Modulation of nuclear factor b signaling attenuates allergic airway inflammation 2

Upon re-encounter with allergen, or during an asthma attack, the effector memory T cells interact with antigen presenting dendritic cells and can rapidly release pro-inflammatory cytokin

1 Introduction

1.1 Asthma

1.1.1 Epidemiology of Asthma and impetus to develop novel anti- inflammatory

agents

Globally, 300 million people suffer from asthma and the prevalence of asthma still continues

to increase With this rising trend, it is predicted that there will be at least 400 million asthmatic patients by 2025 (Masoli et al., 2004) The prevalence is highest in developed countries — UK (> 15%), USA (~11%) and, Australia (~15%) (Figure 1.1) (Pawankar et al., 2012) The increase in prevalence of asthma could be attributable to urbanization and shift away from “naturalistic” diet and lifestyle as explained by hygiene hypothesis (Fishbein and Fuleihan, 2012) According to the hygiene hypothesis, excessive Th2 response is mediated by the absence of recurrent microbial infection, while Th1 response is mediated by microbial infection Allergies are less common in children growing up in rural environment, particularly

in the farms, as compared to those growing up in urban environment (Ege et al., 2011) Protection against allergy correlates positively with the level of exposure to bacterial and fungal microbes In addition, allergy in children is inversely related to the habit of drinking unpasteurized milk and the exposure to high level of endotoxin in house dust mite (HDM) during infancy (Waser et al., 2007) However, hygiene hypothesis theory has oversimplified the nature of allergic asthma (Fishbein and Fuleihan, 2012; Haschke and Klassen, 2009) Although exposure to microbes may offer children protection against allergy, viral infection predisposes children to wheezing and asthma Rhinovirus-induced wheezing in the first three years of life is the greatest risk factor for developing asthma by six years of age (Holgate, 2012) Several postulations have been made to explain for this controversy These postulations shall be elaborated in later sections Briefly, it is postulated that viral infection increases the sensitivity of airway epithelial cells to allergens (Monick et al., 2003) A disturbed immune regulation involving T-regulatory (Treg) cells, rather than a mere shift towards Th2 immunity, was the key behind allergic asthma (Haschke and Klassen, 2009)

Although asthma is not considered to be a life-threatening disease, it is the key factor behind one out of every 250 deaths worldwide Complexity and severity of asthma continue to increase in children and young adults Severe or uncontrolled asthma presents high socio-economic burden on the countries The healthcare costs correlate positively with severity of asthma The financial burden of asthma ranges from US$300 to US$1, 300 per patient per year in the developed countries In the United States, 23 million people including seven million children suffer from asthma As a result of asthma attacks, these seven million children miss 14 million days of school each year Caregivers of these children would have to take time off work to attend to them, which would result in lost wages for the caregivers (Pawankar et al., 2011) In developing countries like Vietnam, with gross domestic product per capita of US$1, 411, the financial burden of asthma estimates to be US$184 per patient per year In India, the medication for an asthmatic child can cost a third of the family’s income (Table 1.1) (Pawankar et al., 2012)

In summary, the rising prevalence, mortality, and high economic burden of asthma are having

a huge impact on the health-care systems worldwide Although current therapies for asthma are relatively safe and effective at controlling symptoms, these therapies do not change the chronic course of disease Currently, there is no established method to prevent asthma The major unmet needs of this area include better management of the severe forms of the disease and the developments of curative therapies (Akdis, 2012) Therefore, much research has been done to better understand the pathophysiology of asthma and to explore novel therapies for this asthma One attractive target for therapeutic intervention would be the nuclear factor (NF)-κB signaling pathway, which plays an important role in Th2-mediated inflammation (Edwards et al., 2009)

Figure 1.1: World map of the prevalence of clinical asthma (Adapted from Matthew Masoli 2004) (Matthew Masoli, 2004)

Country Year cost

calculated

Population (2010) (million)

Cost estimate

Table 1.1 The economic burden of asthma (Adapted from Pawankar et al., 2011)

1.1.2 Pathophysiology/ Development of asthma

Asthma is considered to be a heterogeneous disease with numerous distinct clinical phenotypes The most common form of asthma — allergic asthma — affects 60 percent of the asthmatics (Kim et al., 2010)

Allergic asthma is a chronic airway inflammatory disease (Fishbein and Fuleihan, 2012; Lambrecht and Hammad, 2012; Pawankar et al., 2012) The inflamed airway, similar to a chronic wound, is susceptible to a wide range of environmental insults (for example, biologically active allergens, viruses, air pollutants, certain drugs, and chemicals) and has an altered repair response that involves growth factors secretion, which induces goblet cell metaplasia (GCM), smooth muscle proliferation, angiogenesis, fibrosis, and nerve proliferation (Barnes, 2011; Holgate, 2012; Lambrecht and Hammad, 2012)

Allergic asthma is often initiated when one is sensitized to inhaled allergens from the environment such as HDM, cockroaches, animal danders, fungi, and pollen (Barnes, 2011; Holgate, 2012; Lambrecht and Hammad, 2012)

During initial sensitization, the inhaled allergens interact with epithelial cells and result in the release of endogenous danger signal, including chemokine ligand (CCL)-2 and CCL-20 to recruit more dendritic cells progenitors and dendritic cells from the bone marrow The role of epithelial cells in the pathogenesis of asthma shall be discussed in details in section 1.1.2.1 on

“Airway Epithelial Cells” Besides interacting with the airway epithelium, the inhaled allergens also interact with the dendritic cells Degradation of airway epithelium by proteolytic activity of allergens breaches the epithelium barrier function and allows the allergen to gain access to the dendritic network (Wan et al., 1999)

These antigens presenting dendritic cells express pattern recognition receptor (PRR) and takes

up the allergens The interaction between PRR and allergens initiates the migration of

dendritic cells to the T cell area of the draining regional lymph nodes During migration, dendritic cells undergo further maturation and process the allergens into small peptides (Lambrecht and Hammad, 2010) In the T cell area of the lymph node, mature dendritic cells arrest and select the rare nạve antigen-specific T cells and present the processed peptides in the context of major histocompatibility complex (MHC) class II to T cell receptor (TCR) (Stoll et al., 2002) The antigen presentation leads to differentiation of nạve T-cells to antigen-specific Th-2 cells (Figure 1.2) (Holgate, 2012) The mechanisms responsible for Th-

2 polarisation during initial allergen sensitization remain poorly understood The possible mechanisms shall be discussed in section 1.1.2.2 on Th2 cells (Holgate, 2012)

The differentiated antigen specific Th2 cells that mediate pathophysiology of subsequent allergen exposure consist of two subsets: effector memory T-cells or resident memory T cells and central memory T cells

The effector memory T cells have reduced expression of lymph node homing receptor cluster

of differentiation (CD)-62L and migrate to the site of inflammation and serves as surveillance for future re-exposure to allergen (Figure 1.2) Upon re-encounter with allergen, or during an asthma attack, the effector memory T cells interact with antigen presenting dendritic cells and can rapidly release pro-inflammatory cytokines into the airway (Robinson et al., 1992)

Unlike the effector Th2 cells, the central memory T cells express CD62L but lack immediate effector function (Iezzi et al., 2001) These central memory T cells are localized to the lymph node Upon allergen re-exposure, dendritic cells release signals to the central memory cells in the lymph nodes Consequently, the memory cells proliferate and differentiate into effect T cell and migrate to the site of allergen challenge to mediate airway inflammatory response (Sallusto and Lanzavecchia, 2001)

Figure 1.2 Inflammatory and immune cells involved in allergic airway inflammation (Adapted from Lambrecht and Hammad, 2012)

