From Department of Medicine Solna, Respiratory Medicine Unit Karolinska Institutet, Stockholm, Sweden

Innate and Adaptive Immune Responses in Pulmonary

Maria Wikén

Stockholm 2010

The figure on the front page shows a lung with a and two T cells hit by laser beams, and was made by help from Victor Wikén.

All previously published papers were reproduced with permission from the publisher. Published by Karolinska Institutet. Printed by Larserics Digital Print AB.

© Maria Wikén, 2010 ISBN 978-91-7409-894-5

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ABSTRACT

Sarcoidosis is an inflammatory disease characterized by formation of in various organs and tissues. Although it is a systemic disorder, the lungs are commonly affected, and an infiltration of T cells, in particular CD4pos T helper 1 (Th1) cells, is seen in the lower airways. HLA-DRB1*0301pos (HLA-DR3pos) patients commonly present with acute disease onset, typically with Löfgren´s syndrome, accumulation of lung CD4pos T cells expressing the T cell receptor (TCR) gene segment AV2S3 (AV2S3pos T cells) and good prognosis. In contrast, HLA-DR3neg patients show insidious disease onset and are at risk of developing lung fibrosis. The aetiology of sarcoidosis is still unknown; however, several studies indicate an infectious cause, suggesting a role for innate immune receptors, including toll-like receptors (TLRs) and nod-like receptors (NODs). Recently, the mycobacterial protein mKatG was identified in sarcoidosis tissues but not in healthy subjects, and T cell responses have been reported to mKatG. However, their specificity and profile, as well as the phenotype of lung-accumulated T cells in general, has not been that well described. The aim of this thesis was to examine differences between patients and healthy subjects, as well as between distinct patient subgroups, regarding innate and adaptive immune responses in the affected organ, i.e. the lung, and in the . Sarcoidosis patients had, as expected, an enhanced Th1 mediated immune response in their lungs, as compared to healthy subjects. HLA-DR3pos patients had a lower IFNγ and TNF gene expression in BAL cells, and tendencies to lower IL-2 and TNF protein levels in BAL fluid, than HLA-DR3neg patients, suggesting that a less pronounced Th1 immune response in HLA-DR3pos patients may be related to their good prognosis. Blood of patients had higher baseline TLR2 and TLR4 expression, as compared to healthy subjects, which could have consequences for host-pathogen interaction. Stimulation with TLR2 and NOD2 ligands in combination resulted in a much higher production in blood cells from patients of IL-1β and TNF, which could promote the initiation of inflammatory responses. In addition, combined TLR2 and NOD2 stimulation gave rise to a synergistic induction of IL-1β in patients, whereas IL-10 was synergistically induced in healthy subjects. Total BAL CD4pos T cells of patients with Löfgren´s syndrome (including HLA-DR3pos patients) had a more pronounced multifunctional cytokine profile, i.e simultaneous production of IFNγ and TNF, after stimulation with mKatG, whereas total BAL CD4pos T cells of non-Löfgren patients exhibited a predominantly single-functional cytokine profile, i.e. production of IFNγ alone. This indicates that the quality of T cell responses may be of critical importance for the clinical presentation and disease outcome. BAL and blood TCR AV2S3pos T cells of HLA-DR3pos patients produced more IFNγ in response to mKatG, as compared to TCR AV2S3neg T cells, whereas the opposite was seen in BAL after stimulation with PPD. This, together with our previous knowledge of HLA-DR3pos patients having a good prognosis, implies that the role of TCR AV2S3pos T cells is to eliminate an offending antigen. HLA-DR3pos patients had a reduced frequency of FoxP3pos regulatory T cells among their BAL CD27pos and CD27neg memory T cell subsets, as compared to HLA-DR3neg patients. HLA-DR3pos patients had a higher frequency of naïve CD25negCD27pos BAL T cells, as compared to HLA-DR3neg patients, implying that the total T cells are less activated in HLA-DR3pos patients, which is in line with the finding of a less pronounced Th1 response in the lungs of these patients. BAL TCR AV2S3pos T cells, that exhibited a CD27pos memory phenotype, were much less positive for the regulatory marker FoxP3, as compared to the TCR AV2S3neg T cells, further supporting that TCR AV2S3pos T cells are effector cells rather than regulatory cells. BAL and blood TCR AV2S3pos T cells were more activated and differentiated than TCR AV2S3neg T cells, indicating their encountering with a postulated sarcoidosis antigen in vivo. Taken together, the findings presented in this thesis reveal that patients with good prognosis in their lungs exhibit an efficient multifunctional cytokine profile of mKatG-specific T cells, a reduced number of FoxP3-expressing memory T cells, an increased number of naïve T cells, and highly activated TCR AV2S3pos effector T cells, which we suggest all contribute to an efficient immune response, focused towards elimination of the sarcoidosis antigen(s), followed by spontaneous recovery.

POPULÄRVETENSKAPLIG SAMMANFATTNING

Sarkoidos är en inflammatorisk sjukdom som vanligtvis drabbar icke-rökare i åldrarna 20- 40 år, och i Sverige rapporteras årligen 1500-2000 nya fall. Sjukdomen är systemisk och kan angripa i stort sett alla organ i kroppen, dock engageras lungorna i huvudsak. Sarkoidos kännetecknas av ett inflöde av inflammatoriska celler som kallas T-celler. Deras uppgift är att känna igen och reagera på främmande ämnen, så kallade antigen, som härrör t.ex. från bakterier och virus. Dessa förekommer som CD8pos T-celler (så kallade mördarceller) och som CD4pos T- celler (så kallade T-hjälparceller; Th), där främst de senare påträffas vid sarkoidos. T- cellsaktiveringen startar då en antigenpresenterande cell, via en så kallad HLA-molekyl, presenterar en peptid (kort fragment av ett protein) för T-cellsreceptorn på en CD4pos T-cell. Detta resulterar i bildandet av olika lösliga signalsubstanser, så kallade cytokiner, vilka kan delas in i Th1- och Th2-typ. Vid sarkoidos har man sett förhöjda nivåer av bl.a. Th1-cell cytokinerna IFNγ, som är viktig för aktivering av makrofager, de så kallade ätarcellerna, samt TNF liksom av IL-1β. Tillsammans gynnar dessa cytokiner utvecklingen av granulom bestående av makrofager omgivna av aktiverade CD4pos T-celler. Granulombildning är ett sätt för immunsystemet att avskärma icke-eliminerbara antigen. Vid sarkoidos återfinns granulom i olika organ och vävnader och har ett för sjukdomen typiskt utseende. T-celler kan vidare vara minnes- eller effektorceller, eller uppvisa regulatorisk immunhämmande egenskap. De kan dessutom vara mer eller mindre aktiverade och differentierade. För att avgöra deras egenskaper används antikroppar, vilka är märkta med fluorescerande molekyler som möjliggör detektion i ett instrument som kallas flödecytometer. Antikropparna binder till cellytemolekyler eller molekyler inuti cellen. Patienternas genetiska uppsättning, framförallt de gener som är involverade i antigenpresentation, de så kallade HLA-generna, har visat sig vara viktiga för sjukdomsutvecklingen. Patienter med HLA-typ DR3 (HLA-DR3pos) har vanligtvis en akut sjukdomsdebut med Löfgrens syndrom (en kombination av typiska symtom från lungor, leder och hud), en ansamling av CD4pos T-celler med en speciell T-cellsreceptor, så kallade AV2S3pos T-celler, samt god prognos. Patienter med andra HLA-typer (HLA-DR3neg) uppvisar istället en smygande sjukdomsdebut och löper risk att utveckla lungfibros (bindvävsomvandling). Idag vet man inte vad det är som orsakar sarkoidos, men studier tyder på att sjukdomen skulle kunna vara infektionsutlöst. Detta medför att så kallade ”pattern-recognition”-receptorer (PRRs), t.ex. Toll-lika receptorer (TLRs) och Nod-receptorer (NODs), skulle kunna vara involverade vid sjukdom. Dessa uttrycks på exempelvis makrofager samt deras föregångare i blod, monocyter, och används till att binda upp främmande ämnen. Nyligen identifierades det mykobakteriella proteinet mKatG i sarkoidosvävnad men inte hos friska individer, och T-cellssvar mot mKatG har rapporterats. T-cellernas specificitet och cytokinprofil, samt egenskaperna hos de lung-ackumulerade T-cellerna i allmänhet, har dock inte karakteriserats. Syftet med studierna i denna avhandling har varit att undersöka skillnader i immunsvar mellan patienter och friska individer, samt mellan distinkta subgrupper av patienter (de som tillfrisknar spontant och de som har risk för kronisk sjukdom). Vi har studerat celler och lösliga signalsubstanser i blodet och i lungsköljvätska, där den senare erhölls genom så kallad lungsköljning (bronkoalveolärt lavage; BAL). BAL är en undersökningsmetod som används i diagnostiskt syfte, och går ut på att ett flexibelt fiberoptiskt instrument förs ned i luftvägarna via näsan, varefter koksaltlösning sprutas ned och därefter samlas upp.

Resultaten som presenteras i denna avhandling visar att sarkoidospatienter hade ett kraftigare Th1-medierat immunsvar i lungan än friska individer. HLA-DR3pos patienter hade ett lägre genuttryck av IFNγ och TNF i lungceller, och tendenser till lägre proteinnivåer av IL-2 och TNF i lungsköljvätska, jämfört med HLA-DR3neg patienter. Detta indikerar att ett mindre uttalat Th1-immunsvar i HLA-DR3pos patienter kan vara relaterat till deras goda prognos. Blodmonocyter från patienter hade ett högre basalt uttryck av TLR2 and TLR4 än friska individer, vilket skulle kunna ha konsekvenser för värd-patogen-interaktion. Kombinerad TLR2- och NOD2-stimulering resulterade i en mycket högre produktion i patienters blodceller av TNF och IL-1β, vilket kan gynna initieringen av inflammatoriska respons. De CD4pos T-cellerna i lungsköljvätska hos patienter med Löfgrens syndrom (inklusive HLA-DR3pos patienter) uppvisade en simultan produktion av IFNγ och TNF, en så kallad multifunktionell cytokinprofil, efter stimulering med mKatG, medan CD4pos T-celler i lungsköljvätska hos patienter utan Löfgrens syndrom producerade i huvudsak IFNγ, en så kallad singelfunktionell cytokinprofil. Detta indikerar att kvaliteten på T-cellssvaret kan vara av stor vikt för symptombilden samt sjukdomsförloppet. AV2S3pos T-celler i både lungsköljvätska och blod från HLA-DR3pos patienter visade sig reagera på mKatG, och producerade mer IFNγ i respons till mKatG, jämfört med de AV2S3neg T-cellerna. Detta, tillsammans med vår tidigare kunskap om att HLA-DR3pos patienter har god prognos, indikerar att AV2S3pos T-celler verkar för att eliminera ett hotande antigen. HLA-DR3pos patienter hade en minskad nivå av regulatoriska T-celler i lungsköljvätska, jämfört med HLA-DR3neg patienter. Vidare hade HLA-DR3pos patienter fler omogna T-celler än HLA-DR3neg patienter i lungsköljvätska, vilket tyder på att T-cellerna totalt sett är mindre aktiverade i HLA-DR3pos patienter. Detta är i linje med fynden av ett mindre uttalat Th1- respons i lungorna hos dessa patienter. AV2S3pos T-celler i lungsköljvätska var effektor- och inte regulatoriska T-celler, och mer aktiverade och differentierade än de AV2S3neg T-cellerna, vilket indikerar att de har mött ett möjligt sarkoidosantigen i lungan.

Sammanfattningsvis visar fynden i denna avhandling att sarkoidospatienter med god prognos uppvisar en effektiv multifunktionell cytokinprofil hos mKatG-specifika T-celler, ett reducerat antal regulatoriska T-celler, ett ökat antal omogna T-celler, samt aktiverade AV2S3pos effektor-T-celler, i sina lungor. Detta föreslår vi kan leda till ett effektivt immunrespons som är fokuserat på att eliminera sarkoidosantigen och leda till spontan utläkning. Denna kunskap hoppas vi ska kunna användas för att manipulera immunsvaret hos patienter med kronisk sjukdom, i syfte att utveckla nya terapier.

LIST OF PUBLICATIONS

The present thesis is based on the following papers, which will be referred to in the text by their Roman numerals.

I. Idali F, Wikén M, Wahlström J, Mellstedt H, Eklund A, Rabbani H, Grunewald J. Reduced Th1 response in the lungs of HLA-DRB1*0301 patients with pulmonary sarcoidosis. European Respiratory Journal 2006; 27:451-459.

II. Wikén M, Grunewald J, Eklund A, Wahlström J. Higher expression of TLR2 and TLR4, and enhanced pro- inflammatory synergy of TLR2 with NOD2 stimulation in sarcoidosis. Journal of Clinical 2009; 29(1):78-89.

III. Wikén M, Ostadkarampour M, Willett M, Chen E, Eklund A, Moller D, Grunewald J, Wahlström J. Multifunctional T cell responses against the mycobacterial protein mKatG in sarcoidosis patients with Löfgren´s syndrome. Submitted

IV. Wikén M, Grunewald J, Eklund A, Wahlström J. Phenotyping of lung and blood CD4pos T cells in patients with pulmonary sarcoidosis. Manuscript

CONTENTS

1 INTRODUCTION ...... 1

1.1 GENERAL INTRODUCTION...... 1 1.2 THE IMMUNE SYSTEM...... 2 1.2.1 Innate immunity...... 2 1.2.1.1 Innate immune cells ...... 2 1.2.1.2 Innate immune recognition...... 4 1.2.2 Adaptive immunity...... 7 1.2.2.1 MHC molecules and antigen presentation...... 7 1.2.2.2 Adaptive immune cells ...... 8 1.2.2.3 T cell markers...... 14 1.2.2.4 Inflammatory mediators...... 15 1.3 THE RESPIRATORY SYSTEM ...... 18 1.3.1 Lung immunity ...... 19 1.3.2 Interstitial lung disease...... 19 1.3.2.1 BAL - the procedure for collecting lung fluid and cells...... 20 1.4 SARCOIDOSIS ...... 21 1.4.1 Epidemiology...... 21 1.4.2 Clinical features...... 21 1.4.3 Diagnosis and treatment ...... 22 1.4.4 Aetiology...... 23 1.4.4.1 Infectious and non-infectious triggers...... 23 1.4.4.2 Genetic factors...... 23 1.4.4.3 Autoimmunity...... 24 1.4.5 Immune pathology...... 24 1.4.5.1 Granuloma formation...... 25 1.4.5.2 Inflammatory cells ...... 27 1.4.5.3 Models for sarcoidosis...... 29 2 COMMENTS ON SUBJECTS & METHODS...... 30

2.1 STUDY SUBJECTS...... 30 2.2 SAMPLING...... 30 2.3 METHODOLOGY...... 31 2.3.1 Soluble proteins in supernatants...... 31 2.3.2 Gene expression...... 32 2.3.3 In vitro stimulation...... 33 2.3.4 Flow cytometry ...... 35 2.4 STATISTICAL ANALYSIS ...... 36 3 AIMS...... 37

4 RESULTS & DISCUSSION ...... 38

4.1 BAL FLUID CHARACTERISTICS ...... 38 4.2 INNATE IMMUNITY IN SARCOIDOSIS ...... 39 4.2.1 Role of TLR and NOD in sarcoidosis (Paper II) ...... 39 4.3 ADAPTIVE IMMUNITY IN SARCOIDOSIS ...... 44 4.3.1 Cytokine profile in BAL fluid (Paper I) ...... 44 4.3.2 Role for mycobacterial proteins in sarcoidosis (Paper III)...... 46 4.3.3 T cell phenotyping (Paper IV) ...... 51 5 CONCLUDING REMARKS...... 58

6 SUMMARY FIGURE...... 59

7 FUTURE PERSPECTIVE...... 60

8 ACKNOWLEDGEMENTS ...... 61

9 REFERENCES...... 64

ABBREVIATIONS

ACE Angiotensin-converting enzyme AM Alveolar APC Antigen presenting cell AV2S3 Variable gene segment 2.3 of the T cell receptor α chain BAL Bronchoalveolar lavage BAL fluid Bronchoalveolar lavage fluid BCR B cell receptor BFA Brefeldin A BHL Bilateral hilar CBA Cytometric bead array CBD Chronic beryllium disease CCR Chemokine receptor CD Cluster of differentiation CDR Complementarity determining region CTLA-4 Cytotoxic T -associated antigen 4 DC

DLCO Diffusing capacity of the lung for carbon monoxide EN Erythema nodosum ER Endoplasmatic reticulum FACS Fluorescence-activated cell sorter

FEV1 Forced expiratory volume in 1 second FITC Fluorescein isothiocyanate FoxP3 Forkhead box protein 3 FSC Forward scatter (cell size) FVC Forced vital capacity HLA Human leukocyte antigen IFNγ Interferon-γ Ig Immunoglobulin IL Interleukin ILD Interstitial lung disease iTreg Inducible regulatory T cell LPS Lipopolysaccharide mAb MFI Median (or mean) fluorescence intensity MHC Major histocompatibility complex mKatG Mycobacterial catalase-peroxidase NK cell Natural killer cell NKT cell Natural killer T cell NOD Nucleotide-binding oligomerization domain receptor nTreg Natural regulatory T cell PBL Peripheral blood

PBMC Peripheral blood mononuclear cells PBS Phosphate-buffered saline PCR Polymerase chain reaction PE Phycoerythrin PGN Peptidoglycan PPD Purified protein derivative PRR Pattern recognition receptor RT-PCR Reverse transcriptase-polymerase chain reaction SEA Staphylococcus enterotoxin A SEB Staphylococcus enterotoxin B SSC Side scatter (cell granularity) TCR T cell receptor TGFβ Transforming growth factor-β Th cell T helper cell TLR Toll-like receptor TNF Tumor factor Tr1 cell Regulatory T cell type 1

Introduction 1 INTRODUCTION 1.1 GENERAL INTRODUCTION

Sarcoidosis was first described in 1899 by the Norwegian physician Caesar Boeck, and was at that time considered to be a skin disease. Today we know that almost any organ in the body can be affected, however, the lungs are involved in more than 90% of all cases. The hallmark of disease is granuloma formation, as well as infiltration of T cells in the lower airways. In order to study these cells, bronchoalveolar lavage is performed.

Although sarcoidosis has been known for more than 100 years, the cause is still unknown. Familial clustering and racial differences suggest a genetic predisposition, and findings of seasonal clustering indicate that also environmental factors are of significance for disease development. The clinical presentation and outcome varies; some patients present with acute disease onset and good prognosis, whereas other show insidious onset, persistence, and are at risk of developing lung fibrosis.

The overall aim of this thesis was to get a better understanding of immunopathogenic mechanisms in sarcoidosis, with focus on distinct patient subgroups, by studying both innate and adaptive immune responses.

Hopefully, this thesis will bring some light into the mystery of sarcoidosis.

1 Maria Wikén

1.2 THE IMMUNE SYSTEM

The immune system is a network of organs, tissues and cells that work together with the purpose to protect the host from harmful pathogens, i.e. bacteria, viruses, fungi and parasites, as well as other foreign substances. In the absence of an effective immune defence, even minor infections can be very serious and in some cases even cause death. The immune system is divided into innate (non-specific) and adaptive (specific) immunity [1], each with distinct cell types and functions. However, secreted soluble molecules, a well as cell-cell interactions, bridge these two systems.

