Modulation of nuclear factor b signaling attenuates allergic airway inflammation 2

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 socioeconomic 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 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) USA UK Australia 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 South Korea Israel Mexico USA 2005 2007 2007 50 103 310 US$2 billion US$250 million US$0.35 billion US$20 billion 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 naïve 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 naïve T-cells to antigen-specific Th-2 cells (Figure 1.2) (Holgate, 2012) The mechanisms responsible for Th2 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, GMCSF, 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 naïve 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 naïve 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 naïve 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, nucleotidebinding 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], Ctype 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 Ecadherin 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 10 does not Experimental results indicate that card domain is essential for Rip-2’s participation in various signaling pathways (Navas et al., 1999) Rip-2 was first described by three independent groups as a novel Rip-1-like kinase that has a role in NF-κB activation and apoptosis (Inohara et al., 2000; McCarthy et al., 1998; Thome et al., 1998) Many molecules that interact with Rip-2 are involved in the activation of NF-κB Evidence was provided that mitogen-activated protein/extracellular signal-regulated kinase kinase (MEKK)-4, a MAPK, binds to Rip-2 and inhibits Rip-2 from mediating nucleotidebinding oligomerization domain (NOD)-2 induced NF-κB activation (Clark et al., 2008) Recent study indicates that Rip-2 also interacts with NOD-like receptor protein (NLRP)-10, which is a receptor capable of activating NF-κB (Eisenbarth et al., 2012) Corroborating well with this observation, another study shows that ablation of NLRP-10 attenuates Th2 allergic inflammation, which is associated with persistent NF-κB activation (Lautz et al., 2012) Furthermore, Rip-2 has been demonstrated to mediate T cell receptor signaling to NF-κB activation, cytokine production and T cell proliferation; notably, serine/threonine kinase activity is not required for NF-κB response (Kobayashi et al., 2002; Ruefli-Brasse et al., 2004; Zhang et al., 2010) It was subsequently reported that Rip-2 deficient cells have reduced IL-1, IL-18, and Toll receptor- induced cytokine production (Chin et al., 2002; Inohara et al., 2000) Rip-2 is an inducible transcriptional product of NF-κB activation, and serves as a positive regulator of NF-κB pathway by binding to the IKK complex (Yin et al., 2010) It has been demonstrated that Rip-2 serves as a scaffolding structure by directly interacting with IKKγ (NEMO) In addition, Rip-2 polyubiquitinates NEMO at lysine-285 The interaction between Rip-2 and NEMO renders the IKK complex functional The functional IKK complex subsequently phosphorylates IκB and allows NF-κB to translocate into the nucleus (Figure 1.15) (Hasegawa et al., 2008; Inohara et al., 2000) 60 Rip-1 Rip-2 Rip-3 Rip-4 Figure 1.14 Network of the Rip family members in the multiple cellular signaling pathways (taken from Zhang et al, 2010) Rip-1 is the central mediator of several signaling pathways that result in the activation of MAPKs and NF-κB, as well as cell death Rip-2 is essential for signaling from NOD-like receptors (NLR) and can trigger MAPKs and NF-κB activation Rip-3 is mainly involved in necrosis; nevertheless, it may participate in the process of apoptosis and regulation of NF-κB signaling pathway as well Rip-4 is generally involved in activation of NF-κB and MAPK signaling pathways Abbreviations: ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; NF-κB , nuclear factor-κB; NOD, nucleotidebinding oligomerization domain; Rip, receptor-interacting protein 61 Given the role that Rip-2 play in NF-κB and the association between NF-κB and allergic airway inflammation, we hypothesize that down-regulation of Rip-2 may afford protective effect against asthma In addition, an associate study using linkage disequilibrium mapping has linked Rip-2 gene to severe childhood asthma (Nakashima et al., 2006) 1.4 RPS-3 and NF-κB signaling pathway in lung cell lines RPS-3 regulates the transcription of a subset of