Genetics of Inflammatory Mediators of Atopy and Asthma in Adults

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Genetics of Inflammatory Mediators of Atopy and

Asthma in Adults

KATI ÅDJERS

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Tampere University Dissertations 441

KATI ÅDJERS

Genetics of Inflammatory Mediators of Atopy and Asthma in Adults

ACADEMIC DISSERTATION To be presented, with the permission of

the Faculty Council of the Faculty of Medicine and Health Technology of Tampere University,

for public discussion in the Jarmo Visakorpi auditorium of the ARVO building, Arvo Ylpön katu 34, Tampere,

on 13 August 2021, at 12 o’clock.

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ACADEMIC DISSERTATION

Tampere University, Faculty of Medicine and Health Technology Finland

Responsible

supervisor Professor (emeritus) Mikko Hurme Tampere University

Finland

Supervisor Docent Jussi Karjalainen Tampere University Finland

Pre-examiners Docent Terttu Harju University of Oulu Finland

Docent Varpu Elenius University of Turku Finland

Opponent Professor Johannes Savolainen University of Turku

Finland

Custos Professor Olli Silvennoinen Tampere University Finland

The originality of this thesis has been checked using the Turnitin OriginalityCheck service.

Cover design: Roihu Inc.

ISBN 978-952-03-2028-7 (print) ISBN 978-952-03-2029-4 (pdf) ISSN 2489-9860 (print) ISSN 2490-0028 (pdf)

http://urn.fi/URN:ISBN:978-952-03-2029-4 PunaMusta Oy – Yliopistopaino

Joensuu 2021

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To my family

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ACKNOWLEDGEMENTS

This study was carried out at the Department of Microbiology and Immunology of the Medical School of the University of Tampere, Finland.

I owe a debt of gratitude to my supervisor Professor Mikko Hurme for granting me the opportunity to prepare this dissertation. The elaboration and implementation of this project would have been impossible without him.

I want to express my most profound gratitude to Docent Jussi Karjalainen, also my supervisor, for friendly guidance, patience, and the learning opportunities he has provided me. I would never have completed this project without him and cannot thank him enough!

I want to thank Professor Olli Silvennoinen for his valuable contribution. I also express my thanks to Professors Heikki Hyöty and Hannu Kankaanranta for their collaboration.

I am most grateful to the official reviewers of this thesis, Docents Terttu Harju and Varpu Elenius. Thank you for your invaluable time. Your comments and concerns have improved this thesis immeasurably.

I also thank Docent Sanna Toppila-Salmi, Docent Tanja Pessi, Miia Virta, PhD, Carita Eklund, PhD, and all the co-writers of the studies for their valuable contributions. I also express my sincere thanks to Heini Huhtala, MSc, for her guidance in statistical matters. Further, I thank Ms Sinikka Repo-Koskinen and Ms Eija Spåre or their work in the laboratory at the Department of Microbiology and Immunology.

My sincere thanks go to all colleagues and friends at the Department of Ophthalmology of Tampere University Hospital and other locations for encouraging me to complete this dissertation.

I also want to thank Carolyn Brimley Norris, PhD, for language revision of my thesis and for patiently teaching me academic writing.

I am happy to thank my mother Eija and father Jukka for their endless support.

I also want to thank our beloved daughters Emilia and Matilda for bringing joy and happiness to my life during these years. I finally express my deepest gratitude to my husband, Pasi Ylitepsa, MD, for his love, patience, and support.

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This study received financial support from the Finnish Medical Society Duodecim, the Finnish Society of Allergology and Immunology, the Jane and Aatos Erkko Foundation, the Finnish Cultural Foundation, the Tampere Tuberculosis Foundation, the Väinö and Laina Kivi Foundation, and the Yrjö Jahnsson Foundation, the Finnish Anti-Tuberculosis Association Foundation, the Ida Montin Foundation, the Research Fund of Tampere University Hospital, the Academy of Finland, and the Rehabilitation Fund of the Finnish Social Insurance Institution.

Pirkkala, May 2021

Kati Ådjers

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ABSTRACT

The prevalence of atopic disorders such as asthma, allergic rhinitis and allergic conjunctivitis in many populations is close to 30% and has continued to increase in many developed countries, causing a major burden on the individuals, their health care systems, and on society. The biological bases of these disorders and of atopy in general have undergone investigation to a great extent, but many aspects still need clarification, for example in order to target novel therapies to patients who would benefit.

Our studies examined whether functional single nucleotide polymorphisms of candidate genes IL1A, IL4RA, TLR4, IL4, and NOS3 exert their effects individually on the risk or severity of atopy or asthma, or whether two polymorphisms of different candidate genes show an interactive effect. The aim was also to observe factors associated with polymorphism rs20541 of IL13 and other factors with or without allergic comorbidities such as subject-reported allergic rhinitis and allergic conjunctivitis symptoms in adult asthma patients.

A total of 1,156 asthma patients and 1,792 non-asthmatic subjects as controls participated in our studies, all of them having participated in a Finnish population- based case-control study conducted to investigate the risk factors and predictors of the outcome of adult asthma. Inclusion criteria for subjects with asthma were age over 30 years and entitlement to special reimbursement for asthma medication from the Social Insurance Institution of Finland. Allergic rhinitis and conjunctivitis were defined by questionnaire. Of the participants 245 asthma patients and 405 matched control subjects were tested for atopic status by skin prick tests and by measurement of serum IgE levels, and we also determined variety of polymorphisms of candidate genes previously linked to atopy or asthma.

We demonstrated in our non-asthma control group, which represented the general population reasonably well, an epistatic effect on atopy between IL1A and IL4RA genes. The predisposing combination was the carrier status of genotype TT of IL4RA +22446 and genotype GG of IL1A +4845. An increased risk for asthma appeared in the female carriers of allele G of TLR4 +896 and allele T of IL4 -590.

Additionally, an epistatic effect of allele T of NOS3 +894 and genotype GG of IL1A +4845 influenced degree of atopy. We also showed that allele A of IL13 rs20541

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affected the risk for allergic rhinitis and conjunctivitis in asthma patients. In addition, carriers of allele A of IL13 rs20541 were predisposed towards multisensitization in asthma.

Our findings suggest that gene-gene interactions affect susceptibility to atopy and asthma as well as severity of atopy in Finnish adults. Even if single nucleotide polymorphisms of candidate genes show no individual effects, two separate polymorphisms can potentiate the effects of each other, predisposing carriers of certain allele combinations to clinical manifestations. An effect of TLR and IL4 genes was apparent only in female subjects, indicating sex-dependent differences in the body’s defense mechanisms. Our results indicate that key features of adult atopic asthma phenotype may be high prevalence of the allele A of IL13 rs20541, a multisensitization pattern, and allergic rhinitis and conjunctivitis symptoms.

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TIIVISTELMÄ

Atooppisten sairauksien kuten astman, allergisen nuhan tai sidekalvotulehduksen esiintyvyys on monissa väestöissä jopa 30 % ja ainakin kehittyvissä maissa edelleen lisääntymässä. Kyseisistä sairauksista aiheutuu sekä yksilölle että yhteiskunnalle merkittäviä haittoja ja kustannuksia. Sairauksien genetiikkaa, immunologiaa, patofysiologiaa ja ympäristötekijöitä on tutkittu laajalti, mutta edelleen lisää tietoa tarvitaan, jotta osataan esimerkiksi kohdentaa uusia hoitomuotoja niistä eniten hyötyville.

Tutkimuksissamme selvitimme, vaikuttavatko funktionaalisten kandidaattigeenien IL1A, IL4RA, TLR4, IL4 ja NOS3 yhden nukleotidin polymorfismit joko yksin tai yhdessä atopiataipumukseen, atopian vaikeusasteeseen tai riskiin sairastua astmaan. Tavoitteena oli myös selvittää, liittyvätkö IL13 geenin polymorfismi rs20541 ja allerginen nuha sekä allerginen sidekalvotulehdus aikuisiän astmaan.

