University of Groningen Novel therapeutic targets for the treatment of asthma van den Berg, M.P.M.

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Novel therapeutic targets for the treatment of asthma

van den Berg, M.P.M.

DOI:

10.33612/diss.143959597

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Publication date:

2020

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van den Berg, M. P. M. (2020). Novel therapeutic targets for the treatment of asthma: Focus on Arginase

and TRPA1. University of Groningen. https://doi.org/10.33612/diss.143959597

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Preface

Allergic asthma is a chronic airway disease characterized by airway hyperresponsiveness,

airflow obstruction, inflammation and remodeling. Current treatment options are

not sufficient for a large group of asthma patients, especially patients suffering from

severe asthma. The objective of this thesis is to study novel therapeutic options for the

treatment of asthma, thereby focusing on two specific targets: arginase and transient

receptor potential ankyrin 1 (TRPA1).

Asthma prevalence and perspective

Asthma is considered to be a heterogeneous disease and can be both allergic and

non-allergic in nature, dependent on the trigger that induces asthma symptoms (1,

2). Although, in the recent years more clinical phenotypes have been characterized

based on asthma symptoms (Figure 1) (3-5). Allergens are considered to be the main

provokers of allergic asthma, whereas non-allergic asthma can be induced by stimuli

such as cold air, exercise, air pollution, stress and viral respiratory infections (1). In

both allergic and non-allergic asthma these triggers cause (chronic) airway obstruction,

resulting in limited airflow to the lungs and thus shortness of breath. Other commonly

observed symptoms are wheeze, chest tightness and cough (6). Asthma is commonly

diagnosed based on identification of a characteristic pattern of respiratory symptoms

(e.g. shortness of breath, wheezing, chest tightness and cough)(7). The initial diagnosis

can be supported by diagnostic tests, such as spirometry and responsiveness to

bronchodilators, allergy tests (skin tests, presence of allergen-specific IgE in serum),

and in more severe cases, a bronchial provocation test (6).These tests can also aid in

determining the asthma phenotype of a patient (8).

Typically, asthma symptoms vary in intensity and over time, and may resolve

spontaneously or in response to medication (9). Asthma patients can be

symptom-free for weeks or months at a time before experiencing sudden episodic

flare-ups of their disease, or exacerbations, which can be life-threatening. The airway

hyperresponsiveness (AHR) to direct or indirect airway smooth muscle stimuli and the

chronic inflammation associated with asthma usually persist to some degree, even

during the periods when patients are free of symptoms, but can be reduced with

treatment (9). Asthma severity can be classified in 5 different treatment steps according

to the Global Initiative for Asthma (GINA) guidelines (9). These steps are based on the

level of treatment that is necessary to achieve control of symptoms and exacerbations

(10-12) and will be elaborated on in more detail later on.

Worldwide more than 300 million people are estimated to be suffering from asthma

(13). People of all ages are affected by this chronic disease and it contributes to high

societal costs (14). While remission is common in puberty, it occurs less often in adults

(15). Although the prevalence of non-allergic asthma has remained on a stable level the

last 25 years, the prevalence of allergic asthma has increased (16).

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Figure 1: Asthma symptom heterogeneity based on clinical phenotype. The most frequently used asthma categories based on asthma symptoms and history. Disease history can be classified as childhood or adult onset, and both categories can be further divided based on allergic testing (allergic vs. non-allergic), cytokine expression profile (Th2-high vs Th2-low), main airway inflammatory cell type and the presence of airway wall remodeling (197). Th2, T-helper2.

Large variations in asthma symptom prevalence exist between different countries, even

when the population shares both genetic and ethnic backgrounds, suggesting a role for

environmental factors, and especially western lifestyle, in these differences (17, 18).

Asthma pathogenesis

The asthmatic reactions in asthma

Allergic asthma is characterized by allergen-specific immunoglobin E (IgE)-mediated

reactions to common aeroallergens, such as house dust mite, molds and animal dander.

A complex interaction between cells of the innate and adaptive immune systems and

structural lung cells paginin response to these allergens, results in among others an

early asthmatic reaction (EAR), a subsequent late asthmatic reaction (LAR) caused by

infiltration of inflammatory cells in the lung, and airway remodeling (19).

