Syk inhibition attenuates airway hyperresponsiveness in a murine model of asthma and exacerbation by air pollution

by Patricia Castellanos Penton A thesis submitted in conformity with the requirements for the degree of Master in Science Institute of Medical Science University of Toronto © Copyright by Patricia Castellanos Penton 2012

Syk inhibition attenuates airway hyperresponsiveness in asthma and exacerbation by air pollution Patricia Castellanos Penton Master in Science Institute of Medical Science University of Toronto 2012 Abstract Airway hyperresponsiveness (AHR) is a cardinal feature of asthma that is aggravated by environmental air pollution (EAP). Splenocyte tyrosine kinase Syk has been associated with asthma pathogenesis. Therefore, we sought to investigate the effect of Syk inhibition on AHR and its exacerbation by EAP. For this purpose, we examined Syk protein expression in lung homogenates from three murine models of ovalbumin (OVA)-induced asthma expressing different pathophysiological features of the disease: airway inflammation, AHR and remodeling. Increased Syk expression was observed only in the chronic model of airway inflammation and remodeling. In vivo Syk inhibition attenuates AHR in this model, and further augmentation induced by EAP without affecting the underlying airway inflammation. We demonstrated, for the first time, that Syk inhibition effectively reverted AHR in an already established chronic model of asthma. These findings highlight the therapeutic potential of targeting Syk for the treatment of asthma and its exacerbations by EAP.

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Acknowledgments Foremost, I would like to express my sincere gratitude to my supervisors Dr. Chung-Wai Chow and Dr. Jeremy Scott for their guidance and patience. Thank you for your support and encouragement over these years. I would also like to thank the other committee members, Dr, Jane Batt and Dr. Haibo Zhang, for their comments, questions, and assistance throughout the development of this thesis. I would like to thank the members of my laboratory, who have aided in this work in many ways. I especially want to thank Xiaoming Wang, who performed the in vitro experiments included in this study. Sincere thanks to Sepehr Salehi for his support during these years. I also want to acknowledge Michelle North for her contribution with samples, assistance with pulmonary function testing in mice, and for providing constructive advice during my research. I would like to acknowledge Dr. Nivedita Khanna and Ms. Hajera Amatullah for their assistance with mice sensitization and challenge and quantitative real-time PCR. I would like to show my gratitude to Dr. Krystal Godri and Josephine Cooper for their support during Luminex assay. I am also sincerely grateful to Jui Jani who helped during pulmonary function testing. My sincere thanks to Mike Fila in Dr. Silverman’s laboratory for assisting with air pollution exposures. Finally, I would express a deep sense of gratitude to my family, especially to my parents who even in the distance have always supported and encouraged me. I wish to express my sincere thanks to my husband for his spiritual and emotional support, and endless patience. Many thanks to my friends for all the support and encouragement.

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Table of Contents List of Abbreviations ........................................................................................................ vii List of Tables ..................................................................................................................... ix List of Figures .................................................................................................................... x Chapter 1: Literature Review ............................................................................................ 1 1.1.

Asthma ................................................................................................................. 1

1.1.1.

Characteristics, prevalence and burden of asthma. ....................................... 1

1.1.2.

Development, expression and triggers of asthma ......................................... 2

1.1.3.

Diagnosis of asthma ...................................................................................... 5

1.1.4.

Molecular mechanisms of asthma ................................................................. 6

1.1.5.

Current therapies for asthma ....................................................................... 16

1.2.

Effect of environmental air pollutants in human health ..................................... 18

1.2.1.

Particulate matter (PM) ............................................................................... 19

1.2.2.

Ozone .......................................................................................................... 23

1.3.

Animal models of asthma................................................................................... 25

1.3.1. 1.4.

Mouse models of asthma ............................................................................ 26

Spleen Tyrosine Kinase (Syk) ............................................................................ 28

1.4.2.

Syk in disease pathogenesis ........................................................................ 40

1.4.3.

Syk inhibition in allergic disease and asthma ............................................. 41

Chapter 2: Hypothesis and research strategy ................................................................. 43

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2.1.

General Hypothesis ............................................................................................ 43

2.2.

Research objectives ............................................................................................ 43

2.2.1.

Expression levels and functional role of Syk in three murine models of

airway inflammation .................................................................................................. 43 2.2.2.

Role of Syk in air pollution-induced asthma exacerbation ......................... 44

Chapter 3: Materials and Methods.................................................................................. 45 3.1.

In vivo methods .................................................................................................. 45

3.1.1.

Murine OVA-sensitization and -challenge model of allergic airway

inflammation .............................................................................................................. 45 3.1.2.

PM2.5+O3 exposure, Syk Inhibition, pulmonary function testing and

methacholine challenge ............................................................................................. 47 3.2.

