8 by ELISA. In addition, the lysates were analyzed by immunoblotting for pyrin, IL- 1β and actin. The data represents mean ±SEM for three independent experiments. ND indicates cytokine levels not detectable by ELISA. The blots shown are from one representative experiment.

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Figure 3.9: Knockdown of pyrin suppresses IL-1β release in peripheral blood monocytes. Peripheral blood monocytes were nucleofected with control or pyrin siRNA and allowed to incubate for 18 h before stimulation with LPS (1.0 µg/ml) for 6 h. Cell culture media and lysates were analyzed as in Figure 6. The data represents mean ± SEM of 7 donors for IL-1β and mean ± SEM of 4 donors for IL-8. The blots shown are from one representative donor.

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3.4 Discussion

Of the novel intracellular sensors and regulators of the innate immune response

pyrin is one that has been genetically linked to human disease. Pyrin/marenostrin was

Mediterranean fever (FMF) (174,175). FMF is a genetically recessive disease

characterized by recurrent self limited episodes of fever and localized inflammation affecting the serosal membranes, joints and skin and sometimes leading to systemic AA amyloidosis (220). In this context, the function of pyrin remains controversial although it

is generally held to be an inhibitor of the function of the inflammasome via its ability to disrupt ASC/caspase-1 interactions (186,192). Therefore we sought to study the role of

pyrin as a potential regulator of caspase-1 activation in monocytes and macrophages which differ dramatically in their relative abilities to activate caspase-1 and process and release IL-1β (207,209,215).

As background information it is important to summarize what is known about

pyrin function. Yeast two hybrid analysis of pyrin indicates that pyrin interacts with an

adaptor protein, apoptosis-associated speck-like protein with a CARD (abbreviated as

ASC) (221). The N-terminal PYD domains of ASC and pyrin mediate the interaction.

ASC causes the oligomerization of caspase-1 which induces its activation and the

cleavage of procaspase-1 to mature caspase-1(78,146,167). It has previously been shown

that pyrin deficient macrophages produce more IL-1β when stimulated with IL-4, LPS or

the combination as compared to the wild type (186). Pyrin deficient macrophages had

higher levels of activated caspase-1. Pyrin interacts with ASC (an activator of caspase-1)

through its N-terminal pyrin domain. One hypothesis is that wild type pyrin blocks the

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caspase-1- ASC interaction and thus prevents the activation of caspase-1 and subsequent cleavage of pro-IL-1β. We initially hypothesized that if pyrin is an inhibitor of caspase-1 activation then macrophage levels of pyrin should be high compared to monocytes. We show that both monocytes and macrophages produce the caspase-1 substrate, proIL-1β, and we have previously documented that, although macrophages contain sufficient caspase-1, they are deficient in processing IL-1β (215). Therefore, we predicted high pyrin levels in the macrophages. Unexpectedly, pyrin levels by quantitative PCR and by immunoblots of cell lysates were low in monocyte derived macrophages but easily detectable in fresh human monocytes and monocytic cell line THP-1 cells. As previously shown, monocyte derived macrophages were shown to be limited in their ability to process and release IL-1β, the classic product of caspase-1 activation. Thus, we modified our original hypothesis to ask whether pyrin may function to enhance the activation of caspase-1, a concept that has recently been proposed (215). To test this we utilized pyrin overexpression in HEK293 cells and THP-1 cells and pyrin knockdown experiments in

THP-1 cells and human monocytes to analyze the role of pyrin in caspase-1 activation.

The model tested these systems in response to the TLR4 stimulus, phenol extracted E. coli lipopolysaccharide.

In agreement with the role of pyrin as a positive regulator of caspase-1, HEK293

cells transfected with increasing amounts of pyrin showed an up regulation of caspase-1

activity and IL-1β release that correlated with the pyrin dose (Figures 3.5 and 3.6).

Consistent with the concept of pyrin as an activator, a decrease of IL-1β release was seen

in both THP-1 cells and human monocytes in which pyrin had been knocked down with

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pyrin siRNA (Figures 3.8 and 3.9). However, it must be noted that this enhancing effect

of pyrin was not substantiated in THP-1 cells over expressing pyrin (Figure 3.7). This apparent discrepancy may be explained by the possibility that THP-1 cells have more than sufficient baseline pyrin levels whose function cannot be further enhanced by further increases. Thus, in summary, pyrin levels decrease with differentiation of monocytes to macrophages and IL-1β processing and release decreases in conjunction with these changes in pyrin.

We are the first to demonstrate a function for pyrin in fresh human monocytes and

to document pyrin changes with monocyte differentiation. At the very least, in

mononuclear phagocytes that are responding to a TLR4 stimulus, our data suggest that

pyrin is not an inhibitor of IL-1β processing and release. However, that pyrin can

activate IL-1β release differs from the prior report that pyrin is suppressive (186). In this

context, it should be noted that murine pyrin differs substantially from human pyrin.

Murine pyrin lacks the C-terminal B30.2 domain of human pyrin which may at least

partially explain this difference (222).

It is noteworthy that the magnitude of the pyrin change with monocyte to

macrophage differentiation is large and correlates well with the magnitude of the change

seen with IL-1β release during differentiation. However, the magnitude of the IL-1β

change seen with pyrin overexpression in HEK293 cells is relatively modest. This raises

the distinct possibility that there are other additional changes with inflammasome related

proteins during monocyte to macrophage differentiation that may further complicate the

issue. For example, using quantitative PCR we have studied the expression of other

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inflammasome genes including NALP1, NALP3 and ASC in monocytes and

macrophages. In addition, we have generated a polyclonal antibody to ASC in an effort

to delve more deeply into this question. By qPCR we find a decrease in mRNA of

NALP1 and NALP3 from monocyte to macrophage differentiation. In contrast, the level

of ASC mRNA is increased in macrophages compared to monocytes, but, there was no

significant difference in ASC protein expression (as shown in Figure 3.3A). Recently,

Kummer et al. have shown that NALP1 and NALP3 protein levels are very low in

monocytes but high in THP-1 cells (223). Therefore, if pyrin is cooperating with

NALP1 or NALP3 or ASC for the activation of caspase-1, the situation may be complex.

That is, pyrin levels are likely to be only a part of the monocyte and macrophage difference. Furthermore, the complexity is further documented by the finding that pyrin has been suggested to be a suppressor of IL-1β (192).

