Andrius Januškevičius

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Doctoral Dissertation

Natural Sciences,

Biology (N 010)

Andrius Januškevičius

VIABILITY AND EFFECT OF

EOSINOPHILS ON PROLIFERATION

OF AIRWAY SMOOTH MUSCLE CELLS

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LITHUANIAN UNIVERSITY OF HEALTH SCIENCES

Andrius Januškevičius

VIABILITY AND EFFECT OF

EOSINOPHILS ON PROLIFERATION

OF AIRWAY SMOOTH MUSCLE CELLS

AND PULMONARY FIBROBLASTS

IN ASTHMA

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The Dissertation has been prepared at the Laboratory of Pulmonology of Department of Pulmonology of the Faculty of Medicine of Lithuanian University of Health Sciences during the period of 2016–2020.

Scientific Supervisor

Prof. Dr. Kęstutis Malakauskas (Lithuanian University of Health Sciences, Natural Sciences, Biology – N 010).

The Dissertation is defended at the Biology Research Council of the Lithuanian University of Health Sciences:

Chairperson

Prof. Dr. Vilmantė Borutaitė (Lithuanian University of Health Sciences, Natural Sciences, Biology – N 010).

Members:

Prof. Dr. Rasa Banienė (Lithuanian University of Health Sciences, Natural Sciences, Biology – N 010);

Assoc. Prof. Dr. Jurgita Skiecevičienė (Lithuanian University of Health Sciences, Natural Sciences, Biology – N 010);

Prof. Dr. Sonata Jarmalaitė (Vilnius University, Natural Sciences, Biology – N 010);

Prof. Dr. Peter Howarth (University of Southampton, Medical and Health Sciences, Medicine – M 001).

Dissertation will be defended at the open session of the Biology Research

Council of the Lithuanian University of Health Sciences at noon on the 22nd

of April, 2021 in the Great Auditorium at the Hospital of Lithuanian Univer-sity of Health Sciences Kauno Klinikos.

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LIETUVOS SVEIKATOS MOKSLŲ UNIVERSITETAS

Andrius Januškevičius

EOZINOFILŲ GYVYBINGUMAS

IR POVEIKIS BRONCHŲ LYGIŲJŲ

RAUMENŲ LĄSTELIŲ BEI PLAUČIŲ

FIBROBLASTŲ PROLIFERACIJAI

SERGANT ASTMA

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Disertacija rengta 2016-2020 metais Lietuvos sveikatos mokslų universiteto Medicinos fakulteto Pulmonologijos klinikos Pulmonologijos laboratorijoje. Mokslinis vadovas

Prof. dr. Kęstutis Malakauskas (Lietuvos sveikatos mokslų universitetas, gamtos mokslai, biologija – N 010).

Disertacija ginama Lietuvos sveikatos mokslų universiteto biologijos mokslo krypties taryboje:

Pirmininkė

prof. dr. Vilmantė Borutaitė (Lietuvos sveikatos mokslų universitetas, gamtos mokslai, biologija – N 010).

Nariai:

prof. dr. Rasa Banienė (Lietuvos sveikatos mokslų universitetas, gamtos mokslai, biologija – N 010);

doc. dr. Jurgita Skiecevičienė (Lietuvos sveikatos mokslų universitetas, gamtos mokslai, biologija – N 010);

prof. dr. Sonata Jarmalaitė (Vilniaus universitetas, gamtos mokslai, biolo-gija – N 010);

prof. dr. Peter Howarth (Sautamptono universitetas, medicinos ir sveikatos mokslai, medicina – M 001).

Disertacija ginama viešame biologijos mokslo krypties tarybos posėdyje 2021 m. balandžio 22 d. 12 val. Lietuvos sveikatos mokslų universiteto ligo-ninės Kauno klinikų Didžiojoje auditorijoje.

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ABBREVIATIONS

AA – allergic asthma

APC – allophycocyanin

ASM – airway smooth muscle

BSA – bovine serum albumin

CD – cluster of differentiation

CysLTs – cysteinyl leukotrienes

D. pteronyssinus – Dermatophagoides pteronyssinus

DMEM – Dulbecco’s modified Eagle’s medium

ECM – extracellular matrix

ECP – eosinophil cationic protein

EDN – an eosinophil-derived neurotoxin

EDTA – ethylenediaminetetraacetic acid

EPO – eosinophil peroxidase

FeNO – fractional exhaled nitric oxide

FEV₁ – forced expiratory volume in one second

FITC – fluorescein isothiocyanate

FVC – forced vital capacity

GINA – Global Initiative for Asthma

GM-CSF – granulocyte-macrophage colony-stimulating factor

GRADSP – Gly-Arg-Ala-Asp-Ser-Pro peptide

HS – healthy subjects

ICAM-1 – intercellular adhesion molecule-1

iEOS – inflammatory eosinophils

IFN-γ – interferon γ

Ig – immunoglobulin

IL – interleukin

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o-PD – o-phenylenediamine

PBS – phosphate-buffered saline

PCR – polymerase chain reaction

PDGF – platelet-derived growth factor

PSGL-1 – P-selectin glycoprotein ligand-1

rEOS – lung-resident eosinophils

RGDS – Arg-Gly-Asp-Ser peptide

RNA – ribonucleic acid

ROS – reactive oxygen species

SNEA – severe non-allergic eosinophilic asthma

T_1/2 – half-life

TGF-β₁ – transforming growth factor β₁

Th – T helper cells

TNF-α – tumor necrosis factor-α

TSLP – thymic stromal lymphopoietin

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INTRODUCTION

Asthma is a heterogeneous and complexive disease with imbalanced air-way tissue repair associated with airair-way inflammation and hyperresponsive-ness, leading to recurrent coughing episodes, wheezing, breathlesshyperresponsive-ness, and chest tightness [1]. The morbidity of asthma reached the epidemic level and got worse every year [2], leaving behind a substantial economic burden to the countries.

Chronic airway inflammation rich in eosinophils is a critical feature seen in asthma. Eosinophilia is associated with increased asthma exacerbations and more intense treatment [3, 4]. Eosinophils are in the bone marrow mat-urated granulocytes that circulate in the bloodstream and can be recruited to inflammation sites after immunological or inflammatory responses [5]. Eo-sinophils release a high amount of cytokines, growth factors, chemokines, and lipid mediators that affect pulmonary structural cells activity and disturb lung homeostasis [6].

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in-Without chronic inflammation, allergic and non-allergic phenotypes of asthma are characterized by structural changes in the lungs called airway re-modeling [24]. Airway rere-modeling in asthma includes neoangiogenesis, sub-epithelial and airway smooth muscle (ASM) thickening, sub-epithelial changes [25]. These changes develop after repetitive cycles of tissue injury and abnor-mal repair processes because of chronic inflammation. Airway remodeling develops mainly due to disturbed ASM cells and pulmonary fibroblast pro-liferation that determine the increase in tissue mass because of enhanced cell number and the release of the extracellular matrix (ECM) components [26]. Eosinophils might promote ASM cells and pulmonary fibroblast proliferation after producing pro-proliferative mediators, as transforming growth factor

β-1 (TGF-β₁) or cysteinyl leukotrienes [27, 28]. However, we hypothesized

that direct contact after stable adhesion through eosinophils integrins might also affect ASM cells and pulmonary fibroblasts proliferation. It could be affected due to direct signal transduction or increased eosinophils viability leading to a prolonged influence at inflammation sites. Moreover, recently was revealed the existence of two distinct eosinophils subtypes – one subtype is called lung‐resident eosinophils (rEOS), which maturate independently to IL-5 and have the primary function to maintain tissue homeostasis, and in-flammatory eosinophils (iEOS), which maturate in IL‐5‐dependent manner and are mainly involved in immune responses [29]. There is lack of informa-tion about biological differences of eosinophils subtypes, however, their dif-ferent localization in airways suggest about their distinct adhesive properties and adhesion-related survival.

Eosinophilic airway inflammation and its effect on airway remodeling re-ceives many scientists’ attention; however, there are still a lot of unanswered questions. Studies to find new therapeutic targets for inhibition of eosinophils quantity and activity are essential for eosinophilic asthma treatment. Further-more, there is a lack of information on how eosinophils’ adhesion affects their survival and how prolonged eosinophils’ viability could affect the develop-ment of airway remodeling.

