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Therapeutic potential of rhesus theta (θ) defensin-1 in cystic fibrosis airway infection and inflammation

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Therapeutic potential of rhesus theta (θ) defensin-1 in cystic fibrosis airway infection and inflammation

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Content 1 Therapeutic Potential of Rhesus Theta (q) Defensin-1 in Cystic Fibrosis Airway Infection and Inflammation Timothy Jay Bensman A Dissertation Presented to the Faculty of The University of Southern California Graduate School In Partial Satisfaction of the Requirements for the Degree Doctor of Philosophy December 2016 Clinical and Experimental Therapeutics Dissertation Committee: Advisor: Professor Paul Beringer, Chair Co-Advisor: Professor Wei-Chiang Shen Professor Michael Selsted Professor Kathleen Rodgers 2 TABLE OF CONTENTS Page LIST OF FIGURES ........................................................................................................................ 4 LIST OF TABLES. ........................................................................................................................ 5 DEDICATION….. .......................................................................................................................... 6 ACKNOWLEDGMENTS ............................................................................................................... 7 ABSTRACT OF THE DISSERTATION ......................................................................................... 8 CHAPTER 1: Introduction ......................................................................................................... 11 Cystic Fibrosis (CF) Background .................................................................................. 11 Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). ............................... 11 CF Lung Disease .......................................................................................................... 12 Obstruction ..................................................................................................... 13 Infection .......................................................................................................... 14 Inflammation ................................................................................................... 15 Cellular Mechanisms of Airway Inflammation ............................................................... 16 Respiratory Epithelium and Immune Dysfunction in CF ................................. 17 Airway Neutrophils and Immune Dysfunction in CF ....................................... 19 Host Defense Peptides ................................................................................................. 23 Lung Host Defense Peptides .......................................................................... 24 a-, b-,and q-Defensins .................................................................................... 25 Antimicrobial Mechanisms .............................................................................. 27 Immunomodulatory Mechanisms .................................................................... 28 The CF Microenvironment and Host Defense Peptides ................................................ 31 Inactivation and Predisposition to Infection .................................................... 31 q-Defensins as a Potential CF Therapeutic ................................................................... 32 The Current State of the CF Lung Armamentarium ........................................ 32 Rhesus q-Defensin-1 ...................................................................................... 33 Expanding the Arsenal: The Duality and Promise of RTD-1. ......................... 35 Aims and Summary of Chapters ................................................................................... 36 CHAPTER 2: Rhesus Theta-Defensin-1 (RTD-1) Exhibits In Vitro and In Vivo Activity Against Cystic Fibrosis Strains of Pseudomonas aeruginosa ............................................ 38 Abstract .......................................................................................................... 38 Introduction ..................................................................................................... 40 Methods and Materials ................................................................................... 42 Results ............................................................................................................ 50 Discussion ...................................................................................................... 59 References ..................................................................................................... 64 3 CHAPTER 3: Anti-Inflammatory Effects of Rhesus Theta (q)-Defensin-1 in a Murine Model of Endotoxin-Induced Acute Lung Injury ............................................................................... 70 Abstract .......................................................................................................... 71 Introduction ..................................................................................................... 72 Materials and Methods ................................................................................... 74 Results ............................................................................................................ 82 Discussion ...................................................................................................... 98 References ................................................................................................... 103 CHAPTER 4: Therapeutic Potential of the Host Defense Peptide Rhesus Theta (q)- Defensin-1 in Cystic Fibrosis ................................................................................................ 112 Abstract ........................................................................................................ 113 Introduction ................................................................................................... 114 Methods and Materials ................................................................................. 117 Results .......................................................................................................... 132 Discussion .................................................................................................... 152 References ................................................................................................... 157 CHAPTER 5: Research Summary .......................................................................................... 167 General Conclusions ................................................................................................... 167 Antipseudomonal Activity ............................................................................. 167 Anti-inflammatory Activity ............................................................................. 168 Bench to Bedside ......................................................................................... 169 Future Directions ......................................................................................................... 170 Dose Optimization and Long-Term Inhalation Toxicology ............................ 170 Airway Mucus Obstruction and Drug Delivery .............................................. 171 Comparative Physiology, HDPs and extrapolations ..................................... 172 References .................................................................................................................. 174 APPENDIX…………… .............................................................................................................. 198 4 LIST OF FIGURES FIGURE 1-1. Classification of CFTR Mutations. ........................................................................ 12 FIGURE 1-2. Progressive Airway Damage. ............................................................................... 12 FIGURE 1-3. CFTR Dependent Mucoesculatory Impairment And Airway Obstruction. ............. 13 FIGURE 1-4. Prevalence of Respiratory Bacteria by Age With Focus On P. Aeruginosa. ........ 15 FIGURE 1-5. Pathological Effects of Host-Pathogen Interactions in CF. ................................... 16 FIGURE 1-6. Role of Metalloproteinases in CF Lung Disease. ................................................. 22 FIGURE 1-7. Defensin Genes and Peptides. ............................................................................. 25 FIGURE 1-8. Conserved g-Core Motif Across Diverse Disulfide-Containing Host Peptides. ..... 29 FIGURE 1-9. Pathophysiology and Drug Treatment in CF Airway Disease. .............................. 32 FIGURE 2-1. Concentration-Dependent Killing of RTD-1 Against PA01. .................................. 53 FIGURE 2-2. Activity of RTD-1 Against PA01 Biofilms. ............................................................. 54 FIGURE 2-3. RTD-1 Concentrations in Epithelial Lining Fluid (ELF) Versus Time After a Single Aerosolized Dose of RTD-1 in Non-Infected C57BL/6 Mice. ................................... 55 FIGURE 2-4. Aerosolized RTD-1 Significantly Reduces Lung CFU in a Murine Model of Chronic P. Aeruginosa Lung Infection. ................................................................................. 57 FIGURE 2-5. Phagocytosis of Zymosan Particles by THP-1 Macrophages. .............................. 58 FIGURE 3-1. Dose Dependent Effects of RTD-1 on In Vitro dHL-60 Migration or LPS-Induced MH-S Leukocyte Inflammation. ............................................................................... 83 FIGURE 3-2. Plasma RTD-1 Concentration Vs Time Profile. .................................................... 85 FIGURE 3-3. Dose and Time Dependent Effects of RTD-1 on Lung Neutrophilia, Neutrophil Activation, and Microvascular Disruption. ............................................................... 89 FIGURE 3-4. Dose and Time Dependent Effects of RTD-1 On BAL Cytokines. ........................ 91 FIGURE 3-5. Time Dependent Effects of RTD-1 on Soluble Receptor Shedding. ..................... 94 FIGURE 3-6. RTD-1 Effects on Severity of Acute Lung Injury. .................................................. 97 FIGURE 4-1. Nose Only Inhalation Exposure System Equipment Schematic. ........................ 126 FIGURE 4-2. RTD-1 Reduces Inflammatory Response in CF Epithelium. .............................. 133 FIGURE 4-3. RTD-1 Reduces Spontaneous Inflammatory Secretions in Airway Leukocytes. 135 FIGURE 4-4.RTD-1 Inhibits MMP-9 And ADAM-17 Metalloproteinases, but Not Serine Proteinase NE. ..................................................................................................... 137 FIGURE 4-5. RTD-1 Inhibits MMP-9 Activity. ........................................................................... 138 FIGURE 4-6. RTD-1 Does Not Inhibit the Serine Proteinase NE. ............................................ 139 FIGURE 4-7. RTD-1 Improves CF Sputum Pour-Ability Ex Vivo. ............................................ 140 FIGURE 4-8. RTD-1 is Robust to CF Sol Airway Proteolysis Ex Vivo. .................................... 142 FIGURE 4-9. Rtd-1 Does Not Induce Airway Hyper-Responsiveness In Vivo. ........................ 143 FIGURE 4-10. Aerosolized Properties of RTD-1. ..................................................................... 144 FIGURE 4-11. Characterization of Aerosolized RTD-1 In Vivo. ............................................... 145 FIGURE 4-12. Aerosolized RTD-1 Does Not Incite Leukocyte Recruitment. ........................... 148 FIGURE 4-13. RTD-1 Anti-Infective And Anti-Inflammatory Treatment Effects In Vivo. .......... 150 5 LIST OF TABLES TABLE 1-1. Antimicrobial Products in ASL ................................................................................ 24 TABLE 1-2. Examples of Immunoregulatory Functions of HDPs And IDRs a ............................. 30 TABLE 2-1. Activity of RTD-1 Against CF Clinical Isolates of P. Aeruginosa ............................ 51 TABLE 2-2. Activity of RTD-1 Against Colistin-Resistant Clinical Strains of P. Aeruginosa with Known Resistance Mutations in PhoPQ and/or PmrAB .......................................... 52 TABLE 3-1. Total Exposure of Inflammatory Markers in Mice with LPS-Induced Pulmonary Inflammation Treated with RTD-1 ........................................................................... 93 TABLE 4-1. CF Patient Characteristics and Sputum Cell Analysis .......................................... 118 TABLE 4-2. CF Patient APE Characteristics and Sputum Analysis. ........................................ 134 TABLE 4-3. RTD-1 Non-Compartmental PK Analysis in ELF .................................................. 146 TABLE 4-4. 7 D BALF Cytokines in Chronic P. Aeruginosa Lung Infected Mice Treated with RTD-1. ................................................................................................................... 151 6 DEDICATION This dissertation is dedicated to my family who through actions have taught me the meaning of unconditional, sacrificial love. Your heart is the soil from which this work bears its fruit. 7 ACKNOWLEDGMENTS I owe a great debt to Dr. Paul Beringer who stands as a testament to what a mentor truly is: role model, counselor, friend. Thank you for leading me down the exciting path of clinical and experimental therapeutics and opening a door to a future never before conceptualized. I will be forever grateful and indebted to you. I owe a great thank you to Dr. Wei-Chiang Shen for his dedication to my professional development through his exceptional wisdom: Your insights made a profound impact. I also owe a great thank you to Dr. Michael Selsted for his generous dedication to my training: Incorporation into your team has made this a truly thoughtful, insightful and rewarding journey. Thank you to Dr. Kathleen Rodgers for her time, efforts, and guidance: I learned much through our conversations even if the science didn’t always go “my way”. Also, I must thank Dr. Dat Tran for his remarkably kind and limitless time, assistance, and mind: I not only learned science, but also how to Macgyver it! Others whose advice and collaboration have been essential to my technical and professional development include: T-90 fellowship director: Dr. Michael Paine. Lab colleagues: Dr. Justin Schaal, Jordana Jayne, Patty Tran, Dr. Jason Yamaki, Dr. Melissa Agnello, Kristy Trinh. The USC Centers for Adult Cystic Fibrosis and Advanced Lung Disease (Dr. Adupa Rao, Dr. Kamyar Afshar, Dr. Santhi Iyer Kumar, Debbie Benitez, Lynn Fukushima) and their associated patients. The USC School of Pharmacy for their commitment to support student scientists. Last but certainly not least, I want to thank my Heart and my Soul. To my wife, Joyce: You are my Heart. Your patient understanding, unshakable support, and reassuring belief in me have kept me moving forward on this grand adventure. To my daughter, Elle: You are my Soul. Your beauty and joy in exploration and discovery inspires me to stay curious and hungry -not to take life for granted. 8 ABSTRACT OF THE DISSERTATION Therapeutic Potential of Rhesus Theta (q) Defensin-1 in Cystic Fibrosis Airway Infection and Inflammation by Timothy Jay Bensman Doctor of Philosophy in Clinical and Experimental Therapeutics University of Southern California Professor Paul M. Beringer Professor Wei-Chiang Shen Cystic fibrosis (CF) is the most common life-limiting genetic disorder in Caucasians. The majority of the morbidity and mortality is attributed to bronchiectasis and progressive loss of lung function. Despite improvements with aggressive antibiotics and airway clearance therapies, the life expectancy for children born and diagnosed with cystic fibrosis (CF) in 2010 is 40 years. The central feature of pulmonary disease is a chronic cycle of airway infection, neutrophilic inflammation, and obstruction. In particular, airway inflammatory markers (e.g. airway neutrophilia and neutrophil elastase) are strongly associated with disease severity. The importance of lung inflammation is reinforced by controlled clinical trials which demonstrated a reduced rate of lung function decline in patients receiving chronic prednisone or high-dose ibuprofen (Eigen et al., 1995; Konstan et al., 1995). However, serious adverse effects limit their long-term clinical use (Eigen et al., 1995; Konstan et al., 2007). Rhesus q-defensin-1 (RTD-1) is a novel host defense peptide with antimicrobial and anti-inflammatory actions. This dissertation sets out to explore the therapeutic potential of RTD-1 for treatment of CF airway disease. 9 The principal pathogen responsible for chronic lung infections in patients with CF is Pseudomonas aeruginosa. Based on published data demonstrating broad spectrum antimicrobial activity we hypothesized that RTD-1 would exhibit antipseudomonal activity. To test this hypothesis, we designed experiments to test the efficacy of RTD-1 using in vitro and in vivo models. Using in vitro assays of planktonic P.aeruginosa we demonstrate bactericidal activity against mucoid, non- mucoid, and multi-drug resistant CF isolates. Within the airways of patients with CF, P. aeruginosa exists within biofilm aggregates which resist host defense mechanisms and antimicrobial agents. Using an in vitro model of adherent P. aeruginosa, we demonstrate that RTD-1 significantly reduces biofilm mass. Based on these encouraging in vitro data we performed pharmacokinetic and preliminary efficacy studies of aerosol administration of RTD-1 in a murine model of chronic P. aeruginosa infection in CF mice. Airway pharmacokinetics (PK) studies demonstrated concentrations exceeded the minimum inhibitory concentration (MIC) for 12 hrs. Preliminary efficacy studies showed treatment significantly reduced bacterial burden in infected mice. Chronic lung infections in CF are associated with an intense neutrophilic inflammatory response. Targeting neutrophil migration and disrupting the release of proteases and oxidants is an important therapeutic strategy. Data from in vitro and animal models of sepsis and SARS demonstrate that RTD-1 exhibits immunomodulatory activity. To determine the feasibility of targeting neutrophilic airway inflammation we tested the efficacy of systemic administration of RTD-1 in a murine model of LPS-induced acute lung injury. We demonstrate that LPS-induced pulmonary inflammation in mice treated with RTD-1 exhibit reduced lung injury as measured by pulmonary edema and histopathologic changes. RTD-1 treated mice also demonstrate dose and time dependent reductions in airway neutrophils and pro-inflammatory cytokines, while augmenting anti-inflammatory activity through increased soluble gp-130 levels. In vitro assays suggest direct inhibition of neutrophil migration and reduced cytokine/chemokine production by 10 alveolar macrophages may be responsible for in vivo effectiveness. Plasma PK data, demonstrate concentrations necessary for direct neutrophil inhibition are achieved in vivo. To further determine RTD-1’s therapeutic potential in CF, additional investigational models were employed, first we tested the efficacy of aerosolized RTD-1 in F508del homozygous mice with chronic P. aeruginosa lung infection. Descriptive in vivo PK of aerosolized RTD-1 demonstrate the achievement of ELF concentrations necessary for antibacterial and anti-inflammatory effects. In line with PK data, we observed antibacterial and anti-inflammatory activity in the lungs of chronically infected CF mice receiving aerosolized RTD-1. Furthermore, the safety of aerosolized RTD-1 was supported by airway leukocyte counts, body weight, and lung histology data, as well as airway hyper-responsiveness studies in naïve uninfected mice. Second, in vitro assays demonstrate specific and potent metalloproteinase inhibition with some selectivity for ADAM17 over MMP-9. Third, ex vivo CF airway leukocytes, under RTD-1 treatment, resulted in reductions in spontaneous secretion of pro-inflammatory products. Using, in vitro CF bronchial epithelial cells stimulated with P. aeruginosa diffusible material we found TLR and adapter protein mRNA downregulation associated with reduced inflammatory cytokine release. Collectively, this body of work supports the further development of RTD-1 for use in CF. 11 CHAPTER 1 Introduction Cystic Fibrosis (CF) Background CF is the most common lethal Mendelian disease in Caucasians (Foundation, 2013). It is characterized by abnormal fluid and electrolyte transport across epithelia. Both absorptive and secretory processes are affected, resulting in a pathologic inspissation of organ secretions that include the pancreas, lung, liver, gastrointestinal tract, sweat glands, and reproductive tract (Reddy and Stutts, 2013). Hallmarks of disease include condensed, viscous mucosal secretions, as well as chronic infection and inflammation. Failure to thrive is the earliest feature (Giglio et al., 1997). Reasons for this manifestation include meconium ileus, distal intestinal obstruction syndrome, and pancreatic insufficiency. While a multi-organ disease, the pulmonary pathology is responsible for the high morbidity and mortality. Early, patients experience chronic coughing with productive expectoration of mucopurulent material. Over a variable period of time chronic airway infection is established and its persistence despite antibiotics leads to respiratory insufficiency and eventual failure. Aggressive therapy targeting the airways has been associated with an increase in life expectancy. Despite these attempts, > 90% of patients succumb to pulmonary failure with the median age of death at 29.1 years (Foundation, 2014). Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). The basic defect is located on chromosome 7 and established that mutations of the CFTR gene (most common variant DF508) cause CF (Rommens et al., 1989). To date, this ABC binding cassette protein has over 2000 variants (Cutting, 2015). Disease causing variants are grouped into classes at the cellular level based on CFTR production, trafficking, function, and stability, which provides a useful framework for understanding the basic defect (Fig. 1-1). Lack of expression at the membrane results in a multitude of biological effects including reduced chloride 12 and bicarbonate secretion, enhanced sodium absorption and mucin secretion, which leads to acidic microenvironments, impaired host defense peptide activity, defective phagocytic activity, and increased airway surface liquid viscosity. Not surprisingly, several drugs targeting this primary defect have reached patients. However, this precision medicine approach is currently only applicable for approximately 28% of the CF population. In addition, the tempered clinical effects (albeit beneficial) and reported drug interactions between potentiators and correctors demonstrate that the search for the cure continues (Cholon et al., 2014; Veit et al., 2014). CF Lung Disease While CF is a multi-organ disease, greater than 90% of the morbidity and mortality are secondary to the respiratory condition (Foundation, 2014). Pathological hallmarks of CF lung disease include airway obstruction, as well as, a continuous cycle of chronic infection and neutrophilic inflammation. Thus by adulthood most patients have severe bronchiectasis that ultimately results in pulmonary failure and death (Fig. 1-2). Figure 1-1. Classification of CFTR Mutations. Reproduced from (Foundation, 2014) with permission from CF Foundation Figure 1-2. Progressive Airway Damage. Images of 10-year old female. (i-ii); (i-ii); arrows point to decreases in central mucous plugging and increases in central bronchiectasis, circles show decreases in central mucous plugging and an increase in the bronchiectatic region in the lower lobe. Reproduced from (de Jong et al., 2006) with permission from BMJ Publishing Group Ltd. 13 Obstruction CFTR works as a cAMP-dependent Cl - channel that in conjunction with the Ca +2 – activated Cl - channel (CaCC), secrete Cl - allowing for proper hydration of mucins and other secreted molecules that form airway mucus (Quinton, 2007). In addition, to Cl - secretion, normal CFTR also limits Na + and fluid absorption via the amiloride-sensitive epithelial Na + channel (ENaC) (Mall et al., 2004; Mall, 2008; Rubenstein et al., 2011). Thus, the regulation of NaCl plays a critical role in fluid balance. Airway surface liquid (ASL) depletion has been observed in CF as the result of an imbalance between Cl - and EnaC mediated Na + absportion (Rubenstein et al., 2011). In addition, CFTR dependent luminal bicarbonate deficiency impairs the maturation of secreted mucins (e.g. carbohydrate expansion, hydration) and results in airway aggregates that are poorly soluble and less transportable (Quinton, 2008; Shah et al., 2016). Airway mucins play an important role in innate immunity. Secreted mucins provide a barrier to repel foreign invaders, while cell surface mucins provide a shield to prevent pathogen adhesion and maintain mucosal homeostasis. Importantly, a gel on brush model has been proposed that accounts for the proper ciliary movement and biophysical network that clears properly hydrated mucus (Button et al., 2012). In CF, impaired ciliary beating and mucus transport promote mucus stasis, adhesion, and airway obstruction (Fig. 1-3). Beyond abnormal lung function (i.e. FEV 1 ), a primary innate defense mechanism is severely impaired and dysregulated (Knowles and Boucher, 2002). Figure 1-3. CFTR Dependent Mucoesculatory Impairment and Airway Obstruction. Abnormal CFTR function results in thick mucus with heavy cellular infiltration that obstructs and distends bronchiole. Reproduced from 2013 NACFC with permission from Bonnie Ramsey www.nacfconference.org/art/plenaryarchives/2013%20Ramsey 14 Infection CF pulmonary disease reflects a chronic endobronchial infection of the conducting airways. Mutations in the CFTR gene cause important functional impairments of the innate host defense systems. Importantly, CFTR dysfunction results in reduced epithelial bicarbonate secretion leading to an acidic ASL. The acidic pH impairs antimicrobial peptide activity allowing bacteria to establish in the airways as demonstrated in animal and human CF lung disease.(Pezzulo et al., 2012; Shah et al., 2016) Host defenses are further impaired as heavy protease burden degrades respiratory antimicrobial peptides such as lactoferin, elafin, b defensin-2, and human cathelicidin (Forde et al., 2014; Hiemstra et al., 2016). The clinical result is invasion of Staphylococcus aureus and non-typeable Haemophilus influenzae early in life (i.e. infancy) and the development of persistence by childhood (Lyczak et al., 2002)(Fig. 1-4a). In children and young adults acquisition of opportunistic pathogens including P. aeruginosa and Burkholderia cepacia complex (Bcc) are associated with progressive loss of lung function and premature death (Henry et al., 1992; Isles et al., 1984; Kosorok et al., 2001; Muhdi et al., 1996). During the course of chronic infection in the lungs, these pathogens acquire a number of virulence and resistance mechanisms which allow them to persist despite robust host defenses and intense antibiotic therapy. In particular, biofilm- like structures provide a physical barrier to host defense mechanisms and many antibiotics. Repeated exposures to antibiotics results in inducible drug resistance mechanisms (e.g. beta- lactamase, topoisomerase, efflux pump, porin, and altered membrane architecture). These as well as other known bacterial adaptations contribute to the persistence of infections within the lungs of patients with CF. (Caceres et al., 2014; Cigana et al., 2011; Livermore, 2002; Lyczak et al., 2002; Staudinger et al., 2014). Thus by adulthood ~70% of patients are chronically infected with P. aeruginosa with approximately 20% representing multidrug resistant infections (Foundation, 2014)(Fig. 1-4b). 15 Inflammation Persistent neutrophilic inflammation in the airways is a hallmark of CF pulmonary disease. In vitro and ex vivo work demonstrate a dysregulated and exaggerated inflammatory response in relation to pathogen burden (Hartl et al., 2012). Exuberant levels of oxidants, as well as imbalanced protease/anti-protease and pro-inflammatory/anti-inflammatory axes exist in the CF airway (Fig. 1-5.). These mediators attack and destroy the bronchial elastin matrix, as well as other lung structures, impairing the body’ removal of infected bronchial secretions. Chronic inflammation and infection further erode lung integrity until a permanent irreversible dilatation or ballooning of the bronchi develops. This cyclical pathology and associated lung impairment is defined clinically as Figure 1-4. Prevalence of Respiratory Bacteria by Age with Focus on P.aeruginosa. A) Age Cohorts and corresponding prevalence of bacterial isolates in the airways. B) Rates of multi-drug resistant P. aeruginosa and association with maturity. Reproduced from (Foundation, 2014) with permission from CF Foundation. 16 bronchiectasis. The development of bronchiectasis in CF occurs early (infancy) and is progressive. In fact, free neutrophil elastase levels in CF infants (3 months old) can be detected and are associated with early and persistent bronchiectasis (Sly et al., 2013). Cellular Mechanisms of Airway Inflammation Bronchoalveolar lavage studies performed in patients with CF reveal that neutrophils account for > 90% of the cells. Since neutrophils express little, if any, CFTR protein, in addition to growing evidence indicative of an imbalanced environment-driven immune response, it is likely that the altered immune homeostasis is a downstream consequence of the primary defect (Sorio et al., 2016). The dysregulated recruitment of neutrophils can be caused by several cellular mechanisms including the lung epithelium, T-helper lymphatics, and monocyte/macrophage responses (Conese et al., 2003). While data on the contribution of lymphocytes and monocytes/macrophages to the chronic lung inflammatory state is emerging, their role in the pathogenesis of CF lung disease remains unclear. In particular, investigations have revealed Th 17 cells, the main producers of an IL-17-driven neutrophil recruitment response, are present in lung Figure 1-5. Pathological Effects of Host-Pathogen Interactions in CF. Collectively representing a frustrated acute inflammatory response to infection that is chronically prolonged and fatal. Reproduced with permission from (Davis and Ferkol, 2013), Copyright Massachusetts Medical Society. 17 tissue of CF patients compared to healthy controls and associated with disease severity (Tan et al., 2011). In vitro and ex vivo data from CF patients have identified a monocyte-selective adhesion deficiency trapping these cells in the lung parenchyma resulting in their subsequent activation and chemotactic factor release which may contribute to the persistent lung neutrophilia (Blohmke et al., 2008; Sorio et al., 2016). Therefore, we have chosen to focus our research on examining the effects of RTD-1 on lung epithelia and neutrophilic responses. Respiratory Epithelium and Immune Dysfunction in CF The airway epithelium is in direct contact with the external environment and serves as the first point of approach between host and invader. As both sentry and soldier, it is not surprising the vast arsenal bestowed upon these cells. These wardens utilize barriers, blockades, detection systems and chemical weaponry to protect themselves. As noxious agents (e.g. particles, pathogens) are inhaled first line barriers such as the epithelium and the mucus secreted by these cells provide for entrapment and effective removal through the mucocilliary apparatus. In addition, this blockade allows for the early detection of harmful molecules through specific receptors on the epithelium such as those that recognize pathogen associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs). The most notable of these pattern recognition receptors (PRRs) include Toll-like receptors (TLRs) and nucleotide oligomerization domain-like receptors (NODs). Upon activation these cells produce chemical molecules that comprise antimicrobial and immune modulating actions. The production of cytokines can augment pathogen clearance through activation of resident phagocytes as well as recruit immune cells such as neutrophils to the site of infection. Additionally, antimicrobial molecules (i.e. lysozyme, lactoferrin, mucins, and cationic peptides) are secreted to directly attack and kill invading pathogens. Moreover, if tissue damage should occur, molecules to activate the immune system (alarmins) are actively secreted (Cohen and Prince, 2012). 