Lung epithelium expresses pattern recognition receptors (PRRs) such as toll-like receptors (TLRs), nod-like receptors (NLR), C-type lectins, and protease-activated receptors (PARs) PRRs when bound to allergens result in activation of signaling pathways, which would cause the release of endogenous danger signals (uric acid, ATP, LPA, TSLP, IL-25, IL-33, GM-CSF, IL-1 members) Epithelial cells also release CCL-2 and CCL-20 to attract dendritic cell progenitors — monocytes — to the lung These signals from epithelial cells activate dendritic cells Activated dendritic cells migrate to T cell region in the draining lymph nodes In the lymph nodes, dendritic cells interact with nạve T cells and induce T cell differentiation Depending on the signals released by dendritic cells, T cells can differentiate into Th1, Th2,

or Th17 cells For example, interaction between OX40L of dendritic cells and OX40 of T cell would enhance differentiation of nạve T cells to Th2 cells Differentiated and activated Th2 cells would activate B cells and result in IgE production by B cells Notably, basophil is considered to be an antigen presenting cell and has been reported to be an important source of IL-4, which supports differentiation of nạve T cell to Th2 cell

Abbreviations: CCL, chemokine (C-C motif) ligand; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL, interleukin; LPA, lysophosphatidic acid; NOD 1/2, nucleotide-binding oligomerization domain; ROS, reactive oxygen species; TGF-β, transforming growth factor-β; and TSLP, thymic stromal lymphopoietic protein

1.1.2.1 Airway Epithelial Cells

The airway epithelial cells are located at the interface between the host and the environment; therefore, they are the first line of defense against foreign antigens (Xiao et al., 2011) Epithelial cells express many PRRs (Toll-like receptor [TLR], NOD-like receptor [NLR], C-type lectins, and protease-activated receptor [PAR]) that interact with pathogen associated molecular pattern (PAMP)s and danger-associated molecular pattern (DAMP)s Activation of PRR by PAMPs or DAMPs would result in the activation of NF-κB signaling (Lambrecht and Hammad, 2012) Studies showed that tonic activation of NF-κB in airway epithelial cells is sufficient to activate dendritic cells, breach inhalation tolerance and enhance sensitization to innocuous inhaled allergen, such as OVA (Ather et al., 2011; Poynter et al., 2004) On the other hand, the inhibition of NF-κB in airway epithelial cells attenuates Th2 cell infiltration and airway remodeling (Das et al., 2001) Activated NF-κB signaling pathway results in the release of an array of cytokines and chemokines, such as TSLP and IL-8 (Edwards et al., 2009; Lambrecht and Hammad, 2012) These cytokines contribute to pathogenesis of allergic airway inflammation TSLP is hought to mediate polarization of Th-2 immune response (Holgate 2012) Its role shall be discussed in details in section 1.1.2.2 on Th2 cells On the other hand, IL-8 contributes to airway inflammatory cell infiltration (Kunkel et al., 1991; Lampinen et al., 1999) The roles of these cytokines in the pathophysiology of asthma shall be discussed in the following paragraphs

IL-8 is a pivotal chemoattractant for neutrophils as well as eosinophils (Kunkel et al., 1991; Lampinen et al., 1999) This chemoattractant is a marker for asthma because it can be detected in the serum of asthmatic patients but not in control subjects (Shute et al., 1997) It was also reported that asthmatic patients with severe neutrophilic asthma phenotype have elevated IL-8 levels in the supernatant of their sputum as compared to mild asthmatic patients

or control subjects (Bonnans et al., 2002) In addition, IL-8 also contributes to mucus hypersecretion (section 1.1.2.5) (Bautista et al., 2009)

Activation of NF-κB signaling pathway in epithelial cells triggers the release of endogenous danger signals, which in turn activate dendritic cells (Lambrecht and Hammad, 2009) The threshold for activation of epithelial cells is dependent on the expression of PRRs and the expression of signaling proteins downstream of PRRs The greater the number of PRRs, the greater the sensitivity of the epithelial cells is to the allergen For example, viral infection of the airway epithelial with respiratory syncytial virus (RSV) increases the expression of TLR-4 This increased sensitivity of airway epithelial cells may in part explain how RSV infection predisposes children to wheezing and asthma (Monick et al., 2003)

Besides functioning as a receptor for foreign particles, the airway epithelium is also a physical barrier that prevents the access of allergens to lung dendritic cells (Lambrecht and Hammad, 2012) Based on bronchial biopsy studies, the airways of subjects with asthma have fragile epithelial (Lackie, 1997; Lambrecht and Hammad, 2012) The integrity of airway epithelial is maintained by apical tight junctions and adherent junctions, which keep the cells together and maintain their apicobasal polarity (Xiao et al., 2011) The main component of adherent junction is E-cadherin, which constantly releases inhibitory signals to dendritic cells and thereby suppresses dendritic cell-mediated allergic sensitization As compared to normal individuals, asthmatic patients have lower expression of E-cadherin; this is possibly due to epithelial-to-mesenchymal transitions (Jiang et al., 2007; Nawijn et al., 2011) The loss of E-cadherin may be caused by exposure to inhaled allergens — HDM, cockroaches, pollen, fungi, respiratory viruses, or environmental pollutants (cigarette smoke, ozone) (Lambrecht and Hammad, 2012) Therefore, exposure to these allergens induces disruption of epithelial junctional proteins and the barrier function of the airway epithelium Once the integrity of the epithelial cells has been destroyed, inhaled allergens can gain access to the dendritic cell network and activate immune responses (Lambrecht and Hammad, 2012) In particular, HDM results in epidermal growth factor receptor (EGFR)-induced tyrosine phosphorylation and delocalization of junctional protein Degradation of E-cadherin and destruction of the intact epithelial barrier function subsequently allows for the EGF on the basolateral side of the

epithelium to bind with of EGF on EGFR on the epithelial of the apical side of the epithelium (Heijink et al., 2007; Heijink et al., 2010; Lambrecht and Hammad, 2012) Activation of EGF signaling pathway results in goblet cell metaplasia, where goblet cells increase in number, and thus causes mucus hypersecretion The role of epithelial cells in mucus hypersecretion shall

be elaborated in section 1.1.2.5

Fifteen years ago, based on the observations of asthma biopsies and cultured bronchial epithelial cells, Stephen Holgate was the first to suggest epithelial cell as a major culprit in asthma Years of intense research that follows confirm that airway epithelium controls many aspects of allergic sensitization and is a central player in allergic airway inflammation, remodeling and bronchial hyperreactivity

1.1.2.2 T cells

T cells constitute the majority of lung lymphocytes in normal individuals and could be found within the airway, alveolar epithelium, and interstitium (Baraldo et al., 2007; Barnes, 2011) Nạve T cells require three sources of signals for activation: (1) signals from antigen-MHC class II; (2) signals from costimulatory molecules; and (3) signals from autologous IL-2 TCR interacts with peptide antigen MHC class II on the surface of antigen presenting cells The T cell also interacts with inducible co-stimulatory molecules (ICOS) such as B7 and CD40 expressed on antigen presenting cell At the same time, these activated T cells synthesizes IL-2 along with α chain of the IL-2 receptor (also known as CD25) Unlike activated T cell IL-2 receptors, resting T cells IL-2 receptors only composed of β and γ chains This form of receptor binds IL-2 with moderate affinity only However, in activated T cells, α chain synthesized associates with the existing β and γ heterodimer to create a receptor with much higher affinity for IL-2 Binding of IL-2 to the receptor triggers the activation, differentiation and proliferation of T cells (Janeway et al., 2005) Once activated, T cells can differentiate and proliferate into mature Th1, Th2, Th17, Th9, Treg, natural killer (NK)T cells,

or γδ T cells depending on the cytokines released by antigen presenting cells (Kaiko et al., 2008) These different subtypes of T cells contribute to development of allergic asthma in various ways (Figure 1.3) (Lloyd and Hessel, 2010)

