THE ROLE OF TH9 CELLS AND EOSINOPHIL APOPTOSIS IN ALLERGIC ASTHMA

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LITHUANIAN UNIVERSITY OF HEALTH SCIENCES MEDICAL ACADEMY

Deimantė Hoppenot

THE ROLE OF TH9 CELLS

AND EOSINOPHIL APOPTOSIS

IN ALLERGIC ASTHMA

Doctoral Dissertation Biomedical Sciences, Medicine (06B) Kaunas, 2016 1

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Dissertation has been prepared at the Department of Pulmonology and Immunology, Medical Academy of Lithuanian University of Health Sciences during the period of 2011–2016.

Scientific supervisor:

2011–2015 Prof. Dr. Raimundas Sakalauskas (Lithuanian University of Health Sciences, Biomedical Sciences, Medicine – 06B)

2015–2016 Prof. Dr. Kęstutis Malakauskas (Lithuanian University of Health Sciences, Biomedical Sciences, Medicine – 06B)

Consultant

2015–2016 Prof. Dr. Raimundas Sakalauskas (Lithuanian University of Health Sciences, Biomedical Sciences, Medicine – 06B)

Dissertation is defended at the Medicine Research Council of the Medical Academy of Lithuanian University of Health Sciences.

Chairperson

Prof. Dr. Habil. Vaiva Lesauskaitė (Lithuanian University of Health Sciences, Biomedical Sciences, Medicine – 06B)

Members:

Assoc. Prof. Dr. Marius Žemaitis (Lithuanian University of Health Sciences, Biomedical Sciences, Medicine – 06B)

Prof. Dr. Vilmantė Borutaitė (Lithuanian University of Health Sciences, Biomedical Sciences, Biology – 01B)

Prof. Dr. Saulius Šatkauskas (Vytautas Magnus University, Biomedical Sciences, Biology – 01B)

Prof. Dr. Elisabeth Bel (University of Amsterdam, Biomedical Sciences, Medicine – 06B)

The dissertation will be defended in the open session of the Medicine Research Council on May 4, 2016, at 1:00 pm in the Large Auditorium at the Hospital of Lithuanian University of Health Sciences Kauno Klinikos.

Address: Eivenių 2, LT-50009 Kaunas, Lithuania. 2

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LIETUVOS SVEKATOS MOKSLŲ UNIVERSITETAS MEDICINOS AKADEMIJA

Deimantė Hoppenot

9 TIPO T LIMFOCITŲ PAGALBININKŲ

BEI EOZINOFILŲ APOPTOZĖS

VAIDMUO SERGANT ALERGINE

ASTMA

Daktaro disertacija Biomedicinos mokslai, Medicina (06B) Kaunas, 2016 3

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Disertacija rengta 2011–2016 metais Lietuvos sveikatos mokslų universiteto Medicinos akademijos Pulmonologijos ir imunologijos klinikoje.

Moksliniai vadovai:

2011–2015 m. prof. dr. Raimundas Sakalauskas (Lietuvos sveikatos mokslų universitetas, biomedicinos mokslai, medicina –06B)

2015–2016 m. prof. dr. Kęstutis Malakauskas (Lietuvos sveikatos mokslų universitetas, biomedicinos mokslai, medicina – 06B)

Konsultantas

2015–2016 m. prof. dr. Raimundas Sakalauskas (Lietuvos sveikatos mokslų universitetas, biomedicinos mokslai, medicina – 06B)

Disertacija ginama Lietuvos sveikatos mokslų universiteto Medicinos akademijos biomedicinos mokslų srities medicinos krypties taryboje. Pirmininkė

Prof. habil. dr. Vaiva Lesauskaitė (Lietuvos sveikatos mokslo universi-tetas, biomedicinos mokslai, medicina – 06B)

Nariai:

Doc. dr. Marius Žemaitis (Lietuvos sveikatos mokslo universitetas, bio-medicinos mokslai, medicina – 06B)

Prof. dr. Vilmantė Borutaitė (Lietuvos sveikatos mokslo universitetas, biomedicinos mokslai, biologija – 01B)

Prof. dr. Saulius Šatkauskas (Vytauto Didžiojo universitetas, biome-dicinos mokslai, biologija – 01B)

Prof. dr. Elisabeth Bel (Amsterdamo universitetas, biomedicinos moks-lai, medicina – 06B)

Disertacija ginama viešame Lietuvos sveikatos mokslų universiteto Me-dicinos akademijos MeMe-dicinos krypties tarybos posėdyje 2016 m. gegužės 4 d. 13 val. Lietuvos sveikatos mokslų universiteto ligoninės Kauno klinikos Didžiojoje auditorijoje.

Addresas: Eivenių 2, LT-50009 Kaunas, Lithuania. 4

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ABBREVIATIONS

AA – allergic asthma

ADAM33 – a disintegrin and metalloproteinase domain-containing protein 33 AJ – adherent junctions

APC – antigen-presenting cells AR – allergic rhinitis

ARIA – Allergic Rhinitis and its Impact on Asthma ASM – airway smooth muscle

BAL – bronchoalveolar lavage

BHR – bronchial hyperresponsiveness CCL – C-C chemokine ligand

CCR – C-C chemokine receptor CD – cluster of differentiation

CGRP – calcitonin-gene-related peptide

CTLA-4 – cytotoxic T-lymphocyte-associated protein 4 DCs – dendritic cells

DTT – dithiothreitol

DAR – dual asthmatic response EAR – early asthmatic response ECP – eosinophil cationic protein EDN – eosinophil-derived neurotoxin

ELISA – enzyme-linked immunosorbent assay EP – eosinophil peroxidase

ERS – European Respiratory Society ETS – environmental tobacco smoke EDTA – ethylene diamine tetra-acetic acid FcεRI – high-affinity IgE receptor

FcɣRIII – low affinity IgG receptor FEV1 – forced expiratory volume in 1 sec. FITC – fluorescein isothiocyanate

Foxp3 – forkhead box P3

GBD – Global Burden of Disease Study GINA – Global Initiative for Asthma

GM-CSF – granulocyte macrophage-colony stimulating factor GPRA – G protein-coupled receptor for asthma

HDM – house dust mites

ICAM – intercellular adhesion molecule IFN – interferon

Ig – immunoglobulin IL – interleukin

iNOS – inducible form of nitric oxide synthase IQR – interquartile range

JAK – Janus kinase 1

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K3EDTA – ethylene diamine tetra acetic acid tribasic potassium salt LAR – late asthmatic response

LBP – lipopolysaccharide binding protein LPS – lipopolysaccharides

LT – leukotrien

mAb – monoclonal antibody MBP – major basic protein

MHC – major histocompatibility complex MIP – macrophage inflammatory protein MMP – matrix metalloproteinase

NaCl − natrium chloride NO – nitric oxide

NF-κB – Nuclear factor kappa-light-chain-enhancer of activated B cells transcription factor

NK – natural killer

NOD – nucleotide binding oligmerization domain OVA – ovalbumin

PAF – platelet activating factor PBS – phosphate-buffered saline

PD20− provocative dose of metacholine in mg causing a 20% fall in FEV1

PS – phosphatidyleserine

PU.1 – purine box transcription factor 1

RORγt – retinoic acid receptor–related orphan receptor-γt ROS – reactive oxygen species

RSV – respiratory syncytial virus RV – rhinovirus

SEM – standart error mean

STAT – signal transducer and activator of transcription T-bet – T-box transcription factor

TCR – T-cell receptor

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

TJ – tight junctions TLR – Toll-like receptor TNF – tumor necrosis factor Treg – regulatory T cells

TSLP – thymic stromal lymphopoietin SNP – single nucleotide polymorphism TX – tromboxane