The aim

To investigate the viability of eosinophils and their effect on airway smooth muscle cells and pulmonary fibroblast proliferation in asthma.

Objectives:

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2. To determine the adhesion-related eosinophils effect on airway smooth muscle cells and pulmonary fibroblasts proliferation and apoptosis; 3. To estimate the changes of airway smooth muscle cells proliferation

after the suppression of eosinophils integrins with Arg-Gly-Asp-Ser peptide;

4. To evaluate the viability of blood eosinophils subtypes in relation to their adhesion properties in non-severe allergic asthma and severe non-allergic eosinophilic asthma patients;

Research novelty

This study provides novel evidence on (1) enhanced adhesion of eosin-ophils in asthma and the importance of direct eosineosin-ophils interaction with pulmonary structural cells for their viability; (2) effect of eosinophil on ASM cells and pulmonary fibroblasts proliferation and apoptosis in relation to their adhesion properties; (3) the impact of eosinophils integrins suppression to their adhesion-related effect on ASM cells proliferation; (4) blood quantity of distinct eosinophils subtypes – rEOS and iEOS, and their adhesion-related viability properties during different asthma phenotypes.

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1. LITERATURE REVIEW

1.1. Asthma 1.1.1. Definition and epidemiology of asthma

Asthma is a heterogeneous disease usually characterized by chronic airway inflammation. It is defined by the history of respiratory symptoms, imbal-anced airway tissue repair associated with airway inflammation, and hyper-responsiveness, leading to recurrent coughing episodes, wheezing, breath-lessness, and chest tightness [1, 30]. Asthma is a major non-communicable disease worldwide and the most common type of disease among children. It is an incurable disease that lasts for the whole patients’ life, but the intensity of asthma symptoms and expiratory airflow limitation can vary over time.

World Health Organization counts that approximately 340 million people

are suffering from asthma [31]. By the data of 2016th_{, a total of 0.4 million}

deaths occurred due to asthma, and 24.8 million deaths were attributable to asthma at the global level. However, no present data are provided, but it is stat-ed that morbidity and mortality of asthma increase dramatically every year. It nearly reached the global epidemic level; however, it has been acknowledged as an epidemic in individual sections of societies and countries [2].

1.1.2. Asthma phenotypes and endotypes

The heterogeneity of the disease could be described according to distin-guished distinct asthma phenotypes and endotypes. A current phenotype and endotype manifest in the relation of an individual’s underlying genetics that may change in response to a new predominant environment. It is important to divide patients into subgroups because it may significantly affect the choice of diagnostic tests, long-term prognosis, and predict responsiveness to spe-cific pharmacotherapies. In recent years many different clinical subgroups of asthma have been characterized [31] based upon clinical, physiologic, and pathologic characteristics. However, there is no unified, continuously used classification. A coordinated global agreement is a priority, and would signif-icantly facilitate the disease’s concept, let easier predict its course, and select the most optimal treatment.

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mecha-nism) were identified as well. Used parameters for classifying asthma into the categories were based upon variables as patients’ atopic status, symptomatic triggers, patterns of airflow obstruction, and disease severity [32]. Three ex-tensive cluster analysis made in Europe and United States was the first which provided the basis for asthma classifications [33–35], according to the age of asthma onset, gender, allergic status, asthma symptoms, and lung function. Moreover, now for asthma identification, seven complex variables are used: clinical characteristics, biomarkers, lung physiology, genetics, histopatholo-gy, epidemiolohistopatholo-gy, and treatment response.

Based on the official Global Initiative for Asthma (GINA) recommenda-tions, asthma is divided into five main phenotypes: allergic asthma, non-aller-gic, adult-onset, asthma with persistent airflow limitation, and obesity-related asthma [31]. Allergic asthma phenotype is the most common – it includes almost all children cases and about 50% of adults. However, it usually occurs in early life, while non-allergic – later in life and is often distinguished by a more severe course of the disease [36]. After the extension of variables, asth-ma could be divided into more subgroups: endotypes – T helper 2 (Th2)-me-diated asthma (low/high) and non-Th2 asthma; phenotypes – eosinophilic, non-eosinophilic, early-onset, late-onset, aspirin-exacerbated respiratory dis-ease, steroid-insensitive, allergic bronchopulmonary mycosis, cross country skiing-induced asthma, exercise-induced asthma [32, 37–43].

1.1.3. Pathogenesis of asthma

There is no single precise mechanism describing the development of asth-ma. However, it is thought that the disease arises in the combination of ge-netics and environmental factors, as allergens and solid particles due to air pollution [44, 45]. The characteristics can vary among patients and be related to obesity, infectious respiratory diseases, constant exposure of particles in the air, and organism reaction into the non-steroid anti-inflammatory medi-cations [44].

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responses [47, 48]. Not sufficient maternal immune system adaptation could be one of the triggers for asthma development in the child.

The main asthma characteristic is chronic airway inflammation, basical-ly determined by Th2 responses. Earbasical-ly immune development is affected in children with asthma, which leads to the deficiency of released interferon γ (IFN-γ) and changes in innate immunity, which determines inclination to uncontrolled Th2 inflammation [49, 50]. Moreover, a detailed transcriptom-ic analysis of three distinct asthma phenotypes revealed the differently ex-pressed genes among phenotypes related to immune defense, inflammatory responses, responses to stimuli, wounding, and stress [51]. However, it is still unclear the proportion of the environmental and genetic factors that influence asthma development.

Many cells are involved in asthma pathogenesis, including mast cells, neu-trophils, T lymphocytes, macrophages, epithelial cells, and eosinophils. These cells produce inflammation mediators important in asthma pathogenesis, like histamine, leukotrienes, prostaglandins, and bradykinins [52]. Since the be-ginning of asthma research, Th2 inflammation is held in the leading position keeping asthma as the hallmark of Th2 disorder of the lungs. Nowadays, it is also the most studying part of the disease pathogenesis, as recently found that most asthma endotypes fall into the Th2-low and Th2-high inflammation clusters [53].

1.1.3.1. Airway remodeling in asthma

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Fig. 1.1. Changes in asthmatic airways

ASM – airway smooth muscle; ECM – extracellular matrix, (adapted from Prakash Y., [59]).

1.1.3.2. Airway smooth muscle cells in asthma

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prolifer-ASM cells are also a vital contributor to the ECM protein pool in the lungs – they can produce various ECM proteins contributing to the tissue structure and elasticity, which are unbalanced in asthma. ASM cells can produce a wide range of ECM components, as fibronectin, collagens, matrix metalloprotein-ases (MMP), and their tissue inhibitors. Moreover, they release pro- and anti-inflammatory cytokines, growth factors, and angiogenetic factors [59] (Fig. 1.2).

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Fig. 1.2. Airway smooth muscle remodeling

PPAR – peroxisome proliferator-activated receptor; TSLP – thymic stromal lymphopoietin; ET1 – endothelin 1; PGs – prostaglandins; MMP – matrix metalloproteinase; IL – interleu-kin; TIMP – tissue inhibitors of metalloproteinases; VEGF – vascular endothelial growth factor; CysLTs – cysteinyl leukotrienes; BDNF – brain-derived neurotrophic factor; Abl – nonreceptor tyrosine kinase; TGF-β – transforming growth factor β_l; Cav1 – caveolin 1, PPARγ – peroxisome proliferator-activated receptor gamma, (adapted from Prakash Y., [59]).

ASM cells can differentiate between phenotypes. The current phenotype’s dominance is related to environmental conditions, as cell-cell interaction, cy-tokines, growth factors, and mechanical strength. One of the main media-tors regulating differentiation to proliferative phenotype is platelet-derived growth factor (PDGF) and TGF-β, responsible for specific gene expression regulation through extracellular signal-regulated p70S6 kinase. Eosinophils are a significant source of PDGF and TGF-β and can contribute to the differ-entiation of ASM cells [68].

