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Title:
Modulation of monocyte-derived dendritic cell maturation and function by cigarette smoke condensate in a bronchial epithelial cell co-culture model
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Book
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English
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Montpetit, Alison J
Publisher:
University of South Florida
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Tampa, Fla
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Subjects / Keywords:
Immune
Cytokine
Lung
Tobacco
Pulmonary
Dissertations, Academic -- Nursing -- Doctoral -- USF   ( lcsh )
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non-fiction   ( marcgt )

Notes

Summary:
ABSTRACT: Lung airway epithelium is the first line of defense against inhaled particulates such as cigarette smoke. Subepithelial dendritic cells (DC) survey the airway epithelial lining and represent the link between innate and adaptive immune response. No study has investigated the effect of cigarette smoke on DC in the presence of epithelial cells (EC). The purpose of this 4x2 factorial design study was to co-culture normal human bronchial epithelial (NHBE) cells with monocyte-derived dendritic cells (MDDC) and examine the effect of cigarette smoke condensate (CSC) and poly I:C stimulation (TLR3 ligand that mimics viral infection) on MDDC homotypic clustering, phagocytosis ability, surface marker expression, DC cytokine response, T cell (TC) proliferation and TC cytokine response. Experiments were performed with MDDC and TC derived from four individual donors.Two planned comparisons (DMSO vehicle control compared to CSC high dose in both the no poly I:C and poly I:C stimulated groups) were analyzed. In MDDC stimulated with CSC, there was a significant increase in homotypic clustering, reduced phagocytosis, increased CD54, increased CD83 and CD86 maturation marker expression. Although no significant changes were observed in MDDC cytokine production, IL10 exhibited a trend to increase with CSC exposure but failed to reach statistical significance. Despite evidence that CSC exposed MDDC are maturing, there was no increase in TC proliferation; however in poly I:C stimulated co-cultures, CSC exposure increased IL2, IL5, IL10 and IL13 expression while non-stimulated co-cultures exhibited an increase in IL10 following CSC exposure.This study indicates that cigarette smoke has the ability to decrease phagocytosis ability of DC and increases co-stimulatory maturation markers without inducing TC proliferation and potentiating a Th2 environment. These findings suggest that DC of smokers may be less likely to mount a normal immune response to invading pathogens thus providing evidence for smoker susceptibility to infection and how DC of smokers may respond to viral infection. In addition, these findings are particularly important in patients with allergic airway disease since increasing Th2 cytokines would increase the risk for disease exacerbation.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2008.
Bibliography:
Includes bibliographical references.
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Mode of access: World Wide Web.
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by Alison J. Montpetit.
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Title from PDF of title page.
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Document formatted into pages; contains 81 pages.
General Note:
Includes vita.

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aleph - 002000373
oclc - 318895548
usfldc doi - E14-SFE0002478
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ABSTRACT: Lung airway epithelium is the first line of defense against inhaled particulates such as cigarette smoke. Subepithelial dendritic cells (DC) survey the airway epithelial lining and represent the link between innate and adaptive immune response. No study has investigated the effect of cigarette smoke on DC in the presence of epithelial cells (EC). The purpose of this 4x2 factorial design study was to co-culture normal human bronchial epithelial (NHBE) cells with monocyte-derived dendritic cells (MDDC) and examine the effect of cigarette smoke condensate (CSC) and poly I:C stimulation (TLR3 ligand that mimics viral infection) on MDDC homotypic clustering, phagocytosis ability, surface marker expression, DC cytokine response, T cell (TC) proliferation and TC cytokine response. Experiments were performed with MDDC and TC derived from four individual donors.Two planned comparisons (DMSO vehicle control compared to CSC high dose in both the no poly I:C and poly I:C stimulated groups) were analyzed. In MDDC stimulated with CSC, there was a significant increase in homotypic clustering, reduced phagocytosis, increased CD54, increased CD83 and CD86 maturation marker expression. Although no significant changes were observed in MDDC cytokine production, IL10 exhibited a trend to increase with CSC exposure but failed to reach statistical significance. Despite evidence that CSC exposed MDDC are maturing, there was no increase in TC proliferation; however in poly I:C stimulated co-cultures, CSC exposure increased IL2, IL5, IL10 and IL13 expression while non-stimulated co-cultures exhibited an increase in IL10 following CSC exposure.This study indicates that cigarette smoke has the ability to decrease phagocytosis ability of DC and increases co-stimulatory maturation markers without inducing TC proliferation and potentiating a Th2 environment. These findings suggest that DC of smokers may be less likely to mount a normal immune response to invading pathogens thus providing evidence for smoker susceptibility to infection and how DC of smokers may respond to viral infection. In addition, these findings are particularly important in patients with allergic airway disease since increasing Th2 cytokines would increase the risk for disease exacerbation.
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Modulation of Monocyte-Derived Dendrit ic Cell Maturation and Function by Cigarette Smoke Condensate in a Bronchi al Epithelial Cell Co-Culture Model by Alison J. Montpetit A dissertation submitted in partial fulfillment of the requirement s of the degree of Doctor of Philosophy College of Nursing University of South Florida Major Professor: Mary E. Evans, Ph.D. Co-Major Professor: Sh yam S. Mohapatra, Ph.D. Maureen Groer, Ph.D. Jason Beckstead, Ph.D. Stuart Brooks, Ph.D. Date of Approval: June 27, 2008 Keywords: immune, cytokine, lung, tobacco, pulmonary Copyright 2008, Alison J. Montpetit

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Dedication This dissertation is dedicated to my family for their continued support and sacrifice during my PhD work and to our unborn baby, Weston Louis who unknowingly motivated me to persevere.

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Acknowledgements I would like to acknowledge my major professor, Dr. Mary Evans who always supported my ambitions and my co-major professor, Dr. Shyam Mohapatra who gave me the opportunity to explore the basic sciences. To the committee members, Dr. Maureen Groer for being a wonderful mentor, Dr. Jason Beckstead for always taking time and giving great advice and Dr. Stuart Brooks for continued encouragement and many wonderful articles. I would also like to thank Dr. Richard Lockey and the Joy McCann Culverhouse Airway Disease Center for the opportunity and wonderful lab experiences and a special thank you to Shawna Shirley for her mentorship, friendship and encouragement during many late nights in the lab. I would also like to acknowledge my family (Doug and Shelia Free, Tony, Andr ea, Alex, Tyler and Ashley Jones, Kerry, Erica, Kulyn and Adley Hubbard, Frances Osborne and Letha Nix) and my friends (Kristin and Kyle Crawford and Shawna and Karl Gilman) for their love, inspiration, support, prayers, patience and understanding throughout my time at the University of South Florida. To my superman husband Marty Montpetit, PhD, we struggled through our PhD work together and survived. Thank you for your unconditional love, understanding and words of affirmation. I woul d also like to acknowledge the National Institute of Nursing Research (F31 NRSA) and the Joy McCann Culverhouse endowment for providing fellowship and funding support throughout my pre-doctoral training.

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i Table of Contents List of Figures iv List of Tables v List of Abbreviations vi Abstract ix Chapter One Introduction 1 Chapter Two Review of Literature 5 Bronchial Epithelial Mucosa as First Line of Defense 5 Dendritic Cells: The Professional Antigen Presenting Cell 6 Epithelial and Dendritic Cell Interaction 9 Cigarette Smoke as an Oxidant Stressor 10 Effects of Cigarette Smoke on Humans in vivo 11 Effects of Cigarette Smoke on Murine Dendritic Cells in vivo 12 Effects of Cigarette Smoke on Human Dendritic Cells i n vitro 13 Polyinosinic-Polycytidylic Acid as an Immunological Stimulant 14 Chapter Three Method 17 Purpose 17 Hypothesis 18 Specific Aims and Research Questions 19 Specific aim 1 20 Specific aim 2 21 Materials 22

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ii Normal human bronchial epithelial cell culture 22 Monocyte-derived dendritic cells culture 23 Co-culture of NHBE and MDDC cells 24 Treatment of cell cultures with CSC 25 Homotypic clustering of DC 26 Quantification of cytokines by multi-plex bead assay 28 Determination of viability and phenotypic maturation of MDDC by immunofluorescence labeling and flow cytometry 30 Determination of functional maturation of MDDC by endocytosis assay 31 Proliferation and polarization of allogeneic CD4+ TC in response to CSC exposed and/or poly I:C stimulated MDDC 32 Data Analysis 34 Chapter Four Results 36 Characteristics of the Sample 36 Experimental Conditions Do Not Affect DC viability 38 CSC Induces Homotypic Clustering in Non-Stimulated Co-Cultures 39 CSC Reduces Phagocytosis in Non-Stimulated Co-Cultures 41 CSC Induces Maturation-Associated Co-Stimulatory and Adhesion Molecules 42 CSC Failed to Significantly Affect Dendritic Cell Cytokines 46 CSC Failed to Induce TC Proliferation but Enhances Th2 Cytokine Production 48 Chapter Five Discussion 55 Summary of Findings 55

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iii Limitations 60 Future Directions 61 Significance to Nursing 62 References 63 Appendices 75 Appendix A: Definitions 76 About the Author End Page

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iv List of Figures Figure 1 Structure of poly I:C 16 Figure 2 Conceptual model 18 Figure 3 Working model 19 Figure 4 Aim 1 experimental schema 20 Figure 5 Aim 2 experimental schema 22 Figure 6 Generation of the co-culture model 25 Figure 7 Homotypic cluster scoring 28 Figure 8 Pairwise comparisons for dependent variables 35 Figure 9 Dendritic cell viability 38 Figure 10 Dendritic cell homotypic clustering 40 Figure 11 Dendritic cell phagocytosis 42 Figure 12 Dendritic cell surface markers: Controls, CD11c, HLA-DR 44 Figure 13 Dendritic cell surface markers: CD80, CD83, CD86, CD54 45 Figure 14 Dendritic cell cytokines and chemokines 47 Figure 15 T cell proliferation: Flow cytometry 50 Figure 16 T cell proliferation: Percent proliferation 51 Figure 17 T cell cytokines 52 Figure 18 Summary of findings 59

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v List of Tables Table 1 Homotypic clustering scoring system 27 Table 2 Cytokine and chemokine analysis 30 Table 3 Dendritic cell surface marker analysis 32 Table 4 Descriptive statistics of all dependent variables 37 Table 5 Dendritic cell viability 38 Table 6 Dendritic cell homotypic clustering 41 Table 7 Dendritic cell phagocytosis 42 Table 8 Dendritic cell surface markers 46 Table 9 Dendritic cell cytokines and chemokines 48 Table 10 T cell proliferation: Percent proliferation 51 Table 11 T cell cytokines 53 Table 12 Th1/Th2 cytokine ratios 54

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vi List of Abbreviations 7-AAD: 7-amino-actinomycin APC: allophycocyanin BALF: bronchoalveolar lavage fluid BDCA: blood dendritic cell antigen CCL20: C-C motif ligand 20 CFSE: carboxyfluoroscein succinimidyl ester CSC: cigarette smoke condensate CSCH: cigarette smoke condensate high dose, 50 g/ml CSCL: cigarette smoke condensate low dose, 10 g/ml CSE: cigarette smoke extract CD: cluster of differentiation COPD: chronic obstructive pulmonary disease DC: dendritic cells DEP: diesel exhaust particulate DMSO: dimethyl sulfoxide: EC: epithelial cells eta2: eta squared ETS: environmental tobacco smoke FITC: fluorescein isothiocyanate GMCSF: granulocyte macrophage colony stimulating factor HLA-DR: human leukocyte derived antigen:

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vii ICAM1: intracellular adhesion molecule 1, also known as CD54 IFN : interferon gamma IP10: interferon-inducing protein 10 IL: interleukin LN: lymph nodes Log: logarithm base 10 LPS: lippolysaccharide g: microgram MFI: mean fluorescence intensity MHC: major histocompatibility complex ml: milliliter MLR: mixed lymphocyte reaction mDC: myeloid dendritic cells MDDC: monocyte-derived dendritic cells NHBE: normal human bronchial epithelial cells OS: oxidant stress: PBMC: peripheral blood mononuclear cells pDC: plasmacytoid dendritic cells PE: phycoerythrin PFA: paraformaldehyde pg: picogram PHA: phytohemmaglutinin Poly I:C: polyinosinic-polycytidylic acid SEM: standard error of the mean siRNA: short interfering ribonucleic acid

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viii TC: T cells TCR: T cell receptor TGF : transforming growth factor beta Th0: T helper type 0, T regulatory Th1: T helper type 1 Th2: T helper type 2 TLR3: toll-like receptor 3 TNF : tumor necrosis factor alpha Treg: T regulatory, Th0 TSLP: thymic stromal lymphopoietin

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ix Modulation of Monocyte-Derived Dendritic Cell Maturation and Function by Cigarette Smoke Condensate in a Bronchial Epithelial Cell Co-Culture Model Alison J. Montpetit ABSTRACT Lung airway epithelium is the first line of defense against inhaled particulates such as cigarette smoke. Subepithelial dendrit ic cells (DC) survey the airway epithelial lining and represent the link between innate and adaptive immune response. No study has investigated the effect of cigarette smoke on DC in the presence of epithelial cells (EC). The purpose of this 4x2 factorial design study was to co-culture normal human bronchial epithelial (NHBE) cells with monocyte-derived dendritic cells (MDDC) and examine the effect of cigarette smoke c ondensate (CSC) and poly I:C stimulation (TLR3 ligand that mimics viral infection) on MDDC homotypic clustering, phagocytosis ability, surface marker expression, DC cytokine response, T cell (TC) proliferation and TC cytokine response. Experiments were perform ed with MDDC and TC derived from four individual donors. Two planned comparisons (DMSO vehicle control compared to CSC high dose in both the no poly I:C and poly I:C stimulated groups) were analyzed. In MDDC stimulated with CSC, there was a significant increase in homotypic clustering, reduced phagocytosis, increased CD54, increased CD83 and CD86 maturation marker expression. Although no significant changes were observed in MDDC cytokine production, IL10 exhibited a trend to increase with CSC exposure but failed to reach statistical significance. Despite evidence that CSC exposed MDDC are maturing, there was no increase in TC proliferation; however in poly I:C stimulated co-cultures, CSC

PAGE 13

x exposure increased IL2, IL5, IL10 and IL13 expression while non-stimulated co-cultures exhibited an increase in IL10 following CSC exposure. This study indicates that cigarette smoke has the ability to decrease phagocytosis ability of DC and increases costimulatory maturation markers without inducing TC proliferation and potentiating a Th2 environment. These findings suggest that DC of smokers may be less likely to mount a normal immune response to invading pathogens thus providing evidence for smoker susceptibility to infection and how DC of smokers may respond to viral infection. In addition, these findings are particularly important in patients with allergic airway disease since increasing Th2 cytokines would increas e the risk for disease exacerbation.

