IMMUNE MARKERS AND PROTEASOMAL GENE POLYMORPHISMS SPECIFIC FOR ASTHMA PHENOTYPES

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LITHUANIAN UNIVERSITY OF HEALTH SCIENCES

Edita Gasiūnienė

IMMUNE MARKERS

AND PROTEASOMAL GENE

POLYMORPHISMS SPECIFIC

FOR ASTHMA PHENOTYPES

Doctoral Dissertation Biomedical Sciences,

Medicine (06B)

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Dissertation has been prepared at the Department of Pulmonolgy and Immu-nology during the period of 2011–2016, and at the Department of Immuno-logy and AllergoImmuno-logy, during the period of 2016–2018, Medical Academy of Lithuanian University of Health Sciences.

Scientific Supervisor:

Prof. Dr. Brigita Šitkauskienė (Lithuanian University of Health Sciences, Medical Academy, Biomedical Sciences, Medicine – 06B). Dissertation is defended at the Medical Research Council of the Medical Academy of Lithuanian University of Health Sciences:

Chairperson

Prof. Dr. Habil. Virgilijus Ulozas (Lithuanian University of Health Scien-ces, Biomedical ScienScien-ces, Medicine – 06B).

Members:

Prof. Dr. Asta Baranauskaitė (Lithuanian University of Health Sciences, Biomedical Sciences, Medicine – 06B);

Dr. Vacis Tatarūnas (Lithuanian University of Health Sciences, Biome-dical Sciences, Biology – 01B);

Prof. Dr. Saulius Šatkauskas (Vytautas Magnus University, Biomedical Sciences, Biophysics – 02B);

Prof. Dr. Leif Hilding Bjermer (Lund University, Biomedical Sciences, Medicine – 06B).

Dissertation will be defended at the open session of the Medical Research Council on November 16, 2018, at 1:30 pm in the Large Auditorium at the Hospital of Lithuanian University of Health Sciences Kauno klinikos.

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LIETUVOS SVEIKATOS MOKSLŲ UNIVERSITETAS

Edita Gasiūnienė

IMUNINIAI

ŽYMENYS IR

PROTEOSOMINIŲ GENŲ

POLIMORFIZMAI

, BŪDINGI

ASTMOS FENOTIPAMS

Daktaro disertacija Biomedicinos mokslai, medicina (06B) Kaunas, 2018

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Disertacija rengta 2011–2016 metais Lietuvos sveikatos mokslų universiteto Pulmonologijos ir imunologijos klinikoje. 2016–2018 metais Lietuvos svei-katos mokslų universiteto Imunologijos ir alergologijos klinikoje.

Mokslinė vadovė

prof. dr. Brigita Šitkauskienė (Lietuvos sveikatos mokslų universitetas, Medicinos akademija, biomedicinos mokslai, medicina – 06B).

Disertacija ginama Lietuvos sveikatos mokslų universiteto Medicinos akademijos medicinos mokslo krypties taryboje:

Pirmininkas

prof. habil. dr. Virgilijus Ulozas (Lietuvos sveikatos mokslų universi-tetas, biomedicinos mokslai, medicina – 06B).

Nariai:

prof. dr. Asta Baranauskaitė (Lietuvos sveikatos mokslų universitetas, biomedicinos mokslai, medicina – 06B);

dr. Vacis Tatarūnas (Lietuvos sveikatos mokslų universitetas, biomedi-cinos mokslai, biologija – 01B);

prof. dr. Saulius Šatkauskas (Vytauto Didžiojo universitetas, biomedi-cinos mokslai, biofizika – 02B);

prof. dr. Leif Hilding Bjermer (Lundo universitetas, biomedicinos mokslai, medicina – 06B).

Disertacija bus ginama viešame Lietuvos sveikatos mokslų universtiteto Medicinos akademijos medicinos mokslo krypties tarybos posėdyje 2018 m. lapkričio 16 d. 13.30 val. Lietuvos sveikatos mokslų universiteto ligoninės Kauno klinikų Didžiojoje auditorijoje.

Disertacijos gynimo vietos adresas: Eivenių g. 2, LT-501619 Kaunas, Lietuva.

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ABBREVIATIONS

ACO – asthma COPD overlap

ADAM33 – a disintegrin and metalloproteinase domain-containing protein 33 AEs – asthma exacerbations

AHR – airway hyperresponsiveness BAL – bronchoalveolar lavage BCR – B cell receptor

BHR – broncial hyperreactivity BMI – body mass index CCL – C-C chemokine ligand CCR – C-C chemokine receptor CD – cluster of differentiation cDCc – classical dendritic cells

COPD – chronic obstructive pulmonary disease DCs – dendritic cells

ECP – eosinophil cationic protein

ELISA – enzyme-linked immunosorbent assay EPO – eosinophil peroxidase

EPX – eosinophil protein X

FEV1 – forced expiratory volume in 1 sec.

FVC – forced vital capacity

GATA3 – trans-acting T cell-specific transcription factor GINA – Global Initiative for Asthma

GM-CSF – granulocyte/macrophage-colony stimulating factor GOLD – Global Innitiative for Chronic Obstructive Lung Disease HDM – house dust mite

hRV – rhinovirus

ICAM – intercellular adhesion molecule ICS – inhaled corticosteroids

Ig – immunoglobulin IFN – interferon IL – interleukin

ILCs – innate lymphoid cells IS – induced sputum LABA – long-acting β2 agonist

LRIs – lower respiratory tract infections MBP – major basic protein

MCs – mast cells

MIP – macrophage inflammatory protein MMP – matrix metalloproteinase

moDCs – monocyte derived dendritic cells NF-kB – nuclear factor kappa B

NOD – nucleotide binding oligomerization domain PPRs – pattern recognition receptors

RARα – related orphan receptor alpha RIG-I – retinoic acid inducible gene I ROS – reactive oxygen species

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8 RSV – respiratory syncytial virus SNP – single nucleotide polymorphism

STAT – signal transducer and activator of transcription TCR – T cell receptor

Tc1 – cytotoxic T cell

TGF-β – transforming growth factor-β Th – T helper cells

TLRs – Toll-like receptors TNF – tumor necrosis factor

TSLP – thymic stromal lymphopoietin VC – vital capacity

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INTRODUCTION

Asthma is one of the most common chronic respiratory disease in the world, affecting nearly 10% of adults and about 14% of children [1]. The other chronic respiratory disease – chronic obstructive pulmonary disease (COPD) usually affects about 10% of adults over 40 years of age [2]. Although both cases are described as chronic respiratory diseases, the mechanism of inflammation is different. Asthma is usually characterized by allergic inflammation, with increased production of IgE, mast cell degra-nulation and eosinophil infiltration orchestrated by type 2 T helper (Th2) cells. COPD has a different pattern of inflammation – with a predominance of neutrophils and macrophages, increased number of cytotoxic T (Tc1) cells and Th1 cells. However, the described immunological mechanisms of these diseases do not always explain the mechanisms of airway inflam-mation as well as the variety of symptoms and the clinical course of the disease, or even the different response to treatment. Therefore, scientists are continually looking for new causative factors in asthma and COPD patho-genesis.

One of the recently discovered mediators, interleukin (IL)-32, secreted by epithelial and endothelial cells, fibroblasts and macrophages, is involved in the pathogenesis of various types of cancer and autoimmune diseases [3-5]. Therefore it is assumed that this interleukin is also important in the pathogenesis of chronic obstructive airway diseases. It is believed that IL-32 can act both as inflammatory and as an anti-inflammatory marker in certain conditions.

Recently, there is increasing data on the immunomodulatory effects of vitamin D in the pathogenesis of various diseases including asthma. Vitamin D can increase secretion of pro-inflammatory cytokines (IL-5, IL-6, IL-8), which are very important in asthma pathogenesis; however, Vitamin D deficiency can reduce the secretion of anti-inflammatory cytokine – IL-10 and enhance proliferation of smooth muscle cells in the respiratory tract [6].