Allergic airway reactions start with the sensitization to specific allergens. Initial

recognition of inhaled allergens by allergen presenting cells, such as dendritic cells, in

the airway epithelium and submucosa, leads to their presentation to naïve T cells in

lymph nodes and subsequent activation and differentiation of naive CD4+ T-cells into

(6)

Type 2 helper T (Th2)-cells (Figure 2) (19). These Th2-cells and their cytokines, driven

by interleukin (IL)-4 and IL-13, induce the formation of allergen-specific IgE-molecules

by B-cells, which bind to the high affinity IgE (FcεRI)-surface receptors of mast cells,

basophils and eosinophils (20-22). Together with the Th2-cytokines (e.g. IL-9), these

IgE-molecules are able to prime mast cells, resulting in the sensitization to the specific

allergen. Besides inducing IgE formation and priming mast cells, Th2-cytokines also play

a crucial role in other aspects of allergic inflammation. IL-5 is involved in B-cell growth

and activation, and survival and recruitment of eosinophils, whereas IL-13 impacts on

airway smooth muscle constriction, mucus production and maturation of epithelial cells

into goblet cells, and the generation of extracellular matrix proteins (19).

On a subsequent encounter with the allergen, the allergen will bind to the IgE on FcεRI

on the surface of mast cells leading to receptor cross-linking (23, 24). This regulates

intracellular Ca

2+

_{concentrations and mast cell activation. Upon activation, mast cells}

will degranulate and release pre-made and stored proinflammatory mediators from

their granules (e.g. histamine, serotonin, tryptase, chymase) (25). Within minutes,

these molecules induce airway smooth muscle contraction, mucus production and

vascular leakage (26, 27). In addition, de novo synthesized mast cell mediators (e.g.

leukotrienes, prostaglandins, cytokines) are released and further enhance the allergic

airway response (26). Although the mast cell mediator-induced acute airway obstruction

generally resolves within 1 hour, it does play an important role in the subsequent LAR

by inducing AHR (28).

The LAR follows 4 to 6 hours after the EAR. During this phase of the allergic airway

response, the inflammatory mediators that were induced during the EAR have caused

the recruitment of inflammatory cells into the airways (29). These inflammatory cells,

(mainly eosinophils, but also macrophages, neutrophils and basophils), as well as also

structural airway cells (e.g. epithelial cells, endothelial cells, fibroblasts) are able to

produce large quantities of inflammatory chemokines and cytokines (27, 29), that can

act either directly or indirectly (through neuronal pathways) on effector cells involved

in airway obstruction, such as airway smooth muscle cells and mucus-producing cells

(30-32). After 24 to 48 hours, infiltrating Th2-cells can be found in the inflamed airway.

The cytokines (e.g. IL-4, IL-5, IL-13, granulocyte-macrophage colony-stimulating factor

(GM-CSF)) excreted by these Th2-cells further enhance IgE production and promote

chemotaxis, the expression of vascular adhesion molecules, and activation of eosinophils

and macrophages (33). Via these IgE-driven, so-called Th2-high, processes, and under

the influence of ongoing allergen exposure, chronic airway inflammation is promoted

which contribute to increased AHR and airway obstruction (34).

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Figure 2: Inflammatory pathways in allergic and non-allergic asthma. In the allergic, IgE-mediated pathway (left), inflammation and airway and bronchial hyperresponsiveness are mainly caused by aeroallergens that are taken up and presented by dendritic cells to naive T-cells that mature into Th2-cells. Cytokine pro-duction by Th2-cells promotes IgE propro-duction by B-cells. Together with these cytokines, IgE induces mast cell sensitization to the allergen. Furthermore, the Th2-cytokines contribute to airway eosinophilia, mucus hypersecretion and airway hyperreactivity. In the non-allergic pathway (right), environmental irritants stim-ulate induce the release of IL-33, IL-25 and TSLP by epithelial cells, leading to ILC2 activation. The cytokines produced by ILC2s contribute to mucus hypersecretion, eosinophilia and airway hyperresponsiveness (50). FcɛRI, high-affinity receptor for IgE; IgE, immunoglobulin E; IL, interleukin; ILC2, type 2 innate lymphoid cell; NKT, natural killer T cell; Th, T-helper; TSLP, thymic stromal lymphopoietin.

Non-allergic mechanism

In non-allergic asthma (so-called Th2-low asthma), and also in allergic asthma,

mechanism other than IgE-mediated responses that contribute to the disease can be

identified, mediated by Th1- and Th17-cells, airway nerve activation, epithelial responses

and type 2 innate lymphoid cells (ILC2s). A mixed Th1- and Th17-inflammatory response

is associated with neutrophilic asthma (35-37), however much is still unknown about

the exact mechanism underlying this asthmatic phenotype (38).

