Investigating the Role of Amino Acid Transporter SLC6A14 As a Modifier of Pseudomonas Aeruginosa Lung Infection in Cystic Fibrosis

Investigating the Role of Amino Acid Transporter SLC6A14 as a Modifier of Pseudomonas aeruginosa Lung Infection in Cystic Fibrosis

Michelle Di Paola

A thesis submitted in conformity with the requirements for the degree of Master of Science Biochemistry University of Toronto

Investigating the Role of Amino Acid Transporter SLC6A14 as a Modifier of Pseudomonas aeruginosa Lung Infection in Cystic Fibrosis

Michelle Di Paola

Master of Science

Biochemistry University of Toronto

2016

Abstract

The most common disease causing mutation in CF is F508del, however there is considerable variability in the clinical phenotype of patients homozygous for this mutation. To address this variation, genetic modifiers are studied. Li et al found that SNPs in the putative promoter region of SLC6A14 were significantly associated with severity and age of first Pseudomonas aeruginosa lung infection in CF. Experiments were aimed at studying if SLC6A14-mediated amino acid uptake is regulated by bacterial pathogens and in turn regulates bacterial growth. Arginine uptake from the ASL of non-CF and CF primary bronchial cultures was measured, after treatment with purified flagellin from Pseudomonas. This treatment resulted in a 26.4% (n=5; p<0.01) and 16.6% (n=6; p<0.0204) increase in arginine uptake, in non-CF and CF cultures respectively. Currently, the impact of SLC6A14 function on Pseudomonas growth in co-culture is being investigated, to better understand the role which SLC6A14 plays in modulating infection.

Acknowledgments

I first need to extend my deepest gratitude to my supervisor, Dr. Christine Bear, for providing me with endless support and guidance. Your scientific inputs and patience has truly been instrumental to this work, and I could not imagine a supervisor who is more kind, helpful and understanding to their students. It has been a true joy to work in your lab. Thank you to my supervisory committee members, Dr. Patricia Brubaker and Dr. Igor Stagljar, for taking the time to provide me with helpful inputs and questions, which have pushed me to think more critically about this project.

Next, I need to thank my family, especially my mother Antonietta and my father Antonio, for their unwavering support and belief in me and my abilities, in addition to my beautiful baby sister Jessica and grandmother Gerarda, for being amazing and understanding always. No words can express how lucky I am to have all of your love and support.

I also need to thank many researchers at SickKids, specifically members of the Bear Lab (past and present), which is filled with many of my friends and mentors. Firstly, thank you to Dr. Saumel Ahmadi, for being not only a great friend, but someone who is always willing to help with any problem, providing endless amounts of ideas and energy, no matter what the situation. Thank you Sunny Xia, for being an amazing friend, someone who I can laugh with in times of stress, and who has always been helpful and kind. Thank you to all the students of the Bear Lab, Dr. Steven Molinski, Stephanie Chin, Maurita Hung, Dr. Stan Pasyk, Andrew Lloyd-Kuzik, Onofrio Laselva, Donghe Yang, Wilson Wu, Ida Szárics, Randolph Kissoon, and Alec Popa, for making each day in the lab enjoyable and full of energy. I know you all will have bright and successful futures. Also, thank you to Ling Jun Huan, for being a great lab technician and friend, in addition to all remaining members of the Bear Lab and the SickKids community, Dr. Mohabir Ramjeesingh, Dr. Danny Li, Dr. Paul Eckford, Catherine Luk, Dr. Leigh Wellhauser, Angela Skoutakis, Dr. Kai Du, Dr. Zoltán Bozóky and Natalie Workewych, for helping to make the lab like a second home. Thank you to Dr. Cezar Khursigara, Dr. Amber Park and the rest of their group at Guelph University, for their great work and enthusiasm on this project. Lastly, I need to thank my friends outside the lab, Jonathan Desmond, John Kedzierski, Josie Vu and Harishni Ramesha, for their continuing friendship and support over these past years.

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Table of Contents

Abstract ...... ii

Acknowledgments ...... iii

Table of Contents ...... iv

List of Tables ...... vi

List of Figures ...... vii

List of Appendices ...... ix

List of Abbreviations ...... x

Chapter 1: Introduction ...... 1

1.1 General Airway Physiology ...... 1 1.1.1 Airway Surface Fluid ...... 1 1.1.2 Importance of CFTR in Regulating Airway Surface Fluid ...... 2

1.2 Cystic Fibrosis ...... 5 1.2.1 Cystic Fibrosis Transmembrane Conductance Regulator ...... 5 1.2.2 CFTR Genotypes ...... 7 1.2.3 Lung Infection in CF ...... 7 1.2.4 Linking Infection to Inflammation ...... 8 1.2.5 Heterogeneity in CF ...... 9 1.2.6 Genetic Modifiers ...... 12

1.3 Solute Carrier Family 6 (Amino Acid Transporter) Member 14 ...... 13

1.4 Alternative Arginine Transporters ...... 15

1.5 Project Rationale ...... 18

1.6 Hypothesis and Specific Aims ...... 19

Chapter 2: Methods ...... 21

2.1 Cell Culture ...... 21

2.2 Lentiviral Infection ...... 23 iv

2.3 Quantitative Real-Time Polymerase Chain Reaction ...... 23

2.4 Western Blotting ...... 25

2.5 Amino Acid Uptake Studies ...... 25 2.5.1 L-[2,3-3H]-arginine Uptake ...... 25 2.5.2 Treatment and Collection of Airway Surface Fluid ...... 27

2.6 Reagents ...... 28

2.7 Statistical Analysis ...... 28

Chapter 3: Modulation of SLC6A14 messenger RNA expression in lung epithelium ...... 29

3.1 SLC6A14 expression is enhanced by an immune activating component from Pseudomonas aeruginosa ...... 29

3.2 SLC6A14 expression can be modified by shRNA-mediated knock-down ...... 33

Chapter 4: SLC6A14 plays an important role in regulating amino acid uptake in lung epithelium ...... 34

4.1 Inhibition of SLC6A14 causes a significant reduction in L-[2,3-3H]-arginine uptake..34

4.2 Inhibition or up-regulation of SLC6A14 significantly modifies uptake of arginine in non-CF and CF primary airway cells ...... 36

4.3 Changes in arginine uptake positively correlates with up-regulation of SLC6A14 expression following treatment with purified flagellin ...... 43

Chapter 5: Discussion ...... 44

References ...... 55

Appendix ...... 62

List of Tables

Table 1.1: Expression of arginine transporters in the lung………………………………...……16

Table 2.1: List of primer sequences used of quantitative real-time PCR experiments……...…..24

Table 2.2: Cycling conditions for quantitative real-time PCR reactions………………………..24

List of Figures

Figure 1.1: Constituents of the apical and basolateral membrane play an important role in maintaining fluid homeostasis in the airways……………………………………………………..4

Figure 1.2: An illustration highlighting the major organs impacted in CF disease..……………..6

Figure 1.3: Proportions which CFTR genotype and modifier genes play in modifying the major CF-impacted tissues……………………………………...………………………………………11

Figure 1.4: Expression of arginine transporters in polarized airway epithelium………………..17

Figure 1.5: Proposed role of SLC6A14 as a modifier of lung infection in CF epithelial cells….20

Figure 2.1: Post-transplant bronchial tissue form a well-differentiated monolayer when seeded onto transwell inserts…………………………………………………………………………….22

Figure 3.1: Treatment with flagellin significantly increases SLC6A14 expression in Calu-3 cells lung epithelial cells………………………………………………………………………………30

Figure 3.2: Relative expression levels of SLC6A14 do not differ between non-CF and CF cultures…………………………………………………………………………………………...31

Figure 3.3: Treatment with flagellin significantly increases SLC6A14 expression in non-CF and CF primary bronchial epithelial cells…………………………………………………………….32

Figure 3.4: Lentiviral infection with shRNA causes significant knock-down of SLC6A14 message expression………………………………………………………………………………33

Figure 4.1: L-[2,3-3H]-arginine uptake significantly decreases with the addition of alpha-MT in Calu-3 cells………………………………………………………………………………………34

Figure 4.2: Percentage of L-[2,3-3H]-arginine uptake significantly decreases with the addition of alpha-MT in Calu-3 cells…………………………………………...……………………………35

Figure 4.3: Representative chromatographic traces from the ASL of a non-CF airway culture..37

Figure 4.4: Percentage of arginine uptake is significantly modified, upon treatment with alpha- MT or FLA-PA, in non-CF primary airway cells………………………………………………..38

Figure 4.5: Percentage of glutamic acid uptake is not significantly modified, upon treatment with alpha-MT or FLA-PA, in non-CF primary airway cells…………...……………………….39

Figure 4.6: Percentage of arginine uptake is significantly modified, upon treatment with alpha- MT or FLA-PA, in CF primary airway cells………...…………………………………………..40

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Figure 4.7: Percentage of glutamic acid uptake is not significantly modified, upon treatment with alpha-MT or FLA-PA, in CF primary airway cells…...……..……………………………..41

Figure 4.8: Percentage of arginine uptake does not differ significantly when comparing non-CF and CF-affected airway epithelium………………………………………………………………42

Figure 4.9: Percentage increase in arginine uptake as a function of fold change in SLC6A14 expression………………………………………………………………………………………..43

Figure 5.1: Co-culture of Pseudomonas aeruginosa and primary lung epithelium……………..49

Figure 5.2: SLC6A14 expression increases following incubation with Pseudomonas aeruginosa in CF primary nasal cells………………………………………………………………………...51

Figure 5.3: L-[2,3-3H]-arginine uptake significantly decreases following slc6a14 knock-out in murine tracheal tissue…………………………………………………………………………….52

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List of Appendices

Figure A1: Examining SLC6A14 expression via western blotting.……………………...……..62

Figure A2: The concentration of arginine remaining in the ASL is significantly modified, upon treatment with alpha-MT or FLA-PA, in non-CF primary airway cells…………………………63

Figure A3: The concentration of glutamic acid remaining in the ASL is not significantly modified, upon treatment with alpha-MT or FLA-PA, in non-CF primary airway cells………..64

Figure A4: The concentration of arginine remaining in the ASL is significantly modified, upon treatment with alpha-MT or FLA-PA, in CF primary airway cells……………………………...65

Figure A5: The concentration of glutamic acid remaining in the ASL is not significantly modified, upon treatment with alpha-MT or FLA-PA, in CF primary airway cells……………..66

List of Abbreviations

ALI: air-liquid interface alpha-MT (or α-MT): alpha-methyl-DL-tryptophan, blocker of SLC6A14

ANOVA: analysis of variance

Arg: arginine

ASL (or ASF): airway surface liquid/fluid

ATP: adenosine triphosphate

CaCC: Calcium-activated Chloride Channel

Calu-3: human bronchial submucosal gland adenocarcinoma cell line cAMP or GMP: cyclic adenosine or guanosine monophosphate cDNA: complementary DNA

CF: Cystic Fibrosis

CFTR: Cystic Fibrosis Transmembrane Conductance Regulator

CPM: counts per minute, determined via scintillation counting

CT: cycle threshold in quantitative real-time polymerase chain reaction studies ddH2O: double-distilled water

EDTA: ethylenediaminetetraacetic acid

ENaC: Epithelial Sodium Channel

ER: endoplasmic reticulum

F508del-CFTR: CFTR protein containing deletion of phenylalanine at position 508

FLA-PA (or flagellin): purified flagellin protein from Pseudomonas aeruginosa eGFP: (enhanced) Green-Fluorescent Protein