Th2 cells

Twenty years ago, studies showed that there is an increased presence of Th2 cells in the airways of patients with asthma Since then, asthma has been categorized as a Th2 cell associated inflammatory disease (Robinson et al., 1992) Subsequent studies on human samples confirmed that the number of Th2 cells present in the airway correlates positively with the severity of asthma These data suggest an essential role for Th2 cells in human asthma (Larche et al., 2003) Although Th2 response plays an important role in development

of asthma, the mechanism responsible for initiating Th2 response remains to be elucidated Nonetheless, recent studies have shown that Th2 response is likely to be initiated by IL-25, IL-33, and TSLP (Figure 1.2) (Kaiko and Foster, 2011; Lambrecht and Hammad, 2012; Lloyd and Hessel, 2010) These Th2 priming cytokines are postulated to be released by epithelium upon protease allergen or parasitic antigens stimulation (Kaiko et al., 2010)

IL-25 is also known as IL-17E It is a member of the IL-17 family (Kim et al., 2010) IL-25 was originally thought to be released by Th2 cells only However, recent studies confirm that this cytokine is also released by epithelial cells upon allergen challenge Upon exposure to allergen, both human and animal cell lines have dysregulated IL-25 expression (Hammad et al., 2009) Studies have shown that IL-25 amplifies Th2 cytokine production (Wang et al., 2007) In addition, administration of soluble IL-25 receptor fusion protein or antibody to mouse asthma model before allergen sensitization and challenge results in attenuation of airway inflammation (Tamachi et al., 2006) More importantly, administration of recombinant IL-25 results in the production of Th2 cytokines by innate cells — nuocytes — and leads to expulsion of helminths in both wildtype and recombination-activating-gene-deficient mice (Fallon et al., 2006) These innate helper cells can be found in the lungs (Moro et al., 2010)

Figure 1.3 T cells involved in the induction of allergic phenotype (Lloyd and Hessel, 2010)

Asthma is a heterogeneous disease that is associated with AHR, inflammatory cell infiltration, goblet cell metaplasia, and tissue remodeling An array of T cell subsets are proposed to influence the nature and severity of allergic airway inflammation

Abbreviations: AHR, airway hyperresponsiveness; IFN-γ, interferon-γ; and TH, Th or T helper

IL-33 is another cytokine released by epithelial cells (Figure 1.2) IL-33 is a member of the IL-1 cytokines Its expression is increased in airway epithelium of asthmatic patients and correlates positively with the severity of asthma (Prefontaine et al., 2010) Studies have shown that IL-33 activates cell through its receptor ST2, which is expressed on cell surface of dendritic cells, mast cells, and epithelial cells (Besnard et al., 2011; Kurowska-Stolarska et al, 2009; Smithgall et al., 2008) Activation of ST2 results in activation of NF-κB and mitogen-activated protein kinase (MAPK) signaling pathway (Milovanovic et al., 2012) In a study by Besnard et al., using OVA-induced mouse asthma model, IL-33 was shown to plays a critical role on dendritic cell activation, maturation, and initiation of Th-2 mediated allergic airway inflammation (Besnard et al., 2011c) This suggests the potential role that IL-33 may play in the initiation of Th-2 immune response Antibodies that block IL-33 or ST2 are currently in clinical development (Barnes, 2011)

Compelling evidence implicates TSLP as a potential initiator of Th-2 bias allergic airway inflammation Blockade of TSLP has been shown to reduce airway dendritic cells migration and thus diminishes CD4+ T cell priming (Fernandez et al., 2011) In line with this observation, another study demonstrated that TSLP activates dendritic cells by inducing the expression of OX40L, enhancing Th2 inflammatory response (Ito et al., 2005) Furthermore,

in the bone marrow, TSLP promotes the growth and differentiation of basophils, which produce IL-4 that enhances Th2 inflammation (Figure 1.2) (Siracusa et al., 2010; Lambrecht and Hammad, 2012) Corresponding well with the data obtained, it has been demonstrated that over-expression of TSLP and GM-CSF in mice lungs results in spontaneous Th2 sensitization to harmless inhaled allergen OVA (Stampfli et al., 1998; Zhou et al., 2005) Genetic polymorphisms in the promoter region of TSLP are linked to increased risk of asthma (Harada et al., 2011) However, there is no strong evidence that dendritic cells release IL-4 in response to TSLP activation, such discrepancy suggests the existence of an alternate

hematopoietic antigen presenting cell source for IL-4, which is needed to initiate the differentiation of nạve T cells to Th2 cells (Kaiko and Foster, 2011)

The alternative source of IL-4 has been postulated to be basophils Basophils have been reported to rapidly release IL-4, IL-9, IL-25, and TSLP upon activation (Lambrecht and Hammad, 2012; Siracusa et al., 2010) Recent reports demonstrate that basophil-derived IL-4

is essential for the induction of Th2 response to papain In addition, basophil, rather than dendritic cell, is a crucial antigen presenting cells for the Th2 response Furthermore, an observational study shows that basophil number is higher in peripheral blood of asthmatic patients as compared to healthy individual (Gilmartin et al., 2007) However, these observations contradict the conventional idea that dendritic cells play an important role as an antigen presenting cell Therefore, the mechanism behind the initiation of Th2 response remains to be verified Nonetheless, 20 years after the discovery of Th2 subsets, the mechanism behind the synthesis of Th2 cytokines by T cells is clearer now

Upon activation by IL-4 released by antigen presenting cells, Janus Kinase (JAK) molecules

in T cells are activated, leading to dimerization and nuclear translocation of transcription factor signal transduction and activator of transcription (STAT)-6 In the nucleus, STAT-6 results in the expression of GATA-3, a transcription factor of Th2 cytokines (Nelms et al., 1999) In addition to mediating the expression of Th2 cytokines, GATA-3 also inhibits the activation of T-Bet, a transcription factor of Th1 cytokines (Ouyang et al., 1998) Notably, the expression of GATA-3 activation is dependent on NF-κB signaling pathway (Das et al., 2001) The role of NF-κB in the pathophysiology of asthma shall be discussed in section 1.2.2 Activation of IL-4 receptor signaling, in turn, induces T cell to release the Th2 cytokines IL-4, IL-5, and IL-13 (Figure 1.4) These Th2 cytokines initiate and maintain key pathophysiological features of the disease (Lloyd and Hessel, 2010)

IL-4 is a key cytokine that influences nạve T cells to differentiate into Th2 cells (Figure 1.4) (Long, 2009) Other sources of IL-4 include certain population of invariant natural killer (NKT) cells (Lai et al., 2011), eosinophils (Piehler et al., 2011), basophils (van Panhuys et al., 2011), and mast cells (Gessner et al., 2005) The IL-4 receptor consists of two chains: (1) high affinity IL-4 binding chain (p140, α chain) which binds to IL-4 and (2) IL-2Rγ chain which amplifies the signaling of IL-4 receptor (IL-4R) Notably, expression of high affinity IL-4Rα binding chain is localized to the airway epithelial cells, lymphocytes, and mast cells in the airway mucosa As compared to normal individuals, asthmatic patients are reported to have higher expression of high affinity IL-4Rα binding chain (Slager et al., 2012) Also, IL-4 is essential signal for isotype switching of B-cells for IgE production In addition, IL-4 has been reported to promote the expression of low-affinity IgE receptors (FcεRII) and FcεRI on mast cells and basophils Finally, IL-4 contributes eosinophilia (Figure 1.4) (Long, 2009; Palmer-Crocker and Pober, 1995) Results from mouse asthma model suggest that removal or blockade of IL-4 attenuates eosinophilia, mucus hypersecretion, and airway hyperresponsiveness (AHR) (Brusselle et al., 1994; Long., 2009)