1.2.1 INNATE IMMUNITY

The first line of defense towards invading pathogens is the innate, or non-specific, immune system. Innate immunity is always active and responds immediately when foreign microbes enter the lung or other tissues. There is no requirement for prior exposure, and it cannot be strengthened with subsequent exposure [2]. The includes three barriers; the anatomic barriers, which involves skin, tears, saliva, mucus and cilia in the intestinal and respiratory tract; the humoral barriers with the , i.e. a number of small proteins found in the blood (generally synthesized by the liver) that mark pathogen for destruction through ; the cellular barriers, which involves recruitment of inflammatory cells (see 1.2.1.1 Innate immune cells, p. 2) [1].

1.2.1.1 Innate immune cells PHAGOCYTES Phagocytes are a group of white blood cells specialized in finding, internalizing and digesting foreign microorganisms, with a process called [1].

Mononuclear cells (Monocytes and ) Monocytes are immune effector cells derived from the . They circulate in the blood, and upon infection, they migrate into the tissues, using their chemokine receptors and adhesion molecules. In addition to their ability to engulf cells and toxic molecules, they can also differentiate into dendritic cells or macrophages during depending on the inflammatory milieu [3].

Macrophages are derived from blood monocytes and are found in most tissues of the body [1]. They are long-lived and can survive within the body from a couple of months up to several years. Macrophages are large mononuclear phagocytic cells that clear approximately 2×1011 erythrocytes each day, as well as other cells and cellular debris, thus they are called the “big eaters”. Phagocytosis of pathogens usually leads to macrophage activation and thereby release of pro-inflammatory , e.g. inter-

2 Introduction leukin (IL)-1, IL-6 and (TNF). Macrophages also phagocytize apoptotic cells [4], which is a process that prevents secondary necrosis from occurring, and instead leads to production of anti-inflammatory cytokines, e.g. transforming growth factor (TGF)-β [5], thus helping maintain tolerance to self. Macrophages also act to modify the immune responses of other cells. For example, macrophages induce proliferation of T cells [6], and they can down-regulate immunoglobulin (Ig)-G and IgM production by B cells [7].

Macrophages exhibit a remarkable plasticity that allow them to efficiently respond to environmental signals, resulting in change in their phenotype [8]. Different cytokines produced by immune cells give rise to different macrophage subtypes. Classically activated macrophages arise in response to IFNγ and possess microbicidal activity, alternatively activated macrophages develop in response to IL-4 and play an essential role in tissue repair, and regulatory macrophages are generated in response to IL-10 and have anti-inflammatory activity [9].

Neutrophils are short-lived innate immune cells, normally found circulating in the blood stream. Neutrophils recognize pathogens covered with antibodies or complement proteins, i.e. opsonized pathogens, or via pattern-recognition receptors [1] (see 1.2.1.2 Innate immune recognition, p. 4). Upon activation, neutrophils can eliminate microorganisms by using a range of different mechanisms, including phagocytosis, of oxygen radicals, or releasing cytotoxic peptide- or protein-containing granules [2].

Dendritic cells Dendritic cells (DCs) are derived from the bone marrow, and function as sentinels of the immune system [10]. Immature DCs circulate as precursors in the blood before migration into peripheral tissues, especially into those areas that provide an environmental interface, such as the draining lymph node of the lungs. Immature DCs in the skin are called Langerhans cells [1]. They contain large granules, named Birbeck granules, which function as phagosomes. Upon infection, these cells migrate to regional lymph nodes, where they rapidly lose the ability to engulf and process antigens, yet they synthesize new MHC molecules that present pathogenic peptides. In addition to high MHC molecule expression, DCs also express CD80/CD86 (B7.1/B7.2) that co- stimulate naïve T cells and induce immune responses.

NATURAL KILLER CELLS Natural killer (NK) cells are a bone-marrow derived small distinct subset of lymphocytes in the first line of defence against viral infection and cancer [11, 12]. NK cells circulate in the blood, where they become activated by cytokines, including type I interferons and IL-12 [13, 14], or by cells expressing ligands for NK cell receptors [15]. Upon activation, NK cells lyse target cells by exocytosis of perforin and granzyme, as

3 Maria Wikén well as mediate immune responses by secreting cytokines, such as IFNγ, IL-5 and IL- 10 [16].

NATURAL KILLER T CELLS Classical natural killer T cells, i.e. invariant NKT (iNKT) cells, are a subset of T cells that are suggested to bridge the gap between innate and adaptive immunity. Similar to NK cells, iNKT cells express the surface marker NK1.1 (CD161), and similar to T cells they express CD3, as well as an αβ T cell receptor (TCR). iNKT cells are defined as being restricted for CD1d, i.e. a non-polymorphic MHC class I-like protein, and in humans they express the invariant TCR Vα24Jα18 chain combined with Vβ11 [17]. They are more limited in their recognition, but are specific for glycolipid antigens, e.g. the marine sponge derived α-galactosylceramide (α-GalCer) presented by CD1d molecules [18]. iNKT cells are immune-regulatory T cells that modulate immunity towards infectious organisms, including bacteria and viruses, as well as in autoimmunity and allergy [17, 19], by rapidly producing a broad range of cytokines of both Th1 type (IFNγ) and Th2 type (IL-4) within minutes to hours after antigenic stimulation.

1.2.1.2 Innate immune recognition Cells of the innate immune system express pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs) [20]. PAMPs are conserved motifs unique to microorganisms and essential for their metabolism and survival. The principal function of PRRs includes activation of complement, opsonization, phagocytosis, induction of inflammatory cytokines and induction of programmed cell death (). PRRs are functionally divided into two classes; those involved in cell recognition, i.e. the endocytic PRRs, and those that are involved in cell signaling [21].

ENDOCYTIC PRRs Endocytic PRRs are found on the phagocytic cell surface and promote attachment of microorganisms. The mannose receptor, a receptor belonging to the C-type lectin family, binds carbohydrate motifs on several pathogens, e.g. bacteria, viruses, fungi and parasites, and the scavenger receptor binds bacterial surface cell wall components. Opsonin receptors are soluble molecules, that includes acute phase proteins, complement pathway proteins and surfactant proteins, which are produced as part of the host´s immune defenses [22].

SIGNALING PRRs Signaling PRRs recognize microorganisms and trigger signaling pathways that lead to production of inflammatory cytokines. The best studied signaling PRRs are Toll-like

4 Introduction receptors (TLRs) and Nod-like receptors (NLRs) [23], which recognize different classes of pathogens, such as bacteria, viruses and protozoa.

Toll-like receptors The Toll receptor, discovered in Drosophila (fruit-flies) in 1985, was found to be essential in early fruit-fly development [24], and it was later revealed that the Toll pathway played a critical role in the immune defence, e.g. towards fungal challenge [25]. The mammalian homologue, then called the Toll-like receptor (TLR), was later identified [26]. To date, 10 TLRs have been found in humans, and 13 have been found in mice [27]. TLRs are differentially expressed (inducible or constitutively) in distinct cell types and tissues. In humans, TLR1, 2, 4, 5, 6 and 11 are expressed on the cell surface, whereas TLR3, 7, 8 and 9 are localized in endosomal compartments.

There are three general categories of TLR ligands: proteins, nucleic acids (DNA and RNA), and lipid-based elements (cell wall components) [2, 28] (Figure 1).

Figure 1. The 10 known human Toll-like receptors (TLRs), their cellular localization and the respective ligands. Inspired by Kaufmann [29].

Since TLRs cooperate with each other they provide combinatorial mechanisms, thus exhibiting wide recognition. Among all TLRs, TLR2 has the broadest specificity, since

5 Maria Wikén it can form a homodimer with another TLR2 molecule, or form a heterodimer with either TLR1 or TLR6. TLR2/TLR1 binds triacylated lipopeptides, whereas TLR2/TLR6 recognizes diacylated lipopeptides [30]. TLR2 is expressed on monocytes, mature macrophages and dendritic cells (DCs), and mast cells, and can bind various microbial pathogens, such as lipoprotein from Gram-negative bacteria [31], peptidoglycan (PGN) or lipoteichoic acid from Gram-positive bacteria [32], as well as lipoarabinomannan from mycobacteria [33, 34]. TLR4 is expressed predominantly on monocytes, mature macrophages and DCs, mast cells and the intestinal , and is the main receptor for lipopolysaccharide (LPS) from Gram-negative bacteria [35]. In addition, TLRs can also be found on for example T cells [36], B cells [37], neutrophils, natural killer cells [38], as well as on airway [39] and alveolar type II epithelial cells [40].

TLRs are characterized by three common structural features; a ligand binding extracellular domain with leucine-rich repeats, a short transmembrane region, and a highly homogenous cytoplasmic Toll/interleukin-1 receptor (TIR) domain [41]. The extracellular domain is thought to be involved in the recognition of pathogens [42]. Upon binding of ligands, the TLRs dimerize and undergo the conformational changes required for recruitment of downstream signaling molecules [43]. TLR activation results in activation of transcription factors, such as nuclear factor-kappaB (NFκB), which controls the expression of inflammatory cytokines.

Humans with mutations in TLR2 show increased susceptibility to staphylococcal infection [44], and TLR4 mutations are associated with septic shock [45]. In addition, TLR2- and TLR4-knockout mice exhibit impaired clearance of and thereby increased mortality [46, 47]. Although the roles for TLRs in human disease are not completely understood, TLRs are considered to be potential therapeutic targets. For example, inhibitors of TLR2, TLR4, TLR7 and TLR9 could be used in treatment of sepsis and inflammatory diseases [48].

Nod-like receptors The Nod-like receptors (NLRs) are a family of more than 20 soluble cytoplasmic proteins that recognize intracellular pathogens. Among all NLRs, NOD (nucleotide- binding oligomerization domain) 1 (CARD4) and NOD2 (CARD15) have primarily been studied. NOD1 is expressed in nearly all tissues and cells, whereas NOD2 is constitutively or inducible expressed in monocytes, macrophages, T cells, B cells and dendritic cells (DCs) [49]. NOD1 senses PGN that is commonly found in Gram- negative bacteria [50], whereas NOD2 binds muramyl dipeptide (MDP) [51] that serves as the largest PGN motif present in both Gram-negative and Gram-positive bacteria. However, the mechanism of how PGN reaches the cytosol is still unknown.

NODs have been found to be genetically linked to inflammatory diseases. NOD1 polymorphisms have been demonstrated in Japanese sarcoidosis patients [52], and mutations in NOD2 predispose to granulomatous diseases, including Crohn´s disease

6 Introduction

(i.e. inflammation in the intestine) [53], Blau syndrome (i.e. an autosomal dominantly inherited syndrome characterized by granulomatous arthritis and skin granulomas) [54] and early-onset sarcoidosis (i.e. a rare disease that appears in children younger than four years of age, and affects skin, joint and eyes, but without pulmonary involvement) [55], but not in adult sarcoidosis. In addition, polymorphisms in NOD1 and NOD2 have also been reported in non-granulomatous disorders, such as asthma [56] and cancer [57].

1.2.2 ADAPTIVE IMMUNITY

When a pathogen breaks through the innate immune defence barrier, the adaptive immune system takes over. The adaptive immune system is more complex than the innate immune system, and needs several days to become protective. It is characterized by specificity, which includes recognition of different structures of foreign processed antigens, by antigen-specific receptors on B cells and T cells. Adaptive immunity is also characterized by memory. When the antigen has been removed, most antigen- specific cells undergo apoptosis. However, some cells persist, leading to a more rapid and effective immune response upon re-encountering the same antigen.

1.2.2.1 MHC molecules and antigen presentation T cells recognize antigenic peptides bound to major histocompatibility complex (MHC) molecules [1]. MHC molecules are highly polymorphic glycoproteins that in humans are referred to as human leukocyte antigen (HLA) molecules. The HLA complex is located on chromosome 6, and consists of more than 200 genes divided into three classes. Commonly, HLA genes involved in immune responses fall into HLA class I and II, whereas HLA class III contains genes coding for immune modulating functions, e.g. complement. HLA class I involves HLA-A, HLA-B and HLA-C, and HLA class II involves HLA-DP, HLA-DQ and HLA-DR. More than 100 different diseases have been associated with various alleles of the HLA genes.

MHC class I molecules are expressed on all nucleated cells, and are involved during infection with intracellular pathogens, such as viruses or intracytosolic bacteria. Proteins of the intracellular organisms are fragmented into peptides in the proteasome (a protein complex with proteolytic activity), and are then transported to the endoplasmatic reticulum (ER), in which these peptides, usually 8-10 amino acids long, bind to the MHC class I molecules. The peptide:MHC class I complex is transported to the cell surface, where it is presented for the TCR of a CD8+ T cell [1].

MHC class II molecules are presented on a limited group of cells, i.e. the professional antigen presenting cells (APCs): macrophages, dendritic cells and B cells. The MHC class II molecules are constitutively expressed by these cells, but they can also be induced on different cells by cytokines, in particular by interferon-γ (IFNγ). Proteins of

7 Maria Wikén extracellular organisms, e.g. bacteria, are engulfed by phagocytosis or endocytosis and end up in an endocytic vesicle. The vesicles fuse with lysosomes to form phagolysosomes, and the proteins are degraded into peptides by proteases. MHC class II molecules are transported to the phagolysosome via ER and the Golgi apparatus, in which they capture their peptides, usually longer than 13 amino acids, whereupon the peptide:MHC class II complex is transported to the cell surface, and are presented for the TCR of a CD4+ T cell.

1.2.2.2 Adaptive immune cells The two major effector cells in the adaptive immune response are B cells and T cells. They distinguish one microorganism from another by the use of unique cell-surface receptors, B cell receptors (BCR) and T cell receptors (TCR). The antigen receptors are clonally distributed, with each B cell or T cell only having one unique receptor- specificity. Therefore, the selectively of immune responses relies on a constant availability of a large and diverse lymphocyte pool. The number of unique BCRs or TCRs is estimated to be approximately 2.5×107 in each individual. However, the human genome is considered to contain only 20-25 thousand genes. This equation is possible to solve due to gene segment rearrangements (discussed in section T cells, p. 9). Binding to the unique receptors results in an antigen-induced clonal expansion of B cells and T cells, which explain how the adaptive immune response can be very focused and specific [1].

B cells B cells are derived from the bone marrow, were they remain until matured. They are then found circulating in the blood, from which they migrate to various parts of the body, yet they concentrate in lymph node, spleen and liver. The B cells represent about 5-15% of the circulating lymphoid pool, and of all cells in the lung of healthy non- smokers, B cells account for 1-1.5% [58]. Each B cell has its unique surface expressed B cell receptor (BCR) (binds intact proteins), which is a membrane-bound immunoglobulin, consisting of the heavy chains V (variable) segment, D (diversity) and J (joining) segments, as well as the light chains V and J segments [59]. The BCRs are generated through somatic rearrangement of the gene segments, generating a great diversity among B cells. The BCRs are found in over a thousand identical copies scattered over the entire cell surface.

Before activation, the surface-bound BCRs are of IgM and IgD type, however, activation promotes isotype switching, resulting in development of antibodies with different heavy chains, each having distinct effector functions. B cells can be activated in a T cell-dependent or T cell-independent manner [1]. Most antigens are T cell- dependent, i.e. the first signal is delivered through binding of an antigen to the BCR, and the second signal comes from co-stimulation, i.e. via interaction between CD40 on the B cell and CD40-ligand on a T helper cell. In the T cell-independent activation, the

8 Introduction second signal can come from the antigen itself, e.g. direct binding of an antigen-motif to an innate immune receptor.

After activation, the B cells differentiate into plasma cells or memory cells. Plasma cells are short-lived and produce large amounts of specific antibodies that are released into the blood, lymph, linings of lungs and gut. The antibodies can function in several ways; antibodies bind microbes, making them easier targets for phagocytosis by APCs, such as macrophages; antibodies are capable of neutralizing viruses and toxins, thus preventing the viruses to infect other cells, as well as preventing toxins to cause further harm; antibodies can cover pathogens, leading to a more efficient elimination by complement proteins. Memory B cells, which comprise a small fraction of the B cell clone, remain in the circulation for a long time, and if a re-infection take place, these cells divide rapidly resulting in more plasma and memory cells [1, 60].

T cells T cells are derived from the bone marrow, but migrate as immature thymocytes to the thymus, were they mature. In the thymus the T cell undergoes gene arrangements, leading to development of its membrane-bound unique T cell receptor (TCR). Each T cell expresses approximately 3000 identical TCR on its surface [61]. The TCR consists of two disulphide-bond connected polypeptide chains, α and β, of which the β chain is formed first. The TCR consists of a constant (C) region, as well as a variable (V) region. The V region is responsible for the antigen recognition, and it is formed through rearrangements of V and J (joining) gene segments in the α chain, and V, J and D (diversity) gene segments in the β chain. Each V domain further includes three variable regions, the complementarity determining regions (CDR1, CDR2 and CDR3). CDR1 and CDR2 make contact with the MHC molecule outside the peptide-binding groove, whereas CDR3 shows the highest variability and diversity, and is responsible for the main contact between the TCR and the peptide in the peptide-binding groove of the MHC molecule [1].

In addition to αβ TCR, a minority (<5% of total T cells in peripheral lymphoid organs) of TCRs is composed of γ and δ chains. The primary function of the γδ T cells is not well understood [62], however they are mainly found in epithelial-containing organs, e.g. lung and intestine, suggesting involvement in immune responses towards microbial pathogens and toxic substances [63]. Upon bacterial or viral infections their number is increased [64].

Approximately 98% of the thymocytes that develop in the thymus die there, during the processess of positive and negative selection [1]. Immature thymocytes are negative for CD3, CD4 and CD8, and give rise to the two T cell lineages: the minor γδ T cell subset, which are CD4 and CD8 negative, and the major αβ T cells that are CD4 and CD8 positive, i.e. double positive thymocytes. These double positive thymocytes go through the positive selection, in which αβ T cells that have a moderate affinity to MHC

9 Maria Wikén molecules are selected, whereas T cells with no or low affinity to MHC molecules are removed. Thereafter, the negative selection occurs; in which αβ T cells with too strong affinity for self antigens are depleted through apoptosis. Around 2% of all αβ T cells survive the positive and negative selection process. If the T cell binds to MHC class I, CD4 expression is lost and the cell becomes a CD8+ T cell, and if the T cell binds to MHC class II, CD8 expression is lost and the cell becomes a CD4+ T cell. The T cells can now leave thymus as mature, but naïve cells, ready to be primed by antigens. Depending on cytokine milieu, the T cells will differentiate into distinct T cell subsets [1].

Activation of the naïve T cells requires two independent signals delivered by the same APC. Signal 1 is mediated via the CD3 subunits when the TCR binds a foreign peptide presented on the MHC molecule on an APC. The co-receptors, CD4 on the CD4+ T helper cell or CD8 on the cytotoxic CD8+ T cell, help to stabilize this binding. Signal 2 is mediated when the co-stimulatory proteins CD80 (B7.1) or CD86 (B7.2) on the APC binds the CD28 molecule on the naïve T cell. This second signal permits the T cell to respond to the antigen [1].