Molecular methods ............................................................................................. 50

3.2.1.

Lung homogenization and Western blotting ............................................... 50

3.2.2.

Histology and immunofluorescence staining of lung tissue sections. ........ 51

3.2.4.

Laser Capture Microdissection ................................................................... 53

3.2.3.

Real Time-PCR ........................................................................................... 53

3.2.5.

Bronchoalveolar lavage .............................................................................. 54

3.2.6.

Luminex Analysis ....................................................................................... 55

3.2.7.

Cell culture and PM stimulation ................................................................. 55

3.2.8.

Enzyme Linked Immunoabsorbance Assay (ELISA) and Luminex Analysis 56 v

3.2.9. 3.3.

Electrophoretic Mobility Shift Assay (EMSA)........................................... 56

Statistical Analysis ............................................................................................. 57

Chapter 4: Results ............................................................................................................ 58 4.1.

Syk expression in the OVA model of allergic airways inflammation................ 58

4.2.

Localization of Syk expression within the lung ................................................. 60

4.3.

Acute administration of NVP-QAB-205 attenuates airways responsiveness to

MCh in established disease ........................................................................................... 62 4.4.

PM2.5+O3 augmented airways responsiveness to methacholine in OVA/OVA

mice, a response that is abrogated by NVP-QAB-205. ................................................. 66 4.5.

Effect of NVP-QAB-205 on airway inflammation ............................................ 68

4.6.

PM2.5 + O3 induced KC and VEGF expression in the BALF of mice. .............. 72

4.7.

PM2.5 activates human airway epithelial cells in vitro and induces Syk-

dependent expression of IL-6 and IL-8 ......................................................................... 73 4.8.

PM2.5 induces Syk-dependent transcriptional factor activation in primary human

airway epithelial cells .................................................................................................... 74 Chapter 5: Discussion ...................................................................................................... 76 Chapter 6: Conclusions ................................................................................................... 90 Chapter 7: Future directions ........................................................................................... 91 Chapter 8: References ...................................................................................................... 93

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List of Abbreviations AHR

airways hyperresponsiveness

ASM

airway smooth muscle

ASO

antisense oligo nucleotides

BAL

bronchoalveolar lavage

BALF

bronchoalveolar lavage fluid

COPD

chronic obstructive pulmonary disease

DEP

diesel exhaust particles

EAP

environmental air pollution

ECM

extracellular matrix

FA

HEPA-filtered air

FEV1

forced expiratory volume in one second

FVC

forced vital capacity

G

tissue damping

HDM

house dust mite

HRV

Human rhinovirus

i.p.

intraperitoneal

Ig

immunoglobulin

IL

interleukin

ITAM

immunoreceptor tyrosine-based activation motif

LCM

laser capture microdissection

MCh

methacholine

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NO2

nitrogen dioxide

NOx

oxides of nitrogen

O3

ozone

OVA

ovalbumin

PBS

phosphate buffered saline

PM

particulate matter

PM0.1

ultrafine particles

PM10

coarse particles

PM2.5

fine particles

RN

newtonian resistance or resistance of the central airways

Rrs

resistance of the total respiratory system

RSV

Respiratory syncytial virus

Th

T-helper cells

VOC

volatile organic compounds

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List of Tables Table 1. The role of different cell populations in the inflammatory response in asthma. 12 Table 2. Functional role of Syk in different cell types ..................................................... 35 Table 3. Syk inhibitors and their applications. ................................................................. 39 Table 4. Ambient monitoring data during in vivo exposures ........................................... 48 Table 5. Exposure and drug treatment conditions of chronic model (OVA/OVA) and control (OVA/PBS) groups. ...................................................................................... 50 Table 6. Primers sequences of Syk and cyclophilin A used for qRT-PCR....................... 54 Table 7. NVP-QAB-205 does not affect respiratory mechanics in the OVA-model........ 64 Table 8. PM2.5 and O3 peak concentrations recorded in real-world settings .................. 81