VCAM – vascular cell adhesion molecule VEGF – vascular endothelial growth factor

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INTRODUCTION

Asthma is a heterogeneous disease usually with predominant chronic airway inflammation. It is often triggered by allergen exposure, change in weather, viral respiratory infections or exercise. During the past decades the prevalence of asthma is increasing in many countries, especially among children [1]. Factors and mechanisms which affect asthma development are very complex. It is proved that inhalant allergens especially those derived from house dust mites, cockroaches, animal dander, fungi and pollens are an associate of lung function and airway hyper-responsiveness in adults. Exposure to aeroallergens has been identified as a major environmental risk factor for the sensitization and later on for the development of asthma in children [2, 3]. The components of innate and adaptive immunity regulate asthma pathogenesis. In most cases, asthma occurs through the selective expansion of T lymphocytes, particularly of the T helper cells (Th) type 2 that secrete many proinflammatory cytokines. These cytokines conduct the allergic inflammatory cascade in asthma, including Th2 cell survival, B cell isotype switching to IgE synthesis, mast-cell differentiation and maturation, eosinophil maturation and survival and basophil recruitment [4]. Lately it was discovered that asthma is much more heterogeneous and complex disease than suggested by the Th2 theory and by mouse models of allergic asthma [5] with new adaptive immunity participants – T cells belonging to the Th17 and Th9 lineages. Th17 cells produce interleukin (IL)-17 that have a positive impact on neutrophil recruitment into the airways and might participate in pathogenesis of asthma exacerbations [6, 7]. Thus Th9 cells belong to the newest and less well characterized cluster of differentiation (CD) 4+ subset. Different studies proved that Th9 cells express IL-9 but not the key signature cytokines of other Th cell subsets [8, 9]. Recent reports have showed that Th9 cells are important in human atopic diseases. Different researchers found that patients with allergy have higher numbers of circulating T cells which secrete IL-9 in response to allergen (such as pollen, cat dander, peanuts or house dust mite (HDM) extract) compare to control individuals [10–12]. It is believed that Th9 cells develop under stimulation of IL-4-activated signal transducer and activator of transcription protein-6 (STAT6) [13]. Purine box transcription factor 1 (PU.1) promotes switching between Th2 and Th9 phenotypes. PU.1 plays an important in the development of Th9 memory cells and Th9 immunity [14].

IL-9 induces mucus production, goblet cell hyperplasia and participates in airway remodeling. IL-9 promotes mast cell growth and function and

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prolongs survival of eosinophils during allergic airways inflammation [15, 16]. Eosinophils are the central effector cells in ongoing airway infla-mmation. Enhanced eosinophil survival is considered critical for the accumulation of eosinophils in the lungs of asthmatics. Delayed apoptosis of blood and tissue eosinophils in patients with asthma is found compared to healthy individuals [17] but little is known about eosinophil apoptosis during late phase allergen induced inflammation. In most cells activation of nuclear factor kappa-light-chain-enhancer of activated B cells transcription factor (NF-κB) protects from apoptosis, through induction of survival genes. NF-κB activation is enhanced in asthmatic tissue. It is believed that inhibition of NF-κB results in dramatic increase in eosinophil apoptosis [18, 19].

There are very few data, concerning Th9 cells investigations in mice and even less in humans. In this study peripheral Th9 cells role and eosinophil apoptosis was investigated in allergic asthma patients after inhaled allergen challenge. During this study the role of the transcription factors PU.1, STAT6, and NF-κB in allergen-induced inflammation in adult atopic asthmatics were investigated.

Study aim

The aim of this study was to evaluate the role of Th9 cells and eosinophil apoptosis in allergic asthma patients.

Study objectives

1. To investigate peripheral blood Th9 cell count, interleukin-9 level in allergic asthma and compare to allergic rhinitis patients and healthy individuals.

2. To evaluate the expression of transcription factors STAT6 and PU.1 in peripheral blood Th9 cells and NF-κB in eosinophils.

3. To investigate peripheral blood Th9 cell count, interleukin-9 level and expression of STAT6 and PU.1 during allergen-induced late-phase airway inflammation.

4. To determine the changes of peripheral blood eosinophil apoptosis and expression of NF-κB in eosinophils during allergen-induced late-phase airway inflammation.

5. To compare peripheral blood Th9 cell count, interleukin-9 level and eosinophil apoptosis between asthma patients with dual asthmatic response and with isolated early asthmatic response.

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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 [1]. It is often triggered by allergen exposure, change in weather, viral respiratory infections or exercise.

Asthma may affect different age individuals. It is an important and growing health problem all over the world. Asthma is the 14th most im-portant disorder in the world in terms of the extent and duration of disability.

During the past decades the prevalence of asthma is increasing in many countries, especially among children [1, 20]. Factors responsible for increas-ing asthma rates are not fully understood, but environmental and lifestyle changes play the key roles.

Global prevalence of asthma is found to be 1–18 percentage of the population in different countries [1, 20, 21]. According to the most recent comprehensive analyses which were done analyzing data during 2008–2010 years by the Global Burden of Disease Study (GBD) asthma affects about 334 million of people in the world. GBD states that 14% of the world’s children and 8.6% of young adults (aged 18–45) experience asthma symp-toms. These numbers are not precise; rather they are estimated from the best data available. The historical view of asthma being a disease of high-income countries no longer holds: most people affected are in low- and middle-income countries, and its prevalence is estimated to be increasing fastest in those countries. Asthma symptoms became more common in children from 1993 to 2003 in many low- and middle-income countries which previously had low levels, according toInternational Study of Asthma and Allergies in Childhood. However, in most high-prevalence countries, the prevalence of asthma changed little and even declined in a few countries. 235 million of asthma cases world-wide were mentioned in Global Asthma Report 2011. That time the numbers came from the analysis which underwent from 2000-2002 and the conclusion was made from the best data available but it is difficult to say if incidence of asthma in the world has increased from 235 to 334 million between 2011 and 2014 reports.

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1.2. Factors affecting asthma development

Factors and mechanisms which affect asthma development are very complex. Interaction of some genes, maturation of immune response, aller-gens, and infections especially during the early childhood might influence asthma development especially for individuals with genetic predominance for asthma [22, 23].

Genes

Markers in 19 chromosomal regions have shown some evidence of linkage to asthma, atopy, or related phenotypes in multiple independent genome-wide searches. Linkages to five of these regions (5q, 6p, 11q, 12q, and 13q) have also been reported in non-genome-wide screens. In addition, at least two independent studies have reported linkages to markers on 16p. Numerous candidate genes in these regions have shown varying levels of association to asthma or atopic phenotypes, potentially implicating them as disease susceptibility loci. These include the IL4, CD14, and B2ADR genes on 5q, the HLA-DRB1 and TNF genes on 6p, the FCERB1 and CC16 genes on 11q, and the IL4RA gene on 16p [24]. More than 20 genome-wide linkage screens have been reported in different populations investigating chromosomal regions that are linked to asthma and atopy, or related phenol-types like elevated immunoglobulin E (IgE levels), wheezing, and bronchial hyperresponsiveness (BHR). A number of chromosomal regions of different genes which may lead to asthma development were identified in different studies. For example the cytokine cluster on chromosome 5q which contains interleukin 3 (IL-3), IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF), FCER1B on 11q, interferon-γ (IFN-γ) and STAT6 on 12q, and interleukin-4 receptor α (IL4R; the IL-4Rα chain, also part of the IL-13R) on 16p [25]. On chromosome 2q32-q33 cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is the most prominent candidate gene. CTLA-4 is involved in T-cell activation and regulation of IgE. Chromosome 5q31 contains several candidate genes like IL-4, IL-13, IL-5, cluster of differentiation (CD14) and granulocyte macrophage colony stimulating factor (GM-CSF), which are possibly important for the development and progression of inflammation associated with allergy and asthma. On chromosome 6, the major histocompatibility complex (MHC) region is located containing many molecules involved in innate and specific immunity. Genes of the MHC class II have been shown to influence the

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ability to respond to particular allergens [26]. The β chain of FcεRI, the high affinity receptor for IgE, is localised on chromosome 11q13; variation of the β chain may affect the stability of the receptor during surface expression and possibly modify receptor function [27]. On chromosome 12q13 signal transducer and activator of transcription 6 (STAT6) is one of many candidate genes because it plays an important role in IL-4/IL-13 signaling. The chromosomal region 13q14 has been demonstrated to be linked to total serum IgA and IgE levels [28]. Findings of G protein-coupled receptor for asthma (GPRA) [29, 30] and a disintegrin and metallopro-teinase domain-containing protein 33 (ADAM33) [31, 32] are expressed in airway smooth muscle (ASM) cells [33] and are believed to be related with BHR and so playing important role in asthma pathogenesis [34]. GPRA mediated regulation of ASM tone and ASM growth is one of the main impulses of acute and chronic features of asthma [35].

