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Therapeutic potential of host defense peptides for the treatment of cystic fibrosis lung disease

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Therapeutic potential of host defense peptides for the treatment of cystic fibrosis lung disease

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Therapeutic Potential of Host Defense Peptides for the
Treatment of Cystic Fibrosis Lung Disease

By

Mansour Awwad Dughbaj

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
in Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(CLINICAL AND EXPERIMENTAL THERAPEUTICS)

May 2022

Copyright 2021 Mansour Awwad Dughbaj
ii

Acknowledgements
I want to express my deep gratitude to my advisor, Dr. Paul Beringer, for his continuous support
and guidance throughout my doctoral studies. His patience, motivation, and insightful feedback
have aided me greatly and sharpened my thinking as a better scientist.
I would also like to extend my appreciation to my dissertation committee members, Dr. Annie
Wong-Beringer and Dr. Stan Louie, for their encouragement, advice, and guidance through the
completion of my Ph.D.
I would also like to thank my family and friends for their support. My father always motivated
me during tough times, and I greatly appreciate his wisdom. Allah yerhamo.
Thank you to my wife Michelle for her boundless love and encouragement. I am forever grateful
for her endless support and motivation.
All of this was possible because of you all, and I genuinely thank you.

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Table of Contents
Acknowledgements ....................................................................................................................... ii
List of Figures ................................................................................................................................ v
List of Tables ............................................................................................................................... vii
Abstract ....................................................................................................................................... viii
Preface ........................................................................................................................................... xi
Chapter 1: Introduction 1
Cystic Fibrosis (CF) Background ................................................................................................ 1
Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) ............................................. 2
The role of the CFTR in CF Disease ........................................................................................... 3
CFTR Variants and Their Impact on CF Disease Severity ......................................................... 6
Clinical Manifestations of General CF Disease .......................................................................... 8
Clinical Manifestations of CF Lung Disease .............................................................................. 9
CF Microbial Infections ............................................................................................................ 10
Inflammation in CF Airways .................................................................................................... 13
Cellular Pathophysiology of CF Lung Disease ......................................................................... 14
Epithelial Dysfunction in CF Airways ...................................................................................... 14
Role of Macrophages in CF Lung Disease................................................................................ 15
CF Lung Neutrophilia ............................................................................................................... 18
The Inflammasome Relation to CF ........................................................................................... 20
Therapeutic Limitations for CF Lung Disease .......................................................................... 22
Host Defense Peptides ............................................................................................................... 24
HDPs as Antibiotic Adjuvants .................................................................................................. 28
Resolving Linear HDP activity through Cyclotide Scaffolds ................................................... 29
Cathelicidins .............................................................................................................................. 31
Rhesus Theta Defensin .............................................................................................................. 34
Summary ................................................................................................................................... 37
Chapter 2: Engineered Antimicrobial Cyclotides for Treatment of
P. aeruginosa Microbial Infections 39
iv

Introduction ............................................................................................................................... 39
Materials and Methods .............................................................................................................. 42
Results ....................................................................................................................................... 52
Discussion ................................................................................................................................. 74
Chapter 3: Anti-Inflammatory Effects of RTD-1 in a Murine Model of
Chronic Pseudomonas aeruginosa Lung Infection: Inhibition of NF-κB,
Inflammasome Gene Expression, and Pro-IL-1β Biosynthesis 78
Introduction ............................................................................................................................... 78
Materials and Methods .............................................................................................................. 81
Results ....................................................................................................................................... 88
Discussion ............................................................................................................................... 113
Chapter 4: Evaluation of Host Defense Peptides as Antibiotic Adjuvants
for Cystic Fibrosis Lung Disease 116
Introduction ............................................................................................................................. 116
Materials and Methods ............................................................................................................ 122
Results ..................................................................................................................................... 126
Discussion ............................................................................................................................... 137
Chapter 5: Summary and Future Directions 140
References 145

v

List of Figures
Figure 1-1. The Molecular Structure of the Human CFTR Protein. ............................................... 3
Figure 1-2. The pathogenesis of CFTR dysfunction. ...................................................................... 5
Figure 1-3. The Types of CFTR Mutations. ................................................................................... 8
Figure 1-4. Pathogenesis of CF. .................................................................................................... 10
Figure 1-5. Respiratory Pathogen Prevalence in Cystic Fibrosis Patients in 2020. ...................... 12
Figure 1-6. Different hypothetical models of HDP antimicrobial activity. .................................. 25
Figure 1-7. Immunomodulatory Activities of HDPs. ................................................................... 27
Figure 1-8. Structure of cyclotides................................................................................................ 31
Figure 1-9. The various roles of LL-37 in immunity. ................................................................... 33
Figure 1-10. The immunomodulatory mechanisms of RTD-1. .................................................... 36
Figure 2-1. Analytical reverse-phase C18-HPLC traces and ES-MS spectra
for reduced linear PG-1, cyclization/folding crude and purified PG-1. .........................................51
Figure 2-2. Scheme depicting the approach used to design the different .........................................
MCo-PG antimicrobial cyclotides. ............................................................................................... 53
Figure 2-3. Analytical reverse-phase C18-HPLC traces and ES-MS spectra of
MCo-PG linear precursor thioesters, cyclization/folding crudes and purified
folded cyclotides. ......................................................................................................................... 56
Figure 2-4. Chemical synthesis and characterization of cyclotide MCo-PG2. ............................. 58
Figure 2-5. 2D TOCSY Spectra of Cyclotides. ............................................................................ 59
Figure 2-6. 2D NOESY Spectrum of MCo-PG2. ......................................................................... 60
Figure 2-7. Cytotoxic activities of cyclotide MCo-PG2. .............................................................. 68
Figure 2-8. Stability of cyclotides MCo-PG2, MCoTI-I, and protegrin PG-1 to
human serum at 37° C. .................................................................................................................. 70
Figure 2-9. Preliminary toxicological data for antimicrobial cyclotide MCo-PG2
and PG-1. ...................................................................................................................................... 72
Figure 2-10. Evaluation of cyclotide MCo-PG2 against P. aeruginosa (Schroeter)
Migula (ATCC 27853) in a P. aeruginosa-induced bacterial peritonitis model. ........................ 73
Figure 3-1. Pathophysiological Studies of Chronic Pseudomonas aeruginosa Lung
Infection in Mice Treated with Aerosolized RTD-1. 89
vi

Figure 3-2. Effect of Aerosolized RTD-1 on BALF Cytokines in Murine Model
of Chronic Pseudomonas aeruginosa Lung Infection. ………………………………………….91
Figure 3-3. Microarray analysis of Lung Tissue and BALF Cells of RTD-1-treated
and Saline Control Mice with Chronic Pseudomonas aeruginosa Lung Infection. .................... 95
Figure 3-4. Top Up- or Downregulated Genes in Lung Tissue and BALF cells in
RTD-1-treated Mice with chronic P. aeruginosa lung infection. ..................................................97
Figure 3-5. Inflammasome-associated Gene Expression Changes in Lung Tissue
and BALF in RTD-1-treated Mice with Chronic Infection. ......................................................... 98
Figure 3-6. Inhibition of NF-κB by RTD-1. ............................................................................... 100
Figure 3-7. RTD-1 Treatment Reduces CXCL10 Transcription and Translation in
Different Cell Types. .................................................................................................................. 102
Figure 3-8. Effect of RTD-1 Treatment on Inflammatory Gene Expression. ............................. 103
Figure 3-9. Effect of RTD-1 Treatment on Inflammatory Cytokine Release. ............................ 105
Figure 3-10. Inhibition of Inflammasome-associated Gene Expression by RTD-1
Treatment. ................................................................................................................................... 107
Figure 3-11. RTD-1 Does Not Destabilize NLRP3 mRNA Transcription in THP-1
Macrophages. .............................................................................................................................. 108
Figure 3-12. Inhibition of IL-1β Protein Expression. ................................................................. 110
Figure 3-13. Inhibition by RTD-1 Treatment. ............................................................................ 112
Figure 4-1. The classes of Antibiotic Adjuvants. ....................................................................... 120
Figure 4-2. Acute P. aeruginosa pneumonia model. .................................................................. 125
Figure 4-3. Time-kill kinetics of RTD-1- and PG-1-colistin combination. ................................ 134
Figure 4-4. Pathophysiological Studies of Acute Pneumonia Pseudomonas
aeruginosa Lung Infection in Mice Treated with RTD-1 and colistin combination. ................. 136

vii

List of Tables
Table 2-1. Susceptibility profile of P. aeruginosa and S. aureus clinical isolates. ...................... 48
Table 2-2. Molecular weight, cyclization/folding yields for the MCo-PG grafted
cyclotides. ..................................................................................................................................... 57
Table 2-3. Tabulation of chemical shifts between MCo-PG2 and MCoTI-I. ............................... 61
Table 2-4. Minimum inhibitory concentrations (MIC) of antimicrobial peptide
PG-1 and MCo-PG2 through MCo-PG5 cyclotides. ................................................................... 63
Table 2-5. Full list of minimum inhibitory concentration (MIC) of antimicrobial
peptides MCo-PG2 and PG-1 against clinical isolates. ................................................................ 65
Table 2-6. Compiled minimum inhibitory concentrations of antimicrobial
peptides MCo-PG2 and PG-1 against clinical isolates. ................................................................ 66
Table 3-1. Forward and reverse primer sequences used in qRT-PCR
experiments. ................................................................................................................................. 85
Table 4-1. v ................................................................................................................................. 127
Table 4-2. Fractional inhibitory concentration indices of HDP-antibiotic
combinations. .............................................................................................................................. 127
Table 4-3. Fractional inhibitory concentrations of each combination. ....................................... 128
Table 4-4 ..................................................................................................................................... 129
Table 4-5. Checkerboard Assay Results of RTD-1 and colistin combinations
against CF clinical isolates......................................................................................................... 131
Table 4-6. Checkerboard Assay Results of RTD-1 and colistin combinations
against CF clinical isolates......................................................................................................... 132
Table 4-7. Complied Checkerboard assays with P. aeruginosa clinical isolates. ...................... 133

viii

Abstract
Cystic fibrosis (CF) is the most common autosomal recessive genetic disorder among
Caucasians and leads to chronic pulmonary infection and inflammation, the principal causes of
CF morbidity and mortality. The central feature of CF is lung disease which includes airway
obstruction, neutrophil-dominated inflammation, and microbial infection that leads to
progressive loss of lung function and eventual death. An increasing prevalence of multidrug-
resistant (MDR) bacteria, including MDR Pseudomonas aeruginosa and methicillin-resistant
Staphylococcus aureus, pose life-threatening risks for CF patients, as these pathogens are
associated with an accelerated decline in lung function and shortened survival. As these
pathogens are increasing worldwide, the lack of antimicrobial agents to combat them are
diminishing. Patients with CF are also predisposed to lung infection due to their mutation in the
CFTR gene, which leads to inactivation of innate antimicrobial proteins and host defense
peptides (HDPs) in the airways and the inability to clear pathogens. Due to the lack of safe and
effective therapeutic options to alleviate this lethal airway disease, our overall goal is to assess
HDPs as therapeutic options for CF lung infection and inflammation and to elucidate whether
HDPs can act as antibiotic adjuvants to synergistically combat pathogens with conventional
antibiotics.

To establish whether HDPs can be replenished and utilized to combat microbial
pathogens, we have developed a novel approach for engrafting HDPs onto non-immunogenic
cyclotides to improve the peptides’ stability and safety. Cyclotides are cyclized microproteins
stabilized by three disulfide bonds that form a cystine knot, thus protecting the compounds from
ix

chemical, thermal, and biological degradation. Here, the development of a novel engineered
cyclotide with effective broad-spectrum antibacterial activity against several ESKAPE bacterial
strains and clinical isolates is reported. The most active antibacterial cyclotide was extremely
stable in serum, showed little hemolytic activity, and provided protection in vivo in a murine
model of P. aeruginosa peritonitis. These results highlight the potential of the cyclotide scaffold
for the development of novel antimicrobial therapeutic leads that could fundamentally alter the
clinical management of CF airway disease.

To treat CF inflammation, Rhesus theta defensin-1 (RTD-1), a macrocyclic HDP with
known antimicrobial and immunomodulatory properties may be utilized. Our objective was to
investigate the anti-inflammatory effect of RTD-1 in a murine model of chronic P. aeruginosa
lung infection. Mice received nebulized RTD-1 daily for 6 days. Bacterial burden, leukocyte
counts, and cytokine concentrations were evaluated. Microarray analysis was performed on
bronchoalveolar lavage fluid (BALF) cells and lung tissue homogenates. In vitro effects of RTD-
1 in THP-1 cells were assessed using quantitative reverse transcription PCR, enzyme-linked
immunosorbent assays, immunoblots, confocal microscopy, enzymatic activity assays, and NF-
κB-reporter assays. RTD-1 significantly reduced lung white blood cell counts on days 3
(−54.95%; p = 0.0003) and 7 (−31.71%; p = 0.0097). Lung tissue homogenate and BALF cell
microarray analysis revealed that RTD-1 significantly reduced proinflammatory gene expression,
particularly inflammasome-related genes (nod-like receptor protein 3, Mediterranean fever gene,
interleukin (IL)-1α, and IL-1β) relative to the control. In vitro studies demonstrated NF–κB
activation was reduced two-fold (p ≤ 0.0001) by RTD-1 treatment. Immunoblots revealed that
x

RTD-1 treatment inhibited proIL-1β biosynthesis. Additionally, RTD-1 treatment was associated
with a reduction in caspase-1 activation (FC = −1.79; p = 0.0052). RTD-1 exhibited potent anti-
inflammatory activity in chronically infected mice. Importantly, RTD-1 inhibits inflammasome
activity, which is possibly a downstream effect of NF-κB modulation. These results support that
this immunomodulatory peptide may be a promising therapeutic for CF-associated lung disease.

Lastly, to establish the antibiotic adjuvant property of HDPs, we screened six HDPs with
five different conventional antibiotics against P. aeruginosa. We identified that HDPs were
highly synergistic with colistin and imipenem. We further validated that RTD-1 and colistin is
highly synergistic against several P. aeruginosa clinical isolates. RTD-1 and colistin were shown
to eradicate P. aeruginosa PAO1 by 4 hours without any bacterial regrowth in time kill kinetics.
In vivo acute pneumonia models of P. aeruginosa infection further elucidated the bactericidal
activity as there was a significant reduction in lung bacterial burden and leukocyte counts
compared to the vehicle control. There was a further reduction in white blood cell counts and
bacterial burden compared to colistin and RTD-1 monotherapy; however, it is not significant.
These results highlight the potential of HDPs to act as antibiotic adjuvants to combat CF lung
inflammation and infection.

xi

Preface
The aim of this dissertation is to provide evidence of the therapeutic potential of HDPs
for CF lung disease as antimicrobial and immunomodulatory agents and antibiotic adjuvants. We
demonstrated that utilizing MCoTI-I as a peptide scaffold is an effective method to stabilize
HDPs to act as antimicrobial peptides in an in vivo model of P. aeruginosa peritonitis. The
antimicrobial peptide was found to increase survival in the infected mice comparable to 10
mg/kg colistin. In addition, RTD-1, a cyclic peptide, was found to have immunomodulatory
activities in infection models. In our study, we determined that RTD-1 did not reduce bacterial
burden; however, did reduce pro-inflammatory cytokines and chemokines and was found to
downregulate several inflammasome-related genes. We further elucidated that its activity is most
likely due to its ability to modulate NF-κB signaling. Lastly, we wanted to illustrate HDPs
activities as antibiotic adjuvants for P. aeruginosa infection. We screened several HDPs and
conventional antibiotics and found that RTD-1 and colistin is a potent synergistic combination
for P. aeruginosa infections. In vivo acute pneumonia murine models demonstrated that
treatment with colistin and RTD-1 significantly reduced bacterial burden, white blood cell
infiltration and pro-inflammatory cytokines and chemokines. Thus, this thesis provides evidence
that HDPs have many roles in infection and inflammation that can be utilized for the treatment of
CF lung disease.
This thesis is adapted from the following manuscripts:
• Dughbaj MA, Ganesan R, Ramirez L, Beringer S, Aboye TL, Shekhtman A, Beringer PM,
Camarero JA. “Engineered Cyclotides with Potent Broad in Vitro and in Vivo Antimicrobial
Activity.” Chemistry. 2021 Sep 1;27(49):12702-12708.

xii

• Dughbaj MA, Jayne JG, Park AYJ, Bensman TJ, Algorri M, Ouellette AJ, Selsted ME,
Beringer PM. “Anti-Inflammatory Effects of RTD-1 in a Murine Model of Chronic
Pseudomonas aeruginosa Lung Infection: Inhibition of NF-κB, Inflammasome Gene
Expression, and Pro-IL-1β Biosynthesis.” Antibiotics (Basel). 2021 Aug 26;10(9):1043.

• Dughbaj MA, Kim SW, Beadell B, Beringer PM. “Host Defense Peptides as Antibiotic
Adjuvants for CF Lung Disease.” In preparation.

Chapter 1: Introduction
Cystic Fibrosis (CF) Background
CF is the most widespread Caucasian life-limiting genetic disorder that affects about
30,000 children and adults in the United States of America and 70,000 individuals worldwide.[1]
CF is triggered by an autosomal recessive mutation in the cystic fibrosis transmembrane
conductance regulatory gene, or CFTR[2, 3]. This gene encodes for a protein that serves as a
chloride channel throughout the body, most notably in the lungs and digestive system.[3, 4] As a
result, the median predicted survival age is approximately 45 between 2014-2018, while we are
now seeing a survival age of 50 this last year.[1] The improved life expectancy is due to the
increased knowledge and treatment of the disease for these patients that include neonatal
screening, antibiotic treatment, anti-inflammatory therapies, pancreatic enzyme replacement,
respiratory physiotherapy, and mucolytics.[3]

The majority of the morbidity and mortality is attributed to the lung disease severity. CF
lung disease is characterized by a vicious cycle of chronic microbial infection, endobronchial
airway obstruction, and neutrophil-dominated inflammation in the lungs.[5] In comparison to
normal airways, CF airways are widened and secrete thick and sticky mucus that allow for
bacteria to adhere.[6, 7] Inflammatory cells, including neutrophils and macrophages, infiltrate the
airways and contribute significantly to tissue damage and eventual bronchiectasis and pulmonary
function decline.[8, 9] Bronchiectasis results from protease burden leading to the degradation of
collagen and elastin and subsequent decline in airway structural integrity.[5] This chronic cycle
2

of inflammation and infection also leads to intermittent acute exacerbations, which lead to
significant loss of pulmonary function and eventually respiratory failure that these individuals
succumb to.

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)
Lap-Chee Tsui identified the CFTR gene in 1989, where it is located in chromosome 7,
location 7q31.2.[10] The CFTR protein is positioned at the apical surface of epithelial cells to
regulate ion transport and fluid homeostasis. This member of the adenosine triphosphate (ATP)-
binding cassette (ABC) transporter superfamily consists of two transmembrane domains (TMBs)
that form a pore to control the transportation of specific anions, primarily chloride. As the CFTR
is an ABC transporter, it contains two ATP nucleotide-binding domains (NBD) and a
cytoplasmic regulatory domain (R domain) and allows for activation of the ATP-gated ion
channel.[11, 12] Phosphorylation of the R domain is mediated through protein kinase A (PKA),
which is a prerequisite for the NBDs to bind and hydrolyze ATP. The active confirmation of the
CFTR opens and actively pump chloride anions across the membrane through its electrochemical
gradient.[12] Bicarbonate transport occurs directly across the CFTR, which influences the pH of
the apical surface of epithelial cells and mucosa.[13, 14] As the CFTR influences the
concentration of chloride and bicarbonate ions on the apical membrane of epithelial cells, it
strongly regulates other channels including epithelial sodium cation channels (ENaC),
aquaporins, other ion transport systems (i.e., bicarbonate channels).[15-17]
3

Figure 1-1. The Molecular Structure of the Human CFTR Protein. The dephosphorylated
CFTR in this ribbon diagram shows the CFTR with its different domains, the two
transmembrane domains, the regulatory domain, and the two nucleotide-binding domains.
Through cAMP-dependent phosphorylation of the R domain, the two NBDs bind and hydrolyze
ATP to allow chloride and bicarbonate to pass down their electrochemical gradient. Image from
Ratjen et al., 2015. [4]

The role of the CFTR in CF Disease
The CFTR acts as an ATP-gated anion channel, increasing the conductance for certain
anions (i.e., chloride and bicarbonate) to pass down their concentration gradient. In cystic
fibrosis, this protein is defective so that anions cannot pass to the apical membrane.[18] As the
4

defective CFTR does not allow bicarbonate to move across the cell membrane, the depletion of
bicarbonate in the apical membrane leads to acidification of the airway surface liquid.[14] In
addition, the CFTR defect affects epithelial sodium channels located on the apical membrane of
epithelial cells to reabsorb sodium cations. As NaCl plays a crucial role in tonicity, the
deficiency of ions on the apical membrane curbs the osmotic pressure into the basolateral side of
the epithelial cells. The irregular osmotic pressure leads to water reabsorption through
aquaporins, ultimately depleting the water content in the airway surface liquid.[19, 20]

This water loss, in turn, leads to mucus hyperviscosity in the apical membrane of
exocrine glands and the lungs. Mucin maturation is reduced due to bicarbonate insufficiency
leading to poor mucin solubility, immobility, and accumulation.[13, 14] To further inhibit mucus
clearance, epithelial ciliary beating frequency is diminished due to the viscous mucus that creates
plugs in the airways. These mucus plugs contribute to tissue damage and an increase of
inflammation. This mucosal obstruction is the root cause of CF-associated pathology.[21, 22]
The viscous mucus in the lungs is sufficient for pathogens including Pseudomonas aeruginosa
and methicillin-resistant Staphylococcus aureus to adhere to.[23, 24] These pathogens are
correlated with significant loss of pulmonary function as they contribute to airway disease by
releasing microbial enzymes and toxins to damage the epithelial tissue and affect the lung
immune response. The microbes upregulate the pro-inflammatory state of the lungs and cause
intense neutrophil recruitment.[22] These neutrophils lead to inflammation and release proteases,
including elastase and MMP9, that contribute to the structural integrity decline of various organs.
For instance, the significant protease burden in CF lung disease leads to bronchiectasis.[22]
5

During this release or degranulation, other factors are released, including host defense peptides
(HDPs). However, due to the acidification and significant protease burden in the lungs, HDPs are
inactivated and diminished.[14, 25-27] Figure 1-2 illustrates the vicious cycle of CF
pathogenesis led by the CFTR genetic defect in epithelial tissue, primarily in CF lungs. This
cascade of CFTR dysfunction is known as the “low volume” hypothesis and is considered the
most widely accepted model for CF disease.[28, 29]

Figure 1-2. The pathogenesis of CFTR dysfunction. The CFTR leads to a cascade of tonicity
dysfunction and acidification of the apical membrane of epithelial tissue that eventually advances
to mucus hyperviscosity, inflammation, protease burden, infection, tissue remodeling, and
fibrosis that these patients succumb to. Image from Lopes-Pacheco (2016).[30]

6

CFTR Variants and Their Impact on CF Disease Severity
The CFTR is transcribed and translated inherently in normal individuals, where the
immature CFTR protein is appropriately folded in the endoplasmic reticulum. The CFTR is then
conveyed to the Golgi apparatus for post-translational modifications and packaged into transport
vesicles to be trafficked to the cell surface for expression on the apical membrane of epithelial
cells.[30] However, in CF, mutants of the CFTR gene can impact different steps in protein
synthesis, where the mutants are categorized into six different classes.(Figure 1-3)[31] The
former three classes are associated with more severe disease, while the latter three are not.