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List of Figures Figure 1. Mechanisms of inflammation and remodeling in asthma................................. 15 Figure 2. Molecular structure of Syk and isoform Syk B ................................................. 28 Figure 3. Mechanisms of Syk mediated-signaling. ........................................................... 30 Figure 4. Induction of the three OVA-exposure models of allergic airways inflammation and timeline of exposure and interventions. .............................................................. 46 Figure 5. Syk expression is upregulated in the chronic OVA-exposure model of allergic airways inflammation. ............................................................................................... 59 Figure 6. Syk expression is upregulated in the airway epithelia in the chronic OVAexposure model. ......................................................................................................... 61 Figure 7. Laser capture microdissection of lung sections ................................................. 62 Figure 8. Syk inhibition abrogates the airways hyperresponsiveness to methacholine in the chronic OVA-challenge model. ........................................................................... 65 Figure 9. Syk inhibition decreased central airway resistance (RN) but did not affect tissue damping (G). .............................................................................................................. 66 Figure 10. PM2.5+O3 augmented AHR to MCh in OVA/OVA, a response that is abrogated by NVP-QAB-205. ................................................................................... 67 Figure 11. BALF total and differential cell counts were not affected by treatment with NVP-QAB-205 Syk inhibitor. ................................................................................... 69 Figure 12. Neither exposures to PM2.5+O3 nor Syk inhibitor treatment affected inflammatory infiltrates nor presence of PAS-positive cells in the lungs of the OVA/OVA mice. ....................................................................................................... 71 Figure 13. PM2.5+O3 increased KC and VEGF production in BALF ............................. 72

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Figure 14. PM2.5 activates airway epithelial cells and induces Syk-dependent expression of IL-6 and IL-8. ........................................................................................................ 73 Figure 15. PM2.5 induces Syk-dependent transcriptional factor activation in airway epithelial cells. ........................................................................................................... 74 Figure 16. Hypothetical model of Syk- mediated NFkB and NFAT activation in airway epithelial cells in response to PM stimulation. .......................................................... 88

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Chapter 1: Literature Review 1.1.

Asthma

1.1.1. Characteristics, prevalence and burden of asthma. Asthma is a chronic inflammatory disorder characterized by variable and repeated episodes of dyspnea, wheezing, chest tightness and coughing. These symptoms occur as a consequence of a reversible and variable degree of airway obstruction, airway hyperresponsiveness (AHR), and underlying inflammation, sometimes associated with structural changes of the airways [1, 2]. Asthma can be attributable to atopy [3]. However, other factors may contribute to the development of the disease. Over the past decades there has been a dramatic increase in global prevalence, mortality, morbidity, and economic burden associated with asthma, especially in children. Asthma is a major public health concern with an estimated of 235 million affected individuals worldwide [4]. The highest prevalence is observed in western industrialized countries including United Kingdom, Canada, New Zealand, Australia and the Republic of Ireland [5]. In Canada, about 2.5 million adults have asthma [6] while at least 10% of Canadian children are affected with the disease [7]. Although asthma is more common in developed countries, the prevalence of the disease is rapidly increasing in developing countries, with an apparent correlation with increasing urbanization and the adoption of more westernized life styles. In Africa, for example, more than 50 million people suffer from asthma, while in South and Central America and East Asia/Pacific region asthma affects over 40 million individuals respectively [5].

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Significant morbidity and mortality are observed among asthmatic patients. Current worldwide statistics show increasing rates of hospital admission for asthma and about 250,000 deaths annually are attributable to the disease [8]. In Canada, asthma accounts for about 500 deaths each year [9]. The economic costs associated with asthma such as hospital admissions and pharmacological expenditures exceed those of tuberculosis and HIV/AIDS combined [5]. In Canada, for example, asthma exacerbations account for more than 146,000 emergency department visits [10]. The Conference Board of Canada estimates that in 2010 chronic lung diseases, including asthma, cost $3.4 billion in direct health care costs, and 8.6 billion in indirect costs. The latest includes costs associated with absenteeism from school and work [11]. In summary, asthma has a significant burden in patient quality of life and health care cost. Thus it is important to find new treatments to alleviate and control asthma symptoms.

1.1.2. Development, expression and triggers of asthma The origins of asthma and the factors that make some persons more susceptible to its effect are still under investigation. However, asthma is a condition that likely result from the complex interactions between host and environmental factors. Host factors comprise genetic predisposition, obesity and sex, while environmental factors include airborne pollutants, allergens and respiratory infections [12]. A number of studies have indicated that the susceptibility to asthma has an inheritable component. Genetics may also determine the responsiveness of asthmatic

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patients to therapies. To date several genes have been associated with asthma and some specific features. However, linkage studies have shown associations with certain phenotypic characteristics but not necessarily to the pathophysiologic disease process or clinical picture itself [13]. The prevalence of asthma before the age of 14 is higher in boys than in girls [14]. In contrast, during puberty and adulthood, females exhibit greater frequency and severity of asthma episodes [15]. In Canada, for example, asthma affects about 9.8% females and 7.1% males in population aged 12 and over; while in children aged 2-7the prevalence is 11.4 % in males versus 7.9% in females [16]. Apparently, events during puberty drive these changes, but the precise mechanisms have not been fully elucidated. It has been hypothesized that the female sex hormones influence the development and severity of asthma [17, 18]. Other possible explanations for the disparities include increased perception of asthma symptoms [19] and increased AHR [20] in women, as well as differences in asthma management between women and men [21]. Epidemiological investigations have shown that obesity is a risk factor for asthma. Asthma occurs more frequently and is difficult to control in obese individuals. However, to date, the mechanisms that link obesity with the development of asthma are poorly understood [22]. Both outdoor and indoor aeroallergens trigger asthma symptoms [23]. There is evidence of causal relationships between allergens, such as house dust mite [24], cockroach [25], cat [26], and dog [27] dander and mold [28] and the development of asthma in young children. Sensitization to these allergens depends of the length of the exposure, allergen dose, genetics and the child’s age [29]. However, new studies have

