Examination of the Role of Zip8 and Cadmium in the Development of Chronic Obstructive Pulmonary Disease

EXAMINATION OF THE ROLE OF ZIP8 AND CADMIUM IN THE DEVELOPMENT OF CHRONIC OBSTRUCTIVE PULMONARY DISEASE

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Jessica Rose Napolitano, B.A.

Biomedical Sciences Graduate Program

The Ohio State University

2014

Dissertation Committee:

Daren L. Knoell, PharmD., Advisor

Estelle Cormet- Boyaka, PhD.

Mark Failla, PhD.

Joanne Turner, PhD.

Jessica Rose Napolitano

2014

ABSTRACT

Chronic obstructive pulmonary disease (COPD) is a complex respiratory disease primarily caused by cigarette smoking. Cadmium (Cd), a toxic metal abundantly present in cigarette smoke, has been implicated in the development of disease, and accumulates in the bodies of smokers. It was recently discovered that a zinc (Zn) transporter, SLC39A8 (ZIP8), is responsible for the primary import of Cd into cells. Our lab discovered ZIP8 is under the transcriptional regulation of the central inflammatory NF-κB pathway. We hypothesize that inflammation in the lung created by smoke exposure increases the expression of ZIP8 thereby facilitating Cd uptake and pathology associated with COPD.

The first aim of our work addressed the role of ZIP8 in Cd-mediated epithelial cell toxicity using the adenocarcinomic alveolar epithelial A549 cell line.

Cd-induced toxicity was enhanced by TNFα in an NF-κB-dependent manner, which stimulated expression of ZIP8. Use of an NF-κB (p65) inhibitor (Bay11-

7082) or ZIP8 siRNA resulted in a significant decrease in cell toxicity. Cell death was also reversible with increasing concentrations of the micronutrient Zn.

Immunohistochemical analysis of primary human upper airway epithelial cells revealed preferential ZIP8 expression on the environmentally-facing apical

ii membrane. Analysis of lung tissue from GOLD stage 0 cigarette smokers and non-smoking controls revealed ZIP8 mRNA and protein to be significantly increased in the lungs of smokers.

We translated these findings into a mouse model of chronic cigarette smoke exposure using a transgenic ZIP8 overexpressing mouse line. ZIP8 overexpression dramatically increased emphysematic pathology, compared to smoke exposed C57/Bl6 control mice. In line with previous studies, our epidemiologic analysis of the 2011-2012 National Health and Nutrition

Examination Survey revealed blood Cd levels of smokers correlated with lower

Zn serum levels. Based on our findings, we contend ZIP8 is a potential mediator of COPD pathogenesis by creating an imbalance of the toxicant Cd and micronutrient Zn.

Lastly, we investigated the contribution of Cd to macrophage dysfunction in COPD. We found Cd to significantly reduce a macrophages ability to respond to an endotoxin challenge, specifically by inhibiting NF-κB activity, an effect not observed in monocytes, a closely related cell type. Atomic absorption spectroscopy revealed a greater accumulation of Cd within macrophages than monocytes, suggesting fundamental differences in Cd metabolism. We postulate this may be an important mechanism by which Cd contributes to impaired immune responses observed in COPD patients. Taken together, this novel body of work suggests Zn metabolism may inadvertently contribute to COPD pathogenesis by facilitating cadmium import and thereby lung pathology.

iii

DEDICATION

This document is dedicated to my future husband and best friend, Andrew.

ACKNOWLEDGMENTS

First, I would like to thank my advisor, Dr. Daren Knoell. You constantly challenged and encouraged me to become a better scientist, and I am deeply appreciative of your mentorship. I am grateful for your unwavering support in my pursuit of opportunities to diversify my training, as this has made all the difference as I begin to establish my career. You are an outstanding teacher and

I am proud to have completed my training under your tutelage. Thank you.

The members of my lab, past and current, have all played a significant role in my training. Ying Bao, thanks for always being so patient and taking the time to teach me nearly every bench technique I have learned. Mingjie Liu, I feel fortunate to have worked with a scientist as talented and meticulous as you, as you’ve set an outstanding example for me to follow. Charlie Pyle, thank you for all of your scientific discussion in lab meetings and over cups of Brennen’s coffee, they undoubtedly improved my work. Many thanks to my collaborators on the 4th floor of the DHLRI and beyond for your invaluable assistance and expertise. I would like to thank Drs. Amy Ferketich and Susan Olivo-Marston for their collaboration and encouragement to discover and pursue the science I love.

I would also like to thank my committee members Dr. Estelle Cormet-Boyaka, Dr.

Mark Failla and Dr. Joanne Turner whom have been incredibly generous with

v their time, support and guidance. Your input has greatly improved this body of work and I sincerely appreciate your assistance.

I would also like to thank my friends and family for their constant encouragement through graduate school. Ashley Bowers, I am so grateful for your friendship and all of our time spent together outside of the lab, you helped keep me balanced. To my sisters, Alyssa and Kelsey, you’ve been my biggest cheerleaders and I’m so appreciative of your friendship. To my parents, Thomas and Christine, thank you for recognizing my love of learning early, and nurturing my passion for science. You gave me the courage to pursue my Ph.D. and I cannot thank you enough for always believing in me, no matter the circumstances. And last but not least, thanks to my favorite classmate and husband-to-be, Andrew. Thank you for all of your love, patience and encouragement on this journey. I am so grateful to have found such a wonderful teammate in you, and I am so excited to see what life has in store for us.

VITA

March 13, 1987 ...... Born – New York, New York

June 2005 ...... Mineola High School

May 2009 ...... B.A. Biology, College of the Holy Cross

July 2009 to present ...... Graduate Research Associate, Department of Pulmonary, Critical Care, Allergy and Sleep Medicine, The Ohio State University

PUBLICATIONS

1. Jessica R. Napolitano, Mingjie Liu, Shengying Bao, Estelle Cormet-Boyaka, Patrick Nana-Sinkam, Melissa Crawford, Daren L. Knoell. Cadmium toxicity requires NF-kB-mediated transcriptional activation of the human zinc transporter ZIP8 in lung epithelia. Am J Physiol Lung Cell Mol Physiol. 2012 May;302(9):L909-18

2. Liu M-J, Bao S, Napolitano JR, Burris DL, Yu L, et al. (2014) Zinc Regulates the Acute Phase Response and Serum Amyloid A Production in Response to Sepsis through JAK-STAT3 Signaling. PLoS ONE 9(4): e94934. doi:10.1371/journal.pone.0094934

FIELDS OF STUDY

Major Field: Biomedical Sciences Graduate Program

vii

TABLE OF CONTENTS

Abstract ...... ii

Dedication ...... iv

Acknowledgments ...... v

Vita ...... vii

List of Tables ...... xii

List of Figures ...... xiii

List of Abbreviations………………………………………………………………………...... xv

Chapter 1: Introduction ...... 1

1.1 General Introduction………………………………………………………..1

1.2 COPD……..………………………………………………………………….1

1.2.1 Clinical Pathology ………………………………………………..1

1.2.2 Cellular responses to tobacco smoke………………………….4

1.2.3 Tobacco constituents, cadmium and immune activation……..6

1.2.4 Inflammatory signaling…………………………………………...8

1.2.5 Hypothesized disease mechanisms………………………...... 12

1.3 Tobacco smoking………………………………………………………….14

viii

1.3.1 Epidemiology…………………………………………………….15

1.3.2 Cadmium toxicity………………………………………………..17

1.4 Zinc nutrition and metabolism……………………………………………19

1.4.1 Zinc biochemistry………………………………………………..19

1.4.2 Dietary zinc………………………………………………………20

1.4.3 Zinc metabolism………………………………………………...22

1.4.4 ZIP8 and inflammation…………………………………………23

1.4.5 ZIP8 and cadmium…………………………………………...... 25

1.5 Specific aims……………………………………………………………….26

1.6 Tables………………………………………………………………………29

1.7 Figures……………………………………………………………………...30

Chapter 2: Cd-mediated toxicity of lung epithelia is mediated through ZIP8

expression in vitro………………………………..……………………………30

2.1 Summary…………………………………………………………………...30

2.2 Results……………………………………………………………………...32

2.3 Discussion………………………………………………………………….37

2.4 Materials and methods……………………………………………………43

2.5 Figures……………………………………………………………………...50

Chapter 3: The contribution of Cd and ZIP8 to COPD pathogenesis in vivo……54

3.1 Summary…………………………………………………………………...54

3.2 Results……………………………………………………………………...56

3.3 Discussion………………………………………………………………....64

3.4 Materials and methods……………………………………………………72

3.5 Figures……………………………………………………………………...80

Chapter 4: Determining the influence of Cd upon monocytes and macrophage

immune function……………………………………………………………….86

4.1 Summary…………………………………………………………………...86

4.2 Results……………………………………………………………………...89

4.3 Discussion………………………………………………………………….96

4.4 Materials and methods………………………………………………….102

4.5 Figures…………………………………………………………………….108

Chapter 5: Discussion……………………………………………………………….117

Bibliography…………………………………………………………………………..133

LIST OF TABLES

Table 1.1 GOLD Staging of COPD…………………………………………………..29

LIST OF FIGURES

Figure 1.1 Hypothesized model………………………………………………………30

Figure 2.1 A549 cell toxicity in response to Cd and TNFα treatment…………….50

Figure 2.2 ZIP8 inhibition reduces Cd-mediated toxicity…………………………..51

Figure 2.3 ZIP8 increases Cd and Zn uptake in lung epithelia…………………...52

Figure 2.4 ZIP8-mediated uptake induces apoptosis and necrosis………………53

Figure 2.5 Polarized ZIP8 expression in primary lung epithelia increases cell

toxicity in response to TNFα and Cd treatment…………………………….54

Figure 3.1 ZIP8 mRNA and protein expression is elevated in smokers…………78

Figure3.2 Cigarette smoke exposed BTZIP8-3 mice have increased

emphysematic lung tissue…………………………………………………….79

Figure 3.3 Cd transporter expression is similar between cigarette exposed

C57/Bl6 and BTZIP8-3 mice………………………………………………….80

Figure 3.4 Metal accumulation in the lungs of C57/Bl6 and BTZIP8-3 mice does

not differ………………………………………………………………………...81

Figure 3.5 Cigarette smoke exposed BTZIP8-3 mice exhibit a trending increase

in MMP mRNA expression……………………………………………………82

Figure 3.6 Expression of inflammatory markers does not differ in cigarette

exposed C57/Bl6 and BTZIP8-3 mice……………………………………….83

xii

Figure 3.7 Loss of BTZIP8-3 genotype corresponds with loss of emphysematic

phenotype after chronic cigarette exposure………………………………...84

Figure 3.8 Cd is elevated in the blood of cigarette smokers………………………85

Figure 4.1 Toxicity profiles in Cd-treated monocytes and macrophages………108

Figure 4.2 Cytokine release profiles in Cd-treated monocytes and

macrophages…………………………………………………………………109

Figure 4.3 Cytokine mRNA profiles of Cd treated monocytes and

macrophages…………………………………………………………………111

Figure 4.4 Differences in NF-κB protein phosphorylation in Cd-treated monocytes

and macrophages…………………………………………………………….113

Figure 4.5 NF-κ activity is decreased in Cd-treated macrophages…………...... 114

Figure 4.6 Cd inhibits IKKβ kinase activity………………………………………...115

Figure 4.7 Cd accumulation and transporter profiling differences between

monocytes and macrophages………………………………………………116

xiii

LIST OF ABBREVIATIONS

AAT α1 anti-trypsin deficiency

AV annexin V

BIGM103 Bacillus calmette-gueirin-inuced gene in monocyte clone 103

BTZIP8-3 Bac-transgenic line ZIP8 – 3 inserted copies

Cd cadmium

COPD Chronic obstructive pulmonary disease

DAMP danger-associated molecular patterns

DAPI 4',6-diamidino-2-phenylindole

DDT dichlorodiphenyltrichloroethane

DELFIA Dissociation-Enhanced Lanthanide Fluorescent Immunoassay

FBS fetal bovine serum

FDA Food and Drug Administration

FEV1 forced expiratory volume in 1 second

FVC forced vital capacity

GATS Global Adult Tobacco Survey

GOLD Global Initiative for Chronic Obstructive Lung Disease

HUAEC human upper airway epithelial cells

ICP-MS inductively coupled plasma mass spectrometry

ICP-OES inductively coupled plasma optical emission spectrometry xiv

IL interleukin

IKK IκB kinase

Km Michaelis constant

LDH lactate dehydrogenase

LPS lipopolysaccharide

M-CSF macrophage colony stimulating factor

MMP matrix metalloproteases mRNA messenger RNA

NHANES National Health and Nutrition Examination Survey

NHLBI National Heart Lung and Blood institute

NDMA N-Methyl-D-aspartic acid

NEMO NF-kappa-B essential modulator

NE neutrophil elastase

NLR Nod-like receptors

Nramp2 natural resistance-associated macrophage protein 2

SLC39a8 ZIP8

SLC29a14 ZIP14

PAMP pathogen-associated molecular patterns

PRR pattern recognition receptors

PMA phorbol myristate acetate

PI propidium iodine

ROS reactive oxygen species

RDA recommended daily allowance siRNA small interfering RNA

TIMP tissue inhibitors of metalloproteases

TLR Toll-like receptors

TEER transepithelial electrical resistance

TNFα tumor necrosis factor α

WHO World Health Organization

Zn zinc

ZnT zinc transporter

ZIP Zrt- Irt- like protein

xvi

CHAPTER 1: INTRODUCTION

1.1. General Introduction

The goal of this dissertation is to elucidate mechanisms by which the toxic metal cadmium (Cd), highly abundant in cigarette smoke, contributes to the development of chronic obstructive pulmonary disease (COPD), specifically by exploitation of the zinc (Zn) transporter ZIP8 (Slc39a8). This unique study is an intersection of three distinct fields not previously considered altogether in the context of COPD: environmental toxicology, Zn nutrition, and immunology. The purpose of this chapter is to serve as a source of pertinent background information regarding the pathology of COPD, Cd toxicity and cigarette composition, as well as Zn nutrition and metabolism. We believe this body of work fills a critical gap in understanding how the dynamic interplay between an environmental toxicant and essential micronutrient may influence disease outcomes.

1.2. COPD

1.2.1 Clinical Pathology

COPD is a complex respiratory condition primarily caused by tobacco smoking. It is characterized by obstructed airflow that is not fully reversible and usually associated with an abnormal inflammatory response of the lung to

1 noxious chemicals abundant in cigarette smoke [1]. COPD patients typically present with a constellation of symptoms that include chronic cough, dyspnea, and fatigue that can be categorized into two general phenotypes, referred to as chronic bronchitis and/or emphysema. Chronic bronchitis is characterized by a productive cough for 3 months in each of two successive years [2]. Usually, excess mucus is produced, airway linings become thickened and a cough develops [3]. Emphysema is pathologically defined as the permanent enlargement of airspace in the distal bronchioles [4]. The alveolar spaces of the lung contain alveoli where carbon dioxide derived from circulating blood is exchanged for inspired oxygen to be delivered throughout the body. In a healthy lung, alveoli are intact and oxygen and carbon dioxide are rapidly exchanged between the blood and alveoli. In an emphysematic patient, the alveoli are destroyed resulting in a loss of surface volume for gas exchange to occur thereby impairing an individual’s capacity to respire.

In 2001, the National Heart Lung and Blood Institute (NHLBI) and the

World Health Organization (WHO) formed the Global Initiative for Chronic

Obstructive Lung Disease (GOLD) [1]. This committee brought forth a clear and agreed upon definition of COPD and methods by which to diagnosis the disease.

Diagnosis is made by a thorough medical exam followed by spirometric measurements made after administration of a short-term acting bronchodilator.

Spirometry is used to determine the forced expiratory volume in 1 second (FEV1) and the forced vital capacity (FVC). FVC measures the total volume of air that

2 can be forced out after inspiration. These measures are then used to help stage the patient based on predicted values for their age, gender and ethnicity so that an appropriate course of treatment and management can be prescribed. Table

1.1 lists the current GOLD staging of COPD that serves as the standard used by clinicians worldwide [1].

Unlike chronic bronchitis, there are several subtypes of emphysema, the most common being centrilobular emphysema [4]. In this scenario, emphysema originates in the central bronchioles and moves outwards into the lobes, typically the upper lobes, a common site for cigarette smoke and environmental toxicant exposure. Distribution of emphysematic spaces throughout the lobes is usually heterogeneously distributed [5]. Panlobular emphysema is less common in smokers, though it does occur. The pattern of emphysematic lesions is more uniformly distributed and typically observed in the lower lobes [6, 7]. This subtype of emphysema is most common in individuals that suffer from emphysema as a consequence of 1 anti-trypsin deficiency (AAT), a heritable disease that leads to early onset of emphysema, typically beginning the 4th to 5th decade of life [8]. Disease is usually more severe in subjects with AAT deficiency if they smoke tobacco products. Paraseptal emphysema refers to degradation of airspace adjacent to visceral pleura [3]. Regardless of subtype, these patients present with dyspnea without significant sputum production. Most importantly, lung tissue cannot be regenerated once lost, and there are no cures for the disease, only management of symptoms.

While 99% of COPD patients have a history of tobacco use, only 20% of smokers develop COPD [9]. Similarly, 87% of all lung cancer deaths in the US are attributable to smoking, yet only 10% of smokers develop lung cancer [10].

These disproportionate figures reflect significant heterogeneity in the population that remains to be elucidated. This is further reflected by the multitude of proposed mechanisms that drive tissue destruction in emphysema. Hypotheses include imbalance in protease/anti-proteases, dysregulated oxidative stress, and destructive inflammatory responses coupled to changes in immune function [11].

Given the heterogeneity within the COPD population, whereby patients can have symptoms consistent with both chronic bronchitis and emphysema, it is unlikely that any of these mechanisms act alone in driving disease given many of these processes overlap. Unfortunately, though great advances have been made in revealing pathogenic mechanisms that account for COPD, it remains unclear to what extent biological and environmental risk factors can be used to distinguish a chronic smoker that will never develop COPD from one who will.

1.2.2 Cellular responses to tobacco smoke

Cigarette smoke enters the lung where it first encounters epithelial cells that continuously line the airway from the nasopharynx down to the alveoli. The epithelial barrier acts as the first defense against these antagonistic compounds, and is aided by the clearance of the mucociliary escalator and humoral factors like complement. Immune cells are also abundant within the lungs, the most relevant being macrophages, neutrophils and dendritic cells, all of which can

4 increase in disease states, although macrophages more so in COPD [12]. These cells are key components of the innate immune system that in part defines their generalized role in defending the host against pathogens, noxious chemicals, and response to tissue injury. Macrophages are resident tissue cells that are highly abundant in the airspace and constantly monitor the environment. Their primary function is to phagocytose pathogens, foreign debris, and byproduct dead cells. Macrophages are derived from circulating monocytic cells that migrate into tissues. In the context of COPD, the number of macrophages within the lung dramatically increases, typically 5-10 fold compared to a healthy smoker, and positively corresponds with disease severity [13]. This cell type is critical in signaling to other cell types of potential harmful changes within the microenvironment, depending on what debris or pathogens they may phagocytose.

Neutrophils are another phagocytic cell important to this initial response.

Although not highly abundant in the lung of nonsmokers, smoke exposure results in the recruitment of neutrophils from the bone marrow, through the blood compartment, and into the lung. Neutrophil numbers increase 2-4 fold in COPD patients compared to healthy smokers [13]. Neutrophils degranulate upon activation, a process by which antimicrobial proteins and chemical mediators are released to combat pathogens. Neutrophils, along with macrophages, produce the serine protease neutrophil elastase (NE). AAT-deficient individuals are

5 susceptible to emphysema because they are unable to properly counter the production of NE, the target of AAT, resulting in destruction of lung tissue [14].

Dendritic cells, which reside within the surrounding lung tissue, also phagocytose debris and pathogens akin to macrophages. Dendritic cells typically double in COPD patients compared to healthy smokers [15]. Their primary function is to present foreign antigen within surrounding lymphatic tissue, thereby serving as a messenger that links the initial innate immune response to the more sophisticated adaptive immune system. This leads to the recruitment of

B and T lymphocytes into the lung, eliciting a Th1 immune response [16]. Th1 immune responses coordinate cell-mediated immunity which is elaborated by the infiltration of cytotoxic CD8+ T cells that accumulate within peripheral airspaces and correlate with disease severity [17, 18]. This response further activates macrophages to aggressively resolve any insult or injury to the environment.

1.2.3 Tobacco constituents, cadmium, and immune activation

The toxic composition of tobacco smoke incites an aggressive response of the immune system. Manufactured cigarettes contain 599 additives, as confirmed by the Food and Drug Administration (FDA), that upon combustion produce an estimated 7,357 chemicals [19]. After a cigarette is ignited, two forms of smoke are generated. Mainstream smoke is inhaled from the butt end of the cigarette, and side stream smoke is generated from the burning end of the cigarette. The two forms of smoke have different chemical composition and concentrations and both are considered dangerous to humans [20]. In 2003, a significant study

6 compiled relative toxicity data for all studied constituents of tobacco smoke, and determined their individual risk of contributing to cancer, respiratory disease and cardiovascular disease. Categorically the identification of leading harmful components was as follows: aldehydes and small organics (i.e. acrolynitrile), metals (i.e. arsenic, Cd), nitrosamines (i.e. NDMA), polycyclic aromatic hydrocarbons (i.e. benzo(a)pyrene), and unclassified agents (i.e. DDT) [21].

This study was key in identifying the major contributors to disease sourced in mainstream cigarette smoke. Based upon these findings, 1,3-Butadiene posed the greatest cancer risk, acrolein the greatest risk for respiratory disease and hydrogen cyanide the greatest cardiovascular disease risks. Relevant to our investigations, inorganic metals were also identified as leading constituents that have substantial toxicity in humans. Arsenic, hexavalent chromium and Cd fell into the top ten list of all cigarette components that have the highest risk of cancer. Additionally, arsenic had the second greatest potential to cause cardiovascular effects in smokers. Cd, hexavalent chromium and nickel were within the top constituents possessing the greatest associated risk of respiratory disease. This study has substantially impacted the manner by which the FDA and NIH investigates and accordingly regulate, how tobacco products are manufactured, advertised, and sold to consumers in the U.S [19, 21].

Our interests lie in understanding how the inorganic metal Cd may aggravate the lung microenvironment and cause subsequent disease, in part by damaging epithelial cells and altering normal immune responses. Cd, and

7 smoke as a whole, causes a provocation of resident cells and inflicts irreversible injury to lung tissue. Over time, chronic exposure leads to recruitment of more immune cells that can perpetuate injury by damaging the host in failed efforts to restore health. Perturbations in cellular function and viability are in part coordinated through specific PRRs (pattern recognition receptors). These receptors include Toll-like receptors (TLRs), which are present on the cell surface, and NOD-like receptors (NLRs), which are present intracellularly.

Collectively, these two families of receptors can specifically recognize a broad constellation of pathogen- and danger-associated molecular patterns, known as

PAMPs and DAMPs, respectively. PAMPs refer to bacteria and virus-specific components that trigger recognition of our immune system to pathogen invasion, such as bioactive endotoxin found within smoke. DAMPs refer to endogenous molecules derived from host cells that are released consequent to cell damage and death. Importantly, the toxic components that are abundant in cigarette smoke have the capacity to provoke necrotic and apoptotic cell death [22-26].

Upon death, molecules typically retained within lung cells are then released into the extracellular space thereby triggering immune activation. ATP and uric acid are both examples of DAMPs elevated in the lungs of COPD patients compared to non-COPD subjects [27].

1.2.4 Inflammatory signaling

Following PAMP or DAMP recognition and phagocytosis, several signaling pathways are stimulated through the binding of cell surface and internal

8 receptors. This leads to the release of various pro- and anti-inflammatory cytokines and chemokines by epithelial cells and macrophages, amongst others cell types. Cigarette smoke, as well as Cd, has the capability to modulate several of the pathways responsible for the transcription of these mediators that go on to coordinate the immune response. Inflammatory pathways activated in

COPD patients include but are not limited to the PI3K/Akt, p38, ERK1/2, and NF-

κB pathways [28, 29]. These pathways are central in the coordination of the inflammatory response of smoke-exposed cells, and often engage in cross talk.

As a whole these pathways are responsible for transcribing cytokines and resolving cell stress, though when dysregulated can also be a source of pathogenesis.

Of particular interest to our lab is the NF-κB pathway, a central innate immune signaling pathway whose activity is increased in the lungs of COPD patients [29, 30]. Our focus is specifically on the canonical NF-κB pathway, which is activated by inflammatory signals such as tumor necrosis factor α

(TNFα), interleukin-1 (IL-1), and lipopolysaccharide (LPS). Briefly, these external stimuli, amongst others, converge upon the central IKK complex of the pathway.

The IKK (IκB kinase) complex is composed of three proteins: IKKα, IKKβ, and

NEMO (IKK). Phosphorylation of these proteins by upstream messengers result in phosphorylation of the IκB proteins. Specifically, phosphorylation of IKKβ activates it to go on to phosphorylate IκBα. This is important because IκBα naturally binds to the p65:p50 transcription factor heterodimer complex, retaining

9 it as an inactive complex in the cytoplasm. However, following phosphorylation,

IκBα is rapidly ubiquitinated and degraded, releasing p65:p50 to translocate into the nucleus and induce the expression of targeted inflammatory genes. Retention of p65:p50 within the cytosol as inactive precursors positions the NF-κB pathway to act as a first responder to stress and injury. The thousands of genes that it regulates are involved in removing noxious substances and pathogens, and reestablishing cell homeostasis [31]. We have chosen to evaluate the impact of

Cd upon the NF-κB pathway in monocytic cells given its relevance to COPD and immune function, work that is presented in chapter 4.