First and foremost, class 1 CFTR mutations categorize mutants that affect protein
production, During translation, a shortened or dysfunctional protein is formed due to a premature
stop codon. Class 1 mutations are responsible for approximately 10% of CF cases
worldwide.[32] For instance, the G542X mutation is a nonsense mutation that results in a
dysfunctional truncated CFTR protein.[31] Class 2 CF mutants are missense mutations or amino
acid deletions that lead to protein processing defects. Class 2 CFTR defects have folding or
maturation defects that result in premature CFTR degradation through the proteasome. The most
common CF mutation, F508del, is one such class 2 defect that had a single amino acid removed
from the CFTR protein. The defective CFTR does not form the correct confirmation, where the
cell will recognize it as an improperly folded protein and degrade it.[31, 33] Class 3 mutants are
recognized as gating mutations where there is a defect in channel regulation. Mutants, such as
Gly551Asp and Gly178Arg, lock the CFTR in a closed confirmation so that chloride cannot pass
through its electrochemical gradient.[33]
7

Compared to Classes 1-3, classes 4-6 are considered less severe. Class 4 CFTR mutations
have decreased conductance where chloride movement is impaired due to a faulty channel.
Arg117His, Arg347Pro, and other conduction mutations are due to missense mutations where the
protein moves to the cell surface, but the channel is faulty so that chloride cannot move quickly
and easily.[31] Also, some mutants may cause the CFTR to be produced at a lower than
sufficient amount, known as Class V mutations. These mutations include the 3849+10 kb C→T
mutant, where missense or splicing mutations decrease chloride trafficking to cause CF
pathogenesis.[30, 34] Lastly, Class VI mutants such as the 4326delTC and Gln1412X are
associated with a decreased stability of the CFTR protein.[34] Even though these proteins are
functional, they are less stable and more susceptible to lysosomal degradation after being
removed from the plasma membrane.[30, 35]

In recent years, treatment for the CFTR defect has been progressing where CFTR
modulators have entered the market. These CFTR modulators have different roles as
potentiators, correctors, and premature stop codon suppressors.[34] Potentiators are utilized to
keep the CFTR in an open confirmation so that chloride can flow down their electrochemical
gradient. Ivacaftor is utilized as a potentiator to enhance the function of CFTR gating and
conduction mutants. In addition, correctors aid the protein to create the correct structure.[30]
Elexacaftor and tezacaftor are potent correctors to influence the CFTR to form the correct
confirmation and be trafficked to the membrane bilayer. Elexacaftor is considered a second-
8

generation CFTR modulator as it corrects an extra abnormalities to form the correct CFTR
structure that tezacaftor cannot.[36] Lastly, premature stop codon suppressors are being
investigated to bind to the ribosome, allowing the ribosome to continue translation after it reads
the stop codon. The CF Foundation has awarded Eloxx Pharmaceuticals to investigate their
potential stop codon suppressor ELX-02.[37]

Figure 1-3. The Types of CFTR Mutations. CFTR mutations are categorized into six different
classes, the latter three classes being less severe. Image from Elborn (2016).[22]

Clinical Manifestations of General CF Disease
CF is manifested throughout the body as various organs are affected throughout the body.
One of these manifestations, excessive sodium chloride secretion in sweat glands, is used to
9

diagnose whether an individual has the disease.[38] As lung dysfunction is predominantly
associated with CF mortality and morbidity, other comorbidities are manifested, including heat
shock, malabsorption due to pancreatic insufficiencies, biliary cirrhosis, endocrine dysfunction,
and male infertility.[22, 38] CF malabsorption is a multifactorial dysfunction that is mainly due
to pancreatic insufficiencies where bicarbonate and digestive enzymes are not secreted. In
addition, to pancreatic insufficiencies, bile secretion is reduced, leading to diminished absorption
of nutrients. Sticky and thick bile may obstruct the biliary duct and cause inflammation and
irritation that eventually leads to scarring in the liver (i.e., biliary cirrhosis).[21] The reduced
volume of mucosal secretions not only affects the digestive system and lungs but can affect the
endocrine system, where cystic fibrosis-related diabetes can occur. Insulin-secreting cells are
reduced and replaced with fibrotic tissue in the pancreas, which leads to diabetes in these
patients. Lastly, CF patients are observed to have an increased rate of heat shock due to sweat
gland obstruction and infertility in males due to the congenital absence of the vas deferens.[22]

Clinical Manifestations of CF Lung Disease
CF lung disease is characterized by a chronic cycle of obstruction, infection, and
inflammation. As previously discussed, the CFTR affects various channels throughout the body,
most notably in the lungs. The increased accumulation of viscous mucus creates plugs in the
airways that contribute to tissue damage and contribute to an increase in inflammation. In normal
lungs, mucins are utilized as a barrier to inhibit bacteria from establishing colonies. However, in
CF, the weakened ciliary beating is incapable of stopping mucus plug formation, airway
10

obstruction, polymicrobial colonization, and inflammation that subsequently leads to loss of
pulmonary function and eventual failure.[2, 22, 29]

Figure 1-4. Pathogenesis of CF. The CFTR defect leads to mucus hyperviscosity, inflammation,
infection, where defects in innate immune responses are observed. Image from Marteyn et al.,
(2017).[39]

CF Microbial Infections
The microbiology of CF airways is vastly dynamic as the environment changes
significantly with age. Children are predominantly infected with S. aureus and Haemophilus
11

influenzae.[6] As CF patients age, other opportunistic pathogens, including P. aeruginosa,
methicillin-resistant S. aureus, and Burkholderia cepacian complex (Bcc), tend to colonize the
lungs of these patients. As a result, these opportunistic pathogens perpetuate an accelerated
decline in lung function and premature death.[22, 40] A recent set of pathogens that have been
increasing in the population is nontuberculous mycobacterium.[22, 40] Since CF lungs are
always under chronic infection by various pathogens, treatment consists of antimicrobial agents.
Pathogens are known to combat these therapies by inducing antibiotic resistance mechanisms,
including biofilm formation, mucoidal phenotypes, efflux pumps, porin mutations, enzymes to
degrade antibiotics, and various other methods.[41, 42] Regarding P. aeruginosa infection, a
pathogen with a mucoidal (alginate-producing) phenotype is nearly impossible to eradicate, even
with rigorous antibiotic regimens. These sessile pathogens combat antibiotic treatment by
creating biofilms that limit their penetration.[22] In addition, these pathogens release various
virulent factors in the airways that cause an immense inflammatory response.[40, 41] To make
matters worse, CF epithelial cells release more pro-inflammatory cytokines and significantly
reduce IL-10 levels, an anti-inflammatory cytokine, when exposed to P. aeruginosa compared to
normal epithelium.[43] Also, P. aeruginosa can bind to a highly expressed epithelial cell
receptor, asialo GM1, to active NF-κB signaling and induce IL-8 release.[44] The dysregulation
of cytokines due to P. aeruginosa stimulation is known to accelerate the decline of pulmonary
function in CF patients. MRSA infections are also seen to increase the decline in FEV1 and
mortality in CF patients; thus, they are treated with aggressive therapies a well.[6]

12

Figure 1-5. Respiratory Pathogen Prevalence in Cystic Fibrosis Patients in 2020. The
prevalence of multiple pathogens was sorted by age. The prevalence of (B) P. aeruginosa
(including multidrug-resistant P. aeruginosa) and (C) methicillin-susceptible and methicillin-
resistant S. aureus (MSSA and MRSA, respectively) was also sorted by age in 2020. Data
sourced from the CF Foundation 2020 Registry.[6]
13

In all, greater than 70% of patients tend to be chronically infected with P. aeruginosa by
adulthood, where 20% of this population has a multidrug-resistant P. aeruginosa infection.[6,
22] For MRSA, greater than 20% of patients tend to be chronically infected with this type of
pathogen.[6] However, the prevalence of P. aeruginosa continues to decrease in the general CF
population. As treatment for CF lung disease is advancing, the prevalence of infection with
multidrug-resistant P. aeruginosa dropped from 7.3% in 2019 to 4.2% in 2020 as well. In
addition, there has been a plateau regarding the prevalence of MRSA for the past decade;
however, there has been a sharp decline in 2020. The resulting decline of all these pathogens
may be caused by infrequent culture screening during the pandemic.[6]

Inflammation in CF Airways
Not only is chronic microbial infection a contributor to CF lung disease severity, but
inflammation plays a pivotal role as well. Scientists previously thought that CF patients were
first colonized with bacteria that caused inflammation. One study in 2005 examined 70 CF
patients between the ages of 1.5-71 months and assessed inflammatory markers.[45] They stated
that CF patients in their ‘pristine’ category had lower inflammation signs, and infected patients
had elevated inflammatory markers in the BALF. As a result, they concluded that in CF,
infection precedes inflammation; however, recent reports indicate that inflammation precedes
infection. In the recent Australian Respiratory Early Surveillance Team for Cystic Fibrosis
(AREST CF) publication, researchers conclude that early CF lung disease harbors increased
14

mucus secretion and inflammatory markers without infection or lung structural changes.[46]
They postulate that in early CF, newly secreted mucins are developed due to common airway
insults such as viruses or aspiration. These mucins are not normally hydrated because of the
CFTR defect. Thus, the mucus secretions become hyper viscous, altering the mucociliary
clearance negatively, which leads to mucus plugs. The mucus plugs become hypoxic and trigger
a neutrophilic inflammatory response.[46, 47] The initial insult of viral infection or aspiration
may resolve; however, the muco-inflammatory-cycle will persist in allowing pathogens
including P. aeruginosa to adhere, establish biofilms and further contribute to the decline in
pulmonary function.[46, 48-50]

Cellular Pathophysiology of CF Lung Disease
Innate immune cells, including airway epithelium, macrophages, and neutrophils, play a
significant role in the pathogenesis of CF. All these cells contribute to the elevated
concentrations of pro-inflammatory cytokines found in the BALF leading to dysfunctional
clearance of bacteria in CF airways.[22] These pro-inflammatory cytokines include tumor
necrosis factor (TNFα), interleukin-1 (IL-1), IL-6, IL-8, and IL-17A, while anti-inflammatory
cytokines including IL-10 are diminished.[43]

Epithelial Dysfunction in CF Airways
The epithelium secretes various factors, including antimicrobial peptides/proteins,
cytokines, enzymes, and eicosanoids.[51] Compared to the healthy epithelium, CF epithelial
15

cells have diminished expression of IL-10, where its reduced levels tend to allow for pro-
inflammatory cytokine expression when encountering external stresses such as viral or P.
aeruginosa infection.[43, 52] In-depth, various mechanisms for epithelial cytokine dysfunction
are at play. ICAM-1, an essential pro-inflammatory adhesion molecule, is largely expressed in
lung epithelial tissue, which allows for neutrophils and macrophages to localized and infiltrate
into the airways which is regulated by IL-8 production.[53] In vitro results have confirmed that
CFTR-defective lung epithelial cells have elevated production of pro-inflammatory cytokines
and increased activation of NF-κβ signaling. The number of cytokines includes TNFα, IL-1β, IL-
8, IL-9, IL-10, IL-12, IL-15, and IL-17A, which are produced at a higher level in CF epithelium
when compared to healthy epithelial tissue.[43] CF epithelial cells also contribute to the pro-
inflammatory status of CF airway disease by diminishing the activity of pH-sensitive and salt-
sensitive antimicrobial proteins/peptides by altering the composition of the airway surface
fluid.[14, 54] Lastly, airway epithelial overexpresses asialo-GM1 receptors in response to CFTR
mutations that overproduce the R domain, consequently facilitating P. aeruginosa binding.[55]
Thus, CF epithelial cells contribute to the local inflammation and facilitate bacterial colonization
in the airways.

Role of Macrophages in CF Lung Disease
Macrophages orchestrate immune surveillance by coordinating inflammatory responses
and promoting inflammation resolution in tissue repair. In the lungs, there are two types of
macrophages: alveolar macrophages that interact with alveolar epithelial cells and interstitial
macrophages found in the parenchyma. Alveolar macrophages are tissue-resident immune cells
16

that participate in lung development and immune response in maintaining lung structural
integrity and combat environmental challenges. They are derived from fetal monocytes that
populate the alveoli shortly after birth and self-renew for the duration of the person’s life.[56]
These cells are vital for lung homeostasis, inflammation, tissue repair, phagocytosis of microbes,
and efferocytosis to resolve inflammation.[57] Alveolar macrophages terminally differentiate
through granulocyte macrophage-colony stimulating factor, and their phenotype is modulated by
the microenvironment of the airways.[58] Macrophages may elicit two distinct phenotypes: the
classically activated (M1) or the alternatively activated (M2) phenotype. Immature macrophages
can be stimulated with pro-inflammatory cytokines TNFα, IL-17A, and IFN-y to induce an M1
phenotype to further establish a pro-inflammatory state in the lungs. M1 macrophages
phagocytose microbes rapidly and secrete a vast array of pro-inflammatory cytokines.
Macrophages can also be transition into the M2 phenotype through IL-4 and IL-13 modulation to
resolve inflammation. In response, these M2 macrophage will elaborate anti-inflammatory
cytokines such as IL-10 to reduce pro-inflammatory cytokine production and downregulate NF-
κβ signaling.[56] M2 macrophages can also clear apoptotic cells and other cellular debris
through efferocytosis and release other factors, including transforming growth factor (TGF)-β to
mediate fibroblast recruitment and promote collagen production for tissue repair.[59]

In CF, alveolar macrophages are hyperinflammatory and suboptimal phagocytes. One
hypothesis is that alveolar macrophages have an enhanced expression of immune receptors
known as pathogens through receptors (PRRs) on their surface (i.e., TLR4 and TLR5).[60] PRR
overexpression occurs in response to increased secretion of pro-inflammatory cytokines (i.e.,
17

TNFα) and other mediators. An in vivo murine F508del model elicited abnormally high levels of
IL-1β, which suggests that the TLR4 overexpression in macrophages may play a role. Also, PRR
overexpression is due to defects in endosomal trafficking. The decreased degradation of the
protein is caused by the downregulation of Rab-proteins and microtubule formation that support
endosomal trafficking for lysosomal degradation of the PRRs. Pro-inflammatory mediators are
released in CF lungs due to obstruction (known as damage-associated molecular patterns
[DAMPs]) and infection (pathogen-associated molecular patterns [PAMPs]).[56] Due to the
unfolded protein response (UPR), other inflammatory mediators are released. This response is
caused by the X-box-binding protein-1 (XBP-1), which acts as a transcription factor that
stimulates endoplasmic reticulum protein folding and secretory pathways that increase
inflammatory mediator production.[61] However, not all mediators are released. Heme
oxygenase-1 (HO-1) trafficking is inhibited in activated CF macrophages. This inflammatory
mediator has robust immunomodulatory activities promoting the transition of macrophages into
M2-like phenotype.[62] As HO-1 is not trafficked, no carbon monoxide is produced to kill
microbes, and the resolution of infection cannot occur.[62] As a result, pro-inflammatory
mediators are continuously released, such as reactive oxygen species and proteases that lead to
sustained signal transduction of pro-inflammatory pathways, including NF-κβ and MAPK
signaling. In addition, defective expression of sphingolipids and cholesterol affects signaling
platforms known as lipid rafts.[57] Negative feedback of immune signaling pathways is also
blunted as CF macrophages have an irregular expression of nuclear receptors, including liver X
receptors (LXRs) and peroxisome proliferator-activated receptors (PPARs) that influence fatty
18

acid metabolism. Lastly, CF macrophages have altered transmigration capabilities as the CFTR
defect impairs β-1 and β-2 integrin-mediated monocyte adhesion and chemotaxis.[57, 63]

CF Lung Neutrophilia
Neutrophils are the key components in response to pathogenic intrusion. They represent
most of the leukocytes found in the blood and have various roles in facilitating inflammation and
combating infection. Upon exposure to environmental stimulants or tissue damage, pro-
inflammatory mediators, cytokines, and chemoattractants such as IL-8 are released from
epithelial cells and macrophages.[64] Neutrophils will extravasate from blood vessels and into
the extracellular space found in the airways. Its presence in this highly reactive
microenvironment allows them to react and eliminate potential pathogens or cellular debris
found in the lungs. For instance, neutrophils will clear and uptake cellular debris and microbes
through phagocytosis using their Fc (Fragment, crystallizable) receptor.[65] These cells utilize
complement and antibody binding to facilitate microbial recognition and removal through
neutrophil binding onto Fc region. In addition, these cells can utilize their PRRs to mediate
uptake of the unwanted material into phagosomes that utilize endosomal trafficking.[65] Once
the phagosome is trafficked to a granule, it fuses with the lysosome to form phagolysosome.
These granules consist of antimicrobial molecules and proteases. These antimicrobial molecules
include antimicrobial peptides (i.e., lactoferrin and lysozyme), antimicrobial peptides (i.e.,
cathelicidins and defensins), and reactive oxygen species to destroy intracellular pathogens.[64,
66] In addition, neutrophil granules have proteases including neutrophil elastase, proteinase 3,
and cathepsin G that assist with the antimicrobial molecules to digest the microbes in the
19

phagolysosome.[67] Neutrophils also combat extracellular pathogens through degranulation.
These cells will release their granules by fusing the granules with the plasma membrane to
release the various antimicrobial molecules and proteases and kill them.[64, 66] The proteases
and antimicrobial molecules also have inflammatory properties to induce inflammation and
endothelial cell apoptosis to resolve inflammation. Neutrophils can also form extracellular DNA
network containing antimicrobial factors and proteases called neutrophil extracellular traps
(NETs).[68] During this process known as NETosis, the neutrophil nuclei can rupture releasing
nuclear and mitochondrial DNA web that can trap intracellular granules contents allowing it to
bind bacteria and eliminate them [69, 70] If the bacteria do not die from the entrapment, then
other neutrophils will easily target them as the web-like structures trap the bacteria in place. As a
result, non-viable neutrophils can sustain inflammation to combat infection as enucleated
neutrophil cytoplasts.[71]

In CF, neutrophils have impaired phagocytic capabilities and inflammatory processes.
Generally, a large abundance of neutrophils in the airways is associated with IL-8 production and
pulmonary function decline.[45, 72, 73] IL-8 is the central inflammatory mediator that acts as a
chemoattractant to recruit neutrophils into the airways. In CF, serum IL-8 is highly upregulated
and CF neutrophils spontaneously secrete high levels of IL-8 compared to normal
individuals.[74] As CF neutrophils spontaneously release high levels of IL-8 in comparison to
healthy neutrophils, they traffic into the lungs, releasing their intracellular proteases that can
indiscriminately destroy pathogens and normal tissues.[64] This protease burden, mainly
neutrophil elastase-associated, impairs CXCR1, a receptor for IL-8, leading to bactericidal
20

defects and sustained inflammation.[75] Not only does the abundance of neutrophil elastase
impair IL-8 signaling, but it also impact elafin and the secretory leucoprotease inhibitor.[64]
Impairment of these antiproteases leads to a downregulation of antimicrobial and anti-
inflammatory processes that further contribute to CF disease. Subsequently, neutrophil elastase
can cause an overproduction of pro-IL-8 by upregulating the expression of TLR4 and EGFR.[64]
As a result, neutrophils are endlessly migrating into the airways and releasing NETs and
proteases that cause the lungs to become fibrotic.

The Inflammasome Relation to CF
Inflammasomes are a set of protein complexes that sense infection or tissue damage that
can trigger an inflammatory response and activate several different cytokines. Inflammasome
complexes can arise from one of five known intracellular sensors (Nod-like receptors [NLRs]),
the inflammasome adapter protein apoptosis-associated speck (ASC), and procaspase-1.[76]
These NLRs are a subset of PRRs, including AIM2, Pyrin, NLRC4, NLRP1, and NLRP3.[77]
Regarding the innate immune system, the NLRP3 inflammasome is expressed mainly in
macrophages to establish a pro-inflammatory response when it senses imbalances.

The NLRP3 inflammasome is initially activated by cytokines or PAMPS, which lead to
the upregulation of NLRP3 inflammasome components via NF-κB signaling. The inflammasome
is primed by various PAMPs or DAMPs that activate upstream signaling events, including ROS
production.[76] Inflammasome assembly occurs where it activates caspase-1, which in turn
21

cleaves IL-1β and IL-18 precursors into their active forms. These cytokines are released to
initiate their pro-inflammatory signal transduction, while caspase-1 is critical for programmed
cell death in response to intracellular pathogens or pyroptosis.[76]

Multiple reports have observed that high levels of IL-1β plague CF patients. CF sputum,
plasma, and BAL samples have been shown to have high IL-1β cytokine production, and IL-1β
polymorphisms are linked with disease severity [78-82]. Also, chronic P. aeruginosa infection
can further elevate IL-1β levels. Studies in CF children have shown that IL-1β levels are
diminished when P. aeruginosa is cleared from their lungs. To further corroborate this notion,
CF adults colonized with P. aeruginosa have raised IL-1β protein expression compared to
individuals without chronic P. aeruginosa.[83] Emerging data indicates the inflammasome may
play an essential role in CF inflammatory disease as the CFTR defect may increase nod-like
receptor protein 3 (NLRP3) stimulation and maturation of IL-1β [83, 84]. Recently, one report
demonstrates that the CFTR regulates TNFα signaling by enhancing the degradation of TRADD,
a key signaling intermediate between TNFα and NF-kB signaling.[15] This TRADD protein
forms a complex with TNFα to drive NF-kB signaling. The CFTR plays a role in a negative
feedback loop to inhibit TRADD by lysosomal degradation, suppressing NF-kB activation. In
CF, the defective CFTR cannot regulate TRADD, leading to endless NF-kB signaling.[15]

22

Therapeutic Limitations for CF Lung Disease
CF lung infection and inflammation is mainly treated with antibiotics and anti-
inflammatory therapies, respectively. Antibiotic therapy is used to control, eradicate and clear
pathogens from the lungs. Antibiotics can be used as prophylactic treatments to reduce the
prevalence of S. aureus infection and prevent secondary bacterial infection during viral
respiratory infection by Haemophilus influenzae or other pathogens.[85] For chronic use, inhaled
or intravenous antibiotics are used commonly. Two standard inhaled therapies for P. aeruginosa
infection include colistin and tobramycin, where the latter drug is given on a monthly cycle.[86]
Also, if the patient has chronic P. aeruginosa infection, patients are prescribed ciprofloxacin to
prevent P. aeruginosa-associated loss of pulmonary function.[87] Although this is a chronic lung
disease, CF patients present with periodic acute pulmonary exacerbations. Acute pulmonary
exacerbations are defined as increased respiratory symptoms (including increased cough, sputum
production, and shortness of breath), accompanied by an acute reduction in lung function. For
these acute events, intravenous (IV) antibiotics are utilized as inhaled therapies cannot be utilized
effectively.[86, 87] During this intense period, IV antibiotics do not guarantee that they will
return to their baseline lung function. A study by Sanders et al. (2010) observed that patients who
had two or more exacerbations in one year are at an increased risk of experiencing a 5% decline
in FEV1 compared to patients who had one or no exacerbations that year.[88] IV antibiotics are
used that have different mechanisms of action to reduce the possibility of antibiotic resistance to
one and for the possible benefit of synergistic activities. However, antibiotic resistance is making
these antibiotics less effective. Recent reports indicate that antibiotic-resistant pathogens killed
23

more people than HIV or malaria in 2019, further illustrating the need for novel antimicrobial
agents.[89]

Anti-inflammatory agents are also given to CF patients to limit lung inflammation. For
instance, patients can be given oral nonsteroidal anti-inflammatory drugs (NSAIDs) or
corticosteroids. High dose ibuprofen has been shown to reduce neutrophil recruitment and the
rate of pulmonary function decline.[90-92] Prednisone has also been shown to reduce the decline
of FEV1 in CF patients over four years. Even though these treatments are promising, a complex
pharmacokinetic profile and adverse events limit their use.[92, 93] High-dose ibuprofen is
associated with an increased risk of serious gastrointestinal issues, including bleeding, ulceration,
and perforation of the stomach or intestines. Prednisone is linked with an increased risk of
cataracts, infection, high blood sugar, osteoporosis, fractures, suppressed adrenal gland hormone
production, thinning skin, bruising, and slower wound healing.[90-93] Currently, azithromycin is
prescribed as an anti-inflammatory drug for CF patients. The mechanism of action of
azithromycin is not well understood, but it is known to decrease the production of several pro-
inflammatory mediators and cytokines. It also acts as an antimicrobial agent against P.
aeruginosa. Studies conducted with azithromycin illustrated that it is highly promising as an
anti-inflammatory compound.[5] In CF adults, azithromycin treatment proved to maintain lung
function, reduce intravenous antibiotic regimens, C-reactive protein levels, and pulmonary
exacerbations while improving the quality of life in these patients.[94] Two pediatric studies
have also demonstrated that azithromycin treatment significantly reduced acute pulmonary
exacerbations and the rate of pulmonary decline with no significant adverse events compared to
24

the placebo group.[95] To corroborate these findings, large, multicenter studies assessed the
safety and efficacy of azithromycin in CF patients. This study found a reduction in the loss of
pulmonary function, pulmonary exacerbations, and an improvement in body weight.[96, 97]
Even though azithromycin is safe and efficacious, its use as an anti-inflammatory compound is
modest. As a result, the decline in pulmonary function is not halted, and on average, a loss of
approximately 2.2% FEV1 annually is still observed.[98]

Host Defense Peptides
Host Defense Peptides (HDPs) are diverse short cationic amphiphilic peptides that play
an crucial role in innate immune function in essentially all living organisms.[99] As “jacks of all
trades,” these peptides have various roles in innate immune function to target microbes and assist
with the inflammatory process. They can elicit antimicrobial activity through ion and
hydrophobic interactions to combat fungi, viruses, and bacteria in infection.[99] Due to this
activity, these HDPs can combat established biofilms and prevent biofilm formation.[14, 54, 99,
100] For instance, these peptides bind strongly to the negatively charged bacterial membranes
and do not bind strongly to mammalian membranes as they are zwitterionic.[101] The HDPs’
antimicrobial action is based on the peptide concentration to lipid ratio, which can be described
by four models: barrel stave, toroidal, disordered toroidal-pore, and carpet models.[101, 102] At
low peptide to lipid ratios, these peptides will bind to the surface of membranes. Once a
sufficient peptide to lipid ratio is reached, these peptides reorient themselves into a
transmembrane orientation.[101] They will insert themselves and form pores to cause
depolarization of the bacterial membrane and induce cytoplasmic leakage. These peptides can
25

form ordered cylindrical water pores (barrel stave) to depolarize the membrane.[101, 102] The
peptides can also depolarize the membrane through the toroidal model by thinning the membrane
where it induces a bend in the bilayer so that the upper and lower membrane leaflets meet,
allowing for the peptides to form an orderly transmembrane pore.[102] Abnormal pores can also
be formed to create a disordered toroidal pore.[102] Lastly, they can aggregate or “carpet” the
membrane of the bacterial membrane. In this carpet model, the peptides depolarize the
membrane leading to membrane disintegration and micellization to kill a pathogen.[101]

Figure 1-6. Different hypothetical models of HDP antimicrobial activity. HDPs may perturb
lipid membranes through four different methods, including (A) barrel stave, (B) carpet, (C)
toroidal, and (D) disordered toroidal. Image adapted from Sengupta et al. (2008).[102]

HDPs are also immunomodulatory agents that the innate immune response elicits. During
the initiation of infection, HDPs will release HDPs through their granules to bind to
26

lipopolysaccharides (LPS) to dampen pro-inflammatory responses.[99, 103] During this early
stage of inflammation, HDPs can promote the chemotaxis of monocytes/macrophages and
neutrophils.[104] HDPs may act as chemoattractants via formyl-peptide receptors. This
mechanism of HDPs is not fully supported as the effects occur at concentrations above what is
physiologically relevant.[99, 103, 104] Moreover, HDPs can modulate the immune system by
promoting cellular differentiation. HDPs can activate immature dendritic cells and macrophages.
It has been seen that some HDPs can differentiate macrophages to an intermediate phenotype
between an M1 and M2 state.[103] Another immune function of HDPs is that they can modulate
adaptive immunity and recruit T cells to the site of infection. HDPs can alter IgG1 production
and sensing of CpG oligodeoxynucleotides in B cells.[99, 104] They can also promote wound-
healing by promoting keratinocyte migration by acting through epidermal growth factor
receptors (EGFRs).[99, 104, 105] Another component of HDP immune modulation is enhancing
autophagy, a mechanism to degrade and salvage dysfunctional molecules. HDPs were also found
to modulate apoptosis in different cell types.[103] In epithelial cells, HDPs can enhance
apoptosis to clear pathogens by activating of caspase 3 and 9; however, in neutrophils, it can
inhibit apoptosis from promoting further pathogen clearance. HDPs can modulate pyroptosis to
inhibit caspase-1 activation and reduce an excessive inflammatory response.[103] Another
mechanism that these peptides take to target pathogens is promoting NETosis; however, the role
that these peptides take to enhance NETosis is not fully understood. As the bacterial membrane
cannot be altered significantly, resistance to this antimicrobial mechanism is uncommon.[103,
106] These mechanisms are depicted in Figure 1-7.
27

Figure 1-7. Immunomodulatory Activities of HDPs.HDPs play a pivotal role in inflammation
and infection by depolarizing the membrane of pathogens, promoting the recruitment,
differentiation, and activation of leukocytes, suppressing pro-inflammatory cytokine production,
and regulating autophagy and NETosis. Image from Mansour (2014).[103]

In CF, active HDPs are largely reduced. Reports indicate that the CF airways have high
levels of HDPs; however, this abnormal level of HDPs are most likely associated with their
inactive counterpart.[27, 107] Due to the lack of active HDPs, bacterial clearance and biofilm
prevention and eradication are impaired.[108] Excessive inflammation may play a role in
diminishing HDPs as several factors can bind them up, including virulent factors, DNA, F-actin,
and glycosaminoglycans.[26, 27] Another factor that diminishes these peptides is the massive
28

protease burden in the lungs. Many HDPs are linear peptides which are susceptible to proteolytic
degradation and rendered useless to assist with inflammation and microbial infection.[107, 108]
HDPs also face the acidic CF lung environment. To illustrate this issue, there are two reports that
assessed the bactericidal activity of airway surface liquid (ASL) of normal and CF-pigs against
microbes (both P. aeruginosa and S. aureus).[14, 54] One report indicated that the ASL of CF
pigs had impaired antimicrobial activity.[14] They postulated that two antimicrobial proteins,
lactoferrin and lysozyme, were deficient due to the acidic pH environment caused by the CFTR
impairing bicarbonate secretion.[14] They showed that as the pH decreases in ex vivo ASL
samples, its antimicrobial activity is impaired. They also examined the antimicrobial activity of
lactoferrin and lysozyme in various pH environments and observed that an acidic pH
environment could decrease the activity of these proteins.[14] Additionally, the second paper
indicated that two HDPs, LL-37 and human beta-defensin-1, have impaired antimicrobial
activity against S. aureus in conditions similar to the acidic CF environment.[54] These reports
show that the proteins and peptides are not stable in the acidic environment of the CF
airways.[54] Thus, replenishing the endobronchial airways with acid and protease stable HDPs
may be therapeutically beneficial to increase the number of active HDPs to combat the bacterial
load seen in CF lungs.