Tutkimuksiin osallistui 1156 iältään yli 30-vuotiasta astmaa sairastavaa ja 1792 kaltaistettua vertailuhenkilöä. Astmakriteerinä käytettiin Kelan myöntämää astmalääkkeiden erityiskorvausoikeutta. Allergisen nuhan ja sidekalvotulehduksen esiintyminen selvitettiin oirekyselyllä. Perinnöllisten tekijöiden vaikutusta selvitettiin kliinisesti tutkittujen 245 astmapotilaan ja 405 kaltaistetun vertailuhenkilön väestöpohjaisessa otoksessa. Tutkituille tehtiin ihopistotestit ja määritettiin seerumin IgE-tasot. Genotyypitykset aiempien tutkimusten mukaan mahdollisista astman tai atopian riskigeeneistä tehtiin aikaisemmin kuvattuja menetelmiä käyttäen.

Tutkimuksissamme selvisi, että melko hyvin normaaliväestöä edustavien vertailuhenkilöiden IL1A ja IL4RA geeneillä oli yhteisvaikutus (ns. epistaattinen vaikutus) atopiariskiin. IL1A-geenin polymorfismin +4845 genotyypin GG ja IL4RA-geenin polymorfismin +22446 genotyypin TT todettiin altistavan atopialle.

Naisilla havaittiin vastaavasti astmariskin lisääntyminen TLR4-geenin polymorfismin +896 alleelin G ja IL4-geenin polymorfismin -590 alleelin T suhteen. NOS3-geenin polymorfismin +894 alleeli T ja IL1A polymorfismin +4845 genotyyppi GG vaikuttivat vastaavalla tavalla atopian vaikeusastetta lisäten. Totesimme myös IL13- geenin polymorfismin rs20541 alleelin A lisäävän allergisen nuhan ja

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sidekalvotulehduksen riskiä astmapotilailla. Näillä potilailla myös esiintyi herkistymistä useammille allergeeneille ihopistokokeissa.

Löydöksemme suomalaisilla aikuisilla astmapotilailla tukevat ajatusta, että eri geenit vaikuttavat yhdessä atopiaan ja astmaan. Vaikka yksittäinen geeni ei vaikuttaisi taudille altistavasti, kahden eri geenin polymorfismit voivat vahvistaa toistensa vaikutusta ja altistaa molempien riskialleelien kantajat taudille. TLR4- ja IL4-geenien yhteisvaikutus havaittiin vain naisilla, mikä vahvistaa löydöksiä sukupuoliriippuvaisista eroista elimistön puolustusmekanismeissa. Tutkimuksemme mukaan IL13 geenin polymorfismin rs20541 alleeli A, herkkyys useille allergeeneille ihopistokokeissa sekä allergisen nuhan ja sidekalvotulehduksen oirekuva saattavat olla tärkeitä aikuisten atooppisen astman fenotyyppiin liittyviä tekijöitä.

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ABBREVIATIONS

AC Allergic conjunctivitis

AD Atopic dermatitis

AKC Atopic keratoconjunctivitis

AR Allergic conjunctivitis

ARC Allergic rhinoconjunctivitis CD Cluster of differentiation

DNA Deoxyribonucleic acid

eNOS Endothelial nitric oxide synthase FeNO Fraction of exhaled nitric oxide

FEV1 Forced expiratory volume in one second GWAS Genome-wide association scan

IFN Interferon

Ig Immunoglobulin

IL Interleukin

IL4R Interleukin 4 receptor

ILC Innate lymphoid cells

ILL Innate-like lymphocytes

LAR Local allergic rhinitis

LPS Lipopolysaccharide

NERD NSAID-exacerbated respiratory disease

NO Nitric oxide

NOS Nitric oxide synthase

NP Nasal polyposis

PAC Perennial allergic conjunctivitis

PCR Polymerase chain reaction

PEF Peak expiratory flow

SAC Seasonal allergic conjunctivitis

SPT Skin prick test

Th T helper

TLR Toll-like receptor

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TNF Tumor necrosis factor

Treg Regulatory T cell

TSLP Thymic stromal lymphopoietin VKC Vernal keratoconjunctivitis

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ORIGINAL PUBLICATIONS

The thesis is based on the following original publications, which are referred in the text by the Roman numerals I-IV.

Publication I Ådjers, K., Pessi, T., Karjalainen, J., Huhtala, H., & Hurme, M.

(2004). Epistatic effect of IL1A and IL4RA genes on the risk of atopy. Journal of allergy and clinical immunology, 113(3), 445–447.

Publication II Ådjers, K., Karjalainen, J., Pessi, T., Eklund, C., & Hurme, M.

(2005). Epistatic effect of TLR4 and IL4 genes on the risk of asthma in females. International archives of allergy and immunology, 138(3), 251–

256.

Publication III Pessi, T., Ådjers, K., Karjalainen, J., Rontu, R., Hurme, M. (2006) The interaction of IL1A and endothelial nitric oxide synthase polymorphisms is associated with the degree of atopy. Journal of allergy and clinical immunology, 118(1), 246-8.

Publication IV Ådjers, K., Luukkainen A., Pekkanen, J., Hurme, M., Huhtala H., Renkonen R., Wang, D., Mäkelä, M., Karjalainen J., Toppila-Salmi, S. (2017) Self-reported allergic rhinitis and/or allergic conjunctivitis associate with IL13 rs20541 polymorphism in Finnish adult asthma patients. International archives of allergy and immunology, 172(2), 123-128.

The original publications are reprinted with the permission of the copyright holders.

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AUTHOR’S CONTRIBUTION

I Kati Ådjers has performed the statistical analyses, interpreted the results, and written the manuscript.

II Kati Ådjers has participated in planning and performing the laboratory examinations, performed the statistical analyses, interpreted the results, and written the manuscript.

III Kati Ådjers has performed the statistical analyses, interpreted the results, and participated in writing the manuscript.

IV Kati Ådjers has participated in planning the study, performing some of the statistical analyses, interpreting results and writing the manuscript.

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1 INTRODUCTION

The prevalence of atopic disorders, underlying bronchial asthma, allergic rhinitis (AR), allergic conjunctivitis (AC) and atopic dermatitis (AD), is close to 30 % in many populations and continues to increase in many developed countries, causing for those affected and their society a major burden. Determinants of atopic disorders are genetic and environmental factors. (Cookson, 1999; Rosenwasser, 1996) The gene–environment relationship can be captured in the phrase “genetics loads the gun and the environment pulls the trigger” (Turner, 2017). The genetics, immunology, pathomechanisms and environmental effects of atopic disorders and atopy in general have been under considerable investigation, with much left unknown. In addition, most of the studies have been performed in children with not all the phenotypes considered.

An interesting approach to learning about the mechanisms of allergic diseases involves searching for associations between the diseases and the functional polymorphisms of various genes, especially the ones coding for molecules involved in immune responses. Genome-wide association scans (GWAS) compare the DNA of individuals with the disease to DNA of individuals without it in order to discover which genetic variants are associated with the disease. Several GWASs on asthma, AR, atopy, allergy, and IgE have appeared. (Welter et al., 2014). GWASs do not, however, identify the causal variants associated with a disease, and the estimated cumulative genetic risk of the variants identified with asthma alone has been under 15% (Mathias, 2014).

Despite the large number of candidate genes identified for asthma by GWAS and basic research, few of those discoveries have been rigorously replicated. Several candidate genes examined have still failed later replication. This may be due to a variety of endotypes of asthma.

