Monocyte Activation and Membrane Disruption Mediated By

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MONOCYTE ACTIVATION AND MEMBRANE DISRUPTION MEDIATED BY

HUMAN β-DEFENSIN-3

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

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

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

Works Cited ...... 107

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

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 .. 49

Figure 2.14: Lactadherin protects monocytes from hBD-3-induced membrane damage. 50

Figure 2.15: Lactadherin binding and PI uptake in staurosporine treated B cells and T cells ...... 51

Figure 3.1: Blocking P2X7 inhibits CD86 induction on monocytes ...... 66

Figure 3.2: Increased expression of CD86, but not CD80, on monocytes by hBD-3 is mediated by P2X7 ...... 68

Figure 3.3: ATP up-regulates CD86 on monocytes by activating P2X7 ...... 69

Figure 3.4: Lowering ATP concentration abrogates CD86 up-regulation ...... 70

Figure 3.5: Overnight up-regulation of CD86 occurs with ATP stimulation of between 15 and 30 minutes ...... 71

Figure 3.6: Increased expression of CD86 occurs more than 6 hours after ATP stimulation ...... 72

Figure 3.7: HBD3 and ATP stimulation increased CD86 mRNA expression ...... 73

Figure 3.8: Inhibition of hBD-3 mediated CD86 induction by apyrase ...... 74

Figure 3.9: Apyrase treatment abrogates LL37-induced CD86 expression while having little effect on Pam3CSK4-induced CD86 expression ...... 75

Figure 3.10: Evidence for ATP release into the supernatant 1 hour post hBD-3 stimulation ...... 76

Figure 4.1: Mutant hBD-3 molecules cause less PI uptake than the native molecule ..... 94

Figure 4.2: Differential effects of mutant hBD-3 molecules on CD80 and CD86 induction …………………………………………………………………………………………... 95

Figure 4.3: Blockade of P2X7 inhibits mutant induced up-regulation of CD86 ...... 97

Figure 4.4: HBD-3, but not Pam3CSK4 or LPS, causes capping of CD14 ...... 98

Figure 4.5: Due to activation by the isotype control, it is not possible to determine if CD14 neutralization affects hBD-3 activation of monocytes ...... 99

Figure 4.6: Different doses of sCD14 have different effects on hBD-3 induction of CD86 …………...... 100

Figure 4.7: Enhanced allogeneic stimulation of T cells by hBD-3 treated monocytes .. 101

Figure 4.8: Overnight incubation of monocytes with ionomycin induces CD86 up- regulation ...... 102

Figure 4.9: Proposed model of hBD-3 interactions in inflammatory settings ...... 104

vii

LIST OF ABBREVIATIONS

AF700 - AlexaFluor 700

AMP - Antimicrobial peptide

APC – Allophycocyanin

APCs – Antigen presenting cells

APC Cy7 - Allophycocyanin-Cy7

ATP - Adenosine triphosphate

AZ - AZ 10606120 dihydrochloride

BSA - Bovine serum albumin

CFSE - Anionic carboxyfluorescein, succinimidyl ester

CM - Complete medium

CRE – cAMP responsive element

CREB - cAMP response element binding protein

DAMP – Damage-associated molecular pattern molecules

DC - Dendritic cell

EDTA - Ethylenediaminetetraacetic acid

ELISA - Enzyme-linked immunosorbent assay

FBS - Fetal bovine serum

FITC - Fluorescein isothiocyanate

FMO - Fluorescence minus one

FSC-A - Forward scatter area

FSC-H - Forward scatter height hBD-2 - Human beta-defensin-2

viii hBD-3 - Human beta-defensin-3

HIV – Human immunodeficiency virus

IGF-I - Insulin-like growth factor I

LAMP1 - Lysosomal associated membrane protein 1

LPS – Lipopolysaccharide mBD-2 – Mouse β-defensin-2 mBD-14 – Mouse β-defensin-14 mDC - Myeloid dendritic cells

MFI - Mean fluorescence intensity

MPL - monophosporyl lipid A

Pam3CSK4 - N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]- seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysine x3 HCl

PAMP – Pathogen associated molecular pattern molecules

PBMC - Peripheral blood mononuclear cell

PBS - Phosphate buffered saline pDC – Plasmacytoid dendritic cells

PI - Propidium iodide

PMA - Phorbol 12-myristate 13-acetate

PS - Phosphatidylserine

RP-HPLC - Reversed phase high performance liquid chromatography

RPMI - Roswell Park Memorial Institute medium

SDF-1 – Stromal cell-derived factor 1

SPR - Surface plasmon resonance

SSC-A - Side scatter area

TAM – Tumor-associated macrophage

TLR – Toll-like receptor

VEGF – Vascular endothelial growth factor

ACKNOWLEDGEMENTS

I would first like to thank my family for all the support that they have given me over the past few years. Mom, Dad, Matthew, Andrew, Sam and everyone else, thank you so very much. The love and support you offered helped me to work hard, plus you provided me a place I could return to when I needed to recharge.

I would also like to thank my advisor, Dr. Scott Sieg, for all of the help and guidance you gave me. I could always come to you when I had a question or a problem.

Thank you for making me the scientist I am today.

I want to thank my committee members as well. You always kept me pointed towards the finish line and gave me suggestions that always made me a better scientist.

Thank you.

Also I wish to thank all of my friends for everything they've done for me.

Unfortunately, there are too many of you to name but know I appreciate you all. Whether it was a last minute trip to Winking Lizard for dinner and a drink when I needed people to talk to, or giving me advice when I needed help with experiments, all of you have done so much for me. Thanks.

Finally, I want to thank my coworkers. Doug, you were my partner-in-crime as well as a good friend. Thank you. And Nick, without your work none of my work would have been possible. Thank you. Everyone else, you made the environment I worked in amazing. Thank you.

Monocyte Activation and Membrane Disruption Mediated by Human β-Defensin-3

Abstract

by ANTHONY BRUNO LIOI

Human β-defensin-3 (hBD-3) is a cationic antimicrobial peptide produced by epithelial cells. The expression of hBD-3 is markedly increased in inflammatory and disease settings including psoriasis and oral carcinoma. HBD-3 can not only kill bacteria, it can also recruit monocytes into tissues. Previous studies from our group have demonstrated that hBD-3 can activate monocytes to express the co-stimulatory molecules CD80 and

CD86 in addition to various cytokines. Monocyte activation by hBD-3 has been linked to a TLR1/2-dependent signaling mechanism. Here we provide evidence that the same concentrations of hBD-3 that cause monocyte activation can also cause membrane damage in these cells via a mechanism that is dependent on cell surface expression of negatively charged phospholipids. In addition, structure/function studies of mutant hBD-

3 and native hBD-3 molecules led us to discover a novel pathway of monocyte activation that may involve membrane damage and repair processes in these cells. This pathway involves the potential leakage of ATP from damaged cells that results in the activation of monocytes via the ATP-gated P2X7 ion channel. These observations define molecular mechanisms through which hBD-3 causes activation and maturation of monocytes.

Further studies of these mechanisms in vivo may help to establish the potential role for hBD-3 in shaping inflammatory responses in tissues and in serving as a bridge between the innate and adaptive defenses.

CHAPTER 1:

INTRODUCTION

1.1 Antimicrobial peptides and innate defense

The immune system consists of innate and adaptive immunity. The innate immune system includes physical barriers to infection as well as rapid response mechanisms, while the adaptive branch acts more slowly but is capable of specifically targeting the invading pathogen. Adaptive immunity also entails "memory" responses that permit quicker and more efficient immune responses upon secondary challenge from the same pathogen.

Antimicrobial peptides are proteins that directly combat invading pathogens and therefore play an important role in innate defenses. Small, cationic proteins with antimicrobial function, called thionins, were first identified in plants in 1972 [1]. These thionins were capable of inhibiting the growth of many plant pathogens as well as being toxic to animal cells. Soon after this, antimicrobial peptides from insects and frogs were isolated and characterized [2-4]. Antibacterial peptides isolated from rabbit lung macrophages helped explain how engulfed bacteria were killed by phagocytic cells [5], though it was not until later that the term “alpha-defensin” was coined to describe them

[4]. Beta-defensins were first discovered in cows [6], and it was not until 1995 that the first human beta-defensin was isolated [7].

A variety of antimicrobial peptides exist in humans. For example, histatins, defensins, cathelicidins, lactoferrin, dermicidin, and lysozymes are all proteins which have been shown to have antimicrobial properties. For instance, lactoferrin is known to be antibacterial, antiviral, antifungal, and antiparasitic. Its main role is to sequester free

2 iron, thereby depriving pathogens of an essential nutrient; but it can also directly bind bacteria, and through a not fully understood mechanism, kill the bacteria [8]. The antifungal histatins, on the other hand, cross into the cytoplasm of the fungal cell where they interact with mitochondria and cause the release of ATP eventually killing the cell

[9].

In the studies that follow, we investigate a particular antimicrobial peptide, human beta defensin-3 (hBD-3) for its potential to serve a new role in immunity. In particular, we investigate the ability of hBD-3 to modulate the activation of antigen-presenting cells

(APCs) as a potential mechanism to bridge innate and adaptive defenses. This, in turn, could lead to various usages (vaccine adjuvant) in human therapeutics.

1.2 Structure and antimicrobial activity of hBD-3

β-defensins are small, cationic molecules that have a β-pleated sheet core structure [10]. This structure is stabilized by three disulfide bonds arranged in a C1-C5,

C2-C4, C3-C6 disulfide motif [11]. β-defensins have a wide range of activity against both gram-positive and gram-negative bacteria, viruses, and even against some fungi

[12]. In addition, they have immune-modulatory effects such as chemotaxis of various cell types including keratinocytes, mast cells, and monocytes and induction of activation markers on monocytes [13-15].

Of the first three human β-defensins discovered, hBD-3 is the most salt tolerant and also has the widest range of antimicrobial activity [16]. It also has the greatest overall positive charge [17]. Like hBD-2, hBD-3 expression by epithelial cells is inducible by a variety of stimuli. In addition to stimuli like IL-1β, TNF-α, IFN-γ and bacterial species such as P. aeruginosa and S. aureus, hBD-3 can also be induced by

3 insulin-like growth factor I (IGF-I), and TGF-α [16, 18-21]. Both IGF-I and TGF-α bind to receptor tyrosine kinases and can signal through an AKT signaling pathway, a pathway shown to be important in hBD-3 transcription [22]. TGF-α is very potent growth factor produced by keratinocytes and macrophages as part of the wound healing response [23,

24]. In addition, macrophages activated with LPS are known to release TGF-α as well

[25]. Therefore induction of hBD-3 can occur during the host response to damage/infection when an inflammatory response is warranted. Evidence of hBD-3 has been found in a variety of locations including epithelial cells of the skin, airway, and gastrointestinal tract, the heart, liver, fetal thymus and placenta [16, 18, 21].

HBD-3, like most antimicrobial peptides, is able to preferentially target pathogens because of a very important difference in the make-up of membranes between microbe and host. The outer leaflet of microbial membranes contains high numbers of negatively charged phospholipids while the outer leaflets of eukaryotic cells contain mostly neutral phospholipids and cholesterol. HBD-3 utilizes this charge difference to target the negatively charged membranes of bacteria and other pathogens, while leaving host cells relatively unscathed [26]. When hBD-3 interacts with the surface of its target, it is able to disrupt the membrane and kill the pathogen. The method of this action, though, is under contention. One model, the Pore-Forming model, suggests that oligomers of hBD-

3 interact with the bacterial membrane via charge interactions, then hBD-3's hydrophobic residues allow the defensin to be inserted into the membrane, forming a pore [27].

Evidence that hBD-3 forms dimers suggests that this model is plausible [17]. A second model, the Carpet model, suggests that charge interactions bring the hBD-3 molecule into contact with the microbial membrane and then, acting like a detergent, the molecule

4 disrupts the membrane [27]. A third model proposes that when the antimicrobial peptide interacts with the outer leaflet, lipid displacement occurs causing the formation of holes in the membrane [26].

Several studies have been performed in an effort to map the antimicrobial activity of hBD-3 to specific structures or residues. Disulfide bridging is not necessary for antimicrobial activity as linear molecules are just as effective against E. coli, P. aeruginosa, S. aureus, B. cereus, C. albicans, and E. faecium as the natural molecule [12,

28, 29]. A study that created linear analogues, in which the cysteines of hBD-3 were replaced with other amino acids, showed that increasing the hydrophobicity of the molecule can increase the antimicrobial activity, but at the same time, it can make the molecule more cytotoxic to eukaryotic cells [29]. The overall charge of hBD-3 is also very important. Shortened peptides containing fewer positive charges, or peptides where positively charged amino acids are substituted with neutral ones, have reduced antimicrobial activity compared to native hBD-3 [12, 30]. There also appears to be a structural requirement for some antimicrobial activity because only the small peptide that corresponded to the N-terminus of hBD-3, which contains an α-helix structure, maintained activity against C. albicans and S. aureus while truncations that removed this section exhibited diminished activity [12].

Interestingly, hBD-3 also has antiviral properties. Expression of hBD-3 can be induced by HIV and human rhinovirus [31, 32]. Fusion of influenza haemagglutinin to host cellular receptors can be blocked by hBD-3 [33]. In addition, hBD-3 can inhibit

HIV infection both directly and indirectly [31, 34]. It must be noted that in some of the assays, hBD-3 treatment decreased cellular proliferation which may have played a role in

5 its apparent antiviral activity [34]. Because the inhibition was more effective against

CXCR4-tropic viruses, it was hypothesized that this inhibition was due to hBD-3/CXCR4 interactions [31]. It has been shown that hBD-3 can act as a competitive antagonist to stromal cell-derived factor 1 (SDF-1) by interacting with CXCR4 to block SDF-1 binding and triggering its internalization without inducing a calcium flux or initiating a signaling cascade [35]. The CXCR4 receptor is present on T cells, monocytes, and epithelial cells and is well known as an important co-receptor in HIV infection of T cells [36]. During normal immune function, the peptide SDF-1 binds to CXCR4 and initiates a signaling cascade which plays a role in vascular and neuronal development, haematopoiesis, and T cell migration and activation [37]. The six cysteine residues, the N- and C-terminal acidic residues, the first 10 N-terminal residues, and three surface exposed basic residues

(Lys8, Lys32, and Arg36) of hBD-3 are all integral for this CXCR4 antagonism [38].

1.3 Immune modulatory activity of hBD-3

Not only are defensins antimicrobial, they also regulate chemotaxis (the movement of a cell along a chemical concentration gradient). HBD-2 and hBD-3 are chemotactic to immature DCs and memory T cells and achieve this through binding with

CCR6 [27, 28, 39, 40]. Both hBD-2 and hBD-3 are capable of signaling through the chemokine receptor CCR2 on monocytes and macrophages to induce the chemotaxis of these cell types [41]. HBD-3 is also chemotactic to human epidermal keratinocytes, mast cells, immature DCs, and memory T cells [13, 14, 21, 27]. HBD-3's chemoattraction requires the presence of the disulfide bridges, as monocytes treated with an hBD-3 which has had its cysteines replaced with α-aminobutyric acid (it is considered to have similar properties to cysteine but does not disulfide bond) did not migrate [28].

Defensins can stimulate cells to release cytokines as well. HBD-3 and hBD-4 are capable of inducing the degranulation of mast cells [14]. HBD-2,-3, and -4 can stimulate epidermal keratinocytes to produce IL-6, IL-10, IP-10, MCP-1, MIP-3α, RANTES, and

IL-18 [13, 42]. Activation of monocytes by hBD-3 also leads to a release of cytokines including IL-1β, IL-6, and IL-8 [43]. Thus, hBD-3 is an important component of both the innate immune system and the adaptive immune system, acting as a bridge to ensure proper functioning of both.