Looking for new inflammatory markers in the pathogenesis of chronic respiratory diseases, as possible candidates – periostin and IL-33 are descry-bed. Periostin is important in the proliferation of inflammatory cells and air-way remodeling. IL-33 is a nuclear cytokine, which can act as an alarmin as well as a transcriptional regulator [7]. IL-33 might also serve as a bridge between innate and acquired immune cells to promote Th2 mediated inflam-mation [8]. It has been shown that genetic factors are also important in asthma development. Previous studies have demonstrated that proteasomes coding genes are involved in the pathogenesis of inflammatory and

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mune diseases [9]. Proteasomes within the ubiquitin-proteasome system are one of the main regulators of nuclear factor kappa B (NF-kB). The NF-kB signaling pathway regulates many inflammatory mediators. Thus proteaso-mes may participate in the regulation of inflammation and pathogenesis of asthma. Therefore proteasomal gene polymorphisms may be associated with airway inflammation.

Study aim

This study aimed to evaluate immune markers and proteasomal gene polymorphisms specific for different asthma phenotypes.

Study objectives

1. To identify patterns of immune cells and IL-32 levels in different tissue compartments (serum, induced sputum, bronchoalveolar lavage) of asthma in comparison to COPD and healthy subjects. 2. To determine asthma phenotypes according to clinical features in

relation to immunological markers (IL-33, periostin, vitamin D). 3. To analyze the polymorphisms of proteasomal genes PSMA6,

PSMA3 and PSMC6 in different asthma phenotypes.

4. To identify asthma endotypes according to multiple demographic, clinical and immunological characteristics.

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1. REVIEW OF LITERATURE

1.1. Definition and prevalence of asthma

Asthma is a heterogeneous disease, usually characterized by chronic airway inflammation. It is defined by the history of respiratory symptoms such as wheeze, shortness of breath, chest tightness and cough that vary over the time and in intensity, together with variable expiratory airflow limi-tation [10]. Asthma symptoms may be triggered by allergens, viral respira-tory infections, airborne irritants, changes in weather or exercise. Asthma is one of the most common chronic respiratory disease with an increasing prevalence worldwide [11]. This disease affects individuals all over the world regardless of ethnic group and country of residence; however, it has been observed that the incidence of asthma varies between different regions [12, 13]. The incidence of asthma has increased over the last years, espe-cially among children [1]. According to the Global Burden of Disease Study (GBD) data 334 million people have asthma worldwide. 14% of the world’s children and 8.6% of young adults experience asthma symptoms (aged 18– 45). Interestingly, the International Study of Asthma and Allergies in Child-hood (ISAAC) revealed that in high-income countries, the incidence of asthma has remained the same or even decreased, but in low-income countries, it has increased. It has been shown that 5–15% European citizens are asthmatics and the prevalence is higher in Northern Europe. According to the GBD asthma is the 14th most important disorder in the world in terms of the extent and duration of disability. Asthma is usually compared to another chronic respiratory airway disease – chronic obstructive pulmonary disease (COPD). According to Global Initiative for Chronic Obstructive Lung Disease (GOLD), COPD is a common, preventable and treatable disease that is characterized by persistent respiratory symptoms and airflow limitation that is due to airway and/or alveolar abnormalities usually caused by significant exposure to noxious particles or gases. COPD is now the fourth-ranked cause of death worldwide, affecting approximately 10% of persons older than 45 years [14]. Both diseases are characterized by chronic inflammation in the lung, but the nature of the inflammation as well as triggering factors differ between diseases [2]. The exact factors leading to persistent airway inflamemation, as well as the type of inflammation are not well known. That is why during the last decade a lot of studies were performed and are still ongoing in this field.

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1.2. Factors affecting the development of obstructive airway diseases The most commonly referred causes of asthma are genetic heredity, the influence of harmful environmental factors, chronic respiratory infection, allergy. The exact cause of chronic inflammation of the respiratory tract is still unclear. In recent scientific literature states that the most important are genetic and environmental factors, their interactions [15].

Gender affects the development of asthma in a time-dependent manner. Asthma is far more common in boys than girls during early childhood. The prevalence equalizes between the genders during adolescence and then switches to female predominance in adulthood [16]. Sex-specific effects of a few candidate genes associated with asthma and atopy phenotypes have been reported [17]. Whereas, the main risk factor for another chronic respi-ratory disease – COPD – is smoking. Therefore, COPD now affects women as often as men, reflecting the equal prevalence of smoking [18].

Obesity is a significant risk factor in the development of asthma [19, 20]. The prevalence of asthma is higher in obese than in lean adults [21, 22] and obesity increases the incidence of asthma by 2.0- and 2.3-fold in children and adults, respectively [22]. Moreover, significant dose-dependent effects of elevated body mass index on asthma are observed [19-21]. Obese asthmatic patients are often described as severe and poorly controlled [23, 24] perhaps because they are less responsive to corticosteroids and exhibit a less atopic inflammatory phenotype [25].Obesity is associated with chronic low-grade systemic inflammation [26]. It is known that adipose tissue can regulate systemic inflammation through the production of a variety of adipokines which may link the two disorders mechanistically [27]. Recent studies demonstrate that adipokines (leptin and adiponectin) can regulate the survival and function of eosinophils, affecting eosinophil trafficking from the bone marrow to the airways [20, 28]. It was also shown that clearance of dead cells (efferocytosis) by airway macrophages or blood monocytes appears impaired in obese asthma patients and is inversely correlated with glucocorticoid responsiveness [20]. Weight loss improves asthma control and should be involved in asthma treatment regimen in obese patients [29].

Body weight in COPD is a matter of discussion. On the contrary to asthma, low body mass index (BMI) is associated with increased mortality risk when compared to overweight and even obese COPD patients with moderate to severe airway obstruction [30, 31]. However, obese COPD pa-tients tend to have more comorbid diseases, such as cardiovascular disease, diabetes, metabolic syndrome. There are studies, showing that obesity in mild-to-moderate COPD is associated with increased all-cause mortality [30].

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There is evidence that viruses and frequent infections are associated with a higher risk of developing asthma. A lot of prospective birth control studies tracking community-based population through the school age and beyond, have identified lower respiratory tract infections (LRIs) occurring in the first few years of life as independent risk factors for the development of asthma [32-34]. The respiratory viruses associated with asthma include the same viruses that cause the common cold, influenza-like illness and wheeze and bronchiolitis in children [35]. Respiratory viruses are the main triggers of asthma exacerbations (AEs) in adults and school-aged children [36, 37]. Viruses associated with increased risk of asthma development and AEs include a respiratory syncytial virus (RSV), influenza viruses, and human rhinoviruses [35]. Independent prospective cohort studies published that RSV has been associated with wheeze in infants, asthma inception and increased linkage to allergic sensitization [38–40]. Besides, LRIs can predispose to the development of early asthma, conferring a slightly higher risk than atopy alone [41]. However, the most severe childhood asthma is encountered when LRIs occur against a background of pre-existing aeroallergen sensitization [42]. Rhinovirus (hRV) induced LRIs are also very common in infancy and preschool years and are strongly associated with the risk of subsequent asthma [43]. Particularly strong association with hRV subtype C was noticed [37].

hRV infection in the asthmatic airway in vivo induces the release of IL-33 and IL-25, therefore, activating type-2-driven inflammation through the increased secretion of type-2 cytokines (IL-4, IL-5, IL-13) [44, 45]. Cell and tissues from asthmatic patients can respond to infection with antiviral response represented by the delayed or deficient production of innate interferons: type I (IFN-α/β) and type III (IFN-λ) [46, 47], which are the first-line defence against viral infection [48].

In the case of COPD, there is little data about viral infection as a causal factor for a COPD development. There is much more data about hRV, RSV and influenza virus as significant triggers of COPD exacerbation [49].

The hygiene hypothesis proposes that a lack of early life exposure to microbes alters early life immune system priming and, consequently, increa-ses susceptibility to atopic diseaincrea-ses [50]. David Strachan in the late 1980’s developed the hygiene hypothesis [51]. It states that the lack of exposure to microbes and other pathogens (viruses, gut flora, parasites) increases the susceptibility to allergic diseases, including asthma. Subsequently, these findings were based on T lymphocyte helper studies by an explanation of the endotoxin, gram-negative bacterial membrane lipopolysaccharide, and immune response modifying effects [52, 53].