CD4+ T helper cells CD4+ T helper (Th) cells are key players in the adaptive immune response. Presently, the naïve CD4+ Th cells are divided into four lineages, i.e. Th1, Th2, Th17 and regulatory T cells [65] (Figure 2). Key elements controlling the CD4+ T cell differentiation include the cytokine environment during priming, as well as the transcription factor activation.

Figure 2. Naïve CD4+ T cells differentiate into Th1, Th2, Treg or Th17 cells, each characterized by a distinct transcription factor (Tbet, GATA3, FoxP3 or RORγt), depending on the cytokine milieu. Inspired by Kaufmann [29].

10 Introduction

Th1 and Th2 cells The differentiation of naïve CD4+ T cells into Th1 and Th2 effector T cells is controlled by several factors, including APCs, cytokine milieu, means of infection, as well as antigenic dose [66]. The activation status of the APC, i.e. DC, has been suggested to determine the ability to selectively prime the naïve T cells to develop into distinct T cell subtypes [67, 68]. This activation is mediated by PAMPs that bind to PRRs expressed on the DCs [69]. TLR ligands, such as LPS, bacterial DNA or viral double-stranded RNA can promote IL-12 production, and activate DCs that promote differentiation of Th1 cells [70, 71]. The Th1 cells are characterized by the expression of the transcription factor Tbet [65], as well as the production of IFNγ, IL-2 and lymphotoxin. These cells are associated with inflammation, tissue injury [72], mediate cell-mediated immunity, and are involved in clearance of intracellular pathogens [1]. Other TLR ligands, including products of parasites, yeast and cholera toxins, can promote production of IL-4, and IL-2 or IL-7, and activate DCs that direct the induction of Th2 cells [71, 73, 74]. The Th2 cells are characterized by the expression of transcription factor GATA3 [65], as well as secretion of IL-4, IL-5, IL-6, IL-10 and IL-13. These cytokines promote antibody production, including an immunoglobulin (Ig) class switch to IgG4 and IgE. Th2 cells mediate humoral immunity and are involved in clearance of extracellular pathogens, parasites and toxins [1].

Th1 and Th2 subsets regulate each other in that Th1 cell-produced IFNγ may inhibit the proliferation and function of Th2 cells, and Th2 cell-produced IL-4 and IL-10 suppress the cytokine production of Th1 cells. An imbalance in Th1/Th2 subsets is seen in various disorders. In Crohn´s disease and sarcoidosis, Th1 cell-dominance is seen, whereas Th2 responses are involved in atopic disorders. In the airways of asthmatics, increased levels of Th2 cells, as well as Th2-associated cytokines (IL-4 and IL-5), are found.

Regulatory T cells Regulatory T cells (Tregs) are a subset of T cells with the function of balancing inflammation and antigen specific immune responses, including prevention of autoimmune disease by maintaining immunologic self-tolerance [75, 76] and suppression of allergy and asthma [77]. Some markers used to define Tregs are CD25 [75], CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) [78], GITR (glucocorticoid-induced TNF receptor family-related gene) [79] and CD127low [80]. However, these markers are not strictly Treg-specific, since they can become up- or down-regulated upon T cell activation [81, 82]. Tregs are usually divided into natural thymic derived Tregs (nTreg) and peripherally induced Tregs (iTregs).

It has been proposed that CD4+ thymocytes that bear an autoreactive TCR with high affinity for self-peptides are induced to become CD4+CD25+ Tregs [83]. These so called natural Tregs are a distinct T cell lineage [84-87], and constitute 5-10% of CD4+ T cells in human blood. The natural Tregs are defined as CD4+CD25high cells that

11 Maria Wikén express the X-linked forkhead/winged-helix transcription factor box P3 (FoxP3). FoxP3 is critically important for Treg cell development and function [88], and has been suggested to be the most reliable marker for these cells [89, 90]. FoxP3 expression is highly restricted to αβ T cells, and is almost undetectable in other immune cells [91].

However, it has been shown that almost every T cell that gets activated goes through a phase of FoxP3 expression [81, 92-95], thus making it harder to use it as a distinct natural Treg marker. Mutations in the human FOXP3 gene may lead to multi-organ autoimmune disease [96, 97] and abnormalities in the Treg cell regulation have been described in many chronic inflammatory and autoimmune disorders, including atherosclerosis and rheumatoid arthritis [98-100].

Tregs can also be generated in the periphery from conventional naïve CD4+ T cells, e.g. under experimental conditions this has been shown after intravenous or oral antigenic administration [101], or after allograft transplantation [102], giving them the name inducible regulatory T cells (iTregs). Depending on disease setting and the site of regulatory activity they have a variable expression of CD25 [103]. Examples of iTregs are the IL-10-secreting Tr1 cells [104] and TGFβ-secreting Th3 cells [105].

Tregs can suppress activation and immune cell expansions by using different mechanisms (Figure 3).

Figure 3. Regulatory T cell (Treg) mechanisms for suppression, including suppression by inhibitory cytokines, granzyme-mediated suppression, or suppression of APCs. Inspired by Shevach [106].

Suppression by cytokines: Tregs secrete IL-10 and TGFβ that directly inhibit the function of the conventional T cells [104, 105].

12 Introduction

Suppression by cytolysis: It was previously thought that only NK cells and CD8+ T cells exhibited cytotoxic activity, i.e. perforin-granzyme mediated killing [107]. However, it was later shown that CD4+ T cells in humans, but not in mice, also displayed this capacity. Activated human FoxP3+CD4+ Tregs have been found to express granzyme A [108, 109], and can thereby directly kill effector cells, such as activated CD4+ and CD8+ T cells.

Suppression by affecting APCs: Tregs can suppress the function of APCs and indirectly prevent activation of naïve T cells. CTLA-4 is constitutively expressed only on Tregs, and can interact with the co-stimulatory molecules CD80 and CD86 on DCs, and thereby down-regulate or prevent their up-regulation [110, 111]. In addition, the CD4 homolog LAG-3, expressed on Tregs, can interact with MHC class II on immature DCs with high affinity [112]. Upon binding, an inhibitory signal is induced that suppresses DC maturation, as well as immune stimulation.

Th17 cells Th17 cells are induced in parallel with Th1 cells, but develop in response to TGFβ, and IL-6 or IL-21. In addition, IL-23 is essential for maintenance of Th17 effector functions [113]. Th17 cells are characterized by the expression of the transcription factor retinoid orphan receptor gamma T (RORγt) [65], and these cells provide protection against extracellular bacteria and fungi, particularly at epithelial surfaces [113, 114]. Th17 cells are characterized by production of IL-17A and IL-17F, both belonging to the IL-17 family [115], as well as IL-21 [116] and IL-22 [117]. IL-17 (IL-17A) recruits neutrophils to the site of infection [118], acts on macrophages to promote their recruitment and survival [119], and stimulate production of pro-inflammatory cytokines and anti-microbial peptides [117, 120]. IL-17 has also been shown to enhance the proliferation of conventional T cells and Tregs [121]. In autoimmune diseases, such as rheumatoid arthritis [122], multiple sclerosis [123] and uveitis [124], an increased expression of IL-17 expression has been demonstrated. In addition, Th17 cells are involved in the defence system of pulmonary infections [125].

CD4+ cytotoxic T cells A subpopulation of the CD4+ T cells, presenting at low frequency in the circulation of most healthy individuals, carry granzyme B and perforin-containing granules. This gives them direct cytotoxic capabilities in a peptide-specific and MHC class II- restricted manner [126]. The cytotoxic CD4+ T cells are characterized by having lost their expression of the co-stimulatory molecule CD28 [126, 127]. The number of cytotoxic CD4+ T cells are increased in chronic inflammatory conditions, such as inflammatory bowel disease (IBD) [128], indicating a pathogenic role in inflammatory diseases.

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CD8+ cytotoxic T cells The role of CD8+, or cytotoxic, T cells is to eliminate in particular viral intracellular infectious. When CD8+ T cells encounter its antigen, it starts to secrete cytotoxins, such as perforin, granzymes and granulysin. Perforin forms pores in the plasma membrane of the target cell, allowing granzymes to enter the cell. Granulysin has antimicrobial function and creates holes in the target cell membrane and thereby destroys it. Upon stimulation with IL-12 and IL-18, or in response against chronic virus infection, CD8+ T cells rapidly produce IFNγ [129]. Although FoxP3 expression is associated with CD4+ Tregs, several studies have shown that also regulatory FoxP3-expressing CD8+ T cells exists [130-132]. For example, FoxP3+CD8+ T cells have been found after in vitro stimulation with human hepatitis C virus [133]. The FoxP3-expressing CD8+ T cells are mainly found within the human blood [134].

1.2.2.3 T cell markers The cluster of differentiation (CD) is a nomenclature used for identification and investigation of cell surface molecules present on leukocytes. The CD molecules can act as adhesion molecules, receptors, or play a role in cell signaling. To date, there are approximately 350 known CDs. For example, CD3 is used to define T cells, CD4 is expressed on T helper cells and CD8 is expressed on cytotoxic T cells.

CD25, or the α chain of the IL-2 receptor, is a type I transmembrane protein important for differentiation and proliferation. CD25 was first known as an early activation marker, however, a high expression of CD25 was later found to define regulatory T cells. Importantly, also non-regulatory conventional T cells have a transient expression of CD25 upon activation [95].

CD27, a member of the TNF receptor family, is expressed on naïve and subsets of memory T cells [135]. CD27 is therefore commonly used in combination with the memory T cell markers CD45RA or CD45RO. Following activation of T cells via TCR/CD3, expression of CD27 is highly induced. In addition, there is a release of a soluble extracellular part of the molecule [136]. After prolonged antigenic in vitro stimulation the CD27 expression is gradually down-regulated [137]. CD27 can also be studied in combination with CD25, thus identifying natural Tregs, since it has been shown that CD25+CD27+ T cells are highly enriched in FoxP3+ T cells [138, 139].

CD45, a transmembrane tyrosine phosphatase, is expressed on all hematopoietic cells, and plays an essential role in antigen-induced lymphocyte activation [140]. CD45 exists in different isoforms, and among human T cells there are two major subsets, i.e. those T cells expressing the high-molecular weight-isoform CD45RA (defining naïve T cells), and those that express the low-molecular weight-isoform CD45RO (defining central memory and effector memory T cells) [141]. Whereas CD45RA T cells are small

14 Introduction resting cells that divide only rarely, the CD45RO T cells are activated to a higher degree, and divide relatively frequently [142].

CD69 is up-regulated within 1-2 hours after TCR engagement [143], and is said to be an early activation marker.

FoxP3 is the transcription factor for natural Tregs, and is considered to be the most reliable marker for this T cell subset [89, 90]. The expression of FoxP3 is highly restricted to αβ T cells, and is almost undetectable in B cells, γδ T cells, NK cells, macrophages and dendritic cells [91]. FoxP3 is essential for Treg function, and FoxP3 deficiency results in severe autoimmune diseases [96, 97]. Due to the intracellular localization, isolation of live cells to be used for functional assays, by using the FoxP3 marker, is impossible.

1.2.2.4 Inflammatory mediators Cytokines are short-lived soluble proteins produced by leukocytes, as well as other cells, with a molecular weight between 8-40 kD. These molecules act via specific receptors and upon binding, signaling cascades are promoted leading to up-regulation or down-regulation of genes in target cells. Some cytokines promote inflammation, and are called pro-inflammatory cytokines, whereas other cytokines suppress inflammation, and thus are called anti-inflammatory cytokines. In addition, cytokines can also be divided into e.g. Th1 or Th2 types (as previously described, see Th1 and Th2 cells, p. 11). An imbalance between effector cytokines, including Th1/Th2-, Th17- or anti- inflammatory regulatory T cell-cytokines, may determine the outcome of disease [144]. In addition, chemokines belong to a super-family of small chemotactic proteins that facilitate trafficking and re-circulation of immune cells from the circulation and tissue, into secondary lymphoid organs and different peripheral tissues [145]. Chemokines mediate their activity by binding to receptors on the cell surface of the target cells.

Listed below are selected cytokines and chemokines that are assumed to be of importance in sarcoidosis, as well as other inflammatory lung disorders;

IFNγγγ is a Th1 and pro-inflammatory cytokine produced by T cells and NK cells. It is important for activating macrophages and to increase the MHC class II expression on APC. By stimulating the macrophages to produce IL-12, IFNγ can inhibit the activities of a Th2 response.

TNF(ααα) is a Th1 cytokine produced by T cells, macrophages and NK cells. After stimulation with IL-2, it enhances the proliferation of T cells, and acts on the endothelium to promote local inflammation.

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IL-1βββ is a pro-inflammatory cytokine, which is produced by epithelial cells and macrophages, and has the capacity to activate T cells and macrophages.

IL-2 is a Th1 cytokine produced by T cells and it functions as a growth factor for the T cell in an autocrine manner, as well as for other T cells.

IL-4 is a Th2 cytokine, produced by T cells and mast cells, that activates B cells (promoting class-switching to IgE), inhibits the development of Th1 cells, increases MHC class II expression on macrophages, and acts, together with IL-10, in an immune regulatory way to decrease the activity of activated macrophages.

IL-5 is a Th2 cytokine produced by T cells and mast cells that has an essential role in asthma, where it stimulates growth and differentiation of .

IL-6 is a pro-inflammatory cytokine produced by T cells, macrophages and endothelial cells that stimulates T cell growth and differentiation. IL-6 is always found in increased levels at sites of inflammation.

IL-8 (CXCL8) is a pro-inflammatory chemokine produced by, among others, macrophages and endothelial cells. IL-8 attracts neutrophils, promotes their binding to vascular endothelial cells and thereby facilitates for the neutrophils to enter tissue where they are needed, for instance during inflammation.

IL-10 is a Th2 and anti-inflammatory cytokine produced by T cells and macrophages. It reduces macrophage functions and down-regulates Th1 cells and MHC class II expression on APCs. Together with IL-4, IL-10 acts in order to decrease inflammatory activities of macrophages.

IL-12 is a pro-inflammatory cytokine produced by macrophages, B cells and DCs that induces Th1 immune responses. IL-12 is composed of two subunits, p35 and p40, and when these are combined, the bioactive IL-12p70 is formed. However, p40 can also associate with p19 to form IL-23.

IL-13 is considered to be a Th2 cytokine, although it can be secreted by various cell types. IL-13 is primarily associated with the induction of allergic airway disease, and elevated levels have been reported in asthmatics.

IL-17, the family name of the isoforms IL-17A-F, is a proinflammatory cytokine produced by Th17 cells. IL-17 promotes the production of several cytokines (e.g. IL- 1β, IL-6, TNF and TGFβ) and chemokines (e.g. IL-8) from various cell types, and has been shown to be involved in several immune- and autoimmune associated diseases, such as asthma and rheumatoid arthritis.

16 Introduction

IL-23 is a heterodimeric cytokine that consists of the subunits p40, which is shared with IL-12, and p19. IL-23 is produced by macrophages and dendritic cells and acts to stimulate naïve CD4+ T cells to differentiate into Th17 cells.

TGFβββ exists in three isoforms, TGFβ1, TGFβ2 and TGFβ3. TGFβ1 is produced by a subset of regulatory T cells, i.e. Th3 cells, and acts to prevent activation of other cells, inhibit secretion and activity of cytokines, such as IFNγ and TNF, and down-regulates cytokine receptors, e.g. the IL-2-receptor. TGFβ2 plays a role in embryonic development, and TGFβ3 is involved in cell differentiation and development, for example in controlling lung development in mammals.

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1.3 THE RESPIRATORY SYSTEM

The main function of the respiratory system is to supply the body with oxygen and to remove carbon dioxide from the blood stream. The respiratory tract can be divided into a lower and an upper part. The upper part, which functions to filter, warm and humidify inhaled air, includes the nose, nasal cavity, pharynx (throat) and larynx (voice box). The lower part consists of the trachea (windpipe), bronchi and bronchial tree.

The respiratory system begins with the nose and mouth inhaling air, continues down through the pharynx and larynx. The larynx is covered with elastic cartilage (epiglottis) that stays open during breathing, and closes during swallowing, and thereby protects the connecting trachea from solid particles and liquids. The trachea is a 10-12.5 cm long tube that branches into right and left bronchi, and further divides approximately 23 times into smaller and smaller subdivisions, making up the bronchial tree. The right bronchus subdivides into three lobes/secondary bronchi (upper, lower and middle), whereas the left bronchus subdivides into two (upper and lower). These bronchi further divide into segmental/tertiary bronchi, terminal bronchioles (the smallest airways), and finally end up in the alveoli (Figure 4). Alveoli are small air-filled sacs in which exchange of oxygen and carbon dioxide occurs between the lungs and the bloodstream.

Diseases associated with the respiratory system include infections in the upper respiratory tract (common cold) or in the lower respiratory tract (pneumonia), obstructive lung diseases (bronchi become narrowed, resulting in airflow limitations), and restrictive lung diseases, i.e. interstitial lung diseases (incomplete lung expansion and increased lung stiffness).

Figure 4. Illustration of the human respiratory system.

18 Introduction

1.3.1 LUNG IMMUNITY

The lung contains the largest epithelial surface of the body, which serves as the first line of contact for harmful agents, including pathogenic microorganisms, allergens and pollutants. Failure of the local host defense may result in microbial colonization and subsequently infection of the airways and lung parenchyma. The airway epithelium expresses TLRs that sense microbial products, such as bacteria and viruses. Upon binding to TLRs, the airway epithelial cells increases their production of antimicrobial proteins, e.g. defensins. For example, alveolar epithelial cells express TLR2 mRNA and protein [146]. TLR2 bind products of Gram-positive bacteria and mycobacteria, thus mediating inflammatory responses. In addition, this stimulates epithelial cells to produce chemokines that recruit neutrophils into the airway lumen [147]. Usually, neutrophils represent <2% of the cells in the lungs, however, upon encounter with infectious agents, a massive influx of neutrophils occur. Both innate and adaptive immune cells will be activated that help to immobilize and kill invading pathogens.

Particles of 1µm in size or smaller, which is the size of bacterial and viral elements, are carried to the alveolar surface where they interact with soluble components in the alveolar fluid (e.g. complement proteins, IgG) and alveolar macrophages (AM). AMs normally account for 90-95% of airspace leukocytes, whereas lymphocytes represent 5- 10%, and neutrophils only 1%. AMs ingest all types of inhaled particles that reach the alveolar spaces, and the TLRs in the phagosomal membrane provide discrimination among the various microbial products entering these cells [148]. The pro-inflammatory cytokines and chemokines, in particular IL-8, produced by AMs, initiate a localized inflammatory response by recruiting neutrophils and other immune cells from the lung into the alveolar space.

1.3.2 INTERSTITIAL LUNG DISEASE

Interstitial lung diseases (ILDs) are a group of pulmonary disorders [149], including more than 100 members that all have similar clinical, radiographic, physiologic or pathologic manifestations. ILDs can be caused by a number of factors, including lung infection, environmental toxins, certain medications, and chronic autoimmune diseases. In most cases a disruption is seen in the lung parenchyma, resulting in damage of epithelial cells. In addition, basal membranes will lose their integrity, leading to influx of several inflammatory cells and increased expression of extracellular matrix [150].