18 In CF, elevated levels of pro-inflammatory molecules, including IL-1b, IL-6, IL-8, and TNF, and reduced anti-inflammatory molecules, IL-10, IL-1b receptor antagonist, and soluble TNF, promote a noxious inflammatory environment in the lung (Courtney et al., 2004). CF epithelium produce large amounts of IL-6 and IL-8 (Berube et al., 2010; Courtney et al., 2004; Greene et al., 2005). Moreover, bronchial epithelial-derived IL-10 in CF is thought to be significant (Bonfield et al., 1999; Bonfield et al., 1995). While levels of IL-1b and TNF, as well as detectable levels of soluble TNF receptors, and IL-1 receptor antagonist have been observed in airway epithelia, they are not thought to be the primary producers of these molecules (Coulter et al., 1999; Hartl et al., 2012; Saperstein et al., 2009; Tang et al., 2012). Importantly, the lung epithelium in CF has been shown to have increased TLR-dependent NF-kB activity and the significant reduction in IL-10 removes a natural brake on this immune response allowing for expression of these molecules. (Barnes and Karin, 1997; McIsaac et al., 2012). TLRs are important sentinels in recognizing pathogens and activating an immune response. Through in vitro work, two TLRs have been the mainstay focus of study and therapeutic target. TLR 4 appears to have reduced expression on the apical surface in bronchial epithelial cells from patients with CF and is not responsive to LPS stimulation. This impairment results in reduced MyD88 and TRIF-dependent signaling that is hypothesized to impair host defense (Hartl et al., 2012). TLR 5 recognizes flagellin- a product of P. aeruginosa and B. cenocepacia- and therefore represents the primary TLR for immune signaling in these cells. Importantly, clinical evidence from CF patients with a premature stop codon mutation in TLR5 (c.117C<T), which would reduce flagellin responsiveness, showed improved clinical health when looking at weight, but not lung function (FEV 1 ) (Blohmke et al., 2010). TLR5 antibody antagonism was shown to abolish CF epithelial cell IL-6 production after P. aeruginosa stimulation. However, another study with P. aeruginosa diffusible material suggest that neutralization of TLR5 was insufficient in inhibiting IL- 6 synthesis completely supporting the presence of alternative pathways (Berube et al., 2010). 19 In addition to resident pathogens, TLRs found on the lung epithelium can be stimulated by several other factors in the CF microenvironment. Neutrophil elastase through activation of meprin and/or tumor necrosis factor alpha-converting enzyme (TACE) indirectly simulates TLR 4 on CF lung epithelium leading to exaggerated IL-8 release in vitro (Bergin et al., 2008). Additionally, high airway levels of alarmins such as high mobility group box -1 (HMGB1), S100A8/S100A9, and LL- 37 are thought to amplify the exuberant dysregulated immune response in CF (Chen et al., 2004; Lorenz et al., 2008; Rowe et al., 2008; Tirkos et al., 2006). The endocrine system also seems to have a role; as higher circulating estrogen levels increase secretory leucoprotease inhibitor (SLPI) via estrogen-receptor activation. A TLR hyporesponsiveness occurs through SLPI inhibition of NF-kB activation resulting in increased cytokine expression. The clinical consequences observed for this gender gap phenomena are poorer lung function, more frequent exacerbations, and earlier acquisition of the mucoidy P. aeruginosa phenotype in CF women compared with CF men (Chotirmall et al., 2010). Airway Neutrophils and Immune Dysfunction in CF While the bronchial epithelium is a key target of airway therapeutics, neutrophils are recognized as active players in CF lung inflammation (Laval et al., 2016). Sampling of the airways by expectorated sputum or bronchoalveolar lavage (BAL) demonstrate a well-established chronic airway neutrophilia- a CF hallmark (Hartl et al., 2012; Henig et al., 2001). While neutrophilia is considered advantageous when fighting an acute infection, a chronic lung neutrophilia can cause significant lung tissue damage from accumulation of granular contents (i.e. serine- and metallo- proteases, oxidants). Clinical evidence from 4 randomized controlled trials show a strong negative correlation between neutrophil numbers and lung function. Neutrophil elastase is also associated with disease severity and is the strongest correlate to pulmonary function – further strengthening the importance of neutrophils in CF pathology (Mayer-Hamblett et al., 2007). The long held dogma has been that blood neutrophils, having been terminally differentiated after leaving the bone 20 marrow and programmed for death (apoptosis), cannot escape their fate. However, recent ex vivo observations from CF patient airway neutrophils demonstrate surface and intracellular changes suggestive of an anabolic reprogramming process that involves glucose and amino acid nutrient transport, danger associated molecular patterns (sRAGE, S100A12), the CREB and mTOR pathways, and CXCR4 supporting the view of an active, rather than passive, and coordinated response to the CF lung microenvironment (i.e. high oxidant, DAMP, and ATP levels) (Laval et al., 2016; Makam et al., 2009). Phenotypically, these live neutrophils undergo active degranulation of all granule types in the lung compartment and particularly impressive is the high degree of exocytosis of primary granules and cleavage of CD16 a phagocytosis associated receptor (Tirouvanziam et al., 2008). Chronic neutrophilia contributes to the ongoing cycle of infection, inflammation, and airway obstruction through granular release, impaired phagocytosis, and NET formation. As a boneyard, the luminal surface of the chronically infected lung is exposed to a heavy proteolytic burden via natural turnover of neutrophils. Proteases can be released into the lungs by passive and active means. First, necrotic neutrophils with leaky membranes dump proteases in an unregulated fashion. Alternatively, active neutrophils can perform degranulation, or exocytosis, of vesicle contents in a highly sequential and regulated fashion to prevent excessive degranulation. Regulation of exocytosis occurs through a binary signal system (adhesion then immune receptor activation) to minimize aberrant degranulation. (Sheshachalam et al., 2014). Secretory vesicles (containing membrane-associated receptors and cytokines for early inflammatory response), then gelatinase granules (rich in MMP9, a protein involved in transmigration and ECM proteolysis) are the first to be released upon receptor activation. Prolonged immune receptor stimulation results in specific granule release (predominately antimicrobials – lactoferin, cathelicidine). Finally, chronic stimulation or direct pathogen contact recruits azurophil granules (predominately MPO 21 and serine proteases such as NE) to the phagosomes for release. Thus the accumulation of liberated protease through passive and active mechanisms, result in an exuberant protease burden in the CF airway microenvironment (Sheshachalam et al., 2014). Because of the extent and swathe of proteases released in the CF lung the stoichiometric imbalance is augmented by proteolytic reductions in anti-proteases creating a pathological proteinase/anti-proteinase imbalance. Examples include MMP cleavage of a-1 antitrypsin (Vissers et al., 1988), degradation of serine leukocyte protease inhibitor (SLPI) by cysteine cathepsins, and the activation of pro- MMP-9 and inactivation of the tissue inhibitor of metalloproteinases-1 (TIMP-1) via NE (Jackson et al., 2010; Voynow et al., 2008) These unchecked proteolytic systems then impair normal immune functions. For example, NE targets surface receptors such as the CXCL8/IL-8 receptor (CXCR1), and complement receptor 1 (CR1) resulting in reduced bacterial killing (Hartl et al., 2007; Tosi et al., 1990). This dysregulated axis represents a significant therapeutic target as demonstrated by its association with lung disease severity (i.e. pulmonary function)(Mayer- Hamblett et al., 2007). In addition, host metal-dependent proteinases play an important role as regulatory triggers. In particular, they can modulate the activity of chemokines, cytokines, and growth factors, shed pro- inflammatory molecules, cytokine receptors and adhesion molecules, degrade extracellular matrix (ECM) and chemokine binding proteins, as well as activate surface receptors and intracellular signaling responses via proteolysis (Greenlee et al., 2007; Leduc et al., 2007; Murphy et al., 2008). MMP-9 specifically, can damage innate defenses such as surfactant proteins A and D, immunoglobulins, and CD4 on lymphocytes dampening the ability of the host to respond to bacterial airway infection (Griese et al., 2008; Hartl et al., 2007; Leduc et al., 2007). Furthermore, MMP-9 augments IL-8 potency via protease cleavage (Xu et al., 2011), and generates the matrikine proline-glycine-proline from collagen amplifying neutrophil recruitment to the lung (Hartl 22 et al., 2012). Indeed, sputum from children with CF show elevated MMP-9 and an unbalanced molar ratio with endogenous inhibitor TIMP1 in favor of MMP proteolysis. This imbalance is associated with increased matrix breakdown products (e.g. elastin, collagen) and pulmonary disease severity representing an important therapeutic target (Sagel et al., 2005) (Hilliard et al., 2007). An additional proteinase, A disintegrin and metalloproteinase (ADAM17) acting at the cell surface liberates soluble TNF and is regulated by its endogenous tissue inhibitor of matrix metalloproteinases (TIMP3) (Horiuchi et al., 2007). Clinical data show elevated airway soluble TNF in CF vs healthy controls and an inverse association with pulmonary function severity (Greally et al., 1993; Karpati et al., 2000). And a 4 year longitudinal study observed sustained elevation of airway TNF in CF patients supporting the therapeutic targeting of this enzyme (Sagel et al., 2012). Collectively the literature suggest that these systems work together to promote a self-perpetuating cycle of neutrophil influx, activation, inflammation, proteolysis, and lung scaring. Importantly, in chapter 4 we disclose RTD-1 mediated effects on these CF relevant drug targets (Fig. 1-6). Neutrophil phagocytosis is also suggested to be altered in CF. In addition to the proteolytic loss of host chemokine and Fc receptors, the presence of bacterial biofilms reduce phagocytosis (Hartl et al., 2012; Hartl et al., 2007). Intelligently, using the resources available to it, P. aeruginosa exploits the abundant DNA (from apoptotic and activated neutrophils) to form an extracellular polymeric matrix to which a community of adherent microbes live. These biofilms allow microbes to resist host defenses and antibiotics through the regulation of global response elements and Figure 1-6. Role of Metalloproteinases in CF Lung Disease. ROS activates ADAM17 resulting in increased sTNF which binds to TNFR 1/2 activating transcription of CXCL-8. Neutrophil infiltration results in release of MMP9 which causes matrix degradation. RTD-1 inhibits the metalloproteinases (ADAM17, MMP9). 23 quorum sensing, as well as production of an exopolysaccharide (alginate) which beyond providing a physical barrier for leukocyte migration and phagocytosis, also scavenges hypochlorite, and inhibits complement (Jesaitis et al., 2003). Thus in CF, impairment of this system leads to inefficient or ineffective killing and phagocytic host defense. After failure to eradicate pathogens by phagocytic and granule attacks, neutrophils sacrificially employ their own DNA (wrapped in antimicrobial products) as an all-out assault on the foreign invaders. These neutrophil extracellular traps (NETs) are a formation of histone and granule proteins and extracellular DNA that entangle, immobilize, and kill microbes. Unfortunately the mucoidy phenotype (commonly present in CF) has been correlated with resistance to NET killing and the increase in cellular debris to the already thick and tenacious mucosal layer likely exacerbates airway obstruction (Young et al., 2011). At the crossroads of the CF microbial-host interface are airway epithelial and neutrophilic host defenses. Impairment of these systems precipitate, if not cause, the hallmark pathologies and contribute to disease progression. At the molecular level, dysregulated inflammatory processes seem to be pivotal in CF lung pathology and prognosis. Collectively, it represents a frustrated acute inflammatory response to infection that persists. Host Defense Peptides (HDP) There are close to 3,000 HDPs identified to date (Wang, 2016). Traditionally, these endogenous antimicrobial peptides have been thought to serve as a first-line chemical barrier against pathogen invasion in plants and animals (vertebrates and invertebrates) (Jarczak et al., 2013). In conjunction with the adaptive immune system, peptide concentrations of ~100 µg/mL demonstrate broad-spectrum antimicrobial activity against gram positive and negative bacteria, mycobacteria, fungi, and enveloped viruses providing for rapid killing or neutralization -shielding the host from the constant threat of infection (Mansour et al., 2014). While first recognized as microbicides in 24 the lab, it is now appreciated that these host defense peptides also possess immunomodulatory properties (Mansour et al., 2014). Due to their importance in pulmonary infections, we will focus on the human host defense peptides (HDPs) in the lung. Lung Host Defense Peptides As part of the ancient innate immune system, HDPs are highly conserved and “ever ready” in the lung. The relative airway surface liquid concentrations of common lung antimicrobial peptides are listed in table 1-1 (Schutte and McCray, 2002). Of particular interest because of their relative abundance are the defensins, LL-37, lysozyme, and phospholipase A2. Human a-defensins are primarily produced in neutrophils and classically referred to as human neutrophil peptides (HNPs). In contrast, b- defensins are mainly produced in the airway epithelium. In contrast to these smaller peptides (<6 kDa), the only human cathelicidin, LL-37 a 18 kDa polypeptide, can be found expressed by both neutrophils and epithelial cells (Hiemstra et al., 2016; Schutte and McCray, 2002). Lysozyme was the first recognized human antimicrobial protein, discovered by Alexander Flemming in 1922 (Skerrett, 2004; Wang, 2014). Its abundance in the lung is quite pronounced as it is secreted by lung epithelium and also released upon degranulation of neutrophils and macrophages. Phospholipase A2 can be found in soluble, cytosolic or lysosomal forms. It is primarily secreted upon neutrophil and macrophage degranulation, however, epithelial and Paneth cells also produce this protein. Interestingly the liver manufactures a soluble acute phase form (Schaloske and Dennis, 2006). In contrast to the defensins and LL-37 both lysozyme and phospholipase A2 Reproduced from (Schutte and McCray, 2002) with permission from Annual Reviews Table 1-1. Antimicrobial Products in ASL 25 are enzymes that hydrolyze the cell wall of microbes. Host defense peptide and protein production is both constitutive and inducible by a variety of stimuli (Choi et al., 2012). Inflammatory stimulation and increased neutrophil emigration are primarily responsible for elevated levels of most of these peptides and proteins (Sagel et al., 2009; Schaloske and Dennis, 2006). However, recently there have been several disease states where risk alleles, in promoter activity regions, or HDP copy numbers variation are associated with altered HDP production (Gallo and Hooper, 2012). a-, b-,and q-Defensins Defensins are characterized by their 18 to 45 amino acids (~2 – 4 kDa), an arginine rich cationic charge (+1 to +11), as well as, parallel b sheet folds and tri-disulfide framework (Selsted and Ouellette, 2005). Sub classification into a, b, and q families is based on peptide length, disulfide bridge location, and tertiary structure and are further described below and represented in Fig. 1- 7 (Selsted and Ouellette, 2005). Defensins are ubiquitous in mammals, although each species displays distinct profiles (Selsted and Ouellette, 2005). Germaine to this work, both a- and b- defensins are found in humans and mice, however human defensins are expressed predominately in leukocytes, while mice lack this phenotype (Mestas and Hughes, 2004). Defensins are richly expressed in the crypts of the small intestine by Paneth cells in the mouse (Ouellette and Selsted, 1996). Human a-, b-defensins (DEFA and DEFB) are cationic non-cyclic host defense peptides with both antimicrobial and immune regulating properties. Human a-defensins, consisting of 29-35 Figure 1-7. Defensin Genes and Peptides. Left, defensin genes. Tan and blue represent residues in the mature defensin. Right, three-dimensional structures of rabbit a- defensin RK-1 (top), human b-defensin-1 (middle) and q-defensin RTD-1 (bottom) with disulfide arrangements. Reproduced from (Selsted and Ouellette, 2005) with permission from Nature Publishing group. 26 amino acids with disulfide pairings of I-VI, II-IV, III-V, are located in myeloid and enteric tissues (Selsted and Ouellette, 2005). They are single gene products (chromosome 8p23) and their maturation involves the removal of the signal peptide from the ‘prepropeptide’ as well as, proteolytic removal of an anionic 40 amino acid ‘propiece’ that provides a net neutral charge that may reduce intracellular toxicity (Selsted and Ouellette, 2005).The human neutrophil peptides 1- 4 (DEFA 1-4) are expressed and packaged in primary granules of leukocytes (~50% of protein content) while “cryptidins” human defensins-5 and -6 (i.e. DEFA-5, DEFA-6) are manufactured in Paneth cells and secreted into the lumen of the gastrointestinal tract from the base of the crypts of Lieberkühn (Ouellette, 2006). Disulfide pairing patterns differentiate the larger human b- defensins (~45 aa) from the a-defensin family with a I-V, II-IV, III-VI arrangement. b-defensins are abundantly expressed at the mucosal surfaces of the host. Like the a-defensins, they are single gene products, and initially expressed as prepropeptides (Selsted and Ouellette, 2005) . Production and expression are found primarily in epithelial cells, but also leukocytes (Schutte and McCray, 2002). To date, 31 human b-defensins genes (DEFB) located on chromosome 8, 6, and 20 have been identified however, only DEFB-1, DEFB-2, DEFB-3 and DEFB-4 have been found expressed at the protein level (Jarczak et al., 2013).The localized expression of these peptides and current data suggest a strong mucosal immune response that bridges innate and adaptive immune functions (Schutte and McCray, 2002). Last, the most recently reported member of the defensin family are the q-defensins. Uniquely, these are the only completely cyclized peptides found in animals. These macrocyclic octadecapeptides (18 amino acids) are stabilized by a tri- disulfide array with I-VI, II-V, and III-IV arrangement (Selsted and Ouellette, 2005). They are thought to be created from the binary ligation of 2 precursor alpha defensin paralogs that have been trimmed to a 9 amino acid segment after post-translation processing of the 12 amino acid propiece (Selsted and Ouellette, 2005). Splicing arrangements from the 3 known rhesus macaque DEFT genes provides the potential for 6 different cyclic peptides. To date only 3 have been 27 isolated from rhesus granulocytes (Selsted and Ouellette, 2005). However, other q-defensins have been found in Old World Monkeys (e.g. baboon) and orangutans (Selsted and Ouellette, 2005). While humans possess the q-defensins pseudogene (yDEFT) the premature stop codon in the mRNA signal sequence causes the abortion of translation and thus a peptide product is never realized. This mutation in the stop codon is thought to have occurred between the divergence of orangutan and hominoid lineages (Lehrer et al., 2012). Reasons for the evolutionary loss are currently unknown. The structure of q-defensins, specifically RTD-1 are shown in Fig. 1- 7. Antimicrobial Mechanisms HDPs contribute to innate lung defense directly by antimicrobial actions (Gallo and Hooper, 2012). Because of their multiple modes of action (cell wall disruption and intracellular translocation) and highly conserved targets it is thought that these peptides are less prone to resistance than current antibacterials (Lo and Lange, 2015; Nijnik and Hancock, 2009). Differentiation between eukaryotic and prokaryotic membranes is required for proper host homeostasis and effective bacterial lysis. Differences in composition, transmembrane potential, and structural features between mammalian and bacterial membranes are critical (Melo et al., 2009). Human membranes are relatively net neutral as they are composed predominantly of phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin. Alternatively, the bacterial membrane carries a general negative charge as it is composed largely of phosphatidylglycerol, cardiolipin, and phosphatidylserine (Melo et al., 2009; Yeaman and Yount, 2003). This charge difference is likely important for arginine rich cationic HDP contact via van der Waals interactions. Another compositional difference is the lack of sterols (i.e. cholesterol in humans and ergosterol in fungi) in bacterial membranes and the energetically favorable tryptophan rich HDP interaction (Yeaman and Yount, 2003). In regards to transmembrane potential (Dy) differences, the lower bacterial Dy facilitates cationic peptide interactions and help drive membrane insertion/translocation (Yeaman 28 and Yount, 2003). In regards to anionic structural components, putative ligands such as lipopolysaccharide (LPS), lipoteichoic acids, and peptidoglycan have been shown to be targeted by cationic HDPs and upon membrane disruption allow electrostatic interactions with phospholipid head groups (Chan et al., 2006; Melo et al., 2009). Furthermore, HDP characteristics such as cationic charge, hydrophobicity, and amphipathicity improve van der Waals interactions, stronger partitioning, and polar non-polar residue segregation improve membrane interactions and perturbation respectively (Takahashi et al., 2010). Beyond membrane lysis, microbial killing mediated through intracellular mechanisms such as inhibition of cell wall-, nucleic, and protein synthesis, as well as intracellular enzymatic activity have been suggested (Brogden, 2005). Beyond antibacterial actions, anti-viral and anti-fungal mechanisms exist and include the above lytic mechanisms, prevention of viral entry, replication and direct neutralization (Gwyer Findlay et al., 2013; Silva et al., 2014; Wiens et al., 2014). Immunomodulatory Mechanisms In addition to antimicrobial effects, HDPs have demonstrated themselves as multifunctional immune regulating effector molecules (Mansour et al., 2014). HDPs cover the entire inflammatory response from induction of acute inflammation to resolution and repair (Table 1-2). They display pro- and anti-inflammatory properties that are concentration dependent and mediated through membrane and intracellular signaling pathways, cytokine release, chemotactic recruitment, polarization of dendritic and macrophage cells through maturation and differentiation, as well as, direct cell death (i.e. apoptosis, pyroptosis, autophagy) and wound repair. An archetypical example is LL-37. Originally, endotoxin neutralization was believed to be the mechanism for reduced TLR-mediated inflammation (Pulido et al., 2012; Rosenfeld et al., 2006). However, more recent work demonstrate direct and selective effects from TLR stimulation on NF-kB nuclear translocation (Mookherjee et al., 2006). Additional research has described GAPDH as an intracellular cognate receptor for LL-37 that modulates p38 mitogen-activated protein kinase (MAPK) activity (Mookherjee et al., 2009). Furthermore, LL-37 enhanced IL-1b induced cytokines 29 selective for monocyte and macrophage recruitment (i.e. MCP-1 and MCP-3). While strengthening these inflammatory effects in blood mononuclear cells, LL-37 counterbalanced this through the upregulation of the anti-inflammatory cytokine IL-10, demonstrating specific effector function in the innate immune response (Yu et al., 2007). In regards to chemoattraction, generally HDPs promote migration either directly or indirectly (Mansour et al., 2014). The induction of chemokines such as CXCL-8 and CCL-2 by LL-37 in keratinocytes is mediated through Src family kinases (SFKs) and the purinergic receptor P2X7 (Nijnik et al., 2012). Direct migration by HDPs (i.e. LL-37 and hBd-2) have also been described at supra-physiological levels (less potent than endogenous chemokines) but may suggest another arm between innate and adaptive immunity (Choi et al., 2012). Interestingly, data comparing kinocidins (microbicidal chemokines) and HDPs support the hypothesis that both evolved through a related structural signature and effector determinant motif (i.e. gamma-core) to defend a very complex and diverse microenvironment (Yount and Yeaman, 2006). While some contain the chemotactic amino acid sequences (e.g. CC, CXC), it is likely that similarities in size/shape, cysteine bonding, and cationic charge likely mediate the chemotactic activity of HDPs (Fig. 1-8) (Durr and Peschel, 2002; Yount and Yeaman, 2006). Adaptive immunity has also been demonstrated to be modulated by LL-37 and other HDPs. For example, dendritic cell maturation is upregulated with LL-37 (Davidson et al., 2004). Adjuvant-like properties of LL-37 allowed for more sensitive detection of unmethylated CpG’s such that LL-37 promoted recognition of prokaryotic CG rich DNA (but not eukaryotic DNA), in lymphocyte and dendritic cells faster and at much lower concentrations (Hurtado and Peh, 2010). Resolution phase effects such as in Figure 1-8. Conserved g-core motif (red) across diverse disulfide-containing host peptides. Antimicrobial peptides (APS) Reprinted from (Yount and Yeaman, 2006) with permission from Elsevier. 30 wound healing, demonstrate LL-37 dependent repair mechanisms through EGFR transactivation and metalloproteinases resulted in wound re-epithelialization, while its absence was associated with chronic ulcers (Heilborn et al., 2003; Tjabringa et al., 2003). Additionally, controlled cell turnover is critical for resolution and frustrated catabolic metabolism of cellular components (autophagy) can exacerbate inflammation and impair intracellular pathogen killing (Levine et al., 2011). Interestingly, LL-37 with vitamin D3 show enhanced autophagy through Beclin-1 (Yuk et al., 2009). Furthermore, LL-37 demonstrates cell type specific turnover by promoting caspase 3 and 9 in infected epithelial cells, or inhibiting P2X7 in neutrophils (Barlow et al., 2010). Reductions in excessive pyroptosis (caspase-1 dependent cell death), and thus inflammation, have also been demonstrated with LL-37 (Hu et al., 2014). Finally, pathogen clearance/neutralization have been reported through HDP dependent mechanisms. Neutrophil extracellular traps (i.e. DNA network fibers) which assist in killing extracellular microbes are augmented by HDPs (e.g. HNP-1 and LL-37) through direct killing or improved stability of NETs through nuclease inhibition mechanisms (de la Fuente-Nunez et al., 2014; Neumann et al., 2014; von Kockritz-Blickwede et al., 2008). The importance of HDP dependent enzyme inhibition against pathogen virulence has also been demonstrated in anthrax-infected Table 1-2. Examples of Immunoregulatory functions of HDPs and IDRs a a Immune defense regulator (IDR) peptide Reproduced from (Choi et al., 2012) with permission from Karger Publishers 31 mice. Most notably q-defensin inhibition of the Zn +2 dependent metalloprotease in the anthrax lethal factor complex protects mice from otherwise certain death (Gould et al., 2012; Wang et al., 2006). Intriguingly, other host defense peptides such as LL-37 and serine leukocyte protease inhibitor (SLPI) have demonstrated host cysteine and serine anti-peptidase activity respectively and serve as a counter measure to limiting collateral damage from exuberant inflammation (Andrault et al., 2015; Sallenave et al., 1997). Their role in limiting excessive inflammation will be explored later. It should be noted that these effector functions typically occur at lower concentrations than their microbicidal killing and experts view immune regulation as the principal action of HDPs (Mansour et al., 2014). The CF Microenvironment and Host Defense Peptides In CF lung disease, human neutrophil peptides (HNP) 1-3, LL-37 (a cathelicidin), lysozyme, and lactoferrin can all be found elevated in airway secretions; the result of a massive neutrophil presence in the infected lungs (Hiemstra et al., 2016; Sagel et al., 2009). However, despite this enhanced presence, infection persists. Furthermore, not all HDPs are elevated. The human b defensin (DEFB2)-2 is observed to be reduced in bronchoalveolar lavage fluid despite no evidence of transcriptional or translational abortions in CF (Hiemstra et al., 2016). Underscoring the importance of HDPs to innate defense, is the negative linear relationship with DEFB2 and lung function (Chen and Fang, 2004). Reasons for a weakened innate defense in the context of CF will be explored below. Inactivation and Predisposition to Infection A number of unfavorable conditions exists in CF airway disease that may account for the dysregulation and inactivity of HDPs. First, the heavy protease burden- derived from host and pathogen- in CF suggest proteolytic degradation of HDPs. Elevated CF airway soluble cysteine cathepsins have been reported to degrade b-defensin-2,3, lactoferin, SLPI, and LL-37 (Andrault et al., 2015; Rogan et al., 2004; Taggart et al., 2003; Taggart et al., 2001). The host serine 32 protease neutrophil elastase and pathogen metalloproteinase pseudolysin have been shown to cleave lactoferin and elafin (Guyot et al., 2010; Guyot et al., 2008; Rogan et al., 2004). Furthermore, host neutrophil elastase has also been shown to cleave SLPI (Weldon et al., 2009). Second, the high polyanionic nature of sputum (e.g. DNA, F-actin, etc.), likely complexes and inactivates HDPs (Hiemstra et al., 2016). Third, the acidic pH of ASL in CF reduces bacterial killing of HDPs such as lysozyme, lactoferin, LL-37, and b-defensin-3 (Abou Alaiwa et al., 2014; Pezzulo et al., 2012). Lastly, the ionic strength of the local environment regulates the activity of some HDPs (Ganz, 2003). While, the “Salt Wars” debate in the CF community has largely concluded that ionic compositions in the lung don’t change -it is still a theoretical possibility. Collectively, it appears most of the HDPs are susceptible to the inactivating conditions in the airways of CF and at this time data support altered expression secondary to environmental cues, not modifier gene activity in CF. q-Defensins as a Potential CF Therapeutic The Current State of the CF Lung Armamentarium Currently, Chronic CF airway disease management is aimed at controlling ongoing infection, inflammation, and obstruction. Figure 1-9 summarizes the current standard of care for these patients. Recently, clinical trial data have established disease defining airway neutrophilia, neutrophil elastase (NE), and hyper- inflammation as the strongest markers of disease severity (Mayer-Hamblett et al., 2007). Importantly, glucocorticosteroids and ibuprofen therapies have shown improved disease prognosis through slowing the rate of lung function decline demonstrating inflammation as an important therapeutic target Figure 1-9. Pathophysiology and Drug Treatment in CF Airway Disease. Reproduced from (Worlitzsch et al., 2002) with permission from American Society for Clinical Investigation. 