NF-κB target genes This NF-κB regulator is a non-Rel subunit of NF-κB DNA binding complexes RPS-3 contains heterogenous nuclear protein K (hnRNP K) homology (KH) domain, which is a structural motif that binds to singlestranded RNA and DNA with some sequence specificity The KH domain is also essential for the interaction between RPS-3 and p65 (Siomi et al., 1993) RPS-3 regulates the activity of NF-κB by mediating the recruitment of NF-κB p65 to the selected κB sites In the absence of stimuli, RPS-3 associates with the p65-p50-IκBα complex in the cytoplasm In the presence of stimuli, RPS-3 is translocated to the nucleus and binds to κB sites in a number of NF-κB dependent genes (Figure 1.12) Studies have shown that there is a significant association between RPS-3 dependence and p65-dependence in transcription of a subset of NF-κB target genes induced by T cell receptor activation RPS-3 exerts a dramatic synergistic effect on DNA-binding activity of both p65 homodimer and p50-p65 heterodimer, but not of p50 homodimer complexes in electrophoretic mobility shift essay (EMSA) (Wan et al., 2007) As a DNA binding component, RPS-3 dramatically stabilises NF-κB’s association with selective κB sites The role that RPS-3 plays in mediating NF-κB signaling pathway activation explains the extremely high affinity of native NF-κB complexes for κB DNA Although more than 100 proteins have been reported to interact with p65, no other p65 interacting protein can integrate into NF-κB complexes or can dramatically increase the DNA-binding affinity of NF-κB (Wan and Lenardo, 2009b) Furthermore, the essential role of RPS-3 in regulating NF-κB transcription has been highlighted in certain key 62 NF-κB activating signals Cytoplasm P Rip-2 P Nemo P IκBα/β p65 IKK Complex Positive Regulation ub IκBα/β IκBα/β p50 p65 IκB-NF-κB Complex p50 p65 p50 p65 Nucleus Degradation NF-κB molecule p50 κB Rip-2 gene transcription Figure 1.15 NF-κB mechanism of action In the presence of stimulation, Rip-2 serves as a scaffolding molecule and polyubquitinates Nemo Such polyubiquitination is essential for functional IKK complex The functional IKK complex phosphorylates the inhibitory protein IκB, which would result in the ubiquitination and proteosomal degradation of IκB As NF-κB molecules are released from IκB, NF-κB molecules translocate into the nucleus where they bind to the κB site of the NF-κB target genes and mediate gene transcription Rip-2 is a transcription gene of NF-κB; therefore, it positively regulates NF-κB signaling pathway Abbreviations: IKK, IκB kinase; Nemo, NF-κB essential modulator; NF-κB, nuclear factor κB; P, phosphorylate; Rip-2, receptor interacting protein-2; ub, ubiquitinated 63 physiological processes, including B cell induction of Ig-κ light chain gene expression, and T cell proliferation and cytokine secretion (IL-2 and IL-8) (Wan et al., 2007) However, RPS-3 is only essential for selected κB-regulated genes to be activated under certain conditions It is not known whether RPS-3 subunit is essential for other NF-κB activation-inducing stimuli, and whether RPS-3 targeting of NF-κB complexes to specific κB site is cell context specific These issues deserve further investigations and lead us to consider the possibility that RPS-3 is involved in the transcription of NF-κB dependent target genes, which are associated with asthma We therefore investigate the role of RPS-3 in NF-κB activity in the context of TNF-α stimulated lung epithelial cell lines 1.5 siRNA mechanism RNA interference (RNAi) is a natural process in which cells turn down, or silence, the activity of specific genes This process was first demonstrated in nematode (Caenorhabditis elegans) Briefly, a long double-stranded RNA (dsRNA) was delivered into the C elegans, in which the dsRNA enters the cytoplasm and results in degradation of the complementary mRNA, which codes for a myofilament protein As a consequence of mRNA degradation, the gene expression of the myofilament protein was silenced (Kole et al., 2012) The mechanism that mediates gene silencing involves breaking down of the long dsRNA into siRNA by an enzyme known as DICER siRNA is a dsRNA fragment that is 21 to 22 nucleotides long The siRNA interacts with a multimeric protein RNA-induced silencing complex (RISC) to target gene silencing Within RISC, the double-stranded siRNA is unwound by an ATP-dependent RNA-helicase Following that, the sense strand of the siRNA is discarded; while the remaining antisense strand, which