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1 Chapter One Introduction Cigarette smoking has been identified as the most important cause of preventable morbidity and mortality in the United States. Over 438,000 Americans die annually from diseases directly related to smoking (American Lung Association, 2006). In the United States, conservative estimates of the economic burden due to smoking are $167 billion in direct healthcare costs, $92 billion in lost productivity and $75 billion in excess medical expenditures (American Lung As sociation, 2006). Approximately 8.6 million Americans have at least one serious illness caused by smoking (American Lung Association, 2006). For people suffering with lung disease, breathing is often a daily challenge. Symptom management of lung disease is a vital component of therapy. Relieving dyspnea and breathlessness by encouraging sm oking cessation is one of the most effective strategies. Despite efforts of nurses and other health care professionals to educate smokers of the risk of pulmonary disease, many continue to smoke and approximately 15 billion cigarettes are sm oked worldwide every day (American Lung Association, 2003). In addition, over 50% of teenagers aged 13-15 who claim to have never smoked are exposed to secondhand smoke in the home (Centers for Disease Control and Prevention, 2007). There is a need to conduct research that explores immune responses to cigarette smoke and potential use of immune modulating therapies and their effects on the pulmonar y system in smokers and nonsmokers.

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2 Lung airway epithelium is the first line of defense against inhaled particulates such as cigarette smoke and represents a component of the innate immune response. Dendritic cells (DC) in the respiratory tract are professional antigen presenting cells that reside within the epithelium, submucosa and lung parenchyma (Lambrecht & Hammad, 2003; Upham & Stick, 2006). One function of the DC is to survey the lung environment for antigens, microbes and dead or dying cells (Rossi & Young, 2005). DC also link the innate immune response with the adaptive immune response and play a key role in not only antigen presentation but also in determining Th1 and Th2 immune responses. Cigarette smoke has been shown to suppress DC function and lead to an induction of Th2 immune response thus potentiating the allergic response (Vassallo & Chen, 2004; Vassallo, Tamada, Lau, Kroening, & Chen, 2005). In addition, the crosstalk between epithelial cells (EC) and DC has been shown to be especially important in airway inflammation and asthma (Lambrecht & Hammad, 2003; Upham & Stick, 2006). Several studies have shown that smokers and people exposed to secondhand smoke are at increased risk for infections (Carroll et al., 2007) and at high risk for the development of chronic obstructive pulmonary disease (COPD) and asthma (Goodwin, 2007; Halken, 2004; Teramoto, 2007). Asthmatics who smoke are less responsive to corticosteroid therapy than non-smoking asthmatics (Thomson, Shepherd, Spears, & Chaudhuri, 2006), making treatment of patients who smoke quite challenging for health care providers. In addition, patients wi th asthma and/or COPD who smoke or are exposed to secondhand smoke are more prone to disease progression and exacerbation (Alvarez, Schulzer, Jung, & Fitzgerald, 2005; Eisner & Iribarren, 2007; Eisner et al., 2005; Kanner, Anthonisen, & Connett, 2001; Koga, Oshita, Kamimura, Koga, & Aizawa, 2006; Wark & Gibson, 2006).

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3 Lung disease exacerbation is linked with a decline in lung function, decreased quality of life and increased morbidity and mortality (Bai, Vonk, Postma, & Boezen, 2007; Cote, Dordelly, & Celli, 2007; Donaldson, Seemungal, Bhowmik, & Wedzicha, 2002; Rogers, Adelroth, Hattotuwa, Dewar, & Jeffery, 2007). Studies indicate that cigarette smoke has the ability to modulate the immune response most likely, dampening the Th1 type immune response and favoring a Th2 type immune response. Despite recent advances in research related to the immune response to cigarette smoke exposure, the exact mechanisms of smoker vulnerability remains unclear. One possible explanation for the increased risk of infection, inflammation and exacerbation of pulmonary disease in smoker s is that smokers have fewer numbers of DC in the bronchial mucosa. This finding was observed in bronchial biopsies of patients with COPD who smoke compared with nonsmoking normal, asthmatic and COPD controls (Rogers et al., 2007). In addition, one study reported observing significantly less CD83+ mature DC in the airways of smoking asthmatics when compared to nonsmoking asthmatics and healthy non-smoking controls (Tsoumakidou et al., 2007). This phenomenon has also been observed in cord blood from neonates of smoking mothers (Pachlopnik Schmid et al., 2007). This study reported a significant decrease in all leukocytes including CD11c+ myeloid and CD123+ lymphoid DC. By decreasing the number of DC in the airways, the innate i mmune system's ability to respond is hindered; however, the potential mechanism requires further inquiry as this observation may merely be explained by migration of DC to the lymph nodes, other tissues or downregulation in surface marker expression. Despite overwhelming evidence that smoking is harmful, it cannot be ignored that evidence suggests a protective effect of tobacco smoking against certain diseases including, but not limited to: Parkinsons' and Alzheimer's disease (Fratiglioni & Wang,

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4 2000), sarcoid arthritis (Visser et al., 2002), and ulcerative colitis (Mahid, Minor, Soto, Hornung, & Galandiuk, 2006). Interestingly, these diseases have an inflammatory or autoimmune component and particulates in cigarette smoke and/or nicotine may suppress the immune response and potentially pr otect against these diseases. Overall, it is clear that cigarette smoking modul ates the immune system by activation and suppression making research inquiry complex. People who smoke face often face challenges in the life-dependent act of breathing. Nursing care is guided toward symptom assessment and management as well as playing a critical role in educ ating patients and family regarding symptom management, smoking cessation, diseas e pathophysiology and progression, and prevention of exacerbations. No study has investigated the effect of cigarette smoke in DC in the presence of epithelial cells (EC). By specifically studying the interaction of DC and EC exposed to cigarette smoke particulates, this study will provide focused insight into the immune effects of smoke and the link between innate and adaptive immunity. This will add to the body of knowledge regarding smoke immunomodulation enabling nurses and other health care providers to improve current methods of diagnosis, symptom management and monitoring in order to prevent disease exacer bation. In chapter two, a review of the literature will be presented in order to further develop the concepts to be investigated in this study.

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5 Chapter Two Review of Literature Bronchial Epithelial Mucosa as First Line of Defense Bronchial EC play a critical role in protecting submucosal tissue from inhalation injury by entrapment and removal of particulates by mucous secreting goblet cells, ciliated cells, and intact tight junctions. Although this is a key function of the bronchial lining, recent advances in immunology research have indicated that EC play an important role in linking the innate and adaptive immune systems. This is evident in the ability of EC to secrete cytokines and chemokines in response to inhaled allergens, pollutants and chemicals that lead to infla mmatory cell recruitment, airway remodeling and development or potentiation of airway disease such as asthma, emphysema and chronic bronchitis (Gershwin, 2007; Kato & Schleimer, 2007; Lambrecht & Hammad, 2003; Schleimer, Kato, Kern, Kuperman, & Avila, 2007; Upham & Stick, 2006). The immune system is composed of two major branches: innate and adaptive immunity. The innate immune response is the body's first line of defense against infection and hinges on the ability to recognize self from non-self. EC in the pulmonary system are heavily equipped with numerous systems that protect the body from microbial, viral, chemical and particulate invasion. Some of these mechanisms include but are not limited to the complement sy stem, collectins, mucins, reactive oxygen species and toll-like receptors (Ezekowitz & Hoffman, 2003; Schleimer et al., 2007). In addition to the innate immune system, EC play a role in the adaptive immune system.

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6 The adaptive immune system response is characterized by antigen processing/presentation and induction of a memory response to specific antigens and involves many cells including T and B cells. This review will focus primarily on one type of antigen presenting cell, DC. Dendritic Cells: The Professional Antigen Presenting Cell DC represent 1-2% of peripheral blood mononuclear cells and approximately 0.5% of cells found in bronchoalveolar lavage fluid (BALF) (Donnenberg & Donnenberg, 2003; Tsoumakidou, Tzanakis, Papadaki, Koutala, & Siafakas, 2006; van Haarst et al., 1994). In humans, there are two major types of DC, myeloid (mDC) and plasmacytoid (pDC). Myeloid DC express the surface marker CD11c and capture antigen by phagocytosis and macropinocytosis. Plasmacytoid DC are derived from a lymphoid lineage, do not express CD11c and are inefficient in phagocytosis (Schuurhuis, Fu, Ossendorp, & Melief, 2006). Both mDC and pDC have the ability to induce Th1 and Th2 type responses but the response is primarily determined by environmental stimuli such as pulmonary inflammation. Both types of DC exist in the lungs; however, mDC are predominate and they are located in the conducting airway, intersti tium and alveoli (de Heer, Hammad, Kool, & Lambrecht, 2005; Iwasaki, 2007). One study reported 0.06% of pDC in BALF and 0.47% mDC and demonstrated an increase in both pDC and mDC following a segmental allergen challenge (Bratke et al., 2007). In addition, one study investigated DC in lung digests and the presence of blood DC antigen (BDCA), a marker used to subtype blood DC. This study showed four subtypes of DC in the lungs: mDC1 (BDCA1+/HLA-DR+), mDC2 (BDCA3+/HLA-DR+), pDC (BDCA2+/CD123+) and CD1a+ DC in the conducting airway epithelium (Demedts, Brusselle, Vermaelen, & Pauwels, 2005). Another study

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7 confirmed the presence of the three subtypes (mDC1, mDC2, pDC) in BALF (Tsoumakidou et al., 2006). It is apparent that with advances in flow cytometry techniques and identification of surface markers, more pulmonary DC subtypes will be elucidated. DC are key in directing immune responses throughout the body, but for the purposes of this review, only pulmonary DC are examined. In the lungs, DC are located within the lung epithelium and interstitium and perfectly placed for sampling the bronchial airway for inhaled antigens and particulates (Grayson, 2006; Lambrecht, Prins, & Hoogsteden, 2001). Studies have shown that DC that reside underneath and within the epithelium are able to extend their dendrites through epithelial tight junctions in order to sample the airway. The DC then migrate to draining lymph nodes where they confer antigen-specific responses. Pulmonary inflammation plays a key role in the maturation process and trafficking of DC. Immature DC phagocytose/endocytose bacteria, allergens, pollutants and cellular debris in the airway. They then process the antigen and carry immune signals from the periphery or site of capture to the secondary lymphoid tissue where, after maturation, they present antigen to TC (Cook & Bottomly, 2007). Pulmonary DC are the sentinels of the lung and they direct clonal expansion of TC by cross-presentation of allergens to CD4+ T helper cells, CD8+ cytotoxic TC and are the only cells capable of priming nave TC (Cools, Ponsaerts, Van Tendeloo, & Berneman, 2007; Novak & Bieber, 2008). The response of naive T helper cells after contact with pathogen-specific, mature DC is determined by three main signals: 1) antigen specific (by engagement of the TC receptor by MHC class II), 2) co-stimulation and 3) polarization. Signal 1 is characterized by antigen specificity. Signal 2 provides protective immunity by co-

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8 stimulatory molecules and if signal 2 is not present, it leads to TC anergy and potentially tolerance. Signal 3 is important in determining the balance of cytokine production from primed TC. There is a functional difference in DC and TC depending on the microenvironment of the third signal. It is unclear where all of these third signals originate but the micro-environment, character of antigen, and the affected tissue all play a critical role. Since DC are in close proximity to EC, it is hypothesized that polarizing signals may be driven by EC (Kalinski, Hilkens, Wierenga, & Kapsenberg, 1999; Kapsenberg, 2003; Schuurhuis et al., 2006). Nave TC generally have four broad fates including: Th1, Th2, Th17 and Treg. For the purposes of this review, only CD4+ TC subsets will be discussed. Th1 is primarily characterized by IFN and TNF release; Th2 is primarily characterized by IL4, IL5 and IL13 release, Th17 by IL17 and Treg by IL10 and TGF An imbalance towards one type has been implicated in many dis eases for example, Th1 in autoimmune diseases, Th2 in allergic diseases, Th17 in chronic inflammatory and autoimmune disorders and Treg in cancer and immunosuppression (Hammad & Lambrecht, 2006; Romagnani, 2006). Many antigens induce DC maturation following uptake resulting in TC activation; however, most antigens fail to induce maturation requiring secondary signals from the micro-environment (Kalinski et al., 1999; Kapsenberg, 2003). This is a critical balance since approximately 10,000 L of air circulate through the lungs every day exposing EC and DC to many particles (Cook & Bottomly, 2007). In fact, the outcome of inhalation of most harmless antigens results in tolerance. Under steady state conditions, DC remain in an immature or semi-mature state during which they are continue to phagocytize antigen and have high expression of receptors for pro-inflammatory chemokines and cytokines (Novak & Bieber, 2008). This active process is characterized by tolerogenic