Examples of ILDs are bronchiolitis (usually caused by viruses), silicosis (caused by inhalation of crystalline silica dust), allergic alveolitis (caused by exposure for bacteria or mould), chronic beryllium disease (exposure for beryllium), Wegener´s granulomatosis (unknown aetiology), and idiopathic pulmonary fibrosis (unknown aetiology). Among all ILDs in the western world, sarcoidosis is the most common.

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1.3.2.1 BAL - the procedure for collecting lung fluid and cells The bronchoalveolar lavage (BAL) technique was introduced as a research tool in 1974 by Reynolds and Newball [151], and was shortly found to be very useful for collecting cells and proteins from the lower respiratory tract [152]. The procedure involves a bronchoscope that is passed through the nose (or mouth) into the lungs, whereupon saline solution is instilled and recollected for examination.

BAL may be used as a diagnostic tool, however, one should be aware that different factors may affect the results. For example, the BAL results can be affected by the volume of instilled fluid. With larger volumes, there may be better mixing and therefore sampling of terminal bronchioles and alveoli. The BAL fluid recovery should preferentially be between at least 50-60% [153]. A condition that may affect recovery is loss of lung tissue elasticity, which is seen in patients with emphysema (destruction of alveolar walls).

20 Introduction

1.4 SARCOIDOSIS

1.4.1 EPIDEMIOLOGY

Sarcoidosis affects primarily non-smoking people [154], throughout the world, yet the incidence varies in different countries and ethnical groups, between age and gender, and with season. The annual incidence in the western world is between 10 and 25 per 100 000 [155, 156]. People in northern European countries are most commonly affected, with 5 up to 40 cases per 100 000 people [157]. A well-cited study reported that the annual incidence in Sweden was 64 per 100 000 [156]. In the United States, African-Americans are more frequently affected compared to Caucasians, and they also tend to have a more severe and chronic disease course [158]. People at all ages can develop disease, however, there is a peak incidence between 20-39 years of age [159]. Sarcoidosis is thought to be more common among females [156, 160-162], yet this difference is not that well established. Furthermore, a seasonal clustering has been described with most newly diagnosed acute patients in early spring [163]. A subgroup of Scandinavian patients, with the haplotype HLA-DRB1*0301 (discussed below, see 1.4.4.2 Genetic factors, p. 23), had peaks of incidence in January, as well as in April and May [164]. Taken together, this indicates that genetic factors, as well as environmental factors, could play a role in disease susceptibility.

1.4.2 CLINICAL FEATURES

Sarcoidosis is a heterogeneous disorder, with a considerable variation in the clinical manifestations and disease outcome. The disease is said to be systemic and affect most organs, including eyes, skin, liver, heart and the nervous system, however, the lungs are involved in more than 90% of all cases [165]. In Scandinavia, the majority of patients have a good prognosis and approximately 50% recover spontaneously within two years. Among them, Löfgren´s syndrome is commonly seen. Löfgren´s syndrome was originally described in 1952 by Sven Löfgren [166, 167], and is characterized by a combination of distinct symptoms that are easy to identify; acute disease onset with , bilateral hilar lymphadenopathy (BHL) with or without lung parenchymal infiltration, and erythema nodosum and/or ankle arthritis [168, 169]. These patients commonly have good prognosis and recover within a couple of years [170]. However, a recent study from our group showed that almost every patient with Löfgren´s syndrome that had haplotype HLA-DRB1*0301 (discussed below, see 1.4.4.2 Genetic factors, p. 23) recovered within two years, whereas 49% of Löfgren´s syndrome patients, presenting with other haplotypes, showed prolonged disease course [164]. Sarcoidosis may also present with an insidious disease onset, with dry cough, fatigue, shortness of breath and weight loss, and these patients may be at risk of developing chronic disease with pulmonary fibrosis [171]. The mortality rate of sarcoidosis is 1-5%, and death is mostly caused by heart or nervous system involvement [165].

21 Maria Wikén

1.4.3 DIAGNOSIS AND TREATMENT

The diagnosis is based on a combined evaluation of clinical symptoms, chest radiography, pulmonary function tests, findings of non-caseating epithelioid granulomas in biopsies from various tissues, and laboratory parameters [165]. The clinical symptoms vary depending on mode of onset, but dry cough, fatigue and fever are common, and in more advanced cases also weight-loss and shortness of breath. If the onset is acute, there may also be erythema nodosum mainly on the lower limbs (mostly women) and/or ankle arthritis (mostly men). Conventional chest X-ray is performed and the findings are staged 0-IV according to degree of involvement (see Table 1). Lung function is evaluated by static and dynamic spirometries to reveal signs of restrictive and/or obstructive functional changes. Diffusion capacity for carbon monoxide (DLCO) is done to measure the gas exchange, which is often reduced [172]. Findings of non-caseating granulomas in biopsies are supportive for the diagnosis. Intra-thoracic specimens may be recruited from the bronchial mucosa or lung parenchyma by endobronchial or transbronchial biopsies taken during bronchoscopy. BAL fluid lymphocytosis and a BAL CD4/CD8 T cell ratio > 3.5 will strongly support the diagnosis [173]. A laboratory parameter that further strengthens the diagnose sarcoidosis is elevated serum angiotensin converting enzyme (ACE) activity [174].

A recent study demonstrated a decreased expression of CD103 on BAL CD4+ T cells of sarcoidosis patients as compared to other interstitial lung diseases [175]. CD103 is an integrin that mediates binding to E-cadherin (a transmembrane protein important for cell adhesion, i.e. ensuring that cells within tissues are bound together), and is expressed by T cells within the bronchial epithelium [176-178], by cells in the alveolar wall and by T cells in BAL fluid [179, 180]. However, whether CD103 can be used for diagnostic evaluation of sarcoidosis is under investigation.

Table 1. Chest radiographic staging Stage Finding 0 Normal chest radiograph I Bilateral hilar lymphadenopathy (BHL) II Parenchymal infiltrations with BHL III Parenchymal infiltrations without BHL IV Volume reduction and signs of fibrosis

Since the cause of sarcoidosis is unknown, there is no specific treatment available. However, a large number of patients, in particularly those with Löfgren´s syndrome, recover spontaneously and are usually not in need for medication (except perhaps non- steroid anti-inflammatory drugs). In contrast, patients with chronic disease course, i.e. non-Löfgren´s syndrome patients or patients with extra-pulmonary involvement, may be given long-term oral corticosteroids, with or without additional cytotoxic agent (e.g. methotrexate), in order to dampen the inflammatory immune response [181].

22 Introduction

Furthermore, since TNF is critical for the maintenance of the granulomatous inflammation, anti-TNF treatment is under evaluation [182].

1.4.4 AETIOLOGY

The aetiology of sarcoidosis is still unknown, and researchers have over the past decade been trying to find new insights in potential sources leading to disease, which include infectious and non-infectious triggers, genetic factors, as well as involvement of autoimmunity. The causative agent must be capable of inducing the histological hallmarks, i.e. non-necrotizing granulomas, and the heterogeneous clinical features. Alternatively, there are several different aetiologies.

1.4.4.1 Infectious and non-infectious triggers Microbial agents Several microorganisms have been proposed to be associated with disease. Based on the histological similarities between sarcoidosis and tuberculosis, one of the most discussed agent is Mycobacterium tuberculosis [165, 183-187]. Mycobacterial antigens have been found in sarcoid granulomas [188], and T cells specific for Mycobacteria have been found expanded among peripheral blood mononuclear cells and BAL cells of sarcoidosis patients [185, 189-192]. In addition, circulating antibodies towards Mycobacterium tuberculosis have been found in serum samples of sarcoidosis patients [183-185]. Some studies instead show an association between sarcoidosis and Propionibacterium acnes [193, 194]. Treatment of P. acnes has been reported to be effective in a small subset of sarcoidosis patients with skin involvement [195].

Environmental agents Due to the global variability in prevalence and incidence of sarcoidosis, there could also be involvement of environmental agents. There is a predisposition for fire-rescue workers and health-care workers, and an increased risk has been found for workers in agriculture or when exposured to mould or pesticides. In contrast, there is a strong negative association between tobacco smoking and sarcoidosis [196], but no association between the use of moist snuff and sarcoidosis [197].

1.4.4.2 Genetic factors Findings of familiar clustering, and differences in disease outcome in different ethnical groups, support a role for the genetic background in the development of sarcoidosis.

HLA genes Several studies demonstrate the importance of genes located in the MHC region, and a couple of HLA variants, in particular of HLA class II, have been shown to associate

23 Maria Wikén with sarcoidosis [198]. The HLA-DRB1*0301 allele is strongly associated with acute disease onset and good prognosis with spontaneous recovery within two years. In contrast, haplotypes HLA-DRB1*14 or 15 are associated with chronic disease.

The HLA class I alleles have also been shown to be involved in the disease course [199, 200]. For example, in Swedish patients, HLA-A*01 was more common among those with resolving disease as compared to healthy subjects, and HLA-A*03 was shown to be associated with persistent disease when compared to controls [199]. HLA- B*07 was more common in patients, and further biased towards persistent disease.

Non-HLA genes In addition to the HLA genes, there are also non-HLA candidate genes reported to be associated with sarcoidosis [158, 201], including those that influence antigen processing and presentation, as well as macrophage and T cell activation. The Butyrophilin-like 2 gene (BTNL2) is located close to the HLA genes, and has been shown to associate with sarcoidosis, yet independently of HLA-DRB1 variations [202]. Polymorphisms of BTNL2 may influence T cell activation and regulation.

Since infections, including involvement of Mycobacteria, may be a risk factor for developing sarcoidosis, some studies propose that genetic mutations of TLR genes could be involved in the pathogenesis. However, the evidence for whether TLR2 polymorphisms are associated with sarcoidosis is contradictory [203]. Polymorphisms in TLR4 have been shown to associate with German chronic sarcoidosis patients [204], yet there was no such association in a Dutch population [205]. NOD2 (CARD15) is a susceptibility gene for the granulomatous disorder Crohn´s disease [206]. Very recently, NOD2 polymorphisms was shown to be associated with severe pulmonary sarcoidosis [207].

1.4.4.3 Autoimmunity There is evidence indicating an autoimmune component in sarcoidosis. For example, sarcoidosis is associated with specific HLA antigens and there is an infiltration of T lymphocytes in affected organs, although these features are not unique for autoimmunity. A study from our laboratory recently showed that in vitro stimulation with peptides derived from the cytoskeletal protein vimentin, resulted in a strong IFNγ response in blood of HLA-DRB1*0301+ patients [208]. Vimentin has been suggested to be an auto-antigen in rheumatoid arthritis [209].

1.4.5 IMMUNE PATHOLOGY

The immune mechanisms of sarcoidosis are still incompletely understood, however, we know that the first manifestation of disease is an accumulation of inflammatory cells, including monocytes/macrophages and T cells, in involved organs. We may hypothe-

24 Introduction size that the sarcoid stimulus is presented for the T cell receptor (TCR) on a CD4+ T cell, via MHC on an APC, and T cells and macrophages are then activated and secrete cytokines promoting recruitment, activation and proliferation of mononuclear cells, resulting in alveolitis. By orchestration of various cytokines and chemokines, granuloma formation is promoted and some patients will end up with a fibrotic lung.

1.4.5.1 Granuloma formation Granuloma is an organized structure that arises in response to continued antigenic stimulation, and functions to prevent spreading of harmful and infectious microorganisms. Their main function is to limit the inflammation and thereby protect surrounding tissue from damage. However, in sarcoidosis, as well as in other multi- systemic disorders, such as Wegener´s granulomatosis, the granulomas are not believed to be protective, and instead contribute to tissue pathology. Non-caseating epithelioid cell granuloma formation is the pathological hallmark of sarcoidosis, and these granulomas are often found in the larger airways.

A hypothesis for the granuloma formation in sarcoidosis Although the aetiology is unknown, most data support that the granuloma formation is initiated by one or several infectious agents that are presented as peptides by the MHC (HLA in humans) class II molecule of an antigen-presenting cell (APC) to the T cell receptor (TCR) of a CD4+ T cell. Resting T cells and monocytes/macrophages are activated by T cell-produced IL-2 and IFNγ, respectively, and the number of inflammatory cells, in particular T cells, increase in the lower airways, resulting in a state called alveolitis [210]. Cytokines, in particular IFNγ and TNF, stimulate monocytes to differentiate into epithelioid cells that fuse to form multinucleated giant cells with the property of being bactericidal. The central core of the granuloma consists of monocytes and macrophages at different activation and differentiation states. The core is surrounded by CD4+ T cells of Th1 type, but also a few CD8+ T cells and B cells are found in the periphery [211-214] (Figure 5).

If the infectious factors can be eliminated inside the granuloma, one would expect the inflammatory response to decline and finally be cleared. In some cases the granulomas do disappear and these patients display a limited clinical course with spontaneous resolution. However, other patients exhibit massive development of granulomas and do not recover, but instead develop fibrosis and respiratory failure, despite strong immunosuppressive therapy [215].

25 Maria Wikén

Figure 5. A hypothesis for the sarcoidosis pathogenesis. The HLA class II molecule presents an antigen for the TCR of a CD4+ T cell. IL-2 and IFNγ activate resting immune cells that initiate the granuloma formation.

Different types of granulomas The sarcoidosis granulomas (Figure 6) are said to be non-necrotizing and tightly formed, and they look very similar to granulomas found in patients with chronic beryllium disease (CBD). However, granulomas of CBD patients are formed in response to beryllium salts, and it is therefore of most importance to set the anamneses carefully [216]. Granulomas found in patients with tuberculosis are necrotizing.

Figure 6. A non-caseating epithelioid cell granuloma compatible with sarcoidosis.

26 Introduction

1.4.5.2 Inflammatory cells Alveolar macrophages In sarcoidosis, BAL fluid contains approximately 60-80% alveolar macrophages (AMs). AMs are found in the lower airways were they play a key-role in the inflammatory process by engulfing pathogens or harmful particles [217]. Activated AMs secrete large amounts of IL-12, which induce Th1 immune responses [218]. In addition, AMs release high amounts of chemoattractant molecules, such as CXCL9 and CXCL10 that interact with the Th1 chemokine receptor CXCR3 and promote these cells to migrate and accumulate in the lung [219]. AMs also produce pro-inflammatory cytokines, including IL-1β, IL-6 and TNF, which contribute to the inflammatory process in the alveolar spaces. TNF is a key cytokine for the granuloma formation, and the level of TNF is significantly higher in patients with active disease [220], as well as in patients with progressing disease [221].

T cells Sarcoidosis is characterized by an accumulation of T cells, in particular CD4+ T cells, in the lungs but also at other sites of disease activity, such as lymph nodes, spleen and skin [213]. The accumulation of CD4+ T cells in the alveolar space, results in T cell alveolitis, i.e. inflammation in the air sacs of the lung, which give rise to an elevated BAL fluid CD4/CD8 ratio (usually above 3.5, as compared to a ratio of 2:1 in healthy subjects) [173].

The cytokine milieu, i.e. large amount of the macrophage-derived cytokines IL-12 and IL-18, induces the differentiation of naïve CD4+ T cells into active Th1 T cells [218, 222]. The Th1 predominance is further confirmed by increased expression of the chemokine receptors CXCR3 and CCR5, as well as decreased expression of the Th2 chemokine receptor CXCR4 [223]. In addition, there is also an increased spontaneous production of the Th1 cytokines IL-2 and IFNγ, both responsible for initiation of the sarcoidosis inflammatory process [224, 225].

Some patients develop lung fibrosis in the areas of granulomatous inflammation. Although the mechanisms are not fully understood, it is suggested that this is due to a switch from Th1 into Th2 phenotype, with production of Th2 cytokines IL-4 and IL-13 that stimulate extracellular matrix protein production and function as chemo-attractants for [226].

CD4+ TCR AV2S3+ T cells T cells involved in the immune response of sarcoidosis are mainly recruited from the periphery, however, there are studies indicating that a local proliferation takes place [227-230]. A so called lung-expansion of T cells, which express a unique T cell receptor (TCR) gene segment, supports the hypothesis that the sarcoid pathogenesis is an antigen driven process. Several studies reveal a restricted TCR usage by T cells in

27 Maria Wikén sarcoidosis. For example, an early study showed a preferential usage of TCR Vβ8 on T cells in BAL and blood of sarcoidosis patients [231]. Scandinavian sarcoidosis patients, presenting with haplotype HLA-DRB1*0301 almost always have a dramatic lung accumulation of CD4+ T cells expressing the TCR variable (V) gene segment AV2S3 (in some studies referred as Vα2.3) (>10.5% of total CD4+ T cells; for definition of what constitutes a T cell expansion, see [232]). Interestingly, this accumulation is normalized after recovery [233], and is also absent in patients with other inflammatory pulmonary conditions, e.g. allergic alveolitis or asthma.

The TCR AV2S3+ T cells are found to be more activated than other CD4+ T cells [234], indicating that they have recognized and proliferated in response to a particular antigen. In addition, the number of TCR AV2S3+ T cells in patients´ lung correlates with disease activity [233], and a higher number of TCR AV2S3+ T cells correlates with better prognosis [235]. However, TCR variable chain-usage analysis revealed that the α-chain could pair with different β-chains, so that the TCR AV2S3+ T cell population is oligoclonal, and not monoclonal. The TCR Vβ chains, most often associated with AV2S3, are Vβ7 or Vβ18 [236].

An accumulation of TCR AV2S3+ T cells is also seen in certain HLA-DR13+ patients [237, 238]. This was found to occur in those HLA-DR13+ patients who also carry the HLA-DRB3*0101 allele, which is structurally similar to the HLA-DRB1*0301 molecule and thus have the ability to present similar antigenic peptides [239] (Figure 7).

Figure 7. The HLA-DRB1*0301 and HLA-DRB3*0101 molecules can present identical antigenic peptides, resulting in a lung CD4+ TCR AV2S3+ T cell accumulation.

Oligoclonal T cell expansions have been demonstrated also in other disorders, such as Wegener’s granulomatosis, in which elevated numbers of circulating T cells with a restricted TCR-usage have been found [240], indicating triggering of a particular conventional antigen. Wegener’s granulomatosis is similar to sarcoidosis in that it is a granulomatous disease with unknown aetiology, and it may involve the lungs, kidney, heart, skin and eye. However, in contrast to sarcoidosis the granulomas seen in Wegener’s granulomatosis are necrotizing.

28 Introduction

B cells B cells are found to be activated in sarcoidosis, probably due to the increased cytokine production by T cells. As a result, an elevated secretion of auto-antibodies is seen, e.g. leading to formation of immune complexes that is associated with erythema nodosum [241].

1.4.5.3 Models for sarcoidosis Although the aetiology of sarcoidosis is unknown, attempts have been made to generate models that mimic disease with respect to the inflammatory cell profile and granulomatous inflammation.

Human model The Kveim extract, i.e. homogenates derived from spleen or lymph nodes of sarcoidosis patients, are characterized as being resistant to acid, heat and proteases [242]. Upon intradermal injection with Kveim extract in humans, well-shaped epithelioid granulomas that are indistinguishable from spontaneously formed granulomas, are found within 2-4 weeks [243].