Environmental tobacco smoke

About 4000 chemicals which are generated during burning of tobacco products are released when smoking. More than 250 are known to be toxic or carcinogenic. The nicotine and other chemical compounds which are found in cigarette can cross the barrier of placenta [36]. In pregnant women who smoke or use nicotine replacement therapy, nicotine concentrate is found in fetal blood and amniotic fluid also it is detectable in breast milk during lactation [37, 38]. There is strong evidence that maternal smoking during pregnancy is a very important risk factor for asthma development in children [39, 40]. In one genome-wide linkage analysis which was made by S. Collila and the group multigenerational families with asthma were analyzed testing for the presence of a gene-environment interaction with environmental tobacco smoke (ETS) exposure [41]. The result of this genome-wide linkage analysis showed that genes in 3 chromosomal regions (1p, 5q, and 17p) might interact with ETS to increase the risk of asthma in early childhood. One of the most recent and biggest systematic review and meta-analysis where prenatal and passive smoke exposure and incidence of asthma and wheeze where analyzed showed that the effects of passive smoking on the incidence of wheeze and asthma are substantially higher than previously estimated, particularly for the effect of maternal postnatal smoking exposure. The authors found the strongest significant effect for prenatal maternal smoking and incidence of asthma in children aged ≤2 years. Asthma incidence became lower with children age but remained

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significantly associated with asthma onset between the ages of 5 and 18 years. Postnatal maternal passive smoking did not increase asthma risk in children aged ≤2 years but demonstrated a borderline significant association with incidence of asthma in children aged 5 to 18 years. To conclude with, ETS 20% to 85% increases the risk of incidence of asthma, also ETS increases the risk of incidence of wheeze from 28% to 70% [42]. There are very few studies analyzing risk for asthma development due to paternal smoking and with no data for children aged ≤2 and with conflicting results [42, 43]. But many studies agreed by have shown a clear prenatal effect of maternal smoking; this effect is increased when combined with postnatal smoke exposure (even stronger when both parents are smoking).

Viral infections

Viral infections of the lower respiratory tract often affect early childhood wheezing and might lead to asthma development [44, 45]. Early respiratory syncytial virus (RSV) [46-48], rhinovirus (RV) [49, 50] infections increase the risk for allergic asthma. Whether lower respiratory tract infection promotes sensitization to aeroallergens causing asthma remains still contro-versial. One big and important study performed by N. Sigurs at al. showed that severe primary RSV bronchiolitis in the first year of life is frequently followed by allergic asthma persisting into early adulthood. Predisposition to both early severe RSV bronchiolitis and allergic sensitization share the interleukin (IL)-13/IL-4 gene locus [50]. Genetic studies for atopy and asthma have implicated the same locus at IL13–IL4 [51, 52]. From the other hand there are data from some studies showing that viral infection (RSV) do not impact asthma and allergy development [53]. Bacterial infection in the airways can also cause acute wheezing episodes in young children indepen-dently from viral infection [54].

Two decades ago a hygiene hypothesis appeared [55]. It states that the lack of exposure to microbes and other pathogens (viruses, gut flora, parasites) increases susceptibility to allergic diseases. Hygiene hypothesis propose the mechanism of action with insufficient Th1 cell mediated and overactive Th2 cell immunological responses and so increasing the risk for developing allergies. There are some data saying that some infections might even protect from developing asthma or allergy. For example, one prospec-tive birth cohort study showed that croup and repeated ear infections in the first year of life were inversely associated with atopy [56]. Results of one cross sectional studies where Vietnamese secondary schoolchildren were

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investigated showed that some helminth infections, such as hookworm or Ascaris [57] independently protect against allergic sensitization, particularly to HDM and are associated with a strong protective effect against atopy. Another big analytic cross-sectional study in Ecuador showed also that high level of total serum IgE or anti-Ascaris lumbricoides (A. lumbricoides) IgG4 are independent protective factors against allergen skin test reactivity living in an endemic region [58]. Another analysis was made where 6–13 years of age children from rural areas of Austria, Germany and Switzerland participated. The data demonstrated that A. lumbricoides is not protective for allergy and asthma in a rural, partially farming population [59]. One large study which was conducted in Ecuador showed that long-term periodic antihelmintic treatment was associated with an increased prevalence of allergen skin test reactivity and recent eczema symptoms but not those of asthma or rhino-conjunctivitis [60]. Very similar results were presented by other independent studies [61–63]. Several studies have found consistently low prevalence of allergies and asthma in farmers’ children in both high-income and low-high-income countries [64–66]. Long-term farm exposure for adults is associated with reduced asthma symptoms but not with hay fever and eczema [67]. The hygiene hypothesis has been proposed to explain the increases in asthma and allergy prevalence observed over the last few decades but is unlikely to be the only explanation for this increase [68], so this hypothesis still continues to be investigated.

Sex

Sex specific associations with asthma are found. There are many data that asthma prevalence is higher in boys than in girls in childhood but this reverses around puberty, with higher prevalence in adult women than in men, and it is again higher in men than in women after menopause [69, 70]. There are also some genetic findings related with sex and asthma. In one recent study linkage findings and bivariate association analysis of quantitative score of positive skin test response and forced expiratory volume in 1 second (FEV1) with markers in 5q31 showed significant association with two single nucleotide polymrphisms (SNPs), rs2069885 and rs2069882, within IL9 gene in males. The data of that study say that the IL9 gene encodes IL-9, a cytokine produced by T-helper cells, which plays an important role in immunological and allergic processes [70]. Another new finding – the thymic stromal lymphopoietin (TSLP) gene encodes thymic stromal lymphopoietin, an IL-7-like cytokine that regulates allergic

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asthma in mouse models and shows increased expression in human airway epithelial cells from patients with asthma compared to controls [71]. Expression of TSLP in mice leads to a more severe phenotype in females compared to males [23, 72]. New data were presented by the first genome-wide meta-analysis of genotype by-sex interaction study of asthma. It was discovered two asthma risk loci. The validated associations are for single-nucleotide polymorphism near the IRF1 gene on 5q31.1 in European American males and transcriptional activation of type I interferon (IFN) genes SNPs in the 3′ UTR of the Rap1 GTPase-activating protein 2 (RAP1GAP2) encoding gene in Latino females [73]. RAP1GAP2 is expressed in the lung where it is involved in regulating the secretion of dense granules from platelets at sites of endothelial damage and it is be-lieved that allergen exposure can result in recruitment of platelets to the airways [74].