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9 DC presenting antigen to nave TC which in turn acquire regulatory TC (Treg) properties including Foxp3+, production of IL10 and TGF the ability to suppress Th1 and Th2 processes. There is some debate about characteristics of tolerogenic DC but generally these DC are considered semi-mature, are inefficient at phagocytosis of antigen, produce IL10 and are less motile but may express maturation/co-stimulatory molecules (Cook & Bottomly, 2007; Morelli & Thomson, 2007; Reis e Sousa, 2006; Steinman, Hawiger, & Nussenzweig, 2003). Epithelial and Dendritic Cell Interaction EC are the first contact with inhaled particulates and they play a critical role in regulation of immune responses through interactions with DC, TC and B cells among others. This review will focus specifically on EC and DC interactions. EC may play a role in recruitment of DC into the airways via macrophage inflammatory protein 3 alpha also known as chemokine (C-C motif) ligand 20 (CCL20) and possess the ability to polarize DC to a Th1 or Th2 directing phenotype (Kao et al., 2005; Thorley, Goldstraw, Young, & Tetley, 2005). One example, EC-derived thymic stromal lymphopoietin (TSLP) secretion directs DC to activate nave TC to Th2 cells that produce IL4, IL3, TNF but not IL10 or IFN (Kato, Favoreto, Avila, & Schleimer, 2007; Lee & Ziegler, 2007; Liu et al., 2007). One example of the importance of EC-DC interaction is shown by the effect of diesel exhaust particulates (DEP) to induce DC maturation marker CD80, CD83, CD86 and TC proliferation when DC and EC are co-cultured and this effect is not observed when DC are cultured alone (Bleck, Tse, Jaspers, Curotto de Lafaille, & Reibman, 2006). In this study, supernatant from DEP-exposed EC induced DC maturation and was equivalent to DC cultures with DEP-exposed EC, suggesting that soluble mediators

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10 rather than direct cell-to-cell contact are critical for DC maturation and function. In addition, when a blocking antibody (Ab) and short interfering RNA (siRNA) to the growth factor, granulocyte-macrophage colony-stimulating factor (GMCSF) was administered to DEP-exposed EC cultures, DC failed to induce TC proliferation (Bleck et al., 2006). This finding suggests the critical role of EC-derived GMCSF in DC maturation and function. This study did not evaluate the cytokine production of TC from the MLR; therefore, it is unknown if DEP-exposed EC co-cultured with DC induce TC to produce Th1 or Th2 cytokines. Based upon this study, it is reasonable that cigarette smoke-exposed EC culture may modulate patterns of DC maturation and TC proliferation when cultured with DC since cigarette smoke and DEP are both pro-inflammatory and potent oxidant stressors. Cigarette Smoke as an Oxidant Stressor The pathogenesis of many lung diseases such as chronic obstructive pulmonary disease (COPD) and asthma may be a function of free oxygen radicals or oxidant mediators which can act as immune adjuvants, enhancing the response to inhaled allergens or antigens (Nouri-Shirazi & Guinet, 2006; Siafakas & Tzortzaki, 2002; Sopori, 2002; Tsoumakidou et al., 2007). A number of environmental stimuli or oxidant mediators induce cellular oxidative stress (OS) that stimulates several biological effects such as, increased inflammation and mucous production, decreased cilia function, impaired surfactant activity, promotion of epithelial permeability, inhibition of glucocorticoid responses and many others (R epine, Bast, & Lankhorst, 1997). All these effects compromise the effectiveness of the innate immune system. Conceptually, OS has been defined as a loss of balance in which oxidation exceeds the antioxidant systems in the body. One cause of OS in the lungs is cigarette

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11 smoke. Cigarette smoke has approximately 1017 oxidants/free radicals and 4,800 chemical compounds which increases the oxidant burden in smokers and the risk of developing pulmonary disease through chronic inflammatory processes and increasing airspace permeability (American Lung Association, 2006; MacNee, 2000; Marwick et al., 2002; Moodie et al., 2004). The respiratory tract is extremely differentiated and its ability for gas exchange is dependent on thin epithelial cell membranes in the respiratory tract and are the first contact with air particulates, making them more susceptible to the environment and cigarette smoke than other tissues; thus, they are more vulnerable to reactive oxygen species that can cause cellular damage and alter in the immune response by increased susceptibility to inflammation and infection (Jyonouchi, 1999; Lambre et al., 1996). Effects of Cigarette Smoke on Humans in vivo Several studies have investigated the effect of cigarette smoke in humans. Smokers have dramatically fewer numbers of DC in the bronchial mucosa which was observed in bronchial biopsies of patients with COPD who smoke compared with nonsmoking normal, asthmatic and COPD non-smoking controls (Rogers et al., 2007). In fact, COPD ex-smokers had similar numbers of DC as asthmatics; therefore, the reduction in DC is not a function of the disease process. The location of these DC were determined by transmission electron microsco py images which revealed the presence of DC primarily in the bronchial mucosa epithelium and subepithelium (Rogers, Adelroth, Hattotuwa, Dewar, & Jeffery, 2008). One study reported significantly less CD83+ mature DC in the bronchial biopsies of smoking asthmatics compared to non-smoking asthmatics and healthy non-smoking controls (Tsoumakidou et al., 2007). However, another study did not find a difference in

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12 CD83+ DC in bronchoalveolar lavage fluid (BALF) of smokers versus never smokers. They did observe a difference in CD80+ and CD86+ DC in smokers. In addition, they reported decreased expression of CCR7, a lymph node homing receptor that is important in migration, suggesting that the reduction in DC is not due to DC migration. In a randomized, placebo-controlled trial, non-smoking allergic subjects were exposed to two hours of environmental tobacco smoke (ETS) or clean air then subjected to a nasal challenge with ragweed allergen or saline and nasal lavages were obtained. Twenty-four hours after ETS exposure with allergen challenge, there was a significant increase in ragweed-specific IgE, histamine, IL4, IL5 and IL13 and a significant reduction in IFN when compared with clean air exposure with allergen challenge (Diaz-Sanchez, Rumold, & Gong, 2006). This study was the first to show evidence in humans that cigarette smoke can directly exacerbate allergic disease. Effects of Cigarette Smoke on Murine Dendritic Cells in vivo Several studies have investigated the effect of smoke in murine models. One study exposed mice to two cigarettes per day five days a week for two months. They reported a significant decrease in the num ber of CD11c+/MHC-II+ in smoke exposed mice compared with controls. In addition, this trend preceded emphysematous lesions in the lungs and was lung specific as there was no reduction in DC in regional lymph nodes (Robbins et al., 2004). This provides evidence that smoke effects on lung DC are not merely a result of DC migration. Another murine study where mice were exposed to 6 cigarettes a day, five days a week for one month confirmed this reduction in pulmonary CD11c+ DC and reported that antigen induced migration to draini ng lymph nodes was not affected by smoke exposure. In addition, they reported a reduction in MHC-II, CD80 and CD86 but no

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13 effect on CD40 in DC within the lymph nodes. They also observed a reduction in TC proliferation and significantly less CD4+ TC IL2 production from smoke exposed mice when compared to control (Robbins, Franco, Mouded, Cernadas, & Shapiro, 2008). Effects of Cigarette Smoke on Human Dendritic Cells in vitro Clinical evidence described previously suggests that smokers are more prone to Th2 type response. Since DC have the ability to sample the bronchial airway, take up smoke particulates and are key mediators of immune response, it is reasoned that DC function and DC-induced TC response may be affected by cigarette smoke. Only two studies have evaluated the effects of cigarette smoke on human DC function. The first study investigated the role of cigarette smoke extract (CSE), the particulate phase of smoke on DC co-stimulatory markers (CD40, CD80, CD83, CD86), TC proliferation (BrdU assay) and polarization of TC cytokine response (IL2, IL4, IL10, IFN ). This study provided evidence that CSE suppresses maturation markers (CD40, CD80, CD86) and suppresses IL12 and enhances IL10 production. In addition, CSEexposed DC inhibit TC proliferation and pr omote Th2 (IL4) cytokine release from TC. Together, this suggests that smoke suppresses DC functions and potentiates a Th2 response (Vassallo et al., 2005). The second study investigated TC proliferation (CFSE assay) and cytokine response (IL12p70, IL4, IFN ) in response to DC cultured with nicotine and matured under non-polarized or polarized Th1 and Th2 conditions (Nouri-Shirazi & Guinet, 2006). The study provided evidence that nicotine exposed DC are able to induce TC proliferation but not Th2 differentiation (IL4 secretion) unless under a Th2 polarizing condition described as exposure to IL1 TNF and prostaglandin E2.

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14 In these two studies, DC were cultured alone. As discussed previously, DEPtreated DC did not induce TC proliferation when DC were cultured alone. This is the same observation with cigarette smoke in the first study but not in the nicotine study. This discrepancy may be a function of particulates in smoke-exposed EC. It has been shown that smoke-exposed bronchial EC produc e pro-inflammatory cytokines including, IL6 and IL8; however, it has also been shown that smoke induces downregulation of GMCSF in lippolysaccaride (LPS)-treated EC (Glader et al., 2006; Li, Xu, & Shen, 2007; Phillips, Kluss, Richter, & Massey, 2005). GMCSF was shown to have significant effects on DC maturation in DEP-exposed co-cultures of DC and EC (Bleck et al., 2006). By culturing DC with smoke-exposed EC, it will take into account the impact of the tissue micro-environment on environment al exposures. Therefore, the proposed study will provide insight into EC-DC interactions in smoke exposure and may provide evidence for disease and infection susceptibility in cigarette smokers. Polyinosinic-Polycytidylic Acid as an Immunological Stimulant A major signal for DC priming are toll-like receptors (TLR) which are highly conserved immune receptors responsible for recognition of pathogen-associated molecular patterns (PAMP) of bacteria and viruses thereby initiating the immune response (Duez, Gosset, & Tonnel, 2006; Guillot et al., 2005; Kapsenberg, 2003). Specifically, TLR3 recognizes double stranded RNA (dsRNA) of viruses such as influenza and rhinovirus and induces a potent antiviral response (Doyle et al., 2003; Guillot et al., 2005; Hewson, Jardine, Edwards, Laza-Stanca, & Johnston, 2005). In both epithelial and monocyte-derived DC, TLR3 is primarily located in the cytoplasm and endoplasmic vesicles (Ciencewicki, Brighton, Wu, Madden, & Jaspers, 2006; Duez et al., 2006).

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15 Poly I:C (Figure 1) is a TLR3 ligand and a dsRNA that mimics the effects of viral infections (Marshall-Clarke et al., 2007). EC stimulated with poly I:C have been shown to up-regulate numerous pro-inflammatory cytokines (IL1 IL6), growth factor (GMCSF), chemokines (IP10, IL8) and adhesion molecules such as ICAM-1 (Matsukura et al., 2006; Ritter, Mennerich, Weith, & Seither, 2005). DC stimulated with poly I:C exhibit stable maturation and produce high levels of IL12, low levels of IL10 and a strong TC proliferative response (Verdijk et al., 1999). In addition, poly I:C-stimulated DC retain the ability to produce IL12p70 even after the initial maturation stage, thus driving a Th1 response (Rouas et al., 2004; Verdijk et al., 1999). One study revealed that diesel ex haust enhanced poly I:C-induced expression of TLR3, interferon beta and IL6 in lung EC providing mechanistic evidence that inhalation of particulates affect the immune response to viral infection (Ciencewicki et al., 2006). Dysregulation of the innate immune response could lead to enhancement of inflammatory cell recruitment, DC activation, adaptive immune response and potentially an auto-immune or allergic type reaction. No studies have been conducted investinging modulation of poly I:C effects by cigarette smok e; however, it is important to determine if smoke down-regulate cytokine production and push the Th2 response despite the Th1 poly I:C effects. This will mimic the clinical scenario of cigarette smokers exposed to viral infection.

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16 Figure 1. Structure of poly I:C. Reprinted from (Chemblink.com, 2007) with permission. It is evident that smoke modulates the immune response and impacts disease development and progression. Unfortunately, t he exact mechanism by which this occurs remains unclear. By culturing MDDC with smoke-exposed NHBE, it takes into account the impact of the tissue micro-environment on the response of MDDC. This study provides insight into EC-DC interactions in smoke exposure and may provide evidence for disease and infection susceptibility in cigarette smokers. In addition, by investigating the effect of smoke in poly I:C stimulated co-cul tures, this study also provides insight into how a smoker may respond to viral infection. In chapter three, the purpose, models and methods of this study is described.