Sensory and motor neurons that innervate the airways control smooth muscle

contraction (39-41) and in asthma increased release of neurotransmitters (e.g.

neurokinin A and substance P), contributing to neurogenic inflammation, can be

observed (42). Furthermore, there is increasing evidence suggesting neural plasticity

in asthma, as substantiated by studies showing enhanced excitability and outgrowth of

neurons in asthma (43, 44). Activation of afferent airway nerves will result in defensive

(8)

reflexes such as bronchoconstriction, mucus secretion, cough and inflammation (45,

46).

Airway epithelial cells function as the first physiological barrier in the lung and will

release IL-25, IL-33 and thymic stromal lymphopoietin (TSLP) in response to damage

signals caused by allergens or environmental irritants (e.g. cigarette smoke, cold air).

These so-called alarmins activate among others Th2-cells, mast cells and ILC2s. In

response to these alarmins, ILC2s produce an array of cytokines similar to Th2-cells,

including IL-5, IL-9 and IL-13 (45, 46). These cytokines are able to, among others, promote

IgE production, increase eosinophilia and mucus production, and induce AHR (47-49).

As ILC2s are able to drive severe eosinophilic inflammation in the absence of

allergen-specific IgE-mediated reactions, they are seen as important regulators of non-allergic

eosinophilic asthma (Figure 2) (50). Furthermore, ILC2s are involved in IL-33-mediated

asthma exacerbations in response to respiratory viral infections (51, 52). Viral infection

are considered as important triggers for the development of childhood asthma (53),

and assumed to worsen inflammation in both allergic and non-allergic asthma (54).

Airway remodeling

Airway remodeling is a typical feature of asthma. It may be the result of continuous

injury and incomplete healing of airway tissue in response to airway inflammatory

responses, but can also occur as an unrelated phenomenon under the influence of

mechanical stress or early-life events (55, 56). Airway remodeling is characterized

by structural changes in the airway wall, such as smooth muscle cell hyperplasia,

hypertrophy of submucosal gland mass, angiogenesis, thickening of the basement

membrane and subepithelial fibrosis due to deposition of matrix proteins (57, 58).

Epithelial damage may lead to easier exposure of underlying cells (e.g. airway smooth

muscle cells, ILC2s, airway nerves) to environmental irritants, thereby enhancing, among

others, AHR, airway remodeling and inflammation. Over time, airway remodeling may

result in persistent chronic AHR and may lead to gradual loss of lung function (57, 59,

60).

The role of the mast cell in asthma

As one of the initiators of the allergic response and sentinels at the interface of lung

tissue and the external environment, mast cells play a key role in asthma (25). Mast

cells are highly granulated, tissue resident inflammatory cells that develop from specific

myeloid progenitor cells (61, 62). These partially differentiated progenitor cells are

released from the bone marrow into the bloodstream. Via well-organized integrin/

receptor-mediated trafficking they enter the peripheral tissue, where they mature and

differentiate into mast cells under the influence of local growth factors and cytokines

(62, 63). In healthy individuals, the number of mast cells that can be found in tissues is

relatively low and constant. However, the size of the mast cell population can increase

dramatically in pathophysiological conditions (64, 65), such as those seen in patients

suffering from allergic asthma (66). Stem cell factor (SCF) is the main growth factors

(9)

involved in mast cell development and their subsequent accumulation, maturation and

survival in tissue (67). This is demonstrated by the finding that a deficiency in the SCF

receptor c-kit, has dramatic impact on mast cell viability and may result in partial or

almost complete absence of mast cells in peripheral tissue (63, 68). Other mediators

involved in mast cell survival and growth include IL-3, IL-4, IL-9, IL-10, IL-33, transforming

growth factor (TGF)-β, chemokine (C-X-C motif) ligand (CXCL)-12 and nerve growth

factor (NGF) (63).

Based on their granule protease content, mature human mast cells were originally

divided in two subsets, which both can be found in the lung. The tryptase/chymase mast

cell subset has tryptases, chymases and carboxypeptidase prestored in its granules and

resides in the pulmonary mucosa, whereas the tryptase subset only contains tryptases

and is located in the submucosa of the lung (69-71). Later a third mast cell phenotype,

containing tryptase and carboxypeptidase A3, was found in airway epithelium of asthma

patients (72).

Mast cells are able to express a large variety of receptors (e.g. FcεR1, pattern

recognition receptors, complement receptors), that enable them to respond to

numerous environmental and inflammatory signals (73). It has become clear that the

local microenvironment directly affects mast cell maturation, phenotype and function,

all factors that have a large impact on mast cells’ ability to recognize and respond to

stimuli by releasing a range of biologically active mediators (74). This phenotype is,

however, highly flexible and mast cells have even shown to be able to switch between

subsets during practically all stages of their life-cycle (75). Various factors can induce

phenotypic changes in mast cells, and several changes combined may influence mast cell

homeostatic of pathophysiological responses (76). This can, for example, be observed

during mast cell priming and sensitization by IgE and IL-4 in allergic asthma, where

IL-4 promotes both functional as well as morphological maturation, including FcεRI

upregulation, and IgE-binding anchors those FcεRI on the cell surface (77, 78).