is complementary to the target mRNA, guides RISC to the mRNA Upon binding of siRNA antisense strand to its target mRNA, the endonuclease argonaute (a component of the RISC) cleaves the mRNA at 10 and 11 nucleotides downstream from the 5′ end of the antisense strand (Figure 1.16) (Kole et al., 2012; Meinicke et al., 2009) 64 Currently, siRNA are available commercially and are used as experimental or therapeutic tool to study or regulate physiological and disease processes (Dillon et al., 2005) Table 1.3 and 1.4 summarize the application of siRNA as therapeutic tool or experimental tool Their simple and small structure allows for easy chemical modifications to improve on their existing properties for efficient gene silencing (Dykxhoorn and Lieberman, 2006) With increasing interest in RNAi research, some companies are now offering siRNA in chemically modified format to increase their specificity and stability In the past, choosing a suitable siRNA molecule has been described as an empirical process Although this process has identified numerous effective siRNA, many of these siRNA have yet to be tested for their knockdown efficacy Such incomplete information could have contributed to the failure and instability of most of the confirmed siRNA when tested in vivo Research has now moved towards to new rational siRNA design outlines, which have been made possible due to better understanding of RNAi biochemistry, particularly the RISC mechanism of strand selection (Meinicke et al., 2009; Mittal, 2004) Many studies involving the RISC mechanism have been conducted and systemic analyses of 180 siRNAs have been performed Results from these studies summarized the design concepts that should be considered in order to create the most effective and specific siRNA molecules (Mittal, 2004; Wu et al., 2011) For example, it was found that more effective siRNA can be generated by decreasing the stability of the antisense siRNA This reduction in stability can be achieved by incorporating an A•U base pair at the 5’ end of the antisense strand (Schwarz et al., 2003) In addition, 5’ of the antisense strand should be phosphorylated and the antisense-RNA-target duplex should form an A-helical structure (Jackson et al., 2006) These rules have helped to lower the concentration of siRNA needed to silence target genes of interest Nonetheless, using the above methods to predict a single siRNA that silences the chosen target gene without the highest precision may not always work There are many other factors that affect the design of the most specific siRNA for the given target Screening methods such as RNAi 65 Figure 1.16 Mechanism of siRNA silencing (http://www.gene-quantification.de) Abbreviations: Ago, endonuclease argonaute 2; RISC, RNA induced silencing comples 66 Gene/mRNA targeted Delivery approach Biological action in mouse allergic/ asthma model GATA-3 IN Attenuated airway inflammation, eosinophilia and Th2 cytokine production in the (Finotto et al., 2001) lungs of OVA sensitized mice SyK Aerosolization Suppressed OVA-induced IL-5 expression, eosinophilia, TNF-α production, ICAM (Stenton et al., 2002) expression and AHR in mouse asthma model STAT-1 IN Suppressed IL-5 expression and eosinophilia in mouse lung tissue NPRA TD Suppressed IL-4, IL-5 production, decreased inflammatory cell infiltration, reduced mucus hypersecretion and goblet cell metaplasia, and attenuated AHR in mouse (Wang et al., 2008) asthma model IL-5 IT Suppressed eotaxin and IL-5 expression and attenuated AHR in mouse asthma (Huang et al., 2008) model CD40 IP Decreased IgE and IgG1antibodies in serum, suppressed Th2 cytokine production in (Suzuki et al., 2008) allergic mouse model SphK IN Suppressed airway inflammation, mucus hypersecretion, and AHR in mouse asthma (Lai et al., 2009) model STAT-6 IN Suppressed Th2 cytokine production, inflammatory cell infiltration, and attenuated (Darcan-Nicolaisen et al., AHR in mouse asthma model 2009) Reference (Quarcoo et al., 2004) Table 1.4: siRNA in animal studies Abbreviations: IN, intranasal; IP, intraperitoneal; IT, intratracheal;NPRA, natriuretic peptide receptor A; SphK, sphingosine kinase; SyK, spleen tyrosin kinase; GATA-3, STAT, Signal transducer and activator of transcription; TD, transdermal 67 microarray has been employed to select the most efficient siRNA molecule (Kumar et al., 2003) In order to overcome the barriers of in vivo siRNA delivery, packaging methods and chemical modifications have also been employed