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17 Chapter Three Method Purpose The purpose of this preliminary 4x2 fact orial design study was to examine the effect of cigarette smoke condensate (CSC) on monocyte derived DC (MDDC) clustering, cytokine response, maturation, endocytosis ability, TC proliferation and polarization. This study was guided by the conceptual model in Figure 2. This concept model shows the in vitro model model (NHBE, MDDC co-culture), four levels/doses (control/resting, DMSO/vehicle control, 10 g/ml and 50 g/ml) of cigarette smoke condensate (CSC) exposure, two levels/doses (0 and 25 g/ml) of poly I:C stimulation. The dashed arrows and box, indicates the theoretical concept of DC maturation and function followed by the categories of dependent variables to be measured.

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18 Figure 2. Conceptual model. In vitro study of CSC and poly I:C modulation of dendritic cell maturation and function in NHBE cell and MDDC co-c ulture model, DMSO = dimethyl sulfoxide. Hypothesis It was hypothesized that CSC will suppresses DC maturation and redirect DCinduced CD4+ T lymphocyte differentiation, leading to augmentation of T helper type 2 (Th2) cytokine secretion. The working m odel (Figure 3) depicts the innate and adaptive immune responses following exposure to CSC. EC up-regulate cytokine secretion and MDDC take up CSC particulates, modulating maturation and function. Stimulated MDDC undergo functional maturation and secrete T helper lymphocyte Th2 polarizing type cytokines. Theoretically, the DC then mi grate to the lymph nodes (LN), promoting differentiation and proliferation of CD4+ TC into Th2 cells linking the innate immune response with the adaptive immune response. De p endent Variables Inde p endent Variables Mode l CSC Exposure: 1-Control (resting cells) 2-DMSO (vehicle control) 3-CSC low dose (10 g/ml) 4-CSC high dose (50 g/ml) Poly I:C Stimulation: 1-No stimulation (medium) 2-With stimulation (25 g/ml) MDDC Viability Homotypic Clustering Phagocytosis Cytokines Surface Markers MDDC Maturation Function NHBE MDDC co-culture TC Proliferation Cytokines

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19 Figure 3. Working model. Depicts CSC expos ed co-culture of EC and MDDC. Specific Aims and Research Questions This proposed study has two main aims. The first objective was to characterize the co-culture model and exposure systems and to determine the effect of cigarette smoke exposure and poly I:C stimulation on MDDC viability, homotypic clustering and cytokine secretion. The second objective was to determine the effect of cigarette smoke exposure and poly I:C stimulation on MDDC surface marker expression and phagocytosis. In addition, the effect of CSC exposed and poly I:C stimulated MDDC on TC proliferation and polarization was examined through mixed lymphocyte reaction and cytokine analysis. to LN Innate DC MDDC T EC Adaptive Th2 polarizationMDDC CSC

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20 Specific aim 1. Characterize and optimize CSC exposure and poly I:C stimulation of the NHBE cells, MDDC and a co-culture of NHBE cells and MDDC (Figure 4) Can NHBE cells, MDDC and the co-culture model be cultured effectively? Is there a difference in homotypic clustering of MDDC co-cultured with NHBE cells following exposure to CSC and poly I:C stimulation? What are the cytokine profiles of NHBE cells and MDDC co-cultured with NHBE cells following exposure to CSC and poly I:C stimulation? Does CSC exposure modulate poly I:C-stimulated cytokine/chemokine/growth factor expression of NHBE cells and MDDC co-cultured with NHBE cells? Figure 4. Aim 1 experimental schema. Depicts the experimental schema for aim 1 establishing and characterizing the model, g/ml = microgram per milliliter. MDDC Viability Homotypic Clustering Cytokine Analysis NHBE CSC Exposure Dose (0, DMSO, 10, 50 g/ml) Time (12 hours) Model Poly I :C Stimulation Dose (0, 25 g/ml) Time (1-2 hours) NHBE + MDDC (co-culture) Time (24 hours) Independent Variables Dependent Variables

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21 Specific aim 2. Determine the effect of CSC and poly I:C stimulation on DC maturation, function and the ability of the exposed/stimulated DC to induce TC proliferation and priming. Compare the response of DC cultured alone or with bronchial EC (Figure 5). Does CSC exposure and poly I:C stimulation induce phenotypic DC maturation in MDDC co-cultured with NHBE cells as evidenced by maturation markers (CD80, CD83, CD86, and CD54) within the CD11c+ population? Does CSC exposure and poly I:C stimulation induce functional DC maturation in MDDC co-cultured with NHBE cells as evidenced by a lack of endocytic ability? Do CSC-exposed and poly I:C-stimulated MDDC co-cultured with NHBE cells induce CD4+ TC proliferation in MLR as evidenced by the CFSE proliferation assay? Do CSC-exposed and poly I:C-stimulated MDDC co-cultured with NHBE cells induce CD4+ cell proliferation towards a Th2 phenotype in the MLR as evidenced by a decrease in Th1 cytokines and an increase in Th2 cytokines?

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22 Figure 5. Aim 2 experimental schema. Depicts the experimental schema of aim 2 designed to test the effects of CSC and poly I:C expos ure on MDDC surface marker expression, phagocytosis, TC proliferation and cytokine production. Materials Normal human bronchial epithelial cell culture. Primary NHBE cells were chosen for this study since they may more accurately mimic in vivo cell division characteristics as compared to transformed cell lines. NHBE cells were purchased from Clonetics/Cambrex (Lonza, Switzerland) which obtained the cells from healthy human volunteers after informed consent. Cells from one donor were obtained for replication of experiments. NHBE cells were grown according to the distributor guidelines. Briefly, primary NHBE cells (Passage 1) were grown to 75-80% confluency in an atmosphere of 5% CO2 and 95% air at 37oC. Cell culture medium consists of bronchial epithelial growth medium supplemented with bovine pitu itary extract, insulin, hydrocortisone, NHBE CSC Exposure Dose (0, DMSO, 10, 50 g/ml) Time (12 hours) Model Poly I :C Stimulation Dose (0, 25 g/ml) Time (1-2 hours) MDDC + TC (MLR) Time (4-5 days) TC Proliferation Cytokine Analysis NHBE + MDDC (co-culture) Time (24 hours) MDDC Surface Markers Phagocytosis Independent Variables Dependent Variables

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23 human epithelial growth factor, epinephrine, transferring, retinoic acid and triiodothyronine (Lonza, Switzerland). Cells were subcultured, aliquoted into freezing tubes (50,000 cells/vial), and stored in liquid nitrogen following passage 1. Cells used in these experiments were at a passage of 2 and 3. For NHBE only experiments, NHBE cells were seeded onto 12 well plates and grown to confluency. Confluent cultures will be exposed to CSC depending on experimental group (0, 10 or 50 g/ml) for 12 hours. Poly I:C was then be added to CSC-conditioned medium depending on experimental group (0 or 50 g/ml) for 1-2 hours. Following CSC exposure and poly I:C stimulation, DC were added to NHBE cultures. Supernatants were collected for subsequent cytokine analysis. Monocyte-derived dendritic cells culture. Peripheral blood monocytes (PBMC) were isolated from buffy coats obtained from healthy volunteers (Florida Blood Services) after isolation by Ficoll-Paque (GE Healthcare/Amersham Biosciences, USA) density gradient centrifugation. CD14+ PBMC were isolated by positive selection using magnetically labeled CD14 antibodies and a m agnetic separator (Miltenyi Biotec). PBMC can differentiate toward an immature, myeloid DC phenotype, CD11c+, HLADRlo, E-cadherin-, langerin(de Heer et al., 2005) by an established method of culturing with IL4 and GMCSF as described previously in the literature (Sallusto & Lanzavecchia, 1994). Briefly, CD14+ PBMC were cultured in RPMI 1640 medium supplemented with 2% L-Glutamin, 1% penicillin/streptomycin and 10% fetal bovine serum, 10 ng/ml IL-4, 20ng/ml GMCSF (Peprotech), 1% nonessential amino acids and 1% sodium pyruvate (Sigma). Cultures were kept for 5-6 days in an atmosphere of 5% CO2 and 95% air at 37oC. Immature MDDC (iMDDC) were harvested on day 5 or 6 and characterized by flow cytometry. One study compared the phenotype of DC using this method with lung

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24 DC and reported similar phenotypic (low expression of CD40, CD80, CD83, CD86) and functional patterns (high capacity for the uptake of dextran and the ability to stimulate TC), making this an appropriate model for the in vitro study of pulmonary DC (Cochand, Isler, Songeon, & Nicod, 1999). Co-culture of NHBE and MDDC cells. Methods for the co-culture of NHBE cells and MDDC are based on methods described in the literature (Bleck et al., 2006). Briefly, NHBE cells at passages 2 or 3 were seeded at a density of 2x104 cells/cm2 on 12-well culture plates and maintained in an atmosphere of 5% CO2 and 95% air at 37oC approximately 7 days or until 100% confluency is reached (Figure 6). Day 5 or 6 iMDDC were harvested and re-suspended in complete medium. MDDC (5x105 cells/well) to confluent NHBE cells in 12 well plates and exposed to CSC or poly I:Cconditioned medium depending on experimental group assigned. Following 24 hours of culture, light microscope images were taken with a 4x objective for homotypic clustering analysis and cells were harvested, washed and aliquoted for subsequent flow cytometry, endocytosis assay and mixed lymphocyte reactions. In addition, supernatants were collected for cytokine analysis.

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25 Figure 6. Generation of the co-culture model. A. culture of NHBE cells submerged in culture medium; B. exposure of confluent NHBE cells to CSC conditioned medium for 12 hours; C. stimulation of confluent NHBE cells with polyinosini c:polycytidylic acid (poly I:C) for 1-2 hours; D. addition of MDDC and co-cultured with confluent NHBE cells for 24 hours. Treatment of cell cultures with CSC. Cigarette smoke consists of two components, the gas or vapor phase and smoke particulates. This study focused on the effects of smoke particulates by utilizing commercially available CSC. CSC purchased from Murty Pharmaceuticals (Lexington, KY, USA) was prepared by the manufacturer by using a Phipps-Bird 20 channel smoking machin e. The particulate matter from Kentucky standard cigarettes (IR3F; University of Kentucky, KY, USA) was collected on Cambridge glass fiber filters and the amount obtained was determined by weight increase of the filter. CSC was prepared by dissolving the collected smoke particulates in dimethyl sulfoxide (DMSO) to yield a 4% solution (w/v). The average yield of CSC was 26.1 mg/cigarette. One ml of CSC is equivalent to 2 standard Kentucky IR3F cigarettes and each cigarette contains 1.3 mg of nicotine. CSC was added to culture medium in two doses (0 and 50 g/ml) for 12 hours. A C D B 12 hours 1-2 hours 24 hours

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26 Treatment of cell cultures with poly I:C (Amersham Biosciences, Pittsburgh, PA, USA) was reconstituted in phosphate buffered saline (PBS) to 2 mg/ml by heating to 50oC to solubilize and allowing to slowly cool to room temperature in order to ensure reannealing. Poly I:C was added to cell culture medium at two doses (0 or 50 g/ml) for 12 hours. Poly I:C stimulated cultures were a positive control from MDDC maturation and mimic the effects of viral infection. Homotypic clustering of DC. Homotypic and heterotypic clustering of DC, is critical for antigen presentation and correlates with DC maturation (Delemarre, Hoogeveen, De Haan-Meulman, Simons, & Drexhage, 2001; Lehner et al., 2003). In order to assess cluster formation following CSC exposure and poly I:C stimulation, an aggregation assay was performed prior to supernatant collection and harvesting of cells for flow cytometry analysis, endocytosis assay and mixed lymphocyte cultures. The homotypic cluster scoring system (Table 1) is a modification of methods reported previously (Lehner et al., 2003; Majdic et al., 1994). Figure 7 presents a light microscopy image of DC is various cluster states: single, small and large. Small and large clusters were discriminated by the density/darkness and size. In order to be considered large, over 75% of clusters had to be dark/dense and greater than 10 times the size of a single DC. Clusters were randomly counted by two blind investigators utilizing an inverted light microscope with a 4X objective and average scores were reported. The degree of aggregation or clustering was scored on a scale from 0 to 8 where a score of 0 indicates less than 10% of cells were in small clusters, a score of 1 indicates 10-50% of the MDDC were clustered in small clusters, a score of 2 indicates 10-50% of the MDDC were clustered in large clusters, a score of 3 indicates 51-75% of the MDDC were in small clusters, a score of 4 indicates 51-75% of the MDDC were in

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27 large clusters, a score of 5 indicates 76-99% of the MDDC were clustered in small clusters, a score of 6 indicates 76-99% of the MDDC were clustered in large clusters, a score of 7 indicates 100% of the MDDC were clustered in small clusters, and a score of 8 indicates 100% of the MDDC were clustered in large clusters. Table 1 Homotypic cluster scoring system Score Percent of Clusters Size of Clusters 0 < 10 % small 1 10-50% small 2 10-50% large 3 51-75% small 4 51-75% large 5 76-99% small 6 76-99% large 7 100% small 8 100% large

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28 Figure 7. Homotypic cluster scoring. Light microsco py images depicting single cells, small and large clusters. Quantification of cytokines by multi-plex bead assay. Cell culture supernatants from all experiments were collected, centrifuged and stored at -20oC until cytokine analysis was completed. Supernatant samples were analyzed using human LINCOplex kits (Millipore, Billerica, MA, USA) multi-plex bead technology, which allows the analysis of multiple cytokines from one sample. The assay is based on characterization beads with a distinct emitting fluorescence pattern that are coated with capture antibodies specific for individual cytokines. A total of 7 cytokines were measured in NHBE and MDDC supernatants; they include: interleukin 6 (IL6), IL8, IL10, IL12p70, IL13, IFN inducible protein 10 (IP10) and tumor necrosis factor alpha (TNF ). A total of 6 cytokines were measured in mixed lym phocyte reaction (MLR) supernatants; they Large cluster Small cluster Single cell

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29 include: IL2, IL4, IL5, IL10, IL13, and IFN Table 2 depicts each cytokine and the experiment in which it was collected along with the basic functional classification. Cytokines were measured according to the manufacturer's instructions. Briefly, 25 L aliquots of medium samples were incubated with antibody coated capture beads for 1 hour, washed and incubated with biotin-labeled anti-human cytokine antibodies for 2 hours at room temperature followed by inc ubation with streptavidin-phycoerythin for 30 minutes. Samples were analyzed using Lum inex 100 IS System and IS 2.3 software (Luminex, Austin, TX, USA). Complete culture medium was used as the blank and added to all standards and quality controls. Each sample was assayed in duplicate and data were presented as the average. Standard curves of known concentrations of recombinant human cytokines (provided by the manufacturer) were used to convert mean fluorescence intensities to cytokine concentration in pg/mL by using 4 or 5 parameter logistic regression.