Current treatment options

To date there is no cure for asthma. Instead, treatment focusses on relief, symptom

control and prevention of exacerbations. Two main categories of asthma medication can

be distinguished: controller and reliever medication. The GINA provides clear guidelines

on asthma therapy (Figure 3) (9). The golden standard for controller medication consists

of the combination of an inhaled corticosteroid (ICS) and a long-acting β2-adrenoceptor

agonist (LABA), to counteract inflammation and bronchospasms, respectively. Low dose

ICS-formoterol or a short-acting β2-adrenoceptor agonist (SABA) can be used as reliever

medication, when acute bronchodilation is needed. For most patients the combination

of these medications is sufficient. However, there is a group of patients, classified as

GINA category 5 asthmatics, in which, despite good adherence to this therapy and good

inhalation technique, asthma symptoms remain uncontrolled and add-on therapy is

necessary (12).

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Reliever

(as-needed)

Category 1

Category 2

Daily low dose ICS or as-needed ICS +formoterol

Category 5

High dose ICS-LABA + Add-on therapy dependent on phenotype Category 4 Medium dose ICS-LABA Category 3

Low dose ICS-LABA

Mild Moderate Severe

Preferred controller

Alternative controler

Low dose ICS-formoterol

or SABA

or SABA LTRA or

low-dose ICS when SABA is taken

Medium dose ICS or low dose

ICS +LTRA

High dose ICS, add-on tiotropium

or LTRA

Add low dose OCS As-needed

low dose ICS-formoterol Low dose ICS whenever SABA

is taken

Figure 3: GINA 2020 treatment guidelines for asthma. Based on symptom frequency and severity, five disease categories can be distinguished ranging from mild intermittent to severe persistent asthma. Adapted from (9). ICS, inhaled corticosteroids; LABA, long-acting β2-adrenoceptor agonist; LTRA, leukotriene receptor antagonist; OCS, oral corticosteroids; SABA, short-acting β2-adrenoceptor agonist.

In the Netherlands, this group of GINA category 5 asthmatics comprises around

approximately 3.7% of the asthma patient population (79). The constant symptoms

and recurrent exacerbations have a substantial impact on the daily-life of severe

asthmatics and the morbidity among this patient group is high (80, 81). Moreover,

uncontrolled asthma is correlated with reduced physical and mental health-related

quality-of-life scores (80, 81), and higher social-economic impact due to medications

cost, hospitalization and indirect costs, such as disability and work absenteeism (82).

Category 5 asthmatics are prescribed with high-dose ICS-LABA, possibly supplemented

with low dose oral corticosteroids (OCS). Long-term use of oral corticosteroids by

these patients is often necessary, but undesirable, as risk of systemic adverse event,

such as infections and cardiovascular, metabolic, bone-related and gastrointestinal

implications, is high (83). Furthermore, severe asthma patients are referred for

phenotypic assessment. Based on whether the disease presents high or low blood

eosinophil levels, as a marker of Th2-type inflammation, next steps in asthma treatment

can be undertaken (84). The Th2-high phenotype is more common in patients with

severe asthma and in these patients anti-inflammatory therapies, such as biologicals

aimed at inhibiting IgE (e.g. omalizumab), IL-4 receptor (dupilumab), or IL-5/IL-5 receptor

(e.g. mepolizumab, benralizumab), can be prescribed as add-on treatment. Patients

presenting with Th2-low asthma have less therapeutic options as they are usually

(11)

irresponsive to treatment with ICS and other anti-inflammatory drugs (85, 86). High

dose ICS combined with LABAs or long-acting muscarinic receptor antagonists (LAMAs),

such as tiotropium, are likely the preferred controller medication for these patients, as

they target bronchoconstriction through alternative pathways, however their effects

on airway inflammation has not been fully evaluated yet in randomized controlled

trials (87).

Safety and effectiveness of novel treatments are still not completely clear. In fact,

around 1/3 of severe asthma patients is non-Th2 high and even among Th2-high asthma

patients, efficacy of novel biologicals is not always optimal. Furthermore, since also

mechanisms besides inflammation (e.g. structural changes in the airway wall, airway

nervous system, altered cough reflex) play a crucial role in asthma pathology, research

into additional mechanisms and treatment options is still necessary, particularly for

severe asthma (9). This thesis focuses on two novel potential therapeutic targets:

arginase and TRPA1.