These packaging methods and chemical modifications include viral-vector based and non-viral based vectors Viral-vector based siRNA delivery method allows for siRNA to be delivered to a wide range of cells and tissue types both in vitro and in vivo; however, it is associated with safety issues as well as issues regarding bulk production and quality control For example, lentiviruses have been observed to insert their genomes at random sites in the chromosome of their host Such genomic integration may disturb cellular functions and result in cancer (Hacein-Bey-Abina et al., 2003) To overcome these drawbacks, and safety issues in particular, “artificial viruses” are under development (Mastrobattista et al., 2006) Non-viral mediated vectors generally consist of cationic charged packaging molecules — liposome/lipid-like carriers, cationic polymers, and cationic peptides The following paragraph shall discuss about the clinical applications of liposome carrier since it is the only non-viral mediated vector that is being tested in clinical trial so far Nonetheless, using animal models, it is shown that the use of cationic charged packaging molecules improves the chance of successful interactions between the siRNA and its target cell surface, while facilitating endosomal intake of the gene silencing molecule These cationic particles not only help transport the siRNA but also protect it from degradation by nucleases, thereby prolonging the siRNAs’ overall gene silencing activities (Durcan et al., 2008; Kim et al., 2009) Liposomes are successful siRNA carriers in various in vitro and in vivo animal models with their cationic nature being beneficial for local and mucosal delivery sites Their structure is also advantageous due to the ease in which chemical modifications can be incorporated to the carrier molecule These modifications include polyethylene glycol (PEG), antibodies, and other cell-targeting ligands, all of which have shown to increase the circulating half-life and cell specificity of the vehicle 68 molecule (Kim et al., 2009) Increasing the cell specificity of vehicle molecule is of particular importance as cationic lipid-like carriers are usually toxic in systemic delivery, so specific targeting would help reduce the toxic systemic side effects (Lam et al., 2012) Nevertheless, there have been breakthroughs in the use of liposome complexes in cystic fibrosis patients, for example 1,2-Dioleoyl3-trimethylammonium-propane (DOTAP) liposomes complexed with the cystic fibrosis transmembrane conductance regulator (CFTR) gene has been successfully administered and shown to upregulate the gene expression of CFTR in patients (McLachlan et al., 1996) Although liposome/lipid carrier is a potential vector for siRNA delivery, there are still problems associated with such vector Even though the airway epithelial cells can be relatively easy to target through intranasal / intratracheal routes, the variable nature of the siRNA transfection efficiency can also affect the silencing efficiency This variable nature is due to sequence differences between the siRNA used in the experiments These differences cause a wide range of results from improved significant silencing of a particular gene to no silencing or inhibition of the target gene (Griesenbach et al., 2006) Further development in this group of siRNA carriers includes the use of stable nucleic acid-lipid particles (SNALPs), which enhances the endosomal release of siRNA particles via cellular uptake due to an overall structure of cationic and fusion lipids (Morrissey et al., 2005) In addition, a new chemically synthesized lipid-like carrier known as lipidoids has been developed to overcome the disadvantageous systemic toxicity that can be found in some synthetically developed liposome/lipid like carrier structures (Akinc et al., 2008; Kim et al., 2009) These lipidoids consist of base alkyl acrylates and amides which are joined to primary and secondary amines Lipidoids have shown promising effect in delivery of siRNA to the livers and lungs of the mice (Santel et al., 2006) The development of lipidoids indicates the progress in overcoming the unfavorable off-target effect that can cause systemic immunological side effects (Kim et al., 2009) 69 Taken together, siRNA is regarded as one of the most recent techniques for powerful and effective gene silencing Therefore, in this study, siRNA was used as a tool to down-regulate the expression of Rip-2 and RPS-3 in our mouse asthma model We