33 (Eigen et al., 1995; Konstan et al., 1995; Konstan et al., 2007). However, serious adverse effects limit their long-term clinical use. To date only azithromycin is used as anti-inflammatory agent. Furthermore, ibuprofen has only demonstrated long-term benefits in a narrow patient population (6-12 years of age with FEV 1 ³ 60%) with drug utilization therefore at a dismal 3% (Foundation, 2014; Konstan et al., 1995; Konstan et al., 2007). Chronic low dose azithromycin is the most prominent anti-inflammatory taken (68% utilization) (Foundation, 2014). However, a modest 4% improvement in lung function (FEV 1 ) over 6 months paired with retrospective data demonstrating that these benefits were not maintained beyond a year indicates this therapy provides only modest effects (Fleet et al., 2013; Southern et al., 2012). Thus, there is a clinical need for safe anti- inflammatory drugs to control the persistent neutrophilic inflammation, and tissue destruction of the lung that accounts for 90% of deaths in CF (Foundation, 2014). Rhesus q-Defensin-1 Antimicrobial Actions Rhesus q-defensin-1 (RTD-1) is a macrocyclic peptide with broad spectrum antimicrobial activity. The cyclic structure appears to be important for antimicrobial activity (Conibear et al., 2013; Tang et al., 1999). Early investigations found this peptide to be a salt-insensitive (150 mM NaCl) microbicidal agent with activity against Escherichia Coli, Staphylococcus aureus, and Candida albicans, (Tran et al., 2008). In contrast to the a- and b-defensins which inhibit peptidoglycan biosynthesis via impairment of conserved precursors on the cell wall (e.g. lipid II), RTD-1 impairs or disrupts the membrane barrier which is dependent on the membrane potential of the bacteria. This disruption of the membrane allows for the release of peptidoglycan lytic enzymes (referred to as autolysins) that are critical for bacterial killing (Wilmes et al., 2014). RTD-1 also appears to have anti-viral actions through inhibition of host cell entry by binding to viral proteins gp120 and CD4, as well as, downregulating CXCR4 thus blocking viral entry and replication (Seidel et al., 2010). Subsequent work has expanded our understanding of the spectrum of activity of RTD-1 34 including activity against methicillin-resistant S. aureus and ciprofloxacin resistant P. aeruginosa isolates from patients with bacteremia, pneumonia, wound, or urinary tract infections (Tai et al., 2015). Our laboratory has demonstrated that RTD-1 exhibits rapid bactericidal activity against CF clinical isolates of P. aeruginosa including multi-drug resistant and colistin (cationic peptide) resistant strains (Chapter 2) (Beringer et al., 2015). Anti-inflammatory Actions In addition to these antimicrobial properties, a growing number of investigations disclose that RTD-1 is a multifunctional immune effector peptide. Work in macrophage cell lines and blood leukocyte cultures have shown RTD-1 significantly tempers TLR agonist stimulation that is independent of direct neutralization of the agonist (Schaal et al., 2012). Keystone molecules such as TNF, IL-1b, IL-6 and IL-8 were reduced at 4 hours. Interestingly, RTD-1 analogs exhibited differential effects in blocking release of TNF that suggest a structure activity relationship. Data from LPS stimulated monocytes and macrophages confirmed reductions in the early release/secretion of TNF, IL-1b, and IL-8 (Tongaonkar et al., 2015). Anti-inflammatory activity appears to be mediated by inhibition of cell signaling. In particular, cyclic, but not acyclic, RTD-1 was shown to inhibit NF-kB. Treatment also inhibited LPS stimulated phosphorylation of p38 MAPK and JNK. Importantly, RTD-1 alone did not reduce phosphorylation of these kinases, rather it increased phosphorylation of AKT in both RTD-1 alone and RTD-1/LPS conditions. PI3K inhibitor studies demonstrated that RTD-1 dependent AKT phosphorylation mediates NF- kB activity and pro-inflammatory cytokine production (Tongaonkar et al., 2015). Thus, RTD-1’s anti- inflammatory actions are at least in part through induction of AKT phosphorylation. Elucidation of other mechanisms is currently under investigation. Preclinical investigations of RTD-1 reveal therapeutic promise in models of acute infection/inflammation. Data from two murine models of bacteremic sepsis showed 35 subcutaneously administered RTD-1 improved survival outcomes and reduced circulating levels of pro-inflammatory mediators (Schaal et al., 2012). Additionally, RTD-1 given intranasally as a prophylactic agent in a model of severe acute respiratory disease in mice conferred a survival benefit that was independent of any anti-viral mechanism as viral titers were unchanged in the treatment groups (Wohlford-Lenane et al., 2009). Global reductions in cytokines and chemokines in treated animals suggest the survival benefit was related to the immunomodulatory activity of RTD-1. (Wohlford-Lenane et al., 2009). Safety A series of in vitro and animal data from multiple species demonstrate the excellent safety profile of RTD-1. In vitro, the peptide ( up to 100 µg/mL) was found to be non-hemolytic and non-cytotoxic in erythrocyte and fibroblast assays containing 10% FBS. In vivo, subcutaneous RTD-1 therapy in mice, rats, and chimpanzees demonstrated no immunogenicity or toxicity as measured by serial serum chemistries, hematological parameters, and dot blots after multiple injections (Schaal et al., 2012). Formal preclinical toxicology studies are currently being performed (Selsted personal communication). Of potential concern with the therapeutic development of aerosolized RTD-1 is the recent clinical failure of an aerosolized protegrin-1 (PG-1) derivative (iseganan) due to toxicity concerns. However, recent in vitro work from anionic lipid monolayer interaction studies suggest that RTD-1 would interact poorly with zwitterionic lipid membranes (characteristic of eukaryotes) compared with the more amphiphilic PG-1 which may explain the contrasting safety profiles observed between the two antimicrobial peptides (Gordon et al., 2005; Knyght et al., 2016). Expanding the Arsenal: The Duality and Promise of RTD-1. Collectively, the unique characteristics of RTD-1 in terms of its structure, antimicrobial spectrum, and immune regulating properties support a pre-clinical investigation into the potential application of RTD-1 in CF airway infection and inflammation. Enclosed in subsequent chapters is the investigation and further description of RTD-1’s antimicrobial-immunomodulatory duality and potential therapeutic promise in this “lost” peptide. 36 Aims and Summary of Chapters Pulmonary disease in CF is characterized by chronic lung infections, inflammation, and airway obstruction leading to progressive loss of lung function and eventual respiratory failure of the patient. Excessive neutrophil derived proteases in the airspace disrupt immune cell function and cause degradation of the lung matrix which release matrikines that further augment neutrophil homing to the lung. Clinical data disclose strong relationships between these activities and the severity of lung disease in CF. RTD-1 is a macrocyclic peptide, that was lost to man, with broad spectrum antimicrobial and anti-inflammatory activities. Previously published results have shown improved survival in experimental models of sepsis and SARS under RTD-1 treatment. While accumulating data supports the utility of RTD-1 in treating disorders involving systemic infection and inflammatory disorders, it is not known whether these benefits would translate into models of acute and chronic lung infection/inflammation. This dissertation aims to address the safety and efficacy for RTD-1 in experimental models of CF lung disease. In chapter 2, we describe the CF relevant anti-pseudomonal activity of RTD-1 using CF P. aeruginosa isolates in in vitro microbiology assays and a murine chronic lung infection model to recapitulate the infectious hallmark of the lung disease. In chapter 3, we describe the CF relevant anti-inflammatory activity of RTD-1 using cell culture techniques and an aseptic LPS –induced murine model of pulmonary inflammation to recapitulate the intense neutrophil dominate inflammatory hallmark of the lung disease. In chapter 4, we expand upon the CF relevant anti-inflammatory and anti-pseudomonal activities of RTD-1 using CF bronchial epithelium, airway leukocytes isolated from expectorated sputum, as well as, a chronic lung infection model in CF mice to recapitulate the inflammation, infection, and lung remodeling hallmarks of the lung disease. Furthermore, we describe the pulmonary 37 stability, safety and tolerability of RTD-1 using ex vivo CF secretions and murine lung experiments. Taken together, the results of these investigations suggest that RTD-1 is safe and efficacious in several CF models of investigation. The therapeutic development of RTD-1 would provide an important new approach to the management of chronic lung disease in CF. 38 CHAPTER 2 Rhesus theta-defensin-1 (RTD-1) exhibits in vitro and in vivo activity against cystic fibrosis strains of Pseudomonas aeruginosa Paul M. Beringer. 1* Timothy J. Bensman, 1 Henry Ho, 1 Melissa Agnello, 1 Nicole Denovel, 1 Albert Nguyen, 1 Annie Wong-Beringer, 1 Rosemary She, 2 Dat Q. Tran, 2 Samuel M. Moskowitz, 3 and Michael E. Selsted. 2, 4 1 School of Pharmacy, University of Southern California, 1985 Zonal Avenue, Los Angeles, CA 90033, USA 2 Department of Pathology & Laboratory Medicine, Keck School of Medicine, University of Southern California, 2011 Zonal Avenue, Los Angeles, CA 90033, USA 3 Department of Pediatrics, Massachusetts General Hospital and Harvard Medical School, 275 Cambridge St, Boston, Massachusetts, USA 4 USC Norris Comprehensive Cancer Center, 1441 Eastlake Ave, Los Angeles, CA, USA. * Corresponding author. Tel: 323-442-1402, Fax: 626-628-3024, Email: beringer@usc.edu Running Title Antipseudomonal activity of RTD-1 Keywords: Antimicrobial peptides, Pseudomonas aeruginosa, cystic fibrosis In Press: January 1 st , 2016 in the Journal of Antimicrobial Chemotherapy 39 Abstract Objectives: Chronic endobronchial infections with Pseudomonas aeruginosa contribute to bronchiectasis and progressive loss of lung function in patients with cystic fibrosis. This study aimed to evaluate the therapeutic potential of a novel macrocyclic peptide, RTD-1, by characterizing its in vitro antipseudomonal activity and in vivo efficacy in a murine model of chronic Pseudomonas lung infection. Methods: Antibacterial testing of RTD-1 was performed on 41 clinical isolates of P. aeruginosa obtained from cystic fibrosis patients. MIC, MBC, time kill, and post-antibiotic effects were evaluated following CLSI recommended methodology but with anion depleted Mueller Hinton broth. RTD-1 was nebulized daily for 7 days to CFTR F508del-homozygous mice infected using the agar bead model of chronic P. aeruginosa lung infection. In vivo activity was evaluated by change in lung bacterial burden, airway leukocytes, and body weight. Results: RTD-1 exhibited potent in vitro bactericidal activity against mucoid and non-mucoid strains of P. aeruginosa, (MIC 90 = 8 mg/L). Cross resistance was not observed when tested against multidrug- and colistin-resistant isolates. Time-kill studies indicated very rapid, concentration-dependent bactericidal activity of RTD-1 with ≥ 3 log 10 cfu/mL reduction at concentrations ≥ 4´ MIC. No post-antibiotic effect was observed. In vivo, nebulized treatment with RTD-1 significantly decreased lung P. aeruginosa burden (mean difference of -1.30 log 10 cfu; p=0.0061), airway leukocytes (mean difference of -0.37 log 10 ; p=0.0012), and weight loss (mean difference of -12.62 %; p<0.05) when compared with controls. Conclusion: This study suggests that RTD-1 is a promising potential therapeutic agent for cystic fibrosis airway disease. 40 Introduction Chronic airway infection and inflammation are hallmarks of cystic fibrosis (CF) and contribute significantly to the morbidity and mortality in this population. Pseudomonas aeruginosa is the most common pathogen, chronically present in the lungs of nearly 80% of adults with CF. Epidemiological studies demonstrate a strong association between airway infection with mucoid P. aeruginosa and the ensuing progressive loss of lung function and shortened survival in these patients (Bragonzi, 2010; Paroni et al., 2013). Treatment of P. aeruginosa in patients with CF is challenging due to the development of resistance and adaptations that favor persistence over time within the lung environment of these patients. In particular, P. aeruginosa forms biofilms within the airways, which confer resistance to host defenses and many antibiotics (Staudinger et al., 2014). In addition, resistance to existing antibiotics is of growing concern considering the lack of novel therapies for treatment of this organism. Approximately 25% of adults with CF are chronically infected with multidrug resistant P. aeruginosa. While chronic inhaled therapy with colistin or tobramycin has been shown to decrease hospitalization for acute pulmonary exacerbations, the emergence of P. aeruginosa strains resistant to these agents underscores the need for new drugs that are active against multidrug resistant (MDR) P. aeruginosa without cross-resistance to existing antibiotics (Merlo et al., 2007; Miller et al., 2011; Moskowitz et al., 2012). Theta defensins are cyclic cationic peptides present in leukocytes of Old World monkeys. Because of a premature termination mutation in the theta defensin gene that appeared before the evolutionary appearance of hominids, theta defensin expression is limited to Old World monkeys (Nguyen et al., 2003). Thus it has been suggested that theta defensins expressed in nonhuman primates might be exploited for use as therapeutics for human diseases (Lehrer et al., 2012) Rhesus theta defensin-1 (RTD-1) exhibits potent microbicidal activity broadly against many 41 bacteria, fungi, herpes simplex virus, and human immunodeficiency virus type 1 (Tongaonkar et al., 2011; Tran et al., 2008). Of particular relevance to CF is the potent activity of RTD-1 against methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa (Tai et al., 2015). Systemic administration of RTD-1 in vivo demonstrated significantly improved survival in preclinical models of peritonitis and sepsis (Schaal et al., 2012). Previously published data demonstrate RTD-1 does not cause hemolysis or cytotoxic effects against fibroblasts (Tran et al., 2008). Based on these promising data, the aim of the current study was to test the hypothesis that RTD-1 has therapeutic potential in CF airway infection. To accomplish this, the antipseudomonal activity of RTD-1 was characterized in vitro, and its efficacy was evaluated in vivo through the use of a CFTR F508del-homozygous murine model of chronic Pseudomonas lung infection. 42 Methods and Materials Peptide synthesis The synthesis of RTD-1 (>98%) was performed using FMOC chemistry as previously described (Tran et al., 2008). Stock solutions of RTD-1 were prepared in 0.01% acetic acid for in vitro assays or in 0.85 M NaCl for administration to animals. Susceptibility Testing Study strains included a total of 41 strains of mucoid and non-mucoid P. aeruginosa from patients with CF, of which 29 were from patients at the Keck Medical Center of University of Southern California and 12 were colistin-resistant strains from patients in Denmark (Miller et al., 2011; Moskowitz et al., 2012). Agar disc diffusion susceptibility results for the 29 isolates were provided by the Keck Clinical Microbiology Laboratory. The Institutional Review Boards of the University of Southern California and Massachusetts General Hospital approved the use of clinical materials for this study. The colistin-resistant strains have been well characterized with respect to the mechanism of resistance, which include phoPQ and/or pmrAB mutations (Miller et al., 2011; Moskowitz et al., 2012). Control strains were PAO1 (supplied by Dr. Edith Porter, California State University Los Angeles) and RP73 (supplied by Dr. Alessandra Bragonzi, San Raffaele Scientific Institute). MICs and MBCs of RTD-1 were determined by broth microdilution assays according to the guidelines of the CLSI with some modifications as described below. The components of Mueller Hinton Broth (MHB) (e.g. beef extract and casein) have substantially greater concentrations of negatively charged amino acids relative to the protein composition of human tissues (Turner et al., 1998). Thus, positively charged antimicrobial peptides such as RTD-1 (+5) can be complexed and inactivated by long-chain polyanions present in MHB . In order to provide a more physiologic environment to facilitate in vitro investigations of cationic antimicrobial peptide 43 activity, anion exchange columns have been used to remove these polyanions (Turner et al., 1998). Cation-adjusted MHB (CAMHB; BBL 212322; Becton, Dickinson and Company, Sparks, MD) was prepared according to the manufacturer’s instructions. Anion-depleted MHB (ADMHB) was prepared by pumping 20 mL of CAMHB through a series of three Sep-Pak Plus NH2 anion- exchange cartridges (WAT020535; Waters, Milford, MA) as previously described, (Turner et al., 1998) followed by passage through a 0.22 µm filter (Whatman; Clifton, NJ). Both CAMHB and ADMHB supported luxuriant growth of Pseudomonas aeruginosa (data not shown). Bacteria were grown overnight at 37°C at 200 rpm in CAMHB and were then diluted with additional CAMHB to a 0.5 McFarland standard as measured by spectrophotometry at 600 nm. Bacteria were further diluted 1:200 in either CAMHB or ADMHB and 0.1 mL of a standardized inoculum was dispensed into 96-well plates in triplicate (corresponding to 0.5-1 x 10 5 cfu/well). Experiments were performed using MASTERBLOCK 96-well deep well polypropylene microplates (Greiner bio-one; Frickenhausen, Germany) to reduce potential binding of RTD-1 to the plasticware. RTD-1 concentrations ranging from 0.5 to 256 mg/L at serial two-fold dilutions were then added to the plates. The antipseudomonal colistin sulfate (Sigma; St. Louis, MO) was used as a reference antibiotic. Since most clinical strains tended to grow slower, strains that did not show visible turbidity in the negative control wells were incubated for another 24 h and re-inspected. The minimum bactericidal concentration (MBC) was determined by placing 10 µl of each well onto Pseudomonas isolation agar (PIA) plates and incubating at 37°C for 24-48 h. The MBC was defined as the lowest concentration at which no visible colonies were observed (> 3 log 10 reduction relative to input inoculum). Time-kill kinetics The kinetics of RTD-1 microbicidal activity was evaluated by broth macrodilution against PAO1 using modified MHB as described above. Briefly, an inoculum of 1x10 5 cfu/mL was exposed to a 44 range of RTD-1 concentrations at 0.25´, 1´, 4´, 16´, and 64´ MIC and incubated at 37°C. Aliquots were taken following RTD-1 exposure for 0, 0.5, 1, 1.5, 2, 3, and 4 h, then serially diluted, and plated on Pseudomonas Isolation Agar (PIA). The plates were incubated at 37°C and cfu were counted after 24 h. Killing curves were constructed by plotting the log 10 cfu/mL versus time. Post-antibiotic effect (PAE) The in vitro PAE was determined using PAO1 by standard methods (Li et al., 2001). Briefly, PAO1 in logarithmic growth phase was exposed for 15 minutes in modified MHB to RTD-1 at concentrations of 0.25´, 1´, 4´, 16´, and 64´ MIC. Unbound RTD-1 was removed by twice centrifuging the suspension at 3,000 X g for 10 minutes, decanting the supernatant, and resuspending bacteria in pre-warmed broth. Viable counts were performed at 0, 1, 2, 3, 4, 5, 6, 7, and 24 h on PIA. A growth control was performed similarly, without exposure to RTD-1. The colonies were counted after 24 h of incubation at 37°C. The PAE was defined as the difference in time required for a 1-log increase in cfu between RTD-1 exposed and control curves. Biofilm Growth and Scanning Electron Microscopy (SEM) We investigated the antibacterial effects of RTD-1 on Pseudomonas biofilms using PAO1 in a static colony biofilm assay. Biofilm growth and susceptibility testing was performed as described with some modifications (Schaber et al., 2007). Briefly, for biofilm formation, M63 media (Amresco, Solon, OH), a nutritionally deficient media, was prepared according to manufacturer’s instructions, and supplemented with 0.5% (w/v) casamino acids after autoclaving. For solid M63 media, sterile agarose solution (15 g/L) with 0.25% Tween20 was added to the M63 broth. Biofilms were grown on sterile 25 mm diameter, 0.22 µm pore-size polycarbonate membrane filters. For biofilm growth, PAO1 was inoculated from frozen stock into LB broth and grown overnight, then 45 subcultured 1:100 in M63 broth and incubated at 37°C for about 3 h, until OD 600 = 0.2. Filters (shiny side up) were gently pressed onto the surface of M63 agarose plates and 10 µl of the culture was dropped onto the center of each membrane. Membranes were allowed to dry before inverting and incubating at 37 °C. After 24 h, filters were aseptically transferred to a fresh plate, and incubated for an additional 24 h. Biofilms were imaged by scanning electron microscopy (SEM) at the USC Norris Comprehensive Cancer Center Cell and Tissue Imaging Core as previously described (Schaber et al., 2007). Briefly, bacteria were harvested and fixed in half-strength Karnovsky’s fixative, post-fixed in 2% OsO4, followed by ethanol dehydration and hexamethyldisilazane (HMDS) drying. Air dried specimens were mounted on stub using silver paste and sputter-coated with gold-palladium according to standard procedures. Specimens were visualized by SEM on a JEOL JSM-6390LV (JEOL MA, USA) operated at 10 kV accelerating tension. Biofilm Susceptibility For susceptibility testing, RTD-1 was added to the M63 agarose solution to prepare plates containing 16, 32, 64, or 128 mg/L. After 48 h of growth on M63 agarose alone, biofilms were transferred to the RTD-1-containing plates and incubated for 24 h, then transferred to a fresh RTD-1-containing plate, and incubated for an additional 24 h. Biofilms were aseptically removed, placed in 10 mL of sterile PBS, and subject to sonication for 10 minutes in a solid state ultrasonic FS-9 sonicator (Fisher Scientific Inc., Pittsburg, PA) at 4°C for 10 min with an additional 2 min of vortexing, until complete removal of biofilms from the filters. The solution was then serially diluted and plated on Pseudomonas isolation agar to obtain cfu counts, calculated as number of cfu per square centimeter of the membrane. 46 Pharmacokinetic Study Design All animal studies were conducted with approval of the University of Southern California Institutional Animal Care and Use Committee (protocol #20157). Animals were cared for in accordance with the National Research Council recommendations. Sixteen uninfected C57BL/6N male mice (Charles River, Holister, CA), 8 -12 weeks, were administered RTD-1 (10 mg/mL) in 0.83 M NaCl over 1 h via jet nebulization (6 L/min) using a BANG nebulizer coupled to a 16-port nose-only inhalation exposure system (CH Technologies, Westwood, NJ). The nebulizer produces a relatively small particle size (1 micron) to allow for maximum penetration into the airways and enables simultaneous and uniform aerosol delivery to multiple mice.(Paroni et al., 2013; Tran et al., 2008) The delivered dose to the mouse was calculated to be 167 μg/kg using standard methods. At timed intervals (0.25, 0.5, 1, 8 h), cohorts of four mice were sacrificed for bronchoalveolar lavage fluid (BALF) sample collection. BALF samples were stored acidified with 5% acetic acid (AcOH) at 4°C until analysis. BALF RTD-1 concentration was determined by UPLC-MS/MS (see below) with correction using BALF urea concentration as previously described.(Tongaonkar et al., 2011; Tran et al., 2008) BALF sample preparation and LC-MS/MS quantification BALF samples from RTD-1 exposed animals and controls (1.5 mL) were acidified to 5% AcOH and spiked with 10 μL of internal standard (IS). After vortexing for 1 min, mixtures were loaded onto C18 SPE (Waters Corp., Milford, MA) columns. Column packing bed was hydrated with 100% MeCN and equilibrated with 5% AcOH centrifuged. After loading desalting of the sample was performed with 5% MeOH and 1% AcOH in water. Elution was performed with 70% MeCN and 5% AcOH in water. Samples were collected in siliconized serum top tubes (BD, Franklin Lakes, NJ) and speed evacuated overnight, washed with 1 mL water, frozen and lyophilized using 47 a VirTis Freezemobile 25EL lyophilizer (SP Scientific, Warminster, PA). Samples were re- suspended in 100 μL of 10% AcOH and 5% MeCN and RTD-1 concentrations were determined by LC-MS using an ACQUITY UPLC (Waters Corp., Milford, MA, USA) connected to a Micromass Quattro Ultima mass spectrometer (Micromass UK Limited, Manchester, UK). Samples were resolved using a linear gradient of water/acetonitrile/1% formic acid from 85/5/10 to 35/55/10, v/v, over 10 minutes on a C18 X-Bridge BEH column (2.5 µm, 2.1 x 150 mm XP; Waters Corp., Milford, MA) fit with a VanGuard Pre-Column BEC C18 (1.7 μM; Waters Corp., Milford, MA). RTD-1 was analyzed in positive ionization mode with declustering potential and collision energy optimized for RTD-1. The inter-assay precision in BALF ranged from 4.4% to 11.1% for calibration standards. The precision and accuracy in BALF for a quality control sample was 6% and 106%, respectively. Murine chronic P. aeruginosa lung infection model The chronic murine model of agar bead coated P. aeruginosa infection was used to assess the effect of RTD-1 on lung bacterial burden, airway inflammatory cells, and weight loss. Chronic infection was established in 8-12 week old CFTR F508del-homozygous gut-corrected mice [B6.Tg(FABPhCFTR)-Cftr tmUth1 ; Case Western University] by intratracheal instillation of a mucoid strain of P. aeruginosa (RP73) embedded within agarose beads as previously described. This model establishes chronic P. aeruginosa airway infection with stable colony counts of 10 4 -10 5 cfu/lung 7 days following instillation (Bragonzi, 2010). Animal body weight was assessed daily during the study period. Mice were euthanized on day 7, and bronchoalveolar lavage was performed. Total and differential cell counts were determined and lungs were processed for quantitative culture. 48 Aerosol administration of RTD-1. RTD-1 (10 mg/mL) in 0.83 M NaCl or saline alone was administered daily over 1 h via jet nebulization for 7 days starting 24 h after pulmonary challenge with agar bead coated bacteria (see above). This RTD-1 dose was selected based on preliminary pharmacokinetic and dose finding studies in vivo. Phagocytosis Assay We conducted in vitro experiments designed to evaluate the effect of RTD-1 on phagocytosis as prior studies have shown that defensins reduce bacterial load in mouse models of infection by enhancing macrophage phagocytosis (Schaal et al., 2012; Welkos et al., 2011). THP-1 human monocytic cells were grown in RPMI-1640 medium (Lonza; Walkersville, MD) with 0.05 mM 2- mercaptoethanol (Amresco; Solon, Ohio), 1% penicillin-streptomycin (Sigma; St. Louis, MO), and 10% fetal bovine serum (Lonza; Walkersville, MD) at 37°C and 5% CO 2 . For experiments, THP- 1 cells were resuspended in complete cell culture medium with 100 nm phorbol 12-myristate 13- acetate (Sigma; St. Louis, MO) and incubated for 72 h to allow differentiation into macrophages. Cells were washed and replenished with complete cell culture medium for 48 h to return to a resting state. Effects on phagocytosis were measured colorimetrically using the CytoSelect TM assay kit (Cell Biolabs; San Diego, CA) containing pre-labeled zymosan particles according to the manufacturer’s instructions as previously described.(Tongaonkar et al., 2011) Briefly, THP-1 macrophage cells at 5 x 10 5 cells/mL were pre-incubated for 15 min with RTD-1 over several logs of concentration (0.01-10 mg/L), cytochalasin D (phagocytosis inhibitor), and no treatment. Zymosan particles were added in duplicate, and the samples were incubated for 2 h. Samples were washed, fixed with formaldehyde, blocked, and permeabilized with multiple wash steps in between. An enzyme-labeled detection reagent was added followed by colorimetric substrate. 49 Absorbance was measured at 405 nm, and results are presented as percentage relative to no treatment. Pharmacokinetic Analysis Pharmacokinetic parameters were derived from the concentration time profiles using non- compartmental analysis in accord with statistical moment theory and the linear trapezoidal rule. Statistical analysis Data that follow a normal distribution are reported as mean ± standard deviation. Log 10 transformed data were found to be near normal by measures of central tendency as well as skewness and kurtosis and reported as geometric mean ± 95% confidence interval. Comparisons between two groups of normally distributed data were made using the unpaired two-sample student’s t test or 2-way ANOVA. A two-sided P value of ≤0.05 was considered significant. Bonferroni post-test was used when appropriate. All statistics were performed with GraphPad Prism 6.0 (San Diego, CA). 50 Results RTD-1 exhibits potent in vitro antibacterial activity against P. aeruginosa isolates from patients with CF. Susceptibility testing was performed on 41 clinical strains of P. aeruginosa. For 29 of the isolates the phenotype (mucoid or nonmucoid) as well as agar disc diffusion susceptibility results were available from the Keck Clinical Microbiology Laboratory (Table 2-1). About half (55%) of the isolates were mucoid; 38% were multidrug-resistant (defined as resistance to agents belonging in 2 of the 3 major classes of antipseudomonal antibiotics: meropenem, tobramycin, ciprofloxacin). Antimicrobial activity of RTD-1 against the panel of clinical isolates was approximately 16-fold lower using CAMHB compared with ADMHB; MIC 50 and MIC 90 values were 64 and 128, and 4 and 8 mg/L using CAMHB versus ADMHB, respectively. In contrast, only modest media-specific effects were observed with colistin: MIC 50 and MIC 90 values were >256 and >256, and 64 and 256 mg/L when using CAMHB versus ADMHB, respectively. RTD-1 exhibited antibacterial activity against clinical strains of P. aeruginosa regardless of mucoid (MIC 90 8 mg/L versus 4 mg/L for nonmucoid) or multidrug-resistant phenotype (MIC 90 8 mg/L for MDR versus 8 mg/L for all isolates), demonstrating a lack of cross-resistance to existing classes of antipseudomonal antibiotics. The MBC values for RTD-1 against the clinical strains were on average within 1 tube dilution of the MIC indicating RTD-1 is bactericidal with a mean MBC:MIC ratio of ≤ 4. (Tongaonkar et al., 2011) Additionally, RTD-1 activity was tested in ADMHB against 12 strains of P. aeruginosa that showed high-level colistin resistance caused by mutations in phoPQ and/or pmrAB (Miller et al., 2011; Moskowitz et al., 2012) which confer cross-resistance to other cationic antimicrobial peptides (McPhee et al., 2003) (Table 2-2). RTD-1 exhibited excellent antibacterial activity against the colistin-resistant isolates with MIC 50 and MIC 90 values of 3 and 4 mg/L respectively, in the same range as that of the colistin-susceptible isolates (MIC 50 and MIC 90 values of 4 and 8 mg/L respectively). 