In this dissertation, we investigated the effects of certain candidate genes previously associated with asthma or atopy in a study group of Finnish adult asthma patients and non-asthmatic control subjects. We examined the polymorphisms of Interleukin (IL) 1A, Interleukin 4 receptor (IL4R) A, Toll-like receptor (TLR) 4, IL4

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and endothelial nitric oxide synthase (eNOS, NOS3) and their interactions in atopy and asthma because of these genes’ biological importance. Factors associated with polymorphism rs20541 of IL13 and other factors with or without allergic comorbidities such as subject-reported AR and AC symptoms we also observed in adult asthma patients.

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2 REVIEW OF THE LITERATURE

2.1 Atopy

2.1.1 Definition

Atopy can be defined (according to Joint Allergy Academies for ICD-11) as a personal or familial tendency to become sensitized and produce Immunoglobulin (Ig)E antibodies in response to ordinary exposures to allergens (Tanno et al., 2016).

The skin prick test (SPT) serves as a means to examine IgE-mediated allergic responses, and the results are quite well in accordance with anamnestic data on atopy (Burrows et al., 1989).

On many occasions, terminology is ambiguous, with the words “atopy” and

“allergy” incorrectly used as synonyms. Allergy is a hypersensitivity reaction initiated by proven or strongly suspected immunologic mechanisms; it can be IgE-mediated or non-IgE-mediated and triggered by substances to which the subject has been exposed and sensitized (Tanno et al., 2016).

Atopic disorders typically affect the nose, eyes, skin, and lungs. These include allergic asthma, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, IgE- mediated drug allergy, IgE-mediated insect bites, urticaria and angioedema, and anaphylactic shock (Justiz Vaillant et al., 2020).

2.1.2 Prevalence

The prevalence of atopic diseases such as eczema and hay fever, determined by the symptoms, is almost 30% in many populations in spite of the use of self-reported diagnosis of different definitions (Asher et al., 2006; Barnish et al., 2015). Until recently, the prevalence of asthma, AR, and AD in many developed countries has been increasing (de Marco et al., 2012; Duggan et al., 2012; Gershon et al., 2010;

Hansen et al., 2013). Hygiene level and socioeconomic conditions have improved simultaneously, alongside a decrease in the incidence of several infectious diseases

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and biodiversity and an increase in the consumption of fossil fuels (Bach, 2002;

Hanski et al., 2012; Shafiee et al., 2009).

An association of adult-onset asthma and allergic multimorbidity decreases with age: the proportion of allergic multimorbidity and allergic polysensitization among asthma patients increases with later decades of birth. More studies are essential to investigate whether this results from a cohort effect, i.e. change in host-microbiome- environmental interactions during development over time, or from an aging effect.

(Lynch et al., 2016; Toppila-Salmi et al., 2019).

2.1.3 Pathogenesis and phenotypes

Research had already generated theories linking autoimmune disease and hygiene, but in 1989, Strachan proposed a theory, the “hygiene hypothesis,” based on studies of the relationship between hay fever and microbial infections in early childhood and adolescence (Blackley, 1959; Leibowitz et al., 1966; Strachan, 1989). Infectious diseases, especially gastrointestinal infections, during early childhood have reduced the risk of development of atopic diseases in later life. Conversely, the absence of infectious diseases increases risk. This has been assumed to explain the rapid increase in atopic diseases occurring in developed countries during recent decades. (Wills- Karp et al., 2001). The hygiene hypothesis holds that decreased biodiversity and improved hygiene in early life reduce microbial exposures essential in priming the immune response and protective against atopic disorders. This hypothesis has been expanded to cover asthma and autoimmune diseases. (Hanski et al., 2012; Okada et al., 2010).

At cellular level, T helper (Th)1–Th2 deviation has served to explain the protective influence of infectious agents of immunological disorders. These Th cells comprise two subsets, Th1 and Th2 based on the cytokine pattern they produce (Romagnani et al., 1997). Th1 T cells produce inflammatory cytokines such as IL-2, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α; all function in cell-mediated immunity, whereas Th2 T cells that produce IL-4, IL-5, IL-6, and IL-13 contribute to IgE production and allergic responses. Differentiation of Th cells is under the influence of factors derived from infectious agents, factors such as bacterial lipopolysaccharide (LPS), and by the cytokine profile induced. The presence of these factors favors differentiation towards the Th1 direction; the absence of any stimuli, towards the Th2 direction. (Perussia et al., 2003). Considering the reciprocal down- regulation of Th1 and Th2 cells, the lack of microbial burden in early childhood in

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developed countries, normally favoring a Th1-biased immunity, redirects the immune response towards the Th2 phenotype. As a consequence, this predisposes the host to allergy. (Okada et al., 2010).

These observations are reasonably well in accordance with the concept of a common mechanisms underlying microbiome-mediated protection against allergy and autoimmunity. Many hypotheses can explain the aspects of these mechanisms, including antigenic competition, immunoregulation, nonantigenic ligands, and gene- environment interactions. (Okada et al., 2010).

Today, immune responses are classified into type 1, type 2, and type 3 immunity, which refer to both the innate and the adaptive arms of the immune response. Type 1 effector responses can be defined by Th1 and Th17 cells, cytotoxic T cells, innate lymphoid cells (ILCs) 1 and 3, and IgM, IgA and specific IgG antibody classes. These mediate immunity to many microorganisms such as bacteria, viruses, fungi, and protozoa and help to maintain tumor immune surveillance. (Annunziato et al., 2015).

On the other hand, type 2 immune responses include CD4+ Th2 cells, ILC2s, eosinophils, basophils, mast cells, IL-4- and IL-13-activated macrophages, IgE, and IL-4, IL-5, IL-9, IL-13, thymic stromal lymphopoietin (TSLP), IL-25 and IL-33.

Type 2 immunity protects against extracellular parasites by promoting barrier defences, helps to maintain metabolic homeostasis, and enhances tissue remodeling following injury. (Gause et al., 2013; Heredia et al., 2013; Nguyen et al., 2011; Urban et al., 1998; Wu et al., 2011; Wynn, 2004; Wynn et al., 2013). Although type 2 responses have important functions protecting the host, when dysregulated, chronic, or hyperreactive, they can contribute to the development of diseases, such as allergic disorders (Palm et al., 2012).

Type 3 immunity seems to protect from extracellular bacteria and fungi. Type 3 immune responses consist of RORgt+ lymphocytes, CD4+ Th17 cells, CD8+ TC17 cells, ILC3s, IL-17 and IL-22. (Annunziato et al., 2015).

Recently, what has become clear is that the commensal microbes colonizing the gut, lung, and skin mediate regulatory effects on type 2 immune responses. In the absence of microbes, germ-free mice present with increased type 2 immune responses with elevated levels of IL-4, circulating basophils, serum IgE, and with higher susceptibility to allergy. (Cahenzli et al., 2013; Herbst et al., 2011; Hill et al., 2012; Mccoy et al., 2006). Cellular and molecular pathways are poorly known.

Regulatory T cells (Tregs) seem to dampen the induction of type 2 immunity.

They are characterized by their capacity to secrete both IL-10 and transforming growth factor β (Chen et al., 1994). Tregs suppress the allergen-induced specific T cell activation and effector cells included in allergic inflammation, ones such as mast

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cells, basophils, and eosinophils, and Tregs also inhibit IgE production (Akdis et al., 1998; Jutel et al., 2008; Meiler et al., 2008; Verhagen et al., 2006). The induction of IL-10-producing allergen-specific Tregs that express transcription factor FOXP3 seems to be the most important mechanism of allergen-specific immunotherapy. It plays an important role in the peripheral T cell allergy development against specific allergens that occurs during the traditional subcutaneous route of allergen immunotherapy (Akdis et al., 1998; Francis et al., 2003; Jutel et al., 2003; Savolainen et al., 2004). Synthesis of Treg-type cytokines such as IL-10 also occurs during sublingual immunotherapy (Bohle et al., 2007; Burastero et al., 2008; Cosmi et al., 2006; Savolainen et al., 2006).