GAPDH: Glyceraldehyde 3-phosphate Dehydrogenase

Glu: glutamic acid

GUSB: Glucuronidase Beta

GWAS(-HD): (hypothesis-driven) genome-wide association study

HEPES: 4-(2-hydroxyethyl)piperasine-1-ethanesulfonic acid, buffering agent

HPLC: high-performance liquid chromatography iNOS: inducible nitric oxide synthase

L-[2,3-3H]-arginine: radiolabeled (tritium) arginine

LeuT: bacterial leucine transporter, crystallized homolog of the SLC6 family

Km: Michaelis-Menten constant, substrate concentration at half maximal transport (Vmax)

MI: meconium ileus mRNA: messenger ribonucleic acid

NO: nitric oxide ns: “not significant,” changes between control and test sample were not significantly different

PCL: periciliary layer

PKA: Protein Kinase A qRT-PCR: quantitative real-time polymerase chain reaction

RT: reverse transcriptase enzyme

SDS: sodium dodecyl sulfate, detergent shRNA: small/short hairpin RNA

SLC6A14: Solute Carrier Family 6 (Amino Acid Transporter) Member 14

SNP: single-nucleotide polymorphism

TLR5: Toll-like Receptor 5

WT or WT-CFTR: wild-type or non-mutated CFTR protein

Chapter 1: Introduction

1.1 General Airway Physiology

The gross anatomy of the lung can be divided into two major parts. The first is the extrathoracic (superior) airway, containing the supraglottic, glottic and infraglottic regions. The second is the intrathoracic (inferior) airway, containing the trachea, mainstream bronchi and multiple bronchial generations, whose main function is to conduct air to the alveolar surface. At the microscopic level, the trachea is comprised of many layers, one of which is the mucosa. Mucosa is composed of ciliated pseudostratified columnar epithelial cells and mucus-secreting goblet cells, which sit on the basement membrane (mainly composed of collagen). The remaining layers are the submucosa (containing seromucous glands), fibrocartilage layer and adventitia (which are composed of cartilaginous rings interconnected by connective tissue)[1]. The epithelium of the bronchus is similar to that of the mucosa, but transitions from columnar to cuboidal as it branches out to the smaller bronchioles (0.5-1.0 mm in diameter), which lack cartilage support, causing the muscle layer to become the dominant structure. The terminal bronchioles are the sites of gas exchange, which branch out into alveoli and alveolar sacs. Squamous epithelium comprises the alveolar wall, which is coated by a thin layer of surfactant fluid, whose purpose is to reduce surface tension between opposing alveolar surfaces[2].

1.1.1 Airway Surface Fluid

In the distal lung, as the surface area increases, providing more available sites for gas exchange, so does the potential for lung infection, due to increased exposure and contact with inhaled microbes and particles[3]. This provides the necessity for a defense system, which can effectively clear these agents from the epithelium. This defense is provided by the airway surface liquid/fluid (ASL/ASF), which lines the surface of luminal airway cells and is comprised of a periciliary layer (PCL), which sits directly on top of the epithelium covering cilia, and a viscous gel layer which sits atop the PCL and is referred to as the superficial mucus layer[4]. Optimal height for the periciliary layer is 7 µm, which allows for cilia to beat easily, without contacting the superficial mucus layer[1, 5]. Submucosal glands secrete molecules such as lactoferrin,

2 secretory leukocyte protease inhibitor, lysozyme and β-defensins into the ASL[6, 7]. Ionic composition of the ASL and osmolarity of the cell, are regulated by electrochemical gradients generated due to the function of many constituents expressed on both the apical and basolateral membranes[5].

Efflux of chloride through function of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) promotes the efflux of water through AQP1, AQ3, AQP4 and AQP5, which are paracellularly and transcellularly located Aquaporins[8]. Fluid secretion is also regulated via chloride flux by calcium-activated chloride channels (CaCC). The role of these channels is acute, as they respond to the release of nucleotides such as ATP or UTP located in the lumen of the cell[9]. Like CFTR, the apically localized Epithelial Sodium Channel (ENaC) plays an important role in maintaining fluid homeostasis. It promotes water absorption via sodium transport into the cell and can be blocked using the inhibitor amiloride[10].

Maintenance of the electrochemical gradients required to support fluid homeostasis can be attributed to the presence of sodium-potassium ATPases (Na+/K+-ATPases) and NKCC (Na+/K+/2Cl- co-transporter), on the basolateral membrane. Na+/K+-ATPases exchange 3 sodium for 2 potassium ions, causing the cytosol (in comparison to the lumen) to become more negatively charged, providing a gradient for sodium reabsorption by ENaC. Chloride ions, which will be secreted by CFTR, are provided through the activity of NKCC[8, 11].

1.1.2 Importance of CFTR in Regulating Airway Surface Fluid

As previously mentioned, CFTR-mediated chloride flux leads to the transport of sodium and water across the apical membrane, allowing the epithelial surface to remain well-hydrated. This in turn regulates the volume of airway surface fluid, as changes occur in osmotic pressure of the lumen. This is important for mucociliary clearance, as cilia require an optimal airway surface liquid height in order to beat effectively, allowing for clearance of mucus and microbes[4, 7, 12].

CFTR function has also been reported to inhibit function of ENaC. When CFTR function is impaired (such as in patients with F508del-CFTR, to be discussed in further detail in the following section), sodium becomes hyper-absorbed. This leads to a reduction in the height of airway surface fluid, leaving mucus secretions to become weakly hydrated, making them prone

3 to infections and causing obstruction of the luminal space[13]. When the ASL becomes too viscous, bacteria, viruses and inhaled particles become trapped, increasing their contact time with the epithelium and promoting inflammation[14].

Additionally, CFTR function plays an important role in bicarbonate transport. Therefore, lack of protein function has been associated with a reduction in the pH of the ASL. The acidic pH of the ASL in Cystic Fibrosis patients interferes with many components of the innate immune system. For example, acidic pH destroys the activity of antimicrobial peptides, proteases and alters the rheological properties of mucins, components which play a key role in bacterial defense. This ultimately results in recurrent incidences of infection, inflammation and fibrosis, causing severe organ damage[15].

In patients with Cystic Fibrosis (CF), the balance of the ASL shifts from water to mucus secretion. In CF, water secretion by the gland serous cells of the epithelium is impaired due to lack of CFTR channel function on the apical membrane, which leads to poor movement of the cilia, altered salt and water transport and an accumulation of mucus, which are all major contributing factors to disease phenotype (figure 1.1)[4].

Non-CF Airway CF Airway

- epithelial dehydration and increased mucus production - reduction in ASL height (from 7 µm to 3 µm)

Figure 1.1. Constituents of the apical and basolateral membrane play an important role in maintaining fluid homeostasis in the airways. This figure illustrates expression of apically localized channels such as CFTR, ENaC and CaCC which play a key role in the flux sodium and chloride ions across the membrane, maintaining fluid homeostasis and regulating airway surface fluid. Function of basolateral expressed Na+/K+-ATPases and NKCC also play a key role in the maintenance of electrochemical gradients across the cell. Loss of CFTR-mediated chloride secretion in CF causes a reduction in water secretion, mucus buildup and a reduction in the height of airway surface fluid.

1.2 Cystic Fibrosis

Cystic Fibrosis (CF) is the most common autosomal recessive genetic disorder in the Caucasian population, affecting 1 in 3600 Canadian-born children (Canadian Cystic Fibrosis Registry 2011 Annual Report). It is a fatal disorder, having primarily pulmonary and intestinal manifestations, and also affecting the pancreas, liver, reproductive organs, sweat and salivary glands[16]. Airway obstruction/chronic pulmonary disease is the leading cause of morbidity and mortality (approximately 80%) in CF. This is due to excessive mucus accumulation, which leads to persistent infections and inflammation, causing blockage of the airways. As disease progresses, patients can develop more serious issues, such as pneumothorax, where gas build-up occurs in the pleural space and can cause the lung to collapse or interfere with blood flow. Bronchitis and pneumonia also commonly occur in the lungs of CF patients. Patients can develop polyps in the nasal epithelium and infection of the sinuses commonly referred to as sinusitis[16, 17]. Mucus build-up can also interfere with digestion, as it can block ducts of the pancreas, preventing secretion of enzymes necessary for the absorption and digestion of fats and proteins in the intestines. Pancreatitis (inflammation of the pancreas), inflammation of the bile ducts (leading to liver disease), diabetes, gallstones, and rectal prolapse can also occur[18-20] (figure 1.2).

1.2.1 Cystic Fibrosis Transmembrane Conductance Regulator

Cystic Fibrosis is caused by mutations in the CFTR gene, which is located on the seventh chromosome, at position q31.2 in humans[21]. This gene encodes for The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR, also referred to as ABCC7), a protein which is a member of the ATP-Binding Cassette (ABC) transporter superfamily. The CFTR protein is comprised of two membrane spanning domains (MSD1 and MSD2), two nucleotide binding domains (NBD1 and NBD2) which bind and hydrolyze ATP, and a disordered regulatory (R) domain[22]. There is no known crystal structure of the CFTR protein, however several homology models exist, most based upon the related bacterial ABC transporter, Sav1866[23, 24]. CFTR functions as a cAMP-dependent ion transporter, responsible for transporting chloride and bicarbonate ions across the membrane. Opening of the CFTR channel has been linked to ATP binding, and closure of the gate due to ATPase activity, as confirmed by electrophysiological studies[25]. Phosphorylation by protein kinase A (PKA) enhances ATP hydrolysis and channel

gating[26]. Additionally, protein kinase C has been reported to increase surface expression of CFTR and enhance PKA-stimulated channel gating[27]. CFTR can also be activated by PKGII phosphorylation, which is an isoform of protein kinase G (a protein kinase dependent on cGMP), localized near the cell surface[28].

Figure 1.2. An illustration highlighting the major organs impacted by CF disease. The right panel demonstrates the differences between a healthy and CF lung, which is the main organ impacted by CF disease. In CF, the airway wall widens, blood is present in the mucus, and thick and sticky mucus lines the epithelium, promoting bacterial infections (image acquired from The National Heart, Lung, and Blood Institute, National Institutes of Health; https://www.nhlbi.nih.gov/health/health-topics/topics/cf/signs).

1.2.2 CFTR Genotypes

2009 disease causing mutations in the CFTR gene have been reported to date (CF mutation database). Of these mutations, approximately 40% are proposed to be missense (substitution of a single amino acid). However, the most common and prevalent mutation in the CF population is deletion of phenylalanine at position 508 in the amino acid sequence (F508del). In Canada, 91.5% of patients express this mutation on at least one allele (Canadian Cystic Fibrosis Registry 2011 Annual Report). This mutation is located in the region of CFTR encoding NBD1, and results in the production of a misfolded protein, which cannot traffic to the cell surface. Subsequently, this protein becomes trapped in the endoplasmic reticulum (ER) thereby causing ER stress. F508del-CFTR, sequestered in the ER, is targeted for ubiquitin-dependent degradation through the proteasome, resulting in decreased surface expression and function of this protein[29, 30]. Surface expression and function of this protein can be partially rescued using either low- temperature incubation (at 27°C)[31] or incubation with chemical modulators such as correctors and potentiators. Correctors work to increase the abundance of mature protein present at the cell surface, whereas potentiators are designed to enhance CFTR-mediated ion transport (once the protein reaches the cell surface)[32]. The corrector/potentiator combination therapy ORKAMBI™ (lumacaftor/ivacaftor) has recently become FDA-approved to treat patients over the age of 12, who bear the F508del mutation on both alleles (Vertex Pharmaceuticals Incorporated, 2015).