Arginase

The manganese-containing enzyme arginase plays an important role in the urea cycle, as

it catalyzes the final step in which ʟ-arginine is metabolized to urea and ʟ-ornithine. Two

isoforms of arginase can be distinguished: arginase 1, which is located in the cytosol, and

the mitochondrial expressed arginase 2 (88). The enzyme is thought to have originated

in bacteria, and has persisted through the evolutionary process. Thus, it can be found in

cells of bacteria, yeasts, plants, invertebrates and vertebrates. As most bacteria, yeasts,

plants and invertebrates only express the mitochondrial arginase 2, it is presumed

that the transfer of the arginase enzyme from bacteria to eukaryotic cells occurred via

mitochondria. Organisms that convert excess nitrogen to urea also express arginase 1. In

mammals, arginase 1, due to its role in the ammonia detoxification, is the main arginase

subtype expressed in the liver, whereas arginase 2 is highly expressed in the kidney,

small intestine, prostate and lactating mammary gland. However, both isoenzymes can

also be found in other tissues and their expression is induced by many different stimuli

and in a variety of conditions (89).

In humans the arginase 1 and arginase 2 enzymes are encoded by two separate genes,

that are located on different chromosomes. Human arginases have a protein homology

of around 60%. However, the mechanism of action of both isoenzymes is similar, the

same metabolites are formed and the areas critical for enzyme function are 100%

homologous (90). The expression of arginase 1 and arginase 2 is regulated by different

transcription factors. Transcription factors for arginase 1 include signal transducer and

activator of transcription (STAT)-6/STAT-3, Forkhead box O4, hypoxia-inducible factor

(HIF)-1, CCAAT-enhancer-binding protein-β and activator of iron transcription-2 (91-93),

whereas arginase 2 is regulated by cyclic adenosine monophosphate (cAMP) response

element-binding protein, HIF-2 and interferon regulatory factor-3 (94-96).

(12)

Arginase expression and regulation in the airways

In the airways, arginase 1 and arginase 2 are constitutively expressed, in particular

in epithelial and endothelial cells, inhibitory nonadrenergic noncholinergic neurons,

macrophages and fibroblasts (97-99). Furthermore, the expression of both isoenzymes

may be induced by various stimuli, including Th2-cytokines (e.g. IL-4 and IL-13), TGF-β,

bacterial lipopolysaccharide (LPS) and allergens (100, 101). In mouse macrophages,

the Th2-cytokines IL-4 and IL-10 are able to induce arginase activity, while in human

macrophages IL-4 combined with agents increasing cAMP reaches the same effect

(102-104). In the different animal models of asthma that reported increases in arginase

activity and expression, especially the expression of arginase 1 was found to be

elevated, whereas an elevation in arginase 2 expression was less pronounced or even

completely absent (128, 139).

ʟ-Arginine does not only function as a substrate for arginase, it is also used by nitric

oxide synthases (NOS) for the production of nitric oxide (NO) and ʟ-citrulline (105).

NO is an important biological mediator involved in a wide range of physiological and

pathophysiological processes (106). In the airways, NO is among others involved in

the regulation of pulmonary vascular and airway tone via smooth muscle relaxation,

inflammatory processes and lung development (107-109).Three isoforms of NOS can be

distinguished, two constitutively expressed NOS-isoforms (cNOS) consisting of neuronal

NOS (nNOS) and endothelial NOS (eNOS), and an inducible NOS (iNOS). cNOS generates

a relatively low amount of NO in response to an increase in intracellular calcium

concentration, caused by the action of a receptor agonist or membrane depolarization.

The activity of iNOS, on the other hand, is dependent on changes in gene expression

by for example inflammatory cytokines, and produces large amounts of NO (107). In

conditions of low arginine availability, for example when arginase activity is elevated,

NOS is uncoupled and superoxide is also formed. Superoxide rapidly reacts with NO,

leading to the formation of peroxynitrite, that often has detrimental effects in the tissue

because of nitration of tyrosine residues (110).