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Cigarette smoke is a complex of gaseous and particulate compounds. Nicotine is the principal additive component of tobacco. International Agency for Research on Cancer (IARC) states that there are more than 5,000 compounds identified in cigarette smoke. Of these, more than 250 are known to be toxic or carcinogenic. It has been recognized for some years that asthma and wheezing are more common in children passively exposed to cigarette smoke [54]. In pregnant women who smoke or use nicotine replacement therapy, nicotine crosses the placenta, concentrates in fetal blood and amniotic fluid, and is detectable in breast milk during lactation [55]. One of the most prominent and recent systematic review and meta-analysis on smoking and asthma association proved undeniably that smo-king is a crucial risk factor for asthma development in childhood [54]. Smoking is even more critical in the pathogenesis of COPD than in asthma. Several studies showed that early exposure to parental smoking as well as passive smoking in adult age conveys as much risk for COPD as does smoking in adult life [56, 57]. Smoking causes irreversible lung damage by activating surface macrophages and airway epithelial cells to release multip-le chemotactic mediators, which attract neutrophils, monocytes and lympho-cytes into the lungs [58], leading to emphysema.

Summarizing the role of factors causing chronic obstructive airway diseases shows a necessity for further, more detailed investigations.

1.2.1. Genetic factors

First studies in asthma genetics were performed in 1916 and 1924 esta-blished an increased occurrence of asthma in relatives of subjects having the disease and have shown higher concordance between monozygotic twins in contrast to dizygotic twins [59]. Since the first genome-wide link-age screen for asthma susceptibility loci was published in 1996, >20

independent chromosomal regions have been identified [60].

Approxima-tely 20 genome-wide linkage screens have been reported in different popu-lations investigating chromosomal regions that are linked to asthma and atopy, or related phenotypes like elevated IgE levels, wheezing, and bron-chial hyperresponsiveness. A number of chromosomal regions have been repeatedly identified across multiple studies that contain genes of biological relevance to asthma and allergic disease, including the cytokine cluster on chromosome 5q (containing IL-3, IL5, and granulocyte/macrophage colony-stimulating factor (GM-CSF), FCER1B on 11q, interferon γ (IFNγ) and

STAT6 on 12q, and IL-4R (the IL-4Rα chain, also part of the IL-13R) on 16p

[61]. When the linkage is observed, the association testing of single nucleo-tide polymorphisms (SNPs) across the linked regions was started to define

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those variants and their corresponding haplotype blocks that show strong

genetic association with disease [59]. ADAM33 (a disintegrin and

metallo-proteinase domain-containing protein 33) was the first report of a cloned asthma gene on chromosome 20p13 [62]. Demonstration of ADAM33 expre-ssion in lung cell types yielded the most reliable evidence for associations to variants within the ADAM33 gene identifying it as the most likely gene from a set of ~40 within the linkage peak [62, 63] and was proved to play an essential role in the pathobiology of asthma and pulmonary allergic disease [64].

It has been identified more than 100 genes loci to be associated with asthma development [13, 65]. The most recently discovered genes candi-dates are fillagrin, IL-13 and IL-17F [65]. Polymorphisms of the thymus stromal lymphopoietin (TSLP) gene was shown to be associated with aller-gic asthma [66].

Early studies showed a possible linkage of chromosome 14q11-24 geno-me region to asthma [67, 68]. Chromosogeno-me 14 contains several genes enco-ding proteasome subunits, incluenco-ding PSMA6 gene, which encodes alpha type 6 proteasome subunit, PSMC6 gene encoding 26S proteasome ATPase subunit 6 and PSMA3, which encode alpha type 3 proteasome subunit. Polymor-phisms in these and other proteasome-encoding genes are associated with a variety of inflammatory diseases, diabetes mellitus, and cardiovascular disor-ders [69–71]. Taking this into account, there are certain assumptions that 14q proteasomal genes could participate in asthma pathogenesis.

1.2.2. Allergy

Exposure to aero-allergens has been identified as one of the significant contributors to sensitization and asthma development in children [72]. It was observed that sensitization to indoor aero-allergens is generally more critical to the development of asthma than sensitization to outdoor allergens. There is a direct relationship between allergen exposure and the prevalence and severity of asthma symptoms [73].

House dust mites (HDM) is one of the significant causes of asthma [74]. The most common species are Dermatophagoides pteronyssinus and

Dermatophagoides farinae. HDM is also very closely related to asthma

severity [75] and exacerbations especially in sensitized patients [76]. Sen-sitization to HDM allergens in the first years of life has a significant clinical effect on lung function, wheezing in a pediatric population with poorer outcomes in respiratory health [77]. Results from various pediatric cohort studies, suggest that sensitization to HDM in children less than 5 years of age is significant risk factor for asthma later in childhood [78, 79];

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tization to HDM at the age of 8, increased the risk of respiratory disease in 87% of the Manchester Asthma and Allergy Study cohort [80].

There are some disagreements in the assessment of sensitization to pets on the asthma occurrence. Some studies showed that exposure to pets is associated with increased risk of sensitization to pets [81, 82], and of asthma and wheezing [83, 84]. By contrast, some studies have demonstrated a decreased risk of allergy and asthma development with early exposure to pets [85, 86]. Overall, the review of over 22,000 school-age children from 11 birth cohorts in Europe found no correlation between pets in the homes early in life and lower or higher prevalence of asthma in children [87].

Pollen exposure can trigger asthma exacerbations or worsen symptoms in sensitized individuals according to their flowering period. Pollen is responsible for seasonal or intermittent asthma symptom exacerbation. In Lithuania, there are three significant pollen groups, which can influence asthma and allergic rhinitis symptoms: birch trees, grasses and weeds.

Fungal spores are responsible for both seasonal and perennial allergy symptoms. Outdoor spores peak in the mid-summer and diminish with the first hard frost in regions that experience cold winter seasons [88].

Alterna-ria is the most common mold in dry, warm climates. It is commonly found

in soil, seeds, and plants. Several studies have shown associations between

Alternaria and severe asthma [89, 90]. Cladosporium is the most prevalent

spore in temperate regions and is the most commonly identified outdoor fungus [88]. Molds (Alternaria, Cladosporium, Aspergillus, Penicillium) can cause not only asthma, asthma exacerbations but also allergic rhinitis and hypersensitivity pneumonitis [91].

1.3. Phenotypes of asthma

Asthma heterogeneity has been revealed with the recognition of multip-le pathways, mediators, and systems involved in triggering the characteristic airway inflammation and variable airflow limitation of asthma. Currently, the most commonly isolated asthma phenotypes reflect the heterogeneity of the disease. The dictionary defines a phenotype as “the observable proper-ties of an organism that are produced by the interactions of the genotype and the environment” [92]. Asthma can be divided into various distinct pheno-types based on clinical characteristics, physiological findings, triggers, or inflamematory markers (Table 1.3.1). Usually, this way of phenotyping is a hypothesis-driven univariate approach. The other approach to phenotyping is utilizing computer algorithms to evaluate the hypotheses-free relationship between many clinical and biological characteristics.

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Table 1.3.1. Asthma phenotypes

Category Phenotype

Trigger-induced asthma

Allergic Non-allergic

Aspirin-exacerbated respiratory disease (AERD) Infection

Exercise-induced Clinical presentation

of asthma

Pre-asthma wheezing in infants: • Episodic (viral) wheeze • Multi-trigger wheezing Exacerbation-prone asthma

Asthma associated with apparent irreversible airflow limitation Asthma associated with obesity

Inflammatory markers of asthma Eosinophilic Neutrophilic Pauci-granulocytic Th2-associated asthma

Adapted according to Asthma Phenotypes Task Force recommendations, 2009.