Within the lungs of COPD patients, stimulated cells release multiple cytokines and chemokines transcriptionally regulated in part by the NF-κB pathway [32]. These small signaling proteins are typically divided into a pro- or anti-inflammatory classification dependent on their role in perpetuating or inhibiting inflammatory signals. Inflammatory cytokines increase blood flow to the site of injury or infection, modulate body temperature and activate additional cell types to perpetuate the signal. TNFα, IL-1β, IL-6, and IL-8 are classically described as pro-inflammatory while IL-10 and IL-13 are thought of as anti- inflammatory mediators. Anti-inflammatory mediators are described as such due to their inhibition of the production of pro-inflammatory cytokines. Though this dichotomous classification is simple, the overall response within the lung microenvironment is quite complex. The complexity of signaling is reflected by

10 the inconsistencies reported for cytokine signatures within the lungs of COPD patients across different studies [12, 29, 33-36].

The prevalent hypothesis in the field is that the immune systems of COPD patients reside in a hyperactive state, in which elevated cytokine production leads to perpetual recruitment and overstimulation. By doing so, the hyperinflammatory state of the lung results in immune-mediated damage to the host. Mechanisms of damage include but are not limited to macrophage oxidative burst, excess production of proteases and development of antibodies to endogenous peptides [11]. In addition to the consistent observation of an influx of immune cells into the lung, multiple clinical studies have reported an increase in IL-1β, TNFα, IL-6 and IL-8 expression in sputum, bronchoalveolar lavage fluid, and blood samples from COPD patients compared to smokers [33, 37, 38].

However, within the past decade the inflammation hypothesis has been challenged. This has been predicated in part on the fact that COPD patients do not typically succumb to emphysema or bronchitis, but due to repeated respiratory infection with chronic bronchitis-type patients being the most vulnerable to community and nosocomial acquired infections [39, 40]. In particular, studies have shown that GOLD staging correlates with the extent of hospitalization and mortality due to infection [41]. The inability of COPD subjects to defend themselves from repeated infection has cultivated an alternative hypothesis whereby immune dysfunction, as opposed to exclusive pro- inflammation, may account for the morbidity and mortality of COPD. In support

11 of this concept, multiple studies have reported that alveolar macrophages isolated from the lungs of smokers and COPD patients subject to activation via endotoxin secrete lower levels of cytokines than do healthy donors [34, 36].

This finding runs counter to the classic paradigm of COPD, but also supports the notion that patients have compromised immune systems that may be unable to properly clear infection. While it is significant that long-held ideas in the field are being challenged, it also reflects the complexity of the disease.

1.2.5 Hypothesized disease mechanisms

Cd is a ubiquitous metal with several toxic mechanisms of action, many of which are not surprisingly also observed in the lung tissue of COPD patients. A prevalent Cd-mediated toxic mechanism is increased oxidative stress, a leading hypothesized cause of COPD pathogenesis [42, 43]. Cd plays an indirect role in facilitating chemical reactions that lead to the production of damaging oxygen radicals by depleting antioxidant defenses, such as glutathione [44]. Cd is able to disrupt much of normal cellular function through its ability to tightly bind thiol groups within proteins, including many antioxidants, and render them inactive

[45]. Acute Cd exposure can stimulate the NF-κB pathway, and it is believed secondary reactive oxygen species (ROS) production is the mechanism by which

Cd increases this activity [46]. Like Cd, whole cigarette smoke acts as a source and stimulant of oxidative stress. COPD smokers have a significant increase in oxidative markers both within their lung tissues and systemically [47, 48]. As an example, malondialdehyde (MDA), a product of lipid peroxidation, has been

12 shown to be elevated within the blood stream of COPD patients and inversely correlates with FEV1 [49].

The ROS perpetuated by chronic cigarette smoke, and Cd, may also be compounded by an observable decrease in the antioxidant defenses of COPD patients. The central antioxidant nuclear factor-erythroid 2-related factor (Nrf2) pathway is one of several that are altered by cigarette smoke [48]. This is specifically evident by reduced levels of heme oxygenase-1 (HMOX1) and catalase, two critical proteins in the pathway, in COPD patients compared to healthy smokers [11]. Additional genetic studies have identified several polymorphisms in the superoxide dismutase (SOD) family of proteins responsible for the metabolism of superoxide [50, 51]. Several polymorphisms in SOD3 have been identified as both protective and promotional in the development of COPD

[52, 53]. These studies highlight how disruption of the delicate balance between oxidants and antioxidants may increase risk of disease.

One of the most established hypotheses regarding COPD pathogenesis relates to dysregulation of protease and anti-protease balance. In COPD excessive protease production and release by immune cells overwhelms the antiprotease repertoire, leading to tissue destruction. A well-studied example of protease imbalance centers on the influx of MMPs. MMPs are Zn-dependent endopeptidases that cleave bioactive molecules and extracellular matrix proteins, leading to destruction of the fragile alveolar architecture. An influx of macrophages and neutrophils respond to the release of endogenous peptide

13 fragments and can produce more MMPs, recruiting more immune cells and further perpetuating injury. Chronic cigarette smoking has been shown to induce persistent MMP activity that may lead to tissue destruction and a relative deficiency in anti-proteases such as tissue inhibitors of metalloproteases

(TIMPS) [54, 55]. This is supported by epidemiological studies that have revealed genetic predisposition to excessive MMP activity consequent to polymorphisms in genes for MMP-9 and -12 [56, 57]. Further, it is has been proposed that prolonged smoking may further modify gene expression due to epigenetic-mediated alteration in protease and antiprotease gene expression and function [57, 58]. Interestingly, Cd has been implicated in inducing the expression of lung MMPs in a rat model of inhaled Cd [59]. It is plausible that Cd contributes to this imbalance by influencing signaling pathways regulating MMP expression, such as AP-1 and NF-κB.

To date, no preventive or curative medication-based treatments have been developed to significantly reduce the mortality associated with COPD.

Smoking cessation remains the best approach to significantly decrease morbidity and mortality. Accordingly, medication-based treatment is focused on symptomatic management with an overarching goal to reduce exacerbations through the use primarily of bronchodilators to improve pulmonary mechanics and corticosteroids to temper airway inflammation.

1.3 Tobacco Smoking

1.3.1 Epidemiology

There are an estimated 42.1 million Americans that smoke, which is

18.1% of the adult population in our country [60]. Cigarette smoke is more prevalent among men (20.5%), though the gender gap has narrowed over time

(15.8% of women) [60]. Of this group, nearly 16 million Americans suffer from disease caused by smoking including cancer, cardiovascular, and pulmonary diseases [61]. Smoking is the leading cause of preventable death in the US, and can be attributed to 1 in every 5 deaths [61, 62]. An aging population of smokers has caused COPD to move from the 4th to the 3rd leading cause of death in the

United States [63]. Smoking has become one of the greatest public health crises of our time, which has drawn the attention of public policy makers. Since the publication of the original Surgeon General’s Report in 1964 first warning of dangerous health risks of tobacco use, smoking rates have dramatically declined in our country [60]. More recently, smoking rates have declined from 20.6% in

2009 to 18.1% in 2013, the first statistically significant decrease since the late

90s to mid-2000s [64]. The implementation of high taxes on tobacco products, the creation of workplace smoking bans, and aggressive public service announcement campaigns have all contributed to this decline [61]. This has led to the implementation of more widespread smoking bans in restaurants and bars, parks and college campuses. The reduction of male cigarette smoking prevalence from 54% in the 1960s to the current 20.5% is a testament to the

15 tremendous public health efforts made to educate our nation on the dangers of tobacco.

Despite these significant advancements, the challenge to curb tobacco use in order to further improve public health is far from over. The burden of tobacco-related disease is disproportionately associated with the most vulnerable populations. These groups, who are typically socioeconomically challenged, are the most difficult to engage through public policy and have the least access to adequate health care, making the potential to quit smoking more challenging. As a testament to this, 41.9% of adults with a GED diploma and 27.9% of individuals that live below the poverty line are smokers, well above the national average.

Additionally, smoking rates are highest in minority populations of mixed race

(26.1%) and American Indians (21.8%). It is anticipated that these populations will continue to produce the next generations of aging Americans that suffer from tobacco-related disease [60].

Outside of the U.S. exists an even greater tobacco-fueled public health crisis. While overall global smoking prevalence has declined over the past few decades the total number of smokers has increased from 721 million to 967 million [65]. This distribution varies between different countries. The Global

Adult Tobacco Survey (GATS) conducted in 2010 revealed smoking prevalence in Russian men to be as high as 60%, and 53% in Chinese men, rates similar to those observed in the US in the 1960s. Rates of smoking among women topped at 30% in Greece, the highest for women anywhere. Similar to what we observe

16 in the US population, 80% of tobacco users live in low- and middle- income countries. As these rates continue to increase in some parts of the world and plague our nation’s most vulnerable populations, it is imperative that more resources be devoted to addressing this public health emergency [66].

1.3.2 Cadmium toxicity

As previously described, the thousands of chemicals present in tobacco smoke, including Cd, act as potent instigators of disease pathogenesis. The toxicity of tobacco is due in part to the soil it is grown in. Inorganic metals, such as Cd, are present within the soil through both natural occurrence in the earth’s crust in conjunction with the use of fertilizers and/or pesticides [67, 68]. The tobacco plant is very efficient at absorbing metals thereby effectively increasing the toxicity of tobacco smoke. The concentrations of metals vary between cigarettes, typically by brand. This is due in part to the species of tobacco plant and the geographical location of its growth relative to metal content in soil, as well as processing methods used to produce the cigarette [68].

Cd is one of the most toxic metals found within cigarette smoke as now acknowledged by its associated high risk as a cause of respiratory disease [21].

While most metals in cigarette smoke pose a serious health risk to the smoker,

Cd is unique due to its unusually long half-life in the human body. Reflective of this, cigarette smokers have two to three times the amount of Cd in their bodies compared to non-smokers [69]. Typically, about 0.5-1.5 µg of Cd is present within each cigarette. Of this, about 10% is transferred into the human body

17 following inhalation of smoke [70]. Upon consumption, approximately 50% is absorbed through the lungs into the blood stream [71]. Human studies have shown that Cd efficiently accumulates in the kidney, liver and bone, with a biological half-life of 10-30 years [72-74]. Inhaled Cd also accumulates within alveolar macrophages, although the amount of Cd that deposits in other lung cell types relative to the amount that is absorbed into the bloodstream and then onto other organs remains unknown [75].

Based largely on Cd kinetic uptake studies with candidate transporters conducted over the past decade, five transporters have emerged as bona-fide Cd transporters [76]. Megalin and cubilin are endocytic receptors found in the kidney that bind circulating metallothionein and in turn import its bound Cd into renal cells [77, 78]. Natural resistance-associated macrophage protein 2 (Nramp2), an iron transporter most prominent in the gastrointestinal tract, is another confirmed

Cd transporter [79, 80]. More recently, the Zn transporters ZIP8 and ZIP14 have been identified as importers of Cd [81, 82]. Based upon our longstanding interest in ZIP8 relative to Zn metabolism and human health, the final portion of this introductory chapter will describe significant features of ZIP8 that distinguish it as a significant mediator of Cd-driven disease in the context of cigarette smokers.

1.4. Zinc nutrition and metabolism

1.4.1 Zinc Biochemistry

Zn is an essential trace metal necessary for cell maintenance, growth, and survival. Several key features of Zn distinguish it as essential for cell function. It

18 has unique coordination geometry within its outer shell that enables it to become an integral component of hundreds of proteins thereby regulating both structure and function [83]. It is necessary to multiple families of proteins, including metalloproteases, Kruppel like factor proteins and Zn finger motif domains of transcription factors. As an example, Zn finger motifs function by binding to targeted regions of DNA to initiate gene transcription, and accordingly, Zn is critical for normal function [84]. Zn also possesses antioxidant properties by directly bind sulfhydryl groups in addition to indirect mechanisms. For example,

Zn induces the expression of antioxidant genes, including metallothioneins and glutathione, that go on to scavenge oxidant species within the cell [85]. This is a distinguishing feature of Zn compared to other biological metals, such as iron and copper that do not engage in these reactions.

Zn and Cd are both in Group 12 of the periodic table, meaning that they share several atomic properties despite their dramatically different impact on human health. Both are electropositive, meaning they are good reducing agents, with a constant oxidation state of +2. They are both calcophiles, referring to their preference to chemically interact with sulfides as opposed to oxides, which also accounts in part for why both metals are most commonly found in the earth’s crust as Zn- or Cd-sulfide. Coincidentally, Cd was first discovered as a contaminant of Zn ore, further demonstrating their close proximity in nature [86].

1.4.2 Dietary Zinc

In 1974, the Food and Nutrition board of the US National Academy of

Sciences declared Zn an essential micronutrient [87]. Recognition, surprisingly late compared to other essential micronutrients, launched the development of recommended dietary allowance or intake guidelines. Formation of new policy was largely driven by the work of Ananada Prasad, the scientist considered to be the father of Zn nutrition research. Before initiating studies in 1961, Zn was not considered a required nutrient or cause of disease in subjects that suffered from inadequate dietary intake. However, his work in Iran and Egypt during the ‘60s unquestionably demonstrated that Zn deficiency in humans not only existed but was widespread [88]. He observed that Zn deficient subjects consistently presented with dwarfism, hypogonadism, hepatosplenomegaly, dry skin, geophagia and impaired cognition. While many of these symptoms overlap with other nutritional deficiencies thereby confounding diagnosis, growth retardation and hypogonadism were unique and provided the fundamental clues that Zn was the cause of the phenotype observed. Additionally, these patients also had similar diets consisting of low animal protein intake, and high cereal intake. It was later recognized that cereal is high in phytates, a compound that acts to chelate Zn within the gastrointestinal system thereby preventing Zn absorption into the body. Dr. Prasad and colleagues provided Zn supplementation to these patients and observed a marked reversal of symptoms with normal health being fully restored [88-90]. These findings not only led to the development Zn

20 recommended daily allowances (RDA) but the inclusion of Zn supplemental guidelines for maternal and neonatal nutrition.

Severe Zn deficiency as previously described is not abundant in Western civilization. However, epidemiological studies have revealed that specific populations are at higher risk for mild to moderate Zn deficiency. Similar to

COPD patients, the impoverished and elderly are at higher risk for nutritional deficiencies. Analysis of NHANES III revealed that 35-45% of adults over 60 had

Zn intakes lower than the RDA [91]. Another study reported that individuals from food insecure homes also had Zn intakes below recommended amounts [92].

This may be related to poor diet and other comorbidities that decrease Zn absorption and consumption.

1.4.3 Zinc metabolism

Zn metabolism is tightly regulated within the human body, 90% of which is bound to proteins. The remaining 10% is weakly bound to other proteins, such as glutathione and albumin, and is termed labile Zn due to its high bioavailability

[93]. This pool of labile Zn is tightly trafficked by two distinct families of transporters: ZnTs and ZIPs. The solute-linked carrier 30 (SLC30a1-10, corresponding to ZnT1-10) gene family contains 10 ZnT (Zn transporter) proteins that are largely responsible for efflux of Zn from the cell. While some of these proteins are expressed on the plasma membrane to facilitate Zn transport out of the cell, others are expressed on vesicles and organelles within the cell to regulate Zn compartmentalization within the cell [94]. The expression of ZnTs as

21 well as ZIPs is tissue-specific and influenced by the biochemical environment including intracellular Zn content, cytokines, and hormones [95].

The solute-linked carrier 39 (SLC39A1-14, corresponding to ZIP1-14) gene family, referred to as ZIPs, functions to increase intracellular Zn levels through the import of Zn into the cytoplasmic space, from either the extracellular space, organelles or vesicles. These 14 metal transporters are 8-transmembrane spanning proteins that import divalent metals, primarily Zn but also Cd. All 14

ZIPS contain a histidine-rich loop region between transmembrane domains 3 and

4 that function to sense and bind Zn [96]. Like ZnTs, each ZIP has its own expression pattern. As an example, ZIP1 is ubiquitously expressed on the plasma membrane of all tissues, while ZIP4 is expressed primarily along the gastrointestinal tract and making it principally responsible for the absorption of dietary Zn. Dietary Zn deficiency results in an increase in the expression of ZIP4 within the gastrointestinal tract [96].

1.4.4 ZIP8 and Inflammation

In 2002, a new gene referred to as BIGM103 (Bacillus calmette-guerin- induced gene in monocyte clone 103), was identified in a cDNA screen of monocytes stimulated with Mycobacterium bovis BCG. An increase in the accumulation of intracellular Zn was observed when this protein was expressed

[97]. This was the first study that identified the novel response of ZIP8 (a.k.a.

BIGM103) to a pathogen initiated danger signal. Having published that Zn is vital to lung epithelial survival during inflammatory stress, our lab next sought to

22 examine whether Zn transporters are important in Zn-mediated protection [98,

99]. Specifically, primary human upper airway epithelial cells (HUAECs) were stimulated with TNFα and a messenger RNA (mRNA) screen of all 24 Zn transporters was performed. Strikingly, the only Zn transporter whose expression was induced by TNFα was ZIP8 which we then observed was essential for Zn-mediated uptake and cell survival [100]. Accordingly, our group went on to conduct translational studies in animal models and humans which collectively substantiated the importance of ZIP8 as an immunomodulatory protein that is required for normal innate immune function in response to infection

[101, 102]. In short, we discovered that ZIP8 is expressed during times of physiological stress so that Zn may be imported into the cell to restore homeostasis and act as a cytoprotectant. Consistent with our findings, others have identified similar responsiveness of ZIP8 within epithelial cells and T-cells

[103].

Most recently, our lab identified an important mechanism by which ZIP8 modulates the immune response. This work revealed ZIP8 as a transcriptional target of the NF-κB pathway, a central innate immune signaling pathway.

Further, ZIP8-mediated import of Zn inhibits the NF-κB pathway due to Zn’s capacity to directly bind and consequently prevent the phosphorylation of IKKβ.

Based on this, our group was the first to directly demonstrate that Zn metabolism is directly coupled to regulation of immune function [104]. This is important

23 because the canonical NF-κB pathway is a major regulator of immune function and responsible for the expression of many mediators of inflammation.

Establishing that ZIP8 regulates the NF-κB pathway through Zn as a negative regulator of the immune system opened new doors. We hypothesized that this system evolved to carefully balance immune function and in doing so, prevent hyperinflammation that can cause excessive damage to the host.

Accordingly, our findings also helped to explain how Zn deficiency may increase risk for more morbidity and mortality through immune dysregulation. Our studies in both animals and humans have shown that Zn deficiency leads to more severe sepsis [101, 102]. The regulation of ZIP8 by the NF-κB pathway is a distinguishing feature of this Zn transporter that makes it an attractive candidate for therapeutic targets in inflammatory diseases, such as COPD.

1.4.5 ZIP8 and Cadmium

Despite the essential and beneficial function of ZIP8 relative to host defense, it may also have the capacity to inadvertently contribute to disease.

Nearly a decade ago, our collaborator Dr. Daniel Nebert observed that certain strains of mice exhibited severe testicular necrosis in response to Cd ingestion, while other strains were resistant to this phenotype [82]. Mapping of the genomes of these strains identified that ZIP8 was responsible for this difference.

Remarkably, kinetic studies of this Zn transporter demonstrated that it had a relatively high affinity for Cd (Km = 0.48) compared to its endogenous ligand Zn

(Km = 0.26) [105]. Based largely upon these findings we began to question

24 whether ZIP8 and Zn metabolism play a significant role in smoking related lung disease knowing that Cd is highly abundant in cigarette smoke and that a substantial portion of smokers develop chronic lung disease.

An epidemiologic study conducted in 2010 evaluated the relationship between dietary Zn intake, Cd burden and obstructive pulmonary disease. The retrospective analysis involved over 6700 study subjects and revealed that regardless of smoking status, people with the lowest dietary Zn intake also had the highest risk of pulmonary disease. Risk was, however, highest in current smokers with the lowest Zn intake. Relevant to our interests, those with the lowest Zn intake also had the highest levels of Cd present in urine. Most importantly, the study showed that the highest risk of smoking related lung disease occurred when Zn levels were low and Cd levels were high. When the balance between these two metals favored Zn, the risk of developing lung disease was considerably lower [106]. Based on the retrospective nature of this study, the mechanisms that account for these relationships were not possible to elucidate. Based on our own findings, we postulate that cigarette smoke is a primary source of Cd that once inhaled, modifies the lung microenvironment in favor of an inflammatory state. Further, inflammation in the lung drives the expression of ZIP8 thereby increasing the uptake of Cd into lung cells and even more so when Zn levels are deficient. In summary, our central hypothesis is that

Inflammation driven by smoke exposure will induce the expression of ZIP8 in the

25 lung microenvironment thereby increasing Cd uptake and lung pathology, illustrated in figure 1.1.

1.5 Specific Aims

The work in this dissertation presents three specific aims all focused on examining our central hypothesis. The first aim is to determine the role of ZIP8 in

Cd-mediated lung epithelia toxicity in vitro, presented in Chapter 2. In this aim we present data using a human lung epithelia cell line and primary human upper airway epithelial cells (HUAECs) to characterize the induction of ZIP8 by TNF, and the subsequent impact that this has upon Cd-mediated toxicity.

The second aim is to determine the extent by which ZIP8 increases COPD progression in vivo, presented in chapter 3. This is achieved through three separate studies. The first sought to characterize ZIP8 mRNA and protein expression in tissue samples obtained from chronic cigarette smokers matched in comparison to healthy controls. The second study utilized a murine model of chronic cigarette smoke exposure in conjunction with transgenic ZIP8- overexpressing mice. Upon completion of the study, the animals were assessed for ZIP8 expression, Cd accumulation and any alterations in the microenvironment of the lung tissue. The last study conducted is an epidemiological assessment of the NHANES datasets from 2010-2011 to further substantiate relationships between Zn and Cd within cigarette smokers. This work sought to advance our understanding of the relationship between biological markers of Cd and Zn as it relates specifically to smoking status.

The third aim is to determine the role of ZIP8 and Cd on macrophage immune function as presented in chapter 4. The aim of this work was predicated upon the known pro-inflammatory effects of Cd, as well as the heterogeneity of macrophage immune profiles within the lungs of COPD patients. We employed monocyte and macrophage cell lines and primary human cell culture models to understand the impact of Cd on monocyte and macrophage function. Following

Cd exposure, cells were then challenged with endotoxin. The transcription and release of a panel of pro-inflammatory cytokines, as well as NF-κB activity was critically evaluated and compared between cell types. Further, we determined whether differences in Cd accumulation and metabolism between the two cell types exist and how this may contribute to observed differences in cell profiles and phenotype.

In summary, we contend that the work presented in this dissertation narrows a critical gap in our understanding of how tobacco-source-Cd toxicity may be influenced by dietary Zn intake and contribute to the pathogenesis of

COPD. Specifically, we believe our finding is novel in that we specifically identify

ZIP8 as a critical mediator juxtaposed at the interface between micronutrient metabolism and metal-induced dysregulation of immune function. We believe that this collective body of work provides a compelling case for Cd’s involvement in promoting a dysfunctional immune environment within the lung thereby increasing susceptibility to infection and tissue dysfunction. These findings are also relevant to advancing the field in terms of dietary interventions that may

27 temper the harmful effects of tobacco products thereby possibly mitigating, or prolonging the onset and severity of COPD.

Tables 1.6

STAGE Description FEV1 FEV1/FVC Symptoms

0 At risk Normal Normal May have chronic symptoms such as cough I Mild COPD ≥ 80% predicted < 70% predicted May or may not have chronic symptoms IIa Moderate 50% ≤ FEV1 < 80% < 70% predicted May or may not have COPD predicted chronic symptoms IIb Moderate 30% ≤ FEV1 < 50% < 70% predicted May or may not have COPD predicted chronic symptoms III Severe COPD < 30% predicted < 70% predicted May have respiratory or right heart failure Table 1.1: GOLD Staging of COPD. Adapted from Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease. NHLBI/WHO Global Initiative for Chronic Obstructive Lung Disease (GOLD) Workshop Summary [1].

Figures 1.7

Figure 1.1: Hypothesized Model. In our epithelial cell model, we hypothesize inflammation driven by smoke exposure activates the NF-κB pathway. In turn, this drives ZIP8 expression that goes on to import Cd from cigarette smoke and cause epithelial cell toxicity. In our monocytic cell models, we hypothesize cigarette smoke, pathogens and DAMPs drive the expression of ZIP8 through NF-κB signaling, in turn facilitating uptake of Cd that leads to subsequent immune dysfunction.

CHAPTER 2: CD-MEDIATED TOXICITY OF LUNG EPITHELIA IS MEDIATED THROUGH ZIP8 EXPRESSION IN VITRO

2.1 Summary

Cd is a toxic heavy metal and carcinogen abundantly present in cigarette smoke, and has been identified as a causative agent of tobacco-related lung disease [19]. ZIP8, a Zn transporter, has recently been identified as an avid Cd transporter [82]. More recently, our laboratory discovered that ZIP8 is under the transcriptional regulation of the NF-κB pathway, a distinct feature of this Zn transporter compared to others [104]. The NF-κB pathway is a central inflammatory signaling pathway known to be active in the lungs of cigarette smokers [29]. The high level of activity is thought to be a consequence of the toxic and immunogenic attributes of components of cigarette smoke that are inhaled into the lung, resulting in damage and inflammation. Further, we have shown that Zn directly suppresses this pathway by inhibiting IKKβ. This is significant because IKKβ is a key protein in the IKK complex that is critical for transmitting signals within the NF-κB pathway. Based on this, we hypothesize that cigarette-smoke induced inflammation would increase ZIP8 expression in lung epithelia, leading to enhanced Cd-mediated uptake and cell toxicity. This

31 chapter describes our use of the A549 cells (a human alveolar epithelial cell line) and HUAEC to investigate this proposed phenomenon.