HDPs as Antibiotic Adjuvants
As antibiotic resistance is ongoing, one way to resuscitate old antibiotics is through
HDPs.[109] Several reports have indicated that antimicrobial peptides can be utilized with
conventional antibiotics to improve the effects of both compounds synergistically.[109-112]
29

Many attribute this observation to the fact that the antimicrobial peptides can cause bacterial
membrane disruption, facilitating the entry of antibiotics to accumulate in the cytosol of the
bacteria, leading to their deaths.[109] For instance, regarding MDR-P. aeruginosa infection, an
antimicrobial peptide called B2088-AMP was used in combination with different conventional
antibiotics (chloramphenicol, gentamicin, imipenem, and tobramycin), and all combinations
were found to be synergistic against P. aeruginosa in vitro.[110] Also, in vivo models of MRSA-
infected burn wounds show that WRL3 is synergistic with ceftriaxone.[111] As a result, both
outer and inner membranes are affected, which explains the synergy between the two molecules.
Lastly, a combination of HDPs with antibiotics further reduced abscess size and improved
clearance of colony-forming units of ESKAPE (Enterococcus faecium, Staphylococcus aureus,
Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter
cloacae)-related abscesses in vivo compared to antibiotics alone.[113]

Resolving Linear HDP activity through Cyclotide Scaffolds
As Linear HDPs have various issues regarding their stability in CF airways, cyclotides
present a unique method to assist with stabilizing their structure. Cyclotides are a family of plant
proteins of the Violaceae, Rubiaceae, Cucurbitaceae, and Fabaceae families that are
characterized by a cyclic backbone and a knotted disulfide topology.[114, 115] These
compounds have a broad array of biological activities, such as protease inhibition, antimicrobial,
and insecticidal action. Due to their three disulfide bonds that produce a cystine knot, they are
exceptionally stable to chemical, thermal and proteolytic degradation and poorly
immunogenic.[114] These cyclotides are relatively small (28-37 amino acids), enabling a more
30

straightforward chemical synthesis. MCoTI-I is one such cyclotide that is nontoxic, functions as
a trypsin inhibitor, and can cross cellular membranes through micropinocytosis.[115] Other
cyclotides have various functions because they do not share significant sequence homology with
other cyclotides beyond the presence of the three cysteine bridges.[114] As these peptides are
exceedingly tolerant to sequence variations, cyclotides can be utilized as pioneering frameworks
for HDP drug design. The Camarero group has previously utilized MCoTI-I as a scaffold to
efficiently target the CXCR4 receptor and the p53 pathway both in vitro and in vivo.[116] Thus,
the incorporation of HDPs onto cyclotide scaffolds can be a solution to combat the growing
number of antibiotic-resistant infections among the CF population.
31

Figure 1-8. Structure of cyclotides. The cyclized backbone of MCoTI-II is presented where a
cysteine knot can be seen in the center of the peptide. The primary structure of MCoTI-II
indicates the cystine bonds (in red) that are formed to stabilize the cyclotide structure.

Cathelicidins
Cathelicidins are a group of HDPs that is expressed in epithelial and immune cells, where
they play an important role in inflammation and infection. These peptides are characterized by
their cathelin structural domain, a highly conserved N-terminal domain.[99, 117, 118] The
cathelin domain serves as a signal peptide to target the precursor protein to granule storages or
the extracellular space. Once the domain is cleaved, the cathelicidin domain is liberated to elicit
its biological functions.[99, 117, 118]
32

Animals produce a variety of cathelicidins to serve under the innate immune response. In
pigs, there is a family of cathelicidin peptides called protegrins. There are five members of this
class of proteins, labeled protegrin-1, 2, 3, 4, and 5. The most abundant and well-characterized
protegrin is protegrin-1.[119, 120] The β-hairpin peptide has potent antimicrobial activity and
immunomodulatory functions. This peptide has been used in various in vivo models of bacterial
peritonitis and has demonstrated its efficacy to combat these pathogens, including bacteria
associated with CF lung disease (P. aeruginosa and S. aureus).[121] In addition, this peptide has
immunomodulatory activity as its cationicity allows it to bind to LPS to blunt LPS-induced
inflammation.[120] However, this peptide is limited in its therapeutic potential because of its
linear backbone, which can be degraded easily through proteolytic degradation.

Humans also express one cathelicidin known as LL-37/hCAP18. This HDP was named
based on its close relationship to the CAP18 protein found in rabbits.[122] In this report, LL-37
was first found to bind to LPS, which can suppress LPS-induced inflammation.[122] It was later
found to also transcriptionally suppress NF-kB signaling to reduce virulent factor-mediated
inflammation.[117, 118] Studies have shown that LL-37 is a moderate antimicrobial agent
against various pathogens. LL-37 can prevent biofilm formation from P. aeruginosa and S.
aureus and possibly disrupt established biofilms.[117, 118] This protein is expressed by the
activation of the vitamin D receptor on epithelial and immune cells.[117, 118] In vitro studies
with human macrophages have shown that stimulation of the vitamin D receptor enhanced
33

phagocytosis.[123] Lastly, this peptide is well known for its immunomodulatory properties as it
can influence the activity of the immune response. Figure 1-9 depicts the known antimicrobial
and immunomodulatory properties of LL-37.

Figure 1-9. The various roles of LL-37 in immunity. LL-37 is the only known human
cathelicidin that plays an important role in immunity. It can neutralize LPS to inhibit exaggerated
inflammatory processes. It can act as a chemokine and differentiate and activate several
leukocytes. It can act intracellularly to modulate inflammatory cytokines and mediators.

The hCAP18 proprotein is cleaved by proteinase 3 to form the active LL-37 peptide.[67]
Also, seminal hCAP18 was found to be processed by gastricsin to ALL-38.[124] Also, LL-37
can be further processed by other serine-proteases to yield KR-20, RK-21, and KS-20, which
34

were observed to elicit increased antimicrobial activity.[125] For this reason, it was found that
further cleavage of LL-37 leads to a more potent antimicrobial effect. Reports demonstrate that
antimicrobial activity is found on the GF-17 fragment.[126] The GF-17 fragment has been used
by others to synthesize new compounds. The human cathelicidin LL-37 has been utilized to
produce a novel antimicrobial peptide 17BIPHE2.[127] 17BIPHE2 is designed on GF-17, the
active region of LL-37. It contains three D-leucine amino acids that distort the regular helical
structure of the peptide to improve stability to a select set of proteases. Also, 17BIPHE2 contains
two biphenylalanine amino acids to correct a hydrophobic defect to increase its antimicrobial
properties.[127] Previous literature demonstrates that 17BIPHE2 has potent broad-spectrum
antimicrobial and antibiofilm activity.[128, 129] In all, 17BIPHE2 shows promise as a potential
novel therapeutic for CF bacterial infection.

Rhesus Theta Defensin
Humans express alpha and beta-defensins constitutively in the lungs. Not only are there
alpha and beta-defensins, but there are also theta defensins which is are not expressed in humans.
These defensins are expressed endogenously in Old World monkeys. Rhesus macaques express a
family of these proteins known as Rhesus-theta defensin-1, 2, and 3.[130] These Old-World
monkeys express two genes for the three peptides. Once these peptides are cleaved, two nine
amino acid precursor peptides can be spliced together to form the three different 18 amino acid
cyclic peptides: homodimers of processed RTD-1 and RTD-2 and heterodimers of each RTD-1
and RTD-2 fragment to produce RTD-3.[130] As RTD-1 is a cyclic peptide, it exhibits superb
plasma stability without hemolytic or cytotoxic activity.[131]
35

RTD-1 is found to have potent broad-spectrum antimicrobial activity.[132-136] Similar
to other HDPs, RTD-1’s mechanism of action is through membrane depolarization and inhibition
of β-galactosidase activity in the cytosol.[131] RTD-1 is found to have optimal antimicrobial
activity in physiological salt concentrations of 150 mM NaCl.[131] The linear form of RTD-1
has impaired antimicrobial activity compared to the native cyclic peptide. The peptide can target
CF-related pathogens S. aureus and P. aeruginosa, and other microbes, including E. coli, C.
albicans and, human immunodeficiency virus-1.[132-134] In vivo studies corroborate these
findings where P. aeruginosa bacterial burden was significantly reduced.[135] Also, it was
found that RTD-1 supports long-term survival of murine systemic C. albicans infection, further
corroborating its therapeutic potential as an antimicrobial agent.[136]

RTD-1’s immunomodulatory activity is another reason for its use as a therapeutic. In
vitro studies have shown that RTD-1 has intracellular targets for its anti-inflammatory effects,
which are not mediated through LPS binding.[133, 137] These studies depicted RTD-1 to act as
an anti-inflammatory molecule by inhibition of NF B and MAPK signaling by activation of
AKT phosphorylation.[137] As AKT is phosphorylated, IκBα is degraded, allowing for nuclear
translocation of p50/p65 subunits.[137] Also, studies have shown that RTD-1 inhibits TNFα
converting enzyme (TACE/ADAM17), which in turn will inhibit TNFα release.[138] In vivo
studies corroborate these anti-inflammatory effects in murine acute lung injury and chronic P.
36

aeruginosa models as RTD-1 reduces pro-inflammatory cytokine release (TNFα, IL-1β, and IL-
6) and neutrophil recruitment.[139, 140]

Figure 1-10. The immunomodulatory mechanisms of RTD-1. As TLRs are activated to
induce inflammation, RTD-1 has multiple mechanisms to modulate inflammation. RTD-1 can
inhibit inflammation by TLR-1, -2, -4 and -9 via inhibiting NF‐κB activation, MAPK signaling,
and ERK pathways. Image from Tongaonkar et al. (2015).[137]

37

Summary
In summary, vicious cycles of chronic airway obstruction, lung infections, and
neutrophil-dominated inflammation contribute to morbidity and mortality in cystic fibrosis (CF)
patients. CF patients are consistently bombarded with infection, where P. aeruginosa and S.
aureus negatively affect the rate of pulmonary decline in these patients. In addition, neutrophil
infiltration and invasion contribute to pulmonary decline as the cells release various factors
including proteases that fibrose the lungs leading to bronchiectasis. Antibiotics are
predominantly prescribed for these patients; however, their effectiveness is dwindling as
antibiotic resistance is surging. In addition, new anti-inflammatory therapies are needed to
alleviate neutrophil-dominated inflammation as chronic use of corticosteroids and NSAIDs have
significant adverse effects and azithromycin use is modest. Host defense peptides can bridge the
gap in therapeutics as they possess a wide variety of characteristics to possibly treat CF lung
disease.

Therefore, the aim of this thesis is to examine the wide variety of therapeutic capabilities
that HDPs manifest for CF lung disease.

In chapter 2, we demonstrate the antimicrobial activity of engineered antimicrobial
cyclotides for the treatment of P. aeruginosa peritonitis infection.

38

In chapter 3, we investigate the immunomodulatory properties of RTD-1 as an anti-
inflammatory agent for chronic P. aeruginosa lung infection.

In chapter 4, we explore the possibility of HDPs to act as antibiotic adjuvants against P.
aeruginosa acute pneumonia.

39

Chapter 2: Engineered Antimicrobial Cyclotides for Treatment
of P. aeruginosa Microbial Infections
Introduction
The rise in antibiotic resistance coupled with the slow development of novel antibiotics is
a serious threat to global public health. The CDC reports that antibiotic resistance is a major health
challenge, as deaths related to antibiotic resistant infections are projected to surpass those from
cancer by 2050.[141] Specifically, ESKAPE pathogens (Enterococcus faecium, Staphylococcus
aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and
Enterobacter species) are six notorious pathogens that are capable of ‘escaping’ the microbicidal
activity of multiple antibiotics that can shorten the survival of individuals with chronic
infections.[142] For instance, cystic fibrosis, which caused by an autosomal recessive genetic
defect in the cystic fibrosis transmembrane conductance regulator gene (CFTR), leads to a vicious
cycle of airway obstruction, inflammation and infection.[22] Two ESKAPE pathogens, P.
aeruginosa and methicillin-resistant S. aureus (MRSA), commonly infect CF airways and
contribute to a rapid decline of pulmonary function and shortened survival of this patients.[6, 7]
These chronic pathogens can establish biofilms within CF airways and survive this harsh
environment and exposure to intense antibiotic regimens due to biofilm impenetrability. Moreover,
these pathogens cause progressive loss of pulmonary function through the secretion of toxins and
virulence factors, including cell wall molecules that activate pro-inflammatory signaling.[7, 143]
Decreased pulmonary function results from a robust yet unproductive pro-inflammatory state
during which an intense neutrophilic migration and a sustained cyclic release of proteases lead to
40

lung injury, increased progressive loss of lung function, and decreased survival.[143] Thus,
developing novel antimicrobial therapies is vital to the treatment of CF lung disease.

Host defense peptides (HDPs) are short cationic amphiphilic peptides with both
antimicrobial properties that are essential to the innate immune systems of organisms, ranging
from plants to complex animals.[99] Most HDPs have broad-spectrum antimicrobial activity via
membrane disruption. In addition, HDPs combat existing biofilms and prevent biofilm formation
at doses lower than its minimum inhibitory concentration.[144] During CF, mammalian cells
release HDPs, such as cathelicidins, into the lung airway surface liquid; however, the inactivation
of HDPs in this environment reduces the ability of CF patients to clear various pathogens and
inhibit biofilm formation.[14, 54, 99, 100] HDPs are inactivated in CF airways due to the acidic
environment, LPS binding, and protease degradation.[14, 25-27] Thus, replenishing the
endobronchial airways with acid- and protease-stable HDPs may be therapeutically beneficial by
increasing levels of active HDPs to modulate bacterial load and inhibit neutrophil infiltration in
CF lungs.

Prior studies have concluded that β-hairpin HDPs have potent antimicrobial activity and
cell selectivity.[120, 145-147] For instance, the porcine cathelicidin protegrin-1 (PG-1) is a
promising therapeutic candidate that disrupts anionic bacterial membranes and biofilms and has a
wide range of in vivo immunomodulatory properties, including inhibition of LPS and increase
neutrophil clearance. [120, 146] The distinct antimicrobial mechanism of action of PG-1 limits
41

potential cross-resistance while providing synergy in combination with other locally produced
HDPs and conventional antibiotics. [147] Their effectiveness in several different animal infection
and inflammation models demonstrate that HDPs may represent a new class of antibiotic and
immunomodulatory agents for treating CF. Although these peptides are mechanistically
promising, their therapeutic use is currently limited by their high cytotoxicity, hemolytic activity
and suboptimal stability in CF airways.

Cyclotides are a family of plant proteins of the Violaceae, Rubiaceae, Cucurbitaceae, and
Fabaceae families that are characterized by a cyclic backbone and a knotted disulfide
topology.[114, 115] These compounds have a broad array of biological activities, such as protease
inhibition, antimicrobial and insecticidal action. Due to their three disulfide bonds that produce a
cystine knot, it is extremely stable to chemical, thermal and proteolytic degradation and poorly
immunogenic.[114] These cyclotides are relatively small (28-37 amino acids), which enables more
straightforward chemical synthesis. One such cyclotide is known as MCoTI-I that is nontoxic,
functions as a trypsin inhibitor and can cross cellular membranes through micropinocytosis.[115]
Other cyclotides have various functions because they do not share significant sequence homology
with other cyclotides beyond the presence of the three cystine bridges.[114] As these peptides are
exceedingly tolerant to sequence variations, cyclotides can be utilized as pioneering frameworks
for HDP drug design. Our group has previously utilized MCoTI-I as a scaffold to efficiently target
the CXCR4 receptor, and the p53 pathway both in vitro and in vivo.[116] Thus, our incorporation
of HDPs onto cyclotide scaffolds can be a solution to combat the growing number of antibiotic-
resistant infections among the CF population.
42

Materials and Methods
High-performance liquid chromatography (HPLC)
Analytical HPLC was performed on a HP1100 series instrument with 220 nm and 280 nm
detection using a Vydac C18 column (5 mm, 4.6 x 150 mm) at a flow rate of 1 mL/min. All runs
used linear gradients of 0.1% aqueous trifluoroacetic acid (TFA, solvent A) vs. 0.1% TFA, 90%
acetonitrile in H2O (solvent B). UV-vis spectroscopy was carried out on an Agilent 8453 diode
array spectrophotometer, and fluorescence analysis on a Jobin Yvon Flurolog-3
spectroflurometer. Electrospray mass spectrometry (ES-MS) analysis was performed on an
Applied Biosystems API 3000 triple quadrupole electrospray mass spectrometer using software
Analyst 1.4.2. Calculated masses were obtained using Analyst 1.4.2. All chemicals involved in
synthesis or analysis were obtained from Aldrich (Milwaukee, WI) or Novabiochem (San Diego,
CA) unless otherwise indicated.

Preparation of Fmoc-Aaa-OH
Fmoc-Phe-F was prepared using diethylaminosulfur trifluoride DAST as previously
described[148] and quickly used afterwards. Briefly, DAST (160 L, 1.2 mmol) was added drop
wise at 25° C under nitrogen current to a stirred solution of Fmoc-Phe-OH (387.4 mg, 1 mmol)
in 10 mL of dry dichloromethane (DCM), containing dry pyridine (81 µL, 1 mmol). After 20
minutes, the mixture was washed with ice-cold water (3 x 20 mL). The organic layer was
separated and dried over anhydrous MgSO4. The solvent was removed under reduced pressure to
give the corresponding Fmoc-amino acyl fluoride as white solid that was immediately used.
43

Chemical synthesis of the cyclotides
All cyclotides were synthesized by solid-phase synthesis on an automatic peptide
synthesizer ABI433A (Applied Biosystems) using the Fast-Fmoc chemistry with 2-(1H-
benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
(HBTU)/diisopropylethylamine (DIEA) activation protocol at 0.1 mmole scale on a Fmoc-
Tyr(tBu)-sulfamylbutyryl AM resin. Side-chain protection compatible with Fmoc-chemistry was
employed as previously described for the synthesis of peptide -thioesters by the Fmoc-
protocol, except for the N-terminal Cys residue, which was introduced as Boc-Cys(Trt)-OH.
Following chain assembly, the alkylation, thiolytic cleavage and side chain deprotection were
performed for individual peptides in 1 mL polypropylene columns as previously described [149].
Briefly, ≈100 mg of protected peptide-resin were first alkylated two times with ICH2CN (17.4
µL, 0.24 mmol; previously filtered through basic alumina) and DIEA (8.2 µL, 0.046 mmol) in N-
methylpyrrolidone (NMP) (0.22 mL) for 24 h. The resin was then washed with NMP (3 x 5 mL)
and DCM (3 x 5 mL). The alkylated peptide resin was cleaved from the resin with
HSCH2CH2CO2Et (20 µL, 0.18 mmol) in the presence of a catalytic amount of sodium
thiophenolate (NaSPh, 0.3 mg, 2.2 µmol) in DMF:DCM (1:2 v/v, 0.12 mL) for 24 h. The resin
was then dried at reduced pressure. The side-chain protecting groups were removed by treating
the dried resin with trifluoroacetic acid (TFA):H2O:tri-isopropylsilane (TIS) (95:3:2 v/v, 0.5 mL)
for 3-4 h at room temperature. The resin was filtered and the linear peptide thioester was
precipitated in cold Et2O. The crude material was dissolved in the minimal amount of
H2O:MeCN (4:1) containing 0.1% TFA and characterized by HPLC and ES-MS as the desired
44

grafted MCoTI-I linear precursor -thioester (Fig. 2-1 and Table 2-1). Cyclization and folding
were accomplished by flash dilution of the linear -thioester TFA crude to a final concentration
of ≈25 µM into freshly degassed 0.1 mM EDTA, 1 mM reduced glutathione (GSH), 0.1 M
HEPES buffer at pH 7.2 containing 25% isopropanol for 72-96 h. Folded peptides were purified
by semi-preparative HPLC using a linear gradient of 25-45% solvent B over 30 min. Pure
peptides were characterized by HPLC and ES-MS (Fig. 2-1 and Table 1).

NMR spectroscopy
NMR samples were prepared by dissolving cyclotides into 80 mM potassium phosphate
pH 6.0 in 20% (v/v) d4- MeOD, 80% (v/v) 5 mM potassium phosphate buffer at pH 6.0 (v/v) to a
concentration of approximately 0.5 mM. All
1
H NMR data were recorded on either Bruker
Avance III 500 MHz or Bruker Avance II 700 MHz spectrometers equipped with TCI
cryoprobes. Data were acquired at 298 K, and 2,2-dimethyl-2-silapentane-5-sulfonate, DSS, was
used as an internal reference. The carrier frequency was centered on the water signal, and the
solvent was suppressed by using WATERGATE pulse sequence.
1
H,
1
H-TOCSY (spin lock time
80 ms) and
1
H,
1
H-NOESY (mixing time 150 ms) spectra were collected using 4096 t2 points and
256 t1 of 64 transients. Spectra were processed using Topspin 2.1 (Bruker). Each 2D-data set was
apodized by 90
0
-shifted sinebell-squared in all dimensions, and zero filled to 4096 x 512 points
prior to Fourier transformation. Assignments for H (H-C ) and H’ (H-N ) protons of folded
MCo-PG2 (Table 2) were obtained using standard procedures [150, 151].

45

Human serum stability
Peptides were dissolved in water at 10 mg/mL concentration. 150 µg of peptides (15 µL)
were mixed with 500 µL of human serum and incubated at 37° C. Samples (30 µL) were taken at
various time intervals (0 – 120 h) and serum proteins were precipitated using 180 µL of
acetonitrile containing 0.1 % TFA. After centrifugation the pellet was dissolved in 8 M GdmCl
and the supernatant was lyophilized and re-dissolved in 5 % acetonitrile in water containing
0.1% formic acid. Both the supernatant and solubilized pellet fractions were analyzed by HPLC
and LC-MS/MS. Each experiment was done in triplicate.

