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.