Our results demonstrate that TNFα induces ZIP8 expression in A549 cells and primary HUAECs and results in increased Cd accumulation and cell death.

Through the use of pharmacological inhibitors and a small interfering RNA

(siRNA), we have shown toxicity and Cd accumulation to be dependent on the expression of ZIP8. Further, we investigated the potential cytoprotective effect of

Zn against Cd toxicity. Zn is a critical micronutrient and a natural endogenous ligand for ZIP8. Our results reveal a dose-dependent reduction in Cd-mediated toxicity in the presence of Zn. From these studies, we conclude that ZIP8 expression is induced in lung epithelia in an NF-κB-dependent manner thereby resulting in increased cell death due to Cd accumulation. Based on these findings, we contend that ZIP8 plays a critical role at the interface between micronutrient (Zn) metabolism and toxic metal exposure (Cd) in the lung microenvironment following cigarette smoke exposure. Further, dietary Zn intake, or a lack thereof, may be a contributing factor in smoking-induced lung disease.

2.2 Results

TNFα enhances Cd toxicity in lung epithelia

ZIP8 expression is typically low in lung epithelia but highly induced by pro- inflammatory mediators. Based on this and knowing that ZIP8 is a transporter of

Cd, we predicted that induction of ZIP8 expression by TNFα, a relevant pro-

32 inflammatory factor present in the lung of smokers, would increase Cd toxicity in lung epithelia. To investigate the transporter’s contribution to Cd-induced toxicity, A549 cells were first stimulated with TNFα for a time sufficient to increase ZIP8 expression and then exposed to increasing concentrations of Cd for 24 hours. A549 cells stimulated with TNFα prior to Cd challenge had a significant increase in cell death, as determined by LDH release, in comparison to cultures that were exposed only to Cd (Figure 2.1.A). Western analysis confirmed a 6.5-fold induction of membrane-bound ZIP8 following TNFα stimulation (Figure 2.1.B). These results indicate that lung epithelia become more vulnerable to Cd-mediated toxicity following activation by TNFα.

NF-κB and ZIP8 inhibition decrease Cd-induced cell toxicity

We next determined whether Cd-induced toxicity in lung epithelia is dependent on the induction of ZIP8 expression. Our group recently reported that ZIP8 expression is transcriptionally activated by NF-κB (p65). Knowing this, we stimulated lung epithelia cultures with TNFα but in the presence of Bay 11-7082, a compound that prevents IκB-α phosphorylation, thereby inactivating the canonical NF-κB pathway. Inhibition of the NF-κB pathway resulted in a significant decrease in cell toxicity in cultures exposed to a combination of TNFα and Cd when compared to the DMSO vehicle control treatment group (Figure

2.2.A). Knowing that Cd-mediated toxicity is NF-κB-dependent, we then pursued a similar study in conjunction with siRNA designed to suppress the induction of

ZIP8 expression. A549 cells were first treated with ZIP8-specific siRNA or a

33 corresponding scrambled siRNA control, then stimulated with TNFα and then once again subject to increasing concentrations of Cd. Cultures in which ZIP8 expression was inhibited exhibited a significant decrease in cell toxicity relative to cultures treated with the siRNA control (Figure 2.2.B). Western blotting confirmed a ~70 % knockdown of ZIP8 in cultures treated with either Bay 11-

7082 or siRNA (Figure 2.2.C). Collectively, these results support our hypothesis that the induction of ZIP8 expression by a pro-inflammatory factor associated with chronic cigarette smoke exposure significantly increases lung epithelial vulnerability to Cd.

Zn decreases Cd-induced cell toxicity

ZIP8 was first identified as a Zn importer and then subsequently discovered to also be an avid transporter of Cd. Knowing that Zn acts as a cytoprotectant in lung epithelia, we wanted to determine whether physiologically relevant concentrations of extracellular Zn can prevent Cd-mediated toxicity. A549 cells were again stimulated with TNFα and then exposed to a constant concentration of Cd but in the presence of increasing concentrations of Zn. Strikingly, lung epithelial cell toxicity induced by Cd was decreased in the presence of increasing concentrations of Zn which became apparent when the molar ratio between Cd and Zn was in favor of Zn (Figure 2.3.C). Cell toxicity was completely inhibited by a 4-fold molar excess of Zn relative to Cd. Consistent with these findings, we observed an increase in intracellular Cd concentrations in TNFα treated cells, and a decrease in intracellular Cd concentrations when treated in conjunction

34 with a ZIP8 siRNA (Figure 2.3.A, 2.3.B). Taken together these findings further imply that ZIP8 is an important regulator of Cd uptake and that the vital micronutrient Zn plays an important role in preventing Cd uptake into lung epithelia, which we believe to be relevant when considering that a substantial number of COPD subjects are also malnourished.

Cd induces apoptosis and necrosis

Having established that Cd induces toxicity in lung epithelia in a ZIP8-dependent manner we also wanted to determine whether cell death was a consequence of necrosis, apoptosis, or both under these conditions. We first evaluated cells for the presence of caspase-cleaved cytokeratin-18 to identify apoptotic cells following combined TNFα and Cd exposure using the M30 apoptotic marker antibody and nuclear DAPI stain. Cells were considered apoptotic only in the presence of diffuse M30 staining throughout the cytosol and in the presence of a condensed nucleus (Figure 2.4.A). Combined TNFα and Cd exposure increased the frequency of apoptotic cells, achieving statistical significance at the highest

Cd exposure (Figure 2.4.B). Using flow cytometry in conjunction with annexin V

(AV) and propidium iodide (PI) staining, we next determined the extent of both necrotic and apoptotic cell populations. Briefly, cells that did not stain positive for either dye were considered viable. AV-positive/ PI-negative cells were considered early apoptotic, AV-positive/PI-positive were identified as mixed late apoptotic/necrotic and PI-positive/ AV-negative cells were considered to be necrotic. Consistent with previous findings, the combination of TNFα and Cd

35 exposure resulted in an increase in both the necrotic and apoptotic populations

(Figure 2.4.C). Combined TNFα and Cd exposure increased the frequency of PI and AV positive cells, achieving statistical significance at both Cd concentrations

(Figure 2.4.D).

ZIP8 is preferentially expressed at the apical surface and mediates Cd- induced toxicity in primary human lung epithelia

Initial studies were conducted in fully differentiated and polarized HUAEC monolayers to determine whether ZIP8 preferentially translocates to the apical or basolateral membranes following transcriptional activation by TNFα. Confocal analysis of TNFα stimulated HUAECs cultures established that ZIP8 protein preferentially but not completely localized to the apical membrane upon cell activation (Figure 2.5.A) whereas, little if any ZIP8 was observed in nonstimulated cultures. Next, we determined whether polarized cultures were more sensitive to Cd following apical or basolateral exposure. Treatment conditions were similar to past studies; however, TNFα was administered basolaterally, as previously reported by our group, followed by Cd exposure at either the apical or basolateral surface [98]. Consistent with past observations, basolateral and apical Cd exposure resulted in increased cell toxicity and more so in TNFα stimulated cells as measured by LDH release. Strikingly, Cd exposure at the apical surface resulted in significantly more cell damage when compared to basolateral exposure (Figure 2.5.B). A similar significant reduction in transepithelial electrical resistance (TEER; a.k.a Rt) was also observed

36 demonstrating that apical Cd exposure in TNFα activated cultures resulted in the largest reduction in epithelial barrier function (Figure 2.5.C). Taken together, our findings demonstrate that ZIP8 protein expression is rapidly induced in primary lung epithelia and preferentially localizes to the apical membrane, an anatomical location ideally suited for Cd uptake following cigarette exposure.

2.3 Discussion

Cigarette smoking is responsible for 90% of all COPD cases in the US.

Cigarette smoke contains over 2000 xenobiotic compounds that can damage lung tissue resulting in chronic bronchitis and emphysema [19]. Specifically, chronic smoke inhalation creates an inflammatory environment within the lung by activating resident cell populations. These cells go on to perpetuate inflammation through the production of chemokines and cytokines, including

TNFα [38, 107]. This chronic inflammation is consistent with the elevated NF-κB activity observed within the lungs of cigarette smokers [29, 30]. Cd, a toxic heavy metal abundant in cigarette smoke, has been identified as a causative agent of tobacco-related lung disease, though the mechanism(s) by which it is able to do so is unknown. In this chapter we present compelling evidence that

Cd causes lung epithelia toxicity by utilizing the inflammation-responsive Zn transporter ZIP8. In addition, we contend that the extent of cellular toxicity may be compounded under Zn-deficient conditions.

Our collaborator Dr. Daniel Nebert first identified the Zn transporter ZIP8 as the primary Cd transporter [82, 108]. ZIP8s affinity for Cd (Km = 0.48) is

37 similar to that of Zn (Km = 0.27), its endogenous ligand. This was a significant finding in the field of toxicology as it long remained unclear how a foreign, nonessential metal was able to efficiently gain access into cells. We were able to rescue lung epithelia from Cd toxicity using siRNA techniques to knockdown

ZIP8, confirming ZIP8s role in this process in lung epithelia. Intracellular Cd accumulation was also dependent on ZIP8 expression, as illustrated in figure 2.3.

Interestingly, our lab recently discovered that ZIP8 is under the transcriptional control of the NF-κB pathway, an element that sets it apart from the other 24 known Zn transporters [104]. Based on this finding, we first revealed that an endogenous function of ZIP8 is to import the critical micronutrient Zn specifically during times of cell stress to help reestablish homeostasis. However, when considered in the context of the inflammatory environment of the lung of a smoker, it becomes plausible that ZIP8 is facilitating disease through Cd import.

This off-target function paired with its specific responsiveness to inflammation targets ZIP8 as a potential central player in the development of COPD in cigarette smokers.

ZIP8 is primarily expressed in the kidney, lung and testis. Our microscopic analysis of HUAECs showed that ZIP8 expression increases in response to TNFα administration. More importantly, we revealed that increased

ZIP8 expression was specifically localized to the apical membrane, as seen in figure 2.5. It is believed that apical expression of ZIP8 evolved in kidney tubule cells to optimally position transporters for improved filtration of metals. This has

38 important clinical implications for cigarette smokers, as it is the apical surface of cells that cigarette smoke comes in direct contact with. The apical expression of

ZIP8 likely provides greater opportunity for Cd to transport into lung tissue with subsequent redistribution and accumulation to other tissues, including the lung, thereby increasing the risk of developing Cd-induced toxicity.

Epidemiological studies of the nutritional status of cigarette smokers have identified that deficiencies in Zn intake positively correlate with the severity of obstructive lung disease and Cd body burden [106]. This raises questions as to whether Zn supplementation can protect lung epithelia from Cd toxicity. We took initial steps toward answering this question by utilizing a cell culture model of lung epithelia. Cd was kept at a constant concentration and Zn was increased relative to Cd at molar ratios of 1:1, 1:2 and 1:4. Remarkably, cells supplemented with Zn survived despite the presence of Cd, as seen in figure

2.3.C. This is an important first step in understanding the ability of Zn to potentially act as a cytoprotective agent against Cd within the context of chronic cigarette smoke exposure. We were limited in our ability to assess the mechanism by which Zn is able to protect cells from Cd. It remains unclear whether differences in metal uptake, in which Zn is preferred and Cd is not imported as efficiently, promote survival. It is also likely that both Zn and Cd are being imported into the cell whereby Zn excess is triggering survival pathways that counter the toxic effects of Cd. Radio-labelling of Cd and Zn would help to

39 elucidate the potential differences in metal uptake and further focus in on the mechanism underlying this phenomenon.

The mechanisms that account for cellular Cd toxicity are multiple [26, 74].

Cd increases ROS production, disrupts mitochondrial function and can trigger necrotic and apoptotic pathways. Consistent with this, emphysema patients exhibit increased ROS burden and apoptotic lung tissue [30, 109]. It is hypothesized that these processes play an important part in the pathogenesis of the disease through destruction of alveolar tissue. As shown in Figure 2.4 A549 cells subjected to Cd following TNFα stimulation exhibited significantly more toxicity. We identified potential modes of cell death initiated by Cd using flow cytometric methods as shown in figure 2.4.B. Cd caused a significant increase in

AV+/PI+ cells, a unique mixed population that exhibits characteristics of the programmed apoptotic pathway (AV increase), as well as the more damaging necrotic pathway in which the cell membrane is disrupted (PI increase). Moving forward, it will be important to determine the extent to which Cd contributes to apoptosis, necrosis and perhaps other modes of cell death since removal of dead and dying cells and their debris play a critical role in perpetuating inflammation, immune function, and tissue recovery.

The goal of this work was to determine the extent by which ZIP8 contributes to cell toxicity. We have clearly demonstrated Cd is dependent on this transporter in lung epithelia, and that ZIP8 provides new pathogenic insight into a potential mechanism that accounts for pathogenesis in the lungs of

40 cigarette smokers. It is important to consider the strengths and limitations of our models in the context of the lung microenvironment of a cigarette smoker. Our experimental approach focused specifically on the potential role of ZIP8. ZIP8 expression was induced in lung epithelia with the pathophysiologically relevant cytokine TNFα which was the followed by Cd exposure. This approach may not fully emulate the lung environment of a chronic smoker whereby Cd concentrations likely accumulate and persist as inflammation and infections repeatedly occur over time. Nevertheless, our model to an extent emulates what may occur in a naive smoker where inflammation occurs at the onset of initial smoke exposure. In addition, COPD subjects have repeated bacterial infections whereby inflammation is triggered (e.g. TNFα release) leading to further changes in Cd metabolism and uptake. With respect to chronic smokers, Cd body burdens are twice that of non-smokers, which would strongly suggest that lung epithelia are persistently loaded with Cd perhaps before they encounter the inflammatory stimulus of their next cigarette [69]. In the future it would be interesting to investigate a model in which cells are chronically exposed to Cd before cytokine exposure. It is plausible that chronic Cd exposure may alter the capacity of lung epithelia to express ZIP8 thereby changing the extent of Cd- mediated toxicity in favor or disfavor of the host.

Our work demonstrates a clear dependency of Cd on ZIP8 to initiate toxicity in vitro. The use of primary cells (HUAECs) strengthens our findings: however, it is important that we expand our studies into in vivo models of COPD

41 that can more accurately resemble the human condition. The development of models to investigate ZIP8 and Cd in the context of disease is possible, though notably difficult, given the natural progression of COPD that takes decades to manifest in humans. Additionally, only 20% of cigarette smokers go on to develop COPD, an important statistic but one that has eluded researchers [9].

Established murine models of chronic smoke exposure exist and are available.

Using these models in conjunction with genetically engineered mice that either do or do not contain an excess of ZIP8 should provide greater insight into contributions of ZIP8 relative to prolonged Cd exposure, via cigarette smoke exposure, in the lung. Animals models can also be manipulated in a manner that will allow us to understand the role of dietary Zn in preventing or slowing lung pathogenesis. Further, repositories of samples of COPD patient lungs can be utilized to understand patterns of ZIP8 expression and Cd accumulation in comparative analysis of nonsmokers, healthy smokers and smokers with COPD.

In conclusion, we have identified that ZIP8 is a major regulator of Cd- mediated lung toxicity in the setting of inflammation. The expression of ZIP8 is upregulated by the NF-κB pathway that in turn enhances Cd uptake into lung epithelia causing apoptosis and necrosis. We believe that these findings fill a current gap in our understanding of Cd-mediated toxicity in the lung through interaction with a unique Zn transporter thereby advancing our understanding of

COPD pathogenesis. Additionally, it has provided important insight as to how

42 dietary Zn may play a significant role in attenuating the toxic impact of Cd in this disease.

2.4 Materials and Methods

Cell Culture

The human lung epithelial A549 cell line (catalog no. CCL-185, American Type

Culture Collection) was maintained under standard culture conditions in DMEM supplemented with 10% fetal bovine serum (FBS), 0.1 mg/ml streptomycin, 100

IU/ml penicillin and 1% non-essential amino acids at 37°C in a 5% CO2- humidified incubator. All cells were used between passages 6 and 20. Cells were maintained in serum-free conditions 24 hours before and throughout the duration of experiments to minimize absorptive Cd loss due to serum protein binding. Primary, differentiated, polarized HUAECs were isolated and cultured on collagen-coated cell culture inserts. Results in this investigation are derived from three different donors. HUAECs were maintained in 1:1 DMEM and Ham’s

F-12 media (DMEM-F-12) supplemented with 2% Ultroser G (BioSepra,

Villeneuve, France) and antibiotics unless otherwise stated. Human lungs were collected with approval from The Ohio State University Institutional Review

Board.

Cd Exposure and Analysis of Cytotoxicity

A549 cells were seeded and then serum starved for 24 hours followed by overnight treatment with 100 ng/ml TNFα or vehicle control. Cultures were then exposed to increasing concentrations of CdCl2 for 24 hours. All experiments,

43 unless otherwise stated, were performed in triplicate. Culture supernatants were then collected and lactate dehydrogenase (LDH) activity was measured using the

Cytotoxicity Detection Kit (Roche Diagnostics, Mannheim, Germany). Samples were compared to a positive control treatment group, generated for each experiment by treatment with 2% Triton-X (Sigma Chemical Company, St. Louis,

MO) in DMEM for 10 minutes to yield 100% cell death. The same protocol was conducted in primary cultures however, TNFα was administered to the basolateral surface of polarized cultures whereas Cd was administered either basolaterally or apically. In addition to LDH release, the integrity of primary cultures was monitored by measuring transepithelial electrical resistance (TEER) using a portable ohmmeter (Millicell-ERS, Millipore). For TEER measurements,

200 µl of media was placed on the apical surface and then immediately removed following recording. Baseline TEER measurements were determined at the beginning of each experiment and then following Cd exposure. In our model, fully differentiated cultures that maintain an air-to-liquid interface typically exhibit

TEER measurements > 400 Ω.

Inhibition of NF-κB and ZIP8

A549 cells were initially seeded and serum starved for 24 hours followed by treatment with 20 μM Bay 11-7082, a pharmacologic inhibitor of the NF-κB pathway that irreversibly binds to the phosphorylation site of IκB-α, or DMSO as a vehicle control. Following a one hour exposure, fresh medium was then replaced containing Bay 11-7082 or DMSO and TNFα and then incubated for an

44 additional 24 hours. Cultures were then exposed to increasing concentrations of

CdCl2 (0 to 25 µM) for an additional 24 hours after which cytotoxicity was determined by measuring LDH release.

ZIP8 expression was inhibited using a 21-mer short interfering (si)RNA target sequence (QIAGEN, Valencia, California) following transfection into A549 cells using HiPerFect transfection reagent (QIAGEN). ZIP8 expression was typically decreased by > 70% following TNFα stimulation. ZIP8 siRNA treated cultures were compared to a nonsilencing control siRNA that did not affect ZIP8 expression. Following siRNA treatment, cultures were exposed to TNFα and then increasing concentrations of CdCl2 (0 to 25 µM) for an additional 24 hours after which cytotoxicity was determined by measuring LDH release.

Zn competition study

Cells were seeded and serum starved for 24 hours, then stimulated with TNFα or vehicle control for 24 hours. Cultures were then exposed to a fixed concentration of CdCl2 (10 µM) with or without increasing concentrations of ZnCl2 (0, 10, 20, and 40 µM) and then incubated for an additional 24 hours. Cytotoxicity was again determined by measuring LDH release.

Western Analysis

Membrane protein fractions were generated by suspending cells in a lysis buffer composed of Buffer A (20 mM Tris/HCl pH 7.5, 5 mM MgCl2, 1 mM ethylene glycol tetraacetic acid, 20 mM β-glycerophosphate, 1 mM phenylmethanesulfonylfluoride, 2 μg/ml aprotinin, 2 μg/ml leupeptin, 1 mM

45 sodium vanadate) followed by sonication for 5 seconds four times while on ice.

The lysate was then centrifuged at 2900 rpm for 5 minutes. The supernatant was then centrifuged at 55,000 rpm for 30 minutes with a TLA 120.2 rotor using the

Optima TLX-120 Ultracentrifuge (Beckman Coulter, Brea, California). The pellet was resuspended in Buffer A and centrifuged for 5 minutes at 2900 rpm. The remaining pellet was resuspended in Buffer A containing 1% NP-40 and agitated for 1 hour at 4°C followed by centrifugation at 55,000 rpm for 30 minutes giving yield to the membrane extract in the resulting supernatant. The whole cell and membrane lysates were quantified using a protein assay (Bio-Rad, Hercules, CA) and then mixed in Laemmli buffer (Bio-Rad) containing 5% (vol/vol) 2- mercaptoethanol, boiled for 5 minutes, separated on 10% SDS-PAGE gel (Bio-

Rad), and then transferred to a nitrocellulose membrane (Amersham

Biosciences, Little Chalfont, UK). Membranes were blocked with 5% milk (w/v) in

PBS 0.1% Tween 20 (PBS-T) for 1 hour at room temperature and then incubated with primary antibody overnight at 4°C. After washing, the membranes were incubated with secondary antibody for 1 hour at room temperature. The signal was detected with an ECL Kit (Amersham Biosciences) and a Fluor-S Multi-

Imager Max/Bio-quantity one (Bio-Rad). The following antibodies were used in our experiments: rabbit anti-ZIP8 (1:2000, Covance, Princeton, NJ), mouse anti-

β-actin (1:2000, MP Biomedicals, Aurora, OH), goat anti-rabbit IgG-HRP (1:3000,

Cell Signaling), and horse anti-mouse IgG-HRP (1:3000, Cell Signaling).

Intracellular Cd measurements

A549 cells were subjected to 24 hour TNFα stimulation with or without ZIP8 siRNA, and then treated with increasing concentrations of Cd as previously described. Cell supernatants were then collected and centrifuged to further collect detached cells. The Measure-iT™ Lead and Cd Assay Kit (Invitrogen,

Carlsbad, CA) was used to measure intracellular Cd concentration. DMSO was added directly to each well and then cells were scraped and combined with pelleted cells in the supernatant, followed by vortexing to lyse the cells. Then 10

μl of sample was added to a 96 well plate followed by 200 μl of the Measure-iT™ kit reagent. The fluorescence intensity was recorded for each well at 520 nm

(λex: 490). Samples were analyzed in triplicate and three readings were performed for each sample. A Cd calibration curve with a range between 5 to

200 nM CdCl2 was used per manufacturer guidelines to determine intracellular

Cd concentration within each sample and then standardized to protein content, as measured in the lysates using the Pierce BCA Protein Assay Kit (Thermo

Scientific, Rockford, Illinois).

Analysis of Apoptosis and Necrosis

A549 cells were detached with trypsin and pooled with cells already suspended during culture, and then fixed using 100% methanol and stained with an M30

CytoDEATH, Fluorescein conjugated antibody (Boehringer Mannheim,

Indianapolis, IN), a monoclonal antibody that specifically detects caspase- cleaved human cytokeratin-18 (CK-18). Concomitant nuclear staining was also

47 conducted using 0.5 mg/ml 4’6-diamidino-2-phenylindole dihydrochloride (DAPI;

Roche Molecular Biochemicals, Indianapolis, IN). Upon staining, cells were collected and cytospun onto slides and analyzed by fluorescent microscopy.

Apoptotic cells (M30-positive cells with fragmented nuclei) were enumerated by a blinded observer who randomly selected 14 fields of view per treatment condition. Data are presented as the average percentage of apoptotic cells divided by the total number of cells per viewing area. Lung epithelial cultures were also evaluated by flow cytometric analysis to evaluate cell death. Briefly,

A549 cells were detached with trypsin and combined with cells already suspended during culture and then pelleted. The pellet was resuspended and washed with PBS and again pelleted. Pellets were then resuspended in AV binding buffer with an AV antibody and then incubated in the dark for 15 minutes.

An additional volume of AV binding buffer was added and then samples were filtered through 0.2 μm filters into tubes suited for flow cytometry. Propidium iodide was added immediately before flow analysis.

Immunohistochemical Analysis of ZIP8 expression

The apical surface of HUAEC cultures grown on 24-well transwell inserts were first washed with PBS to remove debris and then fixed with 4% formaldehyde.

Monolayers were then blocked with 10% goat serum for two hours at room temperature in permeabilizing buffer and then incubated with primary rabbit anti-

ZIP8 antibody overnight at 4°C. Following washing, membranes were incubated with secondary antibody (Alexa Fluor 488 goat anti-rabbit antibody, Invitrogen)

48 for one hour. Nuclear DNA was detected with DAPI. Slides were mounted with

Citifluor antifadent mounting medium (AF1, Electron Microscopy Science) and then examined using a disk scanning confocal microscope at 600x (Olympus

BX61). The magnification of all images was performed using X10 (WHN10X) and X60 (Olympus 60X/1.42 Oil PlaneApon or 60X/0.90N LUMPLANF1) objectives. The z-section images were obtained and analyzed using Slidebook

(Intelligent Imaging Innovations Inc., Denver, CO) software.

Statistical analysis

All data are expressed as mean ± standard error (SE). For comparisons involving multiple variables and observations, a two- and three-way ANOVA

(GraphPad, La Jolla, California) were used. Having passed statistical significance by ANOVA, individual comparisons were made with the Bonferroni multiple-comparison test. Statistical significance was defined as a p value <

0.05.