Animal model Another attempt includes intravenous injection of sepharose beads coated with the mycobacterial protein mKatG (found in tissue of sarcoidosis patient, but not in healthy subjects [184]) in the tail-vein of rodents, which resulted in a granulomatous inflammation within a couple of days (D. Moller, personal communication). In addition, very recently, Swaisgood et al, developed a pulmonary sarcoidosis mouse model by using mycobacterial superoxide dismutase A (SodA) peptides isolated from granulomas of sarcoidosis patients, but not found in patients with tuberculosis [244]. Four days after challenge, granulomas were found in the mouse lung.

29 Maria Wikén 2 COMMENTS ON SUBJECTS & METHODS

All results presented in this thesis are based on human materials. The subjects included gave their written informed consent to participate, and the studies were performed after approval by the Regional Ethical Review Board. The corresponding papers will be referred to with roman numerals (I-IV).

2.1 STUDY SUBJECTS

Sarcoidosis patients (Papers I-IV) Sarcoidosis patients included are described in detail in each paper. The majority of patients were referred to the Respiratory Medicine Unit in Solna (Karolinska University Hospital, Stockholm, Sweden) for primary diagnostic investigation, whereas a minor number of patients (four non-Löfgren´s syndrome patients included in Paper III) had been followed for 3-20 years because of earlier being diagnosed with sarcoidosis. In addition, a minor number of patients (included in Paper III) were referred to the Respiratory Medicine Unit at Södersjukhuset, Stockholm, Sweden. The diagnosis was established using the criteria by the World Association of Sarcoidosis and other Granulomatous Disorders (WASOG) [165], including chest radiographic picture, pulmonary function tests (vital capacity, forced vital capacity, forced expiratory volume in one second, and diffusing capacity of the lung for carbon monoxide), histology (positive biopsies), bronchoalveolar lavage (BAL) findings, and clinical symptoms compatible with sarcoidosis.

The patients were subdivided in Papers I, III, IV into HLA-DRB1*0301+ (having an accumulation of BAL CD4+ TCR AV2S3+ T cells) and HLA-DRB1*0301-, or in Paper III into patients with or without Löfgren´s syndrome.

Healthy subjects (Papers I, II) Healthy subjects were included in Papers I and II. None of them were smokers, and none had signs of infections at time of BAL.

2.2 SAMPLING

Bronchoalveolar lavage (Papers I, III, IV) Patients underwent bronchoscopy with bronchoalveolar lavage (BAL) in the morning. Morphine-scopolamine was injected intramuscularly, and topical anesthesia was sprayed into the nose and throat immediately before the BAL procedure. BAL was performed during flexible fiberoptic bronchoscopy after wedging the bronchoscope in a middle-lobe segmental bronchus. Sterile phosphate-buffered saline (PBS) solution was instilled in five aliquots of 50 mL. The solution was gently aspirated, using a negative

30 Comments on Subjects & Methods pressure, into a plastic bottle and kept on ice until use. In the laboratory, the BAL fluid was filtered to remove debris and mucus and the recovered volume was determined. By centrifugation, BAL cells were separated from BAL fluid and counted in a Bürker chamber in order to obtain the total cell number. Trypan blue staining was used to assess cell viability.

The BAL cell differential count, including percentages of macrophages, lymphocytes, neutrophils, eosinophils, and mast cells, were determined using May- Grünwald-Giemsa staining of cytospin slides.

Peripheral blood mononuclear cells (Paper II) Whole blood was drawn in the same morning of bronchoscopy and collected in heparinized tubes. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Hypaque density gradient separation (Pharmacia, Uppsala, Sweden). Equal volumes of blood and room-tempered (RT) PBS solution (pH 7.4) were mixed and 8 ml was transferred to tubes containing 4 mL of RT Ficoll-PaqueTM Plus (> 0.12 EU/ml lymphocyte isolation sterile solution, Amersham Bioscience AB, Uppsala, Sweden). The tubes were centrifuged (400g for 25 min at RT) and the PBMCs formed a whitish layer just beneath the plasma. The PBMCs were carefully transferred into a new tube and 30 mL of cold PBS solution was added after which the mixture was centrifuged (400g for 10 minutes at 4ºC). The supernatant was removed, the cell pellet was re- suspended and diluted in 45 mL of cold PBS solution, and the centrifugation procedure was repeated. The obtained pellet was diluted in PBS solution to obtain a total volume of 5 mL. PBMCs were counted in a Bürker chamber in order to obtain the total cell number, as well as the number of lymphocytes/mL. Trypan blue staining was used to assess cell viability.

Whole blood (Papers III, IV) Whole blood was drawn in the morning, collected in heparinized tubes and kept in room-temperature until use (maximum one hour).

2.3 METHODOLOGY 2.3.1 Soluble proteins in supernatants

Concentration of BAL fluid supernatants (Paper I) In order to measure cytokines in BAL fluid, a concentration procedure was required. Frozen BAL supernatants (45 mL) were thawed and centrifuged (3939g for 20 min at 4°C) to discard mucus. Thereafter, 3×15 mL BAL supernatant was centrifuged (45 min) in 15 mL Amicon Ultra-filters (Millipore, Sweden), whereupon obtained volumes were centrifuged in 4-5 mL Amicon Ultra-filters (70 min). To concentrated samples with volumes less than 70 µL cold PBS solution was added, in order to obtain a final

31 Maria Wikén volume of 70 µL, required for CBA analysis (see Cytometric bead array, p. 32). By using our protocol, the BAL fluid was concentrated approximately 640X. The Amicon Ultra-filters give high recovery (>90%), and soluble molecules that are trapped in the filter have a molecular weight of >5 kD.

Cytometric bead array (Papers I, II) Cytometric bead array (CBA) (BD, Sweden) is a multiplexed bead assay, enabling measurement of several analytes, such as cytokines and chemokines, in one single serum, plasma, cell lysate or tissue culture supernatant sample. The technique involves the use of beads, with a diameter of 7.5 µm, internally labelled with different concentrations of a dye. When hit by a laser, these beads emit fluorescence with different intensities and can so be distinguished by their mean fluorescent intensity (MFI) value. Each bead serves as a capture bead through covalently coupled antibodies directed against a specific cytokine. Because of this specificity, the amounts of individual cytokines are measured simultaneously. Bound cytokine is detected using a PE-conjugated anti-cytokine antibody [245].

The concentrations of IFNγ, TNFα, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10 and IL- 12p70 were measured by using a 12 point standard curve (i.e. recombinant protein mix) (5 pg/ml – 5000 pg/mL, plus a negative control (i.e. without recombinant protein mix)). One standard curve was done for each experiment. Mixtures containing standard/concentrated BAL supernatant, capture bead mix and PE-conjugated detection antibody were prepared and incubated on shake at RT, protected from light exposure. After washing and re-suspension the samples were analysed using flow cytometry. Before each assay, optimal instrument settings for the FACSCalibur instrument (BD, Sweden) were determined.

2.3.2 Gene expression

Real-time PCR (Paper I) Real-time polymerase-chain-reaction (PCR) is the most sensitive technique for mRNA detection and quantification. Using PCR, short DNA sequences (usually 100-600 bases) can be exponentially amplified into large quantities within a few hours. Real- time-PCR means that this reaction progresses in real time, and the concentration of the PCR product is correlated to fluorescence intensity.

The number of PCR cycles required to obtain fluorescence intensity greater than background fluorescence is defined as the cycle threshold (Ct). Ct is inversely proportional to the amount of target nucleic acid in the sample, given that a greater quantity of target DNA in the starting material results in faster increase in fluorescence signal, yielding a lower Ct. The PCR reaction includes 40 cycles; Ct=35=1 DNA copy, Ct=37=0 DNA copy. In our experiments we set the Ct threshold to 36.

32 Comments on Subjects & Methods

Total RNA was isolated from BAL cells through the guanidium thiocyanate phenol- chloroform technique [246], using RNAbee (Nordic Biosite, Sweden), and cDNA was then synthesized using random hexamer primers, dNTP mix and RNAsin (Pharmacia Biotech, Sweden), SuperscriptTMII RNase H- Reverse transcriptase (Invitrogen, Sweden) and buffers. All procedures were according to the manufacturer´s protocol.

The mRNA expression of IFNγ, TNFα, IL-4, IL-10, IL-12p40 and TGFβ1 was quantified by TaqMan real-time PCR, using ABI Prism 7700 Sequence Detection System (Applied Biosystem, CA, USA), and the relative quantification of expression of genes was determined using the arithmetic formula 2-∆∆CT [247]. By using the 2-∆∆CT method, the expression of the target gene is presented as fold change normalized to an endogenous reference gene and relative to a control group (in our case non-treated healthy subjects). The expression of target gene was normalized to the housekeeping gene β-actin.

Choice of house-keeping gene A housekeeping gene is constitutively expressed in all human cells, and required for the maintenance of cellular function. However, some housekeeping genes are expressed at relatively constant levels, whereas other may vary depending on experimental conditions. To choose a reliable housekeeping gene when studying cells from the respiratory compartment is not easy, since only a few reference genes have been evaluated.

In the majority of studies, target genes have been normalized against GADPH or β- actin, and our choice in year 2005 (when all our experiments were done) became β- actin. However, in a recent study, Kriegova and colleagues investigated the mRNA expression by quantitative realtime-PCR of a range of housekeeping genes in BAL cells from sarcoidosis patients and healthy subjects [248]. It was found that the housekeeping genes PSMB2 and RPL32 were constantly expressed in BAL cells from both cohorts, irrespectively of gender, smoking, immune cell infiltration, Löfgren´s syndrome or multi-organ involvement. On the contrary, the expression of β-actin varied a little. Also illustrating variation in results depending on housekeeping genes, Kriegova et al further showed that there was a significant increase in IFNγ mRNA expression in sarcoidosis patients compared to healthy subjects when normalizing towards PSMB2, whereas β-actin normalization only gave rise to a tendency to higher expression [248].

2.3.3 In vitro stimulation

TLR and NOD PBMC stimulation (Paper II) After isolation from whole blood, PBMC were stimulated with the TLR2 ligands peptidoglycan (PGN; cell wall component of Gram positive bacteria) and Pam3Cys-

33 Maria Wikén

Ser-(Lys)4 (Pam3CSK4; synthetic agonist), the TLR4 ligand lipopolysaccharide (LPS; derived from Gram negative bacteria), and the NOD2 ligand muramyl dipeptide (MDP; component of PGN). Optimal concentrations were determined after testing a range of concentrations, and the chosen stimulation time of 16 hours was determined after testing cytokine concentration after a range of different time points. LPS is commonly used to in vitro stimulate TLR4, whereas PGN often is used for TLR2 stimulation. However, since there might be a risk of LPS contamination, the synthetic TLR2 ligand

Pam3CSK4 was used in addition. Supernatants of stimulated cells were collected and placed in -20°C until further use (CBA analysis).

Antigenic T cell stimulation (Paper III) During antigen stimulation, whole blood was co-stimulated with αCD28 and αCD49d. BAL cells and whole blood were thereafter in vitro stimulated with recombinant mycobacterium tuberculosis catalase-peroxidase (mKatG), purified protein derivative (PPD) or superantigens (Staphylococcus enterotoxin A (SEA) and Staphylococcus enterotoxin B (SEB)) in combination.

The recombinant mKatG protein was isolated and prepared at the John Hopkins University School of Medicine, using an Escherichia coli strain [249]. Each new mKatG batch was tested for purity, using western blot.

Purified protein derivative (PPD) is an extract of Mycobacterium tuberculosis and is a diagnostic tool for tuberculosis. PPD is extensively used for in vitro stimulation throughout the world, however, little is known about the active components. Yet, approximately 170 different proteins have been identified [250].

Superantigens SEA and SEB, produced by Staphylococcus aureus, were used as positive control for in vitro stimulation. Superantigens stimulate T cells by bridging the constant region of the MHC molecule on the APC and the Vβ region of the TCR, irrespective of the Dβ and Jβ regions, or the TCRα chain [251], thus in a physiological way of stimulating a large fraction of T cells (20-30%).

As negative control, BAL cells were incubated in media alone, and whole blood was kept unstimulated. After deduction of spontaneous cytokine production (cytokine production in BAL cells after incubation in media alone, or in unstimulated whole blood cells), some values ended up negative. This could depend on the random variation between different tubes of in vitro stimulated cells being larger than any antigen-induced cytokine production, in the cases where only few antigen-specific cells are present.

Brefeldin A (BFA, i.e. GolgiPlug; derived from Penicillum), is a protein transport inhibitor that was added to the cells during the last four hours of in vitro antigen stimulation, in order to block the intracellular protein transport process. The result is an

34 Comments on Subjects & Methods accumulation of cytokines and/or proteins inside the Golgi complex, and enables intracellular cytokine staining with monoclonal antibodies for analysis in the flow cytometer.

2.3.4 Flow cytometry

Flow cytometry, using a Fluorescence Activated Cell Sorter (FACSCalibur, with the capacity of three-color analysis; Papers I, II, or FACSCanto II, with the capacity of eight-color analysis; Papers III, IV; both purchased from BD, Sweden), were applied for measuring soluble proteins, for counting of cells, and for phenotypic characterization of cells. Color compensation was set for each T cell marker. In addition, for some experiments, a fluorescence minus one (FMO) control was performed. In each experiment, anti-human monoclonal antibodies were used (purchased from BD, Sweden, unless otherwise stated).

Routine BAL test For all patients, a routine flow cytometric test was performed to determine the BAL fluid CD4/CD8 T cell ratio, as well as the percentage of BAL TCR AV2S3+ of total CD4+ T cells. A BAL fluid CD4/CD8 ratio >3.5 was used to support the diagnosis (awaiting a positive biopsy), and patients with a BAL CD4+ TCR AV2S3+ T cell- frequency >10.5% (defined as 3 times median AV2S3% of CD4+ T cells in peripheral blood of healthy subjects, i.e. 3×3.5% [232]) was determined to have an AV2S3- expansion.

Analyzing TLRs (Papers II) After FACS gating on CD14+ monocytes, the mean fluorescence intensity (MFI), which is directly related to the quantitative number of receptors on a per-cell basis [252], of TLR2 and TLR4 was measured, after deduction of background, i.e. matched isotype controls.

Analyzing intracellular cytokines (Paper III) After antigen in vitro stimulation, the percentage of cells expressing intracellular IFNγ, TNF and IL-2 was measured, by successive gating on 1) lymphocytes, 2) CD3+ T cells, 3) CD4+ or CD8+ T cells, 4) CD4+ TCR AV2S3+ or AV2S3- T cells (only in HLA- DRB1*0301+ patients), and finally 5) the cytokine of interest. In addition, the median fluorescence intensity (MFI) of each cytokine was calculated for single IFNγ, single TNF, or single IL-2 production, as well as for production of simultaneous IFNγ and TNF production or simultaneous IFNγ and IL-2 production. Matched isotype controls were used for each cytokine.

35 Maria Wikén

Analyzing T cell surface markers (Paper IV) Unstimulated BAL or whole blood cells were stained with a range of different monoclonal antibodies, and thereafter successively gated on 1) lymphocytes, 2) CD3+ T cells, 3) CD4+ or CD8+ T cells, 4) CD4+ TCR AV2S3+ or AV2S3- T cells (only in HLA-DRB1*0301+ patients), and finally on the T cell markers of interest. Matched isotype controls were used for each marker.

Analyzing intracellularly expressed transcription factor FoxP3 (Paper IV) After staining for surface markers, cells were fixed, permeabilized and stained intracellularly for FoxP3 (clone PCH101-PE) and a matched isotype control (both purchased from eBioscience, San Diego, CA, USA).

2.4 STATISTICAL ANALYSIS

In all papers, data are assumed to be not normally distributed, and are therefore presented as median values. Analysis was performed using GraphPad Prism 4.03 (GraphPad Software Inc., San Diego, CA, USA) and a p value <0.05 was considered to be significant.

Paper I The non-parametric Mann-Whitney U-test was used for comparisons between patients and healthy subjects, or between patient subgroups. The Spearman´s rank correlation test was used for correlations between different parameters.

Paper II The non-parametric Mann-Whitney U-test was used for comparison between patients and healthy subjects. The non-parametric Wilcoxon´s signed rank test was used for within group comparisons.

Paper III The non-parametric Mann-Whitney U-test was used for comparison between patient subgroups. The non-parametric Wilcoxon´s signed rank test was used for comparisons between BAL and blood, or between various cell types in the same individual.

Paper IV The non-parametric Mann-Whitney U-test was used for comparison between patient subgroups. The non-parametric Wilcoxon´s signed rank test was used for comparisons between BAL and blood, or between various cell types in the same individual.

No correction was done for multiple comparisons.

36 Aims 3 AIMS

The general aim of this thesis was to study innate and adaptive immune responses in pulmonary sarcoidosis.

Specific aims:

I. Characterize Th1/Th2 and pro/anti-inflammatory cytokine patterns in the lungs of sarcoidosis patients, with focus on different patient subgroups, as well as in healthy subjects.

II. Determine the basal TLR2 and TLR4 expression on blood monocytes, as well as TLR expression and cytokine secretion after TLR and NOD2 in vitro stimulation, in sarcoidosis patients and healthy subjects.

III. Elucidate the ability of T cell subsets in lung and blood of different patient subgroups to respond with cytokine production on mycobacterial antigen stimulation, as well as determine whether these T cells displayed a single- or multifunctional cytokine profile.

IV. Phenotypically characterize T cells in lung and blood, with regard to markers for differentiation, activation and regulatory capacity, in different patient subgroups.

37 Maria Wikén 4 RESULTS & DISCUSSION 4.1 BAL FLUID CHARACTERISTICS

The BAL cell analysis revealed the following;

Total cell concentration was higher in patients than in healthy subjects (Paper I), higher in HLA-DR3- than in HLA-DR3+ patients (Paper I, IV), and similar between patients with or without Löfgren´s syndrome (Paper III).

Percentage of macrophages was lower in patients than in healthy subjects (Paper I), and the same in patient subgroups (Paper I, III, IV).

Percentage of lymphocytes was higher in patients than in healthy subjects (Paper I), and the same in patient subgroups (Paper I, III, IV).

BAL fluid CD4/CD8 ratio was the same between patient subgroups (Paper I, III, IV). BAL fluid TCR AV2S3% was higher in patients with Löfgren´s syndrome than in patients without Löfgren´syndrome (Paper III) and higher in HLA-DR3+ patients than in HLA-DR3- patient (Paper IV).

These characteristics are in agreement with those previously reported from our laboratory.

Cell viability was >92% for each study subgroup.

38 Results & Discussion

4.2 INNATE IMMUNITY IN SARCOIDOSIS

The aetiology of sarcoidosis is still unknown, although there is evidence for an infectious cause. DNA from Mycobacteria [253] and Propionibacteria [254] has been found in sarcoidosis tissue and lymph nodes, and among the first molecules involved in bacterial antigen recognition are the pattern-recognition receptors (PRRs), including TLRs and NODs. TLR2 and TLR4 have been proposed being involved in mycobacterial recognition on the cell surface and endosomally, whereas NOD2 is a suitable candidate for intracellular mycobacterial recognition [255, 256], although there are contradictory data [46, 257-260].

4.2.1 Role of TLR and NOD in sarcoidosis (Paper II)

Peripheral blood mononuclear cells (PBMC) were isolated from a total of 24 sarcoidosis patients (n=10 classified as HLA-DR3+, of which seven had Löfgren´s syndrome; n=11 classified as HLA-DR3-, of which none but one had Löfgren´s syndrome; n=3 not done) and 22 healthy subjects.