1.1.3.3. Pulmonary fibroblasts in asthma

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contractility, and cellular differentiation to myofibroblasts phenotype with an up-regulated rate of ECM production [70]. In turn, activated fibroblasts differentiate into the more active myofibroblasts phenotype and hyper-pro-duce ECM components, cytokines IL-1β, IL-33, chemotactic and inflamma-tory family cytokines (CXC), CC family chemokines [69], various types of MMPs as well as reactive oxygen species (ROS) [71, 72]. These mediators allow fibroblasts to contribute to the activation and recruitment of resident immune cells, including eosinophils, and endow fibroblast roles in chemical and cell-mediated immunity, acute and chronic inflammation, extravasation of immune cells into the connective tissue of the lungs [69].

While mostly fibroblasts determine ECM protein composition in the lungs, the ECM can affect the structural cell activity in lung tissue [73]. ECM re-modeling is also an important feature related to airway rere-modeling in asthma [74] and is closely associated with fibroblast differentiation into the activated myofibroblasts. ECM dysregulation is described as altered quantitative and qualitative ECM composition, changes in the activity of molecular signaling pathways that are responsible for triggered ECM production, variations in MMPs synthesis, increased oxidative stress [75]. ECM connects the airways and lung parenchyma. It plays a crucial role in maintaining pulmonary struc-ture and functions, affecting the adhesion and distribution of inflammatory cells, fluid balance, and elasticity. It can act as a reservoir for inflammatory mediators [74]. In asthma, predominant eosinophilic airway inflammation due to highly produced various mediators and ROS can result in ECM dys-regulation. Eosinophils are the most source of the TGF-β in airways that are confirmed to be essential for fibroblasts proliferation, differentiation, and ECM protein production [76].

1.2. Eosinophils in asthma 1.2.1. The biology of eosinophils

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Eosinophils are distinguished from other granulocytes (neutrophils and basophils) by their specific morphological features – bilobed nuclei and pink, about 12–17 μm in diameter cytoplasmic granules. It is the main distinctive feature, as no unique surface markers for eosinophils identification are found so far. Four main eosinophils cytotoxic cationic proteins are packed in gran-ules: major basic protein, an eosinophil-derived neurotoxin, eosinophil per-oxidase (EPO), eosinophil cationic protein (ECP) [81]. These proteins are the basis for maintaining eosinophils’ primary function – to participate in the host defense, including parasites, viruses, fungi, or bacteria. However, granules store other cytokines, enzymes, and growth factors, revealing their different part of the biological role [82].

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Fig.1.3. Cellular features of eosinophils

APRIL – a proliferation-inducing ligand; CCL – CC-chemokine ligand; CCR – CC-chemo-kine receptor; CXCL – CXC-chemoCC-chemo-kine ligand; CXCR – CXC-chemoCC-chemo-kine receptor; EGF – epidermal growth factor; EPX – eosinophil peroxidase; FPR1 – formyl peptide receptor 1; GM-CSF – granulocyte–macrophage colony-stimulating factor; IFN – interferon; IL – inter-leukin; MBP – major basic protein; NGF – nerve growth factor; NOD – nucleotide-binding oligomerization domain protein; PAR – proteinase-activated receptor; PDGF – platelet-de-rived growth factor; PIRB – paired immunoglobulin-like receptor B; PPARγ – peroxisome proliferator-activated receptor-γ; PRR – pattern-recognition receptor; PSGL1 – P-selectin glycoprotein ligand 1; RAGE – receptor for advanced glycation end-products; RIG-I – re-tinoic acid-inducible gene I; TGF – transforming growth factor; TLR – Toll-like receptor; TNF – tumour necrosis factor; SCF – stem cell factor; VEGF – vascular endothelial growth factor, (adapted from Rosenberg H.F et al., [82]).

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Fig. 1.4. Eosinophil-derived mediators and their functions

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Most of these mediators are packaged and stored in crystalline core-con-taining granules. Eosinophils-specific granules are important for eosinophil’s development and survival as well. It is indicated that impaired granules bio-genesis during eosinophils formation and not safe cationic proteins packaging into the granules are lethal for eosinophil’s survival [87–89]. Eosinophils can generate mediators based on transcriptional activation, de novo protein syn-thesis, or by releasing preformed proteins that do not need de novo transcrip-tion [90]. Several mediators, including IFN-γ, IL4, IL6, TNF-α, IL10, IL12 (p70), and IL13, are found to be stored in eosinophils granules as a preformed protein permanently [91]. Eosinophils-derived proteins are involved in sev-eral main processes: cell-mediated immunity (T cell activation and polariza-tion, recruitment of dendritic and T cells); humoral immunity (maintenance of bone marrow plasma cells and secretory immunoglobulin (Ig)A in the in-testine); tissue regeneration, repair, angiogenesis, and fibrosis (eosinophil-ic airway inflammation); metabol(eosinophil-ic homeostasis (adipose tissue); host pro-tection (cationic proteins production); steady-state development (mammary gland and intestine); cell-cell interaction (connection of immune and nervous systems) (Fig. 1.4).

1.2.2. Eosinophilic airway inflammation

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Fig. 1.5. Two different pathogenetic pathways of eosinophilic airway

inflammation in asthma

CRTH2 – chemoattractant receptor-homologous molecule expressed on TH2 cells; ALX/ FPR2 – receptor for lipoxin A4; FcεRI – high-affinity receptor for IgE; GATA3 – GATA-bin-ding protein 3; PGD2 – prostaglandin D2; RORα – retinoic acid receptor-related orphan receptor α, (adapted from Brusselle G.G. et al., [97]).

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TSLPR. In turn, activated ILC2 over-produce high amounts of 5 and IL-13 and causes severe eosinophilia, hypersecretion, and airway hyperreactivity [95, 96]. Insufficient non-allergic patients’ response to corticosteroids can be explained by a lower effect on ILC2 than TH2 cells.

IL-5 became the interest area for drug targets because increased levels of eosinophils in blood and airways were related to increased rates of asthma exacerbations [98, 99]. Eosinophil-based therapies trying to control the eosin-ophilia in asthma patients are based on anti-IL-5 targeted biological therapy. These biologics are monoclonal antibodies against IL-5 (mepolizumab, resli-zumab) or IL-5 α part of the receptor (benraliresli-zumab); however, the clinical trials with these medications achieved limited success. Even in studies with IL-5 deficient mice, there is reduced allergen-induced airway eosinophilia after absolute IL-5 depletion; however, there are still basal levels of eosin-ophils left in the blood. It was a perspective treatment, as IL-5 receptor-tar-geted treatment suppresses IL-5-related eosinophils activation and promotes natural killer cells to induce eosinophils apoptosis [100]. Anti-IL-5 treatment significantly affected the frequency of asthma exacerbations, airway eosino-philia, and usage of medications [99]. However, studies showed that for non phenotyped asthma patients who used the inhaled corticosteroids, inhibition of IL-5 pathways had limited effect.

1.2.3. Eosinophils adhesion properties

Recruitment of eosinophils to the site of inflammation and their accumula-tion in the airway wall and lumen is a critical step in asthma. Therefore, medi-ators that regulate eosinophil’s development and recruitment are perceived as appropriate targets for therapeutic ablation in asthma [101]. Moreover, there is evidence that eosinophils contribute to ASM remodeling through direct contact via integrin-ligand interaction [102]. The migration of eosinophils into the airways is related to both – the adhesion process and directed move-ment in the presence of locally generated chemotactic mediators. Several ag-onists are named that can modulate eosinophils recruitment into the lungs – CC chemokine family proteins (eotaxins, regulated on activation – normal

T cell expressed and secreted protein); complement factor 5; leukotrienes B₄

and C₄; lipid mediators, such as PDGF; prostaglandin D2 [103, 104].

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integ-rins, adhesion molecules P-Selectin, P-selectin glycoprotein ligand (PSGL-1), very late activation antigen, and VCAM-1 [107, 108]. After recruitment to the lung tissues, they can interact with resident cells, as airway smooth muscle cells, pulmonary fibroblasts, epithelial cells, or ECM proteins [108].