In a major report that contradicts our findings, published since our manuscript was submitted. Chae et al. focused their attention to pyrin experiments in cells that do not contain ASC (i.e. PT67 cells derived from fibroblasts). This choice was to avoid the complication that ASC interacts with both pyrin and caspase-1(192). These ASC deficient cells do show a depression of IL-1β release in response to high levels of pyrin

transfections (192). Since our model was done with ASC sufficiency, we suspect that the

ratio of ASC to pyrin and caspase-1 may be critical to determine the ultimate fate of pyrin

in the ASC mediated caspase-1 activation that is central to most caspase-1 activation

reports (146,224).

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It is important to relate our findings to the condition of patients with familial

Mediterranean fever (FMF). Since FMF is characterized by spontaneous bouts of

inflammation and fever, and since our data suggest that pyrin regulates IL-1β in a

positive fashion, the mutations associated with FMF may be gain of function mutations.

The mutations may provide a selective advantage that increases the inflammatory

response to a particular infectious challenge. Just as cryopyrin has recently been linked

to ATP, bacterial DNA, Staphylococcus, Listeria, and uric acid crystals (132,134,135),

pyrin may have its own specificities. In this context, it is important to keep in mind that

our data is centered only upon one type of infectious challenge, the response to

exogenous LPS. It is possible that pyrin represents a positive regulator in the context of

LPS but a negative regulator of inflammation in the context of other types of infectious

challenges, e.g., from an intracellular pathogen. Our future studies are directed at

addressing this question.

Another interpretation of our results is that pyrin’s regulation of IL-1β processing may not be pyrin’s main function. Indeed, pyrin may regulate other aspects of inflammation. It is well known that ASC, pyrin’s binding partner, can regulate apoptosis and also may determine, in part, the relative activation of NFκB (170). Thus pyrin may be able to modify inflammation independently of IL-1β via its ability to activate NFκB levels and hence the transcription of many inflammatory cytokines. Another potential function of pyrin is related to pyrin as a member of the TRIM family of proteins. It is possible that pyrin interacts directly with intracellular pathogens as has been implied by the pyrin homologue TRIM5α which impairs transcription of HIV-1 (225). Lastly, pyrin

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may play important roles in other aspects of pathogen recognition such as phagocytosis.

Pyrin is known to interact with actin and microtubules (189) and recently pyrin has been

shown to be part of the WASP complex via its interaction with PSTPIP1 (193). Thus,

pyrin may have multiple functions and the FMF mutations may provide a number of

modifications in the innate host response beyond the regulation of caspase-1.

In summary, pyrin protein levels are down regulated in monocytes as they mature

into monocyte derived macrophages. These changes occur at the same time that IL-1β processing and release is also down regulated. Since knockdown of pyrin levels in monocytes is associated with a decrease in IL-1β processing and release, pyrin may function to augment caspase-1 activation events in some innate host response settings.

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CHAPTER 4

FAMILIAL MEDITERRANEAN FEVER IS NOT DUE TO

ENHANCED CASPASE-1 ACTIVATION

4.1 Introduction

Familial Mediterranean fever (FMF) is the most common of all the autoinflammatory diseases. The gene is named after MEditerranean FeVer causing gene

(MEFV) and codes for the protein pyrin/marenostrin. This gene was identified by positional cloning by two independent consortiums in 1997 (174,175). FMF is an autosomal recessive disease characterized by recurrent, self-limited episodes of fever, serositis, erythematous skin lesions, and localized inflammation. The patients suffering from FMF have abdominal pain due to peritonitis and arthritis. There is a massive influx of neutrophils into the affected tissues (158,226). Attacks begin suddenly without any precipitating symptoms and are characterized by high levels of acute phase proteins.

Acute FMF attacks are characterized by nonspecific rise in the inflammatory mediators. Patients during attack have increased levels of serum amyloid A protein

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(SAA), C reactive protein (CRP), IL-6, IL-8, soluble ICAM-1, and soluble TNF receptors

p55 and p75. The measurement on cytokines among various studies tends to vary

depending upon the time of collection of the samples. Depending on the study, the level

of IL-1β measured in patients tends to vary. Gang et al. 1999 found no difference in

level of IL-1β in patients during attacks (227). However, Notarnicola et al. 2002 reported

increased level of IL-1β, TNF, IL-6, and IL-8 in leukocytes of attack free patient

compared to the healthy control (228). Another study found a decreased expression of

MEFV mRNA in patients with FMF (229).

This disease mostly occurs in the people originating from the Mediterranean

basin. The carrier frequency for FMF mutations is high among the people living there. It

is speculated that the heterozygote carrier population are selected for a pathogen or a

group of pathogen as they may confer resistance to a specific pathogen or class of

pathogens like Brucella abortus (230). Colchicine is the traditional treatment of patients

with FMF and in fact the response to colchicine differentiates patients with FMF to other

hereditary periodic syndromes. Colchicine inhibits the influx of neutrophils to the

affected area by inhibiting microtubule cytoskeleton rearrangement.

More than 80 different mutations have been identified in the gene and most of the mutations are in the B30.2 domain of the protein which is encoded by a single exon. The

disease is mainly caused by four mutations namely M694V, M694I, V726A and M680I.

The role of E148Q in causing the disease is controversial (231,232). It is interesting to note that 16% of FMF patients have no identifiable mutations (233). The variants of pyrin which cause the disease in human are wild type pyrin protein in other species. The

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allele frequency is extremely high in people living in the Mediterranean indicating that

the people might have some selective advantage (221).

As most of the mutations causing the disease are found in the B30.2 domain of

pyrin, it is reasonable to speculate that B30.2 domain is important in the function of

pyrin. So far, it is not clear as to how the mutations affect the function or pyrin in

causing the disease. It is believed that mutations may affect protein-protein interaction

leading to the disease phenotype. Recently, the structure of B30.2 domain was solved by

three different groups (69,182,234). The β sandwich of B30.2 domains consists of two layers of β sheets, sheet A and Sheet B, each of which consists of 8 and 7 strands arranged antiparallaly. A database search by using program Dali revealed that B30.2 domain is similar in folding and topology to carbohydrate recognition domain of galectin-

3 (234) . Goulielmos et al. 2006 did a mutational analysis of the B30.2 domain of pyrin based on the recent molecular data from patients with FMF (235). Based on their model they placed 13 out of 24 mutations in the binding cavity. Mutations that cause severe phenotypes are found in the binding cavity. This indicates that the mutations affect the binding of a protein or a ligand. Based on their model, they predicted the size of the ligand as the size of 5 α helices with a hydrophobic surface (235).