It has been hypothesized that regulation of NO levels via substrate competition with

NO synthases (NOS), is one of the biological functions of extrahepatic arginase (102,

111, 112). Although ʟ-arginine has an approximately 1000-fold higher affinity for NOS

than for arginase, there is still competition between arginase and NOS for ʟ-arginine,

since the V

_max

(maximal enzymatic rate) for arginase is around 1000 times higher than

for NOS (88, 113, 114). Besides direct substrate competition between arginase and

NOS, the two metabolic pathways may also interact at other levels. The intermediate

NOS metabolite N

ω

_{-hydroxy-ʟ-arginine functions as an endogenous arginase inhibitor}

(115-117). Furthermore, via a feedback mechanism ʟ-ornithine is able to inhibit arginase

(113, 118, 119), and ʟ-arginine uptake by cells producing NO (120-122). These are all

indications of the existence of a fine balance in ʟ-arginine homeostasis. A disrupted

balance, by changes in NOS and arginase expression and activity, has shown to play a

role in many pathologies, including asthma (figure 4) (108, 123).

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Figure 4: The role of ʟ-arginine metabolism by arginase and NOS in asthma. NOS and arginase compete for ʟ-arginine as substrate. Conversion of ʟ-arginine by arginase results in the production of urea and ʟ-ornithine. ʟ-Ornithine, that can inhibit arginase activity, is further metabolized to ʟ-proline and polyamines, that are involved in collagen formation and cell proliferation, respectively. NOS converts ʟ-arginine to ʟ-citrulline and NO. During this process the endogenous arginase inhibitor NOHA is formed. NO induces smooth muscle relaxation, decreases inflammation and forms metabolites (e.g. nitrate, nitrite) in the airway. When ʟ-argi-nine levels are low, uncoupling of NOS leads to the additional formation of O2-, which rapidly reacts with NO to form ONOO-. Adapted from (109, 125). NO, nitric oxide; NOHA, Nω-hydroxy-L-arginine; NOS, nitric oxide synthases; O2-, superoxide anion; ONOO-, peroxynitrite.

Arginase in asthma

In asthma, increased arginase activity, for example under the influence of Th2-cytokines

after allergen challenge, leads to a decreased substrate availability for NOS. As a result,

less bronchoprotective NO is produced. Decoupling of NOS, due to low ʟ-arginine

levels, results in the production of peroxynitrite, that further enhances smooth muscle

contraction and airway inflammation (Figure 4)(108, 123). The interaction between

arginase activity and NO production was first demonstrated in perfused guinea pig

tracheal preparations, in which arginase inhibition decreased methacholine-induced

airway narrowing (124). Addition of exogenous ʟ-arginine or coincubation with a NOS

inhibitor, abolished the effect of arginase inhibition cholinergic airway narrowing,

indicating that arginase activity indeed inhibits NO production by limiting substrate

for NOS (124). Using perfused tracheas of allergen-sensitized and challenged guinea

pigs, it was later shown that increased arginase activity, as a result of allergen challenge,

contributes to allergen-induced deficiency of cNOS-derived NO and AHR after EAR,

thereby proving the involvement of arginase in asthma pathophysiology (125). Similarly,

it was shown that allergen challenge caused an increased arginase activity after LAR

(126, 127). Furthermore, arginase expression was found to be increased in response to

allergen challenge in two different mouse models of allergic asthma (101).

(14)

The role of arginase in asthma pathology could be validated in asthma patients, in

whom associations were found between asthma exacerbations and, respectively, serum

arginase activity, arginase expression in bronchial brushings, and plasma ʟ-arginine and

metabolite concentrations (101, 128). Alleviation of asthma symptoms corresponds with

a temporary reduction of arginase activity and an increase in plasma ʟ-arginine levels

and the ʟ-arginine/ʟ-ornithine ratio (128). Moreover, gene association studies identified

links between polymorphisms of arginase 1 and arginase 2 with asthma, asthma risk,

asthma severity and reduced responsiveness to ICS and β

₂

-agonists (129-132). .

Several experimental models of asthma show that increased arginase activity contributes

to allergen-induced airway obstruction, hyperresponsiveness and inflammation by

causing a shortage of anti-inflammatory and bronchodilatory NO (133-135). This is

supported by the finding that administration of an arginase inhibitor can counteract

the observed effects on NO in both guinea pigs (125, 136, 137) and mice (138, 139).

Furthermore, in severe asthma patients, lung function (FEV

₁

and FEV

₁

/FVC ratio) shows

a direct correlation with ʟ-arginine bioavailability, and an inverse correlation with serum

arginase activity, which indicates that serum arginase activity decreases ʟ-arginine levels

in the circulation and, in this way, contributes to NO deficiency in the airways (140).

Arginase inhibition was able to reduce methacholine-induced contractions ex vivo in

perfused tracheal preparations of allergen-sensitized guinea pigs (125, 127) and AHR to

methacholine in central and peripheral airways of allergic mice (135). Furthermore, in

guinea pig models the arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH) was

found to reduce the allergen-induced AHR, EAR and LAR and eosinophilic inflammation

in acute allergic asthma (133), and airway remodeling in chronic allergic asthma (141).

