HUMAN β DEFENSIN 3: LINKING INNATE AND ADAPTIVE IMMUNE RESPONSES By: NICHOLAS FUNDERBURG Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Advisors: Michael Lederman, MD. Scott Sieg, PhD. Department of Molecular Biology and Microbiology CASE WESTERN RESERVE UNIVERSITY January 2008 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of ______________Nicholas Funderburg_____________________ candidate for the Ph.D. degree *. (signed)_________David McDonald______________________ (chair of the committee) ______________Michael Lederman__________________ ____ _Scott Sieg________________________ ________Aaron Weinberg___________________ Thomas McCormick___ __________ ________________________________________________ (date) _____7-17-07__________________ *We also certify that written approval has been obtained for any proprietary material contained therein. 2 Table of Contents Table of Contents 3 List of Tables 7 List of Figures 8 Acknowledgements 11 Abstract 12 Chapter 1: Introduction 14 Innate Immunity 17 Antimicrobial Activity of Beta Defensins 19 Human Defensins: Structure and Expression 21 Linking Innate and Adaptive Immune Responses 23 HBDs in HIV Infection 25 Summary of Thesis Work 26 Chapter 2: Human β defensin-3 activates professional antigen-presenting cells via Toll-like Receptors 1 and 2 31 Summary 32 Introduction 33 3 Results 35 HBD-3 induces co-stimulatory molecule expression on APC 35 HBD-3 activation of monocytes occurs through the signaling molecules MyD88 and IRAK-1 36 HBD-3 requires expression of TLR1 and 2 for cellular activation 37 Discussion 40 Materials and Methods 43 Reagents 43 Primary cells and culture conditions 44 HEK293 cell transfectants 44 Flow Cytometry 44 Western Blots 45 TLR Ligand Screening 45 Murine macrophage studies 45 CHO Cell experiments 46 Statistical Methods 46 Acknowledgments 47 Chapter 3: Induction of Surface Molecules and Inflammatory Cytokines by HBD-3 and Pam3CSK4 are regulated differentially by the MAP Kinase Pathways. 57 Summary 58 4 Introduction 59 Results 61 HBD3 induces homing molecule expression on monocytes 61 HBD-3 induces inflammatory cytokine production by monocytes 61 Exposure to different TLR 1/2 ligands generates unique responses from monocytes 62 Differential effects of MAP kinase inhibitors on the induction of CD80 by hBD-3 or Pam3CSK4 63 Discussion 65 Materials and Methods 69 Reagents 69 Cell Preparation and Culture 69 Flow Cyotmetry 70 Quantification of Inflammatory Cytokines 70 Chapter 4: Discussion and Future Directions 86 Discussion 87 Future Directions 90 Hypothesis: HBD-3 will increase the ability of antigen presenting cells to induce T cell responsiveness to antigen. 90 Hypothesis: HBD-3 will enhance the in vivo T cell responsiveness to antigen by activating antigen presenting cells. 92 5 Hypothesis: HBD-3 can interact with TLRs 1 and 2 because it forms dimers and has a high cationic charge. 93 Hypothesis: The Leucine Rich Repeats (LRRS) found in TLR1, but not TLR6, are important for interaction with hBD-3. 95 Hypothesis: Antigen presenting cells from HIV infected patients will have decreased responsiveness to hBD-3 stimulation and lowered levels of hBD-3 production at mucosal sites. 97 Conclusions 100 Appendix: Toll like receptor ligands induce human T cell activation and death, a model for HIV pathogenesis 114 Bibliography 139 6 List of Tables Table 4.1. Summary of HIV+ Donors. 108 7 List of Figures Figure 1.1 Toll-like receptors can signal in MyD88-dependent and independent pathways. 29 Fig. 2.1. Overnight culture with hBD-3 results in increased expression of CD80, CD86, and CD40 on mDCs and monocytes, but not on pDCs. 48 Fig. 2.2. MyD88I inhibits induction of CD80 expression by hBD-3. 50 Fig. 2.3. HBD-3 induces phosphorylation of IRAK-1 in purified monocytes. 51 Fig. 2.4. HBD-3 does not stimulate HEK293 cell lines expressing single Toll-like Receptors. 52 Fig. 2.5. HBD-3 activates cells via TLR1 and TLR2. 53 Fig. 2.6. Human β defensin-3 does not activate mouse Bone Marrow-derived Macrophages. 55 Fig. 2.7. Disruption of the structure of shBD-3 results in loss of activity. 56 Figure 3.1. HBD-2 does not induce Inflammatory Cytokine Expression from Human Peripheral Blood Mononuclear cells. 72 8 Figure 3.2. Overnight exposure to hBD-3 results in increased surface expression of CCR7, CD62L and HLA-DR on monocytes. 73 Figure 3.3. Inflammatory cytokine production is increased from purified monocytes exposed to hBD-3. 75 Figure 3.4. IL-10 production and CD86 expression are differentially affected by exposure to the TLR 1/ 2 ligands hBD-3 and Pam3CSK4 78 Figure 3.5. Inhibition of p38, JNK, and ERK, prevents induction of CD80 by hBD-3 but enhances Pam3CSK4 induced expression of this molecule. 80 Figure 3.6. Inhibition of p38, JNK, and ERK signaling decreases inflammatory cytokine production from monocytes stimulated with hBD-3. 83 Figure 4.1. Human β Defesnsin-3 links the innate and adaptive immune responses at mucosal sites. 