IL-4 and IL-13 share the same receptor subunit IL-4Rα Therefore, these two cytokines share many functional properties For example, IL-4 and IL-13 mediate the migration of eosinophil (Hogan et al., 2008; Stone et al., 2010; Wegmann, 2011) These Th2 cytokines up-regulates the expression of: (a) eotaxin (CCL11 and CCL26), which are chemoattractant for eosinophils; and (b) endothelial cell vascular cell adhesion molecule (VCAM) - 1, adhesion molecule that interacts with integrin (very late antigen [VLA]-4) expressed on eosinophils, resulting in eosinophilia (Kotowicz et al., 1996; Long, 2009 Matsukura et al., 2001; Palmer-Crocker and Pober, 1995) (Figure 1.4) Also, both IL-4 and IL-13 play central role in IgE production;

Figure 1.4 Components of airway allergic inflammation (Taken from Long, 2009)

Allergens stimulate and activate epithelial cells to mediate the release of these cytokines TSLP is a product of NF-κB activation and is known to activate dendritic cells and upregulate the expression of OX40L, which plays an important role in mediating Th-2 inflammatory response Dendritic cells internalise and process the antigen into fragments, then present the processed fragments to antigen specific T-cells in the context of MHC class II Differentiation

of nạve T-helper cells to Th2 cells is favoured by the presence of 4 Th-2 cells release

IL-4, IL-13, and IL-5 IL-4 and IL-13 mediates the production of IgE IgE plays an important role in the activation and degranulation of mast cell and basophil Degranulation of these cells release biologically active mediators that act on airway smooth muscle and cause

PRR Agonist

PAR Agonist

Protea se

PRR Agonist PAR Agonist

Dendritic cell

Differentia ting cytokines

1 MHC-Ag-TCR

2 Co-stimulatory Molecules

Immunological synapse

TSLP, GM-CSF, IL-25, IL-33

nạve T-cell

IFN-γ, IL-12 TGF-β, IL-2

IL-5,TGF-β,

Th-1 cell

Th-17 cell IL-17TSLP

IL-4

IL-5, IL-9, GM-CSF, IL-3

Eosinophil

IgE

IL-4

bronchoconstriction IL-13 mediates mucus hypersecretion; while IL-5 enhances the proliferation and survival of eosinophils

Abbreviations: PRR, pattern recognition receptor; PAR protease activated receptor; TSLP thymic Stromal Lymophopoietin; GM-CSF, granulocyte-macrophage colony stimulating factor Arrows indicate the direction in which the identified signal is acting Red lines indicate the discharge of cellular mediators (including granular contents, lipid mediators and cytokines)

goblet cell metaplasia, which results in mucus hypersecretion; and AHR development (Figure 1.4) (Long, 2009) IL-4 and IL-13, together with TGF-β and IL-6, induce airway remodeling (Cosmi et al., 2011; Long, 2009) Nonetheless, IL-4 and IL-13 also have distinct functions For example, IL-13 is also involved in the recruitment of pro- inflammatory cells such as monocytes, macrophages, and T cells (Long., 2009) In animal studies, blockade of IL-13 has been shown to result in the attenuation of airway inflammation, airway responsiveness, and mucus hypersecretion (Yang et al., 2004) Monoclonal anti-IL-13 antibodies are currently under clinical trial phase I and II (Holgate, 2011)

IL-5 is an eosinophilopoietin; therefore, IL-5 is crucial for the differentiation of hematopoietic progenitor cells to eosinophil In addition, it also plays an important role in mediating the maturation of eosinophil and the survival of eosinophil (Figure 1.4) Eosinophil express transforming growth factor (TGF)-β, a pro-fibrotic growth factor; and therefore, IL-5 has also been linked to airway remodeling and development of AHR (Long, 2009; Wegmann, 2011)

Although these Th2 cytokines are mainly released by Th2 cells, the sources of these Th2 cytokines are not limited to Th2 cells Recent reports show that these Th2 cytokines can also

be produced by nuocytes The function of nuocyte is inferred based on the observation that Th2 response still develops in mice in the absence of T and B cells (Moro et al., 2010)

In order to suppress the unwanted Th2 response in asthma, studies have also focus on the inhibitory effect that Th1 response might have on asthma The deletion of Th1 cell master transcription factor T-bet in mice resulted in spontaneous development of AHR and eosinophilia (Finotto et al., 2002) However, the administration of Th1 associated cytokine interferon (IFN)-γ resulted in no improvement of disease symptoms (Boguniewicz et al., 1995) Recent cluster analysis of asthma clinical phenotypes demonstrates that eosinophilic and non-eosinophilic subtypes of asthma exist (Holgate, 2012) Although atopic asthma has a substantial Th2 cell component, this disease comprises of different subtypes, and recent

evidence suggests that other T cells are contributors to different stages and different phenotypes of asthma (Lloyd and Hessel, 2010)

Th17 cells

Th17 is CD4+ T cell that is developed from a different T helper cell lineage The development of Th17 cell in mice is mediated by TGF-β and inflammatory cytokines IL-6, IL-21, and IL-23 However, it is still unclear which cytokines prime the differentiation of nạve T cells to Th17 cells Th17 cells express key transcription factors orphan retinoic acid nuclear receptor (ROR)γt and RORα, as well as pro-inflammatory cytokine IL-17 IL-17 also mediates the recruitment of neutrophils (Figure 1.3) (Lloyd and Hessel, 2010) In line with these functions, there are reports showing that there is marked Th17 cell infiltration in the lungs of asthmatic patients with high neutrophil counts (Al-Ramli et al., 2009) The role that Th17 plays in the pathogenesis of asthma is supported by other evidence Although allergen sensitization via the airway primes only modest Th2 responses, such route of sensitization primes strong Th17 cell responses that promote airway neutrophilia and acute AHR (Wilson

et al., 2009) In addition, Th17 cells also exacerbate Th2 mediated eosinophilic airway inflammation (Wakashin et al., 2008) Also, the transfer of Th17 cells promotes steroid resistant airway inflammation and AHR in mice (Figure 1.3) (McKinley et al., 2008) Moreover, a polymorphism in IL-17F that that leads to a loss-of-function mutation is inversely related to asthma risk (Kawaguchi et al., 2006) Finally, abrogation of IL-17 during allergen sensitization provides protection against allergic airway inflammation (Nakae et al., 2002) However, administration of exogenous of IL-17A during allergen re-challenge attenuates airway inflammation This attenuation suggests that under certain conditions there

is a regulatory role for IL-17 in the allergic lung (Murdoch and Lloyd, 2010; Candrian et al., 2006) Therefore, whether IL-17 mediates inflammatory or suppress inflammation would depend on its cellular source, which includes γδ T cell (Murdoch and Lloyd, 2010)