HBD-3 is also capable of increasing the expression of co-stimulatory molecules such as CD80, CD86, and CD40 on the surface of monocytes and myeloid dendric cells

(mDC) [15]. CD80 and CD86 induction by hBD-3 may play an important role in subsequent T cell/APC interactions [44]. These molecules both bind to CD28 and

CTLA-4 on T cells with different effects [45-47]. Depending on the expression kinetics of these two molecules, different T cell responses may ensue. For instance, lack of CD80 binding to T cells results in much lower IL-2 production [48]. In vitro blockade of CD86 enhances the suppressive function of CD25+ Tregs while blockade of CD80 decreases the suppressive function of these Treg cells [49]. This agrees with an in vivo mouse model in which blockade of CD86 increases peripheral regulatory T cell expansion as well as shifting the T cell response to a Th2 bias [50]. It is also thought that CD86 is more important for the activation of resting memory T cells with recall antigens than

CD80 [51].

1.4 Toll-like receptors play an important role in monocyte activation by hBD-3

Toll-like receptors are a class of pattern recognition receptors which recognize pathogen associated molecular patterns (PAMPs) - molecules shared by microbes which

7 differentiate the pathogen from host cells. Toll receptors were identified in Drosophila in the 1980s, but it wasn’t until the 1990s that Toll-like receptors were identified in mammals [52]. Many TLRs utilize MyD88 adaptor molecules and IRAK signaling cascades to cause cellular responses, but others, like MAL/TIRAP, TRIF, or TRAM can also be recruited for signaling [52].

The first evidence that TLRs might be interacting with antimicrobial peptides came from studies in mice in which TLR4 on DCs was shown to be directly acted upon by mouse β-defensin-2 (mBD-2) [53]. This activation leads to up-regulation of co- stimulatory molecules and maturation of the DC. In humans, the activation of monocytes by hBD-3 is at least partially dependent on TLR1 and TLR2 expression [15]. Antibodies against TLR1/2 can block activation of monocytes caused by hBD-3. Moreover, only cell lines that express TLR1/2 as opposed to those cell lines that express TLR2/6 or the

TLRs alone, responded to hBD-3 stimulation. This signaling by hBD-3 through TLR1/2 requires MyD88 and includes the rapid phosphorylation of IRAK1 [15]. It can induce the activation of p38 and ERK1/2 (parts of the MAP kinase pathway) but not p52 (part of the alternative NFκB pathway) [43].

The capacity of hBD-3 to cause APC activation via TLR1 and 2 has important implications. TLR2 is a pattern recognition receptor which, in complex with either TLR1 or TLR6, binds to microbial products such as LTA (lipoteicholic acid) or peptidoglycan to initiate signaling cascades [20]. TLR2 signaling has been implicated in the immune response to bacteria such as Mycobacterium tuberculosis and Group B Streptococci [54,

55]. Depending on specific TLR1, 2, or 6 polymorphisms present, an individual may have increased or decreased susceptibility to tuberculosis [56]. But TLR2 signaling is not

8 always a good thing. It is believed that TLR2 activation and signaling during brain ischemic/reperfusion injury has a pro-inflammatory effect which increases damage to the brain [57]. In addition, TLR2 signaling has been linked with systemic inflammatory response syndrome, which is a potentially lethal complication arising during severe acute pancreatitis [58].

Although hBD-3 utilizes TLR1/2 signaling to mediate monocyte activation, the biological effects of hBD-3 and a well-characterized TLR1/2 agonist, Pam3CSK4, are clearly distinct. For example, whereas both molecules are capable of inducing the release of IL-1β, IL-6, and IL-8, only Pam3CSK4 is capable of inducing IL-10 [43].

The mechanism behind the differential effects of hBD-3 and Pam3CSK4 are not clear but may be related to either unique signaling effects on TLR1/2 receptors or potential interactions with other receptors. The latter seems more likely in the case of hBD-3 since this molecule is known to interact with a variety of receptors including

CXCR4, CCR2, MC1R, and CCR6 [28, 35, 41, 59, 60]. In Chapter 3, the potential for hBD-3 to mediate activation of monocytes via an alternative receptor, P2X7, is investigated.

1.5 The P2X7 Receptor

Extracellular ATP and other nucleotides are very potent signals from apoptotic cells and necrotic cells to phagocytic cells [61-63]. Monocytes and macrophages will respond to these signals by engulfing the cellular debris, and based on the concentration of the nucleotides, either release pro-inflammatory or anti-inflammatory cytokines [62,

63]. One of the ATP sensing receptors present on monocytes and macrophages is P2X7

[64]. P2X7 is a member of the P2X receptor subfamily of membrane proteins. Members

9 of this receptor family are ligand-gated ion channels whose specific ligand is ATP [65].

Seven members of this family have thus far been pharmacologically classified. P2X family members are thought to be made up of subunits which harbor two hydrophobic transmembrane domains (both N- and C-termini are present in the cytoplasm) and an extracellular region which contains ten cysteines and between two and six N-linked glycosylation sites [66]. Their size range is from 379 amino acids to 595 amino acids in length. P2X7 differs in that its C-terminal tail extends farther into the cytoplasm than the other six subunits [67]. All members signal through sodium and calcium influx and potassium efflux which leads to membrane depolarization and increased cytosolic calcium concentration [65]. P2X7 is found to different degrees on a variety of cell types including monocytes/macrophages, dendritic cells, T cells and B cells [64, 65, 68-72]. In the case of P2X7, prolonged activation by ATP causes the channel to further open, allowing the traversal of molecules up to 900 Da [73]. Activation of this receptor has been linked to a variety of cellular responses, such as IL-1β release, cell death via apoptosis or necrosis (dependent on the amount of time P2X7 is activated by ATP), phosphatidylserine “flipping”, phagosome maturation, exocytosis of secretory lysosomes, plasma membrane blebbing, and surface protein shedding [74-84]. In addition, IL-18 processing and release has been linked to activation of P2X7 in monocytes [68].

1.6 Monocyte maturation and model of hBD-3 activity in tissues

In the absence of infection, monocytes tend to circulate throughout the body.

When damage or infection occurs, cells in the immediate vicinity will release chemokines and cytokines to signal passing immune cells. When immune cells such as monocytes sense these chemoattractant signals, they will home to the site of inflammation. Once the

10 monocytes arrive, through the process of leukocyte extravasation, they will infiltrate the site from circulation while maturing into macrophages.

Macrophages are long-lived phagocytic cells which also function as antigen- presenting cells, and it is thought that they can be one of two main phenotypes. M1 macrophages are thought to be pro-inflammatory while M2 macrophages are considered anti-inflammatory. In vitro differentiation of monocytes into M1 macrophages is induced by IFN-γ or bacterial compounds and these macrophages contribute to continued inflammation by releasing cytokines such as tumor necrosis factor (TNF) or IL-1β [85].

Too much inflammation, though, can eventually cause damage and inhibit wound repair.

Induction of M2 macrophages, on the other hand, is accomplished by incubating monocytes with IL-4/IL13 and IL-10. These macrophages can stimulate angiogenesis and dampen immune responses [85]. HBD-3 can act as one of these chemoattractant signals for CCR2+ monocytes [59]. CCR2 is considered a definite M1 marker for monocytes [86] and these cells tend to maintain their inflammatory phenotype when they mature into macrophages [87], though evidence suggests that these cells can still become

M2 macrophages if the tissue environment contains IL-4 [88]. So while hBD-3 is chemotactic to the M1 inflammatory monocytes, depending on the rest of the tissue environment, either macrophage phenotype could be present to contribute to the overall immune response.

Because hBD-3 is an inducible peptide, concentrations vary depending on location and disease status. Saliva has been reported in the literature to contain around

112 pg/ml of hBD-3 [89] or 326 µg/L (326 ng/ml) [90]. This disparity can be explained by the masking effect of other proteins present in saliva which can be hinder hBD-3

11 detection [90]. Forte et al. did not perform their ELISA assays in the presence of 250 mmol/L CaCl2 [90] which likely explains their lower observed hBD-3 concentration.

Diseased tonsils contain between 117 and 232 pg/ml of hBD-3 [91] though this is likely a low estimate due to the ELISA assays not being performed in the presence of CaCl2.

Psoriatic scales contain almost one hundred times as much hBD-3 as skin from healthy controls [16]. Serum concentrations of hBD-3 have been reported at 159 pg/ml, though this increases to 239 pg/ml during the acute phase of bacterial pneumonia [92]. The concentrations of hBD-3, therefore, can vary greatly according to tissue and inflammation state.

Based on the inducible expression of hBD-3 as well as hBD-3's chemotactic properties and potential to activate APCs, we propose a model whereby hBD-3 can serve as a link between innate and adaptive immune responses. Monocytes encountering low concentrations of hBD-3 in the periphery could be recruited into tissues [21, 41], and once there, higher concentrations of hBD-3 could induce activation and potentially influence their maturation into macrophages [15].

Although hBD-3 functionality has been largely defined in vitro, several in vivo conditions suggest that hBD-3 may have similar activity in vivo. For example, cells from oral carcinomas have been shown to produce high levels of hBD-3 by immunohistochemistry [59]. High levels of hBD-3 production in this condition are associated with recruitment of CCR2+ macrophages into the tumor site. These cells may then contribute to an environment that benefits tumor cells by aiding in cancer cell growth [59]. Another example of a condition with high levels of hBD-3 expression in affected tissues is psoriasis [16]. Psoriasis, as an inflammatory disease marked by

12 dysregulation of the immune system, is known to have an increase in T cell and dendritic cell infiltration [93]. Beta-defensins are known to be chemotactic to memory T cells and immature dendritic cells though CCR6 [39], and hBD-3 is known to be chemotactic through CCR6 as well [28]. Activation of both plasmacytoid dendritic cells (pDCs) and myeloid dendritic cells (mDCs) has been linked to psoriasis [93]. HBD-3 may play a role in the activation of both cell types in this disease because it is known that mDCs can be activated by hBD-3 [15], and it has recently been shown that hBD-3, in complex with self-DNA, can activate pDCs via a TLR9 dependent mechanism [94], a mechanism known to activate pDCs in psoriasis [93]. So it appears that hBD-3 plays an important role in vivo.

1.7 Potential to develop hBD-3 as a vaccine adjuvant

With the ability of hBD-3 to activate antigen presenting cells as well as being chemotactic to those same APCs and T cells, hBD-3 has the potential to be a very potent adjuvant in vaccines [95]. An adjuvant is a molecule or compound which when added with a vaccine will boost the immune response to that vaccine. This means the dose of vaccine administered or amount of antigen in the vaccine can be lowered, potentially lowering the cost of the vaccine and increasing the manufacturing capacity for the vaccine. Currently the United States Food and Drug Administration has approved for human use the following vaccine adjuvants: aluminum salts (alum, aluminum hydroxide, aluminum phosphate, or mixed aluminum salts) and AS04 (combination of aluminum hydroxide and monophosphoryl lipid A) [96]. Alum was thought to function by creating an “antigen depot,” trapping the antigen at the site of injection and allowing for its slow release to continually stimulate the immune system, but more recent studies suggest that

13 alum allows for antigens to be more efficiently internalized by APCs and that it can activate the inflammasome [97]. As a shortcoming, alum alone tends to induce a Th2 immune response without much in the way of cytotoxic T cell stimulation (which is important in diseases caused by intracellular pathogens), but when alum is paired with monophosporyl lipid A (MPL), a more balanced response is achieved [52]. The inclusion of MPL, a Toll-like receptor ligand, in vaccine formulations increased the immunogenicity of, and the immune response to, the vaccine antigens [52]. As vaccine development attempts to tackle more complicated diseases like HIV and malaria, more effective adjuvants must be developed to keep pace. Emulsions, liposomes, saponins and

Toll-like-receptor agonists are all being studied as potential adjuvants [97].

Defensin molecules may also be considered for development as vaccine adjuvants based on their ability to modulate immune responses and their potential usefulness in mucosal vaccines. Studies in mice have found that the addition of defensins (murine β- defensin-2, Human Neutrophil Protein-1,-2,-3, hBD-2, or hBD-3) as adjuvants can increase both the cellular and humoral immune response to influenza, HIV-1 (nasal delivery), tuberculosis, melanoma, and lymphoma antigens [98-102]. HBD-3 is of particular interest because it is capable of recruiting and activating APCs [15, 59] and it is a host-derived molecule that naturally exists in mucosal tissues.

Although studies of PHA blasts suggest that hBD-3 may be harmful to cells [34], high concentrations of hBD-3 do not damage human red blood cells [16]. In Chapter 2, we hypothesized that hBD-3 damage to human cells is cell-type dependent and we found that while monocytes are susceptible to hBD-3-induced membrane disruption, T cells and

B cells are not. This differing susceptibility is likely the result of differences in the

14 amount of negatively charged phospholipids present on the surface of monocytes compared to T cells or B cells. These studies provided the first evidence that certain primary human cells can be directly damaged by hBD-3.

Damaged cells release molecules such as Heat-shock proteins, uric acid, or ATP which are known as damage-associated molecular patterns (DAMPs). These molecules can contribute to immune responses by interacting with receptors on leukocytes and activating these cells [63]. In Chapter 3 we test the hypothesis that ATP release caused by hBD-3 may contribute to activation of the ATP-gated ion channel, P2X7. Our results suggest that hBD-3-mediated up-regulation of the co-stimulatory molecule, CD86, is at least partially dependent on P2X7 receptor signaling. This receptor activity is at least partially dependent on extracellular ATP since apyrase, an enzyme that breaks down

ATP, was able to partially block the up-regulation of CD86 induced by hBD-3.

Moreover, we found that ATP alone is capable of causing the up-regulation of CD86.

These data suggest that ATP release and signaling through P2X7 is important for hBD-3- mediated induction of CD86.

The potential for hBD-3 to influence APC maturation suggests that this molecule may also have effects on the downstream activation and maturation of T cells. In Chapter

4, preliminary experiments describe the potential for hBD-3 to enhance the ability of monocytes to activate T cells responding to alloantigen. These experiments are an important step in determining the potential use of hBD-3 as a vaccine adjuvant.

To determine the structural requirements that are important for the diverse biological activities of hBD-3 on monocyte activation and membrane disruption, we have begun to study hBD-3 variant molecules (recombinant hBD-3 molecules with N-terminal

15 deletions or with substituted cysteine residues). Preliminary studies of these molecules suggest that the N-terminus of hBD-3 may be important in the potential for this molecule to cause membrane damage and to induce co-stimulatory molecule expression. In addition, the three-dimensional structure of hBD-3 may not be critical for the ability of this molecule to induce CD86 expression via P2X7-dependent mechanisms since hBD-3 molecules with cysteine mutations maintain the capacity to induce CD86 but not CD80 expression in monocytes and since the induction of CD86 by hBD-3 variant molecules is blocked by a specific P2X7 inhibitor. These reagents demonstrate that hBD-3 molecules can be designed that vary in their ability to activate monocytes, to induce specific co- stimulatory molecules (CD80 vs CD86), and to cause membrane disruption.

CHAPTER 2:

Membrane damage and repair in primary monocytes exposed to human beta

defensin-3 [103]*

Anthony B. Lioi1, Angel L. Reyes-Rodriguez1, Nicholas T. Funderburg2, Zhimin Feng3,

Aaron Weinberg3, Scott F. Sieg2

1Department of Molecular Biology and Microbiology

2Department of Medicine, Division of Infectious Diseases, Center for AIDS Research

3Department of Biological Sciences, Case School of Dental Medicine

Case Western Reserve University, Cleveland OH 44106

*Copyright release found on page 106

2.1 ABSTRACT

Interactions of antimicrobial peptides with plasma membranes of primary human immune cells are poorly characterized. Analysis of propidium iodide exclusion as a measure of membrane integrity indicated that human beta defensin-3 (hBD-3) caused membrane perturbations in monocytes but not T or B cells at concentrations typically used to kill bacteria or to induce activation of antigen-presenting cells. Bleb-like structures were observed in monocytes exposed to hBD-3. These cells also increased surface expression of LAMP1, a membrane repair marker after exposure to hBD-3. Furthermore, cell death was enhanced by adding an inhibitor of membrane repair. Removal of cholesterol from membranes resulted in greater susceptibility of cells to hBD-3 but cholesterol content was not different between the cell types as assessed by filipin staining. Freshly isolated monocytes expressed higher levels of the negatively charged phospholipid, phosphatidylserine, on their outer leaflet compared to B or T cells. Pre-incubation of monocytes with molecules that bind phosphatidylserine protected these cells from hBD-

3-induced membrane damage, suggesting that outer membrane phosphatidylserine expression can at least partially explain monocyte susceptibility to hBD-3. The potential for membrane disruption caused by antimicrobial peptides should be evaluated in various cell types when considering these molecules for therapeutic applications in humans.