Microbial colonization induces intestinal Tregs (Atarashi et al., 2013; Geuking et al., 2011; Lathrop et al., 2011). This happens through production of the short-chain fatty acids butyrate and propionate (Arpaia et al., 2013; Furusawa et al., 2013; Smith et al., 2013). Mice deficient in the FOXP3 enhancer conserved noncoding sequence 1 generate no microbially induced Tregs. As a consequence, they develop increased Type 2 responses and mucosal inflammation in the gastrointestinal tract and the lungs. (Josefowicz et al., 2012).

Microbial control of type 2 immune responses seems also to involve other cell types like epithelial cells, dendritic cells, ILCs, and other innate cell types. The amount of ILC2 in the lungs of germ-free and of colonized mice seems to be equal, indicating that their recruitment may not require microbial signals. (Monticelli et al., 2011) Instead, microbial control of intestinal macrophages and ILC3 plays a role in the induction of Tregs and is required to induce tolerance and to control reactivity to food antigens (Mortha et al., 2014).

Several experiments indicate that infectious agents are able to protect against allergic diseases through mechanisms independent of their constitutive antigens.

This leads to stimulation of receptors that are not antigen specific. TLRs can illustrate this reasonably well. Considering the capacity of TLRs to stimulate cytokine production and immune responses, it can be assumed that stimulating them by infectious ligands would cause or exacerbate allergic responses. (Lang et al., 2005;

Okada et al., 2010; Schaub et al., 2004).

For a long time, allergic diseases and their associated clinical traits have been recognized as being heritable. Twin and migration studies performed on families provide the earliest evidence for genetic contributions. (Coca et al., 1923; Davis et al., 1961; Duffy et al., 1990; Manolio et al., 2003; Palmer et al., 2000). Twin studies of eczema and hay fever suggest that hereditary factors explain up to 80% of these atopic conditions (Thomsen et al., 2006, 2007). Genetic factors play a major role in

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predisposition to atopy. They regulate the total IgE synthesis and produce IgE antibodies to specific epitopes. The tendency to overproduce IgE depends on inheritance of several genes. However, the full inheritance pattern is known to be multigenic. (Qi et al., 2018).

Atopic diseases sometimes develop as an atopic march. This gradually development describes the progression of atopic disorders in children from AD in infants to AR and asthma later in childhood (Spergel et al., 2003). Some patients may have long-lasting disease whereas others may improve as they age (Spergel, 2010).

The risk for developing atopic diseases is complex, and the pattern described in the atopic march is not always a simple progression; it applies to only some patients and has been under investigation predominantly in childhood asthma. Although these disorders share risk factors, the development of disease varies among individuals depending on genetics and environment (Cookson, 1999; Rosenwasser, 1996).

2.2 Asthma

2.2.1 Definition

Asthma is a heterogeneous disease commonly characterized by chronic airway inflammation. It is defined by the history of respiratory symptoms underlying wheeze, shortness of breath, chest tightness, and cough that vary over time and in intensity, together with variable expiratory airflow limitation. This definition has been based on consideration of the characteristics that are typical of asthma before the commencement of controller treatment. These features distinguish it from other respiratory conditions. Nevertheless, one possibility is that airflow limitation becomes persistent later on in the course of the disease. (Global Initiative for Asthma, 2020).

2.2.2 Prevalence

Asthma is a chronic respiratory disease affecting 1% to 18% of the population and all age groups among countries. Its prevalence is still increasing. Some countries have, however, seen a decline in asthma hospitalizations and deaths (Haahtela et al., 2017). In Finland, the prevalence of asthma in adults has been estimated to be around 10% (Jousilahti et al., 2016). Asthma still imposes an unacceptable burden on

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individuals, on health care systems, and on society. Despite the maximal medical therapy, the percentage of asthma patients who have severe disease and are symptomatic reaches 5% to 10 %. (Global Initiative for Asthma, 2020; Wenzel, 2006).

The childhood prevalence of asthma is higher in boys. Nevertheless, the prevalence is approximately 20% higher in women than in men, demonstrating a shift after puberty. (Almqvist et al., 2008; Carey et al., 2007; Leynaert et al., 2012).

Moreover, potential genetic and hormonal contributors, as well as sex differences in concomitant conditions such as obesity and cigarette smoking may lead increased asthma risk (Raghavan et al., 2016). Besides asthma, the protective role of puberty has been apparent also in other allergic diseases, for instance, in vernal keratoconjunctivitis mainly occurring in boys and disappearing after puberty (Leonardi et al., 2012). Androgens produced by post-pubertal men generally suppress immune cell reactivity in a variety of systems, although the exact pathomechanisms are still quite poorly understood (Kissick et al., 2014; Klein et al., 2016; Kurukulaaratchy et al., 2011).

2.2.3 Phenotypes and pathogenesis

Asthma can be divided into many phenotypes traditionally defined on the basis of their relation to specific triggers, to immunopathology, or to clinical phenotype such as frequency of exacerbations or treatment resistance (Brightling et al., 2012). During the last few years, a strategy has been evolving to associate molecular mechanisms with phenotype: dividing asthma into endotypes which describe these distinct functional or pathophysiologic mechanisms at a cellular and molecular level (Kuruvilla et al., 2019). Cluster analyses have recently revealed considerable information on various asthma phenotypes and endotypes (Hsiao et al., 2019;

Ilmarinen et al., 2017; Newby et al., 2014; Schatz et al., 2014; Wu et al., 2019).

According to data from twin studies, data regarding phenotypic variability in asthma severity, genetic factors determine approximately 25%. Nongenetic factors include environmental and psychosocial factors, behavioral traits, and comorbidities.

(Thomsen et al., 2012). Various environmental exposures are associated with asthma.

In the case of childhood asthma, these include respiratory viruses, especially rhino- and RSV viruses, exposure to second-hand smoke, to inhaled chemicals, mold, and ambient air pollutants, and also include some deficiencies in maternal diet (Bergroth et al., 2020; Dick et al., 2014; Jartti et al., 2019, 2020). In adult asthma, factors

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associated with asthma risk include smoking, obesity, allergic conditions, inflammatory upper airway diseases, occupational exposure agents, residential dampness and mold, outdoor pollutants, psychosocial factors, and depression (Guarnieri et al., 2014; Jarvis et al., 2012; Karjalainen et al., 2002; Quansah et al., 2012; Siroux et al., 2019; Toppila-Salmi et al., 2015; Toppila-Salmi et al., 2019).

Asthma has long been associated with atopy; it has traditionally been regarded as an atopic condition, but factors unrelated to atopy have also emerged, ones proving to be important in disease development (Anderson, 2005; Zimmerman et al., 1988).

Important causes of this non-atopic asthma include smoking, pollutants, and occupational-exposure agents, and non-atopic asthma can easily be more persistent than atopic asthma, with few obvious triggers other than infection. Viral respiratory infections can also trigger asthma in adulthood. (Anderson, 2005; Diamant et al., 2007; Rees et al., 2010). Atopic asthma generally starts in childhood or adolescence, with certain triggers that provoke wheezing. It is also frequently associated with a family history of allergic diseases. (Diamant et al., 2007; Townshend et al., 2007).

The disease typically results from an allergic response to specific allergens such as house-dust mite, grass and tree pollen, and dander from domestic pets (Ward et al., 2010). This corticosteroid-dependent asthma has further been divided into two subtypes according to the presence or absence of airway eosinophilia (Wenzel et al., 1999).

The broad consensus supports dividing severe asthma phenotypes according to their tendency to type 2 inflammation (Figure 1). Allergic asthma in the majority of patients is associated with type 2 inflammation (Holgate, 2015). Such lung inflammation is mostly affected by the overproduction of type 2 cytokines such as IL-4, IL-5, and IL-13. IL-4 produced by Th2 cells is critical for allergen-specific production of IgE, which releases inflammatory mediators upon cross-linking of the high-affinity IgE receptors on the surfaces of mast cells and basophils. IL-5 supports the development of eosinophils in the bone marrow and enrolls eosinophils into the lung mucosa and interstitium. Enzymatic activity of the allergens damages the epithelial cell layers and activates mucosal dendritic cells that stimulate allergen- specific naive CD4 T cells in lymph nodes to differentiate into type 2 cytokine- producing Th2 cells. (Lambrecht et al., 2014).