chose direct intracheal application of siRNA because this method allows for siRNA molecules to be delivered into the animals noninvasively whilst targeting local lung cells and tissues (Durcan et al., 2008) 1.6 Pharmacology of Fisetin There is an increased interest on the use of plant based polyphenols Studies have shown that these polyphenols possess varied biological properties such as anti-inflammatory, anti-oxidative, antimicrobial, and anti-carcinogenic (Khan et al., 2008) These polyphenols, especially those from dietary sources, are perceived as non-toxic and are widely accepted (Surh, 2003) Currently, several nonnutritive, macronutrient phyotchemicals are being tested for management of various inflammatory diseases Flavonoids are polyphenolic macronutrients that are present in large number of plants These polyphenolic macronutrients have been shown to affect cellular signaling (Adhami et al., 2012) Fisetin (3,7,3′,4′-tetrahydroxyflavone) (Figure 1.17) is a bioactive flavonol that falls under the subgroup of flavonoids (Howells et al., 2010) This naturally occurring bioactive flavonol can be isolated from plants like the smoke tree (Cotinus coggygria), and fruits and vegetables such as strawberry, apple, persimmon, grape, onion and cucumber at concentrations ranging from to 160 µg/g Notably, among the fruits and vegetables analyzed, strawberry contains the highest concentration of fisetin per gram (Sung et al., 2007) Fisetin exhibits a broad array of biological properties such as anti-bacterial, anti-oxidative, neuroprotective, anti-inflammatory, and anti-cancer In 1966, fisetin was identified to be an antimicrobial agent (Gabor and Eperjessy, 1966) It was later shown to prevent oxidative stress-induced nerve cell death Fisetin also exhibits neurotrophic activity; it enhances nerve cell differentiation through activation of ERK, making it an attractive therapeutic molecule for Huntington’s disease (Maher et al., 2011) It is shown that oral administration of fisetin to mice enhanced ERK-dependent long term potentiation and promoted memory (Maher, 2006) In other studies it is demonstrated that 70 Figure 1.17 The structure of fisetin (Taken from Howell et al, 2010) 71 fisetin reduces cytotoxicity of LPS-stimulated microglia towards B35 neuroblastoma cells in a coculture system This observation suggests that fisetin has strong anti-inflammatory properties in brain microglia, and is a potential therapeutic agent for treatment of neuroinflammatory disease (Zheng et al., 2008) Fisetin is reported to be a dual inhibitor of PI3K/AKT and mTOR signaling pathways These signaling pathways are mediators of cancer In line with this inhibition, fisetin has been shown to enhance apoptosis of prostate cancer cell lines (LNCaP, CWR22Rν1 and PC-3 cells) (Suh et al., 2010) Many of the anti-cancer properties of fisetin are closely linked to its anti-inflammatory properties Numerous studies show that fisetin exhibits anti-inflammatory activities More specifically, fisetin was found to suppress the level of TNF-α, IL-6, IL-8 and MCP-1 in a murine rheumatoid arthritis model and in human synovial membrane samples from patients with rheumatoid arthritis (Lee et al., 2009) In addition, fisetin could attenuate LPS-induced lung inflammation through reduction of iNOS and TNF-α levels in mice (Geraets et al., 2009) The exact molecular mechanism that mediates these anti-inflammatory effects of fisetin has been unequivocally determined Nevertheless, there is concrete evidence pointing to the inhibition of NF-κB transcriptional activity (Suh et al., 2010; Sung et al., 2007) It has been shown that fisetin may inhibit the upstream kinase transforming growth factor (TGF)-β-activated kinase (TAK1) and IKKγ which are vital for the activation of the NF-κB canonical pathway (Sung et al., 2007) 1.7 The mouse asthma models Experimental animal models of asthma provide an excellent system for understanding the pathogenesis of asthma and for the development of new therapies in the past decades (Bates et al., 2009) Many species of animals are commonly used to study asthma, including horses, pigs, dogs, cats, ferrets, monkey, guinea pig, rats and mice In general, every species has its advantages and disadvantages, and species-specific differences which include anatomical properties, and physiological and immunological responses (Zosky and Sly, 2007) Nevertheless, the steps involved in generating the asthma models are generally similar in most animals Basically, animals