Hemolysis assays
Hemolytic activity of the peptides was tested against human red blood cells (h-RBC).
Single donor human red blood cells were purchased from Innovative research
(IWB3ALS40ML). Prior to the experiment, h-RBC were washed three times with phosphate-
buffered saline (PBS) by centrifugation for 10 min at 1,000 x g and resuspended in PBS.
Different concentrations of the peptide solutions were then added to 50 μL of h-RBC in PBS to
give a final volume of 100 μL and a final erythrocyte concentration of 4% (v/v). The plate was
incubated with agitation for 1 h at 37 °C. The samples were then centrifuged at 1,000 x g for 10
min. Release of hemoglobin was monitored by measuring the absorbance of the supernatant at
405 nm with a UV spectrophotometer. Controls for no hemolysis (blank) and 100% hemolysis
consisted of human red blood cells suspended in PBS and 0.1% Triton X-100, respectively.

46

Cytotoxicity Assays
Cyclotide cytotoxicities against human HEK293T and A549 epithelial cells were evaluated
using Resazurin (alamarblue
TM
; Thermo Fisher Scientific, Waltham, MA). HEK293T cells were
grown in minimum essential media (MEM), while A549 cells were grown in Dulbecco’s minimum
essential medium (DMEM) supplemented with 10% FBS and maintained at 37°C with 5% CO 2.
For each cell line, 1 × 10
4
cells were added to each well in a 96-well polystyrene plate and
incubated for 16 hours. Peptides were serially diluted two-fold at concentrations ranging from
100 - 0.2 µM in each cell lines’ respective medium containing 1% FBS in polystyrene 96 well
microtiter plates (Corning). Medium was replaced with each cell line’s respective medium
containing the serially diluted peptide concentrations and 1% FBS. After 22 hours of incubation,
the medium was replaced with fresh growth medium containing resazurin and incubated for
another 2 hours. Cell viability was quantified spectrophotometrically and cells lacking peptides
and containing 2% Triton X-100 (Sigma) served as controls.

Antimicrobial activity of MCo-PG cyclotides
Broad-spectrum antimicrobial activity was evaluated using the broth microdilution assays
against two different pathogens from four different species of bacteria (P. aeruginosa, S. aureus,
K. pneumoniae, and E. coli) to determine if the cyclotides retained its potent activity. Broth
microdilution assays were utilized to determine the minimum inhibitory concentrations (MICs) of
47

our cyclotides according to the CLSI (formerly NCCLS) guidelines modifications as described
below [121]. The assay utilized cation-adjusted Mueller-Hinton broth (CAMHB) (Becton,
Dickinson and Company, Franklin Lakes, NJ) which was prepared according to the manufacturer's
instructions. Cyclotide solutions were prepared as 10× solutions in 0.01% acetic acid. Cyclotide
were serially diluted two-fold at concentrations ranging from 25 - 0.05 µM that contained CAMHB
and 0.04% bovine serum albumin (BSA) in polypropylene microtiter plates (Corning, Corning,
NY). All bacteria were incubated overnight at 37°C at 200 rpm in CAMHB and bacterial inoculum
was adjusted with additional CAMHB to 0.5 McFarland standard through spectrophotometry at
600 nm. Bacteria was then further adjusted 1:100 in CAMHB and dispensed into 96-well
polypropylene microtiter plates (Corning, Corning, NY) in triplicate (corresponding to 0.5–1 × 10
5

CFU/well) and incubated for 24 h to determine the MIC. Using the most potent cyclotide, we
further evaluated its potency against cystic fibrosis isolates of P. aeruginosa and S. aureus. Study
strains included a total of 20 P. aeruginosa and 20 methicillin resistant S. aureus strains from
patients with cystic fibrosis at the Keck Medical Center of the University of Southern California.
As many clinical isolates grow at slower rates, they were incubated for 48 h total and inspected for
their MIC. Colistin sulphate (Sigma-Aldrich, St Louis, MO) was used as a reference antibiotic for
P. aeruginosa and E. coli, while vancomycin hydrochloride and meropenem trihydrate were used
as reference antibiotics for S. aureus and K. pneumoniae. Additional susceptibility assays were
conducted with various antibiotics against the clinical isolates (Table 2-1) and determined that
30% of the P. aeruginosa clinical isolates were multi-drug resistant (MDR) and 65% of them were
mucoid.

48

Table 2-1. Susceptibility profile of P. aeruginosa and S. aureus clinical isolates. Green
denotes isolates that are susceptible to the antibiotic, yellow denotes intermediate susceptibility
to the antibiotic, and red denotes isolates that are resistant to the antibiotic. For tigecycline, some
S. aureus pathogens were listed in a nonsusceptible category.

PA
Isolates
Colistin
Levofloxacin
Ceftazidime
Meropenem
Imipenem
Tobramycin
MRSA
Isolates
Vancomycin
Linezolid
Ceftaroline
Tigecycline
200174 30138-2
864684 38878-2
894925 10630-1
0.5
119133 30493-1
219686 32120-1
678449 766
857950 20021-1
167482 87180
446055 10337-1
486442 28310
618154 28283
815159 87066
844265 902674
95302 28355
298473 87112
894496 20457-1
254831 4084-1
262309 4094
900719 88531
774248 6151-1
PAO1 USA300
Legend:
Susceptible
Intermediate
Resistant
nonsusceptible category
49

Time kill assay
We determined our cyclotide’s bactericidal kinetics against a laboratory strain of P.
aeruginosa (PAO1) was through broth microdilution using CAMHB as described previously
[135]. Briefly, bacteria inoculums of 1 × 10
5
CFU/mL were exposed to a range of MCo-PG2
cyclotide concentrations (0.25×, 1×, 4× and 16× MIC) over time and incubated at 37°C over time.
Aliquots of the inoculum were taken following peptide exposure at 0, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6
and 24 h post treatment, then serially diluted two-fold and plated onto tryptic soy agar. The plates
were incubated at 37°C for 16 hours and CFUs were counted.

Maximum tolerated dose (MTD) toxicology Studies
The MTD was determined using two different endpoints: weight loss and clinical scoring.
Clinical scores were evaluated through activity, appearance and body condition, similar to
previous published literature [152]. The starting doses were based on prior literature except for
MCo-PG2, which we set the starting dose at 1 mg/kg. Single-dose administration was escalated
two-fold until the any mice met the endpoint of ≥15% weight loss or a clinical score ≥2. Any
dose escalation that leads to moderate toxicity (clinical score of ≥2) was ceased and the dose
prior served as the MTD. Mice were monitored every hour for 4 h after injection on the first day.
After 24 hs, mice were monitored twice daily for another two days until weight and clinical
scores were returned to normal. Mice that met the criteria for moribund included a ≥20% weight
loss and clinical score of ≥3 and were euthanized.

50

P. aeruginosa-induced peritonitis murine model
All animal experiments were evaluated and approved by the Institutional Animal Care
and Use Committee at the University of Southern California (Protocol 20994). Eight to 10-week-
old Balb/c mice (Jackson Laboratory, Bar Harbor, ME) were housed five in a cage with standard
chow and water ad libitum. To establish the model, 1.5 × 10
7
CFU of log-phase P. aeruginosa
ATCC 27853 was injected intraperitoneally. Mice were intraperitoneally treated immediately
with either 10 or 25 mg/kg MCo-PG2, 5 mg/kg PG-1, 15 mg/kg colistin sulphate or isotonic
PBS. Mice were monitored for 7 days and/or euthanized if any mice presented signs of
moribundity.

Protegrin PG-1
Protegrin PG-1 was synthesized and folded as previously described [153]. Briefly,
protegrin PG-1 was synthesized by solid-phase synthesis on an automatic peptide synthesizer
ABI433A (Applied Biosystems) using the Fast-Fmoc chemistry with HBTU/ DIEA activation
protocol at 0.1 mmole scale on a Rink-amide resin. Side-chain protection compatible with Fmoc-
chemistry was employed as previously described for the synthesis of peptides, Cys residues were
introduced as Fmoc-Cys(Trt)-OH. Following chain assembly, side chain deprotection and resin
cleavage were performed by acidolytic treatment with TFA as previously described for
cyclotides [149]. Briefly, side-chain protecting groups were removed by treating the dried resin
with trifluoroacetic acid (TFA):H2O:tri-isopropylsilane (TIS) (95:3:2 v/v, 0.5 mL) for 3-4 h at
room temperature. The resin was filtered and the linear peptide thioester was precipitated in cold
51

Et2O. The crude material was dissolved in the minimal amount of H2O:MeCN (4:1) containing
0.1% TFA and characterized by HPLC and ES-MS as the desired PG-1 reduced linear precursor
(Figure 2-1). Oxidative folding was accomplished by flash dilution of the linear PG-1 TFA
crude to a final concentration of ≈25 µM into freshly degassed 0.1 mM EDTA, 1 mM oxidized
glutathione, 100m M HEPES buffer at pH 7.4 containing 25% isopropanol for 24 h. Folded PG-1
was purified by semi-preparative HPLC using a linear gradient of 18-35% solvent B over 30
min. Pure PG-1 was characterized by HPLC and ES-MS (Figure 2-1) and biological activity.

Figure 2-1. Analytical reverse-phase C18-HPLC traces and ES-MS spectra for reduced
linear PG-1, cyclization/folding crude and purified PG-1. HPLC analysis was performed
using a linear gradient of 0-70% solvent B over 30 minutes. The peaks marked with “*” denotes
the expected product. The peak marked with “#” corresponds a non-peptide impurity from the
TFA acidolytic cocktail. Expected molecular weights are shown in parenthesis.
52

Results

In order to produce a cyclotide with PG-1 antimicrobial activity, we employed the
naturally-occurring cyclotide MCoTI-I as molecular framework (Fig. 2-2). MCoTI- cyclotides
are potent trypsin inhibitors isolated from the seeds of Momordica cochinchinensis[154] and
show very low toxicity in human cells,[116, 149] and therefore represent a desirable molecular
scaffold for engineering new cyclotides with minimal toxicity and novel biological
activities.[116, 155-159]

53

Figure 2-2. Scheme depicting the approach used to design the different MCo-PG
antimicrobial cyclotides. A circular permuted version of porcine protegrin PG-1, where the
original Arg
1
residue in PG-1 was moved to its C-terminus, was grafted into onto of cyclotide
loop 6 of cyclotide MCoTI-I between Ser1 and Ser33 residues. The backbone cyclized structure
of the cyclotide is shown a connecting bond in green. Cys residues are highlighted in yellow and
disulfide bonds are indicated in red. The ribbon structures of cyclotide MCoTI-II (PDB:
1IB9)[160] and porcine protegrin PG-1 (PDB: 1PG1)[161] are shown for reference.

According to the solution structure of PG-1,[161] its N- and C-termini are very close in
space although the N-terminus is slightly more extended (Fig. 2-2). Therefore, a modified
version of PG-1, where the N-terminal Arg residue was moved to the C-terminal position of the
54

PG-1 sequence, was grafted into loop 6 of the cyclotide MCoTI-I (cyclotide MCo-PG2, Fig. 2-
2). We also explored the effect of adding an extra disulfide to the grafted PG-1-derived sequence
to further stabilize the grafted b-hairpin structure. This was accomplished by replacing both
residues Arg
4
and Gly
17
in the original PG-1 sequence with Cys residues (MCo-PG3, Fig.2-2).
Two more cyclotides were also designed with longer and shorter versions of the PG-1-based
grafted sequence to explore the effect of the distance of the grafted sequence from the cyclotide
and to minimize the size of the PG-1-derived graft (Fig. 2-2). The elongated version (MCo-PG4)
was obtained by adding two extra Gly residues to the N- and C-terminal positions of the
modified PG-1 sequence. The shorten version (MCo-PG5) removed the N- and C-terminal Gly
and Arg residues from the modified PG-1 sequence, respectively. The different sequences were
grafted onto loop 6 by replacing residue Asp
32
(Fig. 2-2) as loop 6 of MCoTI-cyclotides has been
shown to be less rigid in solution[162, 163] and quite tolerant to sequence grafting of relatively
long peptide sequences.[116, 158, 159, 164-166]

All grafted MCo-PG cyclotides were chemically synthesized on a sulfonamide resin
using an Fmoc-based solid-phase peptide synthesis protocol.[149] The corresponding fully
deprotected linear peptide α-thioesters were obtained by alkylation of the sulfonamide linker
followed by thiolytic cleavage of the alkylated sulfonamide linker and acidolytic deprotection of
the side-chain protecting groups. Cyclization and oxidative folding were accomplished in a one-
pot reaction under thermodynamic control using sodium phosphate buffer at pH 7.2 in the
presence of 1 mM reduced glutathione (GSH). In all the cases, the cyclization/folding reactions
were complete in 72-96 h (Figs. 2-3 and 2-4A). The yields for the cyclization/folding reactions
55

ranged from 16% (MCo-PG3) to 40% (MCo-PG2) (Table 2-2). All folded cyclotides were
purified by reverse-phase HPLC and characterized by ES-MS (Figs. 2-1 and 2-3, Table 2-2). In
addition, cyclotide MCo-PG2 was characterized by
1
H-NMR. The ∆d values for most of the
backbone protons for the common part shared with the parent cyclotide MCoTI-I were smaller
than 0.1 ppm indicating that MCo-PG2 adopts a native cyclotide fold (Fig. 2-4C and 2-5, Table
2-3). Analysis of the amide-amide region 2D NOESY spectrum of cyclotide MCo-PG2 also
revealed long-range NOEs between the backbone H’ protons from residues Leu
37
and Val
48
, and
residues Tyr
39
and Val
46
showing that the protegrin-derived graft in cyclotide MCo-PG2 adopts a
native b-hairpin fold (Fig. 2-6).

56

Figure 2-3. Analytical reverse-phase C18-HPLC traces and ES-MS spectra of MCo-PG
linear precursor thioesters, cyclization/folding crudes and purified folded cyclotides. HPLC
analysis was performed using a linear gradient of 0-70% solvent B over 30 minutes. The asterisk
denotes the peak of the corresponding product.

57

Table 2-2. Molecular weight, cyclization/folding yields for the MCo-PG grafted cyclotides.
Expected molecular weights are shown in parenthesis.
Peptide Name Molecular weight (Da) Cyclization/folding
Linear thioester Cyclized/folded yield (%) time (h)
a

MCo-PG2 5636.1 ± 1.3 (5634.5) 5504.7 ± 0.6 (5504.5) 40 72
MCo-PG3 5627.5 ± 0.4 (5627.5) 5495.5 ± 0.4 (5495.5) 16 96
MCo-PG4 5748.1 ± 0.2 (5748.6) 5618.6 ± 0.4 (5618.6) 18 96
MCo-PG5 5490.7 ± 1.2 (5490.3) 5360.4 ± 0.7 (5360.3) 20 96

a
Time for efficient cyclization

58

Figure 2-4. Chemical synthesis and characterization of cyclotide MCo-PG2. A. Analytical
HPLC traces of for the linear thioester precursor, GSH-induced cyclization/folding crude after 72
h and purified cyclotide. An arrow indicates the desired peptide. B. ES-MS characterization of
pure MCo-PG2. The expected average molecular weight is shown in parenthesis. C. Chemical
shifts differences of the backbone, H′ and H
α
protons between the common sequence (residues 1
through 34) of MCoTI-I [162, 163] and MCo-PG2.

59

Figure 2-5. 2D TOCSY Spectra of Cyclotides. Overlaid 2D TOCSY spectra of MCoTI-I (red)
and MCo-PG2 (blue) in 20% (v/v) d4-MeOD and 80% (v/v) 5 mM potassium phosphate buffer,
pH 6.0.

60

Figure 2-6. 2D NOESY Spectrum of MCo-PG2. Amide-amide region of the 2D NOESY (150
ms mixing time) spectrum for MCo-PG2 in 20% (v/v) d4-MeOD, 80% (v/v) 5 mM potassium
phosphate buffer at pH 6.0. Long range NOE cross peaks are indicated for amide protons of
Y39/V46 and L37/V48. These H’-H’ connectivities were also observed in PG-1 [161]. Other
long-range NOEs detected in PG-1 that were not observed in MCo-PG2 include: H’R41/H’F44
(R41 signal broadened beyond detection), H’R41/H’R43 (R41 and R43 signals broadened
beyond detection), H
α
R36/H
α
G49 (NOEs may be missing due to water suppression) H
α
C40/H
α

C45 (NOEs may be missing due to water suppression).

61

Table 2-3. Tabulation of chemical shifts between MCo-PG2 and MCoTI-I. δ
1
H’ and δ
1
H
α

proton shifts for the common residues between cyclotides MCo-PG2 and MCoTI-I and their
respective chemical shift differences.
Residue
a
δ
1
H’ in
MCo-PG2
(ppm)
δ
1
Hα in MCo-
PG2
(ppm)
δ
1
H’ in
MCoTI-I
(ppm)
δ
1
Hα in
MCoTI-I
(ppm)
Δδ
1
H’
(ppm)
Δδ
1
Hα
(ppm)
C1 8.809 5.426 8.780 5.391 0.029 0.035
G2 9.905 4.569 9.902 4.559 0.003 0.01
S3 N/A
c
N/A
c
8.715 4.502 - -
G4 9.008 4.238 9.201 4.403 -0.193 -0.165
S5 N/A
c
N/A
c
8.783 4.542 - -
G6 8.289 4.083 8.237 4.095 0.052 -0.012
G7 8.289 4.083 8.213 4.015 0.076 0.068
V8 8.425 4.198 8.434 4.058 -0.009 0.14
C9 8.530 5.338 8.565 5.122 -0.035 0.216
P10 N/A
b
N/A
b
N/A
b
N/A
b
- -
K11 8.220 4.330 N/A
d
N/A
d
- -
I12 7.744 4.460 7.713 4.425 0.031 0.035
L13 8.798 4.587 8.693 4.525 0.105 0.062
Q14 9.057 4.602 N/A
d
N/A
d
- -
R15 8.810 4.500 8.749 4.517 0.061 -0.017
C16 8.411 4.841 8.393 4.841 0.018 0
R17 9.529 4.461 9.538 4.461 -0.009 0
R18 8.085 4.757 8.097 4.777 -0.012 -0.02
D19 N/A
c
N/A
c
N/A
d
N/A
d
- -
S20 8.146 4.280 8.150 4.331 -0.004 -0.051
D21 7.745 4.625 7.776 4.625 -0.031 0
C22 8.113 4.992 8.065 4.990 0.048 0.002
P23 N/A
b
N/A
b
N/A
b
N/A
b
- -
G24 8.540 3.831 8.520 3.809 0.02 0.022
A25 8.287 4.463 8.496 4.474 -0.209 -0.011
C26 8.295 4.653 8.201 4.682 0.094 -0.029
I27 9.059 4.437 8.998 4.450 0.061 -0.013
C28 9.468 4.968 9.486 4.992 -0.018 -0.024
R29 8.157 4.350 8.150 4.331 0.007 0.019
G30 N/A
c
N/A
c
N/A
d
N/A
d
- -
N31 7.814 4.724 7.814 4.724 0 0
G32 8.478 4.027 8.496 4.023 -0.018 0.004
Y33 7.358 5.268 7.345 5.297 0.013 -0.029
a
Sequence numbers are based on Fig. 1.
b
Not available. P10 and P23 do not have amide protons.
c-d
H’/H cross peaks were broadened beyond detection for the following residues: S3 ,S5 ,D19,
G30 in MCo-PG2 (c) and K11, Q14, D19, G30 in MCoTI-I (d).
62

Next, we screened the broad-spectrum antimicrobial activity of the different PG-1-grafted
cyclotides against different strains of four ESKAPE pathogens, P. aeruginosa, S. aureus, K.
pneumoniae, and E. coli (Table 2-4). The naturally occurring cyclotide MCoTI-I and the porcine
protegrin PG-1 were used as negative and positive controls, respectively. The minimum
inhibitory concentration (MIC) values for the different peptides were determined by broth
microdilution assay using a cation-adjusted Mueller-Hinton broth (CAMHB).[167] This growth
medium contains 128 mM NaCl supplemented with calcium and magnesium salts providing very
similar ionic strength to that of physiological conditions. As expected, protegrin PG-1 exhibited
potent and strong activity against Gram-negative and Gram-positive laboratory and clinical
bacteria, with MIC values ranging from 0.03 µM (E. coli DS377) to 0.4 µM (S. aureus USA300
and HH35, both methicillin resistant strains; and K. pneumoniae BAA1705 and K6) (Table 2-4).
This is an agreement with published data for this protegrin.[167] Interestingly, all grafted PG-1
grafted MCoTI-based cyclotides showed antibacterial activity against P. aeruginosa, with MIC
values from 25 µM for the less active cyclotide (MCo-PG3) to 1.6 µM for the most active
cyclotides (MCo-PG2 and MCo-PG4) (Table 2-4). Cyclotide MCo-PG3 showed no observed
activity against S. aureus, K. pneumoniae and E. coli, with MIC values in all the cases above 25
µM, indicating that addition of an extra-disulfide bond to the grafted peptide significantly
reduced its antimicrobial activity. Shortening the grafted PG-1-derived sequence had a
detrimental effect on the antimicrobial activity of cyclotide MCo-PG5 although the effect was
not as pronounced as the observed for cyclotide MCo-PG3. Elongation of the grafted sequence
by adding extra Gly residues had little impact on the antimicrobial activity, with cyclotides
MCo-PG2 and MCo-PG3 practically showing the same antibacterial activity. As shown in Table
63

2-4, MCo-PG2 was slightly more active than MCo-PG4 against P. aeruginosa, S. aureus and E.
coli, but slightly less active against K. pneumoniae. As expected, the naturally-occurring
cyclotide MCoTI-I did not show any antibacterial activity in this assay up to a concentration of
200 µM (Table 2-4), indicating that the antimicrobial activity of PG-1 grafted cyclotides was
specific and comes from the grafted sequence.

Table 2-4. Minimum inhibitory concentrations (MIC) of antimicrobial peptide PG-1 and
MCo-PG2 through MCo-PG5 cyclotides. Naturally occurring protegrin PG-1 and cyclotide
MCoTI-I were used as a positive and negative controls, respectively. Antimicrobial activities
were performed by broth microdilution assays using cation-adjusted Mueller-Hinton broth
(CAMHB). This growth medium contains 128 mM NaCl supplemented with Ca
2+
and Mg
2+
salts
providing a very similar ionic strength to that of physiological conditions.

64

Based on the better profile activity of cyclotide MCo-PG2 against three of the four
ESKAPE pathogens tested in our study, and in particular P. aeruginosa and S. aureus which two
ESKAPE pathogens that commonly infect the airways of patients with cystic fibrosis, we
decided to further test the antimicrobial activity of cyclotide MCo-PG2 against 20 different
clinical isolates of P. aeruginosa and S. aureus collected from patients suffering from cystic
fibrosis at the Keck Medical Center, University of Southern California (Tables 2-1, 2-5, 2-6).
Remarkably, MCo-PG2 retained its antimicrobial activity against P. aeruginosa and S. aureus
clinical isolates, with MIC values ranging from 0.4 µM to 12.5 µM (Table 2-6). The median
MIC (MIC50) and MIC 90% (MIC90) values for the P. aeruginosa population (n=20) were 1.5
µM while and 3.1 µM, respectively. For the S. aureus isolates (n=20), the MIC50 and MIC90 were
6.25 µM and 12.5 µM, respectively, indicating that MCo-PG2 shows four times better
antimicrobial activity (MIC90 values) against P. aeruginosa than to S. aureus stains. In
comparison to protegrin PG-1, cyclotide MCo-PG2 was around four and ten times less active
(MIC90 values) against P. aeruginosa and S. aureus than the natural protegrin peptide (Table 2-
6). These results were extremely encouraging indicating cyclotide MCo-PG2 was able to
maintain good MIC values against pathogenic clinical isolates. It is important to remark that 30%
of the P. aeruginosa clinical isolates were multidrug resistant strains (MDR), while 100% of the
S. aureus clinical strains were methicillin-resistant, hence further highlighting the significance of
MCo-PG2 MIC values against these pathogens.

65

Table 2-5. Full list of minimum inhibitory concentration (MIC) of antimicrobial peptides
MCo-PG2 and PG-1 against clinical isolates. P. aeruginosa (n=20) and methicillin-resistant S.
aureus (MRSA) (n=20) were collected from patients suffering from cystic fibrosis at the Keck
Medical Center, University of Southern California. Broth microdilutions were used to assess the
potency of MCo-PG2. Colistin and vancomycin were used as positive controls for P. aeruginosa
and S. aureus, respectively.

66

Table 2-6. Compiled minimum inhibitory concentrations of antimicrobial peptides MCo-
PG2 and PG-1 against clinical isolates. P. aeruginosa (n=20) and methicillin-resistant S.
aureus (n=20) were collected from patients suffering from cystic fibrosis at the Keck Medical
Center, University of Southern California. MIC50 and MIC90 were determined by compiling the
MICs of MCo-PG2 against the clinical isolates. Antimicrobial activities were performed as
described in Table 1. Colistin and vancomycin were used as positive controls for P. aeruginosa
and S. aureus, respectively.

MIC (µM)
PG-1 MCo-PG2 Colistin
P. aeruginosa MIC 50 0.2 1.5 ≤ 0.2
P. aeruginosa MIC 90 0.8 3.1 0.4
P. aeruginosa MIC range 0.05-1.5 0.4-12.5 ≤ 0.2-0.4
PG-1 MCo-PG2 Vancomycin
S. aureus MIC 50 0.4 1.5 0.7
S. aureus MIC 90 0.8 3.1 0.4
S. aureus MIC range 0.2-0.8 3.1-12.5 0.5-1.4

Next, we used a time-kill kinetic assay to establish the bactericidal activity of cyclotide
MCo-PG2 against P. aeruginosa PAO1 (Fig. 2-7A). This was accomplished by using different
MCo-PG2 concentrations ranging from 0.25 x MIC to 16 x MIC values. The results indicated a
rapid and concentration dependent killing kinetics against P. aeruginosa PAO1 at MCo-PG2
with greater than 3 log10 CFU/mL bactericidal activity at concentrations of 4 times the MIC
value. It is important to highlight that by using 16 times the MIC value of MCo-PG2 no regrowth
of P. aeruginosa after 24 h was observed (Fig. 2-7A).