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Figure 1. Severe asthma endotypes (modified from Wenzel, 2012 with permission of the publisher).

The involvement of innate lymphoid cells and innate-like lymphocytes (ILLs) in the pathogenesis of asthma has recently inspired valuable insight into asthma research.

ILCs are innate immune cells lacking the T cell and B cell receptors, and they are able to rapidly secrete various cytokines, especially effector cytokines IL-5, IL-13, and IL-17, in response to stimuli. (Vivier et al., 2018). The ILLs are subpopulations of T or B cells expressing T and B cell receptors but functioning very similarly to ILCs (Lanier, 2013; Spits et al., 2013). Both of them lack antigen-specificity, reside in tissues, and are located at mucosal sites where infection and inflammation frequently occur, the important one in asthma being the respiratory tract (Chou et al., 2018). ILLs and ILCs are involved in the pathogenesis of asthma, and therefore, as the treatment targets of both Th2 and non-Th2 asthma, they offer potential strategies (Huang et al., 2019).

ILC2s in particular are potent sources of IL-13 and IL-5. ILC2 activation promotes eosinophil infiltration, mucus secretion, and airway hyperreactivity, but does not affect IgE production (Martinez-Gonzalez et al., 2015). ILC2-derived IL- 13 promotes the migration of lung dendritic cells into the draining lymph node.

There it initiates Th2 cell differentiation. (Halim et al., 2014).

In allergic airway inflammation, allergens seem to provoke the release of alarmins such as IL-33. These stimulate lung-resident ILC2 into secreting type 2 cytokines, which cause allergic inflammation and promote Th2 cell activation and subsequently

Non Type 2

Early-onset allergic

NSAID-exacerbated respiratory disease Allergen

specific IgE

Neutrophilic

Paucigranulocytic Obesity Smoking

Type 2

ASA or NSAID sensitivity Late-onset eosinophilic

Eosinophilia Lack of inflammation

in airways Sputum

neutrophils

Antibiotics

Lifestyle and behavior changes Biomarker testing

Targeted therapy

Pollutants Occupational exposure agents

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develop adaptive B cell responses and IgE production. (Martinez-Gonzalez et al., 2015). Female ILC2s can produce significantly higher amounts of IL-5 and IL-13 than do male ILCS2 cells. Type 2 cytokine gene expression is higher in IL-33- stimulated female ILC2s than in male ILC2s. (Warren et al., 2017).

The majority of asthma patients represent type 2 inflammation, which is associated with certain cytokines, especially IL-4, IL-5, and IL-13, and inflammatory cells such as eosinophils, mast cells, basophils, type 2 Th lymphocytes, and plasma cells producing IgE. Airway epithelial cells play a large role in regulating type 2 inflammation, and cytokines IL-25, IL-33, and TSLP seem to be involved here.

(Fahy, 2015).

The suggested biomarkers of type 2 immune response in asthma comprise blood eosinophilia, specific IgE, serum periostin level, serum dipeptidyl peptidase 4, sputum eosinophilia, IL-13 levels in induced sputum, and fraction of exhaled nitric oxide (FeNO) in exhaled breath (Berry et al., 2016). IgE can be targeted by anti-IgE biologicals such as omalizumab. Prediction of response to corticosteroids and novel anti-IL-4/IL13 and anti-IL-5 treatments (dubilumab, mepolizumab, reslizumab, benralizumab) depend on measurement of blood eosinophilia (Cavkaytar et al., 2013;

Ortega et al., 2014; Pavord et al., 2012). Sputum eosinophil levels can serve in prediction of the response to anti-IL-13 and anti-IL-5 therapy (Bel et al., 2014;

Ortega et al., 2014; Green et al., 2002; Haldar et al., 2009; Nair et al., 2009).

Measuring serum periostin, serum dipeptidyl peptidase 4 levels, and sputum IL-13 levels has also revealed them to be valuable markers when one is considering anti- IL-13 therapy (Brightling et al., 2015; Corren et al., 2011; Piper et al., 2013). FeNO values indicate eosinophilic airway inflammation in steroid-naive asthma patients (Hanania et al., 2013). Higher baseline FeNO can also predict a greater reduction in frequency of exacerbations upon anti-IL-4/IL-13 treatment (Castro et al., 2018).

The discovery of non-type 2 asthma has illuminated new ways to understand this disease. Asthma patients without strong type 2 inflammation frequently exhibit a poor response to corticosteroids; their disease is often more challenging to manage.

(Mims, 2015). In non-type 2 asthma, sputum neutrophils have served as an indicator of neutrophilic endotype. Other suggested biomarkers are adipokine, IL-8, and IL- 17. (Bullens et al., 2006; Gounni et al., 2001) For the time being, these have no clinical applications in asthma, although there do exist biologicals targeting cytokines of non-type 2 inflammation, such as IL-12, IL-23, IL-17, IL-1, IL-31, and TNF-a.

These have been useful against some other allergic conditions. (Tan et al., 2016).

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2.3 Allergic rhinitis

2.3.1 Definition

Allergic rhinitis is an IgE-mediated inflammation of the nasal mucosa resulting from inhaled allergen introduction in a sensitized individual (Bousquet et al., 2001; Maurer et al., 2007). AR was defined in 1929 as a process which included three cardinal symptoms: sneezing, nasal obstruction, and mucus discharge (Hansel, 1929).

Although frequently regarded as merely a seasonal nuisance, AR can involve minimum persistent mucosal inflammation which can synergize with infective inflammation. Hence, individuals with AR sometimes have additional difficulties with viral colds. (Ciprandi et al., 1995; Cirillo et al., 2007) AR also affects one’s social life, school performance, and work productivity, particularly for patients with severe forms of the disease (Bousquet et al., 2001; Bousquet et al., 2006; Canonica et al., 2007).

2.3.2 Prevalence

The prevalence of AR ranges from 10% to 40%, depending on geographic location.

The highest incidence occurs in school-aged children, and it does not typically manifest until the second year of life at the earliest. (Asher et al., 2006; Bauchau et al., 2004). In Finland, AR prevalence estimates are around 30% and still increasing (Jousilahti et al., 2016).

Up to 80% of asthma patients also have AR (Bousquet et al., 2008). On the other hand, individuals with AR have a 5– to 6-fold greater risk for developing asthma (Leynaert et al., 2004). A case-control study of French adult asthma patients showed that 92.4% of AR patients reported AC symptoms (Burte et al., 2015). In a Cameroonian community-based survey, 15.9% of adult asthma patients reported AC symptoms, but only 5.2% of the non-asthma controls reported ARC symptoms (Pefura-Yone et al., 2015). One Finnish population-based study in adults discovered that for incident asthma, ARC doubles the risk (Pallasaho et al., 2011).

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AR has traditionally been characterized by presumed cause and seasonal or perennial presentation. At present, AR is divided into two types based on symptom duration.

Intermittent AR shows symptoms for less than 4 days per week or for fewer than 4 consecutive weeks, whereas persistent AR patients have symptoms occurring more than 4 days per week for at least 4 consecutive weeks. Persistent AR patients are therefore symptomatic most of the time. (Wallace et al., 2008). Intermittent forms of AR are typically a reaction to pollen produced by trees, grasses, and weeds;

persistent forms, on the other hand, are most often caused by animal dander, dust mites, and molds (Bousquet et al. 2008). AR can be defined as mild when patients experience no impairment of sleep, of daily activities, of work or school performance, or if they suffer any troublesome symptoms; it is moderate to severe when these problems accumulate (Bousquet et al., 2001).