51 Table 2-1. Activity of RTD-1 against CF clinical isolates of Pseudomonas aeruginosa All (n=29) M/NM (n=16/13) CIP R (n=13) MEM R (n=13) TOB R (n=5) MDR (n=11) MIC 50 a 4 4/4 4 4 4 4 MIC 90 b 8 8/4 6.4 8 8 8 MIC range 2-8 2-8/2-4 2-8 2-8 4-8 2-8 MBC 50 c 4 4/4 4 4 4 4 MBC 90 d,e 16 30/13 42 42 8 53 MBC range 2-64 2-64/2-16 2-64 2-64 4-8 2-64 MBC/MIC f 1.8 1.8/1.8 2.1 2.1 1.1 2.2 Note: M = Mucoid, NM = Nonmucoid, R = resistance determined by agar based disc diffusion according to CLSI criteria, MEM = meropenem, CIP = ciprofloxacin, TOB = tobramycin, MDR = multidrug-resistant a,b MIC 50 and MIC 90 ; MIC (mg/L) inhibiting 50 and 90% of the strains tested respectively. c,d MBC 50 and MBC 90 ; MBC (mg/L) killing 50 and 90% of the strains tested respectively. e One isolate (mucoid, mer and cip resistant) exhibited an MBC of 64 mg/L f Media 52 Table 2-2. Activity of RTD-1 against colistin-resistant clinical strains of P. aeruginosa with known resistance mutations in phoPQ and/or pmrAB Isolate Colistin RTD-1 MIC (mg/L) MIC (mg/L) MBC (mg/L) 1019 4 4 4 1565 8 4 4 1995 8 2 4 1033 16 2 4 1603 32 4 4 1571 64 1 2 1581 64 2 4 1582 64 2 4 1016 128 4 4 1018 128 2 4 1020 >256 4 4 1597 256 4 4 53 RTD-1 exhibits rapid, concentration-dependent bactericidal activity against PAO1 Based on the observation that RTD-1 exhibits bactericidal activity, we sought to further characterize its pharmacodynamic properties by performing time-kill and postantibiotic effect experiments. Results of the time-kill studies indicated very rapid, concentration-dependent bactericidal activity of RTD-1 against PAO1 (Fig. 2-1). Greater than a 3 log 10 cfu/mL reduction was achieved with RTD-1 at concentrations equal to or greater than 4 times MIC (MIC = 2 mg/L). No appreciable post-antibiotic effect was observed since bacterial regrowth ensued within 60 minutes following short exposures to all concentrations tested (data not shown). Figure 2-1. Concentration-dependent killing of RTD-1 against PA01. After overnight growth in CAMHB the bacterial suspension (PAO1 RTD-1 MIC = 2 mg/L) was diluted 1:200 in ADMHB and incubated with RTD-1 at various concentrations. Aliquots were taken at specified times, serially diluted, incubated for 24 h at 37°C for cfu counting 54 Activity of RTD-1 against P. aeruginosa biofilms Pseudomonas aeruginosa is known to adapt to the lung environment in patients with CF and resist host defenses as well as the action of antibiotics through the formation of biofilm aggregates (Staudinger et al., 2014). Several cationic peptides have demonstrated activity against P. aeruginosa biofilms when tested at concentrations exceeding the MIC against planktonic bacteria (Dosler and Karaaslan, 2014). We therefore tested the activity of RTD-1 against PAO1 biofilms using a colony biofilm assay. Aggregates of P. aeruginosa on polycarbonate membranes were visible by scanning electron microscopy (Fig. 2-2). Treatment of established biofilms with RTD-1 demonstrated a modest but statistically significant reduction in cfu when compared to negative control at all concentrations tested (Figure 2). RTD-1 exerted maximum effect at 32 mg/L, decreasing biofilm abundance by an average of 74.2% compared to control. However, there was no increase in effect with increasing concentrations (64 and 128 mg/L respectively). A. B. Figure 2-2. Activity of RTD-1 against PA01 biofilms. Biofilms were grown on polycarbonate membranes placed on M63 agarose plates (A) EM image of PAO1 aggregates on polycarbonate membranes. After 48 h growth, the membranes were transferred to fresh agarose plates containing RTD-1. (B) Activity of RTD-1 against PAO1 Biofilms. After exposure to RTD-1 at various concentrations (8-64 X MIC) for 48 hours, biofilms on the membranes were resuspended in sterile PBS, diluted and plated to determine cfu. Values represent an average of 3 independent experiments ± SEM. * P < 0.05 for comparison 55 PK of Aerosolized RTD-1 The epithelial lung fluid (ELF) concentration-time plot is shown in (Fig. 2-3). The area-under-the ELF concentration versus time curve of RTD-1 over 8 hours (AUC 0-8 ) was 200.9 ± 16.1 mg/L × h, with a maximum concentration (C max ) in the ELF of 50.64 ± 10.49 mg/L at 30 minutes post nebulization. The AUC 0-8 : MIC was 50.2 ± 4.0 and the peak ELF concentration to MIC was 12.7 ± 2.6. The half-life of RTD-1 was 3.2 ± 0.7 hours in the airway with the time above the MIC estimated to be 11.8 ± 3.1 hours. Figure 2-3. RTD-1 concentrations in epithelial lining fluid (ELF) versus time after a single aerosolized dose of RTD-1 in non-infected C57BL/6 mice. Data represent mean ± SEM from mice (n=4 each timepoint) with linear regression line. The dashed line indicates the in vitro MIC of the P. aeruginosa isolate (RP73) used in the chronically infected mice. 56 Aerosolized RTD-1 is efficacious in a murine model of chronic P. aeruginosa lung infection. The agar bead model of chronic P. aeruginosa airway infection has been used for evaluating the activity of antibacterial agents for potential application in the treatment of CF lung disease(Bragonzi, 2010; Paroni et al., 2013). This model was predictive of the therapeutic failure of a leukotriene B4 antagonist in patients with CF and has been recommended as a preclinical screening tool for potential anti-inflammatory therapies in CF (Sagel, 2015). A comparator group of ten cystic fibrosis transmembrane conductance regulator (CFTR) F508del-homozygous mice was exposed to aerosolized saline. A significant reduction in log 10 cfu of P. aeruginosa was observed when compared with control at the end of a 7-day treatment (mean difference of -1.30, p=0.0061; 95% CI: -2.18 to -0.42) (Fig. 2-4a). These results are consistent with the in vitro activity of RTD-1 in ADMHB against the infecting strain RP73 (RTD-1 MIC = 4 mg/L). RTD-1 treatment also produced significant reduction in airway leukocytes (mean log 10 difference of -0.37 cells, p=0.0012; 95% CI: -0.57 to -0.16) (Fig. 2-4b) as well as significantly less weight loss at days 6 (mean difference of -12.25%; p<0.05) and 7 (mean difference of -12.62%; p<0.05) (Fig. 2-4c). Mortality was not observed in this chronic infection model. 57 C A B Figure 2-4. Aerosolized RTD-1 significantly reduces lung cfu in a murine model of chronic P. aeruginosa lung infection. Total lung cfu were counted 7 days after intratracheal inoculation with P. aeruginosa (RP73 MIC = 4 mg/L) in ΔF508 mice. Nebulized treatment with a delivered dose of 167 μg/kg RTD-1 or saline began 24 hours post inoculation. Data were pooled from 2 independent experiments (Saline n=10)(ΔF508 n=12). Treatment with aerosolized RTD-1 was associated with a significant reduction in (A) log 10 cfu of P. aeruginosa (mean difference of -1.30 [95%CI: -2.18 to -0.42]; **p-0.0061), (B) log 10 airway leukocytes (mean difference of -0.37 [95% CI: -0.57 to -0.16] ; **p-0.0012 ), data represent mean ± SEM, and (C) weight loss at days 6 (mean difference of -12.25; *p<0.05) and 7 (mean difference of -12.62; * p<0.05) when compared with control. Two sample Student’s t-test two tailed with alpha =0.05. 58 Phagocytosis Cationic peptides have been reported to enhance phagocytosis of bacteria in vivo. Therefore, we conducted experiments designed to determine if RTD-1 has an effect on phagocytosis, which could account for the reduction in lung bacterial counts in the murine model of chronic P. aeruginosa infection. Using an in vitro model with zymosan particles, we found that treatment of THP-1 macrophages with RTD-1 did not have any appreciable effect on phagocytosis, which suggests a direct bactericidal activity of RTD-1 (Fig. 2-5). Figure 2-5. Phagocytosis of zymosan particles by THP-1 macrophages. THP-1 macrophages cells at 5 x 10 5 cells/mL were preincubated with RTD-1 (10-0.01 mg/L), the known phagocytosis inhibitor cytochalasin D, or no treatment for 15 min. Zymosan particles were added in duplicate, and the samples were incubated for 2 h. An enzyme-labeled detection reagent was added followed by colorimetric substrate. Absorbance was measured at 405 nm, and results are presented as percentage relative to baseline. 59 Discussion The relatively high prevalence of antibiotic resistance in P. aeruginosa and adaptations, including the propensity to form biofilm aggregates in CF lung disease, highlights the critical need for novel therapies to combat this pathogen. Theta defensins are cationic macrocyclic antimicrobial peptides isolated from leukocytes of Old World monkeys including rhesus macaques and Olive baboons (Tang et al., 1999). The peptides are not expressed in humans, great apes, or other hominids. Six rhesus macaque theta defensin isoforms have been identified, with RTD-1 being the most abundant, accounting for nearly 50% of the RTD content in polymorphonuclear neutrophils (PMNs) (Tongaonkar et al., 2011). The cyclic backbone structure of theta defensins is unique among peptides produced in the animal kingdom and provides remarkable stability (e.g. heat, low pH) and increased microbicidal activity when compared with their acyclic versions (Schaal et al., 2012; Tang et al., 1999). Importantly, the cyclic structure confers stability to proteases (unpublished data; P. Beringer, T. Bensman), which is relevant since the airways of patients with cystic fibrosis are known to contain high concentrations of serine and metalloproteases. RTD-1 exhibits potent broad-spectrum microbicidal activity against Escherichia coli, Staphylococcus aureus, and Candida albicans (Tongaonkar et al., 2011; Tran et al., 2008). Of particular relevance to CF is the potent activity of RTD-1 against clinical isolates of methicillin- resistant Staphylococcus aureus and fluoroquinolone-resistant Pseudomonas aeruginosa (Tai et al., 2015). The mechanism of the antibacterial effect of RTD-1 was investigated in studies on S. aureus and E. coli and is attributed to peptide-induced permeabilization/impairment of the cytoplasmic membrane of both organisms (Tran et al., 2008; Wilmes et al., 2014). 60 In this study, we evaluated the antibacterial activity of RTD-1, against CF clinical isolates of P. aeruginosa, including isolates with mucoid, multidrug-resistant, and colistin-resistant phenotypes. RTD-1 was bactericidal against a panel of antibiotic-resistant clinical isolates including several strains with phoPQ and/or pmrAB mutations that confer high resistance to colistin. In vitro activity was present only when tested in ADMHB, consistent with other studies that have evaluated the antibacterial activities of cationic antimicrobial peptides (Giacometti et al., 2000; Turner et al., 1998). ADMHB, described by Lehrer and colleagues, eliminates RTD-1 binding polyanions from CAMHB, but still allows for luxuriant bacterial growth (Turner et al., 1998; Yu et al., 2000). Importantly, the antipseudomonal activities of RTD-1 in vitro in ADMHB correlated with efficacy of the peptide in vivo. The low MBC/MIC ratio and rapid bactericidal activity in the time-kill experiments demonstrate that RTD-1 exhibits highly potent antipseudomonal activity. These properties are characteristic of several cationic antimicrobial peptides in vitro, which exert their antimicrobial activity by disrupting the bacterial cell membrane and gaining access to critical intracellular functions. In particular, the rapid concentration-dependent bactericidal activity observed with RTD-1 very closely mirrors that of colistin with nearly complete bacterial killing within 90 minutes at RTD-1 concentrations as low as 4 times the MIC (Li et al., 2001). RTD-1 did not demonstrate any appreciable post-antibiotic effect in vitro. This contrasts with the aminoglycosides and colistin, which exhibit in vitro post-antibiotic effects (Craig and Ebert, 1990; Li et al., 2001). Knowledge of these pharmacodynamics characteristics suggests that dosing of RTD-1 should be optimized by maximizing the peak concentration while minimizing the length of 61 time that concentrations are below the MIC. Ongoing pharmacokinetic studies will help define the optimal dose and frequency of administration to maximize antibacterial activity. The antibacterial activity of RTD-1 was compared with colistin, a cationic peptide currently in clinical use for treatment of infections involving multidrug-resistant P. aeruginosa in CF. Colistin exerts its antibacterial activity through binding to LPS, the major constituent of the Gram-negative outer membrane, resulting in membrane permeabilization. Intracellularly, colistin disrupts cellular respiration resulting in cell lysis and death. Mutations in phoPQ and pmrAB confer high level resistance to colistin as a result of modifications to the phosphate groups within the lipid A and core oligosaccharide moieties of LPS leading to reduced binding (Miller et al., 2011; Moskowitz et al., 2012). Due to widespread use of chronic inhaled colistin in patients with CF, it is not surprising that resistance has emerged to this agent (Miller et al., 2011; Moskowitz et al., 2012). RTD-1 retained excellent activity against CF clinical isolates that are colistin-resistant phoPQ and/or pmrAB mutants as well as those with a multidrug-resistant phenotype indicating no cross- resistance to existing antipseudomonal therapies. The MICs for all isolates were between 2-8 mg/L with no outliers’ indicative of resistance. Consistent with these findings, RTD-1 was shown to have little affinity to LPS, especially compared to polymyxin B, an analog of colistin (polymyxin E) (Schaal et al., 2012). To further assess the therapeutic potential of RTD-1 for CF, we determined its activity against P. aeruginosa biofilms. Chronic infections within the CF airways occur in part due to bacterial adaptations, specifically the formation of biofilm aggregates. The presence of biofilms is significant due to their resistance to antibiotics and host defenses (Alhede et al., 2014). Several cationic peptides have demonstrated activity against P. aeruginosa biofilms in vitro (Dosler and 62 Karaaslan, 2014; Hirt and Gorr, 2013; Kapoor et al., 2011; Pompilio et al., 2012). Using a static colony biofilm assay, we found that RTD-1 exhibits moderate activity against P. aeruginosa biofilms. Significant activity was demonstrated starting at concentrations of 8 times the MIC; however, higher concentrations did not result in greater killing. The basis for this plateau phenomenon is not known, but potentially reflects phenotypic variability among organisms within the biofilm. Studies are underway to delineate the mechanistic basis for the observed plateau in the dose-response against biofilm bacteria. We evaluated the efficacy of RTD-1 in vivo using a CFTR F508del-homozygous murine model of chronic P. aeruginosa lung infection. RTD-1 was administered via nebulization to maximize concentrations within the airways. Seven-day treatment of infected mice with nebulized RTD-1 significantly reduced lung infection burden, the number of airway neutrophils, and weight loss when compared with saline control. Pharmacokinetic studies demonstrated achievement of therapeutic concentrations in the airways for approximately 12 hours. RTD-1 administered via aerosol represents an attractive therapeutic option for the CF patients due to the potential of achieving high local lung concentrations. The results of the phagocytosis assay indicate that RTD-1 has no effect on phagocytosis. These data suggest the killing activity of RTD-1 against P. aeruginosa in vitro and in vivo is a direct antibacterial effect. 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Selsted 2* , Paul M. Beringer 1* 1 University of Southern California, School of Pharmacy, 1985 Zonal Avenue, Los Angeles, CA 90033 2 University of Southern California, Keck School of Medicine, 2011 Zonal Avenue, Los Angeles, CA 90033. 3 State University of Maringáfits, Maringá, Paraná, Brazil * Corresponding author. Tel: 323-442-1402, Fax: 626-628-3024, Email: beringer@usc.edu * Co-Corresponding author. Tel: 323-442-1180, Fax: 323-442-3314, Email: selsted@usc.edu Short Title: Anti-inflammatory effect of RTD-1 in ALI Abbreviations ALI, acute lung injury; SARS, severe acute respiratory syndrome; BALF, bronchoalveolar lavage fluid; RTD-1, rhesus theta defensin-1;; MH-S, murine alveolar macrophage cell line; HL-60; human promyelocytic leukemia cell line; NCA, non-compartmental analysis; AUC, area under the curve. In Preparation: Pharmacological Research –Pharmacol Res 71 Abstract BACKGROUND AND PURPOSE: Acute lung injury (ALI) is a clinical syndrome characterized by acute respiratory failure and is associated with substantial morbidity and mortality. Rhesus theta defensin-1 (RTD-1) is an antimicrobial peptide with immunomodulatory activity. This study evaluated the therapeutic efficacy of RTD-1 in a pre-clinical model of ALI. EXPERIMENTAL APPROACH: In vitro experiments were performed with differentiated HL-60 and LPS-stimulated MH-S cells to assess the effect of RTD-1 on neutrophil chemotaxis and early leukocyte-driven pulmonary inflammation. In vivo, single escalating doses of RTD-1 were administered subcutaneously in a murine model of endotoxin induced ALI. Lung injury was assessed by histopathology, pulmonary edema, inflammatory cell recruitment, and inflammatory cytokines/chemokines in the bronchoalveolar lavage fluid. KEY RESULTS: In vitro studies demonstrated that RTD-1 suppressed chemotaxis of differentiated HL-60 cells and MH-S alveolar macrophage activity. Pharmacokinetic studies demonstrated RTD-1 administered subcutaneously at 5- and 25 mg kg -1 achieved serum concentrations predicted to inhibit neutrophil chemotaxis in vivo. Treatment with RTD-1 significantly inhibited LPS-induced ALI by reducing pulmonary edema and histopathological changes. RTD-1 was associated with dose- and time-dependent inhibition of proinflammatory cytokines (TNF, IL-1b, and IL-6), peroxidase activity, neutrophil recruitment into the airways, and induction of the anti-inflammatory cytokine soluble gp130. CONCLUSIONS AND IMPLICATIONS: These studies demonstrate that RTD-1 is efficacious in an experimental model of ALI possibly by inhibiting neutrophil chemotaxis and attenuating keystone responses of the resident alveolar macrophage. 72 Introduction Acute lung injury (ALI) is a clinical syndrome characterized by lung edema, impaired gas exchange, and respiratory failure (Matthay & Zimmerman, 2005). The principal cause of ALI is the excessive and prolonged activation of neutrophils, in response to infection or aspiration, which cause damage to the pulmonary parenchyma and microvasculature (Johnson & Matthay, 2010). Proinflammatory cytokines are associated with the severity of ALI, underscoring the importance of inflammation as a therapeutic target (Park et al., 2001). Despite improvements in the understanding of the pathophysiology of ALI, outcomes remain poor with significant morbidity and mortality at 40% (Johnson & Matthay, 2010). Some have proposed a paradigm shift toward prevention or early treatment of ALI with the goal of improving outcomes (Levitt & Matthay, 2012). Defensins are cationic antimicrobial peptides that promote key innate and adaptive immune responses within the lungs. In humans, the a-defensins are primarily expressed in neutrophils while the b- defensins are expressed in epithelial cells lining the respiratory tract. While first studied for their broad spectrum activity against bacteria, fungi, and viruses; defensins also contribute to recruitment of inflammatory cells and activation of adaptive immune responses. While the a- and b- defensins are critical to mounting an effective immune response to a pathogen, the a-defensins have been shown to contribute to the pathogenesis of ALI by disrupting the capillary-epithelial barrier (Bdeir et al., 2010). Theta (θ) defensins are macrocyclic antimicrobial peptides found in leukocytes of Old World Monkeys (Lehrer, Cole & Selsted, 2012; Selsted, 2004). They are 18 amino acids long and are formed by the dimeric head-to-tail ligation of two α-defensin related precursors and further stabilized with three disulfide bonds. Similar to the human defensins, Rhesus Theta Defensin 1 73 (RTD-1) exhibits broad spectrum microbicidal activity against viruses, bacteria, and fungi (Beringer et al., 2015; Tai et al., 2015; Tran et al., 2008; Tran, Tran, Tang, Yuan, Cole & Selsted, 2002). In contrast to the a-defensins which are pro-inflammatory, RTD-1 exhibits anti- inflammatory activity in vitro (Schaal et al., 2012; Tongaonkar et al., 2015). In murine models of polymicrobial sepsis and severe acute respiratory syndrome (SARS), RTD-1 treatment significantly reduced inflammatory cytokines (Schaal et al., 2012; Wohlford-Lenane et al., 2009). Importantly, early pre-clinical investigations in mice, rat, and chimpanzee suggest RTD-1 to be safe and non-immunogenic (Schaal et al., 2012). With its dual antimicrobial and anti-inflammatory properties, RTD-1 may be well suited to the treatment of infection, a frequent predisposing factor for ALI, and the inflammation that directly causes ALI. In this study, we sought to investigate whether the immunomodulatory potential of RTD-1 mitigates the ALI properties of cellular airway influx and leukocyte-induced lung injury. (Proudfoot, McAuley, Griffiths & Hind, 2011). To achieve this we treated mice with RTD-1 before or immediately after initiation of pulmonary inflammation using the well-established murine model of intranasal LPS-induced acute lung neutrophilia (Matute-Bello et al., 2011b; Proudfoot, McAuley, Griffiths & Hind, 2011). In vitro investigations using neutrophil chemotaxis assays, as well as LPS-induced alveolar macrophage inflammation provide supportive details on the target cell population. The effects of subcutaneous RTD-1 on airway neutrophil burden and activation, cytokine/chemokine release, pulmonary vascular leakage, and extent of lung injury were evaluated. We provide evidence that RTD-1 reduces LPS-induced lung injury by inhibiting neutrophil recruitment and alveolar macrophage pro-inflammatory cytokine production. 74 Methods and Materials Human and murine cell cultures The human promyelocytic leukemia cells, (HL-60, a kind gift from Dr. Wei-Chiang Shen), were maintained in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO) containing 10% fetal bovine serum (FBS). Cells were cultured to a density of 2 × 10 6 cells ml -1 . Previously described methods for DMSO-driven neutrophilic differentiation (dHL-60s) were followed (Millius & Weiner, 2009). The murine (BALB/c) alveolar macrophage cells, MH-S, were purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in RPMI 1640 (Sigma-Aldrich, St. Louis, MO) containing 10% FBS (Sigma-Aldrich), 1% Penicillin-Streptomycin (Amresco, Dallas, TX), and 0.05 mM 2-Mercaptoethanol (Amresco). In vitro chemotaxis Neutrophil migration was assessed using dHL-60 cells (5 × 10 5 in 200 uL HBSS w/o Ca +2 or Mg +2 ) in a 3 μm pore size Thincert transwell system (Greiner-Bio-one, Monroe, NC) under a 10% FBS chemotactic gradient. dHL-60s were pre-incubated in triplicate with or without RTD-1 at a range of concentrations for 15 to 20 min. Inserts were then placed in media containing 600 μL of RPMI and 10% FBS in a 24 well plate and allowed to migrate for 1 h at 37°C and 5% CO 2 . To detach adherent migrated cells, 15 uL of a 0.5 mM EDTA solution was added to the lower chamber and placed at 4°C with gentle tapping every 5 min for a total of 15 min. Migrated dHL-60s were quantified by a Coulter Z2 automated particle size counter (Beckman-Coulter, Brea, CA). Percentage of maximal chemotaxis was calculated as previously described (Ha, Bensman, Ho, Beringer & Neamati, 2014). Briefly, the number of migrated cells in the presence of 75 chemoattractant alone was set to 100% after correcting for random migration (chemokinesis). The response of treated cells under chemoattractant were normalized to this positive control. In vitro airway leukocyte inflammation Alveolar macrophage inflammation was assessed using MH-S cells ( 1.25× 10 5 in 250 µL) in a 48 well plate. The cells were incubated at 37°C and 5% CO 2 for 24 hours then the media changed and the cells treated with 100 µg mL -1 RTD-1 and 100 ng mL -1 LPS. There was no pre-incubation step. After 24 hours of incubation, the supernatant was collected for cytokine analysis. Animals and ethical statement All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Southern California (Protocols 11676 and 11956). The animal studies are reported as recommended by the ARRIVE guidelines (McGrath, Drummond, McLachlan, Kilkenny & Wainwright, 2010; McGrath & Lilley, 2015). Male, 8-10 week old BALB/c mice (Charles River Laboratories, CA) weighing 20-25 g, were housed under specific pathogen- free conditions with a temperature of 22-24C and humidity of 60-65%, with 12 h light/dark cycles. The animals were provided standard laboratory chow and water ad libitum. Experimental procedures Bacterial LPS applied topically to the airways has been shown to induce acute neutrophilic airway inflammation and lung injury both in mice and humans and serves as a well-established model for investigations of the cellular influx and pulmonary disruption hallmarks of the disease (Matute- Bello et al., 2011a; Proudfoot, McAuley, Griffiths & Hind, 2011). The type and amount of LPS 76 selected have previously demonstrated lung injury secondary to neutrophil migration and therapeutic evaluations (Balloy et al., 2014; Szarka, Wang, Gordon, Nation & Smith, 1997; Wieland, Siegmund, Senaldi, Vasil, Dinarello & Fantuzzi, 2002). Mice were sedated with a single intraperitoneal injection of 100 µL of ketamine 80 mg·kg −1 (Hospira, Lake Forest, IL, USA) and xylazine 10 mg·kg −1 (Lloyd, Shenandoah, IA, USA) Acute lung inflammation was induced by intranasal administration of 10 µg of LPS in 50 μL of PBS. In the first set of experiments (dose ranging study), mice were randomly assigned to receive a subcutaneous injection of RTD-1 in 1 mL saline at 0, 0.2, 1, 5, or 25 mg kg -1 body weight or saline alone 0.5 h before LPS challenge. After 24 h following LPS administration, the mice were euthanized by sodium pentobarbital. In a separate set of experiments (time course study), peptide was administered at 5 and 25 mg kg -1 in 1 mL of saline immediately following intranasal LPS administration. After 0.5, 1, 3, 7, 24, 48, and 72 h, the mice were euthanized by sodium pentobarbital. Pharmacokinetic studies in mice Plasma concentrations of RTD-1 at 0.25, 0.5, 1, 7, 24, 48, and 72 h were measured by an LC/MS assay in LPS challenged mice receiving a single dose of 5 or 25 mg kg -1 RTD-1 subcutaneously. Samples were obtained from the above time course study as well as a separate kinetic experiment in LPS challenged mice at 0.25, 0.5, 1, 7, and 24 h. Data were expressed as ng ml -1 and plasma 77 concentration-time profiles were subjected to a non-compartmental analysis (NCA) with parallel sampling. The area under the curve (AUC) was calculated with the linear trapezoidal rule. LC/MS analysis of RTD-1 Approximately 400 µL of blood or 2.6 mL Bronchoalveolar lavage fluid (BALF) was collected at 0.25, 0.5, 1, 7, 24, 48, and 72 h after RTD- 1 administration. Blood was collected by cardiac puncture into EDTA-tubes and plasma and BALF prepared by a two-step centrifugation (200 x g for 10 min followed by 23,000 x g for 15 min). Samples were transferred to siliconized tubes and stored at -80° C. Concentrations of RTD-1 were determined after solid phase extraction (SPE) and quantitation by C18 RP-HPLC, photodiode array (PDA) detection, and electrospray-ionization mass spectroscopy (ESI-MS). BALF quantification used exact methods previously described (Beringer et al., 2015). Plasma quantification used a method described previously with slight modification(Tongaonkar et al., 2011). Briefly, 100 μl of plasma was diluted 1:10 with 4% H3PO4, 10% ACN containing 50 ng of RTD-2 as internal standard. This was loaded on a 3cc Oasis WCX cartridge (Waters, Milford, MA, USA) equilibrated in water. Two ml each of 5% NH4OH, 20% ACN, 1% TFA, and 10% ACN + 1% TFA washed the cartridge. Elution was performed with 2 ml 1% TFA + 40% ACN. Eluents were lyophilized, suspended in 100 μl of 10% HOAc, 5% ACN, and chromatographed at 0.3 ml/min on a Waters C18 X-Bridge BEH C18 2.5 μm 2.1 x 150mm XP column fitted with a VanGuard Pre-Column BEC C18 (1.7 µm; Waters) using a linear 5 to 55% water-acetonitrile gradient containing 0.1% formic acid. Chromatography was performed on an Acquity H-class UPLC with an analytical PDA detector using Empower 3 software (Waters, Milford, MA, USA). Quantitative mass spectrometry was performed on post PDA eluent using a Micromass Quattro Ultima mass spectrometer with Mass Lynx 4.1 (Waters, Milford, MA, USA). 78 The assay proved to be linear and acceptable with an r >0.98 for the two standard curves. Accuracy and precision were 99.3% and 0.23% for a middle quality control sample, respectively. Total and differential cell counts and evaluation of lung edema BALF was collected by flushing the lungs three times (1, 0.8 and 0.8 mL) with PBS (w/o Ca +2 and Mg +2 ) and cOmplete protease inhibitor cocktail (Roche, IN) as suggested by the manufacturer. Mean recovery of BAL fluid via syringe aspiration was 86%. Collected BAL samples were kept on ice until clarified by centrifugation at 360 ×g for 10 min at 4°C; supernatant was stored at -80°C. Airway total cell counts were determined for each BAL sample using Turk blood diluting fluid and a hemocytometer. Approximately 5 x 10 5 cells were added to a slide chamber, spun in a cytocentrifuge (Shandon, Thermo Scientific, Waltham, MA), and stained using the Diff Quick Stain kit per commercial protocol (Polysciences, Warrington, PA). Cell counts were conducted on 200 cells per slide. Lung edema was assessed by measurement of total protein concentration in supernatants of BALF using the Bradford assay (Bio-Rad, Hercules, CA) (Matute-Bello et al., 2011b). BALF and cell culture cytokine multiplex Multiple analyte levels (TNF, IL-1a, IL-1b, IL-6, MCP-1, KC, MIP1a, and MIP1b), were determined at 0.5, 3, 7, 24, and 48 h using Milliplex MAP multiplex kits (EMD Millipore, Billerica, MA) with bead fluorescence readings analyzed on a Bioplex 200 with HTF (BioRad, Hercules, CA). Times were determined based on RTD-1’s treatment effects on total airway cell counts. Frozen BAL samples were thawed on ice and processed according to the overnight protocol according to the manufacturer’s instructions. For the in vitro 24 h MH-S experiment, soluble cytokines TNF, IL-6, KC, MCP-1, and MIP-2 were determined from clarified cell culture supernatant as described 79 above. All plates met reasonable precision (CV ≤ 20%) and samples below the lower detection limit were extrapolated in order to avoid statistical analysis of zero values. No values were found above the upper limit of detection. BALF MMP and peroxidase activities BALF obtained from 24 h LPS alone, RTD-1 treated, or PBS untreated mice was assayed for total MMP activity using a fluorogenic peptide substrate BML-P128 [Dnp-Pro-ß-cyclohexyl-Ala-Gly- Cys(Me)-His-Ala-Lys(Nma)-NH2] (Enzo life sciences, Farmingdale, NY). A 100 μL reaction mixture containing 50 μL BAL fluid plus 10 μM substrate was read kinetically over 2 h. Enzymatic reactivity was determined at 340 nm excitation and 440 nm emission on a Synergy H1 Hybrid plate reader (Biotek, Winooski, VT). BAL peroxidase activity was measured as previously described (Abdel-Latif, Steward, Macdonald, Francis, Dinauer & Lacy, 2004). Briefly, 150 μL TMB substrate solution was added to 50 μL of BALF and incubated at room temperature for 30 minutes prior to termination with 50 μL 1M H 2 SO 4 . Plates were read spectrophotometrically at 450 nm (Tecan, CA, USA). Lung histopathology In a separate study mice were treated with LPS + Saline, LPS + 25 mg kg -1 RTD-1, or PBS + Saline for 24 h. Murine lungs were infused with 10% formalin buffered solution (Thermo Fisher Scientific, Waltham, MA) and inflated at 25 cm 2 as recommended by an official ATS workshop report (Matute-Bello et al., 2011b). Staining was conducted by the USC Pathology lab for hematoxylin-eosin (H&E) on 5 μm slices. Photomicrographs were captured at 10X magnification using a Nikon Microphot-FXA microscope and images captured using SPOT insight 4.0 MP CCD color digital camera and software under 10X objective lens. Scientifig software was used to 80 construct figure and scale bar (Aigouy & Mirouse, 2013). Lung injury scores were evaluated by an investigator who was blinded to the identity of the slides following a previously described lung injury scoring system (Matute-Bello et al., 2011b; Matute-Bello, Winn, Jonas, Chi, Martin & Liles, 2001). Data and statistical analysis This was a pilot study to characterize the pharmacokinetics and preliminary efficacy in an experimental model of ALI to evaluate the dose-dependent effects of RTD-1. Treatment groups were not blinded, nor sample size/power analysis performed. For statistical analyses, raw data was used except in the case for the POD and migration assays which have been normalized to untreated controls to minimize the variance. Group sample sizes less than 5 per condition can be found in the time course and histology studies (n=3 per group) and should be considered exploratory. Mean biological values were obtained from at least 2 technical triplicates in all assays. Statistical and graphical analysis were carried out using GraphPad Prism version 6.0 (GraphPad Software, San Diego, CA), and STATA 13 (StataCorp, College Station, TX). Residual errors of univariate data was inspected for near normal shape distribution (skewness and kurtosis ±2, histogram) and central tendency (mean, median) (Westfall P, 2013). Non-normal data was subsequently log-transformed and evaluated as described above. The parametric ANOVA with post-hoc analysis P-values were calculated with the Bonferonni corrections between LPS treated and untreated groups and reported as either mean ± SD or geometric mean ± 95% confidence interval. A normalized response sigmoidal E max model was applied to the chemotaxis data to estimate IC 50 defined as the concentration associated with a 50% inhibition of the maximal chemotactic response. A variable Hill slope model was selected based on the F-test criteria. The time course of inflammatory markers modulated by RTD-1 were evaluated by computing the area under the concentration-time curves (AUCs) from 0.5 to 48 h and their standard deviation using 81 the sparse sampling method in Kinetica 5.1 (ThermoScientific, Waltham, MA) using the Bailer- Satterhwaite method to estimate the mean AUC and its variance. Non-compartmental analysis of RTD-1 pharmacokinetics was computed using the sparse AUC method in Kinetica 5.1. All studies, experimental design, data analysis, and statistical procedures comply with recommendations (Curtis et al., 2015) Endotoxin, RTD-1, and general reagents. Pseudomonas aeruginosa lipopolysaccharide was purchased from Sigma (Pa-LPS, MO, USA, Serotype 10, ATCC strain 27316, Phenol extracted). The hydrochloride salt of RTD-1 (>98%) was synthesized using fmoc chemistry as described previously (Garcia, Osapay, Tran, Yuan & Selsted, 2008; Tang et al., 1999; Tran, Tran, Tang, Yuan, Cole & Selsted, 2002). A stock solution was prepared in sterile water and 0.22 μm filter-sterilized. Working solutions were further prepared in 0.9% Sodium Chloride for injection, USP (Hospira, Lake Forest, IL) for administration to animals. Turks blood diluting fluid for leukocyte counts was obtained from Ricca Chemical Co (Arlington, Tx). PBS w/o Ca +2 , Mg +2 was purchased from Lonza (Walkersville, MD). Otherwise, unless specified, materials were purchased from VWR (Radnor, PA). 