Allergic asthma is identified as the most common phenotype, especially among children. About 60% of asthma population is considered allergic [93], while non-allergic asthma occurs in 10% to 33% of individuals with asthma [94]. The distribution of patients to allergic and non-allergic asthma-tics is quite rough and not very accurate. Allergic asthma is typically identi-fied based on sensitization, as determined by at least one positive skin prick test to perennial and/or clinically relevant allergen in vitro testing for IgE [92]. The definition of non-allergic asthma includes that subset of subjects with asthma with whom allergic sensitization cannot be demonstrated [94]. These individuals should have a negative skin prick test or in vitro specific-IgE test. Sensitivity should be checked to a panel of seasonal and perennial allergens, including local pollens (grass, tree, weed), molds (Alternaria,

Aspergillus, Cladosporium), house dust mites (Dermatophagoides farinae, Dermatophagoides pteronyssinus), cockroach, cat, and dog. Allergic asthma

can present at any age, but allergic asthma patients are found to be younger when compared to non-allergic asthmatics [95-98]. Romanet-Manent et al. discovered that allergic asthma is more common in male patients, whereas non-allergic asthma is more common in female patients [95]. It was also noted that among allergic asthma patients, familial history of asthma was more common [96], although other study did not confirm the fact [99].

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Studies showed that allergic asthma is associated with allergic rhinocon-junctivitis and atopic dermatitis [95] while in non-allergic asthma nasal polyps, chronic rhinosinusitis and gastroesophageal reflux disease are more prevalent [96, 97]. Studies report that allergic versus non-allergic asthma is less severe [100–102] and that there is no association between severity and atopic status [103]. Although the main distinguishing feature of allergic asthma is allergic sensitization, however, it is a more important factor in triggering asthma exacerbation than induction of disease. Total IgE levels are usually higher in allergic asthma group than non-allergic [96]. Zoratti

et al. demonstrated higher levels of peripheral eosinophilia in patients with

allergic asthma versus non-allergic asthmatics [104].

The mass of the inflammatory phenotyping studies in asthma has iden-tified eosinophilic asthma as one of the most prominent. This phenotype of asthma could be termed as “allergen exacerbated asthma”. Usually, patients are sensitized to one or more aeroallergens (atopy), have concomitant aller-gic rhinitis, report worsening symptoms on exposure, and are more common in childhood-onset [105, 106]. This phenotype also includes patients in whom the central pathology remains eosinophils yet who do not demonstra-te atopy, and it is called idiopathic eosinophilic asthma [106]. The latdemonstra-ter phenotype can present at any age but much more often begins in adult age and have a more severe asthma phenotype. The third presentation of eosino-philic asthma comprises individuals with aspirin-exacerbated respiratory disease [107]. The defining features include the presence of chronic rhinosi-nusitis with nasal polyps and the sensitivity to aspirin and other non-selective inhibitors of cyclooxygenase [108].

Eosinophilic asthma phenotype is defined according to sputum eosino-phils (>2% or > 3%) or blood eosinoeosino-phils (≥0.3×109/L) [109]. Sputum reflects local inflammation, and that is why sputum eosinophil use has been validated method of guiding inhaled corticosteroid therapy and is assumed as a predictive marker of response to many biologic therapies [110, 111]. Sputum eosinophils have some disadvantages because not all patients can provide the adequate samples (especially children). The induced sputum test can be technically challenging and not universally available in all centers. Therefore, serum eosinophils are much easier to measure. Blood eosinophils correlate with sputum eosinophilia, poor asthma control and can predict exacerbations [112, 113]. However, varying cutoffs of serum eosinophils used in research raise many discussions about a wider application in prac-tice. Majority of clinical trials have used blood eosinophil levels ranging from 0.15×109/L [114, 115] to 0.4×109/L [116].

Obese asthma phenotype is described when the BMI is higher than 30 kg/m2, and asthma is diagnosed. It was observed that obese asthma

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notype patients are less atopic, lack the eosinophilic airway inflammation, have the reduced lung function, and disease is usually difficult to control [23]. Cluster analysis has shown that this phenotype is not a uniform group. Two major groups were identified: early-onset obese and late-onset obese asthma phenotypes. Early-onset obese-asthma phenotype is associated with male gender, atopy, severe decrease in lung function, significant airway hy-perresponsiveness, eosinophilic airway infiltration and high Th2 biomarkers [117, 118]. Late-onset obese-asthma phenotype is more prominent in fema-les, usually non-atopic, with minimal airway obstruction, less airway hyper-responsiveness, neutrophilic infiltration and low Th2 biomarkers [118, 119]. It is believed that the identifying the phenotypes of asthma with the help of cluster analysis (identifying so-called endotypes) and so understanding in-depth of underlying pathophysiology, will allow to develop more effective and targeted treatment options for patients with asthma, as well as may facilitate the development of primary disease prevention and disease modification. Emerging new therapeutic options allows us quite precisely select the treatment very individually according to the different predominant pathological mechanism.

1.4. Asthma immunology 1.4.1. Innate immune system in asthma

The innate immune system comprises a range of host defense systems that generate nonspecific responses to environmental triggers (pollutants, allergens, etc.). It encompasses cellular and non-cellular components. The airway epithelium and mucosal layer also provide innate immune functions beyond serving as mechanical barriers. Innate immune cells include dendri-tic cells (DCs), innate lymphoid cells (ILCs), and leukocytes such as macrophages, neutrophils and eosinophils [120]. Pattern recognition recep-tors such as Toll-like receprecep-tors (TLRs), retinoic acid inducible gene I (RIG-I)-like receptors and nucleotide-binding oligomerization domain (NOD)-like receptors recognize different ligand motifs and are important in mediating early immune responses that subsequently shape adaptive immunity [121]. Non-cellular components of innate immunity are also important in host defense and include a variety of secreted factors such as lactoferrin, interferons, defensins, secretory leukocyte protease inhibitor and cathelicidin-derived antimicrobial peptide (LL-37).

The airway epithelium forms a continuous, highly regulated physical barrier that lines the airway lumen, separating the underlying tissue from inhaled environmental antigens [122]. Intercellular epithelial junctions –

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tight junctions, adherent junctions and desmosomes – form the structural adhesive forces that maintain the airway epithelial barrier [123, 124]. One of the most important molecules involved in junction formation is E-cadherin, which belongs to adherens junction group. E-cadherin is also a ligand for the cognate receptor CD103, expressed on innate and adaptive immune cells, including CD8+ T cells, a fraction of CD4+ T cells and regulatory CD4+CD25+Foxp3+ T cells (Tregs) [125]. HDM, cockroach, molds can lead to the proteolytic degradation of airway epithelial intercellular adhesions inducing a reduction in epithelial resistance [126, 127]. Mutations in the E-cadherin gene are associated with airway hyperresponsiveness [128].

Lung epithelial cells are activated through pattern recognition receptors (PPRs), including TLRs. Chronic inhalation of irritants (cigarette smoke, biomass fuel smoke, air pollutants) activates TLRs, resulting in activation of innate immune response, i.e. increased number of neutrophils and macro-phages [129]. Activated epithelial cells also can influence type 2 mucosal immune response, through the production of thymic stromal lymphopoietin (TSLP), IL-25 and IL-33.

Mucus hypersecretion is an important component of asthma as well as of COPD leading to mucous plugging [130] and even AHR in asthma. Hyperplasia of submucosal glands and increased number of epithelial goblet cells exist in both asthma and COPD. IL-13 is a potent to induce mucus hypersecretion in the asthma model, while in COPD epidermal growth factor receptor (EGFR) plays an important role [131].