2.2 INTRODUCTION

Human β-defensin-3 (hBD-3) is a cationic molecule produced by epithelial cells that has antimicrobial as well as immunomodulatory activity [13, 14, 21, 39]. At nanomolar concentrations, hBD-3 provides chemotactic signals to a variety of cells including antigen-presenting cells [21, 28, 59]. At low micromolar concentrations, hBD-3 has microbicidal activity against bacteria, fungi and encapsulated viruses but also is able to induce maturation of antigen-presenting cells, including monocytes and myeloid dendritic cells [12, 27]. The mechanisms by which hBD-3 mediates membrane damage in microbes while avoiding toxicity against host cells at these high concentrations are not fully understood but may involve differences in cell membrane electrostatic attraction as the outer leaflet of bacterial cell membranes are enriched in negatively charged phospholipids or may be related to differences in cholesterol content between eukaryotic and prokaryotic organisms [26, 104].

Previous studies have demonstrated that human cells can tolerate high concentrations of antimicrobial peptides (AMPs). For example, red blood cells are resistant to lysis by hBD-3 at concentrations up to 500 µg/ml [16]. Nonetheless, high concentrations of hBD-3 may impede cell proliferation in PHA activated peripheral blood mononuclear cells (PBMC) [34], and there is relatively little data concerning the susceptibility of primary cells and cell subsets to the potential toxicity of hBD-3.

In these studies, we investigated the hypothesis that hBD-3 may be cytotoxic to primary immune cells such as monocytes, T cells, or B cells. We found that hBD-3 caused membrane damage in primary human monocytes at concentrations similar to those reported to promote monocyte maturation and to kill bacteria [15, 16, 21].

Exposure of monocytes to hBD-3 at concentrations up to 5 micromolar tended to result in membrane damage, although most monocytes survived by undergoing membrane repair.

Higher concentrations of hBD-3 resulted in greater tendency for cell lysis. Our studies suggest that expression of phosphatidylserine on the surface of human monocytes may, in part, explain the enhanced susceptibility of these cells to hBD-3 compared to B or T cells.

It is possible that membrane damage caused by hBD-3 could affect the viability of monocytic cells that infiltrate inflammatory tissues.

2.3 MATERIALS AND METHODS

2.3.1 Reagents: Complete medium (CM), that was used to maintain cells, consisted of

RPMI 1640 (BioWhittaker, Walkersville, MD) supplemented with 10% Fetal bovine serum (FBS) (Sigma Aldrich, St. Louis, MO), 0.4% L-Glutamine (BioWhittaker), 0.4%

Penicillin/Streptomycin (BioWhittaker), and 0.4% HEPES (BioWhittaker). Wash buffer for flow cytometry consisted of BBL TTA Hemagglutination Buffer (BD, Sparks, MD),

1% Bovine serum albumin (BSA) (Sigma Aldrich, St. Louis, MO), and 0.1% Sodium

Azide (Sigma Aldrich). THP-1 cells were provided by Dr. Hameem Kawsar (Case

Western Reserve School of Dental Medicine, Cleveland, OH). Synthetic hBD-3 was purchased from Peptides International (Louisville, KY). RosetteSep Monocyte

Enrichment cocktails were purchased from StemCell Technologies (Vancouver, BC,

Canada). Antibodies used were anti-CD14-APC Cy7 (BD Pharmingen, San Diego, CA), anti-CD3-APC (BD Pharmingen), anti-CD20-eFluor450 (eBioscience, San Diego, CA), anti-CD14-FITC (BD Pharmingen), mIgG1-FITC (BD Pharmingen), and anti-

LAMP1/CD107a-FITC (BD Pharmingen). Bovine lactadherin and lactadherin-FITC were purchased from Haematologic Technologies (Essex Junction, Vermont). Unlabeled

Annexin V, Annexin buffer, Annexin V-FITC, anti-CD45RA-biotin, and streptavidin-

FITC were purchased from BD Pharmingen. Methyl-β-cyclodextrin and filipin complex were purchased from Sigma Aldrich. BAPTA-AM was purchased from Invitrogen

(Eugene, OR). PMA and staurosporine aglycone were purchased from Sigma Aldrich.

2.3.2 Isolation of PBMC: PMBC were isolated from blood drawn in heparin-coated tubes (for RosetteSep isolation experiments, EDTA coated tubes were used instead).

Briefly, 20 ml of blood was mixed with RPMI (up to 30 ml total volume) and centrifuged

21 for 25 minutes at 461 x g over 10 ml of Ficoll-Paque PLUS (GE Healthcare, Uppsala,

Sweden). Cells in the buffy coat were harvested and washed with RPMI before being re- suspended in CM and the cells counted.

2.3.3 HBD-3 treatment: Cells were plated in 24-well plates at a density of 1.0 x 106 cells per well in 500 µl of CM. HBD-3 was added to wells at a concentration of 20 µg/ml

(3.9 µM) and then the cells were incubated at 37°C overnight. In some experiments, cells were re-suspended in PBS (HyClone Laboratories, Logan, UT) and plated in 24-well plates at a density of 1.0 x 106 cells per well in 500 µl. HBD-3 was added at 20 µg/ml and incubated at 37°C for 1 h. In some assays, cells were treated with BAPTA-AM (80

µg/ml) at 37°C for 1 h prior to the addition of hBD-3. In other assays, cells were stained with CFSE (Molecular Probes Invitrogen, Grand Island, NY) prior to hBD-3 treatment.

THP-1 cells were plated in 24-well plates at a density of 1.0 x 106 cells per well in 500 µl of CM. To mature THP-1 cells into macrophage-like cells, these cells were incubated at

37°C overnight with PMA (20 ng/ml), washed, re-suspended in CM and allowed to incubate an additional 2 d [59]. Matured THP-1 cells or THP-1 cells pre-incubated in medium alone were treated with hBD-3 (20 µg/ml) and incubated at 37°C for 1 hour.

2.3.4 PI staining and flow cytometry: After 1 hour or overnight incubation, cells were transferred to Falcon tubes (BD Biosciences, Bedford, MA) and washed twice in wash buffer. Cells were stained with anti-CD14-APC Cy7, anti-CD3-APC and anti-CD20- eFluor450 for 10 minutes in the dark. In some assays, cells were also stained with anti-

LAMP1-FITC. Cells were then washed and re-suspended in 400 µl wash buffer along with 10 µl of PI (BD Pharmingen). After an additional 10 minute incubation in the dark, cells were analyzed on an LSRII flow cytometer (Becton Dickinson, San Jose, CA) and

22 analyzed using FACSDIVA (Version 6.1; BD Bioscience, San Diego, CA) or FlowJo

(Version 7.6, Tree Star, Ashland, OR) software.

2.3.5 Immunofluorescence: Monocytes were isolated from whole blood by negative selection using RosetteSep Monocyte Enrichment Cocktail. Isolated cells (approximately

80% CD14+ cells) were incubated in complete medium at 37°C overnight in the presence or absence of hBD-3 (20 µg/ml). Cells were then transferred to falcon tubes and stained with anti-CD14-FITC and PI. The cells were transferred to small petri dishes whose bottoms were replaced with coverslips and coated with Poly L-Lysine (Sigma-Aldrich).

Live cell imaging was accomplished using a Deltavision RT epifluorescent microscope system fitted with an automated stage and images were captured in z-series on a CCD digital camera (Applied Precision, Issaquah, WA). 3-D volume projections were generated using the Softworx analysis program (Applied Precision).

2.3.6 Amnis ImagestreamX studies: Monocytes isolated using RosetteSep Monocyte

Enrichment Cocktail were incubated in PBS at 37°C for 1 h in the presence or absence of hBD-3 (20 µg/ml) before being stained with anti-CD14-FITC and PI. Cells were analyzed using the Amnis ImagestreamX instrument (Amnis, Seattle, WA).

2.3.7 Phosphatidylserine expression: Freshly isolated PBMC were stained with anti-

CD20-eFluor450, anti-CD14-APC Cy7, anti-CD3-APC and either Annexin V-FITC in

Annexin buffer or lactadherin-FITC in PBS. Controls for the Annexin V stain included cells incubated without Annexin V and cells incubated with annexin V in the absence of the calcium supplemented annexin buffer. Negative controls for lactadherin-FITC included cells that were not stained with lactadherin and cells that were stained with streptavidin-FITC only.

2.3.8 Annexin V and lactadherin protection assays: PBMC were re-suspended in annexin buffer and were incubated with unlabeled Annexin V (25µg/ml) for 15 minutes at room temperature. Cells were then treated with hBD-3 (20 µg/ml) at 37°C for 1 h, washed, and then stained with anti-CD14-APC Cy7, anti-CD3-APC, anti-CD20- eFluor450, and PI as described for flow cytometric analysis. In some studies cells were incubated with unlabeled lactadherin (20µg/ml) in PBS at room temperature for 15 minutes after which hBD-3 was added and the cells were incubated at 37°C for 1 h. The cells were then stained as above for flow cytometry. For staurosporine experiments, cells were incubated at 37°C overnight in medium alone or medium + staurosporine (20 µM).

The next day, cells were washed with PBS and in some wells hBD-3 (20 µg/ml) was added before being incubated at 37°C for an additional hour. After this, the cells were stained with anti-CD3-APC, anti-CD20-eFluor450, lactadherin-FITC, and PI.

2.3.9 Cholesterol removal and cholesterol staining: PBMC were incubated with cyclodextrin (2.5 mM) at 37°C for 2 h in CM. HBD-3 was then added and the cells were incubated for an additional hour at 37°C. Afterwards, the cells were stained with anti-

CD14-APC Cy7, anti-CD20-eFluor450, anti-CD3-APC, and PI and then assessed by flow cytometric analysis. To assess free cholesterol content in cell membranes, 1 million

PBMCs were incubated with filipin (0.05 mg/ml in PBS/10% FBS) for 2 h at room temperature in the dark. The cells were then washed and stained with anti-CD14-APC

Cy7, anti-CD20-eFluor450, and anti-CD3-APC as above and analyzed on an LSRII flow cytometer (UV laser for filipin detection).

2.3.10 Human studies: Human subject studies have been reviewed and approved by a

University Hospitals Case Medical Center Institutional Review Board.

2.3.11 Statistical Methods: Nonparametric Wilcoxon matched pairs test was used to assess differences in PI staining comparing monocytes incubated in medium with monocytes incubated with hBD-3. This test was also used to assess the capacity of lactadherin to inhibit membrane damage in hBD-3-treated monocytes.

2.4 RESULTS

2.4.1 Evidence for membrane damage in monocytes but not T or B cells exposed to hBD-3.

To determine if hBD-3 could cause membrane damage in primary human cells,

PBMCs were incubated with various concentrations of hBD-3 and subsequently assessed for PI dye exclusion. We found that at an hBD-3 concentration of 20 µg/ml (3.9µM), which is an optimal concentration for monocyte activation [15], monocytes (CD14+), but not B cells (CD20+) or T cells (CD3+) stained positively with PI (Figure 2.1F, G, and H).

PI staining in most cells treated with hBD-3 was low to intermediate, but a small percentage of cells exhibited bright PI staining (Figure 2.1F). The PI uptake was dose- dependent and nearly absent at 2.5µg/ml (0.48µM) (Figure 2.1F and I-K). At very high concentrations of hBD-3 (60-80 µg/ml), monocytes appeared to undergo lytic cell death as indicated by the high frequencies of PI bright cells (Figure 2.2).

As hBD-3 at 20µg/ml has been used to induce APC activation, we chose to use this concentration for most of our analyses. Our observations indicated above were confirmed by experiments performed with cells from 14 different donors and the percentage of monocytes that exhibited dim PI staining and bright PI staining was significantly increased compared to monocytes incubated in medium alone (median %PI dim cells = 22.9 and 3.8, and median %PI bright cells = 7.9 and 1.4 in monocytes that were treated with hBD-3 compared to those cells treated in medium alone, respectively;

P<0.002; Wilcoxon test for paired samples).

Cells were then treated with LPS or Pam3CSK4, two well known TLR agonists and activators of monocytes, to determine if cellular activation was the basis for the

26 observed PI uptake. Neither treatment caused the PI staining pattern observed when cells were treated with hBD-3 (Figure 2.3). In addition, treatment with two other cationic antimicrobial peptides (LL-37 and hBD-2) in similar conditions did not result in increased PI staining (Figure 2.4), suggesting that hBD-3 has unique properties which cause these effects. To determine if the PI uptake is the result of the phagocytosis of apoptotic cells by monocytes, PBMCs were isolated from whole blood and separated into two groups. One group was stained with CFSE and then incubated overnight in medium alone, while the other group was incubated overnight in medium supplemented with 20

µg/ml hBD-3. The following day, those cells treated with hBD-3 were stained with PI.

Half of these cells were then mixed with half of the CFSE labeled cells for 1 hour before being stained for flow cytometry. Of the mixed population, those cells that were CFSE positive did not exhibit any PI staining (Figure 2.5D) while those CFSE negative cells were positive for PI (Figure 2.5C).

We noted that the PI dim/intermediate staining pattern in monocytes was similar to PI staining described previously in cells exposed to sublytic concentrations of perforin, which induced membrane damage followed by membrane repair and cell survival [105].

In these previous studies, PI staining was shown to be confined to bleb-like structures associated with damaged cells.

Because flow cytometry does not provide information regarding compartmentalization or subcellular staining characteristics, we examine the PI staining of monocytes that had been incubated overnight with or without hBD-3 by fluorescent microscopy. Monocytes that died spontaneously in culture exhibited a bright, nuclear staining pattern (Figure 2.6A) whereas those cells exposed to hBD-3 demonstrated a

27 vesicular-like staining pattern where the PI did not gain entry to the nucleus (Figure

2.6B). We then used ImagestreamX, which combines microscopy with flow cytometric output to confirm that the PI dim staining corresponded with the vesicular staining and the PI bright staining corresponded with the nuclear staining. Monocytes were purified from whole blood and exposed to hBD-3 for 1 h in PBS. Cells with the intermediate level of PI staining on the dot-plot histogram exhibited vesicular PI staining and those cells with bright PI staining on the dot-plot displayed nuclear PI staining (Figure 2.7).

These observations confirm that the intermediate PI staining observed in monocytes exposed to low micromolar concentrations of hBD-3 corresponds to PI uptake but exclusion from the nucleus.

To ensure that monocytes were damaged directly by hBD-3 and to assess the potential for macrophage-like cells to be damaged by hBD-3, we treated the THP-1 monocytic cell line and THP-1 cells which were matured with PMA into macrophage- like cells with hBD-3 overnight and then stained them with PI. Both the monocytic-like cells and the mature macrophage-like THP-1 cells were damaged and killed by hBD-3

(Figure 2.8), confirming that hBD-3 directly mediates these effects on monocytes and also suggesting that macrophages could be susceptible to hBD-3 damage.

Next, we considered the possibility that more cell death would occur in primary monocytes exposed to hBD-3 for a longer incubation period. At a concentration of 20

µg/ml of hBD-3, PI staining was assessed for up to 5 days of incubation. This long-term incubation did not lead to marked differences in the PI staining pattern (data not shown), suggesting that those cells damaged by hBD-3 were able to survive even prolonged periods of incubation.

2.4.2 Membrane repair is induced in monocytes exposed to hBD-3.

Because our data suggested that low micromolar concentrations of hBD-3 damaged monocytes without killing them, we explored whether membrane repair mechanisms might allow monocytes to withstand hBD-3 mediated damage. We measured the surface expression of LAMP1 (CD107a) on monocytes exposed to hBD-3.

LAMP1 surface expression can be used as a marker of lysosomal membrane donation as a result of the cellular membrane repair response [106]. Monocytes treated with hBD-3 overnight exhibited an increased level of LAMP1 surface expression compared to monocytes incubated in medium alone (Figure 2.9A). This increased LAMP1 expression was also observed on cells treated with hBD-3 in PBS, and in these culture conditions,

LAMP1 expression could be observed as early as 10-20 min after hBD-3 exposure

(Figure 2.9D-F).