Allergen exposure

Exposure to aeroallergens has been identified as a major environmental risk factor for the sensitization and later on for the development of asthma in children [2]. Very often exposure and sensitivity follow a dose dependent relationship [75]. It is proved that there is a linear relationship between allergen exposure and the prevalence and severity of asthma symptoms [76]. Exposure of the genetically predisposed adult to a critical level of allergen may result in sensitization, and then further exposure of the sensitized individual leads to the development of airway inflammation and BHR. The results of one cross-sectional study confirm that sensitization to inhalant allergens is an associate of lung function and BHR in adults. The authors of that study say that amongst the adults there is a quantitative relationship between the level of allergen-specific IgE or the size of skin prick test reaction and the level of lung function as well as the BHR [3].

House dust mites (HDM) is one of the major causes of asthma [77, 78], asthma severity [79] or asthma exacerbations especially in sensitized individuals [80]. Sensitization to cat and dog dander [81, 82], as well as sensitization to Alternaria mold (the latter one – independently) increase asthma risk and also may be the cause of asthma exacerbations [83–85]. It is believed that families with low socioeconomic status are associated with a high likelihood of cockroach allergen in home dust at moderate level concentrations to cause sensitization to high levels to impact asthma exacerbation. These families have low likelihood of having dust mite

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Dermatophagoides pteronyssinus (D. pteronyssinus), cat, and dog allergen levels to cause atopy or asthma exacerbation. In contrast, the families of high socioeconomic status are strongly associated with having a high likelihood of elevated levels of dust mite D. pteronyssinus, cat, and dog allergens and a low risk of elevated concentrations of cockroach allergen [2].

Two decades ago it was concluded that in addition to genetic factors, exposure in early childhood to HDM allergens is an important determinant of the development of asthma [86, 87]. The data of many studies show that there is a clear relationship between allergen exposure and the prevalence also the severity of sensitization (allergen specific IgE response) [76]. More than 50% of children and adolescents with asthma are sensitized to D. pteronyssinus [88]. Nevertheless the evidence for HDM exposure leading to asthma development has debating results [86, 89, 90].

Cat allergen might appear in dust samples where a cat does not live [91]. Cat allergen exposure is associated with the development of sensitivity and asthma in dose-dependent [92, 93] or non-linear [94] manner. Interesting data revealed one study which was conducted in Boston and during which home allergen levels within the first three months of life were measured and repeated wheeze in the first year of life were taken in account. There were no associations found between symptoms and levels of cat or dog allergens, but a significant association was observed with cockroach allergen levels greater than 0.05 U/g in the family room [90, 95]. Another resent popu-lation-based study was performed in Sweden which showed that asthma in childhood was associated with higher levels of dog, cat or horse component sensitization, and sensitization to more than one component from the same animal [96]. There are data that most of children admitted to the hospital for asthma exacerbations are sensitized to multiple indoor allergens. In another population-based cohort study 478 children aged 4–16 years hospitalized for asthma exacerbation were examined. The study concluded that more than a half of the study enrolled children (68 perc.) admitted to the hospital were sensitized to multiple allergens (such as dog, cat dander, dust mite, Asper-gilus, Alternaria, cocroach) [97].

The presence of IgE-specific mold sensitivity in children approaches 50%. Alternaria alternata is the best described in relation to immunolo-gically based respiratory symptoms in children and adults, though Clado-sporium, aspergillus and penicillium have also been implicated to varying degrees [75, 98]. Longitudinal study assessing increased exposure to indoor fungi before the development of asthma symptoms suggests that

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cillium, Aspergillus, and Cladosporium species pose a respiratory health risk in susceptible populations. Increased asthma exacerbation rate in both children and adults were associated with increased levels of Penicillium, Aspergillus, Cladosporium, and Alternaria species [99].

Obesity

Increasing epidemiological data identify a link between obesity and asthma incidence and severity [100]. The prevalence of asthma is higher in obese (body mass index >30 kg/m2) than in lean adults [101, 102]. Obesity increases the incidence of asthma by 2.0 fold in children [103] and 2.3 fold in adults [102]. Obesity makes asthma control more complicated with lower response to corticosteroids [104]. Still it is not clear why asthma develops more in obese but this might be associated with different (e.g., less atopic) inflammatory phenotype and with obesity induced changes on lung me-chanics, more co-morbidities, hormonal and neurological changes. Weight loss improves asthma control and should be included in asthma management plan of obese patients [105].

1.3. Innate immune system in asthma

Despite many human and murine studies performed asthma pathogenesis remains not fully understood. Asthma is very complex disease. A long-standing debate in the asthma field is whether asthma is a single disease with a variable presentation, or several diseases that have variable airflow obstruction as a common feature [106]. Even though many discussions are ongoing about different possible asthma phenotypes and endotypes [107, 108] it is clear that innate and adaptive immune systems are indispensable in the pathogenesis of asthma.

Humans are exposed to many potential pathogens every day. The ana-tomic barriers are fixed defenses against infection.

The human trachea, bronchi and bronchioles are lined mainly by a pseudo-stratified epithelium whose surface is dominated by ciliated. Respi-ratory epithelial cells create multiple barriers. One of them is apical junc-tional complexes which connect neighboring cells and consist of the most apical tight junctions (TJ) and the underlying adherent junctions (AJ). TJ are most apical and regulate paracellular transport of ions and certain mole-cules. They consist of three major types of transmembrane proteins: mem-bers of claudin family, tight junction–associated MARVEL protein family

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members (i.e., occludin, tricellulin, and MARVELD3) and immuno-globulin-like proteins, such as junctional adhesion molecule and coxsackie adenovirus receptor. E-cadherin and members of the nectin family represent the major trans-membrane proteins of epithelial AJ which are important for initiation and maintenance of cell–cell adhesion. Barrier dysfunction also allows penetration of luminal particles and antigens into the sub-epithelial space, where they encounter innate immune cells that initiate inflammation and immune reactions. Defects in junctional integrity are associated with asthma and may predispose allergen-induced mucosal inflammation and microbial persistence. Also virus-induced epithelial permeability may faci-litate the translocation of inhaled aeroallergens [109–112].

Another component of the respiratory epithelial cells barrier is secretory molecules. Human β-defensin, lysozyme, lactroferrin, cathelicidin LL37 and surfactant proteins A and D are expressed by airway epithelial cells and are regulated by exposure to pathogens, toxicants and cytokines. They help to defend from pathogens [113–117]. Epithelial surface also contains membrane-associated mucins (for example, MUC4, MUC13, MUC16 and MUC21) which serve as a direct host defense barrier. Secreted airway mucins MUC5B, MUC5AC and MUC2 form a mucous gel that disrupts bacterial aggregation. Mucins bind microbial pathogens thus preventing them from adhesion to cell surfaces [113, 118].

Goblet cells are a primary source of mucus within the airway. Surface goblet cells produce MUC5AC and MUC5B. Submucosal glands produce MUC5B alone. In asthma mucous metaplasia, hyperplasia, and hypertrophy appear with mucin hyper-secretion which often cause mucous plugging of the airways and there are some debating data that it might induce BHR [119–121].

Structural changes reported in the airways of asthmatics include epi-thelial fragility, goblet cell hyperplasia, enlarged sub-mucosal mucus glands, angiogenesis, increased matrix deposition in the airway wall, increased airway smooth muscle mass, wall thickening and abnormalities in elastin [119].

Airway epithelial cells might produce a broad range of cytokines; some of them such as IL-25, IL-33 and TSLP are believed to play important roles in the regulation of local innate and adaptive immunity. There are scientific data that IL-25 might enhance the activity of TSLP [122]. It is known that allergen activated epithelial cells produces greater amount of IL-25 and IL-33 which increase Th2 cytokine production. In the mouse IL-25 is related with airway eosinophilia and greater BHR [123, 124]. IL-33 levels

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are reportedly increased in human asthmatic subjects. IL-33 can influence maturation and activity of dendritic cells (DCs) leading to their enhanced expression of MHC-II, CD86, and IL-6 [125]. IL-33 as well as TSLP can stimulate mast cell cytokine production and promote maturation of CD34+ mast cell precursors especially after allergen challenge [126, 127]. TSLP has been associated with allergic inflammation in the airways and in the skin in mice and in humans. In mice model over-expression of TSLP in the skin and airways leads to expression of allergic dermatitis and allergic asthma phenotypes.[128]. Increased expression of TSLP has been observed in patients with allergic dermatitis and those with allergic asthma as well [129].