A phenotype of local allergic rhinitis (LAR) can be characterized by nasal Th2 allergic response and local production of specific IgE. LAR is the diagnosis in any case with a positive response to a nasal provocation test or with specific IgE in the nasal mucosa with no evidence of systemic atopy. (Arasi et al., 2016). In LAR, biomarkers can be detectable, ones such as nasal IL-5 and thymic stromal lymphopoietin (Zicari et al., 2016).

2.4 Allergic conjunctivitis

2.4.1 Definition

The term “allergic conjunctivitis” refers to hypersensitivity disorders affecting the lid, conjunctiva, or cornea or any combination of these. When rhinitis is also present, the condition may be known as rhinoconjunctivitis or conjunctivorhinitis depending on the main symptoms. Ocular allergy has been defined as an anterior ocular surface inflammatory disorder mediated primarily by triggering of the IgE-mast cell system.

(Bielory et al., 2007).

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2.4.2 Prevalence

Because conjunctival symptoms often go unreported in medical interviews or in questionnaire-based epidemiologic studies on rhinitis or asthma, AC prevalence has been difficult to define, presumably leading, in many epidemiologic studies, to underestimations. A variety of studies suggest a high comorbidity of conjunctivitis and rhinitis. Conjunctival symptoms are often considered a minor problem, although that is not always how ophthalmologists consider it. The surveys that target specifically ocular symptoms do not indicate conjunctival symptoms being minor, either. (Palmares et al., 2010).

The epidemiology of ocular allergy is mostly based on allergic rhinoconjunctivitis (ARC) studies. Seasonal ARC may affect 3% to 42% and perennial ARC affect 1%

to 18% of various populations, varying by climate and age (Brozek et al., 2010).

Most recent estimates suggest that in the United States, ocular allergies affect as high as 15% to 25% of the population (Miraldi Utz et al., 2014; O’Brien, 2013; Ono et al., 2005). The prevalence of AC is similar in Europe, Japan, and Australia, and is increasing worldwide (Rosario et al., 2011). One American survey discovered that 35% of families interviewed experienced allergies, and half reported associated eye symptoms (Stahl et al., 2004). According to another American study, ocular symptoms were the only manifestation of allergy in approximately 25% of allergic adults (Singh et al., 2010). A European survey reported that among patients with AR, itchy, red eyes afflicted 71% and watery eyes afflicted 67% (Canonica et al., 2007).

The prevalence of AC was higher in female subjects (Geraldini et al., 2013).

Ocular allergy can be classified into several clinical forms. These fall into two main groups, the first including the more frequent, seasonal (SAC) and a perennial allergic conjunctivitis (PAC). Since the definitions of seasonal and perennial do not include specific duration, international consensus panels have recently suggested the terms

“intermittent”, i.e. less than 4 weeks in duration, or “persistent”, i.e. more than 4 weeks in duration, these being in accordance with the definitions of AR. (Bielory et al., 2020).

The second group involves the reasonably rare chronic, severe forms of ocular allergy: vernal keratoconjunctivitis (VKC) and atopic keratoconjunctivitis (AKC).

These conditions can include corneal findings and require ophthalmologic management. (Leonardi et al., 2008). AKC and VKC show clinical and

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pathophysiological traits differing from those of SAC and PAC, although these share some common markers of allergy (Bielory et al., 1992).

At times, contact-lens-associated or ocular-prosthesis-associated giant papillary conjunctivitis can be included among ocular allergies. Nonetheless, they are not true allergic diseases, but chronic ocular microtrauma-related disorders. (Leonardi et al., 2008).

Seasonal and perennial AC are typically caused by a IgE-mediated reaction to environmental airborne allergens. The most common allergens are grass and tree pollens, mites, mold, and animal dander. SAC is usually acute or subacute and can be determined by peaks of self-limiting signs and symptoms. In repeated allergen stimulations during pollen season, the symptoms of itching, redness, and lid swelling become persistent. Tearing, mucous discharge, and burning are also typical. When redness, burning, itching, and chemosis persist for months, the disease can be considered PAC. (Ono et al., 2005). Traditionally, AC has been considered an ocular manifestation of type I allergy mediated by IgE antibodies. Nonetheless, a recent study also suggests the existence of local AC, a phenotype of AC, in which only the local levels of IgE is elevated. (Yamana et al., 2019).

Although not lifethreatening, ocular allergy symptoms make a remarkable impact on the productivity and quality of life (Virchow et al., 2011). AKC or VKC are quite rare, but can be severe and sight-threatening (Chen et al., 2014; De Smedt et al., 2013).

The most prevalent symptom of ocular allergy is tearing; about three-fourth of these patients suffer from it. Approximately half the patients have photophobia and itching, and more than one-third have foreign-body sensation. (Geraldini et al., 2013). In another study, itching occurred in 90% and redness in 85%. Lid-skin problems were involved in 22% of cases and keratitis in 11%. (Leonardi et al., 2015).

In the pathogenesis of ocular allergy, mast cells play a key role. During the pollen season, one study found, median mast cell numbers in the lamina propria were 61%

higher in SAC patients than in normal subjects. In allergic patients, they also remained increased out of season. (Anderson et al., 1997).

Histamine from degranulated mast cells binds histamine receptors 1 to 4 on vascular endothelial cells, neuronal fibers, goblet cells, immune cells, and on the conjunctival epithelium (Bielory et al., 2005). Selective agents which bind to these receptors offer possibilities for therapeutic effects (Wade et al., 2012). Histamine receptor subtype agonists influence many processes of allergic inflammation, ones such as stimulation and release of adhesion molecules, chemokines, and cytokines, and activation of dendritic cells, all of these leading to maturation of antigen-

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presenting cells and activation of CD4 Th2-lymphocytes. Such lymphocytes and also mast cells seem to be the most important immune cells involved in allergic inflammatory responses of the ocular surface. (Bielory et al., 2020).

Activated mast cells can release several cytokines affecting the mucosa and can induce chemokines and adhesion molecules contributing to the recruitment of inflammatory cells (Anderson et al., 2001). Furthermore, mediators released during the late phase of allergic inflammation of the ocular surface, ones such as lipid mediators formed from the mast cell membrane arachidonic acids and cytokines recruit and activate eosinophils, lymphocytes, monocytes, and neutrophils. These mediators offer targets for therapeutic interventions (Leonardi et al., 2006).

A variety of ophthalmologic studies report local cytokine dynamics in the eye, including the palpebral conjunctiva and lacrimal fluid (Fukagawa et al., 2000;

Kumagai et al., 2000; Matsuda et al., 2009; Sugita et al., 2017; Uchio et al., 2000). As a source of biomarkers in the development of clinical assays for ocular allergies, tears show potential. Neutrophils, eosinophils, and lymphocytes with different cellular profiles are evident in tears in acute and chronic ocular allergies. (Bonini et al., 1990;

Pelikan, 2012) T cells and B cells, as well as CD4:CD8 T-cell ratios in tears are reportedly higher in AKC than in control subjects, whereas increased Th2 cell levels in tears appear in VKC (Avunduk et al., 1998; Leonardi et al., 1999). Increased tear levels of IgE, tryptase, histamine, and eosinophil cationic protein may serve as biomarkers of ocular allergy (Bourcier et al., 1998; Leonardi, 2013).

VKC is an example of a gender-specific protective action by pubertal factors.

This disease occurs predominantly in childhood and adolescence and almost always in boys. It disappears after puberty in 90% of patients and is quite rare in Europe and much more frequent in subtropical countries. (Bremond-Gignac et al., 2008;

Leonardi et al., 2012). Half the patients represent hyper IgE. VKC has been associated with T cell-mediated responses and immense eosinophil infiltration; a trigger can be non-specific hyper-activity such as sunlight, wind, or dust. (Laffont et al., 2017).