are 72 systemically sensitised to a particular allergen After a period of time, the animals are challenged with the same allergen administered via the airway (Bates et al., 2009) Among the animal models, mouse asthma models are the most widely used Not only are mice easy to breed, handle, and maintain, the immunology of mice is well described and many immunological tools and genetically altered strains are available (Holmes et al., 2011; Pichavant et al., 2007; Zosky and Sly, 2007) Furthermore, the mouse model mimics many features of human asthma (Zosky and Sly, 2007) In most studies, OVA has been used as a standard allergen to systemically sensitize and aerosol challenge the mice Nonetheless, other antigens being used include ragweed and the recombinant major cat allergen, FelD1 In addition to systemic sensitization to allergen followed by airway challenge, mice can be sensitized exclusively via the airways For example, in the HDM model, a preparation of HDM antigen is normally administered in the airway for several consecutive days (Blanchet et al., 2012) In the OVA model, mice are typically immunized twice with chicken OVA coupled to aluminium hydroxide as adjuvant The sensitizations typically occur seven days to 14 days apart to allow for the development of OVA-specific IgEs (Bao et al., 2009; Cheng et al., 2011) In order to induce an allergic inflammatory response, the mice are challenged with aerosolised OVA Several challenges are needed to increase the severity of airway inflammation In response to challenges, mice develop airway inflammation characterised by eosinophilia, mast cell infiltration, and AHR These features closely mimic the inflammatory responses that occur in human (Bates et al., 2009) However, this model does not allow for the evaluation of more chronic features of asthma because of its short nature In order to observe airway remodelling in mouse asthma model, the mice must be exposed to further allergen challenges for a period of two to three weeks (Yamagata et al., 2008) Nonetheless, such chronic model is still not severe enough Although the OVA model is considered to be an ideal model to study allergic airway inflammation, it is not an ideal model to study airway remodelling (Blanchet et al., 2012) 73 Although the HDM model is a less studied mouse model of asthma, it is now gaining popularity HDM model is emerging as the gold standard for the study of the onset of asthma because of the following reasons: (1) the priming is performed through the natural (intranasal) route of airway exposure; (2) administration HDM interferes with the epithelium, causing local sensitization of the airways to antigen; and (3) HDM occurs naturally in the environment and does not require adjuvant to mediate Th-2 polarised immune response, which makes it a more physiologically relevant model of antigen exposure than OVA (Blanchet et al., 2012) HDM induces inflammatory effect by disrupting airway epithelial tight junctions to gain access into the dendritic network Such disruption could be achieved through the proteases activity of HDM (Wan et al., 2000) In addition, HDM’s proteolytic component also activates PAR on the epithelium and result in the secretion proinflammatory cytokines such as IL-6, IL-8, Rantes, and eotaxin (Jacquet, 2011) HDM contains microbiological glucose structures, for instance β-glucan, which can activate epithelial C-type lectin receptors, including dectin-1(Nathan et al., 2009) Activation of C-type lectin receptors induces Ca2+ fluxes and may lead to activation of the endogenous protease calpain and cleavage of cell-cell contact proteins (Chun and Prince, 2009) Although TLR4 activation is essential for HDM induced airway inflammation, activation of TLR4 by LPS (a ligand for TLR4 that is commonly found in HDM) does not affect the epithelial barrier function (Post et al., 2012) 74 ... al., 20 09; Lambrecht and Hammad, 20 12) These cytokines contribute to pathogenesis of allergic airway inflammation TSLP is hought to mediate polarization of Th -2 immune response (Holgate 20 12) Its... (Lambrecht and Hammad, 20 12) Based on bronchial biopsy studies, the airways of subjects with asthma have fragile epithelial (Lackie, 1997; Lambrecht and Hammad, 20 12) The integrity of airway epithelial... IL-33 or ST2 are currently in clinical development (Barnes, 20 11) Compelling evidence implicates TSLP as a potential initiator of Th -2 bias allergic airway inflammation Blockade of TSLP has been shown