Higher monocytic TLR2 and TLR4 expression in sarcoidosis Using flow cytometry, the TLR expression on blood monocytes was measured as mean fluorescence intensity (MFI; reflecting the number of receptors expressed on a per-cell basis [252]). The monocytes were defined using forward scatter (FSC; cell size) and side scatter (SSC; cell granularity), as well as with a monoclonal CD14-Pe-Cy5 antibody. We found that the TLR2 and TLR4 expression at baseline was significantly higher on monocytes from patients compared to healthy subjects, and this difference was sustained after culture in medium for 16 hours, yet with an increase in MFI in both groups. One explanation for the increased TLR expression after incubation could be plastic adherence. It has been shown that genes encoding inflammatory cytokines can be induced upon adherence of monocytes to plastics [261, 262].

The TLR expression is modified in human diseases. For example, the surface expression of TLR2 and TLR4 was higher on CD14+ blood monocytes of patients with malaria than healthy subjects [263]. In addition, both TLR2 and TLR4 expression was strongly up-regulated in the intestinal epithelium of patients with inflammatory bowel disease when compared to controls [264, 265]. Mutations in certain TLRs have been shown to be linked to disease susceptibility, and the genetic variation may result in an altered immune response toward pathogenic challenging, and thus influencing the pathogenesis or outcome of disease. The higher TLR expression in sarcoidosis patients could be a result of genetic influence, but whether sarcoidosis is associated with genetic mutations in the TLR2 or TLR4 genes remains unclear, as different studies have shown conflicting results. In a Dutch cohort there was no association between TLR2 polymorphisms (Pro631His or Arg753Gln) and the whole group of sarcoidosis patients [203]. However, these polymorphisms were found to be crucial in patients with chronic

39 Maria Wikén diseases, as compared to patients with Löfgren´s syndrome or otherwise remitting disease. Additional supportive evidence for TLR2 being a susceptibility gene for sarcoidosis comes from studies showing that a TLR2 polymorphism (Arg753Gln) increases the risk of developing tuberculosis [266], as well as affecting disease phenotype in patients with inflammatory bowel disease [267]. TLR4 polymorphisms (Asp299Gly and The399Ile) leads to diminished airway response to inhaled LPS in healthy subjects [268], resulting in an increased risk for Gram-negative infections [269]. Pabst et al found a strong tendency of higher prevalence for mutations in a German cohort of sarcoidosis patients compared to healthy subjects [204]. When patients were subdivided into acute and chronic, a highly significant association was found with TLR4 polymorphisms and chronic disease. However, Veltkamp and colleagues showed no such association in a Dutch cohort, even though the allele frequency was almost the same in both study groups [205].

In addition to a possible genetic variation, the higher monocytic TLR expression in patients could be a result of environmental exposure to specific sarcoidosis antigens. It has been shown that children growing up on farms have a higher expression of TLR2 mRNA in blood cells as compared to non-farmer children [270], which the authors of that particular study suggest could explain the fact that farmers´ children are at decreased risk of developing allergies. In addition, Droemann et al showed that smokers had a lower TLR2 expression on their alveolar macrophages as compared to healthy non-smokers [271]. In our study, four of the patients were smokers and four were ex-smokers. Among healthy subjects, seven were ex-smokers. However, we believe that this bias would not affect our results, since Droemann´s study further showed that there was no difference in TLR2 or TLR4 expression on monocytes between smokers and non-smokers [271]. We did not observe any difference between non-smokers and smokers.

It has further been shown that cytokines, such as IFNγ and TNF, or corticosteroids enhance the mRNA expression of TLR2 in respiratory epithelial cells [272]. In a recent study it was found that the corticosteroid Budesonide, i.e. treatment for asthma and chronic obstructive pulmonary disease, in combination with LPS, TNF or swine dust, increased the TLR2 mRNA expression in activated bronchial epithelial cells [273]. TLR expression can also be modified by chronic infection. It was previously shown that the expression of TLR2 was up-regulated on monocytes from the pleural fluid of tuberculosis patients, when compared with peripheral blood mononuclear cells, indicating the involvement of this receptor at site of infection [274]. None in our study population had an ongoing infection. It may be speculated, however, that remaining proteins of the postulated infectious sarcoidosis agent is causing the up-regulation of TLR expression.

We did not find any differences in TLR expression between different subgroups of patients, indicating that the TLR expression per se may be involved in the development of sarcoidosis, yet have no impact on the clinical presentation.

40 Results & Discussion

Since the lung is the active site of disease, as well as a major site for TLR expression, we were interested to study TLR2 and TLR4 expression on alveolar macrophages of sarcoidosis patients and healthy subjects. However, due to high background staining we were unable to measure the TLR expression on alveolar macrophages with flow cytometry. Nevertheless, a low TLR expression on alveolar macrophages, when compared to the expression on monocytes, was found by others [271, 275]. Instead we measured the mRNA expression of TLR2 and TLR4 in FACS-sorted alveolar macrophages (gated on cell size). We found that the TLR2 mRNA expression was significantly lower in patients compared to healthy subjects, and this down-regulation was most pronounced in patients with Löfgren´s syndrome (Figure 8). However, also a low TLR expression could be sufficient to induce cytokine production. The TLR4 expression did not differ between groups. The contrasting results of TLR expression on monocytes and for mRNA in alveolar macrophages indicate that different maturation stages of the cells, as well as different microenvironments in the source tissues, may affect TLR expression differently.

TLR2 3.0 ** 2.5

2.0

1.5

1.0

0.5

Relative gene expression gene Relative 0.0 HC Löfgren Non-Löfgren

Figure 8. TLR2 mRNA expression in FACS-sorted alveolar macrophages of healthy controls (HC) and patients with or without Löfgren´s syndrome. The TLR2 expression was normalized to the housekeeping gene (β-actin), and the relative expression of TLR2 was calculated in relation to the mean values of TLR2 expression in the healthy control group. Horizontal lines depict median TLR2 expression. One-way Anova (Kruskal-Wallis), followed by Dunn’s post-test, ** p<0.01.

Altered TLR expression after TLR2, TLR4 and NOD2 stimulation in vitro Peripheral blood mononuclear cells (PBMC) of sarcoidosis patients and healthy subjects was stimulated for 16 hours with ligands for TLR2 (PGN or Pam3CSK4), TLR4 (LPS) or NOD2 (MDP). The TLR2 and TLR4 expression was thereafter measured. Compared to baseline, the TLR2 expression was increased after stimulation

41 Maria Wikén

with the TLR2 ligand Pam3CSK4, but not after stimulation with the TLR2 ligand PGN. The contradictory data could be a result of different ligand specificity. Pam3CSK4, a synthetic triacylated lipoprotein, binds the TLR2/TLR1 heterodimer [30], whereas PGN, derived from Staphylococcus aureus, binds the TLR2/TLR6 heterodimer [32]. Stimulation with the TLR4 ligand LPS up-regulated the TLR2 expression in both patients and healthy subjects, yet with no difference between groups. It is known that stimulation of one PRR can alter the expression of one other, and it has been postulated that this is a mechanism to fine-tune innate immune recognition of various pathogens. For example, LPS-stimulation of alveolar macrophages from healthy non-smokers increased both mRNA and protein expression of TLR2 [271].

Stimulation with the NOD2 ligand MDP, i.e. a component of PGN, gave rise to an up- regulated TLR2 expression, and a down-regulated TLR4 expression, in both patients and healthy subjects. Yet, the significant difference was sustained between the groups. Since NOD2 is a susceptibility gene for the granulomatous disorder Crohn´s disease [53, 206], and the two rare diseases Blau syndrome [54], and early-onset sarcoidosis [55], all with clinical features similar to sarcoidosis, its involvement in sarcoidosis has been an intriguing subject of several studies. Various studies have previously suggested that there are no associations between NOD2 mutations and sarcoidosis [276-278]. However, in a very recent study by Sato et al, it was shown that NOD2 polymorphisms in fact are associated with a severe course of pulmonary sarcoidosis [207].

Synergistic induction of cytokines after TLR2 and NOD2 stimulation in vitro We further elucidated whether functional differences could be found between patients and healthy subjects. After TLR and NOD stimulation, the protein concentrations of a range of selected cytokines, including TNFα, IL-1β, IL-6, IL-8, IL-10 and IL-12p70 in supernatants were analysed using cytometric bead array and flow cytometry. There were no differences between patients and healthy subjects in cytokine production after stimulation with individual TLR2, TLR4 or NOD2 ligands. However, combined TLR2 and NOD2 stimulation gave rise to a higher production of TNFα and IL-1β in patients. TNFα and IL-1β are both pro-inflammatory cytokines and important for initiation of inflammatory responses, as well as for the granuloma formation. In addition, TNFα and IL-1β are produced by cells within the granulomas [279, 280]. Interestingly, the combined TLR2 and NOD2 stimulation gave rise to a synergistic induction of cytokine production, with different cytokine patterns in patients and healthy subjects. Patients had a synergistic induction of IL-1β, whereas the immune regulatory IL-10 was synergistically induced in healthy subjects. This finding supports an involvement of NOD2 in the pathogenesis of sarcoidosis.

There is a suggested interplay between TLRs and NODs, in that intact PGN could signal through TLR2, and after uptake and release into the cytosol, the PGN- component MDP would signal through NOD2. In that way, different PRRs complement each other when recognizing PAMPs, with providing the immune system

42 Results & Discussion with an integrated defence mechanism against invading microbes. There are, however, conflicting results whether these receptors share signaling pathways, since contamination with LPS sometimes serves as a problem. However, several studies in humans and mice show that NOD2 stimulation can synergize with different TLRs, including TLR2, TLR3, TLR4 or TLR9. Netea and colleagues stimulated mononuclear cells of healthy subjects with MDP in combination with the TLR2 agonist Pam3Cys and found a synergistic induction of TNF, IL-1β and IL-10 [281]. In addition, Uehara et al demonstrated a synergistic effect of MDP with synthetic TLR2, TLR4 and TLR9 ligands [282].

It has, however, recently been shown that the synergistic effect is dose dependent [283]. When stimulating human monocytes with TLR ligands in combination with a low concentration of MDP (1-25 µg/mL), a positive synergistic effect on TNF production was seen, whereas this induction was lost when using a high MDP dose (100 µg/mL). MDP has been found in sarcoidosis granulomas [284], where it may function to activate macrophages [285, 286]. The actual MDP concentration within the granulomas is difficult to establish, but is has been shown that a MDP concentration of 1 up to 1000 µg/mL can induce multinucleated- formation from human monocytes [287], and thus our choice of MDP dose (2 µg/mL) can be considered as physiologically relevant. The different synergistic cytokine patterns we observed in patients compared to healthy subjects, in combination with the above mentioned TLR2 and NOD2 interaction, indicate that different PRRs might be involved and co-operate in the pathogenesis of sarcoidosis. There were no differences in cytokine secretion after stimulation between patient subgroups, again indicating that alterations in TLR and NOD2 pathways may be associated with a general susceptibility to sarcoidosis, but not to clinical phenotype of disease.

43 Maria Wikén

4.3 ADAPTIVE IMMUNITY IN SARCOIDOSIS

Although the underlying cause of sarcoidosis is unknown, T cells are known to play an essential role in mediating the inflammatory process. CD4+ T cells are accumulating in the airways, and these cells are highly active and produce large amounts of cytokines.

4.3.1 Cytokine profile in BAL fluid (Paper I)

Several studies indicate that there is an imbalance of T helper (Th)-1 and Th2 cytokines produced by alveolar cells, and this disproportion would affect the outcome of the pulmonary immune response seen in sarcoidosis [288, 289]. We therefore measured the gene expression and protein concentrations of Th1 (IFNγ, IL-2) and Th2 cytokines (IL- 4, IL-5, IL-6), as well as pro-inflammatory (TNFα, IL-1β, IL-6, IL-12) and anti- inflammatory cytokines (IL-10, TGFβ1), in BAL cells and BAL fluid with focus on different subgroups of patients, but also in healthy subjects.

A total of 40 sarcoidosis patients, all with active disease, participated in this study. For gene expression analysis, we included 12 HLA-DR3+ patients (all had acute onset and an accumulation of CD4+ TCR AV2S3+ BAL T cells, and eight had Löfgren´s syndrome), and 13 HLA-DR3- patients (12 had an insidious disease onset, none had an accumulation of CD4+ TCR AV2S3+ BAL T cells, and none but one had Löfgren´s syndrome). For protein expression analysis we included 14 HLA-DR3+ patients (all had acute onset and an accumulation of CD4+ TCR AV2S3+ BAL T cells, and 12 had Löfgren´s syndrome), and 20 HLA-DR3- patients (12 had an insidious disease onset, none had an accumulation of CD4+ TCR AV2S3+ BAL T cells, and none but one had Löfgren´s syndrome). In addition, 11 healthy subjects were included as a control group for both mRNA and protein analysis.

Th1 mediated immune response in sarcoidosis Our data showed that sarcoidosis patients had an elevated mRNA expression in total BAL cells of the Th1 cytokines IFNγ and IL-2, as compared to healthy subjects. This is in line with previous studies showing a spontaneous production of IFNγ and IL-2 from alveolar macrophages and lung T cells [218, 224, 225, 290-292]. The alveolar cells are also the major sources of mRNA of these cytokines in pulmonary tuberculosis [293]. In addition, the mRNA expression of the Th2 cytokine IL-4 was undetectable, which also is in line with previous studies showing very low or no expression of IL-4 [218, 290, 291].

There were no differences in gene expression between patients and controls regarding TNFα or IL-12p40, which was a bit surprising. TNFα is essential for granuloma formation, and an increased level in BAL fluid has been shown to correlate with prolonged disease course [288, 294]. In addition, IL-12 promotes naïve T cells to differentiate into Th1 cells, as well as induces these cells to produce IFNγ [295]. Our

44 Results & Discussion data was, however, in line with some studies [222, 296], whereas other studies showed the opposite [218, 297]. Discrepancies in results between studies could be due to variation in patient characteristics, including X-ray stage, smoking history and treatment, or be a result of different methodology.

Gene expression of IL-10 was elevated in patients, which is in line with previous studies [291, 296]. IL-10 is produced by regulatory T cells, and function to dampen immune responses, including mediating a down-regulation of TNFα and IL-12.

Furthermore, the protein concentration of the Th1 cytokine IL-2, and the pro- inflammatory cytokines TNFα, IL-1β, IL-6 and IL-12p70 was increased in BAL fluid of patients as compared to controls, and thus our result, concerning BAL fluid levels of TNFα and IL-12, differ as compared to the gene expression in BAL cells. However, mRNA and protein expression of cytokines do not always correlate, since the protein concentration could be affected by several parameters, such as synthesis and cleavage. The observed differences could be due to regulation on translational level, be a result of intracellular storage of cytokines, or be a result of that other cells, e.g. epithelial cells that are not collected during BAL, produce these cytokines [291].

Less pronounced Th1 mediated immune response in HLA-DR3+ patients The cytokine profile could influence the clinical course of disease, and since the HLA type has been shown to associate with prognosis, we were interested to study the cytokine patterns in HLA-DR3+ and HLA-DR3- patients.

We found that the mRNA levels of IFNγ and TNFα in total BAL cells was lower in HLA-DR3+ patients, as compared to HLA-DR3- patients, and that there were tendencies to higher levels of TGFβ1 in these patients. TGFβ1 acts, in the same way as IL-10, as an anti-inflammatory mediator and has been shown to correlate with good prognosis, possible by suppressing IL-2-producing T cells [298]. In addition, the HLA- DR3- patients had higher protein levels of TNFα, IL-2 and IL-12p70 compared to controls, whereas HLA-DR3+ patients did not.

The clinical features of pulmonary sarcoidosis can be divided into active and non-active stages, and earlier studies have been focusing on the cytokine profile in active disease [218, 299, 300]. Individuals with active disease have higher percentage of BAL cells expressing mRNA for IL-2, IL-10, IL-12 and IFNγ, as compared to non-active patients [291].

Less pronounced Th1 mediated immune response in Löfgren patients Worldwide, a subdivision of patients into those with or without Löfgren´s syndrome is more established than subdivision based on HLA type. For gene expression analysis, nine out of 25 patients were defined as having Löfgren´s syndrome, and for protein expression analysis, 13 out of 34 were defined as having Löfgren´s syndrome. Our data

45 Maria Wikén showed the same differences between patients with and without Löfgren´s syndrome, as when subdivided into HLA-DR3+ or HLA-DR3-, i.e. higher mRNA expression of IFNγ and TNFα in non-Löfgren´s syndrome patients. There was also a tendency of higher TGFβ1 mRNA expression in patients with Löfgren´s syndrome. New-found differences between patient subgroups included decreased protein levels in BAL fluid of TNFα, IL-2 and IL-6 in patients with Löfgren´s syndrome, which further support our findings of a diminished Th1 mediated response in the lungs of patients with favourable prognosis.

4.3.2 Role for mycobacterial proteins in sarcoidosis (Paper III)

The mycobacterial protein Mycobacterium tuberculosis catalase-peroxidase (mKatG) was identified in tissue from sarcoidosis patients, but not in healthy subjects [184]. mKatG is an important virulence factor for Mycobacteria that provides protection from harmful effects of peroxides, and also allows survival of mycobacterial organisms inside macrophages. As a poorly soluble tissue antigen, mKatG was found to have the capacity of triggering adaptive immune responses in patients [184]. We and others have previously shown that peripheral blood mononuclear cells (PBMCs), in a majority of Swedish [192] and U.S. [185, 192] patients, as well as PPD+ healthy subjects, respond to mKatG with IFNγ production, whereas PPD- healthy subjects do not. Therefore we were interested to further investigate the cellular response towards mKatG in distinct subgroups of sarcoidosis patients. In addition, we aimed to compare the T cell response from CD4+ and CD8+ T cells looking at more cytokines.

We divided our study population into HLA-DR3+ patients with Löfgren´s syndrome (n=13, all had acute disease onset and an accumulation of CD4+ TCR AV2S3+ BAL T cells), and HLA-DR3- patients without Löfgren´s syndrome (n=10, all had an insidious disease course and none had an accumulation of CD4+ TCR AV2S3+ BAL T cells). It has previously been shown that HLA-DR3+ patients, presenting with Löfgren´s syndrome, have the most favourable prognosis [164]. Therefore, our choice of patient subdivision enabled us to study clinically very distinct patient subgroups.

T cell responses towards mycobacterial proteins We chose to measure the production of IFNγ, TNF and IL-2. IFNγ and TNF are both effector cytokines important in bacterial clearance [301-303]. IL-2 has, on the other hand, little direct effector function, but promotes the expansion of CD4+ and CD8+ T cells, giving an amplification of effector T cell responses.

BAL and whole blood were stimulated with the mycobacterial proteins mKatG or purified protein derivative (PPD), or superantigens (SEA+SEB; positive control). Stimulation with mKatG resulted in a positive cytokine response in BAL and blood CD4+ T cells in patients with or without Löfgren´s syndrome (as compared to spontaneous cytokine production, i.e. in BAL cells cultured in media alone, or in un-

46 Results & Discussion stimulated whole blood cells). T cells play a vital role in the elimination of Mycobacterium tuberculosis by their production of IFNγ [302, 304-306]. As potent IFNγ-producers, CD4+ T cells have long been known to be essential effector cells for controlling mycobacterial infections [307]. This is also supported by the finding that humans with IFNγ receptor deficiency tended to have CD4+ T cells with reduced ability to kill mycobacterial-infected cells [308].