Dendritic cells (DCs) are specialized macrophage-like cells in airway epithelium, which are central antigen-presenting cells in lungs [132]. There are three major subsets of DCs: classical, plasmacytoid, and monocyte-deri-ved DCs [133]. DCs are activated by epithelial cytokines, including IL-25, IL-33, TSLP, and GM-CSF. Classical DCs (cDCs) can be further divided into subgroups, cDC1s which can recognize intracellular pathogens and play a role in type 1 immune responses [134], whereas cDC2s induce type 2 immune responses against parasites by activating Th2 cells [135] as well as initiate type 3 immune responses through the activation of Th17 and type 3 ILCs [136, 137]. Plasmacytoid DCs can produce high levels of type 1 INFs in response to viral infection [134, 138]. Monocyte-derived DCs (moDCs), also known as inflammatory DCs, are differentiated from monocytes under inflammatory circumstances [139]. In a murine model, it was shown that cDC2s, also known as CD11b+, and moDCs are capable of inducing Th2 cytokine production and eosinophilic airway inflammation [140]. Activated moDCs at the site of eosinophilic inflammation express costimulatory mole-cules such as OX40L, CD80, etc.; stimulate effector T cells and T resident memory cells, thus inducing airway hyperreactivity (AHR). Summarizing

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given information about DCs, in asthmatic inflammation DCs are respon-sible for the allergen processing and transporting to local lymph nodes, thus inducing the production of allergen-specific T cells, Th2 cytokine produc-tion and eosinophilic airway inflammaproduc-tion. While in COPD, in response to cigarette smoke DCs tend to produce IL-8 and prostaglandin E2. The effect on DCs appears to be mediated via both oxidative stress and the direct effects of nicotine [141].

Innate lymphoid cells (ILCs) exhibit similar morphologies to T cells/B cells but do not express TCR/BCR or other lineage-specific markers [133] and are therefore antigen nonspecific, but can react rapidly to a wide range of innate signals [142]. There are three types of ILCs – type 1(ILC1s), type 2(ILC2s), and type 3(ILC3s). ILC1s is the natural killer (NK) cell, which produces INF-γ and expresses T-bet (T-box transcription factor) and shows antimicrobial activity [143]. Recent studies have shown that NK cells play an important role in the development of several forms of asthma. NK cells are responsible for lung inflammation during viral infections and may regulate the pathogenesis of virus-induced asthma [144]. In experimental allergic asthma model, NK was responsible for the AHR [145]. Taken toge-ther, studies suggest potential roles of ILC1s in asthma pathogenesis. ILC2s were previously called natural helper cells or nuocytes, require trans-acting T cell-specific transcription factor (GATA3) and RAR-related orphan recep-tor alpha (RORα) for their differentiation and maintenance [146], and secrete IL-4, IL-5, IL-9, and IL-13 [147]. Human ILC2s reliably express CD45, ST2, GATA3, inducible T cell costimulator (ICOS) etc. [148], though their phenotype varies among mucosal, pathological, and non-patho-logical tissues [149]. ILC2s in addition to IL-33, IL-25, TSLP [150], HDM allergen [151], influenza infection [152], produce large amounts of IL-5 and IL-13, leading to activation of eosinophils and induction of AHR. Studies on ILC2s in humans show the frequency of ILC2s is dramatically increased in asthmatic patients [147, 153]. Lombardi et al. found that ILC2s is increased in allergic vs non-allergic subjects [154].

ILC3s express ligand-dependent nuclear hormone receptor RORγt [155]. ILC3s are the IL-17 and IL-22-producing cells and play a role in driving neutrophilic inflammation in COPD patients [156]. Recent studies indicated that IL-17 could directly cause AHR by inducing contraction of smooth muscle cells [157]. Increased level of IL-17A was found in obese patients with asthma [158]. In bronchoalveolar lavage fluid of severe asthmatics, IL-17-producing ILC3s were found [158]. Therefore it can sug-gest that ILC3s might play a role in obese asthma phenotype. Further studies are needed to clarify the importance of IL-22-producing ILC3s in asthma.

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Basophils are phenotypically and functionally related to MCs and are recruited into inflammatory sites under allergic conditions [159, 160]. Baso-phils express FcεRI and produce various mediators including histamine, Th2-associated cytokines, and lipid mediators [161]. IL-3 is the main basophil development driving cytokine, and it is also responsible for baso-phil recruitment to lymph nodes [162]. IL-33 alone can induce the produc-tion of IL-4 in basophils as well as promote cell-adhesion and a CD11b expression on basophils [163]. TSLP also causes the migration of basophils to the inflammatory sites and promote further Th2 cytokine-mediated in-flammation [164]. The number of basophils is elevated in the lungs of asthmatic patients [165] as well as found in large number in post-mortem biopsies of fatal asthma [166]. In asthma patients, administration of dupilu-mab (an antibody against IL-4 receptor α-chain), which blocks IL-4 and IL-13 signalling, reduces the frequency of exacerbation in moderate to seve-re cases and improves lung function [167]. Theseve-re is no evidence of basophil recruitment in COPD inflammation.

Mast cells (MCs) arise in the bone marrow but classically reside in tis-sues, where they can survive for months. Human MCs express a broad range of receptors, allowing them to respond to a diverse range of stimuli, inclu-ding via IgE-independent mechanisms. In asthma pathogenesis the main receptors are c-Kit, FcεRI and ST2. MCs release cytokines and biochemical compounds stored in their cytosolic granules after activation of the high-affinity FcεRI with allergens, superantigens, autoantibodies [159]. The cyto-solic granules contain cytokines (IL-4, IL-5, IL-6, and IL-13), biogenic amines (histamine and serotonin), serglycin, proteoglycans, mast cell-deri-ved proteases (chymase, tryptase), and lipid mediators (platelet-activating factor (PAF), leukotrienes, prostaglandins, and sphingolipids) [159]. Degra-nulation of these MCs and basophil associated substances induce recruit-ment of inflammatory cells into the airways, smooth muscle constriction, and increased vascular permeability. IL-33 is the only one interleukin which can provoke cytokine/chemokine release without MCs degranulation [10]. Mast cells do not seem to play a significant role in COPD.

The role of macrophages in asthma is currently uncertain. There are some data that macrophages may play a dual role in asthma and allergic inflammation. After the allergen access the immune system, macrophages migrate into the airway of allergic asthmatics. These cells secrete cytokines (IL-1, IL-1β, IL-6, TNFα) and chemokines (CXCL8, CCL2, CCL3), which activate other inflammatory cells. Macrophages are also known to produce PAF, reactive oxygen species (ROS) and NO that affect vascular smooth muscle tone and bronchial epithelial cells. Furthermore, macrophages may have an anti-inflammatory effect by secreting IL-10 and prostaglandin E2

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[168]. However, macrophages play an essential role in orchestrating the inflammatory response in COPD pathogenesis [169, 170]. In patients with COPD, there is the increased recruitment of monocytes from the circulation and as a consequence increased number of macrophages in lung paren-chyma, BAL fluid and sputum [171]. Activated macrophages from COPD patients can release high levels of inflammatory mediators (IL-1β, IL-6, TNFα, CXCL1, CXCL8, CCL2 and LTB4) and ROS as well as elastolytic enzymes (matrix metalloproteinase (MMP)-2, MMP-9, MMP-12, cathepsins K, L and S) leading to destruction of lung tissue [2, 172-174].

The precise role of neutrophils in asthma pathogenesis is still unclear. There are data that neutrophils can produce a wide range of products, inclu-ding lipids (PAF, thromboxane A2, etc.), cytokines (IL-6, TNFα, TGFβ, CXL8), proteases (MMP9, elastase), reactive oxygen intermediates and nitric oxide. This variety of mediators can cause airway narrowing, increa-sed mucus production, BHR [175]. Neutrophils can be found in sputum and airways in some patients with asthma. Usually, it can be found in smoking asthmatics, severe asthmatics or during exacerbations of asthma, as well as in neutrophilic and obese asthma phenotypes [176]. The mechanisms of neutrophilic inflammation in asthma is not precise and could be related to the use of high doses of inhaled CS which prolong neutrophil survival in the airways or due to infection [177] as well as the anti-neutrophilic therapies have so far been ineffective clinically.

In the other obstructive airway disease – COPD inflammation is gene-rally characterized as neutrophilic, since the increased number of activated neutrophils are found in sputum and BAL fluid, and this number correlates with disease severity [2]. Neutrophils secrete neutrophil elastase, cathepsin G, proteinase-3, MMP-8, MMP-9, which contribute to alveolar destruction as well as to mucus hyperproduction [178]. An increased neutrophil number was observed in BAL fluid of patients with exacerbated COPD [179] and in induced sputum from COPD patients [180].