Schnyder-Th9 cells

IL-9 was originally thought to be released by Th2 cells However, recent studies show IL-9 is produced by a discrete population of CD4+ T cells — Th9 cells (Lloyd and Hessel, 2010) In addition, studies have also shown that other sources of IL-9 include eosinophils and mast cells (Angkasekwinai et al., 2010; Chang et al., 2010) Th9 cells depend on IL-4 and TGF-β for their differentiation (Veldhoen et al., 2008) TGF-β re-programmes Th2 cells to Th9 cells, which enforces the responsiveness of Th9 cells to IL-25 IL-25 is essential for IL-9 production (Angkasekwinai et al., 2010) Notably, Th9 cells produce IL-9 and IL-10, and not Th2 cytokines (Wong et al., 2010) Generation of Th9 cells requires the expression of PU.1 transcription factor (Lloyd and Hessel, 2010) There is evidence indicating that IL-9 is involved in allergic inflammation in lungs IL-9 is detected in lung biopsies of asthmatic patients and IL-9-transgenic mice have eosinophilia and develop AHR (Figure 1.3) (McLane

et al., 1998; Shimbara et al., 2000) Although PU.1 deficient T cells mice (mice with functional Th9 cells) were reported to develop normal Th2 responses after allergen exposure, these PU.1 deficient T cells mice have reduced pulmonary inflammation IL-9 is postulated to

non-be involved in the remodeling processes of asthma due to the role of TGF-β in Th9 cells development In addition, IL-9 is known to induce differentiation and proliferation of mast cells (Barnes, 2008) (Figure 1.3) Recently, a humanized monoclonal antibody for blockade

of IL-9 is undergoing phase I clinical trial as a potential treatment for moderate to severe asthma (Antoniu, 2010)

CD8+ T cells

CD8+ T cells are present in the airway and sputum of asthmatic patients and are reported to release IL-4, IL-5 and IFN-γ (Ying et al., 1997) The number of CD8+ T cells correlates positively with the decline in (forced expiratory volume in 1 second) FEV1 (van Rensen et al., 2005) Interestingly, CD8+ cells in the airways are different from CD8+ cells in the peripheral blood Briefly, airway CD8+ cells produce IL-5 and IFN-γ in the sputum The amount of cytokine released correlates positively to the severity of asthma However, such

trend could not be observed in peripheral blood CD8+ cells (Cho et al., 2005) It is noted that patients who died from asthma have higher proportion activated CD8+ T cell as compared to asthmatic patients who died from other causes (O'sullivan et al., 2001) In addition, virus specific CD8+ T cells, in the presence of Th2 cells, switch off the production of IFN-γ and secrete IL-5, resulting in eosinophilia (Coyle et al., 1995)

Results from animal studies show that adoptive transfer of CD8+ αβ T cells results in worsening of airway inflammation (Isogai et al., 2004) In addition, CD8 knockout mice are

less susceptible to allergic airway inflammation The transfer of in vitro generated

antigen-primed effector memory T cells into sensitized CD8 knockout mice increased AHR, eosinophilic inflammation, and levels of IL-13 in bronchoalveolar lavage fluid (BALF) (Miyahara et al., 2004a) However, the transfer of CD8+ T cells from IL-13 knockout mice or non-challenged mice failed to result in airway inflammation, indicating that the CD8+ T cells produced IL-13 locally and are activated within the airway (Miyahara et al., 2004b)

Treg

Treg cells play a crucial role in modulating and regulating immune responses by promoting tolerance, suppressing inflammatory reactions, and maintaining homeostasis It is well established that the number and functions of Treg cells are impaired or altered in allergic patients as compared with healthy individuals, providing possible explanation for the inappropriate immune response to innocuous allergens observed in asthmatic patients (Lloyd and Hessel, 2010; Thorburn and Hansbro, 2010) Evidence suggests the protective effect of Treg cells in asthma To date, two Treg cell subsets have been identified — (1) forkhead box

(FOX)P3+CD4+CD25+ cells and (2) inducible Treg cells (Lloyd and Hessel, 2010) In vivo

transfer of FOXP3+CD4+CD25+ Treg cells attenuates the development of airway inflammation and AHR and prevents the allergen induced activation of dendritic cells in the airways (Kearley et al., 2005) In addition, as compared to healthy children, Treg in the airways of asthmatic children have reduced expression of FOXP3 (Lin et al., 2008)

Furthermore, the reduced chemotactic response of CD4+CD25+ Treg correlates positively with FEV1 (Nguyen et al., 2009) Finally, glucocorticoid resistant asthmatic patients have non-functional CD4+CD25+ Treg cells, i.e their Treg cell fail to produce IL-10 upon stimulation (Xystrakis et al., 2006)

Treg cells exert their regulatory effect through releasing IL-10 and TGF-β IL-10 prevents the synthesis of pro-inflammatory cytokines and down-regulates the expression of effector T cells cytokines as well as antigen presentation and costimulatory properties of antigen presenting cells (Figure 1.3) (Saraiva and O'Garra, 2010) On the other hand, TGF-β inhibits the proliferation and differentiation of T cells, B cells, and macrophages; while promotes the apoptosis of these cells TGF-β helps to maintain the regulatory functions of Treg cells and promotes the differentiation of adaptive Treg (Yoshimura et al., 2010)

The regulatory effect of Treg is also achieved by expressing CTLA-4 CTLA-4 closely resembles T cell costimulatory molecules (CD28) However, as compared to CD28, CTLA-4 has higher ligand binding affinity to CD80/86 on dendritic cell Consequently, CTLA-4 effectively competes with CD28 for CD80/86 binding site, inhibiting dendritic cells mediated

T cells activation and resulting in formation of anergic T cells (Thorburn and Hansbro, 2010) The ligation of CTLA-4 to CD80/86 on dendritic cells induces the production of indoleamine

2, 3 dioxygenase, causing dendritic cells to be more immune-suppressive Indoleamine 2, 3 dioxygenase is an enzyme that degrades tryptophan and induces cellular apoptosis, leading to immunosuppressive activity (Fallarino et al., 2003; Thorburn and Hansbro, 2010)

Finally, Treg ameliorate effector responses by competing with effector cells for essential growth factor — IL-2, which is essential for both Treg and effector cell function Effector cells deprived of IL-2 undergo apoptosis (Chen et al., 2011)

NKT cells

Majority of the NKT cells express an invariant TCR and recognize CD1d, while a small population of NKT cells express do not express the invariant TCR but still recognize CD1d-associated antigens Activation of the invariant TCR on NKT cells results in the production of pro-inflammatory cytokines, including IL-4 and IFN-γ (Iwamura and Nakayama, 2010) These pro-inflammatory cytokines help to activate dendritic cells, macrophages, T cells, and

B cells, driving the development of adaptive immunity (Bendelac et al., 2007; Iwamura and Nakayama, 2010) NKT cells have been demonstrated to play an important role in the pathogenesis of asthma Although mice deficient in NKT cell had some degree of eosinophilia, these mice failed to develop AHR (Umetsu and DeKruyff, 2010) Nonetheless,

it was reported that pulmonary instillation of NKT cell activating glycolipid galactosylceramide) promoted production of IL-13 and TSLP and promoted AHR (Figure 1.3) In addition, exposing the mice to the same glycolipid together with another antigen enhanced NKT-dependent allergen sensitization in the subsequent allergen exposure (Akbari

(α-et al., 2003) Similar results were obtained in non-human primate asthma model (Matangkasombut et al., 2008)

Although the studies of NKT cells in animal models have been compelling, the role of NKT cells in the development of human asthma has been controversial Up till now, there are at least 14 studies that examine the presence of NKT cells in BALF, endobronchial biopsy specimens, or both from patients with asthma (Matangkasombut et al., 2009).Although most

of these studies (10 reports) found that NKT cell numbers were increased in the lungs of patients with asthma, four of these studies concluded that NKT cell numbers were not increased (Umetsu and DeKruyff, 2010) These differences are possibly caused by variation

in experimental techniques and patient population (Lloyds and Hessel, 2010)