82 Results RTD-1 inhibits neutrophil-like HL-60 cell migration and dampens leukocyte-induced pulmonary inflammation Given the importance of airway neutrophilia in the pathogenesis of ALI we began by testing RTD- 1’s activity using in vitro methods. First, we investigated RTD-1 mediated cell migration using neutrophil-like differentiated human HL-60 (dHL-60) cells in a transwell migration assay. RTD-1 treatment significantly reduced FBS stimulated dHL-60 chemotaxis when compared with control with 50% inhibition at 250 ng mL -1 (Fig. 3-1a). At 10 µg mL -1 RTD-1 chemotaxis was completely inhibited. To determine the possible contributions of RTD-1 on the acute leukocyte-induced pulmonary inflammation response, we utilized an in vitro LPS-induced alveolar macrophage model using MH-S cells and profiled the macrophage relevant cytokines MIP-2, KC, MCP-1, IL- 6, and TNF. MH-S cells treated with RTD-1 resulted in ~ 5-, 3-, 154-, and 4-fold reductions in MIP- 2 (p<0.0001), MCP-1 (p<0.01), TNF (p<0.001), and IL-6 (p<0.001) respectively when compared with LPS stimulated MH-S cells alone (Fig. 3-1b,d,e,f). There was no difference in KC levels between groups (p>0.05) (Fig. 2c). 83 Figure 3-1. Dose dependent effects of RTD-1 on in vitro dHL-60 migration or LPS- induced MH-S leukocyte inflammation. (A) dHL-60 neutrophil (5 x 10 5 ) cells in RPMI were pretreated with RTD-1 for 15 minutes then placed in the apical chamber of their respective transwells. Inserts were then placed in the presence of 10% FBS in RPMI to induce chemotaxis. After 1 h, cells were collected and counted via automated cell counter. n=4/group and represented as Mean ± SD; gray band represents the 95% confidence interval. MH-S (1.25 x 10 5 ) alveolar macrophage cells were plated 24 hours before experimentation. 24 h cellular supernatant from LPS induced cells in the presence or absence of RTD- were collected and analyzed for (B) MIP-2, (C) KC, (D) MCP- 1, (E) TNF, (F) IL-6 by multiplex ELISA. n=3 per group and represented as Mean ± SD. Statistically significant groups have filled symbols (*) p<0.05, (**) p<0.01, (***) p<0.001, (****) 84 RTD-1 pharmacokinetics in plasma following single dose s.c. administration The mean plasma concentration versus time plot following single subcutaneous RTD-1 doses at 5 or 25 mg kg -1 in diseased mice are depicted in Figure 3-2a. The observed mean maximum RTD- 1 concentrations of 342.5 and 1169.9 were achieved at 1 h indicating rapid absorption. The disposition of RTD-1 appears to be biphasic with a short distribution phase followed by a prolonged elimination phase. A less than proportional increases in the Cmax (3.4-fold) and AUC (2.3-fold) was observed when comparing the disposition between the 5 and 25 mg/kg doses (Fig. 3-2b). Airway concentrations were detectable but not quantifiable by our assay methods. 85 0 7 24 48 72 10 100 1000 2000 HOURS RTD-1 [ng/mL] 5 mg/kg 25 mg/kg A. B. 5 25 RTD-1 [mg/kg] AUC 0-72 3503 (348) 1 (0) 8157.1 (367.4) 1 (0) C max T max 342.5 (130.3) 1169.9 (133.8) Hr ng⋅mL -1 ng⋅h⋅mL -1 Figure 3-2. Plasma RTD-1 concentration vs time profile. (A) RTD-1 distribution in plasma after sc injection at 5 and 25 mg kg -1 . Plasma was collected at 0.25-, 0.5-, 1-, 7-, 24-, 48-, and 72- h and quantified by LC/MS. Data is represented as median and IQR. N= 3-7 per group. (B) Sparse NCA methods using the Bailer-Satterhwaite approach were performed to evaluate the AUC. 86 RTD-1 reduces airway neutrophil burden and pulmonary edema Following LPS exposure at 24 h, total inflammatory cell counts in BAL fluid were increased approximately 32-fold (p<0.0001) in LPS untreated control mice compared to sham mice. Cellular differentials confirmed that neutrophils were the main infiltrative leukocyte (>90% of total cells) (Fig. 3-3a,b). RTD-1 treatment resulted in a significant decrease in neutrophils in RTD-1 treated mice at 5 mg kg -1 (p<0.01) and 25 mg kg -1 (p<0.001). Treatment with RTD-1 was not associated with any increases in airway macrophages when compared to LPS untreated mice (p>0.05) (Fig. 3-3b). Survival was 100%. In the time course experiments, RTD-1 treatment resulted in a significant dose dependent decrease in inflammatory cell infiltrate in response to LPS as early as 3 h and persisted out to 48 h post endotoxin challenge (p<0.01) (Fig. 3-3c). A single RTD1 dose of 5 mg kg -1 significantly reduced airway neutrophils at 7- (p<0.0001), 24- (p<0.0001) and 48- h (p<0.0001) when compared with LPS untreated mice. Greater potency was observed for 25 mg kg -1 RTD-1 treated mice with a significant reduction at 3- (p<0.01), 7- (p<0.0001), 24- (p<0.0001), and 48 h (p<0.0001) when compared with LPS untreated mice. Cumulative or total exposure of neutrophils to the airways (area under the concentration versus time curve) was significantly reduced in 5 mg kg -1 and 25 mg kg -1 RTD-1 treated mice compared with LPS untreated mice (p<0.0001) (Table S1). Survival was 100%. Airway neutrophil activation status was measured by cell free BAL fluid peroxidase (POD) activity and global MMP activity. RTD-1 diminished airway peroxidase (Fig. 3-3d) and MMP9 activity (Fig. 3-3e) in a dose response fashion in the 5 mg kg -1 and 25 mg kg -1 treatment groups when compared with LPS control mice (p<0.01). A strong positive association was observed between 87 the total neutrophil count and POD activity (p<0.05) (Fig. 3-3f), or global MMP activity (p<0.05) (Fig. 3-3g). Data from 0.2 and 1 mg/kg RTD-1 treated mice are absent secondary to sample misplacement. Total protein concentrations in BAL fluid were quantified in order to assess disease severity and more specifically, alveolar-capillary barrier leakage induced by LPS. BAL fluid protein levels were increased 3.8-fold (p<0.001) after LPS challenge in untreated mice compared to sham mice. Compared with LPS untreated mice, RTD-1 treatment resulted in significant reductions in protein levels in the 1 mg kg -1 (p<0.05), 5 mg kg -1 (p<0.05), and 25 mg kg -1 (p<0.001) RTD-1 groups (Fig 3-3h). 88 0 20 40 60 80 100 120 BAL POD Activity (%) ** LPS RTD-1 - - 0.2 1 5 25 + + + + + - *** 0 200 400 600 800 1000 Total BAL WBCs (x10 4 ) LPS ** RTD-1 + + + + + - - - 0.2 1 5 25 **** 0 200 400 600 BAL MMP Activity (RFU) LPS RTD-1 - - 5 25 + + + - *** *** 0 200 400 600 0 200 400 600 800 1000 Global MMP Activity Neutrophils (x10 4 ) r = 0.87 p<0.05 0 50 100 150 0 200 400 600 800 1000 POD Activity Neutrophils (x10 4 ) r = 0.78 p<0.05 0 200 400 600 800 BAL protein [µg/mL] LPS RTD-1 - - 0.2 1 5 25 + + + + + - * * *** A. B. C. D. E. F. G. H. 0.5 1 3 7 24 48 0 200 400 600 800 1000 HOUR Total BALF WBCs (x10 4 ) 25 mg/kg 5 mg/kg LPS ** **** **** **** **** **** **** 0 200 400 600 800 1000 Total BALF WBC (x10 4 ) PMN MAC LPS RTD-1 + + + + + - - - 0.2 1 5 25 ** *** 89 RTD-1 regulated LPS-induced inflammatory cytokines/chemokines Given the observed effects of RTD-1 on lung neutrophilia, 24 h airway levels of pro-inflammatory cytokines were measured from BAL fluid to further evaluate immunomodulatory activity (Fig. 3- 4a-c). TNF, IL-1a, IL-1b, IL-6, MCP-1, KC, MIP1a, MIP1b, were elevated in response to LPS challenge in mice compared to sham controls (p<0.0001). RTD-1 treatment at 5 mg kg -1 and 25 mg kg -1 achieved significant reductions in IL-1 β (p<0.05) and TNF (p<0.01) when compared with LPS untreated mice respectively (Fig. 3-4a,b). In addition, RTD-1 at the highest treatment dose resulted in significant reductions in IL-6 (p<0.05) (Fig. 3-4c). No treatment differences were observed in the levels of IL-1a, KC, MCP-1, MIP1a, MIP1b, when compared to LPS untreated controls. Figure 3-3. Dose and time dependent effects of RTD-1 on lung neutrophilia, neutrophil activation, and microvascular disruption. In a dose response study intranasal LPS treated mice received subcutaneous pretreatment with 0, 0.2, 1, 5 or 25 mg kg -1 RTD-1 and BAL was obtained at 24 hours. Alternatively, in a time course study intranasal LPS treated mice followed immediately with 0, 5 or 25 mg kg -1 RTD-1 and BAL was obtained at serial times. Cells were collected, manually counted (A,C) and applied to slide by cytospin centrifugation, dried and DiffQuick stained and counted for differential populations (B). BAL was evaluated for peroxidase (POD) activity (D), global MMP activity (E) and total protein (H). Strength and direction of association between total neutrophils and POD activity (F) or MMP activity (G) were quantified by pearson correlation. Dose response study n= 6 mice per group and represented as geometric mean ± 95% CI; Time course study n = 3 mice per group and represented as Mean ± SD. (*) p<0.05, (**) p<0.01, (***) p<0.001, (****) p<0.0001. 90 In the time course experiments, expression of MIP-2, KC, IL-1b, TNF, and IL-6 were increased in response to LPS challenge and exhibited variable -time and concentration - dependent RTD-1 treatment differences. Based on the lack of treatment response noted in the dose ranging studies, we excluded IL-1a, MCP-1, MIP1a, and MIP1b, a priori from the cytokine panel for the time course study. At the early time point (3 h), MIP-2, KC, and TNF levels were reduced in a dose-dependent manner (p<0.05) (Fig. 3-4d-f). At later time points (24 h, 48 h) IL-1β was significantly reduced by treatment with RTD-1 (p<0.05) (Fig. 3-4g). In addition, 24 h TNF showed dose-dependent reduction that were not statistically significant (p>0.05) (Fig 4-4f) while no observable trend was seen for IL-6. 91 0.5 3 7 24 48 0 2000 4000 6000 8000 HOURS MIP-2 [pg/mL] 25 mg/kg 5 mg/kg LPS **** ** 0.5 3 7 24 48 0 10 20 30 40 HOURS IL-1β [pg/mL] * * 0.5 3 7 24 48 0 1000 2000 3000 4000 5000 HOURS KC [pg/mL] **** *** 0.5 3 7 24 48 0 500 1000 1500 2000 HOURS TNF [pg/mL] *** * F. D. G. E. 0 1000 2000 3000 4000 5000 IL-6 [pg/mL] LPS RTD-1 + + + + + - - - 0.2 1 5 25 * 0 100 200 300 400 500 600 TNF [pg/mL] LPS RTD-1 + + + + + - - - 0.2 1 5 25 ** *** 0 10 20 30 40 50 60 IL-1β [pg/mL] LPS RTD-1 + + + + + - - - 0.2 1 5 25 * *** A. B. C. Figure 3-4. Dose and time dependent effects of RTD-1 on BAL cytokine and chemokines. In a dose response study intranasal LPS treated mice received subcutaneous pretreatment with 0, 0.2, 1, 5 or 25 mg kg -1 RTD-1 and BAL was obtained at 24 hours. Alternatively, in a time course study intranasal LPS treated mice followed immediately with 0, 5 or 25 mg kg -1 RTD-1 and BAL was obtained at serial times. Treatment effects on 24 h dose response cytokines (A) IL-1b, (B) TNF, and (C) IL-6 and time course cytokines (D) MIP-2, (E) KC, (F) TNF, (G) IL-1b, and (H) MCP-1 were quantified by multiplex ELISA. Dose response study n= 6 mice per group and represented as geometric mean ± 95% CI; statistically significant groups have filled symbols. (*) p<0.05. Time course study n = 3 mice per group and represented as Mean ± SD. (*) p<0.05, (**) p<0.01, (***) p<0.001, (****) p<0.0001. 92 To evaluate the relationship between RTD-1 treatment effects and cumulative local airway cytokine exposure, we compared AUCs between LPS- treated and -untreated mice. Overall MIP- 2, TNF, and IL-1β exposures were reduced for 5 mg kg -1 and 25 mg kg -1 RTD-1 treated mice compared to LPS untreated controls (p<0.01) (Table 3-1). No treatment effect on cytokine exposures for KC, MCP-1, or IL-6 was observed. To determine the effect of RTD-1 on proteolysis we measured BALF concentrations of the membrane bound MMP substrates gp130, IL-6R, TNFRI, TNFRII, and IL1RII. A biphasic response in soluble glycoprotein 130 (sgp130) levels was observed in RTD-1 treated mice compared to LPS untreated mice. At 0.5 h post LPS challenge, RTD-1 (25 mg kg -1 ) treated mice showed significantly less soluble ligand as compared to LPS untreated controls by 2.3-fold (p<0.05). However, at 3 h, significant elevations in local sgp130 in 5 mg kg -1 (p<0.05) and 25 mg kg -1 (p<0.001) RTD-1 treated mice compared to LPS untreated controls was observed (Fig. 3- 5a). Total exposure of soluble gp130 as measured by AUC analysis revealed significant increases in overall airway exposure of sgp130 in 5 mg kg -1 and 25 mg kg -1 RTD-1 treated mice compared to LPS untreated controls (p<0.05) (Table 3-1). No treatment effects were observed for IL-6R, TNFRI, TNFRII, and IL1RII (Fig. 3-5b-e). 93 Table 3-1. Total Exposure of Inflammatory markers in mice with LPS-induced pulmonary inflammation treated with RTD-1 n=3/group RTD-1 [mg/Kg] VARIABLE 0 5 25 WBC 2,571,220.0 (62,570.9) 1,267,870.0 # (121,595.0) 814,981.0 # (59,138.8) MIP-2 48,972.2 (5,274.1) 30,368.0 ! (6,506.6) 28,412.9 ! (818.6) KC 57,789.1 (3,398.9) 51,719.6 (8,313.2) 46,748.4 (1,502.1) TNF 27,549.5 (1,453.8) 22,543.7 $ (1,062.9) 18,627.2 $ (769.9) IL-1b 761.5 (51.5) 516.4 $ (48.8) 395.6 # (18.8) sgp130 186.9 (49.7) 641.4* (110.0) 801.7 ! (246.5) sIL-6R 20,980.3 (634.6) 25,119.5 (2,924.4) 22,338.0 (2,420.6) sTNFRI 20,872.4 (671.6) 25,511.8 (3,549.9) 23,974.6 (2,849.9) sTNFRII 192,113.0 (8,930.2) 198,590.0 (14942.0) 152,459.0 (9117.3) sIl-1RII 15,942.8 (1645.9) 16,327.7 (1298.2) 14,067.9 (668.9) a AUC analysis using the Bailer-Satterhwaite method to estimate the mean (standard deviation) from sparse sampling. * p<0.05, ! p<0.01, $ p<0.001, # p<0.0001 94 Figure 3-5. Time dependent effects of RTD-1 on soluble receptor shedding. Intranasal LPS treated mice received subcutaneous pretreatment with 0, 5 or 25 mg kg -1 RTD- 1. BAL was obtained at 0.5, 3, 7, 24, and 48 h after the single dose. (A) Soluble receptors sgp130, (B) sIL-6R, (C) sTNFRI, (D) sTNFRII, and (E) sIL1RII were quantified by multiplex ELISA. n= 3 mice per group; Mean ± SD. (*) p<0.05, (**) p<0.01, (***) p<0.001, (****) p<0.0001. 0.5 3 7 24 48 0 50 100 150 HOUR sgp130 [pg/mL] 25 mg/kg 5 mg/kg LPS * *** * 0.5 3 7 24 48 0 200 400 600 800 1000 HOUR sTNFRI [pg/mL] 0.5 3 7 24 48 0 2000 4000 6000 8000 10000 HOUR sTNFRII [pg/mL] 0.5 3 7 24 48 0 200 400 600 800 HOUR sIL1RII [pg/mL] 0.5 3 7 24 48 0 200 400 600 800 1000 HOUR sIL-6R [pg/mL] A. C. E. B. D. 95 RTD-1 protects against LPS induced acute lung injury Considering the maximal immunomodulatory effects observed at the higher dose, we selected the 25 mg kg -1 RTD-1 dose to evaluate the treatment effect on 24 h disease severity as measured by histology and lung pathology scoring. We compared the presence of congestion, interstitial and alveolar leukocytes, and alveolar hyaline membranes between RTD-1 treated, LPS untreated and sham mice (Fig. 3-6). RTD-1 exhibited a protective effect against LPS-induced lung injury as evidenced by the lack of alveolar leukocyte infiltrates, absence of hyaline membranes and normal alveolar wall thickness (Fig. 3-6a-i). Standardized lung injury scores confirmed the apparent gross histology (p<0.01)(Fig 6j). 96 J. A 100 μm B 100 μm C 100 μm D 100 μm E 100 μm F 100 μm G 100 μm H 100 μm I 100 μm LPS ALONE LPS + 25 mg/kg RTD-1 PBS SHAM 97 Figure 3-6. RTD-1 effects on severity of acute lung injury. Intranasal LPS treated mice received subcutaneous pretreatment with 0 or 25 mg kg -1 RTD-1. H&E staining of formalin inflated lungs and are representative photomicrographs of each mouse and their respective treatment groups (A-C) LPS alone, (D-F) LPS +25 mg kg -1 RTD-1, or (G-I) PBS sham. (J) Pathological scoring of slides for acute lung injury and inflammation. Parameters included the presence of congestion, interstitial and alveolar leukocytes, and alveolar hyaline membranes. n=3 mice per group; Mean ± SD. (*) p<0.05, (**) p<0.01, (***) p<0.001, (****) p<0.0001 98 Discussion ALI is a disorder of acute inflammation characterized by loss of alveolar-capillary membrane integrity, excessive neutrophil infiltration, release of pro-inflammatory mediators, and pulmonary edema (Johnson & Matthay, 2010). Lung infections and/or sepsis are frequently the underlying cause of ALI. Limited therapies are currently available for treatment of ALI. Due to the significant morbidity and mortality associated with ALI, current strategies being investigated include anti- inflammatory therapies for prevention or early treatment of ALI (Cornelio Favarin et al., 2013; Gong et al., 2014; Zhang et al., 2015). Cationic antimicrobial peptides participate in multiple phases of the lung immune response and include bacterial killing, as well as initiation and resolution of acute inflammatory responses (Hancock & Sahl, 2006; Tecle, Tripathi & Hartshorn, 2010). Several cationic antimicrobial peptides have demonstrated efficacy in murine models of ALI (Bals, Weiner, Moscioni, Meegalla & Wilson, 1999; Simpson et al., 2001; Tasaka et al., 1996; Vandermeer et al., 1994). The beneficial effects of these peptides are in part due to LPS neutralization or disruption of LPS binding protein complex formation which block TLR signal transduction. RTD-1 is a cationic peptide with broad spectrum antimicrobial and immunomodulatory activities. The immunomodulatory property of RTD-1 appears to be mediated through several mechanisms and is independent of antimicrobial actions (Wohlford-Lenane et al., 2009) or LPS neutralization (Schaal et al., 2012). RTD-1 inhibits NF-kB activation by blocking IkBa (Tongaonkar et al., 2015). Additionally, in vitro data suggests RTD-1 inhibits pericellular proteolysis given its fast temporal inhibition of soluble TNF (Schaal et al., 2012; Tongaonkar et al., 2015). In vivo, RTD-1 treatment significantly improved survival in murine models of sepsis and SARS (Schaal et al., 2012; 99 Wohlford-Lenane et al., 2009). The survival benefit in the SARS model was attributed to the anti- inflammatory activity since RTD-1 treatment had no effect on viral burden. The effect of RTD-1 in a model of direct lung injury has not previously been investigated. Our study provides the first evidence that the q-defensin, RTD-1, has protective effects in mitigating ALI. In vitro experiments showed dose-dependent reductions in migration of the neutrophil-like (dHL-60) cells and dampening of pro-inflammatory cytokine levels in LPS- stimulated alveolar macrophage-like (MH-S) cells. In vivo, we found that RTD-1 reduces airway neutrophil recruitment, activation, and vascular permeability and blunted pro-inflammatory cytokines in a murine model of LPS-induced ALI. Neutrophil activation and recruitment play a key role in the pathogenesis of ALI. LPS causes TLR4 dependent signal activation on alveolar macrophages and lung epithelia resulting in chemokine production (e.g. CXCL-8) (Guillot et al., 2004). Elevated chemokines cause neutrophil activation followed by transmigration into the lung. It is well documented that KC and MIP-2 (murine homologs of human CXCL8) are pivotal in regulating migration and activation of neutrophils in murine LPS-induced pulmonary inflammation (Huang, Paulauskis, Godleski & Kobzik, 1992; Jeyaseelan, Chu, Young & Worthen, 2004). RTD-1 inhibited neutrophil (dHL-60) chemotaxis in vitro. In addition, alveolar macrophage (MH-S) responses to LPS induced inflammation and neutrophil chemoattractant release were significantly blunted by RTD-1. In vivo, RTD-1 treatment reduced airway neutrophilia and activation in a dose-dependent manner. Taken together, this data supports both cellular mechanisms may be responsible for RTD-1’s treatment effects in vivo. 100 RTD-1 exhibited favorable pharmacokinetics following subcutaneous administration. Plasma concentrations of RTD-1 exceeded the in vitro IC 50 for inhibition of neutrophil chemotaxis. At the highest dose (25 mg kg -1 ), concentrations were sustained above the IC 50 for » 5 h. The area under the plasma concentration curve (AUC) did not increase in proportion to the dose when comparing between the 5- and 25 mg kg -1 doses. One possible explanation is saturable absorption from the injection site. This observation explains the less than proportional increase in anti-inflammatory activity at the higher dose. Additional formulations work is necessary to enhance absorption of subcutaneous administration to improve bioavailability. While neutrophils are critical to host defense against invading pathogens, excessive and sustained neutrophilia can be detrimental. Migrated neutrophils release proteolytic enzymes (NE, MMP-9) and reactive oxygen species which damage the lung epithelium and endothelium leading to increased permeability of the alveolar-capillary barrier and pulmonary edema (Grommes & Soehnlein, 2011). In vitro RTD-1 inhibits MMP-9 activity (unpublished data). In vivo, RTD-1 reduced POD and MMP-9 activity in a dose-dependent manner. The strong association between neutrophil counts and POD or MMP activity supports further these dose-dependent effects in the airways since they are predominantly found in neutrophils and released upon inflammatory stimulation. RTD-1 reduced total protein in BALF indicating treatment reduced pulmonary edema. Release of cytokines from lung immune cells and epithelia augment the acute inflammatory process in the lung. We found that the total airway exposure of several monocyte/macrophage related cytokines (i.e. MIP-2, TNF, IL-1b) were reduced in a dose-dependent manner with RTD- 1 treatment. In particular, we observed a distinct and early reduction in soluble TNF. Therefore, we hypothesized RTD-1 may inhibit early shedding events given the time course of neutrophil 101 burden in the air space. To further explore this in vivo, we used endogenous substrates of metalloproteinases and measured their levels in BAL in a time dependent fashion. While sTNFRI, sIL-1RII, and sIL-6R levels were unchanged, sgp130 expression was found to be paradoxically increased in RTD-1 treated compared to LPS untreated mice. Soluble gp130 has been reported to be a negative regulator of IL-6 mediated trans-signaling and thus anti-inflammatory with demonstrated therapeutic potential in murine models of inflammatory disease such as antigen- induced arthritis, cecal ligation puncture induced polymicrobial sepsis, and Type 1 T-helper cell mediated experimental colitis models (Barkhausen et al., 2011; Chalaris et al., 2010; Jostock et al., 2001; Mullberg et al., 1993; Nowell et al., 2003; Nowell et al., 2009). Taken together, these data support an overall picture of a reduced inflammatory response to noxious lung challenge in RTD-1 treated mice. Given the significant blunting of alveolar macrophage cytokines in vitro and the mixed treatment response of airway neutrophils at 3 h in vivo. We hypothesize that the 3 h in vivo attenuation of inflammation support addition of the macrophage as a cellular target of RTD- 1. Importantly, in both independent studies, neutrophils and IL-1b were found to be significantly reduced and exhibited dose dependence demonstrating robust treatment effects. In addition, TNF demonstrated dose dependent reductions across both studies, but sample size issues limited showing statistical significance in the time course study. Interestingly, at the 7 h time point RTD- 1’s effects on airway cytokines waned, despite a persistent reduction of airway neutrophils. Given, RTD-1’s concentration-time profile, concentrations necessary to suppress cytokine release may not have been sustained at 7 hours. Alternatively, direct injury from LPS causes bronchial cell apoptosis independent of neutrophil presence (Vernooy, Dentener, van Suylen, Buurman & Wouters, 2001). Since, RTD-1 does not neutralize LPS it is possible that the direct insult on the airway epithelium explains our observation (Schaal et al., 2012). However, more work is needed 102 to clarify this. Furthermore, future investigations should evaluate RTD-1’s efficacy at different stages of the acute inflammatory phase of ALI possibly by altering the time interval between LPS insult and peptide administration. Following the acute inflammatory phase some patients continue into a fibroproliferative phase of ALI and RTD-1’s therapeutic potential should be examined in models recapitulating this disease characteristic. In conclusion, data described here demonstrates that the q-defensin RTD-1 exhibited anti- inflammatory effects in a murine model of LPS-induced ALI. In vitro findings suggest that RTD-1 reduced in vivo lung injury and airway neutrophilia possibly by directly inhibiting neutrophil migration and attenuating keystone responses of the resident alveolar macrophage. These observations suggest RTD-1 may provide a potential new therapeutic approach to the treatment of ALI. 103 References Abdel-Latif D, Steward M, Macdonald DL, Francis GA, Dinauer MC, & Lacy P (2004). 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Respir Res 16: 43. 112 CHAPTER 4 Therapeutic Potential of the Host Defense Peptide Rhesus Theta (q)-Defensin-1 in Cystic Fibrosis Timothy J. Bensman 1 , Jordanna Jayne 1 , Meiling Sun 1 , Elza Kimura 2 , Niklas Werner 3 , Joshua Meinert 1 , Joshua C. Wang 1 , Justin B. Schaal 4 , Dat Tran 4 , Adupa P Rao 5 , Omid Akbari 6 Michael E. Selsted 4* , Paul M. Beringer 1* 1 University of Southern California, School of Pharmacy, 1985 Zonal Avenue, Los Angeles, CA 90033 2 State University of Maringá, Maringá, Paraná, Brazil 3 University of Gothenburg, Gothenburg, Sweden 4 University of Southern California, Keck School of Medicine, 2011 Zonal Avenue, Los Angeles, CA 90033. 5 University of Southern California, Keck School of Medicine, 2020 Zonal Avenue, Los Angeles, CA 90033. 6 University of Southern California, Keck School of Medicine, 1450 Biggy Street, Los Angeles, CA 90033 * Corresponding author. Tel: 323-442-1402, Fax: 323-442-1395, Email: beringer@usc.edu * Co-Corresponding author. Tel: 323-442-1180, Fax: 323-442-3314, Email: selsted@usc.edu Short Title: RTD-1 treatment in experimental models of CF airway disease In Preparation: Antimicrobial Agents and Chemotherapy - Antimicrob Agents Chemother 113 Abstract While the advent of drugs combating the underlying defect in cystic fibrosis (CF) has been realized, at this time effects on airway inflammation have not been demonstrated. Therefore, development of anti-inflammatory drugs against Pseudomonas aeruginosa initiated neutrophilic airway inflammation is a priority. Furthermore, rugged molecules able to withstand a noxious environment are required. Rhesus theta (q)-defensin-1 (RTD-1) is a macrocyclic antimicrobial peptide that has demonstrated immune regulating properties in murine sepsis and severe acute respiratory syndrome (SARS). RTD-1 has also shown to be a broad spectrum antimicrobial agent with antipseudomonal activity. Here we describe the therapeutic potential of RTD-1 in several in vitro, ex vivo, and in vivo models of CF investigations. In vitro and ex vivo studies demonstrate direct MMP-9 and ADAM-17 inhibition. RTD-1 treated CF airway leukocytes exhibited reduced spontaneous secretion of inflammatory cytokines ex vivo. Peptide treated CF bronchial epithelial cells stimulated with P. aeruginosa material exhibited TLR and adapter protein downregulation associated with reduced inflammatory cytokines. Aerosolized RTD-1 demonstrated mouse epithelial lining fluid (ELF) concentrations associated with in vitro effects. In vivo efficacy was tested in a CF murine model of chronic endobronchial P. aeruginosa reducing global airway inflammation and MMP-9 protease burden at 167 µg/kg. Further escalations in dose supported antimicrobial activity. Pulmonary delivery data in mice suggests it to be safe, tolerable, and well within the respirable range of humans. Airway pharmacokinetics are favorable and peptide stability in CF airway secretions robust. Collectively, these studies support development of RTD- 1 in CF airway disease. 114 Introduction Cystic Fibrosis (CF) is characterized by a chronic cycle of airway obstruction, infection, and inflammation leading to progressive loss of lung function and eventual respiratory failure (Chmiel et al., 2002). Patients with CF are susceptible to lung infections attributable in part to loss of the cystic fibrosis transmembrane conductance regulator (CFTR) protein in lung epithelia which causes reduced bicarbonate secretion leading to acidified airway surface liquid and reduced activity of antimicrobial peptides (e.g. lactoferrin and lysozyme) (Pezzulo et al., 2012; Stoltz et al., 2010). In addition, CFTR loss in macrophages results in impaired phagocytosis and intracellular killing of pathogens (Bonfield et al., 2012; Hartl et al., 2012). Pseudomonas aeruginosa is the most prevalent organism in adults and chronic infection with P. aeruginosa is associated with an increasing rate of lung function decline and reduced survival (Emerson et al., 2002; Henry et al., 1992; Li et al., 2005; Rosenfeld et al., 2001). Lung infections incite an intense inflammatory response leading to injury and progressive loss of pulmonary function (Cohen and Prince, 2012; Stoltz et al., 2010). Inflammation is characterized by excessive neutrophils and release of proteases (neutrophil elastase (NE) and matrix metalloproteinase-9 (MMP-9)) which are strongly associated with matrix breakdown products (e.g. elastin, fibronectin, collagen), lung remodeling, and loss of pulmonary function (Hilliard et al., 2007; Mayer-Hamblett et al., 2007; Sagel et al., 2005). In addition, excessive release of proteases cleave surface receptors on immune effector cells (e.g. CXCR1, CR-1) leading to impaired bacterial killing (Davis and Ferkol, 2013). Therefore, targeting infection and infection- induced inflammation is an important therapeutic strategy to slow the progression of CF lung disease and prolong survival. 