1.2.3 Lung Infection in CF

The most prevalent pathogen found in the CF airways is Pseudomonas aeruginosa. Pseudomonas aeruginosa is a gram-negative rod-shaped bacterium, found in moist areas. In most clinical situations it exhibits low virulence, as it can be rapidly cleared in the airways. However, it causes opportunistic pulmonary infections in the lungs of CF patients[33]. Early infection in CF is mostly caused by Staphylococcus aureus and Haemophilus influenza. However, approximately 20-25% of infants with CF will have a bacterial culture which tests positive for Pseudomonas aeruginosa, and the probability of infection increases with age. More than 80% of adult CF patients (who are 25 years or older) suffer from chronic lung infection with Pseudomonas aeruginosa[34-36].

In chronic infection, Pseudomonas aeruginosa forms organized membrane-associated structures known as biofilms. During this process, the bacteria undergo genotypic and phenotypic changes, altering them from their planktonic (individual free-moving) to a membrane-associated state. When this transition occurs, the bacteria reduce expression and secretion of toxins and virulence factors (which are common hallmarks of acute infection), and promote biofilm formation and secretion of exopolysaccharides[37]. Due to the robust formation of biofilms, bacterial persistence and antibiotic resistance are two primary consequences of chronic infection. Antibiotic resistance is an ongoing issue for CF patients, as biofilm bacteria can exhibit up to a 1000-fold greater antibiotic resistance than planktonic bacteria[38]. Additionally, CF patients have been reported to have higher concentrations of amino acids present in their sputum compared to non-CF, and it has been proposed that these amino acids might promote growth of auxotrophic Pseudomonas aeruginosa[39].

At the cellular level, infection with Pseudomonas aeruginosa has been reported to decrease the chloride secretory capacity of both WT and surface expressed F508del-CFTR, following correction and potentiation with the compounds VX-809 (lumacaftor) and VX-770 (ivacaftor). It has been proposed that co-culture with Pseudomonas aeruginosa on human bronchial epithelial cells causes a decrease in surface expression of WT-CFTR protein due to secretion of a CFTR inhibitory factor, a virulence factor secreted by gram-negative bacteria which causes ubiquitination and degradation of CFTR[40].

1.2.4 Linking Infection to Inflammation

Infections in the lung, with bacteria such as Pseudomonas aeruginosa, trigger inflammatory responses within the cell, leading to tissue damage which further worsens patient health. Of the immune cells, neutrophils are the most abundant found in bronchoalveolar fluid and sputum of CF patients, and are an important contributor to the development of lung disease in CF[41]. Neutrophils migrate into the lung due to an increase in chemokines, such as IL-8, IL-7, LTB4 and PGP[42, 43]. Due to the robust formation of biofilms and adherence to mucus, neutrophils are unable to recognize and subsequently phagocytose bacteria in the airways. In addition to this, neutrophils themselves contribute to the pro-inflammatory state of the lung, through production of pro-inflammatory cytokines and other inflammatory mediators[43].

Anti-inflammatory therapies have been used as a method of treatment for CF patients, as these therapies have been shown to improve lung function. One component essential to the innate immune response, which mediates inflammatory responses, is Toll-like Receptor 5 (TLR-5), expressed on the surface of lung epithelia. TLR-5 is a receptor for the flagellin protein expressed on bacterial flagella[44]. This interaction, between flagellin and TLR-5, has been implicated as a major factor in the inflammatory responses triggered by Pseudomonas aeruginosa[45]. It is proposed that this interaction leads to the activation of NF-kB, which is a transcription factor that works to increase production of pro-inflammatory agents. Work by Blohmke et al showed that patients with mutations in the gene encoding TLR-5 showed improved lung function, implicating this receptor as a modifier of CF phenotype, in addition to an anti-inflammatory target[46, 47].

1.2.5 Heterogeneity in CF

Approximately 95% of patients who are homozygous for F508del have insufficient exocrine pancreatic function. However, patients with the same F508del mutation on both alleles also have been reported to have significant variation in multiple CF disease phenotypes, especially those impacting the lung, such as airway obstruction and infection. Forced-expiratory volume (FEV), which measures the amount of air exhaled during a forced breath, is a common measure used to determine lung function in CF patients, and demonstrates high patient-patient variability (even amongst those patients with the same genotype)[48-50]. This observation has been validated by twin/sibling studies, showing that there is a strong non-CFTR contribution (ranging from 0.54-1) to lung function[51].

This variability is also observed in the intestine, and was first characterized by Rozmahel et al, who demonstrated that CF mice with different genetic backgrounds show differences in their intestinal phenotype. Some strains of mice, such as 129/Sv, DBA/2J/129/Sv were unable to survive from birth whereas other strains, such as C57BL/6J/129/Sv, were able to survive for several months after birth. In addition, they observed that severity of the intestinal pathology of the cftr-/- mouse was more severe than the cftrf508del/f508del[52].

This phenotypic variability, seen between CF patients, can be due to both environmental and genetics factors[50, 51]. A recent review by Cutting outlines how CFTR genotype, environment and genetic modifiers all play a critical and varying role in determining the phenotype displayed by

10 many of the CF-impacted organs. With the exception of the exocrine insufficiency, genetic modifiers appear to play a crucial role in modifying CF phenotypes, particularly in the lung and gastrointestinal tract, and thus are an important area of CF research[53]. Specifically, airway obstruction, infection, intestinal obstruction, risk and age at onset of CF-related diabetes, and Body Mass Index (BMI) are all highly dependent on genetic modifiers and the environment, and not simply determined by CFTR genotype alone, thus providing a crucial need to study genetic modifiers as an alternative mechanism for developing CF therapies (figure 1.3)[53].

Figure 1.3. Proportions that CFTR genotype and modifier genes play in modifying CF- impacted tissues. This figure outlines some key genetic modifiers that have been implicated to play a role in modifying CF-affected phenotypes in the lung, pancreas and intestines. From this graphic it is evident that many CF phenotypes, especially those in the lung such as airway obstruction and infection, are predicted to be highly influenced by genetic modifiers, and are not simply determined by CFTR genotype (image adapted from Cutting, 2015 and The National Heart, Lung, and Blood Institute, National Institutes of Health; https://www.nhlbi.nih.gov/health/health-topics/topics/cf/signs)[53].

1.2.6 Genetic Modifiers

The discovery of genes which may play a role in modifying CF disease phenotypes requires genome-wide search approaches to be used, allowing for large patient cohorts to be examined. The first genome-wide association study (GWAS) performed by Wright et al in 2011, analyzed the genomes of patients with homozygous expression of the F508del mutation. This study identified two loci, 11p3 and 20q13.2, which were implicated to significantly impact the severity of lung disease in CF[54]. The first loci encodes for the epithelial-specific-Ets transcription factor EHF, which is expressed in bronchial epithelial cells, smooth muscle cells and fibroblasts. Expression and induction of EHF-dependent pathways has been proposed to improve folding, protein glycosylation and trafficking to the cell surface of the F508del-CFTR protein[55]. The second loci encodes for APIP, an inhibitor of apoptosis, robustly expressed in the lung and the trachea. Increased expression of APIP is associated with a reduction of lung function in CF patients, which implies that blocking apoptotic pathways negatively impacts lung disease in CF[54].

Following this, through hypothesis-driven genome-wide association studies (GWAS-HD) performed by Sun et al, multiple constituents of the apical plasma membrane were discovered to have a significant association with CF patient susceptibility to meconium ileus (MI)[56]. MI is a form of bowel obstruction which occurs when the first stool of a newborn is thick and sticky, causing blockage in the ileum. MI is highly heritable (>88%), minimally impacted by the environment and easily diagnosable at birth[57-59]. From these studies, it was discovered that single-nucleotide polymorphisms (SNPs) in the promoter region of SLC6A14, located on the X chromosome, were most significantly associated with a severe MI phenotype (p = 1.28x10-12). In addition to SLC6A14, other top modifiers from this study included SLC26A9 and SLC9A3[56].

SLC26A9 encodes for an anion transporter, which can act as a chloride/bicarbonate exchanger (in addition to playing a role in sodium-anion co-transport), expressed in lung epithelium, sweat glands and the gastrointestinal tract. SLC26A9 has been reported to physically interact with CFTR and enhance expression of both WT and F508del-CFTR in human bronchial epithelial cells[60, 61]. Knock-out of SLC6A9 function in cftr-/- mice has been shown to reduce survival, due to changes fluid absorption and bicarbonate secretion in the mucosa of the proximal duodenum. These changes are thought to impact digestive functions, as lack of secretions will cause mucus

13 build-up in sites which are commonly obstructed in CF[62]. It has also been implicated as a modifier of CF-related diabetes[20].

SLC9A3 (or NHE3) encodes for a sodium/proton exchanger which is highly expressed in the intestinal tract, and also expressed in the kidneys and lungs[63]. In cftr-/- mice, loss of one or more copies of NHE3 decreased amounts of sodium reabsorption, causing increased fluidity of the intestinal contents. This increased fluidity prevented intestinal obstruction, therefore reducing expression of this transporter is thought to positively impact CF intestinal phenotypes[64].

Given that CF is a multisystem disorder, Li et al were prompted to determine if the same polymorphisms which were implicated in modifying MI in the colon, also impact CF phenotypes in the lung. Interestingly, they determined that the above polymorphisms in SLC6A14 were also associated with the age of first lung infection with Pseudomonas aeruginosa and the severity of this infection in CF patients[65]. This implicated SLC6A14 as an important target to study as a modifier of lung infection in CF.

1.3 Solute Carrier Family 6 (Amino Acid Transporter) Member 14

Solute Carrier Family 6 Member 14 (SLC6A14) belongs to the SLC6 (Solute Carrier 6) family. This family consists of transporters for amino acids, neurotransmitters, osmolytes, and energy metabolites, whose function is dependent on sodium and/or chloride. Altered expression or lack of function in these transporters has been linked to a variety of diseases, such as hyperekplexia, autism, depression and Tourette Syndrome[66]. At the biochemical level, the determination of SLC6 transporter structures has been aided by the crystallization of LeuT, a bacterial homolog from Aquifex aeolicus. SLC6 transporters contain 12 transmembrane helices, 10 of which comprise the transporter core[67].

Specifically, SLC6A14 demonstrates the properties of an ATB0,+ transporter, amino acid transporter responsible for B0,+. It has broad substrate selectivity, as it able to transport neutral and cationic amino acids, and is up-regulated by protein kinase C (contrary to other SLC6 family members, which are down-regulated by the activity of PKC)[68]. Function of this transporter group was first functionally characterized by Galietta et al, who identified the presence of an amino acid transporter (now considered to be SLC6A14), following Michaelis-Menten kinetics

and demonstrating a Km (concentration at half-maximum transport) for arginine of approximately 80 ± 8.9 µM, when measuring short-circuit currents in the presence of the ENaC blocker amiloride[69].