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30 Table 2 Cytokine and chemokine analysis Name Th1/Th2 Experiment Interleukin 2 (IL2) Th1 MLR Interleukin 4 (IL4) Th2 MLR Interleukin 5 (IL5) Th2 MLR Interleukin 6 (IL6) Pro-inflammatory NHBE+MDDC-EC Interleukin 8 (IL8) chemokine NHBE+MDDC-EC Interleukin 10 (IL10) Th2 NHBE+MDDC-EC MLR Interleukin 12p70 (IL12p70) Th1 NHBE+MDDC-EC Interleukin 13 Th2 MLR Interferon gamma (IFN ) Th1 MLR IFN -inducible protein-10 (IP10) or CXCR3 Th1 Chemokine NHBE+MDDC-EC Tumor necrosis factor alpha (TNF ) Th1 or Th2 NHBE+MDDC-EC Determination of viability and phenotypic maturation of MDDC by immunofluorescence labeling and flow cytometry. For phenotypic characterization of MDDC and NHBE cells following co-culture, MDDC were discriminated from EC by morphology (forward and side scatter). Flow cytometry was used to determine viability and the expression of cell surface maturation markers in MDDC and co-cultures of MDDC and NHBE cells exposed to CSC or poly I:C. Viability was determined by the addition of 7-Amino-Actinomycin D, 7-AAD (BD Biosciences Pharmingen) stain to identify non-viable cells during immunostaining. In order to phenotype MDDC, cells were stained with fluorochrome conjugated cell surface antibodies including: fluorescein isothiocyanate (FITC)conjugated CD80 and CD83, phycoerythrin (PE)-conjugated

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31 CD11c and CD86, allophycocyanin (APC)-conjugated HLA-DR and CD54 (BD Biosciences Pharmingen; Milteynyi Biotec). Table 3 depicts surface markers used to phenotype mature or activated MDDC. Harvested MDDC were aliquoted into two tubes at approximately 2x105 cells per tube and washed. Each tube was stained with three conjugated Abs at the concentration recommended by the manufacturer. After surface staining, MDDC were washed with and fixed in PBS containing 4% paraformaldehyde (PFA), resuspended in stain buffer, protected from light and refrigerated at 4oC until flow analysis was performed. Flow cytometry was performed on a Becton Dickinson Canto II and LSR II Immunocytometry System. Expression of cell surface markers was determined by average mean fluorescence intensity (MFI). Poly I:C treatment (50 g/ml for 24 hours) served as a positive control for MDDC maturation. Unstained cells and single stained cells were used to determine nonspecific binding and background auto-fluorescence. Determination of functional maturation of MDDC by endocytosis assay. Since mature DC exhibit a loss of endocytic acti vity, an endocytosis assay was performed to confirm MDDC maturation. MDDC (5x105/mL) cultured alone or in co-culture with NHBE cells were exposed for 24 hours to CSC, poly I:C, CSC and poly I:C or untreated. Following exposure, MDDC were harvested by pipetting, washed, re-suspended in complete RPMI medium and incubated on ice for 30 minutes. FITC-dextran (molecular weight 70,000, Molecular Probes) was added at a final concentration of 1mg/mL and exposed MDDC were incubated for 1 hour at 37oC or on ice (background staining). Following incubation, cells were immediately put on ice, washed and fixed with 4% paraformaldehyde, re-suspended in staining buffer and analyzed by flow cytometry (BD

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32 FACS Canto). Immature MDDC will endocytose the FITC-dextran and mature MDDC will not. Data were expressed as MFI after subtracting for background staining. Table 3 Dendritic cell surface marker analysis. Biological markers used for dendritic cell characterization Marker Immature Mature Function Class CD11c +++ +++ dendritic cell marker phenotypic HLA-DR ++ +++ T cell receptor ligand phenotypic CD80 + +++ co-stimulatory molecule phenotypic CD83 + ++ co-stimulatory molecule phenotypic CD86 + +++ co-stimulatory molecule phenotypic CD54 (ICAM1) ++ +++ adhesion molecule phenotypic Phagocytosis +++ -antigen uptake functional MLR -+++ T cell proliferation functional Note. CD = cluster of differentiation; HLA-DR = human leukocyte antigen; + = minimal expression; ++ = moderate ex pression; +++ = high expression; -= no expression. Proliferation and polarization of allogeneic CD4+ TC in response to CSC exposed and/or poly I:C stimulated MDDC. To determine the effect of CSC and poly I:C on DC-induced TC proliferation, MLR were performed with allogeneic (different donors) responder CD4+ TC confirmed by flow cytometry (FACS Canto II). Untouched human T helper cells were isolated from human blood buffy coats (Florida Blood Services)

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33 following isolation by Ficoll-Paque and density gradient centrifugation and MACS cell suspension kit, CD4+ T cell isolation kit II (Miltenyi Biotec). The PBMCs were depleted of non-CD4+ TC including, CD8+ TC, gamma delta ( / TC, B cells, natural killer cells, DC, monocytes, granulocytes, and erythroid cells. In order to deplete these cell types, PBMCs were indirectly magnetically labeled using a cocktail of biotin-conjugated antibodies against CD8, CD14, CD16, CD19, CD36, CD56, CD123, TCR / and CD235a (glycophorinA) according to the manufacturer's instructions. The negative fraction was confirmed as the CD4+ population by double positive staining for CD3 and CD4 by flow cytometry analysis. Immature MDDC (5x105) were exposed alone or in co-culture with NHBE cells for various culture times and doses of CSC and Poly I:C in complete medium supplemented with IL4 and GMCSF. Exposed MDDC were washed in fresh medium and treated with mitomycinC (20 g/mL, 45 min at 37oC) to prevent proliferation. TC were treated with carboxyfluoroscein succinimidyl ester (CFSE; Molecular Probes, Invitrogen) in order to determine proliferation. The protocol for CFSE staining is based on methods described previously (Quah, Warren, & Parish, 2007). In order to determine optimal MDDC to TC ratios, 3 different ratios of MDDC in 100 L of medium were analyzed while the number of TC remains constant (2x105). Ratios were assayed in triplicate, including 1:8, 1:16, and 1:32. MDDC were co-cultured with responder allogenic TC (100 L of 1x105 cells/mL) for 4-5 days in 96-well round bottom microplates. MLR cell culture supernatants were collected for cytokine analysis (as described previously) in order to determine TC polarization as Th1 (IL2, IFN TNF ) or Th2 (IL4, IL5, IL10) as described in Table 1. TC were harvested and analyzed by flow cytometry.

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34 Controls for this assay included: unstained (no CFSE) treated TC, TC cultured alone, TC-treated with mitomycin C. The positive control was MDDC cultured with TC and stimulated with a mitogen, phytohemmaglutinin (PHA) at a concentration of 5 g/mL. The assay works by CFSE passively diffusing into the TC. The CFSE undergoes esterase cleavage of the acetate groups and diffuses throughout the cytoplasm where it reacts with amines forming highly fluorescent conjugates that are well retained. As the cells divide, the CFSE is split equally between the daughter cells resulting in diminished CFSE signal detection that occurs with each subsequent cell division allowing for a quantitative determination of the number of cell divisions. Data Analysis In this 4x2 factorial design study, experiments were performed with MDDC and TC derived from at least four individual donors. Data were reported as mean and standard error of the mean. Each of the dependent variables were transformed by base 10 logarithm except for DC viability (percent live), homotypic clustering (cluster score 08) and TC proliferation (percent proliferation); in these three cases, raw data was used. Log transformation was performed in order to normalize data. Each donor exhibited similar trends in data but a variation in magnitude between donors was observed. Paired ttests were conducted for two planned comparisons (see Figure 8), including DMSO vehicle control versus CSC high dose (CSCH) in non-stimulated and poly I:C stimulated co-cultures. In order to determine level of significance and reduce the likelihood of a Type I error, critical alpha values were determined using a modified Bonferroni correction (Holland & Copenhaver, 1988). Briefly, each test of significance was ranked. The smallest observed p value was compared against a critical alpha value of p = 0.025 (alpha 0.05 divided by the number of planned comparisons = 2). The

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35 second test was compared against a critical alpha value of p = 0.05. In addition, eta squared ( t2 / t2 + degrees of freedom) values are reported to display the effect size for each dependent variable. All statistics were performed with the Statistical Package for Social Sciences (SPSS version 15) and fi gures constructed with GraphPad Prism (Version 3.03). In chapter four is a summary of results obtained in this study is presented. Pairwise Comparisons MediumPoly I:C 0 1 2Control DMSO CSCL CSCH A BDependent Variable Figure 8. Pairwise comparisons for dependent variables.

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36 Chapter Four Results Characteristics of the Sample Buffy coats from a total of four individual donors were used in this study to isolate MDDC and TC. Identifying data about each donor was de-identified by Florida Blood Services and only age and gender were given. Two of the four donors were female and the average age was 38 and ranged from 21 to 60 years (21, 21, 50 and 60 years). Identifying data about the NHBE donor was de-identified by Lonza (Walkersville, Inc.) and only age and gender were given. The NHBE donor was a 2 year old male. Overall descriptive statistics of the raw data for each dependent variable are displayed in Table 4.

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37 Table 4 Descriptive statistics of all dependent variables Unit of Measure N Minimum Maximum Mean SEM Medium Viability % Live 16 86.20 98.90 97.05 0.78 Cluster Score 0-8 32 0.00 5.00 2.78 0.22 Phagocytosis MFI 16 17223 54312 30055 2912 CD11c MFI 16 40783 67670 54132 2026 HLA-DR MFI 16 995 2573 1640 137 CD80 MFI 16 10893 39214 19305 2016 CD83 MFI 16 6535 20412 11142 969 CD86 MFI 16 13740 43733 25712 2301 CD54 MFI 16 28699 115052 69096 6603 Interleukin 6* pg/ml 16 0.00 16.13 6.76 1.22 Interleukin 8* pg/ml 16 0.00 1287 123 86.13 Interleukin 10* pg/ml 16 0.00 178 52.77 12.79 Interleukin 12p70* pg/ml 16 0.00 6.68 0.79 0.54 Tumor Necrosis Factor pg/ml 16 0.00 7.70 2.18 0.84 IFN-Inducing Protein 10* pg/ml 16 0.00 17.43 2.23 1.20 T Cell Proliferation % Proliferation 16 23.47 55.82 37.82 2.94 Interleukin 2 pg/ml 16 11.88 89.02 39.78 5.99 Interferon pg/ml 16 35.27 3841 591 239 Interleukin 4 pg/ml 16 32.80 725 173 43.60 Interleukin 5 pg/ml 16 11.27 286 88.79 20.69 Interleukin 10 pg/ml 16 140 3613 986 233 Interleukin 13 pg/ml 16 56.63 2329 860 205 Poly I:C Viability % Live 16 73.25 96.77 90.70 1.69 Cluster Score 0-8 32 3.00 6.00 4.81 0.18 Phagocytosis MFI 16 457 7952 2531 509 CD11c MFI 16 44360 72549 62684 2399 HLA-DR MFI 16 1092 2457 1741 109 CD80 MFI 16 29630 92959 51408 5295 CD83 MFI 16 4458 39609 30029 224 CD86 MFI 16 11005 109085 81333 5939 CD54 MFI 16 74106 131152 100183 4475 Interleukin 6* pg/ml 16 529 3131 1837 211 Interleukin 8* pg/ml 16 0.00 5178 1891 363 Interleukin 10* pg/ml 16 15.57 3176 1009 262 Interleukin 12p70* pg/ml 16 50.14 5935 1199 450 Tumor Necrosis Factor pg/ml 16 86.75 1957 492 137 IFN-Inducing Protein 10* pg/ml 16 0.00 8797 2329 716 T Cell Proliferation % Proliferation 16 33.44 64.92 49.02 2.60 Interleukin 2 pg/ml 16 21.31 286 100 22.01 Interferon pg/ml 16 130 5022 1432 395 Interleukin 4 pg/ml 16 30.5 611 187 43.35 Interleukin 5 pg/ml 16 8.85 392 119 25.00 Interleukin 10 pg/ml 16 261 3984 1424 283 Interleukin 13 pg/ml 16 63.89 3667 1128 253 Note. = Co-culture values minus EC only cultur e values; SEM = standard error of the mean.