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2.5 Genetics of atopy and asthma

2.5.1 Associated genes

An interesting approach to the discovery of mechanisms of allergic diseases is to search for associations between the diseases and polymorphisms of various genes, especially those coding for molecules involved in immune responses. However, seeking the genetic factors involved in asthma and atopic diseases can be likened to trying to find a needle in a haystack. (Barnes, 2015).

Since the arrival of the polymerase chain reaction, genetic fingerprinting has involved amplifying deoxyribonucleic acid (DNA) fragments in the human genome, trying to find the restriction-fragment-length polymorphisms. These microsatellite markers spanning the human genome have enabled family based GWAS.

Technology has led to many GWAS on asthma and its associated phenotypes (CSGA, 1997; Daniels et al., 1996; Dizier et al., 2000; Hakonarson et al., 2002;

Laitinen et al., 2001; Malerba et al., 1999; Ober et al., 1998; Ober, et al., 2000; Van Eerdewegh et al., 2002; Wjst et al., 1999; Yokouchi et al., 2000, 2002). Multiple chromosomal regions have been identified as candidate loci, with six novel asthma genes identified by positional cloning (Allen et al., 2003; Laitinen et al., 2004; Nicolae et al., 2005; Noguchi et al., 2005; Van Eerdewegh et al., 2002; Zhang et al., 2003).

GWAS have functioned similarly for AR (Dizier et al., 2005; Haagerup et al., 2001;

Kruse et al., 2012; Kurz et al., 2005; Yokouchi et al., 2002).

By March 2020, 135 GWAS on asthma, 12 on AR, 38 on allergy, and 10 on IgE, had appeared (NHGRI-EBI GWAS Catalog, 2020). However, GWAS do not identify the causal variants associated with a disease, and the estimated cumulative genetic risk of the variants identified for asthma alone has been under 15% (Mathias, 2014). Early-onset asthma has a stronger genetic component and greater heritability than does adult-onset of asthma. It has been associated with more than 2.5 times the number of genome-wide significant loci compared with adult-onset asthma, despite the greater sample sizes for the latter. (Ferreira et al., 2019; Pividori et al., 2019).

Despite the large number of candidate genes for asthma identified in GWASs and in basic immunological research, few of those discoveries have been rigorously replicated. Ot the variety of candidate genes examined, all have failed in subsequent replication studies (Barnes, 2015; Bønnelykke et al., 2014; Ober et al., 2006; Vercelli, 2008; Zhang et al., 2008). The most strongly linked and also replicated genes include the ORMDL3-GSDMA-GSDMB locus on chromosome 17, as well as DENND1B,

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GAB1, HLA, IKZF4, IL1RL1, IL33, IL2RB, IL6R, LRRC32, PDE4D, RORA, SLC30A8, SMAD3, TLR1, TSLP, and cadherin-related family member (CDHR) 3 (Barnes, 2015; Bønnelykke et al., 2014; Moffatt et al., 2007, 2010; Portelli et al., 2015).

These genes are involved in epithelial barrier function and innate and adaptive immune responses (Moffatt et al., 2007, 2010; Portelli et al., 2015). CDHR3, a childhood asthma susceptibility gene product, is a recognized rhinovirus C receptor (Basnet et al., 2019; Bochkov et al., 2015). Nevertheless, the effects of all these loci are weak, accounting for only a minor proportion of asthma heritability (Barnes, 2015). Many studies consider asthma to be one disease, even though it includes several endotypes, and this may in part explain the weakness of the effects found.

Genes also been quite extensively replicated include the beta 2 adrenergic receptor gene, IL4, IL4RA, IFNG, IFNGR1, STAT6, GATA3, and TBX21 (Basehore et al., 2004; Haller et al., 2009; Liggett, 1995; Munthe-Kaas et al., 2008;

Pykäläinen et al., 2005). In addition, genes involving in the cellular responses characterizing atopic disease, genes such as IL13 and its receptor and the FCER1B gene have been replicated (Howard et al., 2002; Kabesch et al., 2006;

Potaczek et al., 2009; Vladich et al., 2005; Wu et al., 2010).

Loci associated with total IgE concentrations overlap quite poorly with those related to asthma, indicating that atopy may not be the primary driver of susceptibility (Noguchi et al., 2005; Zhang et al., 2003). Quantitative genetic scores of the combined effect of numerous common SNPs separately show a weak influence on asthma risk, indicating that asthma has a strong polygenic component (Laitinen et al., 2001). This accords with findings of familial segregation of this disease (Haagerup et al., 2001).

Examples of genes associated with atopy in general are cytokine gene cluster IL3, IL4, IL5, IL13, CD14, beta-2-adrenergic receptor, and GM-CSF. IL-4 and IL-13 promote IgE switching, whereas IL-5 stimulates eosinophil growth and activation.

Beta-2-adrenergic receptors regulate contraction of bronchial smooth muscles. In addition, MHC class II alleles regulate T cell responses to environmental antigens, and the high-affinity IgE receptor beta-subunit is involved in mast cell activation.

Genes for stem cell factor intervene in mast cell growth and differentiation, and IFN-g inhibits IL-4 synthesis, whereas STAT6 mediates IL-4 signal transduction.

Other genes associated with atopy are IL4RA, DPP10 (a protein that regulates chemokine and cytokine activity), ADAM33 metalloproteinase (an enzyme involved in airway remodeling), and CD80/CD86, RANTES, and PHF11, all three of which encode for a transcriptional regulator of the clonal expansion of B cells and

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immunoglobulin expression. (Barnes et al., 1999; Blumenthal, 2005; Pinto et al., 2008; Weidinger et al., 2004).

The genetics of asthma severity is under investigation in association studies.

Polymorphisms in the ADRB2, ARG1, ARG2, CTLA4, IL4, IL4R, IL18, TGFB1, TLR4, SERPINE1, and ADAM33 genes seem to play a role in asthma severity in several populations (Chiang et al., 2007; Harada et al., 2009; Holloway et al., 2000;

Lee et al., 2002; Pulleyn et al., 2001; Tripathi et al., 2011; Vonk et al., 2010; Wenzel et al., 2007; Zhang et al., 2011).

2.5.2 Interaction

On many occasions, the effect of a gene mutation has been dependent on the presence or absence of mutations of one or more other genes. This phenomenon in genetics is called epistasis. The effect of the mutation therefore depends on the genetic background in which it appears. (Gros et al., 2009; Phillips, 2008).

The concept of gene-gene interaction has recently gained attention in genetic studies (Lee et al., 2012; Liu et al., 2012). Applying an epistatic analysis to clinical genetic research makes it possible to examine the interactions of two or more genes involving the whole system of the human body. Such analysis can reveal the reality of dynamic molecular mechanisms and possibly provide a breakthrough to overcome the GWAS limitations. (Yoshikawa et al., 2014). As an example, FUT2 and ABO genes have recently been found to have an epistatic effect in raising the risk of childhood asthma (Ahluwalia et al., 2020).

2.6 Some inflammatory mediators previously associated with atopy and asthma

2.6.1 Interleukin-1

The innate immune system is the first step in defense of the body, a response initiated by nonspecific, nondirected phagocytosis, complement activation, or by the activation of innate immune receptors. The immune receptors react to structures conserved in many pathogens rather than against certain antigens or peptides. The link between innate and adaptive immune immunity can be demonstrated by

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vaccination: supporting innate immune activation is a usual means to generate an adaptive immune response. (Janeway et al., 2002).

Resident and invading immune cells mediate the initial immune response.

Resident tissue macrophages and epithelial cells carry an excess of immune receptors; these cells can produce innate cytokines to attract invading macrophages or support neutrophils by producing cytokines or chemokines (Akira et al., 2006) The most important cytokines associated with the innate immune response are IL- 1α, IL-1β, IL-6, IL-18, IL-33, and TNF-a, all of which can act both locally and systemically (Schroder et al., 2010).