Integrins are dimeric transmembrane receptors that are the link for cell-cell and cell-cell–ECM interactions. Cell adhesion through integrins can trigger signal transduction and control cell growth, division, survival, cellular differ-entiation, and apoptosis [20, 109]. Seven integrin heterodimers expressed by eosinophils (α₄β₁, α₆β₁, α_Lβ₂, α_Mβ₂, α_Xβ₂, α_Dβ₂, and α₄β₇) interact with adhesion molecules, laminin, fibrinogen/fibrin, vitronectin, and periostin and medi-ate diverse functions, including eosinophil rolling, stable adhesion, migra-tion, respiratory bursts, degranulamigra-tion, and viability [21-23]. ASM cells and pulmonary fibroblasts express VCAM-1 and ICAM-1 adhesion molecules, which can act as ligands for eosinophils integrins. The adhesion ability of eosinophils in asthma patients might be different than those of healthy peo-ple. On this basis, the blocking of eosinophil surface integrins to suppress the impact of inflammation on airway structural cells and airway remodeling has emerged as a potential therapeutic approach for asthma [110].

1.2.4. Eosinophils viability-promoting factors

Mature eosinophils can not divide; thus, their count in peripheral blood and tissues depends on their release from the bone marrow and received sur-vival signals. Several anti-apoptotic factors exist for eosinophils viability, which acts in different locations – bone marrow, peripheral blood, and tis-sues. IL-5 is the most important and specific survival factor for eosinophils [111], but important mediators also include GM-CSF and IL-3 [11], TNF-α [12], leptin [13], CD40 engagement [14]. Moreover, there is evidence that p38 mitogen-activated protein kinase is important in regulating eosinophil’s survival [112, 113].

It is known that circulating eosinophils in steroid-free asthma patients demonstrate increased survival compared with healthy subjects. Moreover, apoptosis of eosinophils increased in the presence of steroid treatment to a similar level of eosinophils from healthy subjects [114, 115]. Allergen-in-duced late asthma responses are also related to prolonged eosinophil’s surviv-al and delayed apoptosis [116].

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eosino-tion, activaeosino-tion, survival, and apoptosis [118–121]. These cytokines receptors share the same β-chain, essential for signal transduction that explains these cytokines’ overlapping activities. The importance of these cytokines for eo-sinophil’s behavior during inflammation responses puts them first for new therapeutic approaches to asthma management.

It is known that combined culturing with pulmonary structural cells en-hances eosinophils viability [15, 16, 122, 123]; however, the precise mecha-nisms remain unknown, and there are no recent studies about direct adhesion effect on eosinophils survival. It is thought that integrins as transmembrane molecular mechanosensors may change their activation states under asthmat-ic conditions and transduce the signal through the cytoskeleton, thus regulat-ing eosinophil activity and viability [17, 18].

1.2.5. Eosinophils subtypes

Mature eosinophils are blood circulating cells, which migrate into the tar-get tissues after the appropriate stimulus, including the gastrointestinal tract, uterus, mammary gland, adipose tissue, or lungs [124]. They are related to many different disorders, which are interrelated with the severity of blood, tissues, and organs eosinophilia [125]. Eosinophils that residue the specif-ic tissue are called tissue-resident or homeostatspecif-ic eosinophils. Historspecif-ically eosinophils were described as a critical player in host defense [126, 127]; however, it became clear that steady-state eosinophils can contribute to im-munoregulation and tissue homeostasis as well [127-129].

Steady-state eosinophils half-life (T_1/2) is approximately 3-24 h in the cir-culation [8, 9, 130]. However, tissue-resident eosinophils prolong their viabil-ity, and their half-life is about 36 h in the lungs and six days in the intestine, thymus, or uterus [9] (Fig. 1.6). Studies show that increased eosinophils sur-vival is associated with CD11c expression; however, only intestine, uterus, and thymus tissue-resident eosinophils express CD11c, while lung and blood eosinophils not [9, 29]. At birth, eosinophils do not residue in the lungs, but their count gradually increases in time [71] that has a relation with the de-velopment of microbiota. It is seen only in airways, as in the gastrointestinal tract, where microbiota are most developed, tissue-resident eosinophils re-cruitment is independent of the microbiota.

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regulate their survival [133]. Tissue-resident eosinophils maturate in the bone marrow from eosinophils progenitor cells independently of IL-5, and it is their main difference from inflammatory (iEOS) eosinophils.

Exist differences in morphological and phenotypic features among tis-sue-resident eosinophils; however, most studies are done in mice. They can be characterized by the most typical features of eosinophils – specific gran-ules with cytotoxic cationic proteins, expression of CCR3, Singlec-F, CD125 [9, 82, 134] (Fig. 1.6). They also express CD11b (adipose tissue, thymus, in-testine), F4/80 (adipose tissue, mammary glands, and lungs), CD69 (thymus and intestine) [9, 29, 124, 135]. However, among tissues, they differ in T_1/2, the shape of the nucleus, and current homeostatic functions [124].

Fig. 1.6. Overview of the tissue-resident eosinophils

CD – cluster of differentiation; T_1/2 – half-life; IL – interleukin; Th17 – T-helper cell 17; Th2 – T-helper cell 2; IgA – immunoglobulin A; F4/80 – EGF-like module-containing mucin-like hormone receptor-mucin-like; AAMs – alternatively activated macrophages (adapted from Ma-richal T. et al., [136]).

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mature in an IL-5-dependent manner and are mainly involved in immune responses [137].

Blood iEOS infiltrate the airways mainly after the environmental stimulus like allergen and leave the airways with bronchial secretions. Furthermore, the existence of rEOS and iEOS in peripheral blood is confirmed, and pri-mary research for eosinophils subtypes surface markers was made according to the data of human peripheral blood eosinophils [29, 138]. It suggests that eosinophil differentiation occurs in the blood before migration into the lungs. rEOS reside in lung tissue for their entire lifetime regulating local immunity [137]. Peribronchial area was usually considered as eosinophils localization in asthma [139]; however, eosinophils subtypes differ by their locus – only iEOS are found peribronchial, while rEOS are localized in the lung parenchy-ma. In mice, the model was shown that blood rEOS quantity remains stable, while iEOS number increases after Dermatophagoides (D.) farinae-induced airway inflammation [137], indicating a different role of eosinophils subtypes in allergic conditions.

The ring-shaped nucleus presented by rEOS is usually considered as a sign of immaturity [140]. However, they are characterized by the whole fea-tures and functionality as mature eosinophils, including piecemeal degran-ulation and phagocytosis [29]. The only iEOS was named to be involved in the immune responses because of their localization, morphological, pheno-typic, and transcriptomic features changes during allergic airway inflamma-tion [29]. It suggests that iEOS and rEOS, as distinct eosinophils subtypes, represents a different biological role in asthma. Blood iEOS are recruited to the lung sites of inflammation during allergic responses and are defined as

segmented nucleus SiglecFhi_CD62L−_CD101hi_{cells, however in sputum}

sam-ples – Siglec-8+_CD62Llo_{IL-3Rhi cells. Steady-state blood rEOS are defined}

as round-shape Siglec-Fint_CD62L+_CD101lo_{cells, digested from lung tissue –}

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2. MATERIAL AND METHODS

2.1. Ethics statement

The study protocol was registered to the Kaunas Regional Biomedical Research Ethics Committee of the Lithuanian University of Health Scienc-es (Protocol no. BE-2-13). Each participant gave his/her written consent af-ter being informed about ongoing research. The study was regisaf-tered in the U.S National Institutes of Health trial registry. Trial registration: ClinicalTri-als.gov with identifier NCT03388359. The study subjects were recruited in 2014-2020th.

2.2. Study design and population 2.2.1. Study population and inclusion/exclusion criteria

The study population was composed of newly recruited, not studied in-dividuals. The study population consists of severe non-allergic eosinophil-ic asthma (SNEA) patients with high doses of inhaled steroids, steroid-free allergic asthma (AA) patients, and healthy subjects (HS) who comprised the control group. All participants were non-smokers. The participants were women and men between the ages of 18–75 years old. The patients were from the Department of Pulmonology at the Hospital of the Lithuanian University of Health Sciences Kaunas Clinics. A summary of the inclusion and exclusion criteria are provided in Fig. 2.1.

We included a total of 118 investigated subjects into the study completing all dissertation objectives: 46 AA patients, 28 patients with SNEA, and 44 healthy control subjects. For 21 AA group subjects and 19 HS subjects, the bronchial allergen challenge was performed.

AA group:

Inclusion criteria: the AA group was newly-established and untreated non-severe patients, approved with symptoms and medical history for at least 12 months, having a positive skin prick test to D. pteronyssinus allergen and positive bronchial challenge with methacholine.