115 Clinical trials of anti-inflammatory therapies have demonstrated a significant impact on pulmonary disease progression, but are fraught with serious adverse effects. In particular, long-term studies with oral prednisone (Auerbach et al., 1985) and high-dose ibuprofen (Konstan et al., 1995; Konstan et al., 2007; Lands et al., 2007) resulted in higher pulmonary function when compared with placebo; however, safety concerns including growth retardation and impaired glucose metabolism with prednisone and gastrointestinal discomfort, bleeding, elevated hepatic transaminases, and acute renal insufficiency with high-dose ibuprofen have limited the use of these agents. The results of these studies highlight the need to identify novel anti-inflammatory agents with improved safety profiles. Furthermore, since topical drug administration to an affected target site typically limits systemic exposure and thus reduces toxicity and/or immune paralysis concerns, pulmonary delivery is usually advocated. Defensins are small cysteine-rich cationic peptides with broad-spectrum antimicrobial and immunomodulatory activities(Ganz, 2003; Mansour et al., 2014; Yang et al., 2007). Theta- (q) defensins are unique cyclic mammalian peptides expressed exclusively in non-human Old World primates (Selsted, 2004) (Lehrer et al., 2012). Rhesus theta defensin-1 (RTD-1) exhibits broad- spectrum antibacterial activity including activity against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and P. aeruginosa (Tai et al., 2015). In vivo, RTD-1 improved survival in murine models of bacteremic sepsis and severe acute respiratory syndrome (SARS) (Schaal et al., 2012; Wohlford-Lenane et al., 2009). Interestingly, RTD-1 treatment had no effect on viral load, but significantly reduced inflammatory cytokines suggesting that the improvement in survival in the SARS model was due to immunomodulatory activity. The mechanism of the anti- inflammatory action of RTD-1 appears to be mediated through inhibition of MAPK and NF-kB pathways (Tongaonkar et al., 2015). Previously, we have shown in vitro and preliminary in vivo 116 data supporting RTD-1’s antimicrobial activity against CF P. aeruginosa isolates (Beringer et al., 2015). In this investigation, we utilized in vitro, ex vivo, and murine models of infection-induced CF airway inflammation to advance our understanding of the therapeutic potential of RTD-1 in CF. Based on the importance of pathogen interactions with resident immune cells (e.g. macrophages and neutrophils) as well as airway epithelia (Hartl et al., 2012; Laval et al., 2016) we assessed the anti-inflammatory effect of RTD-1 in these distinct populations by performing ex vivo testing in sputum and leukocyte cultures obtained from P. aeruginosa positive CF patients, in vitro testing in P. aeruginosa stimulated human bronchial epithelial cells (hBEC), and finally DF508 homozygous mice with chronic P. aeruginosa airway infection. 117 Methods and Materials Materials The CF P. aeruginosa RP73 clinical strain (multi-drug resistant, non-mucoidy phenotype) was a kind gift from Alessandra Bragonzi (Bragonzi et al., 2006; Jeukens et al., 2013). The hydrochloride salt of RTD-1 (>98%) was synthesized using fmoc chemistry as described previously (Garcia et al., 2008; Tang et al., 1999; Tran et al., 2002). A stock solution was prepared in sterile water and 0.22 μm filter-sterilized. Working solutions were further prepared in 0.9% Sodium Chloride for injection, USP (Hospira, Lake Forest, IL) for administration to animals. Turks blood diluting fluid for leukocyte counts was obtained from Ricca Chemical Co (Arlington, Tx). PBS w/o Ca +2 , Mg +2 was purchased from Lonza (Walkersville, MD). Otherwise, unless specified, materials were purchased from VWR (Radnor, PA). Human Subjects Patients attending the adult CF Clinic at the University of Southern California in Los Angeles, CA, were invited to participate in a cross-sectional study investigating lung inflammatory biomarkers in CF. The inclusion criteria were: Adults (>18 y/o) diagnosed CF and able to spontaneously expectorate sputum. Eighteen patients provided spontaneously expectorated sputum, 12 were obtained during stable outpatient visits and 6 were from patients admitted for the treatment of an acute pulmonary exacerbation (APE). Age, body mass index (BMI), percent predicted Forced Expiratory volume in 1 second (ppFEV 1 ) and anti-inflammatory therapy at the time of sampling were recorded (Table 4-1). Leukocyte populations from the APE subset of sputum samples were prepared for cell culture experiments (Table 4-2). The study received ethical approval (HS-12- 118 00320) from the institutional review board at the University of Southern California and all participants provided written informed consent. Table 4-1. CF patient characteristics and sputum cell analysis Pt Demographics (ALL) N=18 Age (yrs) 33 (13.5) Male (n, %) 14/18 (88) BMI (kg/m 2 ) 23.4 (2.8) ppFEV 1 (%) 43 (24.75), P. aeruginosa sputum culture (n, %) 18/18 (100) Mucoid phenotype (n, %) 16/18 (89) Azithromycin (n, %) 14/18 (88) Results are expressed as median (IQR), unless otherwise stated ppFEV 1 = percent predicted forced expiratory volume in 1 second Animals All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Southern California (Protocols 11676, 11956, 20252, and 20157). Male, 8-10 week old BALB/c mice (Charles River Laboratories, CA) weighing 20-25 g, male and female Cftr∆F508/∆F508 mice with C57BL/6 background (Case Western Reserve University, Cleveland, OH) 10 to 12 weeks of age weighting 17-22 g, and Male 10 to 12 week C57BL/6N mice (Charles River Laboratories, CA) weighing 24-28 g were housed under specific pathogen- 119 free conditions with a temperature of 22-24°C and humidity of 60-65%, with 12 h light/dark cycles. The animals were provided standard laboratory chow and water ad libitum. In Vitro and Ex Vivo Models to Assess the Anti-inflammatory effect and Stability of RTD-1 CF bronchial epithelial cells CuFi cells (ΔF508/ΔF508 Bronchial epithelial cell line) were maintained at 37°C, 5% CO2, 100% humidity, in Bronchial Epithelial Growth Medium (Lonza) supplemented with SingleQuots (without gentamicin-amphotericin B, Lonza) and seeded on plates coated with human placental collagen type IV substratum (Sigma). All experiments were conducted between passage 14 and 18. CuFi cells were stimulated with 20% (v/v) P. aeruginosa diffusible material in 24-hour conditioned BEGM as previously described (Berube et al., 2010; Wu et al., 2005). Briefly, filtrates of late stationary phase of the P. aeruginosa isolate RP73 were grown in bronchial epithelial cell growth medium containing bronchial epithelial basal medium (Cat# CC-3171, Lonza) and SingleQuot supplements and growth factors (Cat# CC-4175, Lonza). The filtrate was clarified, filter sterilized (0.45 µM) and heat-inactivated for 10 min at 95 °C before use. Treatment with 1- or 10 μg/mL RTD-1 was based on previous in vitro work (Schaal et al., 2012; Tongaonkar et al., 2015). No cytotoxicity was observed over 24 hours with or without RTD-1 as determined by LDH release (biological relevant threshold defined as >1.2-fold change of treated/untreated) (Berube et al., 2010; Wu et al., 2005). 120 CF sputum leukocyte culture Expectorated CF sputum samples (n=6) were obtained from APE inpatients within 48 hours of starting IV antibiotics and were processed as previously described (Marjanovic et al., 2011). Cellular viability after processing was >82% which is consistent with published data. (Marjanovic et al., 2011; Profita et al., 2003). Differential cell counts were determined by placing approximately 5 x 10 5 cells onto a slide chamber, spinning in a cytocentrifuge (Shandon, Thermo Scientific, Waltham, MA), and staining using the Diff Quick Stain kit per commercial protocol (Polysciences, Warrington, PA). Cell counts were conducted on 200 cells per slide. Cells were suspended in RPMI + L-glutamine and 10% FBS (Sigma Aldrich, St. Louis, MO) at 2 x 10 6 /mL. A 0.5 mL aliquot of the cell suspension was placed in a 48 well cell plate (Greiner-Bio one, Monroe, NC). Wells were spiked with 100 μg/mL RTD-1 or cell culture media and incubated for 24 hours. This increase in dose relative to the CuFi cell line was based on in house data suggesting significant protein binding in the presence of serum and safety data previously published demonstrating no hemolytic or cytotoxicity concerns at this dose (Tran et al., 2008). Clarification of cell supernatants were aliquoted and stored at -80°C until analysis.(Marjanovic et al., 2011; Profita et al., 2003) No cytotoxicity was observed over 24 hours with or without RTD-1 as determined by trypan blue exclusion and LDH release (biological relevant threshold defined as >1.2-fold change of treated/untreated). (Marjanovic et al., 2011; Scheicher et al., 2007). The sampling of lower airways is supported by <20% squamous epithelial cell contamination (Table 4-2). Inflammatory gene and protein expression RNA was extracted from CuFi hBECs and sputum leukocytes using the RNeasy mini kit per the manufacturer with the optional DNAse treatment (Qiagen). Quality and Quantity of RNA extracted was assessed using UV-Vis (Nanodrop, Thermo). A 260/A280 >2.0 and A 260/A230 >1.8 was achieved 121 for all samples ran. cDNA synthesis was performed using the iScript cDNA synthesis kit (Bio- Rad). SSO Universal SYBR Green Supermix (Bio-Rad) and 0.5 µg cDNA/plate. Samples were analyzed using a PCR array containing 84 paneled genes (RT 2 Profiler PCR Array, Cat# PAHS- 052Z, Qiagen). Spontaneous release of IL-1b, TNF, IL-8 and IL-6 from CuFi or sputum cells was quantified using ELISA (MSD, Rockville, MD) 24 hours post RTD-1 incubation. Antiprotease activity of RTD-1 In vitro MMP-9 activity was measured using a fluorogenic substrate assay. A final concentration of 3 µM active recombinant human MMP-9 from P. pastoris ( Enzo life sciences, Farmingdale, NY) was added to increasing concentrations (0.2 to 25 µM) of Fluorogenic MMP substrate Dnp- Pro-ß-cyclohexyl-Ala-Gly-Cys(Me)-His-Ala-Lys(Nma)-NH2 (Enzo life sciences) with or without increasing concentrations of RTD-1. Additionally, in pooled soluble phase expectorate sputum (SOL) from stable outpatients (n=7) MMP-9 activity was captured using the above fluorogenic substrate assay. MMP-9 activity was quantified by continuous measurement of fluorescence intensity in a microplate fluorimeter (Synergy H1 Hybrid; Bio-tek Instruments Inc., Winooski, VT) at 37 ºC and at λex 340 nm and λem 440 nm in accordance with the manufactures suggestion. Data were acquired with Gen5 data analysis software (Bio-tek Instruments Inc.). Initial velocity was computed and Ki estimated by the competitive enzyme inhibition model. Alternatively, the IC 50 estimate was calculated using the normalized response sigmoidal E max model with fixed Hill slope. Neutrophil Elastase (NE) activity was measured using a kinetic, fluorogenic microtiter plate assay using MeOSuc-Ala-Ala-Pro-Val-AMC (InnoZyme, EMD Millipore) as a substrate. SOL samples were assayed at a 200-fold final dilution with or without test compound (RTD-1, Sivelestat, or 122 saline) and the reaction initiated with 400 µM final substrate concentration in a 110 µL final volume. Activity was measured by continuous measurement of fluorescence intensity in a microplate fluorimeter (Synergy H1 Hybrid; Bio-tek Instruments Inc., Winooski, VT) at 37 ºC and at λex 380 nm and λem 460 nm in accordance with the manufacturers protocol. Data were acquired with Gen5 data analysis software (Bio-tek Instruments Inc.). Initial velocity (O.D./min) was computed and percent change from baseline (Sol phase + saline) calculated and plotted for comparative analysis. ADAM17 activity was determined in live airway leukocyte culture (n=2). Adult samples were collected from routine clinical visits (25 and 53 years of age) with chronic P. aeruginosa lung infections. Both patients were receiving long-term azithromycin treatment and had mild and severe lung disease respectively (84 and 22 ppFEV 1 ) . Sputum leukocytes were processed as described, then washed and re-suspended in assay buffer (HBSS). Cells were plated in duplicate at 1 x 10 6 cells/well with 5 μm TACE II fluorogenic substrate (Enzo Life Sciences, Farmingdale, NY). ADAM17 enzymatic activity was quantified by continuous measurement of fluorescence intensity in a microplate fluorimeter (Synergy H1 Hybrid; Bio-tek Instruments Inc., Winooski, VT) at 37 ºC and at λex 485 nm and λem 535 nm in accordance with the protocol described previously (Alvarez-Iglesias et al., 2005). Data were acquired with Gen5 data analysis software (Bio-tek Instruments Inc.). Initial velocity was computed and imported in the dose-response analysis. A normalized response sigmoidal E max model with variable Hill slope was chosen to estimate the IC 50. 123 Mucoactivity of RTD-1 Macrorheological analysis was conducted within 3-4 hr of sputum collection. Samples (n=6) were stored on wet ice until sputum aliquots (~0.2 g) were treated for 1 hr at 37°C with vehicle (10% v/wt normal saline), rh-DNAse (50 μg/ml)(Qiagen, Valencia, CA) or RTD-1 (10 μg/mL). The RTD- 1 concentration was based on previously published anti-inflammatory work in vitro (Schaal et al., 2012). Then a modified pourability assay was performed as follows. Treated samples were placed into a vertically positioned 2 mL graduated pipette and run under gravity and the quantification of sputum velocity (mm/s) was calculated over a distance of 390 mm (Devereux et al., 2015; Keal, 1971). This test is an indirect measure of sputum elasticity and adhesion (Rubin, 1992; Shak et al., 1990). RTD-1 stability in CF sputum Stability of RTD-1 in sputum was monitored by ultra-performance liquid chromatography (UPLC) (210 nm) and identity confirmed by mass spectrometry (MS) (M/z 521.65). Briefly, pooled CF sputum samples (n=7) were processed as previously described (Rees et al., 1997). Undiluted soluble phase sputum (SOL) containing extracellular enzymes was spiked with saline or RTD-1 (100 μg/mL) (5%v/v), vortexed for 5 sec and placed in a 37°C orbital shaker. At 0 and 24 hr, 10% acetic acid was added to stop all enzymatic activity. Samples were clarified by centrifugation prior to UPLC-MS and analyzed as under BALF LC/MS methods. 124 Experimental Models Airway tolerability Airway hyper-responsiveness was assessed, in naïve BALB/c mice exposed to 3 mg/kg RTD-1 via intranasal administration, by methacholine-induced (Mch) airflow obstruction. The dose of RTD-1 administered via this route was based on previous published in vivo work demonstrating efficacy and minimal airway toxicity concerns (Wohlford-Lenane et al., 2009). Mice were anesthetized by administration of ketamine (80 mg/kg; Hospira, Lake Forest, IL) and xylazine (10 mg/kg; Lloyd, Shenandoah, IA). An incision was made to access the trachea and mice were mechanically ventilated. Lung mechanics were measured by restrained plethysmography (Buxco Electronics, Troy, NY). Aerosolized methacholine was administered in increasing concentrations and lung resistance (R L ) and compliance (C dyn ) were continuously computed by fitting flow, volume, and pressure to an equation of motion. Nose-only aerosol drug delivery RTD-1 solution (RTD-1 in 0.45% NaCl) was delivered via a syringe pump (flow rate 0.1 mL/min) to an aerosol inhalation exposure system consisting of the bio-aerosol generator (BANG) and 16- port nose-only inhalation chamber with clear plexiglass nose-only mouse restraint holders. House air was dried by passing through a desiccator tube prior to entering the BANG nebulizer. A sampling flow rate of 0.5 L/min (comparable to operational flow rate of exposure port) was used in experiments designed to measure aerosol particle size using a 7-stage cascade impactor (Intox; Moriarty, NM), and aerosol concentrations using a teflon liquid impinger (Fig 4-1a-e). All items, unless specified, were purchased from CH Technologies (Westwood, NJ). The estimated delivered dose was based upon the concentration of RTD-1 in sampled air, respiratory minute 125 volume of a 25 g mouse, duration of exposure, inhalable fraction, and body weight and calculated as described (Alexander et al., 2008; Tuttle et al., 2010). Operating parameters included: Exposure time = 1 hour for 49, 167 and 360 µg/kg delivered doses and 15 minutes for the 6.8 µg/kg delivered dose. Total system flow rate: 2- (6.8 and 49 ug/kg), 4- (167 µg/kg), and 6-(360 µg/kg) L/min. 126 Air supply House air Desiccator Nebulizer Exposure tower Aerosol Exhaust Syringe pump System control unit PSI L/min Dry air Vacuum source Impinger aerosol sampler Impactor particle sizer Mouse restraint tube Figure 4-1. Nose only inhalation exposure system equipment schematic. Arrows show direction of air and vacuum. Drug solution is delivered via syringe pump to the nebulizer where a forced airstream collides to cause an aerosol. Mice were restrained in a nose only exposure tower for drug delivery. To determine particle size a 7 stage impactor was attached and sample drawn by vacuum. To determine the aerosol concentration in the exposure tower a liquid impinger was attached and vacuum applied. 127 Aerosol pharmacokinetics and safety Single-dose pharmacokinetics were determined in C57BL/6NCrl mice following aerosol doses of 6.8, 49, 167, or 360 μg/kg. Bronchoalveolar lavage (BAL) was performed at 0.5, 1, 4, and 8 hours post aerosolization (n=4/time point). RTD-1 concentrations from BAL fluid (BALF) samples were measured by LC/MS as described below. The urea correction method was utilized to determine RTD-1 concentrations in epithelial lining fluid (ELF) as previously described (Rennard et al., 1986). Pharmacokinetic parameters were derived from the concentration– time profiles using non- compartmental analysis. The maximum concentration (Cmax) and time to maximum concentration (Tmax) were obtained from the observed data. The area under the concentration- time curve (AUC) was determined using the linear trapezoidal rule. In addition to respiratory tolerance, total BALF leukocyte counts and body weight, as described under the infection model, served as surrogates of pulmonary safety. Furthermore, lungs were inflated and infused with 10% formalin buffered solution (Thermo Fisher Scientific, Waltham, MA) as recommended by ATS (Matute-Bello et al., 2011). Hematoxylin-eosin (H&E) staining on 5 μm slices was performed by the USC Pathology lab. Photomicrographs at 10X magnification using a Nikon Microphot-FXA microscope under 10X objective lens were captured using SPOT insight 4.0 MP CCD color digital camera and software. Histopathological evaluation of lung inflammation was performed by a single nonblinded investigator based on neutrophil involvement in the lung, presence of hyaline membranes, proteinaceous debris, and alveolar wall thickening (Matute-Bello et al., 2011). 128 BALF LC/MS RTD-1 concentrations from BALF samples were measured by LC/MS as previously described. (Beringer et al., 2015). Briefly, samples were acidified with 5 % acetic acid, spiked with an internal standard, extracted using C18 SPE columns (Waters, Milford, MA), lyophilized, and re-suspended in 100 µL of 10% acetic acid, 5% acetonitrile. Samples were then resolved on a C18 X-Bridge BEH column (Waters) using an ACQUITY UPLC system (Waters). RTD-1 concentrations were determined from post PDA eluent using a Micromass Quattro Ultima mass spectrometer under electrospray ionization. RTD-1 treatment effect in chronic P. aeruginosa lung infection model Chronic P.aeruginosa airway infection. Cftr∆F508/∆F508 mice, 10 to 12 weeks of age, were infected with P. aeruginosa (RP73) by intratracheal instillation (2.5 to 5 x 10 5 starting inoculum) (Bragonzi et al., 2006; Facchini et al., 2014). After 24 hours of infection the animals received a 1-hour daily treatment with either aerosolized half normal saline (n=15), or RTD-1 (49,167, and 360 μg/kg) (n=8,12, 11) for 7 days. Three independent experiments with treatment and control groups were performed (n=16/group). 129 Controls were then pooled across experiments. Work regarding the antibacterial effects of aerosolized RTD-1 at 167 µg/kg were previously published. (Beringer et al., 2015) These observations are included to compare with new doses, as well as, to provide new data on the biological effect of each dose on inflammation markers (e.g. cytokines and proteases). Lung lavage collection. Lungs were washed with 0.8 mL NS twice on day 7 of infection. Cells were pelleted at 1,000 rpm for 10 minutes at 4°C. Pelleted cells were re-suspended and washed twice in PBS for analysis of total and differential cell counts.  An aliquot of the BALF was used to quantify the BALF was removed and stored at -80°C for later analysis of inflammatory cytokines/chemokines and proteases. Total and Differential Cell Counts were performed by diluting cells in Turks blood diluting fluid. Total cell count was performed using a hemocytometer while cell differential was performed using the commercially available Romanowsky stain variant (Diff-Quick). Lung bacterial burden. Bacterial counts were performed as previously described (Facchini et al., 2014). Briefly, an aliquot of BAL and homogenized lung tissue were serially diluted and plated on TSA plates. Total lung bacterial burden was calculated from the sum of the colony counts in the BAL and lung homogenate.   130 Anti-inflammatory effects of RTD-1 in chronic endobronchial P.aeruginosa infected mice. Inflammatory mediators from BALF were quantified using Milliplex MAG (Millipore, Billerica, MA) kits for soluble receptors and inflammatory mediators. Analytes included IL-6, MCP-1, TNF, IL- 17, IL-1β, KC, MIP-2, TIMP-1(total), and Amphiregulin. Quantification was performed using Luminex XMAP technology and Luminex 100 platform (Luminex, Austin, TX). All samples were neat and incubated overnight with antibodies of interest. Lung proteases were also measured. Total murine MMP-9 (pro-, active, and TIMP-complexed) and NE activity were quantified from BALF. The Quantikine Mouse MMP-9 ELISA (R&D, Minneapolis, MN) was used following the manufacturer’s protocol to determine total MMP-9. NE activity was measured by a fluorogenic substrate assay using MeOSu-AAPV-AMC (Cayman Chemical, Ann Arbor, MI) as previously described (Kossodo et al., 2011). In addition to inflammatory marker the general clinical response was quantified. Weight and survival were monitored daily. Change from baseline was calculated as [(Daily weight/Initial weight) -1] * 100. Statistical methods and analysis Statistical and graphical analysis were carried out using GraphPad Prism version 6.0 (GraphPad Software, San Diego, CA). Residual errors of univariate data was inspected for near normal shape distribution (skewness and kurtosis ±2, histogram) and central tendency (mean, median) (Westfall P, 2013). Non-normal data was subsequently log-transformed and evaluated as described above. The parametric ANOVA with post-hoc analysis P-values were calculated with the Bonferroni corrections between P. aeruginosa treated and untreated groups and reported as either mean ± SD or geometric mean ± 95% confidence interval. However, multiple testing adjustment using the Dunn test was performed when unequal sample sizes existed. The ratio paired t-test was performed on sputum leukocyte samples as the ratio (treated/control) but not difference was a 131 more consistent measure of effect. RT-PCR array analysis was performed using the online Data Analysis Center (Qiagen, Valencia, CA) with multiple internal control normalization genes. The DD Ct method with fold expression of each gene treated with RTD-1 compared to control was performed. Changes were Log 2 normalized and parametric testing performed for statistical significance without post-hoc adjustments. Significance was determined at p £ 0.05. In regards to model building, the most appropriate model was selected based on the F-test criteria. Non- compartmental analysis of RTD-1 pharmacokinetics was computed by building 4 complete concentration time profiles and applying the linear trapezoidal rule and statistical moment theory in Excel for Mac 2016 (Microsoft, Redmond. WA). 132 Results Inflammatory gene and protein expression in RTD-1 treated CF bronchial epithelium Exposure of CuFi cells to 10 ug/mL RTD-1 resulted in approximately a 2 fold reduction in NLRP3, IL-1b, and CD14 gene expression (p<0.05) (Fig. 4-2a). P. aeruginosa filtrate-induced the release of IL-1b, TNF, CXCL8, and IL-6 by approximately 8-, 30-, 35-, and 17-fold compared to unconditioned CuFi cells. Under RTD-1 treatment, we observed reductions in IL-1b (p<0.001), TNF (p<0.01), IL-6 (p<0.01), and CXCL8 (p<0.01) at both 1 and 10 µg/mL compared to untreated P. aeruginosa filtrate stimulated cells (Fig. 4-2b-e). At the highest dose tested, IL-1b, IL-6, and IL-8 were reduced by approximately 1.3-fold, while TNF was observed to be reduced by approximately 2-fold. None returned to non-stimulated baseline levels. 133 -1.2-0.8-0.4 0.0 0.4 0.8 0.0 0.5 1.0 1.5 2.0 2.5 -Log 10 (p-value) Log 2 (FC RTD-1/Cont) NLRP3 CD14 IL-1β 0 10 20 30 IL-1β [pg/mL] Pa Filt **** **** RTD-1 + + + - - - µg/mL 0 50 100 150 200 IL-6 [pg/mL] Pa Filt ** *** RTD-1 + + + - - - µg/mL 0 2000 4000 6000 8000 10000 IL-8 [pg/mL] Pa Filt ** ** RTD-1 + + + - - - µg/mL 0 10 20 30 TNF [pg/mL] Pa Filt ** ** RTD-1 + + + - - - µg/mL B. C. D. E. A. Figure 4-2. RTD-1 reduces inflammatory response in CF epithelium. CF hBEC’s stimulated with Pa filtrate in the presence or absence of 0, 1 or 10 µg/mL RTD-1 for 24 hours (A) RTD-1 down regulates 24 hour NLRP3 inflammasome and accessory receptor. Innate and adaptive immune response of CuFi mRNA transcription profiles by RT- PCR were plotted according to the log2 fold change (X axis) and log10 unadjusted p-value. N=3/group. Horizontal significance line (-) indicates a threshold cutoff of 0.05. Closed circles () indicate p<0.05. (B-E) RTD-1 reduces pro-inflammatory cytokine secretion. Cytokine levels of (B) IL-1b (C) TNF, (D) IL-6, (E) IL-8 were quantified by ELISA. N=3/group. Treatment differences were analyzed by ANOVA. Statistically significant groups have filled symbols *p<0.05, **p<0.001, ***p<0.0001. 134 RTD-1 reduces protein secretion of Inflammatory mediators in CF sputum leukocyte culture Six patients with CF who were hospitalized for treatment of an acute pulmonary exacerbation participated in the sputum inflammation study. Admission demographics were as follows: mean age was 28 years, with normal weight (BMI of 23.4), and moderate lung disease (ppFEV 1 39%). All patients had chronic P. aeruginosa positive respiratory cultures, and were receiving treatment with azithromycin. Total and differential cell counts in sputum demonstrated a neutrophil dominant airway inflammation (Table 4-2). RTD-1 at 100 μg/mL for 24 hours significantly reduced spontaneous secretion of IL-1b (p<0.001), TNF (p<0.05), and CXCL8 (p<0.01) on average by approximately 2.5-, 2-, 1.3- fold respectively (Fig. 4-3a-e). Although IL-6 was also reduced, variability around the point estimate was large and did not reach significance (p=0.14). Table 4-2. CF patient APE characteristics and sputum analysis. Pt Demographics (APE) N=6 Age (yrs) 28 (6.3) Male (n, %) 5/6 (83) BMI (kg/m 2 ) 23.4 (4.5) ppFEV 1 (%) 39 (25.8) P. aeruginosa sputum culture (n, %) 6/6 (100) Mucoid phenotype (n, %) 5/6 (83) Azithromycin (n, %) 6/6 (100) Cell Analysis of Sputum Samples N=6 Sputum Weight (g) 1.6 (3.6) Viability (%) 94.4 (3.7) Squamous cells (%) 1 (1.5) Total Cells (10 6 /g sputum)* 36.8 (13.4) Neutrophils (%) 93.5 (3) Monocyte/Macrophages (%) 4.5 (1) Eosinophil (%) 0 (0) Lymphocyte (%) 1 (0.75) Results are expressed as median (IQR), unless otherwise stated ppFEV 1 = percent predicted forced expiratory volume in 1 second 135 Control RTD-1 0 200 400 600 800 1000 IL-1β [pg/mL] Control RTD-1 0 1000 2000 3000 4000 IL-6 [pg/mL] Control RTD-1 0 20000 40000 60000 80000 IL-8 [pg/mL] Control RTD-1 0 200 400 600 800 TNF [pg/mL] -2.0 -1.5 -1.0 -0.5 0.0 0.5 IL-6 IL-8 TNF IL-1β Fold Change (Log 2 ) *** * ** A. B. C. D. E. Figure 4-3. RTD-1 reduces spontaneous inflammatory secretions in airway leukocytes. Expectorated CF sputum was processed and isolated CF leukocytes cultured in the presence or absence of 100 µg/mL RTD-1 for 24 hours. (A) IL-1b, (B) TNF, (C) IL-8, (D) IL-6 were quantified by ELISA. Paired samples were plotted in a before-after fashion; Log2 fold change (RTD1/Baseline) and 95% confidence intervals of cytokine release in sputum leukocytes in (E). The vertical dashed line indicates no difference. Treatment differences (Log2) were analyzed by paired t-test. N=6/group. Statistically significant groups have filled symbols *p<0.05, **p<0.001, ***p<0.0001. 136 RTD-1 inhibits lung proteases RTD-1 inhibited MMP-9 enzyme activity with an IC 50 = 551 nM in a dose-dependent manner (Fig 4-4a). RTD-1 exhibited competitive inhibition with a Ki = 571 nM (95% C.I. 513.9 to 628.9; r 2 = 0.99) based on additional studies performed with varying substrate concentrations (Fig. 4-5). Peptide concentrations of 600 nM only slightly inhibited (~20%) neutrophil elastase activity as measured by fluorescent intensity (Fig. 4-4c). Increasing the dose of RTD-1 did not result in improved enzyme inhibition suggesting that RTD-1 does not inhibit NE (Fig. 4-6). In comparison, the well described NE inhibitor silvelestat completely blocked NE activity at a similar concentration. In airway leukocytes isolated from CF sputum (n=2), we observed that RTD-1 inhibited 50% enzyme cleavage of the ADAM17 exogenous substrate at 312 nM (Fig. 4-4b). Together, the inhibitory activity against both MMP-9 and ADAM17 suggests RTD-1 shows specificity for the MMP catalytic domain and may be a biologically relevant inhibitor of metalloproteinase activity. 137 0 25 50 75 100 % ACTIVITY * *** RTD-1 SIVELESTAT - - 600 - 400 - [nM] NEUTROPHIL ELASTASE 10 100 1000 10000 0 20 40 60 80 100 120 RTD-1 [nM] % ACTIVITY IC 50 = 312 nM (95 CI: 264 to 369) r 2 = 94% ADAM-17 0 100 1000 10000 0 25 50 75 100 RTD-1 [nM] % ACTIVITY IC 50 = 551 nM (95% CI: 499 to 609) r 2 = 0.99 MMP-9 A. B. C. Figure 4-4.RTD-1 inhibits MMP-9 and ADAM17 metalloproteinases, but not the serine proteinase NE. (A) Substrate-reaction plot of recombinant human active MMP-9 in the presence or absence of RTD-1 at varying concentration. (B) Dose response curve of RTD-1 and Surface ADAM17 activity in CF airway leukocytes by fluorgenic substrate reporter. N=2/group. Alternatively, (C) NE activity was measured using a fluorogenic substrate assay with Sol phase sputum with saline, RTD-1, or Sivelestat. Mean ± SD are reported; treatment differences were analyzed by ANOVA and statistically significant groups have filled symbols *p<0.05, **p<0.001, ***p<0.0001. 138 0 5 10 15 20 25 0.0 0.1 0.2 0.3 0.4 Substrate [µM] VELOCITY [RFU/sec] RTD-1 [nM] 0 96 240 960 1900 19000 Ki = 571 nM (95 CI 513.9 to 628.9) R 2 = 0.99 MMP-9 Figure 4-5. RTD-1 inhibits MMP-9 activity. Substrate-reaction plot of recombinant human active MMP-9 in the presence or absence of RTD-1 at varying concentrations. Data are represented as mean ± SD and the competitive inhibition model provided best fit by F-test criteria. N=2 independent experiments in technical duplicates. 139 0 25 50 75 100 % ACTIVITY * **** RTD-1 SIVELESTAT - - 600 - 400 - [nM] NEUTROPHIL ELASTASE *** 2500 - Figure 4-6. RTD-1 does not inhibit the serine proteinase NE. NE activity was measured using a fluorogenic substrate assay with Sol phase sputum with saline, RTD-1, or Sivelestat. Data are represented as mean ± SD. N=3 per group. Statistical analysis was performed using ANOVA with Bonferroni adjustment *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 140 Sputum pourability is improved by RTD-1 The effect of RTD-1 on sputum pourability was tested in the six APE participants. Treatment with either 10 µg/mL RTD-1 or 50 μg/mL human recombinant DNAse significantly mobilized CF sputum samples compared with no treatment (p<0.01) (Fig 4-7). On average, DNAse improved pourability to a greater extent than RTD-1. Vehicle RTD1 DNAse 0 20 40 60 Sputum Velocity [mm/sec] ** ** Figure 4-7. RTD-1 improves CF sputum pour-ability ex vivo. Sputum pour-ability as measured by velocity was determined in normal saline, RTD-1 and DNAse treatment groups. N= 6 per group; Geometric mean ± 95% CI. Treatment differences were analyzed by ANOVA. Statistically significant groups have filled symbols. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. 