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38 Experimental Conditions Do Not Affect DC Viability In order to determine that CSC effects were not related to a loss in DC viability experiments were conducted using 7AAD staining, analyzed by flow cytometry and percent viable/live was calculated (Figure 9). Descriptively, it appears there is no difference in DMSO vehicle versus CSCH in both non -stimulated and poly I:C stimulated co-cultures (Table 5). MediumPoly I:C 0 25 50 75 100Control DMSO CSCL CSCH % Live Figure 9. Dendritic cell viability. Data presented as mean + SEM. (7AAD staining) Table 5 Dendritic cell viability Comparison (DMSO vs CSCH) Mean 1 (DMSO) Mean 2 (CSCH) Medium 98.4 + 0.27 94.2 + 2.7 Poly I:C 93.9 + 0.28 88.4 + 3.8 Note. Data are presented as mean percent live + SEM.

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39 CSC Induces Homotypic Clustering in Non-Stimulated Co-Cultures None of the previous human in vitro studies described DC homotypic clustering following smoke exposure. Homotypic clustering is the ability of DC to up-regulate adhesion molecules and cluster together and is an indicator of DC maturation (Delemarre et al., 2001). In order to determine if smoke exposure induced homotypic clustering, light microscopy images (4x) were obtained following smoke and poly I:C exposures and scored as previously descri bed. A representative donor was chosen and typical clustering can be seen in Figure 10B. A total of 8 individual donors were used to calculate homotypic clustering and two blinded investigators scored each condition based on a scoring system (Table 1) and scores were averaged. Co-cultures that were not stimulated with poly I:C had a significant increase in cluster score secondary to CSC exposure (2.31 + 0.47 versus 3.62 + 0.26), t (3) = -3.19, p <0.025 (twotailed) (Figure 10C and Table 6). In poly I:C stimulated co-cultures, clusters were larger and accounted for a higher percentage of DC compared to no poly I:C stimulation; however, there was no effect of CSC exposure (4.88 + 0.44 versus 4.75 + 0.37), t (3) = 0.73, p >0.050 (two-tailed), as depicted in Figure 10C and Table 6. A two-tailed test was performed because it was unknown if CSC or poly I:C would reduce or increase clustering.

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40 Figure 10. Dendritic cell homotypic clustering. Light microscopy images: A. Single dendritic cell (40x); B. small cluster (20x); C. large cluster (10x); D. Light microscopy images (4X) of all experimental conditions from a representative donor ; E. Cluster score, horizontal bar represents mean, = p value < critical al pha, 0.025 (two-tailed), n = 8. Control Medium Poly I:C DMSO CSCL CSCH Medium ControlDMSOCSCLCSCH 0 1 2 3 4 5 6 7 8 *Cluster ScoreD E Poly I:C ControlDMSOCSCLCSCH 0 1 2 3 4 5 6 7 8Cluster ScoreA B C

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41 Table 6 Dendritic cell homotypic clustering Comparison (DMSO vs CSCH) Mean 1 (DMSO) Mean 2 (CSCH) eta2 t Critical p Observed p Medium 2.31 + 0.47 3.62 + 0.26 0.59 -3.19 0.025 0.015* Poly I:C 4.88 + 0.44 4.75 + 0.37 0.02 0.73 0.050 0.732 Note. Data presented as mean cluster score + SEM. = p value < critical alpha 0.025, (twotailed), n = 8. CSC Reduces Phagocytosis in Non-Stimulated Co-Cultures None of the previous human in vitro studies described phagocytosis of DC following smoke exposu r e. Phagocytosis or the ability for DC to take up antigen is another indicator of DC maturation and function. When a DC has taken up antigen and is mature, it will no longer phagocytize antigen or particulates; therefore, mature or maturing DC will not take up the FITC-dextran and will have lower mean fluorescence intensity (MFI). In order to determine if smoke exposure affected phagocytosis, FITCdextran uptake was analyzed following CSC and poly I:C exposure. The evaluation of FITC-dextran phagocytosis demonstrated an i nhibition following CSC exposure in nonstimulated co-cultures (4.50 + 0.08 versus 4.34 + 0.08), t (3) = 6.13, p<0.025 (two-tailed) (Figure 11 and Table 7). Poly I:C stimulated co-cultures served as a positive control for MDDC maturation and exhibited very low MFI compared to non-stimulated co-cultures and there was no significant effect of CSC in poly I:C stimulated co-cultures (3.25 + 0.12 versus 3.41 + 0.22), t (3) = -1.38, p<0.025 (two-tailed). A two-tailed test was performed because it was unknown if CSC or poly I:C would reduce or increase phagocytosis.

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42 MediumPoly I:C 3 4 5Control DMSO CSCL CSCH *log Phagocytosis MFI Figure 11. Dendritic cell phagocytosis. Data we re transformed by base 10 logarithm and presented as mean + SEM. = p value < critical alpha (two-tailed), n = 4. Table 7 Dendritic cell phagocytosis Comparison (DMSO vs CSCH) Mean 1 (DMSO) Mean 2 (CSCH) eta2 t Critical p Observed p Medium 4.50 + 0.08 4.34 + 0.08 0.93 6.13 0.025 0.009* Poly I:C 3.25 + 0.12 3.41 + 0.22 0.39 -1.38 0.050 0.261 Note. Data were transformed by base 10 logarithm and presented as mean + SEM. = p value < critical alpha (two-tailed), n = 4. CSC Induces Maturation-Associated Co-Stimulatory and Adhesion Molecules Surface markers or co-stimulatory markers are essential in inducing a TC response and are often used as an indicator of DC maturation. Only one study has investigated the effects of smoke on DC surface expression markers in vitro and in that study, DC were cultured alone (Vassallo et al., 2005). This study explored the effect of smoke on surface expression markers in MDDC and NHBE co-cultures. To address this

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43 question, co-cultures were exposed to two doses of smoke, stimulated with poly I:C and surface marker expression was observed wi thin the CD11c+ MDDC population. The percent of CD11c+ MDDC ranged from 98.94% to 99.88% in all experimental conditions and all donors (n = 4). As shown in Figure 12 and Table 8, smoke did not have an effect on CD11c or HLA-DR expression in either stimulated or non-stimulated cultures. Although the previous study found a reduction in surface maturation markers when DC were exposed to smoke, another study investigating DEP reported an increase in CD80, CD83 and CD86 when DC were co-cultured with EC (Bleck et al., 2006). Therefore, it was speculated that maturation markers may be increased in co-culture conditions. As shown in Figure 13, smoke exposure signi ficantly increased markers CD83 (3.96 + 0.02 versus 4.34 + 0.03), t (3) = -11.86, p<0.025 (two-tailed) in non-stimulated co-cultures. CD80 and CD86 followed a similar pattern but failed to reach statistical significance (Table 8). This upregulation of surface maturation markers was confirmed in MDDC derived from four different donors. No study has examined the effect of smoke on DC expression of CD54 in vitro but previous reports demonstrated a r eduction in CD54 expression in alveolar macrophages and circulating monocytes of smokers (Lensmar, Elmberger, Skold, & Eklund, 1998; Luppi, Lain, Jeyabalan, & DeLoia, 2007; Skold, Lundahl, Hallden, Hallgren, & Eklund, 1996). Therefore, it was speculated that smoke exposure may have an effect on DC CD54 expression. As shown in Figure 12 and Table 8, smoke exposure significantly increased CD54 expression in non-stimulated co-cultures (4.68 + 0.08 versus 4.98 + 0.05), t (3) = -9.15, p<0.025 (two-tailed). In all poly I:C stimulated cocultures, smoke did not induce significant changes of any DC surface markers (Table 8). A one-tailed test was performed because it was unknown if CSC or poly I:C would reduce or increase clustering.

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44 Figure 12. Dendritic cell surface markers: Controls, CD11c and HLA-DR. A. Unstained controls for each fluorochrome (FITC, APC, PE), B. Flow cytometry histograms fr om a representative donor; C. MFI data were transformed by base 10 logarithm and presented as mean + SEM, n=4. Medium Poly I:C CD11c HL A -DR DMSO US CSCH US DMSO US DMSO CSCH A B C MediumPoly I:C 4.5 5.0log CD11c MFI MediumPoly I:C 2.5 3.0 3.5 4.0log HLA-DR MFIControl DMSO CSCL CSCH

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45 Figure 13. Dendritic cell surface markers: CD80, CD 83, CD86 and CD54. A. Flow cytometry histograms from a representative donor; B. MF I data were transformed by base 10 logarithm and presented as mean + SEM, = p value < critical alpha (two-tailed), n=4. CD80 CD83 CD86 CD54 Medium Poly I:C DMSO US DMSO CSCH A B Control DMSO CSCL CSCH MediumPoly I:C 4 5log CD80 MFI MediumPoly I:C 3 4 5log CD83 MFI MediumPoly I:C 4.0 4.5 5.0log CD86 MFI MediumPoly I:C 4.0 4.5 5.0 5.5log CD54 MFI

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46 Table 8 Dendritic cell surface markers Comparison (DMSO vs CSCH) Mean 1 (DMSO) Mean 2 (CSCH) eta2 t Critical p Observed p Medium CD11c 4.73 + 0.05 4.78 + 0.03 0.38 -1.36 0.050 0.265 HLA-DR 3.17 + 0.04 3.22 + 0.03 0.63 -2.27 0.025 0.108 CD80 4.18 + 0.06 4.42 + 0.09 0.72 -2.78 0.025 0.069 CD83 3.96 + 0.02 4.23 + 0.03 0.98 -11.86 0.025 0.001* CD86 4.33 + 0.08 4.51 + 0.04 0.78 -3.24 0.025 0.048 CD54 4.68 + 0.08 4.98 + 0.05 0.97 -9.15 0.025 0.003* Poly I:C CD11c 4.78 + 0.09 4.82 + 0.06 0.63 -2.25 0.025 0.110 HLA-DR 3.25 + 0.06 3.23 + 0.06 0.27 1.05 0.050 0.372 CD80 4.68 + 0.08 4.70 + 0.12 0.02 -0.25 0.050 0.819 CD83 4.50 + 0.04 4.53 + 0.05 0.10 -0.60 0.050 0.595 CD86 4.93 + 0.04 4.92 + 0.05 0.02 0.26 0.050 0.815 CD54 5.00 + 0.04 4.99 + 0.05 0.34 1.26 0.050 0.298 Note. MFI data were transformed by base 10 logarithm and presented as mean + SEM. = p value < critical alpha (two-tailed), n = 4. CSC Failed to Significantly Affect Dendritic Cell Cytokines Poly I:C stimulated co-cultures produced substantial amounts of all cytokines and chemokines assayed as compared to non-st imulated cultures. Although none of the DC cytokines were statistically different, two cytokines approached statistical significance, IL10 in both poly I:C and non-stimulated co-cultures and interferon inducing protein 10 (IP10) in poly I:C stimulated co-culture s (Figure 14 and Table 9). Smoke exposure increased IL10 suggesting that smoke induces DC Treg polarizing signal that could contribute to immunosuppressive effects. IP10 was also increased in poly I:C stimulated cultures which is an important chemoattractant of Th1 lymphocytes and is an appropriate response to a viral stimulus.

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47 Figure 14. Dendritic cell cytokines and chemokines. Data were transformed by base 10 logarithm and presented as mean + SEM, n=4. MediumPoly I:C 0 1 2 3 4log IL6 pg/ml MediumPoly I:C 0 1 2 3 4log IL8 pg/ml MediumPoly I:C 0 1 2 3 4log IL12p70 pg/ml MediumPoly I:C 0 1 2 3 4log IP-10 pg/ml MediumPoly I:C 0 1 2 3log TNF pg/mlControl DMSO CSCL CSCH MediumPoly I:C 0 1 2 3 4log IL10 pg/ml

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48 Table 9 Dendritic cell cytokines and chemokines Comparison (DMSO vs CSCH) Mean 1 (DMSO) Mean 2 (CSCH) eta2 t Critical p Observed p Medium Interleukin 6 0.91 + 0.14 0.98 + 0.09 0.23 -0.95 0.025 0.207 Interleukin 8 0.69 + 0.69 0.78 + 0.78 0.00 -0.07 0.050 0.475 Interleukin 10 1.36 + 0.25 1.71 + 0.35 0.69 -2.61 0.050 0.040 Interleukin 12p70 0.00 + 0.00 0.00 + 0.00 ----Tumor Necrosis Factor 0.44 + 0.26 0.23 + 0.23 0.23 0.94 0.050 0.208 Interferon-Inducible Protein 10 0.32 + 0.32 0.41 + 0.25 0.07 -0.46 0.050 0.338 Poly I:C Interleukin 6 3.17 + 0.14 3.21 + 0.11 0.05 -0.43 0.050 0.349 Interleukin 8 2.78 + 0.37 3.54 + 0.08 0.54 -1.88 0.025 0.079 Interleukin 10 2.37 + 0.47 2.74 + 0.39 0.75 -2.97 0.025 0.029 Interleukin 12p70 2.54 + 0.38 2.56 + 0.38 0.03 -0.31 0.025 0.389 Tumor Necrosis Factor 2.33 + 0.26 2.63 + 0.21 0.37 -1.33 0.025 0.138 Interferon-Inducible Protein 10 1.97 + 0.70 3.82 + 0.04 0.70 -2.62 0.025 0.040 Note. -= unable to calculate; Data were transformed by base 10 logarithm and presented as mean + SEM; critical p value (two-tailed). CSC Failed to Induce TC Proliferation but Enhances Th2 Cytokine Production TC proliferation is an indicator of DC ability to induce an immune response to invading pathogens. Smoke exposed and poly I:C stimulated MDDC were co-cultured with allogeneic, CFSE stained CD4+ TC in mixed lymphocyte reactions (DC:TC ratios of 1:8, 1:16, 1:32) for 4-5 days. Figure 15A shows stained, unstained, non-proliferated and proliferated (PHA stimulated) controls and Figure 15B displays a representative donor of TC proliferation (1:16 ratio). This study showed that despite evidence that CSC exposed DC are maturing (increased clustering, decreased phagocytosis, increased maturation markers), they do not significantly increase TC proliferation (Figure16). Figure 16 and Table 10 represent pooled data from four experiments at a ratio of 1:16 (1:8, 1:32 data