Using the intracellular calcium chelator BAPTA-AM, it is possible to block the membrane repair process by interfering with the calcium sensors which initiate membrane repair [105]. Monocytes were treated with BAPTA for 1 h before being exposed to hBD-3. These monocytes demonstrated increased frequencies of PI bright stained cells compared to monocytes treated with hBD-3 alone (Figure 2.10). This suggests that a calcium-dependent repair process was contributing to the survival of monocytes treated with hBD-3.

2.4.3 Cholesterol protects cells from membrane damage caused by hBD-3.

Cholesterol in eukaryotic cell membranes is known to be protective against the damage caused by some AMPs [107]. We therefore considered the possibility that cholesterol could play a role in protecting cells from hBD-3. PBMCs were pre-incubated

29 with cyclodextrin prior to hBD-3 treatment. Cyclodextrin is a molecule which removes cholesterol from the plasma membrane of cells. Cells treated with cyclodextrin were more susceptible to cell death caused by hBD-3 treatment than cells incubated without cyclodextrin. An enhanced susceptibility to hBD-3 was observed in monocytes, B cells, and T cells (Figure 2.11), suggesting that cholesterol can play a role in protection against hBD-3-mediated damage in various cell types.

To ascertain if these cell types might have differences in cholesterol membrane content, PBMCs were stained with filipin, a molecule that binds to free cholesterol and provides a fluorescent emission under UV light [108]. Using UV laser flow cytometric analyses, we observed similar staining patterns between monocytes, B cells and T cells which suggest that there is no marked difference in cholesterol content amongst these cell types (Figure 2.12). While our studies illustrate an important protective role for cholesterol in protecting cells from membrane damage caused by hBD-3, any differences in cholesterol content between monocytes, B cells, and T cells are modest and therefore not likely to explain the greater susceptibility of monocytes to hBD-3 compared to B cells or T cells.

2.4.4 Freshly isolated monocytes express phosphatidylserine on their outer leaflet.

Negatively charged phospholipids on the outer leaflet of bacterial cells, phosphatidylglycerol, may play an important role in attracting positively charged AMPs

[107]. Phosphatidylserine (PS) is a negatively charged phospholipd which normally resides on the inner leaflet of eukaryotic membranes, but it will be flipped out to the outer leaflet during apoptosis where it will be used as a receptor for phagocytosis. Previous studies suggest that myeloid cells may have high content of PS on their cell surfaces even

30 in the absence of apoptosis [109]. Therefore, we hypothesized that there may be differences in basal levels of PS on the outer leaflet of monocytes and T cells or B cells and that this could contribute to differences in susceptibility to hBD-3.

Freshly isolated PMBCs were stained with fluorescently tagged Annexin V, a molecule that binds to PS [110], and examined by flow cytometry. Compared to background fluorescence (Figure 2.13A) or staining done in the absence of calcium (data not shown), monocytes clearly stained with Annexin V whereas T cells and B cells did not (Figure 2.13B and C). To confirm this observation, freshly isolated PBMCs were also labeled with fluorochrome-conjugated lactadherin, which is another PS binding molecule [111]. The staining pattern of lactadherin binding to monocytes was distinguishable from both background fluorescence as well as the staining pattern of lactadherin binding to B cells and T cells (Figure 2.13D-F). As a further control, streptavidin conjugated with the matched fluorochrome was used to stain PBMCs. The fluorescence pattern of monocytes stained with streptavidin was low and distinguishable from lactadherin (Figure 2.13D). The THP-1 monocytic cell line also exhibited a staining pattern of lactadherin and Annexin V, suggesting that these cells also express moderate levels of surface PS (data not shown). Together, these observations provide evidence that monocytes intrinsically express PS on their cell surface even in the absence of apoptosis induction.

2.4.5 Pretreatment with lactadherin or Annexin V protects monocytes from the effects of hBD-3.

We considered the possibility that interactions of hBD-3 with PS could contribute to the increased susceptibility of monocytes to membrane damage caused by hBD-3. To

31 test this, cells were pre-treated with lactadherin before being incubated with hBD-3 to block binding to PS. Those monocytes which were treated with lactadherin were protected from damage when compared to cells that were treated with hBD-3 alone

(Figure 2.14A-C). There was a significant decrease in the percentage of cells that stained

PI bright and a significant decrease in the percentage of cells that stained PI dim when incubated in lactadherin prior to hBD-3 treatment (Figure 2.14E and F). When Annexin

V was used in place of lactadherin, similar results were observed (data not shown).

These data suggest that the level of PS expression on monocytes is important in determining their sensitivity to membrane damage caused by hBD-3.

One prediction from these data is that increasing PS on the surface of B and T cells could make these cells more susceptible to hBD-3. To increase PS on the surface, we treated PBMCs with staurosporine aglycone to induce apoptosis. With staurosporine treatment, both B cells and T cells had increased surface expression of PS and both cell types became more susceptible to hBD-3-induced membrane damage (Figure 2.15).

2.5 DISCUSSION

Monocytes are recruited to inflammatory settings where they mature to mediate effector functions and act as antigen presenting cells. Members of our investigative team and others have demonstrated that nanomolar concentrations of hBD-3 can recruit monocytes via CCR2 [41, 59] while micromolar concentrations are required to induce monocyte maturation [15]. In some inflammatory conditions [16, 59], hBD-3 is found at high concentrations in tissues and is likely to reach concentrations capable of causing membrane disruption.

HBD-3 kills organisms by interacting with the surface of its target and disrupting the plasma membrane. There are three models that have been suggested to explain this effect. In the “pore-forming” model, cationic oligomers of hBD-3 electrostatically interact with anionic sites in the bacterial membrane. Then, because of hBD-3’s amphipathic structure, the oligomers aggregate to form pores which lead to microbial destruction [27]. Evidence that hBD-3 molecules have the potential to interact and form dimers suggests that this model may be feasible [17]. The “carpet” model proposes that charge interactions bring the AMP into contact with the membrane where it acts like a detergent to disrupt the membrane and kill the bacterium [27]. The third model posits that interactions between the AMP and the outer leaflet of the membrane cause lipid displacement and membrane hole formation [26]. While our studies do not provide evidence supporting one of these possibilities over the others, we have implicated PS, a negatively charged phospholipid, as playing a role in hBD-3 susceptibility, suggesting an importance of electrostatic interactions.

We provide evidence that freshly isolated monocytes express negatively charged

PS on their surface. This observation, consistent with other reports, reflects PS levels which are not likely to induce phagocytosis which is generally the fate of apoptotic cells

[109, 112]. These PS levels are below the levels observed in apoptotic monocytes [109].

A functional role for spontaneous PS expression on monocytes is not entirely clear, but it may be related to a role in coagulation since PS can serve as a docking site for thrombin

[113] or it may be a mechanism for scavenging apoptotic cells similar to matured macrophages which utilize surface PS for phagocytosis of apoptotic targets [114-116]. In any case, our studies suggest that this intrinsic expression of PS on monocytes confers greater susceptibility to hBD-3-mediated membrane damage and may also contribute to susceptibility of macrophages to hBD-3 as suggested by our studies of matured THP-1 cells.

Two other AMPs, LL-37 and hBD-2, did not induce membrane damage in monocytes suggesting that this effect was specific for hBD-3. This is likely because the net charge of these molecules (+6 for LL-37 and hBD-2) is almost half as positive as hBD-3 (+11). However, it is certainly possible for other, as yet undetermined, factors to contribute to this difference. Further structure/function studies of hBD-3 may be useful in determining these potential factors.

We implicate cholesterol as being an agent which protects cells from hBD-3- induced membrane damage. Removing cholesterol from primary cells with cyclodextrin resulted in much greater susceptibility to hBD-3. Because these observations hold true for multiple cell types, it appears cholesterol plays an important role in protecting primary human cells from hBD-3. Cholesterol is known to provide protection from other

AMPs, like magainins [107], though the exact protective mechanism is not understood.

Because our studies imply similar levels of cholesterol in monocytes, B cells, and T cells, it appears that some other difference between the cells will explain the differing susceptibility of these cell types to hBD-3-mediated membrane damage.

Other studies report that sublytic concentrations of perforin, a pore-forming molecule released by cytotoxic T cells, can induce similar membrane damaging effects as we report here for hBD-3. Similar to monocytes exposed to low micromolar concentrations of hBD-3, cells treated with intermediate levels of perforin are capable of repairing themselves without dying [105]. Surface staining of these cells reveals increased levels of LAMP1 as a consequence of lysosomal membrane donation. As we have shown here with cells treated with hBD-3, these perforin treated cells have more necrotic death if they are also treated with a calcium chelator to block membrane repair.

According to models of membrane damage and repair, blebbing occurs when the damaged lipid bilayer detaches from the cytoskeleton [117]. The membrane protrusions

(blebs) that form from this detachment contain cytoplasm. Then calcium-sensitive proteins, including annexins, will rapidly form a protein "plug" by sealing the damaged region from the rest of the cell [118]. This could explain why the PI fails to diffuse throughout the cell and is only contained to the blebs in some hBD-3 treated cells. In this case, the PI staining is potentially dependent on the presence of RNA molecules [119].

Monocytes exposed to hBD-3 may undergo membrane damage that could affect the survival of these cells as well as their activation, maturation, and function.

Nonetheless, it is clear that low micromolar concentrations of hBD-3 can induce monocytes to release cytokines and up-regulate co-stimulatory molecules which implies that membrane damage and repair do not inhibit all cellular functions [15, 43]. Further

35 studies to identify structural components of hBD-3 which mediate these diverse effects could provide insight into the interactions between hBD-3 and APCs, and also provide further insight into the role this molecule plays in adaptive immunity.

Figure 2.1. HBD-3 causes detectable PI staining in monocytes, but not B cells or T cells. PBMC were isolated from whole blood, incubated overnight in medium alone or in medium + hBD-3 (20 µg/ml) and then stained with CD14, CD20, CD3 antibodies and PI. Cells were analyzed by flow cytometry. Cellular debris was gated out via forward scatter-area (FSC-A) vs. side scatter-area (SSC-A) (A). Doublets were gated out via FSC-A vs. forward scatter-height (FSC-H) (B). Cell types were identified as CD14+ monocytes (C), CD20+ B cells (D), and CD3+ T cells (E). Monocytes (F), B cells (G), and T cells (H) incubated in medium alone were compared to cells incubated in medium + hBD-3 for PI staining. PI staining in monocytes cultured in medium with varying concentrations of hBD-3; 10 µg/ml (I), 5 µg/ml (J), and 2.5 µg/ml (K) were compared to monocytes cultured in medium alone. Pictured are data representative of seven experiments.

Figure 2.2. Increasing concentrations of hBD-3 increases the number of PI bright monocytes. PBMC were isolated and incubated overnight in medium alone (red histogram, A-D), medium + hBD-3 (20 µg/ml; blue histogram, A), medium + hBD-3 (40 µg/ml; blue histogram, B), medium + hBD-3 (60 µg/ml; blue histogram, C), or medium + hBD-3 (80 µg/ml; blue histogram, D). Cells were stained with CD14 antibodies as well as PI and then analyzed by flow cytometry using the gating strategy outlined in Figure 1.

Figure 2.3. PI uptake is not caused by canonical monocyte activators. PBMC were incubated in medium alone (black histogram – A, B, C), medium + hBD-3 (20 µg/ml; gray histogram - A), medium + LPS (50 ng/ml; gray histogram - B), or medium + Pam3CSK4 (500 ng/ml; gray histogram - C) overnight and were stained with PI (x-axis). CD14+ cells were selected for analysis of PI staining.

Figure 2.4. Neither hBD-2 or LL-37 increased PI staining in monocytes. PBMC were isolated and incubated for 3 hr in medium alone (red histogram, A-C), medium + hBD-3 (20 µg/ml - 3.9 µM; blue histogram, A), medium + hBD-2 (16.77 µg/ml - 3.9 µM; blue histogram, B), or medium + LL-37 (19.11 µg/ml - 3.9 µM; blue histogram, C). Cells were stained with CD14 antibodies as well as PI and then analyzed by flow cytometry using the gating strategy outlined in Figure 1. Above histograms are representative of four experiments.

Figure 2.5. Phagocytosis of apoptotic blebs does not appear to be the cause of the observed PI staining pattern. PBMC were isolated and half were labeled with CFSE and incubated overnight in medium alone. The other half were incubated overnight in hBD-3 (20 µg/ml). The next day, these hBD-3 treated cells were stained with PI and allowed to mix with the CFSE labeled cells for 1 hour before being stained for flow cytometry. CD14 positive cells were gated on (A), followed by a separation based on CFSE staining between those cells that are CFSE positive or CFSE negative (B). Of the CFSE negative cells (C), PI uptake comparisons were made between background PI staining in unstimulated cells (black, dashed line), hBD-3 treated cells which were not mixed with CFSE labeled cells (grey, solid line), and cells mixed with CFSE labeled cells (black, solid line). Of the CFSE positive cells (D), PI uptake comparisons were made between the PI background of unmixed, CFSE labeled cells (grey, solid line) and those cells that were mixed with hBD-3 treated cells (black, solid line). Above histograms are representative of three experiments.

Figure 2.6. HBD-3 induces bleb-like PI staining in monocytes. Monocytes were enriched by RosetteSep and incubated overnight in medium alone or in medium plus hBD-3 (20 µg/ml). Deconvolution microscope pictures of PI staining is shown for 3 cells that spontaneously died in untreated cultures. These cells are representative of bright nuclear staining (A). Typical PI staining of monocytes that were treated with hBD-3 overnight (B). green = CD14; red = PI; scale bar = 15 µm.

Figure 2.7. Imagestream analysis of hBD-3-induced PI staining in monocytes demonstrates differences in nuclear staining and bleb-like staining patterns. Monocytes were enriched using RosetteSep, re-suspended in PBS plus or minus hBD-3 (20 µg/ml) for 1 hour, and analyzed on an Amnis ImagestreamX. A dot-plot histogram is shown for CD14+ cells (y-axis) treated with hBD-3 and stained for PI (x-axis). Representative pictures of cells in box A (PI negative cells), box B (PI intermediate cells), and box C (PI bright cells) are shown. Box A was drawn for PI negative cells and was based on the PI staining of untreated cells. The division of box B and box C was based on identification of cells with bleb-like staining (box B – arrows indicate bleb-like structures) or nuclear staining (box C) as demonstrated in the representative pictures. Each number corresponds to the cell labeled in the dot plot.

Figure 2.8. PI uptake in THP-1 cell line. After being incubated in medium alone for 3 days, THP-1 cells were incubated in medium alone (black histogram), or medium + hBD-3 (20 µg/ml; gray histogram) for 1 h before being stained with PI (A). Macrophage-like THP-1 cells were generated by stimulating cells overnight with PMA, washing the cells and incubating for 2 additional days in CM as described previously [59]. PI staining of macrophage-like THP-1 cells that were incubated in medium alone (black histogram), or medium + hBD-3 (20 µg/ml; gray histogram) for 1 hour are shown (B).

Figure 2.9. Evidence of membrane repair in monocytes treated with hBD-3. PBMC were incubated overnight (A, B, C) in medium alone or medium + hBD-3 (20 µg/ml). Cells were stained with CD14, CD20, CD3, and LAMP1 and analyzed by flow cytometry using the gating strategy outlined in Figure 1. Medium alone (black, solid line histograms) was compared to medium + hBD-3 (grey, solid line histograms) for LAMP1 staining in monocytes (A), B cells (B), and T cells (C). Isotype controls were also present (black, dashed line histograms). PBMCs were incubated in PBS alone (black histograms) or PBS + hBD-3 (gray histograms). Pictured are monocytes treated for 10 minutes (D), 30 minutes (E), or 1 hour (F).

Figure 2.10. Inhibition of membrane repair enhances hBD-3 mediated PI uptake in monocytes. PBMC were isolated and incubated for 1 hour in PBS alone (A), PBS + hBD-3 (20 µg/ml) (B), PBS + BAPTA-AM (80 µg/ml) (C), or PBS + BAPTA-AM (80 µg/ml) + hBD-3 (20 µg/ml) (D). Cells were stained with CD14, CD20, and CD3 antibodies as well as PI and then analyzed by flow cytometry using the gating strategy outlined in Figure 1. Pictured are CD14+ cells.

