MONOCYTE ACTIVATION AND MEMBRANE DISRUPTION MEDIATED BY HUMAN β-DEFENSIN-3 by ANTHONY BRUNO LIOI Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Adviser: Dr. Scott Sieg Department of Molecular Biology and Microbiology Molecular Virology Program CASE WESTERN RESERVE UNIVERSITY January, 2014 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of _______________________Anthony Bruno Lioi___________________________ candidate for the ______________Doctor of Philosophy_____________ degree *. (signed) ____________________David McDonald_________________________ (chair of the committee) _______________________Scott Sieg_____________________________ _______________________Michael Lederman______________________ _______________________George Dubyak_________________________ _______________________Aaron Weinberg________________________ _______________________Alan Levine___________________________ (date) _____11/20/2013_________ *We also certify that written approval has been obtained for any proprietary material contained therein. TABLE OF CONTENTS Table of Contents ................................................................................................................ i List of Tables ..................................................................................................................... v List of Figures ................................................................................................................... vi List of Abbreviations ...................................................................................................... viii Acknowledgments ............................................................................................................ xi Abstract .............................................................................................................................. 1 Chapter 1: Introduction ...................................................................................................... 2 1.1 Antimicrobial peptides and innate defense ………………………………….. 2 1.2 Structure and antimicrobial activity of hBD-3 ………………………………. 3 1.3 Immune modulatory activity of hBD-3 ……………………………………… 6 1.4 Toll-like receptors play an important role in monocyte activation by hBD-3 . 7 1.5 The P2X7 Receptor ………………………………………………………...... 9 1.6 Monocyte maturation and model of hBD-3 activity in tissues …………….. 10 1.7 Potential to develop hBD-3 as a vaccine adjuvant …………………………. 13 Chapter 2: Membrane damage and repair in primary monocytes exposed to human beta defensin-3 ......................................................................................................................... 17 2.1 Abstract .......................................................................................................... 18 2.2 Introduction .................................................................................................... 19 2.3 Materials and Methods ................................................................................... 21 2.3.1 Reagents .......................................................................................... 21 2.3.2 Isolation of PBMC .......................................................................... 21 i 2.3.3 HBD-3 treatment ............................................................................. 22 2.3.4 PI staining and flow cytometry ....................................................... 22 2.3.5 Immunofluorescence ....................................................................... 23 2.3.6 Amnis ImagestreamX studies .......................................................... 23 2.3.7 PS expression .................................................................................. 23 2.3.8 Annexin V and lactadherin protection assays ................................. 24 2.3.9 Cholesterol removal and cholesterol staining ................................. 24 2.3.10 Human studies ............................................................................... 24 2.3.11 Statistical Methods ........................................................................ 25 2.4 Results ............................................................................................................ 26 2.4.1 Evidence for membrane damage in monocytes but not T or B cells exposed to hBD-3 .................................................................................... 26 2.4.2 Membrane repair is induced in monocytes exposed to hBD-3 ....... 29 2.4.3 Cholesterol protects cells from membrane damage caused by hBD-3 ....................................................................................................... 29 2.4.4 Freshly isolated monocytes express PS on their outer leaflet ......... 30 2.4.5 Pretreatment with lactadherin or Annexin V protects monocytes from the effects of hBD-3 ........................................................................ 31 2.5 Discussion ...................................................................................................... 33 Chapter 3: HBD-3 increases CD86 expression on monocytes by activating the ATP-gated channel P2X7 ………………………………………………………………………...… 52 3.1 Abstract .......................................................................................................... 53 3.2 Introduction .................................................................................................... 54 ii 3.3 Materials and Methods ................................................................................... 56 3.3.1 Reagents .......................................................................................... 56 3.3.2 Isolation of PBMC .......................................................................... 56 3.3.3 HBD-3 treatment …………………………………………………. 56 3.3.4 PI staining and flow cytometry …………………………………... 57 3.3.5 Analysis of Extracellular ATP ………………………………….... 57 3.3.6 qPCR analysis of CD86 mRNA expression …………….………... 57 3.3.7 Statistical methods ……………………………………………...... 58 3.3.8 Human studies ……………………………………………………. 58 3.4 Results ............................................................................................................ 59 3.4.1 Induction of CD86 expression on monocytes by hBD-3 is dependent on P2X7 ………………………………………………………………... 59 3.4.2 Activation of P2X7 by ATP alone is sufficient to cause induction of CD86 expression on monocytes ………………………………………... 59 3.4.3 Induction of CD86 expression by hBD-3 is at least partially explained by release of ATP from hBD-3-treated cells ………………... 61 3.5 Discussion ...................................................................................................... 63 Chapter 4 General Discussion and Future Directions ………………………………...... 77 4.1 Structure/Function activity of hBD-3 variant molecules ...……………....… 77 4.2 Possible interaction between CD14 and hBD-3 ………………………......... 80 4.3 Downstream T cell activation by hBD-3-primed APCs ................................ 83 4.4 Activation of P2X7 by hBD-3 ....………………………………………....... 85 4.5 HBD-3 as a link between innate and adaptive immunity ………………….. 88 iii Copyright Release ...………………………………………………………………...… 106 Works Cited ................................................................................................................... 107 iv LIST OF TABLES Table 4.1: Wild-type and Mutant Sequence ..…………………………………….….… 93 Table 4.2: Predicted structure of mutant hBD-3 molecules ...………………………..… 96 Table 4.3: Cellular receptors hBD-3 can interact with ……..………………………… 103 v LIST OF FIGURES Figure 2.1: HBD-3 causes detectable PI staining in monocytes, but not B cells or T cells ........................................................................................................................................... 37 Figure 2.2: Increasing concentrations of hBD-3 increases the number of PI bright monocytes ........................................................................................................................ 38 Figure 2.3: PI uptake is not caused by canonical monocyte activators ........................... 39 Figure 2.4: Neither hBD-2 nor LL-37 increased PI staining in monocytes ..................... 40 Figure 2.5: Phagocytosis of apoptotic blebs does not appear to be the cause of the observed PI staining pattern ............................................................................................. 41 Figure 2.6: HBD-3 induces bleb-like PI staining in monocytes ...................................... 42 Figure 2.7: Imagestream analysis of hBD-3-induced PI staining in monocytes demonstrates differences in nuclear staining and bleb-like staining patterns .................. 43 Figure 2.8: PI uptake in THP-1 cell line .......................................................................... 44 Figure 2.9: Evidence of membrane repair in monocytes treated with hBD-3 ................. 45 Figure 2.10: Inhibition of membrane repair enhances hBD-3 mediated PI uptake in monocytes ........................................................................................................................ 46 Figure 2.11: Removal of cholesterol increases cellular susceptibility to hBD-3 induced membrane damage ........................................................................................................... 47 Figure 2.12: Cholesterol levels are similar between cell types ........................................ 48 Figure 2.13: Phosphatidylserine expression is detected on freshly isolated monocytes