The innate immunity also involves the immune system cells such as granulocytes (neutrophils eosinophils, basophils and mast cells), phagocytes (neutrophils, dendritic cells, macrophages) and natural killer (NK) cells [130]. Basophils express a variety of surface receptors that can lead to their activation: e.g. FcεRI, CD123, TLR4, low affinity IgG receptor (FcɣRIII) [131, 132]. Basophils participate in acute and chronic allergic inflammation and usually circulate within the blood stream. FcεRI stimulation of basophils, as in the mast cells, leads to the immediate release of histamine and proteases. Later basophils can secrete leukotrienes, cytokines and chemokines such as IL-4, IL-5, IL-8, IL-10 and IL-13. Allergen stimulation leads to increased basophil numbers in the blood. Although basophils are not usually detected within healthy human tissues, this cell type has been identified in a variety of inflammatory tissue reactions in situ by using a range of indirect techniques during allergic inflammation (airways of asthmatic patients, skin of patients with atopic dermatitis, nasal mucosa of patients with allergic rhinitis) [133–137].

Mast cells unlike other leukocytes mature in the tissue. Tyrosine kinase receptor c-kit, which is expressed on the surface of mast cells and their precursors, the c-kit ligand and stem cell factor are essential for mast cell development and survival both in mice and rats and in humans [138, 139]. It is believed that mast cells are very important in acute allergic reactions, such as anaphylaxis or the acute wheezing provoked by allergen challenge in subjects with allergic asthma. Mediators stored in the cytoplasmic granules of mouse or human mast cells include histamine, proteoglycans, serine proteases, carboxypeptidase A, small amounts of sulfatases and exoglycosidases. The various mediators associated with the proteoglycans dissociate at different rates – histamine very rapidly but tryptase and chymase much more slowly. Mast cells were known to produce multiple

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cytokines and interleukins: IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-13 and IL-16 following IgE-dependent activation [140]. There are some data that mast cells also express Toll-like receptors (TLR) that recognize various microbial components [141]. Airway remodeling plays an important part in asthma pathogenesis. Plasminogen activator inhibitor (PAI)-1 is the main inhibitor of the fibrinolytic system and is known to play an essential role in tissue remodeling. Mast cells were found to be a major source of PAI-1 by using microarray technology. A large number of mast cells expressing PAI-1 are found in the airways of patients with severe asthma [PAI-140, PAI-142, PAI-143].

Neutrophils play an essential role in the immune system. They defend organism from bacterial and fungal infections by phagocytosis and the release of enzymes and other cytotoxic substances. It is also known that neutrophils release some mediators that play important role in allergic processes in general, and particulary asthma [144]. Increased neutrophil levels are more often seen in patients with low numbers of eosinophils and poor response to inhaled corticosteroids [145, 146].

The precise role of neutrophils in the pathogenesis of asthma is difficult to understand but there are data that neutrophils can contribute to early and late asthma responses. Mediators involved in early responses are released within 30 minutes of the in vitro allergen challenge of neutrophils [144]. One of them is matrix metalloproteinase-9 (MMP-9). Elevated levels of MMP-9 have been found in both bronchoalveolar lavage (BAL) fluid and sputum from patients with asthma [147]. Neutrophilic elastase levels might be increased in nasal lavage fluid of patients with allergic rhinitis and in the bronchi of allergic asthma patients [148, 149]. Study conducted by J. Monteseirin et al. has demonstrated that lactoferrin is secreted by the neutrophils of asthmatic patients through an IgE-dependent mechanism [144]. Elevated levels of myeloperoxidase (MPO) released from neutrophils have also been found to be elevated in the BAL fluid of asthma patients. MPO release has been seen to be greater in pollen-atopic patients at the end of spring than at times when these patients are asymptomatic [150]. In the absence of a stimulus, neutrophils have been seen to produce more superoxide in atopic than in nonatopic individuals. Neutrophils have also been found to produce more toxic oxygen radicals in asthmatic patients [151, 152]. Eosinophil Cationic Protein (ECP) is a powerful cytotoxic molecule which several years ago was thought to be released only from eosinophils. There are some studies which found that ECP could be released and synthesized from the neutrophils of asthmatic patients [153].

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Activated neutrophils produce several chemokines, including IL-8, growth-related oncogene-α (GRO-α), macrophage inflammatory protein 1-α (MIP-1α) and MIP-1ß. IL-8 and GRO-α are chemoattractive for neutrophils and therefore form a positive feedback loop that induces the accumulation of large numbers of neutrophils. They recruit immune cells such as T cells and DCs to the inflammation site, instruct these cells directly, and induce adaptive immune responses. During inflammation, neutrophils are able to travel from the inflammation site to the nearest lymph node where they undergo apoptosis and are taken up by DCs. As a consequence, DCs can present neutrophil-derived antigen to T cells. Finally, they can directly transfer antigens to DCs, which subsequently activate T cells [154]. A possible role for bacterial or viral infections, the activation of neutrophil elastase and an impaired nuclear recruitment of histone deacetylase 2 have been proposed to explain the presence of neutrophils in a subset of asthmatics and the resistance of these patients to glucocorticoids [155].

Many microbes interact with the host through receptors that identify a specific pathogen or microbe-derived molecules. Microbes may limit the development of the allergic adaptive immune response through interaction with a group of these innate immune re ceptors, including receptors termed Toll-like receptors (TLRs), early in life [156]. Thus, the innate immune system appears to play a critical role in determining the phenotype of the adaptive immune response. In addition to TLRs, cellular receptors for innate molecules include NOD proteins, Dectin, CD14, and collectins. TLRs not only recognize bacteria (at least TLR1, 2, 4, 5, and 9), but also fungi (TLR6), protozoa, and viruses (TLR3 and 9) [157]. Lipopolysaccharide (LPS), a ligand of TLR4, is also termed endotoxin, and a significant component of gram-negative enteric bacterial cell walls. LPS exposure appears to both protect against [156, 158] and promote asthma. Adults that get exposed to very high concentrations of LPS in occupational settings (grain workers, farming occupations) develop asthma.

After inhalation, endotoxins interact with macrophages in the airway. LPS binds to the lipopolysaccharide binding protein (LBP). The LBP-LPS complex is delivered to the cell surface protein CD14. CD14 then transfers the LPS to the myeloid differentiation protein 2 and toll-like receptor 4 (MD2/TLR4) complexes. The activation of TLR4 initiates the signal transduction cascade within the cell that leads to activation of NF-κB. This signals make the cell to start production of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukins 1 and 8 (IL-1, IL-8) [159]. Nucleotide binding oligmerization domain (NOD) proteins are

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intracellular, cytoplasmic receptors that are characterized structurally by caspase activation and recognition domain, a centrally located NOD and multiple C-terminal leucine-rich repeats [160]. There are two NOD proteins that are known to exist in humans: NOD1 and NOD2. NOD1 gene is on chromosome 7p14 and this region has been strongly linked to asthma in multiple linkage analyses performed in humans [161].