SLC6A14 is expressed on the apical surface of polarized epithelia, and its function is coupled to both sodium and chloride gradients, in addition to membrane potential[70]. It is estimated to co- transport 1 amino acid along with 1 chloride and 2 sodium ions across the membrane[71]. In a variety of different cancers, such as colon, cervical and estrogen-receptor positive breast cancer, the expression of this transporter is up-regulated. It has been proposed that this up-regulation is due to an increasing demand for essential amino acids by the tumour cells, as a mechanism to sustain their rapid growth[72].

Additionally, it has been shown that transporter function may be blocked with the selective inhibitor alpha-methyl-DL-tryptophan (α-MT). Karunakaran et al demonstrated that at a concentration of 2.5 mM, alpha-methyl-DL-tryptophan was able to block approximately 85% of glycine uptake. It has been proposed that blockage of SLC6A14 leads to amino acid starvation, decreased proliferation and apoptosis in cancer cells, demonstrating the considerable impact which SLC6A14 has on the transport of solutes and nutrient provision in proliferating epithelial tissue[73, 74].

Recent studies conducted by Babu et al demonstrated the importance of SLC6A14 in the development and progression of breast cancer using a slc6a14-/- mouse model. Expression of slc6a14 was knocked-out in mice of the PyMT and MMTV/Neu (mouse mammary tumour virus promoter-Neu)-Tg backgrounds, which generate spontaneous mammary tumour growth. From these studies, it was first noted that knock-out of transporter expression did not affect viability of the mice or cause changes in expression of alternative/compensatory amino acid transporters, such as transporters which are members of the SLC1, SLC3, SLC6, SLC7 and SLC38 families. Importantly, the slc6a14-/- mice showed decreased cell proliferation and evidence of amino acid starvation, decreasing mammary tumour growth and tumour incidence rate[75].

Furthermore, SLC6A14 dependent leucine transport has been implicated as a potent activator of the cell growth and proliferation regulator, the mammalian target of rapamycin (mTOR), which plays a crucial role in regulating cell cycle, proliferation and cancer[76]. In addition to cancer, SLC6A14 is up-regulated in patients with ulcerative colitis, a chronic mucosal inflammation of

15 the large intestine. It was proposed that SLC6A14 might play a role in recovering fluid loss or enhancing host antimicrobial response, aimed at restoring the integrity of the intestinal mucosa[77].

1.4 Alternative Arginine Transporters

One main focus of the studies outlined in the proceeding chapters is to determine the importance of SLC6A14 function in transporting the substrate arginine, from the airway surface fluid of polarized lung epithelia into the cell. Therefore, it is important to discuss some other relevant transporters (either expressed on the apical or basolateral membrane of lung epithelia), which also function to transporter cationic (and/or neutral) amino acids into the cell.

Table 1.1 and figure 1.5 provide a summary of additional cationic amino acid transporters which are expressed in polarized lung epithelia (and other tissues), the affinity each transporter has for arginine, whether this transport is dependent on sodium gradients, and their localization on either the apical or basolateral membrane[78-82].

Transporter Amino Acid Km for Arginine Arginine Tissue Expression Substrates Transport Transport (mmol/L) Na+-dependent?

SLC6A14 neutral or 0.08-0.15 Yes (also Small intestine, cationic dependent on Cl-) colon, lung, mammary gland, pituitary gland, salivary gland

SLC7A1 cationic 0.10-0.16 No Ubiquitously (CAT-1) expressed (but not in the liver)

SLC7A2 cationic 0.038-0.38 No Inducible in a (CAT-2B) variety of cell types (by inflammatory cytokines and LPS)

SLC7A6 exchangers 0.12-0.14 Yes, for neutral Heart, kidney, brain, (y+LAT2) + of neutral (independent of amino acid small intestine, lung, SLC3A2 and cationic [Na+]) transport only thymus, testis (4F2hc)

SLC7A7 exchangers 0.34 (0.93 Yes, for neutral Small intestine, (y+LAT1) + of neutral without Na+ amino acid kidney, spleen, lung SLC3A2 and cationic present) transport only leukocytes, placenta (4F2hc)

SLC7A9 exchangers 0.08-0.2 No Lung, kidney, small (b0,+AT) + of neutral intestine, placenta SLC3A1 and cationic (rBAT)

Table 1.1. Expression of arginine transporters in the lung. This table provides a summary of additional arginine transporters expressed in lung epithelia, the Km values for arginine transport and the dependence of this transport on sodium gradients. Many Km values for these transporters appear to overlap with that SLC6A14, however, SLC6A14 appears to be the only transporter listed above whose function is dependent on both sodium and chloride gradients within the cell (summary table adapted from Closs et al, 2004)[78-82].

Figure 1.4. Expression of arginine transporters in polarized airway epithelium. This figure provides an illustration of the localization of arginine transporters expressed on the membranes of lung epithelial tissue.

1.5 Project Rationale

It has been shown that lung infection, which is the leading cause of morbidity and mortality in CF patients, is heavily impacted by genetic modifiers and the environment, and cannot be addressed by simply examining CFTR genotype alone. This infers the need to study other molecules, in addition to CFTR, as potential therapeutic targets for treatment of CF airway disease, particularly infection[48]. It was Galietta et al who first proposed the presence of an electrogenic transporter on the surface of airway epithelial tissue and the idea that this transporter should be studied in patients with respiratory distress syndrome. They proposed that if you were to remove function of this transporter, the extracellular concentration of amino acids would increase, providing a source of nutrients for pathogens[69].

This was followed by GWAS-HD studies performed by Sun et al, implicating polymorphisms in SLC6A14 as a modifier of CF phenotypes in the colon[56]. This transporter was also confirmed by Li et al to be a potential modifier of CF lung infection with Pseudomonas aeruginosa[65]. Currently, the mechanism underlying how SLC6A14 modifies CFTR function and CF phenotypes remains unknown, but it continues to be a modifier of interest, especially when considering the role it may play in modifying intestinal obstruction and pulmonary infections with Pseudomonas aeruginosa[83]. Current hypotheses implicate SLC6A14 to have an important role in modifying health and nutrient status of the epithelium and regulation of fluid in the airways[70]. This provides a good basis to mechanistically determine if SLC6A14 does play a significant role in modifying infection in CF patients, and if it can be used as a future therapeutic target for those patients with severe CF phenotypes in the lung.

Additionally, the adverse effects of Pseudomonas aeruginosa infection on the function of F508del-CFTR protein, rescued using the chemical corrector VX-809, has caused an increase in the need for discovery of alternative therapeutics and therapeutic targets, aimed at ameliorating defects observed in the CF lung[40].

1.6 Hypothesis and Specific Aims

Since SLC6A14 is has been shown to be up-regulated in inflammation, and has been implicated as a modifier of Pseudomonas aeruginosa infection in the CF lung, it may play a role in counteracting the pro-inflammatory pathways initiated by bacterial interactions with the epithelium, specifically through TLR-5. Chronic and severe infections and inflammation are a predominant hallmark in the environment of the CF lung, therefore I hypothesize that SLC6A14 expression is regulated by bacterial pathogens and that in turn, SLC6A14 expression modifies bacterial infections in CF.

The goal of this project is to determine if SLC6A14 function plays an important role in recovering amino acids from airway surface fluid in the lung, causing depletion of a nutrient source for Pseudomonas aeruginosa, preventing bacterial growth and colonization (figure 1.6). Therefore, this project is aimed at determining if SLC6A14 expression and function are affected by Pseudomonas aeruginosa and whether SLC6A14 function modifies the composition of the airway surface fluid.

The specific aims of this project are:

I. To characterize SLC6A14 function and expression in respiratory epithelium, and to determine if SLC6A14 expression is altered in the presence of immune activating components from Pseudomonas aeruginosa

II. To investigate if up-regulation or inhibition of SLC6A14 causes significant changes to the composition of amino acids in the airway surface fluid

Figure 1.5. Proposed role of SLC6A14 as a modifier of lung infection in CF airway epithelial cells. This figure provides a graphical representation of the hypothesis described above. It highlights the prediction that in the airways, the presence of Pseudomonas aeruginosa or bacterial components (such as flagellin, which will contact the epithelium) will bind to TLR-5 and may lead to downstream up-regulation of SLC6A14 expression, ultimately causing an increase in amino acid uptake at the apical membrane and a decrease in bacterial growth and/or colonization.

Chapter 2: Methods

2.1 Cell Culture

The human bronchial (submucosal gland) adenocarcinoma cell line (Calu-3 cells) was purchased from the American Type Culture Collection (ATCC, Virginia, USA), and were grown and maintained in 75 cm2 flasks. Media was changed every 2-3 days and cells were passaged upon reaching 90-100% confluency, at a dilution of 1:2. Cells were cultured in Eagle’s Minimum Essential Medium (EMEM) supplemented with 10% fetal bovine serum and 1% penicillin- streptomycin (stock solution of 10000 IU penicillin and 10000 µg/mL streptomycin). Media for Calu-3 cells which were infected with lentivirus was additionally supplemented with 10 µg/mL puromycin, to select for infected cells.

For quantitative real-time polymerase chain reaction, western blotting and L-[2,3-3H]-arginine uptake studies, cells were seeded (at high density) onto 12-well flat bottom, tissue culture-treated plates, where media was changed every 2-3 days until reaching confluency. Assays were completed 1-2 days post-confluency. For fluid uptake studies, primary airway cultures were obtained from non-CF and CF post-transplant bronchial tissue. This tissue was provided through collaboration between Dr. Shaf Keshavjee (University of Toronto), Dr. Phillip Karp and Dr. Michael Welsh (NIH Iowa Culture Facility). Cells were plated on collagen IV coated polyester membrane inserts for 12-well plates (with a 0.4 µM pore size). Seeding on these inserts allows for cell polarization and gives access to both apical and basolateral compartments, allowing cells to be grown at an air-liquid interface (figure 2.1). Post-seeding, media was not present on the apical compartment, and media in the basolateral compartment was changed every 2-3 days, maintaining cultures at an air-liquid interface. Media was comprised of a 1:1 mixture of Dulbecco’s Modified Eagle’s Medium and Ham’s F12 Medium (DMEM/F12), supplemented with 2% Ultroser G Serum Substitute (PALL France SAS; Saint Germain-en-Laye, France). Trans-epithelial resistance was measured using a volt-ohm meter (World Precision Instruments, Florida, United States), and functional studies were done once cultures reached and maintained a trans-epithelial resistance of approximately >800 ohms.

Non-CF

10 µm

Figure 2.1. Post-transplant bronchial tissue forms a well-differentiated monolayer when seeded onto transwell inserts. The left panel provides a graphical illustration of the way in which these cells are grown and maintained within the transwell insert. The right panel displays immunofluorescence images (staining performed by Dr. Kai Du, Dr. Christine Bear Laboratory, Hospital for Sick Children) of these cultures, taken from both a non-CF patient and a CF patient homozygous for F508del expression. From these images, it is evident that CFTR protein is absent on the apical surface of the CF epithelium (green), and it retained in the cytosol. It is also clear that this culturing method allows for apical to basolateral polarization and differentiation of the epithelium, visible by staining for zona occludin-1 (ZO-1), a marker of tight junction formation (red). Nuclei are also visible (blue).