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emerged suggesting that under certain circumstances, early exposure to dogs may protect against the occurrence of asthma [30]. Other studies have shown an inverse correlation between exposures to microorganisms and the risk of childhood asthma [31]. To date, there is no conclusive explanation for these diverse outcomes. Viral infections early in life constitute a risk factor for the development of an asthmatic phenotype. The most significant asthma-associated viruses are respiratory syncytial virus (RSV) and human rhinovirus (HRV) [32] which are also linked to disease exacerbation [33]. Tobacco smoke is a well–recognized risk factor for asthma. Prenatal exposure to tobacco smoke may increase the risk of developing asthma early in the childhood [34]. In addition, tobacco smoking triggers exacerbation of asthma [35], while smokers exhibit poorer disease control [36], and response to conventional treatments [37]. Diet and exposure to air pollutants also influence the development and clinical course of asthma. Some studies have suggested that diet during the first years of life plays a role in the development of asthma and atopy. For example, breast fed children appear to have less risk to develop asthma compare to alternative milk fed children [38]. The effect of air pollution on asthma development and exacerbation is reviewed in detail further in this thesis. In summary, complex interactions between host and environmental factors will determine the development of asthma and worsening of symptoms.

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1.1.3. Diagnosis of asthma The diagnosis of asthma requires the evaluation of the patient’s medical and family history. Symptoms, such as episodic dyspnea, wheezy, persistent cough and chest tightness, along with family history of allergies or asthma, strongly suggest an asthma diagnosis. Physical examination of the respiratory function could further evidence the presence of airflow limitation or other features such as hyperinflation if the exam is performed during symptomatic periods [29, 35]. International guidelines recommend both the evaluation of symptoms and objective measurement of variable airway obstruction for an appropriate diagnosis of asthma [29]. Spirometry is the preferred method to measure airflow limitation and reversibility to establish a diagnosis of asthma [29]. During the spirometry test, air flow and volume are measured during a forced expiratory maneuver to determine the forced expiratory volume in one second (FEV1) and forced vital capacity (FVC). Reversibility is assessed by the improvement in FEV1 after inhalation of a short-acting bronchodilator, or following a more effective controller treatment such as glucocorticoids; while variability is assessed by measuring the changes in lung function during the day, from month to month or seasonal [39]. A 12% improvement (or > 200 ml) in FEV1 post bronchodilator therapy is accepted as reversible airflow obstruction and therefore indicative of the presence of asthma [29, 39]. In addition, spirometry is also useful to differentiate between restrictive and obstructive lung disorders. Asthma, for example, is an obstructive lung disease. Therefore, in contrast with restrictive lung diseases, FVC remains close to normal while FEV1 decreases and the ratio FEV1/FVC is decreased [39]. The FEV1/FVC

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ratio of about 0.75-0.80 or higher in children (0.90) is considered normal, while any lower value indicates airflow obstruction [39]. Measuring airway responsiveness to direct challenges, such as inhaled methacholine and histamine [40], and indirect challenges, such as inhaled mannitol [41], or exercise [42], can be used to confirm a diagnosis in patients with asthma symptoms but normal lung function [29]. Methacholine is the agent approved for human use in nonspecific bronchoprovocation challenge testing since it displays lower risk of systemic side effects and less variability [43]. Methacholine responsiveness is calculated as the provocative dose (PC20) that induces a 20% decrease in the force expiratory volume (FEV1) [44]. In healthy subjects PC20 is about 100 mg/ml of methacholine whereas in mild and severe asthma typical PC20 values are 4 and 1 mg/ml of methacholine, respectively [45].

1.1.4. Molecular mechanisms of asthma Airway inflammation, AHR and remodeling are central issues in the pathogenesis of asthma. The underlying molecular mechanisms are complex and involve numerous cell types and their interaction, inflammatory mediators, cytokines, chemokines and growth factors that in turn cause the pathophysiological features of the disease.

