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Immunomodulatory properties, mechanisms, and therapeutic potential of macrocyclic theta defensins

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Immunomodulatory properties, mechanisms, and therapeutic potential of macrocyclic theta defensins

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Content i

Immunomodulatory Properties, Mechanisms, and Therapeutic Potential
of Macrocyclic Theta Defensins

A dissertation submitted in partial satisfaction of
the requirements for the program of Medical Biology and the degree
Doctor of Philosophy in Medical Biology

by

Justin Blaine Schaal

The USC Graduate School
University of Southern California
August 2016

Dissertation Committee:
Professor André Ouellette, Chair
Professor Michael Selsted
Professor Paul Beringer
ii

TABLE OF CONTENTS

Page

LIST OF FIGURES iii

LIST OF TABLES iv

ACKNOWLEDGEMENTS v

CURRICULUM VITAE vi

ABSTRACT OF THE DISSERTATION viii

GENERAL INTRODUCTION 1

CHAPTER 1: Rhesus Macaque Theta Defensins Suppress Inflammatory Cytokines and
Enhance Survival in Mouse Models of Bacteremic Sepsis 6
Abstract 7
Introduction 8
Materials and Methods 10
Results 15
Discussion 30
References 34

CHAPTER 2: Macrocyclic Theta Defensins Modulate TNF Shedding by Inhibition of
Tumor Necrosis Factor-α Converting Enzyme 38
Abstract 39
Introduction 40
Materials and Methods 44
Results 49
Discussion 68
References 76

CHAPTER 3: A Retroevolutionary Strategy for Inducing Remission of Autoimmune
Arthritis using Macrocyclic θ-Defensins from Nonhuman Primates 82
Abstract 83
Introduction 84
Materials and Methods 87
Results 95
Discussion 110
References 114

GENERAL CONCLUSIONS 120

iii

LIST OF FIGURES
Page
Figure 1-1. Effects of RTD-1 on stimulated release of cytokines/chemokines. 17
Figure 1-2. RTD-1 inhibits the release of soluble TNF in whole blood. 19
Figure 1-3. RTD-1 is ineffective in neutralizing LPS. 20
Figure 1-4. Temporal analysis of TNF release from E. coli stimulated blood. 22
Figure 1-5. RTD isoforms differentially inhibit TNF release by E. coli- and
LPS-stimulated human blood and THP-1 monocytes. 24
Figure 1-6. High-dose RTD-1 injection site reaction in Balb/c mice. 26
Figure 1-7. RTD-1 increases survival in E. coli peritonitis and modulates
cytokines/chemokines. 28
Figure 1-8. RTD-1 increases survival in a mouse model of polymicrobial sepsis. 29
Figure 2-1. Covalent structure of θ-defensins. 43
Figure 2-2. RTD-1 suppress TNF release from buffy coat cells stimulated with
TLR agonists. 50
Figure 2-3. Inhibition of TNF release is dependent on extracellular RTD-1 and is
reversible by removal of the peptide. 52
Figure 2-4. TNF suppression by RTD-1 is independent of protein synthesis and
disruptions in soluble TNF signaling. 55
Figure 2-5. θ-defensins inhibit TACE and suppress TNF release from LPS
stimulated THP-1 monocytes. 58
Figure 2-6. TACE inhibition correlates with suppression of TNF release from
stimulated THP-1 monocytes and whole blood. 60

iv

Figure 2-7. RTD-1 in a non-competitive, fast binding inhibitor of TACE. 62
Figure 2-8. RTDs inhibit cellular TACE and ADAM10 sheddase activities. 65
Figure 2-9. θ-defensins inhibit cleavage of a TNF-peptide substrate in a continuous
real-time measurement of TACE activity in THP-1 and HT-29 cells. 67
Figure 3-1. Biosynthesis and structural features of θ-defensins. 86
Figure 3-2. Efficacy of RTD-1 in rat PIA. 96
Figure 3-3. Effects of RTD-1 on gross pathology and histology of rat PIA. 97
Figure 3-4. Efficacy of RTD-1 in severe PIA and comparison with RA drugs
and θ-defensin isoforms. 100
Figure 3-5. Dose-dependent anti-arthritic effects and pharmacokinetics of RTD-1. 102
Figure 3-6. RTD-1 is non-immunogenic following multiple subcutaneous challenges. 104
Figure 3-7. Effects of RTD-1 on arthritogenic mediators. 107
Supplementary Figure 3-1. Additional examples of effects of RTD-1 on
gross pathology and histology of rat PIA. 98
Supplementary Figure 3-2. θ-defensin stability in human and rat plasma. 103

LIST OF TABLES
Table 3-1. RTD-1 inhibition of proteinases associated with RA pathogenesis. 109

v

ACKNOWLEDGEMENTS

Special thanks to Dr. Michael Selsted for being a supportive, engaging, and encouraging mentor.
His dedication to science and education made this dissertation possible.

I would like to thank Dr. Dat Tran for being a remarkable colleague, mentor, and friend.

Thanks to Patti Tran for making the Selsted Lab a wonderful place to conduct research.

Thanks to Dr. André Ouellette for always having an open door and ear.

Thanks to my committee members for their time, effort, wisdom, and guidance.

And finally I would like to thank my friends and family for supporting me on this challenging
endeavor. Thank you to my best friend, Kennedy. I couldn’t ask for a more understanding and
supportive partner.
vi

CURRICULUM VITAE
Justin Blaine Schaal
Education and Employment
2003-2007 B.S. Biological Sciences, University of California, Irvine
2004-2009 Laboratory Assistant, Department of Pathology, University of California, Irvine
2009-2012 Laboratory Technician, Department of Pathology, University of Southern California
2012-2016 Ph.D. Medical Biology (May 2016), University of Southern California

Publications
1. Tran D, Tran P, Roberts K, Osapay G, Schaal J, et al. (2008) Microbicidal properties and
cytocidal selectivity of rhesus macaque theta defensins. Antimicrob Agents Chemother
52: 944-953.

2. Tongaonkar P, Tran P, Roberts K, Schaal J, Osapay G, et al. (2011) Rhesus macaque theta-
defensin isoforms: expression, antimicrobial activities, and demonstration of a prominent
role in neutrophil granule microbicidal activities. J Leukoc Biol 89: 283-290.

3. Schaal JB, Tran D, Tran P, Osapay G, Trinh K, et al. (2012) Rhesus macaque theta defensins
suppress inflammatory cytokines and enhance survival in mouse models of bacteremic
sepsis. PLoS One 7: e51337.

4. Wilmes M, Stockem M, Bierbaum G, Schlag M, Gotz F, et al. (2014) Killing of staphylococci
by theta-defensins involves membrane impairment and activation of autolytic enzymes.
Antibiotics (Basel) 3: 617-631.

5. Tongaonkar P, Trinh KK, Schaal JB, Tran D, Gulko PS, et al. (2015) Rhesus macaque theta-
defensin RTD-1 inhibits proinflammatory cytokine secretion and gene expression by
inhibiting the activation of NF-kappaB and MAPK pathways. J Leukoc Biol 98: 1061-
1070.
6. Schaal JB, Tran D, Subramanian A, Patel R, Laragione T, et al. (2016) A retroevolutionary
strategy for inducing remission of autoimmune arthritis using macrocyclic theta defensins
from nonhuman primates. (submitted to Science Translational Medicine)
7. Schaal JB, Tran D, Tran P, Tongaonkar P, Maretzky T, et al. (2016) Macrocyclic theta
defensins modulate TNF shedding by inhibition of tumor necrosis factor-α converting
enzyme. (manuscript in preparation for submission to Journal of Biological Chemistry)

vii

Presentations and Published Abstracts
Schaal JB, Tran D, Tran P, Osapay G, Trinh K, et al. Anti-Inflammatory Properties of θ-
Defensins. Antimicrobial Peptides Gordon Research Conference, Ventura, California.
February, 2013 (abstract).
Schaal JB, et al. Anti-Inflammatory Properties of Rhesus Macaque Theta Defensins. University
of Southern California Keck School of Medicine Department of Pathology Conference,
Long Beach, California. February, 2014 (presentation).
Schaal JB, Tran D, Bensman T, Beringer P, Tran P, et al. Rhesus Theta Defensins: A Retro-
Evolutionary Therapeutic Approach for Rheumatoid Arthritis. University of Southern
California Keck School of Medicine Department of Pathology Conference, Long Beach,
California. February, 2015 (abstract and presentation).
Schaal JB, et al. Rhesus Theta Defensins: A Transphylogenetic Therapeutic Approach for
Rheumatoid Arthritis. University of Southern California Keck School of Medicine
Department of Pathology Conference, Long Beach, California. February, 2016
(presentation).

viii

ABSTRACT OF THE DISSERTATION

Immunomodulatory Properties, Mechanisms, and Therapeutic Potential
of Macrocyclic Theta Defensins
by
Justin Blaine Schaal
Doctor of Philosophy in Medical Biology
University of Southern California
Professor Michael E. Selsted

Defensins are a class of antimicrobial peptides that function as part of the innate immune
system. The mammalian defensin family is divided into three subfamilies known as α-, β-, and
θ-defensins. Peptides from each of the respective defensin families were initially discovered for
their antimicrobial properties, and have been shown to kill or neutralize bacteria, fungi, protozoa,
and viruses. More recently, members of the α- and β-defensins have been demonstrated to
possess immune modulating activities which include the induction of cytokine/chemokine
signaling, immune cell recruitment/activation, and initiation of adaptive immune responses. θ-
defensins, being the most recently discovered family, have only just recently been evaluated for
their potential roles in immune responses. The goal of this dissertation was to discern the
immune modulating properties θ-defensins, what molecular mechanisms they utilize, and if these
unique molecules could be used as future therapeutics in the context of human diseases.
The results of our studies presented here demonstrate that θ-defensins are potent
agents of immune modulation. The macrocyclic peptides significantly suppressed
proinflammatory cytokines/chemokines induced by a multitude of bacterial, fungal, and viral
ix

agonists in vitro. Each of the rhesus θ-defensin isoforms profoundly inhibited the keystone
cytokine tumor necrosis factor (TNF) in stimulated human whole blood,
monocytes/macrophages, and buffy coat preparations. Single dose treatment in murine models
of E. coli bacteremia and polymicrobial sepsis resulted in dramatic reductions in lethality.
Survival in E. coli induced peritonitis was associated with reductions in pathogenic levels of
proinflammatory cytokines/chemokines.
The molecular mechanisms θ-defensins utilize to modulate proinflammatory
responses includes the inhibition of tumor necrosis factor-α converting enzyme (TACE).
Inhibition of TACE was shown to prevent the cleavage of membrane bound TNF, contributing to
reduced soluble TNF signaling and potential down-stream reductions in global inflammatory
responses. Characterization of TACE inhibition by RTD-1, our prototype isoform, indicated the
peptide was a non-competitive, fast-binding, reversible inhibitor of TACE with an IC
50
in the
low nanomolar range.
Further evaluation of the therapeutic potential of θ-defensins utilized pristane
induced arthritis in rats, a non-infectious disease of inflammation and a model of human
rheumatoid arthritis. θ-defensin treatment resulted in remarkable attenuation and reversal of
disease pathology, with restoration of limb function, joint architecture, and reductions in gross
pathology. Resolution of arthritis correlated with reductions in IL-1β in joint tissues. In vitro
analyses indicated that RTD-1 was capable of altering the pathogenic characteristics of
fibroblast-like synoviocytes, including modulation of secreted proinflammatory cytokines,
proliferation, and invasiveness.
Additionally, θ-defensins were shown to be non-toxic and non-immunogenic in
mice, rats, and chimpanzees. Furthermore, the pharmacokinetic profile of RTD-1 indicated a
x

prolonged circulating half-life with remarkable peptide stability in blood and plasma. The
implications of this dissertation strongly support the further evaluation of θ-defensins as a novel
transphylogenetic therapeutic approach for the treatment of human diseases.

1

GENERAL INTRODUCTION
Antimicrobial peptides (AMP) are vital components of the innate immune response in
host defense. They provide a mechanism for the rapid detection and neutralization of microbial
pathogens in nearly all multicellular organisms including fungi, plants, insects, and vertebrates
[1]. To date more than 7,000 unique AMPs have been discovered, and while they possess
significant differences in their sequences and structures, most are small, amphipathic, cationic
polypeptides. AMPs were first reported in the late 19
th
century [2], and since then most AMPs
have been initially characterized by their antimicrobial activities. As the field of immunology
has advanced it has become apparent that these ancient components of the immune system can
function as pleiotropic molecules active not only as microbicides but also as immunoregulators
[3].
The most widely expressed family of AMPs are the defensins. Initially discovered within
neutrophil cytoplasmic granules of rabbits and humans, today defensins are known to be
expressed in a variety of organisms, including plants, insects, and vertebrates [4]. The
mammalian defensins are composed of three subfamilies which include α-, β-, and θ-defensins.
These peptides are 18-45 amino acids in length (2-4 kDa), and share several structural features
including; a tri-disulfide array, cationic net charges, a β-strand secondary structure, and arginine-
rich sequences. Each subfamily of defensins has been shown to kill or neutralize bacteria, fungi,
protozoa, and viruses [5]. The antimicrobial mechanisms of defensins are not fully understood,
however evidence suggests defensins act by binding to and disrupting target cell membranes.
Binding of the cationic defensins to the negatively charged membranes of commensals as well as
pathogens is thought to induce depolarization or the formation of pores resulting in membrane
permeabilization [6]. In addition to membrane disruption, members of the defensin family can

2

directly neutralize pathogenic components such as LPS and anthrax lethal toxin [7].
Additionally, defensins use multiple anti-viral mechanisms including the prevention of viral
entry into host cells as well as direct viral neutralization [8]. While the microbicidal mechanisms
of defensins have yet to be fully elucidated, continuing studies indicate these versatile peptides
utilize multiple effector functions to kill/neutralize such a wide variety of commensals and
pathogens.
Mammalian α- and β-defensins evolved from a common ancestral β-defensin gene after
they diverged from other vertebrates. They have since rapidly evolved through duplication,
diversification, and chromosomal translocations. Defensin expression is tissue/cell type specific
and is highly varied among mammals [5]. Defensins are most commonly found within cells of
myeloid or epithelial origin. Most defensin expression is constitutive, though the expression of
certain defensin genes is inducible in certain tissue types. All mammals examined to date
possess defensins, however expression of the defensin subfamilies is species-specific. For
example, rabbits only express α-defensins, while humans, rats, and mice possess both α- and β-
defensins [5].
Humans have two types of α-defensins of either myeloid or enteric origin. There are four
myeloid α-defensins, human neutrophil peptides 1-4 (HNP 1-4). They are found in abundance
packaged into azurophilic granules of leukocytes, where they function by killing or neutralizing
phagocytosed pathogens [9]. Human defensins 5 and 6 (HD-5 HD-6) are of enteric origin and
expressed in Paneth cells of the small intestine where they are secreted into the lumen. Their
roles are thought to include the defense of mucosal surfaces as well regulating the intestinal
microbiome [10]. There have been 31 β-defensin genes identified in humans, however only 11
of the encoded peptide products have been isolated. Human β-defensins (HBD) are expressed in

3

both leukocytes and a wide variety of epithelial tissues [11]. They have diverse functions
ranging from the protection of epithelial surfaces from microbial colonization to vital roles in
sperm fertility [12].
In addition to their antimicrobial activities, human α- and β-defensins have been
demonstrated to orchestrate multiple immune responses. Extensive studies have linked these
peptides to inflammation, initiation of adaptive immune responses, lymph- and angiogenesis,
wound healing, fertility, and cancer [4]. Their roles in immune modulation, specifically in
inflammatory responses, are reviewed more extensively in Chapters 1, 2, and 3 with a greater
evaluation of the known literature.
First reported in 1999, θ-defensins are the most recently discovered subfamily of
defensins. They are structurally dissimilar to α- and β-defensins, despite being encoded by
truncated α-defensin genes [13]. They are unique to Old World monkeys and are found nowhere
else in the animal kingdom. θ-defensins present in rhesus macaques and olive baboons have
been well characterized for their expression, structure, and antimicrobial activities, which are
described in greater detail in the following chapters. Humans do not possess θ-defensins. The
stop codon mutation that prevents humans from producing the peptides is thought to have
occurred approximately 10 million years ago when the orangutan and hominoid lineages
diverged [14]. Therefore, the great apes including chimpanzees, bonobos, and gorillas possess θ-
defensin pseudogenes containing the same mutation found in humans. It is not known what
evolutionary force selected for the loss of θ-defensins in higher primates.
The unique immune modulating properties of θ-defensins will be presented in the
following chapters and will propose the reintroduction of humans and their lost θ-defensins.

4

This general introduction was intended to provide a brief background on defensins. Each
of the following chapters contains a detailed introduction and relevant background and literature
reviews which will provide additional information. The chapters contained in this thesis are
presented in manuscript form. They have been formatted to fulfill stylistic requirements of the
University of Southern California. Chapter 1 was published in February 2012 in the journal
PLOS1. Chapter 2 is a manuscript in preparation for submission to the Journal of Biological
Chemistry. Chapter 3 is a copy of the manuscript submitted to the journal Science Translational
Medicine.
REFERENCES
1. Zasloff M (2002) Antimicrobial peptides of multicellular organisms. Nature 415: 389-395.
2. Stec B (2006) Plant thionins--the structural perspective. Cell Mol Life Sci 63: 1370-1385.
3. Oppenheim JJ, Biragyn A, Kwak LW, Yang D (2003) Roles of antimicrobial peptides such as
defensins in innate and adaptive immunity. Ann Rheum Dis 62 Suppl 2: ii17-21.
4. Suarez-Carmona M, Hubert P, Delvenne P, Herfs M (2015) Defensins: "Simple" antimicrobial
peptides or broad-spectrum molecules? Cytokine Growth Factor Rev 26: 361-370.
5. Selsted ME, Ouellette AJ (2005) Mammalian defensins in the antimicrobial immune response.
Nat Immunol 6: 551-557.
6. Tran D, Tran PA, Tang YQ, Yuan J, Cole T, et al. (2002) Homodimeric theta-defensins from
rhesus macaque leukocytes: isolation, synthesis, antimicrobial activities, and bacterial
binding properties of the cyclic peptides. J Biol Chem 277: 3079-3084.
7. Kudryashova E, Quintyn R, Seveau S, Lu W, Wysocki VH, et al. (2014) Human defensins
facilitate local unfolding of thermodynamically unstable regions of bacterial protein
toxins. Immunity 41: 709-721.
8. Wiens ME, Wilson SS, Lucero CM, Smith JG (2014) Defensins and viral infection: dispelling
common misconceptions. PLoS Pathog 10: e1004186.
9. Selsted ME, Ouellette AJ (1995) Defensins in granules of phagocytic and non-phagocytic
cells. Trends Cell Biol 5: 114-119.

5

10. Ouellette AJ, Selsted ME (1996) Paneth cell defensins: endogenous peptide components of
intestinal host defense. FASEB J 10: 1280-1289.
11. Jarczak J, Kosciuczuk EM, Lisowski P, Strzalkowska N, Jozwik A, et al. (2013) Defensins:
natural component of human innate immunity. Hum Immunol 74: 1069-1079.
12. Selsted ME, Tang YQ, Morris WL, McGuire PA, Novotny MJ, et al. (1996) Purification,
primary structures, and antibacterial activities of beta-defensins, a new family of
antimicrobial peptides from bovine neutrophils. J Biol Chem 271: 16430.
13. Tang YQ, Yuan J, Osapay G, Osapay K, Tran D, et al. (1999) A cyclic antimicrobial peptide
produced in primate leukocytes by the ligation of two truncated alpha-defensins. Science
286: 498-502.
14. Conibear AC, Craik DJ (2014) The chemistry and biology of theta defensins. Angew Chem
Int Ed Engl 53: 10612-10623.

6

Chapter 1

Rhesus Macaque Theta Defensins Suppress Inflammatory Cytokines
and Enhance Survival in Mouse Models of Bacteremic Sepsis

Justin B. Schaal
1
, Dat Tran
1
, Patti Tran
1
, George Ösapay
3
, Katie Trinh
1
, Kevin D. Roberts
1
,
Kathleen M. Brasky
4
, Prasad Tongaonkar
1
, André J. Ouellette
1,2
and Michael E. Selsted
1,2*

1
Department of Pathology and Laboratory Medicine, Keck School of Medicine, and the
2
Kenneth
Norris Comprehensive Cancer Center, University of Southern California, Los Angeles,
California, U.S.A.