2.2 Lentiviral Infection

Calu-3 cells were seeded at a density of 50,000 cells per well in a 24-well plate, and were then transduced with lentiviral particles encoding pGIPZ shRNA targeting SLC6A14 (sequence: 5’- AGATTGTCACAAAAATGGA-3’) or a non-silencing hairpin control (both containing a puromycin resistance gene; sequences provided by Dr. Saumel Ahmadi, Dr. Christine Bear Laboratory). Lentiviral transduction was performed by Christopher Fladd and Diane Ly (SPARC BioCentre, Hospital for Sick Children). Media was replaced with a serum-free media containing 8 µg/mL of hexadimethrine bromide (polybrene). Virus was infected with a titre of approximately 2.5x106 transducing units per millilitre, at a multiplicity of infection of 200. Cells were incubated with virus for 4 hours, which was then replaced with media containing serum (lacking antibiotics) and incubated overnight. After 24 hours, media was replaced with standard Calu-3 cell culture media (lacking puromycin), and incubated for an additional 48 hours. After 48 hours, cells were washed 3 times with PBS (containing calcium and magnesium), to remove any residual virus, and media was replaced with media containing puromycin (10 µg/mL) and maintained in this media, for selection of infected cells.

2.3 Quantitative Real-Time Polymerase Chain Reaction

Cells were lysed and RNA extraction was performed following the protocol outlined by the RNeasy® Plus Mini Kit (Qiagen, Germany). Following cell lysis and extraction, RNA concentration was measured using a Nanodrop 2000 (Thermo Fisher Scientific, Massachusetts, USA). Samples were used with concentrations higher than >100 ng/µL, with a 260/280 ratio of 2.0-2.2. iSCRIPT cDNA Synthesis Kit (Bio-Rad, California, USA) containing reverse transcription (RT) enzyme, was used to make 1 µg of cDNA for each sample (control samples were RNA incubated with water in place of RT). Quantitative real-time PCR was performed using the EvaGreen fluorophore (SsoFast EvaGreen Supermix with Low Rox, Bio-Rad) in a 96- well plate format using a CFX96 TouchTM Real-Time PCR Detection System (Bio-Rad). Table 2.1 shows the primers used for amplification, sequences were provided from Dr. Johanna Rommens’ Laboratory (Hospital for Sick Children). The fluorophore binds newly synthesized DNA molecules and gives a real-time read-out of production of the DNA molecule of interest.

Data were output as cycle threshold values (CT), which is the number of PCR cycles required for

24 the fluorescence signal to cross threshold. Low values indicate high amounts of the target cDNA molecule. Table 2.2 outlines the cycling parameters used for each qRT-PCR experiment.

Gene Forward Primer Reverse Primer

CFTR 5'-GCATTTGCTGATTGCACAGT-3' 5'-CTGGATGGAATCGTACTGCC-3'

GAPDH 5'-CAAGAGCACAAGAGGAAGAGAG-3' 5'-CTACATGGCAACTGTGAGGAG -3'

GUSB 5'-CCCATTATTCAGAGCGAGTATG-3' 5'-CTCGTCGGTGACTGTTCAG-3'

SLC6A14 5'-TATGGCGCAATTCCATACCC-3' 5'-CCAGGTATGGACCCCAGTTA-3'

Table 2.1. List of primer sequences used for quantitative real-time PCR experiments.

Cycling Step Temperature Time Number of Cycles

Enzyme Activation 95°C 2 minutes 1

Denaturation 95°C 10 seconds 40

Annealing/Extension 60°C 30 seconds

Table 2.2. Cycling conditions for quantitative real-time PCR reactions.

-∆∆C [84] Fold-change in transcript levels was calculated using the 2 T (Livak) Method . This is calculated by taking the CT difference of the target gene (either SLC6A14 or CFTR) to that of the reference gene (GUSB or GAPDH) for both the control and treated sample: ∆CT = CT(target, treated) -

CT(reference, treated) ; ∆CT = CT(target, control) - CT(reference, control)

Next, the ∆CT of the treated was normalized to that of the control: ∆∆CT = ∆CT(treated) - ∆CT(control)

-∆∆C Then, the expression ratio was calculated: 2 T = normalized expression ratio

This result is equivalent to the fold increase (or decrease) of the target gene following treatment relative to the control and normalized to the reference (housekeeping) gene. GUSB was used for qRT-PCR studies done with Calu-3 and primary bronchial epithelial cells, whereas GAPDH was used for studies done with primary nasal epithelial cells.

2.4 Western Blotting

Cells were lysed using a buffer containing 50 mM tris-hydrochloride, 150 mM sodium chloride, 1 mM EDTA (pH 7.4), with 0.1% (v/v) SDS, 1% (v/v) Triton X-100 and 1% (v/v) protease inhibitor cocktail (Amresco), then incubated at 4 °C for 15 minutes while shaking. Following incubation, lysates were centrifuged at 14000 rpm for 5 minutes. After centrifugation, the supernatants were collected from each cell sample and solubilized in Laemmli sample buffer (at a 1:5 dilution) and run on an 8% SDS-PAGE gel (Life Technologies, California, USA). Protein was then transferred onto a nitrocellulose membrane and blocked with 5% (w/v) skim milk. Following blocking, the membrane was probed with either an antibody against human SLC6A14 (raised in rabbit, added in a 1:1000 dilution, Abgent, California, USA) or human β-actin (raised in mouse, added in a 1:10000 dilution, Sigma-Aldrich), and then incubated with a horseradish peroxidase conjugated secondary antibody which was raised in goat, against rabbit or mouse (added in a 1:5000 dilution). The membrane was then washed and chemi-luminescence was used to detect signals from the respective antigens.

2.5 Amino Acid Uptake Studies

2.5.1 L-[2,3-3H]-arginine Uptake

Cells were washed and then incubated for 30 minutes in HEPES buffer at 37°C, to remove cell culture media and reduce the intracellular pool of amino acids present. Cells were then treated with HEPES buffer supplemented with 1 µCi/mL of L-[2,3-3H]-arginine (specific activity of 54.6 Ci/mmol) and 100 µM arginine, in the presence or absence of the pharmacological blocker alpha-methyl-DL-tryptophan (at a final concentration of 2 mM) for 15 minutes. Control samples consisted of HEPES buffer supplemented with 1 µCi/mL of L-[2,3-3H]-arginine and 10 mM arginine, to outcompete the uptake of the radiolabeled amino acid. Following this, cells were

26 washed three times using ice-cold HEPES buffer supplemented with 10 mM arginine and lysed with 0.5 sodium hydroxide for 10-15 minutes, shaking on ice. 12.5 µL of each lysate was added to 2 mL of EcoScint A Scintillation Fluid (Diamed, Switzerland) and counts were read using a Beckman Scintillation Counter (LS-6000IC), which gives a read-out of counts per minute (CPM). The total amount of protein in each sample was determined using a Bio-Rad protein assay. Amount of protein is determined by absorbance (at 595 nm) of a Coomassie Brilliant Blue

G-250 dye. Diluted protein samples (diluted 1:2 in ddH2O) were added to this dye, and protein concentration was determined using a bovine serum albumin standard curve.

To calculate the amount of arginine uptake into the cell, the following equation was used:

Arginine Uptake (mol/minute/mg protein) =

(CPM - CPMBackground) × (moles arginine/CPMMax) × (µL sample counted/µL scintillator fluid added) × minutes-1 × [protein]-1

3 CPMBackground detects the amount of L-[2,3- H]-arginine in the presence of 10 mM (unlabelled) arginine (maximal uptake competition, either by non-specific transport or diffusion into the cell) and is considered to be the background control. CPMMax represents the CPM value acquired from counting the buffer alone, which allows for normalization to the total amount of radiolabeled substrate in each buffer. Moles of arginine represents the moles of unlabelled (not tritiated) arginine present in the buffer.

Normalized arginine uptake was calculated as follows:

% total arginine uptake = ((R – MT)/R) × 100%, where R is equal to uptake of the control and MT is equal to uptake of the tritiated arginine in the presence of 2 mM alpha-MT (blocking SLC6A14 function)

For studies completed using mouse tracheal tissue, CPM values obtained from each reading were taken as a ratio of whole-tissue lysate from each sample (expressed as CPM/[protein]).

2.5.2 Treatment and Collection of Airway Surface Fluid

Transwell inserts were apically pre-treated with either HEPES buffer or HEPES buffer supplemented with 1 µg/mL of purified flagellin protein (FLA-PA) for 6 hours, in the presence of 500µM arginine. In the secondary protocol (examining a shorter time frame for amino acid uptake), inserts were pre-treated with HEPES buffer or HEPES buffer supplemented with FLA- PA for 5 hours, lacking amino acids. Following this pre-treatment, either DMSO (control), arginine (added to final concentration of 80-110µM, concentration determined by examining t=0 samples), and/or alpha-MT (at a final concentration of 2 mM), all of which were dissolved in HEPES buffer, were additionally placed onto the apical membrane for 1-hour. Glutamic acid was also supplemented into the fluid as a control for all experiments (concentrations varied according to each protocol). Following these treatments, the fluid was collected and amino acid content was determined via high-performance liquid chromatography (HPLC), using a reverse phase C18 column, performed by Reynaldo Interior (SPARC BioCentre, Hospital for Sick Children). Data is displayed as chromatographic traces, with the area under each curve representing the picomoles (which was used to calculate µM, given the sample volume) of each amino acid present in the sample.

Percent uptake was calculated as follows (analysis dependent on experimental set-up):

% uptake = ((amount of argininecontrol – amount of argininetreatment with FLA-PA)/(amount of

argininecontrol)) × 100%, this method of calculation used when examining uptake over 6-hours

% uptake = ((amount of amino acid added at t=0 of assay – amount of amino acid remaining after 1-hour)/t=0) × 100%, where t=0 is equal to the concentration of the amino acid added at the start of the assay (taken as an independent sample), this method of calculation used when examining uptake over 1-hour

To assess differences in arginine uptake between non-CF and CF cultures, following treatment with FLA-PA, the following calculation was done for each sample:

% increase in uptake = (% uptake following FLA-PA treatment – % uptake of control)

2.6 Reagents

All tissue culture reagents, including culturing media, puromycin, penicillin-streptomycin, and fetal bovine serum were purchased from Wisent Bioproducts (Quebec, Canada). All amino acids used were of the ‘L’ stereoisomer, and were purchased from Sigma-Aldrich, including alpha- methyl-DL-tryptophan (Oakville, Ontario, Canada). L-[2,3-3H]-arginine was purchased from The American Radiolabeled Chemicals Incorporated (Missouri, USA). HEPES buffer was made using 25 mM HEPES, 140 mM sodium chloride, 5.4 mM potassium chloride, 1.8 mM calcium chloride, 0.8 mM magnesium sulfate, and 5 mM glucose (in ddH2O) and buffer pH was brought to 7.4 using sodium hydroxide. Regents for this buffer were purchased from either Sigma- Aldrich or BioShop Canada (Burlington, Ontario, Canada). Purified flagellin from Pseudomonas aeruginosa was obtained from InvivoGen (San Diego, California, USA) and was dissolved in endotoxin-free water.

2.7 Statistical Analysis

Data are presented as means +/- standard error of the mean (SEM), for a minimum of n=3 biological replicates for each data set. Quantitative real-time PCR experiments and percentage amino acid uptake from airway surface fluid was analyzed using one-way ANOVA and the Tukey’s multiple comparison test. To account for day-to-day variability in magnitude of amino acid uptake, uptake was normalized to the value obtained for the t=0 sample (amino acids added at the start of the assay, for 1-hour uptake experiments). Data sets comparing non-CF to CF donors were analyzed using unpaired two-tailed t-test. To assess if there is a correlation between message expression and amino acid uptake, both a Pearson-product moment correlation coefficient (in addition to linear regression) and a Spearman rank correlation were computed, with a two-tailed analysis done to determine if these correlations were significant. Data were analyzed using GraphPad Prism (version 6.01).