As pyrin binds to ASC and caspase-1 and is involved in the regulation of caspase-

1 activation, it is reasonable to speculate that the mutations of pyrin may affect

interaction and assembly of these inflammasome proteins and also the co-localization of

pyrin and inflammasome proteins. To understand the mechanism of the role of mutations

of pyrin in the cause of FMF we wanted to test the ability of wild type pyrin and mutants

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of pyrin to co-localize and interact with ASC. Upon analysis, we find that pyrin co-

localizes with ASC in ASC formed specks when expressed in HEK293 cells.

Interestingly we find no difference in co-localization of the two mutant form of pyrin

E148Q and M694V with ASC. We found no differences in interaction between wild type

and mutant form of pyrin with ASC. In support of co-localization and interaction

experiments, we find that there is no difference in caspase-1 activation and IL-1β processing and release between wild type pyrin and mutants of pyrin. Our data suggests that mutations in pyrin may affect other functions of pyrin but not caspase-1 activation.

We show that FMF is not due to enhanced caspase-1 activation.

4.2 Materials and Methods

4.2.1 Cell culture and transfection

HEK293 cells were cultured in DMEM low glucose supplemented with 10% fetal

calf serum and 1% Pen/Strep. For transfection, HEK293 cells were plated at a density of

0.8X106 in a 12 well plate. Transfection was carried out with 2.5 μl of Lipofectamine

2000 (Invitrogen) with respective plasmids. Cell lysates were harvested 24 h after

transfection. For co-localization experiments, transfection was done in an 8 well chamber

slide with respective plasmids. Cells were visualized with fluorescence microscope 24 h

after transfection.

4.2.2 Plasmids and constructs

Pyrin was cloned in DSRed2-N1vector (Clontech) with DSRed tag at the N-

terminus. ASC was cloned in pEGFP vector with EGFP tag at the N-terminus. For the

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production of ASC protein, ASC was cloned in pMALC2X with an N-terminal MBP tag.

Pyrin was cloned into pcDNA3.1Myc/His (-) B with an N-terminal Flag tagged. For

generation of mutants of pyrin, Quick Change site directed mutagenesis kit was used

(Stratagene). For pyrin deletion constructs namely ΔPD pyrin and ΔB30.2 domain pyrin

both were cloned in pcDNA3.1 Myc/His (-) B with a C-terminal Myc/His Tag. All

plasmids were sequenced to confirm the integrity of plasmids. Detailed information

about the constructs used in this study is shown in Figure 4.1.

4.2.3 Fluorescence microscopy

Cells were grown and transfected in an 8 well chamber slides. Twenty four hours

after transfection, cells were visualized using Olympus BX40 fluorescent microscope for

the respective fluorescent tags.

4.2.4 MBP pull down assay

ASC was cloned in pMALC2X vector with an N-terminal MBP tag. This

construct was overexpressed in BL21 DE3 codon plus RIL strain (Novagen). The cells

were induced 6 hrs after stimulation with IPTG. Cell lysates were passed over amylase

column. The beads which has MBP or MBP tagged with ASC was washed with the low

maltose buffer and these beads which were bound with MBP or MBP-ASC were used for

the MBP pull down assay. HEK cell lysates expressing vector alone, pyrin wild type,

E148Q pyrin, M694V pyrin, ΔPD pyrin (pyrin domain deleted) or ΔB30.2 domain pyrin

(B30.2 domain deleted pyrin) were used for MBP pull down assay.

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Figure 4.1: Disease associated mutations in pyrin and constructs used in this study. (Adapted from Schaner and Gumucio. 2005 (236) A) Shows the disease associated mutations in pyrin protein. Numbers indicate the exon information of pyrin protein and the corresponding domain information. Two mutations E148Q and M694V are the most common mutations seen and were used in this study are indicated by long arrow. B) Gives the construct information used in this study for MBP-Pull down assay.

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HEK293 cell lysates were incubated with amylose beads bound to either MBP or MBP-

ASC for over night. Next day the beads were washed with RIPA buffer (25 mM Tris HCl

pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate supplemented with cocktail inhibitor (Sigma) and 2 mM PMSF) three times. The pull down samples was eluted by using 5X loading dye. The elutes were separated by SDS-PAGE and immunoblotted with antibodies to pyrin and MBP.

4.3 Results and Discussion

4.3.1 Pyrin or mutants forms of pyrin co-localize with ASC

It has been demonstrated by others that pyrin interacts with ASC and co-localized with ASC in specks (169,176,237). It is believed that mutations of pyrin affect the ability of pyrin to bind to the inflammasome components namely ASC and caspase-1. This idea supports the role of pyrin in sequestering ASC thereby suppressing the assembly of the inflammasome. Since mutations of pyrin causes increased inflammation and it is believed that mutations affect the pyrin’s ability to sequester ASC, we hypothesized that mutant forms of pyrin may not co-localize with ASC in specks. To test this hypothesis, we transfected pyrin or mutant forms of pyrin and analyzed their localization with the help of a fluorescence microscope. Wild type pyrin was diffusedly stained when it was transfected alone (Figure 4.2A). ASC, when transfected alone formed both diffused staining and also localized in specks (Figure 4.2B). When both wild type pyrin and ASC were transfected together, pyrin co-localized with ASC in specks (Figure 4.2 C and D).

When two mutant forms of pyrin, E148Q and M694V were transfected with ASC, both

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mutant forms of pyrin were co-localized with ASC in specks (Figure 4.2 E, F and G, H).

There was no difference in co-localization of wild type or mutant forms of pyrin with

ASC. This proves that mutation of pyrin does not affect ASC-pyrin co-localization. This

finding is agreement with other groups that ASC co-localized with wild type or mutants

forms of pyrin in specks (169,176,237).