3
Department of Pathology and Laboratory Medicine, University of California, Irvine, California
U.S.A.

4
Texas Biomedical Research Institute, San Antonio, Texas, U.S.A.

*Published in PLOS1 December 6, 2012

7

ABSTRACT
Theta-defensins (θ-defensins) are macrocyclic antimicrobial peptides expressed in
leukocytes of Old World monkeys. The peptides are broad spectrum microbicides in vitro and
numerous θ-defensin isoforms have been identified in granulocytes of rhesus macaques and
Olive baboons. Several mammalian α- and β-defensins, genetically related to θ-defensins, have
proinflammatory and immune-activating properties that bridge innate and acquired immunity. In
the current study we analyzed the immunoregulatory properties of rhesus θ-defensins 1-5 (RTDs
1-5). RTD-1, the most abundant θ-defensin in macaques, reduced the levels of TNF, IL-1α, IL-
1β, IL-6, and IL-8 secreted by blood leukocytes stimulated by several TLR agonists. RTDs 1-5
suppressed levels of soluble TNF released by bacteria- or LPS-stimulated blood leukocytes and
THP-1 monocytes. Despite their highly conserved conformation and amino acid sequences, the
anti-TNF activities of RTDs 1-5 varied by as much as 10-fold. Systemically administered RTD-
1 was non-toxic for BALB/c mice, and escalating intravenous doses were well tolerated and non-
immunogenic in adult chimpanzees. The peptide was highly stable in serum and plasma. Single
dose administration of RTD-1 at 5 mg/kg significantly improved survival of BALB/c mice with
E. coli peritonitis and cecal ligation-and-puncture induced polymicrobial sepsis. Peptide
treatment reduced serum levels of several inflammatory cytokines/chemokines in bacteremic
animals. Collectively, these results indicate that the anti-inflammatory properties of θ-defensins
in vitro and in vivo are mediated by the suppression of numerous proinflammatory cytokines and
blockade of TNF release may be a primary effect.
8

INTRODUCTION
Antimicrobial peptides play a major role in host defense functions of mammalian
granulocytes. Defensins, expressed in leukocytes and/or epithelia of most mammals studied, are
2-4.5 kDa cationic peptides that are further divided into three structural families (α-, β-, and θ-
defensins) based on their distinctive tridisulfide motifs [1,2]. - and -defensins, though
genetically distinct, share similar peptide folds and are widely expressed in mammals including
humans [3,4]. Defensins of all three structural families were first recognized for their
antimicrobial properties in vitro. Collectively, the antimicrobial spectrum of defensins includes
bacteria, fungi, protozoa, and viruses [5,6,7]. Defensins also function as “alarmins” which elicit
adaptive responses to infection and tissue injury [8,9,10].
-defensins have only been isolated from Old World monkeys and are absent in higher
primates including gorillas, chimpanzees, and humans [1,2,11]. The peptides have a macrocyclic
backbone that is post-translationally generated by pair-wise excision and head-to-tail splicing of
two nine-residue segments derived from truncated α-defensin-related precursors [12]. The
nonapeptides may be identical (homodimeric splicing) or derived from different precursors
(heterodimeric splicing) thus amplifying the diversity of θ-defensin gene encoded products. Six
rhesus macaque θ-defensin isoforms (RTDs 1-6) are expressed in neutrophils where they are
packaged in cytoplasmic granules. Specific neutralization of RTDs in lysates of macaque
neutrophil granules markedly reduced the antimicrobial activities of this preparation against E.
coli, S. aureus, and C. albicans, indicating a prominent role for these peptides as components of
the PMN antimicrobial armamentarium [13].
Intranasal administration of RTD-1 protected BALB/c mice from lethal infection by a
mouse adapted strain of SARS-coronavirus (SARS-CoV) [14], despite the fact that the peptide
9

did not neutralize the virus in vitro. The protective effect in vivo correlated with the reduction of
pulmonary inflammation and the suppression of several pro-inflammatory cytokines in lung
homogenates. To further characterize the immunoregulatory properties of θ-defensins, we
analyzed the effects of natural θ-defensin isoforms on cytokine/chemokine responses in
stimulated human leukocytes and THP-1 monocytes, and tested the efficacy of RTD-1 in two
mouse models of bacteremic sepsis. The results of these studies also demonstrate that cyclic θ-
defensins have sequence-specific anti-inflammatory properties that distinguish them from human
neutrophil α-defensins.

10

MATERIALS AND METHODS
Ethics statement.
Human subjects. Blood was obtained from healthy adult volunteers who provided
written consent to participate. The study and consent form were approved by the Institutional
Review Board at USC (protocol #HS-09-00280).
Animal studies. All animal studies were approved by the Institutional Animal Care and
Use Committees where studies were performed: UC Irvine (protocol #2451; mouse studies),
University of Southern California (protocol #11355; mouse studies), and Texas Biomedical
Research Institute (protocol #1119PT0; chimpanzee study). Approved anesthetics were used for
surgeries, recommended analgesics were used for post-operative care, and every effort was made
to minimize suffering. Chimpanzees were group housed in social groups in indoor/outdoor
housing and cared for in accordance with the U.S. Public Health Service Guide for the Care and
Use of Laboratory Animals and the U.S. Animal Welfare Act. They are fed standard monkey
chow supplemented with fruits and vegetables twice daily. Potable water is available to each
enclosure using lixit mechanisms. There is a very active environmental enrichment program for
all animals and animal training is implemented to reduce stress and accomplish animal
husbandry procedures cooperatively. For this study blood samples and compound administration
were performed in anesthetized animals so any suffering was mitigated. No animals were
sacrificed on this study. All animals were returned to their respective groups upon recovery from
anesthesia.
Peptide reagents. The hydrochloride salts of RTDs 1-5 (>98%) were synthesized as
described previously [12,15,16]. Human neutrophil α-defensins (physiologic mixture or HNP 1-
3, or purified HNP-2) were purified from human neutrophils as previously described [17]. Stock
11

solutions of each peptide (0.5 – 1.0 mg/ml) were prepared in 0.01% acetic acid (HOAc) for in
vitro analyses or 0.85 M NaCl for administration to animals.
Bacteria and TLR agonists. Staphylococcus aureus 502a and Escherichia coli K12
were obtained from ATCC (Manassas, VA). A clinical isolate of E. coli was obtained from the
clinical laboratory of the University of California Irvine Medical Center. Bacteria were cultured
from single colonies and harvested as previously described [15]. Bacteria were washed and
suspended in either 10 mM piperazine-N,N´-bis(2-ethane-sulfonic acid) (PIPES), pH 7.4 or
phosphate buffered saline (PBS). Bacterial density was determined by spectrophotometric
absorbance at 620 nm and correlated with colony forming units on tryptic soy agar plates. Toll-
like receptor (TLR) agonists were obtained from Invivogen (San Diego, CA) and used as
recommended by the manufacturer.
Cell culture. THP-1 monocytic cells (ATCC, Manassas, VA) were grown and
maintained in RPMI-1640 medium containing 10% fetal bovine serum and
penicillin/streptomycin. Cells were harvested by centrifugation, washed with RPMI-1640
medium and suspended at 5 × 10
5
cells/ml in fresh medium containing 5% human EDTA plasma.
Cytokine/chemokine release assays. EDTA-anticoagulated blood was obtained from
healthy adult volunteers as approved by the Institutional Review Board at USC (protocol # HS-
09-00280). Peripheral blood leukocytes (PBL) were harvested from 10-15 ml whole blood after
centrifugation at 200 g. Cells were washed twice with 3-5 ml of RPMI, counted with a
hemocytometer, and suspended to a cell density of 5×10
5
cells/ml in RPMI + 5% human EDTA-
plasma. PBLs were stimulated for 4 h with either 0.9 µg/ml ssRNA40, 30 ng/ml S. typhimurium
flagellin, 3.3×10
7
CFU/ml heat-killed L. monocytogenes (HKLM), 3 ng/ml E. coli K12 LPS, or
100 CFU/ml of the clinical E. coli isolate. Cytokines/chemokines were quantified using a
12

Milliplex MAP kit on a BioRad Bioplex HTF Luminex reader at the Beckman Center for
Immune Monitoring at USC-Norris Cancer Center.
TNF assay. Peptides (RTDs 1-5, human neutrophil α-defensins) were aliquoted into
wells of pyrogen-free 12- or 24-well plates wherein final peptide concentrations were 0-10 µg/ml.
Wells were inoculated with 1 10
5
CFU/ml S. aureus or 100 CFU/ml E. coli, or 1-3 ng/ml of E.
coli K12 LPS. Samples (5-10 µl) containing peptide or bacteria/LPS were placed on opposite
sides of the plate well. Mixing commenced with the addition of 0.5 to 2.0 ml of 1:10 diluted
blood, PBLs (5 x 10
5
/ml), or THP-1 cells (2-5 x 10
5
cells/ml). Plates were incubated at 37 °C in
5% CO2 for 4 h with gentle mixing. In other experiments, 1:10 blood/RPMI, E. coli cells, and 10
µg/ml of RTD-1 were pre-incubated in binary combinations for up to 120 minutes, followed by
further incubation for 4 h for TNF release. Incubation mixtures were centrifuged at 200 g for
10 min at 22 °C and supernatants were clarified by centrifugation at 23,000 g for 15 min at
4 °C. Supernatant TNF was quantified by sandwich ELISA (OptEIA II; BD Biosciences; Hu
TNF-α, Invitrogen) per suppliers’ directions, using a SpectraMax Plate Reader. In control
experiments, RTD-1 had no effect on TNF ELISA standard curves.
LPS neutralization assay. E. coli 0111:B4 LPS (Lonza), dissolved in endotoxin-free
water (2 effective units/ml) was incubated with 0-10 μg/ml of RTD-1 or 0-10 µg/ml polymyxin
B (Sigma) dissolved in 0.01% HOAc in a 50 μl reaction mixture for 10 min at 37 °C. To each
sample, 25 µl of Limulus amoebocyte lysate (LAL; Lonza) was added and incubated for 10 min
at 37 °C after which 100 μl of LAL substrate was added and incubated for 6 min at 37 °C.
Reactions were quenched with 50 μl of 25% HOAc and read spectrophotometrically at 405 nm.
RTD-1 stability analysis. RTD-1 (50 µg/ml final concentration) was incubated at 37 °C

in freshly prepared human serum, EDTA plasma, or 50 mg/ml human serum albumin in PBS.
13

Aliquots were removed at 24 hour intervals for up to 72 h and acidified by addition of HOAc
(10% final concentration). Peptide was quantified by sequential solid phase extraction on Strata
X resin and quantitative RP-HPLC as described previously [13].
Animal exposure studies. Groups of four BALB/c mice received daily 0.5 ml
subcutaneous injections containing 0, 2.5, 10, 40, or 160 mg of RTD-1 per kg body weight.
Twenty four h after the fourth injection, mice were euthanized with CO 2, blood was collected by
cardiac puncture, and tissues were harvested and fixed in 10% buffered formalin.
Histopathologic examination was performed on sections of heart, lung, liver, spleen, kidney, and
injection site skin and subcutaneous tissue. A basic metabolic panel (UC Davis-William R.
Pritchard Veterinary Medical Teaching Hospital) was performed on serum samples obtained by
cardiac puncture at the time of euthanasia.
Primate studies were conducted using two adult chimpanzees (15 y.o. male, 13 y.o.
female). Animals were infused with escalating doses of RTD-1 (0.02, 0.1, 0.3, 1.0, and 3.0
mg/kg on days 0, 3, 7, 10, and 14, respectively) dissolved in 5 ml of pyrogen-free sterile saline.
Blood specimens were obtained from each animal prior to RTD-1 administration and 30 and 60
min after peptide infusion. A comprehensive metabolic panel and complete blood count with
differential was performed on each specimen. Samples from each animal were also obtained on
days 21, 28, and 60 and similarly analyzed. Serum samples obtained at days 21, 28, and 60 were
also analyzed for anti-RTD-1 antibody by dot blot analysis as described [13] employing anti-
tetanus toxoid immunoreactivity as positive control.
E. coli peritonitis model. Six to 8 week old BALB/c mice (Jackson Labs) were housed
individually and provided with standard chow and water ad libitum. Peritonitis was induced by a
single intraperitoneal injection of 8 × 10
8
CFU of log-phase E. coli K12 in 500 µl PBS [18].
14

Mice were treated immediately with a single subcutaneous injection of 5 mg/kg RTD-1 in 0.5 ml
of normal saline, or normal saline alone (control). Sham challenge was carried out with
intraperitoneal injections of saline. Animals were monitored for 28 days and/or euthanized if
they became moribund (counted as non-survivor). For cytokine/chemokine analyses, groups of
four mice from each treatment group were euthanized at 0, 0.5, 1, 2, 4, and 12 hours following
intraperitoneal challenge. Blood was collected by cardiac puncture into EDTA-tubes and plasma
was prepared by a two-step centrifugation (200 g for 10 min followed by 23,000 g for 15
min). Soluble cytokines/chemokines were quantified using a mouse-specific Milliplex
cytokine/chemokine kit as described above.
Cecal-ligation/puncture (CLP) induced sepsis. Polymicrobial peritonitis was induced
in 6-8 week old BALB/c mice by CLP as described [19,20]. Briefly, laparotomy was performed
on anesthetized animals and the cecum was ligated below the ileocecal valve. Both walls of the
cecum were punctured twice with an 18-gauge needle, and the surgical wound closed with 3-0
nylon suture. All animals fully recovered within 60 min. Four hours following CLP surgery,
each animal received 150 l of normal saline (n=10) or 150 l of normal saline containing 5
mg/kg of RTD-1 (n=11) by tail vein injection. A separate group of mice (n=5) was treated with
RTD-1 as above but the single administration was delayed until 24 h after CLP surgery. Animal
health was evaluated daily for 28 days. As above, mice were euthanized when they became
moribund and scored as non-survivors.
Statistical analyses. All values are expressed as mean +/- S.E.M except in experiments
where n=2 (as indicated in figure legends) where data are expressed as standard deviation.
Significance of peptide effects on cytokines was determined by Student’s t test. Daily
survival/death values were determined by χ
2
test (Figures 1-7A and 1-8).
15

RESULTS

θ-defensin modulation of TLR-induced cytokines/chemokines. In a previous study,
RTD-1 protected mice from lethal SARS-CoV via mechanisms that were independent of an
antiviral effect, as RTD-1 was not virus neutralizing. Rather, RTD-1 administration appeared to
protect infected animals by reducing pulmonary inflammation and suppressing IL-1α, IL-1β, IL-
6, IL-12, CXCL1 (KC), CCL2 (MCP-1), CCL3 (MIP-1α), and CCL5 (RANTES) 2-4 days post-
infection [14]. To determine how RTD-1 modulates inflammatory responses of human cells,
peripheral blood leukocytes (PBL) were incubated with different TLR agonists with and without
10 μg/ml of RTD-1 and evaluated for the release of soluble cytokines/chemokines. We initially
tested the effect of RTD-1 on ssRNA (TLR8 agonist) induced responses of PBLs to compare
human cell responses with those obtained in mouse SARS-CoV (a single stranded RNA virus)
pneumonitis. As shown in Figure 1-1, ssRNA stimulation of human leukocytes induced
cytokines/chemokines similar to those observed in murine SARS-CoV pneumonitis including IL-
1α, IL-1β, IL-6, TNF, CXCL8 (IL-8), CCL2 (MCP-1), CCL3 (MIP-1α), and CCL4 (MIP-1β).
The simultaneous addition of 10 μg/ml of RTD-1 reduced supernatant levels of each of the above
listed cytokine/chemokines with suppression ranging from 68% (IL-8) to 95% (IL-1β).
RTD-1 also down regulated leukocyte release of cytokines/chemokines induced by other
TLR agonists: HKLM (TLR2), flagellin (TLR5), LPS (TLR4) and live E. coli cells (Fig. 1-1).
RTD-1 markedly reduced IL-1α, IL-1β, IL-6, TNF, CXCL8 (IL-8), CCL3 (MIP-1α), and CCL4
(MIP-1β) levels induced by HKLM (TLR2) and LPS (TLR4) and by E. coli cells, similar to the
effect observed with RTD-1 treatment of ssRNA-stimulated cells (Fig. 1-1). As expected, the
effects of RTD-1 on leukocyte responses to LPS and E. coli were similar. The effect of RTD-1
16

on HKLM-stimulated cells differed somewhat from the peptide’s effect on leukocytes activated
by other agonists. Although RTD-1 treatment of HKLM-stimulated cells markedly reduced TNF,
IL-1α, IL-1β, and IL-6 levels, similar to the effects observed with other agonists, there was ~
40% increase in IL-10 and no suppression of induced levels of chemokines CXCL8 (IL-8),
CCL2 (MCP-1), CCL3 (MIP-1α), or CCL4 (MIP-1β). RTD-1 alone had little effect on
cytokine/chemokine release by leukocytes with the exception of VEGF and to a lesser degree
CCL2 (MCP-1).

17

Figure 1-1. Effects of RTD-1 on stimulated release of cytokines/chemokines. Human buffy coat
cells from EDTA-anti-coagulated blood were stimulated for 4 h with a panel of TLR agonists:
0.9 μg/ml ssRNA40, 30 ng/ml S. typhimurium flagellin, 3.3 × 10
7
CFU/ml heat-killed L.
monocytogenes (HKLM), 3 ng/ml E. coli K12 LPS, or 100 CFU/ml E. coli cells. Levels of ten
soluble cytokines/chemokines were measured using a Milliplex MAP kit. Buffy coat cells
stimulated with agonist (yellow), with + 10 µg/ml RTD-1 (green), 10 µg/ml RTD-1 alone (red),
and solvent control 0.01% HOAc (blue).
18

RTD-1 inhibits TNF release in human blood. Of the cytokines evaluated in the
experiments described above, TNF is regarded as the earliest inflammatory response signal and
in this regard functions as a trigger of numerous downstream inflammatory responses [21,22].
To investigate the effect of RTD-1 on TNF release stimulated by bacterial antigens,
anticoagulated human blood was inoculated with E. coli or S. aureus, or stimulated with E. coli
K12 LPS in the presence of varied concentrations of the peptide. In each of these mixtures,
RTD-1 blocked stimulated TNF released in a dose-dependent manner, with ED50’s of ~0.1
µg/ml for S. aureus, ~2 µg/ml for E. coli, and ~5 µg/ml for LPS (Fig. 1-2). RTD-1 alone had no
effect on TNF release by blood leukocytes. Of note, a physiologic mixture of human α-defensins
(HNP 1-3) had no TNF-suppressive effects on E. coli-stimulated PBLs (Fig. 1-2). In keeping
with previous studies [18], no cytotoxicity was detected in these incubations as evidenced by a
lack of Trypan blue staining of leukocytes.

19

Figure 1-2. RTD-1 inhibits the release of soluble TNF in whole blood. Human EDTA-anti-
coagulated blood diluted 1:10 in RPMI, was stimulated for 4 h with live E. coli cells ( ), S.
aureus 502a ( ), or E. coli K12 LPS (□) with simultaneous addition of RTD-1 at the
concentrations indicated. E. coli-stimulated blood was similarly analyzed for effects of a natural
mixture of HNP 1-3 (▼). Blood incubated for 4 h with RTD-1 alone shown as ( ). N = 2-7 for
each experiment.

20

RTD-1 binding to LPS. The inhibition of LPS-induced cytokine/chemokine release by
RTD-1 (Figs. 1- 1 and 2) suggested that the peptide might bind this agonist, as has been
demonstrated for other anti-microbial peptides [23,24]. RTD-1 was ineffective in neutralizing E.
coli LPS, being 50-100 fold less effective than polymyxin B (Fig. 1-3). Moreover, mixing of
RTD-1 with polymyxin B showed no additive or antagonistic effects (Fig. 1-3). These results
indicate that RTD-1 inhibits endotoxin-stimulated release of TNF via mechanisms other than
LPS neutralization. This is consistent with the finding that RTD-1 was a potent inhibitor of TNF
release by PBLs stimulated with multiple TLR agonists (Figs. 1- 1 & 2).

Figure 1-3. RTD-1 is ineffective in neutralizing LPS. The biological activity of LPS in the
presence of 0-10 µg/ml of RTD-1 ( ), polymyxin B ( ), or 1:1 mixtures of RTD-1 and
polymyxin B ( ) was determined by limulus amoebocyte lysate assay. Data are the average on
N=4 experiments.
21

Temporal analysis of RTD-1-mediated blockade of TNF release. In the experiments
summarized in Figures 1-1 and 1-2, RTD-1 modulation of leukocyte responses was analyzed
following simultaneous mixing of peptide, inflammatory stimulus, and leukocytes or whole
blood. To better understand the sequence of peptide-mediated TNF blockade, mixing
experiments were performed wherein blood, peptide, and E. coli cells were pre-incubated in
varied combinations (see Methods), and TNF release was quantified as described above. Pre-
incubation of RTD-1 with blood for up to 2 h had no effect on the magnitude of TNF-release
inhibition which was quite stable (70 - 80%) over the pre-incubation time course (Fig. 1-4A).
This is consistent with the effect of RTD-1 on TNF release when RTD-1, E. coli, and blood were
mixed simultaneously (Fig. 1-2). In contrast, pre-mixing of blood and E. coli cells for up to 2 h,
followed by addition of RTD-1, showed a time-dependent increase of TNF release in both
control and peptide containing mixtures (Fig. 1-4B). However, at each time point the presence
of RTD-1 reduced TNF levels (46-93%; Fig. 1-4B). These results indicate that RTD-1-mediated
blockade of E. coli-stimulated TNF release is very rapid, occurring immediately after addition of
the peptide to leukocyte-containing mixtures (Figs. 1- 2 and 4). When E. coli cells were
incubated with RTD-1 prior to addition of blood, complete blockade of TNF release was
observed at all time-points (Fig. 1-4C), whereas E. coli alone elicited increasing levels of TNF as
a function of pre-incubation time. During the 120 minute pre-incubation interval, bacterial
counts increased 3.2-fold, consistent with the temporal rise in inducible TNF release following
the addition of blood (Fig. 1-4C). On the other hand, the absence of viable bacteria in samples
containing RTD-1 revealed that E. coli was efficiently killed by the peptide within 30 min. The
potent inhibition of E. coli-stimulated TNF release by blood leukocytes at T=0 in this experiment
further demonstrates the rapid blockade of bacteria-stimulated TNF release by RTD-1 (Fig. 1-
22

4C). Moreover, the bactericidal effect of RTD-1 terminates further production of bacterial
antigen which, based on bacterial cell numbers, tripled in 2 h in the absence of RTD-1 (Fig. 1-
4C). Taken together, the data summarized in Figures 1-2 and 1-4 suggest that RTD-1 interacts
with leukocytes to suppress TNF in response to microbial antigens.