Chapter 3: Modulation of SLC6A14 messenger RNA expression in lung epithelium

3.1 SLC6A14 expression is enhanced by an immune activating component from Pseudomonas aeruginosa

In order to determine the role SLC6A14 plays in modifying lung epithelia, it first needed to be determined if transporter expression can be detected, and subsequently modified in lung epithelium.

By measuring mRNA levels in the Calu-3 cell line, it was determined that SLC6A14 expression is detectable in lung epithelium. To determine if transporter expression can be modulated by interactions with bacterial components, these cells were subsequently treated with purified flagellin protein from Pseudomonas aeruginosa. Upon treatment with flagellin, SLC6A14 mRNA levels significantly increased compared to the untreated control. This corresponds to an average reduction in CT values from approximately 28.2 to 24 (for n=5). To demonstrate that this up- regulation was SLC6A14 specific, the response of CFTR mRNA levels to treatment with flagellin was examined in parallel (CT values reduced, on average, from 23.5 to 23 for n=4). Although CFTR expression is slightly enhanced in the presence of flagellin, this increase is not significant when compared to control. This indicates that an innate immune activating component of Pseudomonas aeruginosa, such as flagellin protein, plays a role in modulating SLC6A14, but not CFTR expression, at the mRNA level. Figure 3.1 depicts the average fold increase in transcript levels for both SLC6A14 and CFTR, following treatment with flagellin and normalized to a housekeeping gene.

****

Figure 3.1. Treatment with flagellin significantly increases SLC6A14 mRNA expression in Calu-3 cells lung epithelial cells. Upon 6-hour treatment with FLA-PA, an average fold- increase in SLC6A14 mRNA levels of 26.4 was observed, when normalized to the housekeeping gene GUSB (one-way ANOVA using Tukey’s multiple comparison test, **** p<0.0001 for n=5), compared to untreated control. For comparison, fold-increase of CFTR mRNA levels following FLA-PA treatment was also surveyed, and increased by only 1.8-fold, which was not significant when compared with the control (one-way ANOVA using Tukey’s multiple comparison test, “ns” p=0.7687 for n=4).

Next steps involved confirming that this up-regulation could also be observed in primary bronchial epithelial cells, from both non-CF and CF donors. From the cultures tested, a few observations can be made. The first is that, there is no significant difference in the relative expression levels of SLC6A14 between non-CF (CT average prior to normalization of 24.6 for n=4) and CF (CT average prior to normalization of 24.4 for n=4) epithelia (figure 3.2). The second is that treatment with flagellin causes a significant fold-increase in SLC6A14 expression in primary bronchial epithelial cells, however there is no significant difference in this fold- increase between genotypes (figure 3.3).

Figure 3.2. Relative expression levels of SLC6A14 does not differ between non-CF and CF cultures. When normalized to both the housekeeping gene GUSB and also to non-CF mRNA levels, average expression of SLC6A14 appears to be 1.9-fold higher in cultures from CF donors (before treatment with FLA-PA), however this difference is not statistically significant (unpaired two-tailed t-test, “ns” p=0.2649 for n=4 donors for each genotype).

Non-CF 6 *

0 Control FLA-PA

Figure 3.3. Treatment with flagellin significantly increases SLC6A14 expression in non-CF and CF primary bronchial epithelial cells. Upon 6-hour treatment with FLA-PA, an average fold-increase in SLC6A14 mRNA levels of 3.9 and 4-fold was observed, in non-CF and CF primary epithelial cells, respectively, when normalized to the housekeeping gene GUSB (unpaired two-tailed t-test, * p<0.05 for n=4 donors for each genotype). These fold changes correspond to an average change in CT values of 24.6 to 22.9 and 24.4 to 22.5, for non-CF and CF cultures, respectively.

3.2 SLC6A14 expression can be modified by shRNA-mediated knock-down

In order to test the effect of transporter function on modifying amino acid transport in lung epithelium, a cell line in which there would be (partial) knock-down of SLC6A14, using lentiviral infection with shRNA, was generated. Using this system, the Calu-3 cell line was modified to reduce SLC6A14 expression (figure 3.4). Unfortunately, this partial knock-down, although significant, was not substantial enough to pursue usage of this cell line in functional studies.

Figure 3.4. Lentiviral infection with shRNA causes significant knock-down of SLC6A14 message expression. Upon knock-down, expression levels of SLC6A14 reduced by 0.26-fold, when compared to cells infected with a control (scramble) shRNA and normalized to the housekeeping gene GUSB (unpaired two-tailed t-test, * p=0.0468 for n=3).

Chapter 4: SLC6A14 plays an important role in regulating amino acid uptake in lung epithelium

4.1 Inhibition of SLC6A14 causes a significant reduction in L- [2,3-3H]-arginine uptake

From the above data, it is clear that SLC6A14 is expressed in the Calu-3 lung epithelial cell line, and this expression can be modulated. In addition to expression levels, it is important to confirm that this transporter is also functionally expressed in these cells. To test this, uptake of radiolabeled arginine, L-[2,3-3H]-arginine, was assessed using Calu-3 cells. Over a 15-minute time period, a significant reduction in arginine uptake was observed, following inhibition of SLC6A14 using the blocker alpha-MT (figure 4.1-4.2).

1.2 10 -13

1.0 10 -13

7.5 10 -14

5.0 10 -14 *** 2.5 10 -14

0 Control alpha-MT

Figure 4.1. L-[2,3-3H]-arginine uptake significantly decreases with the addition of alpha- MT in Calu-3 cells. This figure depicts mol/min/mg protein of L-[2,3-3H]-arginine uptake in Calu-3 cells, after 15 minutes of treatment with 100 µM L-arginine in the presence or absence (control) of the inhibitor alpha-MT. Upon alpha-MT treatment, L-[2,3-3H]-arginine uptake significantly reduces (paired two-tailed t-test, *** p=0.0004 for n=3).

***

Figure 4.2. Percentage of L-[2,3-3H]-arginine uptake significantly decreases with the addition of alpha-MT in Calu-3 cells. This figure depicts the percent decrease of mol/min/mg of L-[2,3-3H]-arginine uptake in Calu-3 cells, after 15 minutes of treatment with 100 µM L- arginine in the presence or absence (control) of the inhibitor alpha-MT. Upon alpha-MT treatment, L-[2,3-3H]-arginine uptake reduces approximately 77.5% (unpaired two-tailed t-test, *** p=0.0002 for n=3).

4.2 Inhibition or up-regulation of SLC6A14 significantly modifies uptake of arginine in non-CF and CF primary airway cells

Following the characterization of SLC6A14 expression and function in the Calu-3 lung epithelial cell line, it was imperative to determine if up-regulation or reduction in transporter function would cause significant changes to the composition of airway surface fluid. For these studies, primary airway cultures, grown at an air-liquid interface, were used. Due to sample size and availability, function of SLC6A14 was assessed by monitoring changes in the amino acid content in the ASL, determined by HPLC analysis, rather than using the L-[2,3-3H]-arginine uptake assay. Concentration changes are taken to reflect the amount of uptake of each amino acid into the cell, from the apical surface. Changes in two amino acids were tracked over a 1-hour time period, arginine (SLC6A14 substrate) and glutamic acid (non-substrate).

A key concept to test was the idea that inhibition of SLC6A14, using the blocker alpha-MT, or up-regulation, using purified flagellin protein, can modify the composition of arginine in the ASL of non-CF and CF primary airway cultures, given the observations made in the Calu-3 cell line and primary bronchial cells by qRT-PCR analysis. For this, the composition of arginine and glutamic acid in the ASL was analyzed, after either pre-treatment with FLA-PA or acute inhibition of SLC6A14 with alpha-MT. Figure 4.3 represents chromatographic traces, taken from the ASL of a non-CF airway culture, following either SLC6A14 up-regulation or inhibition. The area under each peak curve was used to determine micromolar concentrations for each amino acid present in the sample. These values were then used to determine amount of arginine and glutamic acid uptake, allowing for the quantification of SLC6A14 function following various treatments, in both non-CF and CF cultures. From these results it was determined that treating non-CF and CF cultures with either FLA-PA or alpha-MT significantly modifies the composition of arginine (figure 4.4, 4.6) but not glutamic acid (figure 4.5, 4.7) in the ASL. In this system, there is no significant difference in the percentage increase of arginine uptake between non-CF and CF cultures, after treatment with FLA-PA (figure 4.8).

Control

FLA-PA

alpha-MT

Figure 4.3. Representative chromatographic traces from the ASL of a non-CF airway culture. This figure depicts three chromatogram traces, taken from samples treated either with amino acids only (arginine and glutamic acid) or amino acids in combination with either FLA- PA or alpha-MT. Peaks representing arginine and glutamic acid from each condition are highlighted (using arrows). A larger peak for arginine, indicating an overall higher concentration of arginine, is present in samples where SLC6A14 function was inhibited (alpha-MT). The reverse can be seen in samples which were treated with FLA-PA, where smaller peaks can be observed, indicating less arginine was present in the ASL and was transported inside the cell, due to an up-regulation of SLC6A14. These trends are not observed for the glutamic acid peaks.

** *

Figure 4.4. Percentage of arginine uptake is significantly modified, upon treatment with alpha-MT or FLA-PA, in non-CF primary airway cells. Upon blockage of SLC6A14 using alpha-MT, average arginine uptake significantly decreases (an average of 33.4% reduction in uptake). Conversely, following pre-treatment with FLA-PA, average arginine uptake significantly increases (an average of 26.4% increase in uptake), which can also be inhibited using alpha-MT (an average of 56.5% reduction in uptake; one-way ANOVA using Tukey’s multiple comparison test, ** p<0.01 and * p=0.0151 for n=5 patients). Corresponding concentrations for each treatment are depicted in supplementary figure A2.

ns ns ns

Figure 4.5. Unlike arginine, glutamic acid uptake is not significantly modified following blockage of SLC6A14 with alpha-MT or up-regulation using purified flagellin protein in identical non-CF airway cultures (one-way ANOVA using Tukey’s multiple comparison test, “ns” p>0.60 for n=5 patients). Corresponding concentrations for each treatment are depicted in supplementary figure A3.

** **

Figure 4.6. Percentage of arginine uptake is significantly modified, upon treatment with alpha-MT or FLA-PA, in CF primary airway cells. As observed in the non-CF cultures, upon blockage of SLC6A14 using alpha-MT, average arginine uptake significantly decreases (an average of 53.9% reduction in uptake). Conversely, following pre-treatment with FLA-PA, average arginine uptake significantly increases (an average of 16.6% increase in uptake), which can also be inhibited using alpha-MT (an average of 61.6% reduction in uptake; one-way ANOVA using Tukey’s multiple comparison test, ** p<0.01 and * p=0.0144 for n=6 patients). Corresponding concentrations for each treatment are depicted in supplementary figure A4.

ns ns

Figure 4.7. Unlike arginine, glutamic acid uptake is not significantly modified following blockage of SLC6A14 with alpha-MT or up-regulation using purified flagellin protein in identical CF airway cultures, replicating those results observed in non-CF cultures (one-way ANOVA using Tukey’s multiple comparison test, “ns” p>0.10 for n=6 patients). Corresponding concentrations for each treatment are depicted in supplementary figure A5.

ns 20

0 non-CF CF

Figure 4.8. Percentage of arginine uptake does not differ significantly when comparing non-CF and CF-affected airway epithelium. When comparing changes in arginine uptake between non-CF and CF cultures, it appears that there is larger increase in percentage of arginine uptake, following FLA-PA treatment, in cultures from non-CF donors, however this difference is not statistically significant (unpaired two-tailed t-test, “ns” p=0.0856 for n=5 non-CF and n=6 CF donors). These percentage changes reflect an average absolute increase in arginine uptake of 32 µM and 20.4 µM in non-CF and CF cultures, respectively.