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SNEA p atients (n = 28) SNEA p atients : -DVWKP D KLVWRU\ \H DU ; -QHJDWLYH VNLQ SULFN WHVW ; -SHULSKHUDO EORRG HRVLQRS KL O [ 9O ; -KLJK GRVHV oI LQKDOHG VWHURLGV + ORQJ -DFWLQJ EHWD DJRQLVW ± ORQJ -DFWL QJ DQWLP XVFDULQL F DJHQW ± HSLVRGL F XVH RI RUDO FRUWLFRVW HUR LG V AA patients : -DVWKP D V\ P SWRP V \H DU ; -non-VHYHU H FRXUVH RI WKH GLVHDVH ; -SRVLWL YH VNLQ SULFN WHVW WR D. pter onyssi nu s; -SRVLWL YH EURQFKL DO FKDOOHQJH Z LWK P HWKDFKROLQH HS : -QR FKURQLF UHVSLUDWR U\ DQG RWKHU GLVHDVHV -QHJDWLYH VNLQ SULFN WHVW -QHJDWLYH EURQFKL DO FKDOOHQJH Z LWK P HWKDFKROLQH In clu sion criteria Exclu si on criteria (for all gr oups ) -FOLQLFDO O\ VLJQLIL FDQW DOOHU J\ V\ P SWRP V; -DFWLYH DLUZ D\ LQIHFWLR Q m onth SULRU to VWXG\ ; -a st hm a H[DFHUEDWLRQ m onth SULRU to VWXG\ ; -XVH RI RUDO VWHURLG V P RQWK SULRU to VWXG\ ; -VP RNLQJ Scr eening visit (history and physical exam inati on ): -CBC -VSLURP HWU\ -P HWDFKROLQH FKDOOHQJH WHVW -VNLQ SULFN WHVW AA patients (n = 46) H S ( n = 44) Re cr ui tme nt : Fig. 2.1.

Exclusion and inclusion criteria of the study population

AA

– aller

gic asthma; HS – healthy subjects; SNEA

– severe non-aller

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SNEA group:

Inclusion criteria were asthma diagnosis for at least 1 year and non-al-lergic phenotype, approved by negative skin prick tests. Peripheral

eosin-ophil counts were ≥0.3 × 109_{/L during the screening visit or ≥0.15 × 10}9_/L

if there was a documented eosinophil count ≥0.3 × 109_{/L in the 12 months}

before the screening. A severe course of the disease was approved with at least a 12-month treatment of high doses of inhaled steroids combined with long-acting beta-agonist ± long-acting antimuscarinic agent ± episodic use oral corticosteroids.

Exclusion criteria: active airway infection 1 month before the study, sig-nificant permanent allergy symptoms, exacerbation ≤ 1 month before study, use of oral steroids ≤ 1 month before examination, and smoking, treatment with biological therapy.

Control group:

The control group consists of HS individuals without allergy and other chronic respiratory diseases, non-smokers.

2.2.2. Study design

SNEA patients were asked to visit the clinic for one time, AA patients and HS – twice (at baseline and 24 hours after bronchial allergen challenge). All study participants were invited into the study not earlier than three days but no later than two weeks after their inclusion and exclusion criteria were confirmed.

During the first visit, for all study subjects, peripheral blood was collected and measured for FeNO. Additionally, AA patients and HS underwent a bron-chial allergen challenge with D. pteronyssinus.

Collected peripheral blood was used for the granulocytes isolation. Gran-ulocytes were counted, and viability was assessed to perform the quality con-trol (viability at least 98% and total granulocytes count of >4 × 107_).

Fur-ther, the eosinophils were enriched from the granulocytes population and second quality control was completed. Samples that passed the requirements (count – >1.5 × 106_{/20 mL blood; viability – >98%; purity – >96%) were}

used for experiments (or eosinophils subtyping).

(33)

(34)

AA patients HS Gr ou p -&ROOHFWLRQ RISHULSKHUDO EORRG -S SLURPH WU\ -)H12 4 h ,V RO DW HG SHUL SKHUDO EORRGHRVLQRSKLOV SXULW\ YLDELOLW\ K 3UHSDUHG FRPELQHG FHOO FXOWXUH V ZLWK $ 60 FH OOV RU SXOPRQDU\ I LEUREODVWV K (RVLQRSKLOVDGKHVLRQ h $60 FH OOV SUROLIH UD WLRQ h F IRS T VIS IT (b ef or e b ro nc hial al lergen ch al le n ge) 6XSSUHVVLRQ of HRVLQRSKLOV LQWH JU LQV Z LWK R GDS or *5$'6 3 SHSWLGHV B S E COND VIS IT (AA an d HS gr oups) (24 af ter br onchial al lergen ch al le n ge) -&ROOHFWLRQ RISHULSKHUDO EORRG -S SLURPH WU\ -)H12 4h ,V RO DW HG SHUL SKHUDO EORRGHRVLQRSKLOV SXULW\ YLDELOLW\ 2h 6HSDUDWHG HRVLQRSKLOV VXEW\ SHV (FRQILUPDWLRQ E\ I ORZ F\ WRPH WH U) (YD OXD WLRQ of U(26 DQG L(26 qua ntity DQG YLD ELOLW\ 3UHSDUHG FRPELQHG FH OO FX OWXUH V ZLWK $ 60 FH OOV (RVLQRSKLOVYLDELOLW\ (RVLQRSKLOVDGKHVLRQ h h h -&ROOHFWLRQ RISHULSKHUDO EORRG -S SLURPH WU\ -)H12 4h ,V RO DW HG SHUL SKHUDO EORRGHRVLQRSKLOV SXULW\ YLDELOLW\ 3UHSDUHG FRPELQHG FHOO FX OWXUH V ZLWK $ 60 FH OOV (RVLQRSKLOVYLDELOLW\ F IRS T VIS IT 2h 6HSDUDWHG HRVLQRSKLOV VXEW\ SHV (F RQILUPD WLRQ E\ I ORZF \WRPH WH U) h (YD OXD WLRQ of U(26 DQG L(26 qua ntity DQG YLD ELOLW\ (RVLQRSKLOVDGKHVLRQ h % URQKLDO DOOHU JHQ FKDOOHQJ H ZLWK D. Pter onyssinus for $$ SDWLHQWV DQG HS h h en ts ie n ts roup K Fig. 2.2. Study design

ASM – airway smooth muscle; iEOS – inflammatory eosinophils; rEOS – lung-resident eosinophils; FeNO – exh

aled

– Gly-Ar

g-Ala-Asp-Ser

-Pro peptide; RGDS –

Ar

g-Gly-Asp-Ser peptide; HS – healthy subjects; SNEA

– severe no

n-al-ility and

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2.3. Clinical examination 2.3.1. Lung function testing

Forced vital capacity (FVC), baseline forced expiratory volume in one

sec-ond (FEV₁), and FEV₁/FVC ratio was recorded as the highest of three

repro-ducible measurements. The results were offset according to the predicted val-ues under the standard methodology based on age, gender, and body height. Pulmonary function was assessed by using a pneumotachometric spirometer “CustovitM” (Custo Med, Germany).

Airway responsiveness was registered using inhaled methacholine via pressure dosimeter (ProvoX, Niederlauer, Germany). Methacholine was in-haled at intervals of 2 min, starting with a 0.0101 mg dose and increasing it up to 0.121, 0.511, and 1.31 mg of the total cumulative amount was achieved,

or until a 20% decrease in FEV₁ from the baseline. The bronchoconstriction

effect of each methacholine dose was expressed as a percentage of reduction

in FEV₁ from the baseline value. The provocative amount of methacholine

causing a ≥20% fall in FEV₁ (PD_20M) was calculated from the log

dose-re-sponse curve by linear interpolation of two adjacent data points. 2.3.2. Skin prick testing

For all study participants, a skin prick test using standardized allergen ex-tracts (Stallergenes, S.A., France) was assessed by using the following aller-gens: Dermatophagoides farina, D. pteronyssinus, dog and cat dandruff, birch pollen, five mixed grass pollens, Alternaria, mugwort, Cladosporium and Aspergillus. The positive control was histamine hydrochloride (10 mg/mL), and diluent (saline) was a negative control. Skin testing was read 15 min after application. The skin prick test results were considered positive if the mean wheal diameter was higher than 3 mm.