γδ T cells

γδ T cells are generally found near the airway epithelium, where they exert an immunosurveillance function, responding to endogenous stress signals and migrating to site

of tissue damage (Wands et al., 2005) Interestingly, evidence from mouse asthma model suggests that γδ T cells have dual contrasting roles in the pathogenesis of asthma γδ T cells can be divided into two subsets — Vγ1+γδ T cell and Vγ4+γδ T cells The Vγ1+γδ T cells release IL-5 and IL-13 into the airway and act together with NKT cells to induce AHR (Hahn

et al., 2004; Lloyd and Hessel, 2010) On the other hand, Vγ4+γδ T cells may release IL-17A (Hahn et al., 2003) Interestingly, the IL-17A released by Vγ4+γδ T cells may exert suppressive effect on established airway inflammation and AHR (Murdoch and Lloyd, 2010) Therefore, γδ T cells derived IL-17 has regulatory role; whereas Th17 cells derived IL-17 has pro-inflammatory role (Figure 1.3) Notably, AHR develops in non-allergic mice that are depleted of γδ T cells, indicating a role for γδ T cells in the regulation of normal airway function (Zuany-Amorim et al., 1998) Taken together, these results indicate that the tissue localization of γδ T cells and their capability to detect stress or damage-associated antigen allows them to exert protective immune responses On the other hand, signals from local inflammatory environment requires the γδ T cells to exert regulatory functions, controlling inflammation and stimulating resolution (Lloyd and Hessel, 2010)

1.1.2.3 B cell IgE production and mast cell activation

B cell is an immune cell that contributes to the pathophysiology of asthma B cells develop and undergo maturation in the bone marrow This process involves nuclear factors such as E-box factors, early B cell factors, and NF-κB signaling (Schutte et al., 2012) In particular, some components of NF-κB signaling influence several stages of late B cell differentiation and maturation For example, p50-deficient mice lack marginal zone V-cells; while c-Rel (v-rel reticuloendotheliosis viral oncogene homolog) deficient mice show reduced numbers of marginal zone B cells (Cariappa et al., 2000; Köntgen et al., 1995) In addition, p50/p52 double knockout mice fail to generate B cells and are unable to produce immunoglobulin, including IgE (Franzoso et al., 1997)

Nạve B cells leave the bone marrow and enter peripheral lymphoid organs, where antigen specific B cell are activated and are involved in isotype switching from IgM to IgE, which is

an important event in pathophysiology of allergic airway inflammation Isotype switching of

B cells to IgE requires the presence of Th2 cytokines — IL-4 or IL-13 These Th2 cytokines bind to their receptors on B cells, together with two other B cell activating signals: (1) MHC class II-associated allergen presentation by B cell to Th2 cells and (2) costimulation involving CD40-CD40L Upon B cell activation, the intracellular molecular sequence undergoes germline gene transcription and DNA homologous recombination Such homologous recombination results in the synthesis and secretion of allergen-specific IgE antibody A subset of the IgE secreting B cells would mature into plasma cells, which produces large amount of allergen specific IgE (Holgate, 2012)

IgE secreted binds to α chain of FcεRI on mast cells and basophils The mast cells could be localized in ASM and epithelium of asthmatic patients (Ammit et al., 1997; Lei et al., 2008) Allergen present mediates crosslinking of IgE bound to FcεRI, resulting in the activation of mast cell The crosslinking can also occur in the form of a bivalent or multivalent antigen crosslinking a number of adjacent FcεRI-bound IgE, forming an aggregate of allergen-IgE-FcεRI complex This aggregation results in the initiation of complex signaling events, leading

to mast cell secretion of a diverse group of biologically active products that act on ASM (Galli and Tsai, 2012; Brightling et al., 2002)

Activation of mast cell results in the release of a diverse group of biologically active products, which may induce infiltration of inflammatory cells, promote Th2 differentiation, result in mucus hypersecretion, change ASM activity, and AHR (He and Walls, 1998; Holgate and Polosa, 2008; Wang et al., 1999; Okumura et al., 2005; Taipale et al., 1995) Some products, such as those stored preformed in the cells’ cytoplasm granules, for example, histamine, proteases (tryptase, chymase and/ or carboxypeptidase A3), and proteoglycans (heparin and/or chondroitin sulfates), as well as leukotrienes (LT)D4 and LTE4 and certain cytokines,

are released by mast cells within minutes of antigen exposure These products induce “early asthmatic response”, characterized by bronchoconstriction and inflammatory cell infiltration (Galli and Tsai, 2012; Holgate and Polosa, 2008; Moiseeva and Bradding, 2011) In line with this induction, increased mast cell population was found in ASM of asthmatic patients (Brightling et al., 2002) Specifically, histamine and cysteinyl leukotrienes increase endothelial expression of P-selectin and E-selectin, which are needed to initiate rolling of leukocytes

Besides releasing preformed products, activated mast cells also synthesize and secrete a diverse spectrum of cytokines; chemokines, CCL2, CCL8, CCL7, and CCL13; monocytes chemotactic protein (MCP)1-4 and; growth factors (e.g vascular endothelial growth factor [VEGF] and basic fibroblast growth factors [bFGF]) These newly synthesized products are only secreted a few hours after initial mast cell activation The growth factors, together with histamine and lipid mediators, could lead to proliferation and remodeling of epithelium, ASM hyperresponsiveness, goblet cell metaplasia, mucus hypersecretion (Galli and Tsai, 2012; Holgate and Polosa, 2008) These observations taken together, suggest that Ig-E activated mast cells contribute to both the early-phase and late-phase reaction in asthma (Galli and Tsai, 2012)

1.1.2.4 Eosinophils

Blood and tissue eosinophilia are hallmark signs of asthma, parasitic infection, allergy, eosinophilic gastrointestinal disorders, and a number of other rare disorders In particular, elevated eosinophil count in tissue, blood, and bone marrow is associated with severity of asthma This association suggests that eosinophil is one of the many pivotal effector cells in the pathogenesis of asthma (Hogan et al., 2008; Wegmann, 2011)

In the presence of Th2 dominated inflammation, eosinophil migrates to the inflammatory site,

in the case of asthma the inflammatory site would be the airway Besides IL-4 and IL-13,

TNF-α also upregulates the expression of adhesion molecule VCAM-1 (Babu et al., 2011; Berry et al., 2007) In addition, potent eosinophil chemotactic factors include PAF, cysteinyl leukotrienes, complement protein C5a, and CCL5 also known as regulated and normal T cell expressed and secreted (RANTES) (Hogan et al., 2008; Stone et al., 2010; Wegmann, 2011) Survival of eosinophils in the tissues might be enhanced by eosinophilopoietins, IL-33, and IFN-γ (Cherry et al., 2008)

Eosinophils function as effector cells of immune response Activation of eosinophils can be mediated by eosinophilopoietins, CC chemokines, and PAF Activated eosinophil degranulates, releasing mediators and cytotoxic products MBP, EPO, ECP, EDN, TGF-β, and

up to 28 cytokines, growth factors and chemokines (Figure 1.5) (Stone et al., 2010; Venge, 2010; Wegmann, 2011) These products are implicated in many aspects of asthma pathogenesis Eosinophils express elevated levels of TGF-β, which has been shown to promote the synthesis and secretion of numerous proteins into the ECM, leading to airway remodelling or subepithelial fibrosis in particular (Figure 1.4) (Ohno et al., 1996; Halwani et al., 2011; Long, 2009) Studies have shown that the degree of eosinophilia in bronchial mucus of severe asthmatics correlates positively with the thickening of the subepithelial basement membrane (Wenzel et al., 1999) Such thickening or remodelling of the airway is likely to result in AHR (Cockcroft and Davis, 2006) MBP, EPO, ECP and EDN are profibrotic and lead to shedding of epithelial cells In addition, lipid mediators, such as leukotriene C4, and PAF are bronchoconstrictors and secretagogues (Kay, 2005; Wegmann, 2011) Such tissue destruction would result in AHR Results from rat and mouse asthma models suggest eosinophil infiltration and the cytotoxic products of eosinophil underlie the pathogenesis of AHR (Coyle et al., 1994; Shen et al., 2003)