In addition, mKatG also stimulated the CD8+ T cells to IFNγ production. It has recently been shown that CD8+ T cells rapidly accumulate within mice lung tissue infected with Mycobacterium tuberculosis aerosols [309]. These CD8+ T cells were further shown to contribute to mycobacterial protection. That both CD4+ and CD8+ T cells respond to mKatG is in line with a previous study showing positive IFNγ response after mKatG stimulation in U.S. patients [192].

As compared to T cells in mice, human CD4+ and CD8+ T cells exhibit a considerable redundancy in effector mechanisms in that both subsets are capable to release potent macrophage activating cytokines, as well as are capable in utilizing the exocytose pathway [310, 311]. This suggests that CD8+ T cells not only have cytotoxic capacity. Our findings that CD8+ T cells exhibit a cytokine profile similar to CD4+ T cells after mKatG stimulation indicate that CD8+ T cells contribute to protective immunity against Mycobacterium tuberculosis infection by a combination of cytotoxic activity and cytokine production. CD8+ T cells are found within granulomas, where they function to prevent spreading of bacteria [312]. They recognize cytosolic antigens presented in the context of HLA class I-molecules, which are present on nearly all nucleated cells, with the result that CD8+ T cells can attack a broad range of target cells, and thus play a role in host defense against a variety of intracellular bacterial and viral pathogens. Although CD8+ T cells most commonly are in a minority among BAL cells from sarcoidosis patients, their relative proportion increases at later stages of disease. They may therefore play part in cytokine release during the alveolitis. Taken together, both CD4+ and CD8+ T cells play a vital role in mycobacterial clearance, which is also supported by other studies [305, 313].

When comparing the T cell reactivity towards mKatG in BAL and whole blood, we found that BAL CD4+ T cells possessed a higher cytokine expression, suggesting that mKatG-specific T cells are accumulating in the affected organ. In a previous study, looking at CD4+ T cells responses towards the Mycobacterium tuberculosis protein ESAT-6 in BAL and peripheral blood of Mycobacterium tuberculosis infected subjects, it was shown that in vitro stimulation resulted in an IFNγ-response of 1.81% of CD4+ T cells in BAL, but only 0.02% of CD4+ T cells in blood [314]. In addition, Chen et al performed flow cytometric analysis and found, in a few individuals, that mKatG or PPD stimulation resulted in higher IFNγ secretion from BAL CD4+ and CD8+ T cells, as compared to the CD4+ and CD8+ T cells in blood [192]. This is further supporting our finding of an enrichment of antigen-specific T cells in the affected organ.

47 Maria Wikén

Furthermore, it has been shown that there is a compartmentalization of cytokine production within the alveolar lymphocytes, resulting in higher cytokine secretion as compared to production from peripheral cells [292, 315]. It is, however, important to remember that the activation level of alveolar cells and peripheral cells differ, with the former being more activated.

Stimulation with PPD resulted in a positive IFNγ response only from CD4+ T cells, and not from CD8+ T cells, which indicates that there might be a selective recognition of the mycobacterial epitopes among T cells.

After mKatG or PPD stimulation, we did not observe any major differences between patient subgroups, which may seem to be in contrast to Paper I, where we saw a reduced Th1-associated profile, with decreased mRNA expression of IFNγ and TNF in HLA-DR3+ patients (and also in patients with Löfgren´s syndrome). However, explanations for such discrepancies could be that we in Paper I measured the cytokine mRNA expression in total BAL cells and in BAL fluid, which involves several cytokine-producing alveolar cells, whereas we in Paper III studied the capacity of T cells to respond to specific antigenic stimulation ex vivo. In addition, we used different techniques, i.e. realtime-PCR and cytometric bead array assay in Paper I, and intracellular cytokine staining in Paper III, that possess different sensitivity.

We also tried to measure the antigen-induced intracellular expression of IL-10, IL-17 and IL-22, however, the levels were undetectable with our experimental conditions.

Patients with Löfgren´s syndrome display a multifunctional T cell profile T cells are functionally heterogeneous and can exhibit multifunctional capacity, i.e. secrete two or more cytokines. It is known that IFNγ production is necessary when combating bacterial infections [301, 302], however TNF is also a potent effector cytokine [303, 316]. When combined, IFNγ and TNF synergize in their capacity to mediate effective killing [317, 318]. Forbes et al showed that T cells, secreting IFNγ, TNF and IL-2 simultaneously, were found to possess the best protection towards Mycobacterium tuberculosis [319].

Among CD4+ and CD8+ T cell subsets, we calculated the percentage of total mKatG- reactive cells that produced single IFNγ, single TNF, or IFNγ and TNF in combination (the same calculation was made for IFNγ in combination with IL-2). We found that mKatG stimulated the BAL CD4+ T cells to less single IFNγ production, but more simultaneous production of IFNγ and TNF, in patients with Löfgren´s syndrome as compared to non-Löfgren´s syndrome patients. In contrast, PPD stimulation gave rise to similar cytokine pattern in both patient subgroups. Taken together with the findings in Paper I, i.e. a less pronounced Th1 response in HLA-DR3+ patients, this could indicate that patients with a better prognosis have a more potent immune response towards a minor number of antigens, in which mKatG could be one of them, leading to

48 Results & Discussion pathogen elimination followed by recovery. We may speculate that patients with non- Löfgren´s syndrome exhibit an epitope spreading within the lungs, leading to involvement of more antigens and more IFNγ production with solid inflammation and prolonged disease.

In patients with Löfgren´s syndrome we also measured the median fluorescence intensity (MFI) of produced cytokines, i.e. the quantitative number of cytokine content on a per-cell basis [252]. We found that the multifunctional CD4+ T cells had the highest MFI values, suggesting that they produce more of the respective cytokine from each cell, compared to single cytokine-producing CD4+ T cells. This indicates that each cell has a more potent effector capacity with regard to each respective cytokine. In vaccine development it is of great importance to generate potent and durable T cell responses. The magnitude, i.e. frequency of T cells that are antigen specific, is most often used as a measurement [320]. The commonly used parameter to assess vaccine responses, and in terms of effector function, is the frequency of IFNγ-producing T cells, because of its role in clearance of bacterial, viral or fungal infections [301, 302, 321]. However, IFNγ-producing T cells are not sufficient in protection [322, 323], and in addition to IFNγ, TNF also has the function of killing intracellular infectious virus and bacteria. Combined production of IFNγ and TNF lead to enhanced killing of Mycobacterium tuberculosis [317, 318, 324], compared with either cytokine alone. There are various CD4+ T cell effector subtypes, ranging from early activated cells making only IL-2, to cells making only IFNγ, to multifunctional cells making IL-2, IFNγ and TNF, and the presence of these cells is associated with protection against for example Leishmania major [252]. In patients with tuberculosis [325], in vaccinated infants [326], as well as in individuals living in high infected areas [327], a great amount of multifunctional T cells have been found. Taken together, this indicates that a large production of different cytokines would benefit the protection against a postulated sarcoidosis pathogen, and could explain the good prognosis for patients with Löfgren´s syndrome.

CD4+ TCR AV2S3+ T cells respond to the mycobacterial protein mKatG One main focus in our research approach has been to characterize and evaluate the role of T cell receptor (TCR) AV2S3+ T cells, accumulating in the lungs (>10.5% of total CD4+ T cells) of HLA-DR3+ patients, i.e. patients usually with acute disease onset and good prognosis [237]. In an early study it was shown that in vitro stimulation with Mycobacterium tuberculosis, resulted in TCR AV2S3+ T cell proliferation among peripheral blood mononuclear cells (PBMCs) from Mycobacterium bovis BCG- vaccinated HLA-DR3+ healthy subjects [328]. Recently, using the ELISPOT technique, we found a correlation between the absolute number of TCR AV2S3+ T cells and the number of IFNγ-spots towards mKatG [192]. However, in that study, the antigen specificity of the TCR AV2S3+ T cells could not be determined. In the present work we show, for the first time, that the mycobacterial protein mKatG triggers a cytokine response in TCR AV2S3+, as well as AV2S3- CD4+ T cells of HLA-DR3+ patients.

49 Maria Wikén

Interestingly, the TCR AV2S3+ T cells responded to a significantly higher extent than the TCR AV2S3- T cells (median 0.65% vs. 0.48%, p=0.016). This indicates that mKatG is a specific disease-related antigen that triggers different subsets of T cells. It is important to keep in mind that mKatG is a large protein, approximately 700 amino acids, and likely contains several T cell epitopes with the capability to bind various TCRs. It is therefore not surprising that both TCR AV2S3+ and AV2S3- T cells can respond to the same protein.

Similar to what was found in BAL, the blood TCR AV2S3+ T cells also responded to mKatG, and to a higher extent than blood TCR AV2S3- T cells. This gives an indication that the TCR AV2S3+ T cells are re-circulating throughout the body. Support for re-circulating T cells comes from a study by Lehmann et al, showing that T cells can leave the lung, migrate across the alveolar epithelium, re-enter the lung tissue, and from the regional lymph node be distributed throughout the systemic circulation [329]. Alternatively, the sarcoidosis antigen that stimulates the TCR AV2S3+ T cells may be distributed systemically.

An intriguing question to ask is whether a cytokine response of approximately 0.5%, carried out by a particular T cell subset, is big or small. Mack et al recently reported that in patients with chronic beryllium disease (CBD), i.e. a granulomatous disease similar to sarcoidosis characterized by infiltration of beryllium-specific CD4+ T cells in the lungs, the frequency of beryllium-specific CD4+ T cells was around 2.3% in BAL and 1.5% in peripheral blood mononuclear cells [138]. The fraction of BAL CD4+ T cells in sarcoidosis, responding to mKatG, is smaller than what is seen with beryllium- stimulation in CBD. However, mKatG is likely only one of a number of mycobacterial antigens, although a dominant one [192].

It is essential to keep in mind that the TCR AV2S3+ T cell subset is not a T cell clone, but instead an oligoclonal T cell subset. We have previously shown that the variable (V) α-chain can bind to different Vβ-chains, yet with a preference for Vβ7 or Vβ18 [236]. Mallone et al revealed that different T cell clones can exhibit different avidity towards a given antigen [330], and that some clones only proliferated, whereas others both proliferated and produced cytokines, in response to a given concentration of the same antigen. One may hypothesize that some TCR AV2S3+ T cells are proliferating, whereas other exhibit effector functions, depending on the Vβ-usage. Oligoclonal T cells expansions, as that seen in sarcoidosis, have also been demonstrated in other inflammatory disorders. For example, in Wegener´s granulomatosis, an abnormal expansion of T cells, with particular TCR Vα- and Vβ-usage, have been shown [240].

We have previously shown that the number of BAL TCR AV2S3+ T cells correlates with disease activity [233], and furthermore that a higher number of TCR AV2S3+ T cells correlated with better prognosis [235]. We know from previous studies, using three-color flow cytometry, that TCR AV2S3+ T cells are more activated and more differentiated than the TCR AV2S3- T cells [237], and also that they are effector cells

50 Results & Discussion

[234] rather than FoxP3+ regulatory T cells, based on analysis of FoxP3 mRNA expression [331]. This was also illustrated by flow cytometry in Paper IV, i.e. among memory T cells there were much fewer FoxP3-expressing cells in the TCR AV2S3+ T cell subset, than in the TCR AV2S3- T cell subset. This support the TCR AV2S3+ T cells to be the “good guys” in disease, and one may speculate that they are associated with good prognosis and spontaneously resolving disease because of their ability to secrete effector cytokines upon mycobacterial stimulation.

Stimulation with superantigens gave rise to differences in cytokine response in blood, but not in BAL, i.e. blood TCR AV2S3+ T cells secreted more IFNγ, TNF and IL-2 as compared to blood TCR AV2S3- T cells. One explanation for this could be that there is different TCR Vβ usage among the TCR AV2S3+ T cells compared to the AV2S3- T cells in BAL and blood.

Diminished CD27 expression on BAL T cells CD27 is a co-stimulatory molecule expressed on naïve and memory T cells [135]. Antigen stimulation via the T cell receptor up-regulates the CD27 expression, which is followed by an irreversible loss upon repeated stimulation [136, 332, 333]. Our data showed that the T cells in BAL exhibited a CD27- phenotype, whereas the blood T cells were to higher extent CD27+, thus there was a more differentiated subset in the affected organ. In addition, the BAL CD27- T cells produced more IFNγ in response to mKatG, which has also been seen in patients with chronic beryllium disease (CBD) [138]. However, in blood the CD27+ T cells were the major cytokine producing cells.

4.3.3 T cell phenotyping (Paper IV) Phenotyping of total CD4+ and CD8+ T cells in BAL and blood T cells are key players in the immunological process of sarcoidosis. Patients are characterized by having an increased number of CD3+ CD4+ T cells in their lungs, leading to an elevated CD4/CD8 ratio [334, 335]. Although not fully diagnostic, this information can be clinically useful, since BAL T cells from healthy subjects typically display a lower CD4/CD8 ratio (2:1). As previously discussed, many parts of the pathogenesis are still unknown. However, by clarifying what distinct T cell subtypes are actually found in the lung and blood of patients, and furthermore, what differs between different patient subgroups, some immunological features could be described. We focused on markers for memory and effector T cells, regulatory T cells, as well as markers of T cell activation and differentiation in 11 HLA-DR3+ and 11 HLA-DR3- patients.

Memory and effector T cells Immunological memory is the result of clonal expansions and differentiation of antigen-specific cells that have the ability to persist for many years, or even during a

51 Maria Wikén whole life time. The maturation from human naïve T cells into central or effector memory T cells is associated with loss of the transmembrane molecule CD45RA and up-regulation of the CD45RO isoform [336]. In addition, the co-stimulatory molecule CD27 is expressed on naïve T cells, however, persistent antigenic stimulation results in an irreversible loss [136, 332, 333]. In a recent study, Okada et al showed that memory T cells that have lost the CD27 expression exhibited better effector function, i.e. produced more cytokines upon antigen stimulation, as compared to CD27+ memory T cells [337]. Using the combination of CD27 and CD45RO markers enabled us to study the most potent effector cells (CD27-CD45RO+, by us considered to be fully differentiated effector cells) (Figure 9). Other authors have previously used CD45RO in combination with the lymph node homing receptor CCR7, to define naïve CCR7+CD45RO- T cells, and so called CCR7+CD45RO+ central memory (CM) T cells and CCR7-CD45RO+ effector memory (EM) T cells. Since CCR7 is lost before CD27 during T cell differentiation, our CD27-CD45RO+ cells is a subset of EM [336].

CD27+ CD27+ CD27- CD27- CD45RO- CD45RO+ CD45RO+ CD45RO- (Naïve)

Figure 9. Schematic diagram of T cell differentiation, using CD27 and CD45RO expression.

Among BAL CD4+ T cells, the majority of cells in both HLA-DR3+ and HLA-DR3- patients, were CD27- (“fully differentiated effector”) memory T cells, followed by CD27+ memory T cells. In addition, only a minority were naïve CD27+CD45RO- T cells. In blood, however, the cell picture were quite different, with CD27+ memory T cells and naïve CD27+CD45RO- T cells being the dominant subsets, followed by a smaller frequency of CD27- (“fully differentiated effector”) memory T cells. These findings are in line with what was seen in patients with chronic beryllium disease [138], a granulomatous disorder very similar to sarcoidosis, illustrating that the affected organ has a the greater frequency of highly differentiated T cells. In BAL CD8+ T cells, the dominating subset was CD27+ memory T cells, followed by CD27- (“fully differentiated effector”) memory T cells, and then naïve CD27+CD45RO- T cells. This demonstrates that BAL CD4+ T cells are more differentiated as compared to the CD8+ T cells, which may indicate that CD4+ T cells, to a higher extent than CD8+ T cells, have encountered their antigen. Blood CD8+ T cells showed similar patterns as blood CD4+ T cells. There was no difference between patient subgroups, implying that the frequency of these T cell subsets do not have an impact on disease course or outcome.

52 Results & Discussion

HLA-DR3+ patients have fewer FoxP3+ regulatory T cells Regulatory T cells suppress proliferation and cytokine production of effector cells [338], prevent development of autoimmune diseases, such as type I diabetes [339], multiple sclerosis [340] or myasthenia gravis [341], and play a vital role in mediating tolerance towards transplants [342]. However, the role of regulatory T cells in sarcoidosis has not been clarified. A constitutive expression of CD25 was for long used as the marker for regulatory T cells. However, since activated T cells also express CD25, there were needs for a better regulatory T cell marker. The transcription factor FoxP3 was later found to be essential for regulatory T cell development and function [89, 90], and is to date considered to be the most reliable marker for regulatory T cells. Mice or humans with FoxP3 deficiency develop severe auto-immune disesase [96, 343].

Since a large frequency of CD25- T cells was positive for FoxP3, we decided to include all FoxP3+ cells when analyzing our regulatory T cells. We examined the FoxP3 expression in CD27+ and CD27- memory CD4+ T cell subsets, and found that HLA- DR3+ patients had fewer FoxP3-expressing cells among both CD27+ and CD27- memory CD4+ T cell subsets in BAL, as compared to HLA-DR3- patients. In Paper III we showed that the BAL CD4+ T cells of HLA-DR3+ patients exhibited a multifunctional cytokine profile, i.e. produced two cytokines simultaneously, upon stimulation with the mycobacterial protein mKatG. These results taken together suggest that the lower number of FoxP3+ regulatory T cells, in combination with multifunctional antigen-specific T cells, allow for a highly efficient immune response in HLA-DR3+ patients, and thereby elimination of the sarcoidosis antigens, leading to recovery within a couple of years.

Furthermore, the frequency of FoxP3-expressing CD27+ memory CD4+ T cells was higher in BAL as compared to blood. This is in line with what was previously reported in a study by our group looking at total CD4+ T cells [331]. In patients with active tuberculosis the highest number of FoxP3+ T cells is found in the pleura [344, 345], or in the lung or lymph node [346], as compared to the frequency in the circulation. In addition, patients with Crohn´s disease have the highest number of FoxP3+ T cells in the mucosal lymphoid tissues [347]. This implies that the regulatory T cells are accumulating at site of disease.

Since FoxP3+ T cells also have been found among the human CD8+ T cells [130-132], we further elucidated whether there were any FoxP3-expressing cells among the CD27+ and CD27- memory CD8+ T cells. Similar to what was demonstrated for BAL CD4+ T cells, the HLA-DR3+ patients also had fewer FoxP3+ T cells in the CD27+ memory CD8+ T cell subset. The frequency of FoxP3-expressing T cells among the CD27- T cells were too few to detect by using our experimental protocol. Since our data regarding FoxP3+ CD8+ T cells are based on the analysis of only few flow cytometric events in each experiment, because of relatively few cells expressing these combin-

53 Maria Wikén ations of markers, the results should be interpreted with causion. Further studies, looking at this T cell subset, would include larger number of cells.