Inflammation Asthma is a chronic airway inflammatory disease characterized by the infiltration to the airways of lymphocytes, mast cells, basophils, dendritic cells, macrophages, eosinophils and neutrophils. Inflammation can affect airflow through the production of

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mucus, through the release of inflammatory mediators, and by enhancing susceptibility to bronchospasms [46]. Dendritic cells are a central component for the sensitization and the subsequent immune response to specific allergens in asthma. Located in the airway epithelium and mucosa of the lung [47], they sample antigens (allergen, pollutants, and viruses) from the environment, which are internalized via the IgE receptors [48], pattern recognition receptors and C-type lectin receptors [49] located on their surface. The internalized antigens are then presented to T cells in the lymph node, initiating sensitization and subsequent immune Th2 reaction to the specific immunogen [50, 51]. Two subsets of dendritic cells have been described: CD11c+ (myeloid) and CD11c- (plasmacytoid) [52], which appear to have opposing roles. Myeloid dendritic cells are important for Th2 sensitization and subsequent immune response while plasmacytoid (regulatory subset) dendritic cells take part in the induction of tolerance to allergens [53]. There is a whole body of evidence regarding the role of T cells in initiating and maintaining the allergic response in asthma [54, 55]. An increased presence of CD4+ T helper (Th) cells in the airways, predominantly of the Th2 subtype, is characteristic of asthmatic patients [56]. These cells have the ability to regulate fate, function and localization of several components of the immune system such as eosinophils, mast cells and macrophages, and orchestrate the inflammatory response [57, 58]. IL-4, IL-5, IL-9 and IL-13 are important Th2-associated cytokines for the pathogenesis of asthma [59]. Whereas IL-4 is important for allergic sensitization and “class switching” from IgG to IgE antibodies production by B lymphocytes, IL-13 is responsible for the development of AHR, goblet cells hyperplasia and remodeling, and IL-5 is essential for eosinophil

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differentiation and survival. Lastly IL-9 is associated with eosinophils and mast cells recruitment and activation [60]. Although asthma is categorized as a Th2-driven disease, a distinct subset of CD4 + T helper cells, named Th17 [61], has recently emerged as an important player in the pathogenesis of asthma [62]. IL-17, a Th-17-associated cytokine, has been linked to the development of airway neutrophilia and increased severity of the disease [63, 64]. B-lymphocytes regulate the humoral immunity as they recognize specific antigens. In asthma, B cells become activated upon antigen recognition. Activated B cells, in conjunction with T lymphocytes, are the source of IgE, a type of antibody that plays a major role in the allergic response [65, 66]. Mast cells have long being considered to play a crucial role in the pathophysiology of asthma due to their ability to degranulate in response to IgEdependent [67, 68] and several other non-immunological stimuli. Mast cells are responsible for the acute asthmatic response observed within the first few minutes following allergen challenge [69]. In chronic asthma, they are permanently activated and the source of continuous mediator release and cytokine synthesis [63]. An important insight into the pathogenesis of asthma was the discovery of an increased number of mast cells in the airway smooth muscle of patients with chronic asthma that may adversely affect the growth and function of the airway smooth muscle [70]. Basophils, similar to mast cells, express the IgE receptor FcεRI and degranulate in response to allergen challenge. They produce a wide range of mediators and are a dominant and rapid source of IL-4 in patients with atopic asthma [71, 72].

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Eosinophils are found in high numbers in the airways, sputum and bronchoalveolar lavage fluid of asthmatic patients and are a rich source of mediators with a wide variety of biological effects [73, 74]. Eosinophils were long regarded as the effector cells responsible for much of the pathology of asthma [75]. However, this concept was challenged when anti-IL-5 therapy had little or no impact on the clinical outcomes, although the number of eosinophils in blood and sputum were significantly reduced [76]. In addition, asthma symptoms are observed in patients regardless of the presence (eosinophilic asthma) or absence (non-eosinophilic asthma) of eosinophils into the airways [77]. Eosinophils recruited to the airway in asthma, contribute to the late asthmatic reaction of congestion and mucus hypersecretion. They are a rich source of cytotoxic proteins, lipid mediators, ROS and cytokines that cause epithelial damage and desquamation of cells, contribute to airway remodeling and potentiate bronchoconstriction and airway hyperresponsiveness to smooth muscle agonists [78]. Neutrophils are the first cells recruited to the sites of allergic reaction, hence play a role in clinical presentation, in the development of severe chronic asthma and its exacerbation. Once neutrophils arrive to the site of inflammation they control the recruitment of other cell type including dendritic cells, T cells and more neutrophils through the secretion of chemoattractants such as IL-8, GRO-α, MIP-α and MIP-β. Airway neutrophilia is a characteristic of more severe and aggressive asthma [79]. Similarly, abnormal accumulation of neutrophils in the airways is observed during acute exacerbations and in steroid-resistant asthma [80]. Recent studies have shown that neutrophilic inflammatory phenotype is associated with systemic inflammation in asthma [81].