Eosinophilic inflammation is a characteristic hallmark of asthmatic air-ways [181]. Eosinophilia in lung tissue is driven mainly by IL-5, which is very important in eosinophil development in bone marrow and is respon-sible for eosinophil recruitment in lung mucosa and interstitium via produc-tion of eotaxins [182]. Other essential cytokines which are important in eosinophil proliferation and activation are granulocyte-macrophage-colony-stimulating factor (GM-CSF) and IL-3 [183]. Upon stimulation, eosinophils can release biologically active substances contained in granules. Four dis-tinct proteins such as eosinophil cationic protein (ECP), eosinophil protein X (EPX), eosinophil peroxidase (EPO) and major basic protein (MBP) results in damage of airway endothelial cells, intracellular matrix and neurons [184].

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Eosinophils also release cysteinyl leukotriene C4, which is a bronchoconst-rictor, and mucus secretion inducer, cytokines IL-1β, IL-6, IL-8, IL-4, TGFβ as well as ROS also contributes to airway remodeling, BHR [185]. Eosi-nophil role in COPD is less certain than in asthma. The presence of eosi-nophils in patients with COPD predicts more favorable therapeutic response to bronchodilators and corticosteroids and may indicate co-existing asthma or asthma-COPD overlap (ACO) [186-188].

There are several innate immune cytokines – IL-25, IL-33 and TSLP – considered to be alarmins. IL-25 is a member of the IL-17 family cytokines [189]. IL-25 can be produced by epithelial and endothelial cells, activated mast cells, alveolar macrophages, eosinophils, basophils and Th2 cells [190, 191]. IL-25 triggers the expression and release of Th2 cytokines [191], enhances ILC2s development and activation, thus leading to IL-5 and IL-13 secretion [192]. Epithelial IL-25 also acts directly on fibroblasts and endo-thelial cells promoting airway remodeling and angiogenesis as well as increase the production of TSLP and IL-33 [193]. Another innate immune cytokine – TSLP is a member of IL-2 cytokine family. The highest TSLP levels are found in the lungs, skin-derived epithelial cells [194]. It is produ-ced by fibroblasts, endothelial cells, mast cells, macrophages/monocytes, granulocytes and DCs [195]. Mechanical injury, viral infection, pro-inflam-matory cytokines and proteases cause TSLP production and release from epithelial cells [196, 197], further activating human DCs and inducing the Th2 cell-attracting chemokine production [198].

IL-33 role in asthma

IL-33 is a tissue-derived nuclear cytokine, a member of the IL-1 family cytokines. IL-33 is emerging as a crucial immune modulator with pleiotro-pic activities in type-1, type-2 and regulatory immune responses [199]. The main cells producing this cytokine are intestinal, and airway epithelial cells, but keratinocytes, endothelial cells, smooth muscle cells are also shown to produce IL-33 [200]. IL-33 is continuously expressed under homeostatic conditions and can be augmented under inflammatory conditions [133], especially in endothelial cells, although macrophages, DCs, mast cells and monocytes can produce IL-33, albeit in lower levels [200]. Genome-wide association studies identified IL33 and IL-1 receptor-like 1 (IL1RL1)/IL18R1 as asthma susceptibility loci [201].

IL-33 has several bioactive forms. IL-33 full-length precursor consists of seven coding exons, which produce a 30 kDa peptide [202]. The IL-33 protein is composed of two domains, exons 1–3 encode the N-terminal nu-clear domain, whereas exons 4–7 encode the C-terminal IL-1-like cytokine

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domain [203]. Inflammatory serine proteases from neutrophils and mast cells (neutrophil cathepsin G, elastase and proteinase 3, mast cell chymase, tryptase and granzyme B) can cleave IL-33 full-length precursor into mature forms of IL-33: IL-3395-270, IL-3399-270, IL-33109-270 [204, 205]. These clea-ved IL-33 forms have 10 to 30 time’s greater potency than the full-length protein. Proteases derived from pathogens, pollens, HDM, fungal allergens are also capable of inducing IL-33 cleavage [206]. IL-33 could be a dual function protein, acting intracellularly as a nuclear factor regulating trans-cription, and extracellularly as a potent cytokine [207]. Endogenous IL-33 full-length precursor is released from endothelial cells after cellular damage or mechanical injury to alert the immune system about the cell or tissue damage [208]. There are two types of IL-33 binding receptor: soluble ST2 (sST2) and membrane ST2 (ST2L). sST2 and ST2L are two main products of ST2 gene – IL-1RL1. sST2 is present constitutively in human serum [209] and acts as a decoy receptor by binding free IL-33 to make it inactive. IL-33 exerts its effects through binding to a heterodimeric receptor complex including membrane-bound ST2L and IL-1 receptor accessory protein (IL-1RAcP) resulting in the production and release of proinflammatory cyto-kines [210]. The IL-33/ST2/IL-1RAcP complex induces signaling through a MyD88 adaptor, IRAK1 and IRAK4 kinases, and TRAF6 that culminates in the activation of MAP kinases and NFƙB transcription factors [199, 211]. Tissue-resident immune cells (mast cells (MCs), ILC2s and Tregs) that express ST2 are significant targets of IL-33 [211]. IL-33 mediated signaling pathway activates MCs, basophils, and induce migration, maturation, adhe-sion, promote survival and production of pro-inflammatory cytokines [212] as well as act directly on Th2 cells to increase secretion of IL-5 and IL-13 [213]. Therefore, IL-33 is assumed as an essential cytokine bridging innate and adaptive immune system (Fig. 1.4.1.1).

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F ig. 1.4.1.1. R ol es of I L -33 i n t he pat hoge ne si s of as thm a by br idgi ng be tw ee n i nnat e and ac qui re d i m m uni ty A d ap ted acco rd in g N ab e T , J P h ar m aco l S ci , 2 0 1 4 . 26

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27 1.4.2. Adaptive immune system

In majority cases, asthma has been considered as Th2 mediated chronic inflammatory airway disease [214]. CD4+_{T cells depending on the cytokine} milieu can differentiate into Th2 (mainly) or other T lymphocytes, including Th1, Th17, Th9, Tregs [215] (Table 1.4.2.1). Th2 cells differentiated from CD4+ T cells via MHCII and antigen presenting cells (DCs, MCs) can secrete Th2 type cytokines, such as IL-4, IL-5, IL-6, IL-9, IL-13, RANTES and eotaxin which further mediate the allergic inflammation response in asthmatics [216]. IL-4 and IL-13 cause the proliferation of B cells as well as differentiation into antibody-secreting B plasma cells and subsequently induce isotype switching from IgM to IgE. IgE is a predominant antibody in allergy and asthma inflammation through the FcεRI receptor enable to induce hypersensitivity reaction [217]. Th17 cells produce IL-17A, which promotes the production of cytokines and chemokines that recruit neutro-phils into the airways and may also assist in enhancing smooth muscle cell contractility and AHR [218]. Th9 cells secrete IL-9, which stimulates MCs proliferation and activation, prolongs ILC2 survival, and may also promote mucus secretion and airway remodeling [182]. Th1 cells produce IFNγ, which inhibits Th2 cell differentiation [215]. Tregs produce IL-10 and TGFβ, which inhibits inflammation, although TGFβ together with ILC2 derived amphiregulin may promote airway remodeling [182].

There is an increase of T lymphocytes in the lung parenchyma and peri-pheral and central airways of patients with COPD, mainly CD8+_{(Tc1) and a} smaller number of CD4+ (Th1) and Th17 T cells (Table 1.4.2.1). As it was mentioned above, CD8+ and CD4+ T cells preferentially express CXCR3, a receptor for CXCL9, CXCL10 and CXCL11, and therefore they are accu-mulated in inflammatory lung tissue [219]. Tc1 cells are cytotoxic because of the release of perforin, granzyme B, TNFα which contribute to alveolar cell apoptosis and the development of emphysema [220]. Th1 express acti-vated STAT4, a transcription factor essential for activation and commitment of the Th1 lineage [221, 222]. Alveolar macrophages release IL-6 and IL-23 and therefore regulates Th17 cell function. In turn, Th17 secrete IL-17A and IL-22 and play a role in neutrophilic inflammation [223, 224]. An ILC3 number is increased in lungs with COPD, which secrete IL-17 and IL-22 and therefore can subsequently play a role in neutrophilic inflammation [156]. An Anti-IL-17 was noticed to protect mice against smoking-induced emphysema [225]. An IL-17 blocking antibody, CNTO-6785, did not improve FEV1 or symptoms in patients with COPD during six month treatment period [226]; therefore, other studies are aimed at more accurately selecting patients with neutrophilic airway inflammation.