4.3 Changes in arginine uptake positively correlates with up- regulation of SLC6A14 expression following treatment with purified flagellin

Another goal of these studies was to test if there is a correlation between the fold change in SLC6A14 expression levels and the percentage increase in arginine uptake, after treatment with purified flagellin from Pseudomonas. From the data below, it appears that within the patient population tested, there is a significant correlation between increase in SLC6A14 transcript levels and SLC6A14 function. SLC6A14 function was taken as a measure of percentage increase in arginine uptake into the cell from the airway surface fluid, over a 6-hour period (figure 4.9).

Figure 4.9. Percentage increase in arginine uptake as a function of fold change in SLC6A14 expression. A Pearson-product moment correlation coefficient was calculated to assess the relationship between percentage increase in arginine uptake from the airway surface fluid and up-regulation of SLC6A14 at the message expression level, following a 6-hour treatment with FLA-PA, for both non-CF (black) and CF (green) samples. A positive correlation was observed between these two variables (r=0.8174, p=0.0470 for n=6 patients; 3 from non-CF and 3 from CF donors). This data is also significant using a Spearman rank correlation (r=0.9429, p=0.0167 for n=6), demonstrating that increases in SLC6A14 expression lead to increases in arginine uptake (indicating expressional changes are correlated to increases in SLC6A14 function).

Chapter 5: Discussion

Modulation of SLC6A14 Expression by Bacterial Flagellin

It has been previously demonstrated, through qRT-PCR analysis, that the Calu-3 cell line expresses cationic amino acid transporters, such as SLC6A14, SLC7A1, SLC7A2, SLC7A6, SLC7A7, and SLC3A2[85]. The findings described above confirm that SLC6A14 is expressed in this cell line and that this expression can be modified by treatment with immune activating components derived from Pseudomonas aeruginosa, such as purified protein from bacterial flagellin of Pseudomonas aeruginosa. Upon treatment with flagellin, there is a significant fold- increase in overall mRNA expression levels of this transporter. This degree of fold-increase is not observed when examining CFTR mRNA levels, following incubation with purified flagellin. On average, SLC6A14 message expression increases 26.4-fold (when normalized to the GUSB housekeeping gene), whereas CFTR message expression levels only increase 1.8-fold (figure 3.1). From this, it is evident that the substantial up-regulation in mRNA levels, observed upon treating airway cells with purified flagellin protein, may be specific to SLC6A14 expression.

Due to the fact that the Calu-3 cell line is derived from a submucosal gland adenocarcinoma, it was important to confirm these results in an alternative cell system, such as post-transplant primary bronchial epithelial cells. These cells might have a modified expression and/or functional demand for arginine because they are derived from cancerous cells, highlighting the importance to confirm these findings in more relevant tissues. Although the magnitude of up- regulation was not as large as what was observed in the Calu-3 cell line, treatment with purified flagellin from Pseudomonas aeruginosa also up-regulates SLC6A14 mRNA levels in primary bronchial epithelial cells, from both non-CF and CF donors. On average, message expression of SLC6A14 increased 3.9 and 4-fold in non-CF and CF cultures, respectively (figure 3.3). It is worth noting that relative expression levels (prior to treatment with flagellin) of SLC6A14 does not significantly differ between non-CF and CF-affected epithelial tissue, despite average SLC6A14 expression appearing to be slightly elevated in the CF cultures (figure 3.2).

These findings are novel and were predicted based on previous evidence demonstrating that SLC6A14 is up-regulated in patients with ulcerative colitis (inflammation of the colon). This up-

45 regulation was predicted to serve as a potential mechanism for host-antimicrobial defense in these patients[77].

Unfortunately, SLC6A14 protein levels could not be assessed, due to lack of a specific antibody for western blotting, and is one limitation to these studies (supplementary figure A1).

Role of SLC6A14 in Amino Acid Transport and Airway Surface Fluid Modulation

These findings (mentioned above), indicate that cellular interactions with bacterial components, such as the TLR-5 agonist flagellin, may be modulating transporter transcript levels. This modulation may play an important role in altering transporter function. By increasing total abundance of SLC6A14 transcript, it is expected that there will be a subsequent increase in the total amount of protein produced, which will lead to an increased abundance of SLC6A14 expressed on the apical membrane of the epithelium. If there is an increased amount of transporter protein present on the apical membrane, it is predicted that there will be an increase in amino acid transport (neutral and cationic) into the cell, from the airway surface fluid.

To address this hypothesis, subsequent studies were aimed at testing the impact of modifying expression or blocking transporter function, on modulating amino acid uptake and the composition of airway surface fluid. This was investigated using two methods. The first method used measured uptake of radiolabeled arginine into the cell. Blocking SLC6A14 function using the inhibitor alpha-MT caused a 77.5% reduction in radiolabeled arginine uptake in Calu-3 epithelial cells (figure 4.1-4.2). This supports previous findings from this cell line, which have reported that approximately 50% of arginine influx (at 100 µM) was contributed to by the activity of a sodium-dependent transporter of the system B0,+[85]. These findings also confirm the function of alpha-MT as an inhibitor of SLC6A14-mediated uptake. Previously, this inhibitor was proposed as a potential anti-cancer therapeutic, which can block amino acid uptake in rapidly proliferating cancer cells[74]. Ideally, these studies should have been performed on permeable inserts similar to those completed with the primary cultures (as opposed to flat- bottomed plastic plates), to allow for access to both the apical and basolateral membranes. However, from these studies it is evident that blockage of SLC6A14 causes a significant and substantial reduction in arginine uptake, and therefore transporter function plays a crucial role in arginine uptake (in airway epithelium).

The second method was aimed at testing if blockage or up-regulation of transporter activity will lead to changes in surface fluid composition. This method used non-CF and CF primary airway cells, derived from post-transplant tissue. This was completed via HPLC analysis of the fluid collected from the apical membrane of these cells. From these cultures, a few important observations can be made. The first is that upon treatment with the SLC6A14 blocker alpha-MT, arginine becomes concentrated in the airway surface fluid of both non-CF and CF cultures. On average, a 33.4% and 53.4% reduction in the percentage of arginine uptake was observed in the non-CF (figure 4.4) and CF (figure 4.6) cultures, respectively. The second observation made is that, treatment with flagellin significantly increases the amount of arginine transported into the cell, in both non-CF and CF airways. An average increase in percentage of arginine uptake of 26.4% and 16.6% in non-CF (figure 4.4) and CF (figure 4.6) cultures was observed, respectively. A third and very important observation that can be made from these cultures is that, these modifications do not significantly impact uptake of glutamic acid, a non-substrate (figure 4.5, 4.7). This data supports the primary observation made in Calu-3 cells (and then further validated in primary bronchial epithelial cells) that treatment with immune activating components derived from Pseudomonas aeruginosa will lead to an increase in the abundance of SLC6A14. From studies conducted with primary tissue, it is evident that increased abundance in SLC6A14 will cause a higher amount of amino acids (specifically arginine) to become transported inside the cell from the airway surface fluid.

Similar to the results seen from quantitative real-time PCR analysis, there is no significant difference in percent change of arginine uptake, following treatment with flagellin between non- CF and CF cultures (figure 4.8). Although it appears that non-CF cultures have a larger response to flagellin, this may be because basal arginine uptake is slightly (but not significantly) higher in the CF cultures, however, the maximal percentage of arginine uptake seen with flagellin treatment is comparable across the two sets of cultures. Therefore, from this data it can solely be concluded that SLC6A14 expression and function is impacted by treatment with immune activating components from Pseudomonas aeruginosa, in both non-CF and CF-affected epithelium. It is worth noting that patient-to-patient variability is a key contributing (and limiting) factor when analysing data from primary cultures.

Furthermore, although these cultures are a useful model for studying SLC6A14 function and expression in primary tissue, because they are maintained within a sterile culturing system

(containing antibiotics), they may not be the best representative of the native environment in the airways. Using these cultures, differences between the non-CF and CF epithelium may not be distinguishable until they are incubated with Pseudomonas aeruginosa, which can be used to model the infection which occurs in the CF lung. This data allows us to conclude that SLC6A14 plays an important role in uptake of amino acids in cultures from both genotypes, however differences between these genotypes must be examined via infection studies or alternative models (see blow).

These studies confirm the findings and mechanism proposed by Galietta et al, of the presence of an electrogenic amino acid transporter of the system B0,+, present on the apical membrane able to transport amino acids into the cell (potentially reducing their intake by colonizing pathogens), in human bronchial epithelial cells, and the lack of difference in arginine uptake between non-CF and CF epithelium[69].

Correlating Functional Changes to mRNA Expression

From the data discussed thus far, it has been concluded that SLC6A14 expression and function are both significantly impacted by treatment with purified flagellin. Interestingly, changes in mRNA expression, following this treatment, positively and significantly correlate with an increase in the percentage of arginine uptake from the airway surface fluid of both non-CF and CF cultures. Cultures from patients with higher fold increases in SLC6A14 mRNA expression (following flagellin treatment) demonstrate higher increases in percentage of arginine uptake, validating that up-regulating transporter expression leads to an increase in transporter function (figure 4.9).

Ongoing and Future Studies

Transporting amino acids (neutral and cationic) inside the cell, from the apical membrane, depletes them from the airway surface fluid of airway epithelium. This provides nourishment to the cell but also depletes a potential nutrient source for colonizing bacteria, such as Pseudomonas aeruginosa. Therefore, patients with down-regulated or diminished SLC6A14 activity would be predicted to have a higher accumulation of amino acids in the fluid lining their airways, readily available to be consumed by colonizing bacteria and subsequently would be expected to experience more severe and recurring infections. To test the hypothesis that

SLC6A14 function impacts bacterial growth, future studies will be aimed at assaying the impact of SLC6A14 inhibition (using alpha-MT) on growth of Pseudomonas aeruginosa, in a co-culture system with primary airway cultures from non-CF and CF patients.

Preliminary studies, in collaboration with Dr. Khursigara’s group in Guelph University have shown that bacteria are able to grow and form biofilms on CF primary airway and nasal cultures, and this growth may be altered following inhibition of SLC6A14 or correction of CFTR protein (unpublished data; experiments in progress). Another preliminary observation that has been made is that there are substantial differences in bacterial adherence between non-CF and CF bronchial cultures. Cultures from non-CF patients are able to clear bacteria more effectively than CF, which is expected since chronic and severe lung infection is a common hallmark of the CF lung. This demonstrates that these co-culture systems are a viable model for testing the impact of SLC6A14 function on growth of Pseudomonas aeruginosa in CF (figure 5.1). These preliminary data address one limitation to the functional data mentioned above, which is that non-CF and CF airways respond differently to infection. Under sterile culturing conditions (as in the cultures used for HPLC studies), there are significant effects of both blockage or up-regulation of SLC6A14 in cultures from both CF patients and healthy controls. Therefore, co-culture studies can be used to survey how SLC6A14 function modulates bacterial growth, but also how this modulation is dependent upon the CFTR genotype.