Figure 1-4. Temporal analysis of TNF release from E. coli stimulated blood. A: Blood diluted
1:10 in RPMI was pre-incubated with 10 µg/ml of RTD-1 ( ) or solvent ( ) for 0, 30, 60, and
120 min after which 100 CFU/ml of live E. coli cells were added and incubated for another 4 h.
B: Diluted blood in RPMI was pre-stimulated with 100 CFU/ml E. coli for 0, 30, 60, and 120
min followed by addition of 10 µg/ml of RTD-1 ( ) or solvent ( ) and incubated for an
additional 4 h. C: 100 CFU/ml E. coli cells were pre-treated with 10 µg/ml of RTD-1 ( ) or
0.01% HOAc ( ) in RPMI for 0, 30, 60, and 120 min after which whole blood (1:10 dilution
final) was added and incubated for 4 h. After the secondary 4 h incubations in A-C, supernatant
TNF levels were determined by ELISA. Data are average of N= 2-3 experiments.

23

Anti-TNF activities of θ-defensin isoforms. RTD-1 is the most abundant of six θ-
defensin isoforms expressed in neutrophils and monocytes of rhesus monkeys [13]. To
determine the relative anti-inflammatory activities of other θ-defensin isoforms (RTDs 2-5; Fig.
1-5A), we analyzed the effects of these peptides on TNF levels in E. coli-blood assays described
above. As shown in Figure 1-5B, all θ-defensin isoforms suppressed supernatant TNF levels
with potencies, based on estimations of IC50, ranging from 1-10 µg/ml (0.5 – 5 µM). RTDs 2
and 5 were substantially more effective than RTD-1, whereas RTDs 3 and 4 were less active.
Analogous experiments were performed to assess the effects of θ-defensin isoforms on LPS-
stimulated THP-1 monocytes. In these experiments RTD 1-5 inhibited release of stimulated
TNF and the IC50s once again varied ca. 10 fold (Fig. 1-5C). Of note, the hierarchy of anti-TNF
potencies was the same as that obtained in E. coli-stimulated blood experiments, i.e., RTD-5 > 2
> 1 > 4 > 3. Human α-defensin HNP-2 had no inhibitory effect on TNF release in either assay
(Fig. 1-5 B & C; also see Fig. 1-2), and a physiologic mixture of HNP 1-3 also lacked TNF
inhibitory activity (data not shown). In each of the in vitro experiments described above, we
confirmed that the reduction of cytokine expression was not the result of cytotoxic effects on the
target cells as trypan blue staining confirmed that cell viability was > 99% at the end of each
incubation interval. Lack of cytotoxicity was further confirmed by the finding that VEGF-A
expression increased following RTD-1 treatment (Fig. 1-1; discussed further below).

24

Figure 1-5. RTD isoforms differentially inhibit TNF release by E. coli- and LPS-stimulated
human blood and THP-1 monocytes. A. Covalent structures of RTD 1-5 are shown with
invariant residues in black, variable Arg (red), variable hydrophobic residues (green), and
variable Thr (blue). B. Human EDTA-anticoagulated blood diluted 1:10 in RPMI was
stimulated for 4 h with live E. coli (100 CFU/ml) and RTD-1 ( ), RTD-2( ), RTD-3 ( ), RTD-
4 ( ), RTD-5 (▼), or HNP-2 ( ) at the indicated concentrations. C. THP-1 cells in RPMI +
5% human EDTA-plasma were stimulated with 1 ng/ml K12 LPS and incubated with RTDs 1-5
and HNP-2 as described in (B). For both (B) and (C), supernatant TNF levels were determined
by ELISA. N=2 experiments.
C.
A.
B.
25

RTD-1 is non-toxic and non-immunogenic. In earlier studies RTD-1 was non-toxic to
host cells in vitro [15] and was well tolerated when administered intranasally to mice [14]. In the
current study, no acute toxicity was observed in animals receiving subcutaneous doses of RTD-1
(up to 160 mg/kg, the highest dose tested), and serum chemistries in RTD-1-treated mice were
indistinguishable from saline-treated controls. Histopathologic examination of lungs, kidneys,
heart, liver, and spleen from RTD-1 treated animals showed no abnormalities after the 4 day
dosing regimen at all peptide levels tested. The only detected tissue effect associated with RTD-
1 administration was focal, non-erythematous swelling that appeared at the injection site (dorsal
midline thoracic skin) in 4/4 mice (by day 3) in the 160 mg/kg group and 2/4 animals (at day 4)
in the 40 mg/kg group. Histologic examination of affected tissue revealed lobular panniculitis
with fat necrosis around the injection site in affected animals (Fig. 1-6).
Escalating doses of RTD-1 (0.2 to 3.0 mg/kg over 14 days) were administered
intravenously to two adult chimpanzees and the animals were evaluated clinically for effects on
serum chemistries and hematologic parameters. No clinical or injection site effects were
observed at any dosing level over the period of the study. Comprehensive metabolic panels and
complete blood counts with differential revealed no abnormalities associated with peptide
administration at any time point during the study, including those obtained after dosing was
halted (day 14), i.e., at days 21, 28, and 60. Serum samples at each time point were also
evaluated for anti-RTD-1 antibody by dot blot immunoassay [13] and for anti-tetanus toxoid
antibody as positive control. No anti-RTD-1 antibody was detected in samples from either
animal.
26

Figure 1-6. High-dose RTD-1 injection site reaction in Balb/c mice. Hematoxylin-eosin
stained sections of normal (A) and indurated (B, C) skin demonstrate that multiple injections
with 40 mg/kg RTD-1 (B) or 160 mg/kg RTD-1 (C) produce a lobular panniculitis with fat
necrosis. These changes were absent in animals receiving multiple injections of 0, 2.5, or 10
mg/kg of RTD-1.
RTD-1 stability. The macrocyclic conformation of RTD-1 confers remarkable resistance
to enzymatic degradation, a property that confounded initial attempts to determine the peptide’s
covalent structure [12]. RTD-1 is completely stable to heating (100
o
C, 30 min) and extended
storage at pH 2.0 (Tran & Selsted, unpublished data). We further evaluated RTD-1 for stability
in human serum, EDTA-anticoagulated plasma, and 50 mg/ml human serum albumin, incubating
the mixtures at 37
o
C for 72 h. Time zero concentrations of RTD-1, determined by quantitative
RP-HPLC, of each mixture were identical within the limits of method precision. After 72 h of
incubation, RTD-1 levels, relative to time zero, were 112% (serum), 93% (plasma), and 81%
(albumin), demonstrating that the peptide is stable in biological fluids. Consistent with these
27

data, RTD-1 is also highly stable in whole blood from humans, rats, and mice for at least 24 h
(data not shown).
Efficacy of RTD-1 in mouse peritonitis. We evaluated the effects of RTD-1 in vivo
using two mouse models of bacterial peritonitis. A single subcutaneous dose of RTD-1 (5
mg/kg) significantly improved survival of mice infected intraperitoneally with live E. coli (Fig.
1-7). Animals in the peptide and saline controls that survived beyond day 3 were clinically
normal and no further deaths occurred over the course of the trial (day 22; Fig. 1-7A). Plasma
cytokine/chemokine levels in RTD-1 treated and untreated bacteremic mice were analyzed in
cohorts of mice from each treatment group euthanized at 2, 4, and 12 hours post
challenge/treatment. Untreated bacteremic animals had marked elevations in IL-1α, IL-1β, IL-6,
IL-10, CXCL1, CCL2, CCL3, CXCL5, TNF, and VEGF (Fig. 1-7B). Treatment with RTD-1
resulted in reductions of all cytokines, but only the decreases of IL-1α, IL-1β, and VEGF were
statistically significant (P < 0.05). Cytokine/chemokine levels in uninfected, RTD-1 treated
animals were unaltered compared to saline treated controls.
We also evaluated the effects of RTD-1 in BALB/c mice rendered septic by cecal ligation
and puncture [20]. While 90% of the saline treated animals died within 5 days of CLP surgery, a
single i.v. dose of RTD-1 (5 mg/ml) administered 4 h after CLP surgery resulted in long term
survival of 10 of 11 mice (Fig. 1-8). Surprisingly, 4 of 5 mice that were not treated until 24 h
post CLP, also recovered and were clinically normal through the end of the trial.
28

Figure 1-7. RTD-1 increases survival in E. coli peritonitis and modulates cytokine/chemokines.
A. BALB/c mice were challenged with 8 x 10
8
CFU E. coli K12 and treated simultaneously with
s.c. injection of saline ( ; n=13) or 5 mg/kg RTD-1 ( ; n=13). Endpoint survival data are
plotted and were subjected to χ
2
analysis and P-value was ≤ 0.017 *) by day 3 or later. B. Plasma
cytokines/chemokines were quantified in blood obtained from animals euthanized (n=4 for each
time point) at 0, 2, 4, and 12 h after i.p. challenge and treatment with saline ( ) or 5 mg/kg
RTD-1 ( ). Sham controls were injected with RTD-1 alone (▼). Cytokines/chemokines were
quantified as described in Methods and results subjected to Student’s t-test; P ≤0.05 (*).
A.
B.
29

Figure 1-8. RTD-1 increases survival in a mouse model of polymicrobial sepsis. CLP was
performed as described in Methods and animals were treated with i.v saline 4 h post CLP
surgery, ( , n=10), 5 mg/kg RTD-1 4 h post-CLP surgery ( , n=11), or RTD-1 24 h after CLP
surgery ( , n=5). Endpoint survival data are plotted and were subjected to χ
2
analysis and
statistical significance are indicated with * for P <0.05 and ** for P < 0.001.
30

DISCUSSION

The results of studies presented here reveal that θ-defensins possess potent anti-
inflammatory properties in vitro and in vivo. In a previous study, RTD-1 suppressed pulmonary
bronchiolitis and the levels of pro-inflammatory cytokines induced by SARS-CoV, an ssRNA
virus. RTD-1 treatment of ssRNA-stimulated PBLs suppressed the levels of inflammatory
cytokines and chemokines (Fig. 1-1), and the anti-inflammatory profile was similar to that
obtained in the SARS-CoV pneumonitis model. RTD-1 also suppressed cytokine/chemokine
release by PBLs stimulated with other TLR agonists, including those for TLRs 2, 4, and 5, and
the peptide suppressed TNF release stimulated by both Gram-positive (S. aureus) and Gram-
negative (E. coli) bacteria. Based on these findings we speculated that the effects of RTD-1 were
due to the modulation of early interactions of leukocytes with inflammatory stimuli. RTD-1 was
ineffective in neutralizing LPS, indicating that peptide binding of this TLR4 agonist is unlikely
to be an important anti-inflammatory mechanism. This differentiates θ-defensins from other
antimicrobial peptides that bind and neutralize LPS [23,24,25].
Pre-incubation experiments described above (Fig. 1-4) revealed that RTD-1 very rapidly
blocked E. coli-stimulated TNF release in human blood, and temporal analyses of these mixing
experiments implicated peptide-leukocyte interactions to be the critical determinant of TNF
blockade. The time scale of the inhibitory effects (minutes) suggests that θ-defensin may disrupt
the mobilization of TNF from the surface of stimulated cells. Since TNF plays a central role in
triggering and sustaining inflammatory cascades [21,22,26], θ-defensin blockade of TNF may
suppress subsequent inflammatory responses, thereby reducing levels of other inflammatory
31

cytokines/chemokines in vitro (Fig. 1-1) and in vivo (Fig. 1-7B). Alternatively, θ-defensins may
interrupt TNF autocrine circuits that amplify the effect of this acute phase cytokine. While
suppression of RTD-mediated TNF release was a common feature of antigen-stimulated
leukocyte responses in vitro, we have yet to identify downstream mechanisms that result in the
down regulation of secondary pro-inflammatory cytokines/chemokines. In this context, the
effects of inflammatory mediator blockade is highly complex and context dependent, and likely
involves crosstalk of signaling factors that are differentially induced by distinct TLR agonists
and other stimuli to produce protective and/or pathologic responses [27,28,29]. Thus it is not
surprising that the immunomodulatory effects of RTD-1 varied as a function of inflammatory
stimuli.
Correlation of in vitro effects, such as those analyzed using whole blood, PBLs, and
monocyte-macrophages, with effects observed in vivo (e.g., sepsis models) must be interpreted
with caution. In this regard, the relatively modest effects of systemically-administered RTD-1
on plasma cytokines in bacteremic mice contrasts with the dramatic down regulation of
cytokines that occurred when leukocytes were treated with θ-defensins in vitro; this apparent
discrepancy is observed in many if not most in vitro/in vivo model comparisons. It is evident
that the pathways induced by θ-defensins in vivo (e.g., Fig. 1-7A and 1-8) require further
investigation to delineate the mechanisms that confer efficacy in these models. In this context,
RTD-1 treatment of PBLs produced a reproducible elevation of VEGF-A. However, the peptide
had no such effect in vivo, and in fact significantly reduced VEGF levels in bacteremic mice (Fig.
1-7B). In the lung, both protective and pathologic roles have been ascribed to VEGF and its
physiologic regulation appears to play a critical role in the outcome of pulmonary acute lung
injury and acute respiratory distress syndrome [30]. The induction of leukocyte VEGF-A by
32

RTD-1 may represent a new mechanism whereby circulating cells are stimulated to release this
vascular growth factor by locally expressed θ-defensin. It is of interest to note the human
neutrophil α-defensins inhibit VEGF-dependent neovascularization [31,32], potentially
highlighting another difference between α- and θ-defensins. Studies are underway to analyze the
mechanisms underlying the induction of VEGF-A in θ-defensin stimulated leukocytes.
Despite the fact that all known θ-defensins have an invariant 10-amino acid core structure
(Fig. 1-5A), five θ-defensin isoforms varied significantly in their blockade of E. coli or LPS-
stimulated inflammatory responses, and the hierarchy of anti-TNF potencies was the same in
these two cellular assays. There was no correlation between peptide charge and anti-TNF
efficacy; in fact the most effective (RTD-5) and least effective (RTD-3) peptides both have a net
charge of +4. Human α-defensins shared none of the anti-inflammatory properties observed with
θ-defensins. This is not surprising, given the lack of structural similarity between α- and θ-
defensins [2], and the fact that α-defensins possess pro-inflammatory properties that include up
regulation of TNF and IL-1β expression by monocytes [33], stimulation of TNF, IL-6, and IL-12
expression in myeloid dendritic cells [34], and induction of IL-8 release by lung epithelial cells
[35,36,37].
The macrocyclic structure of θ-defensins confers remarkable stability. RTD-1 was
unmodified by incubation for up to 3 days in freshly prepared plasma or serum. RTD-1 was also
well-tolerated when administered intravenously or subcutaneously to mice, rats, and
chimpanzees. Following repeated injections, neither of two chimpanzees produced anti-RTD-1
antibody. The biocompatibility of RTD-1 enabled an evaluation of its therapeutic potential in
animal models of systemic inflammation. The discovery of antimicrobial peptides and their
proven roles in host defense has prompted studies to evaluate diverse peptides derived from
33

human cells (LL-37 [38,39]), ungulates (indolicidin [40]) sheep myeloid antimicrobial peptide
(SMAP)-29 [42]), and pigs (protegrins [23,43]) in preclinical bacteremia models. Protection
against lethal bacteremia by LL-37 [39], indolicidin [44], and SMAP-29 [42] was observed
following a single systemic administration of peptide at the time of bacterial challenge and
therapeutic effects in each case were attributed to anti-endotoxic activities of the respective
peptides. In contrast, in CLP sepsis, multiple doses of porcine protegrin PG-1 had little
endotoxin-neutralizing effect and the treatment regimen produced no therapeutic effect compared
to vehicle control [45]. Data presented here suggest that RTD-1 alters the course of disease in
two models of bacteremic sepsis in a manner different from the above examples. Single dose
administration of RTD-1 in either E. coli peritonitis or CLP sepsis produced a therapeutic
response. In the former model, simultaneous but modest reductions in inflammatory cytokines
were observed in surviving animals. Surprisingly, a single dose of RTD-1 in mice rendered
septic by CLP surgery were rescued even when treatment was delayed for 24 after peritonitis
was induced. Efficacy of θ-defensins in these models appears to be independent of direct anti-
endotoxic effects since the peptide was ineffective in blocking the effects of endotoxin in the
limulus amoebocyte assay. Current studies are underway to further characterize the mechanistic
bases for the immunomodulatory activities of θ-defensins in vitro and in vivo. The lack of
immunogenicity and toxicity across species raises the possibility that θ-defensin may have utility
as human therapeutics.