4.3.2 Pyrin or mutants forms of pyrin interact with ASC in MBP pull down

assay

In the co-localization experiments, we did not find any difference between wild type or mutant forms of pyrin in co-localization with ASC. We wanted to test whether

there was any difference between wild type or mutant forms of pyrin in interacting with

ASC. To test this hypothesis, we made use of the MBP pull down assay. Pyin or the

mutant forms of pyrin did not bind to MBP, when MBP was used alone. In contrast

when we used MBP-ASC, we observed binding of both wild type and mutant forms of

pyrin. This interaction was mediated by pyrin domain, as pyrin domain deleted pyrin did

not bind to ASC. We did not observe any appreciable differences between wild type or

mutant forms of pyrin in binding to ASC (Figure 4.3). B30.2 deleted pyrin did bind to

ASC indicating that B30.2 domain is dispensable for ASC-pyrin interaction. This

indicates that ASC-pyrin ASC interaction is mediated by pyrin domains of both the

proteins and B30.2 domain of pyrin is dispensable for this interaction.

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Figure 4.2: Wild type and Mutant forms of pyrin do not differ in co-localization with ASC. HEK293 cells were transfected with EGFP-ASC, with dsRED-pyrin or dsRED-pyrin mutants (EPyrin-E148Qpyrin, Mpyrin-M694Vpyrin). Transfected cells were observed using a fluorescent microscope 24 h after transfection. A) Transfected with pyrin alone. B) Transfected with ASC alone. C) Transfected with pyrin wild type and ASC, observed for ASC and D) Transfected with pyrin wild type and ASC, observed for pyrin. E) Transfected with E148Q pyrin and ASC, observed for ASC and F) Transfected with E148Q mutant pyrin and ASC, observed for pyrin. G) Transfected with M694V pyrin and ASC, observed for ASC and H) Transfected with M694V mutant pyrin and ASC and observed for pyrin.

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Figure 4.3: Interaction of ASC with pyrin wild type or mutants. Pyrin wild type, mutant forms of pyrin, Δ PYD pyrin and ΔB30.2 domain pyrin were incubated with amylose column bound to MBP or MBP-ASC. The column was washed three times and eluted with 5x dye. The eluates were separated and immunoblotted with antibody to pyrin and MBP. The result is a representative of three experiments.

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4.3.3 Pyrin or mutants forms of pyrin have no difference in caspase-1 activity or IL-1β processing and release.

To determine the role of pyrin mutants in caspase-1 activation and thereby IL-1β

processing and release, we used HEK293 cells transfected with pyrin or the different mutants of pyrin along with pro-IL-1β, ASC and caspase-1. In agreement with the interaction experiments there was no difference between wild type or mutant forms of pyrin in caspase-1 activation and IL-1β release (Figure 4.4). All forms of pyrin enhanced

IL-1β release. Also, we did not observe any differences in expression of pyrin wild type or mutant forms of pyrin. Wild type or mutant forms of pyrin do not differ in IL-1β processing and release in HEK293 cell system.

4.4 Conclusions

Familial Mediterranean fever is the most prevalent autoinflammatory disease and

was the first to be linked to the mutations of MEFV gene. The disease is characterized by

bouts of recurrent fever and inflammation. The underlying cause of inflammation is not

known. Since the cloning of MEFV gene in 1997, there has been tremendous progress in

understanding the role of pyrin and mutants in inflammation. However, there is no

conclusive evidence regarding the role of pyrin in caspase-1 activation. The role of pyrin

in caspase-1 activation has been both activating and suppressing based on the model

systems used (131,169,185,186,191). As of now, there are no proper explanations as to

why the mutations in pyrin lead to excess inflammation. As the disease is a recessive

mode of inheritance, it is thought that mutations in pyrin are a loss of function in caspase-

1 activation. Wild type pyrin sequesters ASC and caspase-1 molecule thereby disrupting

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Figure 4.4: Pyrin and mutant forms of pyrin do not differ in IL-1β processing and release in HEK293 overexpression system. HEK 293 cells were transfected with plasmid encoding proIL-1β, caspase-1, ASC and with pyrin or mutant forms of pyrin. IL-1β processing and release were measured in the supernatants by ELISA. The cell lysates were analyzed by immunoblotting with anti pyrin antibody.

125 the assembly of NALP3 inflammasome and mutations in pyrin are loss of function mutations and therefore mutant pyrin will not be able to sequester ASC and caspase-1 thereby have an enhanced IL-1β processing and release. Chae et al. 2006 showed that pyrin interacted with both pro and mature caspase-1 and this interaction was defective in pyrin mutants. This finding explains the enhanced inflammatory response seen in patients with FMF. They did not show the role of pyrin wild type or mutants in interacting with ASC (192). However, subsequent study by Papin et al. 2007 showed that pyrin did interact with caspase-1 through B30.2 domain but it also interacted with other inflammasome player namely NALP3 and ProIL-1β. They found no differences in interaction between wild type or mutant forms of pyrin with caspase-1, NALP3 and

ProIL-1β (191).

Since there was limited information regarding interaction of ASC with mutant forms of pyrin, we wanted to test whether there were any differences in wild type and mutants forms of pyrin in interacting with ASC. We demonstrate that pyrin or mutant forms of pyrin co-localize with ASC in specks. Mutations did not affect ASC co- localization. Pyrin formed a diffused staining when transfected alone but in presence of

ASC, pyrin co-localized in specks. Mutant forms of pyrin were no different that pyrin wild type in co-localization with ASC. In support of our finding, Cazeneuve et al. 2003 also reported that there was no discernible differences in localization of pyrin and mutant forms of pyrin with ASC (237).

We also analyzed interaction of ASC with wild type or mutant forms of pyrin.

We find that there is no difference in interaction of wild type or mutant forms of pyrin

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with ASC. As it very elusive and not very clear that pyrin and mutant forms of pyrin

differ in interacting with caspase-1, our future experiments should be directed in that direction.

We also analyzed for caspase-1 activity and IL-1β processing and release of pyrin

and mutant forms of pyrin in an HEK293 overexpression system. We failed to detect any

appreciable change in IL-1β processing and release between wild type and mutant form

of pyrin in our HEK293 overexpression system.

Since HEK293 cells are distinct from macrophages; and since all the experiments

reported so far used mutants over expressed in HEK293 cells, it will be interesting to

analyze the role of mutant forms of pyrin in cell lines which are more closer to monocytic

lineage and that do not express pyrin. Chae et al. 2003 over expressed wild type and

mutant forms of pyrin in U937 cells. U937 cells expressing mutant form of pyrin had a

slightly elevated IL-1β release compared to the cells expressing wild type pyrin, but the

data did not reach statistical significance. Since the role of wild type and mutant forms of

pyrin has not been studied in a system which mimics monocytes from diseased patients, it

will be interesting to study the role of mutants in monocytic cells that do not have pyrin

in response to different line bacterial or viral stimulus.