Antigen presenting cells (APC), s. DCs serve as an important bridge between the innate and adaptive immune system. The role of DCs in allergic Th2 sensitization is believed to be one of the main risk factors for developing asthma. DCs arise from CD341 bone marrow progenitor cells or CD141 monocytes and differentiate into immature DCs of three types: Langerhan’s cells, myeloid DCs, and plasmacytoid DCs. Immature DCs have the greatest capacity for uptake of antigen; however, DC maturation is associated with greater ability for antigen presentation. They are located above and beneath the basement membrane of the upper and lower airways. Antigen is presented to cells by APCs via MHC II, but requires co-stimulatory molecule expression (e.g., CD80, CD86, and CD40) to generate an adaptive immune response [162]. DCs can activate different T cells subsets and in some situations may lead to the differentiation. There are many suggestions that DCs can establish this link between a non-specific innate immunity, based upon recognition of the type of pathogen, and specific adaptive immunity directed against a non-specific peptide of the allergen. The innate immune responses are based on preserved pathogen-associated molecular patterns (PAMPs) that identify the type of pathogen. The recognition of PAMP acts as a third signal, in addition to the required signal 1 (T cell receptor and MHC II interaction) and signal 2 (co-stimulation by co-stimulatory molecules, like CD80 and CD86). Signal 3 relies on the ligation of pattern recognition receptors, such as Toll-like receptors [163, 164].

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1.4. Adaptive immune system in asthma

Adaptive immunity is the hallmark of the immune system of higher animals. This response consists of antigen-specific reactions through T and B lymphocytes. However, many clinical and experimental observations over the past 5 years have suggested that asthma is much more heterogeneous and complex than suggested by the Th2 theory and by mouse models of allergic asthma [5]. Furthermore, non-allergic forms of asthma, triggered by environmental factors such as air pollutants (for example, smoke and diesel particles), viral infection, stress and obesity, trigger or cause asthma independently of Th2 cells [165–168].

Allergic inflammation often is classified into three temporal phases. Early-phase reactions are induced within seconds to minutes of allergen challenge, and late-phase reactions occur within several hours. By contrast, chronic allergic inflammation is a persistent inflammation that occurs at sites of repeated allergen exposure.

There are data indicating that inflammation caused by allergen results in airway narrowing for sensitized allergic asthma (AA) subjects. Usually if this narrowing develops within 10 to 15 minutes after the allergen inhalation it is called the early asthmatic response (EAR). In some people with EAR the airway narrowing may persists and either does not return to baseline values or recurs after 3 to 4 hours reaching a maximum over 6 to 12 hours. It is called the late asthmatic response (LAR)[169][170].

1.4.1. Early-phase of allergen induced airway inflammation

Most allergens are proteins (some are lipids or carbohydrates), and many, including the major HDM allergen, Der p 1, are proteases [171]. After inhalation of allergen, early-phase airway inflammation is described to develop due IgE-mediated mast cell activation and subsequent bronchial smooth muscle cell constriction [172]. It belongs to I type or immediate hypersensitivity reaction. DCs take up the allergens from the airway lumen or submucosa (allergens with protease activity can reach submucosa layer). Activated DCs mature and migrate to regional lymph nodes or to sites in the local mucosa. There they process allergens into small peptides and then present them through the MHC class II molecules for recognition by naive T cell receptors. GM-CSF which is released from epithelial cells and immune cells in the presence of IL-4 and TNF-α, leads to DCs maturation to a fully competent as APCs. During initial allergen entering to airways to sensitize,

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Th2 lymphocyte differentiation from naïve T cells requires IL-4 release. The cellular source of the IL-4 is still unclear. It is possible that early IL-4 is produced by basophils, mast cells, eosinophils, natural killer T cells and T cells [173]. In the presence of Th2 cells secreted cytokines IL-4 and IL-13 and co-stimulatory molecules (CD40 with CD40 ligand, and CD80 or CD86 with CD28), B cells start to produce IgE. Whereas some of the Th cells make their way to the B-cell follicle to facilitate immunoglobulin class switching from IgM to IgE, others move back to the airway mucosa to elicit the classical Th2 response through the secretion of the proallergic cytokines. Pattern-recognition receptors have a crucial adjuvant role in directing allergen sensitization. There is some knowledge that basophils and mast cells also can produce IL-4 and/or IL-13, and can stimulate B cells through CD40. IgE diffuses locally and enters the lymphatic vessels. It subsequently enters the blood and is then distributed systemically. Then IgE binds to the FcεRI on tissue-resident mast cells, thereby sensitizing them to respond when the host is later re-exposed to the allergen [173, 174]. Antigen sensitization was previously thought to occur primarily in lymphoid germinal centers, but IgE-producing B cells that undergo clonal selection and affinity maturation also can be generated in the respiratory mucosa [175]. It is worth to say that sensitization does not produce any symptoms. In an allergic person, whose tissue mast cells and basophils already have antigen specific IgE bound to FcεRI, re-exposure to the original or a cross-reactive bivalent or multivalent antigen results in the cross-linking of adjacent FcεRI-bound IgE and the consequent aggregation of surface FcεRI. When the FcεRI aggregation is of sufficient strength and duration, it triggers mast cells and basophils to initiate complex signaling events that ultimately result in the secretion of a diverse group of biologically active products [176].

Human mast cells are divided into two major subtypes based on the presence of tryptase or tryptase and mast cell-specific chymase [177]. Activated by allergen mast cells secrete many different mediators after re-exposure to allergen. Mast cells mediators are packaged within secretory granules. After the stimuli the madiators such as histamine, proteases and proteoglycans are released extracellulary within minutes. It is known that histamine causes airway smooth muscle contraction by having direct effect on smooth muscle cells and by stimulating nerve endings also increases venule permeability and mucus secretion [178]. The function of tryptase in vivo is unknown, but in vitro it can cleave complement C3 and C3a, fibrinogen, pro-matrix metalloprotease-3 etc. Also tryptase can activate

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fibroblasts, promote accumulation of inflammatory cells, and potentiate histamine-induced airway bronchoconstriction. Chymase has a procollagen proteinase activity and is probably directly toxic to the airway cells as well it might stimulate mucus production in the bronchi. Mast cells activated through FcεRI rapidly synthesize eicosanoid mediators from endogenous membrane arachidonic acid stores (for example, LTB4 which is chemoatractant for neutrophils and effector for T-cells; cysteinyl leukotrienes and PGD2 which are potent bronchoconstrictors and chemoattractants for eosinophils and/or basophils) [178, 179]. TNF-α is a major cytokine produced by mast cells; it upregulates endothelial and epithelial adhesion molecules and increases BHR. In murine asthma model TNF-α contributes significantly to allergic inflammation and BHR, possibly by enhancing lymphocyte recruitment and Th2 cytokine production. Higher TNF-α concentration was found in exhaled breath condensate from asthmatics compared to healthy controls [4, 180]. Other cytokines produced by mast cells include IL-3, GM-CSF, and IL-5, which are critical for eosinophil development and survival; and IL-6, IL-10 and IL-13. Moreover, they produce chemokines, such as CC-chemokine ligand (CCL) 3 (macrophage inflammatory protein (MIP)-1 α) which has the ability to stimulate histamine release from mast cells and basophils also CCL2 (mo-nocyte chemotactic protein (MCP)-1), and CCL11 (eotaxin-1), which are involved in leukocyte attraction during inflammation [179, 181, 182]. The release of preformed and lipid-derived mediators contributes to the acute signs and symptoms associated with early-phase reaction. These signs and symptoms vary according to the site of the reaction but can include vasodilation (in part reflecting the action of mediators on local nerves, and producing erythema (reddening) of the skin or conjunctiva), markedly increased vascular permeability (leading to tissue swelling and, in the eyes, tear formation), contraction of bronchial smooth muscle (producing airflow obstruction and wheezing), and increased secretion of mucus (exacerbating airflow obstruction in the lower airways and producing a runny nose). Such mediators can also stimulate nociceptors of sensory nerves resulting in sneezing, itching or coughing.