141 RTD-1 retains structural integrity in pooled CF sputum Peptide stability in pooled SOL (n=7 patients) was demonstrated by the signature peak at a retention time (RT) of 4.8 min (Fig. 4-8a-c) The peak AUC for RTD-1 alone compared to acid- treated Sol remained unchanged when examined by UPLC-MS (p=0.91). Ionization mass spectrometry confirmed RTD-1’s identity (m/z 521.68). Overall these data indicate that RTD-1 is a durable peptide able to withstand the high intrapulmonary protease burden in CF. 142 3.6 4.0 4.4 4.8 5.2 MINUTES 0.00 0.04 0.08 0.12 0.16 0.20 0.24 0.28 0.32 0.36 0.40 0.44 AU AU 0.00 0.04 0.08 0.12 0.16 0.20 0.24 0.28 0.32 0.36 0.40 0.44 3.6 4.0 4.4 4.8 5.2 MINUTES 0.00 0.04 0.08 0.12 0.16 0.20 0.24 0.28 0.32 0.36 0.40 0.44 AU 3.6 4.0 4.4 4.8 5.2 MINUTES RTD-1 AUC: 350,706 41,430 M/Z: 521.68 RTD-1 + ACID AUC: 354,978 44,557 M/Z: 521.68 BLANK AUC: -- M/Z: -- C. B. A. ± ± Figure 4-8. RTD-1 is robust to CF Sol airway proteolysis ex vivo. RTD-1 without (A) and with 10% acetic acid (B) was added to Sol for 24 hours and peptide stability was monitored by UPLC. Sol without RTD-1 (C) confirmed signature peak retention time of 4.8 min. Confirmation of intact RTD-1 was identified by ionization mass spectrometry (M/Z: 521.68). Data from pooled SOL (n=7) experiments were performed 3 times on separate days and are represented by mean ± SD. 143 Airway Reactivity is Unchanged by RTD-1 Dose response curves were essentially superimposable for C dyn and R L measures (Fig. 4-9a,b). Reactivity to Mch after 20 µL of a 3 mg/mL RTD-1 solution showed no change in C dyn or R L compared to PBS sham. Given these findings RTD-1 does not appear to enhance airway reactive effects such as narrowing conducting zones or lung unit de-recruitment. 0 10 20 30 40 0 20 40 60 80 100 120 METHACHOLINE (mg/mL) % ARIWAY RESISTANCE PBS SHAM RTD-1 3 mg/kg 10 20 30 40 0 20 40 60 80 100 120 METHACHOLINE (mg/mL) % AIRWAY COMPLIANCE PBS SHAM RTD-1 3 mg/kg A. B. Figure 4-9. RTD-1 does not induce airway hyper-responsiveness in vivo. Pulmonary mechanics (A) resistance and (B) compliance were evaluated by oscillometry at intranasal doses of 0, and 3 mg/kg RTD-1 under methacholine challenge in balb/c mice. N= 3 per group; mean ± SD; statistically significant groups have filled symbols. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. 144 Aerosol Drug Delivery Aerosol particle size-and estimated delivered dose of RTD-1 were determined following drug delivery through the 16-port nose-only nebulizer system. Particle size analysis with a 7-stage cascade impactor allowed for the characterization of the mass median aerodynamic diameter (MMAD) of nebulized RTD-1 as 0.81±0.12 µM with geometric standard deviation of 1.32 ± 0.05 and log-normal distribution (Fig 4-10b-d). MMAD did not appear to change significantly across doses. The estimated delivered doses of RTD-1 were 6.8, 49, 167, 360 µg/kg Figure 4-10. Aerosolized properties of RTD-1. A) Mean mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) for delivered doses of 49 and 360 µg/kg RTD-1. (B) linear regression of cumulative probability vs diameter to determine the MMAD for delivered doses. Histograms of normalized Mass Frequency of (C) 49- and (D) 360- µg/kg and their log-normal distribution. 0.1 0.5 0.9 0.99 0.01 0.1 1 10 Cumulative Probability < Stated Size Diameter [µm] MMAD 49 µg/kg 360 µg/ kg 0.1 1 10 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Cut Diameter [µm] Normalized Mass Frequency [dM/dln(d)] B. C. 0.1 1 10 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Cut Diameter [µm] Normalized Mass Frequency [dM/dln(d)] D. A. MMAD (µm) GSD RTD-1 0.81 (0.12) 1.32 (0.05) 145 Airway PK and safety of RTD-1 The epithelial lung fluid (ELF) concentration–time plot for nebulized RTD-1 after single dose administration is depicted in (Fig. 4-11). Overall, the C max and AUC were not proportional to dose delivered. The half-life of RTD-1 in the airways across doses was similar with a median and IQR of 3.19 ± 0.71 (Table 4-3). Lung histology, inflammation scoring, total airway cells in BALF, and body weight changes did not appear to be affected by airway concentrations achieved (Fig. 4- 12). Plasma levels of RTD-1 following aerosol administration were below the lower limit of quantification. Figure 4-11. Characterization of aerosolized RTD-1 in vivo. Epithelial lining fluid concentration vs time profile of delivered doses of 360-, 167-,49-, and 6.8- µg/kg RTD-1. N=4 per group; data are plotted as the median and interquartile range (IQR). 146 Table 4-3. RTD-1 Non-compartmental PK analysis in ELF NCA Parameters a DELIVERED DOSE [µg/kg] 6.8 49 167 360 AUC (µg•hr/mL) 57.98 (18.88) 183.15 (8.36) 257.24 (55.42) 420.96 (24.24) C max (µg/mL) 20.54 (4.55) 47.79 (12.90) 69.17 (11.49) 137.27 (11.47) T max (hr) 0.5 (0) 0.5 (0) 0.5 (0) 0.5 (0) t 1/2 (hr) 2.50 (0.10) 3.07 (0.31) 3.31 (0.60) 3.36 (0.76) Data are presented as Median (IQR) a determined after a single nebulized dose 147 6.8 μg/kg 49 μg/kg A. B. C. D. 148 Figure 4-12. Aerosolized RTD-1 does not incite leukocyte recruitment. BAL at respective time points were analyzed for total airway leukocytes after naïve C57/BL6N mice received a single aerosolized dose of 6.8-, 49-, 167-, or 360-µg/kg RTD-1. (A) Photomicrographs were taken 4 hours post dose and are independent and representative images of all mice (n=4). (B) Lung injury scores were evaluated by an unblended investigator following a previously described lung injury scoring system . (C) Total leukocyte counts in BALF were determined by manual counting in Turks solution. Data are represented as mean ±SD (n=4/group). The gray band represents the 95% confidence interval of total airway leukocyte values in untreated mice previously published and digitized using Webplot Digitizer v3.6. (Tongaonkar et al., 2011) (D) Changes in body weight were determined as described in methods. Data represent the mean and 95% confidence intervals. The gray band represents ± 2% weight loss. 149 Lung infection/inflammation are reduced by RTD-1 treatment Effect of RTD-1 on lung bacterial burden Chronic P. aeruginosa lung infection was achieved in DF508 mice with a geometric mean bacterial colony forming units (CFU) of 3.98 x 10 6 in untreated control mice at day 7. RTD-1 treatment reduced the total CFU recovered from the lungs and BALF compared with sham controls on average by approximately 1 log at 167 µg/kg (p<0.01) and 360 µg/kg (p<0.05) (Fig. 4-13a). While initial plans were for 16 mice/group, unbalanced treatment groups were the result of mortality either spontaneously during quarantine or surgical complications. These problems have been well described (van Heeckeren and Schluchter, 2002; van Heeckeren et al., 2000). Mice that recovered from surgery, were included in this study and exhibited 100% survival throughout the investigational period. Effect of RTD-1 on airway Inflammation Daily aerosol RTD-1 treatment significantly reduced (~ 0.5 log) airway WBCs at 49 µg/kg (p<0.01) and 167 (p<0.05) µg/kg compared to untreated control mice (Fig. 4-13b). Differential cell counts indicate the reduction in airway WBCs reflects a decrease in airway neutrophils (Fig. 4-13c). No difference was demonstrated at 360 µg/kg aerosolized RTD-1 despite CFU reductions (Fig. 4- 13a-c). The effect of RTD-1 treatment on pro-inflammatory cytokine, chemokine, growth factor, and protease levels are summarized in Table 4-4. We observed that KC, IL-6, TIMP-1, and total MMP- 9 levels were significantly reduced at 167 µg/kg RTD-1 compared to untreated controls (p<0.05). 150 Figure 4-13. RTD-1 anti-infective and anti-inflammatory treatment effects in vivo. (A) Total lung colony-forming units (CFU) and (B) airway leukocytes and (C) cellular differentials were quantified after 6 treatments and 7 days post inoculation in C57/B6 mice. Longitudinally, (D) weight change from baseline was also recorded. Data (A,B) are presented as geometric mean ± 95% CI, (C,D) are presented as mean ± SD. Treatment differences were analyzed by ANOVA; statistically significant groups have filled symbols *p<0.05, **p<0.001, ***p<0.0001. This graph contains previously published data on the 167 µg/kg dose for comparison . Effect of RTD-1 treatment on weight Overall, RTD-1 treatment reduced weight loss in a dose-dependent manner and resulted in a quicker return to baseline weight (Fig. 4-13d). However, at the highest dose delivered no treatment effect in weight recovery was observed. 0 20 40 60 80 100 % Monocyte SALINE * RTD-1 n=15 n=8 n=12 n=11 Neutrophil Lymphocyte 2 3 4 5 6 7 Total Lung CFU (log 10 ) ** * SALINE RTD-1 n=15 n=8 n=12 n=11 0 µg/kg 49 µg/kg 167 µg/kg 360 µg/kg 2 3 4 5 6 7 Total BALF WBCs (log 10 ) ** * SALINE n=15 n=8 n=12 n=11 RTD-1 0 µg/kg 49 µg/kg 167 µg/kg 360 µg/kg 0 1 2 3 4 5 6 7 -35 -30 -25 -20 -15 -10 -5 0 5 10 Days after inoculation Change from initial body weight (%) SALINE RTD-1 167 µg/kg RTD-1 49 µg/kg * * * ** *** ** *** RTD-1 360 µg/kg A. B. C. D. 151 Table 4-4. 7 d BALF cytokines in chronic P. aeruginosa lung infected mice treated with RTD-1. RTD-1 Delivered Dose [µg/kg] Variable (pg/mL) 0 (n=14) 49 (n=8) 167 (n=12) 360 (n=11) IL-6 a 89.5 (46.7 to 171.4) 94.2 (7.3 to 1215.0) 9.7* (2.3 to 40.5) 173.5 (36.4 to 826) TNF a 16.2 (9.3 to 28.1) 17.9 (3.7 to 86.6) 4.8 (1.7 to 14.0) 15.6 (6.3 to 38.7) IL-17 a 4.4 (2.9 to 6.5) 5.2 (2.0 to 13.3) 2.9 (1.2 to 6.7) 1.6 (0.9 to 3.0) KC a 110.9 (83.0 to 148.1) 76.4 (29.5 to 187.6) 34.2* (13.6 to 85.8) 60.5 (41.3 to 88.6) MIP2 a 296.4 (200.9 to 437.4) 116.4 (15.0 to 902.6) 70.6 (16.6 to 299.9) 427.9 (101.9 to 1797.0) Amphiregulin a 26.7 (8.6 to 83.5) 93.9 (29.5 to 298.9) 27.8 (14.8 to 52.2) 91.6 (41.8 to 200.7) TIMP-1 a 1181.0 (501.0 to 2784.0) 918.0 (45.14 to 18670) 54.1* (5.5 to 529.8) 1042.0 (86.4 to 12565.0) Total MMP-9 b 11640.0 (11130.0 to 12160.0) 8990.0 (4390.0 to 17850.0) 6133.0** (2328.0 to 9938.0) 10240.0 (8086.0 to 12390.0) NE Activity b,c 223.40 (66.78 to 380.10) 37.5 (-18.5 to 93.5) 61.2 (19.4 to 103.0) 422.0 (195.0 to 649.0) a Results are expressed as geometric mean (95% confidence interval) b Results are expressed as mean (95% confidence interval) c Relative fluorescent units (RFU) *p<0.05, **p<0.01, ***p<0.001 152 Discussion Persistent infection, inflammation and mucous obstructed airways are hallmarks of CF lung disease. Multiple in vivo models of CF, including the piglet and ferret, demonstrating infection and inflammation are intimately linked (Cohen and Prince, 2012). Therefore, strategies to treat lung bacterial infections, inflammation, and obstruction in CF, remain important therapeutic targets. RTD-1 is a macrocyclic peptide with broad spectrum antibacterial activity. We have previously demonstrated that RTD-1 exhibits activity against P. aeruginosa, including multidrug resistant isolates from patients with CF (Beringer et al., 2016). In addition, RTD-1 has been shown to exhibit anti-inflammatory activity in murine models of peritonitis/sepsis and SARS (Schaal et al., 2012; Wohlford-Lenane et al., 2009). This activity is thought to be mediated in part by inhibition of NF- kB (Tongaonkar et al., 2015). In the current investigation we report on a series of in vitro, ex vivo, and in vivo experiments designed to determine the therapeutic potential of RTD-1 for treatment of CF respiratory disease. Airway inflammation in CF is orchestrated by interactions between invading pathogens and resident immune cells (e.g. macrophages and neutrophils) as well as airway epithelia (Hartl et al., 2012; Laval et al., 2016). Therefore, to test the effects of RTD-1 on these distinct cell populations we performed in vitro evaluations in both human CF bronchial epithelial cells stimulated with P. aeruginosa soluble filtrate as well as leukocyte cultures from expectorated CF sputum. Data demonstrate diminished release of key inflammatory cytokines and chemokines (e.g. IL-1b, CXCL-8). Cellular responses where similar between the populations. Transcriptome profiling of the innate and adaptive immune response in the stimulated CF bronchial cells suggest that RTD- 1 may be working through TLR-mediated inflammation pathways as NLRP-3, CD14, and IL-1b transcription levels were reduced ~2-fold. This is further supported by previous data showing 153 reduction in NF-kB phosphorylation in leukocytes stimulated with specific TLR agonists when treated with RTD-1 (Tongaonkar et al., 2015). Importance of lung proteases to CF disease progression is significant. NE and MMP-9 levels are associated with clinical worsening of pulmonary disease (Mayer-Hamblett et al., 2007; Sagel et al., 2005). Furthermore, proteolytic cleavage by these proteases alters immune regulation/activity (Davis and Ferkol, 2013), augments protease activity (Jackson et al., 2010) and support chronic neutrophilic signaling (Hartl et al., 2007; Xu et al., 2011). While, a number of endogenous protease inhibitors (i.e. TIMPs, elafin, SLPI) are present in the airways, antiproteinase balance is lost because of targeted degradation by the overwhelming proteinase burden (Greene and McElvaney, 2009; Sagel et al., 2005). Given that host defense peptides such as LL-37 and serine leukocyte protease inhibitor (SLPI) have demonstrated cysteine and serine anti-peptidase activity respectively (Andrault et al., 2015; Sallenave et al., 1997), we set up in vitro and ex vivo enzyme assays to test the inhibitory activity of RTD-1 against proteinases. MMP-9 and NE were chosen given their strong correlation with disease severity in CF (Mayer-Hamblett et al., 2007; Sagel et al., 2005). NE actively was largely unaffected in the SOL assay. In contrast, peptide concentrations in the high nanomolar range inhibited recombinant active human MMP-9 in a simple buffer system. Unfortunately, MMP-9 substrate solubility limited our modeling of enzyme inhibition and we were not able to robustly test mechanisms beyond competitive inhibition. It is well characterized that the metalloproteinase family shares homology across their catalytic domains. Furthermore, given the importance of liberated TNF in pro-inflammatory signaling, we chose to focus on the predominant TNF sheddase ADAM17 and hypothesized that RTD-1 would inhibit its activity. Using an ex vivo sputum leukocyte assay which measured ADAM-17 ectodomain shedding, we observed an ~2-fold reduction of the IC 50 for ADAM17 compared with 154 MMP-9 with RTD-1. These data suggest that RTD-1 likely exhibits some selectivity in the ADAM containing enzyme. Inspissated secretions in CF result in severe airway obstruction. Therapies targeting this pathology are routinely prescribed to mobilize secretions and improve lung function (Yang et al., 2016). Prior cationic molecules have demonstrated beneficial effects in compacting sputum DNA and “liquefying” this complex biological matrix (Dubois et al., 2013). We hypothesized that RTD- 1 with its high cationic charge cluster would perform similarly. Thus, we performed a modified sputum pourability assay; a measure of velocity and muco-adhesiveness (Keal and Reid, 1972; Rubin, 1992). Our data demonstrated an increase in the speed of unprocessed sputum traveling down a glass pipette. The magnitude of this effect was less than the 50 μg/mL DNAse condition. however, we did not perform a dose response study to optimize RTD-1. In vitro DNAse testing at this dose predicted clinical trial success and appears to be in the ballpark of what is clinically achieved (Sanders et al., 2006; Shak et al., 1990). While speculative, RTD-1 may provide an additive effect via an alternative mechanism for improving mucus clearability. Further studies are warranted to confirm this early observation with definitive rheological assays. Airway delivery is an attractive option in CF because of the ability to topically deliver large amounts of drug to the afflicted target site while minimizing systemic, whole body, exposure. In preparation for in vivo studies we conducted a series of experiments designed to characterize RTD-1 stability, pharmacokinetics, safety, and tolerability via aerosol administration. Due to the exuberant levels of proteases present in CF sputum, it is important to consider drug stability in preclinical evaluations. Given its unique cyclic backbone, we hypothesized that RTD-1 would be resistant to the soluble proteases present in CF airway sputum. LC-MS analysis revealed that RTD-1 retains 155 its conformation. This integrity is likely secondary to RTD-1’s unique macrocyclic structure with N-to C-terminus linkage which may retard amide bond cleavage by proteases. Single-dose pharmacokinetic studies following aerosol administration demonstrated for all doses, the maximum concentration observed (C max ) in ELF exceeded the IC 50 for MMP-9 and ADAM-17, as well as, concentrations used to inhibit inflammation in CF bronchial epithelial cells. The C max relative to the minimum inhibitor concentration of RP73 (MIC =4) was ~5.1, 11.9, 17.3, and 34.3 for 6.8-, 49-, 167-, and 360- µg/kg doses respectively. These ratios were associated with rapid bactericidal activity in vitro (Beringer et al., 2015).. Assuming linear kinetics, the elimination of RTD-1 from the ELF was relatively rapid with concentrations declining below 10 mcg/mL after ~2.5-, 7-, 9-, and 12.5-hours for 6.8-, 49-, 167-, and 360-µg/kg doses respectively. While a treatment effect was observed in vivo, this data suggest trials with increasing frequency of administration are warranted to further optimize RTD-1’s biological effects. Importantly, after aerosol administration of 360 µg/kg, ELF RTD-1 levels reached peptide concentrations tested in the sputum leukocyte cell cultures. RTD-1 administered via aerosol appeared to be well tolerated based on test showing no changes compared to controls on drug-induced airway hyper- responsiveness in naïve mice. Based on the promising results above, we tested the efficacy of aerosolized RTD-1 in an established model of chronic endobronchial P. aeruginosa infection in CF (DF508/ (DF508) mice. (Cigana et al., 2016a; Doring et al., 2014). Importantly, hallmarks of human CF respiratory disease such as chronic P. aeruginosa lung infection, airway neutrophilia, as well as remodeling with elevated MMP burden were recapitulated in this murine model as previously described (Cigana et al., 2016b) We found aerosolized RTD-1 at doses of 167 and 360 µg/kg reduced day 7 bacterial burden, airway leukocytes, and BALF cytokines, and improved weight. However, while the highest dose reduced lung CFUs it did not show any difference in airway leukocytes, cytokines, or weight. 156 The reduced efficacy at the 360 µg/kg dose may be attributed to a higher infecting inoculum (5 x 10 5 CFU) compared with 167 µg/kg (2.5 x 10 5 CFU) mice. Others have reported grossly, that establishment of chronic infection and immune cell recruitment is not significantly different across a 1 Log 10 range (Facchini et al., 2014). However, the ability to discern treatment differences may be more sensitive to starting inoculums. Alternatively, although our initial safety studies demonstrated no evidence of inflammation associated with aerosolized delivery of RTD-1 it is possible that at higher doses airway toxicity exists. At 167 µg/kg RTD-1, we observed that IL-6, KC, total MMP-9 and TIMP-1 levels declined along with reduction in CFUs in the murine airways. While reduced bacterial burden is a likely explanation, our vitro and ex vivo data suggest alternative immune effector mechanisms. Future work is planned to clarify this dynamic relationship. 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Yang, D., Liu, Z.H., Tewary, P., Chen, Q., de la Rosa, G., and Oppenheim, J.J. (2007). Defensin participation in innate and adaptive immunity. Curr Pharm Des 13, 3131-3139. 167 CHAPTER 5 Research Summary General Conclusions The overall aim of this project was to investigate the therapeutic potential of RTD-1 in CF pulmonary disease utilizing a strategy that included several preclinical models of human disease. The main goals were to demonstrate intrinsic anti-pseudomonal and anti-inflammatory activity and to begin bench to bedside translation. Using in vitro and in vivo models that recapitulates the chronic airway infection and inflammation observed in patients combined with pulmonary drug delivery -the likely route of administration in this patient population – we achieved those goals. Results disclosed in this thesis shape our understanding and direction for further development of this novel peptide to target CF pulmonary infection and inflammation. Antipseudomonal Activity The presence of P. aeruginosa is associated with increased morbidity and mortality with mucoid phenotypes displaying even worse outcomes (Emerson et al., 2002; Henry et al., 1992). Therefore, targeting this pathogen is critical. We utilized several in vitro and in vivo CF investigational models to describe RTD-1’s anti-pseudomonal activity. In vitro experiments defined the MBC/MIC and identified the rapid bactericidal of RTD-1 against CF isolates of P. aeruginosa. The rapid concentration-dependent bactericidal activity with RTD-1 is similar to the FDA approved cationic antibiotic colistin (Li et al., 2001). Due to potential cross-resistance, we tested the activity of RTD-1 against a panel of MDR clinical CF isolates- including several strains with phoPQ and/or pmrAB mutations that confer high resistance to colistin and determined that RTD-1 retained bactericidal activity. Based on the common observation that biofilm aggregates are commonly insensitive to antibiotics, we found that RTD-1 exhibits moderate activity against P. aeruginosa biofilms. Despite data showing reduced potency in complex liquids (e.g. protein binding) the in vitro activity of RTD-1 was confirmed in a murine model in which nebulized treatment significantly reduced chronic lung P. aeruginosa burden. This model mimics the 168 persistent endobronchial infection phenotype observed in patients. A lack of enhanced phagocytic activity in vitro by RTD-1 and PK data demonstrating airway concentrations above the MIC for 12 hours support a direct killing mechanism. Anti-inflammatory Activity While neutrophils are critical to host defense against invading pathogens, excessive and sustained neutrophilia can be detrimental as disease severity is associated with airway neutrophil counts and the neutrophil protein, neutrophil elastase (Mayer-Hamblett et al., 2007). Therefore, targeting a persistent and frustrated acute inflammatory response is critical in disease management. We utilized a preclinical model of LPS-induced acute airway neutrophilia/inflammation, as well as, several in vitro CF models to investigate the intrinsic immunoregulatory potential of RTD-1. In vivo data demonstrate dose dependently reduced, lung neutrophil recruitment, vascular leakage, and a pro-inflammatory cytokine response. In fact, the total airway exposure of several monocyte/macrophage related cytokines (i.e. MIP-2, TNF, IL-1b, MCP-1) were reduced in a dose-dependent manner with RTD-1 treatment. In particular, we observed a distinct and early reduction in soluble TNF. Therefore, we hypothesized RTD-1 may inhibit early shedding events given the time course of neutrophil burden in the air space. To further explore this in vivo, we used endogenous substrates of metalloproteinases and measured their levels in BAL in a time dependent fashion. While sTNFRI, sIL-1RII, and sIL-6R levels were unchanged, sgp130 expression was found to be paradoxically increased in RTD-1 treated compared to LPS untreated mice. Soluble gp130 has been reported to be a negative regulator of IL-6 mediated trans-signaling and thus anti-inflammatory with demonstrated therapeutic potential in murine models of inflammatory disease such as antigen-induced arthritis, cecal ligation puncture induced polymicrobial sepsis, and Type 1 T-helper cell mediated experimental colitis models (Barkhausen et al., 2011; Chalaris et al., 2010; Jostock et al., 2001; Mullberg et al., 1993; Nowell et al., 2003; Nowell et al., 2009). Complementary in vitro studies demonstrate that RTD-1 may work through a chemotaxis mechanism. Reduced chemotactic factor release from alveolar 169 epithelial cells (A549) support an indirect mechanism by which RTD-1 may inhibit neutrophil emigration to the lung in vivo. Alternatively, direct RTD-1 application to neutrophils (dHL-60) inhibited chemotaxis in vitro. Given plasma PK data that demonstrate achievable RTD-1 concentrations that correspond with inhibition of neutrophilic migration in vitro, as well as, its lack of detection in BALF, early peptide mediated effects through direct neutrophil migration are likely responsible for attenuating disease severity when given subcutaneously. However, when delivered as an aerosol, airway PK suggest achievable concentrations that correspond to intrinsic anti-inflammatory actions as observed in the inhibition of spontaneous release of keystone cytokines in airway CF leukocyte and P. aeruginosa diffusible material stimulated CF bronchial epithelial cell cultures. Obtainment of these levels could also target metalloproteinases. We demonstrate inhibition of MMP-9 and cell associated-ADAM-17 in vitro and ex vivo respectively. Therefore, intrapulmonary delivery is likely the preferred route given the potential for dual anti- inflammatory and anti-pseudomonal activity. Bench to Bedside To move this potential peptide therapy closer to the CF clinic, we also set out to describe several pulmonary delivery issues. Owing to the impressively high protease burden in the CF lung, we felt it necessary to determine the peptides stability. Through an ex vivo assay with CF secretions, RTD-1 demonstrated impressive ruggedness with no loss in intact peptide at 24 hours. A mass median aerodynamic diameter of ~1 µM, as well as, unchanged total airway leukocytes and bronchial challenge test in single dose studies demonstrate good respirability properties and limit some concerns of airway toxicity or hyper-responsiveness. Furthermore, the chronic endobronchial infection model has been used previously to demonstrate immune paralysis issues (Doring et al., 2014). Here we did not observe any signs of immune suppression leading to bacterial overgrowth and mortality. 170 Future Directions While the enclosed results are very promising, there are several limitations and therefore experiments that must be carried out for further insight and advancement before clinical studies can be suggested and include dose optimization and long-term inhalation toxicology, airway obstruction and drug delivery, as well as comparative physiology, HDPs and extrapolations. Dose Optimization and Long-Term Inhalation Toxicology Aerosol PK data reported in this thesis are from uninfected naive mice after a single dose. It is highly likely that airway concentrations achieved in infected mice are less given altered inspiratory and expiratory breathing patterns, as well as, increased particle phagocytosis and mucocilliary clearance mechanisms. While in a pilot study we observed only a 2X decrease in airway concentrations at 4 hours subsequent investigations describing airway PK should be conducted in disease animals (data not shown). In addition, a critical evaluation of the concentrations achieved demonstrate a relatively narrow dosing range (~6X). While we were limited by nebulizer efficiency (optimized by flow, humidity, etc.), peptide, and overall costs, further dose escalation studies must be carried out to address dose dependent effects. This could be achieved by increasing the number of nebulizers to the existing exposure tower, or through an endotracheal route with the MicroSprayer™ Aerosolizer device from Penn-Century Inc. which has been shown in the chronic pseudomonas airway infection model to achieve delivered doses in the mg/kg range. The one limitation with this device is the larger aerosol particle size with mass median aerodynamic diameter between 16 and 22 µM. However adequate airway spread and deposition is likely achieved given the beneficial effects of several investigational drugs in the chronic endobronchial P. aeruginosa infection mouse model (Cigana et al., 2016; Sabet et al., 2009). Intranasal and intratracheal instillation techniques are not proposed as airway obstruction by agarose beads makes heterogeneous lung dispersion difficult. Furthermore, because of the repeated nature of administration intratracheal instillation is likely too invasive. Once single dose 171 efficacy has been described including its minimum and maximum effects, further optimization of can occur through multiple-dose escalation studies. After determination of this optimized dosing regimen, long-term inhaled toxicokinetic studies (chronic dosing) should occur to predict its safety and maximum tolerated dose. This information, in addition to histology, pulmonary function testing, and lab data are critical before clinical testing. Airway Mucus Obstruction and Drug Delivery Polymixin E1, an active cyclic peptide metabolite of colistin, has previously been shown in naïve rats to induce a local inflammatory reaction in the lungs after 28-day repeat exposure (Beringer, 2001). The dose given ranged from 3 to 24 mg/kg/day and no dose was found to be tolerable in this preclinical study. However, we regularly administer via nebulizer the prodrug colistimethate sodium (150 mg colistin base) twice a day in CF patients despite the similarity in mg/kg/day exposure. In fact high levels (~10 mg/L) of polymixin E1 and colistin base can be found in sputum through a 12 hour period and reveal very slow airway clearance (S et al., 2014). This prolonged retention is likely because of its high affinity for mucins. This high avidity would also explain tolerability through the drug binding mechanism (i.e. low free drug concentrations present for irritation) (Huang et al., 2015). Taken together, this data would then support efficacy and safety studies in an alternative model of CF with impaired mucocilliary clearance and airway obstruction. Several models exist including the b ENaC overexpressing mouse, CF pig, and CF ferret (Stoltz et al., 2010; Sun et al., 2014; Zhou et al., 2011). These models would also help address concerns of mucous as a barrier to RTD-1 drug delivery and aid in the development of potential pharmaceutical formulations or targeting techniques to overcome this hurdle. Comparative Physiology, HDPs and extrapolations The results presented here should be taken with the appreciation that while human defensins are expressed predominately in leukocytes, mice lack this phenotype (Mestas and Hughes, 2004). Rather, defensins are richly expressed in the crypts of the small intestine by Paneth cells in the 172 mouse (Ouellette and Selsted, 1996). It is becoming well recognized that host defense peptides particularly the defensins are integral in mucosal immune signaling (Oppenheim et al., 2003). Whether compensatory defensin signaling in the human allays RTD-1 treatment effects is unknown. However, in vitro and ex vivo efficacy with RTD-1 in human leukocytes and bronchial epithelium disclosed here, as well as data previously published in human peripheral whole blood somewhat temper this issue (Schaal et al., 2012; Tongaonkar et al., 2015). However, dynamic expressions, regional patterns, and turnover differences can only be tested in vivo. Future pre- clinical investigations will help to untangle the uncertainty regarding this phylogenetic concern and the “uniqueness” of RTD-1 among a broad range of innate chemical protectors in humans. An investigation new drug (IND) application is currently being sought for RTD-1 in rheumatoid arthritis and will ultimately provide us with an answer. If successful, this would allow a parallel track for further development of aerosolized RTD-1 in CF. Another comparative difference between mice and humans is lung architecture. In addition to overall reductions in the diameter of the airways and nasal filtration, the monopodial, compared to human dichotomous, airway branching is characteristic of rodents (Fox, 2007). The geometrical structure of the lung then helps determine inhaled particle deposition. For example, it has been observed that submicron particle deposition shifts from the lower airway generations (e.g. bronchi, terminal bronchi) in humans to the upper airway generations (e.g.) in the mouse (Winkler-Heil and Hofmann, 2016). 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Abstract (if available)