67

We also evaluated the hemolytic activity of cyclotide MCo-PG2. As shown in Fig. 2-7B,
MCo-PG2 exhibited a significantly lower hemolytic activity (HC50 = 88 ± 5 µM) than to that of
protegrin PG-1 (HC50 = 6.3 ± 1.6 µM). As expected, the control cyclotide MCoTI-I did not have
any hemolytic activity up to a concentration of 100 µM. The membranolytic selectivity index
(HC50/MIC) is often used as an indicator of the therapeutic potential of a peptide-based
antibiotic.[168] The HC50/MIC50 values for MCo-PG2 and PG-1 against P. aeruginosa clinical
isolates (Table 2-6) were ≈ 60 and ≈32, respectively. The HC50/MIC50 values for S. aureus
clinical strains (Table 2-6) were found to be similar for PG-1 and MCo-PG2 with value around
15. These results indicate that cyclotide MCo-PG2 has greater therapeutical potential than PG-1
against P. aeruginosa, while shows similar therapeutic potential against S. aureus.

The cytotoxicity profile of cyclotide MCo-PG2 was also studied using two types of
human epithelial cells: HEK293T (transformed kidney epithelial cells) and A549 (lung
carcinoma). As shown in Fig. 2-7C, the cyclotide MCo-PG2 was about three times less toxic
than PG-1. As previously reported,[116] the control cyclotide MCoTI-I did not present any
cytotoxicity in human cells up to 100 µM.
68

Figure 2-7. Cytotoxic activities of cyclotide MCo-PG2. A. Bactericidal activity of PG-1
against log-phase P. aeruginosa PAO1. P. aeruginosa was grown to log phase, and aliquots were
treated with compounds at incremental concentrations relative to MICs, from to 0.25 x MIC to
16 x MIC. B. Hemolytic activity of protegrin PG-1 and cyclotide MCo-PG2. Hemolytic activity
was determined using human erythrocytes in PBS. Peptide concentrations causing 50%
hemolysis (HC50) were derived from the dose-response curves. C. Cytotoxic profile of protegrin
PG-1 and cyclotide MCo-PG2 to various mammalian cells (A549 and HEK293T). Cells were
treated with increasing concentrations of the corresponding peptides. Cell viability was assessed
by using the MTT assay. Cyclotide MCoTI-I was used as control. Data are mean ± SEM for
experiments performed in triplicate.
69

The biological stability of cyclotide MCo-PG2 was explored and compared to that of the
empty scaffold (MCoTI-I) and the grafted peptide sequence (PG-1) (Fig. 2-8). This was
accomplished by incubating the corresponding peptides in human serum at 37° C. The
quantitative analysis of undigested polypeptides was performed using liquid chromatography
coupled with tandem mass spectrometry (LC-MS/MS). MCoTI-cyclotides present a very rigid
structure,[162, 163] which makes them extremely stable to proteolytic degradation. Remarkably,
cyclotide MCo-PG2 showed slightly greater stability in human serum (t1/2 = 60 ± 6 h) than the
parent cyclotide MCoTI-I (t1/2 = 52 ± 5 h, Fig. 2-8). More importantly, cyclotide MCo-PG2
displayed minimal degradation within the first 24 h of the serum stability assay, while 40%
cyclotide MCoTI-I was degraded during the first 24 h of the assay (Fig. 2-8). In contrast,
protegrin PG-1 was degraded significantly faster than MCo-PG2 (t1/2 = 30 ± 3 h) also showing
significant degradation after 24 h of incubation with serum. A linearized, reduced and alkylated
version of MCoTI-I was used as positive control and as expected was rapidly degraded (t1/2 = 18
± 6 min). These results highlight the importance of the circular Cys-knot topology for proteolytic
stability.
70

Figure 2-8. Stability of cyclotides MCo-PG2, MCoTI-I, and protegrin PG-1 to human
serum at 37° C. Linearized reduced cyclotide was used as control for serum activity. Undigested
peptide was quantified by HPLC-MS/MS.

Encouraged by these results, we decided next to explore the biological activity of
cyclotide MCo-PG2 in vivo. We first determined the toxicity profile of MCo-PG2 and PG-1 in
Balb/c mice (n=3) using intraperitoneal (i.p.) administration (Fig. 2-9). Colistin was used as a
control antibiotic.[169] The studies revealed that intraperitoneal doses of 5 mg/kg for PG-1, 25
mg/kg for MCo-PG2 and 15 mg/kg for colistin were well tolerated by mice causing only very
mild toxicity after 1 h of dosing with all mice recovering after 24 h (Fig. 2-9). This maximum
tolerated dose found for colistin is consistent with previously published data.[169] Based on
these results, we decided to use the corresponding compound MTDs to test the antimicrobial
activity in vivo. For this purpose, we employed a P. aeruginosa bacterial peritonitis model.[170]
This animal model is a well-established acute infection model and is commonly utilized as a
common preclinical screening method for new antibiotics.[171] Peritonitis in Balb/c mice (n=10)
71

was established by intraperitoneal injection of 1.5×10
7
colony forming units (CFU) per mouse of
P. aeruginosa (Schroeter) Migula (ATCC 27853). The animals were then immediately treated by
intraperitoneal injection with PBS, PG-1 (5 mg/kg), MCo-PG2 (10 or 25 mg/kg) and colistin (15
mg/kg). As shown in Fig. 2-10, single-dose administrations of 10 mg/kg and 25 mg/kg of
cyclotide MCo-PG2 in the septic mice were associated with high survival rates (hazard ratio
[HR]: 0.0875 and 0.048, respectively; p <0.001) comparable to those obtained in animals treated
with 5 mg/kg PG-1 and 15 mg/kg colistin ([HR]: 0.040; p <0.001). After day 3 post-treatment,
all the animals treated with PBS or the corresponding compound that survived were completely
healthy and no further dead or moribund mice were observed over the course of the seven-day
experiment (Fig. 2-10).

72

Figure 2-9. Preliminary toxicological data for antimicrobial cyclotide MCo-PG2 and PG-1.
The MTD was determined using two different endpoints: weight loss and clinical scoring.
Clinical scores were evaluated through activity, appearance and body condition, similar to
previous published literature [152].

73

Figure 2-10. Evaluation of cyclotide MCo-PG2 against P. aeruginosa (Schroeter) Migula
(ATCC 27853) in a P. aeruginosa-induced bacterial peritonitis model.[170] P. aeruginosa
was administered to mice by intraperitoneal injection 1.5 × 10
7
colony forming units (CFU) per
mouse. The animals were then immediately treated by intraperitoneal injection with PG-1 (5
mg/kg) and MCo-PG2 (10 or 25 mg/kg). Colistin (15 mg/kg) and PBS were used as positive and
negative controls. The numbers of surviving mice were determined daily for 7 days. Single-dose
administrations of MCo-PG2 (10 mg/kg, 1.8 µmol/kg; 25 mg/kg, 4.5 µmol/kg) were associated
with a high survival rate of septic mice (Hazard ratio (HR): 11.4 and 20.8 respectively, p <0.001)
comparable to treatments with PG-1 (5 mg/kg, 2.3 µmol/kg) and 15 mg/kg colistin (15 mg/kg,
12.3 µmol/kg) (HR: 24.8, p <0.001).
74

Discussion
In summary, we report here for the first time the design and synthesis of a novel cyclotide
with broad-spectrum antimicrobial activity in vitro against different ESKAPE pathogens (P.
aeruginosa, S. aureus, K. pneumoniae, and E. coli), including 20 clinical isolates for the human
pathogens P. aeruginosa and S. aureus, and more importantly in vivo using an acute infection P.
aeruginosa bacterial peritonitis model. This was successfully accomplished by grafting a series
of topologically modified peptides based on the porcine protegrin PG-1 sequence onto loop 6 of
the cyclotide MCoTI-I. Structural studies in solution by
1
H-NMR revealed that the new
antimicrobial cyclotides adopts a native cyclotide scaffold, allowing the grafted PG-1-based
sequence to assume a bioactive native conformation. Our observation highlights the tolerance of
this loop in the MCoTI-based cyclotide family for the molecular engraftment of long peptide
sequences.[115, 172, 173] For example, the sequence engrafted in the bioactive cyclotide MCo-
PG2 was 18 residues long. The most active cyclotide, MCo-PG2, displayed good antimicrobial
activity against different ESKAPE pathogen strains, including P. aeruginosa, S. aureus, K.
pneumoniae, and E. coli (Table 2-4), including 20 clinical strains of P. aeruginosa and S. aureus
isolated from patients with cystic fibrosis (Tables 2-5, 2-6 ). All the S. aureus clinical isolates
were methicillin-resistant (MRSA), while around 30% of the P. aeruginosa were classified as
multi-drug (MDR) strains, i.e. showing antimicrobial resistance to at least three or more
antimicrobial agents from different groups of antibiotics. Cyclotide MCo-PG2 showed strong
activity against these clinical strains with MIC50 values of 1.5 µM against P. aeruginosa (n=20)
and 6.25 µM against S. aureus (n=20) indicating its potential therapeutic value (Table 2-6).
More importantly, MCo-PG2 (25 mg/kg, 4.5 µmol/kg; 10 mg/kg, 1.8 µmol/kg) provides a
75

similar level of protection to that of PG-1 (5 mg/kg, 2.3 µmol/kg) and colistin (15 mg/mol, 12.3
µmol/kg) when used as single dose treatment in a murine P. aeruginosa-induced bacterial
peritonitis model(Fig. 2-10). These results reveal that although cyclotide MCo-PG2 was in
general less active than protegrin PG-1 in vitro showed a similar level of activity to that of PG-1
in vivo. Cyclotide MCo-PG2 also exhibited 14 times less hemolytic activity than PG-1, while
was only about three times less cytotoxic than PG-1 to human epithelial cells. In vivo toxicity
studies revealed that cyclotide MCo-PG2 was approximately 4 times less toxic than PG-1 in
mice. These results are extremely encouraging and open the possibility to improve even more the
antimicrobial activity of cyclotide MCo-PG2 in future studies. Cyclotides contain multiple loops
that are amenable to variation using different molecular evolution techniques.[148, 174-177]
Hence, more active cyclotides could be produced by modifying adjacent loops to loop 6 in MCo-
PG2, mainly loops 1, 3 and 5 (Fig. 2-2). It is also worth noting that cyclotide MCo-PG2 showed
remarkable resistance to biological degradation in serum, with a t1/2 value of ≈60 h and not
showing any significant degradation for the first 24 h (Fig. 2-8). In contrast, protegrin PG-1 was
significantly degraded (≈55% degradation) after the first 24 h under the same conditions, hence
revealing the superior proteolytic stability of the circular cystine-knot topology of MCo-PG2
versus the disulfide-stabilized b-hairpin structure of PG-1.

Our results show that engineered cyclotides hold great promise for the development of a
novel type of peptide-based broad spectrum antimicrobial agents that efficiently target emerging
CF clinical pathogens. While HDPs have antimicrobial properties, they can also modulate the
immune system depending on the context.[99] During a pro-inflammatory state, HDPs act as
76

chemoattractant agents to recruit leukocytes and enhance innate immune functions. One of the
most notable functions of HDPs is their ability to modulate inflammation by binding to negatively
charged bacterial lipopolysaccharide (LPS). As LPS interacts with host pattern recognition
receptors including toll-like receptors, the neutralization of LPS reduces inflammation.[99, 133]
Also, HDPs can stimulate inflammation by prolonging the lifespan of neutrophils through novel
caspase-dependent anti-apoptotic properties. To facilitate microbial clearance, HDPs can aid in
micropinocytosis and the formation of neutrophil extracellular traps to combat the pathogens.
Lastly, these HDPs can generate reactive oxygen species to resolve an infection.[178]

The dual activity of HDPs is important in CF as HDPs are essential to combat the diverse
microbial infection and modulate inflammation. HDPs are diminished due to several factors
including microbial toxin neutralization, protease degradation, and an acidic pH environment.[14,
54] Currently, azithromycin is used as an anti-inflammatory drug for CF; however, its effect is
modest. Also, CF clinical trials have shown that glucocorticoids and high-dose ibuprofen slow the
rapid decline in lung function, although significant adverse effects limit their long-term clinical
use.[90, 93] Without new treatment options, the immune system does not modulate itself and
cannot combat infections, leading to a progressive loss of respiratory function that these
individuals succumb to. Thus, we believe that engraftment of HDPs onto a cyclotide will yield a
peptide with both antimicrobial and immunomodulatory properties that could serve as a CF
treatment as it replenishes the airways with stable HDPs.

77

Encouraged by our results, we will improve the bacterial selectively and toxicity profile of
MCo-PG2. Our peptide was good enough to warrant as a “proof of concept” in an acute infection
model. Even though our peptide was efficacious, MCo-PG2’s therapeutic potential needs to be
improved and we will improve the therapeutic index through different strategies. For instance, it
is possible to create synthesis-based peptide libraries to improve the therapeutic potential of our
MCo-PG2 cyclotides.[114] Molecular evolution strategies enable the generation and high-
throughput selection of compounds with optimal antimicrobial, inflammatory and cytotoxicity
activities. We have implemented both synthesis-based and in-cell molecular-evolution strategies
to produce an exceedingly complex set of peptides that could reach 10
9
different cyclotide
sequences.[115, 116] Our approach has been used to produce efficacious compounds for various
diseases. For instance, small chemically synthesized peptide library based on a CXCR4 cyclotide
antagonist was recently created by using a “tea-bag” approach in combination with the “one-pot”
cyclization protocol to concomitantly cyclize and fold the peptides. The approach also includes an
efficient purification step to easily remove non-folded cyclotides from the cyclization-folding
crude, allowing for the possibility to produce an amino acid and positional scanning library to
carry out efficient screens of the large chemical-generated libraries.[116] Once a peptide with an
optimal therapeutic index is identified, we will evaluate the in vivo efficacy of the lead cyclotide
in a P. aeruginosa chronic pneumonia murine model.[135, 140] Altogether, our results
demonstrate that engineered cyclotides hold great promise for the development of a novel type of
peptide-based broad spectrum antimicrobial agents that efficiently target specific CF pathogens.

78

Chapter 3: Anti-Inflammatory Effects of RTD-1 in a Murine Model of Chronic
Pseudomonas aeruginosa Lung Infection: Inhibition of NF-κB, Inflammasome Gene
Expression, and Pro-IL-1β Biosynthesis
Introduction
Cystic fibrosis (CF) is an autosomal recessive disorder characterized by mutations in the
CF transmembrane conductance regulator gene (CFTR). Over 1900 mutations can lead to
defective mucus secretion, predominantly in the respiratory and gastrointestinal systems [179].
CFTR dysfunction results in defects in ion and water transport and increased mucus viscosity
that then leads to destructive cycles of airway obstruction, inflammation, and infection
contributing to bronchiectasis and progressive loss of lung function [22].

Lung disease in CF is characterized by neutrophil-dominated airway inflammation with
excessive concentrations of proteases (e.g., neutrophil elastase) and proinflammatory cytokines
(i.e., tumor necrosis factor [TNF], interleukin (IL)-1, IL-6, and IL-8), which are associated with
lung disease progression and shortened survival [52]. In particular, clinical studies have shown
that IL-1β concentrations are significantly increased in the bronchoalveolar lavage (BAL) and
sputum of people with CF, and IL-1β polymorphisms are linked with disease severity [78-81].
Emerging data indicates the inflammasome may play an important role in CF inflammatory
disease as the lack of extracellular chloride due to a CFTR dysfunction may increase nod-like
receptor protein 3 (NLRP3) stimulation and maturation of IL-1β [83, 84]. Clinical studies with
high-dose ibuprofen and oral corticosteroids have demonstrated reduced rates of pulmonary
79

function decline; however, the clinical use of these drugs is limited due to significant adverse
effects [90, 180, 181]. Azithromycin is frequently prescribed to patients with CF, however, its
anti-inflammatory effects are modest and may not be sustained [182-184]. Therefore, there is a
critical need to develop safe and efficacious anti-inflammatory therapeutics to treat lung disease
in CF.

Cationic host defense peptides (HDPs) exhibit both antimicrobial and
immunomodulatory properties [185, 186]. Several cationic HDPs are effective in murine models
of endotoxin-induced lung injury [139, 187] and in a P. aeruginosa alginate chronic infection
model [135, 140, 188]. Rhesus theta defensin-1 (RTD-1) is a macrocyclic HDP endogenously
expressed in Old World monkey leukocytes. This macrocyclic peptide exhibits broad spectrum
in vitro antibacterial activity that includes activity against multidrug-resistant P. aeruginosa
isolates from people with CF [135, 189]. RTD-1 provides a unique immunomodulatory
mechanism of action compared with other cationic HDPs in which the anti-inflammatory action
is an indirect effect of endotoxin neutralization [133, 137]. Previous investigations found that the
anti-inflammatory activity of RTD-1 is mediated in part by the inhibition of p38 mitogen-
activated protein kinase (MAPK) phosphorylation, NF-κB nuclear translocation via the
phosphorylation of protein kinase B (AKT) and inhibition of IκBα degradation [137], and
inhibition of TNF converting enzyme (TACE/ADAM17) [138]. In vivo studies have indicated
that RTD-1 has potent antimicrobial and immunomodulatory properties in murine models of
acute lung injury [139], chronic lung infection [140], sepsis [133], systemic candidiasis [134],
and rheumatoid arthritis [190].
80

In the current investigation, we sought to further examine the anti-inflammatory activity
of RTD-1 in the context of chronic P. aeruginosa lung infection [191, 192]. We assessed in vivo
anti-inflammatory effects by evaluating white blood cell counts (WBCs) and
cytokine/chemokine secretions in bronchoalveolar lavage fluid (BALF). We also performed
microarray analysis on RNA extracted from murine cells obtained from BALF and lung tissue
homogenates. In vitro mechanistic studies utilizing human and murine monocytes/macrophages
investigated the effects of RTD-1 on cytokine production, inflammasome activation, gene
expression, NF-κB modulation, and transcript stability. These studies confirmed published data
that RTD-1 exhibits anti-inflammatory activity through NF-κB inhibition and provided new
findings that indicate RTD-1 dampens NLRP3 inflammasome activation and related cytokines,
likely through modulation of NF-κB pathways. Overall, these findings support further
investigation of this immunomodulatory peptide as a promising potential therapeutic for the
treatment of CF-associated lung disease.

81

Materials and Methods
Chronic Murine Infection Model
All animal experiments were evaluated and approved by the Institutional Animal Care
and Use Committee at the University of Southern California (Protocol 20252). Male 10- to 12-
week-old C57BL/6 mice (Charles River Laboratories, Wilmington, MA) weighing 23–29 g were
housed in a pathogen-free environment at 22–24 °C and 60–65% humidity with a 12 h light:12 h
dark photoperiod. The mice received ad libitum access to standard normal chow. Chronic lung
infection was established via intratracheal instillation of P. aeruginosa (RP73, mucoid isolate
from a patient with CF) embedded in agarose beads. After overnight storage at 4 °C, 50 µL P.
aeruginosa beads containing 5 × 10
5
colony forming unit in phosphate-buffered saline (PBS)
was instilled intratracheally in each mouse. Previous studies have validated the viability of
overnight stored P. aeruginosa beads (data not shown). After 24 h of infection, each animal
received a 1 h daily treatment with aerosolized saline (n = 8) or a delivered dose of 167 μg/kg of
RTD-1 (n = 8) for 3 or 7 days. Previous dose-ranging studies determined that 167 µg/kg
exhibited the optimal anti-inflammatory effect [140]. Details of the nose-only inhalation
exposure system used, aerosol characterization of the drug, and dose delivery have been
previously published [140].

Mice were euthanized on day 3 or 7. The lungs were washed twice with 0.8 mL PBS
containing mini protease inhibitors (cOmplete™, Millipore Sigma, Burlington, MA, USA).
WBCs isolated from BALF were stained using Turk Blood Diluting Fluid (Ricca Chemical
82

Company, Arlington, TX, USA) and quantified using a hemocytometer. Cell differential counts
were performed after staining using Diff-Quick. Cytokine quantifications were determined from
the BALF using the Luminex Bead Array (Luminex, Austin, TX, USA).

The total lung bacterial burden was quantified by plating serially diluted BALF and
homogenized lung tissue samples onto Pseudomonas isolation agar (agar, 13.6 g/L, magnesium
chloride, 1.4 g/L, peptic digest of animal tissue, 20 g/L, potassium sulfate, 10 g/L, triclosan
(Irgasan), 0.025 g/L) (Millipore Sigma). Total bacterial lung burden was determined from
summation of the BALF and lung tissue homogenate colony counts.

RNA Microarray Analysis
Samples from lung tissue homogenates and BALF cell pellets were stored in an RLT
lysis buffer (Qiagen, Hilden, Germany) containing 1% β-mercaptoethanol (Millipore Sigma).
RNA was isolated using RNeasy Mini kit on-column extraction with DNase-1 digestion
(Qiagen).The Mouse ref 8 v2.0 Expression BeadChip (Illumina, San Diego, CA, USA) was used
to determine sample gene expression. Statistical analysis of differentially expressed genes in the
different tissue samples was performed using the gene expression workflow in Partek GS. To
identify the most significant differentially expressed genes, lists were generated using the criteria
of a false discovery rate corrected using the Benjamini–Hochberg step-up procedure (p < 0.05) to
account for multiple testing and the differences in mean gene transcript levels were required to
be at least 1.5-fold in either direction.
83

Cell Culture
Epithelial cells (NuLi and CuFi [ATCC, Manassas, VA, USA]) were cultured in
Bronchial Epithelial Cell Growth Medium Bulletkit (Lonza, Walkersville, MD, USA) and
inflammation was induced by P. aeruginosa filtrate. THP-1 (ATCC) monocytes and MHS
macrophages (ATCC) were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium,
2 mM L-glutamine, 25 mM HEPES (Corning Inc., New York, NY) + 10% heat-inactivated fetal
bovine serum (HI-FBS; Genesee Scientific, San Diego, CA, USA). THP-1 macrophage-like cell
differentiation was induced by treatment with 100 nM phorbol-12-myristate-13-acetate (PMA;
Millipore Sigma) and cultured for 48 h. Cells were cultured at 37 °C in 5% CO2. Cell lines were
found to be mycoplasma-negative using MycoAlert (Lonza). Experiments were performed using
THP-1 cells cultured between 8 and 18 passages. RTD-1 was solubilized at a concentration of 10
mg/mL in 0.01% acetic acid. At the initiation of each experiment, the cells were cultured in
media containing 1% FBS. Inflammation in THP-1 cells was induced using 100 ng/mL P.
aeruginosa lipopolysaccharide (LPS; InvivoGen, San Diego, CA, USA). Cells were then
immediately dosed with RTD-1 (7.14 μg/mL), azithromycin (50 μg/mL; Millipore Sigma) or
MCC950, an NLRP3 inhibitor, (10 μM; Cayman Chemical Company, Ann Arbor, MI, USA) at
the beginning of the experiment. Both RTD-1 and azithromycin concentrations were determined
based on physiological concentrations observed in the lung epithelial lining fluid in vivo [135,
193]. For RNA collection, cells were seeded at 1 × 10
6
cells per well in 24-well plates and
harvested after 3 h. For secreted protein and NF-κB reporter analysis, cells were seeded at 4 ×
10
5
per well in 24-well plates and harvested after 24 h. For Western blot analysis, cells were
84

seeded at 1 × 10
6
per well in 12-well plates and lysed immediately after the 24 h treatment. To
induce inflammasome activation, THP-1 cells were exposed to 5 mM ATP for the final 30 min
of the experiment. To perform the NLRP3 destabilization assays, THP-1 macrophages were
stimulated and treated with RTD-1 (7.14 μg/mL) for 3 h. A set of cells was exposed to
actinomycin-D (5 μg/mL) for 45 min before the end of the 3 h treatment. All experiments were
conducted in triplicate on three different days.

NF-κB Reporter Assay
The THP1-Lucia™ NF-κB reporter cell line (InvivoGen) was used to assess the effect of
RTD-1 on NF-κB activation. The cells were starved for 4 h in RPMI 25 mM HEPES 1% FBS
before a 16 h overnight treatment period. NF-κB-induced luciferase expression in the cell culture
supernatant was quantified using the luciferase detection reagent QUANTI-Luc™ (InvivoGen).
Luminosity was measured using a Synergy H1 kinetic fluorescent microplate reader (Bio-Tek,
Winooski, VT, USA).

Gene Expression Analysis
Cells (THP-1 monocytes/macrophages, MHS macrophages) were lysed in PureLink
(Thermo Fisher, Waltham, MA) spin column-based kit lysis buffer containing 1% β-
mercaptoethanol and total RNA isolated according to the PureLink manufacturer’s protocol.
cDNA was transcribed using iScript™ Reverse Transcription Supermix for quantitative reverse
85

transcription PCR (RT-PCR; Bio-Rad Laboratories, Hercules, CA, USA). Quantitative realtime
PCR assays were performed using SsoAdvanced™ Universal SYBR
®
Green Supermix (BioRad,
Hercules, CA, USA) per the manufacturer’s instructions on the CFX96™ Real-Time PCR
Detection System (BioRad). Inflammatory cytokines associated with CF-associated lung disease
and inflammasome-related genes were assessed. The primers (Millipore Sigma) used are
presented in Supplementary Materials Table S1 [52]. Transcript copy numbers were normalized
to the housekeeping genes, GAPDH and RPL27, using the ΔΔCt method. Gene expression was
calculated based on relative fold change. Inflammasome-related gene expression profiles were
analyzed with THP-1 macrophages using RT-Profiler Human Inflammasome plates (Qiagen).
The data were analyzed using the  Ct method included in the Qiagen Data Analysis Center.
Table 3-1. Forward and reverse primer sequences used in qRT-PCR experiments.