ACKNOWLEDGEMENT

We thank Tim Bensman for consultations on statistical analysis.
34

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38

Chapter 2

Macrocyclic Theta Defensins Modulate TNF Shedding by Inhibition
of Tumor Necrosis Factor-α Converting Enzyme

Justin B. Schaal
1
, Dat Q. Tran
1
, Patti Tran
1
, Prasad Tongaonkar
1
, Thorsten Maretzky
2
, Carl
Blobel
2
, André Ouellette
1,3
, and Michael E. Selsted
1,3

1
Department of Pathology & Laboratory Medicine, Keck School of Medicine, University of
Southern California, Los Angeles, California, USA
2
Weill Cornell Medical College, Cornell University, New York, New York, USA
3
Norris Comprehensive Cancer Center of the University of Southern California

*Copy of manuscript in preparation for submission to Journal Biological Chemistry

39

ABSTRACT
Theta-defensins (θ-defensins) are macrocyclic pleiotropic peptides expressed in
leukocytes of Old World monkeys. Initial studies identified θ-defensins in rhesus macaque
(RTDs) and Olive baboons (BTDs) as broad-spectrum antimicrobial peptides (AMPs), with
potent activities against bacteria and fungi. More recently, θ-defensins have been shown to
modulate inflammatory responses both in vivo and in vitro. Murine models of sepsis and severe
acute respiratory syndrome (SARS) demonstrated θ-defensins were capable of attenuating
pathogenic inflammatory responses resulting in significant reductions in morbidity and mortality.
Additionally, θ-defensins were efficacious in the treatment of pristane induced arthritis in rats,
with restoration of gross pathology, joint histology, and limb function. Efforts to illuminate the
immune modulating mechanisms of θ-defensins utilized leukocytes stimulated with an array of
TLR agonists and co-incubated with RTDs. These studies showed dramatic reductions in
proinflammatory cytokine/chemokine levels, including remarkable reductions in tumor necrosis
factor-α (TNF), a keystone cytokine in the proinflammatory cascade. Here, we report a
mechanism of θ-defensin TNF suppression via inhibition of tumor necrosis factor-α converting
enzyme (TACE). Proteolytic cleavage of membrane-anchored TNF by TACE is required to
produce soluble TNF. RTDs displayed potent and dose-dependent inhibition of TACE
proteolytic activities, which correlated strongly to suppression of TNF release from stimulated
human whole blood and THP-1 monocytes. RTD-1, our prototype θ-defensin isoform, was
shown to be a fast binding, non-competitive inhibitor of TACE. Additionally, we demonstrate
that suppression of TNF release by RTD-1 is reversible, and independent of protein translation or
TNF paracrine signaling. Collectively, these findings suggest θ-defensins possess a post-
translational mechanism for TNF suppression by the inhibition of TACE proteolytic activities.
40

INTRODUCTION
θ-defensins are a family of antimicrobial peptides expressed exclusively in Old World
monkeys as part of an arsenal of innate effector molecules. θ-defensins comprise one of three
subfamilies of mammalian defensins, which includes α- and β-defensins. Defensins are small (2
- 4 kDa), cationic peptides possessing characteristic tridisulfide motifs [1-4]. While α- and β-
defensins differ in their respective disulfide linkages, they share similar topologies as well as
many in vivo activities. Peptides within these two subfamilies have broad-spectrum
antimicrobial activities and have been shown to possess specific immune modulating activities,
including both anti- and proinflammatory properties as well as roles in immune cell recruitment
and adaptive immune responses [5-10]. θ-defensins, although genetically related, differ from α-
and β-defensins in structure, expression, and immune modulating activities [11-13]. θ-defensins
are the only backbone cyclic peptides known in the animal kingdom (Fig. 2-1), however unlike
α- and β-defensins, they are not expressed in higher primates, hence humans do not possess θ-
defensins. In rhesus macaque monkeys, there are six naturally occurring θ-defensin isoforms
(RTDs 1-6) [14], formed by alternate binary homo- or heterodimeric head-to-tail ligation of two
precursor nonapeptides encoded by three distinct genes (Fig. 2-1) [13]. The peptides are found
in great abundance within granules of neutrophils and monocytes, and have been well
characterized for their anti-infective properties against bacteria, fungi, protozoa, and some
viruses [2-4,13-18]. While θ-defensins possess many of the antimicrobial characteristics as α-
and β-defensins, they are distinct in the mechanisms of their anti-inflammatory properties.
We previously reported θ-defensins to be immune modulators capable of attenuating
proinflammatory signaling pathways both in vivo and in vitro. Rhesus θ-defensin-1 (RTD-1)
treatment in murine models of polymicrobial sepsis, E. coli bacteremia, and SARS corona virus
41

resulted in dramatic reductions in lethality and disease pathology. It was observed that peptide
treatment in each disease model resulted in significant reductions in proinflammatory cytokines
and chemokines [19,20]. To further dissect these phenomena and distinguish between
antimicrobial mechanisms and immune modulation, θ-defensins were tested in a murine model
of rheumatoid arthritis (RA), a non-infectious model of inflammation. Utilizing pristane induced
arthritis in Dark Agouti rats, a RA model characterized by dysregulated TNF, IL-1, and IL-6
cytokine signaling [21], we demonstrated that θ-defensin therapy elicited in an immediate arrest
of disease progression. Treatment with RTD-1 resulted in remarkable attenuation and reversal of
disease pathologies which correlated with significant reductions in proinflammatory cytokines in
joint tissues.
Further investigation into the anti-inflammatory mechanisms of θ-defensins revealed
RTDs to be effective modulators of proinflammatory responses in TLR stimulated human whole
blood, buffy coat cells, peripheral blood mononuclear cells (PBMCs) and THP-1
monocytes/macrophages in vitro [19,22]. Co-incubation of RTD-1 with LPS stimulated human
blood monocytes and THP-1 macrophages affected an inhibition of nuclear factor κB (NF-κB)
and mitogen-activated protein kinase (MAPK) pathways. TLR activation of NF-κB and MAPK
pathways was disrupted by RTD-1 induced phosphorylation of Akt and activation of the
phosphatidylinositol 3-kinase (PI3K) PI3K/Akt pathway. RTD-1 suppression of
proinflammatory signaling pathways correlated with reductions in TNF, IL-1β, and IL-8 mRNA
and protein levels [22]. However, we previously demonstrated that delayed RTD-1 treatment 0.5
- 2 hours after addition of E. coli to human whole blood resulted in immediate blockade of TNF
release [19]. These findings suggest that RTD-1 is capable of rapidly suppressing TNF release
42

after TLR activation of NF-κB and MAPK pathways. We therefore hypothesize a second
mechanism post cellular activation and transcription in which θ-defensins disrupt TNF release.
TNF is first produced as a type-II transmembrane protein that requires cleavage from the
plasma membrane by TACE also known as ADAM17 (a disintegrin and metalloprotease 17)
[23,24]. TACE is a membrane-anchored zinc-metalloprotease and is responsible for “shedding”
the ectodomain of TNF as well as many other cytokines, growth factors, receptors, and adhesion
molecules [25,26]. The precise mechanisms controlling TACE activity are not fully understood,
however previous studies have shown most TACE regulation occurs post-translationally [24].
Dysregulated TACE activity has been associated with disruptions to normal cytokine
homeostasis, resulting in elevated levels of TNF in diseases such as rheumatoid arthritis and
sepsis [25,27-31]. Inhibition of TACE by broad-spectrum metalloprotease inhibitors has been
shown to prevent the release of TNF from cells resulting in suppression of soluble TNF [32].
Here, we report that θ-defensins are a novel class of peptidic TACE inhibitors capable of
attenuating soluble TNF signaling by disrupting its cleavage from the cell membrane.

43

Figure 2-1. Covalent structure of θ-defensins. θ-defensins are macrocyclic nonadecapeptides
containing three disulfide bonds (Top derived from RTD-1 NMR structure). θ-defensin
biosynthesis results from homo or heterodimeric head-to-tail ligation of two nonapeptides
derived from three possible gene precursors. Boxes highlight conserved residues among five
rhesus macaque isoforms, net charge at pH 7.4 is shown. S7 is a chemically synthesized acyclic
analog of RTD-1 with an incomplete cyclic backbone engineered by the incorporation of N- and
C-termini at one of the two possible sites of natural peptide ligation.

44

METHODS
Peptide reagents. RTDs were synthesized as previously described [4]. Human
neutrophil peptides were purified from human buffy coats as described previously [33]. Peptides
were purified (> 98%) by preparative C18 RP-HPLC and paired with HCl. The peptide-
hydochloride was dissolved in either 0.01% acetic acid or H20 and peptide concentration
confirmed by LCMS.
Bacteria and TLR agonists. A clinical isolate of Escherichia coli was obtained from
the clinical laboratory at the University of California Irvine Medical Center. Bacteria were
cultured from single colonies and harvested as previously described [19]. Bacteria were washed
and suspended in phosphate buffered saline (PBS). Bacterial density was determined by
spectrophotometric absorbance at 620 nm and correlated with colony forming units on tryptic
soy agar plates. Toll-like receptor (TLR) agonists were obtained from Invivogen (San Diego,
CA) and used as recommended by the manufacturer.
Buffy coat TNF release assays. EDTA-anticoagulated blood was obtained from healthy
adult volunteers as approved by the Institutional Review Board at USC (protocol # HS-09-
00280). Buffy coat preparations were harvested from whole blood after centrifugation at 200 ×
g. Cells were washed twice with 3-5 ml of RPMI, counted with a hemocytometer, and
suspended to a peripheral blood leukocyte (PBL) cell density of 5×10
5
cells/ml in RPMI + 5%
human EDTA plasma. Buffy coats were co-incubated +/- 15 µM RTD-1 for 4 h with either: 30
ng/ml Pam3CSK4 synthetic triacylated lipopeptide, 10
7
CFU/ml heat-killed L. monocytogenes, 3
ng/ml E. coli K12 LPS, 100 CFU/ml E. coli clinical isolate, 30 ng/ml S. typhimurium flagellin, 3
ng/ml FSL1 synthetic lipoprotein from Mycoplasma salivarium, 0.9 µg/ml ssRNA40, or 1 µM
45

ODN2006 unmethylated CpG oligonucleotide. Cell-free supernatants were prepared by
centrifugation and TNF quantified by ELISA (Life Technologies).
Differentiated THP-1 cell culture. THP-1 (ATCC TIB-202) cells were cultured in
RPMI-1640 10% FBS 100 U/ml Pen/Strep in 5% CO2. Cell differentiation and adhesion was
induced by treatment of 5×10
5
cells/well with 100 nM Phorbol 12-myristate 13-acetate (PMA,
Sigma) for 48 h. Cells were then washed with warm PBS and allowed to incubate 24 h in fresh
PMA-free media before use in assays.
Reversal of RTD-1 TNF suppression. PMA differentiated THP-1 cells (5×10
5
cells/well) were pre-treated with either RTD-1 (5 M), or 0.01% HOAc (peptide solvent) for 1 h
at 37 °C. After pre-incubation the media was removed and cells washed 2x with warm PBS,
followed by adding fresh media back to the wells. Each pre-treatment group was then divided
into one of two secondary treatment groups RTD-1 (5 M) or 0.01% HOAc. After addition of
RTD-1 or 0.01% HOAc, K12-LPS was added to a final concentration of 10 ng/ml, LPS free
controls containing PBS were also included. The cells were then incubated a second time at 37
C 5% CO2 for 4 h, cell-free supernatants were then harvested and analyzed for TNF by ELISA.
TNF responses in cycloheximide treated THP-1 cells. THP-1 cells (5×10
5
cells/ml)
were pre-exposed for 1 h to 10 µM cycloheximide (Sigma) in RPMI-1640 5% human EDTA
plasma. After CHX pre-incubation, RTD-1 was added to final concentrations of 0 – 5 µM. K12-
LPS (10 ng/ml final) was immediately added to cells and samples were incubated 2 or 4 h at 37
°C 5% CO2, after incubation cell-free supernatants were prepared by centrifugation and analyzed
for soluble TNF levels by ELISA. Cell viability was determined by trypan blue staining.
IL-8 responses to soluble TNF stimulation in HT-29 Cells. The colonic epithelial cell
line HT-29 (ATCC) was grown to confluence in a 24-well tissue culture plate in DMEM 10%
46

FBS 100 U/ml Pen/Strep. Growth media was removed, cells washed with PBS, and media
replaced with RPMI-1640 5% human EDTA plasma. RTD-1 was then added to the cells, final
concentrations 0 - 5 µM, immediately followed by the addition of recombinant human TNF (500
pg/ml) (Invivogen). Samples were then incubated at 37 C 5% CO2 for 4 h, centrifuged cell-free
supernatants were analyzed for soluble IL-8 by ELISA (Invivogen).
TACE and ADAM10 inhibition assays. Proteolytic activities of recombinant human
TACE (R&D Systems, 930-ADB) and ADAM10 (R&D Systems, 936-AD) were monitored
using fluorogenic substrates Mca-PLAQAV-Dpa-RSSSR-NH2 (R&D Systems #ES003) and
Mca-KPLGL-Dpa-AR-NH2 (R&D Systems #ES010), respectively. All assays were performed
in F16 black Maxisorp 96 well plates with assay buffer (25 mM TRIS, 2.5 µM ZnCl2, 0.005%
Brij35 vol/vol, pH 9.0). Peptides were added to TACE (0.1 µg/ml) or ADAM10 (0.05 µg/ml) at
final concentrations of 0 – 1.5 µM, followed by the addition of fluorogenic substrate at 10 µM.
Marimastat (Sigma-Aldrich #154036-60-8) was included as a positive control. Enzyme activity
was measured kinetically every 30 seconds for 30 minutes at 22 °C (TACE) or 37 °C
(ADAM10) in a SpectraMax M5e fluorometer (Molecular Devices) with excitation and emission
wavelengths of 320 nm and 405 nm respectively. Maximum velocity of substrate conversion
(Vmax) was calculated for each sample and transformed into % change in product formation
velocity (Mean +/- SEM n = 6). Inhibition curves were fitted with a non-linear variable slope
curve using GraphPad Prism version 5.01 for Windows, GraphPad Software, San Diego
California USA. Half-maximal inhibition concentrations (IC50s) were obtained from fitted
curves.
Michaelis-Menten kinetics. Fluorogenic substrate concentrations of 2.5, 5, 10, and 20
µM were tested across RTD-1 concentrations of 0 - 960 nM. TACE concentration was held
47

constant at 25 ng/ml, and enzyme activity measured every 30 seconds for 90 min. Vo was
plotted against substrate concentration (Ave +/- SD n = 3). Analysis of the Michaelis-Menten
kinetic plot compared best-fit models for competitive, non-competitive, uncompetitive, and
mixed modeling inhibition using Graphpad prism.
Inhibition kinetics of TACE. Inhibition kinetics were measured by adding 0 – 25 µM
RTD-1 to a steady-state (Vs) TACE/substrate reaction in a continuous fluorometric assay at 22
C. Product formation rate was measured every 30 seconds for 10 minutes and then for 25
minutes after the addition of RTD-1.
TNF release from THP-1 monocytes and whole blood. TNF release from THP-1 cells
and human whole blood was performed as previously described [19]. Briefly, THP-1 cells 5×10
5
cells/ml and human EDTA-anticoagulated blood were stimulated with either 1 - 10 ng/ml K12-
LPS or 100 CFU/ml E. coli clinical isolate respectfully. Cells were co-incubated with 0 – 5 µM
of peptide and incubated at 37 C 5% CO2 for 4 h, plasma supernatant was harvested by
centrifugation. Supernatant TNF levels were determined by ELISA (Life Technologies).
Cellular TACE and ADAM10 assays. Cellular TACE and ADAM10 sheddase activity
in African green monkey fibroblast-like kidney COS7 (ATCC) cells was measured by
transfecting cells with alkaline phosphatase-tagged substrates of the respective enzymes. COS7
cells were transiently transfected with (AP)-tagged transforming growth factor-alpha (TGFα) for
6 h and cultured overnight. Cells were washed, starved for 2 - 4 hours, and then pre-treated for
10 minutes with 0 - 5 µM of peptide in opti-mem media. Inducible TACE sheddase activity was
then stimulated by the addition of PMA (25 ng/ml) for 30 - 45 min, after which supernatants
were collected and the cells lysed. Samples of supernatants and cells lysates were added to an
AP activity assay buffer (100 mM Tris, 100 mM NaCl, 20 mM MgCl 2, pH 9.5) and AP activity
48

was then measured by the addition of p-nitrophenyl phosphate (PNPP) substrate (Thermo
Scientific 34045). TACE cleaved AP-tagged-TGFα conversion of PNPP substrate was used to
quantify TACE sheddase activity. Data represented as fold TGFα shedding relative to
constitutive, non-PMA induced, TGFα shedding levels. Cellular ADAM10 sheddase activity
was determined in a similar fashion, however cells were transfected with a AP-tagged ADAM10
substrate, betacellulin (BTC), and ADAM10 sheddase activity was induced with ionomycin (2.5
µM). All samples were performed in triplicate.
Continuous real-time measurement of cellular sheddase activity. THP-1 monocytes
were grown and PMA differentiated, as described above, in Black Clear cellstar 96-well plates
(Greiner). Adherent cells were washed 3x with assay buffer; 20mM TRIS, 154 mM NaCl, 1%
human serum, pH 7.4. Assay buffer was added to the wells, followed by 100x peptide, final
peptide concentrations 0 – 15 M. TNF-like fluorogenic substrate Mca-PLAQAV-Dpa-RSSR-
NH2 (R&D Systems) was then added to each well, final concentration 10 M. The plate was
read every minute at 37 C with excitation and emission at 320 nm and 405 nm respectively.
Maximum velocity of substrate conversion (Vmax) was calculated for each sample and
transformed into % sheddase activity relative to peptide solvent controls. The same protocol was
adapted for adherent colonic epithelial HT-29 cells.

49

RESULTS

RTD-1 suppresses TNF release from TLR stimulated buffy coat cells. Previously, we
reported that human buffy coat cells stimulated with TLR agonists 2, 4, 5, and 8 and co-
incubated with RTD-1 resulted in depressed levels of proinflammatory cytokines, including;
TNF, IL-1α, IL-1β, IL-6, IL-8, IL-10, CCL2, CCL3, and CCL4. Although we measured global
reductions in many proinflammatory cytokines/chemokines, reductions in TNF levels were the
most consistent and profound among each of the various agonists. To expand on these studies
we measured TNF responses in human buffy coat cells stimulated with TLR agonists 1/2, 2, 4, 5,
2/6, 8, and 9 and co-incubated with RTD-1 (Fig. 2). Each agonist elicited a unique TNF
response after a 4 h incubation, with supernatant levels ranging from 33.5 – 1067 pg/ml in
peptide free controls. Co-incubation with 15 µM RTD-1 resulted in significant suppression of
TNF with >87% reduction in TNF levels across all TLR agonists. Reductions in TNF levels
occurred independent of TLR pathway and of similar magnitude when normalized as a % of total
TNF released.

50

Figure 2-2. RTD-1 suppresses TNF release from buffy coat cells stimulated with TLR agonists.
Human buffy coat cells from EDTA-anti-coagulated human blood were incubated for 4 h with a
panel of TLR agonists +/- 15 µM RTD-1. TNF levels of cell free supernatants were measured by
ELISA and are graphed as a % TNF release relative each agonist’s peptide free control. TLR
agonists: 30 ng/ml Pam3CSK4 synthetic triacylated lipopeptide, 10
7
CFU/ml heat-killed L.
monocytogenes, 3 ng/ml E. coli K12 LPS, 100 CFU/ml E. coli clinical isolate, 30 ng/ml S.
typhimurium flagellin, 3 ng/ml FSL1 synthetic lipoprotein from Mycoplasma salivarium, 0.9
µg/ml ssRNA40, 1 µM ODN2006 unmethylated CpG oligonucleotide. Data represents mean ±
SD of two independent experiments containing two technical repeats each.

51

Inhibition of TNF release is dependent on extracellular RTD-1 and is reversible by removal
of the peptide. To evaluate the conditions of RTD-1 and cellular interaction required to suppress
TNF release, the effect of preincubating THP-1 macrophages with RTD-1 and then removing the
peptide before LPS stimulation was tested. THP-1 macrophages were first incubated for 1 h with
either RTD-1 (5 µM) or 0.01% HOAc (peptide solvent) (Fig. 2-3). After a 1 h incubation the
media was removed and the cells washed. Fresh assay media was added to the wells and each
preincubation series then received either RTD-1 (5 µM), or 0.01% HOAc. Immediately
following the addition of peptide or control solvent the wells received K12-LPS (10 ng/ml), non-
LPS controls contained PBS. The THP-1 macrophages were then incubated a second time for 4
h, supernatants were harvested and TNF levels determined by ELISA. Cells preincubated with
0.01% HOAc and then stimulated with LPS showed highly elevated TNF levels (660 ± 198
pg/ml), the presence of RTD-1 during the second incubation reduced TNF to nearly LPS-free
baseline levels. Cells preincubated with RTD-1, washed free of peptide, and then stimulated
with LPS and treated with 0.01% HOAc for the second incubation showed no significant
decrease in TNF levels compared to cells preincubated with 0.01% HOAc (637 ± 162 pg/ml),
suggesting the preincubation with RTD-1 did not result in sustained TNF modulating effects.
However, the addition of RTD-1 for the second incubation period profoundly suppressed TNF
levels. These results show that cells treated with RTD-1 and then washed free of the peptide
retained no anti-TNF activities and that RTD-1 must be in the presence of the cells during TNF
release in order to modulate responses.

52

Figure 2-3. Inhibition of TNF release is dependent on extracellular RTD-1 and is reversible by
removal of the peptide. THP-1 macrophages were pre-treated for 1 h with either 0.01% HOAc
(black bars) or 5 µM RTD-1 (white bars). After 1 h media was removed and the cells were
washed with PBS. A second incubation was initiated by adding fresh media and either 0.01%
HOAc or RTD-1 (5 µM) back to the cells followed by the immediate addition of K12- LPS (10
ng/ml). Cells were then incubated for 4 h and supernatants analyzed for TNF release by ELISA.
Data represents mean ± SD of two independent experiments containing two technical repeats
each.

53

RTD-1 inhibition of TNF release is independent of protein synthesis. Molecules such as IL-
4, IL-13 [34], and Geldanamycin [35] are known to have TNF modulating effects by inducing
disruptions in protein translation, therefore we evaluated if RTD-1 suppression of TNF levels
was associated with interruptions in TNF biosynthesis. Translational elongation was blocked in
THP-1 monocytes by treatment with 10 M cycloheximide (CHX), cells were then stimulated
with LPS (10 ng/ml) and co-incubated with RTD-1 (0 - 5 µM) for either 2 or 4 h, and soluble
TNF levels determined by ELISA (Fig. 2-4A). TNF release in peptide free controls
demonstrated LPS-induced TNF release was possible following CHX treatment and preformed
TNF protein existed within the cells before LPS stimulation. Comparison of RTD-1 free
controls of CHX treated and non-CHX-treated THP-1 cells indicates that interruption of protein
translation reduces the total amount of TNF released by the cells as a result of inhibition of
newly synthesized TNF. Dose-dependent RTD-1 inhibition appears to occur to similar effect in
both CHX treated and non-CHX-treated cells, with nearly complete TNF suppression at 5 M
RTD-1. PMA differentiated THP-1 macrophages and human whole blood were utilized in the
same assay format with similar results (data not shown).