2.3.3 Fractional exhaled nitric oxide measurement

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2.3.4. Bronchial allergen challenge test

For the bronchial allergen challenge, the inhaled D. Pteronyssinus allergen (DIATER, Spain) was used and performed via pressure dosimeter (ProvoX, Ganshorn Medizin Electronic, Niederlauer Germany). The allergen was in-haled every 10 min by histamine equivalent prick (HEP)/mL allergen concen-tration of 0.1, 1.0, 10.0, 20.0, 40.0, 60.0 HEP/mL, interrupting the procedure

after was achieved a 20% decrease in FEV₁ from the baseline. The

provoc-ative dose of allergen causing a ≥20% fall in FEV₁ (PD_20A) was calculated

from the log dose-response curve by linear interpolation of two adjacent data points.

2.3.5. Analysis of peripheral blood cells

Automated hematology analyzer XE-5000™ (Sysmex, Kobe, Japan) was used for a complete blood count test.

2.4. Experimental methods

2.4.1. Granulocyte isolation and eosinophil enrichment

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incubated with a biotin antibody cocktail (biotin-conjugated monoclonal an-tibodies against CD123, CD2, CD19, CD14, CD56, CD16, and CD235a (10

μL per 1 × 107_{cells) for 10 min according to manufacturer recommendations.}

After incubation, 20 μL of anti-biotin microbeads (microbeads conjugated

to monoclonal mouse anti-biotin immunoglobulin (Ig)-G1) per 1 × 107_cells

were added, mixed, and incubated for an additional 15 min at 4 °C. A large separation column (Miltenyi Biotec, USA) was prepared during this time by placing the column in the MACS separator’s magnetic field and moisten it with 2 mL of MACS buffer. The cells were then added to the pre-separation LS column, and the magnetically labeled non-eosinophils were retained on the column in the separator’s magnetic field. At the same time, the eosino-phils are unlabeled cells that passed through the column. Cells were eluted in 10 mL of MACS buffer and centrifuged at 300 × g for 10 min in RT. The pellet was resuspended in 5 mL of PBS. Eosinophils were counted using an ADAM automatic cell counter. To check eosinophil purity, a diluted eosin-ophil suspension was analyzed by flow cytometer FacsCalibur (BD, USA) according to forward and side light scattering whether there were any other cells in the suspension (Fig.2.3). May-Grunwald Giemsa staining was used as an internal control after using new isolation kits to measure eosinophil purity by light microscopy.

A B

Fig. 2.3. Eosinophil enrichment

(A) The fraction of peripheral blood granulocytes after centrifugation and erythrocyte lysis. (B) The fraction of peripheral blood eosinophils after magnetic separation. Collected cell number – 1 × 105_{. Eosinophil quantity expressed from total accumulated cell counts rejecting}

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2.4.2. Eosinophil subtyping

Eosinophils subtyping was performed by using magnetic beads conjugat-ed antibodies (Miltenyi Biotec, Somerville, USA) against CD62L, expressconjugat-ed only on rEOS surface, but not on iEOS [29, 68] and purity confirmed by labe-ling populations with allophycocyanin (APC) conjugated antibodies against CD62L and CD101 (Fig. 2.4). >96 % 99 % A B 98 % 99 % C D

Fig.2.4. Eosinophils subtypes

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USA). 20uL of CD62L-microbeads antibodies were added into the cells

sus-pension (per 1 × 107_{cells) and incubated for 15 min at 2–8°C. An additional}

2mL of MACS buffer was added, centrifuged for 10 min at RT at 300 × g force, and resuspended in 500 uL of a new MACS buffer. The cell suspension was filled on a magnetic column. Unlabeled cells passed through and were identified as iEOS, while labeled rEOS were collected by placing a column out of the magnetic field and adding 500 uL of buffer into the column. Isolat-ed eosinophils were countIsolat-ed, assessIsolat-ed their viability and quantity in periph-eral blood. rEOS purity was confirmed in up to 99% by CD62L, and 98% by CD101 expression, iEOS in up to 99% by CD101 expression. The man-ufacturer affirms that positive separation uses up to 10% of selected surface proteins and does not affect eosinophil activity.

2.4.3. Combined cultures of blood eosinophils and airway smooth muscle cells and pulmonary fibroblasts

Healthy immortalized human ASM cells with stable expression of human telomerase reverse transcriptase [141], and an MRC-5 (Sigma, Ronkonkoma, USA) lung fibroblast line was used for the experiments. The primary cell

lines were grown under standard conditions of 5% CO₂ in air at 37 °C with

medium renewal every 3 days.

Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO, Paisley, U.K.) were used for the cultivation of ASM cells, while minimum essential Ea-gle’s medium (EMEM) (GIBCO, Paisley, U.K.) for MRC-5 cell line. The growth mediums were supplemented with streptomycin/penicillin (2% v/v; Pen-Strep, GIBCO by Life Technologies, Paisley, U.K.), amphotericin B (1% v/v; GIBCO, Paisley, U.K.), and fetal bovine serum (10% v/v; GIBCO, Pais-ley, U.K). Cells were passaged by trypsinization after reaching sufficient con-fluence. Cells were serum-deprived in the antibiotics and insulin, transferrin, and selenium reagent (GIBCO, Paisley, U.K.) supplemented medium before each experiment to stop cell proliferation. The same lines of ASM and MRC-5 cells were used for whole investigating subjects. Preventing a decrease in cell activity and viability after repeated passage times, the mainline’s new cells were unfrozen every time after six passages.

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A B

0,44%

C

Fig. 2.5. Combined cell cultures of eosinophils and ASM cells

(A) The image through 10× objective. (B) The image through 40× objective. (C) Remaining eosinophil count after separation of combined cell culture (collected cell number – 1 × 105_).

Eosinophil number in percentage was expressed from the total accumulated cell count, rejec-ting cell debris, (adapted from Fig.3 in “The enhanced adhesion of eosinophils is associated with their prolonged viability and pro-proliferative effect in asthma”).

Cells were grown in six-well (16 × 104_{cells) or 24-well (4 × 10}4_cells)

culture plates (CytoOne, Brussels, Belgium.). Co-cultures with eosinophils

were made by adding 5 × 104_{or 1.25 × 10}4_{of eosinophils, respectively. To}

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used (10×/22 mm wide-field eyepiece, phase-contrast 10×/0.25 objective and an installed XM10-IR-2 camera (Olympus, Tokyo, Japan)).

2.4.4. Non-specific eosinophils integrins suppression

Whole eosinophils were separated into 3 groups depending on peptides: control eosinophils; eosinophils incubated with integrins suppressing peptide Arg-Gly-Asp-Ser; negative control – eosinophils incubated with integrins non-suppressing peptide Gly-Arg-Ala-Asp-Ser-Pro. The suspension with the required amount of eosinophils in the serum-free growth medium was taken, and the peptide solution was added to the final concentration of 0.125 mg/mL. Eosinophils with peptides were incubated for 1 h at 37°C. After incubation, the growth medium was removed, and eosinophils resuspended in a fresh serum-free growth medium.

2.4.5. Eosinophils adhesion assay

MRC-5 and ASM cells were cultivated for 3 days under the standard con-ditions in 24-well plates until it reached 4 × 104_{cells. 24 h before the}

exper-iments, the medium was rechanged into the serum-free, supplemented with 1% insulin-transferrin-selenium reagent and antibiotics. Eosinophil adhesion was measured after 1 h of co-culturing – a sufficient period for eosinophils to adhere [22]. After incubation, a medium with non-adhered eosinophils was removed, and culture wells were washed twice with warm PBS solution. Eo-sinophil adhesion was determined by measuring residual eoEo-sinophils perox-idase (EPO) activity as described [142]. Intercellular EPO levels decrease in eosinophils from asthmatic individuals due to degranulation [143]; therefore, it was normalized by measuring an EPO activity of fixed eosinophil count in each experiment. To measure EPO activity, 116 μL of phenol red-free DMEM medium and 116 μL of EPO substrate (1 mM o-phenylenediamine (o-PD);

0.1% Triton X-100 in Tris buffer pH 8.0; 1 mM H₂O₂) were added to each

well. To stop the reaction, 68 μL of 4 M H₂SO₄ was added to each well after

30 min of incubation at 37 °C, and the absorbance was measured at 490 nm wavelength by a microplate reader. Results were expressed as % of adhered

eosinophil number from total added. Added 1x eosinophil number–1.25 × 104_.