Recently pyrin has been shown to bind the pro-apoptotic protein siva-1 though the

B30.2 domain. However, in that study, testing mutants of pyrin to siva-1 did not yield

any interesting data. They found no difference between wild type and the different mutant

forms of pyrin in interacting with siva-1. It will be of interest to test whether mutations

of pyrin affect the apoptotic pathway.

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Although the roles of mutation in disease progression of other NLR proteins have been understood, still the role of mutations of pyrin in causing inflammation remains elusive. Generation of pyrin knockout animal and knock-in animals may be able to help us understand the role of pyrin in immunity. Knock-in animals carrying the mutant form of pyrin will help us in understanding the role of mutations in the progress of FMF.

Understanding the role of pyrin mutations in FMF diseases progression is important not only in understanding the function of pyrin but will also help us in treating the patients with FMF. To conclude, our data indicates that FMF is not due to enhanced caspase-1 activation.

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CHAPTER 5

CONCLUSIONS AND FUTURE PERSPECTIVES

The preceding chapters have explored the function of MAIL and pyrin in macrophage innate immunity. I have shown that MAIL, a homolog of IκB proteins is involved in the production of IL-6. Monocytes differ from macrophages in production of

IL-6. This is attributed to the differences in MAIL protein expression between monocytes and macrophages. Similarly, we found that monocytes and macrophages differ in IL-1β processing and release. Expression of pyrin correlated with the ability to process and release IL-1β. Monocytes that had an increased expression of pyrin were able to process and release IL-1β upon LPS stimulation. In this chapter, I will present data from experiments that although in some ways incomplete, may add to future studies in this area. I will start with MAIL as a regulator of COX-2 expression and then I would present some unanswered questions regarding the function of pyrin.

5.1 MAIL is a regulator of COX-2 expression

COX-2 or cyclooxygenase 2, is an enzyme responsible for synthesis of

prostaglandins. Unlike the expression of COX-1 which is constitutive, a

proinflammatory stimulus is required for the expression of isoenzyme COX-2 (238). Both

COX-1 and COX-2 are involved in conversion of arachidonic acid to prostaglandin H2 129

(PGH2). Prostaglandins are involved in lipid mobilization and inflammation by

increasing the vascular permeability and vasodilation and increasing the recruitment of

other cells involved in inflammation (239-243).

The expression of COX-2 is regulated by many transcription factors including

NF-κB, CREB, C/EBP-β, and PU.1 (244-247). COX-2 expression is regulated by NF-κB in RAW264.7 macrophages. Blocking NF-κB activity resulted in decreased COX-2

expression. COX-2 and production of prostaglandins has been shown to be upregulated

by IL-17. IL-17 is a proinflammatory cytokine produced by T cells involved in many

auto inflammatory diseases. IL-17 co-operates with TNF for the production of many

genes involved in host defense like cytokines, chemokines, COX-2, antibacterial protein

24p3, and beta defensins (248,249). It also co-operates with TLR in production of

inflammatory gene expression (250).

Recent work on IL-17 indicates that IL-17 increases prostaglandin E2 production

upstream of COX-2 (251,252). Recently it has been reported that IL-17 induced expression and stabilization of MAIL mRNA (100). It has been reported that MAIL is a positive regulator of NGAL and hBD2 production in epithelial cells (112).

Recent work by Kao et al. 2008 indicates that MAIL is an important regulator of

IL-17 induced hBD2 production. Shen et al. 2006 identified functional transcriptional elements of known IL-17 target genes based on computational analysis. They reported an essential role of NF-κB sites and C/EBP sites in IL-17 target genes (253). Also, it has been reported recently that C/EBP and NF-κB play a crucial role in the genes which are regulated by MAIL (254). COX-2 promoter activity is regulated by many transcription

130 factors that include NF-κB, C/EBPs, AP-1, cAMP response element 1 (CRE-1), and an

E-box that acts a transcription repressor (255). Since IL-17 is a key cytokine involved in expression of MAIL and both the NF-κB and C/EBP sites are present in the promoter of COX-2, we hypothesized that MAIL might regulate the expression of COX-2.

5.1.1 Monocytes express COX-2 upon LPS stimulation

To test the hypothesis that MAIL may be a regulator of COX-2 expression, we analyzed the expression of COX-2 mRNA and protein in monocytes stimulated with

LPS. COX-2 mRNA expression begins at 15 min but the expression peaks only at 8 h after LPS stimulation. In contrast, MAIL mRNA expression is robust at 15 min and the expression was highest at 2 h after stimulation. When we analyzed the protein expression of MAIL and COX-2 in monocytes, MAIL protein peaks at 4 h and after that the expression gradually diminishes, whereas COX-2 expression is peak at 8 h after LPS stimulation (Figure 5.1A).

5.1.2 COX-2 is induced by LPS, IL-1β and not by TNFα in monocytes

As it is known from our own data and data from others that expression of MAIL is induced by LPS, IL-1β, and TNF, we wanted to test whether COX-2 expression followed MAIL expression pattern. Monocytes were stimulated with IL-1β, LPS, TNF or

IL-1β and TNFα in combination for the time points indicated. Cell lysates were analyzed by immunoblotting with antibodies specific for MAIL, COX-2, IL-1β and actin. In accordance with the previously published data, expression of MAIL was induced by LPS, there was little induction by IL-1β but there was no induction by TNFα.