102 Figure 4.2. Activation of moncytes by hBD-3 results in increased T cell proliferation during allogeneic challenge. 103 Figure 4.3. Rhesus Macaque monocytes respond to hBD-3 stimulation by increasing 9 surface co-stimulatory molecules and producing inflammatory cytokines. 104 Figure 4.4. Linearization of the hBD-3 peptide results in decreased ability of this molecule to induce CD80 and CD86 expression on monocytes. 106 Figure 4.5. Comparisons of the amino acid sequences and structures of hBD-2 and hBD-3. 107 Figure 4.6. Monocytes and Dendritic Cells from HIV+ donors may have lower levels of TLR1 expression than cells from HIV- donors. 109 Figure 4.7. Induction of CD80 following hBD-3 exposure appears to be diminished on monocytes from HIV+ monocytes. 111 Figure 4.8. Induction of CD80 by LPS and Pam3CSK4 correlates directly with Viral Load. 113 10 Acknowledgements I would first like to thank Michael Lederman and Scott Sieg, for their support and guidance. Their advice and keen mentorship has been invaluable over the past few years. I’d also like to thank everyone in the Lederman/Sieg lab, especially Kathy Medvik, Doug Bazdar, Janelle Salkowitz-Bokal, and Jiang Wei for helping to train me when I first joined the lab. Angel Luciano, thanks for teaming up with me on the T cell project. I would also like to thank Mary Robertson, Jeanine Agler, and Teresa Szary, for being swell dames and always making sure I arrived where I was supposed to, on time, and with the proper account numbers; ‘ppreciate-ya. I would like to thank my Thesis Committee, Dave McDonald, Tom McCormick, and Aaron Weinberg for their advice and direction. I would also like to thank Zhimin Feng for reagents, Cliff Harding, Don Anthony, and everyone else who attended lab meetings for offering constructive criticism and advice. I would also like to thank everyone in the Department of Molecular and Microbiology for all of the questions, guidance, and support over the course of my training. Thanks to the Cell and Molecular Biology Training Grant and Jo Ann Wise for financial support. A hearty thank you to all the blood donors; without you my research wouldn’t have been possible. I’d like to thank Pete Lalli and Julie Jadlowsky, you’ve both been with me from the start; thanks for everything. Thank you to all of the friends I’ve made here at Case and in Cleveland. John McDowell, for giving me my first real lab experience and helping me realize I wanted to go to PhD school instead of Med school, thank you. And thanks for the continued friendship and counsel. Thanks to my family. 11 Human β Defensin 3: Linking Innate and Adaptive Immune Responses Abstract by NICHOLAS T. FUNDERBURG Human β defensins (hBDs) are cationic antimicrobial peptides that can be produced in response to microbial challenge at mucosal sites. Acting as effector molecules of the innate immune system, hBDs can bind to and lyse microorganisms. Defensins have also been shown to bridge the innate and adaptive immune systems through the chemoattraction of immune cells. Here, we provide evidence that hBD-3 also activates antigen presenting cells. Following overnight exposure, hBD-3 can induce expression of the co-stimulatory molecules CD80, CD86, and CD40 on monocytes and myeloid dendritic cells. Activation of monocytes by hBD-3 is mediated by interaction with TLRs 1 and 2, resulting in signaling that requires MyD88 and results in IRAK-1 phosphorylation. Cell lines (HEK and CHO) that have been engineered to express Toll- like Receptors (TLRs) 1 and 2 could be activated by hBD-3, but cells that express other individual TLRs, or TLRs 1 and 6, could not. Antibodies to TLR 1 and TLR 2 were also able to inhibit the hBD-3 mediated induction of CD80 on monocytes. HBD-3 can also induce expression of inflammatory cytokines (IL-8, IL-6, and IL-1β) from purified monocytes. Exposure to hBD-3 induces the expression of the homing receptors CCR7, CD62L, and a key antigen-presenting molecule - HLA-DR on the 12 surfaces of monocytes. Comparisons of the TLR1, 2 binding elements hBD-3 and Pam3CSK4, demonstrate that cell stimulation by these molecules results in differential outcomes. Exposure to Pam3CSK4 results in heightened production of IL-10 and decreased surface expression of CD86. HBD-3 does not stimulate IL-10 production and induces heightened levels of CD86 on monocytes. Also, induction of co-stimulatory molecule expression (CD80) on monocytes by hBD-3 and Pam3CSK4 is regulated differentially by the MAP kinase pathways. This work demonstrates for the first time, that a human antimicrobial peptide has the potential to stimulate cells in a TLR dependent manner; and that stimulation of TLRs 1 and 2 by unique ligands results in qualitative differences in protein expression. By activating antigen presenting cells through TLRs 1 and 2, HBD-3 may be playing a key role in bridging the innate and adaptive immune systems at mucosal sites.