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49 not shown). There was no effect of smoke on TC proliferation in non-stimulated or poly I:C stimulated co-cultures and no trend was observed at any DC:TC ratio. Multi-plex bead assays were used to assess TC polarization by examining the profile (T regulatory, Th1 or Th2 type) of cytokines that are secreted by the TC cocultured with exposed DC. This study showed no significant effect of CSC on the major Th1 cytokine, IFN but did show a statistically significant increase in the proliferative cytokine, IL2 (1.86 + 0.20 versus 1.94 + 0.29), t (3) = -5.04, p<0.025 (two-tailed) in poly I:C stimulated co-cultures. In addition, several major Th2 cytokines were upregulated secondary to CSC exposure in poly I:C stimulated co-cultures, including: IL5 (1.83 + 0.28 versus 2.13 + 0.25), t (3) = -5.97, p<0.025 (two-tailed), and IL13 (2.75 + 0.28 versus 3.07 + 0.28), t (3) = -5.61, p<0.025 (two-tailed) in poly I:C stimulated cultures. A similar trend was observed in IL4 but it failed to r each statistical significance. One of the primary Treg cytokines, IL10, was upregulated in poly I:C stimulated co-cultures (2.89 + 0.22 versus 3.14 + 0.23), t (3) = -3.30, p<0.050 (two-tailed) and IL10 approached statistical significance in non-stimulated co-cultures (Figure 17 and Table 11). Th1:Th2 ratios were also evaluated and only two significant ratios were observed and only in poly I:C stimulated cultures including, IL2:IL5 and IL2:IL13 (Table 12). CSC treatment significantly increased Th2 type cytokines (IL5 and IL13) despite the Th1 type poly I:C stimulus. In addition, IL10 was increased in both poly I:C stimulated and non-stimulated cultures suggesting smoke exposure induces a Treg and immunosuppressive response. In chapter five, a discussion of the results obtained in this study is presented. In addition, limitations, future directions and the significance to nursing is also discussed.

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50 Figure 15. T cell proliferation: Flow cytometry. A. Proliferated and unproliferated, stained and unstained controls, B. flow cytometry images from a representative donor. Unproliferated Proliferated Unstained Stained Unstained Stained A B Control DMSO CSCL CSCH Medium Pol y I:C

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51 MediumPoly I:C 0 25 50 75 100Control DMSO CSCL CSCH % Proliferation Figure 16. T cell proliferation: Percent proliferation. Data are pres ented as percent proliferation + SEM. Table 10 T cell proliferation: Percent proliferation Comparison (DMSO vs CSCH) Mean 1 (DMSO) Mean 2 (CSCH) eta2 t Critical p Observed p Medium 36.1 + 6.2 38.6 + 5.8 0.58 -2.05 0.025 0.066 Poly I:C 47.8 + 6.5 48.3 + 5.9 0.01 -0.14 0.050 0.899 Note. Data are presented as percent proliferated and presented as mean + SEM; critical p value (two-tailed).

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52 Figure 17. T cell cytokines. Analysis of Th1/T h2 cytokine profiles from mixed lymphocyte reactions. Data were transformed by base 10 logarithm and presented as mean + SEM; = p value < critical alpha (two-tailed), n=4. MediumPoly I:C 1.0 1.5 2.0 2.5 *log IL2 pg/ml MediumPoly I:C 1.5 2.0 2.5 3.0 3.5 log IFN pg/ml MediumPoly I:C 1.5 2.0 2.5 3.0log IL4 pg/ml MediumPoly I:C 1.0 1.5 2.0 2.5log IL5 pg/ml MediumPoly I:C 2.0 2.5 3.0 3.5log IL10 pg/ml MediumPoly I:C 2.0 2.5 3.0 3.5log IL13 pg/ml

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53 Table 11 T cell cytokines: Comparison of T cell cytokine profiles Comparison (DMSO vs CSCH) Mean 1 (DMSO) Mean 2 (CSCH) eta2 t Critical p Observed p Medium Interleukin 2 1.50 + 0.14 1.55 + 0.16 0.24 -0.98 0.050 0.400 Interferon 2.27 + 0.29 2.36 + 0.26 0.69 -2.61 0.025 0.080 Interleukin 4 1.99 + 0.20 2.08 + 0.20 0.26 -1.03 0.050 0.380 Interleukin 5 1.65 + 0.25 1.79 + 0.24 0.54 -1.88 0.050 0.157 Interleukin 10 2.77 + 0.21 2.91 + 0.18 0.80 -3.46 0.025 0.041 Interleukin 13 2.59 + 0.28 2.67 + 0.30 0.15 -0.72 0.050 0.261 Poly I:C Interleukin 2 1.86 + 0.20 1.94 + 0.29 0.89 -5.04 0.025 0.015* Interferon 2.69 + 0.22 3.03 + 0.35 0.51 -1.76 0.050 0.177 Interleukin 4 2.05 + 0.21 2.33 + 0.25 0.74 -2.89 0.025 0.063 Interleukin 5 1.83 + 0.28 2.13 + 0.25 0.92 -5.97 0.025 0.009* Interleukin 10 2.89 + 0.22 3.14 + 0.23 0.78 -3.30 0.050 0.046* Interleukin 13 2.75 + 0.28 3.07 + 0.28 0.91 -5.61 0.025 0.011* Note. Data were transformed by base 10 logarithm and presented as mean + SEM. = p value < critical alpha (two-tailed), n = 4.

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54 Table 12 Th1/Th2 cytokine ratios DMSO (n=4) CSCH (n=4) Contrast p IL2 : IL5 Medium -0.15 + 0.24 -0.24 + 0.22 0.503 Poly I:C 0.03 + 0.27 -0.19 + 0.21 0.012* IL2 : IL13 Medium -1.10 + 0.31 -1.14 + 0.32 0.884 Poly I:C -0.90 + 0.23 -1.14 + 0.27 0.180* IFN : IL5 Medium 0.62 + 0.42 0.58 + 0.24 0.608 Poly I:C 0.87 + 0.27 0.90 + 0.25 0.901 IFN : IL13 Medium -0.33 + 0.51 -0.31 + 0.28 0.894 Poly I:C -0.06 + 0.22 -0.04 + 0.18 0.946 Note. Raw data ratios were transformed by base 10 logarithm and presented as mean + SEM, = p value < critical alpha 0.025, (two-tailed), n = 4.

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55 Chapter Five Discussion Summary of Findings This is the first study to investigate the effects of cigarette smoke in a dendritic/bronchial epithelial cell co-culture model. Since DC posses the ability to sample the bronchial airway, take up smoke particulates and are key mediators of immune response, it was reasoned that DC function and DC-induced TC response may be affected by cigarette smoke. Only two st udies have evaluated the effects of cigarette smoke on human DC function. In these two studies, DC were cultured alone. By culturing DC with smoke-exposed EC, it takes into account the impact of the tissue micro-environment. Therefore, this study provides insight into EC-DC interactions in smoke exposure and may provide evidence for disease and infection susceptibility in cigarette smokers; including how a smoker exposed to viral infection may respond. In the context of smoke exposure only, this study indicates that cigarette smoke has the ability to decrease phagocytosis abi lity of DC and increases co-stimulatory maturation markers without inducing TC proliferation and inducing a potential Treg phenotype (Figure 18A). IL8, a potent neutrophil chemokine was predominantly expressed by EC and was significantly incr eased in smoke exposed, non-stimulated cocultures. This is an important epithelial injury signal that recruits other inflammatory cells into the airway and critical in vivo

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56 DC maturation was significantly affected by CSC through a decrease in phagocytosis ability. No other studies have reported an effect of smoke on phagocytosis ability of DC. However, this reduction in phagocytosis was also observed in another antigen presenting cells, alveolar macrophages of healthy smoking subjects compared with non-smoking controls subjects. In addition, phagocytosis ability was significantly increased in COPD subjects who had quit smoking when compared with COPD current smokers (Hodge et al., 2007). DC maturation was also affected following CSC exposure by an increase in surface co-stimulatory markers, CD83 and CD86. This was not observed in a previous study of the effect s of smoke on MDDC (Vassallo et al., 2005). However, this difference may be attributed to the presence of EC in the co-culture as DEP did not effect maturation markers unless MDDC were co-cultured with EC (Bleck et al., 2006). Together this data suggests that smoke may induce a semi-mature DC phenotype and reduce the ability for DC to sample the airway for antigens. CD54 is another DC surface marker that is a vital component of cell-cell interaction and is also a receptor for rhinovirus. This is the first report that CSC significantly increased CD54 in MDDC. Other studies have investigated the effect of smoke on alveolar macrophages. However, they observed a reduction in CD54 in smokers in BALF and sputum (Lensmar et al., 1998; Skold et al., 1996). It is speculated that CD54 may play a different role in DC versus macrophages. Although CSC did not significantly affect TC proliferation rates, low levels of IL2 may account for the lower TC proliferation observed in non-stimulated versus poly I:C stimulated co-cultures. The lack of TC proliferation and secretion of IL10 by MLR TC suggests a Treg phenotype in non-stimulated, CSC exposed co-cultures. This may be attributed to low IL6, TNF and no IL12p70 expression levels observed in DC cytokine

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57 supernatants. Together, these conditions push nave TC to a Treg phenotype (Hammad & Lambrecht, 2006). One possible explanation for the effects seen in CSC treated MDDC is the concept of tolerance or an alternatively activated DC, which is “a dendritic cell that is activated...resulting in a stable, semi-mature DC that can induce T-cell hyporesponsiveness in vitro ...” (Morelli & Thomson, 2007, p. 612). This would require further investigation of other characteristics of tolerogenic DC such as, expression of programmed cell death ligand 1 (PDL1), production of indoleamine 2,3-dioxygenase (IDO), resistance to innate NK-cell mediated killing and the production of IL10/TGF by TC stimulated by smoke exposed DC (Morelli & Thomson, 2007; Reis e Sousa, 2006). In the context of smoke exposure and poly I:C stimulation, high levels of EC derived IL8 were observed along with high expression of DC maturation markers (CD80, CD83, CD86) adhesion molecule, CD54 and low phagocytosis. All indicating an injured epithelium and activated/mature MDDC capable of producing high levels of IL6, TNF stable IL12p70 and high IFN (Figure 18B). These conditions would normally lead to a Th1 dominant phenotype in MLR TC cytokine expression; however, an overall mixed T helper response was observed. The specific effects of CSC were observed in increased DC IL10 (Treg polarizing) and increased IP-10 (Th1 chemokine). The profile of TC MLR cytokines revealed a mixed picture of high IFN (Th1) and a CSC-induced increase in IL10 (Treg) and Th2 cytokines (IL5 and IL13). Poly I:C is a Th1 stimulus (high IP-10, IL6, TNF and stable IL12p70 expression) capable of inducing IFN production; however, CSC exposure may modulate T hel per cytokine expression to increase both Treg cytokines (IL10) and Th2 cytokines (IL5 and IL13). Although CSC did not significantly affect TC proliferation rates, higher levels of IL2 may account for the overall

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58 higher TC proliferation rate observed in poly I:C stimulated versus non-stimulated cocultures. The mixed presentation in T helper cytokines that was observed in MLR supernatants may be a function of DC to TC ratios. One study reported that high DC:TC ratios (1:4), like the ratio used in this study (1:16), resulted in a mixed Th1/Th2 response while low DC:TC ratios (1:300) resulted in a Th2 phenotype (Tanaka, Demeure, Rubio, Delespesse, & Sarfati, 2000). In addition, MLR reactions were carried out with CD4+ TC, not nave therefore a true picture of TC polarization may not be clear due to the potential presence of all subtypes of T helper cells. Overall, these findings suggest that DC of smokers may be less likely to mount a normal immune response to invading pathogens thus providing evidence for smoker susceptibility to infection. It may also provide insight into how DC of smokers may respond to viral infection. In addition, these findings are particularly important in patients with allergic airway disease since increasing Th2 cytokines would increase the risk for disease exacerbation. Because DC play a critical role in modulating immune response to environmental agents, they have become a primary target for drug development for diseases that are largely mediated by env ironmental triggers, such as asthma and COPD (Kool & Lambrecht, 2007). This study provides basic information that may be used in the development of therapeutic agents.