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Table 1.4.2.1. Differences in the pathogenesis of asthma and COPD

Asthma COPD

Cells Eosinophils Neutrophils

Mast cells Macrophages

ILC2s ILC3s

Th2 Th1, Tc1

Key mediators IL-4, IL-5, IL-13 IL-8

Eotaxin TNFα, IL-1β, IL-6

Adapted according to Barnes PJ, Clinical Science, 2017. 1.4.3. Novelty in asthma pathogenesis

1.4.3.1. IL-32 in chronic airway inflammation

Many cytokines and chemokines that are secreted in both asthma and COPD are regulated by transcription factor nuclear factor-ĸB (NF-ĸB), which is activated in airway epithelial cells and macrophages in both diseases, and may have an important role in increasing airway inflammation [227, 228]. IL-32, a newly discovered proinflammatory cytokine is an important player in innate and adaptive immune response [229, 230], it is implicated in autoimmune inflammatory disorders and some oncological diseases [229, 231, 232]. There are data that our studied chronic obstructive airway diseases, primarily COPD, have a component of autoimmunity [233], though the role of IL-32 in its pathogenesis is not known. It is known that T-lymphocytes, natural killers, monocytes and epithelial cell lines may produce IL-32 when stimulated by IL-2 or IFN-γ [229, 231, 232, 234, 235]. This proinflammatory cytokine strongly stimulates other cytokines such as TNF-α, IL-1β, IL-6 and macrophage inflammatory protein-2 (MIP-2) [231, 232, 236]. Recently, two studies showed the regulation of IL-32 expression in primary nasal epithelial cells by inflammatory cytokines [237, 238]. Several studies demonstrated increased expression of IL-32 in lung tissue of patients with COPD [234] and nasal mucosa of patients with allergic rhinitis [238, 239], thus suggesting that IL-32 is involved in the pathogenesis of these diseases. Bang et al. found that IL-32 level was decreased in patients with asthma and recombinant IL-32 may suppress inflammation in asthma mouse model [240]. However, the role of IL-32 in chronic pulmonary diseases such as asthma and COPD is not well established, and a possible relationship with a risk factor such as smoking is not appropriately studied.

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1.4.3.2. Vitamin D in allergy and asthma

A new theory of increasing allergic diseases appeared – the theory of vitamin D, which states that early vitamin D supplementation is associated with increased newborn’s sensitization to allergens [241]. Recently, there is a keen interest in the importance of vitamin D in the development of asthma and allergy pathogenesis in light of its immune modulating properties [242]. Vitamin D is a fat-soluble nutrient, which is the best known as a critical factor in bone mineralization [243]. Vitamin D3 is converted to 25(OH)D in the liver, and later 25(OH)D is converted into the active form 1,25(OH)2D in kidneys [244]. Over past decades, vitamin D, through the activation of vitamin D receptor (VDR), has been shown to have an immunomodulatory effect on dendritic cells (DCs) [245], macrophages, B and T lymphocytes [242, 246] and structural cells in the airways. In airway smooth muscle (ASM) cells, vitamin D reduced proliferation, production of proinflamma-tory cytokines (TNF-α, TNF-β, PGE2), matrix metalloproteinase (MMP) and mucus secretion [247, 248]. Vitamin D has been shown to decrease costimulatory molecules, C-C chemokine receptor (CCR)-7 expression, ma-turation and antigen presentation in DCs while promoting tolerogenic DCs with enhanced IL-10 expression [245]. In T lymphocytes, this vitamin has been reported to shift the balance from Th-17 cells to T regulatory (Treg), by decreased IL-17 and increased production of IL-10 [242, 246]. Vitamin D inhibits differentiation and proliferation of B cells to plasma cells and is believed to play a role in decreasing antibody production [249]. In innate immune cells involved in asthma, vitamin D has been shown to inhibit differentiation, maturation, homing and cytokine secretion from mast cells, neutrophils, and eosinophils [250]. The overall effects of this immunomo-dulation are decreased airway hyper-responsiveness, inflammation, and remodeling in asthma [249].

1.4.3.3. Role of periostin in asthma

Periostin was first described in 1993 as osteoblast-specific factor 2 in adult mice [251]. Periostin is a 90-kDa member of the fasciclin-containing protein family and is encoded by POSTN gene in humans (GenBank acces-sion no., D13664) [252]. Periostin is a matricellular protein upregulated by IL-4 and IL-13, which causes cell activation by binding to receptors present on the cell surface [253]. It has an essential role in the development of bone, tooth, and heart valves, as well as in healing process after myocardial infarc-tion and tumor development [254]. Moreover, periostin has been associated with atopic conditions such as dermatitis [255, 256] and allergic rhinitis/rhi-nosinusitis [257]. The role of periostin in asthma and type 2 inflammation is

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an area of active research. In mice model, Sehra et al. [258] and Gordon

et al. [259] showed that periostin diminishes airway inflammation, whereas

Blanchard et al. [260] demonstrated that periostin accelerates allergen-indu-ced eosinophil recruitment in the lungs. Takayama et al. were the first to demonstrate the deposition of periostin in the airway subepithelial layer in human asthmatics [261]. Woodruff and research group found that periostin gene expression was increased in airway epithelial cells [262] as well as in sputum cells [263] from patients with asthma compared to healthy subjects. Moreover, periostin expression in airway epithelial cells correlated with airway basement membrane thickness and was found in the serum of patients with asthma [264]. In a BOBCAT study serum, periostin has been identified as best predictor of airway eosinophilia compared to serum eosi-nophil count, FeNO level and serum IgE [265]. Several studies confirmed that periostin concentrations in serum were higher in eosinophilic asthma phenotype [266, 267]. In HDM-treated mice model, periostin was associated with maximal airway hyperresponsiveness and airway inflammation [268]. In humans, periostin has been shown to prolong Th2 exerted inflammation and to aggravate airway remodeling [269, 270]. Periostin is mainly involved in tree mechanisms: eosinophil recruitment, mucus secretion and remode-ling [271]. Periostin expression in epithelial cells is upregulated by IL-4 and IL-13 [261] and causes TGF-β-dependent secretion of collagen by airway fibroblasts [264] and so contributes to tissue remodeling and sub-epithelial fibrosis in asthma. Additionally, periostin enhances eosinophilic adhesion to fibronectin, contributing eosinophilic infiltration [260]. In severe asthma phenotype, periostin can be considered as a systemic biomarker of “Th2-high“ and “Th2-low“ asthma [272]. It was also shown to be associated with higher serum IgE, greater hyperreactivity, subepithelial fibrosis and eosino-philic inflammation [272]. Though, Wagener et al. showed that periostin was not able to distinguish between eosinophilic and non-eosinophilic air-way inflammation [273]. Another interesting study, performed by Matsu-saka et al. found that the “high-periostin” was associated with late-onset eosinophilic asthma, higher prevalence in aspirin intolerance or concomitant nasal disorder, and lower lung function [274]. It was also noted, that periostin in sputum is associated with persistent airflow limitation and airway eosinophilia, despite high-dose ICS treatment [275]. Consequently, periostin is a promising biomarker in predicting response to ICS as well as to the newest biological treatments targeting Th2 inflammation [276, 277]. Discrepancies between various research data should be supported and confirmed by further and more focused studies.

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2. MATERIALS AND METHODS

2.1. The general design of the experiments

The prospective study was performed in the Department of Pulmono-logy and ImmunoPulmono-logy during 2011–2016 and was completed in the Depart-ment of Immunology and Allergology during 2016–2017, in Hospital of the Lithuanian University of Health Sciences Kauno klinikos, Kaunas. The study consisted of two parts.