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Figure 5.1: COX-2 is expressed in human monocytes upon LPS stimulation. A) Comparison of kinetics of MAIL and COX-2 mRNA expression in monocytes. Monocytes were stimulated with 1.0 μg/ml of LPS for the time points indicated for a period of 24h. RNA was isolated by Trizol and analyzed by real time PCR. Cell lysates were immunoblotted with antibody for COX-2. The same membrane was reprobed with antibodies for MAIL and actin. B) LPS and IL-1β induce expression of COX-2. Monocytes were left untreated or treated with IL-1β (10 ng/ml), LPS 1.0 μg/ml, TNFα (10 ng/ml) or a combination of IL-1β and TNFα for 3 and 6h. Cell lysates were immunoblotted with antibodies for COX-2, IL-1β and MAIL and actin using the same membrane. 132

IL-1β in combination with TNFα induced very little MAIL expression. When COX-2 and IL-1β expression were analyzed in the same experiment, their expression followed

MAIL expression pattern. Monocytes are very responsive to LPS and upon stimulation they express MAIL, IL-1β and COX-2. IL-1β is not a good inducer of both MAIL and

COX-2 expression in monocytes. TNFα is a poor inducer of all three genes. Monocytes response to IL-1β and IL-1β and TNfα in combination is diminished when compared to

LPS (Figure 5.1B). However, in other cells such as Beas 2B which is an epithelial cell line, we have shown that MAIL is induced by IL-1β, IL-1β and TNFα in combination but not by TNFα. COX-2 expression followed the expression of MAIL.

5.1.3 Knockdown of MAIL suppresses the expression of COX-2

In order to show that MAIL regulates the expression of COX-2 in monocytes, we decided to make use of the small interference RNA (siRNA) for MAIL. We suppressed the expression of MAIL in monocytes and analyzed the expression of COX-2 mRNA and protein upon LPS stimulation. Suppression of MAIL suppressed the expression of both

COX-2 mRNA and protein but this did not attain statistical significance (Figure 5.2). As a control we analyzed the IκBα mRNA. We did not see any change in IκBα mRNA or protein indicating that siRNA was specific for MAIL. However, these samples had decreased expression of MAIL and IL-6 indicating that siRNA is effective in suppression of MAIL and also the MAIL regulated gene IL-6. With the data presented above we have indirect evidence that MAIL might regulate the expression of COX-2. Both MAIL and COX-2 are induced by LPS and IL-1β but not by TNFα. COX-2 expression occurs at

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Figure 5.2: MAIL suppressed monocytes have decreased COX-2 expression. Monocytes (10x106) were nucleofected with control siRNA or MAIL siRNA. Cells were stimulated 18 h after nucleofection with 1.0 μg/ml LPS for additional 6 h. A) RNA expression was analyzed by real time PCR for MAIL, COX-2 and IκBα. The data represents mean ± SEM for 5 donors. B) Cell lysates were immunoblotted with antibodies to MAIL, COX-2, ProIL-1β, IκBα and actin using the same membrane. Blots from 2 donors are shown.

134 a later time in monocytes upon LPS stimulation in contrast to early MAIL expression.

These data supports a role of MAIL in regulating the expression of COX-2.

To further prove that MAIL is a regulator of COX-2 expression, we have to use

COX-2 promoter constructs and check for COX-2 promoter activity with or without

MAIL overexpression. If MAIL is an important regulator then overexpression of MAIL will lead to increased COX-2 promoter activity. Experiments like electrophoretic mobility shift assay and chromatin immunoprecipitation can also be done to ascertain the binding of MAIL to COX-2 promoter. If all the above experiments failed to give a positive result then it indicates that MAIL regulates the expression of transcriptional factor/s that may be involved in regulating expression of COX-2.

5.2 Role of pyrin in macrophage innate immunity –The unsolved questions

It is appropriate to say that pyrin is a regulator of caspase-1 activation. We do not know for certain whether pyrin is an activator or suppressor of caspase-1 activation. Data that I presented in chapter III indicates the role of pyrin as an activator of caspase-1. We have shown that pyrin expression is dynamic, it is highly expressed in monocytes, and its expression levels are lower in monocyte derived macrophages. This correlates well with the differences in IL-1β processing and release between monocytes and macrophages.

Monocytes tend to release a lot more IL-1β compared to monocyte derived macrophages upon LPS stimulation. It is not clear yet whether the change in model systems lead to such difference in experimental results. As studies about the role of pyrin in caspase-1 activation were done with cell lines it is important to repeat those studies with the help of

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pyrin knock out animal. Here are some important questions to be answered to understand

the role of pyrin in macrophage innate immunity.

5.2.1 Do pyrin over expressing macrophages release IL-1β

A direct link between the differences in pyrin and IL-1β has not been shown so

far. Experiments aimed at over expressing pyrin in macrophages should be done to show

that pyrin is important for the difference in IL-1β processing and release. If the

differences in IL-1β processing and release between monocytes and macrophages is due

to pyrin then pyrin overexpression should restore similar levels of IL-1β processing and

release in macrophages and monocytes. If pyrin over expression does not alleviate the

differences between macrophages and monocytes, then this indicates that other NLR

proteins may be involved in the differences observed. A qPCR analysis or a microarray

analysis should be done to analyze the difference between monocyte and macrophages

globally. This may help in identifying genes that may play an important role in IL-1β processing and release. Another possibility to be considered is the post translational modification of the proteins involved in inflammasome assembly. The differences between monocytes and macrophages may be the post translational modification of inflammasome proteins observed in these two cell types.

5.2.2 Does pyrin interact with other proteins of the inflammasome

In addition to ASC and PSTPIP1 which were identified by yeast 2-hybrid

analysis, recently pyrin has been shown to interact with caspase-1, NALP3, NALP1,

NALP-2, and proIL-1β through the B30.2 domain (176,191-193). It was shown that

pyrin sequesters ASC from NALP3 and thereby preventing NALP3-ASC induced

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caspase-1 activation, NF-κB activation and apoptosis (194). However, this observation was contradicted by observations by Yu et al. 2006. They showed that pyrin did not block IL-1β processing and release even when pyrin was over expressed with NALP3

(169).

So far, it is not clear whether pyrin is a positive or negative regulator of caspase-1 activation. To address this question generation of pyrin knockout mice is required. It will be interesting to evaluate the response of pyrin knockout mouse challenged with endotoxin and other stimulus. Furthermore, since mouse pyrin lacks a B30.2 domain, generation of a human pyrin knock-in mouse is required. The use of human mutant- pyrin knock-in mouse in response to various stimuli may help in understanding the role of mutations of pyrin and may lead to understand the mechanism of FMF. It is not clear

whether mouse-pyrin which lacks the B30.2 domain interacts with caspase-1, NALP3

and proIL-1β. Pyrin may also interact with other NLR proteins through the pyrin-pyrin

domain interaction. Generation of pyrin knockout animal is required to understand the

role of pyrin in inflammation.