A problem arises since T cell receptor-activation transiently can induce FoxP3 expression in both CD4+ and CD8+ T cells. Walker and colleagues induced the expression of FoxP3 by stimulating CD4+CD25- T cells, isolated from human peripheral blood, with anti-CD3 (T cell receptor stimulation) and anti-CD28 (co- stimulation) [92]. Billerbeck et al further showed that FoxP3+ CD8+ T cells could be induced by in vitro stimulating human peripheral blood mononuclear cells with hepatitis C virus [133]. Inducable FoxP3+ T cells do not with certainty possess immunosuppressive function [348, 349]. Since we have not performed any functional studies, we are unable to state whether the FoxP3+ T cells we find have regulatory capacity, and if so, to what extent.

Out of 22 sarcoidosis patients included in Paper IV, five were smokers (three smokers among the HLA-DR3+ patient subgroup and two smokers among the HLA-DR3- patient subgroup). Barceló et al previously showed that smoking could have an impact on the percentage of regulatory T cells, defined as CD4+CD25+ [350]. They demonstrated a significantly higher frequency of CD4+CD25+ regulatory T cells in BAL of healthy smokers, when compared to healthy non-smokers. The frequency in peripheral blood was not affected upon smoke. However, none of the smokers in our patient material tended to be out-liers.

HLA-DR3+ patients a have higher frequency of naïve CD25-CD27+ CD4+ BAL T cells In order to investigate the degree of T cell activation and differentiation, following antigenic stimulation, we combined the CD27 molecule with the early activation marker CD25. This resulted in the following T cell subsets, listed here to reflect the maturation from naïve T cells to fully activated and differentiated T cells; CD25-CD27+ cells, CD25+CD27+ cells, CD25-CD27- cells, and CD25+CD27- cells (Figure 10).

CD25- CD25+ CD25- CD25+ CD27+ CD27+ CD27- CD27- (Naïve)

Figure 10. Schematic diagram of T cell activation and differentiation, using CD25 and CD27 expression.

The dominating subset among BAL CD4+ T cells were the CD25-CD27- T cells, followed by naïve CD25-CD27+ T cells, and then CD25+CD27+ T cells. Our data further revealed that the HLA-DR3+ patients had a significantly higher frequency of naïve CD25-CD27+ CD4+ BAL T cells, as compared to HLA-DR3- patients.

54 Results & Discussion

The increased frequency of naïve T cells in HLA-DR3+ patients is intriguing and may explain our findings in Paper I, i.e. a reduced Th1 mediated response among the HLA- DR3+ patients. In blood, the majority of the CD4+ T cells were naïve CD25-CD27+ cells, followed by CD25-CD27- cells and then CD25+CD27+ cells. Or data indicate that the T cells are more activated and differentiated in the affected organ.

Since FoxP3 has an intracellular localization, and because there are no reliable reagents to be used for isolating live FoxP3+ cells, researchers have been trying to use combinations of surface markers in order to define these cells. Ruprecht et al previously showed that CD4+ T cells, obtained from synovial fluid of patients with juvenile idiopathic arthritis, co-expressing CD25 and CD27, were found to be more positive for FoxP3 than the CD25+CD27- T cells [139]. In eight patients we compared the frequency of FoxP3-expressing cells in the different T cell subsets. Our data revealed that the CD25+CD27+ CD4+ T cells expressed FoxP3 to the same extent as CD25+ CD4+ T cells (Figure 11), so the inclusion of CD27 gave no extra benefit for the identification of regulatory T cells in sarcoidosis.

BAL CD4 T cells 100

80

60

40 %FoxP3 20

0 + 7+ 27- 27- D25+ D2 D C CD27 C CD - + 25- CD25 CD CD25+ C CD25 Figure 11. Comparison of FoxP3 expression in different T cell subsets. The expression of FoxP3 among CD4+CD25+ T cells, as well as in different activated and differentiated T cell subsets, using CD25 and CD27 expression.

In the study of Ruprecht and colleagues it was further demonstrated that the CD25+CD27+ T cells had suppressor function, whereas the CD25+CD27- T cells did not [139]. However, Duggleby et al later found that also non-regulatory T cells express CD27 [351], so one should be careful to considered this molecule to be a distinct regulatory T cell marker.

In the BAL CD8+ T cell subset, the dominating cells were naïve CD25-CD27+ T cells, followed by the CD25-CD27- T cells. In blood, the majority was naïve CD25-CD27+ T cells, followed by CD25-CD27- T cells, and then CD25+CD27+ T cells.

55 Maria Wikén

Both CD4+ and CD8+ T cells require transient exposure to antigen for their induction of an antigen-dependent program of proliferation and differentiation. However, the strength and duration of antigen stimulation, as well as co-stimulation, may affect the differentiation process [352]. For example, naïve CD8+ T cells develop more quickly into effector cells after a short-term primary stimulation, as compared to CD4+ T cells. It is therefore not surprising that the CD4+ and CD8+ T cells exhibit different frequency of the various differentiated T cell subsets.

Within each T cell subset we examined the expression of the early activation marker CD69. Our data showed that the BAL T cells were much more activated than blood T cells. This is in line with what was previously revealed in a study from our laboratory, looking at total CD4+ T cells. There it was shown that BAL cells were more activated than peripheral blood cells (60-80% versus only a few percent) [234].

Phenotyping of CD4+ TCR AV2S3+ and AV2S3- T cells in BAL and blood As already been discussed (see CD4+ TCR AV2S3+ T cells respond to the mycobacterial protein mKatG, p 49), the CD4+ TCR AV2S3+ T cells that compartmentalize in the lungs of HLA-DR3+ patients, are of great interest. Previous studies from our laboratory have been using three-color flow cytometric analysis when studying these cells. So, this is the first time that we phenotype these cells, using a range of different markers characterizing different T cell subsets.

Memory and effector T cells Among both CD4+ TCR AV2S3+ and TCR AV2S3- BAL T cells, the dominating subset was CD27- (“fully differentiated effector”) memory T cells, followed by CD27+ memory T cells, and then naïve CD27+CD45RO- T cells. The TCR AV2S3+ T cells were to higher extent CD27- memory T cells, as compared to the TCR AV2S3- T cells, suggesting that the TCR AV2S3+ T cells are more differentiated, probably due to stimulation with the sarcoidosis antigen. In Paper III we showed that upon stimulation with the mycobacterial protein mKatG, the TCR AV2S3+ T cells produced effector cytokines, including IFNγ and TNF, to a significantly higher extent than the TCR AV2S3- T cells.

TCR AV2S3+ T cells of HLA-DR3+ patients are effector cells and not regulatory cells We then examined the FoxP3 expression in the CD27+ and CD27- memory T cell subsets of the TCR AV2S3+ and TCR AV2S3- T cells. Our data showed that there were much fewer FoxP3-expressing cells among CD27+ memory TCR AV2S3+ T cells, as compared to CD27+ memory TCR AV2S3- T cells. These findings confirms and extends what was previously indicated in three individuals [331], i.e. a lower frequency of FoxP3-expressing cells among TCR AV2S3+ T cells, as compared to TCR AV2S3- T cells. In that study, mainly using PCR analysis of sorted cells, it was also shown that the mRNA level of FoxP3 was lower in the TCR AV2S3+ T cell subset [331]. Taken

56 Results & Discussion together, this clearly demonstrates that the TCR AV2S3+ T cells are effector cells and not regulatory cells.

We also investigated whether there were any indications of antigen-specific regulatory T cells present. Our data showed that a fraction of the FoxP3-expressing CD27- (“fully differentiated effector”) memory T cell subset expressed TCR AV2S3. It has been suggested that regulatory T cells, similar to conventional T cells, requires specific antigen recognition via their T cell receptors, in order to obtain fully suppressive function. For example, in a mice model of type I diabetes, Tang et al recently showed that pancreatic auto-antigen-specific regulatory T cells, defined as CD4+CD25+, were more efficient to prevent disease than polyclonal regulatory T cells [353]. In addition, Tarbell and colleagues showed that auto-antigenic peptides presented by dentritic cells, induced CD4+CD25+ regulatory T cells with better suppressive function than polyclonal CD4+CD25+ T cells [354]. Altogether, our data suggest the existence of antigen-specific regulatory T cells in sarcoidosis, although these cells, if confirmed, only make up a small fraction of the total TCR AV2S3+ T cells. Further studies would include antigenic in vitro stimulation followed by analysis of regulatory T cell- associated cytokines, such as IL-10 and TGFβ.

TCR AV2S3+ T cells of HLA-DR3+ patients are highly activated and differentiated We further investigated the differentiation and activation of the TCR AV2S3+ and TCR AV2S3- T cells, by using CD25 and CD27 markers. Our data showed that the dominating subset in BAL, among both CD4+ TCR AV2S3+ and TCR AV2S3- T cells, were the fully differentiated effector CD25-CD27- T cells, followed by naïve CD25- CD27+ T cells, and then CD25+CD27+ T cells. In contrast, blood had higher frequencies of naïve CD25-CD27+ T cells, followed by fully differentiated effector CD25-CD27- T cells, and then CD25+CD27+ T cells. The frequency of CD25+CD27- T cells was very low in both compartments. According to previous studies from our laboratory, using three-color flow cytometric analysis, the TCR AV2S3+ T cells are less CD25+ and less CD27+, as compared to the TCR AV2S3- T cells [234]. This is, however, the first time we have had the possibility of studying CD25 and CD27 at the same time.

We also examined the degree of activation, by looking at the CD69 expression. Our data showed that both TCR AV2S3+ and TCR AV2S3- T cells in BAL were more activated than those in the blood, implying a higher degree of activation in affected organ. Katchar et al previously showed that the TCR AV2S3+ T cells are more activated than the the TCR AV2S3- T cells [234]. Overall, our present data indicate a more activated phenotype of the TCR AV2S3+ T cells.

By enumeration of these subsets, we also hope to provide foundation for future functional subsets.

57 Maria Wikén 5 CONCLUDING REMARKS

• Sarcoidosis patients displayed an enhanced Th1 associated cytokine profile in BAL, as compared to healthy subjects.

• HLA-DRB1*0301+ (HLA-DR3+) patients had a reduced Th1 associated cytokine profile in BAL, as compared to HLA-DR3- patients, which could be related to their good prognosis.

• Blood monocytes of patients had higher baseline TLR2 and TLR4 expression as compared to healthy subjects, which could have consequences for host-pathogen interaction.

• Stimulation PBMC with TLR2 and NOD2 ligands in combination resulted in a remarkable increase of the granuloma-associated cytokines, IL-1β and TNF, in patients but not in healthy subjects, indicating interplay, as well as a role for these PRRs in sarcoidosis.

• Total BAL CD4+ T cells of patients with Löfgren´s syndrome (including HLA-DR3+ patients) had a more pronounced multifunctional cytokine profile, i.e. simultaneous production two cytokines (IFNγ and TNF), after stimulation with the mycobacterial antigen mKatG, whereas total BAL CD4+ T cells of non-Löfgren patients exhibited a single- functional cytokine profile (IFNγ alone). This indicates that the quality of T cell responses is of importance for the clinical presentation and disease outcome.

• Multifunctional BAL CD4+ T cells had higher median fluorescence intensity (MFI) values, as compared to single cytokine-producing BAL CD4+ T cells, suggesting that single- and multifunctional T cells have distinct abilities to act against a postulated sarcoidosis pathogen.

• BAL and blood TCR AV2S3+ T cells of HLA-DR3+ patients produced more effector cytokines in response to mKatG, as compared to TCR AV2S3- T cells, whereas the opposite was seen in BAL after stimulation with PPD. This, together with our previous knowledge, i.e. HLA-DR3+ patients have good prognosis, implies that the role of TCR AV2S3+ T cells is to eliminate an offending pathogen. This is the first time we show that a specific antigen stimulate the TCR AV2S3+ T cells.

• HLA-DR3+ patients had a lower frequency of FoxP3+ regulatory T cells among their BAL memory T cells, as compared to HLA-DR3- patients, suggesting that HLA-DR3+ patients, in some parts of their immune response, have a reduced suppression of Th1 immune responses.

• HLA-DR3+ patients had a higher frequency of naïve T cells in BAL, as compared to HLA- DR3- patients, implying that the T cells are less activated in HLA-DR3+ patients, which could explain the finding of a less pronounced Th1 response in the lungs of these patients.

• Memory BAL TCR AV2S3+ T cells of HLA-DR3+ patients displayed an effector T cell phenotype rather than a regulatory T cell phenotype. BAL and blood TCR AV2S3+ T cells were more activated and differentiated than TCR AV2S3- T cells, suggesting that they have encountered a postulated sarcoidosis pathogen.

58 Summary Figure 6 SUMMARY FIGURE

Figure 12. A summary of the main findings presented in this thesis.

59 Maria Wikén 7 FUTURE PERSPECTIVE

Listed below are things that I think would be of great interst to further looking into;

• Immunohistochemical analysis of sarcoidosis granulomas in order to measure the expression of TLR2 and TLR4, but also TLR9 (which has been shown to play an essential role in mycobacterial-induced pulmonary granulomatous responses in mice).

• Immunohistochemical analysis of sarcoidosis granulomas, in search for CD4+ TCR AV2S3+ T cells.

• Perform epitope mapping of the whole mKatG protein, in the context of different HLA-DR molecules, in search for novel T cell antigenic peptides to be used for functional analysis. The ultimate goal would be to use such epitopes in vaccine development.

• Elucidate whether regulatory T cells respond to mKatG, as well as other candidate antigens.

• Using fluorescence microscopy to elucidate whether T cells within the sarcoidosis granulomas exhibit a single- or multifunctional cytokine profile, and further compare patient subgroups.

• Assuming that multifunctional T cells are the ones to prefer when combating the sarcoidosis pathogen; isolate multifunctional T cells (however, this would require a surface marker), and inject them into mice having the hallmarks of sarcoidosis (granulomas, T cell infiltration). If the multifunctional T cells are found to be efficient in “curing” the mouse, these cells should be considered as targets when designing antigen-specific therapies. The idea would then be to treat patients with chronic disease course.

60 Acknowledgements 8 ACKNOWLEDGEMENTS

I would like to give my warmest gratitude to colleagues, friends and family for all help and support during the years I have been working on this thesis;

My main supervisor Jan Wahlström, thank you for an amazing journey through all fields of immunology! Thank you for always being so enthusiastic about each and every result and for being a biobank of scientific knowledge. This has been the time of my life and you are truly a source of inspiration.

My co-supervisors; Johan Grunewald, thank you for your big scientific and clinical input during all these years, and for always taking everything to a higher level. Anders Eklund, I am very greatful for your never-ending personal encouragement, for strengthening me as a person, and for always taking time to share your great knowledge in medicine.

My co-authors; Farah Idali, Mahyar Ostadkarampour, Håkan Mellstedt, Hodjattallah Rabbani, Matthew Willett, Edward Chen and David Moller, thank you for sharing your knowledge in science and experimental setups, and for all good discussions.

My colleagues at the lung clinic; in particular, the wonderful research nurses, Heléne Blomqvist, Margitha Dahl and Gunnel de Forest, thank you for your great support, for always being so positive and kind, and for spreading a cheerful atmosphere wherever we are in the world! Emma Karlsson, thank you for help with tricky papers and other stuff, and for all nice chats!

Berit Olsson, thank you for fun times in the FACS room!

My family at Lung lab, present and former members; Thank you for unforgettable years! Charlotta, thank you for all the fun we have had around the world, for always being so helpful, and for all supportive words – it really means a lot! Micke, thank you for all great times at conferences and holidays! Please, come back to Stockholm – I miss you! Abraham, thank you for being my great bicycle-buddy back home from work, and thanks for all crazy times – you really make me laugh (a lot)! Betti, thank you for all fun times at work and outside, and for two amazing trips to my Paradise on Earth – Itacaré♥Brazil! Marija, thank you for being so sensible about things that spin around in my head, and for always having time for a supportive hug! Helena, thank you for being so nice to hang out with, and for always being helpful and kind! Kie, thank you for very good company during late evenings in the lab, and for always being so kind and supportive! Benita E, thank you for all inspiring chats about exercise and other enjoyable things in life! Lotta and Benita D, thank you for very nice company in the

61 Maria Wikén lab! Mahyar, thank you for really good team work! Muntasir, thank you for being such a generous person, and for always having a happy smile! Farah, thank you for everything you have thought me in the lab, and for being such a kind person! Ulrika, thank you for all fun lunches and chats! Caroline, thank you for all help with tricky immunohistochemistry slides, and for always being so nice to talk to! Åsa, Daniel, Pernilla, Ernesto, Magnus S, AnnSofi, Adrian, Lukas, Cecilia, Malin, Tove, Karin, Marianne, Magnus N, Longping, Luca and Magnus L, thank you for making Lung lab the great place to be!

My friends outside Lung lab;

My Lovely Jenny, thank you for lightening up my days by sending parcels, all the way from the U.K., with my favourite liquorice (mumsfilibaba)! You are really great! Mark, you are such a nice person, and little Sam – I am really looking forward to finally meet you!

Ying, Susanne, Hanna, Johanna, Maria L, Andreas, Monika and Jill, thank you for making my holidays and weekends so great!

My singing birds; Sandra, Malin, Cecilia, Kajsa and Camilla, thank you for all lovely tunes and fun times!

Victor C, thank you for your great pep-talk – that was exactly what I needed!

My Kickboxing friends; Fredrik, thank you for all great fighting and big support – you are a true energy source! Diana, thank you for all good times - yummy coffees, music and cheery chats!

Maggie, thank you for being an amazing person, for teaching me about life, and for all big laughs!

Jossan, thank you for being my friend for almost all my life! Now it´s time for lots of catch-up fika! And Leon, you are such a charmer!

Helga, thank you for being a really good friend for all these years! Your big support, all fika and chats, trips around the world, and much, much more, have been the very best!

Maria B, thank you for being such a great friend in each and every way - what would I do without you?! Also thanks to Per, Anna and my favourite little Lydia for very nice gatherings!

62 Acknowledgements

Eva , the best piano teacher ever ♫ Thank you for your great support, for all good coffee and fika, and for all nice discussions about everything! I promise you, I will become a better student from now on ☺!

AnnMari and Kristina, thank you for invaluable support.

My family;

Ylva, my cousin and friend, thank you for all good times!

Bettan, thank you for being like an extra-mummy – you are a wonderful aunt! Nikolai and Aeneas, thank you for all nice gatherings and dinners during my 28 years in life!

Kristoffer, även om det har varit många mil emellan oss har du hela tiden haft en särskild plats i mitt hjärta. Tack för att du finns.

Simba, världens finaste katt, tack för att du hållit mig sällskap när jag suttit och skrivit om nätterna!

Victor, min ögonsten och lillebror – du förgyller verkligen mitt liv med bus och galna upptåg! En jättestor kram till dig för att du hjälpte mig med framsidan till denna bok, och TACK för att du är en toppenbror på alla sätt och vis!

Mamma och pappa, tack för att ni alltid finns vid min sida. Ni betyder verkligen allt för mig!

______

The work in the present thesis was supported by grants from the Swedish Heart-Lung Foundation, King Oscar II Jubilee Foundation, the Swedish Research Council, the U.S. National Institutes of Health, the Mats Kleberg Foundation, Söderberg Foundation, the Stockholm County Council, and Karolinska Institutet.

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