Figure 2.11. Removal of cholesterol increases cellular susceptibility to hBD-3 induced membrane damage. PBMC were isolated and incubated in medium alone (A, B, C), medium + hBD-3 (20 µg/ml; D, E, F), medium + 2.5 mM cyclodextrin (G, H, I), or medium + hBD-3 + cyclodextrin (J, K, L). Cells were first incubated with cyclodextrin for 2 h at 37°C prior to adding hBD-3 for an additional 1 h incubation. The cells were then stained with anti-CD14, anti-CD20, anti-CD3, and PI (x-axis) and analyzed with an LSR II flow cytometer. Percentages of cells that stained PI dim/intermediate and that stained PI bright are shown. Similar results were obtained with cells from 4 other donors.

Figure 2.12. Cholesterol levels are similar between cell types. PBMC were stained with filipin as well as anti-CD14, anti-CD20, and anti-CD3 antibodies and subjected to flow cytometric analysis. Background levels of fluorescence were determined for each cell type with fluorescence minus one staining (FMO, all fluorochromes except filipin) (black, solid line histogram). Levels of cholesterol in monocytes (A), B cells (B), and T cells (C) were determined by brightness of filipin stain (gray, solid line histogram). To ensure specificity of stain, some cells were pre-treated with cyclodextrin (2.5 mM for 2 h) to remove membrane cholesterol (gray, dashed line histogram).

Figure 2.13. Phosphatidylserine expression is detected on freshly isolated monocytes. PBMC were isolated and stained with CD14, CD20, and CD3 antibodies as well as annexin V. Representative levels of annexin V binding (black, dashed line histograms) were analyzed for monocytes (A), B cells (B), and T cells (C). Background levels of fluorescence were determined with FMO (black, solid line histograms). In panels D (monocytes), E (B cells), and F (T cells), cells were stained with lactadherin (black, dashed line histograms) instead of annexin V. Background levels of fluorescence were determined by FMO (black, solid line histogram). As a control for nonspecific binding of a FITC-conjugated molecule, streptavidin- FITC was used in place of lactadherin-FITC (gray, solid line histogram). To ensure the streptavidin-FITC was detectable, some cells were stained with CD45RA biotin + streptavidin-FITC as a positive control (gray, dashed line histogram, F).

Figure 2.14. Lactadherin protects monocytes from hBD-3-induced membrane damage. PBMC were isolated, re-suspended in PBS, and incubated with lactadherin (20 µg/ml) for 15 minutes at room temperature before being treated with hBD-3 (20 µg/ml) for 1 hour at 37°C. Cells were stained with PI and anti-CD14, anti-CD20, and anti-CD3 antibodies and analyzed by flow cytometry using the gating strategy outlined in Figure 1. A representative example of PI staining in monocytes incubated in medium alone (black histograms - A, B, C) is compared to cells incubated in medium + lactadherin (gray histogram - A), cells incubated in medium + hBD-3 (gray histogram - B), or cells incubated in medium + hBD-3 + lactadherin (gray histogram - C). The comparison of five experiments with the effects of lactadherin on the mean fluorescence intensity (MFI) of PI (P<0.05) (D), the percentage of the monocyte population that is PI dim (P<0.05) (E), or the percentage of the monocyte population that is PI bright (P<0.05) (F).

Figure 2.15. Lactadherin binding and PI uptake in staurosporine treated B cells and T cells. PBMCs were incubated overnight in medium alone or medium + staurosporine, then some cells were incubated in hBD-3 for 1 hour before being stained with anti-CD3, anti-CD20, lactadherin and PI. PS on the surface of B cells (A) and T cells (B) incubated in medium alone (black, dashed line histograms) was compared to the amount of PS on the surface of cells incubated in medium + staurosporine (gray, solid line histograms). FMO was used to determine background levels of fluorescence (black, solid line histograms). PI uptake in B cells (C) and T cells (D) was compared between cells which were incubated in medium alone (black, solid line histograms), medium + hBD-3 (black, dashed line histograms), medium + staurosporine (gray, solid line histograms), and medium + staurosporine + hBD-3 (gray, dashed lined histograms). This is representative of four experiments.

CHAPTER 3:

HBD-3 increases CD86 expression on monocytes by activating the ATP-gated

channel P2X7

Anthony B. Lioi1, Brian M. Ferrari2, George R. Dubyak3, Aaron Weinberg4, Scott F.

Sieg2

1Department of Molecular Biology and Microbiology

2Department of Medicine, Division of Infectious Diseases, Center for AIDS Research

3Department of Physiology & Biophysics

4Department of Biological Sciences, Case School of Dental Medicine

Case Western Reserve University, Cleveland OH 44106

3.1 ABSTRACT

Human beta-defensin-3 (hBD-3) is an antimicrobial peptide known to interact with a variety of cellular receptors. TLR1/2 activation by hBD-3 has been shown to up-regulate

CD80 and CD86 expression on monocytes and myeloid dendritic cells. When primary monocytes were pre-incubated with a specific antagonist to the ATP-gated ion channel

P2X7 before they were stimulated with hBD-3, CD86 expression was abrogated whereas

CD80 expression remained unchanged. This appears to be an indirect interaction between P2X7 and hBD-3, as apyrase treatment has the effect of lessening CD86 expression. In addition, treatment with ATP alone was sufficient to cause the up- regulation of CD86 on the surface of monocytes without any change in CD80 expression.

This increased surface expression occurs when ATP stimulates the cells for between fifteen and thirty minutes but does not become apparent until more than six hours have passed. Quantitative PCR studies implicate increased CD86 mRNA transcription as the likely reason for this time frame. Further studies into this mechanism may clarify the ways in which hBD-3 activates monocytes and how this activation affects downstream immune function.

3.2 INTRODUCTION

Human beta-defensin-3 is a cationic, antimicrobial peptide produced by epithelial cells.

HBD-3 has the capacity to activate myeloid antigen-presenting cells causing the induction of co-stimulatory molecules, cytokines and chemokines from these cells [13-

15, 21, 27, 42]. We have demonstrated that hBD-3 activates monocytes in a manner that is at least partially dependent on Toll-like receptors 1 and 2[15]. The biological effects of hBD-3, however, can be clearly distinguished from that of a well-defined TLR1/2 agonist, Pam3CSK4 [43], suggesting that hBD-3’s activity may be more complex than can be explained strictly on the basis of TLR1/2 signaling. More recently, we have found that concentrations of hBD-3 that induce APC activation are also able to cause membrane damage and repair responses in monocytes [103]. It is possible that membrane damage could also contribute to APC activation as a consequence of released damage-associated molecular pattern molecules (DAMPs), which in turn, also activate innate immune receptors. In these studies, we explore the hypothesis that hBD-3 causes release of the

DAMP, ATP, which then contributes to the activation of primary human monocytes.

ATP is released by damaged or dead cells and modulates immune activation in surrounding tissues [63]. Released ATP attracts phagocytic antigen-presenting cells to sites of tissue damage and, at high concentrations, ATP can trigger the P2X7 ion channel, resulting in inflammasome activation and release of pro-inflammatory IL-1β [120].

P2X7 is expressed on many hematopoietic cell types including monocytes, macrophages, dendritic cells, T cells, and B cells [65]. When activated by binding to ATP, P2X7 orients into active ion channels that allow calcium and sodium to move into the cell and potassium to move out of the cell [65]. If ATP is bound for sufficient time, P2X7

54 channels enlarge and permit molecules of up to 900 Da to pass through [73]. This process, given enough time, eventually leads to cell death [74].

Here we explore the possibility that ATP and P2X7 play a role in the activation of monocytes by hBD-3. Our analyses center on the induction of the co-stimulatory molecule, CD86, which is differentially regulated by hBD-3 and Pam3CSK4 in monocytes and that we have shown is induced by another antimicrobial peptide, LL-37 in these cells [121]. Interestingly, LL-37 activation of cells has been linked to P2X7 activation [122], lending feasibility to our hypothesis that activation of this ion channel may play an important role in the CD86 induction caused by hBD-3.

To investigate these possibilities, we have treated human peripheral blood mononuclear cells with ATP, hBD-3, and inhibitors of ATP/P2X7 to uncover a role for this pathway in hBD-3-mediated activation of monocytes. Our studies demonstrate that hBD-3 causes CD86 induction in monocytes in at least a partially indirect manner through P2X7 activation. Moreover, the effects are selective since CD80 induction by hBD-3 is not affected by blocking P2X7. Our results also demonstrate for the first time that short-term exposure to high concentrations of ATP alone is sufficient to cause CD86 induction in monocytes. These findings provide new insights into molecular mechanisms of monocyte activation by hBD-3 and contribute to our understanding of the potential biological effects of ATP on antigen-presenting cells.

3.3 MATERIALS AND METHODS

3.3.1 Reagents: Complete medium (CM) consisted of RPMI 1640 (BioWhittaker,

Walkersville, MD) supplemented with 10% FBS (Sigma Aldrich, St. Louis, MO), 0.4%

HEPES (BioWhittaker), 0.4% Penicillin/Streptomycin (BioWhittaker), and 0.4% L-

Glutamine (BioWhittaker). Antibodies used were anti-CD14-APC Cy7 (BioLegend,

San Diego, CA), anti-CD80-APC (BD Pharmingen, San Diego, CA), and anti-CD86-

AlexaFluor700 (eBioscience, San Diego, CA). The wash buffer used during flow cytometry staining consisted of BBL TTA Hemagglutination Buffer (BD, Sparks, MD),

1% BSA (Sigma Aldrich, St. Louis, MO), and 0.1% Sodium Azide (Sigma Aldrich).

Synthetic hBD-3 was purchased from Peptides International (Louisville, KY). Apyrase was bought from New England BioLabs (Ipswich, MA). AZ 10606120 dihydrochloride was purchased from (Tocris Bioscience, Bristol, United Kingdom). TRIZol was bought from Invitrogen (Carlsbad, CA). RT2 SYBR Green/ROX qPCR Master Mix (PA-012) and predesigned qPCR primers for CD86 and human GAPDH primers were purchased from Qiagen (Valencia, CA). Transcriptor first strand cDNA synthesis kit was from

Roche Applied Science (Indianapolis, IN).

3.3.2 Isolation of PBMC: PMBC were isolated from blood drawn in heparin-coated tubes. Briefly, 20 ml of blood was mixed with RPMI (up to 30 ml total volume) and centrifuged for 25 minutes at 461 x g over 10 ml of Ficoll-Paque PLUS (GE Healthcare,

Uppsala, Sweden). Cells in the buffy coat were harvested and washed with RPMI before being re-suspended in CM and the cells counted.

3.3.3 HBD-3 treatment: Cells were plated in 24-well plates at a density of 1.0 x 106 cells per well in 500 µl of CM. HBD-3 was added to wells at a concentration of 20 µg/ml

(3.9 µM) and then the cells were incubated at 37°C overnight. In some experiments, cells were pre-incubated with AZ for 20 min at 37°C before hBD-3 was added. In other experiments, apyrase (2.5 U/ml) was added to the cells at the same time as hBD-3 and incubated overnight.

3.3.4 PI staining and flow cytometry: After overnight incubation, cells were transferred to Falcon tubes (BD Biosciences, Bedford, MA) and washed twice in wash buffer. Cells were stained with anti-CD14-APC Cy7, anti-CD86-APC, and anti-CD80-

AlexaFluor700 for 10 minutes in the dark. Cells were then washed and re-suspended in

400 µl wash buffer along with 10 µl of PI (BD Pharmingen). After an additional 10 minute incubation in the dark, cells were analyzed on an LSRII flow cytometer (Becton

Dickinson, San Jose, CA) and analyzed using FACSDIVA (Version 6.1; BD Bioscience,

San Diego, CA) or FlowJo (Version 7.6, Tree Star, Ashland, OR) software.

3.3.5 Analysis of Extracellular ATP: Cells were plated as above in a 24-well plate and hBD-3 was added to half of the wells. At designated time points, medium was removed from the wells and spun down. The supernatant was removed and added to small tubes where it was boiled for 5 min before being frozen at -20°C. When quantifying, 50 µl of sample was added to 46 µl ATP assay buffer (Sigma Aldrich) and 4 µl FLAAM (Sigma

Aldrich). Mixture was added to white, round bottom plates (Thermo Scientific,

Waltham, MA). Luminescence was read on Synergy HT (BioTek, Winooski, VT).

Standard curves were generated using known concentrations of ATP added to assay medium, FLAAM, and ATP assay buffer.

3.3.6 qPCR analysis of CD86 mRNA expression: Cells were isolated and plated as above. HBD-3, or ATP, was added to the wells and then the cells were incubated

57 overnight at 37°C. The following day, the supernatant was removed and cells spun down with the resulting supernatant removed. During this time, 1 mL of TRIZol was used to remove the adherent cells which were then mixed in to the cells harvested from the supernatant allowing for extraction of total RNA. Transcriptor First Strand cDNA

Synthesis kit was used to generate first-strand cDNA from the purified RNA. CD86 and

GAPDH transcripts were quantified using a StepOne-Plus Real-Time PCR System

(Applied Biosystems, Grand Island, NY) with reactions performed in 25 µl containing

RT2 SYBR Green/ROX qPCR Master Mix (12.5µl), 1:25 dilutions of RT product, and 1

µM PCR primer pair stock that were run in triplicate. Amplification cycle conditions were 95°C for 10 minutes followed by 40 cycles of (95°C, 15 sec; 55°C, 30-40 sec; and

72°C, 30 sec.). Expression of CD86 was calculated using the ΔΔCt method using

StepOne software v. 2.1 with values normalized to GAPDH expression.

3.3.7 Statistical Methods: A nonparametric paired test was used to assess differences in

CD80 and CD86 staining comparing monocytes incubated with hBD-3 with monocytes incubated with hBD-3 and AZ.

3.3.8 Human studies: A University Hospitals Case Medical Center Institutional Review

Board has reviewed and approved all human subject studies performed.

3.4 RESULTS

3.4.1 Induction of CD86 expression on monocytes by hBD-3 is dependent on P2X7

To determine if P2X7 is involved in monocyte activation caused by hBD-3, we tested the effects of a P2X7 inhibitor, AZ 10606120 dihydrochloride (AZ), on the capacity of hBD-

3 to induce CD80 and CD86 surface expression. PBMCs were pre-incubated with AZ for

20 minutes prior to the addition of hBD-3. AZ remained in the culture with hBD-3 for the duration of an overnight incubation. After this incubation, CD14+ cells were assessed for CD80 and CD86 expression by flow cytometry. Incubation of cells with AZ significantly decreased the capacity of hBD-3 to cause induction of CD86 expression

(Figure 3.1H and Figure 3.2B; Nonparametric paired test, P=0.0087). In contrast, AZ had no effect on the induction of CD80 by hBD-3 (Figure 3.1G and Figure 3.2A;

P=0.1994). Neither LPS nor Pam3CSK4 activation of monocytes was affected by AZ treatment, suggesting that the effects were specific for hBD-3 and not a consequence of toxicity (data not shown).

3.4.2 Activation of P2X7 by ATP alone is sufficient to cause induction of CD86 expression on monocytes

Because P2X7 is an ATP-gated channel but has low affinity for ATP, activation by 3mM

ATP [123] is necessary to activate the receptor. We observed that monocytes treated overnight with 3mM ATP increased CD86 surface expression (Figure 3.1K and Figure

3.3) and that the induction of CD86 expression was blocked when monocytes were incubated with AZ (Figure 3.1K and Figure 3.3). In addition, this CD86 activation was not present when the ATP concentration was lowered (Figure 3.4), consistent with published observations that high concentrations of ATP are needed to activate P2X7

[123]. It must be noted, however, that the monocytes from roughly 40% of our donors died after overnight stimulation with ATP. In addition, when ATP and hBD-3 were added together to the culture for overnight stimulation, essentially all the cells died (data not shown).