5.2.3 Role of mutations of pyrin in phagocytosis.

The involvement of pyrin in phagocytosis comes from the studies that link pyrin

to cytoskeleton remodeling. Pyrin has been shown to interact with microtubules and co-

localize with actin. Chae et al. 2003 showed that pyrin can also interact with PSTPIP1.

PSTPIP is a cytoskeletal associated protein found in centers where actin polymerizes effectively. It is possible that pyrin mutants may have decreased binding to cytoskeletal

proteins and thereby cause decreased phagocytosis. The idea that pyrin may be involved

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in phagocytosis is derived from two indirect observations. First, FMF patients are

prescribed colchicine to decrease the disease manifestation. Colchicine is a microtubule

polymerization inhibitor. This indicates that pyrin’s role in cytoskeletal modeling may be

affected by the mutations in the gene. Second, recently it has been demonstrated that

caspase-1 activation required Rac1 activation. Rac1 knockout cells are deficient in

caspase-1 activation upon LPS stimulation (256,257). This indicates that cytoskeletal

remodeling is associated with inflammasome activation and assembly. As pyrin is

involved in both inflammasome regulation and cytoskeletal remodeling, it will be

interesting to test the role of these mutants in phagocytosis and thus phagocytosis mediated differences in caspase-1 activation.

5.2.4 Is B30.2 domain a pathogen recognition domain

Recent structural analysis predicts B30.2 has a similar structure and sequence to

the carbohydrate binding region of galectin-3. This was done using program DALI

which searches a database for proteins having similar sequence and structure. When they

searched for proteins similar to B30.2 domain in database, most of the proteins that were

identified by the program had carbohydrate recognition sequences. During protein

purification using amylose column, we found that most of the pyrin was bound to the

column even when 10mM maltose was used to elute the protein off the column. In our

purification, we could only identify an N-terminal cleaved portion of pyrin. So based on

this, we hypothesized that B30.2 domain may be involved in binding carbohydrate or

may play a role in identification of PAMPs, which have carbohydrate moieties. So far,

there is very little evidence linking direct binding of PAMPs to any of the TLRs or NLRs.

138

In order to test the hypothesis we used Francisella an intracellular bacteria which causes

tularemia in humans. Francisella novicida activates the inflammasome by an unknown

NLR. Internalization and escape of bacteria from phagosome is required for Francisella

to activate the inflammasome. We used a method similar to a pull down assay, instead of

protein bound to beads we just used the bacteria as a pull down agent. Francisella was

incubated with HEK293 cells expressing EGFP-pyrin or EGFP-ASC. We used ASC as

our control because ASC has a pyrin domain similar to that of pyrin but lacked B30.2 domain. When the bacterial elutes were analyzed by western blot for EGFP, we saw pyrin coming down with Francisella but not ASC (Figure 5.3).

Further experiments needs to be done to confirm this finding. Competitive

binding assay using amylose to compete with the bacteria needs to be done to ascertain

the binding of the bacteria to the B30.2 domain. Use of B30.2 domain deleted pyrin will

help to ascertain the binding of B30.2 to the bacteria.

To support the role of B30.2 domain in innate immunity, recently it was shown

that TRIM21, a B30.2 domain containing protein, was bound to IgG. B30.2 domain of

TRIM21 mediated the interaction with IgG. Recently, Yu et al. 2008 have implicated

that B30.2 domain may be involved in recognition of retrovirus. Interestingly, cryopyrin

from zebra has a fusion of LRR and B30.2 domain to support the hypothesis that B30.2

may be involved in pathogen recognition.

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Figure 5.3: Francisella binds pyrin A). Steps involved in doing a Francisella binding assay. B) F. novicida (F.nov) incubated with cell lysate of HEK293 cells over expressing EGFP-pyrin or EGFP-ASC. The bacteria was spun down and washed three times with PBS. The bacteria with bound protein were lysed in 5x loading dye and separated by SDS-PAGE. The membrane was then probed with antibody to EGFP. Asterisks indicates the lane were Francisella was added.

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5.3 Secretion of inflammasome proteins

IL-1β is a leaderless protein which does not require the golgi secretory vesicles

for its release. It is released in a non classical release pathway. Although we have begun

to understand the mechanisms of inflammasome-mediated caspase-1 activation; the

mechanism of IL-1β release still remains a mystery. We know that the multi-protein

complex inflammasome, which also contains NLR protein, activates caspase-1 and this

activation requires the sensing of the ligands by NLR. When human monocytes are

stimulated with LPS, they express a lot of proIL-1β protein, but very little of this proIL-

1β is processed and released out of the cell. LPS stimulated monocytes need both

priming, which leads to the formation of proIL-1β, and the second stimulation, which is

normally mediated by ATP, and leads to caspase-1 activation. It has been shown by many groups that ATP stimulation activates the P2X7 ion channel and thereby causing ionic imbalances including the efflux of K+ (258-260).

P2X7 are ATP gated ion channels expressed in many cell types including

macrophages. These channels are gated by high extracellular ATP, which is normally

released by dying and injured cells. Upon activation these channels cause a disruption of

cell membranes causing ion imbalances and thereby leading to passage of molecules of

varying sizes. These channels upon opening, can allow molecules up to 900 Da to pass

through. Macrophages from P2X7 knockout mice do not process and release IL-1β upon

stimulation with LPS and ATP. However, recent data suggests that intracellular bacteria

do not require P2X7 receptor activation and therefore K+ efflux for activation of caspase-

1.

141

Recently, it has been demonstrated that pannexin1 is involved in P2X7 receptor mediated large pore formation and this process is upstream of caspase-1 activation.

There are three main models described in literature for the release of IL-1β. The first model and the most recent one describe the release of IL-1β as multi-vesicular bodies and exosomes. IL-1β is secreted along with other proteins involved in the formation of inflammasome in a microvesicular bodies in exosomes The second model which requires calcium independent phospholipase A2, describes IL-1β secretion by secretory lysosomal exocytosis,. The third model is mediated through microvesicles which are formed by calcium dependent membrane evagination or blebbing. These microvesicles are loaded with the proteins of inflammasome and IL-1β (261-266).

Recently the requirement of caspase-1 activity for secretion of non-conventional secretory proteins has been described (116). It was shown that proteins that do not have a secretory sequence either bind to caspase-1 directly or indirectly to facilitate their release.