86

Enzyme-Linked Immunosorbent Assay (ELISA)
Cell culture supernatants were collected at 24 h and stored at −80 °C. Cytokines IL-1β,
TNF, IL-6, and CXCL10 were quantified using the V-Plex ELISA (Meso Scale Diagnostics,
Rockville, MD) according to the manufacturer’s protocol.

Immunoblot Analyses
THP-1 monocytes were harvested, and the cell pellet was washed twice with ice cold
PBS and lysed with Mammalian Protein Extraction Regent lysis buffer (Millipore Sigma)
containing c0mplete™ mini protease inhibitors. Protein contents of cellular lysates were
quantified using a BCA protein assay kit (Thermo Fisher). Standardized total protein (10 μg)
from each treatment was separated on a 4–15% precast polyacrylamide gel (BioRad).
Immunoblots were probed using (1:1000) rabbit monoclonal antibodies that target human IL-1β,
caspase-1, and GAPDH proteins (Cell Signaling, Danvers, MA). The immunoblot was developed
using anti-rabbit HRP-conjugated secondary antibodies and detected by chemiluminescence.
Images were acquired using a Chemidoc MP Imager (BioRad). Western blots were normalized to
GAPDH and divided by the comparator (stimulated cells). Densitometry analyses were
performed using ImageJ software

87

Caspase-1 Activity
THP-1 cells were seeded into 96-well plates at a concentration of 40,000 cells per 100 μL
complete media. To visualize the role of RTD on inflammasome activation, cells were seeded
onto 12 mm glass coverslips (Neuvitro Corporation, Vancouver, WA) in a 24-well plate at a
concentration of 2 × 10
5
cells in 0.5 mL complete medium for confocal imaging. THP-1 cells
were differentiated in the wells for 48 h in 100 nM PMA. The complete medium was aspirated
and replaced with RPMI +1% HI-FBS before addition of LPS (100 ng/mL), RTD-1 (7.14
μg/mL), or azithromycin (50 μg/mL) for 6 h. ATP (5 mM; Millipore Sigma) was added 45 min
before cells were labeled using the FAM-FLICA
®
Caspase-1 Assay Kit (ImmunoChemistry
Technologies, Bloomington, MN, USA) according to the manufacturer’s instructions.
Inflammasome activity was measured using a Synergy H1 kinetic fluorescent microplate reader.
Cells seeded onto the coverslips were fixed and mounted using a Fluoroshield Mounting
Medium (Abcam, Cambridge, UK). Representative confocal images were captured using a
ZEISS LSM 880 with Airyscan (ZEISS, Oberkochen, Germany). The images were analyzed
using ZEN Blue Microscope software (ZEISS). Lastly, RTD-1′s direct effect on caspase-1
inhibition was assessed in vitro using a fluorogenic substrate assay according to the
manufacturer’s instructions (Abcam) using a Synergy H1 kinetic fluorescent microplate reader.

Data and Statistical Analysis
Statistical and graphical analysis were performed using GraphPad Prism version 8.0.1
software. A statistical significance level of p < 0.05 was determined before analysis.
88

Results
In Vivo Efficacy of Aerosolized RTD-1 in a Murine Chronic Pseudomonas aeruginosa
Lung Infection
We initiated an in vivo study to characterize the efficacy of aerosolized RTD-1 by
examining its effects on: (1) immune cell lung infiltration, (2) BALF cytokines/chemokines, and
(3) total lung bacterial burden. The results for the differences in antibacterial and anti-
inflammatory effects between RTD-1-treated and control mice are presented in Fig 3-1. No
differences in antibacterial effects were found for treatment versus control groups on days 3 or 7
(Fig 3-1A). Despite the lack of antibacterial effects, aerosolized RTD-1 treatment led to a
significant reduction in total WBC counts in the BALF on days 3 (−54.95%; p = 0.0003) and 7
(−31.71%; p = 0.0097) (Fig 3-1B). Compared with the control group, mice treated with RTD-1
had reduced weight loss by day 3, but there were no differences on day 7 (Fig 3-1C). WBC
differential counts indicated that neutrophils were predominant on day 3 while an increase in the
proportion of macrophages was evident by day 7. However, there were no significant group
differences (Fig 3-1D).
89

Figure 3-1. Pathophysiological Studies of Chronic Pseudomonas aeruginosa Lung Infection
in Mice Treated with Aerosolized RTD-1. Chronic infection was established via intratracheal
instillation of 5 × 10
5
CFUs of P. aeruginosa (RP73, mucoid CF isolate). 167 mg/kg RTD-1 was
aerosolized 24 h after infection and continued for 6 days. Effects of treatment on: (A) lung
bacterial burden, (B) total immune cell counts, (C) weight, and (D) differential cell counts were
evaluated (** p ≤ 0.01, *** p ≤ 0.001). BALF, bronchoalveolar lavage fluid; CFU, colony
forming unit; PMN, polymorphonuclear leukocyte; WBC, white blood cell.
90

Consistent with the anti-inflammatory effects of RTD-1, peptide treatment was
associated with significant reductions in BALF IL-6, MCP-1, TNF, IL-17, IL-1β, and TIMP-
1 on day 3 (Fig 3-2A). On day 7, there were no significant differences between the treatment
and control groups for most of the inflammatory biomarkers, apart from increases in
KC/CXCL1 and MIP-2/CXCL2 in the RTD-1 treatment group (Fig 3-2B). Cytokine levels
declined in both treated and control mice between days 3 and 7 which is consistent with
previously published data demonstrating a decline in lung bacterial burden after day 3 before
stabilizing at day 7 [192].

91

Figure 3-2. Effect of Aerosolized RTD-1 on BALF Cytokines in Murine Model of Chronic
Pseudomonas aeruginosa Lung Infection. Cytokines were quantified from the BALF of
C57BL/6 mice chronically infected with RP73, a clinical isolate of P. aeruginosa. The mice were
treated with aerosolized saline or RTD-1 (167mg/kg) for 1 h for 6 days, starting 1 day after
bacterial instillation. Cytokines were evaluated on day 3 (A) and on day 7 (B) (* p ≤ 0.05, ** p ≤
0.01). BALF, bronchoalveolar lavage fluid; NT, untreated.
92

Biological Targets of Anti-inflammatory Activity of RTD-1 during Chronic
Pseudomonas aeruginosa Lung Infection
To investigate the anti-inflammatory mechanism of RTD-1 in vivo, we performed a
microarray analysis using RNA isolated from lung tissue homogenates and the BALF cell pellets
of RTD-1-treated and control mice that were treated for 3 days. Using Ingenuity Pathway Analysis
(IPA), we identified networks affected by RTD-1 in lung tissue homogenates and BALF cells. IPA
identified the most relevant biological functions affected by RTD-1 activity. These IPA analyses
indicated that RTD-1 reduced cell-to-cell signaling and interaction, hematological system
development and function, and the inflammatory response. In lung tissue, the highest scoring
networks clustered around TNF. Differential gene expression analysis showed that RTD-1
treatment significantly altered the expression (2-fold up or down; unadjusted p-value < 0.05) of
109 genes in BALF immune cells and 58 genes in lung tissue homogenates. The most affected
were inflammation-associated genes, which were largely downregulated in the RTD-1-treated
group.

Global gene expression analysis of samples from the saline control and RTD-1-treated
mice were compared using volcano plots (Fig 3-3). Although most affected genes were
downregulated, some genes in the lung tissue homogenates and BAL cells were upregulated (>2-
fold change [FC]). The results indicated that RTD-1 upregulated genes encoding cytochrome P450
enzymes, including Cyp2F2 and Cyp2A5 in BALF cells, by 2.555-fold and 2.148-fold,
respectively; Cyp2E1 was upregulated 2.019-fold in lung tissue homogenates. RTD-1 also induced
a 2.307-fold upregulation of the organic anion uptake transporter, SLCO2B1 (OATP2B1) in BALF
93

cell pellets. In lung tissue homogenates, RTD-1 increased expression of BC055107 (FAM107a,
DDR1) by 2.497-fold. This stress-inducible actin-binding protein facilitates regulation of
filamentous actin dynamics [194]. We also found a 2.562-fold increase in NPR3. This gene
encodes a natriuretic peptide receptor that helps to regulate blood pressure, blood volume, and
pulmonary hypertension [195].
94

95

Figure 3-3. Microarray analysis of Lung Tissue and BALF Cells of RTD-1-treated and
Saline Control Mice with Chronic Pseudomonas aeruginosa Lung Infection. Volcano plots
of microarray data for: A) BALF cells and B) lung tissue homogenates. The horizontal line
indicates the cut-off for an unadjusted p-value of 0.05; vertical lines indicate the 2-fold
differences in both directions. BALF, bronchoalveolar lavage fluid.
96

The top 20 genes affected by RTD-1 treatment in BALF immune cells and lung tissue
homogenates are presented in Fig 3-4 (Fig 3-4A). For the lung tissue homogenates, 17 genes were
downregulated, and three genes were upregulated (Fig 3-4B). We found 26 genes in common
between the two tissues that were significantly altered by RTD-1 treatment; 25 genes were
significantly downregulated (Fig 3-4C). RTD-1 induced large fold change reductions in the
proinflammatory cytokines CXCL10 (lung −4.4; BALF −3.45), CXCL9 (lung −2.78; BALF −2.33),
CXCL2 (lung −2.81; BALF −3.22), IL-1β (lung −2.61; BALF −3.27), and IFNγ (lung −2.45; BALF
−2.00) compared to the control treatment group. FKBP5 was the only gene significantly
upregulated in both tissues (BALF 2.68; lung −2.44). FKBP5 is an important stress response
modulator and a cochaperone of heat shock protein-90 (HSP90) [196, 197].

97

Figure 3-4. Top Up- or Downregulated Genes in Lung Tissue and BALF cells in RTD-1-
treated Mice with chronic P. aeruginosa lung infection. The top 20 most highly up- or
downregulated genes in BALF cells and lung tissue homogenates following RTD-1 treatment.
Blue and red squares indicate down- or upregulation of indicated genes in: A) BALF immune
cells or B) lung tissue homogenates of mice treated with aerosolized RTD-1, compared with
saline-treated controls. C) The genes commonly altered by RTD-1 treatment in both lung tissue
homogenate and BALF cells. BALF, bronchoalveolar lavage fluid.

98

Overall, inflammation-associated genes were significantly reduced, including genes
overexpressed in airways of individuals with CF (e.g., TNF and IL-1β). In particular, these results
identified several inflammasome-related genes that were modulated by RTD-1 treatment in both
tissues (Fig 3-5). Inflammasome machinery and cytokines including NLRP3 (lung −1.2; BALF
−2.21), Mediterranean fever gene (MEFV) (lung −2.10; BALF −2.27), IL-1α (lung −2.6; BALF
−3.03), and IL-1β (lung −2.61; BALF −3.27) were significantly downregulated by RTD-1
treatment compared to the control.

Figure 3-5. Inflammasome-associated Gene Expression Changes in Lung Tissue and BALF
in RTD-1-treated Mice with Chronic Infection. Microarray Analysis identified a list of
inflammasome-associated gene expression changes due to RTD-1 treatment. Blue and red
squares indicate down- or upregulation of indicated genes, respectively.
99

RTD-1 Inhibits NF-κB Activity
Due to its importance as a central regulator of inflammatory genes, we evaluated the effect
of RTD-1 treatment on NF-κB activity of THP-1 monocytes and macrophages. Macrophages are
also key contributors to CF-associated lung pathology and epithelial dysfunction via elevated
secretion of proinflammatory cytokines and elastase [57]. A two-fold inhibition of NF-κB activity
occurred after RTD-1 treatment in LPS-stimulated THP-1 monocytes and macrophages (Fig 3-6),
which was consistent with previously published data [137]. Azithromycin appeared to inhibit NF-
κB to a greater extent than RTD-1 in the THP-1 macrophages (Fig 3-6B); however, the difference
was not statistically significant, and was not observed in THP-1 monocytes (Fig 3-6A).

100

Figure 3-6. Inhibition of NF-κB by RTD-1. THP-1 Lucia NF-κB reporter monocytes (A) and
macrophages (B) were treated with LPS (100 ng/mL) and RTD-1 (7.14 µg/mL) or azithromycin
(50 μg/mL) for 16 h. The x-axis denotes the presence (+) or absence (-) of each compound.
Luminescence signal intensity was measured using the Lucia NF-κB reporter gene assay and
calculated as the percentage of treated cells over control cells. Results were normalized to LPS-
stimulated cells (mean ± standard deviation values). Statistical significance between LPS-
challenged and RTD-1 and azithromycin treatments was evaluated using two sample t-tests
(**** p ≤ 0.0001, *** p ≤ 0.001). AZM, azithromycin; LPS, lipopolysaccharide.

101

RTD-1 Inhibits Inflammatory Gene Expression
To further evaluate the anti-inflammatory effects of RTD-1, we conducted in vitro
experiments in LPS-stimulated THP-1 monocytes and bronchial epithelial cells. Study results
suggested that hematopoietic cells are the predominant source of inflammasome-induced IL-1β
secretions within lungs affected by CF [83]; however, pilot experiments using submerged NuLi
and CuFi cell lines revealed limited LPS-induced expression of inflammatory genes (and proteins).
Previously, we observed that RTD-1 downregulated several inflammatory cytokines in murine
MHS macrophages, including macrophage inflammatory protein (MIP)-2, monocyte
chemoattractant protein (MCP)-1, IL-6, and TNF [139]. Thus, we focused the research on human
THP-1 cells. In comparison to untreated cells, RTD-1 significantly increased the transcription of
IL-8, IL-6 and CXCL10 (Fig 3-8D–F). In contrast, RTD-1 significantly inhibited IL-1β, TNF, IL-
1α, IL-8, and IL-6 (Fig 3-8A–E) in LPS-stimulated cells when compared to untreated LPS-
stimulated cells. RTD-1 did not affect CXCL10 expression in LPS-stimulated cells (Fig 3-8F). This
finding contrasts with the in vivo data in this report (Fig 3-4) as well as previous findings that
demonstrate RTD-1 potently inhibited CXCL10 mRNA in murine alveolar macrophages (Fig 3-
7A). This reason for this apparent discrepancy is unknown but appears to be unique to the
immortalized THP-1 cells.

102

Figure 3-7. RTD-1 Treatment Reduces CXCL10 Transcription and Translation in
Different Cell Types. MHS murine alveolar macrophages were stimulated with LPS (100
ng/ml) and RTD-1 (7.14 μg/ml) for 3 h and CXCL10 A) transcription and B) translation was
evaluated through qRT-PCR and ELISA, respectively. C) CuFi bronchial epithelial cells were
stimulated with P. aeruginosa filtrate and cytokine secretion was evaluated. LPS,
lipopolysaccharide; Pa, P. aeruginosa; NT, No treatment.

RTD-1 exhibited activity similar to azithromycin across the cytokine panel with the
exception of CXCL10, where azithromycin treatment resulted in a significant inhibition of
CXCL10 mRNA (3.35-fold reduction) compared to treatment with RTD-1 and LPS-stimulated
cells (Fig 3-8F). Cotreatment with RTD-1 and azithromycin resulted in an additional reduction in
IL-8 and IL-6 mRNA, but not IL-18 (Fig 3-8D,E). The results suggested that RTD-1 and
azithromycin exert anti-inflammatory activities through alternative, but complementary
mechanisms.
103

Figure 3-8. Effect of RTD-1 Treatment on Inflammatory Gene Expression. The effect of
RTD-1 (7.14 μg/ml) on THP-1 monocytes stimulated with 100 ng/ml LPS for 3 h was
determined through gene expression analysis of key inflammatory cytokines: A) IL-1β, B) TNF-
α, C) IL-1α, D) IL-8, E) IL-6 and F) CXCL10. Gene expression was quantified using qRT-PCR
(n = 3). Treatment differences were analyzed using t-tests after normalization to the
housekeeping genes. Mann-Whitney tests were performed for IL-1α and TNF-α (**p ≤ 0.01,
***p ≤ 0.001, ****p < 0.0001). AZM, azithromycin; FC, fold change; LPS, lipopolysaccharide;
NT, No treatment.

104

RTD-1 Treatment Reduces Cytokine Production In Vitro
To determine if the downregulation of inflammatory genes was accompanied by a decrease
in corresponding protein levels, we assessed cytokine production in LPS-stimulated THP-1
monocytes with and without peptide and drug treatment. RTD-1 treatment reduced IL-1β protein
secretion by approximately 10-fold, which was comparable to the effect of RTD-1 on IL-1β gene
mRNA levels (Fig 3-9A). IL-1α protein concentrations were below the lower limit of detection for
these experiments. RTD-1 treatment had similar inhibitory activity when compared to
azithromycin and the NLRP3 inhibitor MCC950 (Fig 3-9A). RTD-1 potently inhibited TNF
secretion with a 5.44-fold reduction (p = 0.0002). When coadministered with azithromycin, there
was a trend towards greater TNF inhibition relative to RTD-1 or azithromycin alone (Fig 3-9B)
suggesting that RTD-1 and azithromycin inhibit TNF protein secretion through different but
complementary mechanisms of action. This mechanistic difference is likely attributable to the
pleiotropic inhibitory activities of RTD-1 against TACE/ADAM17, p38 MAPK phosphorylation
and NF-κB [138]. Although the combination of RTD-1 and azithromycin resulted in additional
inhibition of IL-6 mRNA compared to each treatment alone, the inhibition did not translate at the
protein level (Fig 3-9C). Consistent with the effect on mRNA levels, RTD-1 did not reduce
CXCL10 protein secretion below that of LPS alone (Fig 3-9D). The difference in CXCL10
secretion is unique to THP-1 cells as MHS murine alveolar macrophages and CuFi bronchial
epithelial cells treated with RTD-1 were found to significantly reduce CXCL10 secretion after LPS
or P. aeruginosa filtrate stimulation, respectively (Fig 3-9B-C) which is consistent with the in vivo
data.
105

Figure 3-9. Effect of RTD-1 Treatment on Inflammatory Cytokine Release. The effect of
RTD-1 (7.14 μg/ml) on cytokine expression from THP-1 monocytes stimulated with 100 ng/ml
LPS for 24 h: A) IL-1β, B) TNF-α C) IL-6 and D) CXCL10. Cytokines were quantified using
ELISA (n = 3). Treatment differences were analyzed using parametric t-tests for cytokines IL-1β
and TNF-α. For IL-6 and CXCL10, Mann-Whitney tests were performed. (*p ≤ 0.05, ** p ≤
0.01, *** p ≤ 0.001, ****p ≤ 0.0001). ATP, Adenosine triphosphate; AZM, azithromycin;
ELISA, enzyme-linked immunosorbent assay; LPS, lipopolysaccharide.
106

RTD-1 Downregulates Inflammasome-Associated Gene Expression
Microarray expression analysis from the chronic P. aeruginosa model indicated that
RTD-1 significantly downregulated expression of NLRP3 inflammasome-related genes.
Therefore, we examined the effect of RTD-1 on the inflammasome in LPS-stimulated THP-1
monocytes and macrophages using a PCR array. Preliminary studies in THP-1 monocytes
demonstrated minimal NLRP3 gene expression, compared with THP-1 differentiated
macrophages. Thus, we assessed the differences in mRNA levels of inflammasome-associated
genes in LPS-stimulated THP-1 macrophages. RTD-1 treatment resulted in significantly
decreased fold changes of downstream inflammasome-related genes relative to the control
treatment group, including IL-1β (−8.72), IL-18 (−2.125), CXCL1 (−7.225), CXCL2 (−7.925),
CCL5 (−2.000), and IFNβ1 (−1.665) (Fig 3-10). RTD-1 upregulated mitogen-activated protein
kinase 9 (MAPK9; 2.755) and TNF superfamily member 14 (TNFSF14; 5.725) genes.
107

Figure 3-10. Inhibition of Inflammasome-associated Gene Expression by RTD-1
Treatment. RTD-1 (7.14 μg/mL) inhibited inflammasome-associated genes in LPS (100
ng/mL)-stimulated THP-1 macrophages. RTD-1 reduced expression of downstream
inflammasome molecules. The mRNA transcription profiles obtained using qRT-PCR profiler
array were plotted according to the log2-fold change and a log-adjusted p-value. Significant
treatment differences were assessed using a multiple t-test function in Prism 8.0 software.
Statistically significant gene expression changes are represented by data above the dotted
horizontal line (p < 0.05). FC, fold change; LPS, lipopolysaccharide; qRT-PCR, quantitative
reverse transcription PCR.

The microarray analysis revealed that RTD-1 treatment was associated with a significant
reduction in NLRP3 mRNA transcripts. A previous study observed that azithromycin treatment
108

increased the decay of the NLRP3 transcript over time in THP-1 monocytes [198]. To test the
hypothesis that RTD-1 destabilizes the NLRP3 transcript, the experiment was repeated with LPS-
stimulated THP-1 macrophages treated with RTD-1, with or without a 45 min actinomycin-D (a
transcriptional inhibitor) treatment. RTD-1 reduced NLRP3 mRNA levels (−2.17 FC; p <
0.0001); however, gene expression between treatment groups with or without actinomycin-D
was unchanged, indicating that reduced NLRP3 mRNA levels was not due to transcript
destabilization (Fig 3-11).

Figure 3-11. RTD-1 Does Not Destabilize NLRP3 mRNA Transcription in THP-1
Macrophages. NLRP3 mRNA transcription was determined using THP-1 macrophages
stimulated with LPS (100 ng/ml) and treated with RTD-1 (7.14 μg/ml) for 3 h. A separate set of
cells was also exposed to actinomycin-D (5 μg/ml) for an additional 45 min. ATP, Adenosine
triphosphate; LPS, lipopolysaccharide.

RTD-1 Decreases IL-1β Precursor Formation
109

To determine if the downregulation of inflammasome-associated genes was accompanied
by a decrease in protein expression, we performed immunoblot experiments with RTD-1 in LPS-
stimulated THP-1 monocytes and macrophages. Preliminary experiments in THP-1 macrophages
demonstrated minimal IL-1β precursor expression, compared with THP-1 monocytes; thus,
protein expression was assessed in THP-1 monocytes. RTD-1 and azithromycin significantly
inhibited IL-1β precursor (56.98%, p-value = 0.0015, and 93.24%, p-value ≤ 0.0001, decrease,
respectively), compared with cells treated with LPS and ATP alone (Fig 3-12). The increased
activity of azithromycin relative to RTD-1 against IL-1β precursor is likely attributable to its
effect on the destabilization of inflammatory mRNA transcripts [198]. Cotreatment with RTD-1
and azithromycin resulted in similar levels of inhibition of IL-1β precursor compared to
azithromycin alone. Treatment with RTD-1 or azithromycin, or both, did not result in significant
inhibition of caspase-1 protein expression or any other intermediates (Fig 3-12) indicating that
the inhibition of IL-1β precursor by RTD-1 is not mediated by inhibition of caspase-1 activation.

110

Figure 3-12. Inhibition of IL-1β Protein Expression. THP-1 monocytes stimulated with 100
ng/mL LPS were simultaneously treated with RTD-1 (7.14 μg/mL) for 24 h and lysed
immediately afterwards to determine the effect on expression of (A) IL-1β and (B) caspase-1
precursor formation. Representative Western blot image is shown in (C). Treatment differences
were analyzed using t-tests for IL-1β and caspase-1 (* p ≤ 0.05, ** p ≤ 0.01, *** p < 0.0001).
AZM, azithromycin; LPS, lipopolysaccharide; NT, No treatment.

111

RTD-1 Inhibits Inflammasome Activity
To determine if the downregulation of inflammasome-associated genes was associated
with functional changes in the inflammasome, we performed inflammasome activation assays
(Fig 3-13A). RTD-1, azithromycin, or their combination resulted in significant fold reductions in
caspase-1 activation of −1.79 (p = 0.0052), −2.55 (p = 0.0022), and −2.38 (p = 0.0003),
respectively (Fig 3-13A). Combined treatment with RTD-1 and azithromycin did not result in
any additional reduction in inflammasome activity compared to each treatment alone.
Representative images of these results are presented in Fig 3-13B. To further elucidate whether
RTD-1 inhibits inflammasome activity directly, we utilized an in vitro caspase-1 inhibitor assay
(Fig 3-13C). We observed no direct caspase-1 inhibition at the highest concentration of 41.6
µg/mL of RTD-1, which suggests the reduction in the inflammasome activity by RTD-1 is
possibly a downstream effect of NF-κB modulation.
112

Figure 3-13. Inhibition by RTD-1 Treatment. (A) Caspase-1 activity was determined in THP-
1 macrophages stimulated with LPS (100 ng/mL) and RTD-1 (7.14 μg/mL) or azithromycin (50
μg/mL) for 6 h; ATP (5 mM) was added for an additional 45 min. Cells were labelled with
FLICA caspase-1 specific monoclonal antibodies and fluorescence was measured. (B)
Representative confocal images of inflammasome activation are shown. (C) The direct effect of
RTD-1 on caspase-1 activity using an enzymatic inhibition assay (* p ≤ 0.01, ** p ≤ 0.001).
FLICA caspase-1 specific monoclonal antibody (green), Hoechst 33,342 nuclear stain (blue).
ATP, adenosine triphosphate; AZM, azithromycin; LPS, lipopolysaccharide.
113

Discussion
RTD-1 is an immunomodulatory peptide found to have efficacy in preclinical models of
infection and inflammation [133, 135, 138-140, 190]. In a murine model of LPS-induced acute
lung injury, RTD-1 exhibited a protective effect, as evidenced by the lack of alveolar leukocyte
infiltrates, absence of hyaline membranes, and normal alveolar wall thickness [19]. To better
characterize the immunomodulatory activity of RTD-1 in the context of chronic P. aeruginosa
lung infection, we performed RNA microarray analysis of lung tissue homogenates and BALF
cells isolated from treated and control mice. Overall, we found that RTD-1 inhibited
proinflammatory cytokine gene and protein expression.