2.5 Figures

Figure 2.1: A549 cell toxicity in response to Cd and TNFα treatment. A. A549 cells were first exposed to TNFα (100 ng/ml) for 24 hours and then exposed to increasing concentrations of Cd for an additional 24 hours. A significant increase in cell toxicity was observed in TNFα stimulated cultures exposed to Cd. Toxicity was standardized relative to detergent-lysed control cells (100% cell death). Data is expressed in triplicate and representative of at least 3 experiments (***p < 0.001, Two-way ANOVA). B. A549 cells were exposed to TNFα for 24 hours and cell membrane fractions were analyzed by Western blotting for human ZIP8. Autoradiographs were quantified by standard densitometry and standardized to β-Actin for each sample in order to measure fold induction of ZIP8 expression compared to untreated control samples.

Figure 2.2: ZIP8 inhibition reduces Cd-mediated toxicity. A. A549 cultures were first treated with the NF-κB inhibitor Bay 11-7082 (20 µM) or DMSO as vehicle control for 60 minutes and then exposed to TNFα for 24 hours followed by exposure to increasing concentrations of Cd for an 24 additional hours. NF-κB inhibition resulted in a significant decrease in cell toxicity when compared to the DMSO (vehicle) control in TNFα + Cd treated cultures. Data is expressed in triplicate and representative of 3 separate experiments (***p < 0.001; Two-way ANOVA). B. Similarly inhibition of ZIP8 expression with a ZIP8-specific siRNA resulted in a significant decrease in cell toxicity when compared to the siControl treatment group. Data expressed in triplicate and representative of 3 experiments (***p < 0.001, Two-way ANOVA). C. Membrane fractions were also obtained from samples in A. and B. and analyzed by Western blotting with a primary antibody against ZIP8. Densitometry was standardized to actin and used to determine the percent knockdown of ZIP8.

Figure 2.3: ZIP8 increases Cd and Zn uptake in lung epithelia. A. A549 cells were stimulated with TNFα for 24 hours and then exposed to increasing amounts of Cd (0 - 25 μM) for 24 hours. Intracellular Cd was significantly increased in TNFα treated cells. Data expressed in triplicate and representative of 3 experiments. (***p < 0.001, *p < 0.05, Two- way ANOVA). B. A549 cells were transfected with scrambled or ZIP8 siRNA and then stimulated with TNFα for 24 hours. Cells were then exposed to Cd (0-25 μM) for 12 hours. A decrease in intracellular Cd was observed in cells treated with the ZIP8 siRNA, though not statistically significant. Data expressed in triplicate and representative of 3 experiments. C. A549 cells were first exposed to TNFα and then increasing concentrations of Zn (10 to 40 µM) in combination with a fixed amount of Cd (10 μM). A significant decrease in cell toxicity was observed as Zn concentration increased, relative to Cd, in TNFα stimulated cultures. Data expressed in triplicate and representative of 4 experiments (***p < 0.001, **p < 0.01, Two- way ANOVA).

Figure 2.4: ZIP8-mediated uptake induces apoptosis and necrosis. A. Cells were treated with TNFα and increasing concentrations of Cd (0 to 25 μM) for 24 hours and subsequently stained with an M30 antibody specific for caspase-cleaved cytokeratin 18. Representative photomicrographs of M30 positive A549 cells exposed to TNFα and Cd are shown. Cells were designated as apoptotic if they stained green with condensed nuclei (M30-positive green, DAPI blue). B. An increase in apoptosis was observed in cells exposed to TNFα and Cd. Percent apoptosis was calculated by dividing the number of apoptotic cells by the total number of cells in the field of view. Data is representative of 4 experiments. A statistically significant increase in apoptotic cells was observed in cells exposed to higher Cd concentrations in combination with TNFα (***p < 0.001, Two-way ANOVA). C. A549 cells treated under the same conditions with TNFα and Cd were also stained with AV (AV) and PI (PI) and enumerated by flow cytometry. The incidence of both necrosis (N) (PI+/AV+, top right quadrant) and apoptosis (A) (PI-/AV+, bottom right quadrant) was highest in cells exposed to both TNFα and Cd. Data in each figure is representative of 5 experiments. D. An increase in cell death was observed in cells exposed to TNFα and Cd. There was a statistically significant increase in PI+/AV+ cells following exposure to Cd in combination with TNFα (***p < 0.001, Two-way ANOVA). PI+/AV+ cells represent a mixed population of late apoptotic and necrotic cells.

Figure 2.5: Polarized ZIP8 expression in primary lung epithelia increases cell toxicity in response to TNFα and Cd treatment. A. Immunofluorescent staining was conducted on confluent polarized primary HUAEC cultures using an hZIP8 antibody and then visualized by z-stack using a disc-scanning confocal microscope. TNFα treated cells exhibited an increase in ZIP8 expression that was preferentially localized to the apical membrane whereas nonstimulated cultures showed minimal evidence of ZIP8 expression. ZIP8 staining is indicated by red fluorescence and DAPI nuclear staining is indicated by blue fluorescence. B. Fully differentiated, polarized, primary HUAECs were treated with TNFα for 24 hours, followed by Cd exposure for 48 hours. LDH release was measured and percent cell death was determined using a detergent positive control (100 % cell death). Toxicity was significantly increased in cells treated with TNFα and then apically exposed to Cd. Data expressed in triplicate and representative of four experiments. (*p < 0.05, Two-way ANOVA) C. In a similar experiment, cultures were treated as previously described and transepithelial membrane resistance (TEER; Rt) was measured (Ω). The decrease in Rt, indicative of compromised membrane integrity, was more significant following apical Cd exposure when compared to basolateral Cd exposure. Data expressed in triplicate and representative of three experiments. (*p < 0.05, **p < 0.01, Two- way ANOVAs)

CHAPTER 3: THE CONTRIBUTION OF CD AND ZIP8 TO COPD PATHOGENESIS IN VIVO

3.1 Summary

In chapter 2, we investigated the contribution of ZIP8 and Cd to lung epithelial cell toxicity using in vitro models. Based upon our findings, we chose to expand our studies in a translational manner through the development of in vivo models to test our central hypothesis. To our knowledge, no studies have been conducted that address the clinical impact of ZIP8 and Cd in humans or mouse models in the context of cigarette smoke exposure. Further, examination of whether Zn deficiency augments lung pathogenesis has never been addressed.

We hypothesized that overexpression of ZIP8 in the lungs of mice chronically exposed to cigarette smoke will increase disease pathogenesis through Cd uptake into lung tissue. Further, in humans, we hypothesized that cigarette smokers with COPD will have increased ZIP8 expression, decreased serum Zn and increased blood Cd. This chapter addresses three separate in vivo studies all designed to address our hypothesis: assessment of ZIP8 in GOLD stage 0 patients, a mouse model of combined ZIP8 overexpression and chronic cigarette smoke exposure, and statistical analysis of the NHANES 2011-2012 cohort for correlations between Zn and Cd burden in cigarette smokers.

The work described in this chapter presents compelling in vivo evidence to support our hypothesis that ZIP8 acts as a critical mediator of tobacco related lung disease. Human tissue samples obtained from cigarette smokers demonstrated a significant increase in ZIP8 mRNA and protein throughout lung tissue when compared to non-smoking human controls. This fundamentally important observation served as a stimulus to investigate a model of chronic cigarette smoke exposure in ZIP8 overexpressing mice. Importantly, we observed that mice transgenically engineered to express three extra copies of

ZIP8 developed significantly more emphysema following prolonged smoke exposed when compared to wild-type counterparts. Further, epidemiologic analyses of the NHANES 2011-2012 database revealed that cigarette smokers with the highest blood Cd amounts also had the lowest serum Zn measured. We believe that together, these results provide strong supporting evidence that ZIP8 may contribute to COPD pathogenesis in the context of chronic cigarette smoke exposure and warrants further study.

3.2 Results

ZIP8 is increased in the lungs of chronic smokers

Based upon our findings obtained from human lung epithelial cell models, we sought to evaluate the expression of ZIP8 in human lung tissue. Lung tissue samples were obtained from lifetime non-smokers (n = 5) and chronic smokers (n

= 7) through the NIH sponsored Lung Tissue Research Consortium. These patients were all characterized as GOLD stage 0, meaning no clinical evidence of

COPD disease yet existed in these patients. Quantitative analysis of ZIP8 mRNA levels revealed a consistent and significant increase in ZIP8 mRNA transcripts in the lungs of smokers when compared to non-smokers (Figure

3.1.A). Consistent with RNA findings, immunohistochemical analysis of the same biopsy specimens revealed an increase in ZIP8 protein throughout parenchymal tissue that appeared to be most prominent in upper airway and alveolar epithelia but also in what appeared to be, based on anatomical location and cellular morphology, alveolar macrophages (Figure 3.1.B). The increase of both ZIP8 protein and mRNA transcripts in the lungs of smokers strongly supports our cell culture findings presented in chapter 2, indicating that chronic cigarette smoke exposure increases ZIP8 expression thereby enhancing the capacity of lung tissue to obtain Cd through transporter-mediated uptake.

Emphysema is increased in the lungs of smoke-exposed BTZIP8-3 mice

In our pilot experiment, 8-week old female C57/Bl6 (n = 7) and BTZIP8-3 (n = 5) mice were exposed to cigarette smoke for 16 weeks, a time course sufficient for disease to develop in the wild-type strain as determined by our collaborator Dr.

Michael Borchers. Filtered air controls were unable to be included in the pilot due to limitations in the number of matched littermates at the time of our pilot study. After 16 weeks of cigarette smoke exposure, mice were euthanized and tissues were subject to analysis. mRNA analysis was conducted on lung tissue to determine differences in ZIP8 expression between C57/Bl6 and BTZIP8-3 mice. The analysis showed an average ~2.5 fold increase in the mRNA

57 expression of ZIP8, as expected, in the overexpressing strain when compared to the wild-type strain. This difference closely reflects the variation in gene copies

(2 vs. 5) present between the genome of the two treatment groups and is consistent with previous studies conducted on the expression profile of this line

[23]. Mean linear intercept analysis was conducted by a blinded investigator on

H&E stained sections of both C57/Bl6 and BTZIP8-3 mice exposed to cigarette smoke. Analysis revealed a significant difference in average chord length between the two groups. The average chord length in C57/Bl6 mice was 48.08 ±

1.210 µm, while the average chord length of the BTZIP8-3 mice was 58.05 ±

3.226 µm. The larger chord length reflects the presence of larger air space within the alveolar region of the lungs of these mice, and a decrease in surface area available for gas exchange, which is consistent with pathogenic evidence of emphysema. Photographic examples shown in figures 3.2.A illustrate the differences in air space enlargement between the two treatment groups. This statistically significant difference provides strong evidence that ZIP8 plays a critical role in the development of disease progression in vivo following prolonged exposure to cigarette smoke.

BTZIP8-3 mice Cd transporter profiles are similar

Next, we determined whether any additional changes in the expression of other confirmed Cd transporters was altered as a result of cigarette smoke-exposure and ZIP8 overexpression in the lungs of our two treatment groups. Our rationale was that it was important to establish whether any transporters besides ZIP8

58 could account for the pathologic changes observed in lung tissue. Literature review identified 5 confirmed Cd transporters that included Nramp2, ZIP8, ZIP14, and megalin/cubilin [46]. Nramp2 (Slc11a2) is an iron transporter typically expressed on the apical membrane of enterocytes [79]. ZIP14 (Slc39a14) is expressed in the pancreas [81]. Megalin and cubilin are endocytic receptors expressed in the kidney. [78]. mRNA analysis revealed no significant difference in the expression of these transporters between the wild-type and BTZIP8-3 mice exposed to cigarette smoke, as seen in figure 3.3. Megalin and cubilin exhibited very low expression, which was expected given their specialized kidney expression. ZIP14 was also expressed at very low levels. Nramp2 was expressed at copy numbers similar to ZIP8, though not differentially between the two groups. While it is possible Nramp2 may contribute to the observed Cd accumulation in these mice, the lack of a difference in mRNA expression levels suggest that the contribution would be similar between the two groups and therefore should not account for the observed differences. Protein analysis via immunohistochemical or Western analysis was not conducted.

Disease progression does not correlate with metal accumulation

Based on our hypothesis that ZIP8 contributes to disease by facilitating Cd import, we then quantified the extent of Cd accumulation in lung tissue of smoke exposed mice. Our collaborator Dr. David Killilea conducted inductively coupled plasma optical emission spectrometry (ICP-OES) on the superior right lobe of each mouse. Metal quantification was standardized to dry tissue weight.

Surprisingly, no significant difference in the amount of Cd or Zn lung content was observed when we compared C57/Bl6 and BTZIP8-3 mice that were exposed to cigarette smoke, shown in figure 3.4. This observation does not support our hypothesis. In particular, we anticipated that an increase in Cd accumulation would occur in BTZIP8-3 mice. However, we believe this finding raises important questions regarding the trafficking and compartmentalization of Cd throughout the body, and necessitates future studies that focus on other tissue compartments as well as subcompartments within the lung.

BTZIP8-3 mice exhibit modest increase in MMP mRNA profiles

Emphysema pathogenesis is partially attributed to an increase in MMPs.

Specifically, MMPs -2, -9 and -12 have been shown to play a significant role in the destruction of alveolar space [54, 55, 58, 110]. We hypothesized that the induced expression of these proteins may be altered by an influx of metal accumulation, presumably due to the overexpression of ZIP8. mRNA analysis of these 3 specific MMPs revealed a trend toward an increase in BTZIP8-3 mice compared to C57/Bl6, though it was not statistically significant, as seen in figure

3.5, with MMP-2 expression the closest to achieving significance (p = 0.0743).

The observed increase in pathology in BTZIP8-3 mice may in part be attributed to an increase in MMP expression and presumably activity; however, the modest changes that we observed suggest that larger numbers of animals need to be studied and that other factors may contribute to the significant loss in alveolar tissue and change in lung architecture. In addition, only one time point was

60 examined so it is not yet possible to determine whether MMP proteolytic digestion may be more robust at earlier time points during the 16 week exposure period.

Inflammation profiles do not distinguish BTZIP8-3 smoke-exposed mice

A comprehensive candidate list of inflammatory biomarkers was composed from the COPD literature and primers for RT-PCR analysis were developed to measure mRNA profiles. The markers for inflammation investigated were for macrophage infiltration (CD68, CD80 and CD163), M1-specific macrophage markers (iNOS2, CCR7), M2-specific macrophage markers (arginase1, Mrc1), cytokines (IFNγ, IL-4, IL-13 and IL-10) and chemokines (MCP-1, MIP-1α,

CXCL10, CXCR3). Of the 15 markers studied, we did not observe a significant difference in mRNA expression of any in comparison between B6 and BTZIP8-3 mice, presented in figure 3.6. While this observation was not anticipated, we believe that future studies in this area will require a more rigorous analysis at multiple time points during disease progression.

Loss of ZIP8 overexpression corresponds with loss of observed phenotype

Next, we designed a follow up study based on our pilot experiment. Specifically,

C57/Bl6 and BTZIP8-3 8-week old female mice were exposed to either filtered air

(C57/Bl6; n = 3, BTZIP8-3; n = 9) or cigarette smoke (C57/Bl6; n = 3, BTZIP8-3; n = 8) for 16 weeks. In this experiment, additional tissues besides lung were harvested from each mouse, including blood, kidneys, heart and spleen. The fundamental basis of this study was to add an important control group with a

61 sufficient sample size and to evaluate systemic differences in vital organs other than the lung. Upon initial inspection of treatment groups and much to our frustration, we observed that the mean linear intercepts revealed no difference in emphysema disease progression between C57/Bl6 and BTZIP8-3 mice exposed to cigarette smoke. Upon further inspection, RT-PCR analysis of both lung and kidney tissue revealed no significant difference in ZIP8 expression, which lead us to eventually confirm that the genotype of the BTZIP8-3 mice had been lost during colony breeding by our collaborators as shown in figure 3.7. Confirmatory genotyping conducted by Dr. Nebert’s laboratory confirmed loss of the genotype and subsequent phenotype. While this was a major setback in our study, it unintentionally provided more evidence that expression of ZIP8 is critical for the development of disease in this mouse model.

Cigarette smokers with high blood Cd have low Zn serum

The NHANES 2011-2012 dataset was analyzed using descriptive statistics to compare blood Cd levels and serum Zn levels between smokers and non- smokers. Confirmation of smoker status was first confirmed as exhibited by mean cotinine levels of 106.999 ng/mL, which is more than ten-fold greater than the mean value of 8.987 ng/mL measured in non-smokers. Additionally, there was an approximate 2-fold increase in blood Cd levels in smokers when compared to non-smokers (0.793 vs. 0.333 ng/mL). This is consistent with literature reporting a nearly two-fold increase in Cd body burden of smokers [71].

We then hypothesized that cigarette smokers would also have decreased plasma

Zn levels as a consequence of poor nutrition, a reported phenomenon within this patient population [106]. In contrast to our prediction, we observed that cigarette smokers had a modest increase in mean serum Zn levels (828.33 ng/mL) when compared to nonsmokers (817.241 ng/mL) although this was not statistically significant, shown in figure 3.8. While this finding does not support our central hypothesis, it is consistent with observations obtained from our animal model and indicate that Zn and Cd metabolism may be different between tissue compartments and more complex than originally predicted. Further, our preliminary analysis of this database, we believe, may be limited from a statistical perspective because we did not adjust for confounding variables such as age, ethnicity, socioeconomic status, presence or stage of COPD, and body mass index (BMI). More thorough analyses may be necessary to identify any differences between these populations.

We then focused our analysis on the smoker population to identify a relationship, if any, between serum Zn and blood Cd. Serum Zn was plotted with respect to blood Cd, with an R2 = -0.001. While there was not a statistically significant correlation, we did observe that subjects with the highest measureable blood Cd levels coincided with lower serum Zn levels. While negative correlations between urinary Cd (high levels) and dietary Zn intakes (low intakes) have been noted previously, this is the first evidence of a similar negative relationship between these two factors within the blood compartment.

A recent study measured Cd content following inhalation from a single cigarette while simultaneously measuring blood, urinary and plasma Cd in human subjects [111]. The study revealed that a smaller subset of smokers consistently had high Cd measurements above the average mean of the vast majority of smokers (1.5 ng/mL) despite smoking identical quantities of cigarettes over time.

The authors hypothesized that this observation may have occurred subsequent to heterogeneity within this population caused by a decrease in the capacity to metabolize Cd and hence resulting in more accumulation. Based on this evidence, we reevaluated the NHANES dataset and revealed that ~14% of smokers had blood Cd measures above the threshold value of 1.5 ng/mL.

Though lower than the reported 25% of the first study population, we believe this epidemiological finding strengthens the plausibility that a heterogeneous population of smokers relative to Cd metabolism exists. We further speculate that changes in metabolism could result from genetic heterogeneity within Cd transporters, with ZIP8 as a leading candidate.

3.3 Discussion

Few studies have examined in depth the micronutrient status of cigarette smokers and particularly smokers that develop COPD. A recent study that retrospectively evaluated over 6700 human subjects utilizing NHANES data revealed insufficient Zn intake as a potential risk factor for the development of obstructive lung disease in smokers [106]. COPD patients are typically elderly and of lower socioeconomic status, populations that, perhaps not coincidentally,

64 are also at risk for Zn deficiency [91, 92]. Additionally, COPD patients typically exhibit symptoms such as appetite suppression, muscle wasting, and cachexia that can further compound nutritional deficiencies [3]. Meanwhile, related epidemiologic studies have reported that increased Cd body burden also increases disease risk.

The 2010 epidemiologic study was unique in that it for the first time assessed the relationship between Cd and Zn in a large population of cigarette smokers. In a large patient population of smokers, low dietary Zn intake positively correlated with increased Cd body burden, and when occurring together, significantly increased a smoker’s risk for developing chronic obstructive lung disease. This is the first study that identified potential imbalance between Zn and

Cd, suggesting that the interplay of metabolism between these two metals, or dysfunction therein, may contribute to disease [106]. However, based on the retrospective nature of this study, the authors were limited in their capacity to identify plausible biological mechanisms to account for the observed imbalance that may have contributed to disease. Given the unique expression profile and dual metal import properties of ZIP8, we contend that this protein may play a central role at the interface between Zn and Cd metabolism in the lung of smokers thereby causing imbalance that ultimately leads to disease. The work in this chapter demonstrates for the first time an increased presence ZIP8 in the lung of smokers when compared to age matched, healthy controls. We also show for the first time in a transgenic ZIP8 overexpressing mouse model that this

65 transporter may facilitate the development of emphysema in mice. Additionally, our retrospective epidemiologic analysis of a large human database is the first to identify potential relationships between physiologic measurements of Cd and Zn in smokers. We believe this work identifies potential use of Zn supplementation as an effective strategy to slow or prevent the progression of COPD in this population.

Despite decades of research, much remains to be known regarding the mechanisms that drive COPD pathogenesis. A majority of research in the field has focused on the contribution of specific cell types, inflammatory mediators, and proteases to pathogenesis, but little progress has been made in understanding the contribution of nutrition, or a lack thereof, as a contributor to irreversible obstructive lung disease [112, 113]. We believe our finding that ZIP8 is expressed at significantly higher levels in cigarette smokers provides novel supportive evidence of the potentially critical but underestimated role metal metabolism plays in COPD. We postulate that the inflammatory environment created by cigarette smoke exposure triggers the NF-κB pathway thereby persistently increasing ZIP8 expression and inadvertently leading to more efficient uptake of Cd into cells. Further, we contend that malnourished smokers may experience exaggerated Cd uptake due to a lack of Zn available to compete for uptake into target cells, like lung epithelia and macrophages, in the lung microenvironment. While observations obtained from human samples were revealing, they were limited in terms of providing mechanistic evidence that

66 establishes a role for ZIP8 as a cause of lung disease (Figure 3.1). Based on this, we focused our effort on development of a mouse model in order to determine whether ZIP8 contributes to disease following prolonged cigarette smoke exposure.

The development of genetically engineered mice has led to advances in

COPD in our understanding of how macrophages, MMPs and adaptive immunity drive COPD [110, 114]. Few studies have addressed nutritional strategies, and those that have, have done so in very limited scope [115]. The capacity to utilize the BTZIP8-3 mice provided an opportunity to establish the significance of Zn metabolism, relative to ZIP8, in vivo in the setting of chronic cigarette smoke exposure. Strikingly, BTZIP8-3 mice had a significant loss of alveolar tissue, consistent with emphysematic pathology, after 4 months of smoke exposure in comparison to smoke-exposed WT mice. Additional analysis of mRNA expression levels in lung tissue revealed no significant differences in the expression of 4 validated Cd transporters, in support of our hypothesis and suggesting any differences observed were primarily due to changes in ZIP8 expression. This is a novel finding that we believe begins to narrow a gap in understanding the significance of Zn metabolism relative to COPD progression.

Our original hypothesis that ZIP8 causes disease through Cd uptake is not entirely supported by our pilot study. Specifically, analysis of Zn and Cd levels in the lungs of WT and BTZIP8-3 mice revealed no difference in Cd or Zn accumulation. While this diverges from our expectations, we believe that there

67 are several potential explanations that may account for this finding. ICP-OES is perhaps the most sensitive method to quantify metal content in tissue but measurements were conducted on whole tissue lysates. Our observations are representative of metal content at a macroscopic level but do not delineate margination of metal content within subcompartments between and within cells.

Based on this, we cannot rule out the possibility that ZIP8 is responsible for organelle specific changes in Zn or Cd content. Additionally, the extent to which

Cd is exported via transporters presumably within the zinc transporter (ZnT) family remains largely unknown. Further, alteration of ZIP8 expression may lead to changes in the expression of other metal transporters, including potential Cd exporters that remain to be identified. This could influence Cd trafficking throughout the body within and outside the lung, and alter its biologic half-life.

This highlights an important limitation of our study, in that we only quantified Cd in lung tissue though this metal also accumulates in other tissues. The kidney is a well-known reservoir for Cd with the potential for significant accumulation leading to renal dysfunction. Moving forward, kidney, blood and plasma should be collected and analyzed in future studies in an effort to elucidate the extent of

Cd compartmentalization following chronic cigarette smoke exposure.

Cd has been largely considered a pro-inflammatory agent although some studies have indicated it may also have immunosuppressive effects [22, 25].

Given the complexity of the microenvironment within the lung of smokers, we examined a broad range of biological factors associated with lung inflammation.

This included markers for macrophage infiltration, pro- and anti-inflammatory cytokines and chemokines. mRNA analysis revealed no significant differences between BTZIP8-3 and WT mice. We also observed relatively low expression of most factors in WT mice following cigarette smoke exposure, which was not initially anticipated. Seeking the consultation of experts with mouse smoke exposure models we learned that prolonged exposure results in acclimation whereby an initial burst of inflammation in the first one to two months is followed by a relative and consistent decrease in inflammation despite progressive tissue destruction. In particular, cytokine and chemokine expression peaks around 3 weeks following initial exposure and then begins to decline and plateau. Future studies will need to more rigorously compare earlier time points between treatment groups as well as protein levels to establish a more comprehensive picture of biological events that transpire over time leading up to alveolar destruction.

The animal study also provided us with new insight into the influence that

ZIP8 has on the progression of emphysema in a model of chronic smoke exposure. This study was instrumental in identifying necessary controls for future experiments. A major limitation is that we do not yet have a valid negative control that would consist of mice exposed to filtered air. Based on previous published reports, we anticipate that C57/Bl6 mice exposed to filtered air for 16 weeks will not exhibit abnormal pathology [114]. However, it will also be critical

69 to establish that no further changes in lung architecture occur in filtered air exposed BTZIP8-3 mice.