Other than functioning as an effector cell of immune response, eosinophil can also function as antigen presenting cell Using a mouse model, it is shown that eosinophils from allergic lungs expressed both classes of MHC class I and II peptides and T cell costimulatory molecules —

Figure 1.5 Inflammatory mediators derived from eosinophil (Adapted from Kay, 2005) The eosinophil has the capacity to produce a variety of inflammatory mediators, which contribute to the pathogenesis of asthma They include basic granular proteins, membrane-derived lipids, chemokines, cytokines, fibrogenic and growth factors, in addition to ROS and neuropeptides

Abbreviations: ECP, eosinophil cationic protein; EDN, eosinophil-derived neurotoxin; EPO, eosinophil peroxidase; FGF, fibroblast growth factor; GM-CSF, granulocyte/macrophage-colony stimulating factor; HB-EGF, heparin-binding epidermal growth factor; 15-HETE, 15-hydroxy-eicosatetraenoic acid; IFN, interferon; IL, interleukin; MBP, major basic protein; MCP, monocyte chemotactic protein; MIP, macrophage inflammatory protein; MMP, matrix metalloproteinase; NGF, nerve growth factor; PAF, platelet activating factor; PDGF, platelet-derived growth factor; PG, prostaglandin; RANTES, regulated on activation normal T cell expressed and secreted; SCF, stem cell factor; TGF, transforming growth factor; TNF, tumour necrosis factor; VEGF, vascular endothelial growth factor

CD80 and CD86 (Long, 2009; Wang et al., 2007) Eosinophils migrate to lymph nodes and function as antigen presenting cells to stimulate CD4+ T cells (Barnes, 2011; Jacobsen et al., 2008; Long, 2009; Wang et al., 2007)

Eosinophils are very sensitive to corticosteroid treatment, which induces eosinophil apoptosis and inhibits the response to survival signals such as IL-5 and to GM-CSF Unfortunately, corticosteroid treatment is often associated with side-effects, especially when ingested or inhaled in high doses Nonetheless, advances in the understanding of eosinophil’s function in asthma pathogenesis have suggested therapies that target eosinophil regulators — humanized anti-IL-5 and CCR-3 antagonists (Stein et al., 2008; Ben et al., 2008) These new approaches are promising and could enter a new stage in designing an eosinophil-specific strategy in asthma therapy Nonetheless, there is some debate about the effect of anti-IL-5 antibodies on AHR (Long, 2009)

1.1.2.5 Mucus hypersecretion

The normal airway epithelium comprises of four different groups of cells — ciliated cells, clara cells, basal cells and goblet cells There are few goblet cells in the airway epithelium of healthy individuals However, in subjects with asthma, there are elevated number of goblet cells coupled and excessive mucus production The increase in goblet cell number is termed

as goblet cell metaplasia, which is a result of transdifferentiation of ciliated and clara cells to goblet cells (Gomperts et al., 2007; Lambrecht and Hammad, 2012; Tyner et al., 2006) Goblet cells secrete mucus in response to stimuli, forming a mucus layer that lines the airways This mucus layer normally plays a beneficial role, protecting the host through mucociliary clearance against inhaled pathogens, toxins and other foreign particles (Williams

et al., 2006) However, goblet cell metaplasia is resultant in respiratory disease pathologies (Turner and Jones, 2009) Mucus hypersecretion and inefficient mucociliary clearance result

in mucus accumulates The exact mechanism responsible for poor mucociliary clearance is

not understood (Bennett et al., 2011) However, a recent study showed that excess plasma proteins secreted into the airways of patients with acute asthma inhibit protease-dependent degradation of mucins and suggested that an alteration in viscoelastic properties may impair airway mucus clearance (Innes et al., 2009) Mucus accumulation leads to airway obstruction, which has been reported as a prime mediator of fatal asthma (Kuyper et al., 2003) Airway obstruction and goblet cell metaplasia contribute to AHR (Figure 1.6) (Lai and Rogers, 2010) Pharmacological inhibition of mucin secretion using MARCKS peptide attenuated methacholine-induced AHR in mouse asthma model (Agrawal et al., 2007)

One of the components of mucus includes mucins, which are glycoproteins that make up 2%

of the mucus mass A single mucin monomer comprises of a peptide backbone with hundreds

of O-linked carbohydrate side chains The peptide backbone is encoded by a specific group of mucin (MUC) genes and contains several tandemly repeated serine-rich and threonine-rich regions that serve as glycosylation site (Rose and Voynow, 2006; Williams et al., 2006) The mucins are responsible for the viscous nature of mucus; airway obstruction by highly viscous mucins/mucus is a major cause of morbidity and mortality in asthma (Rose and Voynow, 2006) To date, there are eighteen MUC genes, where 12 are expressed at the mRNA level in the respiratory tract Examples of airway mucins include MUC2, MUC4, MUC5AC, and MUC5B (Voynow and Rubin, 2009) MUC5AC is predominantly expressed in goblet cells

On the other hand, MUC5B is predominantly expressed in mucus cells of submucosal glands (Thornton et al., 2008) Although both mucins can be found in the airway, MUC5AC is the major mucin in the mucus from patients with asthma MUC5AC is one of the genes that are highly expressed by asthma associated signaling pathways (Lai and Rogers, 2010b) Activation of these signaling pathways are mediated by an array of pro-inflammatory mediators — ligands of EGFR, IL-9, IL-13, TNF-α, and IL-1β (Evans et al., 2009; Lai and Rogers, 2010b; Lora et al., 2005b) The following paragraphs describe the roles of these mediators in goblet cell metaplasia and mucus hypersecretion

Figure 1.6 Impact of mucus hypersecretion on airway obstruction in asthma (Taken from Lai and Rogers, 2010)

Necropsy specimen of a lung from a patient who died from acute severe asthma attack mucus (M) could be seen blocking the airway (arrow) (b) Interaction between mucus and bronchoconstriction in acute severe asthma The epithelium (arrow head) is thrown into folds

by airway smooth muscle (ASM) contraction The remaining of the lumen is obstructed by mucus (M) (c-f) Application of Poiseuille’s law to the interaction between bronchoconstriction and mucus Equating the respiratory tract to tubes, the law can be adapted

to describe the impact of mucus on airflow in bronchoconstricted airways Therefore, resistance to flow (R) is proportional to the radius of the airway (r) raised to the fourth power, which in (c) equates 1 unit (d) Reducing the radius by half (as a result of bronchoconstriction) increases airflow resistance to 16 units (e) The presence of a thin film of mucus, with thickness (Tm) 0.1 units, around the internal perimeter of the airway has little significant impact on resistance (increased by 0.5 units compared with c) (f) Bronchoconstriction of this mucus-containing airway causes an exponential increase in airflow resistance (300 units – compare with d)