Basophils may be involved in early phase of allergen induced airway inflammation too. They share many features with mast cells, including expression of FcεR1, secretion of Th2 cytokines, metachromatic staining, and release of histamine after activation. Although basophils have functions similar to mast cells, recent work has highlighted the unique functions of basophils and their role in allergic responses and immune regulation [183,

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184]. Basophils have fewer but larger granules compared to mast cells. Unlike mast cells, basophils have little proliferative capacity. Basophils express a variety of cytokine receptors (e.g. IL-3R, IL-5R, and GM-CSFR), chemokine receptors (CCR2 and CCR3), complement receptors (CD11b, CD11c, CD35, and CD88), prostaglandin receptors (CRTH2), immunoglobulin Fc receptors (FcεRI and FcγRIIb), and TLRs [178]. The major preformed mediator in storage granules of basophils is histamine. Another mediator is heparin and tryptase but they have them less than mast cell granules. After allergen re-exposure basophils may rapidly produce LTC4 and highly peptidolytic LTD4 also LTE4. Also they can produce cytokines such as IL-4, IL-13 and GM-CSF [178, 184].

1.4.2. Late-phase of allergen induced airway inflammation

Late-phase of allergen induced inflammation is usually characterized by excessive inflammation of the airways resulting in structural changes induced by various mediators derived from inflammatory cells (especially from eosinophils and T cells) [169, 185]. Therefore late-phase allergen-induced airway inflammation consists of an increase in airway eosinophils, basophils and less consistently, neutrophils. These responses are mediated by the trafficking and activation of DC into the airways, probably as a result of the release of epithelial cell-derived TSLP, and the release of pro-inflammatory cytokines from Th2 cells. Late phase of allergen induced inflammation is thought to reflect the actions of innate and adaptive immune cells that have been recruited from the circulation, as well as the secretion of inflammatory mediators by tissue-resident cells (the innate immune cells include eosinophils, neutrophils, monocytes and basophils. Other cells that secrete inflammatory mediators include activated mast cells and tissue-resident or recruited T cells). In chronic allergic inflammation repetitive or persistent exposure to allergens has several effects. Innate immune cells including eosinophils, basophils, neutrophils and monocytes/macrophages as well as adaptive immune cells (such as Th2 cells, other types of T cells, and B cells) take up residence in the tissues. More mast cells develop in the tissue more IgE bound to FcεRI and greater mast cell activation occurs. Many interactions are initiated between recruited and tissue-resident innate and adaptive immune cells, epithelial cells and structural cells (such as fibroblasts, myofibroblasts and airway smooth muscle cells) and blood vessels and lymphatic vessels, and nerves [174].

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Eosinophils are the central effector cells in ongoing late-phase airway inflammation. They can be primed by a number of factors, including IL-3, IL-5, GM-CSF, CC-chemokines and platelet activating factor (PAF), resulting in inflammatory mediator release [178] such as major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN) and eosinophil peroxidase (EP). All reliesed mediators results in damage of airway endothelial cells, intracellular matrix and neurons. Actually it is proved that exposure of nerve endings to airway lumina by these active eosinophil derived mediators may result in BHR, airway smooth muscle cell contraction and increased vascular permeability to specific allergens, infection agents or other irritants. It leads to the recruitment of more eosinophils and Th2 cells to the airways [179, 186]. Eosinophils are also a major source of cys-LTs, particularly LTC4 as a potent pro-inflammatory mediator, bronchoconstrictor, and inducer of mucus secretion. Some studies showed increased cys-LTs levels in BAL from atopic asthmatic after allergen challenge during late-phase allergen-induced inflammation that correlated with eosinophil count. Also increased ECP and GM-CSF levels were observed [187]. Several cytokines are released by eosinophils, including some that are stimulatory to eosinophil proliferation and enhance their adhesion to endothelium, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, CXCL8, IL-10, IL-11, IL-12, GM-CSF, CCL3 and CCL5, eotaxin (CCL11), TNF-α, TGF-β, TGF-α. They may also promote eosinophil recruitment and activation through autocrine mechanisms. Eosinophils contribute to airway remodeling, possibly through the ela-boration of TGF-β, through mucus production and through hyperreactivity [188, 189]. Eosinophil level in sputum is associated with the degree of chronic airway obstruction in asthma [190]. The data from the studies where bronchial biopsies were performed after allergic asthmatics bronchial aller-gen challenge show increased level of activated T lymphocytes, eosinophils and increased mRNA expression in IL-5 and GM-CSF. These findings suggest the idea that cytokines/chemokines possibly released by activated T lymphocytes may contribute to local eosinophilic accumulation during late-phase allergen-induced inflammation [191].

Th lymphocytes (mostly Th2) have been identified as another important factor for the development of late-phase allergen-induced inflammation in asthma [192]. Nevertheless co-operative interaction between cytokines produced by Th2 and Th1 cells is also very important. After specific stimuli Th2 cells start to secrete IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13 and GM-CSF. Many of those cytokines have a wide range of inflammatory effects.

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CCL1, CCL11 (eotaxin), CCL17 (thymus and activation-regulated chemokine (TARC)) and CCL22 (macrophage-derived chemokine (MDC)) are Th2 cell-specific chemokines. They further amplify the recruitment of CD4+ Th2 cells, thereby generating a self-sustaining pro-inflammatory cycle [193]. Th1 cells secrete inflammatory cytokines, such as IFN-γ, IL-2, IL-12, IL-18, TNF-α, and TNF-β [4].

Other new Th cells participating in late-phase allergen-induced inflammation in asthma belong to the Th17 lineage (CD4+ T cells producing IL-17 family; IL-17A, IL-17F, IL-22). A decade ago it was discovered that asthmatic individuals, especially those poorly responding to steroid treatment, show airway infiltrations primarily composed of neutrophils. Neutrophils are probably recruited to the airways by IL-17-producing cells. IL-17 induces the expression of a wide range of cytokines and chemokines that have a positive impact on neutrophil recruitment and exacerbation of airway inflammation [6]. Recently the researchers from our university worked on Th17 cells. They challenged allergic asthma (AA) patients with D. pteronyssinus allergen and found that peripheral blood Th17 cells numbers as well as serum IL-17 levels increases after allergen challenge in (AA) and allergic rhinitis (AR) patients [7]. The newest player in late-phase allergen-induced inflammation are Th9 cells. They describet in more detail in Chapter 1.4.3.

Beside eosinophils and neutrophils also have an important role in the late-phase allergen-induced inflammation. They can produce a wide range of products, including lipids (LTA4, LTB4, PAF, thromboxane (TX) A2), cytokines (IL-6, TNF-α, TGF-β, CXCL8), proteases (elastase, collagenase, matrix metalloproteinase (MMP) 9), microbicidal products (lactoferrin, myeloperoxidase, lysozyme), reactive oxygen intermediates (superoxide, hydrogen peroxide, OH−) and nitric oxide (NO). Neutrophil products can cause airway narrowing, increased mucus secretion and increased BHR [197]. The schema of late-phase of allergen-induced inflammation in asthma is shown in Fig. 1.4.2.1.

There are some data that macrophages may play a dual role in allergic responses and inflammation in the airways. Allergen challenge induces their migration to the airway of allergic asthmatics. Macrophages secrete cytokines and chemokines, including IL-1, IFN-γ, TNF-α, IL-6, CCL2, CCL3, and CXCL8. All of them activate other inflammatory cells. Macrophages are known to produce bioactive lipids, PAFs, reactive oxygen species (ROS) and NO that affect vascular smooth muscle tone and bronchial epithelial cells. On the other hand macrophages may induce a

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reversible T cell non-responsiveness during local antigenic stimulation. They may also inhibit allergic inflammation by secreting PGE2 and IL-10 [198].