Part Ⅰ. Patterns of the immune response in chronic obstructive

pulmo-nary diseases – asthma and COPD. The study protocol was approved by the Regional Ethics Committee for Biomedical Research, Lithuanian University of Health Sciences (48/2004), and each participant gave his/her informed written consent. The study is registered with Clinical Trials.gov (Identifi-cation: NCT01378039). A total of 91 adults were recruited for the study: 31 patients with stable mild-to-moderate asthma according to the Global

Initiative for Asthma (GINA), 51 outpatients with stable COPDaccording to

the Global Initiative for Chronic Obstructive Lung Disease criteria, and nine healthy subjects. All subjects were divided into smokers and ex-smokers.

Part Ⅱ. Immune markers and proteasomal gene polymorphisms in asthma. The study was approved by The Kaunas Regional Biomedical Research Ethics Committee (no. BE-2-31). This study is an extended part of the international project “Proteasomal genes alleles as risk factors for bronchial asthma in Latvian, Lithuanian and Taiwanese populations” which was done during 2011–2013.

2.2. Inclusion and exclusion criteria General inclusion criteria for all subjects:

1. Older than 18 years. 2. Male and female.

3. Signed informed consent.

General exclusion criteria for all subjects: 1. Exacerbation of the main disease.

2. Congenital disease and/or any known family predisposition to con-genital disease (except asthma).

3. Acute or chronic infections (more than one months).

4. Any other chronic disease/condition (autoimmune, oncological, etc.). that might have a negative influence on the study parameters.

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32 5. Alcohol or drug abuse.

6. Pregnancy and breastfeeding.

Inclusion criteria for asthma patients in Part Ⅰ–Ⅱ:

1. Diagnosed according to the Global Initiative for Asthma. 2. Clinical history of the disease for ≥1 year.

Additional inclusion criteria for asthma patients in Part Ⅰ:

1. Showed positive reversibility to β2-agonist and/or bronchial

hyper-responsibility to methacholine. 2. Baseline FEV1 >80% of predicted.

3. No use of inhaled, nasal or systemic steroids for at least one month before the study.

Inclusion criteria for COPD patients:

1. Postbronchodilator FEV1/FVC, <0.70; FEV1, <80% of predicted;

2. Negative β2-agonist reversibility test: postbronchodilator FEV1 less than 12% and/or 200 mL.

3. Smoking history, more than 10 pack/years.

4. No history of asthma, bronchiectasis, lung cancer, or other signify-cant respiratory or autoimmune disease.

5. Patients had not been treated with systemic steroids and/or antibio-tics for at least one month before the study.

2.3. Spirometry

Pulmonary function was tested using a pneumotachometric spirometer “CustovitM” (CustoMed, Germany) with subjects in a sitting position, and the recorded highest value of forced expiratory volume in 1 sec (FEV1) and

forced vital capacity (FVC) 85 from at least three technically satisfactory maneuvers differed by less than 5%. Normal values were characterized according to Quanjer and colleagues. Subjects had to avoid using short-acting β2-agonists at least 12 h prior the test and long-acting β2 agonists at

least 48 h prior the lung function test.

2.4. Sputum induction and processing

Subjects inhaled 10 mL of sterile hypertonic saline solution (3%, 4% or 5% NaCl (Ivex Pharmaceuticals, USA)) at room temperature (RT) from an ultrasonic nebulizer (DeVilbiss Health Care, USA). The duration of each inhalation was 7 min and was stopped after expectoration an adequate amount of sputum. Spirometry was performed after each inhalation, to detect a possible decrease of FEV1. Sputum was poured into a Petri dish and

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separated from saliva. A fourfold volume of freshly prepared 0.1% dithio-threitol (DTT; Sigma-Aldrich) was added. The mixture was vortexed and placed on a bench rocker for 15 min at RT. Next, an equal volume of pho-sphate-buffered saline (PBS; Sigma-Aldrich), solution was added to the DTT. The cell pellet was separated using 40 μm cell stainer (Becton Dickin-son, USA). The mixture was centrifuged for 10 min at 4°C; the supernatant was aspirated and stored at –70°C for later assay. The total cell counts, the percentage of epithelial cells and cell viability were investigated using a Neubauer hemocytometer (Heinz-Herenz; Germany) by microscope (B5 Professional, Motic, China), using Trypan blue exclusion method. Cytospin samples of IS were prepared using a cytofuge instrument (Shandon Southern Instruments, USA).

2.5. Bronchoscopy and BAL fluid processing

Bronchoscopy was performed in a week after sputum induction proce-dure. Subjects were not allowed to drink or eat at least 4 h. To perform BAL fluid, the local upper airways anesthesia with 5 mL of 2% lidocaine (Grindex, Latvia) was used. All bronchoscopic examinations were perfor-med in the morning. The bronchoscope (Olympus, USA) was wedged into the segmental bronchus of the middle lobe and 20 mL × 7, a total of 140 mL of sterile saline solution (0.9% NaCl) was infused. The fluid was gently aspirated immediately after the infusion has been completed and was collected into a sterile container. The fluid was immediately filtered using 40 μm cell stainer (Becton Dickinson, USA) and centrifuged at 4°C for 10 min. Supernatants were used for enzyme-linked immunosorbent assay (ELISA). Preparation of BAL fluid cytospins was the same as the prepa-ration of IS samples described above.

2.6. IS and BAL fluid cell analysis

Prepared IS and BAL fluid cytospins were stained by the May-Grünwald- Giemsa method for differential cell counts [278]. Cell differentiation was determined by counting approximately 400 cells in random fields of view under the light microscope, excluding squamous epithelial cells. The cells were identified using standard morphological criteria, by nuclear morpho-logy and cytoplasmic granulation. Cell counts were expressed as percent-ages of total cells and absolute values (106_/L).

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2.7. DNA extraction and genotyping

DNA from peripheral blood samples was extracted using QIAamp DNA blood mini kit (Qiagen, Hilden, Germany) following the manufac-turer’s protocol. Five SNP’s of three proteasomal genes on chromosome 14 were analyzed in this study using allele-specific amplification and cleaved amplified polymorphic sequence methods. SNP’s and genotyping details are listed in Table 2.7.1. Primer sequences and genotyping methods were previously described [279]. PCR was performed in a total volume of 30 ml using the DreamTaq polymerase or DNA Taq polymerase (recombinant) (Fermentas, Vilnius, Lithuania) under the following conditions: denaturation at 94°C for 5 min followed by 35–40 cycles of 94°C for 45 s, appropriate annealing temperature(55–61°C) for 45 s, 72°C for 45 s and a final exten-sion at step at 72°C for 7 min. DNA digestion by restriction enzymes was performed according to the manufacturer’s protocols (Fermentas). RsaI digestion was used to determine the rs1048990 genotype, TspRI digestion was used to determine the rs2348071 genotype, and DdeI (HpYF3I) diges-tion was used to determine the rs2295826 and rs2295827 genotypes. All amplified and digested PCR products were analyzed by electrophoresis in 2–4% agarose gel. For quality control, 16 randomly chosen samples per each marker were genotyped in duplicate in different experiments. Further-more, 20 randomly chosen samples were verified by direct sequencing. The concordance was 100%.

IMMUNE MARKERS AND PROTEASOMAL GENE POLYMORPHISMS SPECIFIC FOR ASTHMA PHENOTYPES

Edita Gasiūnienė

IMMUNE MARKERS

AND PROTEASOMAL GENE

POLYMORPHISMS SPECIFIC

FOR ASTHMA PHENOTYPES

Edita Gasiūnienė

IMUNINIAI

ŽYMENYS IR

PROTEOSOMINIŲ GENŲ

POLIMORFIZMAI

, BŪDINGI

ASTMOS FENOTIPAMS

CONTENTS

ABBREVIATIONS

INTRODUCTION

1. REVIEW OF LITERATURE

2. MATERIALS AND METHODS