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Macrophages are the most abundant cells in the alveolar space and conducting airways that are capable of enhancing the inflammatory response [82] through the secretion of cytokines [83], growth factors [84] and leukotrienes. Macrophages are divided in at least two subsets that are classical (M1) and alternatively activated (M2) [85]. M1 macrophages mediate the inflammatory response by the release of cytokines such as IL-1β and the production of reactive oxygen species; while the M2 subset participates in the resolution of inflammation by supressing lung T cells and IgE production [86, 87]. The airway epithelium is the direct physical contact of the lung with the environment and the first line of defense to environmental triggers [88]. In asthma, the airway epithelium is damaged and the epithelial barrier function is compromised [89]. In these circumstances, the damaged airway epithelium exhibits an inadequate repair capacity leading to the release of cytokines and growth factors [90, 91] that contribute to the existing inflammation. Beyond its role as physical barrier, the airway epithelium integrates innate and adaptive immunity by producing of IL-25, IL-33 and TSLP [92, 93]. These cytokines stimulate a Th2 type response [94, 95] and activate mast cells [96] and dendritic cells [97] in asthma. Airway smooth muscle (ASM) plays a multifaceted role in asthma. This tissue regulates the bronchomotor tone and also contributes to the inflammatory milieu of the disease through the expression of cells surface molecules [98] and Toll like receptors [99] that mediates the release of cytokines upon stimulation with environmental pathogens [100]. In addition, in asthma increased number of mast cells and T lymphocytes are localized within the ASM (myositis) [101]. The interaction between T cells, mast cells

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and ASM enhances smooth muscle function and contribute to remodeling; while at the same time, increase mast cells and T cells survival and activation and thus, perpetuates bronchial inflammation [102, 103]. In summary, both immune and structural cells contribute to airway inflammation in asthma. However, despite of the clinical success with the use of anti-inflammatory drugs for asthma control, the global prevalence of asthma continue to growth [104]. Therefore, future investigations are required in order to identify new targets with therapeutic potential.

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Table 1. The role of different cell populations in the inflammatory response in asthma. Effector cell Dendritic cells T lymphocytes

B lymphocytes Mast cells

Basophils

Eosinophils

Role in asthma

Associated mediators

References

Th2 sensitization, promotes adaptive immunity, tolerance Allergic sensitization and immune response, recruitment of secondary effector cells, AHR, remodeling Humoral immune response IgE-mediated immune response, AHR, remodeling

IL-4, IL-6, IL-12, IL10, cysteinyl leukotrienes IL-3, IL-4, IL-5, IL-9, IL-13, TNF-α, GMCSF

[50, 105]

IgE production

[65, 66]

Th2 cells differentiation

Inflammation, AHR, epithelial damage

Tissue damage, inflammation, Neutrophils remodeling Promote a Th1 response, Macrophages and restore lung homeostasis, tissue damage, inflammatory response Promotes Th2 response, recruits and activates Airway inflammatory cells, epithelium perpetuate inflammation Airway smooth AHR, perpetuates inflammation muscle

Histamine, tryptase, prostaglandin D2 (PGD2), LTC4, IL-4, IL-5, TNF-α, IL-13 Histamine, eicosanoids, cysteinyl leukotrienes, IL-4, IL13, IL-6 Eosinophilic granule proteins, IL-4, IL-5, IL-13, IL-1, TGF-β, IFN-γ, IL-8 MMP-9, myeloperoxidase (MPO), ROS, IL-8

[57-59]

[68, 69]

[72, 106]

[75, 107]

[108-110]

IL-17, IL-6, IL1,TNF-α, NO, IL-1β, IL-13, IL-10

[86, 87]

IL-25, IL-33, TSLP, TNF-α, IL-6, IL-8, GM-CSF, EGF, HBEGF, PDGF, TGF-β IL-8,IL-6, IL-1β, IL11, CXCL10, MCP-1, eotaxin, RANTES

[92, 111, 112]

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[113, 114]

Airway hyperresponsiveness Airway hyperresponsiveness (AHR) is a functional feature of asthma that is often in proportion with the underlying disease severity. The term refers to the ease and the degree of airway narrowing induced by chemical or physical stimuli that has little or no effect in healthy subjects [115]. The airway components that affect AHR may be divided into two interdependent categories: persistent and variable. The persistent component of the AHR has been extensively linked to the structural changes that alter the architecture of the airways to make them thicker, less compliant and more narrowed. These features are associated with higher degree of constriction and airway closure when stimulated with contractile agents [115]. The persistent component is mostly found in chronic stages of the disease. The variable component, in contrast, likely reflects the inflammatory processes that accompany asthma, which can be induced by environmental factors and rapidly improved by treatment [115]. The precise mechanisms of AHR are not completely understood but inflammation and remodeling appear to be involved [116]. However, the variability of AHR provides information on the clinical course of asthma and the mechanisms that regulates this process. For example, environmental agents may enhance underlying AHR while asthma treatments may also modify AHR [115]. Therefore, AHR is a crucial outcome to evaluate the effect of asthma triggers and the effectiveness of therapeutic interventions.