To determine the duration of ATP stimulation required for CD86 up-regulation, 3 mM ATP was added to PBMCs for 15 min, 30 min, 1 h, or 3 h before the cells were washed and re-plated in complete medium for overnight incubation. Incubation of cells with ATP for 30 min was sufficient to cause the induction of CD86 in monocytes (Figure

3.5) indicating that a relatively short duration of ATP exposure is sufficient to induce

CD86 expression.

To determine the kinetics of CD86 induction in cells stimulated continuously with ATP, PBMCs were incubated in medium alone or with 3 mM ATP and tested at four different time points (1 h, 3 h, 6 h, and overnight) for cell surface expression of CD86. In both assays, the cells were stained for flow cytometric analysis at the indicated time.

Induction of CD86 surface expression could not be detected at early time points up to, and including, 6 h of incubation, although it was detectable after overnight incubation

(Figure 3.6). These observations suggest that ATP induction of CD86 expression stems from de novo protein synthesis rather than re-distribution. To confirm this, quantitative

PCR (qPCR) was performed on cells incubated with medium alone, hBD-3, or ATP.

After overnight stimulation, cells were harvested and RNA was isolated. Preliminary analysis of these cells with qPCR showed increases in CD86 mRNA levels in both hBD-3 and ATP stimulated cells compared to the unstimulated controls (Figure 3.7). Additional time-points are still needed to confirm this observation.

3.4.3 Induction of CD86 expression by hBD-3 is at least partially explained by release of ATP from hBD-3-treated cells

To determine if hBD-3 directly interacts with P2X7 or if it acts indirectly to activate

P2X7, we incubated cells with apyrase, an enzyme that breaks down extracellular ATP.

PBMCs were incubated overnight in medium alone, medium + hBD-3, medium + apyrase, or medium + apyrase + hBD-3. LL37, an antimicrobial peptide that is thought to directly activate P2X7 [122], and Pam3CSK4, a TLR1/2 agoinst, were used as controls.

At a concentration of 5 U/ml, the apyrase reagent alone caused increased expression of

CD86 and, therefore, only concentrations below 5 U/ml were used for all additional experiments. Because of this, we titrated the concentration of apyrase down and used the highest concentration that did not cause CD86 up-regulation on its own. CD86 expression was decreased in cells incubated with both apyrase and hBD-3 compared to those cells incubated with hBD-3 alone (Figure 3.8). This implies that the ability of hBD-3 to activate P2X7 is at least partially dependent on indirect mechanism. In addition, we observed a decrease in CD86 expression when cells were incubated with apyrase and LL37 compared with cells incubated in LL37 alone (Figure 3.9A). This suggests that LL37 interacts indirectly with P2X7 in our model. No decrease in

Pam3CSK4 induced CD86 induction was observed when cells were also incubated with apyrase (Figure 3.9B).

We considered the possibility that hBD-3 caused the release of ATP from cells resulting in indirect activation of P2X7. To test this possibility, PBMCs were incubated in medium alone or medium supplemented with hBD-3. Supernatants were removed at various time points and tested for increased levels of ATP. We observed a transient

61 increase of ATP in the supernatant 1 hour after hBD-3 stimulation above the concentration observed in cells incubated in medium alone (Figure 3.10). These data suggest that hBD-3 may cause a transient increase in extracellular ATP that contributes to

P2X7 activation, although further studies are needed to confirm this observation.

3.5 DISCUSSION

Human beta-defensin-3 is an antimicrobial peptide that activates monocytes, leading to increased co-stimulatory molecule expression (CD80 and CD86) on the cell surface. TLR1/2 signaling by hBD-3 has been implicated as being required for this activity [15]. Here, we provide evidence that activation of the P2X7 receptor by hBD-3 is also important for the induction of CD86. The induction of CD86, but not CD80 expression, by hBD-3 is blocked by an inhibitor of P2X7, suggestion that P2X7 is involved in some, but not all aspects of monocyte activation caused by hBD-3. We propose CD80 induction may be more dependent on TLR signaling caused by hBD-3 rather than P2X7-dependent signaling as seems to be the case for CD86 induction.

The ability of hBD-3 to cause P2X7 activation appears to be at least partially explained by ATP release in monocytes exposed to hBD-3. Our previous work demonstrated that hBD-3 can cause membrane damage in monocytes [103] as indicated by increased PI staining in hBD-3 exposed cells. Thus, membrane damage caused by hBD-3 may result in ATP release from cells. It must also be noted that pannexin channel activation by hBD-3 could also explain these observations. Pannexin channel activation has been implicated in ATP release from cells [124]; however, pannexin channels are not known to be permeable to PI. Interestingly, we have not found a tight correlation between PI staining and CD86 induction in monocytes treated with hBD-3, suggesting that either membrane damage, which we have measured with PI uptake, is not reflective of the damage resulting in ATP release, or that there may be an alternative mechanism responsible for ATP release. In our studies, the ATP detected in cell culture supernatants increased 1 h after stimulation of cells with hBD-3 and subsequently declined. The

63 decrease in ATP may be due to the activity of surface ectonucleotidases such as CD39

[61]. Prolonged activation of P2X7 by ATP causes cell death, especially when adding exogenous ATP to cells, it is possible that the activity of those ectonucleotidases would protect cells from hBD-3 induced ATP release and would be the reason why prolonged treatment with hBD-3 did not induce an increase in cellular death [103].

Because CD86 and CD80 are such important co-stimulatory molecules in antigen presentation [44], differences in the accessibility of one versus the other has consequences for adaptive immune function and response [48, 50, 51]. Specifically, blockade of CD86 leads to increased Treg function [49] and a Th2 bias [50]. This would imply that increased CD86 binding to CD28 on Treg cells will inhibit their suppressive functions and allow an inflammatory state to persist.

ATP is released from damaged and apoptotic cells and acts as a chemotactic agent for phagocytic cells [61]. ATP will also enhance the release of IL-1β and IL-18 in monocyte/macrophages that are pre-exposed to LPS. This activity is dependent on ATP signaling through the ligand-gated ion channel P2X7 [68]. Here, we show that activation of the P2X7 receptor by ATP has important biological activity even in the absence of

LPS priming. Exposure of monocytes to ATP appears to result in the induction of CD86 mRNA and cell surface expression. Only a brief exposure (roughly 30 minutes) to high concentrations of ATP is required for this effect. Thus, transient periods of ATP exposure, such as might occur in monocytes infiltrating damaged tissue, could lead to modified maturation and function of these cells.

Another antimicrobial peptide, LL-37, has also been shown to interact with and activate P2X7 [122, 125, 126]. Previous data from our lab show that similar to ATP

64 stimulation, LL-37 induces CD86 but not CD80 surface expression in monocytes [121].

This effect may stem, in part, from the ability of LL-37 to interact directly with P2X7, as suggested by data from other investigators [122]; however, our data indicated that apyrase also partially blocked the activity of LL-37, indicative of ATP-mediated activation. The differences in our results to those previously reported may stem from the different methodologies used in each study. In previous studies, monocytes were positively selected and stimulated with LPS prior to treatment with LL-37. IL-1β processing and release was used in these studies as the outcome. In contrast, we stimulated PBMCs overnight with LL-37 and used CD86 up-regulation as the outcome.

In addition, Elssner et al. used 10-20 µM LL-37 in their experiments compared to the 3.9

µM LL-37 that we used to stimulate cells. It is conceivable differences in LL-37 concentration may have influenced potential interactions with P2X7.

Figure 3.1. Blocking P2X7 inhibits CD86 induction on monocytes. PBMC were isolated from whole blood, incubated overnight in medium alone (solid gray histogram; D, E, G, H, J, and K), in medium + AZ (400 nM; solid black histogram; D and E), in medium + hBD-3 (20 µg/ml; solid black histogram; G and H), in medium + ATP (3 mM; solid black histogram; J and K), in medium + hBD-3 + AZ (dashed black histogram; G and H), or in medium + ATP + AZ (dashed black histogram; J and K) and then stained with CD14, CD80, CD86 antibodies and PI. Cells were analyzed by flow cytometry. Cellular debris was gated out via forward scatter-area (FSC-A) vs. side scatter-area (SSC-A) (A). Doublets were gated out via FSC-

A vs. forward scatter-height (FSC-H) (B). Live monocytes were identified as CD14+ and PIneg/dim (C, F, I). CD80 staining (D, G, and J) and CD86 staining (E, H, and K) of these cells is shown. Pictured are data representative of six experiments.

Figure 3.2. Inhibition of P2X7 partially blocks induction of CD86, but not CD80, in monocytes treated with hBD-3. PBMCs were isolated as above and treated overnight in medium alone, medium supplemented with hBD-3 (20 µg/ml), or medium supplemented with AZ and hBD-3. The following day, cells were stained and analyzed by flow cytometry using an identical gating strategy as Figure 3.1 with CD14+PIneg/dim cells being analyzed for activation. Addition of AZ to cultures had little effect on the hBD-3 induced CD80 induction (A; P=0.1994) whereas it inhibited CD86 induction (B; P=0.0087). Shown are data from six donors.

Figure 3.3. ATP up-regulates CD86 on monocytes by activating P2X7. PBMCs were isolated from whole blood and incubated overnight in medium alone, medium supplemented with ATP, or medium supplemented with AZ and ATP. Cells were then stained for flow cytometric analysis. ATP treatment was capable of inducing CD86 expression which could be blocked by addition of the P2X7 inhibitor, AZ. Shown are data from six donors.

Figure 3.4. Lowering ATP concentration abrogates CD86 up-regulation. PBMC were isolated and incubated overnight in medium alone (gray histogram), or medium supplemented with 3mM ATP (black histogram), 1.5mM ATP (red histogram), or 750µM (blue histogram). The following day, cells were harvested and stained for flow cytometric analysis. Above histogram is representative of two experiments.

Figure 3.5. Overnight up-regulation of CD86 occurs with ATP stimulation of between 15 and 30 minutes. PBMC were isolated and incubated in medium alone (red histograms) or medium supplemented with 3mM ATP (blue histograms). At 15 minutes post stimulation (A) or 30 minutes post stimulation (B) cells were harvested, washed, and re-plated. The next day, cells were harvested and stained for flow analysis. Above histograms are representative of six experiments.

Figure 3.6. Increased expression of CD86 occurs more than 6 hours after ATP stimulation. PBMC were isolated from whole blood and incubated in medium alone (red histograms) or medium supplemented with 3mM ATP (blue histograms) for 1 hour (A), 3 hours (B), 6 hours (C) or overnight (D). Cells were then harvested and stained for flow cytometric analysis. Above histograms are representative of two experiments.

5 Increase ofCD86 0 Fold HBD3 ATP

Figure 3.7. HBD3 and ATP stimulation increased CD86 mRNA expression. PBMC were isolated and incubated overnight in medium alone, medium supplemented with 20 µg/ml hBD-3, or medium supplemented with 3mM ATP. Cells were harvested and RNA was isolated and converted to cDNA. qPCR was used to analyze the amount of expression of CD86 between the culture conditions with GAPDH as the normalization control. Both hBD-3 and ATP increase CD86 RNA.

Figure 3.8. Inhibition of hBD-3 mediated CD86 induction by apyrase. PBMC were isolated from whole blood, incubated overnight in medium alone (solid gray histogram; A and B), in medium + Apyrase (2.5 U/ml; solid black histogram; A), in medium + hBD-3 (20 µg/ml; solid black histogram; B), in medium + hBD-3 + Apyrase (dashed black histogram; B) and then stained with CD14, CD80, CD86 antibodies and PI. Cells were analyzed by flow cytometry. Gating strategy as above with CD86 staining shown. Pictured are data representative of five experiments.

Figure 3.9. Apyrase treatment abrogates LL37-induced CD86 expression while having little effect on

Pam3CSK4-induced CD86 expression. PBMC were isolated and incubated overnight in medium alone (black histograms), medium supplemented with 20 µg/ml LL37 (A, red histogram), medium supplemented with apyrase (2.5 U/mL) and 20 µg/ml LL37 (A, blue histogram), medium supplemented with 500 ng/ml

Pam3CSK4 (B, red histogram), or medium supplemented with apyrase and 500 ng/ml Pam3CSK4 (B, blue histogram). The following day, the cells were harvested and stained for flow cytometric analysis. Above histograms are representative of five experiments.

Figure 3.10. Evidence for ATP release into the supernatant 1 hour post hBD-3 stimulation. PBMCs were isolated and incubated in medium alone (left set of bars) or medium supplemented with 20 µg/ml hBD-3 (right set of bars). At the specified time points, the supernatant was removed. After pelleting the cells, the cell free supernatant was removed and boiled for five minutes to inactivate any ATPases and the ATP concentration was quantified using a luciferase based system. (A) illustrates the results from Donor 1 and (B) illustrates the results from Donor 2.

CHAPTER 4:

GENERAL DISCUSSION AND FUTURE DIRECTIONS

4.1 Structure/Function activity of hBD-3 variant molecules

Data examined in this thesis provide evidence that hBD-3 is capable of activating monocytes as well as inducing membrane damage and repair in these cells. To further understand these processes, it is important to determine the structural requirements of hBD-3 that account for its diverse biological activities. Using variant hBD-3 molecules, it may be possible to dissociate the ability of hBD-3 to activate cells from the ability of hBD-3 to cause membrane damage. Such variants may be useful for future studies of hBD-3 adjuvant activity.

To probe the structural requirements of hBD-3 activity, variant molecules were generated by recombinant technology using published methodology (Table 4.1) [38].

Briefly, variants were generated through site-directed mutagenesis and high fidelity PCR of a plasmid coding for the native hBD-3 molecule. BL21 (DE3) cells were transformed with the resulting plasmid. The coding region was sequenced and verified. Expressed proteins, which disulfide bond in the culture conditions, were then purified via RP-HPLC and their molecular weight was verified with mass spectrometry. Isolated PBMCs were incubated overnight in medium alone, in medium supplemented with variant hBD-3 molecules (Table 4.1), or with either recombinant or synthetic wild-type hBD-3. Cells were examined for PI staining and co-stimulatory molecule surface expression by flow cytometric analysis. In general, variant hBD-3 molecules caused less PI uptake than the native molecule. Variant molecules also tended to cause less induction of CD80 expression compared to wild-type hBD-3. In contrast, CD86 induction was still observed

77 in cells stimulated by several of the hBD-3 variants, particularly those molecules with cysteine mutations (Figure 4.1 and Figure 4.2 respectively). Notably, deletion of the first ten amino acids in the N-terminus, but not the first five amino acids, resulted in nearly complete loss of detectable hBD-3 activity. Also, deletions of amino acids 6-10 also resulted in loss of activity (both induction of co-stimulatory molecules and induction of

PI uptake (Figure 4.2). Secondary structure prediction by PORTER

(http://distill.ucd.ie/porter/) indicated that molecules with 1-10 and 6-10 deletions would be expected to lose the N-terminal helical structure of native hBD-3 (Table 4.2), suggesting that the helix may play an important role in hBD-3 activity.

Interestingly, as Figure 4.2 shows, certain hBD-3 variants (CM1, CM3, CM1-6, and 1-5del) were relatively good inducers of CD86 expression but relatively poor inducers of CD80 expression when compared to native hBD-3. Based on our data in

Chapter 3, we hypothesized that these variants may maintain the capacity to induce activation of monocytes via P2X7. To test this, we incubated cells overnight with these variants in the presence or absence of the P2X7 inhibitor AZ. Flow cytometric analysis indicated that treatment with AZ inhibited the ability of variant hBD-3 molecules to induce surface expression of CD86, providing evidence that P2X7 activation is important in this activity (Figure 4.3). Notably, as we stated in Chapter 3, we did not find evidence that AZ affected CD86 expression that was modulated by LPS or by Pam3CSK4. While we have shown that these molecules do not cause the same amount of PI uptake that native hBD-3 causes (Figure 4.1), it will be important to determine if these variants cause release of ATP, and to ascertain if their ability to induce CD86 expression is affected by extracellular ATP depletion with apyrase treatment.

Notably, even an hBD-3 variant with all six cysteines replaced induced CD86.