In particular, we report new information demonstrating RTD-1 treatment significantly
downregulates the transcription and activation of key inflammasome components. We found that
RTD-1 significantly reduced the expression of proinflammatory cytokines (IL-6, and TNF) and
four inflammasome-related genes (NLRP3, MEFV, IL-1α, and IL-1β). While most genes were
downregulated, FKBP5 was upregulated in both the mouse lung tissue homogenates and the
BALF immune cells. FKBP5 is a molecular cochaperone protein for HSP90 and serves as a
modulator of glucocorticoid receptor sensitivity along with a host of other cellular responses
[196, 197]. HSP90 participates in the regulation of protein kinases, ligases, steroid receptors,
cell-cycle regulation, and nuclear transcription factors [199]. Despite upregulation of FKBP5,
which can induce proinflammatory signaling with HSP90, the overall effect of RTD-1 treatment
in chronically infected mice was reduced lung inflammation.
114

Furthermore, we observed that RTD-1 may have a role in inflammasome regulation.
Treatment with RTD-1 resulted in decreased IL-1β precursor and secreted IL-1β. The gene
expression results indicated that RTD-1 reduced NLRP3 and IL-1β mRNA expression.
Inflammasome and caspase-1 inhibition studies demonstrated that RTD-1 indirectly inhibited
inflammasome activity as caspase-1 activation was reduced. Previous literature indicated NF-κB
inhibition was central to the downregulation of NLRP3 and IL-1β protein expression, but not
caspase-1 expression [200]. These data suggest that RTD-1 inhibits the priming of
inflammasome formation and the subsequent activation of IL-1β and caspase-1, possibly through
disruption of NF-κB signaling.

Accumulating evidence indicates targeting the NLRP3 inflammasome may be an
effective treatment for lung infection and inflammation in individuals with CF [201]. P.
aeruginosa infection induces NLRP3 inflammasome assembly in bronchial epithelial cells
isolated from patients with CF [202], THP-1 macrophages, human monocyte-derived
macrophages [203], and in F508del–CFTR mice [204]. Results using a murine model of
pneumonia indicate that IL-1β and IL-18 participate in the suppression of host capabilities to
clear P. aeruginosa pulmonary infection [205, 206]. In vitro studies found that IL-1β antibody
neutralization and inhibition of inflammasome components improve macrophage-dependent P.
aeruginosa killing and reduce the total bacterial burden [201, 203, 204]; while the addition of
recombinant IL-1β treatment inhibits P. aeruginosa clearance [203]. Results of in vivo and in
vitro studies indicate that IL-1Ra, an inhibitory protein of various IL-1-related immune
115

responses, can mitigate the damaging effects caused by chronic bacterial colonization [207-209].
Inhibition of IL-1β via antibody neutralization [204], small molecule NLRP3 inhibitors including
MCC950 [201], inhibition of the epithelial sodium channel-NLRP3 axis [210], or by the IL-1Ra
agonist Anakinra [207], ameliorates inflammasome-associated inflammation in models of CF via
negative regulation of NLRP3 activity. The results of these studies suggest that P. aeruginosa
exploits the NLRP3 inflammasome pathway to evade immune cell killing and that this
characteristic can be used as a therapeutic target. Clinical studies have shown that IL-1β
concentrations are significantly increased in the BAL and sputum of people with CF and are
negatively correlated to lung function [78-81]. In addition, IL-1β polymorphisms are linked with
disease severity in CF patients [206]. Taken together, the results of this study indicate that RTD-
1 may have therapeutic potential as an inflammasome-modulating agent for the CF lung disease.

In summary, RTD-1 reduced lung neutrophil infiltration and inflammatory cytokine
production in a murine model of chronic P. aeruginosa lung infection. This study also
establishes new findings that RTD-1 significantly downregulated several inflammasome-related
genes (NLRP3, MEFV, IL-1α, and IL-1β) in vivo. In vitro results confirm the dampening of
NLRP3 inflammasome activity of RTD-1 and suggest that the effect is possibly due to a
downstream effect of NF-κB inhibition. The anti-inflammatory properties of RTD-1 offer a
potential unique therapeutic approach to the treatment of CF lung disease.

116

Chapter 4: Evaluation of Host Defense Peptides as Antibiotic Zdjuvants
for Cystic Fibrosis Lung Disease
Introduction
Chronic lung infection and inflammation are the principal causes of morbidity and
mortality in individuals with cystic fibrosis (CF).[22] Cystic fibrosis (CF) is an autosomal
recessive disorder in which the patient’s lungs are highly susceptible to antibiotic-resistant
microbial infections. The majority of CF patients are chronically infected with P. aeruginosa and
Staphylococcus aureus, both of which are associated with a rapid decline of pulmonary function
and shortened survival in these individuals.[6, 7] Further complicating the treatment of CF-
related infections is the emergence of antibiotic resistance and evolving pathogen virulence. For
instance, multidrug-resistant bacteria cause lung infections in up to 30% of CF cases.[6] Such
chronic pathogens can establish biofilms within CF airways and survive this harsh environment
and exposure to intense antibiotic regimens due to biofilm impenetrability. Both planktonic and
sessile pathogens may also render antibiotics ineffective through the use of inactivating enzymes
and efflux pumps.[7] In 2019, antibiotic-resistant pathogens killed more people than malaria or
HIV, highlighting the need for novel antimicrobial agents.[89] Moreover, these pathogens cause
progressive loss of pulmonary function through the secretion of toxins and virulence factors,
including cell wall molecules that activate pro-inflammatory signaling.[7, 143] Decreased
pulmonary function results from a robust yet unproductive pro-inflammatory state during which
an intense neutrophilic migration and a sustained cyclic release of proteases lead to lung injury,
increased progressive loss of lung function, and decreased survival.[143] CF Clinical trials have
shown that glucocorticoids and high-dose ibuprofen slow the rapid decline in lung function,
117

although significant adverse effects limit their long-term clinical use.[6, 22, 90-93] The lungs of
CF patients are predisposed to infections, partly due to deficiencies in the innate immune
response. Innate defense peptides and proteins, including lactoferrin and lysozyme, are
inactivated in the acidic environment of the CF lung and are vulnerable to rapid proteolytic
cleavage.[14, 54, 211] To cope with these growing challenges, there is a critical need to identify
new safe and effective antimicrobial and anti-inflammatory therapies for treatment of CF lung
disease.

Host defense peptides (HDPs) are diverse short cationic amphiphilic peptides that are a
part of the innate immune function in essentially all species from bacteria to mammals.[99, 186]
There are several different types of host defense peptides in the lungs. The key HDPs in the
lungs are the defensins including alpha and beta defensins in humans and there are cathelicidins
including the one human cathelicidin, LL-37, and five porcine cathelicidins known as
protegrins.[99, 118-120] HDPs can modulate immunity depending on the context. They can aid
in facilitating phagocytosis to combat pathogens, but it can also recruit and active immune cells
to control inflammation.[103] During a pro-inflammatory state, they will reduce inflammation by
neutralizing pathogens lipopolysaccharide that stimulates a pro-inflammatory state.[99, 103]
These peptides also have various activities that work through ion and hydrophobic interactions to
kill all kinds of gram-positive and negative bacteria, fungi and viruses.[99, 101, 102] They can
target pathogens through membrane disruption to cause leakage of cytoplasmic contents or
depolarize the membrane In addition, they can “carpet” the membrane of a pathogen where a
certain concentration threshold of the peptides will cause membrane disintegration.[102, 103]
118

However, in CF, these HDPs are depleted as they are largely neutralized by several factors
including virulent factors, DNA, F-actin, and glycosaminoglycans.[26, 27] In addition, these
HDPS are inactivated due to the acidic and protease-rich CF environment.[14, 54] HDP
resistance is uncommon and utilizing them for antibiotic treatment can be beneficial for clearing
bacteria from CF airways.

For protegrin derivatives, researchers identified that a D-Proline—L-Proline template is
stable, where disulfide bridges were not necessary.[212] Using SAR analysis, cyclic 12-amino
acid PG-1 analogs with the D-Proline—L-Proline template were created to produce novel
antimicrobial agents. Two 12-amino acid peptides, L8-1 and L19-45, that had potent
antimicrobial activity were found to have broad-spectrum antimicrobial activity against
Pseudomonas aeruginosa, Staphylococcus aureus, E. coli, and Candida albicans.[212] These
peptides have two faces, an amphipathic and cationic one, and have low hemolytic activity (1%
at 100 µg/mL).[212, 213] The human cathelicidin LL-37 has been utilized to produce a novel
antimicrobial peptide 17BIPHE2.[127] 17BIPHE2 is designed on GF-17, the active region of
LL-37, and contains three D-leucine amino acids that distort the regular helical structure of the
peptide to improve stability to a select set of proteases. Also, 17BIPHE2 contains two
biphenylalanine amino acids to correct a hydrophobic defect to increase its antimicrobial
properties.[127] This HDP has been found to have potent antibiofilm and antimicrobial activity
against P. aeruginosa and S. aureus.[128, 129] In all, 17BIPHE2 shows promise as a potential
novel therapeutic for CF bacterial infection.
119

As these HDPs are known to combat infection, another well-known and recognized
characteristic is their ability to synergize with conventional antibiotics as antibiotic adjuvants.
These adjuvants are categorized into two different classes. Class I adjuvants are separated into
two groups, IA and IB, that target active or passive antibiotic resistance mechanisms,
respectively. Class IA adjuvants include β-lactamase inhibitors (e.g., clavulanic acid and
tazobactam) that inactivate β-lactamase and prevent them from cleaving β-lactam antibiotics.
Loperamide acts as a class IB adjuvant with tetracycline as it can alter the Gram-negative
bacterial proton motive force and consequently, cause the pathogen to become susceptible to
tetracycline antibiotics.[214] As HDPs work through ion and hydrophobic interactions, they may
act as class IB adjuvants and target pathogens indiscriminately and inhibit passive antibiotic
resistance mechanisms.[112] HDPs can also act as class IA adjuvants as they are found to
prevent and eradicate established biofilms in vitro.[110] While antibiotics elicit bacterial stress
pathways such as SOS and rpoS to increase bacterial mutagenesis, HDPs do not.[103] Also, class
II adjuvants can aid antibiotics by reinforcing immunomodulatory pathways to neutralize and
clear pathogens. Streptazolin is one known class II antibiotic adjuvant as it can enhance
macrophage activity by upregulating NF B signaling. HDPs can also act as class II antibiotic
adjuvants as they are immunomodulatory peptides.[99] Evaluation of various peptides for their
synergistic properties in vivo with antibiotics show promise. In one study, HDPs were observed
to decrease abscess size and improve clearance of bacteria in various in vivo abscess murine
models.[113]
120

Figure 4-1. The classes of Antibiotic Adjuvants. Antibiotic adjuvants can assist antibiotics by
various mechanisms including inhibiting active antibiotic mechanisms (I.A) including efflux
pumps and β-lactamases, passive antibiotic mechanisms (I.B) such as bacterial membranes and
enhancing immunomodulatory properties (II) by improving macrophage phagocytosis. Figure
adapted from Wright (2016). [214]

In the current report, we sought to examine the therapeutic potential of host defense
peptides to act as antibiotic adjuvants. We screened the in vitro antimicrobial activity of host
defense peptide and antibiotic combinations by evaluating the susceptibility of P. aeruginosa
laboratory and clinical isolates. We observed many synergistic combinations but wanted to
121

examine the therapeutic potential of Protegrin 1 and RTD-1 with colistin as most HDPs were
synergistic with colistin. Time-kill kinetic assays of HDP-antibiotic combinations against the
laboratory strain PAO1 to validate the increased killing potential of RTD-1 and colistin
combination. We then further sought out to examine the antimicrobial and anti-inflammatory
effects of the HDP-antibiotic combination through an in vivo murine P. aeruginosa pneumonia
model. These findings indicate that RTD-1 and colistin synergistically combat P. aeruginosa by
lowering pro-inflammatory cytokines/chemokines, leukocyte counts and lung bacterial burden.
Overall, these findings support further investigation of the HDP-antibiotic combination as a
potential therapeutic for CF-associated lung disease.

122

Materials and Methods
Broth Microdilution Assays
Antipseudomonal activity was evaluated using broth microdilution assays against a
laboratory strain of P. aeruginosa (PAO1) and 18 P. aeruginosa clinical isolates from patients
with cystic fibrosis at the Keck Medical Center of the University of Southern California. Thirty
percent of the P. aeruginosa clinical isolates were multi-drug resistant (MDR) and 65% of them
were mucoid. Broth microdilution assays were utilized to evaluate the minimum concentration to
inhibit bacterial growth (MIC) of various host defense peptides according to the CLSI (formerly
NCCLS) guidelines with modifications as described below.[121] Briefly, the assay utilized cation-
adjusted Mueller-Hinton broth (CAMHB) (Becton, Dickinson and Company, Franklin Lakes, NJ)
which was prepared according to the manufacturer's instructions. Host defense peptide solutions
were prepared as 10× solutions in 0.01% acetic acid. Host defense peptides were serially diluted
two-fold at concentrations ranging from 25 - 0.05 µM that contained CAMHB and 0.04% bovine
serum albumin (BSA) in polypropylene microtiter plates (Corning, Corning, NY). All bacteria
were incubated overnight at 37°C at 200 rpm in CAMHB and bacterial inoculum was adjusted
with additional CAMHB to 0.5 McFarland standard through spectrophotometry at 600 nm.
Bacteria was then further adjusted 1:100 in CAMHB and dispensed into 96-well polypropylene
microtiter plates (Corning, Corning, NY) in triplicate (corresponding to 0.5–1 × 10
5
CFU/well) and
incubated for 24 h to determine the MIC. Antibiotics including Colistin sulphate (Sigma-Aldrich,
St Louis, MO), ceftazidime, tobramycin, imipenem, and azithromycin and host defense peptides
including Protegrin-1, RTD-1, L8-1, L19-45, GF17, and 17BIPHE2 were used to evaluate their
antimicrobial activities.
123

Synergy Assays
Antibiotics and host defense peptides were used in combination to assess their synergistic
activities against P. aeruginosa by the checkerboard method. Briefly, host defense peptide
solutions were prepared as 10× solutions in 0.01% acetic acid. Host defense peptides were serially
diluted two-fold that contained CAMHB and 0.04% bovine serum albumin (BSA) in
polypropylene microtiter plates (Corning, Corning, NY). Antibiotics were serially diluted two-fold
that contained CAMHB. Overnight bacterial cultures were adjusted to 0.5 McFarland standard.
Combinations of antibiotics and host defense peptides were created and bacteria was further
diluted to 10
5
CFU in each well and incubated for 24 hours to determine the fractional inhibitory
concentration index (FICI). The indexes suggested whether a combination is synergistic (FICI ≤
0.5) additive (0.5 < FICI < 2), no interaction (FICI 2.0). Synergistic combinations were further
evaluated with clinical isolates of P. aeruginosa (n = 18).

Time kill assay
We determined the bactericidal kinetics of our compounds separately and in tandem against
P. aeruginosa (PAO1) through broth macrodilution using CAMHB as described previously [135].
Briefly, bacteria inoculums of 1 × 10
5
CFU/mL were exposed to 0.25× MIC of RTD-1, Colistin,
Protegrin-1, RTD-1 and Colistin or Protegrin-1 and Colistin. During incubation at 37°C, aliquots
of the bacterial inoculum were taken at 0, 1, 2, 4, 6, 8, 12, 16, and 24 h post treatment. The samples
124

were serially diluted two-fold and plated onto tryptic soy agar. The plates were incubated at 37°C
for 16 hours and CFUs were counted.

P. aeruginosa-induced Acute Pneumonia Murine Model
All animal experiments were evaluated and approved by the Institutional Animal Care
and Use Committee at the University of Southern California (Protocol 20936). CD-1 mice
(Charles River Laboratories, Wilmington, MA, USA) were housed three in a cage with standard
chow and water ad libitum in a pathogen-free environment at 22-24°C. Acute pneumonia
infection was established via intubation-mediated intratracheal instillation of 1 × 10
7
CFU of log-
phase P. aeruginosa (PAO1) in 50 μL of PBS. Mice were sedated with isoflurane 2 hours post
infection and treated with either 5 mg/kg RTD-1 intranasal, 10 mg/kg Colistin intraperitoneal, 5
mg/kg RTD-1 and 10 mg/kg Colistin intraperitoneal or vehicle control.

After six hours post infection (4 hours post treatment), mice were euthanized. Lungs were
collected and processed to determine CFU counts and the number of white blood cells in the
lungs as conducted previously. Briefly, the lungs were washed three times with 0.8 mL of PBS
containing mini protease inhibitors (cOmplete™, Millipore Sigma, Burlington, MA, USA).
White blood cells from the bronchoalveolar lavage fluid (BALF) are isolated, stained and
quantified with Turk Blood Diluting Fluid (Ricca Chemical Company, Arlington, TX, USA) and
a hemocytometer. Diff-Quick cell staining solution (Polysciences, Warrington, PA, USA) was
125

used to differentiate the white blood cells. Luminex Bead Array (Millipore-Sigma, Burlington,
MA, USA) were used to determine the cytokine production from the BALF.

Bacterial burden was quantified by serially diluting the BALF and homogenized lung
samples two-fold and plated onto tryptic soy agar. The plates were incubated at 37°C for 16
hours and CFUs were counted. The summation of the BALF and lung homogenates was found to
be the total bacterial lung burden. Figure 4-1 depicts the methods for the study.

Figure 4-2. Acute P. aeruginosa pneumonia model. CD-1 mice were infected with P.
aeruginosa PAO1 and treated with HDPs and/or antibiotics two hours post infection. Six hours
post infection, the mice were euthanized and cytokines, histology, leukocyte counts, and
bacterial burden were assessed.
126

Results
Screening the Synergistic Properties of HDPs with Conventional Antibiotics
To evaluate whether host defense peptide can be used as antibiotic adjuvants, we
screened their ability to synergize with conventional small molecular antibiotics use for P.
aeruginosa. We employed a checkerboard synergy method with modifications for host defense
peptides.[121] We assessed six different peptides, PG-1, RTD-1, L8-1, L19-45, GF-17 and
17BIPHE2 in combination with five different conventional antibiotics (i.e., colistin, ceftazidime,
tobramycin, imipenem, and azithromycin). The antimicrobial activities of these compounds are
summarized in Table 4-1. We noted that there was synergy for most of these host defense
peptides with colistin (Tables 4-2 and 4-3). Only L19-45 was found have additive activity with
colistin, indicating there was potent synergistic properties when combining colistin with these
host defense peptides. In addition, imipenem revealed synergism with three HDPs, L8-1, L19-45
and 17BIPHE2. The combination of imipenem with either of the two PG-1 derivatives, L8-1 or
L19-45, were found to have most potent synergistic activity against P. aeruginosa. Lastly,
ceftazidime was found to have additive activity with five of the HDPs, indicating that these
HDPs have a moderate potential to increase the bactericidal activity of P. aeruginosa.
Tobramycin, and azithromycin were found to have little-to-no antimicrobial shift against P.
aeruginosa when HDPs were used as adjuvants. As majority of the HDPs were synergistic with
colistin, we decided to further elucidate the activity with the two most potent combinations,
RTD-1 and PG-1 with colistin. (Tables 4-2 and 4-3) We did not evaluate the synergistic
capabilities of GF-17 and colistin due to the low antimicrobial activity of GF-17 compared to the
other HDPs. (Table 4-1)
127

Table 4-1. Antimicrobial activities of antimicrobial agents. (A) Peptides and (B) antibiotics
were evaluted for their antimicrobial properties according to CLSI guidelines

Table 4-2. Fractional inhibitory concentration indices of HDP-antibiotic combinations. Six
different HDPs were tested with five different antibiotics against P. aeruginosa PAO1.
Checkerboard assays were used to evaluate whether a combination is synergistic (green),
additive (yellow) and no difference (orange) according to the FICI of the combination against the
laboratory strain.

A. B.
Peptide MIC (μg/ml) Antibiotic MIC (μg/ml)
PG-1 1 Colistin 1
RTD-1 32 Ceftazidime 1
L8-1 20 Tobramycin 0.125
L19-45 1.25 Imipenem 4
GF-17 100 Azithromycin 64
17BIPHE2 6.25
Colistin Ceftazidime Tobramycin Imipenem Azithromycin
RTD-1 0.25 1.25 1.25 2.00 1.50 Legend
PG-1 0.38 0.75 0.75 1.50 0.75 Synergistic
L8-1 0.50 0.63 2.00 0.13 2.00 Additive
L19-45 1.00 0.75 2.00 0.19 2.00 No Difference
GF-17 0.31 1.50 2.00 0.56 2.00
17BIPHE2 0.50 1.00 2.00 0.50 2.00
128

Table 4-3. Fractional inhibitory concentrations of each combination. Six different HDPs
were tested with five different antibiotics against P. aeruginosa PAO1. Checkerboard assays
were used to evaluate whether a combination is synergistic, additive or had no effect. MIC A is the
MIC of the peptide in the presence of an antibiotic in the checkerboard assay. MICB is the MIC
of the antibiotic in the presence of a peptide in the checkerboard assay. MICpeptide and MICAbx are
the MICs observed from broth microdilution assays found in Table 4-1.

Peptide Antibiotic MIC
A
MIC
B
MIC
A
/MIC
Peptide
MIC
B
/MIC
Abx
FICI Outcome
Colistin 4.00 0.25 0.13 0.13 0.25 Synergistic
Ceftazidime 32.00 0.25 1.00 0.25 1.25 Additive
Tobramycin 8.00 0.13 0.25 1.00 1.25 Additive
Imipenem 32.00 4.00 1.00 1.00 2.00 No Difference
Azithromycin 32.00 16.00 1.00 0.50 1.50 Additive
Colistin 0.25 0.25 0.25 0.13 0.38 Synergistic
Ceftazidime 0.50 1.00 0.25 0.50 0.75 Additive
Tobramycin 0.25 0.06 0.25 0.50 0.75 Additive
Imipenem 0.50 2.00 1.00 0.50 1.50 Additive
Azithromycin 0.25 16.00 0.50 0.25 0.75 Additive
Colistin 5.00 0.25 0.25 0.25 0.50 Synergistic
Ceftazidime 10.00 0.13 0.50 0.13 0.63 Additive
Tobramycin 20.00 0.13 1.00 1.00 2.00 No Difference
Imipenem 1.25 0.25 0.06 0.06 0.13 Synergistic
Azithromycin 20.00 64.00 1.00 1.00 2.00 No Difference
Colistin 0.63 0.50 0.50 0.50 1.00 Additive
Ceftazidime 0.63 0.25 0.50 0.25 0.75 Additive
Tobramycin 1.25 0.13 1.00 1.00 2.00 No Difference
Imipenem 0.08 0.50 0.06 0.13 0.19 Synergistic
Azithromycin 1.25 64.00 1.00 1.00 2.00 No Difference
Colistin 6.25 0.25 0.06 0.25 0.31 Synergistic
Ceftazidime 100.00 0.50 1.00 0.50 1.50 Additive
Tobramycin 100.00 0.13 1.00 1.00 2.00 No Difference
Imipenem 6.25 2.00 0.06 0.50 0.56 Additive
Azithromycin 100.00 64.00 1.00 1.00 2.00 No Difference
Colistin 1.57 0.25 0.25 0.25 0.50 Synergistic
Ceftazidime 3.13 0.50 0.50 0.50 1.00 Additive
Tobramycin 6.25 64.00 1.00 1.00 2.00 No Difference
Imipenem 1.57 1.00 0.25 0.25 0.50 Synergistic
Azithromycin 6.25 64.00 1.00 1.00 2.00 No Difference
PG-1 RTD-1 L8-1 L19-45 GF-17 17BIPHE2
129

Table 4-4 Antimicrobial activities of antimicrobial agents against P. aeruginosa clinical
isolates. Peptides and antibiotics were evaluted for their antimicrobial properties according to
CLSI guidelines. These MICs are referenced in checkerboard synergy assays in Table 4-5, and 4-
6.

Clinical Isolates RTD-1 PG-1 Colistin Ceftazidime Tobramycin Imipenem
446055 64.00 0.50 1.00 32.00 2.00 16.00
254831 64.00 0.50 0.50 32.00 8.00 64.00
900719 64.00 1.00 1.00 2.00 16.00 2.00
200174 64.00 0.50 0.50 16.00 16.00 32.00
219686 32.00 1.00 1.00 2.00 2.00 32.00
262309 32.00 1.00 1.00 128.00 8.00 32.00
119133 32.00 0.50 0.50 64.00 2.00 32.00
167482 32.00 1.00 0.50 32.00 2.00 16.00
95302 32.00 16.00 0.25 2.00 2.00 8.00
678447 32.00 1.00 0.13 128.00 4.00 32.00
298473 64.00 0.50 0.50 2.00 0.50 0.50
618154 64.00 1.00 0.50 2.00 0.50 1.00
857950 64.00 2.00 0.50 2.00 2.00 16.00
861684 64.00 1.00 0.25 128.00 2.00 16.00
894496 64.00 1.00 0.50 1.00 0.50 1.00
815158 64.00 0.50 1.00 1.00 0.50 0.25
844265 64.00 1.00 0.25 2.00 0.50 0.50
774248 128.00 0.50 1.00 32.00 0.13 8.00
MIC (μg/ml)
130

Assessing the Synergistic Capabilities of RTD-1/PG-1 with Colistin against CF P.
aeruginosa Clinical Isolates
To validate the synergistic activity of RTD-1 and PG-1 with colistin, we treated 18
clinical P. aeruginosa isolates from Keck Hospital with each combination in checkerboard
assays (Tables 4-4, 4-5, 4-6, 4-7). We determined that 30% of the P. aeruginosa clinical isolates
are multi-drug resistant (MDR) and 65% of them are alginate-producing (mucoidal). In this
study, nine out of the 18 clinical isolates were synergistic (Tables 4-5 and 4-6). The other nine
checkerboard assays using clinical strains were found to be additive, suggesting that there is a
greater potentiation of colistin bactericidal activity with PG-1. For RTD-1, the combination with
colistin was affirmed to be synergistic against 17 of the 18 clinical isolates (Tables 4-4 and 4-6).
Only one assay determined that the combination was additive, indicating that RTD-1 and colistin
have greater synergistic properties compared to PG-1 and colistin.