Non-CF

5 µm

Figure 5.1. Co-culture of Pseudomonas aeruginosa and primary lung epithelium. This figure depicts eGFP-tagged Pseudomonas aeruginosa (PAO1pMF230, laboratory strain of Pseudomonas expressing eGFP) adherent to primary airway epithelial cells, following an 8-hour co-culture (washed to remove planktonic bacteria). Primary airway cultures from a non-CF transplant patient show a reduction in adherent bacteria when compared to CF. Experiments were performed by Dr. Amber Park (in collaboration with Dr. Khusigara’s Laboratory, Guelph University).

In addition to using bronchial epithelial cells, nasal cells can also be used as a model cell system to study CF phenotypes. Dr. Theo Moraes Laboratory (Hospital for Sick Children), has successfully demonstrated that nasal cultures can be grown at an air-liquid interface, similar to bronchial epithelial cells[86]. Cells are acquired from nasal brushings, which is minimally invasive, and allows for brushings to be taken from the same patient multiple times, providing a renewable resource of cells from the same patient.

These cells, when cultured at an air-liquid interface, can be also used in the co-culture system (described above). Preliminary co-cultures experiments done with nasal inserts (provided by Hong Ouyang in Dr. Moraes’ Laboratory) from 3 different CF patients, all with homozygous expression of F508del, show that similar to treatment with purified flagellin, direct incubation with Pseudomonas aeruginosa causes significant increases in SLC6A14 mRNA expression (figure 5.2).

Quantifying message expression changes in SLC6A14 between patients may give insight into potential patient-to-patient variabilities observed when examining levels of bacterial attachment. In addition to characterizing if there are changes in bacterial attachment following inhibition of SLC6A14 function, it will be important to study if fold increase in SLC6A14 expression, following co-culture experiments, correlates to the level of bacterial growth and attachment to the epithelium. It can be predicted, based on previous data showing that treatment with flagellin causes an increase in arginine uptake (figure 4.9), that patients with higher up-regulation in mRNA expression levels of this transporter will have overall less bacterial attachment to their epithelium. This would be due to enhancement of SLC6A14 function, concentrating more amino acids into the cell, removing the pool available for bacterial nourishment.

6 ***

0 Control Pseudomonas aeruginosa

Figure 5.2. SLC6A14 expression increases following incubation with Pseudomonas aeruginosa in CF primary nasal cells. Following co-culture with Pseudomonas aeruginosa for 8 hours, an average increase of 4.5-fold was observed in SLC6A14 expression levels, normalized to GAPDH (average CT value change from 24 to 21.7, prior to normalization; unpaired two-tailed t-test, *** p=0.0002 for n=3 CF patients).

Generation of Models to Further Study SLC6A14

Another limitation to these studies is the use of a single inhibitor to test specificity of SLC6A14- mediated arginine uptake. Due to the lack of availability of other inhibitors/pharmacological agents targeting SLC6A14, attempts to generate a Calu-3 cell line, engineered to express a partial knock-down of SLC6A14, in hopes of using this line in the above functional studies, were also completed (using lentiviral infection with shRNA). This cell line expressed an average of 0.26- fold knock-down in transporter expression levels, compared to the control line (figure 3.4). Despite statistical significance, this percentage of knock-down was too small to pursue functional studies. Generation of a more robust knock-down cell line must be completed (possibly through infection with newly designed shRNA sequences or using CRISPR/Cas9 genome editing), before reliable functional studies can be completed and before these cells can

52 be used as a model to study the impact of transporter activity on composition of airway surface fluid.

Alternatively, our lab has generated mice which are knock-out for slc6a14, on the background of mice expressing either WT or F508del-CFTR (ongoing work done by Catherine Luk and Dr. Saumel Ahmadi). Tissue from these mice can be used in our radiolabeled arginine uptake studies, as an alternative method for examining the impact of SLC6A14 expression and function (or lack of function) on amino acid uptake in both airway and intestinal epithelium. Figure 5.3 highlights preliminary data from these experiments, completed using tracheal tissue from both slc6a14+/y and slc6a14-/y mice (studies completed with the help of Dr. Saumel Ahmadi and Wilson Wu, Dr. Christine Bear Laboratory). This data shows that knock-out of slc6a14 significantly reduced arginine uptake in murine tracheal tissue, indicating that SLC6A14 function is important for arginine transport in tracheal tissue. Further studies will examine the efficacy of alpha-MT in these tissues.

****

Figure 5.3. L-[2,3-3H]-arginine uptake significantly decreases following slc6a14 knock-out in murine tracheal tissue. This figure depicts the ratio of CPM per concentration of whole- tissue lysate (protein) of L-[2,3-3H]-arginine uptake in murine tracheal tissue, after 15 minutes of treatment with 100 µM L-arginine. A significant reduction in L-[2,3-3H]-arginine uptake was observed in slc6a14-/y mice (unpaired two-tailed t-test, **** p<0.0001 for n=3 for each genotype).

Alternative Hypothesis/Possible Mechanisms of Action

Alternatively, SLC6A14 function may be modifying CF infection in the lung through the nitric oxide (NO) pathway. It has been previously reported that CF patients have decreased levels of NO in their airways, due to decreased levels of arginine. By concentrating arginine inside the cell, there will be a subsequent increase in levels of nitric oxide. Increased levels of nitric oxide have been associated with increased expression of CFTR, in addition to relaxation of smooth muscle tissue in the airways. Most importantly, low levels of nitric oxide, like those found in CF patients, is associated with a higher frequency of bacterial colonization with Pseudomonas aeruginosa in the airways. From these results, it is evident that SLC6A14 can effectively transport arginine inside the cell, and hence may be causing activation of the NO pathway as a mechanism to reduce bacterial colonization[87]. A potential way to test this hypothesis would be to test if acute treatment with arginine or inhibition of SLC6A14 with alpha-MT alters inducible nitric oxide synthase (iNOS) levels, the enzyme responsible for production of NO, in cultures which are pre-treated with flagellin[88, 89].

This pathway might also help to give insight as to why no significant differences are observed in in arginine uptake between non-CF and CF cultures (when maintained in sterile cell culturing conditions), but there are observable differences in bacterial attachment between these epithelia. It is a well-understood phenotype that the propensity for infection in CF is greater, due to mucus accumulation and ciliary dysfunction, leading to the inability for clearance of microbes and pathogens. Presence of Pseudomonas aeruginosa infection in the airways has also been reported to cause an increase in arginase activity in CF patients, reducing arginine levels and therefore nitric oxide production, worsening pulmonary function. Increased levels of arginase activity has been reported in both CF patients and cftr-/- mice. Given this, it can be predicted that SLC6A14 expression and levels of transporter up-regulation following treatment with flagellin and/or Pseudomonas aeruginosa may correlate to levels of arginase activity in vivo. Patients with increased SLC6A14 expression (and therefore function), would be predicted to have greater arginine uptake into the cell, higher production of NO and lower arginase activity in the airways, leading to a reduction in bacterial infection and enhanced pulmonary function[90-92].

Conclusions

To date, little is known about the role of SLC6A14 in modulating phenotypes related to CF, however these results give insight into the potential role SLC6A14 may play as a modifier of CF. These data support the hypothesis that SLC6A14 expression and function in the airways is modulated by Pseudomonas aeruginosa, implicating this transporter as a key component in modulating infection in CF airways. These findings are the first which indicate that SLC6A14 expression and function is enhanced in the presence of bacterial products, such as bacterial flagellin derived from Pseudomonas aeruginosa, and therefore may be modified by infection in the CF lung. This gives insight into the mechanism in which SLC6A14 may work to combat infection in CF-affected epithelium, through up-regulation and concentration of amino acids inside the cell, to prevent bacterial colonization and epithelium damage. Identifying the role of SLC6A14 as a modifier of CF infection and inflammation in the lung may be important for the treatment of patients carrying risk SNPs for this gene. Better understanding how this transporter is able to modify the CF phenotype and how risk SNPs impact this modification may play an essential role in discovering potential therapies, targeted at restoring lung epithelia.

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Appendix

Calu -3

MW(kDa)

130

100 SLC6A14 72

35 β-actin

Supplementary Figure A1. Examining SLC6A14 expression via western blotting. The following is a representative western blot, probing lysate from Calu-3 cells following 1-2 days of differentiation (similar time frame for differentiation as cultures used in qRT-PCR studies), for detection of SLC6A14. The antibody used above detects the C-terminus of human SLC6A14 protein. Of those commercially available, this antibody demonstrated the most promising results, in its ability to detect a banding pattern which may be specific to SLC6A14 (presented above 72 kDa, outlined in red). However, many non-specific bands were observed and specificity of this antibody needs to be confirmed using samples which express a significant knock-down of SLC6A14. β-actin was used as loading control.

** **

Supplementary Figure A2. The concentration of arginine remaining in the ASL is significantly modified, upon treatment with alpha-MT or FLA-PA, in non-CF primary airway cells. Upon blockage of SLC6A14 using alpha-MT, the average concentration of arginine remaining in the ASL over 1-hour (112.9 µM) is significantly higher than that of control (76 µM). Conversely, following pre-treatment with FLA-PA, the average arginine concentration significantly decreases (44 µM), which can also be inhibited using alpha-MT (110.69 µM; one- way ANOVA using Tukey’s multiple comparison test, ** p<0.01 for n=5 patients). These results (in addition to t=0 values for each patient) are used to calculate percentage changes in arginine uptake, depicted in figure 4.4.

ns ns ns

Supplementary Figure A3. Unlike arginine, glutamic acid concentrations are not significantly modified following blockage of SLC6A14 with alpha-MT or up-regulation using purified flagellin protein in identical non-CF airway cultures (one-way ANOVA using Tukey’s multiple comparison test, “ns” p>0.40 for n=5 patients). These results (in addition to t=0 values for each patient) are used to calculate percentage changes in arginine uptake, depicted in figure 4.5.

** ***

Supplementary Figure A4. The concentration of arginine remaining in the ASL is significantly modified, upon treatment with alpha-MT or FLA-PA, in CF primary airway cells. Upon blockage of SLC6A14 using alpha-MT, the average concentration of arginine remaining in the ASL over hour (106.6 µM) is significantly higher than that of control (56.3 µM). Conversely, following pre-treatment with FLA-PA, the average arginine concentration significantly decreases (35.9 µM), which can also be inhibited using alpha-MT (97.1 µM; one- way ANOVA using Tukey’s multiple comparison test, * p=0.0283, ** p<0.005, *** p=0.0003 for n=5 patients). These results (in addition to t=0 values for each patient) are used to calculate percentage changes in arginine uptake, depicted in figure 4.6.

ns ns ns

Supplementary Figure A5. Unlike arginine, glutamic acid concentrations are not significantly modified following blockage of SLC6A14 with alpha-MT or up-regulation using purified flagellin protein in identical CF airway cultures, replicating those results observed in non-CF cultures (one-way ANOVA using Tukey’s multiple comparison test, “ns” p>0.09 for n=6 patients). These results (in addition to t=0 values for each patient) are used to calculate percentage changes in arginine uptake, depicted in figure 4.7.