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59 Figure 18. Summary of findings. A. The effect of cigarette smoke exposure dependent variables; B. The effect of cigarette smoke exposure and poly I:C stimulation on dependent variables; arrows indicate statistically significant increase ( ) or decrease ( ). to LNInn ate DC “ Semi-Mature” MDDC T EC Adaptive increasing IL10 “Maturing” MDDCCSC increasing IL10 no IL12 low IP-10 low IL6 low TNF to LN Innate Mature MDDC T EC Adaptive IL10 IL5 IL13 high IFN Mature MDDCCSC increasing IL10 stable IL12 IP-10 high IL6 high TNF B. Cigarette smoke exposur e and poly I:C stimulation Poly I:C CD83 increasing CD80 increasing CD86 CD54 phagocytosis high CD80 high CD83 high CD86 high CD54 low phagocytosis lower proliferation low IL2 A. Cigarette smoke exposure higher proliferation IL2

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60 Limitations A major strength of this study was the use of cells obtained from human subjects; however this is also a major limitation due to human subject variability a repeated measures analysis approach was used in an effort to control for this variability. One of the perplexing questions regarding COPD is why some smokers develop disease while others do not. In addition, there are mixed phenotypes including chronic bronchitis, emphysema and mixed presentations with asthma. Therefore it is not surprising that some subjects would immunologically respond to smoke while others would not. Also, all donor information was de-identified and there was no information regarding health history, medication use or smoking history and due to the limited sample size (n = 4) it is difficult to make general assumptions regarding the effects of smoke. Gender and age of the subjects may have also played a role in the immune responses investigated and the heterogeneous sample may have contributed to the mixed results. However, the small sample size did not permit investigation of the variation due to these demographic variables. Another limitation is that the donor of the NHBE cells is different than the donor of MDDC and TC. This may present an issue of self versus non-self recognition and result in an artificial immune response. Another drawback of this study was the dose and time of CSC exposure. This study used a single acute exposure and NHBE cells were exposed for approximately 36 hours while MDDC were only exposed for 24 hours. Other studies of DEP exposure in EC-DC co-cultures used a 48 hour time course. Since this study used 24 hours, it may have been too early to detect late phase changes. In addition, length of poly I:C exposure may have limited the amount of IFN secreted from MDDC. In this study, MDDC were exposed to poly I:C for 24 hours and a previous study of smoke and poly I:C exposure in human PBMC showed minimal IFN following 24 hours and a significant

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61 increase observed at 48 and 72 hours (Mian, Lauzon, Stampfli, Mossman, & Ashkar, 2008). Time course experiments need to be conducted. Cigarette smoke consists of two components: vapour and particulate phases. CSC provides a model of the particulate phase of smoke. There is some controversy that CSC is an appropriate model for smoke exposure and the need for a particle only control in order to distinguish specific versus non-specific effects of particulates. Most of the chemicals and nicotine associated with smoke are contained in the particulate phase of smoke. It is this component of smoke primarily responsible for the immunosuppressive effects of smoke (Nouri-Shirazi & Guinet, 2006; Sopori, 2002) and is therefore an appropriate model for in vitro study. One cannot argue the value of controls but due to the thousands of chemicals, including tar and nicotine contained in CSC it can be argued that treatment effects observed are most likely due to chemicals rather than particulates. Future Directions Further investigation into the time course effects of cigarette smoke on DC will be important to understand acute versus late phase immune responses. Other surface markers such as CCR7, CD40/OX40L and cytokines such as TSLP/TGF should also be explored to elucidate the effects of migration, TC stimulation and Th2/Treg induction, respectively. In addition, MLR experiments should be conducted with nave TC at various responder ratios in order to determi ne true TC polarization. Findings from this study can inform clinical research. O ne example would be to isolate MDDC from smokers and non-smokers and those isolated cells exposed to cigarette smoke. EC play a critical role in innate immunity by recognizing invading pathogens and secreting antimicrobial substances such as defensins and inflammatory/chemotactic

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62 mediators such as cytokines, chemokines and leukotrienes (Bals & Hiemstra, 2004). Further study should focus on how smoke exposed EC not only regulate inflammation and the immune response but how they interact with DC through cell to cell contact and soluble mediators (Polito & Proud, 1998; Schleimer et al., 2007; Stick & Holt, 2003). This knowledge will be critical in under standing the pathophysiology of many lung diseases and would provide many potential therapeutic targets. Overall, this study provides a sound basis for future investigation into the complex effect of smoke on DC and lung disease. Significance to Nursing Although this study is based on basic science experiments, much of the knowledge obtained can be applied to nursing education, research and practice. Information obtained in nursing and medical research is shared. In nursing education, one of the foundational courses is pathophysiology which includes immunology concepts therefore information obtained in this study applies to the foundation of nursing education. With regards to nursing research, by nurses becoming familiar with immunology techniques they can incorporate t hese concepts in to clinical research and impact the progress of nursing science. Nursing practice is affected by research through the incorporation of research findings into evidence-based practice. The main goal of all research is to increase knowledge in order to improve our understanding of disease and positively affect patient care. By understanding more about the effects of cigarette smoke not only do we increase pathophysiological knowledge of diseases affected by smoking but we indirectly impact patient care through improved diagnostics, therapeutics, interventions and evaluation.

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75 Appendices

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76 Appendix A: Definitions Adaptive immunity: the ability of an antigen to induce antigen-specific responses in lymphocytes. Adaptive immunity is char acterized by an increase in response with exposure, memory and clonal selection of lymphocytes. Allergen: an antigen that has the ability to induce a hypersensitivity or allergic reaction. Allogeneic: different individuals of the same species. Antigen: any molecule that can bind to an antibody. Some have the ability to stimulate antibody production. Chemokine: small proteins that induce the migration and/or activation of cells. Cigarette smoke condensate (CSC): commercially available particulates obtained from burned cigarettes that are dissolved in dimethyl sulfoxide and contains many chemicals including nicotine and tar. In this study, CSC was used to mimic the particulate phase of smoke. CD4: cluster of differentiation 4 is present on thymocytes, monocytes, macrophages and is a co-receptor of MHC Class II. In this study, T cells used in mixed lymphocyte reactions were CD4 positive. CD11c: cluster of differentiation 11c is present on myeloid cells and binds fibrinogen. In this study, CD11c was used to identify dendritic cells in flow cytometry experiments. CD80: cluster of differentiation 80 is present on dendritic cells and is a ligand for CD28 on T cells. It is important in the activation of nave T cells through co-stimulation (signal 2). CD83: cluster of differentiation 83 is present on dendritic, B and Langerhans’ cells. It is a marker for activated dendritic cells and may assist in antigen presentation or cellular interactions that follow lymphocyte activation

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77 CD86: cluster of differentiation 86 is present on dendritic, monocytes and activated B cells and it is a ligand for CD28 on T cells. It is important in the activation of nave T through co-stimulation (signal 2). CD54: cluster of differentiation 54 is present on hematopoietic and non-hematopoietic cells. Also known as intercellular adhesion molecule (ICAM-1) and is involved in adhesion of neutrophils and is a receptor for rhinovirus. Cluster of differentiation (CD): a monocolonal antibody that identifies surface molecules, used to identify various immune cells. Cytokine: proteins made by cells that affect the function of other cells or the cell itself. Dendritic cell (DC): professional antigen presenting cell and the most potent activator of T and B cells. They are involved in linking the innate and adaptive immune systems and located in tissues exposed to the external environment such as the skin, lungs and intestines. In this study, monocyte-derived dendritic cells isolated from human peripheral blood were used in all experiments. Dimethyl sulfoxide (DMSO): a chemical com pound that is a polar aprotic solvent. It is an excellent solvent and cryoprotectant. In this study, CSC is dissolved in DMSO therefore DMSO is used as a vehicl e control in all experiments. Flow cytometry: a research method that uses the principles of light scattering, light excitation, and emission of fluorochrome molecules to generate specific multiparameter data from particles and cells. It is a technique for counting and examining cells or particles based on size, granularity and fluorescence. Homotypic cluster score: clustering of dendritic cells with each other that is an indicator of upregulation of adhesion molecules and chemokines/chemokine receptors. In this study, the scoring of homotypic clustering is a semi-quantitative score of 0-8.

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78 Human leukocyte antigen DR (HLA-DR): human leukocyte antigen encoded major histocompatibility complex, MHC class II, cell surface receptor ligand for T cell receptor. It is present on antigen presenting cells with the primary function to present peptide antigens to the immune system for the purpose of eliciting an immune response. Innate immunity: earliest host response to invading pathogens such as bacteria, virus and allergens that includes various recognition systems such as toll-like receptors. The innate immune response does not increase with repeated exposure. Interferon gamma (IFN ): cytokine that can induce cells to resist viral replication. It is produced by CD4+ Th1 cells and is a type II interferon and has antiviral, immunoregulatory, and anti-tumour properties. Interferon-inducing protein 10 (IP10): chemokine that selectively attracts Th1 lymphocytes and monocytes, and inhibits cytokine-stimulated hematopoietic progenitor cell proliferation. Interleukin 2 (IL2): a T cell derived cytokine that stimulates growth and differentiation of T cells, B cells, NK cells, monocytes, macrophages. A central cytokine in the development of an adaptive immune response. Interleukin 4 (IL4): a cytokine that is se creted by Th2 cells. It enhances both secretion and cell surface expression of IgE and IgG1. An important cytokine in allergic disease and has overlapping functions with IL13. Interleukin 5 (IL5): secreted by Th2 and mast cells that is a key mediator in eosinophil activation. An important cytokine in allergic disease. Interleukin 6 (IL6): both a pro-inflammatory and anti-inflammatory cytokineimportant mediators of fever and of the acute phase response and secreted in response to

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79 activation of the innate immune system. High IL6 It is associated with both Th1 and Th2 responses and low IL6 is associated with Treg responses. Interleukin 8 (IL8): inflammatory chemok ine produced by many cell types. Mainly functions as a neutrophil chemoattractant. Interleukin 10 (IL10): secreted by Th0 cells blocks cytokine synthesis by Th1 cells Interleukin 12p70 (IL12p70): Th1 polarizing type cytokine secreted mainly by dendritic cells. Induces IFN production and proliferation/differentiation of Th1 cells. Interleukin 13 (IL13): secreted by Th2 cells, involved in the upregulation of IgE secretion by B cells. An important cytokine in allergic disease. Ligand: general term for a molecule recognized by another structure such as a receptor. MHC Class II: major histocompatibility complex class II present on antigen presenting cells with the primary function to present peptide antigens, both self and non-self, to lymphocytes for the purpose of eliciting an immune response. Specifically, MHC class II presents antigen fragments to T-helper cells by binding to the CD4 receptor on the T-helper cells. Mixed lymphocyte reaction (MLR): When lymphocytes for two different individuals are cultured together. In this study, CSC/poly I:C exposed dendritic cells were cocultured with allogeneic CD4+ T cells for 4-5 days. Monocyte-derived dendritic cells (MDDC): dend ritic cells obtained by culturing CD14+ monocytes with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL4). Used in this study as the dendritic cell model. Normal human bronchial epithelial cells (NH BE): commercially available primary cell culture of bronchial epithelial cells from human donors. Used in this study as the bronchial epithelial cell model.

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80 Phagocytosis: the engulfment of particles, bacteria, cell debris, etc. by cells. A characteristic function of cells such as macrophages and dendritic cells. In this study, phagocytosis is assessed by FITC -dextran uptake by dendritic cells and analyzed by flow cytometry. Data is presented as mean fluorescence intensity (MFI). A low MFI is associated with a mature dendritic cell and a high MFI is associated with an immature dendritic cell. Polyinosinic-polycytidylic acid (Poly I:C): a commercially available synthetic double stranded RNA. A toll-like receptor 3 ligand that mimics viral infection and induces a Th1 response. In this study, poly I:C is used as a positive control for dendritic cell maturation Polarizing cytokine: a cytokine that induces a specific phenotype in the target cell. T cells (TC): thymic lymphocytes that developed in the thymus. T cell proliferation: In this study, TC proliferation is assessed by CFSE staining and analyzed by flow cytometry. Data are presented as percent proliferated. Nave T helper lymphocyte (Th0): T lym phocytes that have never engaged a specific antigen. They are different from effector and memory T cells. T helper type 1 lymphocyte (Th1): T lymphocyt e characterized by the cytokines they produce (IFN ). An increase in Th1 cytokines are associated with autoimmune diseases. T helper type 2 lymphocyte (Th2): T lymphocyt e characterized by the cytokines they produce (IL4, IL5, IL13). An increase in Th2 cytokines are associated with allergic diseases. T helper type 17 lymphocyte ( Th17): T lymphocyte characterized by the cytokines they produce (IL17).

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81 Toll-like receptor 3 (TLR3): one of the innat e immune systems recogni tions systems. It is one of the toll-like receptors that recognizes double stranded RNA of viruses. T regulatory lymphocyte (Treg): T lymphocyt e characterized by the cytokines they produce (IL10, TGF ). They have the ability to inhibit T cell responses and induce tolerance. Tumor necrosis factor alpha (TNF ): a pleiotropic inflammatory cytokine involved in apoptotic cell death/proliferation, differentiation, inflammation, tumor growth, and viral replication. It is important in both Th1 and Th2 polarization, acute phase reactions and fever. Viability: to be alive. In this study, viability was assessed by 7-AAD staining and analyzed by flow cytometry. Data are presented at percent live.

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About the Author Alison Jones Montpetit was born and raised in Decatur, Alabama. She attended the University of North Carolina at Wilm ington where she obtained a Bachelor’s degree in nursing in 2001. She practiced nursing in the Medical Intensive Care Unit at New Hanover Regional Medical Center. In 2002, she moved to Tampa, Florida to pursue the BS-PhD program at the University of South Florida. During her tenure, she completed a Master’s in nursing (2004) and attended the Summer Genetics Institute at the National Institute of Nursing Research (2004). She is married to Marty Louis Montpetit, PhD and expects her first child in August of 2008.