RTD-1 does not neutralize or disrupt soluble TNF signaling. One of the multiple signaling
pathways for TNF activation and indeed other pro-inflammatory responses is paracrine and
autocrine signaling by soluble TNF. The binding of TNF to its membrane bound receptor
activates the NF-κB pathway inducing downstream cytokine responses, including the stimulation
of TNF release [36]. To evaluate if RTD-1 interferes with soluble TNF signaling we utilized the
colonic epithelial cell line HT-29 to monitor responses to TNF signaling. Studies have shown
HT-29 cells express tumor necrosis factor receptor (TNFRI) and release IL-8 in response to
54

soluble TNF signaling [37]. Briefly, confluent HT-29 cells were pretreated for <2 min with
RTD-1 (0 - 5 µM) with soluble recombinant human TNF subsequently added to a final
concentration of 500 pg/ml. The cells were incubated for 4 h and supernatants were analyzed for
IL-8 responses by ELISA. Incubation with TNF stimulated significant elevations of IL-8 levels
(>250 pg/ml), however the presence of RTD-1 did not reduce the amount of IL-8 released from
the cells (Fig. 2-4B). Interestingly, TNF + 5 M RTD-1 resulted in a modest increase in IL-8
release when no elevations in IL-8 were observed in controls containing 5 µM RTD-1 alone
(data not shown). These findings support the conclusion that RTD-1 does not directly neutralize
TNF or interfere with TNF/TNFRs signaling in HT-29 cells.

55

Figure 2-4. TNF suppression by RTD-1 is independent of protein synthesis and disruptions in
soluble TNF signaling. A, RTD-1 inhibition of TNF release is independent of protein synthesis
in THP-1 monocytes pre-treated for 1 h with 10 µM cycloheximide (solid symbols) or PBS (open
symbols). After pre-treatment cells were co-incubated with RTD-1 (0 - 5 µM) and K12-LPS (10
ng/ml) for either 2 h (circles) or 4 h (squares). TNF levels in supernatants were measured by
ELISA. B, RTD-1 does not affect IL-8 response to soluble TNF signaling. Confluent HT-29
cells were treated with 0 - 5 µM RTD-1 and then stimulated for IL-8 release with human rTNF
(500 pg/ml) for 4 h; supernatants were analyzed for IL-8 by ELISA. Controls containing no
TNF were included (Ctrl), RTD-1 only treatment did not result in an increase in IL-8 baseline
levels (data not shown). Data represents mean ± SD of two independent experiments containing
two technical repeats each.

56

θ-defensins are TACE inhibitors and display a hierarchy of TNF suppression in LPS
stimulated THP-1 monocytes. RTD-1 induced a rapid blockade of TNF release post TLR-4
pathway activation in human whole blood [19] by RTD-1 alluded to a TNF modulating
mechanism post-transcription/translation; these observations guided us to test for inhibition of
the final cellular processing step in the production of soluble TNF. We used purified
recombinant human TACE and an internally quenched fluorogenic peptide substrate
representative of the cleavage site of membrane bound TNF to test for θ-defensin inhibition of
proteolytic activities. Co-incubation of TACE with RTD concentrations as low as 10 nM began
to slow product formation, with 80 - 100% inhibition of enzymatic activity by 1.5 µM (Fig 2-
5A). Each of the RTD isoforms displayed classical dose-dependent inhibition curves, with half
maximal inhibitory concentrations (IC50) ranging from 50 - 265 nM. Despite structural and
sequence similarity among the RTD isoforms (Fig. 1), there were significant differences in
TACE inhibition, which followed an IC50 order of RTD-5 ≤ RTD-2 < RTD-1 ≤ RTD-4 < RTD-3.
A synthetic acyclic version of RTD-1 (S7), engineered with a peptide bond break in RTD-1’s
cyclic backbone (Fig. 2-1), displayed significantly reduced TACE inhibition compared to cyclic
RTD-1 (Fig. 2-5B). Human neutrophil α-defensins HNP-2 and HNP-4, showed no significant
inhibition of TACE. Marimastat, a well-studied broad spectrum metalloprotease inhibitor [38]
was included as a positive control and showed potent inhibition of substrate conversion (Fig. 2-
5B).
θ-defensins have been shown to be effective modulators of TNF release from LPS
stimulated THP-1 monocytes. To expand on previous studies, we utilized LPS stimulated THP-1
monocytes to calculate IC50s for TNF suppression by RTDs 1-5, synthetic acyclic RTD-1 (S7),
human neutrophil peptides (HNPs), and marimastat. Briefly, THP-1 monocytes (5 × 10
5

57

cells/ml) were simultaneously exposed to K12-LPS (1 ng/ml) and peptide (0 - 4.8 µM), and after
a 4 h incubation cell-free supernatants were harvested by centrifugation and TNF levels
evaluated by ELISA. Treatment with RTDs reduced TNF levels in a dose dependent manner,
however there were significant differences in the magnitude of suppression among the natural
isoforms (Fig. 2-5C). RTDs 5 and 2 were the most potent suppressors of TNF levels with nearly
complete suppression of all TNF release by 3.4 µM. RTD-1 was less potent than RTDs 5 and 2
however there was still a ~80% reduction of soluble TNF by 4.8 µM. RTDs 4 and 3 were less
effective at altering TNF levels, with RTD-3 being the least active of the isoforms, reducing TNF
levels by less than 40% at 4.8 µM. The acyclic version of RTD-1 (S7), showed very modest
inhibition of TNF levels when compared to cyclic RTD-1, with a maximal inhibition of 25% at
4.8 µM (Fig. 2-5D). Human α-defensins HNP-2 and a mixture of HNPs 1, 2, 3 lacked any
significant ability to modulate TNF levels. Marimastat showed an IC50 of < 1 µM however the
TACE inhibitor was unable to completely suppress TNF release and maximal inhibition was
limited to 75% despite increasing concentrations of the inhibitor (Fig. 2-5D). Controls
containing peptide or marimastat alone did not elicit TNF release, results not shown.

58

Figure 2-5. θ-defensins inhibit TACE and suppress TNF release from LPS stimulated THP-1
monocytes. A-B, Inhibition of recombinant TACE proteolytic activity in the presence of RTDs
1-5 , acyclic RTD-1 (S7), HNP-2, HNP-4, and marimastat (MRM). Data graphed as % change
in product formation rate relative to a no peptide control. Data represent mean ± SEM of 3
independent experiments containing 2 - 3 technical repeats each. C-D, Suppression of TNF
release from THP-1 monocytes co-incubated with K12-LPS and RTDs 1-5, S7, MRM, HNP-2,
or a mix of HNPs 1, 2, and 3. TNF levels determined by ELISA after a 4 h incubation. Data
represents mean ± SD of 2 – 6 individual experiments containing 2 technical repeats each.

59

TACE inhibition correlates with suppression of soluble TNF release from stimulated THP-
1 monocytes and blood. We previously demonstrated RTDs 1-5 suppress TNF release from
human whole blood stimulated with E. coli. Interestingly, the IC50 hierarchy of TNF suppression
among RTDs 1-5 in E. coli stimulated human blood [19] mirrored those in LPS stimulated THP-
1 monocytes. The calculated IC50 order of TNF suppression in both blood and THP-1 cells is as
follows; RTD-5 ≤ RTD-2 < RTD-1 < RTD-4 < RTD-3. RTD IC50s for TACE inhibition follow a
similar order and when plotted with the IC50s for TNF suppression in blood and THP-1
monocytes, show a significant degree of relation (Fig. 2-6). Statistical analysis of TACE IC50
correlation with blood and THP-1 cells gives Pearson r values of 0.9567 and 0.9639 respectively.

60

Figure 2-6. TACE inhibition correlates with suppression of soluble TNF release from stimulated
THP-1 monocytes and whole blood. IC50 for RTDs 1-5 were calculated for inhibition of in vitro
TACE enzymatic activity and for suppression of soluble TNF level in LPS stimulated THP-1
monocytes ( ) and E. coli stimulated whole blood ( ), numbers in symbols correspond to
RTDs 1-5. Comparison of TACE inhibition and TNF suppression in THP-1 monocytes and
whole blood shows a statistically significant correlation with Pearson r = 0.9639 and 0.9567
respectively, (THP-1 P = 0.0082, Blood P = 0.0107).

61

RTD-1 is a non-competitive, fast binding inhibitor of TACE. Further characterization of θ-
defensin inhibition of TACE focused on RTD-1. Utilizing recombinant TACE and a fluorogenic
TNF-like substrate described above, a Michaelis-Menten kinetics assay was performed to
characterize the nature of TACE inhibition. Substrate concentrations of 2.5, 5, 10, 15, and 20
µM were tested across RTD-1 concentrations of 0 - 960 nM. TACE concentration was held
constant at 100 ng/ml, and enzyme activity measured for 90 min. V
o
was plotted against
substrate concentration (Fig. 2-7A). Computational analysis of the Michaelis-Menten kinetic
plot compared best-fit models for competitive, non-competitive, uncompetitive, and mixed
modeling inhibition; modeling results suggest RTD-1 is a non-competitive inhibitor of TACE.
Binding kinetics of RTD-1 and TACE were evaluated by the addition of peptide to a
steady-state (Vs) TACE/substrate cleavage reaction. RTD-1 (0 - 25 µM) was added to a
continuous steady-state fluorometric TACE/substrate reaction 10 min after initiation. Product
formation rate was monitored every 30 seconds for an additional 20 minutes (Fig. 2-7B).
Product formation velocities respond immediately to RTD-1 addition and maximum inhibition
for each tested concentration is achieved within 30 seconds (limit of detection) after addition of
RTD-1. The characteristics of the binding curve kinetics suggest RTD-1 binds rapidly to TACE
under in vitro assay conditions.

62

Figure 2-7. RTD-1 is a non-competitive, fast binding inhibitor of TACE. A, Michaelis-Menten
kinetics. Substrate concentrations of 2.5, 5, 10, and 20 µM were tested across RTD-1
concentrations of 0-960 nM. TACE concentration was held constant at 25 ng/ml, and enzyme
activity measured for 90 min. Vo was plotted against substrate concentration. Analysis of the
Michaelis-Menten kinetic plot compared best-fit models for competitive, non-competitive,
uncompetitive, and mixed modeling inhibition; modeling results suggest non-competitive
inhibition of TACE. Data represents mean ± SD of a single representative of 3 individual
experiments containing 3 technical repeats each. B, Kinetics of RTD-1 inhibition of TACE was
evaluated in a steady-state TACE/FRET-substrate cleavage reaction. A TACE/FRET-substrate
reaction was initiated and allowed to reach steady state. At 10 min (dotted line) RTD-1 at 0-25
µM was added directly to each reaction. The graph indicates maximum inhibition for each
peptide concentration is achieved within 30 sec upon addition of RTD-1 (limit of detection),
characteristic of a fast binding inhibitor. Data is the mean of a single representative of 2
individual experiments containing 2 technical repeats each.

63

RTDs inhibit cleavage of TGFα by TACE and BTC by ADAM10 in COS7 cells. Evaluation
of cellular TACE and ADAM10 activities in COS7 cells was performed utilizing a reporter
system developed by Blobel et al. COS7 cells, a fibroblast cell line from African green monkey
kidneys, constitutively express both TACE and ADAM10 [39,40]. Sheddase activities can be
individually assessed by quantification of cleaved alkaline phosphatase-tagged substrates
specific to each of the respective sheddases. In separate assays, TACE activity was monitored by
measurement of cleaved transforming growth factor-α (TGFα) following PMA stimulation,
while ADAM10 was assessed by ionomycin induced betacellulin (BTC) shedding.
Peptide free controls show PMA stimulation increased TACE mediated TGFα shedding 2
- 4 fold over constitutive levels (Fig. 2-8 A, B). Addition of RTDs resulted in dose dependent
reductions in TACE cleavage of TGFα. RTDs 1, 3, and 4 reduced TACE activity to below
constitutive levels by 2.5 µM, with near complete suppression of TGFα cleavage by 5 µM.
RTD-5 displayed a steep inhibition curve, with TACE activity reduced to constitutive levels by
1.25 µM, interestingly increasing peptide concentration resulted in plateaued effect with 2.5 and
5 µM RTD-5 reducing TACE activity to 50% of constitutive levels. Treatment with either
acyclic RTD-1 (S7) or HNP-4 did not significantly alter TACE cleavage of TGFα.
In addition to TACE, there are other sheddases known to cleave membrane bound TNF,
however their processing of TNF in leukocytes is thought to be minimal [27,41,42]. ADAM10 is
a related metalloprotease with its own unique substrates, and it has been shown to shed TNF in
certain cell types [43]. Ionomycin treatment increased ADAM10 cleavage of BTC 10 - 20 fold
over constitutive levels. Addition of RTD-1 significantly reduced ADAM10 activity, however
unlike TACE, RTD-1 at 2.5 - 5 µM did not drop activity levels to or below constitutive levels
(Fig. 2-8C). It appears that although RTD-1 is capable of inhibiting ADAM10 activities, the
64

magnitude of the effect is less than that of RTD-1’s effect on TACE. This effect may be due to
relatively higher levels of induced ADAM10 activity compared to TACE. Surprisingly,
treatment with HNP-4 increased ADAM10 shedding of BTC, the mechanism of this effect
remains unknown.

Inhibition of ADAM10. Following a similar fluorogenic assay format as the TACE inhibition
assay described in Figure 2-5 A-B, RTDs were evaluated for ADAM10 inhibition. RTDs
displayed dose-dependent inhibition of ADAM10 enzymatic activity, however significantly
higher amounts of peptide (5 – 10 fold) were required to inhibit ADAM10 compared to TACE
and the hierarchy of potency among the RTD isoforms did not follow the same order (Fig. 2-8D).
HNP-4 did not show any inhibition of the enzyme.

65

Figure 2-8. RTDs inhibit cellular TACE and ADAM10 sheddase activities. A-B, RTDs inhibit
cleavage of TGFα by TACE in COS7 cells. Data is represented as fold TGFα shedding relative
to constitutive non-PMA induced TGFα sheddase levels (n=3). C-D, RTD-1 inhibits cleavage of
BTC by ADAM10 in COS7 cells. Data is represented as fold BTC shedding relative to
constitutive non-PMA induced BTC sheddase levels. Data in panels A-C represent mean ± SD of
1 independent experiment containing 3 technical repeats. D, Inhibition of recombinant ADAM10
proteolytic activity in the presence of RTDs 1-5 or HNP-4. Data graphed as % change in
substrate conversion rate relative to a no peptide control. Data represents mean ± SD of 2
independent experiments containing 2 technical repeats each.
66

RTDs inhibit the cleavage of a TNF-like substrate by macrophages and epithelial cell lines.
Continuous real-time measurement of TACE activity was monitored in live THP-1 macrophages
and in HT-29 colonic epithelial cells. Both cell lines express constitutive levels of active TACE
on their cellular membranes capable of cleaving a fluorogenic peptide substrate representative of
membrane bound TNF [44,45]. Peptides were added to THP-1 macrophages or HT-29 cells
followed immediately by TNF-peptide fluorogenic TACE substrate. Substrate conversion was
monitored at 30 second intervals in a fluorometer at 37 °C for 1 h. After a brief lag period,
substrate conversion in peptide free controls was linear for 60 minutes; additionally there was no
substrate conversion in cell-free wells containing assay media alone. Proteolytic activity was
determined as a function of maximal product formation rate (Vmax) and graphed as % change in
Vmax relative to a no peptide control. In both THP-1 macrophages and HT-29 cells, the addition
of RTDs resulted in dose dependent reductions in substrate conversion rates, however maximum
inhibition was limited to ~75% (Fig. 2-9 A-B). Lack of complete inhibition with increasing
peptide concentration was similar to previously reported studies utilizing small molecule TACE
inhibitors such as GM6001 and BB94 [45]. In THP-1 cells, RTDs 1, 3, and 5 achieved nearly
maximum inhibition by 5 µM while RTDs 2 and 4 showed somewhat lower levels of inhibition
(Fig. 2-9A). In HT-29 cells, RTDs 1, 2, 3, and 5 had similar inhibition curves with RTD-4
having slightly reduced activities compared to the other isoforms (Fig. 2-9B). Maximum
inhibition of substrate conversion was achieved by 15 µM for each isoform in both THP-1
macrophages and HT-20 cells (Fig. 2-9 A, B).

67

Figure 2-9. θ-Defensins inhibit cleavage of a TNF-peptide substrate in a continuous real-time
measurement of TACE activity in THP-1 and HT-29 cells. A, RTD peptides were added to THP-
1 macrophages in 20 mM TRIS, 150 mM NaCl, 1% human serum, pH 7.4, followed immediately
by a TNF-peptide fluorogenic substrate. Substrate conversion was monitored every 30 seconds
in a fluorometer at 37° C for 1 h. Proteolytic activity was determined as a function of % change
in product formation rate relative to a no peptide control. B, Assay for HT-29, a colonic
epithelial cell line, was performed in same manner as THP-1 assay. Data represents mean ± SD
of two independent experiments containing 2-3 technical repeats each.

68

DISCUSSION
The results of the studies presented here reveal that θ-defensins can suppress TNF release
from stimulated cells by the inhibition of TACE sheddase activities. These findings reconcile
previous studies which evaluated the temporal effect of RTD-1 treatment in E. coli activated
blood [19]. The immediate cessation of TNF release from blood by the addition of RTD-1 two
hours after initiation of the TLR-4 signaling pathway and TNF transcriptional activation can be
associated with the rapid suppression of TACE shedding of TNF ectodomain. These findings
suggest that θ-defensins have a second immune modulating mechanism in addition to their
formerly reported NF-κB and MAPK signaling inhibition.
In aforementioned studies, and expanded upon here, we demonstrate that treatment with
RTD-1 can significantly suppress TNF responses in TLR stimulated buffy coat preparations
[19,22]. Alterations in soluble TNF appear to be independent of agonist neutralization as RTD-1
treatment resulted in similar reductions of TNF levels despite chemical, structural, and biological
dissimilarities among the agonists and their toll-like receptors. Furthermore, reductions in TNF
appeared with live E. coli stimulation which is known to elicit proinflammatory responses in
both TLR-dependent and independent pathways (e.g. NOD1, CD36) [46,47]. TNF suppression
also occurred independent of the amount of agonists used; e.g. 100 CFU/ml E. coli versus 10
7

CFU/ml heat-killed L. monocytogenes. In addition, percent reductions in TNF were similar (87 -
99%) despite large variances in the magnitude of agonist-induced TNF release (30 – 1000
pg/ml). Moreover, RTD-1 has extremely limited LPS-neutralizing activities (100-fold less)
compared to LPS-neutralizing antibiotic polymyxin B [19]. Similar effects on TNF across such
a diverse array of stimuli allude to a common mechanism of TNF suppression.
69

Pretreatment of THP-1 macrophages with RTD-1 followed by removal of the peptide
containing media and washing of the cells prior to LPS stimulation resulted in a complete loss of
anti-TNF activities. However, when RTD-1 was added back to the cells during LPS-induced
TNF shedding, the inclusion of peptide caused near total suppression of TNF release. These
findings suggest anti-TNF activities are dependent on the presence of extracellular RTD-1 and/or
the binding of RTD-1 to a cellular receptor, an interaction that must be reversible as evidenced
by loss of anti-TNF activities by washing of the cells with PBS.
Further investigation into the nature of TNF suppression revealed this effect to be
independent of protein translation. Cycloheximide was utilized to terminate all de novo protein
synthesis prior to LPS stimulation of THP-1 monocytes. In keeping with previous reports, CHX
treatment reduced the total amount TNF released by preventing the synthesis of new TNF
protein, however it did not result in a total suppression of TNF responses as the cells were
capable of releasing preformed TNF protein. If RTD-1 mediated anti-TNF affects were via a
disruption in TNF translation or dependent on de novo protein synthesis, there would have been
no additional reductions in TNF levels with RTD-1 treatment. On the contrary, addition of
RTD-1 dose-dependently suppressed the release of preformed TNF from CHX treated cells with
inhibition curves that paralleled non-CHX-treated cells. Therefore, RTD-1 likely possess at least
one anti-TNF mechanism(s) that is not directly related to TNF protein synthesis or the
production of any intermediary protein(s) required to induce TNF suppression.
As a keystone cytokine in the initiation of the proinflammatory cascade, TNF has been a
key molecular target in the development of human therapies [48]. The most common TNF
modulating pharmaceuticals used in clinical settings are either TNF-receptor fusion proteins or
monoclonal TNF antibodies which directly bind and neutralize TNF protein [49-51]. Here, we
70

show that θ-defensins do not share this common mechanism of action. When HT-29 colonic
epithelial cells were stimulated with soluble TNF in the presence of RTD-1, there was no
apparent effect on downstream IL-8 responses. These results suggest the peptide does not
interfere with TNF/TNFRI interactions by either direct TNF neutralization or by blocking TNF
binding to its receptor. In addition, TNF ELISA standard curves were performed with and
without the addition of 5 µM RTD-1, producing essentially identical curves, indicating θ-
defensins do not interfere with the binding of TNF capture or detection antibodies [19]. Lastly,
TNF conditioned cellular supernatants have been percolated through immobilized RTD-1
agarose columns, with no reductions in flow through TNF levels, indicative of a lack of
TNF/RTD affinity (unpublished data Schaal & Selsted). To date, we have no evidence of direct
interaction of θ-defensins and soluble TNF.
In vitro analysis of TACE inhibition revealed a mechanism of TNF suppression common
among TNF signaling pathways and cell types. Analogous to small molecule protease inhibitors,
RTDs dose-dependently inhibited TACE mediated cleavage of a TNF-peptide substrate with
IC50s in the low to mid nanomolar range. Remarkably, despite sharing an invariant 10-amino
acid core structure, the five rhesus θ-defensin isoforms displayed significantly different potencies
in their respective TACE inhibition. These variances did not appear related to peptide charge or
to a homo- or hetero-nonapeptide composition of the peptide. Also of note, acyclic RTD-1 (S7),
which possess the same sequence, charge, and disulfide ladder as RTD-1, but contains a peptidic
break in the cyclic backbone, had significant reductions in potency (IC50 > 10-fold RTD-1). This
suggests that not only is the inhibition of TACE θ-defensin sequence specific, but the unique
cyclic structure may be essential as well. Observed RTD IC50s were 6 - 35 fold higher compared
to that of the broad-spectrum matrix metalloprotease inhibitor marimastat (observed IC50 = 7.55
71