The final portion of this chapter presents an analysis of blood Cd and serum Zn levels of smokers identified in the 2011-2012 cohort of the NHANES.

NHANES field studies are designed to collect comprehensive epidemiological data on the health and dietary patterns of our nation, which are then made publically available for analysis. We selected the 2011-2012 dataset because it was the first to include serum Zn measures, a more accurate measure of patient

Zn status compared to the traditionally used Zn dietary intake measure [91, 92,

106]. From this cohort, we identified a population of 694 smokers and 952 non- smokers for comparison. As expected, smokers exhibited a mean blood Cd measure more than twice that of non-smokers. Serum Zn levels were comparable between smokers and non-smokers. Though unexpected, we believe these raw findings may change upon adjustment for variables that may influence Zn status, such as age, ethnicity, gender and SEC. Further, assessment of Zn serum status with respect to GOLD staging of COPD subjects, based on spirometric analysis that is not yet available but forthcoming, will be particularly helpful to establish whether Zn, or a lack thereof, contribute to COPD susceptibility and disease severity.

The stratification of serum Zn values relative to blood Cd levels revealed an inverse relationship whereby subjects with the highest blood Cd levels also had lower serum Zn values within the lower end of the spectrum which was

70 consistent with a prior study [106]. More importantly, this is the first evidence of the existence of an inverse relationship between biological markers of Zn and

Cd. We believe this to be important because the metabolism and compartmental distribution of inhaled Cd is poorly understood, though we anticipate that it may be complex similar to Zn. Relative to this, one recent study quantified Cd uptake into blood, serum and urine following inhalation of one cigarette [111]. Cd content directly measured from mainstream smoke had a linear correlation with blood Cd levels in 75% of volunteers. However, 25% of volunteers consistently exhibited, nonlinear, higher blood Cd content that exceeded a set threshold of >

1.5 ng/mL, suggesting that significant heterogeneity exists within the population and may account for these differences. Using the parameters set by this study, we found that 13.69% of the NHANES 2011-2012 smoking registrants had Cd blood levels above this threshold, further supporting that heterogeneity exists within the general U.S. population. Based on these findings combined with our own, we contend that ZIP8 may in part account for the heterogeneity observed.

Moving forward we will conduct a larger study that is powered to determine whether functional polymorphic variation exists within ZIP8 and if so, whether it predicts the extent of Cd accumulation in cigarette smokers and risk of developing COPD.

In conclusion, animal and human studies conducted within our laboratory provide significant evidence that ZIP8 is involved in tobacco-related lung disease, in part by mediating a critical balance between Zn and Cd. This is based upon

71 the following observations: 1) a significant increase in ZIP8 expression in early stage COPD patients; 2) transgenic overexpressing ZIP8 mice have a significant increase in alveolar tissue loss following chronic cigarette smoke exposure and;

3) epidemiological analysis revealed that an inverse correlation exists between

Cd content in the body and dietary Zn in smokers. We believe these findings narrow a current gap that has existed within this field by revealing that micronutrient metabolism may play a previously unrecognized role in COPD pathogenesis and that nutrient-based strategies may prevent or delay disease progression.

3.4 Materials and Methods

Description of Human Lung Tissue

Human lung tissue samples were obtained from the Lung Tissue Research

Consortium of the NHLBI that included 7 chronic smokers (COPD Stage 0) and 5 control, life-time nonsmokers. RNA was isolated for quantitative RT-PCR.

Paraffin blocks of these samples were sectioned for staining.

Immunodetection of ZIP8 in Human Lung Tissue

A portion of the same human specimens described above were also fixed and mounted on slides. After deparaffinization, endogenous peroxidases were blocked for 15 minutes with hydrogen peroxide, then blocked with 10% milk in

TBS for 10 minutes, and then with 10% normal goat serum in TBS for 2 hours.

Primary antibody (anti-hZIP8) was then applied in 5% BSA in TBS and incubated overnight at 4°C. Following washing three times with PBS-T, slides were

72 incubated with goat anti-rabbit biotin conjugated antibody for 45 minutes at room temperature. Slides were washed with PBS-T four times and then incubated in avidin-biotin-horseradish peroxidase in PBS-T for 45 minutes. After being washed three times in PBS-T, slides were developed using diaminobencidine

(DAB) diluted in 0.05M Tris-HCl and then rinsed in distilled water and counterstained with hematoxylin. After dehydration with alcohols and xyline, slides were mounted on a permanent coverslip and evaluated by light microscopy. mRNA analysis

Isolated RNA was used to generate cDNA using the ThermoScript RT-PCR kit

(Invitrogen). Primer pairs were designed for GAPDH and target genes using

Primer Express software version 2.0 (Applied Biosystems). The 7900HT Fast

Real-Time PCR system (Applied Biosystems) using SYBR Green Master Mix (2x,

Applied Biosystems, Foster City, CA) was used to perform Real-Time quantitative PCR. All samples were standardized to average cycle threshold number of the GAPDH gene. Message expression was reported as the average

-ΔC relative copy number (RCN) as follows: 2 t ×100, where ΔCt is the Ct value standardized to GAPDH.

BTZIP8-3 Mouse Generation

The BTZIP8-3 mouse line, a constitutive ZIP8 overexpression line, was generated by the laboratory of Dr. Dan Nebert at the University of Cincinnati.

Details regarding the generation of these mice have been previously described

[23]. The established line has a total of 5 expressed copies of the gene: 2 endogenous copies and 3 bacterial artificial chromosome (BAC) copies, resulting in a 2.5 fold increase in mRNA and protein expression. In summary, a BAC encoding only the Slc39a8 gene from a Cd-sensitive 129/SvJ BAC library was microinjected into the heterozygotes of a (B6D1)F1 x B6 cross. Founder females were screened by PCR to quantify Slc39a8 gene copies. Three founder mice were identified as BTZIP8-3, BTZIP8-5 and BTZIP8-6, the final number indicating how many copies of Slc39a8 were successfully included. Each founder female was crossed with a B6 male and her litters were screened for the transgene.

BTZIP8-5 and BTZIP8-6 litters did not produce any viable offspring with the transgene, however the BTZIP8-3 female produced offspring with the transgene at a 1:1 Mendelian ratio.

Mouse model of emphysema

Development and execution of the model was conducted by our collaborator Dr.

Michael Borchers at the University of Cincinnati. C57/Bl6 (n=7) and BTZIP8-3

(n=5) female mice generated by Dr. Dan Nebert (~8 weeks old) were exposed to either filtered-air or cigarette smoke. Cigarettes used were research grade 3R4F

Kentucky Reference cigarettes (University of Kentucky). A TE-10z cigarette smoke machine (Teague Enterprises, Woodland, CA) was connected to an exposure chamber where mainstream-source cigarette smoke was released.

Mice were acclimated to the cigarette smoke for one week before full body smoke exposure. Mice were then given whole body cigarette smoke exposure in

74 the chambers at a concentration of 150 ± 15 mg/m3 total suspended particulates for 4 hours a day, 5 days a week for a total of 16 weeks. Concentration of CO was 400 ± 30 ppm.

Macroscopic Lung Tissue Analysis

Mice were euthanized via isofluorane on a Wednesday or Thursday to control for any variable effects of restarting smoke-exposure following the lapse the weekend created. Immediately after euthanasia, lungs were harvested for analysis as described previously [101]. Briefly, the right main bronchus was cross-clamped, and the left lung was filled with 10% formalin at a constant pressure of 25 cm H2O to preserve the anatomy of the lung. After 48 hours, the left lung was paraffin embedded. The three lobes of the right lung were snap frozen for later mRNA, protein and metal analysis. For each mouse, 5 non- consecutive 5 µm sections were cut from the paraffin block. The first and last sections were stained with hematoxylin and eosin (H&E). The non-stained sections were reserved for alternative staining. Mean linear intercept analysis was performed on the H&E stained slides as previously described [114]. Ten fields per section for two sections per mouse were blinded and evaluated.

Metal quantification

Harvested lung tissue samples were shipped overnight on dry ice to our collaborator Dr. David Killilea at Children’s Hospital Research Institute Oakland.

Samples were desiccated and dissolved in HNO3, and analyzed using ICP-OES.

Metal content (μg) was standardized to dry weight of sample (mg).

RT-PCR

The middle lobe from each mouse was homogenized in Trizol and RNA was isolated. The RNA was reverse transcribed into cDNA using the Platinum

Quantitative RT-PCR Thermoscript One-Step System. The cDNA was then subject to relative quantification using reverse transcriptase-polymerase chain reaction. Primers for housekeeping and target genes were designed and ordered from Integrated DNA Technologies.

NHANES Dataset Analysis

Datasets from the 2011-2012 NHANES conducted by the CDC were accessed using SAS software. The population was selected based on their having answered the question, “Have you smoked at least 100 cigarettes in your lifetime?” A “yes” categorized them as a smoker, while a “no” categorized them as a non-smoker. Those who did not respond or could not accurately answer were not included. The population was then narrowed based on availability of Zn serum measurements. We quantified descriptive statistics of the smoker and non-smoker populations with regards to cotinine levels (ng/mL), blood Cd levels

(ng/mL) and serum Zn levels (ng/mL). Further, we selected the smoker population and plotted serum Zn measurements against blood Cd measurements and surveyed a correlation. Based on literature suggesting differences in Cd metabolism in those with measured blood Cd levels of 1.5 ng/mL, we quantified the percentage of the smoker population that exhibited levels above this threshold [111].

Statistical analysis

All data are expressed as mean ± standard error (SE). Comparisons between groups were made using an unmatched t-test (Graphpad, La Jolla, CA).

NHANES data sets were analyzed using SAS software. Statistical significance was defined as a p value < 0.05.

3.5 Figures

Figure 3.1: ZIP8 mRNA and protein expression is elevated in smokers. A. Total RNA was extracted from human lung tissue samples that were obtained from chronic smokers (stage 0 COPD) and lifetime non-smokers and ZIP8 mRNA expression levels were measured. Quantitative RT-PCR analysis consistently revealed higher ZIP8 mRNA levels in the lung tissue of smokers (n=7) when compared to tissues obtained from non-smokers with the exception of one outlier in the control group (n=5). (Student’s t-test; **p value < 0.01). B. Immunohistochemical staining for ZIP protein was then conducted on tissue sections obtained from two subjects within the chronic smoker group. Consistent with mRNA expression, both specimens exhibited an increase in ZIP8 throughout the lung epithelia (staining indicated by brown areas at 200x and 400x magnification of donor 224671). ZIP8 immunostaining of two lifetime non-smokers did not reveal evidence of increased ZIP8 expression (donor 013011 shown at 200x and 400x magnification). As a negative control, the same specimens were stained with the secondary rabbit IgG control antibody (donors 224671; smoker, and 013011; non-smoker), at 200x.

Figure 3.2: Cigarette-smoke exposed BTZIP8-3 mice have increased emphysematic lung tissue. A. Total RNA was extracted from C57/Bl6 (n=7) and BTZIP8-3 (n=5) mice chronically exposed to cigarette smoke for 16 weeks. Quantitative RT-PCR analysis consistently revealed higher ZIP8 mRNA levels in the lung tissue of BTZIP8-3 mice, confirming their genotype (unpaired t-test, *p < 0.05). B. Mean linear intercept was performed on H&E stained sections of perfused lung tissue of C56/Bl6 and BTZIP8-3 mice after 16 weeks of cigarette smoke exposure. Analysis of 10 photographs of 2 sections of each of the mice revealed a significant increase in average chord length (unpaired t-test, ** p < 0.01). Changes in lung architecture of the lungs were visualized in 200x micrographs of C57/Bl6 (C) and BTZIP8-3 (D) lung sections stained with H&E.

Figure 3.3: Cd transporter expression is similar between cigarette-exposed C57/Bl6 and BTZIP8-3 mice. Total RNA was extracted from the middle right lobes of C57/Bl6 (n=7) and BTZIP8-3 (n=5) mice. Quantitative RT-PCR analysis revealed no significant difference in expression of (A) ZIP14, (B) Nramp2, (C) megalin and (D) cubilin (unpaired t-test, NS).

Figure 3.4: Metal accumulation in the lungs of C57/Bl6 and BTZIP8-3 mice does not differ. ICP-OES was utilized to quantify metal accumulation from the superior right lobes of smoke exposed C57/Bl6 (n=7) and BTZIP8-3 (n=5) mice. Metal (µg) was standardized to dry weight of the tissue (mg). There was no difference in the accumulation of either Zn (A) or Cd (B) in the lungs of either strain (unpaired t-test, NS).

Figure 3.5: Cigarette-smoke exposed BTZIP8-3 exhibit a trending increase in MMP mRNA expression. Total RNA was extracted from the middle right lobes of C57/Bl6 (n=7) and BTZIP8- 3 (n=5) mice. Quantitative RT-PCR analysis revealed a modest increase, though not statistically significant, in the mRNA expression of (A) MMP-2, (B) MMP-9, and (C) MMP-12. This trend was most pronounced in MMP-2 expression (unpaired t-test, NS).

Figure 3.6: Expression of inflammatory markers does not differ in cigarette-smoke exposed C57/Bl6 and BTZIP8-3 mice. Total RNA was extracted from the middle right lobes of C57/Bl6 (n=5) and BTZIP8-3 (n=7) mice. A panel of (A-H) macrophage markers, (I-K) cytokines and (L-P) chemokines was selected based on existing COPD literature. Quantitative RT-PCR analysis revealed no significant difference in the expression of these 16 markers between C57/Bl6 and BTZIP8-3 markers (unpaired t-test, NS).

Figure 3.7: Loss of BTZIP8-3 genotype corresponds with loss of emphysematic phenotype after chronic cigarette exposure. A second study was developed to include filtered-air C57/Bl6 mice (n=3), filtered-air BTZIP8-3 mice (n=9), cigarette smoke-exposed C57/Bl6 mice (n=8), and cigarette smoke-exposed BTZIP8-3 mice (n=8). Total RNA was extracted from the middle lobes of all of these mice. A. Quantitative RT-PCR revealed no significant difference in ZIP8 mRNA expression between these 4 groups of mice (ANOVA, NS). B. Chord length measurements did not vary between these groups of mice (ANOVA, NS). C-F. Micrographs at 200x reveal no visual changes in lung architecture of these 4 groups of mice.

Figure 3.8: Cd is elevated in the blood of cigarette smokers. Data regarding smoking status, cotinine, serum Zn and blood Cd was collected from the NHANES 2011-2012 cycle. A. Descriptive statistics identified a population of smokers (n=694) and non-smokers (n=952). Cotinine and blood Cd (ng/mL) were increased in smokers. Levels of serum Zn were comparable between the two groups. B. The smoker population was selected and serum Zn levels were plotted against corresponding blood Cd levels. Those with the highest blood Cd levels had the lowest serum Zn levels. R2 = -0.001, NS. C. Descriptive statistics revealed that 13.69% of smokers exhibited Cd blood levels above1.5 ng/ml.

CHAPTER 4: DETERMINING THE INFLUENCE OF CD UPON MONOCYTE AND MACROPHAGE IMMUNE FUNCTION

4.1. Summary

To this point our studies have focused on understanding how ZIP8 contributes to COPD pathogenesis by importing Cd into lung epithelia. Based on our findings and knowing that COPD patients suffer from frequent upper respiratory tract infections, we developed an interest to further understand how

Cd may influence the function of other cell types that contribute to disease, specifically cells of the innate immune system. This is important because COPD subjects notoriously suffer from repeated upper respiratory tract infections, a leading cause of morbidity and mortality in this population [40, 116]. Accordingly, we have focused our efforts on macrophages, the principal defense cell within the lower lung whose phenotype and abundance within the alveolar region have also been shown to correlate with disease severity. In particular, the number of macrophages in the lung of smokers substantially increases and it has recently become clearer that the phenotype is also dynamically altered [34-36, 117, 118].

As previously identified, COPD patients are highly susceptible to respiratory infections. Ex vivo stimulation of alveolar macrophages isolated from smokers and COPD patients have demonstrated an inability to mount a proper immune

86 response, as measured by cytokine production [34, 36]. This is relevant to our previous findings because a majority of these relevant immune mediators are under the regulation of the NF-κB pathway.

Studies that have assessed the impact of Cd on NF-κB function have observed an increase in a pro-inflammatory phenotype as a consequence of increased ROS production [46]. Conversely, other studies have reported that Cd has the capacity to inhibit NF-κB signaling [25, 119]. Clearly, more studies are warranted to delineate the mechanisms that cause Cd-mediated immune dysfunction and tissue pathogenesis. Based on our recent findings demonstrating that Zn inhibits IKKβ activity and the remarkably similar atomic properties of both metals, we postulate that Cd may disrupt macrophage function through inhibition of the NF-κB pathway [104]. If correct, provided the long biological half-life of Cd, its detrimental impact on innate immune function could leave an indelible imprint in this microenvironment and enhance the incidence of repeated infection through immune dysregulation.

In chapter 2, we presented data demonstrating Cd to be toxic to lung epithelia in a ZIP8-dependent manner. In this model we introduced the relevant pro-inflammatory stimulus TNFα first to induce the expression of ZIP8, followed by Cd administration. In the monocyte and macrophage models that were utilized in this chapter, we deliberately reversed the order of Cd and inflammatory stimulus exposure. Specifically, cells were first exposed to Cd overnight to allow sufficient exposure and presumably uptake. The following day we introduced an

87 endotoxin challenge and subsequently measured cytokine production and the extent of NF-κB signaling. Our rationale behind this approach was to develop a model that we believe more closely resembles what macrophages experience within the lung of chronic cigarette smokers. In particular, as monocytes are recruited into the alveolar region and differentiate into macrophages, they are continuously exposed to Cd. Consequently, the Cd enriched environment is also subject to inflammatory stimuli through exposure to first hand smoke inhalation as well as airborne pathogens.

We pursued our studies in this chapter using two related cell types: monocytes and macrophages. We chose to compare both models based upon the rationale that 1) monocytes are the precursor cell type to macrophages, 2) both cells have the capacity to respond to similar toxic and inflammatory stimuli and play fundamental roles in innate immunity; and 3) we have previously observed that both cells are capable of ZIP8-mediated Zn uptake. Accordingly, we predict that both cell types would subsequently be vulnerable to Cd uptake and altered function as a consequence of Cd-mediated cellular dysfunction.

Further, we compared both cell types for potential differences, if any, in their response to Cd in an attempt to elucidate mechanisms by which Cd mediates changes in cellular phenotype. Overall, we demonstrate that Cd inhibits NF-κB activity and subsequent cytokine production and release in macrophages, but not monocytes. This fundamental observation raises important questions regarding why differences exist in Cd processing between these two cell types, the role of

ZIP8 (or other Cd transporters) as an uptake portal, and how differences promote pathogenesis in the context of cigarette smoke induced lung disease.

4.2 Results

Cd increases macrophage cytotoxicity

The cytotoxicity of Cd- and LPS-treated THP-1 monocytes and differentiated macrophages was determined by measuring LDH release into cell supernatants as shown in figure 4.1. Monocytes did not exhibit a significant difference in cytotoxicity due to Cd administration, relative to LPS exposure. However, Cd- treated macrophages had high baseline cytotoxicity measures compared to untreated controls that did not further change with LPS administration. The purpose of this experiment was to determine whether excess cytotoxicity may account for differences that we would later assess via cytokine release. Based on these findings, we do not believe that these modest differences will substantially influence our interpretation of cytokine analysis.

Cd suppresses cytokine release in macrophages

THP-1 cells were collected and designated to either remain as suspended monocytes or differentiated into adhered macrophage cultures. We conducted these experiments in parallel from the same passage in an effort to control for any differences between passage numbers. Following overnight Cd exposure, cells were stimulated with 100 ng/ml LPS for either 4 or 12 hours, and then cytokine production was determined at both time points. We observed marked differences in cytokine profiles in response to Cd exposure between THP-1

89 monocytes and THP-1-derived macrophages as illustrated in Figure 4.2. In monocytes, we observed similar amounts of TNFα release at 4 hours and at 12 hours post LPS stimulation (4.2.E). Similar to monocytes, macrophages exhibited an increase in TNFα in response to LPS in Cd unexposed cultures; however, Cd treatment resulted in near complete ablation of TNFα production (4.2.J). Similar to TNFα, IL-6 release by monocytes peaked at 12 hours post LPS exposure which was significantly increased in the presence of Cd (4.2.B). Macrophages followed a similar pattern with IL-6 release at 12 hours but again exhibited decrease production in the presence of Cd (4.2.G). Similar trends between monocytes and macrophages were observed with respect to IL-8 and IL-10 production (4.2.C), (4.2.H). (4.2.D) (4.2.I). We also examined the impact of Cd upon IL-1β, a cytokine that is also relevant to immune defense in the lung microenvironment but is unique to in comparison to the other cytokines examined because it first accumulates intracellularly before being post-translationally processed and released as a mature active peptide subsequent to a secondary stimulus. In contrast to our previous findings with TNFα, IL-6 and IL-10, we observed that in both monocytes and macrophages a significant decrease in the amount of IL-1β release occurred following Cd exposure. Though this finding diverges from the other cytokine patterns, we believe this may be due in part to an additional inhibitory effect of Cd upon the processing and release of IL-1β.

Cd Suppresses Transcription of NF-κB-Dependent Genes in Macrophages

Given the marked observed differences in cytokine expression between cell types, we sought to determine if the Cd-mediated inhibitory effect in cytokine protein production could be accounted for by difference in transcriptional activation. In doing so, qPCR for the mRNA of each cytokine was conducted, in addition to determining the expression pattern of all confirmed Cd transporters. In monocytes, IL-1β transcription was increased at 4 hours, more so in the Cd- treated group, though not statistically significantly. Macrophages also demonstrated a peak in IL-1β transcription at 4 hours, though more so in non-Cd- treated cultures (4.3.F). This difference in transcription that does not correlate with protein further supports the notion there may be a secondary inhibitory effect of Cd upon IL-1β release from monocytes. IL-6 transcription was significantly increased in Cd-treated monocytes at 4 hours, and this trend was replicated at

12 hours (4.3.B). Macrophages saw a statistically significant increase in IL-1 transcription in untreated macrophages at 12 hours post LPS stimulation (4.3.G).

IL-8 production was not different between monocyte groups at 4 hours, though was decreased in Cd-treated cells at 12 hours, one inconsistency in monocyte transcriptional patterns (4.3.C). Macrophages showed statistical decreases in IL-

8 transcription at both 4 and 12 hour time points (4.3.H). IL-10 transcription was significantly increased relative to Cd at 4 hours in monocytes, while it was significantly decreased relative to Cd at 4 hours in macrophages (4.3.E). TNFα transcription did not vary between monocyte groups at any timepoint, though it

91 was significantly decreased relative to Cd at 4 hours in macrophages (4.3.J).

These polarized cytokine profiles are revealing of fundamental differences in transcriptional processes relative to Cd exposure.

Cd Inhibits NF-κB Signaling in Macrophages

Based on the significant decrease in transcription of NF-κB related genes by Cd in macrophages, we then examined deficits, if any, in protein signaling within this pathway. We first examined to what extent differences exist in nuclear p65 phosphorylation and nuclear translocation. Nuclear protein was isolated from cells within one hour following LPS exposure knowing that pathway activation typically occurs within minutes upon activation. Cd-treated monocytes exhibited nuclear translocation of phosphorylated p65 that increased over time following

LPS exposure, which was comparable to non-Cd treated, LPS exposed cultures

(4.4.A). In contrast, Cd-treated macrophages exhibited a decrease in the amount of phosphorylated p65 present in the nucleus when compared to untreated, LPS exposed controls (4.4.G). Western blotting for IκBα, a protein whose phosphorylation and subsequent degradation is required for p65 nuclear mobilization, revealed a substantial decrease in IκBα phosphorylation in Cd- treated macrophages (4.4.I). Consistent with previous findings, Cd-treated monocytes exhibited phosphorylation comparable to non-Cd exposed cultures

(4.4.C). The observed phosphorylation correlated with the extent of degradation of total IκBα protein that occurs upon NF-κB pathway activation (4.4.D). Western blotting of IKKβ, the protein directly upstream of IκBα was also telling. Cd-

92 treated monocytes again demonstrated phosphorylation patterns similar to untreated cells (4.4.D). Cd-treated macrophages exhibited significantly less IKKβ phosphorylation following LPS activation (4.4.K). The cumulative observations via Western blotting provide strong supportive evidence that Cd uptake by macrophages results in significant signaling defects within the NF-κB pathway that may result in the inability of these key immune cells to properly defend the lung microenvironment from pathogens and related danger signals.

Cd Inhibits NF-κB Activity in Macrophages

Our previous findings revealed alteration of phosphorylation and the relative abundance of key proteins involved in NF-κB signaling. Based on this, we next determined whether these changes resulted in defects in p65 protein function following Cd exposure through use of a functional activity assay. Nuclear protein was extracted from both monocytes and macrophages exposed to Cd and LPS and then applied directly to a plate coated with an NF-κB oligonucleotide consensus binding sequence. Only activated p65 that has localized to the nucleus is able to bind this nucleotide, which is subsequently quantified using

ELISA-based methodology. There was no observable difference in p65 activity between normal and Cd-treated monocytes following LPS exposure (4.5.A).