Abstract Cystic fibrosis (CF) is the most common life-limiting genetic disorder in Caucasians. The majority of the morbidity and mortality is attributed to bronchiectasis and progressive loss of lung function. Despite improvements with aggressive antibiotics and airway clearance therapies, the life expectancy for children born and diagnosed with cystic fibrosis (CF) in 2010 is 40 years. The central feature of pulmonary disease is a chronic cycle of airway infection, neutrophilic inflammation, and obstruction. In particular, airway inflammatory markers (e.g. airway neutrophilia and neutrophil elastase) are strongly associated with disease severity. The importance of lung inflammation is reinforced by controlled clinical trials which demonstrated a reduced rate of lung function decline in patients receiving chronic prednisone or high-dose ibuprofen (Eigen et al., 1995

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Asset Metadata

Creator Bensman, Timothy Jay (author)

Core Title Therapeutic potential of rhesus theta (θ) defensin-1 in cystic fibrosis airway infection and inflammation

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Clinical and Experimental Therapeutics

Publication Date 09/28/2016

Defense Date 08/23/2016

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

Tag cystic fibrosis,infection,Inflammation,OAI-PMH Harvest,Preclinical Drug Development

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Beringer, Paul (committee chair), Rodgers, Kathleen (committee member), Selsted, Michael (committee member), Shen, Wei-Chiang (committee member)

Creator Email bensman@usc.edu,bensman09@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-309144

Unique identifier UC11213615

Identifier etd-BensmanTim-4835.pdf (filename),usctheses-c40-309144 (legacy record id)

Legacy Identifier etd-BensmanTim-4835.pdf

Dmrecord 309144

Document Type Dissertation

Format application/pdf (imt)

Rights Bensman, Timothy Jay

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

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