2.4.6. Cell viability assay

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Pulmonary fibroblasts and ASM cells were cultivated in 6-well plates until the confluency of 1.6 × 105_{cells. A co-culture was prepared in the serum-free}

growth medium or 2% (v/v) of investigated subjects’ blood

serum-supple-mented medium by adding 5 × 104_{of isolated eosinophils. After 24 h,}

ana-lyzed eosinophils from co-cultures and control eosinophils incubated alone at the same conditions were collected into 15 mL centrifuge tubes (Corning Inc., New York, USA). Then, pulmonary fibroblasts and ASM cells were detached by trypsinization, collected, and centrifuged at 400 × g for 10 min.

For the pulmonary structural cells viability assay, we used an Annexin V Apoptosis Detection Kit II (BD Bioscience, San Jose, USA) conjugated with fluorescein isothiocyanate (FITC). Before every experiment, we used further controls: unstained cells, cells stained with PI (no annexin V), cells stained with annexin V (no PI). Pulmonary fibroblasts and ASM cells without co-culturing with eosinophils were used as a control for the viability assay of structural cells. Eosinophil’s effect was compared with standard ASM and pulmonary fibroblast apoptosis in the serum-free, insulin-transferrin-seleni-um compound supplemented.

For the eosinophils viability assay, we normalized the data according to the data of growth medium from control structural cells without co-culturing with eosinophils to exclude the possible errors from cellular debris. Pulmo-nary structural cells and eosinophils significantly differ in granularity and size; therefore, appropriate gating on forward and side scattering excludes any remaining culture heterogeneity.

2.4.7. Proliferation assay

Pulmonary fibroblasts and ASM cells were grown in 24-well plates un-der the standard conditions described previously in fetal bovine serum-sup-plemented-supplemented growth medium until confluency of

approximate-ly 4 × 104_{cells/well. 24 h before the experiments, the growth medium was}

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percentage of Alamar blue conversion by pulmonary fibroblasts or ASM cells compared with control cells (without co-culturing with eosinophils), which did not proliferate during the culturing period because a serum-free growth

medium was used. Added eosinophils number – 1.25 × 104_{; 2x amount –}

2.50 × 104_{; 1/2x amount – 0.64 × 10}4_{. Used blood serum volume – 2% v/v.}

2.4.8. RNA isolation and RT-qPCR

ASM cells co-cultured with eosinophils and control ASM cells (cultured under the same conditions, but without eosinophils) were prepared for total ribonucleic acid (RNA) extraction by washing cells with warm PBS. Plates were gently smashed to detach the residual weakly adhered eosinophils.

According to the manufacturer’s instructions, total RNA was extract-ed with a miRNeasy mini kit (Qiagen, Valencia, CA). Reverse transcrip-tion-quantitative polymerase chain reaction (RT-qPCR) was performed

us-ing a Power SYBR® Green RNA-to-C_T™ 1-Step kit (Applied Biosystems,

Foster City, CA) in a 7500 Fast Real-Time PCR system. RT-qPCR was per-formed in these conditions: reverse transcription – 48°C, 30 min; activation of AmpliTaq Gold® DNA polymerase, UP (Ultra-Pure) – 95°C, 10 min; 40 cycles of denaturation – 95°C, 15 s; and annealing and extension – 60°C, 1 min. RT-qPCR data were analyzed using the comparative cycle threshold method (the target gene’s amount was normalized to 18S ribosomal RNA as

endogenous reference gene). A difference in 1 amplification cycle (C_t) after

normalization to the 18S ribosomal RNA indicates a 2-fold higher expression of the target gene. Relative differences were analyzed by normalization of test

sample ΔCt values to control sample ΔC_t values with the equation ΔC_t

(con-trol) – ΔC_t(test), and the differences were expressed by 2ΔΔCt _{value. Primers}

for analyzed genes are shown in Table 2.1.

Table 2.1. Sequences of primers for target genes

Primers: Forward 5′-3′ Reverse 5′-3′

α₄ GCTTCTCAGATCTGCTCGTG GTCACTTCCAACGAGGTTTG

α_M CAGACAGGAAGTAGCAGCTCCT CTGGTCATGTTGATGAAGGTGCT

18S CGCCGCTAGAGGTGAAATTC TTGGCAAATGCTTTCGCTC

β₁ GTGTGGCCCAAGACAGTTCT GGTTACCCCACCCTCTGACT

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2.4.9. Statistical analysis

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3. RESULTS

3.1. Eosinophil adhesion and viability 3.1.1. Characteristics of study subjects

A total of 39 nonsmoking adults (14 men and 25 women) were investi-gated: 10 severe non-allergic eosinophilic asthma (SNEA) patients, 14 ster-oid-free non-severe allergic asthma (AA) patients, and 15 healthy control subjects (HS). Study population demographic and clinical characteristics are shown in Table 3.1.

Table 3.1. Demographic and clinical data of the study population

SNEA Patients AA Patients Healthy Subjects Number, n 10 14 15 Sex, M/F 4/6 7/7 3/12 Age, years 46.5 ± 3.0 #,* _{28.0 ± 2.3} _{28.4 ± 1.8} BMI, kg/m2 _{26.9 ± 2.2} _{24.7 ± 1.6} _{23.2 ± 1.2} PD_20M,geometric mean (range), mg ND 0.08 (0.02–0.26) NR PD_20A, geometric mean

(range), HEP/mL ND 0.84 (0.15–3.04) NR IgE, IU/mL 147.2 ± 30.7 * _{202.8 ± 41.9}* _{29.5 ± 6.0}

FEV₁, L 2.0 ± 0.4 #,* _{3.9 ± 0.2} _{3.9 ± 0.18}

FEV₁, % of predicted 57.6 ± 8.6 #,* _{95.9 ± 3.3} _{106.6 ± 3.7}

Blood eosinophil count,

×109_/L 0.52 ± 0.11 #,* 0.36 ± 0.07 * 0.18 ± 0.02

FeNO, ppb 44.9 ± 10.7 * _{59.5 ± 11.5}* _{12.9 ± 1.5}

SNEA – severe non-allergic eosinophilic asthma; AA – allergic asthma; FEV₁ – forced expi-ratory volume in 1s; F – female; M – male; PD20A – the D. pteronyssinus allergen provo-cation dose causing a 20% decrease in FEV₁; PD_20M – the provocation dose of methacholine causing a 20% decrease in FEV₁; FeNO – fractional exhaled nitric oxide; BMI – body mass index; NR – not responded; ND – not done; IgE – immunoglobulin E. Data presented as the mean ± standard error of the mean; PD_20M and PD_20A are provided as geometric mean (range).

#_{p < 0.05 compared with AA group;}*_{p < 0.05 compared with HS group.}

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3.1.2. Eosinophils adhesion to pulmonary fibroblasts and airway smooth muscle cells

Our data present that 71.7% ± 3.5% of AA patients’ and 66.6% ± 5.8% of SNEA patients’ eosinophils adhere in co-culture with ASM cells after 1 h of incubation, and this count was significantly increased compared to HS (47.2% ± 3.7%, p < 0.05) (Fig. 3.1A). After supplementing the co-culture growth medium with the blood serum of the investigated individuals’ was re-ceived decreased adhered eosinophils ratio to 46.7% ± 7.9% of the added eo-sinophils count only in the SNEA group, p < 0.05, without any effect in other groups. Furthermore, adding double the number of eosinophils in the culture well, we received a significant decrease in AA and SNEA groups adhered eosinophils ratio respectively to 53.8% ± 5.0% and 50.5% ± 6.4% of the to-tal added eosinophil, p < 0.05. It does not affect the HS group (Fig.3.1A). Resembling results were received by measuring eosinophil adhesion to pul-monary fibroblasts–61.2% ± 4.6% of eosinophils number adhered in the AA group, and 37.3% ± 3.7% in the HS group, p < 0.05. Supplementing the me-dium with blood serum had no significant effect in both groups. Finally, the adhered eosinophil ratio decreased to 43.8% ± 7.4% in the AA group after using double the amount of added eosinophils (p < 0.05) (Fig. 3.1B).