Currently, we recognize eleven cytokines in the IL-1 family: IL-1α, IL-1β, IL- 1RA, IL-18, IL-36Ra, IL-36α, IL-37, IL-36β, IL-36γ, IL-38, and IL-33. IL-1, a major pro-inflammatory cytokine, appears in two forms, IL-1α and IL-1β. Several cell types can synthesize these cytokines, which play a major role in the initiation of the immune response. IL-1α is present in the cytosol and on the plasma membrane of cells, and it mediates much of the skin’s inflammatory reaction. (Dinarello, 2018;

Dinarello, 1996; La et al., 2001). IL-1β has plays an important role in contact sensitization (Kermani et al., 2000). IL-1α and IL-1β are coded for by various genes located in the longer arm of chromosome 2 (Cox et al., 1998).

Polymorphisms of these genes influence the strength of inflammation, based on both in vivo and in vitro studies. Several polymorphisms have been associated with the severity of or the susceptibility to many inflammatory diseases. (Bidwell et al., 1999) A G/T base exchange at +4845 (rs17561) in exon 5 of the IL1A gene results in an amino acid substitution of alanine for serine (Velden et al., 1993). This SNP is associated with atopy in non-asthmatic adults, suggesting that allele G predisposes to atopy (Karjalainen et al., 2002; Pessi et al., 2003). The same polymorphism has also been linked to nasal polyposis, a condition often found coexisting with asthma (Erbek et al., 2007; Karjalainen et al., 2003).

2.6.2 Interleukin-4

IL-4 plays an important role in regulating Th2 cell proliferation and survival and IgE synthesis, and is essential in the initiation of allergic airway responses and of humoral responses (Brusselle et al., 1995; Corry et al., 1996; Coyle et al., 1995; Dabbagh et al., 1999; Gavett et al., 1997; Henderson et al., 2000; Tepper et al., 1990).

IL-4, a major anti-inflammatory cytokine, is produced by activated T cells, mast cells, and basophils. It plays a central role in the regulation of B-cell– and T-cell–

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mediated immune responses. (Paul et al., 1994). The IL-4R is an important component of the IL-4 pathway, since IL-4 exerts its biological effects through two types of heterodimeric transmembrane receptor complexes: the type I receptor exclusively binding IL-4 and comprising IL-4Rα and γc subunits, and the type II receptor binding both IL-4 and IL-13 and comprising IL-4Rα and IL-13Rα1 subunits (Gour et al., 2015).

In vitro studies have shown that a variant of the IL4 gene promoter, –590 C/T, associates with IL-4 activity and higher binding activity to nuclear transcription factors, allele T being the high producer of IL-4. (Rosenwasser et al., 1997). Allele T has also been associated in several in vivo studies with high total IgE levels and with asthma and atopy (Amirzargar et al., 2009; Basehore et al., 2004; Beghé et al., 2003;

Berenguer et al., 2014; Davoodi et al., 2015; Hussein et al., 2017; Kabesch et al., 2003; Liu et al., 2003; Marsh et al., 1994; Noguchi et al., 1998, 2001; Rosenwasser et al., 1995; Walley et al., 1996; Zhang et al., 2016).

In the IL4RA gene, coding for IL-4 receptor α and located in the shorter arm of chromosome 16, exist a number of SNPs associating with atopic diseases (Deichmann et al., 1997; Hackstein et al., 2001; Hershey et al., 1997; S. Kruse et al., 1999; Lozano et al., 2001; Mitsuyasu et al., 1998; Ober et al., 2000; Paul et al., 1994). The T/C base exchange at +22446 (known as cys406arg, rs1805012) in exon 9 of the IL4RA gene has been associated with atopy. This base exchange accounts for an amino acid substitution of cysteine for arginine. The allele T coding for cysteine predisposes to atopy. (Howard et al., 2002; Ober et al., 2000).

2.6.3 Toll-like receptor 4

Innate and adaptive immunity are the two types of immunity in humans for overcoming pathogens. The functionality of innate immunity depends on germline- encoded receptors, pattern recognition receptors, to which the TLRs belong. They recognize various pathogen-associated molecular patterns, which are highly conserved structures in micro-organisms and are essential for their survival. (Botos et al., 2011; Mogensen, 2009).

In humans, the toll-like receptors (TLR) include 11 members: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, and TLR11 (Takeda et al., 2003). The TLR4 is the main receptor for bacterial endotoxin, playing a principal role in the innate immune response to gram-negative pathogens and respiratory syncytial virus (Aderem et al., 2000; Lien et al., 2000). Because endotoxin binds to

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TLR4, it initiates an intracellular signaling pathway resulting in the activation of nuclear transcription factors. A single A/G base exchange at +896 (rs4986791) in the TLR4 gene induces an amino acid substitution of glycine for asparagine (at amino acid position 299). This results in reduced cell-surface expression of TLR4, disruption of LPS-mediated signaling and reduced systemic inflammatory response to low-dose inhaled endotoxin. (Arbour et al., 2000; Michel et al., 2003).

As stated in the hygiene hypothesis, early exposure to high endotoxin levels protects from asthma and atopy, whereas low doses cause an increase in disease prevalence (Eisenbarth et al., 2002; Strachan, 1989). Exposure to endotoxin may be a risk factor for asthma; the TLR4 gene seems to modify the endotoxin effects. (Reed et al., 2001; Schwartz, 2001; Tulić et al., 2000; Werner et al., 2003).

Research suggests that bronchial epithelial cells are important in driving naive T cell differentiation towards T-helper 2 (Th2) cells by activating dendritic cells (Hammad et al., 2009; Ryu et al., 2013; Soumelis et al., 2002). This process is mediated by TLR4 signaling through the interaction of epithelial cells and environmental aeroantigens. TLR4 activation seems to stimulate the expression of several epithelium-derived alarmins, ones including TSLP and IL-25 and IL-33 (Allakhverdi et al., 2007; Bartemes et al., 2012). TSLP signaling in airway epithelial cells seems to play a role in initiating airway remodeling by stimulating the Th2 cell response (Chen et al., 2013). Recently, TLR4 antagonist has, in a chronic asthma mouse model, proven a somewhat efficient treatment for airway remodeling, possibly through the inhibition of TSLP overexpression and through Th2 airway inflammation (Li et al., 2017). It has also become apparent in mild asthma patients that tezepelumab, a humanized mAb against TSLP, reduces exacerbation rate and improves FEV1 in patients with moderate to severe asthma and reduced allergen- induced bronchoconstriction and airway inflammation (Corren et al., 2017;

Gauvreau et al., 2014).

The A/G base exchange at +896 (Asp299Gly mutation) inhibits TLR4-mediated signaling. The gene sequence change in receptor responsible for LPS signaling can alter the responsiveness of the host to microbial agents. (Werner et al., 2003). TLR4 SNP at +896 has been associated with asthma in children, G being the predisposing allele (Fagerås Böttcher et al., 2004; Zhao et al., 2017). Negative findings have also appeared as to the association between TLR4 polymorphisms and asthma and atopy (Davoodi et al., 2015; Noguchi et al., 2004; Raby et al., 2002; Yang et al., 2004).

Genetics of Inflammatory Mediators of Atopy and Asthma in Adults

Genetics of Inflammatory Mediators of Atopy and

Asthma in Adults

KATI ÅDJERS

KATI ÅDJERS

Genetics of Inflammatory Mediators of Atopy and Asthma in Adults

To my family

ACKNOWLEDGEMENTS

ABSTRACT

TIIVISTELMÄ

CONTENTS

ABBREVIATIONS

ORIGINAL PUBLICATIONS

AUTHOR’S CONTRIBUTION

1 INTRODUCTION

2 REVIEW OF THE LITERATURE

2.1 Atopy

2.2 Asthma

2.3 Allergic rhinitis

2.4 Allergic conjunctivitis

2.5 Genetics of atopy and asthma

2.6 Some inflammatory mediators previously associated with atopy and asthma