Consistent with previous findings, we observed a highly significant decrease in p65 activity within Cd-treated macrophages as measured by fold-change over untreated controls (4.5.B). We next conducted the same experiment and compared p65 activity between primary human monocytes and macrophages

93 from the same donor. Consistent with THP-1 monocyte cultures, we observed similar changes in p65 activity between primary monocyte treatment groups

(4.5.C) whereas p65 activity was significantly decreased following Cd treatment in primary macrophages (4.5.D). Taken together, we conclude that NF-κB activity is inhibited by Cd in LPS stimulated macrophages but not LPS stimulated monocytes.

Cd Inhibits IKKβ Activity in a Cell Free System

As reported previously by our lab, Zn is able to modulate NF-κB activity by directly binding to and inhibiting IKKβ [104]. Given the similarities between the

Zn and Cd and reports that indicate a strong inhibitory effect of Cd upon this pathway, we hypothesized that Cd may directly inhibit IKKβ [25, 119]. As predicted, IKKβ kinase activity was inhibited by Cd in dose responsive manner with an IC50 in the nanomolar range (~ 130 nM) indicative of a very high affinity and specific interaction, as shown in figure 4.6.

Differential expression of Cd transporters in monocytes and macrophages

To this point, it is unclear why monocyte and macrophages behave differently following Cd exposure. To address this, we sought to determine whether differences exist between monocytes and macrophages in the relative abundance of established Cd transporters. We conducted qPCR on ZIP8,

ZIP14, and Nramp2, as seen in figure 4.7.A-F. There were no detectable levels of megalin and cubilin, which was expected due to their restricted expression in kidney tubule cells. ZIP8 transcription was significantly increased by 4 hours of

LPS exposure in non-Cd treated monocytes (4.7.A). ZIP8 transcription was also elevated in LPS exposed macrophages at this timepoint, though the relative CN is much higher in monocytes (4.7.D). Relative ZIP14 expression was higher in macrophages than monocytes, and peaked significantly at 4 hours (4.7.B &

4.7.E). Nramp2 transcription was elevated in both monocytes and macrophages, though not significantly at any time point relative to Cd exposure (4.7.C & 4.7.F).

Western blotting revealed a significant increase in the expression of ZIP8 (as a glycosylated 140 kDa band) in monocytes; however, ZIP8 was modestly detectable in macrophages. Conversely, ZIP14 was significantly increased in macrophages as a 110 kDa band, with very low detection in monocytes (4.7.G &

4.7.H). Similar to ZIP8, this band is also hypothesized to be the mature form of

ZIP14 found on the cell membrane. We did not probe for Nramp2 due to its primary expression in the brain and gastrointestinal tract, where the environment is acidic enough to allow it properly function as a proton pump. We contend the differences observed between ZIP8 and ZIP14 provides significant insight into potential differences in Cd uptake and metabolism that may account for differences observed in cell function between monocytes and macrophages.

Intracellular Cd accumulation is greater in macrophages compared to monocytes

Based on our finding that Cd transporter expression varies between these two related cell types, we quantified intracellular Cd using atomic absorption.

Utilizing the same models as previously described we measured the extent of Cd

95 accumulation in monocytes and macrophages before and following 4 hour LPS exposure. Figure 4.7.I shows differences in Cd accumulation (nm/μg protein) between these timepoints and cell types. Though the standard deviation of accumulated Cd was quite large, a distinct pattern emerged in which more Cd accumulated in macrophages after LPS exposure. Strikingly, macrophages exhibited a 127% increase in Cd compared to monocytes, as seen in 4.7.J. This finding raises important questions relative to the roles ZIP8 and ZIP14, and perhaps yet to be determined factors, that may play in accounting for this significant difference in metal accumulation.

4.3. Discussion

Respiratory infections are one of the leading causes of morbidity and mortality in COPD. Elderly patients with COPD have a 6-fold increased incidence of pneumonia compared to their healthy peers [39]. Acute exacerbations of the disease, characterized by sudden worsening of dyspnea and respiration, typically correlate with contraction of viral or bacterial infections.

In moderate to severely diseased patients, it is common to suffer from more than

3 exacerbations a year [120]. In line with these observations are multiple studies that report a blunted cytokine response from stimulated macrophages isolated from the lungs of COPD patients and smokers suggesting that immune dysfunction increases the risk of acquiring infection [29, 35].

Macrophages are the principal phagocytic cell in the lung responsible for the removal of pathogens and debris and for coordinating host defense with other

96 cells in part through the release of cytokines. There exists a strong correlation whereby alveolar macrophages accumulate in the lung of smokers and more so in patients with COPD [117]. Despite macrophage accumulation, smokers become more susceptible to infection. Based on this, it is plausible that chronic cigarette exposure alters alveolar macrophage phenotype in a manner that inhibits their capacity to effectively respond to and remove of harmful pathogens.

This is supported by recent studies reporting a shift in the abundance of M2 macrophages within the lung relative to their M1 counterparts [35, 36]. This may be important because the “classically activated” M1 macrophage is an effective mediator of the pro-inflammatory response, whereas the “alternatively activated”

M2 macrophages are known to produce MMPs and have a dissimilar cytokine signature designed to elicit humoral-mediated immunity through the Th2 response. M2 macrophages are postulated to contribute to host injury through the release of MMPs, degradation of lung architecture, and release of DAMPS, thereby inadvertently recruiting more macrophages to the site of injury. Our finding that the pro-inflammatory cytokine response to endotoxin in Cd-treated macrophages is significantly decreased supports past observations from COPD subjects that cigarette smoke toxicants inhaled into the lung, namely Cd, creates an environment that prevents elicitation of the M1 phenotype [36]. Based upon our novel findings, we postulate that Cd may be a potent mediator of macrophage reprogramming through inhibition of a central innate immune signaling pathway central to the M1 response.

We also observed that monocytes, the precursor to tissue macrophages, behave distinctly different and exhibit a pro-inflammatory phenotype when exposed to a PAMP. We believe that this may also be problematic. Systemic inflammation has been documented in COPD patients, supportive of our finding that Cd increases cytokine output by monocytes [33]. Increased inflammatory signaling may perpetuate an influx of monocytes into the lung space that differentiate into dysfunctional macrophages impaired in their ability to mount a proper immune response. Repeated respiratory infections and host injury can then signal for more monocytes to differentiate into alveolar macrophages that are unable to fully neutralize infectious threats, thus creating a dangerous cycle by which more damage is inflicted upon the host. The polarity of response to endotoxin observed between Cd-treated monocytes and macrophages highlights the significance of the versatility of this metal, and how it can drive both pro- and anti-inflammatory responses that synergistically lead to disease.

Cd is a potent disruptor of oxidant balance once inside a cell, resulting in generation of ROS [121]. Acute ROS production is a potent inducer of the NF-κB pathway, thereby coupling intraceullar Cd transport to regulation of this pathway.

Counter to this concept, a number of studies have proposed an inhibitory effect of Cd upon the NF-κB pathway [25, 119]. Based upon findings from our own studies, we observed that Cd-treated macrophages exhibit decreased nuclear p65 translocation into the nucleus with a subsequent decrease in transcriptional activity. Additionally, we observed that this effect is at least in part accounted for

98 through defects in protein phosphorylation of intermediate proteins. The discrepancy that exists between our study and others may be a consequence of study design and choice of model. In particular, evidence that Cd is an activator of NF-κB was derived from acute exposure models that utilized significantly higher Cd concentrations [46]. Our model deliberately utilized more relevant physiologic concentrations of Cd and we allowed sufficient time for cells to uptake Cd and re-equlibrate before challenging with a relevant stimulus. To our knowledge, our study is the first to evaluate the effects of prolonged Cd exposure in primary monocytes compared to macrophages. By doing so, we were able to discover that substantial differences exist in the cellular response between these cell types. In particular, nuclear p65 activity of Cd-treated monocytes was comparable to corresponding non-Cd treated controls following LPS stimulation which is in distinct contrast to the inhibition observed in corresponding macrophages from the same donor.

Despite the similarities that exist between these cells, there are apparent differences in Cd metabolism and its impact on immune function. Currently, it is unknown to what extent, if any, Cd uptake and intracellular trafficking is different.

There currently exist five validated Cd transporters that function as import proteins (ZIP8, ZIP14, Nramp2 and megalin/cubilin). Beyond the plasma membrane, there are as of yet no known Cd transporters designated for cellular organelles or vesicles. However, it has been shown that ZIP8, in addition to Zn import across the plasma membrane, localizes to lysomal membranes thereby

99 promoting Zn transport out of the lysosome and into the cytosol [103].

Accordingly, it is highly plausible that Cd metabolism is more complex than just wholesale transport across the plasma membrane. Further, the intracellular concentration of Cd is likely modulated by other factors, including export proteins and intracellular binding proteins. It is plausible that Cd may be able to utilize one of the known ten Zn exporters, given its likeness to Zn, or be intracellularly bound by a chelating protein, like metallothionein. Based on this, we determined whether changes occur in the expression of ZIP8, ZIP14 and Nramp2 in monocytes and macrophages relative to Cd and/or LPS exposure. We contend that differences in the expression of ZIP8 and ZIP14 are most likely responsible for the differences in Cd accumulation observed between monocytes and macrophages. Remarkably, significantly more Cd accumulated in macrophages than monocytes following LPS exposure (figure 4.7.J). Western blotting was particularly revealing as we determined ZIP8 to be nearly exclusively expressed in monocytes whereas ZIP14 was unique to macrophages. More specifically,

ZIP8 expression due to either Cd and/or LPS exposure did not correlate with modest differences in Cd accumulation in monocytes. (4.7.G) In contrast, ZIP14 expression was abundant under all conditions studied in macrophages but in fact decreased in response to both LPS and Cd expression. One possible explanation to account for these discrepant findings is that we have as of yet only measured total cellular ZIP8 and 14 content but this may not be representative of differences at the cell surface. Accordingly, experiments are currently being

100 conducted to specifically analyze the cell surface fractional change in ZIP8 and

ZIP14 expression between monocytes and macrophages. Additionally, knockdown experiments using an siRNA approach are imperative to determine whether inhibition of transporters expression modulates Cd accumulation, particularly in macrophages. We believe that using both approaches will allow us to determine whether ZIP14 is the principal mediator of Cd accumulation and immune dysfunction in macrophages.

This investigation provides novel insight into a potential mechanism by which Cd could be a cause of immune dysfunction in COPD patients. We postulate that the capacity of macrophages to metabolize Cd is unique relative to its precursor monocyte cell type. Cd potently suppresses the NF-κB pathway in macrophages and by doing so may alter cellular composition toward an M2 phenotype, thereby diverting alveolar macrophages away from the classic M1 phenotype. Having said that, our observations at present have only focused on whether the M1 phenotype exists without fully determining to what extent Cd exposed macrophages transform into an M2 phenotype. Future studies will be designed to more fully characterize and compare features that distinguish the M2 through assessment of PPARγ function, a central signaling pathway that drives this phenotype, and through production of corresponding M2 cytokines and chemokines [118]. Further, while we contend that strength of our findings is that we have conducted a direct comparison of primary monocytes and macrophages relative to Cd exposure, our in vitro approach is limited in that it is difficult to

101 recapitulate the environment within the lung of a COPD patients with an extensive smoking history. Future studies may consider utilizing a long-term, low dose Cd exposure to more closely emulate the lung of smokers.

In conclusion, we have identified major differences in the processing of and response to Cd between monocyte and macrophages. Based on our findings, we contend that Cd renders human macrophages immuno-incompetent and ZIP14 may be a primary importer of Cd in macrophages leading to increased accumulation. Upon cellular entry, Cd is able to prevent macrophages from mounting a proper immune response to endotoxin by directly inhibiting the NF-κB pathway. We believe these findings may provide some insight into the mechanisms that underlie the inability of COPD patients to effectively clear pathogens in the lung leaving them susceptible to repeated upper respiratory tract infections.

4.4. Materials and Methods

Cell culture and maintenance

The human acute monocytic leukemic THP-1 cell line (catalog no. TIP-202,

American Type Culture Collection, Manassas VA) was maintained under standard culture conditions in RPMI supplemented with 10% FBS at 37°C in a

5% CO2-humidified incubator. All cells were used between passages 4 and 20.

Monocytic and Macrophage THP-1 Model

Our cell line model of human THP-1 monocytes were maintained in 2% FBS and

RPMI ± 10 μM CdCl2 over night (~16 hours). Serum concentration was reduced

102 to 2% in order to minimize absorptive loss of Cd while at the same time present to maintain cell viability during Cd exposure. Cells were then stimulated with 100 ng/ml LPS between 15 minutes and out to 12 hours. In order to establish a cell line model of macrophages, THP-1 monocytes were resuspended in 10% FBS and RPMI with 50 ng/ml of phorbol myristate acetate (PMA). After 24 hours, cells were evaluated for adhesion to the plate, and media was replaced with 10%

FBS and RPMI to facilitate differentiation over a period of 5 days. On the fifth day, media was replaced with 2% FBS and RPMI ± 10 μM CdCl2 overnight. Like the monocyte model, the following day cells were stimulated with 100 ng/ml LPS for up to 4 hours.

Primary human monocyte and macrophage culture

Blood was drawn from healthy, non-smoking, human donors with approval from the Ohio State University Institutional Review Board. Briefly, blood was collected into heparin-coated tubes and then diluted with PBS. This mixture was then layered onto Histopaque-1077 (Sigma-Aldrich, St. Louis MO) and centrifuged at room temperature for 20 minutes at 800g. The mononuclear fraction was collected and washed with RPMI. Cells were incubated with CD14+ magnetized beads and run through a magnetic column (Miltenyi Biotec, San Diego CA). After washing, the column was flushed to collect CD14+ cells. Following collection,

CD14+ monocytes, were separated into two groups. Half of the cells were resuspended and maintained in 2% FBS in RPMI ± CdCl2. Freshly isolated monocytes were immediately used in experiments. The other aliquot was

103 resuspended in 10% FBS in RPMI with 20 ng/nl macrophage colony stimulating factor (M-CSF) (Peprotech, Rocky Hill NJ) to initiate differentiation into macrophages. Two days after isolation, cell supernatant was collected and replaced with fresh media containing a second dose of M-CSF. On day 6 following isolation, cells were adherent and fully differentiated macrophages. At this time, identical to treatment of monocytes, media was replaced with 2% FBS and RPMI ± 2 µM CdCl2, and stimulated with 100 ng/ml LPS for up to 4 hours the following day. Utilizing this approach we were able to routinely evaluate inter- individual comparisons between monocyte and macrophage behavior under similar experimental conditions.

Cytotoxicity

Supernatant was collected and centrifuged, and lactate dehydrogenase activity was measured using the Cytotoxicity Detection Kit (Roche Diagnostics,

Mannheim Germany). Samples were compared with a positive control treatment group generated for each experiment by treatment with 2% Triton-X100 for 10 minutes to yield 100% death. Cell death was measured as percentage of positive control.

ELISA

Cells were stimulated with LPS for 4 hours or 24 hours, following overnight Cd incubation. Supernatant was collected and centrifuged to remove debris. ELISA kits were used according to manufacturer’s instructions (BioLegend, Inc. San

Diego CA) for IL-1β, TNFα, IL-6, IL-8 or IL-10. All samples were quantified

104 following comparison to a standard curve with known amounts of recombinant protein. mRNA Analysis

Total RNA was isolated from samples using Trizol reagent. The ThermoScript

RT-PCR kit (Invitrogen, Carlsbad CA) was used to generate cDNA. Primer pairs were designed for IL-1β, TNFα, IL-6, IL-8, IL-10, ZIP8, ZIP14, Nramp2, megalin, cubilin and GAPDH using Primer Express software version 2.0 (Applied

Biosystems, Foster City, CA) as previously reported [122]. The 7900HT Fast

Real-Time PCR system (Applied Biosystems) using SYBR Green Master Mix (2x,

Applied Biosystems) was used to perform real-time quantitative PCR. All samples were standardized to average cycle threshold number of the GAPDH gene. Messenger expression was reported as the average relative copy number

−ΔC as follows: 2 t ×100, where ΔCt is the Ct value standardized to GAPDH.

Western Blotting

Nuclear and cytosolic membranes were harvested using the NE-PER Nuclear

Protein Extraction Kit (Pierce, Rockford IL) according to manufacturer’s instructions. Briefly, cells were washed and harvested, and then subject to cytoplasmic and/or nuclear buffers in concert with sequential centrifugation steps.

Whole cell, nuclear and cytosolic proteins were quantified using a protein assay

(Bio-Rad, Hercules CA) and then mixed in Laemmli buffer (Bio-Rad) containing

5% (vol/vol) 2-mercaptoethanol, boiled for 5 minutes, separated on 10% SDS-

PAGE gel (Bio-Rad), and then transferred to a nitrocellulose membrane

105

(Amersham Biosciences, Little Chalfont, UK). Membranes were blocked with 5% milk (w/v) in PBS 0.1% Tween 20 (PBS-T) for 1 hour at room temperature and then incubated with primary antibody overnight at 4°C. After washing, the membranes were incubated with secondary antibody for 1 hour at room temperature. The signal was detected with an ECL Kit (Amersham Biosciences) and a Fluor-S Multi-Imager Max/Bio-quantity one (Bio-Rad). The following antibodies were used in our experiments: rabbit anti-IKKB, rabbit anti-pIKKβ, rabbit anti-IκBα, rabbit anti-p-IκBα, rabbit anti-p-p65, rabbit anti-p65 (all 1:1000,

Cell Signaling) rabbit anti-ZIP8 and rabbit anti-ZIP14 (1:2000, Covance,

Princeton, NJ), mouse anti-β-actin (1:2000, MP Biomedicals, Aurora, OH), goat anti-rabbit IgG-HRP (1:3000, Cell Signaling), and horse anti-mouse IgG-HRP

(1:3000, Cell Signaling).

NF-κB Activity Assay

Nuclear p65 activity was assessed using the TransAM Transcription Factor

ELISA Kit (Active Motif, Carlsbad CA). Nuclear protein was isolated from samples and analyzed for activity according to manufacturer’s instructions.

Briefly, nuclear lysate was incubated on plates coated with a consensus binding sequence specifically recognized by active p65. Standard ELISA was then used to measure differences in activity between different treatment groups using spectrometric methods. Activity was measured as fold change over baseline

(unstimulated samples).

106

IKKβ Activity Assay

IKKβ kinase activity was measured using the HTScan kinase assay kit (Cell

Signaling). Briefly, recombinant human IKKβ was incubated with a physiologic range of Cd concentrations, and then incubated with ATP and a substrate peptide. Kinase activity was measured using an antibody specific to phosphorylated IκBα and Dissociation-Enhanced Lanthanide Fluorescent

Immunoassay (DELFIA) measures were used to calculate kinase activity. The

IC50 value was calculated using non-linear regression curve fitting method.

Cd Quantification

Cells were thoroughly washed with PBS and a fraction was collected for protein quantification (Bio-Rad). The remaining cell fraction was digested in 1% nitric acid and Cd was quantified using atomic absorptive methods (Varian AA575,

Palo Alto CA) in comparison to known amounts of Cd used to generate a standard curve. Cd measurements were standardized to protein concentration.

Statistical Analyses

All data are expressed as mean ± SD. For comparisons involving multiple variables and observations, a two- and three-way ANOVA (GraphPad, La Jolla,

CA) were used. Having passed statistical significance by ANOVA, individual comparisons were made with the Bonferroni multiple-comparison test. Statistical significance was defined as a p value <0.05.

107

4.5 Figures

Figure 4.1: Toxicity profiles in Cd-treated monocytes and macrophages. A. THP-1 monocytes and B. PMA-derived macrophages were treated overnight with 10 µM CdCl2. The following morning they were stimulated for 4 hours with 100 ng/ml LPS. Supernatant was collected and analyzed for LDH release, an enzyme indicative of cell death. LDH release was compared to 2% Triton-X-lysed control cells. LPS increased cell death in both untreated and Cd- treated cells, and there was no significant difference due to Cd. Macrophages treated with Cd had significantly higher baseline levels of cell death but this did not change with LPS exposure (*** p <0.001, Two-way ANOVA, Bonferroni post-tests). Data shown is from one experiment conducted in triplicate, and representative of 3 independent experiments.

108

Figure 4.2: Cytokine release profiles in Cd-treated monocytes and macrophages. THP-1 monocytes and PMA-derived macrophages were treated overnight with 10 M CdCl2. The following morning they were stimulated for 4 or 12 hr with 100 ng/ml LPS. Supernatants were collected and analyzed by ELISA for the release of IL-1β (A, F), IL-6 (B, G), IL-8 (C, H), IL-10 (D, I) and TNFα (E, J). IL-1β release was significantly decreased in both Cd-treated monocytes and macrophages (*** p < 0.001) at 4 hours compared to untreated samples. IL-6 release was significantly increased in Cd-treated monocytes (** p < 0.01) at 12 hours but significantly decreased in macrophages (*** p < 0.001). IL-8 release was increased in Cd-treated monocytes also at 12 hours, though not significantly, and decreased in macrophages (** p <0.01). IL-10 release was significantly increased at both 4 (* p < 0.05) and 12 hours (** p <0.01) in monocytes, and was significantly decreased at both timepoints in macrophages (*** p <0.001). TNFα release was increased at 12 hours in monocytes (** p < 0.01), but significantly decreased at both 4 (*** p < 0.001) and 12 hours (** p < 0.01) in macrophages. With the exception of IL-1β, a distinct pattern emerged in which Cd tends to increase cytokine release in monocytes over time, and decrease release in macrophages. Figures are representative of 3 independent experiments, all conducted in triplicate. Significance was determined using Two-way ANOVA and Bonferroni post-tests.

109

Figure 4.2

110

Figure 4.3: Cytokine mRNA profiles of Cd-treated monocytes and macrophages. THP-1 monocytes and PMA-derived macrophages were treated overnight with 10 µM CdCl2. The following morning they were stimulated for 4 or 12 hr with 100 ng/ml LPS. Trizol was used to harvest mRNA, which was made into cDNA for RT-PCR analysis for the transcripts of IL-1β (A, F), IL-6 (B, G), IL-8 (C, H), IL-10 (D, I) and TNFα (E, J). IL-1β transcription was increased in Cd- treated monocytes at 4 hours post LPS exposure. At both 4 and 12 hours, its transcription was increased in non-Cd treated macrophages. IL-6 transcription was significantly increased in Cd- treated monocytes (* p < 0.05) at 4 hours but decreased in macrophages at 4 hours, and significantly so at 12 hours (** p < 0.01). IL-8 transcription was significantly decreased in Cd- treated monocytes at 12 hours (* p < 0.05). Cd-exposed macrophage IL-8 transcription was decreased significantly at both 4 (** p < 0.01) and 12 hours (*** p < 0.001). IL-10 transcription was significantly increased at 4 hours (** p < 0.05) in Cd-treated monocytes, though the reverse was observed in Cd-treated macrophages (*** p < 0.001). TNFα release did not vary between monocyte groups but was significantly decreased after 4 hours in Cd-treated macrophages (** p < 0.01). Similar to protein release, a distinct pattern emerged in which Cd increased cytokine transcription in monocytes, but inhibited their transcription in macrophages. Figures are representative of 3 independent experiments. Significance was determined using Two-way ANOVA and Bonferroni post-tests.

111

Figure 4.3

112

Figure 4.4: Differences in NF-κB Protein phosphorylation in Cd-treated monocytes and macrophages. Nuclear and cytosolic protein fractions of Cd-treated THP-1 monocytes and PMA-derived macrophages were harvested following up to 60 minutes of LPS exposure, and then subject to Western blotting. Nuclear phospho-p65 detection was unaltered by Cd following LPS stimulation in monocytes (A,B), though there was a modest decrease in p-p65 within the nuclei of Cd-treated LPS-exposed macrophages (G,H). Phosphorylated IkBα was also unaltered by Cd in monocytes (C,D). However, phosphorylation was decreased in Cd-treated macrophages following LPS exposure, and this was complemented by a relative abundance of total IkBα, which usually degrades upon phosphorylation (I,J). Assessment of IKKβ, a component of the IKK complex that is directly upstream of IkBα, revealed no differences in Cd-treated monocytes (E,F), but inhibition of phosphorylation was observed over time in macrophages (K,L), Phosphorylated and total protein blots are from the same membrane. Blots are representative of 3 independent experiments.

113

Figure 4.5: NF-κB Activity is decreased in Cd-treated macrophages. THP-1 monocytes and PMA-derived macrophages were treated overnight with ± 10 μM CdCl2 followed by 1 to 4 hours of 100 ng/ml LPS. Nuclear lysate was prepared and subject to an ELISA-based p65 activity assay. A. LPS induced activity in THP-1 monocytes but there were no significant differences in changes in p65 activity attributable to Cd exposure at any time point. B. However, Cd-treated macrophages exhibited a significant decrease in activation status at each time point (*** p < 0.001). The graphs are representative of 3 independent experiments, all conducted in duplicate. The same experiment was conducted in freshly isolated human monocytes and M-CSF derived macrophages. C. Similar to THP-1 monocytes, there were no differences in p65 activation between Cd treatment groups. D. However there was a significant decrease in p65 activity at 2 (** p <0.01) and 4 hours (*** p < 0.001) in Cd-treated primary macrophages. Data presented are of one donor, representative of 3 separate donors. Statistical analysis was conducted using a Two-way ANOVA and Bonferroni post-tests.

114

Figure 4.6: Cd inhibits IKKβ kinase activity. Using a cell-free system, we determined whether Cd inhibits IKKβ kinase activity. IKKβ is essential for NF-κB signal transmission and once activated, phosphorylates IκBα. Recombinant human IKKβ was incubated with increasing concentrations of Cd and the substrate peptide. The reaction mixture was then placed in strep- avidin coated 96-well plates and incubated with a phospho-IκBα mouse mAb antibody, followed by a Europium labeled secondary antibody and read with a fluorescent plate reader. From this, we were able to calculate the inhibition curve with an IC50 of ~130 nM. The sub-molar concentration is indicative of a strong inhibitory effect of Cd on IKKβ. Data was obtained from 1 experiment using replicate samples.