± 0.572 nM). Additionally, TACE inhibition by RTDs showed comparable inhibition curves
using a dissimilar TACE/MMP fluorogenic substrate Mca-KPLGL-Dnp-AR-NH2 (data not
shown).
The most analogous human peptides to the rhesus θ-defensins are myeloid derived α-
defensins (HNPs 1-4). Like θ-defensins, these peptides are; arginine-rich, cationic, contain a tri-
disulfide array, packaged into azurophilic granules of neutrophils and monocytes, and have broad
antimicrobial activities [18,52-55]. However, while θ-defensins are produced by truncated α-
defensin genes, structurally the peptides are quite distinct [1,2]. Comparison of the two sub-
families of defensins indicates the human α-defensins do not possess TACE inhibitory activities
or any TNF modulating effects. This is not unexpected as α-defensins have previously been
shown to have pro-inflammatory properties that include the stimulation of TNF from myeloid
dendritic cells [56].
The effect of RTD treatment on LPS-stimulated THP-1 monocytes demonstrated each of
the isoforms possess a unique capacity for suppressing TNF release. In agreement with its
modest TACE inhibition, treatment with acyclic RTD-1 (S7) displayed minimal reductions in
TNF levels. Among the natural RTD isoforms, TNF suppression followed an IC50 order
remarkably similar to previous studies in human whole blood stimulated with E. coli, and mirrors
that seen in TACE inhibition. Correlation analysis comparing relative inhibition of TACE
activity to TNF suppression in THP-1 monocytes and blood, shows a statistically significant
degree of correlation.
The search for TACE inhibitors has been heavily dependent on high through-put screens
of small molecule libraries. Functional TACE inhibitors have included the following; succinate
based compounds, hydroxamate inhibitors, sulfonamide inhibitors, γ-lactam inhibitors, β-
72

benzamido inhibitors, benzothiadiazepine inhibitors, macrocyclic inhibitors, and zinc chelators
[32,57-61]. The majority of these compounds are competitive inhibitors, which bind to the zinc
containing catalytic site of the enzyme with variable binding kinetics and degrees of specificity
[32]. In contrast, Michaelis-Menten kinetics of TACE inhibition by RTD-1 suggests the peptide
is a non-competitive inhibitor of the enzyme, indicative of allosteric regulation. Evaluation of
binding kinetics indicated RTD-1 bound rapidly to TACE upon mixing, and that enzymatic
inhibition concurred with peptide binding. Furthermore the results of the RTD-1 washout
experiment in Figure 2-3 suggest the inhibition of TACE is reversible by washing the cells, a
potentially important function not shared with many of the tight-binding TACE inhibitors.
Despite the usefulness of these in vitro analyses, we recognized these preliminary assessments of
RTD/TACE interaction are not conclusive, and future studies will utilize x-ray crystallography,
in-silico modeling, and isothermal titration calorimetry to enhance our understanding of the θ-
defensin TACE interaction.
Evidence for cellular TACE inhibition was supported by two reporter assays. RTDs were
shown to reduce TACE mediated shedding of TGFα in COS7 cells. Of important note, the
relative inhibition of TGFα release among the RTD isoforms did not parallel those observed in
recombinant TACE inhibition and TNF suppression. This may be due to different TACE
sources, assay conditions effecting peptide/TACE interactions, or the possibility that the
magnitude of TACE inhibition is substrate dependent. TACE is responsible for shedding more
than 30 different membrane bound proteins, however there is no sequence homology among the
various substrates and it is not fully understood how TACE recognizes and cleaves such a
diverse array of proteins [24,26]. Conceivably the allosteric binding of RTDs to TACE may
produce differential inhibitory characteristics based on the enzyme target. This phenomenon
73

could occur through alterations in TACE substrate recognition or peptide induced alterations to
the binding and/or enzymatic clefts. Studies are currently underway to evaluate RTD mediated
effects on additional TACE substrates and may reveal a mechanism of selective inhibition.
Continuous real-time measurement of TACE activity in THP-1 macrophages and HT-29
colonic epithelial cells revealed substantial inhibition of TNF-peptide cleavage at concentrations
of 5 - 15 µM, similar to marimastat and reported hydroxamate-based metalloproteases inhibitors
GM6001 and BB94 [45]. The results of this experiment must be interpreted with the
understanding that there is the potential for other proteolytic enzymes cleaving the TNF-peptide
substrate, such as ADAM10 or various MMPs. However, utilizing a similar assay format,
Alvarze-Inglesias et al, showed that specific inhibition of TACE in THP-1 cells revealed it to be
the principal sheddase responsible for cleavage of the TNF substrate under these assay
conditions. Additionally, removal of the growth media and washing of the cells prior to the
addition of the fluorogenic substrate removed most, if not all, soluble MMPs present in the
extracellular environment. However, a unique advantage of this assay format is that it requires
no stimulus to activate TACE sheddase activity. This provided a means of monitoring cellular
TACE activity independent of agonists, cellular signaling pathways, or TNF transcriptional
activation. Additionally, in the context of TACE inhibition in PMA activated COS7 cells these
results suggest that θ-defensins are capable of inhibiting both constitutive and agonist activated
TACE.
As a member of the ADAM family of proteins, ADAM10 is responsible for shedding its
own unique set of proteins, such as those involved in Notch signaling [27,62]. ADAM10 is the
most closely related ADAM to TACE, and the two sheddases are known to share TNF as one of
their respective substrates [40,43]. RTD-1 was able inhibit the sheddase activities of cellular
74

ADAM10, as ionomycin induced shedding of betacellulin was disrupted by the addition of
peptide. Analysis of recombinant ADAM10 inhibition indicated that each of the RTD isoforms
was capable of ADAM10 inhibition, however IC50s were 5 - 10 fold those of TACE. The
physiologic relevance of this inhibition remains to be determined. The inhibition of ADAM10
was not surprising, as we have previously reported that RTD-1 selectively inhibits other
metalloproteases including multiple members of the MMP family including MMPs 2, 8, 13, and
14. However these effects required significantly higher concentrations of RTD-1 compared to
TACE with MMP IC50s in the low micromolar range (7.8 – 19.6 µM). RTD-1 has also been
shown to inhibit various members of the cathepsin family of cysteine proteases, including
cathepsins B, C, K, L, and V with a wide range of potency (IC50s = 0.027 – 3.7 µM). Therefore,
RTD-1 is a cross class anti-protease capable of selectively inhibiting both zinc metalloproteases
and cysteine proteases with varying degrees of potency.
Previously, we demonstrated co-incubation of RTD-1 reduced proinflammatory cytokine
responses in LPS stimulated THP-1 macrophages. Treatment with RTD-1 significantly reduced
both mRNA levels and secreted protein of TNF, IL-1β, and IL-8. Suppression of transcriptional
activation correlated with observed disruptions in NF-κB and MAPK signaling pathways via
RTD-1 induced pAkt, a negative regulator of the respective pathways [22]. These findings and
those presented above suggest θ-defensins modulate multiple steps in the proinflammatory
cascade. Specific inhibitions of soluble TNF is likely context dependent, with temporal effects,
cell type, cellular conditions, and local peptide concentrations being key factors. It is interesting
that suppression of TNF release from THP-1 monocytes and blood by RTD isoforms 1-5
correlated strongly with their respective TACE inhibition. However, it remains unknown if this
correlation is due to a single mechanism or to a multi-mechanistic effect resulting in suppression
75

of TNF. Studies are underway evaluating NF-κB and MAPK signaling suppression by the
various RTD isoforms in order to further our understanding of TNF suppression.
Dysregulated TACE sheddase activities have been linked to neurodegeneration,
inflammation, cardiovascular diseases, autoimmunity, skin disorders, and cancer [25-27,29-
31,63]. Tissue inhibitor of metalloprotease -3 (TIMP-3) is the only known endogenous inhibitor
of TACE sheddase activities [64-66], however its development as a therapeutic has been
hindered by its strong affinity to extracellular matrix. Several small molecule TACE inhibitors
have entered into human trials, nonetheless none have passed either due to a lack of efficacy or
off-site toxicity [27,32,67,68]. Here, we show that macrocyclic θ-defensins constitute a novel
class of peptidic TACE inhibitors. Multiple in vivo studies have demonstrated remarkable
efficacy in modulating pathogenic inflammation, while showing no toxic or immunoreactive side
effects. The peptides are stable in plasma for up to 72 hours and pharmacokinetic analyses have
indicated a prolonged circulating half-life via multiple routes of administration. With these
unique characteristics, θ-defensins offer a multifaceted therapeutic approach for the treatment of
inflammatory disorders.

76

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82

Chapter 3

A Retroevolutionary Strategy for Inducing Remission of Autoimmune Arthritis using
Macrocyclic θ-Defensins from Nonhuman Primates

Justin B. Schaal
1
, Dat Q. Tran
1
, Akshay Subramanian
1
, Reshma Patel
2
, Teresina Laragione
3
,
Kevin D. Roberts
1
, Katie Trinh
1
, Prasad Tongaonkar
1
, Patti Tran
1
, Dmitriy Minond
4
, Gregg B.
Fields
5,6
, Paul Beringer
7
, André J. Ouellette
1,8
, Percio S. Gulko
3
, & Michael E. Selsted
1,8

1
Department of Pathology & Laboratory Medicine, Keck School of Medicine, University of
Southern California, Los Angeles, California, USA
2
Children’s Hospital Los Angeles, Los Angeles, California, USA
3
Division of Rheumatology, Department of Medicine, Icahn School of Medicine at Mount Sinai,
New York, New York, USA.
4
Torrey Pines Institute for Molecular Studies, Port St Lucie, Florida, USA
5
Department of Chemistry & Biochemistry, Florida Atlantic University, Jupiter, Florida, USA
6
The Scripps Research Institute, Jupiter, Florida, USA
7
School of Pharmacy, University of Southern California, Los Angeles, California, USA
8
Norris Comprehensive Cancer Center of the University of Southern California

*Copy of manuscript submitted to Science Translational Medicine May 2016
83

ABSTRACT
θ-defensins constitute a family of macrocyclic peptides expressed exclusively in Old
World monkeys. The peptides are pleiotropic effectors of innate immunity, possessing broad
spectrum antimicrobial activities and immunoregulatory properties. Here we report that θ-
defensins are highly effective in arresting and reversing joint disease in a rodent model of
rheumatoid arthritis. Parenteral θ-defensin treatment of Dark Agouti rats with established
pristane-induced arthritis (PIA) rapidly arrested evolving joint disease and restored limb mobility
and joint microanatomy. Peptide-mediated efficacy was associated with significant reductions in
joint IL-1β levels in vivo, and inhibition of invasiveness, IL-6 expression, and proliferation of
fibroblast-like synoviocyte (FLS) in vitro. Peptide treatment of rats with PIA also significantly
reduced the fraction of animals in which disease involved all four limbs. θ-defensins were non-
toxic, non-immunogenic, and effective when administered as infrequently as once every five
days, suggesting they have potential as a new class of anti-arthritic biologic agents.
84

INTRODUCTION
Defensins comprise three structural families of evolutionarily related host defense
peptides that participate in innate immunity [1-3]. Peptides of the defensin subfamilies,
designated as α, β, or θ, are expressed in leukocytes and/or epithelia of mammals and were
discovered based on their antimicrobial properties against bacteria, fungi, protozoa, and viruses
[4]. While most mammals express α- and β-defensins, θ-defensins are unique to Old World
monkeys, but are absent in other primates including humans and other hominids [2]. θ-defensins
are the only known cyclic polypeptides in the Animal Kingdom. The peptides are macrocyclic,
tridisulfide-stabilized 18-amino acid molecules produced by post-translational head-to-tail
splicing of two nonapeptides (Fig 3-1a) [5]. This conformation confers resistance to proteolysis
in vitro and in vivo [6,7]. Differential binary pairing of different precursor-derived nonapeptides
produces diverse θ-defensins, of which six and ten isoforms are produced in rhesus monkeys [8]
and baboons [9], respectively. Like α- and β-defensins, θ-defensins were discovered based on
their in vitro antimicrobial activities against bacterial, fungal, and viral pathogens [5,10-12].
The prototype θ-defensin, rhesus theta defensin 1 (RTD-1; Fig. 3-1c) is efficacious in
animal models of infectious disease. The peptide was highly effective in reducing lethality in
mice with experimental severe acute respiratory syndrome (SARS) [13] and E. coli and
polymicrobial sepsis [6]. In these studies the therapeutic effects of peptide treatment appeared to
be mediated, at least in part, by suppression of proinflammatory cytokines as opposed to a
primary microbicidal action. In vitro studies revealed that RTDs suppress expression and
secretion of proinflammatory cytokines including TNF, IL-1β and IL-6 [6,13] by antigen-
stimulated leukocytes, and the anti-inflammatory effects were mediated by regulation of NF-κB
and MAPK signaling pathways [14]. Based on these findings, we hypothesize that θ-defensins
85

are pleiotropic peptides that evolved to neutralize pathogens and modulate inflammation. To test
this concept, we evaluated the effect of RTD-1 and related isoforms on the course of pristane-
induced arthritis (PIA) in Dark Agouti (DA) rats, an autoimmune disorder that shares many
features of rheumatoid arthritis (RA) in humans.
RA is a systemic autoimmune disease that affects 0.5 to 1% the world population [15]. It
most commonly presents as polyarthritis involving the extremities. Affected joints are
characterized by erosive changes mediated by hyperplastic synovium (pannus) and chronic
inflammation [15]. Disease severity is associated with elevated production of inflammatory
cytokines and activities of tissue degrading proteases [15]. While the etiology of RA is poorly
understood, genetic studies in rodent RA models have provided insights into the pathophysiology
of RA that reveal dysregulation of cytokine signaling networks, and the central roles of TNF, IL-
1β, and IL-6 [16-18] have provided rationale for therapies targeting these arthritogenic cytokines.
While these and other biologic interventions have greatly advanced the treatment of RA, up to a
third of all RA patients do not adequately respond to disease-modifying anti-rheumatic drugs
(DMARD) or biologic therapies [19].
Here we report that θ-defensins are effective in treating established pristane-induced
arthritis (PIA) in DA rats. Rhesus θ-defensins (RTDs) arrest disease progression and reverse
joint pathology in animals with pre-existing early or advanced disease. The therapeutic effect
was associated with reduced joint levels of IL-1β in vivo. In vitro, RTD-1 inhibited invasion of
fibroblast-like synoviocytes (FLS), FLS proliferation, and enzymatic activities of joint-damaging
and cytokine activating proteases. These results suggest that θ-defensins, produced in Old World
monkeys but absent in humans, may provide a novel, transphylogenetic approach for the
treatment of RA and other inflammatory diseases.
86

Figure 3-1. Biosynthesis and structural features of θ-defensins. a. RTDs are 18-amino acid,
tridisulfide, macrocyclic peptides produced by head-to-tail splicing of two 9-amino acid
oligopeptides (color-coded) that are excised from corresponding propeptides [5,12]. b.
Comparison of the molecular structure and size of θ-defensins to methotrexate (MTX) and anti-
TNF IgG. c. Amino acid sequences of RTD-1, 2, and 5, color coded to represent the hetero- and
homodimeric splicing of nonapeptide precursors.
87

METHODS
Ethics. Protocols for animal use were approved by the University of Southern California
(USC) Institutional Animal Use and Care Committee (protocol number 11355). Sequential
blood sampling for pharmacokinetic analyses was performed by Bioanalytical Systems, Inc.
(BASi, West Lafayette, IN), an AAALAC-accredited facility. Approved analgesics were used for
pain and every effort was made to minimize suffering throughout the study.
Anti-arthritic agents. Methotrexate (MTX) was obtained from Sigma-Aldrich (St.
Louis, MO) and was dissolved in saline. Etanercept (Enbrel™; Immunex Corp., Thousand Oaks,
CA) was obtained from the USC Investigational Drug Service Pharmacy and was reconstituted
immediately prior to administration per manufacturer’s instructions.
Peptides. The hydrochloride salts of θ-defensins RTD-1, -2, -5 were prepared by solid
phase synthesis, oxidation, cyclization, and ion-exchange as previously reported [5,12]
producing highly pure (> 98%) materials as assessed by analytical HPLC and tandem mass
spectroscopy. Sterile stock solutions of each peptide were dissolved in normal saline and stored
at 4
o
C.
Pristane-induced arthritis (PIA). Female DA/OlaHsd (D blood group allele and
Agouti, also known as Dark Agouti), rats (5-7 weeks of age, 90-100 gm) were obtained from
Harlan Laboratories (Indianapolis, IN). Animals were housed individually, and allowed to
acclimate for 48-72 hours prior to experimental use. Animals were fed standard chow ad lib
during a 12-hour day/night cycle. PIA was induced by intradermal injection of 250 μl pristane
(Sigma-Aldrich, St Louis, USA) divided into 3-5 sites at the base of the tail [20,21]. Animals
were monitored daily for signs of joint swelling. Disease severity of each limb was scored 0 - 4
88

using method of Brand et al. [22]: 0 - no evidence of erythema and swelling; 1 - erythema or
mild swelling confined to the ankle or paw; 2 - erythema and mild swelling from ankle to the
midfoot or the metatarsals; 3 - erythema and moderate swelling extending from ankle to the
metatarsal joints; 4 - erythema and severe swelling encompassing the ankle, foot, and digits. The
observed score of each limb per animal were totaled to determine the rodent’s total Arthritis
Severity (AS; 0 - 16). Disease severity was assessed by at least two scorers and the inter-rater
agreement (kappa) value was 0.806 as determined using MedCalc Statistical Software version
14.8.1 (MedCalc Software, Ostend, Belgium). In three separate trials, treatment was also
blinded to scorers. There was no statistical difference between blinded and unblinded scoring of
any treatment cohort. Treatment with saline (vehicle), RTD-1, or anti-arthritic agents was
initiated when an animal first presented with an arthritis severity score of 3 or more. RTD-1 was
administered subcutaneously with doses ranging from 0.2 to 5.0 mg/kg in an injection volume of
0.5 ml at intervals ranging from once a day (qd) to once every seven days (q7d). Sequential
injections were distributed over different sites of the dorsal midsection. MTX dissolved in saline
was administered at 0.25 mg/kg by intraperitoneal injection once q3d [23]. Etanercept was
administered at 0.4 mg/kg by subcutaneous injection once q3d [24] [25].
Joint histopathology. Limbs from euthanized animals were preserved in buffered
formalin, decalcified, embedded, sectioned, and stained with hematoxylin and eosin (H & E) in
the USC Research Model Pathology Core. Microscopic images were acquired with a Nikon-
Microphot FXA with SPOT 5.1 image capture software.
RTD-1 pharmacokinetics. Eight to 12-week old male Sprague Dawley rats (Harlan)
were fitted with a surgically implanted jugular vein catheter that was externalized over the
scapula for sequential blood collection (BASi, West Lafayette, IN). Animals were individually
89

housed (Culex NxT Automated In Vivo Sampling System; BASi), with ad lib access to water
and standard chow and 12 hour light/dark cycle. RTD-1 dissolved in saline was injected
subcutaneously at 5 mg/kg to 4 animals. Approximately 200 μL of blood was collected prior to
peptide administration and 0.25, 0.5, 1, 2, 4, 6, 8, 12, 18, 24, 30, 36, 48, 60, and 72 h after RTD-
1 injection. Blood was collected into pre-chilled (4° C) vials containing K3EDTA and plasma
was collected after centrifugation for 10 min at 1200 × g at 4°C. Plasma samples were
transferred to sample tubes, snap frozen, and shipped on dry ice to our laboratory where they
were stored at -80° C. Plasma concentrations of RTD-1 were determined after solid phase
extraction (SPE) and quantitation by C18 RP-HPLC, photodiode array (PDA) detection, and
electrospray-ionization mass spectroscopy (ESI-MS) using a modification of a method described
previously [8]. Briefly, 50-100 μl of plasma was diluted 1:20 with 4% H3PO4, 10% ACN
containing 100 ng of RTD-2 and RTD-3 as internal standards. The mixture was loaded on a 3cc
Oasis WCX solid phase extraction cartridge (Waters, Milford, MA) equilibrated in water, and the
cartridge was washed with 2 ml each of 5% NH4OH, 20% ACN, 1% TFA, and (10% ACN + 1%
TFA). Bound material was eluted with 2 ml (1% TFA + 40% ACN) and 2 ml (1% TFA + 60%
ACN). Eluents were lyophilized, suspended in 100 µl of 10% HOAc, 5% ACN, and
chromatographed at 0.3 ml/min on a Waters C18 X-Bridge BEH C18 2.5 µm 2.1 x 150mm XP
column using 5 to 55% water-acetonitrile gradient containing 0.1% formic acid.
Chromatography was performed on an Acquity H-class UPLC with an analytical photodiode
array (PDA) detector using Empower 3 software (Waters, Milford, MA). Quantitative mass
spectrometry was performed on post PDA eluent using a Micromass Quattro Ultima mass
spectrometer with Mass Lynx 4.1 (Waters, Milford, MA). Pharmacokinetic analysis was
performed using parametric population modeling with maximum likelihood estimation via the
90