Remodeling Airway remodeling in asthma refers to the structural changes observed in the airways of asthmatic subjects including epithelial alterations, subepithelial fibrosis,

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angiogenesis and increased airway smooth muscle mass [101]. There is currently no consensus regarding whether chronic inflammation leads to remodeling or whether structural airway alteration induces chronic inflammation [117]. It has been proposed that chronic inflammatory processes may lead to the structural changes in the airways [101]. However, other studies have found features of airway remodeling in young children with asthma [118, 119] suggesting that remodeling may preceded inflammation. Epithelial alterations is a key feature of airway remodeling in asthma that is characterized by shedding of the epithelium, loss of ciliated cells, goblet cell hyperplasia and upregulation of cytokines and chemokines [1]. In addition, the epithelial barrier function is impaired as a consequence of breakdown of epithelial tight junctions and an aberrant repairing response after injury [120]. Two other prominent features of airway remodeling are subepithelial fibrosis and increased airway smooth muscle (ASM) mass [121]. The first occurs as a result of fibroblast differentiation into myofibroblast that are source of mediators and extracellular matrix (ECM) proteins resulting in increased matrix deposition and fibrosis [90]. The second is considered to be the primary cause of airway obstruction. Most likely it occurs due to ASM cell proliferation (hyperplasia) [122], increase in size (hypertrophy) [101] and ASM migration toward the airway epithelium [117]. In addition, the abnormal increase in the number and size of microvessels [123], and mucus hypersecretion of the mucins MUC5AC and MUC5B by goblet cells [1] are other pathophysiologic features of airway remodeling in asthma. Mechanisms of airway remodeling involve cytokines, chemokines and growth factors release by both inflammatory and structural cells in the airway. TGF-β, Th2 cytokines IL-4, IL-5, IL-9 and IL13, Th17 cytokines IL-17and IL-25, and epithelial-

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derived mediators such as RANTES, IL-8 and eotaxin, and MMPs have been shown to promote airway remodeling. In addition, it is been proposed that epithelial-mesenchyme interactions enhances the remodeling processes [117]. Prevent or reverse deleterious remodeling is an unmet need of current asthma therapies. Thus, remodeling requires further studies in human and animal models of asthma.

Figure 1. Mechanisms of inflammation and remodeling in asthma. Adapted from “Allergen-Specific Immunotherapy of Allergy and Asthma: Current and Future Trends” by François Spertini, 2009, Expert Rev Resp Med., 3(1):37-51. Copyright 2009 by Expert Reviews Ltd.

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1.1.5. Current therapies for asthma The purpose of current asthma therapies is to optimize lung function, prevent acute exacerbations and maintain the quality of life of asthma patients while minimizing the side effect of asthma medications. Anti-inflammatory drugs and bronchodilators are the basis of the most common asthma therapies. The first suppresses the underlying inflammation; while the second act by reverting airway smooth muscle contraction [12, 124]. Medication for asthma can be administered in different ways. Inhalational therapies are preferred because treatment is delivery directly into the airways and the risk of systemic side effect is significantly lower [12]. Glucocorticoids are by far the most effective therapy for controlling asthma [125]. Although glucocorticoids do not cure asthma, they reduce airway inflammation, improve lung function, decrease AHR and reduce the frequency of acute exacerbations [125]. Mechanisms of action of glucocorticoids include activation of genes encoding antiinflammatory secretory leukoprotease inhibitor (SLPI) and MAPK phosphatase-1 (MKP1) that inhibits MAPK pathways. Glucocorticoids are also capable of switching off activating inflammatory genes that code for cytokines, chemokines, adhesion molecules, inflammatory enzymes and receptors via attenuation of NF-κB-associated coactivator activity. Furthermore, glucocorticoids are capable of suppressing of Th2 cells and Th2associated cytokines via inactivation of the nuclear transcription factor GATA3, which regulates the transcription of cytokine genes [124]. Glucocorticoids treatment may be poor or non-responsive in smokers and in patients with severe asthma [126]. Studies have