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0 10 20 30 40 50 60 70 80 90 100 _{p < 0.05} p < 0.05 _{p < 0.05}

AA patients HS SNEA patients AA patients HS

AA patients HS SNEA patients

Time (minutes) * * _* 0 10 20 30 40 50 60 70 80 * * 0.0 0.1 0.2 0.3 p = 0.067 p = 0.072 Eosinophils in serum-free medium Eosinophils in serum-supplemented medium 2x of added eosinophils in serum-free medium 30 45 60 120 240 0 20 40 60 80 100 120 140 Adhered EOS number (% of total added) Adhered EOS number (% of total added)

Oxidation of o -phenylendi amine by eosinophils perroxidase (Abs, 492nm) Oxidation of o -phenylendi amine by eosinophils perroxidase (Abs, 492nm) A C D B

Fig. 3.1. Eosinophil adhesion in co-culture with ASM cells and pulmonary

fibroblasts

(A) Eosinophil adhesion to ASM cells. (B) Eosinophil adhesion to pulmonary fibroblasts. (C) EPO substrate activity of 12,500 eosinophils. (D) Asthmatic eosinophils adhesion after different incubation periods with ASM cells. AA – allergic asthma, HS – healthy subjects, SNEA – severe non-allergic eosinophilic asthma, EOS – eosinophils. Results from indepen-dent experiments including AA – n = 14; HS – n = 15; SNEA – n = 10. *_{p < 0.05 compared}

with the eosinophils adhesion of the HS group. Added blood serum: 2% of v/v. Statistical analysis: within one study group – Wilcoxon matched-pairs signed-rank two-sided test, be-tween investigated groups – Mann–Whitney two-sided U-test, (adapted from Fig.4 in “The enhanced adhesion of eosinophils is associated with their prolonged viability and pro-pro-liferative effect in asthma” and Fig.3 in “Eosinophils enhance WNT-5a and TGF-β₁ genes expression in airway smooth muscle cells and promote their proliferation by increased extra-cellular matrix proteins production in asthma”).

We performed the bronchial challenge with D. pteronysinnus allergen for 22 study subjects – 11 subjects from the AA patient group and 11 subjects from the HS group. The effect of in vivo eosinophil activation was determined by comparing the results 24 h after a bronchial allergen challenge to the results at baseline. After bronchial allergen challenge significantly increased the

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to 0.45 ± 0.06 × 109_{/L, while it does not affect HS group eosinophils count.}

Bronchial allergen challenge increased eosinophils adhesion only in the AA group, without an effect on HS eosinophils. Adhered eosinophils number in co-cultures with ASM cells increased from 69.5% ± 5.4% to 87.1% ± 3.1%, while in co-cultures with pulmonary fibroblasts from 59.4% ± 4.3% to 76.2% ± 4.2% of the total added eosinophil count (p < 0.05) (Fig. 3.2).

0 10 20 30 40 50 60 70 80 90 100 * * V1 V2 V1 V2 V1 V2 V1 V2 0 10 20 30 40 50 60 70 80 90 100 * * p < 0.05 p < 0.05 AA patients HS AA patients HS

Adhered EOS number (% of total added) Adhered EOS number (% of total added)

A B

Fig. 3.2. The effect of bronchial allergen challenge on the eosinophil

adhesion

(A) Eosinophils adhesion to ASM cells. (B) Eosinophils adhesion to pulmonary fibroblasts. Results from independent experiments including AA – n = 11, HS – n = 11; AA – allergic asthma, HS – healthy subjects, EOS – eosinophils. *_{p < 0.05 compared with the eosinophils}

adhesion of the HS group. V1 – visit 1 (at baseline); V2 – visit 2 (24 h after bronchial al-lergen challenge). Statistical analysis: within one study group – Wilcoxon matched-pairs signed-rank two-sided test, between investigated groups – Mann–Whitney two-sided U-test, (adapted from Fig.8 in “The enhanced adhesion of eosinophils is associated with their pro-longed viability and pro-proliferative effect in asthma”).

3.1.3. Eosinophils adhesion effect on the viability of airway smooth muscle cells and pulmonary fibroblasts

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0 60 65 70 75 80 85 90 95 100 * # * * * * * # * * * p < 0.05 p < 0.05 p < 0.05

AA patients SNEA patients HS

Eosinophils in serum-free medium

Viable eosinophils number (% after 24 h from isolation)

Eosinophils in serum-supplemented medium Eosinophils + ASM cells in serum-free medium

Eosinophils + ASM cells in serum-supplemented medium Eosinophils + pulmonary fibroblasts in serum-free medium Eosinophils + pulmonary fibroblasts in

serum-supplemented medium

Fig. 3.3. Eosinophil viability

Results from independent experiments including AA – n = 14, SNEA – n = 10, HS – n = 15. AA – allergic asthma, HS – healthy subjects, SNEA – severe non-allergic eosinophilic asth-ma. *_{p < 0.05 compared with the HS group eosinophils viability,}#_{p < 0.05 compared with}

the eosinophils viability of the SNEA eosinophil group. Added blood serum – 2% of v/v. Statistical analysis: within one study group –Wilcoxon matched-pairs signed-rank two-sided test, between investigated groups – Mann–Whitney two-sided U-test, (adapted from Fig.5 in “The enhanced adhesion of eosinophils is associated with their prolonged viability and pro-proliferative effect in asthma”).

(50)

Activated eosinophils in vivo after bronchial allergen challenge positively affected AA patients’ eosinophils viability – the number of non-viable eo-sinophils decreased by 7.6% ± 1.8% if eoeo-sinophils were incubated alone in serum-free growth medium (p < 0.005) but did not affect healthy eosinophils. However, using in vivo allergen-activated eosinophils obtained from the HS group, a positive effect was seen if we used serum-supplemented growth me-dium – non-viable eosinophils number decreased by 6.3% ± 1.8%, p < 0.01. However, serum, collected after the bronchial allergen challenge did not affect the AA patient group more, compared to baseline. Furthermore, AA patients’ allergen-activated eosinophil viability was increased more if they were co-cultured with pulmonary structural cells under standard conditions – in co-culture with ASM cells, non-viable eosinophils’ number decreased by 7.6% ± 2.7% and in co-culture with pulmonary fibroblasts by 8.3% ± 2.1%, p < 0.01. (Fig. 3.4). –4 –2 0 2 4 6 8 10 12 &* &* & & &* & &

Eosinophils in serum-free medium Eosinophils in serum-supplemented medium Eosinophils + ASM cells in serum-free medium

Eosinophils + ASM cells in serum-supplemented medium Eosinophils + pulmonary fibroblasts in serum-free medium Eosinophils + pulmonary fibroblasts in

serum-supplemented medium

AA patients HS

Decrease in non-viable eosinophils number

(%, comparing with eosinophils

before allergene challenge)

Fig. 3.4 Bronchial allergen challenge effect on eosinophils viability Results from independent experiments including AA – n = 11, HS – n = 11. HS – healthy su-bjects, AA – allergic asthma. *_{p < 0.05 compared with the HS group’s eosinophils viability;}

&_{p < 0.05 compared with the eosinophils viability before the bronchial allergen challenge.}

Andrius Januškevičius

Doctoral Dissertation

Natural Sciences,

Biology (N 010)

Andrius Januškevičius

VIABILITY AND EFFECT OF

EOSINOPHILS ON PROLIFERATION

OF AIRWAY SMOOTH MUSCLE CELLS

Andrius Januškevičius

VIABILITY AND EFFECT OF

EOSINOPHILS ON PROLIFERATION

OF AIRWAY SMOOTH MUSCLE CELLS

AND PULMONARY FIBROBLASTS

IN ASTHMA

Andrius Januškevičius

EOZINOFILŲ GYVYBINGUMAS

IR POVEIKIS BRONCHŲ LYGIŲJŲ

RAUMENŲ LĄSTELIŲ BEI PLAUČIŲ

FIBROBLASTŲ PROLIFERACIJAI

SERGANT ASTMA

CONTENTS

ABBREVIATIONS

INTRODUCTION

1. LITERATURE REVIEW

2. MATERIAL AND METHODS

3. RESULTS