EM algorithm with sampling (MLEM) as implemented in the ADAPT 5 (Version 5.0.49) PK/PD
Systems Analysis Software (Biomedical Simulations Resource, University of Southern
California).
Peptide stability in plasma analysis. RTD-1, RTD-1-Cys Ala, and acyclic-RTD-1,
were prepared synthetically as described above. Peptides were dissolved in 0.01% HOAc to 500
µg/ml. 20 µL of peptide was added to 80 µL of either clarified human or Sprague Dawley rat
EDTA plasma. Reactions were incubated at 37° C and samples collected at 0, 3, 24, 48, 144
hours. Samples were quenched and diluted by a 1:10 dilution into 10% HOAc 5% ACN.
Samples were then directly injected onto LCMS and remaining peptide quantified. All samples
were run in duplicate.
Joint tissue cytokine analysis. Saline (control) and RTD-1 (5 mg/kg, s.c., qd) treated
PIA rats were euthanized 4 days after disease onset, defined as arthritis severity scores > 3. The
ankle joints and feet were snap frozen in liquid N2 and pulverized with mortar and pestle as
described [26]. One gram of tissue powder was extracted with 5 ml of ice cold 10 mM sodium
phosphate, 150 mM NaCl, pH 7.4 (PBS) containing protease inhibitor tablets (cOmplete ULTRA
with EDTA; Roche Diagnostics GmbH, Mannheim, Germany) per manufacturer’s directions.
Samples were vortexed for 30 seconds and extracts clarified by centrifugation at 22,000 × g for
10 min, 4°C. Supernatant IL-1β was quantified by ELISA (Life Technologies, Grand Island,
NY).
Isolation of FLS from DA rats. Fibroblast-like synoviocytes (FLS) were isolated by
enzymatic digestion of the synovial tissue from DA rats with PIA as previously described [27].
Briefly, tissues were dissected from ankle joints, minced and incubated in Dulbecco's modified
Eagle's medium (DMEM; Gibco, Invitrogen Corporation, Carlsbad, CA) containing 0.15 mg/ml
91

DNase, 0.15 mg/ml hyaluronidase type I-S, and 1 mg/ml collagenase type IA (Sigma-Aldrich, St.
Louis, MO) for 1 hour at 37°C. Cells were washed and re-suspended in DMEM supplemented
with 10% fetal bovine serum (Gibco), 30 mg/ml L-glutamine (Sigma), 250 μg/ml amphotericin
B (Sigma), and 10 mg/ml gentamicin (Gibco). After overnight culture, non-adherent cells were
removed and adherent cells were cultured to approximately 70-90% confluence and passaged by
detachment with 0.25% trypsin-EDTA for 3 min at 37
o
C. All experiments were performed with
FLS after at least four passages.
Invasion assay. FLS invasiveness was assayed in vitro using Matrigel-coated inserts in a
transwell system (Becton Dickinson, BD) as previously described [27]. Briefly, DA rat FLS
(70–80% confluent) isolated as described above were suspended at 2.0 × 10
4
cells in 500 μl of
serum-free DMEM. Cells were placed in the upper compartment of the Matrigel-coated inserts
to which RTD-1 was added to final concentrations of 0.1, 1, or 10 μg/ml. The lower
compartment contained complete medium and plates were incubated at 37°C. After 24 hours the
upper surface of the insert was wiped with cotton swabs to remove non-invading cells and the
Matrigel layer. The opposite side of the insert was stained with Crystal Violet (Sigma) and the
total number of invading cells was counted at 100X magnification. Experiments were performed
in duplicate, and each of the seven cell lines was generated from individual DA rats.
Stimulation of FLS. FLS were cultured in 12-well plates at 4 - 6 × 10
4
cells/well (1
ml/well) for 24 h (viability > 95% by trypan blue staining). Medium was replaced and FLS were
incubated with TNF or IL-1β at final concentrations of 3 or 10 ng/ml plus RTD-1 (10 μg/ml in
0.01% HOAc) or vehicle alone. After 24 h incubation, FLS culture supernatant was removed
and processed using two-stage centrifugation isolation (900 x g 4°C for 5 min, followed by 6000
x g, 4°C for 5 min). Supernatants were analyzed for IL-6 by ELISA (Invitrogen).
92

FLS proliferation. FLS were seeded at 3 × 10
3
cells/well in a 96-well TC treated plate
(Greiner). Cells were allowed to adhere for 18 h and medium was replaced prior to addition of
peptide or vehicle. RTD-1 was added to wells at final concentrations of 0 – 30 μl/ml in culture
medium and incubated for 0, 24, or 48 h at 37°C in 5% CO2. FLS cell number was determined
by DNA staining using CyQuant Cell Proliferation Assay Kit (Invitrogen) per manufacturer’s
instructions. Cellular toxicity was determined by analyzing FLS supernatants for lactate
dehydrogenase (LDH) activity (CytoTox 96 Cytotoxicity Assay; Promega) per manufacturer’s
instructions. Maximum LDH release was determined by incubating replicate sample with lysis
buffer for 1 h and analyzing LDH release. All assays were performed with ≥ 7 replicates.
Protease inhibition assays. RTD-1 was analyzed for enzymatic inhibition against zinc
metalloproteases ADAM17, ADAM10, matrix metalloproteases (MMPs) 1, 2, 3, 8, 9, 13, and 14,
and cysteine cathepsins (Cats) B, C, H, K, L, S, and V (Table 1). ADAM10 and ADAM17
activities were determined by measuring cleavage of fluorogenic substrates derived from
cleavage sites in pro-TNF (R&D Systems, Minneapolis, MN). ADAM10 was diluted to 0.05
µg/ml and ADAM17 to 0.1 µg/ml with 25 mM TRIS, 2.5 µM ZnCl2, 0.005% Brij35, pH 9.0.
RTD-1 dissolved in 0.01% HOAc was added to wells and serially diluted to final concentrations
of 0.003 to 3 µg/ml. Substrate was added to a final concentration of 10 µM and its conversion
was measured at 37°C (for ADAM10) or 22°C (for ADAM17) every 30 seconds for 1 h in a
SpectraMax M5e fluorometer (Molecular Devices) with excitation (λ ex) and emission (λ em)
wavelengths of 320 nm and 405 nm, respectively.
Cat B was purchased from BPS Bioscience (San Diego, CA) and Cats C, H, K, L, S, and
V were purchased from R & D Systems (Minneapolis, MN). RTD-1 inhibition of Cats B, C, H,
K, L, and V was determined using a 96-well format and enzyme-specific substrates. Hydrolysis
93

of aminomethyl coumarin (AMC) substrates was measured with λ ex = 380 nm and λ em = 460 nm;
the cathepsin S dinitrophenyl (DNP) substrate was measured with λ ex = 320 nm and λ em = 405
nm. Specific assay conditions including substrate and buffer compositions are listed in Table 1.
The activity of each cathepsin was analyzed in the absence or presence of 3 ng/ml to 50 µg/ml
RTD-1.
Inhibition of MMPs by RTD-1 was performed as described [28]. Briefly, 5 µL of 2x
enzyme solution (20 nM) in 50 mM Tris-HCl, 150 mM NaCl, 10 mM CaCl2, 0.05% Brij-35, pH
7.5 was added to solid bottom white 384 plates (Greiner, Monroe, North Carolina). Fifty nL of
peptide (2 ng/ml to 100 µg/ml) was added to corresponding wells using 384 pin tool device
(V&P Scientific, San Diego). After 30 minute incubation at RT the reactions were started by
addition of 5 µL of 2x solutions of substrate (20 µM). Reactions were incubated at room
temperature for 30 minutes, after which the fluorescence was read using a Tecan Safire2
multimode microplate reader (λ ex = 324nm, λ em = 393nm). Inhibition of MMP-3 was performed
using a SensoLyte 490 MMP-3 Assay Kit (AnaSpec) per manufacturer’s instructions. IC50
values were calculated by GraphPad Prism 5.01 utilizing a non-linear curve fit of log [inhibitor]
versus response with variable slope.
RTD-1 immunogenicity analysis. Adult DA rats received single s.c. injections of 5
mg/kg of RTD-1 every day (total of 15 injections) or every other day (total of 8 injections). Two
months later, all animals were re-challenged once s.c. with 5 mg/kg of RTD-1, and two days later
serum was collected from each animal. Nitrocellulose membranes (Amersham Hybond-ECL GE
Healthcare 0.2µm RPN3032D) were dotted with 100 ng of RTD-1 or 100 ng of rat IgG or DA rat
serum (secondary antibody control), blocked with 5% non-fat milk, and incubated overnight in
1:1000 serum from saline or RTD-1 challenged animals. Secondary Ab was 1:50,000 goat-anti-
94

rat IgG-HRP (sc-2006 Santa Cruz Biotech). A positive control for RTD-1 detection utilized
1:500 goat anti-RTD-1 IgG primary overnight incubation, with a 1:50,000 horse anti-goat IgG
horse radish peroxidase secondary. Blots were developed with Supersignal West Pico peroxide
solution & Luminol Enhancer Solution (Thermo 1859674 1859675). Film (Amersham
Hyperfilm ECL GE Healthcare) was exposed to blot for 1 minute and then developed.
Statistics. All statistical analyses were performed utilizing GraphPad Prism 5.01.
Graphs are represented as mean +/- standard error of the mean (SEM) with the exception of
Figure 3-4b represented as + SEM. Unless otherwise indicated, P-values are calculated using
Student’s t-test. Mann-Whitney U-test was utilized for non-parametric sample populations as
indicated in Fig 3-6b. Comparisons of proportions in Fig 3-6g were performed by Chi-squared
analysis. N = number of times experiments were repeated; n = number of replicates in each
experiment or total number of animals included in the graph.
95

RESULTS

Efficacy of RTD-1 in rat PIA. The anti-arthritic effects of θ-defensins were tested in
DA/OlaHsd rats with established arthritis induced by intracutaneous challenge with pristane, a
rodent model that resembles RA in its immunopathology and chronicity [26,29-31]. Animals
with established PIA (Arthritis Severity, AS > 3) were initially treated with daily subcutaneous
(s.c.) injections of saline or 5 mg/kg of RTD-1 for 11 days and scored for gross pathology (Fig.
3-2). Peptide treatment significantly reduced the progression of arthritis severity within 24 h of
the first administered dose (P = 0.0064; Fig. 3-2), and RTD-1 treated rats had markedly reduced
AS scores at the end of the observation period compared to vehicle controls (68.7%; P < 0.0001;
Fig. 3-2). Further, RTD-1 treatment resulted in restoration of limb function and mobility (typical
response shown in Supp. Video 1). Of note, after a disease-free interval ranging from 4-12
weeks, low grade arthritis (average AS score of ~3) recurred in each of three PIA rats that were
observed for 5 months. The recurrence and variability of the disease-free interval is consistent
with previous PIA studies and resembles the clinical course of RA [26,32].

96

0 2 4 6 8 10 12
0
2
4
6
8
10
12
Saline
RTD-1
***
**
*
n = 31
n = 40
Day
Arthritis severity

Figure 3-2. Efficacy of RTD-1 in rat PIA. DA rats with established PIA were treated s.c. daily
with RTD-1 (5 mg/kg) or vehicle (saline). RTD-1 treatment reduced AS within 24 h compared
to control (* P = 0.0064), and therapeutic efficacy increased with duration of treatment (** P <
0.001; *** P < 0.0001) (N = 6).

Effect of RTD-1 on joint gross pathology and histology in PIA rats. RTD-1 treatment
markedly reduced gross pathology in rats with established PIA as evidenced by resolution of
erythema, swelling, and tenderness of affected joints (Fig 3-3 A-C). These finding correlated
with joint histology of limbs from the corresponding cohorts. Joints from vehicle-treated PIA
rats showed extensive synovial hyperplasia, invasive pannus, and erosion of cartilage and bone
(Fig 3-3, panels E, H, & K). In contrast, joints from RTD-1 treated animals (Fig 3-3; panels F, I,
& L) were nearly indistinguishable from those of naïve animals (Fig. 3-3, panels D, G, & J and
Supplementary Fig. 3-1). Further, daily administration of RTD-1 for 5-9 days resulted in
restoration of normal limb function and rat mobility (Supplementary video 1).
97

Figure 3-3. Effects of RTD-1 on gross pathology and histology of rat PIA. Representative
images show gross and histologic features of hind extremities from naïve, saline-treated PIA rats
(daily s.c. x 11 days), and RTD-1 treated PIA rats (daily s.c. 3 mg/kg for 9 or 11 days; see
Supplementary Fig. 3-1 for additional examples of normal, PIA, and RTD-1-treated joint
histology). Joints from saline-treated PIA rats show synovial hyperplasia, invasive pannus, and
disruption of joint architecture (panels e, h, k). RTD-1 treatment of PIA rats resulted in marked
resolution of paw swelling as well as preserved joint architecture (panels c, f, i, l).
98

Supplementary Figure 3-1. Additional examples of joint histology of naïve, PIA, and RTD-1-
treated PIA rats from which representative micrographs were selected. Histology images were
captured at either 10 or 4X magnification (organized by column). The disease severity score of
each individual sectioned limb is indicated as “AS” with possible scores of 0-4.

99

Efficacy of RTD-1 in severe PIA and comparison with RA drugs and θ-defensin isoforms.
RTD-1 treatment was also effective in resolving advanced PIA. Rats with severe arthritis (AS of
>12) received daily 5 mg/kg s.c. injections of RTD-1 or saline for 6 to 9 days. Peptide treatment
produced a rapid reduction in arthritis severity, which was significant within 48 hours of the start
of treatment (P < 0.05), with remission of disease by day 15 (Fig 3-4a). As with animals treated
in early PIA (Fig. 3-2), peptide treatment of animals with severe PIA regained essentially normal
limb function and mobility (Supplementary video 2).
Efficacy of RTD-1 treatment was compared with methotrexate (MTX) and etanercept
(Etan), first line RA drugs that have also been studied in rodent models of arthritis and rat PIA
[15,23-26]. Compared to vehicle controls, both Etan and MTX significantly reduced AS scores
by day 3 of treatment (Fig. 3-4b). Treatment with RTD-1, however, had an even more
pronounced effect than either Etan or MTX, and was the only agent that induced disease
remission (Fig. 3-4b). To our knowledge, θ-defensins are the first agents capable of inducing
disease remission, without concomitant toxicity, in a rodent model of RA.
TNF is one of several cytokines implicated in RA pathogenesis, and biological blockade
of TNF has greatly improved disease management [33]. Further, in several studies TNF has
been implicated in the pathogenesis of PIA in rodents [26,30,34]. In a previous study we showed
that two θ-defensin isoforms, RTD-2 and RTD-5 (Fig. 3-1c), were 2 - 4 fold more potent than
RTD-1 in suppressing TNF release by LPS- or E. coli- stimulated leukocytes [6]. Therefore, we
tested RTDs 1, 2, and 5 for relative efficacy in PIA. As shown in Figure 3-4c, the three peptides
were similarly effective in inducing arrest and remission of PIA. Thus, the differential efficacy of
TNF blockade by the three θ-defensin isoforms in vitro does not directly correlate with
therapeutic potency in PIA.
100

Figure 3-4. Efficacy of RTD-1 in severe PIA and comparison with RA drugs and θ-defensin
isoforms. a. RTD-1 induces remission of advanced PIA (AS > 12 prior to treatment) with
significant (P < 0.05) reduction in AS within 2 d of initiation of daily treatment with s.c. peptide
at 5 mg/kg. b. Efficacy of RTD-1 in rat PIA is superior to that afforded by MTX and etanercept.
Each agent significantly reduced arthritis severity scores (P < 0.05) by day 3 following the start
of treatment. At day 11, RTD-1 treatment resulted in significantly greater disease resolution than
MTX (* P < 0.01) or etanercept (** P < 0.001) treatments (N = 2). c. Anti-arthritic potencies of
RTDs 1, 2, and 5 were tested by daily injection of PIA rats with 5 mg/kg of peptide or saline in
cohorts of 10 animals per treatment arm. Treatment with each RTD isoform resulted in
statistically significant reductions in disease severity by day 4. Relative efficacy of the three
isoforms (RTD-2 > RTD-1 > RTD-5) persisted throughout the observation period but differences
were not statistically significant.

101

Dose-dependent anti-arthritic effects and pharmacokinetics of RTD-1. The anti-arthritic
effects of RTD-1 in PIA were dose-dependent, with AS reductions observed with doses as low as
1 mg/kg (Fig. 3-5a). Further, dosing of RTD-1 at 5 mg/kg was equally effective whether
administered daily or once every 2, 3, or 5 days (Fig. 3-5b). Analysis of single dose
pharmacokinetics (PK) of s.c. administered RTD-1 revealed that the peptide is rapidly distributed
(clearance and half-life of 22.3 ml/kg/h and 0.285 h, respectively), but terminal clearance of
RTD-1 was considerably slower at 3.55 ml/kg/hr (±0.286) with a terminal elimination half-life of
32.3 h (±10.4; Fig. 3-5c). These PK parameters are consistent with the finding that RTD-1 is
highly stable in human [6] and rat plasma (see below) and that q5d dosing is as effective as daily
dosing (Fig. 3-5b).
Previous studies have revealed that the macrocyclic conformation of RTD-1 is essential
for its microbicidal [5,7] and immunomodulatory effects [14]. We hypothesized that the
disulfide-stabilized macrocycle is uniquely configured for stability in biological matrices. RTD-
1 was therefore compared to disulfide-null (all Cys Ala) and open-backbone RTD-1 analogs
(Supplementary Fig. 3-2) for stability in human and rat plasma for 6 days at 37
o
C. Quantitation
of intact peptide at different time points demonstrated that native RTD-1 was the most stable of
the peptides tested (83-87% intact at 144 h), with intermediate stability of the Cys Ala analog,
and only 17-20% of the acyclic analog was intact at the 144 h time point (Supp. Fig. 3-2).
102

Figure 3-5. Dose-dependent anti-arthritic effects and pharmacokinetics of RTD-1. a. PIA rats
receiving daily s.c. injection with 3 or 5 mg/kg of RTD-1 rapidly responded with significant (P ≤
0.0015) and nearly identical reductions in AS by day 3 of treatment. Dosing with 1 mg/kg RTD-
1 resulted in modest reductions in AS, however failed to be statistically significant, whereas 0.2
mg/kg produced no therapeutic effect (N = 2). b. Dose frequency analysis shows that s.c.
injection of PIA rats with 5 mg/kg of RTD-1 daily, or once every 2, 3, or 5 days produced
equivalent disease resolution. (N = 1, n = 5 per treatment group). AS were recorded every day
and depicted on the graph every other day for clarity. c. Pharmacokinetics of s.c. administered
RTD-1 was analyzed in Sprague Dawley rats (n = 4) that received a single 5 mg/kg s.c. dose of
RTD-1. RTD-1 is rapidly distributed with clearance and half-life of 22.3 ml/kg/h and 0.285 h,
respectively. Terminal clearance of RTD-1 was considerably slower at 3.55 ml/kg/hr (±0.286)
with a terminal elimination half-life of 32.3 h (±10.4).
103

Supplementary Figure 3-2. θ-defensin stability in human and rat plasma. RTD-1 (circles) and
structural analogs including disulfide-null RTD-1-Cys Ala (square), and acyclic RTD-1
(diamond) were tested for chemical stability in human (solid) and rat (open) EDTA plasma.
Peptides were incubated in plasma at 37° C for up to 144 hours. Intact peptide was quantified at
each time point by LCMS. Plots represent mean +/- SD of duplicates.
104

RTD-1 is non-immunogenic. The stability of θ-defensins has also been implicated in their lack
of immunogenicity of RTD-1 when employed in antigenic challenge in chimpanzees [6], mice,
and rabbits (unpublished data). To further evaluate the immunogenic potential of RTD-1, we
challenged DA rats with RTD-1 with 8-15 serial s.c. injections over two weeks, followed by a
final s.c. injection two months later. Serum from each animal was analyzed for humoral
response and no anti-RTD-1 Ig was detected in any RTD-1 challenged animals (Fig. 3-6).