Arginase metabolites in asthma

Arginase is involved in multiple downstream metabolic pathways, via its role

in ʟ-ornithine production (Figure 4). Conversion of ʟ-ornithine by ornithine

aminotransferase (OAT) and ornithine decarboxylase (ODC), results in the production

of ʟ-proline and polyamines (putrescine, spermidine, spermine), respectively. Via this

role in ʟ-ornithine metabolism, arginase might also contribute to airway remodeling

by participating in the development of increased smooth muscle mass and fibrosis

in the lung, since collagen formation and increased cell growth and proliferation are

under the influence of ʟ-proline and polyamines (88, 142, 143). In support of such a role

for arginase in remodeling, an involvement of alternatively activated macrophages,

that express arginase 1, has been shown in diseases such as idiopathic pulmonary

fibrosis (144). Moreover, in mouse models the involvement of arginase activity and

expression was found to be involved in several aspects of TGF-β-induced lung fibrosis

(145-147). Altogether, these findings make arginase an interesting potential drug target

for asthma.

(15)

TRPA1

The transient receptor potential ankyrin 1 (TRPA1) ion channel is part of the mammalian

transient receptor potential (TRP) superfamily of cation channels and the only member

of the TRPA subfamily. TRPA1 is a nonselective cation channel (148), and is expressed

on sensory neurons of the nodose, dorsal root and trigeminal ganglia (149-151), where

it aids in the perception of chemically-induced pain, and mechanical and cold stimuli

(150, 152, 153). These sensory neurons innervate various peripheral targets including

the lung and nose (154) and their activation induces respiratory symptoms such as

bronchoconstriction, reflex cough and mucus secretion (42). In addition, TRPA1 channels

are suggested as important sensors of tissue damage and noxious stimuli, and may

mediate the inflammatory reactions in response to specific irritants (e.g. acrolein) and

pro-analgesics agents (e.g. paracetamol) (155, 156). Furthermore, TRPA1 is involved in

the early detection of bacterial LPS, as LPS can directly bind to TRPA1 (157).

TRPA1 can be activated by various noxious exogenous stimuli, such as gingerol,

cinnamaldehyde, mustard oil, chlorine, wasabi, cigarette smoke and scents (158), as well

as endogenous stimuli, including reactive oxygen species, prostaglandins, histamine,

bradykinin and NGF (159). TRPA1 activation at nerve terminals leads to membrane

depolarization and action potential discharge, by passing of Ca

2+

_{and Na}

+

_{through the}

channel, which results in neuropeptide release (e.g. substance P, neurokinin A) (159,

160). Furthermore, endogenous proinflammatory mediators released at the site of

injury (e.g. bradykinin, serotonin, prostaglandins, extracellular proteases) are thought

to be able to modulate TRPA1. These agents can directly activate TRPA1 or induce

TRPA1 phosphorylation via phospholipase C, protein kinase A protein or protein kinase

C pathways. Phosphorylation of TRPA1 affects the activation threshold of the channel,

thereby affection its sensitivity to noxious stimulation (161).

TRPA1 in the airways

TRPA1 can be found on nerve endings throughout the respiratory tract, including the

C-fibers of the vagal and trigeminal ganglia that innervate upper regions of the oral

cavity and oropharynx and on C-fibers in trachea, bronchi, bronchioles and alveoli

(162). TRPA1 activation has been shown to activate vagal bronchopulmonary C-fibers

in rodents resulting in cough in guinea pigs (162-167). Furthermore, in healthy human

volunteers activation of TRPA1 evokes cough and stimulation of TRPA1 channels triggers

isolated human vagal tissue (165).

TRPA1 channels are also expressed on many non-neuronal lung cells, such as fibroblasts,

epithelial cells, smooth muscle cells, mast cells and other inflammatory cells (156).

Activation of non-neuronal TRPA1 has been shown to induce the release of inflammatory

mediators (168-171). Under nonpathological conditions, TRPA1’s main function is

protection of the airways from respiratory irritants by evoking cough and minimizing

exposure by influencing airway tone to cause bronchoconstriction (172). It is unknown

if TRPA1 expression is altered in pathological conditions (149, 166, 173-175).

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TRPA1 in asthma

TRPA1 gene variants are associated with childhood asthma (176). As mentioned earlier

TRPA1 is expressed on neuronal C-fibers, structural and inflammatory airway cells.