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shown systemic side effects associated with long-term use of glucocorticoids including easy bruising, adrenal suppression and decreased bone mineral density [12]. β2 –adrenergic agonist are the most effective bronchodilators for asthma. They act as functional antagonist to induce relaxation of airway smooth muscle [12]. The mechanism of action relies on activation of adenylyl cyclase via the stimulatory Gprotein upon β2-adrenergic receptors engagement, which leads mainly to inhibition of myosin light chain and Ca2+ sequestration into intracellular stores, thus inducing relaxation of the smooth muscle [124]. β2 – receptor agonist may also relief bronchoconstriction indirectly by inhibiting the release of bronchoconstriction agents from inflammatory cells, and neurotransmitter from airway nerves [124]. In despite of the effectiveness of β2 –agonists as bronchodilator agents, they are unable to mitigate the underlying chronic inflammation. Therefore, they are usually combined with glucocorticoids [127]. Improvements in pulmonary function, symptoms, and exacerbation of asthma are achieved by interfering cysteinyl leukotrienes pathways. Cysteinyl leukotrienes are lipid mediators produced in asthma that are well known to induce bronchoconstriction, plasma exudation and mucus hypersecretion [128]. Several cysteinyl leukotrienes receptor antagonist have been developed for the treatment of asthma such as the current drug monteluskat [129]. In addition, pharmacological blockers of cysteinyl leukotrienes pathways 5’-lipoxygenase, for example, the enzyme inhibitor zileuton, have also shown therapeutic benefits for asthma treatment [129]. When use alone as asthma controller, leukotrienes modifiers exhibit less efficacy than low doses of glucocorticoids. However,

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leukotrienes modifiers may reduce the dose of inhaled glucocorticoids and may improve asthma control in glucocorticoids-treated patients with severe asthma [12]. Theophylline is a bronchodilator that was traditionally used for the treatment of asthma and is still in use in developing countries [124]. When used in combination with glucocorticoids it enhances the anti-inflammatory effects. However, lower antiinflammatory and bronchodilator properties than glucocorticoids and β2 –agonists respectively has limited Theophylline use. In addition, significant side effects have been reported hence reducing its usefulness [12]. Anticholinergics, such as ipratropium bromide, are antagonist of muscarinic receptors, which block the effect of endogenous acetylcholine. Although they are less potent as bronchodilator than β2-agonists its combined actions exert an additional bronchodilator effect, especially in patients with more severe disease [130]. Current asthma therapies, especially the combined action of inhaled corticoids and β2 –agonists allows adequate levels of asthma control. However, there are some concerns with the use of inhaled corticoids and long-term side effects. Moreover, corticoids do not cure or modify the course of the disease and inflammation recurs as soon as the medication is interrupted [131]. In addition, in patients with steroid-resistant and severe asthma poor levels of asthma control is achieved with current therapies [132]. Therefore, efforts are required to fulfill these unmet needs.

1.2.

Effect of environmental air pollutants in human health The occurrence of major air pollution episodes in the 20th century created public

awareness of the noxious effect of environmental air pollutants in human health. The

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industrial air pollution incidents in Meuse Valley (Belgium 1930) [133, 134], in Donora Valley (Pennsylvania, United States, 1948) [135] and in London (December 1952) [136] are significant examples: In the first two cases, extreme air pollution levels were caused by excessive smog from domestic and industrial furnaces. In the latter case that lasted for 4 days left an estimated of 4000 excess deaths [136]. The health impact of air pollution primarily affects cardiovascular and respiratory systems [137]. In Canada, short and long term exposure to air pollution is estimated to cause 21000 premature deaths in 2008, 620 000 doctor visits and 30 000 emergency room visits with an annual economic impact associated of $8billion [138]. Thus, it is important to understand the effects of air pollution in human health.

1.2.1. Particulate matter (PM) Air pollution comprises a mixture of gases and particles in the air that modifies the natural composition of the atmosphere. According to the Environmental Protection Agency (EPA), the six more common pollutants are ground-level ozone (O3), particulate matter (PM), carbon monoxide (CO), nitrogen oxide (NO), sulfur dioxide (SO2) and lead. These pollutants can be harmful for both human health and the environment [139]. PM consist of airborne particles in solid or liquid state [140]. The chemical composition of PM varies depending on factors such as combustion sources, climate, season, and type of urban or industrial pollution [141]. The major components include volatile and semi-volatile organic compounds, such as polycyclic aromatic hydrocarbons (PAH), nitro-PAH and quinones; as well as transition metals (iron, vanadium, nickel, chromium, cooper and zinc), reactive gases (ozone, peroxides and aldehydes) along with

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carbonaceous material from combustion processes and vehicular emissions, material from biological origin (bacterias, viruses, plant and animal debris) and minerals (soil dust) [141]. PM may also be classified according to its aerodynamic diameter, and by convention, in PM10 (< 10µM) or coarse, PM2.5 (< 2.5µM) or fine, and PM0.1 (