Thus the tertiary structure of hBD-3 is not likely to be important in the activation of

P2X7. LL-37, which is reported to activate P2X7 directly, requires a helix structure to function [126]. It is possible that the hBD-3 secondary helix structure is important in its ability to activate P2X7 since the variants with predicted structures that retain the N- terminal helix (Table 4.2) also retain the ability to activate CD86 through P2X7 (Figure

4.3). Although our data in Chapter 3, which demonstrate that apyrase partially blocks the induction of CD86 by hBD-3, suggests indirect effects of hBD-3 on ATP release as an important determinant of P2X7 activation, it is still possible that hBD-3 interacts with

P2X7. Thus, we hypothesize that the helical structure of hBD-3 is important for ATP release leading to P2X7 activation.

Further studies with these variants are necessary to confirm the importance of the

N-terminal helix as well as to determine any potential these molecules have as vaccine adjuvants. Based on recombinant molecules that we have tested, we have observed some inter-batch variability in the activity of the hBD-3 variants to activate monocytes (data not shown). This may be the result of different isoforms of hBD-3 variants that may predominate in different batches. The CM1-6 variant (all six cysteines are mutated in this molecule so it cannot disulfide bond and therefore cannot have different isoforms) could be used to test this hypothesis. Multiple batches of CM1-6 would be generated and then tested for their activity. If there is variability in activation, then different isoforms are not likely to be the cause of the observed inter-batch differences. On the other hand, if different isoforms predominating between batches is the reason behind the observed variability, it will become necessary to chemically synthesize these molecules using

79 orthogonal protection of cysteines to ensure proper folding of the molecules and remove this inter-batch variance [28]. In addition, different isoforms of hBD-3 could play a role in vivo in inflammatory settings. Isolating hBD-3 from in vivo settings such as psoriasis or oral carcinoma, and then subjecting these molecules to mass mapping (via mass spectrometry) of the protein fragments generated by proteolytic digestion and Edman degradation, may allow for verification of disulfide topology [28]. Any differences in the types of isoforms present or differences in the relative amounts of each isoform would lend support to the idea that different isoforms play a role in inflammatory settings.

To verify the importance of the N-terminal helix for monocyte CD86 up- regulation, hBD-3 molecules could be designed with mutations that destroy the helix.

These variants could be tested as outlined above for their ability to induce CD86 expression on monocytes after overnight stimulation. As our data suggest that electrostatic charge interactions are important in monocyte membrane disruption mediated by the native hBD-3 molecule, variants that possess more positively charged amino acids than native hBD-3 may have more activity in this model. This could also be tested to ascertain the importance of electrostatic interactions and membrane damage in hBD-3-mediated P2X7 signaling defined above.

4.2 Possible interaction between CD14 and hBD-3

Interestingly, TLR2 signaling by a soluble factor released by Group B

Streptococci requires CD14 [55]. CD14 is a 55 kDa protein best known as being required for LPS/LBP binding and signaling through TLR4 [127]. Despite having no intracellular signaling domain, CD14 has been shown to be associated (via co-immunoprecipitation) with the cytoskeletal signaling protein moesin, even in the absence of stimuli [128]. This

80 hints that CD14 could play a role in signal transduction even though it is a GPI-anchored protein with no intracellular domain. Engagement of CD14 with specific ligands such as ceramide or LPS, will induce its clustering with other receptors in cholesterol rich membrane rafts creating different receptor groups depending on the ligand [129].

Preliminary studies indicated that hBD-3 caused the clustering or "capping" of CD14 on monocytes whereas this capping was not present on cells incubated with LPS or

Pam3CSK4 (Figure 4.4). This observation was replicated in cells from three different donors and subsequent experiments showed that this capping occurred within one hour of hBD-3 stimulation (data not shown). To determine if CD14 played a role in the hBD-3 activation of monocytes, cells were pre-incubated with a CD14 neutralization antibody before overnight incubation with hBD-3. These studies were inconclusive since the isotype control caused activation of on its own, possibly due to Fc receptor cross-linking

(Figure 4.5). Alternative approaches that disrupt CD14 expression on the surface of monocytes such as exogenous treatment with phospholipase C, which cleaves GPI- anchored proteins from the cell surface, may be useful in future studies in order to address this question.

We also tested the effects of adding soluble CD14 to cells treated with hBD-3 to ascertain if this might affect monocyte activation. Previous studies have shown that high levels of soluble CD14 in serum (3-4 µg/ml) inhibits LPS mediated activation of APCs while low levels of soluble CD14 (0.5 µg/ml) enhances LPS induced activation of APC

[130-132]. Presumably, this is related to a direct interaction of sCD14 with LPS that promotes CD14/TLR4 interactions at low concentrations but sequesters LPS from TLR4 at high concentrations. Preliminary dose response studies indicated that treatment with

81 soluble CD14 at a concentration of 10 µg/ml inhibited hBD-3 mediated CD86 expression

(Figure 4.6B) but when cells were treated with lower concentrations of CD14 (5 µg/ml,

2.5 µg/ml, or 1.25 µg/ml), there was an enhancement of CD86 expression (Figure 4.6C-

E). Further experiments need to be performed to confirm these observations and to assess the possibility that hBD-3 may directly interact with CD14 or co-localize with

CD14 on the surface of monocytes.

If the effects described above can be confirmed, it will be important to determine if hBD-3 binds directly to CD14 and if this interaction occurs at high or low affinity. To determine binding affinity, the Kd of the interaction must be known, or because it is the reciprocal for Kd, Ka can also be used. To assess hBD-3/CD14 interactions, accurate measurements of both Ka and Kd can be determined by utilization of the surface plasmon resonance (SPR) technique. CD14 would be immobilized on the surface of an SPR crystal and hBD-3 would be inserted via a microflow system so it can associate with

CD14 for a time which allows a Ka determination to be made (association with the bound

CD14 causes a change in the electromagnetic waves at the crystal's surface which change based on association rate). Buffer would then be inserted to dissociate hBD-3 from

CD14 allowing for Kd to be determined, and binding affinity to be known.

Unfortunately, hBD-3/CD14 interactions may be more complex than binding affinity can illustrate. As described above, soluble CD14 can interact with hBD-3 and modulate monocyte responses, either by inhibiting activation or enhancing activation. This activity complicates experiments designed to probe hBD-3/CD14 interactions.

Nonetheless, the possibility that hBD-3 interacts with CD14 is especially intriguing. It is possible that CD14 could serve as a shuttle for hBD-3, bringing it to

TLR1/2 to initiate the signaling cascade via a mechanism similar to Group B streptococcus/TLR2 interactions [55]. In Chapter 2, we performed experiments in which we removed cholesterol from the membranes of PBMCs before treatment with hBD-3 to test whether cholesterol was protective to cells. It would be interesting to see if the removal of cholesterol would have an effect on monocyte activation by hBD-3. Because cholesterol helps ensure plasma membrane fluidity, its removal could hamper monocyte activation, especially if CD14 is shuttling hBD-3 to either TLR1/2, because the shuttling would be difficult without a fluid membrane. Unfortunately, because up-regulation of

CD86 and CD80 as a readout of monocyte activation requires overnight incubation, there may be an overabundance of cell death in culture because the loss of cholesterol is deadly to cells over a long enough period of time.

4.3 Downstream T cell activation by hBD-3-primed APCs

Activation of the adaptive branch of the immune system requires activation of T cells. This activation is accomplished by presentation of antigens to T cells by antigen presenting cells (APCs) along with binding of certain accessory molecules on T cell surfaces by accessory proteins on the APC's surface and the release of certain cytokines by the APCs. The type of adaptive response is determined by the combination of these released cytokines and up-regulated accessory proteins. Monocytes treated with hBD-3 overnight exhibit an increased surface expression of the co-stimulatory molecules CD80,

CD86, and CD40 [15]. CD80 and CD86 are integral co-stimulatory molecules for the activation of T cells and depending on their levels are capable of skewing T cell responses. CD86 blockade leads to a Th2 response while also increasing CD4+CD25+ regulatory T cells in the periphery [50]. T cells stimulated by cells without CD80 lack a

83 strong IL-2 production response which can be explained by IL-2 promoter transcription activity being greater when stimulated with CD80 as opposed to CD86 [48]. Activation of memory T cells and cytotoxic T lymphocytes also requires CD86 signaling [51, 133].

Thus, it will be important to characterize the effects of native hBD-3 and mutant hBD-3 molecules on APC function and T cell response to antigens.

To examine the effect hBD-3 on downstream immune activation, an allogeneic T cell model was used. Monocytes were isolated from whole blood and some were treated with hBD-3 overnight. The next day, T cells were isolated from a different donor and stained with CFSE. These T cells were then mixed with the monocytes after washing out the hBD-3 and together these cells were incubated at 37°C for 7 days before staining them for flow cytometric analysis to measure CFSE dye dilution (cell proliferation) and

CD25 (cell activation). CD25 is part of the IL-2 receptor and is a marker of T cell activation [134]. Preliminary data suggest that T cells stimulated by monocytes which were incubated with hBD-3 divide more and up regulate CD25 to a greater degree than those T cells stimulated by monocytes that did not get incubated with hBD-3 (Figure 4.7).

Further studies that investigate this activity of hBD-3 and which include studies of antigen recall responses will be important to determine the potential for hBD-3 to enhance APC activation of T cells.

As a component of these studies, it will be important to determine the helper T cell response profile elicited when hBD-3 treated monocytes or myeloid DCs are used to activate T cells. Using the allogeneic method described above, T cells could be exposed to monocytes incubated in medium alone or medium supplemented with hBD-3. After seven days of stimulation, the supernatants could be tested by ELISA for changes in the

84 levels of various cytokines including Th1 cytokines (IFN-γ), Th2 cytokines (IL-4 and IL-

5), and Th17 cytokines (IL-17). Cytokines associated with Treg cells may also be important to investigate (IL-10 and TGF-β).

Eventually, adjuvant testing of hBD-3 will need to be carried out in vivo. As stated above, a mouse model may be useful, particularly for studying the importance of

P2X7 activation in potential adjuvant effects of hBD-3. Mice could be immunized with vehicle alone, antigen alone, antigen plus hBD-3, or antigen plus a well-defined adjuvant

(alum) primed and boosted with intramuscular injections. At various time points, immune responses to the antigen can be measured with ELISPOT assays or other immunological studies. The use of P2X7 knock-out animals for comparison to wild-type mice could provide important insight into the role of this pathway in the adjuvant activity of hBD-3 as defined in vivo.

4.4 Activation of P2X7 by hBD-3

In Chapter 3 above, we have shown that the up-regulation of CD86 that is induced by hBD-3 is dependent upon P2X7 receptor activation. Our results suggest that the activation of P2X7 is at least partially explained by release of ATP into the extracellular space after cells are exposed to hBD-3. This activation then causes the increased CD86 expression that may result from increased mRNA transcription although this observation needs confirmation with additional studies.

From the work that we have described in Chapter 2, we have shown that hBD-3 is capable of causing membrane disruption to monocytes but not B cells or T cells and that this cell-type selectivity is most likely due to electrostatic charge interactions between the positively charged antimicrobial peptide and the negatively charged phospholipid,

85 phosphatidylserine, which is present in greater quantities on the outer surface of monocytes compared to B cells and T cells. When cells were pre-incubated with molecules that bind PS, they were protected from hBD-3-induced membrane damage.

And when B cells and T cells were treated with staurosporine (a molecule that induces apoptosis, and therefore PS flipping), they underwent hBD-3-induced membrane disruption as well. Most damaged monocytes are able to repair themselves, but a subset of cells cannot recover and die. It is possible that this damage to the plasma membrane is what allows the ATP to leak into the extracellular milieu and cause the activation of the

P2X7 receptor on the surviving monocytes.

In the future, it will be important to elucidate how P2X7 activation is able to lead to the increased mRNA of CD86. Both calcium flux and potassium flux have been linked to inflammasome assembly and IL-1β processing and release [135]. We have preliminary data in which we treated cells with ionomycin overnight and then stained for CD86.

Ionomycin is a molecule that induces calcium flux into cells, resulting in increases in intracellular calcium concentration. Those cells treated with ionomycin overnight had an increased expression of CD86 on their surface compared to those cells incubated in medium alone (Figure 4.8). This suggests that calcium flux may be sufficient to cause induction of CD86 expression.

Our preliminary data suggest that induction of CD86 by ATP or by hBD-3 is transcriptionally regulated and can occur in the absence of CD80 induction when cells are stimulated with variant hBD-3 molecules or with ATP alone. Therefore, we searched for differences in the promoters of CD80 and CD86 using the SABiosciences Champion

ChIP Transcription Factor Search Portal

(http://www.sabiosciences.com/chipqpcrsearch.php). One distinction between the CD86 and CD80 promoters are the cAMP response element binding protein (CREB) binding sites which exist for CD86 but not CD80. As a simplified sequence of events, ligand/receptor interactions at the cellular surface lead to the production of a second messenger such as cAMP. This second messenger activates protein kinases which translocate to the nucleus where activation of the CREB protein by the kinase occurs.

The activated CREB binds to cAMP responsive elements (CREs) in gene promoter regions [136]. Ca2+ is also a second messenger in this pathway and CREB activation has been shown to increase CD86 gene transcription in a monocytic cell line [137] which makes this a plausible pathway by which P2X7 activation induces CD86 expression.

As a potential future direction, studies of P2X7 knockout mice may be useful to examine possible in vivo and ex vivo interactions between hBD-3 and P2X7 [120]. Bone marrow derived macrophages could be generated from these mice and treated with hBD-

3. Cells could be assessed for induction of co-stimulatory molecules including surface levels of CD80 and CD86. We would hypothesize that cells from wild-type mice, but not mice lacking P2X7, would respond to hBD-3 stimulation with CD86 induction. It is important to note that there may be species differences that hinder this straightforward testing of hBD-3 with mouse derived cells. A mouse model may not recapitulate all aspects of hBD-3 activity that have been defined in human cells. HBD-3 has been shown to be chemotactic to mouse cells expressing CCR6 and CCR2 [59, 138], but hBD-3 was not able to induce TNF-α expression in mouse cells [15]. In other models, hBD-3 has been shown to have anti-inflammatory activity [139, 140]. In particular, hBD-3, similar to other antimicrobial peptides, can block the activity of LPS. In these studies, hBD-3

87 alone did not cause activation of mouse cells [139, 140]. One clear difference in these models compared to our work is the use of mouse cells for the majority of the data generated in the other studies. The read-outs involved TNF- α production which in our hands is not induced in mouse cells by hBD-3 [15] and inconsistently induced in human cells [43]. Moreover, the concentrations of hBD-3 used in these other studies were lower than the concentrations used in our studies. In addition, they use the human monocytic cell line, THP-1, to measure activation. In our hands, THP-1 cells have inconsistent responses to hBD-3-stimulation (measured by induction of co-stimulatory molecules), which may be related to low levels of TLR1 that we have detected (data not shown).

When using human peripheral blood monocyte derived macrophages, it must be noted that Semple et al. used sodium citrate anticoagulant coated tubes when drawing blood from donors. Our studies have been performed with heparin or EDTA coated tubes, and it is possible that different anti-coagulants may affect subsequent experiments. As an alternative to hBD-3, its mouse ortholog, mouse β-defensin-14 (mBD-14), could be used instead. The mouse protein shares 68% identity with its human counterpart and is also chemotactic to cells expressing CCR6 and CCR2 [41, 138]. Studies of mBD-14 activity in mouse cells will therefore be important to consider. Even if mouse receptors are different enough from human receptors that hBD-3 is unable to activate mouse TLR1/2, if mouse monocytes express enough PS on their surface, and if our hypothesis that membrane damage leads to leaked ATP which activates P2X7 is correct, then it activation of mouse monocytes by hBD-3 is still possible.

4.5 HBD-3 as a link between innate and adaptive immunity

HBD-3 production is increased in inflammatory conditions. Particularly high

88 levels of hBD-3 have been described in vivo in the setting of psoriasis and in oral carcinoma. It is thought that hBD-3 expression may be playing an important role in each of these disease states by mediating a variety of biological effects including the modulation of myeloid cell migration and function.

HBD-3 was originally isolated from the skin of patients with psoriasis, an inflammatory disease characterized by “raised, well-demarcated, erythrematous oval plaques with adherent silvery scales,” an inflammatory infiltrate consisting of T cells, dendritic cells, and macrophages, and an increased number of tortuous capillaries [93].