Figure 3-6. RTD-1 is non-immunogenic following multiple subcutaneous challenges. Animals
were challenged 8-15 times and boosted prior to acquisition of serum samples as described in
Methods. Serum was analyzed for presence of anti-RTD-1 IgG by dot blot. Top Panel: Dot blot
controls for rat IgG and RTD-1 using horse anti-rat-IgG and goat anti-RTD-1, respectively.
Lower panels: Left column membranes were dotted with 1 µL of serum from DA rats
challenged with RTD-1 as indicated, or serum from a saline-only control. Left panel membranes
were probed with horse anti-rat-IgG (positive control). Right column membranes were dotted
with 100 ng of RTD-1, and probed overnight with serum from either challenged or naïve (saline)
rats. All membranes were developed with secondary antibodies (see Methods) and HRP
detection.
105

Effects of RTD-1 on arthritogenic mediators. Studies were performed to identify mechanisms
that mediate the anti-arthritic properties of RTD-1. Because IL-1β plays a central role in the
pathogenesis of RA [35-37] and rodent arthritis [38-41] and because RTD-1 suppresses IL-1β
expression and release by LPS-stimulated monocytes [6,14], we analyzed the effect of RTD-1
administration on joint levels of IL-1β in PIA rats. Consistent with other studies, IL-1β was
markedly elevated in diseased joints of PIA rats, with increased cytokine levels in limbs with
greater disease severity (Pearson r = 0.4923, P = 0.0013; Fig 3-7a); IL-1β was not detectable in
joints of naïve rats. PIA rats with established disease (AS > 3) were treated once a day for four
days with 5 mg/kg RTD-1 or vehicle after which joint tissue from euthanized animals was
analyzed for IL-1β. Peptide treatment reduced joint IL-1β by an average of 44% (P = 0.0054;
Mann-Whitney U-test; Fig. 3-7b), which coincided with the marked reduction in arthritis severity
in RTD-1 treated animals after 4 days of treatment (P = 0.0044; Fig. 3-2).
Because IL-6 is a pleiotropic cytokine that is elevated in RA and implicated in multiple
aspects of RA pathogenesis, including the differentiation of Th-17 cells, induction of
arthritogenic proteases, angiogenesis, synoviocyte invasion [42,43], and bone loss [44], we also
analyzed the effect of RTD-1 on expression of IL-6. IL-6 is induced in fibroblast-like
synoviocytes (FLS) by TNF and IL-1β, and the central role of these cytokines in RA has
provided the rationale for development of FDA-approved biologics that also target IL-1β and IL-
6 [15]. Cultured FLS were stimulated with TNF or IL-1β, both of which induced strong IL-6
secretory responses that were significantly reduced by the addition of RTD-1 (Fig. 3-7c), similar
to the effect of RTD-1 on IL-6 expression by LPS-stimulated monocytes and macrophages [6].
106

Cytokines produced at increased levels by the arthritic synovial tissue stimulate the
proliferation and invasion of FLS, and the resulting pannus and associated invasion factors play a
major role in the erosive joint changes in RA and PIA [27,29,45,46]. The in vitro invasiveness
of FLS correlates with histologic damage in PIA [27] and radiographic damage in RA [47].
Given the efficacy of RTD-1 in rat PIA and its effects on preservation of joint architecture (Fig.
3-3 and Suppl. Fig. 3-1), we analyzed the effect of RTD-1 on FLS proliferation and invasiveness.
RTD-1 dose-dependently suppressed FLS proliferation when analyzed after 24 or 48 hours in
culture (Fig. 3-7d). In addition, RTD-1 dose-dependently suppressed FLS invasion of Matrigel-
coated inserts (Fig. 3-7e). The invasive-suppressive effects were observed even at low
concentrations (100 ng/ml) and were highly reproducible against seven independent DA rat FLS
cell lines (Fig. 3-7f).
Studies have demonstrated that the polyarticular involvement of RA results from the
transmigration of invasive FLS from a primary site of disease via the lymphatics and/or
bloodstream to unaffected sites where FLS disrupt joint tissue homeostasis in otherwise normal
joints [48]. Since RTD-1 suppressed FLS proliferation and invasiveness in vitro, we
hypothesized that RTD-1 would reduce polyarticular disease in PIA. We analyzed the
involvement of all four limbs in PIA rats treated with saline alone or daily treatment with 5
mg/kg of RTD-1. Animals treated with RTD-1 had significantly (P = 8.4 x 10
-7
) reduced
incidence of PIA involving four limbs compared to saline-treated animals (57.1% versus 87.2%,
respectively; Fig. 3-7g). Taken together, these results suggest that RTD-1 treatment limits that
spread and/or proliferation of invasive FLS.
107

Figure 3-7. Effects of RTD-1 on arthritogenic mediators. a. IL-1β levels in joint homogenates
increased with severity of gross pathology (Pearson r = 0.49, P value = 0.0013). Number in
parentheses equals number of paw/ankle joints analyzed. b. RTD-1 treatment significantly (P =
0.005) reduces IL-1β levels in joints of PIA rats after 4 daily 5 mg/kg s.c. treatments compared to
vehicle controls. c. RTD-1 suppresses TNF- and IL-1β- stimulated production of IL-6 by
cultured FLS (* P < 0.05, ** P = 0.0059) (n = 12). d. RTD-1 dose-dependently suppresses
proliferation of cultured DA-PIA-FLS. FLS cell numbers were quantified after 0, 24, and 48 h
incubation with RTD-1 at the indicated concentrations (* P = 0.0023, ** P = 0.0243, *** P <
108

0.0001) (n = 7). e. RTD-1 suppresses invasion of FLS from PIA rats in a dose-dependent
manner, with minimal cytotoxicity (percent viable cells above bars). f. The anti-invasive effects
of RTD-1 on 7 independently isolated rat FLS cell lines were analyzed (as in panel e).
Incubation with 0.1 µg/ml RTD-1 inhibited invasion of all cells lines by 50%. g. Rats treated
daily with RTD-1 (5 mg/kg) in were evaluated on day 11 for the occurrence of polyarticular
arthritis involving all four limbs. By day 11 of treatment, saline control animals had
significantly greater disease frequency (87.2%) in all limbs than those treated with RTD-1
(57.1%); (P = 8.4 x 10
-7
, Chi-squared analysis).

RTD-1 inhibition of proteinases associated with RA pathogenesis. Based on the finding that
RTD-1 is a potent and extremely rapid inhibitor of TNF release by LPS-stimulated leukocytes
[6], we assessed the effects of RTD-1 on proteases implicated in RA and PIA joint disease.
Based on the kinetics of these effects, we hypothesized that RTD-1 inhibits TNF release by
inhibition of TNF-alpha converting enzyme (TACE; ADAM17) [49]. We found that RTD-1 is a
submicromolar inhibitor of ADAM17, as well as ADAM10, a related proTNF sheddase [50]
(Table 1).
With the finding that RTD-1 inhibits ADAMs 17 and 10, two zinc metalloproteinases, we
tested whether the peptide affects the activities of other zinc metalloproteinases implicated in
cartilage degradation in RA. RTD-1 selectively inhibited arthritogenic MMPs 2, 8, 9, 13, and
14, with IC50 values ranging from 2 to 20 µM (Table 3-1), prompting us to test RTD-1 inhibition
on other proteases implicated in RA. Surprisingly, RTD-1 also inhibited cathepsins B, C, K, L,
and V, with its greatest potency against cathepsin K (Table 3-1), the protease primarily
responsible for degradation of bone matrix by osteoclasts [51,52]. These data indicate that RTD-
109

1 is a cross class anti-protease that inhibits TNF activation and matrix degrading proteinases
implicated in RA joint damage.

Buffer
A 25 mM TRIS, 2.5 µM ZnCl2, 0.005% Brij35, 5mM DTT, pH 9.0
B 25 mM 2-[n-morpholino] ethanesulfonic acid (MES), 50 mM NaCl, 5mM DTT, pH 5.0
C 25 mM MES, 50 mM NaCl, 5mM DTT, pH 6.0
D 100 mM Na phosphate, 1 mM EDTA, 5mM DTT, pH 6.8
E 50 mM NaOAc, 2.5 mM EDTA, 0.01% Triton X-100, 5mM DTT, pH 5.5
F 50 mM MES, 5mM DTT, pH 6.0
G 50 mM NaOAc, 250 mM NaCl, 5mM DTT, pH 4.5
H 25 mM NaOAc, 100 mM NaCl, 5mM DTT, pH 5.5
I 50 mM Tris-HCl, 150 mM NaCl, 10 mM CaCl2, 0.05% Brij-35, pH 7.5
J Kit assay buffer (Anaspec SensoLyte AS-71130)
110

DISCUSSION

We hypothesized that RTD-1 would alter the course of disease in a rodent model of RA
model that involves inflammatory networks that overlap with those inhibited by θ-defensins in
infectious inflammation such as those mediated by TNF, IL-1β, and IL-6. In preliminary studies
we found that RTD-1 rapidly reduced joint swelling in collagen-induced arthritis (CIA) in
Sprague-Dawley rats (data not shown). We subsequently undertook studies employing the rat
PIA model since this T and B-cell dependent disease mimics human RA in a number of ways,
including symmetrical involvement of peripheral joints, destruction of cartilage and bone,
dysregulated pro-inflammatory cytokines/chemokines, and its chronic course. PIA is dependent
on MHC class II-restricted T cells [30] and influenced by non-MHC genes, producing cellular
and humoral autoimmunity [29]. Several studies have identified additional non-MHC genetic
loci that regulate rat PIA arthritis severity, revealing the involvement of numerous inflammatory
cytokines, including TNF, IL-1β [53], IL-6, IL-17 [54], and CCL-2, and arthritogenic MMPs
[29,55]. Rat PIA is increasing utilized as a model for preclinical evaluation of human RA drug
candidates.
In this study we demonstrated that RTD-1 arrests and induces remission of pre-existing
and severe PIA in DA rats. The effect of peptide treatment was rapid, dose- and dose-interval
dependent arrest of joint disease which was followed by a reproducible resolution phase that
occurred whether RTD-1 was administered early or late in the disease course. RTD-1-mediated
resolution of PIA signs and symptoms correlated with joint histopathology which revealed
restoration of normal joint architecture in peptide-treated animals. Additionally, RTD-1 treated
animals had remarkable restoration of limb function and significant reductions in discomfort and
111

pain. These results and the corresponding histologic findings indicate that RTD-1 induces
healing of arthritic joint tissues as has been observed in arthritic mice treated with anti-TNF
antibody [56] and in a fraction of RA patients whose joint inflammation has been forestalled by
early, extended pharmacologic intervention [57].
It has been proposed that macrocyclic θ-defensins are endogenous immunoregulators that
contribute to homeostatic resolution of acute or subacute inflammation induced by infection
and/or TLR agonists [6]. Here we provide evidence that these peptides counteract pannus
formation, chronic inflammation, and joint destruction in a model of autoimmune disease.
Systemic treatment with RTD-1 resulted in reduced IL-1β levels in joints of rats with established
arthritis. These findings are consistent with in vitro studies wherein RTD-1 suppressed TNF, IL-
1β, IL-6, and arthritogenic chemokines expressed by human macrophages [14]. Further, RTD-1
inhibits sheddases that mobilize TNF from cell surfaces, as well as matrix degrading proteases.
Inhibition of MMPs and cytokines that activate them may underlie the reduction in polyarthritic
joint involvement observed in RTD-1 treated animals, as it is established that arthritic synovial
fibroblasts spread disease to unaffected joints [48]. We hypothesize that suppression of MMPs,
cysteine proteases and other invasiveness factors, in combination with the anti-proliferative
effect of θ-defensins limit polyarticular spread of disease. While a detailed understanding of
RTD-1 mediated anti-arthritic mechanisms remains to be determined, both in vivo and in vitro
studies suggest that a combination of cytokine suppressive and/or anti-protease effects modulate
disease severity and progression.
The immunomodulating properties of θ-defensins resemble those of pleiotropic
neuropeptides, e.g., vasoactive intestinal peptide, cortistatin, ghrelin, that modulate chronic
inflammation and autoimmunity [58]. Neuropeptides have been evaluated as therapeutic
112

candidates, but those efforts have been thwarted by the rapid clearance and degradation of the
peptides by endogenous proteases [58]. In contrast, the compact cyclic, tri-disulfide
conformation of θ-defensins confer remarkable stability in vivo [6] which likely contributes to
the prolonged circulation time of the peptide in rat PIA (Fig. 3-5c). In this context, the peptides
are also highly stable in plasma and resistance to proteolysis by cysteine cathepsins and zinc
metalloproteases that they inhibit (Table I).
Disease-modifying anti-rheumatic drugs (DMARD) such as methotrexate (MTX) are
commonly first line treatments for patients with RA [59-61] and cytokine-blocking biologics are
routinely added to the treatment regimen when DMARDs prove ineffective. The most successful
biologics include TNF-antagonists which are effective in arresting RA in a majority of patients,
by reducing TNF levels, thereby altering downstream TNF-dependent signaling events [56,62].
Despite these remarkable pharmacological agents, there persists a unmet need for new therapies,
as a third of all RA patients do not adequately respond to DMARDs or biologic therapies, or are
ineligible candidates for their use [19]. Further, biologic therapies can be cost prohibitive and all
carry a “black box” warning for patients who have compromised immune systems or who test
positive for infection with M. tuberculosis [63-66]. We propose that θ-defensins may fill a
unique niche between RA small molecule pharmacologics and large protein biologics. They
possess 3-fold increased circulating half-lives compared to MTX [67] but are still small enough
to be chemically synthesized (Fig. 3-1b). θ-defensins may target multiple RA mediators similar
to MTX. Because RTD-1 also suppresses monocyte and macrophage expression of TNF and IL-
1β activation by inhibiting NF-κB and MAPK pathways [14], it appears that the peptide
suppresses proinflammatory cytokines implicated in RA at multiple levels. We hypothesize that
that θ-defensins modulate arthritogenic molecules/pathways without overly suppressing
113

signaling networks required for appropriate responses to infection [6,9,13]. Finally, θ-defensins
are nonimmunogenic, circumventing the development of neutralizing antibodies, a problem
encountered by a subset of patients in response to biologic therapeutics [68].
θ-defensins are not expressed in humans due to a stop codon mutation that occurred prior
to the emergence of hominids [69]. Multiple θ-defensins are, however, expressed at high levels
in granulocytes and epithelia of several Old World primate species [8,9,70]. The peptides
modulate inflammation without immunocompromise, as evidenced by their efficacy in infectious
disease models [6,9,13], consistent with the concept that they function as immunoregulators in
the natural host. Here and in previous studies [6,9], θ-defensins were shown to be nontoxic,
nonimmunogenic and efficacious as immunomodulators across species. Thus, θ-defensins may
provide a retroevolutionary approach for utilizing macrocyclic peptides from nonhuman primates
to treat and induce remission of RA.
114

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120

GENERAL CONCLUSIONS
1.) Treatment with RTD-1 significantly suppresses proinflammatory cytokine and chemokine
responses from buffy coat preparations stimulated with a wide variety of TLR agonists, including
live E. coli. Reduction of TNF levels among the various TLR agonists conditions appear to be
the most consistent and profound among the cytokines altered by treatment with the peptide.
2.) In addition to its previously reported NF-κB and MAPK signaling inhibition, RTD-1 can
regulate soluble TNF signaling by inhibiting TACE cleavage of membrane bound TNF. RTD-1
is a non-competitive, fast-binding, reversible inhibitor of TACE, with an IC50 in the low
nanomolar range. In addition, RTD-1 selectively inhibits various MMPs and members of the
cathepsin family.
3.) RTD isoforms 1-5 differentially suppress TNF release from stimulated whole blood and THP-
1 monocytes. The relative magnitude of TNF suppression among the isoforms correlates
significantly with their potency of TACE inhibition.
4.) A single treatment with RTD-1 significantly reduces lethality in murine models of
polymicrobial sepsis and E. coli bacteremia. RTD-1 treatment reduced key proinflammatory
cytokines and chemokines in the plasma of bacteremic mice.
5.) RTDs 1, 2, and 5 immediately arrest the progression of pristane induced arthritis in rats. θ-
defensin treatment reduced gross pathology, and restored joint architecture and limb function.
Treatment with RTD-1 was more effective than common human therapies methotrexate and
etanercept, and was the only agent capable of inducing disease remission.
121

6.) In vitro evaluation of PIA rat fibroblast-like synoviocytes indicates RTD-1 effects multiple
pathogenic aspects of the cells. RTD-1 treatment of FLS cells suppresses IL-6 responses to TNF
and IL-1β stimulation, retards cellular proliferation, and inhibits invasive propensities.
7.) Treatment of rat PIA with RTD-1 was effective at dose intervals as great as every five days.
This prolonged therapeutic effect was supported by the remarkable stability of the macrocyclic
peptide in plasma and blood, and by a pharmacokinetic assessment of subcutaneously delivered
RTD-1, which indicated a prolonged circulating elimination half-life.
8.) Multiple deliveries of RTD-1, both intravenous and subcutaneous, were non-immunogenic in
mice, rats, and chimpanzees. Toxicity associated with RTD-1 was limited to lobular panniculitis
with minor adipose necrosis at the site of injection; this was only observed with multiple RTD-1
injections at concentrations 8x therapeutic doses.
9.) The unique immunomodulatory properties of θ-defensins constitute a novel therapeutic
approach for the treatment of inflammatory disorders. The results of this dissertation strongly
support the further evaluation of θ-defensins in the context of human diseases.

Abstract (if available)

Abstract Defensins are a class of antimicrobial peptides that function as part of the innate immune system. The mammalian defensin family is divided into three subfamilies known as α-, β-, and θ-defensins. Peptides from each of the respective defensin families were initially discovered for their antimicrobial properties, and have been shown to kill or neutralize bacteria, fungi, protozoa, and viruses. More recently, members of the α- and β-defensins have been demonstrated to possess immune modulating activities which include the induction of cytokine/chemokine signaling, immune cell recruitment/activation, and initiation of adaptive immune responses. θ-defensins, being the most recently discovered family, have only just recently been evaluated for their potential roles in immune responses. The goal of this dissertation was to discern the immune modulating properties θ-defensins, what molecular mechanisms they utilize, and if these unique molecules could be used as future therapeutics in the context of human diseases. ❧ The results of our studies presented here demonstrate that θ-defensins are potent agents of immune modulation. The macrocyclic peptides significantly suppressed proinflammatory cytokines/chemokines induced by a multitude of bacterial, fungal, and viral agonists in vitro. Each of the rhesus θ-defensin isoforms profoundly inhibited the keystone cytokine tumor necrosis factor (TNF) in stimulated human whole blood, monocytes/macrophages, and buffy coat preparations. Single dose treatment in murine models of E. coli bacteremia and polymicrobial sepsis resulted in dramatic reductions in lethality. Survival in E. coli induced peritonitis was associated with reductions in pathogenic levels of proinflammatory cytokines/chemokines. ❧ The molecular mechanisms θ-defensins utilize to modulate proinflammatory responses includes the inhibition of tumor necrosis factor-α converting enzyme (TACE). Inhibition of TACE was shown to prevent the cleavage of membrane bound TNF, contributing to reduced soluble TNF signaling and potential down-stream reductions in global inflammatory responses. Characterization of TACE inhibition by RTD-1, our prototype isoform, indicated the peptide was a non-competitive, fast-binding, reversible inhibitor of TACE with an IC50 in the low nanomolar range. ❧ Further evaluation of the therapeutic potential of θ-defensins utilized pristane induced arthritis in rats, a non-infectious disease of inflammation and a model of human rheumatoid arthritis. θ-defensin treatment resulted in remarkable attenuation and reversal of disease pathology, with restoration of limb function, joint architecture, and reductions in gross pathology. Resolution of arthritis correlated with reductions in IL-1β in joint tissues. In vitro analyses indicated that RTD-1 was capable of altering the pathogenic characteristics of fibroblast-like synoviocytes, including modulation of secreted proinflammatory cytokines, proliferation, and invasiveness. ❧ Additionally, θ-defensins were shown to be non-toxic and non-immunogenic in mice, rats, and chimpanzees. Furthermore, the pharmacokinetic profile of RTD-1 indicated a prolonged circulating half-life with remarkable peptide stability in blood and plasma. The implications of this dissertation strongly support the further evaluation of θ-defensins as a novel transphylogenetic therapeutic approach for the treatment of human diseases.

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Creator Schaal, Justin Blaine (author)

Core Title Immunomodulatory properties, mechanisms, and therapeutic potential of macrocyclic theta defensins

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Medical Biology

Publication Date 06/22/2016

Defense Date 05/05/2016

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag cytokines,Inflammation,OAI-PMH Harvest,rheumatoid arthritis,sepsis,TACE,theta defensins

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Advisor Ouellette, Andre (committee chair), Beringer, Paul (committee member), Selsted, Michael (committee member)

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