115

Figure 4.7: Cd accumulation and transporter profiling differences between monocytes and macrophages. qPCR was conducted on Cd-treated monocytes and macrophages for analysis of mRNA expression of ZIP8 (A, D), ZIP14 (B, E) and Nramp2 (C, F). The ZIP8 signal was increased in monocytes at 4 hours relative to macrophages. The ZIP14 signal was increased in macrophages compared to monocytes. This led us to then assess Cd-treated monocyte and macrophage protein for changes in ZIP8 and ZIP14 protein expression. Under the conditions studied, monocytes consistently had higher ZIP8 protein expression (140 kDa) than did macrophages, which had low ZIP8 expression (G). Conversely, macrophages had a baseline increase of ZIP14 (110 kDa) in comparison to monocytes (H), a signal that was decreased in the presence of both LPS and Cd. Western blots are representative of three independent experiments. Atomic absorption was employed to quantify intracellular Cd following overnight exposure, as well as an LPS stimulus. I. Quantification (nM) was standardized to sample protein. J. Macrophages had an average 127% increase in intracellular Cd following LPS exposure, compared to almost no change in monocytes (*** p< 0.001, Student’s t-test). Data representative of Cd changes measured from 4 independent experiments.

116

CHAPTER 5: DISCUSSION

We tested the hypothesis that ZIP8 facilitates epithelia toxicity and macrophage dysfunction observed in COPD through Cd import. Based on our findings, we conclude that ZIP8 causes Cd-mediated toxicity in lung epithelial cells, and ZIP8 may also play a vital role in Cd-mediated macrophage dysfunction, although the latter remains a work in progress. Our first key finding showed that Cd-mediated lung epithelia toxicity is dependent on the expression of ZIP8. Consistent with our in vitro findings, we observed significantly increased

ZIP8 expression in the lungs of smokers. The clinical relevance of this finding is complemented by the preferential expression of ZIP8 on the apical membrane.

In vivo studies using a murine model of chronic smoke exposure revealed that

ZIP8 expression correlated with a significant increase in pathology consistent with alveolar tissue loss and emphysema. The in vivo findings support the notion that ZIP8 may be a central mediator of COPD development by importing Cd thereby causing lung pathology. Studies involving monocytes and macrophages of the innate immune system provided surprising results by revealing major differences in the impact of Cd upon these two related cell types. Specifically, Cd caused impairment of NF-κB activity within macrophages but enhanced the pro- inflammatory activity of monocytes. This finding in part contradicts the majority of studies reporting Cd to be a pro-inflammatory agent, yet augments the growing 117 body of literature supporting a dysfunctional immune environment within the lung of a COPD subjects. As a whole, our work has provided significant insight into the role that ZIP8 and Cd may play to synergistically drive COPD pathogenesis in tobacco smokers.

COPD is a devastating disease with no known cure. Hallmarks of this disease include loss of alveolar tissue and excessive mucous production leading to repeat respiratory tract infections. The primary cause of COPD in the U.S. is due to inhalation of cigarette smoke. Cd is a leading environmental toxicant and major component of cigarette smoke. Cd is a known inducer of cell death through both necrosis and apoptosis [23, 123]. It induces necrosis through the disruption of mitochondrial, organelle and cell membranes and it induces apoptosis by increasing ROS stress and diminishing cellular antioxidant capacity

[26, 124-126]. Cd directly induces the activity of caspase-3, a central protein in the apoptotic signaling pathway [124]. Importantly, there are increases in apoptosis within the epithelial lining of COPD patients [127]. Utilizing in vitro models of human lung epithelia, we established that ZIP8 is necessary to induce

Cd toxicity. Our assessment of cell viability following Cd exposure revealed a mixed necrotic and apoptotic population. This is not surprising when considering the ability of Cd to induce both pathways. Additional highlights from our findings were that lung epithelia were rescued when ZIP8 expression was inhibited.

Moreover, intracellular Cd accumulation is dependent on ZIP8 as proven by demonstrating a significant reduction in intracellular Cd following ZIP8

118 knockdown with siRNA. Mechanistically, we revealed that ZIP8 expression is induced by NF-κB, a central signaling pathway that is upregulated in the lung of smokers [30]. Collectively, these findings provided new evidence to explain how

Cd may accumulate in the lung of smokers leading to cell destruction, alveolar loss and emphysema. Prior to our work, studies had not proven but only speculated as to how Cd enters lung parenchymal cells leading to accumulation and cell death [19]. We contend our work to be novel because it directly implicates a specific protein, ZIP8, to be responsible for Cd import and subsequent toxicity to lung epithelia. Our finding that ZIP8 expression is elevated within the lungs of cigarette smokers in comparison to non-smokers is consistent with other studies reporting elevated activity of NF-κB in lung tissue, and to our knowledge, is the only report demonstrating that elevated expression of a Zn transporter correlates with disease. Realizing that ZIP8 is preferentially expressed on the cell surface and knowing that lung epithelia are differentiated, polarized cells, we conducted additional studies in primary human cultures and revealed that upon induction, ZIP8 is preferentially expressed at the apical surface. This observation suggests that ZIP8 is ideally positioned to import Cd into lung epithelia following cigarette smoke inhalation, and further substantiates our central hypothesis that it is a mediator of disease in chronic cigarette smokers.

Cigarette smokers are typically of lower socioeconomic status, which is accompanied by poor nutritional habits and food insecurity [60, 92]. COPD

119 patients have lower blood Zn levels when compared to age-matched, healthy subjects [128]. Cigarette smokers typically also consume more alcohol, a compounding risk factor for malnutrition and Zn deficiency [19]. We observed in our lung epithelial model that cell toxicity is significantly impeded when there is a molar excess of Zn relative to Cd. Based on our findings, it is plausible then that insufficient dietary Zn intake may exacerbate Cd uptake and smoking related lung disease. If this is correct, Zn supplementation may have the potential to reduce the risk of developing disease. An important question that remains to be answered is the mechanism by which Zn is able to protect cells in our model. At present, we speculate that protective effects occurred subsequent to competitive uptake through ZIP8 or by intracellular Zn-mediated effects that initiated protective mechanisms to counter Cd injury. Accordingly, Zn is a well- established antioxidant that is capable of activating defense mechanisms within the cell, along with other factors (i.e. glutathione) [98, 99]. Regardless of the mechanism, if Zn is protecting cells by outcompeting Cd for uptake through ZIP8 and within the cell, it would identify an important attribute of Zn as a micronutrient in the prevention of Cd mediated toxicity. Taken together, we believe that our observations provide supporting evidence that dietary Zn intake is an important variable capable of altering disease susceptibility as well as potentially treating the extent of disease.

Our in vitro findings uncovered an important role for ZIP8 in mediating Cd- toxicity of lung epithelia. Based on this we translated these findings into an in

120 vivo murine model of chronic cigarette smoke exposure, where we expected to observe that BTZIP8-3 mice would accumulate more Cd from cigarette smoke.

Remarkably, we observed a significant increase in lung pathology consistent with emphysema in the lung tissue of smoke-exposed BTZIP8-3 mice compared to smoke exposed WT littermates. Overexpression of ZIP8 as a cause of tissue loss was supported by mRNA analysis on the same samples showing no significant differences in the expression of other confirmed Cd transporters that could potentially contribute to differences in Cd uptake. This finding was supported by additional evidence ZIP8 is a causative agent in COPD in humans because it is abundantly present throughout the lung during the time smoke exposure occurs.

We hypothesized ZIP8 contributes to alveolar septal loss through an increase in Cd accumulation and cell death, thereby making Cd directly responsible for tissue destruction. Surprisingly, quantification of Cd in lung tissue revealed similar quantities of Cd accumulation in WT and BTZIP8-3 mice, a finding divergent from our hypothesis. Despite this unexpected finding, we believe there are alternative potential explanations that may account for this discrepancy. First, it is possible that the insignificant differences in Cd quantities between these two strains is still sufficient to drive toxicity and tissue death in the

BTZIP8-3 mice. The lack of difference may also be due to limitation in the capacity of ICP-OES to accurately quantify subtle differences. In the future we could conduct a more sensitive assay utilizing Inductively Coupled Plasma Mass

121

Spectroscopy (ICP-MS). ICP-OES provides an assessment of total Cd content but may not be sensitive enough to reveal differences within intracellular compartments. Also, we restricted our pilot analysis to lung tissue as a target organ; however, following Cd uptake into the body it redistributes to different tissues, such as the kidney and blood compartments. As a result of our limited analysis, we may have missed the opportunity to observe differences within other organs. Second, ZIP8 may drive disease through endogenous proteolytic activity. ZIP8 possesses an amino acid consensus sequence active site that is homologous to the MMP family proteins [129]. Finally, in our pilot study we did not have sufficient animal numbers to obtain lung tissue from non-tobacco smoked exposed BTZIP8-3 mice to establish whether baseline overexpression of

ZIP8 may alter lung physiology. This is a critical control that must be included in future studies.

In addition to analyzing lung tissue loss and metal content, we characterized the gene expression of multiple well-established cytokines and chemokines that are associated with inflammation in the lung of COPD patients.

We hypothesized that the level of induced expression of these markers would be elevated in BTZIP8-3 mice when compared to WT counterparts. However, similar to Cd exposures in macrophages, we observed only subtle disparities in expression patterns between the two strains of mice. While at first confusing, our collaborator who is an expert in mouse models of smoke exposure, Dr. Michael

Borchers, confirmed that this finding was consistent with his expectations with

122 our smoke exposure model. Mice in response to prolonged smoke exposure rapidly acclimate to the toxicity over time [130]. Differences in inflammation are more robust and detectable in the first weeks of exposure, but then plateau as a result of resident lung cells adapting to the persistent inflammatory stimulus.

This is supported by additional studies which demonstrate that ex vivo stimulation of bronchial cells obtained from the lung of COPD patients are impaired in their capacity to release inflammatory cytokines [131]. Since our only analysis of gene expression occurred at four months following prolonged smoke exposure, it is quite likely we missed the window of opportunity to detect robust changes between treatment groups. Moving forward, we may consider conducting studies where lung tissue is analyzed at earlier and multiple time points during smoke exposure to determine whether Cd accumulation correlates with a downturn in cytokine response. Further, isolating bronchoalveolar lavage fluid from mice for protein detection of cytokines and chemokines will be necessary to determine the biologic consequence of ZIP8 overexpression relative to changes in gene expression profiles.

The findings obtained from animal studies and human lung tissue led us to evaluate blood Cd and Zn levels in a retrospective cohort of the NHANES, 2011-

2012 cycle. Epidemiologic studies using previous cycles reported that decreased

Zn intake in cigarette smokers correlated with an increase in urinary Cd, and when occurring together increased the individual risk for developing obstructive lung disease [106]. Our analysis of the 2011-2012 dataset revealed a greater

123 than two-fold increase in Cd present in the blood of smokers, compared to non- smokers, a finding highly consistent with previous studies [71]. However, we did not observe differences in serum Zn concentration between these two groups.

Because this analysis was not adjusted for relevant demographic factors that could contribute to disparities in serum Zn levels, we cannot yet confirm that our finding is a valid reflection of the status of smokers and nonsmokers. Further, it is difficult to interpret individual nutritional Zn status because it is redistributed from the blood to vital organs in the setting of inflammatory-based comorbid conditions [122]. Not knowing the comorbidities of NHANES subjects limits the extent to which we can confidently make comparisons.

A recent study found that inhaled tobacco-source Cd concentration in smoke correlates well with blood and urine Cd concentrations, which was measured immediately following cigarette smoke inhalation but there did not exist a strong correlation in serum samples [111]. Interestingly, a subset of subjects within this population consistently had higher amounts of Cd, as defined by levels above a threshold value of 1.5 ng/ml, within both the blood and urine. This lead the authors to speculate that genetic heterogeneity within genes that influence

Cd metabolism in the body may account for this difference. Our analysis of the

2011-2012 population revealed that approximately thirteen percent of smokers had blood Cd levels above the 1.5 ng/mL threshold. This percentage was similar to the observed 20% in the previous study further supporting that heterogeneity exists within the smoking population. Interestingly, it appears that heterogeneity

124 was unique to a Cd-specific pathway because similar differences were not observed when comparing blood lead levels. Based on this study and our findings from a much larger cohort, it is plausible that ZIP8 and related genes responsible for Cd metabolism are responsible for the differences in Cd levels observed within smokers. Based upon these collective findings we postulate that genetic polymorphisms within ZIP8 expression may lead to functional changes in

Cd metabolism that ultimately increase intracellular content within a select group of smokers. In doing so, this would increase their inherent risk of developing lung disease. Likewise, it is plausible that genetic heterogeneity may decrease the risk of Cd-mediated lung disease. In fact, this could occur more frequently across the general smoking population when considering that 80% of chronic smokers never develop COPD [9]. Importantly, the work described has established the groundwork for a clinical study that will evaluate the genotype of smokers with and without COPD relative to differences in ZIP8 expression and associated networks.

The damage present in the lungs of cigarette smokers, both in the context of emphysema and chronic bronchitis, is largely driven by the infiltration of macrophages. Significant increases in alveolar macrophage quantity has consistently been shown to correlate with increased disease severity [13, 117].

Using mouse models of chronic cigarette smoke exposure, disruption of macrophage recruitment to the lung has resulted in a marked decrease in alveolar tissue loss likely due to decreased presence of MMPs [110, 132].

125

Macrophages are also critical for the phagocytosis and clearance of respiratory pathogens, of which COPD patients are particularly susceptible to [39, 116]. The observation that increased macrophage infiltration occurs in conjunction with increased respiratory tract infections is counterintuitive and raises important questions regarding the functionality of this cell type. Based on our in vitro findings we contend that Cd, upon entry into macrophages, inhibits the NF-κB pathway rendering the cell incompetent to mount an adequate response against infection. Accordingly, repeated smoke exposure would increase the risk of repeated infections despite an increase in the overall number of alveolar macrophages.

An emerging body of work has revealed that chronic cigarette smoke exposure reprograms alveolar macrophages to be less responsive to invading infection and injury within the lung, thus perpetuating disease [34, 36, 118, 130,

131, 133]. Phenotyping of alveolar macrophages isolated from healthy patients, healthy smokers and COPD patients, revealed a decrease in M1 inflammatory markers (IL-1β, TNFα, IL-6, etc.) of the latter two groups [36]. This corresponded with a modest increase in M2 markers (CD163, IL-10, MMP-2, MMP-9, etc.).

The cytokines associated with the pro-inflammatory M1 phenotype are largely under the transcriptional regulation of NF-κB. This is supported by our results showing macrophage exposure to Cd results in an impaired response, as measured by cytokine release to endotoxin challenge. Taken together it is plausible to consider that Cd may promote a shift in macrophage phenotype

126 observed within the lungs of smokers. Indeed, previous studies revealed an inhibitory effect of Cd upon NF-κB but these findings have been contested by more recent work demonstrating that Cd can activate this pathway [22, 76]. Our findings obtained from monocytes are consistent with these findings, as we observed an increase in cytokine transcription and release despite exposure to

Cd. To our knowledge, direct comparison between monocytes and macrophages in relation to Cd has not previously been determined. Accordingly, our novel findings indicate that Cd has distinctly different effects on innate immune function between monocytes and macrophages. Whether these differences are due to altered Cd metabolism and uptake or changes in Cd-protein interactions within the cell remain to be determined.

While we believe the work presented in this dissertation provides important insight into mechanisms by which Cd may promote lung dysfunction and COPD, a major obstacle that requires further reconciliation is the observed difference in NF-κB activation between epithelial cells, monocytes and macrophages with regard to induction of ZIP8 expression. As previously published by our group, we were the first to reveal that activation of the NF-κB pathway via p65 induces ZIP8 expression that then subsequently increases Cd import [104]. Likewise, we clearly demonstrate that once inside of the cell, Cd is able to directly inhibit p65 nuclear translocation through direct interaction with

IKKβ. Our own evaluation of lung tissue obtained from smokers revealed elevated amounts of ZIP8 mRNA and protein which taken together, would

127 indicate that NF-κB is active despite the presence of Cd. We believe that there exist multiple reasons that may explain this discrepancy. First, the epithelial and mononuclear cell models that we designed had deliberately different exposures relative to Cd and activation with an NF-κB ligand. Second, the immunostaining conducted on human lung samples did not permit us to clearly delineate specific cell types that were ZIP8 positive. Therefore, it remains possible that ZIP8 is specifically expressed within epithelial cells and not alveolar macrophages. It is interesting to speculate that ZIP8 expression may be more robust in epithelial cells thereby facilitating more avid intracellular Cd accumulation leading to cell death. Released DAMPs and cytokines would go on to recruit dysfunctional macrophages unable to properly clear dead and dying cells, due to ZIP8- independent Cd accumulation and resulting inhibition of NF-κB. This hypothesis fits with conflicting literature in which both pro- and anti-inflammatory markers are observed in COPD patients, and emphasizes the idea that we must consider and compare the capacity of all relevant cell types to process Cd, perhaps with different roles in contribution to disease.

It is also likely that other signaling pathways are regulating ZIP8 expression in our model that we did not account for. For example, glutathione is capable of protecting cells from Cd by specifically reducing Sp-1 driven expression of ZIP8, a transcription factor likely active in our models [44].

Therefore, a limitation of our work is that we only examined one signaling impact that has a known regulatory role of ZIP8. As examples, Cd has also been shown

128 to induce Nrf2, ERK1/2, PI3K/Akt, and AP-1 pathways amongst others [46].

Notably, these signaling pathways have also been implicated in COPD pathogenesis. Future studies may examine inhibition and overexpression of other candidates to determine whether alternative pathways influence Cd import and subsequent toxicity. As one example, Cd stimulates the central anti-oxidant

Nrf2 pathway, expressed primarily in epithelia and alveolar macrophages, which is responsible for coordinating cellular responses to oxidant stress [43]. Its expression is decreased in the tissues of COPD patients and contributes to disease through excessive ROS production, a potent inducer of NF-κB [47, 48,

134]. It is plausible in our epithelial model that Cd is impairing Nrf2 signaling, further enhancing ZIP8 expression through ROS activation of NF-κB.

Our work provides what we believe to be important novel mechanistic insight into Cd-mediated toxicity; however, it raises important questions regarding the potential for Zn to prevent or reduce disease. Ideally, we envision developing a study that would prospectively monitor smokers with and without COPD over several decades, with respect to Zn nutritional status, and observe the incidence of COPD progression. Unfortunately, the amount of time, patient number, and resources is prohibitive to conducting a trial of this magnitude. Given these limitations we will continue to explore the in vivo effects of Cd, relative Zn and

COPD in validated animal models. In particular, a previous study reported that smoke-exposed mice maintained on Zn supplemented diets exhibited a significant decrease in macrophage infiltration in the lung [115]. Unfortunately,

129 their analysis did not pursue potential mechanisms to account for their findings.

Using a similar approach, we are currently examining smoke exposure in conjunction with Zn modified diets and intend to first examine differences in emphysema development through chord length measurements. Determining compartmental Zn and Cd content in different body stores, such as BAL, blood, kidneys, spleen and urine, would further help to clarify the effect of Zn, or lack thereof, on Cd accumulation and pathogenesis in vital organs. A similar approach will be taken to focus on the role of ZIP8 relative to Zn diet the BTZIP8-3 overexpressing mice. We would expect animals with more ZIP8 expression may be more responsive to Zn supplementation in prevention of Cd toxicity.

Our most recent discovery is that Cd inhibits the NF-κB mediated pro- inflammatory response in macrophages. Whether this drives alveolar macrophages toward a dysfunctional M2 phenotype remains a work in progress.

At present, we have restricted our assessment to primarily M1 phenotype markers with limited examination of M2 markers in response to Cd. In the future we will conduct rigorous studies that utilize ELISA, qPCR and immunohistochemistical techniques to more thoroughly phenotype these macrophages following Cd exposure. In addition to profiling markers, we will need to evaluate actual changes in cell functionality relative to the detrimental effects of Cd. For example, measurement of macrophage phagocytic capacity would further implicate the role of Cd in immune dysfunction and disease progression. Knowing that macrophages obtained from the lung of COPD

130 patients have decreased phagocytic capacity, we would expect to observe Cd causes similar dysfunction [135, 136].

To our knowledge, no studies have quantified Cd and Zn in the lung tissue of smokers and COPD patients. Metal quantification has focused on the more accessible blood and urine compartments. Elucidation of the compartmentalization of these metals in a disease state should be addressed in a clinical study, measuring Cd and Zn in the bronchoalveolar lavage (BAL) or lung biopsy tissue from patients undergoing diagnostic procedures. Because

BAL acts as a reservoir that houses multiple cell types, separation and quantification of these specific cell populations would be particularly useful. This experiment would fill a critical gap in our current understanding of Cd-induced lung toxicity by providing a more insightful look into how the accumulation or depletion of these metals, potentially within specific cell types, may correlate with disease severity.

This body of work advances the field through the identification of a novel mechanism by which Zn nutrition may modulate COPD susceptibility due to Cd abundance in cigarette smoke inhalation. Previous studies have only identified negative correlations between Zn intake and Cd burden whereas we postulate that ZIP8 is a previously unrecognized mediator of Cd-mediated lung toxicity and that imbalance between Zn relative Cd in favor of the latter, may further contribute to disease susceptibility. We believe this new area of investigation is important because COPD results in 3 million deaths a year worldwide,

131 accounting for 5% of the world’s total mortality [60]. Tobacco consumption is a public health crisis that further burdens already vulnerable populations that suffer from insufficient nutritional intake of micronutrients including Zn [92]. The translational impact of this work is exciting when considering that Zn supplementation is a highly plausible interventional strategy to prevent or slow the progression of COPD. We believe Zn supplementation would be effective in countering not only the toxicity of tobacco-source Cd, but other tobacco-source oxidant stresses that have been implicated in perpetuating disease. Based on our work, we contend ZIP8 is a probable mediator of COPD, and raises important concerns regarding the nutritional habits of tobacco users.

132

BIBLIOGRAPHY

1. Romain A. Pauwels, A.S.B., Peter M. A Calverley, Christine R. Jenkins, and Suzanne S. Hurd, on behalf of the GOLD Scientific Committee, Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease. Am J Respir Crit Care Med, 2001. 163: p. 1256-1276.

2. American Thoracic Society Committee on Diganostic Standards for Nontuberculosis Respiratory Diseases: Chronic Bronchitis, Asthma and Pulmonary Emphysema Arch Environ Health (Chic), 1962. 5: p. 375-382.

3. B.E. Heard, V.K., H. Otto, N.V. Putov, L. Sobin, The morphology of emphysema, chronic bronchitis, and bronchiectasis: definition, nomenclature, and classification. J Clin Pathol, 1979. 32: p. 882-892.

4. Snider GL, K.J., Thurlbeck WM, Bengali ZK, The definition of emphysema: report of a National Heart, Lung and Blood Institute, Division of Lung Diseases, Workshop. Am Rev Respir Dis, 1985. 132: p. 182-185.

5. J.G. Leopold, J.G., The centrilobular form of hypertrophic emphysema and its relation to chronic bronchitis. Thorax, 1957. 12: p. 219-235.

6. C.M. Fletcher, J.G.G., P. Hugh-Jones, J.G. Scadding, Terminology, definitions, and classification of chronic pulmonary emphysema and related conditions: a report of the conclusions of a CIBA guest symposium. Thorax, 1959. 14: p. 286-299.

7. P.C. Pratt, K.H.K., A modern concept of the emphysemas based on correlations of structure and function. Hum Pathol, 1970. 1(443-463).

8. Kueppfers F, B.W., Bearn AG, Hereditary Deficiency of Serum Alpha-L- Antitrypsin. Science, 1964. 146: p. 1678-9.

9. Fletcher C, P.R., Tinker C, Speizer FE, The natural history of chronic bronchitis and emphysema. 1976, Oxford: Oxford University Press.

10. Ahmedin Jemal, M.J.T., Lynn A. G. Ries, Holly L. Howe, Hannah K. Weir, Melissa M. Center, Elizabeth Ward, Xiao-Cheng Wu, Christie Eheman, 133

Robert Anderson, Umed A. Ajani, Betsy Kohler and Brenda K. Edwards, Annual Report to the Nation on the Status of Cancer, 1975–2005, Featuring Trends in Lung Cancer, Tobacco Use, and Tobacco Control JNCI J Natl Cancer Inst, 2008. 100(23): p. 1672-1694.

11. Bernard M Fischer, E.P., and Judith A Voynow, Pathogenic triad in COPD: oxidative stress, protease–antiprotease imbalance, and inflammation. Int J Chron Obstruct Pulmon Dis, 2011. 6: p. 413-421.

12. Baines, K.J., J.L. Simpson, and P.G. Gibson, Innate immune responses are increased in chronic obstructive pulmonary disease. PLoS One, 2011. 6(3): p. e18426.

13. Antonino Di Stefano, A.C., Mirco Lusardi, Piero Balbo, Cinzia Vecchio, Piero Maestrelli, Cristina A. Mapp, Leonardo M. Fabbri, Claudio F. Donner, and Marina Saetta, Severity of Airflow Limitation is Associated with Severity of Airway Inflammation in Smokers. Am J Respir Crit Care Med, 1998. 158: p. 1277-1285.