EGFR ligands (EGF and amphiregulin) bind to EGFR, which is a 170-k-Da receptor tyrosine kinase expressed on the surface of many cell types Studies have shown that EGFR and its ligands are up-regulated in subjects with asthma Activation of EGF signaling pathway leads

to transcription of MUC5AC as well as goblet cell metaplasia, which results in mucus hypersecretion (Fedorov et al., 2005) Activation of EGF signaling pathway also down-regulates forkhead box protein (FOX)A2, which is a forkhead box-family transcription factor involved in suppression of goblet cell metaplasia as well as other pro-inflammatory cytokines (IL-13 and IL-33) and chemokines (CCL17 and CCL-20) (Zhen et al., 2007) Lungs of FOXA2 knockout mice have inflammatory dendritic cells accumulation and Th2 inclined immunity The association between the inhibition of goblet cell metaplasia and Th2 associated pro-inflammatory cytokines suggests that goblet cell metaplasia is closely linked to the regulation of innate and adaptive Th2 immune response (Lambrecht and Hammad, 2012)

Th-2 cytokines IL-4 and IL-13 are prime mediators of mucus production Engagement of IL-4

or IL-13 to IL-4Rα and/or IL-13Rα results in the phosphorylation and activation of STAT-6 Activated STAT-6 binds to a canonical motif, 5’-TTCN4GAA-3’to mediate the pro- inflammatory responses Studies have shown that only STAT-6 signaling in mouse airway clara cells is needed for MUC5AC induction and airway hyperresponsivenes (Kuperman et al., 2002; Zhen et al., 2007) However, the canonical motif is not found in the conserved promoter regions of any MUC5AC orthologs, suggesting that an additional signaling process is present

— FOXA2 Down-regulation of FOXA2 activation by STAT-6 results in the up-regulation of MUC5AC expression Interestingly, FOXA2 is implicated as a common suppressor of both EGFR and IL-13 signaling pathways Therefore, FOXA2 may represent a common signaling node for multiple goblet cell metaplasia and mucus hypersecretion-inducing signaling pathways (Figure 1.7) (Chen et al., 2010)

Although IL-9 is not a Th2 cytokine, IL-9 also causes mucus hypersecretion in patients with asthma IL-9 is produced by Th-9 cells and mediates mucus hypersecretion by up-regulating

Figure 1.7 Molecular control of goblet cell metaplasia in asthma (Taken from Lambrecht and Hammad, 2012)

Transdifferentiation of ciliated and clara cells to goblet cells results in goblet cell metaplasia IL-13, epidermal growth factor (EGF) and amphiregulin induce the transcription factor SAM pointed domain-containing Ets transcription factor (SPDEF) This process requires serpin 3A, which is the mouse homolog of human serpin B3 and serpin B4 Over-expression of SPDEF causes severe goblet cell metaplasia and up-regulates genes expression of following the following: (1) molecules involved in epithelial and goblet cell differentiation; (2) molecules involved in protein glycosylation; (3) members of the forkhead box family (FOXA3 and FOXJ1); (4) Sox17; (5) anterior gradient 2 (AGR2); and (6) N-acetyltransferase 3 mucin type (GCNT3) In healthy individuals, SPDEF activity is suppressed by Nkx2-1 (TTF-1), a developmental transcription factor involved in lung budding from the primitive foregut However, the expression of Nkx2-1 is down-regulated in mucosal biopsies of patients who have asthma Transcriptional activity of SPDEF is also indirectly suppressed by Foxa2, another forkhead-box family transcription factor whose expression is restricted to bronchial epithelial cells Therefore, Foxa2 is known to inhibit goblet cell metaplasia

the human calcium-activated chloride channel (hCLCA)1 (Zhou et al., 2001) Upregulation of hCLCA1 has been associated with mucus hypersecretion (Yasuo et al., 2006)

TNF-α is also a cytokine that is pivotal in mucus hypersecretion This cytokine is elevated in the airway of asthmatic patients The administration of inhaled recombinant TNF-α to normal individuals and asthmatic patients resulted in development of AHR and neutrophilia (Thomas and Heywood, 2002) However, the mechanism behind this outcome has yet to be fully elucidated (Berry et al., 2007) Nonetheless, studies have shown that binding of TNF-α to its receptor results in the activation of NF-κB TNF-α- induced mucus production is mediated by

inhibitor of NF-κB (IκB) and NF-κB both in vitro and in vivo TNF-α stimulated epithelial

cells have up-regulated NF-κB activity and mucus production (Figure 1.8) (Lora et al., 2005a) NF-κB is subjected to additional regulation within the nucleus by kinases, such as MAPK (Lai and Rogers, 2010b) In line with this observation, it is shown that TNF-α can induced MUC5AC expression through MAPK activation in human epithelial cells (Song et al., 2003) The cell signaling pathway of NF-κB shall be elaborated in section 1.2 IL-1β is another stimulant that can result in increased MUC5AC expression (Figure 1.9) This stimulant is a cytokine released by asthmatic bronchial epithelium and activated macrophages (Lai and Rogers, 2010b) IL-1β contributes to mucus hypersecretion through indirect and direct mechanisms Indirectly, IL-1β induces expression of MUC5AC through activation of CD4+ cells and up-regulation of cell surface adhesion molecules (ICAM-1 and VCAM-1) on the blood vessels (Ben-Sasson et al., 2009; Kułdo et al., 2005) On the other hand, IL-1β directly induces the expression of MUC5AC through cyclooxygenase (COX)-2 induction and prostaglandin E2 (PGE2) release IL-1β induces the synthesis of COX-2 through extracellular signal regulated kinase (ERK) and MAPK signaling pathway Blockade of these signaling pathways suppressed COX-2 and MUC5AC expressions COX-2 mediates the production of PGE2 PGE2 binds prostaglandin E2 Receptor (EP-2), a transmembrane G-protein coupled receptor, resulting in the activation of adenylate cyclase and subsequent activation of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) PKA then initiates

transcription of MUC5AC (Gray et al., 2004b; Park et al., 2006) Interestingly, activation of cAMP is also known to result in ASM relaxation and bronchodilation β2 adrenergic receptor agonists up-regulate cAMP activity to relax ASM (Johnston and Edwards, 2009) Therefore,

β2 agonists (for example salbultamol and salmeterol) are associated with side-effects (Edwards et al., 2007; Johnston and Edwards, 2009) Taken together, the above studies suggest that IL-1β contributes to the pathogenesis of asthma Other studies have also shown that IL-1β also plays a role in airway homeostatsis by promoting mucociliary clearance (Gray

et al., 2004a)

Finally, another mucus inducing cytokine — IL-8 is released by lung epithelial cells, macrophages, and ASM upon allergen stimulation This cytokine has recently been shown to mediate increased MUC5AC production by goblet cells IL-8 enhances the interaction between RNA-binding proteins and the 3’-untranslated region (UTR) of MUC5AC Such interaction stabilizes the integrity of MUC5AC mRNA Consequently, mediating increase expression of MUC5AC post-transcriptionally (Bautista et al., 2009)

The production of mucus is a complicated process that requires multiple glycosylation in the endoplasmic recticulum (ER) (Lambrecht and Hammad, 2012) In response to goblet cell metaplasia induction, the lungs epithelial cells increase the activity of (inositol-requiring enzyme) IRE (Martino et al., 2009) Such increase activates transcription factors — X box-binding protein (XBP)-1 and NF-κB — and act synergistically to activate expression of inflammatory gene, including MUC5AC (Martinon et al., 2010) Notably, IRE-1 related gene

— ORMDL3 — has been linked to childhood asthma However it is still unclear how this gene affects the epithelial cell biology or the goblet cell metaplasia differentiation program (Moffatt et al., 2007)

Figure 1.8 Signaling pathways involved in TNF-α-mediated mucin synthesis (Taken from Lai and Rogers, 2010)