131

Table 4-5. Checkerboard Assay Results of RTD-1 and colistin combinations against CF
clinical isolates. Checkerboard assays were used to evaluate whether a combination is
synergistic, additive or had no effect. MICA is the MIC of RTD-1 in the presence of colistin in
the checkerboard assay. MICB is the MIC of colistin in the presence of RTD-1 in the
checkerboard assay. MICRTD-1 and MICColistin are the MICs observed in broth microdilution
assays found in Table 4-4.

Clinical Isolate MIC
A
MIC
B
MIC
A
/MIC
RTD-1
MIC
B
/MIC
Colistin
FICI Outcome
446055 16.00 0.25 0.25 0.25 0.50 Synergistic
254831 8.00 0.06 0.13 0.13 0.25 Synergistic
900719 8.00 0.25 0.13 0.25 0.38 Synergistic
200174 8.00 0.06 0.13 0.13 0.25 Synergistic
219686 4.00 0.25 0.13 0.25 0.38 Synergistic
262309 2.00 0.25 0.06 0.25 0.31 Synergistic
119133 4.00 0.13 0.13 0.25 0.38 Synergistic
167482 1.00 0.13 0.03 0.25 0.28 Synergistic
95302 4.00 0.06 0.13 0.25 0.38 Synergistic
678447 4.00 0.02 0.13 0.13 0.25 Synergistic
298473 4.00 0.13 0.06 0.25 0.31 Synergistic
618154 8.00 0.13 0.13 0.25 0.38 Synergistic
857950 8.00 0.13 0.13 0.25 0.38 Synergistic
861684 8.00 0.06 0.13 0.25 0.38 Synergistic
894496 4.00 0.13 0.06 0.25 0.31 Synergistic
815158 4.00 0.25 0.06 0.25 0.31 Synergistic
844265 4.00 0.06 0.06 0.25 0.31 Synergistic
774248 64.00 0.25 0.50 0.25 0.75 Additive
132

Table 4-6. Checkerboard Assay Results of RTD-1 and colistin combinations against CF
clinical isolates. Checkerboard assays were used to evaluate whether a combination is
synergistic, additive or had no effect. MICA is the MIC of PG-1 in the presence of colistin in the
checkerboard assay. MICB is the MIC of colistin in the presence of PG-1 in the checkerboard
assay. MICPG-1 and MICColistin are the MICs observed in broth microdilution assays found in
Table 4-4.

Clinical Isolate MIC
A
MIC
B
MIC
A
/MIC
PG-1
MIC
B
/MIC
Colistin
FICI Outcome
446055 0.13 0.50 0.25 0.50 0.75 Additive
254831 0.06 0.06 0.13 0.13 0.25 Synergistic
900719 0.13 0.13 0.13 0.13 0.25 Synergistic
200174 0.50 0.25 1.00 0.50 1.50 Additive
219686 0.25 0.25 0.25 0.25 0.50 Synergistic
262309 0.25 0.25 0.25 0.25 0.50 Synergistic
119133 0.06 0.13 0.13 0.25 0.38 Synergistic
167482 0.50 0.13 0.50 0.25 0.75 Additive
95302 4.00 0.13 0.25 0.50 0.75 Additive
678447 0.25 0.03 0.25 0.25 0.50 Synergistic
298473 0.25 0.13 0.50 0.25 0.75 Additive
618154 0.50 0.25 0.50 0.50 1.00 Additive
857950 0.50 0.13 0.25 0.25 0.50 Synergistic
861684 0.06 0.13 0.06 0.50 0.56 Additive
894496 0.13 0.13 0.13 0.25 0.38 Synergistic
815158 0.13 0.25 0.25 0.25 0.50 Synergistic
844265 0.50 0.06 0.50 0.25 0.75 Additive
774248 0.50 0.13 1.00 0.13 1.13 Additive
133

Table 4-7. Complied Checkerboard assays with P. aeruginosa clinical isolates. PG-1 and
RTD-1 were assessed for their ability to act as antibiotic adjuvants with colistin. PG-1 + colistin
was synergistic with 50% of the clinical isolates while RTD-1 + colistin was 94% synergistic.

Bactericidal Activity of RTD-1 and PG-1 combinations with Colistin
To determine whether the antimicrobial activity of RTD-1 and PG-1 in combination with
colistin is bactericidal, we utilized a time-kill kinetic assay against P. aeruginosa PAO1 (Fig. 4-
2). We accomplished this using antibiotic and HDP concentrations of 0.25 x MIC alone or
together. The results indicated that 0.25 x MIC of each HDP did not affect the bacterial growth
of PAO1 significantly. Colistin however, caused a rapid killing kinetic at 0.25 x MIC against P.
aeruginosa PAO1 with greater than 3 log10 CFU/mL. Bactericidal activity was seen up to 8 hours
post treatment, where bacterial growth was comparable to the control by 24 hours. Treatment
with PG-1 and colistin, had a similar effect, but had an extended killing period of up to 12 hours.
The combination of RTD-1 and Colistin was shown to have the highest killing activity as 0.25 x
MIC leading to no observable bacterial counts by 4 hours post treatment (Fig. 4-2). As a result of
Synergistic Additive Indifferent Antagonistic
PG-1 + Colistin 9 (50) 9 (50) 0 (0) 0 (0)
RTD-1 + Colistin 17 (94) 1 (6) 0 (0) 0 (0)
Number of isolates (%) according to FIC Index*
Drug Combination
*Synergy was defined as a FIC index ≤0.5, additive as >0.5 and ≤1.0, indifference as
>1.0 to ≤2.0 and antagonism as >2.0.
134

the checkerboard assays and bactericidal activity, we decided to move forward with RTD-1 and
colistin for our in vivo acute pneumonia model.

Figure 4-3. Time-kill kinetics of RTD-1- and PG-1-colistin combination. Bactericidal
activity of 0.25× MIC of PG-1, RTD-1, colistin, PG-1 + colistin, and RTD-1 + colistin against
log-phase P. aeruginosa PAO1. P. aeruginosa was grown to log phase, and aliquots were treated
with compounds at incremental concentrations relative to MICs, from to 0.25 x MIC to 16 x MIC

135

In Vivo Efficacy of RTD-1 and Colistin Combination in an Acute P. aeruginosa Pneumonia
Murine Model
We have established an in vivo model of acute pneumonia to assess the synergistic
capabilities of RTD-1 and colistin. We examined the synergistic capacity of RTD-1 and colistin
combination treatment through its influence on white blood cell infiltration in the lungs and lung
bacterial burden (Figure 4-3). RTD-1 treatment alone was found to significantly decrease
neutrophil invasion (p < 0.0001); however, total lung bacterial burden was not reduced. Our
observations in the acute pneumonia model are in line with RTD-1 treatment observed in our
chronic P. aeruginosa infection model in Chapter 3. Colistin treatment was found to significantly
reduce neutrophil infiltration (p = 0.0009) and bacterial burden (p = 0.0272). We noted an
approximate 0.9-log reduction in total bacterial burden with colistin treatment compared to the
control mice. For the RTD-1 and colistin treatment mouse group, we also observed a significant
decrease in neutrophil infiltration (p < 0.0001) and bacterial burden (p = 0.0047) compared to the
control group. Even though the dual therapy further reduced bacterial burden by approximately
1.32-log, it was not significantly different from the colistin treatment group (p = 0.89). In
addition, we do see a trend that the dual therapy did further reduce leukocyte counts, but it was
not significant (p = 0.3063).
136

Figure 4-4. Pathophysiological Studies of Acute Pneumonia Pseudomonas aeruginosa Lung
Infection in Mice Treated with RTD-1 and colistin combination. Acute pneumonia infection
was established via intubation-mediated intratracheal instillation of 1 × 10
7
CFUs of P.
aeruginosa PAO1. Two hours post infection mice were treated with either 10 mg/kg colistin IP,
5 mg/kg RTD-1 IN, or 10 mg/kg colistin IP and 5 mg/kg RTD-1 IN and euthanized by six post
infection. Effects of treatment on: (A) lung bacterial burden, (B) total immune cell counts, and
(C) differential cell counts were evaluated

137

Discussion
The combination of HDPs with conventional antibiotics led to synergistic properties in
vitro and in vivo. In this regard, HDPs have both immunomodulatory and antimicrobial
capacities that can act as antibiotic adjuvants. We screened six HDPs with five antibiotics and
observed that many of the antimicrobial peptides were synergistic when combined with either
colistin or imipenem. As majority of the peptides were synergistic with colistin. This was further
validated using clinical isolates when combining colistin with RTD-1 or PG-1. In this analysis,
RTD-1 containing combinations were the most potent and consistent synergistic. No bacteria
growth was detected after four hours in our time kill kinetics studies. PG-1 was not as potent as
RTD-1, where bacterial resistance was detected allowing them to regrow even in the presence of
PG-1. To further determine the impact of RTD-1 and colistin, we employed the combination
using an in vivo acute pneumonia model. Treatment with RTD-1 and colistin two-hours post
infection significantly reduced white blood cell count and lung bacterial burden in comparison to
the control group, supporting our hypothesis that HDPs can act as Class IB and II antibiotic
adjuvants.

HDPs as antibiotic adjuvants have been well recognized for their ability to synergize with
conventional antibiotics. We demonstrate that HDPs can boost the antimicrobial activity of
various antibiotics as several HDPs were found to have additive or synergistic properties with
antibiotics. We hypothesize that the RTD-1 and colistin synergy could be related to previous
reports with LL-37 and colistin against MDR-E. coli.[112] We hypothesize that the improved
bactericidal activity of the combination is due to colistin’s ability to disrupt the outer membrane
138

by binding to LPS while RTD-1 acts as a Class I adjuvant that permeates the lipid outer and
inner membrane through mechanisms that do not involve LPS. PG-1, however, is known to
target the LPS of bacteria in a similar manner to colistin, which explains its lower potentiation to
increase bactericidal activity as an adjuvant compared to RTD-1. Thus, RTD-1 further research
on the synergistic capabilities with other polymyxin antibiotics will be explored. In addition,
three HDPs were found to have synergistic potential with imipenem. The two PG-1 derivatives,
L8-1 and L19-45 were found to have the highest synergistic potential in the panel conducted.
Colistin has been found to have synergy with imipenem and other carbapenems against various
pathogens in the literature. We can postulate that the mechanism in which PG-1 and its
derivatives were found to be synergistic with imipenem could be similar. Future research into the
efficacy of HDPs with carbapenem will be conducted. All tested HDPs were found to have
additive antimicrobial activity with ceftazidime, and most were synergistic or additive with
imipenem and colistin. The improved activity of these antibiotics may be due to destabilization
of pathogens’ outer membrane.[109] Thus, as bacteria reduce membrane permeability to prevent
entry of antibiotics, HDPs are a novel method to improve antibiotic activity of various antibiotics
as class IB antibiotic adjuvants. Also, HDPs can enhance bactericidal activity as class II
adjuvants by boosting the immune response via modulating cytokine/chemokine production and
enhancing macrophage and neutrophil phagocytosis.[109] We indicate that RTD-1 and colistin
therapy can significantly decrease leukocyte counts compared to the control group. Even though
there was a further decline in leukocytes with both compounds, it was not significantly different
from the colistin treatment group. We believe that the moderate reduction in leukocytes is due to
139

colistin’s ability to inhibit LPS-induced inflammation, while RTD-1 acts intracellularly to inhibit
MAPK phosphorylation, NF-κB signaling and TACE/ADAM17 activation.

We plan to further evaluate the mechanism of action. As colistin’s permeability
mechanism is understood to be caused by LPS binding, we will evaluate the mechanism of action
of RTD-1 permeability. Using RTD-1 specific antibodies, we can pull down proteins and lipids
from bacterial lysates and recognize them through mass spectrometry. To mimic inflammation
and infection in CF, we will use the chronic P. aeruginosa lung infection model we used in
chapter 2. There are guidelines that state that CF safety and efficacy should be determined
through this model as it is a great predictor for CF lung therapies. Amelubant, a drug that failed
clinical trials, due to significantly increased risk of adverse pulmonary events was found to have
negative effects in this model. Mice had significantly higher bacteremia rates and lung
inflammation compared to the placebo treated animals, indicating the value of this model to
establish the efficacy and safety of CF lung treatments.[215]

Overall, our results indicated that both RTD-1 and colistin have an enhanced protective
effect on pulmonary infection and inflammation by reducing bacterial burden, BALF white
blood cell counts, and pro-inflammatory cytokines/chemokines compared to colistin and RTD-1
monotherapy. We demonstrate that the dual therapy holds great promise for development and
exploration of antibiotic adjuvants to combat CF lung disease.

140

Chapter 5: Summary and Future Directions
The principal aim of this thesis is to investigate the therapeutic potential of HDPs for CF
lung disease. As “jacks of all trades,” HDPs have diverse properties that can be explored as
therapies for various diseases.[99, 185] CF is an autosomal recessive disorder characterized by a
vicious cycle of chronic pulmonary microbial infection, endobronchial airway obstruction, and
neutrophil-dominated inflammation.[3, 22] As CF airways have diminished HDPs, replenishing
the airways with active and stable HDPs is essential to treat the disease.[14, 54] This thesis
sought to examine the antimicrobial, immunomodulatory, and antibiotic adjuvant properties of
HDPs for use in CF lung disease.

In chapter 2, we hypothesized that linear HDPs can be engrafted onto a cyclotide scaffold
to overcome a linear HDP limitation, proteolytic degradation. We demonstrated that engrafting a
linear HDP into a cyclotide can stabilize its structure and retain its antimicrobial activity. In vivo
peritonitis studies demonstrated that our “proof-of-concept” cyclotide was efficacious to improve
mice survival comparable to colistin treatment. Future iterations of the PG-1 cyclotide could be
synthesized to support our hypothesis further. Cyclotides contain multiple loops that are
amenable to variation through molecular evolution to enhance their antimicrobial activity and
safety profile.[148, 174-177] Hence, more active cyclotides could be produced by modifying
adjacent loops near loop 6 in MCo-PG2, mainly loops 1, 3, and 5, to improve safety and efficacy.
To screen the new cyclotides, we could determine their safety through hemolysis assays and
cytotoxicity assays, while in vitro antimicrobial activity will be assessed through broth
141

microdilution assays, similar to the methods used in Chapter 2. Also, we could affirm our
hypothesis that HDP cyclotides can be used to treat CF lung disease by evaluating the newly
synthesized cyclotides for their immunomodulatory properties, as PG-1 is known to inhibit LPS-
induced inflammation. LPS-stimulated THP-1 macrophages could be treated with our cyclotides
to assess their potential to modulate inflammation through cytokine production and transcription
analysis. Once we find a safe and efficacious cyclotide, we could assess its ability to modulate
inflammation and eradicate the infection in an acute P. aeruginosa pneumonia model, like
Chapter 4. It is a common in vivo screening model for novel lung antimicrobial and anti-
inflammatory compounds.

In chapter 3, we elucidated the immunomodulatory activity of RTD-1 in a chronic P.
aeruginosa infection model. PG-1 and MCo-PG1.2 were not assessed due to their safety
limitations. Anti-inflammatory effects were analyzed by evaluating white blood cell counts and
cytokine/chemokine secretions in the BALF. RTD-1 led to a reduction in various
cytokines/chemokines and leukocyte counts, but not bacterial burden. RTD-1 also downregulates
several inflammasome-related genes in our microarray. To determine the validity of our
microarray, we investigated the effects of RTD-1 in vitro. Inflammasome activation,
inflammasome-associated gene expression, and cytokine production were inhibited by RTD-1.
These studies further support the literature that RTD-1 exhibits diverse anti-inflammatory
activities.[130, 137] Specifically, our new findings support our hypothesis that HDPs have
immunomodulatory activities as RTD-1 dampens NLRP3 inflammasome activation and related
cytokines, most likely through inhibition of NF-κB signaling. Overall, these findings support
142

further investigation of this immunomodulatory peptide as a promising therapeutic for treating
CF-associated lung disease. While many of the intracellular targets of RTD-1 are known, the
upregulation of FKBP5 by RTD-1 is fascinating. The molecular cochaperone is a known
modulator of glucocorticoid receptor sensitivity along with a host of other cellular responses. To
further support our hypothesis, RTD-1’s action on the glucocorticoid receptor and whether a
downstream effect may be therapeutically beneficial for CF lung disease could be elucidated.
Even though we have shown that RTD-1 has anti-inflammatory activity, we see that RTD-1 does
not significantly reduce bacterial burden, which may be due to the challenges in using P.
aeruginosa embedded agar. To determine RTD-1’s efficacy as an antimicrobial agent against P.
aeruginosa, other models, including sepsis peritonitis and acute pneumonia models, could be
utilized to explore its specific antipseudomonal activity.

Previous literature indicated that HDPs can enhance the bactericidal activity of
conventional antibiotics.[99, 109] Chapter 4 examined whether six HDPs can synergize with five
different antibiotics. As Class IB adjuvants, we identified that HDPs could synergize with
colistin and imipenem. These observations are supported as recent reports demonstrate LL-37
can synergize with colistin.[112] Also, the literature shows that colistin can synergize with
imipenem to combat various pathogens as it permeabilizes the membrane of pathogens in a
manner similar to HDPs.[216] Our studies found that RTD-1 and colistin are highly synergistic
in vitro and in vivo. We did not assess the in vivo activity of the PG-1 combination due to its
lower synergistic activity with the clinical strains, lower bactericidal activity, and higher toxicity
profile compared to the RTD-1 combination. Acute P. aeruginosa pneumonia demonstrated that
143

the RTD-1 and colistin combination therapy is significantly more efficacious than the vehicle
control. It moderately reduces bacterial burden compared to colistin; however, it was
insignificant. The addition of RTD-1 enhances immunomodulation and reduces leukocyte count.
As a result, we have explored and supported that HDPs can act as Class IB and II antibiotic
adjuvants, specifically for CF lung disease. We can further improve our hypothesis by
elucidating how RTD-1 acts to permeate lipid membranes. Also, RTD-1 combinations with other
polymyxin derivatives will be used to assess its potential to synergize with polymyxins. Other
combinations that could be explored include PG-1 derivatives with imipenem, as our studies
indicate that PG-1 derivatives have high synergistic capabilities with imipenem. We hypothesize
that the PG-1 derivatives depolarize the outer membrane, allowing carbapenems including,
imipenem, to enter easily and halt cell wall synthesis. We could test other carbapenems with the
PG-1 derivatives and assess the most potent combination in the acute pneumonia model in a
similar manner as outlined in Chapter 4. We could further affirm our hypothesis by assessing the
combinations’ antimicrobial and antibiofilm activity in artificial sputum. P. aeruginosa infection
mainly exists in biofilms and microaerophilic conditions in chronic CF lung infections.[217]
Biofilms in sputum constitute a significant challenge as antibiotics do not distribute well in
sputum, limiting their ability to eradicate the infection. Several studies have shown that HDPs
can inhibit biofilm formation or eradicate biofilms, which suggests that they can improve the
antibiotic activity of these antibiotics.[99, 109] Artificial sputum is a medium that simulates CF
sputum as it consists of amino acids, mucin, and free DNA, which allows P. aeruginosa to
mimic growth in CF airways.[217] We expect to observe antibiotic adjuvant properties of HDPs
in this medium, where the minimum inhibitory concentrations of antibiotics are reduced four-
144

fold. Lastly, efficacy studies determining the potential of RTD-1 to act as an antibiotic adjuvant
with colistin in a chronic P. aeruginosa lung infection model could be performed as this model
can recapitulate chronic lung disease pathology, similar to CF.[192, 215]

In all, we have established and supported evidence that HDPs can be therapeutic leads as
antimicrobial, immunomodulatory, and antibiotic adjuvants for CF lung disease.

145

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Abstract (if available)

Abstract Cystic fibrosis (CF) is the most common autosomal recessive genetic disorder among Caucasians and leads to chronic pulmonary infection and inflammation, the principal causes of CF morbidity and mortality. The central feature of CF is lung disease which includes airway obstruction, neutrophil-dominated inflammation, and microbial infection that leads to progressive loss of lung function and eventual death. An increasing prevalence of multidrug-resistant (MDR) bacteria, including MDR Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus, pose life-threatening risks for CF patients, as these pathogens are associated with an accelerated decline in lung function and shortened survival. As these pathogens are increasing worldwide, the lack of antimicrobial agents to combat them are diminishing. Patients with CF are also predisposed to lung infection due to their mutation in the CFTR gene, which leads to inactivation of innate antimicrobial proteins and host defense peptides (HDPs) in the airways and the inability to clear pathogens. Due to the lack of safe and effective therapeutic options to alleviate this lethal airway disease, our overall goal is to assess HDPs as therapeutic options for CF lung infection and inflammation and to elucidate whether HDPs can act as antibiotic adjuvants to synergistically combat pathogens with conventional antibiotics.
To establish whether HDPs can be replenished and utilized to combat microbial pathogens, we have developed a novel approach for engrafting HDPs onto non-immunogenic cyclotides to improve the peptides’ stability and safety. Cyclotides are cyclized microproteins stabilized by three disulfide bonds that form a cystine knot, thus protecting the compounds from chemical, thermal, and biological degradation. Here, the development of a novel engineered cyclotide with effective broad-spectrum antibacterial activity against several ESKAPE bacterial strains and clinical isolates is reported. The most active antibacterial cyclotide was extremely stable in serum, showed little hemolytic activity, and provided protection in vivo in a murine model of P. aeruginosa peritonitis. These results highlight the potential of the cyclotide scaffold for the development of novel antimicrobial therapeutic leads that could fundamentally alter the clinical management of CF airway disease.
To treat CF inflammation, Rhesus theta defensin-1 (RTD-1), a macrocyclic HDP with known antimicrobial and immunomodulatory properties may be utilized. Our objective was to investigate the anti-inflammatory effect of RTD-1 in a murine model of chronic P. aeruginosa lung infection. Mice received nebulized RTD-1 daily for 6 days. Bacterial burden, leukocyte counts, and cytokine concentrations were evaluated. Microarray analysis was performed on bronchoalveolar lavage fluid (BALF) cells and lung tissue homogenates. In vitro effects of RTD-1 in THP-1 cells were assessed using quantitative reverse transcription PCR, enzyme-linked immunosorbent assays, immunoblots, confocal microscopy, enzymatic activity assays, and NF-κB-reporter assays. RTD-1 significantly reduced lung white blood cell counts on days 3 (−54.95%; p = 0.0003) and 7 (−31.71%; p = 0.0097). Lung tissue homogenate and BALF cell microarray analysis revealed that RTD-1 significantly reduced proinflammatory gene expression, particularly inflammasome-related genes (nod-like receptor protein 3, Mediterranean fever gene, interleukin (IL)-1α, and IL-1β) relative to the control. In vitro studies demonstrated NF–κB activation was reduced two-fold (p ≤ 0.0001) by RTD-1 treatment. Immunoblots revealed that RTD-1 treatment inhibited proIL-1β biosynthesis. Additionally, RTD-1 treatment was associated with a reduction in caspase-1 activation (FC = −1.79; p = 0.0052). RTD-1 exhibited potent anti-inflammatory activity in chronically infected mice. Importantly, RTD-1 inhibits inflammasome activity, which is possibly a downstream effect of NF-κB modulation. These results support that this immunomodulatory peptide may be a promising therapeutic for CF-associated lung disease.
Lastly, to establish the antibiotic adjuvant property of HDPs, we screened six HDPs with five different conventional antibiotics against P. aeruginosa. We identified that HDPs were highly synergistic with colistin and imipenem. We further validated that RTD-1 and colistin is highly synergistic against several P. aeruginosa clinical isolates. RTD-1 and colistin were shown to eradicate P. aeruginosa PAO1 by 4 hours without any bacterial regrowth in time kill kinetics. In vivo acute pneumonia models of P. aeruginosa infection further elucidated the bactericidal activity as there was a significant reduction in lung bacterial burden and leukocyte counts compared to the vehicle control. There was a further reduction in white blood cell counts and bacterial burden compared to colistin and RTD-1 monotherapy; however, it is not significant. These results highlight the potential of HDPs to act as antibiotic adjuvants to combat CF lung inflammation and infection.

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Asset Metadata

Creator Dughbaj, Mansour Awwad (author)

Core Title Therapeutic potential of host defense peptides for the treatment of cystic fibrosis lung disease

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Clinical and Experimental Therapeutics

Degree Conferral Date 2022-05

Publication Date 04/15/2022

Defense Date 03/03/2022

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

Tag cyclotides,cystic fibrosis,host defense peptides,OAI-PMH Harvest,protegrin-1,RTD-1

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Louie, Stan (committee chair), Beringer, Paul (committee member), Wong-Beringer, Annie (committee member)

Creator Email dughbaj@usc.edu,mdughbaj@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC110964899

Unique identifier UC110964899

Document Type Dissertation

Format application/pdf (imt)

Rights Dughbaj, Mansour Awwad

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Source 20220415-usctheses-batch-925 (batch), University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

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