The pathology of the disease includes a lack of T effector cell suppression by CD25+

Treg cells [93]. This effect is likely due to a combination of increased IL-6 secretion, which makes effectors harder to suppress [141], and a lowering of the suppressive ability of Tregs [142]. In psoriasis, hBD-3 may be contributing to the recruitment of myeloid cells into tissues and also modulating the function of these cells to contribute to inflammation. The effects of hBD-3 on myeloid cells may also have implications for

Treg function in psoriasis. HBD-3 can stimulate the secretion of IL-6 from keratinocytes

[13] and monocytes [43], which could contribute to the observed resistance of T effector cells to suppression by Tregs. In addition, it is possible that hBD-3 could have effects on

Tregs via monocyte/Treg interactions. HBD-3 has been shown to increase CD86 and

CD80 on the surface of monocytes [15] and CD86, in particular, is implicated in Treg function since CD86 blockade has been linked to increased Treg suppressive function, both in vivo [50] and in vitro [49]. Thus, binding of CD86 to CD28 on the surface of

Treg cells inhibits their suppressive function. It is possible that hBD-3 stimulation of infiltrating monocytes to express high levels of CD86 could play a role in the impaired

Treg function observed in psoriasis by Soler et al.

In addition, hBD-3, in complex with self-DNA, has been shown to activate plasmacytoid dendritic cells (pDCs) to produce IFN-α through TLR9 stimulation [94].

IFN-α and pDCs both play a prominent role in psoriasis [93]. Clinical observations implicate IFN-α as a potent inducer of psoriasis [143] and IFN-α signaling pathway genes and IFN-α-inducible genes are all highly overexpressed in lesional skin [144]. It has been shown that pDCs, which infiltrate the skin of psoriasis patients, produce IFN-α early during disease [145]. Therefore, activation of pDCs to produce IFN-α is another pathway that hBD-3 may possibly use to contribute to psoriasis disease.

Another example of a disease in which hBD-3 expression is markedly increased is oral carcinoma. Immunohistochemistry studies have demonstrated that hBD-3 is expressed at high levels in oral carcinoma tissue compared to surrounding tissue [22].

This abundance of hBD-3 has been linked with macrophage chemotaxis into the tumor microenvironment [59]. It has been proposed that these tumor-associated macrophages

(TAMs) aid in tumor growth and survival by stimulating angiogenesis [146]. HBD-3 has also been shown to stimulate monocytes to release the angiogenic factor vascular endothelial growth factor (VEGF) [121], so it may contribute to angiogenesis itself. In addition to bringing in TAMs and possibly contributing to angiogenesis, hBD-3 is also known to stimulate the release of IL-6 from macrophages [59]. IL-6 has been linked to tumor promotion and survival [147, 148] so hBD-3 may play role in tumor survival via this mechanism as well.

Part of hBD-3’s diverse biological activities may stem from its promiscuous binding properties, which lead to interactions with a variety of receptors summarized in

(Table 4.3). Several receptors that are ligands for hBD-3 affect cell migration. CCR2, a chemokine receptor on monocytes, macrophages, and neutrophils, is a receptor that hBD-

3 uses to chemoattract these cell types [41]. For immature dendritic cells and T cells,

CCR6 is the chemokine receptor that hBD-3 can signal through to initiate chemotaxis

[28]. HBD-3 can also bind with CXCR4 and cause its internalization without activating receptor signaling [35].

Additional receptors that hBD-3 may interact with include molecules that regulate monocyte/macrophage activation. For example, melanocortin-1 receptor (MC1R) is the receptor for alpha-melanocyte stimulating hormone (α-MSH), and when macrophages are stimulated with it, α-MSH has been shown to have anti-inflammatory properties [149].

HBD-3 is known to be an antagonist for this receptor [60], so it is plausible that hBD-3 is capable of blocking the anti-inflammatory activity of α-MSH. Furthermore, studies from our group have indicated that hBD-3 can activate a TLR1/2 signaling pathway in monocytes and myeloid dendritic cells, although it is not yet clear if this is via a direct or an indirect mechanism. Activation of TLR1/2 leads to signaling via MyD88, IRAK-1, p38, and NFκB to stimulate the induction of CD80, CD86, and CD40 on the cell’s surface [15, 43]. In addition, while hBD-3 treatment has been shown to increase levels of phosphorylated p38 and phosphorylated ERK1/2 in monocytes, only the inhibition of p38 had an effect on CD86 surface levels of hBD-3 treated cells [43]. Overall the findings suggest that the interactions of hBD-3 with various cell subsets are likely to be complex, involving multiple receptors and cell signaling pathways. Our results expand on this understanding as we have now demonstrated yet another mechanism involved in hBD-3- mediated activation of APCs, namely activation of cells via the P2X7 receptor.

We therefore propose the following model of hBD-3 activity (Figure 4.9). HBD-

3 and other chemokines induce monocytes to migrate from the periphery to sites of inflammation. In tissues, these cells may encounter high concentrations of hBD-3 which leads to activation via various receptors including TLR1/2. Tissue damage at these sites, which may also include damage inflicted by hBD-3, could cause the efflux of ATP into the extracellular space. ATP at sufficient concentrations binds to the P2X7 receptors present on monocytes and activates these cells. Cations are able to flow through the opened channel which signal through an as yet undetermined pathway (which may include a protein kinase and CREB) to up-regulate the mRNA levels of CD86. This mRNA is translated into CD86 protein and is expressed on the surface of the monocyte about 8 hours after the initial hBD-3 stimulation. Thus, we propose that P2X7 may play an important role in the biological activity of hBD-3 at inflammatory sites. Moreover, from the work of other groups, hBD-3 may be interacting with extracellular nucleic acids in damaged tissues and causing activation of pDCs through TLR9 stimulation, and production of IFN-α [94].

Table 4.1. Wild-type and Mutant Sequence. The table above shows the sequence of the synthetically produced wild-type hBD-3 (sHBD-3), recombinantly produced hBD-3 (rHBD-3), and all six mutants in addition to their overall charge and the number of hydrophobic residues present in the molecule (red letters indicate substitutions and # indicate deleted amino acids).

Figure 4.1. Mutant hBD-3 molecules cause less PI uptake than the native molecule. PBMCs were isolated from whole blood and incubated overnight in medium alone or medium supplemented with the wild-type or mutant molecules. The next day, cells were stained before flow cytometric analysis. The mutants did not cause the same amount of PI dim cells as the wild-type molecule though some did cause similar amounts of PI high cells. The bar graphs illustrate the percentage of cells that are either PI dim or PI high. Shown is the median of four experiments with interquartile ranges.

Figure 4.2. Differential effects of mutant hBD-3 molecules on CD80 and CD86 induction. PBMCs were isolated from whole blood and incubated overnight in medium alone or medium supplemented with the wild-type or mutant molecules. The next day, cells were stained before flow cytometric analysis. Most of the mutants retained some ability to induce CD86 while losing the ability to induce CD80. The bar graphs illustrate the change in MFI from the un-stimulated control. Shown is the median of four experiments with interquartile ranges.

Name Structure sHBD-3 CCCCCCCCCHHHHHCCEEECCCCCCCEEEEECCCCCCCEEEEECC rHBD-3 CCCCCCCCCHHHHHCCEEECCCCCCCEEEEECCCCCCCEEEEECC CM1 CCCCCCCCCHHHHHCCEEECCCCCCCEEEEECCCCCCCEEEEECC CM3 CCCCCCCCCHHHHHCCEEECCCCCCCEEEEECCCCCCCEEEEECC CM1-6 CCCCCHHHHHHHHHCCEEECCCCCCCEEEEECCCCCCCEEEEECC 1-5del CCCCHHHHHCCEEECCCCCCCEEEEECCCCCCCEEEEECC 6-10del CCCCCCCHCCCEEECCCCCCCEEEEECCCCCCCEEEEECC 1-10del CCCCCCEEECCCCCCCEEEEECCCCCCCEEEEECC

Table 4.2. Predicted structure of mutant hBD-3 molecules. The sequences of the mutant hBD-3 molecules as well as the sequence of the native molecule were imputed into PORTER (http://distill.ucd.ie/porter/) which predicted the secondary structural characteristics of the molecules. C is random coil structure, H is helix structure, and E is beta-sheet structure.

Figure 4.3. Blockade of P2X7 inhibits mutant induced up-regulation of CD86. PBMC were isolated and plated in a 24-well plate. Cells were incubated overnight in medium alone (A, B; grey histogram), medium + CM3 (A; black, solid line histogram), medium + CM3 and AZ (A; black, dashed histogram), medium + CM1-6 (B; black, solid histogram), or medium + CM1-6 and AZ (B; black, dashed histogram). The following day, the cells were stained for flow cytometric analysis. Above histograms are representative of three experiments.

Figure 4.4. HBD-3, but not Pam3CSK4 or LPS, causes capping of CD14. Monocytes were enriched during PBMC prep using RosetteSep Human Monocyte Enrichment Cocktail. Cells were left in PBS alone or PBS + hBD-3 (20 µg/mL), Pam3CSK (500 ng/mL), or LPS (50 ng/mL) for 1 hour before being imaged. Monocytes treated with hBD-3 exhibited capping of CD14 whereas those cells treated with Pam3CSK or LPS did not have this capping. Picture is representative of 3 experiments. Green = CD14; scale bar = 15 µm

Figure 4.5. Due to activation by the isotype control, it is not possible to determine if CD14 neutralization affects hBD-3 activation of monocytes. PBMC were isolated and plated in a 24-well plate. Cells were incubated overnight in medium alone (A, B, C; red histogram), medium + isotype antibody (A;orange histogram), medium + neutralizing CD14 antibody (A; green histogram), medium + 20µg/ml hBD-3 (B; blue histogram), medium + 50ng/ml LPS (C; blue histogram), medium + hBD-3 + isotype (B; orange histogram), medium + LPS + isotype (C; orange histogram), medium + hBD-3 + neutralizing antibody (B; green histogram), or medium + LPS + neutralizing antibody (C; green histogram). Above histograms are representative of three experiments.

Figure 4.6. Different doses of sCD14 have different effects on hBD-3 induction of CD86. PBMC were isolated and incubated overnight in medium alone (A, B; black histograms), medium supplemented with 20 µg/ml hBD-3 (B, C, D, E; red histogram), medium supplemented with 10 µg/ml sCD14 (A, red histogram), medium supplemented with 5 µg/ml sCD14 (A, orange histogram), medium supplemented with 2.5 µg/ml sCD14 (A, green histogram), medium supplemented with 1.25 µg/ml sCD14 (A, blue histogram), medium supplemented with hBD-3 and 10 µg/ml sCD14 (B, orange histogram), medium supplemented with hBD-3 and 5 µg/ml sCD14 (C, blue histogram), medium supplemented with hBD-3 and 2.5 µg/ml sCD14 (D, blue histogram), or medium supplemented with hBD-3 and 1.25 µg/ml sCD14 (E, blue histogram). The following day, the cells were harvested and stained for flow cytometric analysis.

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Figure 4.7. Enhanced allogeneic stimulation of T cells by hBD-3 treated monocytes. Monocytes were purified from whole blood and incubated overnight in medium alone or medium supplemented with 20 µg/ml of hBD-3. The following day, the hBD-3 was washed away and T cells purified from a separate donor were added. These cultures were incubated for seven days before being stained for flow cytometric analysis. Both CD4+ and CD8+ T cells exhibited increased activation by monocytes treated with hBD-3 (far right column) compared to monocytes incubated in medium alone (middle column). Baseline activation of T cells without monocytes in culture is found in the far left column.

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Figure 4.8. Overnight incubation of monocytes with ionomycin induces CD86 up-regulation. PBMC were isolated and incubated overnight in medium alone, medium supplemented with 20 µg/ml hBD-3, or medium supplemented with 0.5 µg/ml ionomycin. Cells were harvested the following day and analyzed via flow cytometry. Monocytes incubated with hBD-3 or ionomycin increased surface expression of CD86.

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Cellular Natural hBD-3 responsive hBD-3 concentration to Mechanism of receptor Tissue distribution hBD-3 signaling response Citation receptor ligand(s) cells elicit response activation by hBD-3

Monocytes, Lipoteicholic Macrophages, Dendritic Monocytes and Up-regulation of CD40, CD80, 5 µg/ml (960 nM) to 20 TLR1/2 acid and cells, T cells, B cells, Myeloid dendritic and CD86. Production and Unknown [15,28] µg/ml (3.9 µM) peptidoglycan Airway epithelial cells, cells release of IL-6, IL-8, and IL-1β and Keratinocytes

Immature dendritic cells CCR6+ HEK293 CCR6 MIP-3α Chemotaxis 10 ng/ml Direct [35] and memory T cells cells

Macrophages, THP-1 CCR2 MCP-1 Monocytes/macrophages cell line, Monocytes, Chemotaxis 100 ng/ml to 200 ng/ml Direct [35,43] Mono-Mac-1 cell line

CEM X4/R5 cell line, Internalization of CXCR4 without T cells, Monocytes, and 2.5 µg/ml (480 nM) to 20 CXCR4 SDF-1 THP-1 cell line, T calcium mobilization, ERK Direct [59] Epithelial cells µg/ml (3.9 µM) cells phosphorylation, or chemotaxis

Melanocytes and MC1R α-MSH Melanocytes Antagonist to α-MSH 100 nM Direct [60] Macrophages

Up-regulation of CD86 P2X7 ATP Monocytes/macrophages, Monocytes (potentially not a direct 20 µg/ml (3.9 µM) Indirect Chapter 3 Dendritic cells, T cells interaction) and B cells

Monocytes/macrophages, Microbial and Plasmacytoid Enhance self-DNA uptake, 2 µM hBD-3 with 10 Indirect (facilitates DNA TLR9 Dendritic cells, T cells [94] self nucleic acids Dendritic cells Promote IFN-α production µg/ml genomic DNA uptake) and B cells

Table 4.3. Cellular receptors hBD-3 can interact with. ATP, Adenosine triphosphate; α-MSH; alpha- melanocyte-stimulating hormone; CCR, CC-chemokine receptor; CXCR4, CXC-chemokine receptor 4; MC1R, Melanocortin 1 receptor; MCP-1, Monocyte chemotactic protein 1; MIP-3α, Macrophage inflammatory protein 3 alpha; SDF-1, Stromal cell-derived factor 1; TLR, Toll-like receptor

103

104

Figure 4.9. Proposed model of hBD-3 interactions in inflammatory settings. (A) HBD-3 acts as a chemotactic molecule for CCR6+ T cells and CCR2+ monocytes, helping to bring them to the inflammatory environment. (B) HBD-3 interacts with TLR1/2 on the surface of monocytes which eventually leads to increased surface expression of CD80, CD86, and CD40 in addition to the production of IL-6, IL-8, and IL-1β. (C) HBD-3 encounters a monocyte and interacts with its plasma membrane. HBD-3 perturbs the plasma membrane, which may allow cellular contents such as ATP to be released, and allows extracellular constituents such as PI into the cell. The released ATP interacts with, and activates P2X7 receptors to become permeable to cations including Ca2+. Through the change in calcium concentration, a protein kinase becomes activated and translocates to the nucleus where it activates CREB. Once activated, CREB binds to the promoter of CD86 and induces the increased transcription of CD86 mRNA which eventually leads to the up-regulation of CD86 on the surface of the monocyte. (D) HBD-3 antagonism of MC1R on the surface of macrophages may block α-MSH-induced suppression of lymphocyte proliferation. (E) Treg cells, which have interacted with hBD-3-activated monocytes (during antigen presentation), may have inhibited ability to suppress T effector cells due to high levels of CD86 on the surface of the hBD-3- activated monocytes. (F) HBD-3, in complex with nucleic acids present in the inflammatory environment, is taken up by plasmacytoid dendritic cells. The hBD-3/DNA complex then activates endosomal TLR9 which leads to increased production of IFN-α by the dendritic cell. Dashed arrows are proposed pathways and interactions.

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Figures 2.1 through 2.15 except 2.2, 2.4, and 2.5

106

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