It functions as a sensor for airway irritants, in which complex interplays between

structural airway cells and nerve fibers are involved that are able to induce among

other neurogenic inflammation (163, 177, 178). Multiple environmental irritants

and noxious chemicals that activate TRPA1 are also known provokers of asthma

attacks (179). The same accounts for various endogenous inducers of TRPA1, such as

prostaglandin PGJ2, bradykinin, 4-hydroxynonenal (4-HNE) and H

₂

O

₂

, that are produced

in lungs of asthma patients. Raised levels of NGF and bradykinin can be detected in

the bronchoalveolar lavage fluid of asthma patients, whereas increases in acrolein

and 4-HNE are found in blood, sputum, lungs, air spaces and breath of patients (179).

Via TRPA1 channels on airway sensory nerve terminals, these inducers can provoke

the release of neuropeptides, proinflammatory cytokines and chemokines that are

involved in early inflammatory responses, such as leukocyte infiltration, and mucus

hypersecretion and airway constriction (149, 174). Moreover, TRPA1 can be activated

directly by inflammatory mediators (180, 181) and inhibiting TRPA1 may prevent mast

cell degranulation (182). In addition, TRPA1 is involved in respiratory infections, which

are known inducers of asthma exacerbation. LPS, the most potent immunostimulatory

signal of gram-negative bacteria, is known to directly bind to TRPA1 (157). Via TRPA1

LPS is able to induce the release of neuropeptides, independently of toll-like receptor

4, in nociceptive sensory neurons resulting in substance P-mediated neurogenic

inflammation (152, 157).

In a murine asthma model it was demonstrated that both TRPA1 deficiency and

TRPA1 antagonism reduce AHR, inflammatory cell infiltration, pro-inflammatory

cytokine production and neuropeptide release in the airways (183). Furthermore,

TRPA1 antagonism with HC-030031 inhibits the allergen-induced LAR in rats (184),

and allergen challenge is suggested to induce airway sensory nerves via the activation

of TRPA1 leading to a central reflex event that results in a parasympathetic cholinergic

airway narrowing response (184). Other TRPA1 antagonist have shown similar results

in different animal models, suggesting a role for TRPA1 in allergen-induced asthmatic

airway inflammation (185).These findings indicate a possible usefulness of TRPA1

antagonist in the treatment of allergic asthma.

Scope of the thesis

It has become apparent that the current therapeutic options are not sufficient for

a substantial group of asthma patients. Research into new treatment options for

asthma is therefore still necessary. This thesis focusses on the role of two potential

targets, arginase and TRPA1, in allergic asthma and identifies novel inhibitors for both

targets.

In the past, arginase was found to be involved in guinea pig models of allergic asthma by

reducing substrate availability for the production of NO by NOS (125, 126, 133, 136, 141).

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In

Chapter 2, we investigate in more detail how arginase inhibition relieves

allergen-induced asthmatic responses in guinea pig models of allergic asthma. Subsequently,

these models are used for the pharmacological evaluation of a novel group of arginase

inhibitors, the SHK-compound series that were developed by the department of Drug

Design (University of Groningen).

A disordered ւ-arginine metabolism by arginase and NOS is known to play a role in the

pathology of asthma and chronic obstructive pulmonary disease (COPD) (186, 187).

Both chronic airway diseases are associated with several co-morbidities (188, 189).

In some of these co-morbidities, clear pathophysiological roles for arginase and NOS

have been identified, while in others the role of an aberrant ւ-arginine homeostasis

is less clear. The review presented in

Chapter 3 focusses on the role of arginase, NOS

and asymmetric dimethylarginine (ADMA) in co-morbidities of asthma and COPD and

speculates on their possible connection with asthma or COPD.

Studies on the role of arginase 1 in animal models of asthma have indicated that

inhibition of this isoenzyme could be beneficial, as it may be involved in airway

hyperresponsiveness and airway inflammation (133, 190-192). The role of arginase 2

in asthma pathology is less clear. In fact, both enhancing and inhibitory roles of this

isoenzyme in allergic asthma have been reported (193-195). Arginase 2 is the main

arginase expressed in mast cells (196). In

Chapter 4, we tried to gain more insight in

the role of arginase 2 in mast cells as initiators of the allergic cascade in asthma.

Chapter 5 focusses on TRPA1 and its role in allergic asthma. Here, we evaluate the

efficacy of a novel TRPA1 antagonist BI01305834 and investigate how this antagonist

may alleviate asthma symptoms in guinea pig models of allergic asthma via neuronal

and non-neuronal pathways.

Finally, in

Chapter 6 the findings presented in this thesis are discussed and placed

into context of current literature, thereby identifying open questions and future

directions for further validation of arginase and TRPA1 as therapeutic targets for allergic

asthma.

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