14. Eriksson, S. Discovery of alpha 1-antitrypsin deficiency. Lung, 1990. 168: p. 523-9.

15. Robert Vassallo, P.R.W., Jeffrey Lamont, Theodore J Kottom, Eunhee S Yi and Andrew H Limper, Cigarette smoke promotes dendritic cell accumulation in COPD; a Lung Tissue Research Consortium study. Respir Res, 2010. 11.

16. Hodge G, N.J., Holmes M, Reynolds PN, Hodge S., Increased intracellular T helper 1 proinflammatory cytokine production in peripheral blood, bronchoalveolar lavage and intraepithelial T cells of COPD subjects. Clin Exp Immunol, 2007. 150(1): p. 22-29.

17. Saetta M, D.S.A., Turato G, Facchini FM, Corbino L, Mapp CE, Maestrelli P, Ciaccia A, Fabbri LM., CD8+ T-lymphocytes in peripheral airways of smokers with chronic obstructive pulmonary disease. Am J Respir Crit Care Med, 1998. 157(3): p. 822-826.

18. Majori M, C.M., Caminati A, Cacciani G, Bertacco S, Pesci A., Predominant TH1 cytokine pattern in peripheral blood from subjects with chronic obstructive pulmonary disease. J Allergy Clin Immunol, 1999. 103(3): p. 458-462.

19. Services, U.S.D.o.H.a.H., How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease: A Report 134

of the Surgeon General. 2010, U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, National Center for Chronic Disease Prevention and Health Promotion, Office on Smoking and Health 2010.: Atlanta, GA.

20. Guerin MR, S.J., Higgins CE, Moneyhun JH, Holmberg RW., Inhalation bioassay chemistry--Walton Horizontal Smoking Machine for inhalation exposure of rodents to cigarette smoke. J Natl Cancer Inst, 1979. 63(2): p. 441-448.

21. J Fowles, E.D., Application of toxicological risk assessment principles to the chemical constituents of cigarette smoke. Tobacco Control, 2003. 12: p. 424-430.

22. Lag, M., et al., Cadmium-induced inflammatory responses in cells relevant for lung toxicity: Expression and release of cytokines in fibroblasts, epithelial cells and macrophages. Toxicol Lett, 2010. 193(3): p. 252-60.

23. Wang, B., et al., Enhanced cadmium-induced testicular necrosis and renal proximal tubule damage caused by gene-dose increase in a Slc39a8- transgenic mouse line. Am J Physiol Cell Physiol, 2007. 292(4): p. C1523- 35.

24. Kwon, K.Y., et al., Expression of apoptotic nuclei by ultrastructural terminal deoxyribonucleotidyl transferase mediated dUTP nick end labeling and detection of FasL, caspases and PARP protein molecules in cadmium induced acute alveolar cell injury. Toxicology, 2006. 218(2-3): p. 197-204.

25. Xie, J. and Z.A. Shaikh, Cadmium-induced apoptosis in rat kidney epithelial cells involves decrease in nuclear factor-kappa B activity. Toxicol Sci, 2006. 91(1): p. 299-308.

26. Lopez, E., et al., Cadmium induces reactive oxygen species generation and lipid peroxidation in cortical neurons in culture. Free Radic Biol Med, 2006. 40(6): p. 940-51.

27. Mortaz, E., et al., ATP and the pathogenesis of COPD. Eur J Pharmacol, 2010. 638(1-3): p. 1-4.

28. Mercer BA, D.A.J., Emerging role of MAP kinase pathways as therapeutic targets in COPD. Int J Chron Obstruct Pulmon Dis, 2006. 1(2): p. 137-150.

135

29. Szulakowski, P., et al., The effect of smoking on the transcriptional regulation of lung inflammation in patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med, 2006. 174(1): p. 41-50.

30. Yagi, O., K. Aoshiba, and A. Nagai, Activation of nuclear factor-kappaB in airway epithelial cells in patients with chronic obstructive pulmonary disease. Respiration, 2006. 73(5): p. 610-6.

31. Hayden, M.S. and S. Ghosh, Shared principles in NF-kappaB signaling. Cell, 2008. 132(3): p. 344-62.

32. Chung, K.F., Cytokines in chronic obstructive pulmonary disease. European Respiratory Journal, 2001. 18(1): p. 50-59.

33. Garcia-Rio, F., et al., Systemic inflammation in chronic obstructive pulmonary disease: a population-based study. Respir Res, 2010. 11: p. 63.

34. Hodge, S., et al., Cigarette smoke-induced changes to alveolar macrophage phenotype and function are improved by treatment with procysteine. Am J Respir Cell Mol Biol, 2011. 44(5): p. 673-81.

35. Kunz, L.I., et al., Smoking status and anti-inflammatory macrophages in bronchoalveolar lavage and induced sputum in COPD. Respir Res, 2011. 12: p. 34.

36. Shaykhiev, R., et al., Smoking-dependent reprogramming of alveolar macrophage polarization: implication for pathogenesis of chronic obstructive pulmonary disease. J Immunol, 2009. 183(4): p. 2867-83.

37. Sarir, H., et al., Cigarette smoke regulates the expression of TLR4 and IL- 8 production by human macrophages. J Inflamm (Lond), 2009. 6: p. 12.

38. Tanni, S.E., et al., Smoking status and tumor necrosis factor-alpha mediated systemic inflammation in COPD patients. J Inflamm (Lond), 2010. 7: p. 29.

39. Marian Ryan, J.A.S., John D. Chapman, William B. Stason, Donald S. Shepard, Cindy Parks Thomas Incidence and Cost of Pneumonia in Older Adults with COPD in the United States PLoS One, 2013.

40. Proud D, C.C., Role of viral infections in asthma and chronic obstructive pulmonary disease. Am J Respir Cell Mol Biol, 2006(35): p. 513-518.

136

41. Benfield T, L.P., Vestbo J., COPD stage and risk of hospitalization for infectious disease. Chest, 2008. 134(1): p. 46-53.

42. Hartwig, A., Mechanisms in cadmium-induced carcinogenicity: recent insights. Biometals, 2010. 23(5): p. 951-60.

43. Liu, J., W. Qu, and M.B. Kadiiska, Role of oxidative stress in cadmium toxicity and carcinogenesis. Toxicol Appl Pharmacol, 2009. 238(3): p. 209- 14.

44. Aiba, I., A. Hossain, and M.T. Kuo, Elevated GSH level increases cadmium resistance through down-regulation of Sp1-dependent expression of the cadmium transporter ZIP8. Mol Pharmacol, 2008. 74(3): p. 823-33.

45. Bertin, G. and D. Averbeck, Cadmium: cellular effects, modifications of biomolecules, modulation of DNA repair and genotoxic consequences (a review). Biochimie, 2006. 88(11): p. 1549-1559.

46. Thevenod, F., Cadmium and cellular signaling cascades: to be or not to be? Toxicol Appl Pharmacol, 2009. 238(3): p. 221-39.

47. Boutten, A., et al., NRF2 targeting: a promising therapeutic strategy in chronic obstructive pulmonary disease. Trends Mol Med, 2011. 17(7): p. 363-71.

48. Malhotra, D., et al., Decline in NRF2-regulated antioxidants in chronic obstructive pulmonary disease lungs due to loss of its positive regulator, DJ-1. Am J Respir Crit Care Med, 2008. 178(6): p. 592-604.

49. Martha Montaño, J.C., Alejandra Ramírez-Venegas, José Pedraza- Chaverri, Daniel Mercado, Carlos Ramos, Raul H. Sansores, Malondialdehyde and superoxide dismutase correlate with FEV1 in patients with COPD associated with wood smoke exposure and tobacco smoking. Inhalation Toxicology, 2010. 22(10): p. 868-874.

50. Young, R.P., et al., Functional variants of antioxidant genes in smokers with COPD and in those with normal lung function. Thorax, 2006. 61(5): p. 394-399.

51. Mak, J.C.W., et al., Polymorphisms and functional activity in superoxide dismutase and catalase genes in smokers with COPD. European Respiratory Journal, 2007. 30(4): p. 684-690.

137

52. Dahl M, B.R., Juul K, et al., Superoxide dismutase 3 polymorphism associated with reduced lung function in two large populations. Am J Respir Crit Care Med, 2008. 178(906-912).

53. Juul K, T.-H.A., Marklund S, Lange P, Nordestgaard BG, Genetically increased antioxidative protection and decreased chronic obstructive pulmonary disease. Am J Respir Crit Care Med, 2006. 173: p. 858-863.

54. PF Mercer, J.S., A Bhowmik, GC Donaldson, JA Wedzicha, JA Warner, MMP-9, TIMP-1 and inflammatory cells in sputum from COPD patients during exacerbation. Respir Res, 2005. 6(1): p. 151.

55. Molet S1, B.C., Lena H, Germain N, Bertrand CP, Shapiro SD, Planquois JM, Delaval P, Lagente V., Increase in macrophage elastase (MMP-12) in lungs from patients with chronic obstructive pulmonary disease. Inflamm Res, 2005. 54(1): p. 31-36.

56. Haq, I., et al., Association of MMP - 12 polymorphisms with severe and very severe COPD: A case control study of MMPs - 1, 9 and 12 in a European population. BMC Medical Genetics, 2010. 11(1): p. 7.

57. Ito I, N.S., Handa T, et al., Matrix metalloproteinase-9 promoter polymorphism associated with upper lung dominant emphysema. Am J Respir Crit Care Med, 2005. 172(1378-1382).

58. Hunninghake GM, C.M., Tesfaigzi Y, et al., MMP12, lung function, and COPD in high-risk populations. N Engl J Med, 2009. 361: p. 2599-2608.

59. Kirschvink N1, V.G., Fiévez L, Onclinx C, Wirth D, Belleflamme M, Louis R, Cataldo D, Peck MJ, Gustin P., Repeated cadmium nebulizations induce pulmonary MMP-2 and MMP-9 production and emphysema in rats. Toxicology, 2005. 211: p. 36-48.

60. Israel T. Agaku, B.A.K., Shanta R. Dube, , Current Cigarette Smoking Among Adults — United States, 2005–2012. Morbidity and Mortality Weekly Report, 2014. 63(2): p. 29-34.

61. Services, U.S.D.o.H.a.H., The Health Consequences of Smoking—50 Years of Progress: A Report of the Surgeon General. 2014, Department of Health and Human Services Centers for Disease Control and Prevention National Center for Chronic Disease Prevention and Health Promotion Office on Smoking and Health: Atlanta.

138

62. Prevention, C.f.D.C.a., QuickStats: Number of Deaths from 10 Leading Causes—National Vital Statistics System, United States, 2010. Morbidity and Mortality Weekly Report, 2013. 62(8): p. 155.

63. Sherry L. Murphy, J.X., Kenneth D. Kochanek, Division of Vital Statistics, Deaths: Final Data for 2010. National Vital Statistics Reports, 2013. 61(4).

64. Services, U.S.D.o.H.a.H., Healthy People 2020 Leading Health Indicators: Progress Update. 2014.

65. Marie Ng, M.K.F., Thomas D. Fleming, Margaret Robinson, Laura Dwyer- Lindgren, Blake Thomson, Alexandra Wollum, Ella Sanman, Sarah Wulf, Alan D. Lopez, Christopher J. L. Murray, Emmanuela Gakidou, Smoking Prevalence and Cigarette Consumption in 187 Countries, 1980-2012. JAMA, 2014. 311(2): p. 183-192.

66. Organization, W.H., WHO report on the global tobacco epidemic, 2011: warning about the dangers of tobacco 2011, WHO.

67. Davis, RD., Cadmium in sludges used as fertilizer. Experientia Suppl, 1986. 50: p. 55-60.

68. Yue, L., Cadmium in tobacco. Biomed Environ Sci, 1992. 5(1): p. 53-56.

69. Ellis, K., et al., Cadmium: in vivo measurement in smokers and nonsmokers. Science, 1979. 205(4403): p. 323-325.

70. Mannino, D.M., Urinary cadmium levels predict lower lung function in current and former smokers: data from the Third National Health and Nutrition Examination Survey. Thorax, 2004. 59(3): p. 194-198.

71. Soisungwon Satarug, J.R.B., Supanee Urbenjapol, Melissa HAswell- Elkins, Paul E.B. Reilly, David J. Williams, Michael R. Moore, A global perspective on cadmium pollution and toxicity in non-occupationally exposed population. Toxicology Letters, 2003. 137: p. 65-83.

72. Suwazono Y, K.T., Nakagawa H, Nishijo M, Honda R, Kobayashi E, Dochi M, Nogawa K., Biological half-life of cadmium in the urine of inhabitants after cessation of cadmium exposure. Biomarkers, 2009. 14(2): p. 77-81.

73. Elinder CG, L.B., Kjellström T, Linnman L, Friberg L., Cadmium in kidney cortex, liver, and pancreas from Swedish autopsies. Estimation of biological half time in kidney cortex, considering calorie intake and smoking habits. Arch Environ Health, 1976. 31(6): p. 292-302. 139

74. Waalkes, M., Cadmium carcinogenesis. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 2003. 533(1-2): p. 107-120.

75. Grasseschi, R.M., Cadmium Accumulation and Detoxification by Alveolar Macrophages of Cigarette Smokers. Chest, 2003. 124(5): p. 1924-1928.

76. Thevenod, F., Catch me if you can! Novel aspects of cadmium transport in mammalian cells. Biometals, 2010. 23(5): p. 857-75.

77. Klassen RB, C.K., Kozyraki R, Verroust PJ, Tio L, Atrian S, Allen PL, Hammond TG., Megalin mediates renal uptake of heavy metal metallothionein complexes. Am J Physiol Renal Physiol, 2004. 287(3): p. 393-403.

78. Wolff NA, A.M., Verroust PJ, Thévenod F., Megalin-dependent internalization of cadmium-metallothionein and cytotoxicity in cultured renal proximal tubule cells. J Pharmacol Exp Ther, 2006. 318(2): p. 782- 791.

79. Bannon DI, A.R., Lees PS, Bressler JP., Effect of DMT1 knockdown on iron, cadmium, and lead uptake in Caco-2 cells. Am J Physiol Cell Physiol, 2003. 284(1): p. 44-50.

80. Ghio, A.J., et al., Divalent metal transporter-1 decreases metal-related injury in the lung. Am J Physiol Lung Cell Mol Physiol, 2005. 289(3): p. L460-7.

81. Girijashanker, K., et al., Slc39a14 gene encodes ZIP14, a metal/bicarbonate symporter: similarities to the ZIP8 transporter. Mol Pharmacol, 2008. 73(5): p. 1413-23.

82. Dalton, T.P., et al., Identification of mouse SLC39A8 as the transporter responsible for cadmium-induced toxicity in the testis. Proc Natl Acad Sci U S A, 2005. 102(9): p. 3401-6.

83. Vallee, B.L. and D.S. Auld, Zinc coordination, function, and structure of zinc enzymes and other proteins. Biochemistry, 1990. 29(24): p. 5647- 5659.

84. Nieto, M.A., The snail superfamily of zinc-finger transcription factors. Nat Rev Mol Cell Biol, 2002. 3(3): p. 155-166.

85. Ho, E., Zinc deficiency, DNA damage and cancer risk. J Nutr Biochem, 2004. 15(10): p. 572-8. 140

86. Buchauer, M.J., Contamination of soil and vegetation near a zinc smelter by zinc, cadmium, copper, and lead. Environmental Science & Technology, 1973. 7(2): p. 131-135.

87. Institute of Medicine, F.a.N.B., Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium and Zinc. 2001, National Academy Press: Washington DC.

88. Prasad AS, M.A.J., Farid Z, Sandstead HH, Schulert AR, Darby WJ., Biochemical studies on dwarfism, hypogonadism, and anemia. Arch Intern Med., 1963. 111: p. 407-428.

89. Prasad AS, S.A., Miale A Jr, Farid Z, Sandstead HH., Zinc and iron deficiencies in male subjects with dwarfism and hypogonadism but without ancylostomiasis, schistosomiasis or severe anemia. Am J Clin Nutr, 1963. 12: p. 437-444.

90. Halstead JA, P.A., Zinc Deficiency in Man. Isr Med J, 1963. 22: p. 307- 315.

91. Ervin RB, K.-S.J., Mineral intakes of elderly adult supplement and non- supplement users in the third national health and nutrition examination survey. J Nutr, 2002. 132(11): p. 3422-3427.

92. Dixon LB, W.M., Radimer KL., Dietary intakes and serum nutrients differ between adults from food-insufficient and food-sufficient families: Third National Health and Nutrition Examination Survey, 1988-1994. J Nutr, 2001. 131(4): p. 1232-1246.

93. Pattison SE, C.R., Zinc uptake and metabolism by hepatocytes. Fed Proc, 1986. 45(12): p. 2805-2809.

94. Kambe, T., et al., Overview of mammalian zinc transporters. Cell Mol Life Sci, 2004. 61(1): p. 49-68.

95. Liuzzi, J.P. and R.J. Cousins, Mammalian zinc transporters. Annu Rev Nutr, 2004. 24: p. 151-72.

96. Eide, D.J., The SLC39 family of metal ion transporters. Pflugers Arch, 2004. 447(5): p. 796-800.

97. Begum, N.A., et al., Mycobacterium bovis BCG Cell Wall and Lipopolysaccharide Induce a Novel Gene, BIGM103, Encoding a 7-TM 141

Protein: Identification of a New Protein Family Having Zn-Transporter and Zn-Metalloprotease Signatures. Genomics, 2002. 80(6): p. 630-645.

98. Bao, S. and D.L. Knoell, Zinc modulates cytokine-induced lung epithelial cell barrier permeability. Am J Physiol Lung Cell Mol Physiol, 2006. 291(6): p. L1132-41.

99. Bao, S. and D.L. Knoell, Zinc modulates airway epithelium susceptibility to death receptor-mediated apoptosis. Am J Physiol Lung Cell Mol Physiol, 2006. 290(3): p. L433-41.

100. Besecker B, B.S., Bohacova B, Papp A, Sadee W, Knoell DL., The human zinc transporter SLC39A8 (Zip8) is critical in zinc-mediated cytoprotection in lung epithelia. Am J Physiol Lung Cell Mol Physiol, 2008. 294(6): p. 1127-1136.

101. Bao, S., et al., Zinc modulates the innate immune response in vivo to polymicrobial sepsis through regulation of NF-kappaB. Am J Physiol Lung Cell Mol Physiol, 2010. 298(6): p. L744-54.

102. Knoell, D.L., et al., Zinc deficiency increases organ damage and mortality in a murine model of polymicrobial sepsis. Crit Care Med, 2009. 37(4): p. 1380-8.

103. Aydemir, T.B., et al., Zinc transporter ZIP8 (SLC39A8) and zinc influence IFN-gamma expression in activated human T cells. J Leukoc Biol, 2009. 86(2): p. 337-48.

104. Liu MJ, B.S., Gálvez-Peralta M, Pyle CJ, Rudawsky AC, Pavlovicz RE, Killilea DW, Li C, Nebert DW, Wewers MD, Knoell DL., ZIP8 regulates host defense through zinc-mediated inhibition of NF-κB. Cell Rep, 2013. 3(2): p. 386-400.

105. Liu, Z., et al., Cd2+ versus Zn2+ uptake by the ZIP8 HCO3--dependent symporter: kinetics, electrogenicity and trafficking. Biochem Biophys Res Commun, 2008. 365(4): p. 814-20.

106. Lin, Y.S., et al., Cigarette smoking, cadmium exposure, and zinc intake on obstructive lung disorder. Respir Res, 2010. 11: p. 53.

107. Churg, A., et al., Tumor necrosis factor-alpha is central to acute cigarette smoke-induced inflammation and connective tissue breakdown. Am J Respir Crit Care Med, 2002. 166(6): p. 849-54.

142

108. He, L., et al., ZIP8, member of the solute-carrier-39 (SLC39) metal- transporter family: characterization of transporter properties. Mol Pharmacol, 2006. 70(1): p. 171-80.

109. Nagai, K., et al., Long term smoking with age builds up excessive oxidative stress in bronchoalveolar lavage fluid. Thorax, 2006. 61(6): p. 496-502.

110. Vlahos, R., et al., Differential protease, innate immunity, and NF-kappaB induction profiles during lung inflammation induced by subchronic cigarette smoke exposure in mice. Am J Physiol Lung Cell Mol Physiol, 2006. 290(5): p. L931-45.

111. Ebert-McNeill, A., et al., Cadmium intake and systemic exposure in postmenopausal women and age-matched men who smoke cigarettes. Toxicol Sci, 2012. 130(1): p. 191-204.

112. Raguso, C.A. and C. Luthy, Nutritional status in chronic obstructive pulmonary disease: role of hypoxia. Nutrition, 2011. 27(2): p. 138-43.

113. Tsiligianni, I.G. and T. van der Molen, A systematic review of the role of vitamin insufficiencies and supplementation in COPD. Respir Res, 2010. 11: p. 171.

114. Motz, G.T., et al., Chronic cigarette smoke exposure generates pathogenic T cells capable of driving COPD-like disease in Rag2-/- mice. Am J Respir Crit Care Med, 2010. 181(11): p. 1223-33.

115. Lang, C.J., et al., Dietary zinc mediates inflammation and protects against wasting and metabolic derangement caused by sustained cigarette smoke exposure in mice. Biometals, 2011. 24(1): p. 23-39.

116. Sethi, S., Bacterial Infection and the Pathogenesis of COPD. CHEST Journal, 2000. 117(5_suppl_1): p. 286S.

117. Tetley, T.D., Macrophages and the Pathogenesis of COPD. CHEST Journal, 2002. 121(5_suppl): p. 156S.

118. Byers, D.E. and M.J. Holtzman, Alternatively activated macrophages and airway disease. Chest, 2011. 140(3): p. 768-74.

119. Shumilla JA, W.K., Barchowsky A., Inhibition of NF-kappa B binding to DNA by chromium, cadmium, mercury, zinc, and arsenite in vitro:

143

evidence of a thiol mechanism. Arch Biochem Biophys, 1998. 349(2): p. 356-362.

120. Soler-Cataluña JJ, M.-G.M., Román Sánchez P, Salcedo E, Navarro M, Ochando R., Severe acute exacerbations and mortality in patients with chronic obstructive pulmonary disease. Thorax, 2005. 60(11): p. 925-931.

121. Jomova, K. and M. Valko, Advances in metal-induced oxidative stress and human disease. Toxicology, 2011. 283(2-3): p. 65-87.

122. Besecker BY, E.M., Hollyfield J, Phillips G, Disilvestro RA, Wewers MD, Knoell DL., A comparison of zinc metabolism, inflammation, and disease severity in critically ill infected and noninfected adults early after intensive care unit admission. Am J Clin Nutr, 2011. 93(6): p. 1356-64.

123. Gunawardana, C.G., et al., Cadmium inhibits both intrinsic and extrinsic apoptotic pathways in renal mesangial cells. Am J Physiol Renal Physiol, 2006. 290(5): p. F1074-82.

124. Coutant, A., et al., Cadmium-induced apoptosis in lymphoblastoid cell line: involvement of caspase-dependent and -independent pathways. Biochimie, 2006. 88(11): p. 1815-22.

125. Ramage, L., A.C. Jones, and C.J. Whelan, Induction of apoptosis with tobacco smoke and related products in A549 lung epithelial cells in vitro. J Inflamm (Lond), 2006. 3: p. 3.

126. Cuypers, A., et al., Cadmium stress: an oxidative challenge. Biometals, 2010. 23(5): p. 927-40.

127. Hodge, S., et al., Increased airway epithelial and T-cell apoptosis in COPD remains despite smoking cessation. European Respiratory Journal, 2005. 25(3): p. 447-454.

128. Afridi, H.I., et al., Evaluation of cadmium, lead, nickel and zinc status in biological samples of smokers and nonsmokers hypertensive patients. J Hum Hypertens, 2010. 24(1): p. 34-43.

129. Taylor, K.M. and R.I. Nicholson, The LZT proteins; the LIV-1 subfamily of zinc transporters. Biochimica et Biophysica Acta (BBA) - Biomembranes, 2003. 1611(1–2): p. 16-30.

144

130. Gaschler, G.J., et al., Cigarette smoke exposure attenuates cytokine production by mouse alveolar macrophages. Am J Respir Cell Mol Biol, 2008. 38(2): p. 218-26.

131. Smith, L.A., et al., Inflammatory response of lung macrophages and epithelial cells to tobacco smoke: a literature review of ex vivo investigations. Immunol Res, 2010. 46(1-3): p. 94-126.

132. Churg, A., et al., Tumor necrosis factor-alpha drives 70% of cigarette smoke-induced emphysema in the mouse. Am J Respir Crit Care Med, 2004. 170(5): p. 492-8.

133. Woodruff, P.G., et al., A distinctive alveolar macrophage activation state induced by cigarette smoking. Am J Respir Crit Care Med, 2005. 172(11): p. 1383-92.

134. Sussan, T.E., et al., Targeting Nrf2 with the triterpenoid CDDO-imidazolide attenuates cigarette smoke-induced emphysema and cardiac dysfunction in mice. Proc Natl Acad Sci U S A, 2009. 106(1): p. 250-5.

135. Kulkarni, R., et al., Cigarette smoke inhibits airway epithelial cell innate immune responses to bacteria. Infect Immun, 2010. 78(5): p. 2146-52.

136. Charles Berenson, M.A.G., Lori Grove, Jane Maloney, and Sanjay Sethi, Impaired phagocytosis of nontypeable Haemophilus influenzae by human alveolar macrophages in obstructive pulmonary disease. The Journal of Infectious Diseases, 2006. 194: p. 1375-84.

145