During late-phase allergen-induced inflammation approximately half of the patients with allergic asthma develop LAR. The recurrent bronchocons-triction usually occurs 2–6 h after allergen exposure and often peak after 6– 9 h. It is not clear enough why LAR do not develop in all sensitized asthmatics. It is known that in some patients there may be no obvious limit between the end of EAR and the beginning of the LAR. LAR is considered to be a better predictor of clinical asthma than the immediate reaction but the mechanisms underlying the LAR are much less clear than in EAR. Some studies present the possible prediction factors for LAR which are: larger wheal diameter in skin prick test [194], greater responsiveness to inhaled methacholine, lower FEV1 at the baseline and greater specific IgE levels [195]. The other major determinant of the LAR is the size of the early response: the greater is the degree of airway narrowing during the EAR, the more likely LAR will develop [169].

Nevertheless despite all existing data, the mechanisms of late-phase allergen-induced inflammation still remain unclear [199].

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Fig. 1.4.2.1. Late-phase of allergen induced airway inflammation

Adapteted according Galli SJ, Tsai M, Piliponsky AM, Nature, 2008 and Soroosh P, Doherty TA, Immunology 2011

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1.4.3. Th9 cells

In 2008 two independent researcher groups found a new CD4+ subset named Th9 cells. During the experiments they discovered that trans-forming growth factor-beta (TGF-β) in combination with IL-4 reprograms Th2 cells to lose their characteristic profile and switch to IL-9 secretion driving to the differentiation of Th-9 cells directly [8, 9]. Thus Th9 cells belong to the newest and less well characterized CD4+ subset. Different studies proved that Th9 cells express IL-9 but not the key signature cytokines of other T helper cell subsets, such as IL-4 (Th2 cells), IFN-γ (Th1 cells) and IL-17 (Th17 cells). Firstly experiments with mouse models showed that Th9 cells exist and also they may be involved in different allergic and other immune diseases. This gave an idea that Th9 cells may be found in humans and play a role also in human diseases. In mouse models of asthma (in which HDM extract, Aspergillus fumigatus or ovalbumin are used as sensitizers) Th9 cells are detectable in the respiratory tract and in the draining lymph nodes, particularly during the early stages of the disease [10]. Mouse models of atopic disease indicate that Th9 cells mediate disease through the production of IL-9. It is believed that Th9 cells promote mast cell and eosinophil accumulation, mucus production, Th2-type cytokine production and bronchial hyperresponsiveness [200, 201]. One recent study with mice showed that in vitro and in vivo generation of Th9 cells is coupled with the upregulation of functional chemokine receptors commonly associated with other effector Th subsets: CCR3 (Th2), CCR6 (Th17/Treg) and CXCR3 (Th1). During this study it was demonstrated that murine Th9 cells use CCR3 and CCR6 to go to allergic inflammation site, whereas migration of these cells to an autoimmune effector site is CCR6- and CXCR3-dependent [202]. Onother recent study with mice showed that after ovalbumin challenge mast cell accumulation in allergic inflammation site was promoted by Th9 cells but not by Th2 cells, i.e. was dependent on IL-9, but not on IL-13 [203].

Recent reports have revealed data that Th9 cells are very important in human atopic diseases. Different researchers found that patients with allergy have higher numbers of circulating T cells which secrete IL-9 in response to allergen (such as pollen, cat dander, peanuts or HDM extract) compare to control individuals. It was also shown that Th9 cells number and serum IL-9 levels correlated with allergen-specific IgE titers in individuals with atopy [10–12, 204, 205].

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Th9 cells also play important role in the pathogenesis of interstitial bowel diseases such as Crohns disease and ulcerative colitis. It is known that patients with Crohn disease are related with altered Th1 type immune response (associated with IFNγ and TNF production), whereas patients with ulcerative colitis have greater Th2 type response. In both diseases, increased Th17 cells numbers are presented. There are some data that Th9 cells and IL-9 directly alter epithelial cell biology within the gastrointestinal tract. The ability of Th9 cells to mediate colitis was shown to be IL-9 dependent and IL-9R expression is seen also elevated in gastrointestinal epithelial cells of ulcerative colitis patients. Negative effect of IL-9 on epithelial cell pro-liferation was from one study showing that that topical administration of recombinant IL-9 reduced epithelial cell tissue repair in vivo. The data also show that IL-9 may be associated with increased intestinal permeability which may be due IL-9 modified TJ protein composition in the layer of gastrointestinal epithelial cells [201, 206, 207].

Moreover Th9 cells may be important in immunity to helminthes. Th9 cells mediate anti-helmintic immunity through the local or systemic pro-duction of IL-9. Th9 cells were able to reduce Nippostrongylus brasiliensis (N. brasiliensis) worm burdens in Il9-mutant mice model. In that study it was shown that Th9 cells increase numbers of infiltrating eosinophils, basophils, mast cells also ILC2 numbers and activity [208]. During Trichuris muris and N. brasiliensis infections, IL-9 is required to destroy the worm infection elimination [209, 210]. Th9 cells are probably protective in certain helminth infections but not always. Still little is known about Th9 cells and IL-9 role in parasitic infections. In one study which included patients suffering from lymphatic filariasis Th9 cell number positively correlated with disease severity [211].

Th9 cells participate in tumor immune response. It has a potent anti-tumor activity, particularly in solid anti-tumors. Numbers of Th9 cells in the blood and the skin are significantly reduced in patients with melanoma compared with in healthy individuals. Through the administration of neutralizing antibody or recombinant protein, researchers have shown that IL-9 has potent anti-tumor effects in a mouse model of melanoma which is mast cell dependent [212, 213]. Th9 cells can directly induce tumor cell death or to limit tumor growth by production of cytokines [214]. It is known that IL-9 is strongly expressed in a subset of patients with anaplastic lymphoma and Hodgkin disease [215]. In vitro studies indicate that IL-9 promotes tumor growth by both enhancing proliferation and inhibiting apoptosis of tumor cells [216].

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Th9 cells participate in experimental autoimmune encephalomyelitis pathogenesis [217]. One very recent study showed that Th9 cells and IL-9 were detected in synovia of rheumatoid arthritis (RA) patients. Also IL-IL-9 was increased in diabetes mellitus patients. Th9 cell role in immune disea-ses still remains with some not answered questions and needs further investigation [218, 219].

1.4.4. The role of PU.1 and STAT6 in Th9 cell development

Differentiation of Th9 cells requires both TGF-β and IL-4 signals. IL-4 signaling activated STAT6 is required in the development of Th9 cells similar to its requirement in Th2 cells. The role of the other STAT mole-cules has not been examined. Although one report has demonstrated that downstream molecules of the TGF-β signaling pathway, Smad2 and Smad3 are probably not required for Il9 expression, another study has documented the requirement of Notch receptors and Smad3 signaling in the induction of Th9 cells [220, 221]. Chang et al. found that the transcription factor, PU.1, promotes the development of IL-9-secreting T cells. PU.1-deficient T cells exhibit attenuated IL-9 production, while ectopic expression of PU.1 in Th2 and Th9 cells can further induce IL-9 production [222]. PU.1 is able to act directly on Il9 gene as it can bind to the Il9 promoter in Th9 cells. PU.1 is also required for IL-9 production in human T cells as inhibiting PU.1 expression in human T cells is associated with diminished IL-9 production. The transcription factor IRF4, required for the development of Th2 and Th17 cells is also essential for Th9 cell development. IRF4-deficient CD4+ T cells display impaired Th9 cell differentiation [223]. STAT6 is the major signalling component of the IL-4 receptor (IL-4R) and is required for the in vitro generation of Th9 cells. IL-4 and STAT6 also have a crucial role in the induction of the Th9 cell developmental programme. Interestingly, the majority of the genes that were enriched in Th9 cells were STAT6 de-pendent. The role of STAT6 and PU.1 for Th9 development shown in Fig. 1.4.4.1.

THE ROLE OF TH9 CELLS AND EOSINOPHIL APOPTOSIS IN ALLERGIC ASTHMA

Deimantė Hoppenot