It has been shown by different groups that the inflammasome components namely ASC, caspase-1 and NALP3 are released from monocytes stimulated with LPS in combination with ATP or by skin keratinocytes upon exposure to UVB radiation (142).

5.3.1 Monocytes secrete ASC.

As it has been shown earlier that the inflammasome proteins are released in the culture media of monocytes stimulated with LPS, we wanted to test whether the released

ASC can be used as a marker for inflammation. To test this hypothesis we used immunoprecipitation followed by immunoblot analysis to quantify the release of ASC.

We found that ASC is released by human monocytes. This release may be further

142

augmented by LPS stimulation. We observed that the release of ASC from monocytes is

spontaneous and independent of caspase-1 activation.

5.3.2 ASC is released from monocytes treated with LPS

As it has been shown by other groups that inflammasome components are

released into the media upon activation (142,146), So we hypothesized that monocytes will release ASC upon LPS stimulation. Monocytes were treated with LPS for time points indicated for a period of 24 h. The supernatants were harvested after 24 h and immunoprecipitated with ASC antibody. The immunoprecipitates were separated on a 4-

12% gradient gel and subsequently immunoblotted with anti ASC antibody. Monocytes released ASC at a very early time point as low as 15 minutes upon LPS stimulation.

There was an increase in ASC release based on the stimulation time. In accordance with the previously published data, we showed that monocytes release ASC upon LPS stimulation (Figure 5.4).

Next we wanted to test whether ASC release was dependent on caspase-1

activation. As it was shown by other groups that activation of inflammasome releases IL-

1β and other proteins of the inflammasome, we hypothesized that caspase-1 activation is

required for ASC release. To test this hypothesis, the monocytes were treated with

caspase-1 inhibitor (YVAD-cmk) 1 h before treating with LPS. To determine whether

ASC is released by dying cells or cells undergoing apoptosis, we treated monocytes with

camptothecin which is an apoptosis inducer.

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Figure 5.4: ASC is released from monocytes Monocytes (2.5x106/ml) were treated with LPS (1.0 μg/ml) for time points indicated for a period of 24 h. Supernatants were harvested and immunoprecipitated with ASC antibody followed by western blot with ASC antibody. Two donor data is represented in the figure.

144

We again used immunoprecipitation followed by immunoblot analysis to quantify

the ASC release. We observed that monocytes without any treatment release ASC

spontaneously. LPS stimulation did not increase ASC release. When we blocked the

caspase-1 activation by using caspase-1 specific inhibitor we did not see any decrease in

the amount of released ASC when compared to control treated cells (Figure 5.5A

compare lane 3 to 4 and 6 to 7). Interestingly, the amount of ASC released by cells treated with apoptosis inducer camptothecin was similar to the control cells. This indicates that ASC release is not due to cells undergoing apoptosis or due to activation of caspase-1.

ASC is an adaptor molecule which is essential for the caspase-1 activation and IL-

1β processing and release. As it has been shown by others that IL-1β is released along

with the inflammasome proteins, we wanted to test whether IL-1β release matched with

the release of ASC. We analyzed the release of IL-1β by ELISA. As expected,

monocytes which were stimulated with LPS had increased IL-1β released in the medium

(3 ng). In contrast, monocytes treated with caspase-1 inhibitor YVAD-CMK had a

reduced level of IL-1β (600 pg) indicating that YVAD-CMK was effective in blocking

caspase-1 activation. Interestingly, when monocytes were treated with camptothecin

alone or in combination with LPS did not release any IL-1β (Figure 5.5B). We analyzed

whether the camptothecin was effective in inducing apoptosis by LDH assay. As

expected, we found an increase amount of LDH released in camptothecin treated cells

compared to the control (data not shown). This indicates that IL-1β release and ASC

145

Figure 5.5: ASC release is independent of caspase-1 activation. Monocytes were pretreated with NT (No Treatment) DMSO, ZFA, YVAD (YVAD-cmk) or CAMP (camptothecin) for 1 h and then left unstimulated or stimulated with LPS for 6 h. Supernatants were harvested after 6 h. A) Supernatants were immunoprecipitated with ASC monoclonal antibody followed by immunoblotting with ASC polyclonal antibody. B) Supernatants were analyzed for IL-1β by ELISA.

146

release are two different events. ASC release in monocytes is spontaneous and does not

require caspase-1 activation. Inflammasome activation may increase the release of ASC

from monocytes which may be dependent on caspase-1 activation.

Further experiments need to be done to determine whether ASC release is

enhanced with the treatment of LPS in combination with ATP compared to LPS alone.

Use of macrophages from caspase-1 knockout animal may be useful in determining the

role of caspase-1 in spontaneous release of ASC.

5.4 Conclusions

This thesis has addressed function of two proteins involved in two different

pathways which are critical in innate immune defense of the host. The signalosome

pathway leads to the production of proinflammatory cytokines and other proteins which

are essential for innate host defense. In chapter 1, we have shown that MAIL is turned on

by the signalosome pathway and is important in regulating IL-6 production in human

monocytes. Monocytes and macrophages differ in the expression of MAIL and therefore

in the production of IL-6. Furthermore, we show that MAIL is important for the

production of IL-6 in response to both intracellular ligands and extracellular ligands.

IL-1β requires both the inflammasome and the signalosome pathway for its production and processing. The processing of IL-1β is more complex and it involves the

activation of a cysteine protease known as caspase-1. Now, we know that caspase-1

activation requires the assembly of a multi protein complex called the inflammasome. In

chapter 3, the role of pyrin in caspase-1 activation is elucidated. Monocytes process IL-

1β but their descendant macrophages lack this ability. Pyrin expression was decreased in

147 macrophages. We show that pyrin is a positive regulator of caspase-1 activation and IL-

1β processing and release. In chapter 4, the role of pyrin mutations in caspase-1 activation was analyzed. We find that FMF is not due to enhanced caspase-1 activation as there was no difference in IL-1β processing and release when wild type or mutant forms of pyrin were over expressed in HEK293 cells. The additional studies mentioned here and elsewhere are needed to completely understand the function of these two proteins which are important in host defense. A complete understanding of MAIL and pyrin will be useful in understanding the mechanism of inflammation and may open new avenues for therapeutic interventions in inflammation and inflammation-induced diseases such as cancer.

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